Phillip J. Robinson

A newly discovered protein export machine in malaria parasites

Publisher: Springer Science and Business Media LLC

Date: 06-2009

DOI: 10.1038/NATURE08104

Altered protein phosphorylation in intact rat cortical synaptosomes after in vivo administration of fluphenazine

Publisher: Elsevier BV

Date: 07-1987

DOI: 10.1016/0006-2952(87)90151-1

Abstract: Phenothiazines such as fluphenazine are able to inhibit calcium-stimulated protein kinases in vitro in both lysed and intact synaptosomes. In this study protein phosphorylation was assayed in intact synaptosomes isolated from the cerebral cortex of rats treated chronically (21 days, 10 mg/kg, i.p.) or acutely (1 hr, 10 mg/kg, i.p.) with fluphenazine. When intact synaptosomes from chronically treated animals were prelabeled with 32Pi, there were two effects on protein phosphorylation: an increase in the basal labeling of many phosphoproteins and a decrease in depolarization-evoked protein phosphorylation. Acute injections had even more pronounced effects, but the direction and nature of the effects were the same. No effects on K+-stimulated calcium entry or on protein phosphatase activity were detected. When lysed synaptosomes from chronically treated animals were labeled in the presence of [gamma-32P]ATP, a small decrease in calmodulin-dependent and cAMP-dependent protein phosphorylation was observed. The results suggest that two different in vivo mechanisms may underlie these effects, and these are discussed. We proposed that intact synaptosomes may be a good model in which to study the in vivo mechanisms of the action of fluphenazine since they appear to retain at least some effects of the drug after subcellular fractionation.

SH3 Domains Differentially Stimulate Distinct Dynamin I Assembly Modes and G Domain Activity

Publisher: Public Library of Science (PLoS)

Date: 10-12-2015

DOI: 10.1371/JOURNAL.PONE.0144609

Dynamin

Publisher: Elsevier

Date: 2009

DOI: 10.1016/B978-008045046-9.01363-2

Synapsin I-associated phosphatidylinositol 3-kinase mediates synaptic vesicle delivery to the readily releasable pool.

Publisher: Elsevier BV

Date: 08-2003

DOI: 10.1074/JBC.M302386200

Syndapin I and endophilin I bind overlapping proline-rich regions of dynamin I: role in synaptic vesicle endocytosis

Publisher: Wiley

Date: 12-03-2007

DOI: 10.1111/J.1471-4159.2007.04574.X

Abstract: Dynamin I mediates vesicle fission during synaptic vesicle endocytosis (SVE). Its proline-rich domain (PRD) binds the Src-homology 3 (SH3) domain of a subset of proteins that can deform membranes. Syndapin I, hiphysin I, and endophilin I are its major partners implicated in SVE. Syndapin binding is controlled by phosphorylation at Ser-774 and Ser-778 in the dynamin phospho-box. We now define syndapin and endophilin-binding sites by peptide competition and site-directed mutagenesis. Both bound the same region of the dynamin PRD and both exhibited unusual bidirectional binding modes around core PxxP motifs, unlike hiphysin which employed a class II binding mode. Endophilin binds to tandem PxxP motifs in the sequence (778)SPTPQRRAPAVPPARPGSR(796) in dynamin, with SPTPQ being an overhang sequence. In contrast, syndapin binding involves two components in the region (772)RRSPTSSPTPQRRAPAVPPARPGSR(796). It required a single PxxP core and a non-PxxP N-terminally anchored extension which bridges the phospho-box and may contribute to binding specificity and affinity. Syndapin binding is exquisitely sensitive to the introduction of negative charges almost anywhere along this region, explaining why it is a highly tuned phospho-sensor. Over-expression of dynamin point mutants that fail to bind syndapin or endophilin inhibit SVE in cultured neurons. Due to overlapping binding sites the interactions between dynamin and syndapin or endophilin were mutually exclusive. Because syndapin acts as a phospho-sensor, this supports its role in depolarization-induced SVE at the synapse, which involves dynamin dephosphorylation. We propose syndapin and endophilin function either at different stages during SVE or in mechanistically distinct types of SVE.

Mechanisms of calcineurin inhibitor-induced neurotoxicity

Publisher: Elsevier BV

Date: 2006

DOI: 10.1016/J.TRRE.2006.02.005

An integrated flow and microwave approach to a broad spectrum protein kinase inhibitor

Publisher: Royal Society of Chemistry (RSC)

Date: 2015

DOI: 10.1039/C5RA09426G

Abstract: The protein kinase inhibitor CTx-0152960 and the piperazinyl analogue CTx-0294885 were prepared using a hybrid flow and microwave approach.

A Cholesterol‐Dependent Endocytic Mechanism Generates Midbody Tubules During Cytokinesis

Publisher: Wiley

Date: 23-09-2015

DOI: 10.1111/TRA.12328

Abstract: Cytokinesis is the final stage of cell ision and produces two independent daughter cells. Vesicles derived from internal membrane stores, such as the Golgi, lysosomes, and early and recycling endosomes accumulate at the intracellular bridge (ICB) during cytokinesis. Here, we use electron tomography to show that many ICB vesicles are not independent but connected, forming a newly described ICB vesicular structure - narrow tubules that are often branched. These 'midbody tubules' labelled with horseradish peroxidase (HRP) within 10 min after addition to the surrounding medium demonstrating that they are derived from endocytosis. HRP-labelled vesicles and tubules were observed at the rim of the ICB after only 1 min, suggesting that midbody tubules are likely to be generated by local endocytosis occurring at the ICB rim. Indeed, at least one tubule was open to the extracellular space, indicative of a local origin within the ICB. Inhibition of cholesterol-dependent endocytosis by exposure to methyl-β-cyclodextrin and filipin reduced formation of HRP-labelled midbody tubules, and induced multinucleation following ICB formation. In contrast, dynamin inhibitors, which block clathrin-mediated endocytosis, induced multinucleation but had no effect on the formation of HRP-labelled midbody tubules. Therefore, our data reveal the existence of a cholesterol-dependent endocytic pathway occurring locally at the ICB, which contributes to the accumulation of vesicles and tubules that contribute to the completion of cytokinesis.

Chapter 22 Depolarization-dependent protein phosphorylation in synaptosomes: mechanisms and significance

Publisher: Elsevier

Date: 1986

DOI: 10.1016/S0079-6123(08)61065-1

Abstract: There are multiple genetic disorders with known or unknown etiology grouped under obesity syndromes. Inspite of having multisystem involvement and often having a characteristic presentation, the understanding of the genetic causes in the majority of these syndromes is still lacking. The common obesity syndromes are Bardet-Biedl, Prader-Willi, Alstrom, Albright's hereditary osteodystrophy, Carpenter, Rubinstein-Taybi, Fragile X, and Börjeson-Forssman-Lehman syndrome. The list is ever increasing as new syndromes are being added to it. One of the recent additions is MOMO syndrome, with about five such cases being reported in literature. Expanding the spectrum of clinical features, we report the first case of MOMO syndrome from India with lobar variant of holoprosencephaly and cryptorchidism, which have not been reported previously.

Protein Composition of Catalytically Active Human Telomerase from Immortal Cells

Publisher: American Association for the Advancement of Science (AAAS)

Date: 30-03-2007

DOI: 10.1126/SCIENCE.1138596

Abstract: Telomerase is a ribonucleoprotein enzyme complex that adds 5′-TTAGGG-3′ repeats onto the ends of human chromosomes, providing a telomere maintenance mechanism for ∼90% of human cancers. We have purified human telomerase ∼10 8 -fold, with the final elution dependent on the enzyme's ability to catalyze nucleotide addition onto a DNA oligonucleotide of telomeric sequence, thereby providing specificity for catalytically active telomerase. Mass spectrometric sequencing of the protein components and molecular size determination indicated an enzyme composition of two molecules each of telomerase reverse transcriptase, telomerase RNA, and dyskerin.

Differential Phosphorylation of Dynamin I Isoforms in Subcellular Compartments Demonstrates the Hidden Complexity of Phosphoproteomes

Publisher: American Chemical Society (ACS)

Date: 07-07-2010

DOI: 10.1021/PR100223N

Abstract: Large-scale comparative phosphoproteomics studies have frequently been done on whole cells or organs by conventional bottom-up mass spectrometry approaches, that is, at the phosphopeptide level. Using this approach, there is no way to know which protein isoforms the phosphopeptide signal originated from. Also, as a consequence of the scale of these studies, important information on the localization of phosphorylation sites in subcellular compartments is not surveyed. As a case study, we investigated whether the isoforms of dynamin I (dynI), at the whole brain and subcellular level, had differential phosphorylation. We first established that the dynI isoforms xa, xb, and xd were expressed in nerve terminals. Our investigation revealed that dynI xa was constitutively phosphorylated to a higher extent than the other isoforms despite identical sequences in the phosphorylated subdomains. DynI xa had a 10-fold higher stoichiometry of diphosphorylation at Ser-774 and Ser-778 than dynI xb and xd combined. Diphosphorylation was 2-fold enriched in nerve terminals relative to whole brain and was preferentially targeted for stimulus-dependent dephosphorylation. Phospho-Ser-851 and Ser-857 were depleted from nerve terminals. Our data reveals major differential phosphorylation of dynI phosphosites in different variants and in different neuronal compartments that would be completely imperceptible to a large-scale phosphoproteomics approach.

Development of quinone analogues as dynamin GTPase inhibitors

Publisher: Elsevier BV

Date: 10-2014

DOI: 10.1016/J.EJMECH.2014.06.070

Abstract: Virtual screening of the ChemDiversity and ChemBridge compound databases against dynamin I (dynI) GTPase activity identified 2,5-bis-(benzylamino)-1,4-benzoquinone 1 as a 273 ± 106 μM inhibitor. In silico lead optimization and focused library-led synthesis resulted in the development of four discrete benzoquinone/naphthoquinone based compound libraries comprising 54 compounds in total. Sixteen analogues were more potent than lead 1, with 2,5-bis-(4-hydroxyanilino)-1,4-benzoquinone (45) and 2,5-bis(4-carboxyanilino)-1,4-benzoquinone (49) the most active with IC50 values of 11.1 ± 3.6 and 10.6 ± 1.6 μM respectively. Molecular modelling suggested a number of hydrogen bonding and hydrophobic interactions were involved in stabilization of 49 within the dynI GTP binding site. Six of the most active inhibitors were evaluated for potential inhibition of clathrin-mediated endocytosis (CME). Quinone 45 was the most effective CME inhibitor with an IC50(CME) of 36 ± 16 μM.

Role of the Clathrin Terminal Domain in Regulating Coated Pit Dynamics Revealed by Small Molecule Inhibition

Publisher: Elsevier BV

Date: 08-2011

DOI: 10.1016/J.CELL.2011.06.025

Abstract: Clathrin-mediated endocytosis (CME) regulates many cell physiological processes such as the internalization of growth factors and receptors, entry of pathogens, and synaptic transmission. Within the endocytic network, clathrin functions as a central organizing platform for coated pit assembly and dissociation via its terminal domain (TD). We report the design and synthesis of two compounds named pitstops that selectively block endocytic ligand association with the clathrin TD as confirmed by X-ray crystallography. Pitstop-induced inhibition of clathrin TD function acutely interferes with receptor-mediated endocytosis, entry of HIV, and synaptic vesicle recycling. Endocytosis inhibition is caused by a dramatic increase in the lifetimes of clathrin coat components, including FCHo, clathrin, and dynamin, suggesting that the clathrin TD regulates coated pit dynamics. Pitstops provide new tools to address clathrin function in cell physiology with potential applications as inhibitors of virus and pathogen entry and as modulators of cell signaling.

The dephosphins: dephosphorylation by calcineurin triggers synaptic vesicle endocytosis.

Publisher: Elsevier BV

Date: 11-2001

DOI: 10.1016/S0166-2236(00)01930-5

Abstract: When nerve terminals in the brain are stimulated, a group of phosphoproteins called the dephosphins are coordinately dephosphorylated by calcineurin, the Ca(2+)-dependent protein phosphatase. Amazingly, the seven presently known dephosphins are not structurally related, yet each has been independently shown to be essential for synaptic vesicle endocytosis (SVE). Nowhere else in biology is there a similar ex le of the coordinated dephosphorylation of such a large group of proteins each sharing roles in the same biological response. This suggests that dephosphorylation and phosphorylation of the dephosphins is essential for SVE. Recent studies in synaptosomes have confirmed this view, with calcineurin-mediated dephosphorylation of the dephosphins essential for triggering SVE. The phosphorylation cycle of the dephosphins might regulate SVE by targeting the proteins to sites of action and by stimulating the assembly of several large essential endocytic protein complexes.

The Clathrin-dependent Spindle Proteome

Publisher: Elsevier BV

Date: 08-2016

DOI: 10.1074/MCP.M115.054809

Dynamin inhibits phosphatidylinositol 3-kinase in hematopoietic cells

Publisher: Elsevier BV

Date: 02-2001

DOI: 10.1016/S0167-4889(00)00130-0

Abstract: Phosphatidylinositol 3-kinase (PI 3-kinase) plays a role in late stages of endocytosis as well as in cellular proliferation and transformation. The SH3 domain of its regulatory p85 subunit stimulates the GTPase activity of dynamin in vitro. Dynamin is a GTPase enzyme required for endocytosis of activated growth factor receptors. An interaction between these proteins has not been demonstrated in vivo. Here, we report that dynamin associates with PI 3-kinase in hematopoietic cells. We detected both p85 and PI 3-kinase activity in dynamin immune complexes from IL-3-dependent BaF3 cells. However, this association was significantly reduced in BaF3 cells transformed with the BCR/abl oncogene. After transformation only a 4-fold increase in PI 3-kinase activity was detected in dynamin immune complexes, whereas grb2 associated activity was elevated 20-fold. Furthermore, dynamin inhibited the activity of both purified recombinant and immunoprecipitated PI 3-kinase. In BaF3 cells expressing a temperature-sensitive mutant of BCR/abl, a significant decrease in p85 and dynamin association was observed 4 h after the induction of BCR/abl activity. In contrast, in IL-3-stimulated parental BaF3 cells, this association was increased. Our results demonstrate an in vivo association of PI 3-kinase with dynamin and this interaction regulates the activity of PI 3-kinase.

Improved Detection of Hydrophilic Phosphopeptides Using Graphite Powder Microcolumns and Mass Spectrometry

Publisher: Elsevier BV

Date: 05-2004

DOI: 10.1074/MCP.M300105-MCP200

Increased levels of SNAP-25 and synaptophysin in the dorsolateral prefrontal cortex in bipolar I disorder

Publisher: Wiley

Date: 15-03-2006

DOI: 10.1111/J.1399-5618.2006.00300.X

Abstract: In order to identify whether the mechanisms associated with neurotransmitter release are involved in the pathologies of bipolar disorder and schizophrenia, levels of presynaptic [synaptosomal-associated protein-25 (SNAP-25), syntaxin, synaptophysin, vesicle-associated membrane protein, dynamin I] and structural (neuronal cell adhesion molecule and alpha-synuclein) neuronal markers were measured in Brodmann's area 9 obtained postmortem from eight subjects with bipolar I disorder (BPDI), 20 with schizophrenia and 20 controls. Determinations of protein levels were carried out using Western blot techniques with specific antibodies. Levels of mRNA were measured using real-time polymerase chain reaction. In BPDI, levels of SNAP-25 (p < 0.01) and synaptophysin (p < 0.05) increased. There were no changes in schizophrenia or any other changes in BPDI. Levels of mRNA for SNAP-25 were decreased in BPDI (p < 0.05). Changes in SNAP-25 and synaptophysin in BPDI suggest that changes in specific neuronal functions could be linked to the pathology of the disorder.

Small molecules demonstrate the role of dynamin as a bi-directional regulator of the exocytosis fusion pore and vesicle release

Publisher: Springer Science and Business Media LLC

Date: 05-05-2015

DOI: 10.1038/MP.2015.56

Abstract: Hormones and neurotransmitters are stored in specialised vesicles and released from excitable cells through exocytosis. During vesicle fusion with the plasma membrane, a transient fusion pore is created that enables transmitter release. The protein dynamin is known to regulate fusion pore expansion (FPE). The mechanism is unknown, but requires its oligomerisation-stimulated GTPase activity. We used a palette of small molecule dynamin modulators to reveal bi-directional regulation of FPE by dynamin and vesicle release in chromaffin cells. The dynamin inhibitors Dynole 34-2 and Dyngo 4a and the dynamin activator Ryngo 1-23 reduced or increased catecholamine released from single vesicles, respectively. Total internal reflection fluorescence (TIRF) microscopy demonstrated that dynamin stimulation with Ryngo 1-23 reduced the number of neuropeptide Y (NPY) kiss-and-run events, but not full fusion events, and slowed full fusion release kinetics. Amperometric stand-alone foot signals, representing transient kiss-and-run events, were less frequent but were of longer duration, similarly to full erometric spikes and pre-spike foot signals. These effects are not due to alterations in vesicle size. Ryngo 1-23 action was blocked by inhibitors of actin polymerisation or myosin II. Therefore, we demonstrate using a novel pharmacological approach that dynamin not only controls FPE during exocytosis, but is a bi-directional modulator of the fusion pore that increases or decreases the amount released from a vesicle during exocytosis if it is activated or inhibited, respectively. As such, dynamin has the ability to exquisitely fine-tune transmitter release.

Glycogen synthase kinase 3 regulates acrosomal exocytosis in mouse spermatozoa via dynamin phosphorylation

Publisher: Wiley

Date: 25-03-2015

DOI: 10.1096/FJ.14-265553

Abstract: The dynamin family of GTPases has been implicated as novel regulators of the acrosome reaction, a unique exocytotic event that is essential for fertilization. Dynamin activity during the acrosome reaction is accompanied by phosphorylation of key serine residues. We now tested the hypothesis that glycogen synthase kinase 3 (GSK3) is the protein kinase responsible for dynamin phosphorylation at these phosphosites in mouse spermatozoa. Pharmacologic inhibition of GSK3 in mature mouse spermatozoa (CHIR99021: IC50 = 6.7 nM) led to a significant reduction in dynamin phosphorylation (10.3% vs. 27.3% P < 0.001), acrosomal exocytosis (9.7% vs. 25.7% P < 0.01), and in vitro fertilization (53% vs. 100% P < 0.01). GSK3 was shown to be present in developing germ cells where it colocalized with dynamin in the peri-acrosomal domain. However, additional GSK3 was acquired by maturing mouse spermatozoa within the male reproductive tract, via a novel mechanism involving direct interaction of sperm heads with extracellular structures known as epididymal dense bodies. These data reveal a novel mode for the cellular acquisition of a protein kinase and identify a key role for GSK3 in the regulation of sperm maturation and acrosomal exocytosis.

Characterization of the urinary albumin degradation pathway in the isolated perfused rat kidney

Publisher: Elsevier BV

Date: 2006

DOI: 10.1016/J.LAB.2005.08.008

Abstract: This study examines the existence of the urinary albumin degradation pathway and the proposed role of receptor-mediated endocytosis in this process using the isolated perfused rat kidney (IPK) model. Albumin-derived peptides in IPK urine are analyzed in terms of their relative size distribution using radioactivity and absorbance at 214 nm, and their susceptibility to trypsin digestion. The effects of perfusing kidneys with concanamycin A and myristoyl trimethyl ammonium bromide (MTMAB), inhibitors of the receptor-mediated endocytosis regulators vacuolar-type H(+) ATPase (v-ATPase) and dynamin GTPase, respectively, are examined. Normal IPK urine contains mildly degraded (defined as approximately 10-40 kDa 43.0 +/- 8.3%) and heavily degraded (defined as <10 kDa 22.6 +/- 7.7%) albumin peptides as well as intact albumin (34.5 +/- 4.1%). The relative size distribution of the peptides is similar by radioactivity and absorbance at 214 nm, and both profiles are reduced to very small peptides following trypsin digestion. Administration of concanamycin A or MTMAB causes a significant increase in the proportion of intact albumin (concanamycin A: 55.8 +/- 11.6% MTMAB: 50.0 +/- 11.9%) excreted compared with normal IPK urine. This coincides with a reduction in the proportion of mildly (concanamycin A: 27.6 +/- 9.8% MTMAB: 39.9 +/- 11.5%) and heavily degraded (concanamycin A: 16.6 +/- 7.4% MTMAB: 10.0 +/- 2.5%) albumin present and is not associated with changes in glomerular permeability to albumin because no significant change is observed in the fractional clearance of Ficoll (radius range 20-60 A) in the presence of concanamycin A. This study demonstrates the existence of albumin peptides in IPK urine and suggests that receptor-mediated endocytosis plays a role in urinary albumin degradation.

Inhibition of dynamin by dynole 34-2 induces cell death following cytokinesis failure in cancer cells.

Publisher: American Association for Cancer Research (AACR)

Date: 09-2011

DOI: 10.1158/1535-7163.MCT-11-0067

Abstract: Inhibitors of mitotic proteins such as Aurora kinase and polo-like kinase have shown promise in preclinical or early clinical development for cancer treatment. We have reported that the MiTMAB class of dynamin small molecule inhibitors are new antimitotic agents with a novel mechanism of action, blocking cytokinesis. Here, we examined 5 of the most potent of a new series of dynamin GTPase inhibitors called dynoles. They all induced cytokinesis failure at the point of abscission, consistent with inhibition of dynamin while not affecting other cell cycle stages. All 5 dynoles inhibited cell proliferation (MTT and colony formation assays) in 11 cancer cell lines. The most potent GTPase inhibitor, dynole 34-2, also induced apoptosis, as revealed by cell blebbing, DNA fragmentation, and PARP cleavage. Cell death was induced specifically following cytokinesis failure, suggesting that dynole 34-2 selectively targets iding cells. Dividing HeLa cells were more sensitive to the antiproliferative properties of all 5 dynoles compared with non iding cells, and nontumorigenic fibroblasts were less sensitive to cell death induced by dynole 34-2. Thus, the dynoles are a second class of dynamin GTPase inhibitors, with dynole 34-2 as the lead compound, that are novel antimitotic compounds acting specifically at the abscission stage. Mol Cancer Ther 10(9) 1553–62. ©2011 AACR.

Pyrimidyn‐Based Dynamin Inhibitors as Novel Cytotoxic Agents

Publisher: Wiley

Date: 10-11-2022

DOI: 10.1002/CMDC.202100560

Abstract: Five focused libraries of pyrimidine‐based dynamin GTPase inhibitors, in total 69 compounds were synthesised, and their dynamin inhibition and broad‐spectrum cytotoxicity examined. Dynamin plays a crucial role in mitosis, and as such inhibition of dynamin was expected to broadly correlate with the observed cytotoxicity. The pyrimidines synthesised ranged from mono‐substituted to trisubstituted. The highest levels of dynamin inhibition were noted with di‐ and tri‐ substituted pyrimidines, especially those with pendent amino alkyl chains. Short chains and simple heterocyclic rings reduced dynamin activity. There were three levels of dynamin activity noted: 1–10, 10–25 and 25–60 μM. Screening of these compounds in a panel of cancer cell lines: SW480 (colon), HT29 (colon), SMA (spontaneous murine astrocytoma), MCF‐7 (breast), BE2‐C (glioblastoma), SJ‐G2 (neuroblastoma), MIA (pancreas), A2780 (ovarian), A431 (skin), H460 (lung), U87 (glioblastoma) and DU145 (prostate) cell lines reveal a good correlation between the observed dynamin inhibition and the observed cytotoxicity. The most active analogues ( 31 a , b ) developed returned average GI 50 values of 1.0 and 0.78 μM across the twelve cell lines examined. These active analogues were: N 2 ‐(3‐dimethylaminopropyl)‐ N 4 ‐dodecyl‐6‐methylpyrimidine‐2,4‐diamine ( 31 a ) and N 4 ‐(3‐dimethylaminopropyl)‐ N 2 ‐dodecyl‐6‐methylpyrimidine‐2,4‐diamine ( 31 b ).

Azido and Diazarinyl Analogues of Bis‐Tyrphostin as Asymmetrical Inhibitors of Dynamin GTPase

Publisher: Wiley

Date: 29-06-2009

DOI: 10.1002/CMDC.200900054

Abstract: Probing the dynamin binding site: Bis-tyrphostin (1, Bis-T), is a potent inhibitor of the phospholipid-stimulated GTPase activity of dynamin I. Analogues of Bis-T have significant potential as a biological probes for the dissection of endocytic pathways. Bis-T-derived compounds were synthesised and evaluated for their ability to inhibit the GTPase activity of dynamin I. Two analogues (23 and 24) represent the first asymmetrically substituted Bis-T analogues to retain dynamin inhibition.Two azidobenzyl amide (4 and 23) and one 3-trifluoromethyl-3H-diazirin-3-ylphenyl (24) analogues of bis-tyrphostin (1, Bis-T) were synthesised as potential photoaffinity labels for the elucidation of the binding site of compound 1 in dynamin I. Of the two azidobenzyl amide analogues (4 and 23), the terminally substituted 23 retained dynamin I GTPase inhibition (IC(50)=6.4+/-2.8 microM) whilst 4, which was substituted on the central carbon of the amide linker, displayed no activity. Analogue 24 also retained inhibitory activity (IC(50)=36+/-9 microM). Photoaffinity labelling experiments did not unequivocally elucidate the binding pocket of compound 1. However, compounds 23 and 24 represent the first asymmetrically substituted Bis-T analogues to retain dynamin inhibitory activity, providing a new direction for analogue synthesis.

Calcium channel agonists and antagonists regulate protein phosphorylation in intact synaptosomes

Publisher: Elsevier BV

Date: 07-1986

DOI: 10.1016/0304-3940(86)90219-3

Abstract: Protein phosphorylation in intact synaptosomes is highly sensitive to alterations in calcium fluxes and was used to probe the possible mechanism of action of the calcium channel agonist BAY K 8644 and antagonists verapamil and nifedipine. These agents (at 1 microM) all increased the basal phosphorylation of a specific set of 4 synaptosomal phosphoproteins termed P139, P124, P96 and P60, but did not alter depolarization-dependent protein phosphorylation. The increases could not be explained by a direct stimulation of protein kinases and appears unrelated to the known effects of these drugs on K+-stimulated neurotransmitter release. This finding may reveal a possible new mechanism of action for drugs which interact with calcium channels.

A Barocycler-based Concurrent Multi-Omics Method to Assess Molecular Changes Associated with Atherosclerosis using Small Amounts of Arterial Tissue from a Single Mouse.

Publisher: American Chemical Society (ACS)

Date: 11-09-2019

DOI: 10.1021/ACS.ANALCHEM.9B01842

Abstract: Atherosclerosis is a complex, multifactorial disease characterized by the buildup of plaque in the arterial wall. Apolipoprotein E gene deficient (

Development of Second-Generation Indole-Based Dynamin GTPase Inhibitors

Publisher: American Chemical Society (ACS)

Date: 18-12-2012

DOI: 10.1021/JM300844M

Abstract: Focused library development of our lead 2-cyano-3-(1-(3-(dimethylamino)propyl)-2-methyl-1H-indol-3-yl)-N-octylacrylamide (2) confirmed the tertiary dimethylamino-propyl moiety as critical for inhibition of dynamin GTPase. The cyanoamide moiety could be replaced with a thiazole-4(5H)-one isostere (19, IC(₅₀(dyn I)) = 7.7 μM), reduced under flow chemistry conditions (20, IC(₅₀(dyn I)) = 5.2 μM) or replaced by a simple amine. The latter provided a basis for a high yield library of compounds via a reductive amination by flow hydrogenation. Two compounds, 24 (IC(₅₀ (dyn I)) = 0.56 μM) and 25 (IC(₅₀(dyn I)) = 0.76 μM), stood out. Indole 24 is nontoxic and showed increased potency against dynamin I and II in vitro and in cells (IC(₅₀(CME)) = 1.9 μM). It also showed 4.4-fold selectivity for dynamin I. The indole 24 compound has improved isoform selectivity and is the most active in-cell inhibitor of clathrin-mediated endocytosis reported to date.

Dynamin regulates specific membrane fusion events necessary for acrosomal exocytosis in mouse spermatozoa

Publisher: Elsevier BV

Date: 11-2012

DOI: 10.1074/JBC.M112.392803

Wiskostatin and other carbazole scaffolds as off target inhibitors of dynamin I GTPase activity and endocytosis

Publisher: Elsevier BV

Date: 02-2023

DOI: 10.1016/J.EJMECH.2022.115001

The dynamin inhibitors MiTMAB and OcTMAB induce cytokinesis failure and inhibit cell proliferation in human cancer cells.

Publisher: American Association for Cancer Research (AACR)

Date: 07-2010

DOI: 10.1158/1535-7163.MCT-10-0161

Abstract: The endocytic protein dynamin II (dynII) participates in cell cycle progression and has roles in centrosome cohesion and cytokinesis. We have described a series of small-molecule inhibitors of dynamin [myristyl trimethyl ammonium bromides (MiTMAB)] that competitively interfere with the ability of dynamin to bind phospholipids and prevent receptor-mediated endocytosis. We now report that dynII functions specifically during the abscission phase of cytokinesis and that MiTMABs exclusively block this step in the cell cycle. Cells treated with MiTMABs (MiTMAB and octadecyltrimethyl ammonium bromide) and dyn-depleted cells remain connected via an intracellular bridge for a prolonged period with an intact midbody ring before membrane regression and binucleate formation. MiTMABs are the first compounds reported to exclusively block cytokinesis without affecting progression through any other stage of the cell cycle. Thus, MiTMABs represent a new class of antimitotic compounds. We show that MiTMABs are potent inhibitors of cancer cell growth and have minimal effect on nontumorigenic fibroblast cells. Thus, MiTMABs have toxicity and antiproliferative properties that preferentially target cancer cells. This suggests that dynII may be a novel target for pharmacologic intervention for the treatment of cancer. Mol Cancer Ther 9(7) 1995–2006. ©2010 AACR.

Cdk1/Erk2- and Plk1-dependent phosphorylation of a centrosome protein, Cep55, is required for its recruitment to midbody and cytokinesis

Publisher: Elsevier BV

Date: 10-2005

DOI: 10.1016/J.DEVCEL.2005.09.003

Abstract: Centrosomes in mammalian cells have recently been implicated in cytokinesis however, their role in this process is poorly defined. Here, we describe a human coiled-coil protein, Cep55 (centrosome protein 55 kDa), that localizes to the mother centriole during interphase. Despite its association with gamma-TuRC anchoring proteins CG-NAP and Kendrin, Cep55 is not required for microtubule nucleation. Upon mitotic entry, centrosome dissociation of Cep55 is triggered by Erk2/Cdk1-dependent phosphorylation at S425 and S428. Furthermore, Cep55 locates to the midbody and plays a role in cytokinesis, as its depletion by siRNA results in failure of this process. S425/428 phosphorylation is required for interaction with Plk1, enabling phosphorylation of Cep55 at S436. Cells expressing phosphorylation-deficient mutant forms of Cep55 undergo cytokinesis failure. These results highlight the centrosome as a site to organize phosphorylation of Cep55, enabling it to relocate to the midbody to function in mitotic exit and cytokinesis.

1,8-Naphthalimide derivatives: New leads against dynamin i GTPase activity

Publisher: Royal Society of Chemistry (RSC)

Date: 2015

DOI: 10.1039/C5OB00751H

Abstract: Fragment-based in silico screening against dynamin I (dynI) GTPase activity identified the 1,8-naphthalimide framework as a potential scaffold for the design of new inhibitors targeting the GTP binding pocket of dynI.

A Direct Fluorescent Activity Assay for Glycosyltransferases Enables Convenient High‐Throughput Screening: Application to O‐GlcNAc Transferase

Publisher: Wiley

Date: 02-04-2020

DOI: 10.1002/ANIE.202000621

Involvement of novel autophosphorylation sites in ATM activation

Publisher: Wiley

Date: 13-07-2006

DOI: 10.1038/SJ.EMBOJ.7601231

FD5180, a Novel Protein Kinase Affinity Probe, and the Effect of Bead Loading on Protein Kinase Identification

Publisher: American Chemical Society (ACS)

Date: 25-07-2017

DOI: 10.1021/ACSOMEGA.7B00020

The in Vivo Phosphorylation Sites of Rat Brain Dynamin I

Publisher: Elsevier BV

Date: 05-2007

DOI: 10.1074/JBC.M609713200

Decreased phorbol ester binding in the parahippocampal gyrus from subjects with schizophrenia is not associated with changes in protein kinase C

Publisher: Springer Science and Business Media LLC

Date: 08-2002

DOI: 10.1038/SJ.MP.4001065

Abstract: Combining in situ radioligand binding with autoradiography, we previously identified a reduction of [(3)H]phorbol 12,13-dibutyrate binding in the parahippoc al gyrus from schizophrenic subjects. To determine whether these changes were due to decreases in the level of protein kinase C, we measured [(3)H]phorbol 12,13-dibutyrate binding, levels of the protein kinase C isoforms alpha, beta, delta, epsilon, gamma, eta and theta, as well as protein kinase C activity in crude particulate membranes from parahippoc al gyri of 15 schizophrenic and 15 control subjects. There was a significant decrease in the density (mean +/- SEM: 6.56 +/- 0.73 pmol mg(-1) vs 9.68 +/- 1.22 pmol mg(-1) P < 0.05) and affinity (mean K(D) +/- SEM: 4.64 +/- 0.34 nM vs 2.95 +/- 0.35 nM P < 0.005) of [(3)H]phorbol 12,13-dibutyrate binding in homogenates from schizophrenic subjects. There were no significant changes in levels of the protein kinase C isoforms which are known to bind phorbol esters or in the activity of protein kinase C in membranes from schizophrenic subjects. These results suggest that there are changes in molecules capable of binding [(3)H]phorbol 12,13-dibutyrate, other than protein kinase C, in the parahippoc al gyrus from subjects with schizophrenia.

Small Molecule Inhibitors of Dynamin I GTPase Activity:  Development of Dimeric Tyrphostins

Publisher: American Chemical Society (ACS)

Date: 29-10-2005

DOI: 10.1021/JM040208L

Abstract: Dynamin I is a GTPase enzyme required for endocytosis and is an excellent target for the design of potential endocytosis inhibitors. Screening of a library of tyrphostins, in our laboratory, against the GTPase activity of dynamin I gave rise to a microM potent lead, 2-cyano-3-(3,4-dihydroxyphenyl)thioacrylamide (1, IC50 70 microM). Our initial investigations suggested that only the dimeric form of 1 displayed dynamin I GTPase inhibitory activity. Subsequent synthetic iterations were based on dimeric analogues and afforded a number of small molecules, low microM potent, inhibitors of dynamin I GTPase, in particular, symmetrical analogues with a minimum of two free phenolic -OHs: catechol-acrylamide (9) (IC50= 5.1 +/- 0.6 microM), its 3,4,5-trihydroxy congener (10) (IC50= 1.7 +/- 0.2 microM), and the corresponding 3-methyl ether (11) (IC50= 9 +/- 3 microM). Increasing the length of the central alkyl spacer from ethyl to propyl (22-24) afforded essentially identical activity with IC50's of 1.7 +/- 0.2, 1.7 +/- 0.2, and 5 +/- 1 microM, respectively. No decrease in activity was noted until the introduction of a hexyl spacer. Our studies highlight the requirement for two free amido NHs with neither the mono-N-methyl (86) nor the bis-N-methyl (87) analogues inhibiting dynamin I GTPase. A similar effect was noted for the removal of the nitrile moieties. However, modest potency was observed with the corresponding ester analogues of 9-11: ethyl ester (90), propyl ester (91), and butyl ester (92), with IC50's of 42 +/- 3, 38 +/- 2, and 61 +/- 2 microM, respectively. Our studies reveal the most potent and promising dynamin I GTPase inhibitor in this series as (22), which is also known as BisT.

Proteomic profiling of idiopathic Parkinson's disease primary patient cells by SWATH-MS

Publisher: Wiley

Date: 07-06-2022

DOI: 10.1002/PRCA.202200015

Abstract: Parkinson's disease (PD) is the second most prevalent neurodegenerative disease. It is generally diagnosed clinically after the irreversible loss of dopaminergic neurons and no general biomarkers currently exist. To gain insight into the underlying cellular causes of PD we aimed to quantify the proteomic differences between healthy control and PD patient cells. Sequential Window Acquisition of all THeoretical Mass Spectra was performed on primary cells from healthy controls and PD patients. In total, 1948 proteins were quantified and 228 proteins were significantly differentially expressed in PD patient cells. In PD patient cells, we identified seven significantly increased proteins involved in the unfolded protein response (UPR) and focused on cells with high and low amounts of PDIA6 and HYOU1. We discovered that PD patients with high amounts of PDIA6 and HYOU1 proteins were more sensitive to endoplasmic reticulum stress, in particular to tunicamycin. Data is available via ProteomeXchange with identifier PXD030723. This data from primary patient cells has uncovered a critical role of the UPR in patients with PD and may provide insight to the underlying cellular dysfunctions in these patients.

Expanding the clinical, pathological and MRI phenotype of DNM2-related centronuclear myopathy

Publisher: Elsevier BV

Date: 04-2010

DOI: 10.1016/J.NMD.2010.02.016

Abstract: Mutations in dynamin-2 (DNM2) cause autosomal dominant centronuclear myopathy (CNM). We report a series of 12 patients from eight families with CNM in whom we have identified a number of novel features that expand the reported clinicopathological phenotype. We identified two novel and five recurrent missense mutations in DNM2. Early clues to the diagnosis include relative weakness of neck flexors, external ophthalmoplegia and ptosis, although these are not present in all patients. Pes cavus was present in two patients, and in another two members of one family there was mild slowing of nerve conduction velocities. Whole-body MRI examination in two children and one adult revealed a similar pattern of involvement of selective muscles in head (lateral pterygoids), neck (extensors), trunk (paraspinal) and upper limbs (deep muscles of forearm). Findings in lower limbs and pelvic region were similar to that previously reported in adults with DNM2 mutations. Two patients presented with dystrophic changes as the predominant pathological feature on muscle biopsies one of whom had a moderately raised creatine kinase, and both patients were initially diagnosed as congenital muscular dystrophy. DNM2 mutation analysis should be considered in patients with a suggestive clinical phenotype despite atypical histopathology, and MRI findings can be used to guide genetic testing. Subtle neuropathic features in some patients suggest an overlap with the DNM2 neuropathy phenotype. Missense mutations in the C-terminal region of the PH domain appear to be associated with a more severe clinical phenotype evident from infancy.

The temporal profile of activity-dependent presynaptic phospho-signalling reveals long-lasting patterns of poststimulus regulation

Publisher: Public Library of Science (PLoS)

Date: 03-2019

DOI: 10.1371/JOURNAL.PBIO.3000170

Dynamin I phosphorylation by GSK3 controls activity-dependent bulk endocytosis of synaptic vesicles

Publisher: Springer Science and Business Media LLC

Date: 06-06-2010

DOI: 10.1038/NN.2571

Syndapin I is the phosphorylation-regulated dynamin I partner in synaptic vesicle endocytosis.

Publisher: Springer Science and Business Media LLC

Date: 30-04-2006

DOI: 10.1038/NN1695

The role of protein kinase C and its neuronal substrates dephosphin, B-50, and MARCKS in neurotransmitter release

Publisher: Springer Science and Business Media LLC

Date: 06-1991

DOI: 10.1007/BF02935541

Potencies of protein kinase C inhibitors are dependent on the activators used to stimulate the enzyme

Publisher: Elsevier BV

Date: 10-1992

DOI: 10.1016/0006-2952(92)90533-O

Abstract: The aim was to examine systematically the potencies of protein kinase C inhibitors as a function of the kinase activator. Protein kinase C is activated by at least four stimulators: calcium plus phosphatidylserine (Ca/PS), phorbol 12-myristate 13-acetate plus PS (PS/PMA), arachidonic acid plus calcium (Ca/AA) and the synthetic peptide activator PCK530-558. With histone or GS1-12 as substrates, protein kinase C was maximally activated by Ca/PS, or to maxima of 62%, 89% or 82% with PS/PMA, Ca/AA or PKC530-558, respectively. One group of inhibitors, including H-7 and staurosporine, were equipotent, regardless of the activator. All other inhibitors showed variable selectivity, dependent upon the activator. A second group of inhibitors, including sphingosine and lipophosphoglycan, were eight or 200 times more potent for inhibition of PS/PMA-stimulated activity (relative to Ca/PS) and a third group, including retinal and palmitoylcarnitine, were 14 or 262 times more potent towards Ca/PS-stimulated activity. A final group (rhodamine 6G) was nine times more potent when Ca/AA was the activator. Similar results were obtained using the endogenous substrates dephosphin or MARCKS in synaptosol. Phosphorylation of MARCKS was stimulated by PS/PMA or Ca/PS, while phosphorylation of dephosphin was stimulated only by Ca/PS. The phosphorylation of either by Ca/PS-activated kinase was nine times more potently inhibited by palmitoylcarnitine, while phosphorylation of MARCKS by PS/PMA-activated kinase was 10 times more potently inhibited by sphingosine. H-7 inhibited both at similar concentrations. A model encompasses these differences in potency if the inhibitors are ided into four groups (A-D) according to their competitive inhibition with the appropriate activator or at the active site. The non-selective inhibitors interact at the active sites of protein kinase C (group A). The compounds which preferentially inhibit PS/PMA-activated kinase (sphingosine and lipophosphoglycan) are competitive inhibitors of PMA and 1,2-diacylglycerol (group B), those selective for Ca/PS-activated kinase (palmitoylcarnitine and retinal) are competitive with PS (group C) and those selective for Ca-AA activation (rhodamine 6G) are likely to be competitive with fatty acid (group D). Therefore, the effectiveness of protein kinase C inhibitors is dependent upon the activator employed.

Rapid Purification of Native Dynamin I and Colorimetric GTPase Assay

Publisher: Elsevier

Date: 2005

DOI: 10.1016/S0076-6879(05)04049-8

Septins and the synapse

Publisher: Wiley

Date: 14-11-2008

DOI: 10.1002/9780470779705.CH11

Dephosphin, a 96 000 Da substrate of protein kinase C in synaptosomal cytosol, is phosphorylated in intact synaptosomes

Publisher: Wiley

Date: 06-05-1991

DOI: 10.1016/0014-5793(91)80520-D

Abstract: A 96,000 dalton phosphoprotein, called dephosphin, is phosphorylated in intact synaptosomes from rat brain and is rapidly dephosphorylated upon depolarisation-dependent calcium entry. A 96,000 dalton phosphoprotein is also a substrate of protein kinase C in synaptosomal cytosol, and the aim of the study was to determine whether the two proteins may be the same. Dephosphin in intact synaptosomes and the 96,000 dalton protein kinase C substrate comigrated on polyacrylamide gels. Both phosphoproteins had identical phosphopeptide maps after digestion with V8 protease. Both phosphoproteins ran on isoelectric focussing gels with a pI of 6.3-6.7 and focussed as a series of 5-6 spots. Both proteins were phosphorylated exclusively on serine. Both proteins could be resolved into a doublet on longer polyacrylamide gels. The two subunits were of 96 and 93 kDa in both phosphorylation conditions and had dissimilar phosphopeptide maps. However, phosphopeptide maps of either the 96 or 93 kDa subunits were identical in intact synaptosomes compared with synaptosomal cytosol. These results show that a phosphoprotein phosphorylated in intact synaptosomes and a 96,000 dalton protein kinase C substrate from rat brain synaptosomal cytosol are the same, and raise the possibility that protein kinase C is the protein kinase responsible for dephosphin phosphorylation in intact synaptosomes.

Dynamin Inhibition Blocks Botulinum Neurotoxin Type A Endocytosis in Neurons and Delays Botulism

Publisher: Elsevier BV

Date: 10-2011

DOI: 10.1074/JBC.M111.283879

Genotoxic stress-induced nuclear localization of oncoprotein YB-1 in the absence of proteolytic processing

Publisher: Springer Science and Business Media LLC

Date: 19-10-2009

DOI: 10.1038/ONC.2009.321

Abstract: Y-box-binding protein 1 (YB-1) is an oncogenic transcription factor whose overexpression and nuclear localization is associated with tumor progression and drug resistance. Transcriptional activation of YB-1 in response to genotoxic stress is believed to occur in the cytoplasm through sequence-specific endoproteolytic cleavage by the 20S Proteasome, followed by nuclear translocation of cleaved YB-1. To study the proteolysis model, we developed a two-step affinity purification of endogenous YB-1 protein species and characterized the products using mass spectrometry. Whereas full-length YB-1 was readily identified, the smaller protein band thought to be activated YB-1 was identified as hnRNP A1. An antibody specific for YB-1 was generated, which revealed only one YB-1 species, even after genotoxic stress-induced nuclear YB-1 translocation. These findings warrant re-evaluation of the mechanism of YB-1 nuclear translocation and transcriptional activation. The relationship between nuclear YB-1 and tumor progression may also have to re-evaluated in some cases.

Cdk5 is essential for synaptic vesicle endocytosis

Publisher: Springer Science and Business Media LLC

Date: 13-07-2003

DOI: 10.1038/NCB1020

Phosphorylation of dynamin I and synaptic-vesicle recycling

Publisher: Elsevier BV

Date: 1994

DOI: 10.1016/0166-2236(94)90179-1

Abstract: In nerve terminals, neurotransmitters are packaged in synaptic vesicles, and released by exocytosis. Empty synaptic vesicles are rapidly recycled for reuse by endocytosis. Much progress has been made in identifying the proteins involved in synaptic-vesicle trafficking, but the mechanism and regulation of endocytosis have largely remained an enigma. One approach to defining regulatory proteins that might be involved is to study stimulus-dependent phosphorylation events in nerve terminals. This has led to the identification of dephosphin, which is quantitatively dephosphorylated by nerve-terminal depolarization. Sequencing reveals that dephosphin is identical with dynamin I, a GTP-binding protein that functions in endocytosis. Phosphorylation and dephosphorylation of nerve-terminal dynamin I/dephosphin regulates its intrinsic GTPase activity in parallel with the regulation of synaptic-vesicle recycling. Therefore, phosphorylation and dephosphorylation of dynamin I might provide a Ca(2+)-dependent switch for endocytosis in the synaptic-vesicle pathway.

Cholinoceptor regulation of cyclic AMP levels in bovine adrenal medullary cells

Publisher: Wiley

Date: 06-1992

DOI: 10.1111/J.1476-5381.1992.TB14341.X

Abstract: 1. The regulation of adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels by cholinoceptors has been studied in cultured bovine adrenal medullary cells. 2. Acetylcholine (100 microM), nicotine (10 microM) and dimethylphenylpiperazinium (20 microM) each increased cellular cyclic AMP levels 2 to 4 fold over 5 min in the absence of phosphodiesterase inhibitors. The muscarinic agonist acetyl-beta-methylcholine (100 microM) had no effect either on its own or on the response to nicotine. The responses to acetylcholine and nicotine were unaffected by atropine (1 microM) but were abolished by mecamylamine (5 microM). 3. Cellular cyclic AMP increased transiently during continuous exposure to nicotine (1-20 microM), with the largest response seen after 5 min, a smaller response after 20 min, and no change in cyclic AMP levels seen after 90 or 180 min. The maximal response after 5 min stimulation was seen with 5-10 microM nicotine and the EC50 was about 2 microM. In contrast, extracellular cyclic AMP levels did not change after 5 or 20 min stimulation with nicotine, but increased slightly after 90 min and further after 180 min. 4. The cellular cyclic AMP response to nicotine (10 microM) was unchanged or weakly enhanced in the presence of the unselective phosphodiesterase inhibitor, isobutylmethylxanthine, and was unchanged in the presence of rolipram. Nicotine did not interact synergistically with low concentrations of forskolin. The response was however completely abolished in the absence of extracellular Ca2+.

A Direct Fluorescent Activity Assay for Glycosyltransferases Enables Convenient High‐Throughput Screening: Application to O‐GlcNAc Transferase

Publisher: Wiley

Date: 02-04-2020

DOI: 10.1002/ANGE.202000621

The Pthaladyns: GTP Competitive Inhibitors of Dynamin I and II GTPase Derived from Virtual Screening

Publisher: American Chemical Society (ACS)

Date: 24-06-2010

DOI: 10.1021/JM100442U

Abstract: We report the development of a homology model for the GTP binding domain of human dynamin I based on the corresponding crystal structure of Dictyostelium discoidum dynamin A. Virtual screening identified 2-[(2-biphenyl-2-yl-1,3-dioxo-2,3-dihydro-1H-isoindole-5-carbonyl)amino]-4-chlorobenzoic acid (1) as a approximately 170 microM potent inhibitor. Homology modeling- and focused library-led synthesis resulted in development of a series of active compounds (the "pthaladyns") with 4-chloro-2-(2-(4-(hydroxymethyl)phenyl)-1,3-dioxoisoindoline-5-carboxamido)benzoic acid (29), a 4.58 +/- 0.06 microM dynamin I GTPase inhibitor. Pthaladyn-29 displays borderline selectivity for dynamin I relative to dynamin II ( approximately 5-10 fold). Only pthaladyn-23 (dynamin I IC(50) 17.4 +/- 5.8 microM) was an effective inhibitor of dynamin I mediated synaptic vesicle endocytosis in brain synaptosomes with an IC(50) of 12.9 +/- 5.9 microM. This compound was also competitive with respect to Mg(2+).GTP. Thus the pthaladyns are the first GTP competitive inhibitors of dynamin I and II GTPase and may be effective new tools for the study of neuronal endocytosis.

Parallel Solution-Phase Synthesis of Targeted Tyrphostin Libraries with Anticancer Activity

Publisher: CSIRO Publishing

Date: 2005

DOI: 10.1071/CH04143

Abstract: The combination of semi-automation, an elegant synthesis, and parallel solution-phase synthesis approaches has allowed the development of five targeted, symmetrical tyrphostin compound libraries. These libraries on average are comprised of 12 compounds. Notwithstanding this, low micromolar potent growth inhibitors against HT29 (colorectal carcinoma) and G401 (renal carcinoma) cell lines were discovered. Additionally, significant SAR data was obtained. We noted that the most potent growth inhibitory activity was consistently observed for those analogues that possessed a 2-chlorophenyl (for 10: GI50 HT29 5.5 ± 0.4 μM, GI50 G401 2.6 ± 0.4 μM for 23: GI50 HT29 2.4 ± 0.2 μM, GI50 G401 1.9 ± 1 μM for 34: GI50 HT29 8.8 ± 3.1 μM, GI50 G401 6.2 ± 2.9 μM for 46: GI50 HT29 5.2 ± 0.9 μM, GI50 G401 3.7 ± 0.6 μM for 57: GI50 HT29 4.6 ± 0.8 μM, GI50 G401 2.1 ± 0.2 μM), a 3-chlorophenyl (for 11: GI50 HT29 3.8 ± 0.7 μM, GI50 G401 1.7 ± 0.7 μM for 48: GI50 HT29 5.9 ± 0.1 μM, GI50 G401 3.4 ± 0.6 μM for 58: GI50 HT29 4.8 ± 0.9 μM, GI50 G401 3.4 ± 0.2 μM), or a 3-methoxyphenyl substituent (for 13: GI50 HT29 7.4 ± 3.8 μM, GI50 G401 2.8 ± 0.5 μM for 26: GI50 HT29 4.5 ± 0.5 μM, GI50 G401 4.9 ± 1 μM for 37: GI50 HT29 3.7 ± 0.2 μM, GI50 G401 1.6 ± 0.2 μM for 49: GI50 HT29 3.7 ± 0.4 μM, GI50 G401 3.4 ± 0.2 μM for 60: GI50 HT29 4.1 ± 0.6 μM, GI50 G401 1.8 ± 0.3 μM). Finally, we noted that increasing the distance between the terminal aromatic rings had only a minimal effect on the 2-, 3-chlorophenyl, and 3-methoxyphenyl analogues, but did have a favourable effect on OH, COOH, and multiply substituted analogues.

Intranuclear rod myopathy: molecular pathogenesis and mechanisms of weakness

Publisher: Wiley

Date: 17-08-2007

DOI: 10.1002/ANA.21200

Abstract: Mutations in the alpha-skeletal actin gene (ACTA1) result in a variety of inherited muscle disorders characterized by different pathologies and variable clinical phenotypes. Mutations at Val163 in ACTA1 result in pure intranuclear rod myopathy however, the molecular mechanisms by which mutations at Val163 lead to intranuclear rod formation and muscle weakness are unknown. We investigated the effects of the Val163Met mutation in ACTA1 in tissue culture and Drosophila models, and in patient muscle. In cultured cells, the mutant actin tends to aggregate rather than incorporate into cytoplasmic microfilaments, and it affects the dynamics of wild-type actin, causing it to accumulate with the mutant actin in the nucleus. In Drosophila, the Val163Met mutation severely disrupts the structure of the muscle sarcomere. The intranuclear aggregates in patient muscle biopsies impact on nuclear structure and sequester normal Z-disc-associated proteins within the nucleus however, the sarcomeric structure is relatively well preserved, with evidence of active regeneration. By mass spectrometry, the levels of mutant protein are markedly reduced in patient muscle compared with control. Data from our tissue culture and Drosophila models show that the Val163Met mutation in alpha-skeletal actin can affect the dynamics of other actin isoforms and severely disrupt sarcomeric structure, processes that can contribute to muscle weakness. However, in human muscle, there is evidence of regeneration, and the mutant protein tends to aggregate rather than incorporate into cytoplasmic microfilaments in cells. These are likely compensatory processes that ameliorate the effects of the mutant actin and contribute to the milder clinical and pathological disease phenotype.

The small GTPases Rab5 and RalA regulate intracellular traffic of P-glycoprotein

Publisher: Elsevier BV

Date: 07-2007

DOI: 10.1016/J.BBAMCR.2007.03.023

Abstract: P-glycoprotein (P-gp) is a plasma membrane glycoprotein that can cause multidrug resistance (MDR) of cancer cells by acting as an ATP-dependent drug efflux pump. The regulatory effects of the small GTPases Rab5 and RalA on the intracellular trafficking of P-gp were investigated in HeLa cells. As expected, overexpressed enhanced green fluorescent protein (EGFP)-tagged P-gp (P-gp-EGFP) is mainly localised to the plasma membrane. However, upon cotransfection of either dominant negative Rab5 (Rab5-S34N) or constitutively active RalA (RalA-G23V) the intracellular P-gp-EGFP levels increased approximately 9 and 13 fold, respectively, compared to control P-gp-EGFP cells. These results suggest that Rab5 and RalA regulate P-gp trafficking between the plasma membrane and an intracellular compartment. In contrast, coexpression of constitutively active Rab5 (Rab5-Q79L) or dominant negative RalA (RalA-S28N) had no effect on the localisation of P-gp-EGFP. Furthermore, the intracellular accumulation of daunorubicin, a substrate for P-gp, increased significantly with an increased intracellular localisation of P-gp-EGFP. These results imply that it may be possible to overcome MDR by controlling the plasma membrane localisation of P-gp.

Phosphorylation of a New Brain-specific Septin, G-septin, by cGMP-dependent Protein Kinase

Publisher: Elsevier BV

Date: 04-2000

DOI: 10.1074/JBC.275.14.10047

Hypertrophy and dietary tyrosine ameliorate the phenotypes of a mouse model of severe nemaline myopathy

Publisher: Oxford University Press (OUP)

Date: 08-11-2011

DOI: 10.1093/BRAIN/AWR274

Abstract: Nemaline myopathy, the most common congenital myopathy, is caused by mutations in genes encoding thin filament and thin filament-associated proteins in skeletal muscles. Severely affected patients fail to survive beyond the first year of life due to severe muscle weakness. There are no specific therapies to combat this muscle weakness. We have generated the first knock-in mouse model for severe nemaline myopathy by replacing a normal allele of the α-skeletal actin gene with a mutated form (H40Y), which causes severe nemaline myopathy in humans. The Acta1(H40Y) mouse has severe muscle weakness manifested as shortened lifespan, significant forearm and isolated muscle weakness and decreased mobility. Muscle pathologies present in the human patients (e.g. nemaline rods, fibre atrophy and increase in slow fibres) were detected in the Acta1(H40Y) mouse, indicating that it is an excellent model for severe nemaline myopathy. Mating of the Acta1(H40Y) mouse with hypertrophic four and a half LIM domains protein 1 and insulin-like growth factor-1 transgenic mice models increased forearm strength and mobility, and decreased nemaline pathologies. Dietary L-tyrosine supplements also alleviated the mobility deficit and decreased the chronic repair and nemaline rod pathologies. These results suggest that L-tyrosine may be an effective treatment for muscle weakness and immobility in nemaline myopathy.

Heat ‘n Beat: A universal high-throughput end-to-end proteomics sample processing platform in under an hour

Publisher: Cold Spring Harbor Laboratory

Date: 28-09-2023

DOI: 10.1101/2023.09.27.559846

The in Vivo Phosphorylation Sites in Multiple Isoforms of Amphiphysin I from Rat Brain Nerve Terminals

Publisher: Elsevier BV

Date: 06-2008

DOI: 10.1074/MCP.M700351-MCP200

Adrenocorticotropin causes vasodilatation in the human fetal-placental circulation

Publisher: The Endocrine Society

Date: 04-1996

DOI: 10.1210/JC.81.4.1406

Clinical applications of mass spectrometry‐based proteomics in cancer: Where are we?

Publisher: Wiley

Date: 26-08-2022

DOI: 10.1002/PMIC.202200238

Abstract: Tumor tissue processing methodologies in combination with data‐independent acquisition mass spectrometry (DIA‐MS) have emerged that can comprehensively analyze the proteome of multiple tumor s les accurately and reproducibly. Increasing recognition and adoption of these technologies has resulted in a tranche of studies providing novel insights into cancer classification systems, functional tumor biology, cancer biomarkers, treatment response and drug targets. Despite this, with some limited exceptions, MS‐based proteomics has not yet been implemented in routine cancer clinical practice. Here, we summarize the use of DIA‐MS in studies that may pave the way for future clinical cancer applications, and highlight the role of alternative MS technologies and multi‐omic strategies. We discuss limitations and challenges of studies in this field to date and propose steps for integrating proteomic data into the cancer clinic.

Protein kinase C-α is multiply phosphorylated in response to phorbol ester stimulation of PC12 cells

Publisher: Elsevier BV

Date: 08-1996

DOI: 10.1016/0167-4889(96)00061-4

Abstract: The protein kinase C (PKC) family consists of a number of closely related isotypes, whose in vivo phosphorylation state is regulated in a dynamic fashion by the enzyme's activators. We have investigated here the changes in PKC phosphorylation in response to phorbol ester. Using a combination of hydroxylapatite chromatography and immunoblot with isotype-specific antibodies, we identified PKC-alpha, -delta, -epsilon, and -zeta as the isotypes expressed in PC12 cells. A two-dimensional immunoblot approach was then developed to measure the changes in the phosphorylation state of PKC-alpha before and after exposure of intact PC12 cells to phorbol ester. We found a pool of four differentially migrating PKC-alpha forms in untreated cells, which undergoes an acidic shift after phorbol ester. Furthermore, a similar shift in the two-dimensional immunoblot profile of PKC-alpha was the result of the enzyme autophosphorylation upon in vitro treatment with a combination of phosphatidylserine and phorbol ester, an effect which was enhanced by co-application of purified bovine lung cGMP-dependent protein kinase-I (PKG-I). These results demonstrate a multiple phosphorylation of PKC-alpha in untreated PC12 cells and suggest that various levels of autophosphorylation and trans-phosphorylation of this isoenzyme may occur in response to phorbol ester.

CORTICOTROPIN-RELEASING HORMONE-INDUCED VASODILATATION IN THE HUMAN FETAL-PLACENTAL CIRCULATION

Publisher: The Endocrine Society

Date: 08-1994

DOI: 10.1210/JC.79.2.666

Autophosphorylation and ATM Activation

Publisher: Elsevier BV

Date: 03-2011

DOI: 10.1074/JBC.M110.204065

Synthesis and protein binding studies of a peptide fragment of clathrin assembly protein AP180 bearing an O-linked β-N-acetylglucosaminyl-6-phosphate modification

Publisher: Royal Society of Chemistry (RSC)

Date: 2012

DOI: 10.1039/C2OB07139H

Abstract: A novel post-translational modification of threonine, β-N-acetylglucosaminyl-phosphate, was recently discovered on assembly protein AP180, a protein which plays a crucial role in clathrin coated vesicle formation in synaptic vesicle endocytosis (SVE). Herein, we report studies aimed at probing the effect of this modification on binding to proteins in rat brain lysate using pull down experiments with peptide fragments of AP180.

A Novel Post-translational Modification in Nerve Terminals: O-Linked N-Acetylglucosamine Phosphorylation

Publisher: American Chemical Society (ACS)

Date: 02-05-2011

DOI: 10.1021/PR1011153

Abstract: Protein phosphorylation and glycosylation are the most common post-translational modifications observed in biology, frequently on the same protein. Assembly protein AP180 is a synapse-specific phosphoprotein and O-linked beta-N-acetylglucosamine (O-GlcNAc) modified glycoprotein. AP180 is involved in the assembly of clathrin coated vesicles in synaptic vesicle endocytosis. Unlike other types of O-glycosylation, O-GlcNAc is nucleocytoplasmic and reversible. It was thought to be a terminal modification, that is, the O-GlcNAc was not found to be additionally modified in any way. We now show that AP180 purified from rat brain contains a phosphorylated O-GlcNAc (O-GlcNAc-P) within a highly conserved sequence. O-GlcNAc or O-GlcNAc-P, but not phosphorylation alone, was found at Thr-310. Analysis of synthetic GlcNAc-6-P produced identical fragmentation products to GlcNAc-P from AP180. Direct O-linkage of GlcNAc-P to a Thr residue was confirmed by electron transfer dissociation MS. A second AP180 tryptic peptide was also glycosyl phosphorylated, but the site of modification was not assigned. Sequence similarities suggest there may be a common motif within AP180 involving glycosyl phosphorylation and dual flanking phosphorylation sites within 4 amino acid residues. This novel type of protein glycosyl phosphorylation adds a new signaling mechanism to the regulation of neurotransmission and more complexity to the study of O-GlcNAc modification.

Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief

Publisher: American Association for the Advancement of Science (AAAS)

Date: 31-05-2017

DOI: 10.1126/SCITRANSLMED.AAL3447

Abstract: Therapeutic targeting of the neurokinin 1 receptor in endosomes provides efficacious and prolonged pain relief.

Activation of Protein Kinase C in Vitro and in Intact Cells or Synaptosomes Determined by Acetic Acid Extraction of MARCKS

Publisher: Elsevier BV

Date: 04-1993

DOI: 10.1006/ABIO.1993.1169

Abstract: MARCKS is a widespread cellular phosphoprotein that migrates at 80-87 kDa on polyacrylamide gels. It is phosphorylated apparently exclusively by protein kinase C (PKC) and its phosphorylation in intact cells can be used as an index of intracellular PKC activation. Most methods for the determination of its phosphorylation state in vitro or in intact cells rely on two-dimensional gel electrophoresis to detect the protein with a pI of 4.6 however, this does not readily allow for multiple s les to be simultaneously and rapidly processed. Here we utilize the acid solubility of MARCKS to develop a novel extraction procedure. MARCKS was found to be soluble in 40% acetic acid and can be extracted quantitatively and rapidly from phosphorylation mixtures in vitro or from labeled intact cells. Acetic acid has the advantage that it is volatile and readily removed and precipitates protein in the presence of SDS, and the extracted protein is more readily resuspended in sodium dodecyl sulfate (SDS) s le buffer. Two extraction methods were developed, one for extraction of MARCKS from [gamma-32P]ATP-labeled subcellular fractions or intact synaptosomes labeled with 32Pi and one for extraction from 32Pi-labeled cultured nucleated cells, the latter requiring an additional protein recovery and DNA removal step. With this procedure MARCKS phosphorylation in intact synaptosomes was shown to be reversibly stimulated upon depolarization and MARCKS phosphorylation was found to be an early event in the activation of cultured smooth muscle cells by angiotensin II.

Ba2+ does not support synaptic vesicle retrieval in rat cerebrocortical synaptosomes.

Publisher: Elsevier BV

Date: 08-1998

DOI: 10.1016/S0304-3940(98)00610-7

Abstract: To investigate whether any specific requirement for extracellular Ca2+ exists in the control synaptic vesicle retrieval, we examined the ability of the alent cation Ba2+ to substitute for Ca2+ in both vesicle exocytosis and endocytosis. Ba2+ stimulated glutamate release from rat cerebrocortical synaptosomes. Ba2+-evoked release was inhibited by bafilomycin A1, indicating release was via exocytosis of synaptic vesicles. However, Ba2+ did not stimulate vesicle retrieval, monitored by a FM2-10-based retrieval assay. Therefore synaptic vesicle retrieval in central nerve terminals has a specific requirement for extracellular Ca2+ and the Ca2+ receptor for retrieval has a different cation specificity to the Ca2+ receptor for exocytosis.

Accelerated Barocycler Lysis and Extraction Sample Preparation for Clinical Proteomics by Mass Spectrometry

Publisher: American Chemical Society (ACS)

Date: 28-11-2018

DOI: 10.1021/ACS.JPROTEOME.8B00684

Abstract: We have developed a streamlined proteomic s le preparation protocol termed Accelerated Barocycler Lysis and Extraction (ABLE) that substantially reduces the time and cost of tissue s le processing. ABLE is based on pressure cycling technology (PCT) for rapid tissue solubilization and reliable, controlled proteolytic digestion. Here, a previously reported PCT based protocol was optimized using 1-4 mg biopsy punches from rat kidney. The tissue denaturant urea was substituted with a combination of sodium deoxycholate (SDC) and N-propanol. ABLE produced comparable numbers of protein identifications in half the s le preparation time, being ready for MS injection in 3 h compared with 6 h for the conventional urea based method. To validate ABLE, it was applied to a erse range of rat tissues (kidney, lung, muscle, brain, testis), human HEK 293 cell lines, and human ovarian cancer s les, followed by SWATH-mass spectrometry (SWATH-MS). There were similar numbers of quantified proteins between ABLE-SWATH and the conventional method, with greater than 70% overlap for all s le types, except muscle (58%). The ABLE protocol offers a standardized, high-throughput, efficient, and reproducible proteomic preparation method that when coupled with SWATH-MS has the potential to accelerate proteomics analysis to achieve a clinically relevant turn-around time.

Krüppel-associated box (KRAB)-associated co-repressor (KAP-1) Ser-473 phosphorylation regulates heterochromatin protein 1β (HP1-β) mobilization and DNA repair in heterochromatin

Publisher: Elsevier BV

Date: 08-2012

DOI: 10.1074/JBC.M112.368381

Septin 3 (G‐septin) is a developmentally regulated phosphoprotein enriched in presynaptic nerve terminals

Publisher: Wiley

Date: 12-10-2004

DOI: 10.1111/J.1471-4159.2004.02755.X

Abstract: The septins are GTPase enzymes with multiple roles in cytokinesis, cell polarity or exocytosis. The proteins from the mammalian septin genes are called Sept1-10. Most are expressed in multiple tissues, but the mRNA for Sept5 (CDCrel-1) and Sept3 (G-septin) appear to be primarily expressed in brain. Sept3 is phosphorylated by cGMP-dependent protein kinase I (PKG-I) and the cGMP/PKG pathway is involved in presynaptic plasticity. Therefore to determine whether Sept3 specifically associates with neurones and nerve terminals we investigated its distribution in rat brain and neuronal cultures. Sept3 protein was detected only in brain by immunoblot, but not in 12 other tissues examined. Levels were high in all adult brain regions, and reduced in those enriched in white matter. Expression was developmentally regulated, being absent in the early embryo, low in late embryonic rat brain and increasing after birth. Like dynamin I, Sept3 was specifically enriched in synaptosomes compared with whole brain, and was only found in a peripheral membrane extract and not in the soluble or membrane extracts. Sept3 was particularly abundant in mossy fibre nerve terminals in the hippoc us. In primary cultured hippoc al neurones Sept3 immunoreactivity was punctate in neurites and predominantly localized to presynaptic terminals, strongly colocalizing with synaptophysin and dynamin I. The specific nerve terminal localization was confirmed by immunogold electron microscopy. Together this shows that Sept3 is a neurone-specific protein highly enriched in nerve terminals which supports a secretory role in synaptic vesicle recycling.

IQGAP1 is associated with nuclear envelope reformation and completion of abscission

Publisher: Informa UK Limited

Date: 10-06-2015

DOI: 10.1080/15384101.2015.1044168

Pan-cancer proteomic map of 949 human cell lines

Publisher: Elsevier BV

Date: 08-2022

DOI: 10.1016/J.CCELL.2022.06.010

Label-Free Quantitation of Phosphopeptide Changes During Rat Sperm Capacitation

Publisher: American Chemical Society (ACS)

Date: 08-01-2010

DOI: 10.1021/PR900513D

Abstract: Before fertilization can occur, ejaculated mammalian spermatozoa must undergo a maturation process known as capacitation, which is dominated by post-translational modifications, particularly phosphorylation. Despite its biological importance, characterization of those proteins targeted for phosphorylation during capacitation remains ill-defined. Here, we report the isolation and purification of 288 phosphorylated peptides from rat spermatozoa using titanium dioxide columns in combination with nanoflow mass spectrometry. This equated to 120 identified phosphorylated proteins present in pure populations of spermatozoa. The MS survey scans of replicate titanium dioxide eluates, derived from both noncapacitated and capacitated sperm lysates, were then compared in silico using a virtual 2D PAGE format and DeCyderMS software. This analysis found 15 differentially phosphorylated proteins during capacitation. Included in this list were sperm qualifiers such as Izumo, a known sperm-oocyte fusion protein. To demonstrate that this label-free quantitative approach to phosphoprotein analysis was viable, we measured the enzymatic activity of 5'-nucleotidase, the phosphorylation status of which changed during capacitation. The results revealed, for the first time, that 5'-nucleotidase activity is up-regulated as sperm capacitate. This change, together with the other protein identifications reported in this study, constitute important new leads in elucidating the biochemical mechanisms by which spermatozoa attain a capacitated state.

Rapid Analysis of Synaptic Vesicle Endocytosis in Synaptosomes

Publisher: Humana Press

Date: 03-10-2014

DOI: 10.1007/978-1-62703-676-4_9

Syndapin – a membrane remodelling and endocytic F‐BAR protein

Publisher: Wiley

Date: 05-07-2013

DOI: 10.1111/FEBS.12343

Abstract: Syndapin [also called PACSIN (protein kinase C and casein kinase II interacting protein)] is an Fes-CIP4 homology Bin- hiphysin-Rvs161/167 (F-BAR) and Src-homology 3 domain-containing protein. Three genes give rise to three main isoforms in mammalian cells. They each function in different endocytic and vesicle trafficking pathways and provide critical links between the cytoskeletal network in different cellular processes, such as neuronal morphogenesis and cell migration. The membrane remodelling activity of syndapin via its F-BAR domain and its interaction partners, such as dynamin and neural Wiskott-Aldrich syndrome protein binding to its Src-homology 3 domain, are important with respect to its function. Its various partner proteins provide insights into its mechanism of action, as well as its differential roles in these cellular processes. Signalling pathways leading to the regulation of syndapin function by phosphorylation are now contributing to our understanding of the broader functions of this family of proteins.

Unique Phosphorylation of Protein Kinase C-α in PC12 Cells Induces Resistance to Translocation and Down-regulation

Publisher: Elsevier BV

Date: 12-1996

DOI: 10.1074/JBC.271.49.31718

Clathrin Terminal Domain-Ligand Interactions Regulate Sorting of Mannose 6-Phosphate Receptors Mediated by AP-1 and GGA Adaptors

Publisher: Elsevier BV

Date: 02-2014

DOI: 10.1074/JBC.M113.535211

Strategies to enable large-scale proteomics for reproducible research

Publisher: Springer Science and Business Media LLC

Date: 30-07-2020

DOI: 10.1038/S41467-020-17641-3

Abstract: Reproducible research is the bedrock of experimental science. To enable the deployment of large-scale proteomics, we assess the reproducibility of mass spectrometry (MS) over time and across instruments and develop computational methods for improving quantitative accuracy. We perform 1560 data independent acquisition (DIA)-MS runs of eight s les containing known proportions of ovarian and prostate cancer tissue and yeast, or control HEK293T cells. Replicates are run on six mass spectrometers operating continuously with varying maintenance schedules over four months, interspersed with ~5000 other runs. We utilise negative controls and replicates to remove unwanted variation and enhance biological signal, outperforming existing methods. We also design a method for reducing missing values. Integrating these computational modules into a pipeline (ProNorM), we mitigate variation among instruments over time and accurately predict tissue proportions. We demonstrate how to improve the quantitative analysis of large-scale DIA-MS data, providing a pathway toward clinical proteomics.

Simian Hemorrhagic Fever Virus Cell Entry Is Dependent on CD163 and Uses a Clathrin-Mediated Endocytosis-Like Pathway

Publisher: American Society for Microbiology

Date: 2015

DOI: 10.1128/JVI.02697-14

Abstract: Simian hemorrhagic fever virus (SHFV) causes a severe and almost uniformly fatal viral hemorrhagic fever in Asian macaques but is thought to be nonpathogenic for humans. To date, the SHFV life cycle is almost completely uncharacterized on the molecular level. Here, we describe the first steps of the SHFV life cycle. Our experiments indicate that SHFV enters target cells by low-pH-dependent endocytosis. Dynamin inhibitors, chlorpromazine, methyl-β-cyclodextrin, chloroquine, and concanamycin A dramatically reduced SHFV entry efficiency, whereas the macropinocytosis inhibitors EIPA, blebbistatin, and wortmannin and the caveolin-mediated endocytosis inhibitors nystatin and filipin III had no effect. Furthermore, overexpression and knockout study and electron microscopy results indicate that SHFV entry occurs by a dynamin-dependent clathrin-mediated endocytosis-like pathway. Experiments utilizing latrunculin B, cytochalasin B, and cytochalasin D indicate that SHFV does not hijack the actin polymerization pathway. Treatment of target cells with proteases (proteinase K, papain, α-chymotrypsin, and trypsin) abrogated entry, indicating that the SHFV cell surface receptor is a protein. Phospholipases A2 and D had no effect on SHFV entry. Finally, treatment of cells with antibodies targeting CD163, a cell surface molecule identified as an entry factor for the SHFV-related porcine reproductive and respiratory syndrome virus, diminished SHFV replication, identifying CD163 as an important SHFV entry component. IMPORTANCE Simian hemorrhagic fever virus (SHFV) causes highly lethal disease in Asian macaques resembling human illness caused by Ebola or Lassa virus. However, little is known about SHFV's ecology and molecular biology and the mechanism by which it causes disease. The results of this study shed light on how SHFV enters its target cells. Using electron microscopy and inhibitors for various cellular pathways, we demonstrate that SHFV invades cells by low-pH-dependent, actin-independent endocytosis, likely with the help of a cellular surface protein.

Phosphorylation of Dynamin I on Ser-795 by Protein Kinase C Blocks Its Association with Phospholipids

Publisher: Elsevier BV

Date: 04-2000

DOI: 10.1074/JBC.275.16.11610

Region and diagnosis-specific changes in synaptic proteins in schizophrenia and bipolar I disorder

Publisher: Elsevier BV

Date: 07-2010

DOI: 10.1016/J.PSYCHRES.2008.07.012

Abstract: Aberrant regulation of synaptic function is thought to play a role in the aetiology of psychiatric disorders, including schizophrenia and bipolar disorder. Normal neurotransmitter release is dependent on a complex group of presynaptic proteins that regulate synaptic vesicle docking, membrane fusion and fission, including synaptophysin, syntaxin, synaptosomal-associated protein-25 (SNAP-25), vesicle-associated membrane protein (VAMP), alpha-synuclein and dynamin I. In addition, structural and signalling proteins such as neural cell adhesion molecule (NCAM) maintain the integrity of the synapse. We have assessed the levels of these important synaptic proteins using Western blots, in three cortical regions (BA10, 40 and 46) obtained post-mortem from subjects with bipolar 1 disorder, schizophrenia or no history of a psychiatric disorder. In bipolar 1 disorder cortex (parietal BA40), we found a significant increase in the expression of SNAP-25, and a significant reduction in alpha-synuclein compared with controls. These changes in presynaptic protein expression are proposed to inhibit synaptic function in bipolar 1 disorder. In schizophrenia, a significant reduction in the ratio of the two major membrane-bound forms of NCAM (180 and 140) was observed in BA10. The distinct functions of these two NCAM forms suggest that changes in the comparative levels of these proteins could lead to a destabilisation of synaptic signalling. Our data support the notion that there are complex and region-specific alterations in presynaptic proteins that may lead to alterations in synaptic activity in both schizophrenia and bipolar disorder.

Steroids from an Australian sponge Psammoclema sp.

Publisher: American Chemical Society (ACS)

Date: 08-01-2009

DOI: 10.1021/NP800688F

Abstract: Investigation of an extract of the Australian marine sponge Psammoclema sp. for dynamin I inhibitory activity led to the isolation of four new trihydroxysterols (1-4) related to aragusterol G. These compounds were largely identified by 1D and 2D NMR spectroscopic methods. While 1 was found to be inactive in the dynamin bioassay, bioassays did reveal that compounds 1-4 inhibited the growth of colorectal, breast, ovarian, and prostate cancer cell lines (GI(50) 5-27 microM). The additional insight that these new compounds give to previous SAR studies is discussed briefly.

Enhanced protein recovery and reproducibility from pull-down assays and immunoprecipitations using spin columns

Publisher: Elsevier BV

Date: 08-2001

DOI: 10.1006/ABIO.2001.5215

Shutting the gate: targeting endocytosis in acute leukemia

Publisher: Elsevier BV

Date: 12-2021

DOI: 10.1016/J.EXPHEM.2021.09.003

Abstract: Endocytosis entails selective packaging of cell surface cargos in cytoplasmic vesicles, thereby controlling key intrinsic cellular processes as well as the response of normal and malignant cells to their microenvironment. The purpose of this review is to outline the latest advances in the development of endocytosis-targeting therapeutic strategies in hematological malignancies.

Dynamin GTPase regulated by protein kinase C phosphorylation in nerve terminals

Publisher: Springer Science and Business Media LLC

Date: 09-1993

DOI: 10.1038/365163A0

Abstract: Dynamin is a microtubule-binding protein with a microtubule-activated GTPase activity. The gene encoding dynamin is mutated in shibire, a Drosophila mutant defective in endocytosis in nerve terminals and other cells. These observations place dynamin into two distinct functional contexts, suggesting roles in microtubule-based motility or in endocytosis. We report here that dynamin is identical to the neuronal phosphoprotein dephosphin (P96), originally identified by its stimulus-dependent dephosphorylation in nerve terminals. Dynamin is a protein doublet of M(r) 94 and 96K arising by alternative splicing of its primary transcript. In the nerve terminal, both forms of dynamin are phosphorylated by protein kinase C (PKC) and are quantitatively dephosphorylated on excitation. In vitro, dynamin is also phosphorylated by casein kinase II which inhibits PKC phosphorylation. Phosphorylation by PKC but not by casein kinase II enhances the GTPase activity of dynamin 12-fold. The dynamins are therefore a group of nerve terminal phosphoproteins whose GTPase is regulated by phosphorylation in parallel with synaptic vesicle recycling. The regulation of dynamin GTPase could serve as the trigger for the rapid endocytosis of synaptic vesicles after exocytosis.

Dynamin regulates L cell secretion in human gut

Publisher: Elsevier BV

Date: 09-2021

DOI: 10.1016/J.MCE.2021.111398

Abstract: The mechanochemical enzyme dynamin mediates endocytosis and regulates neuroendocrine cell exocytosis. Enteroendocrine L cells co-secrete the anorectic gut hormones glucagon-like peptide 1 (GLP-1) and peptide YY (PYY) postprandially and is a potential therapeutic target for metabolic diseases. In the present study, we aimed to determine if dynamin is implicated in human L cell secretion. Western blot was performed on the murine L cell line GLUTag. Static incubation of human colonic mucosae with activators and inhibitors of dynamin was carried out. GLP-1 and PYY contents of the secretion supernatants were assayed using ELISA. s: Both dynamin I and II are expressed in GLUTag cells. The dynamin activator Ryngo 1-23 evoked significant GLP-1 and PYY release from human colonic mucosae while the dynamin inhibitor Dynole 3-42 significantly inhibited release triggered by known L cell secretagogues. Thus, the cell signaling regulator dynamin is able to bi-directionally regulate L cell hormone secretion in the human gut and may represent a novel target for gastrointestinal-targeted metabolic drug development.

Depolarization-dependent protein phosphorylation in rat cortical synaptosomes: The effects of calcium, strontium and barium

Publisher: Elsevier BV

Date: 12-1983

DOI: 10.1016/0304-3940(83)90133-7

Abstract: Depolarization of synaptosomes increases the phosphorylation of a number of proteins in a calcium-dependent manner. The concentration of calcium required for optimum stimulation was 0.1 mM, with higher concentrations up to 2.5 mM being progressively less effective. Calcium was significantly better than strontium at increasing depolarization-dependent protein phosphorylation, while barium had no stimulating effect at concentrations above 0.1 mM. The order of potency of these ions is consistent with a calmodulin-stimulated protein kinase being activated on entry of calcium into synaptosomes, but is not consistent with the known efficacy of these ions in stimulating neurotransmitter release. The data show for the first time that phosphorylation of proteins may not be a prerequisite for neurotransmitter release.

Phosphorylation of dynamin II at serine-764 is associated with cytokinesis

Publisher: Elsevier BV

Date: 10-2011

DOI: 10.1016/J.BBAMCR.2010.12.018

Abstract: Calcineurin is a phosphatase that is activated at the last known stage of mitosis, abscission. Among its many substrates, it dephosphorylates dynamin II during cytokinesis at the midbody of iding cells. However, dynamin II has several cellular roles including clathrin-mediated endocytosis, centrosome cohesion and cytokinesis. It is not known whether dynamin II phosphorylation plays a role in any of these functions nor have the phosphosites involved in cytokinesis been directly identified. We now report that dynamin II from rat lung is phosphorylated to a low stoichiometry on a single major site, Ser-764, in the proline-rich domain. Phosphorylation on Ser-764 also occurred in asynchronously growing HeLa cells and was greatly increased upon mitotic entry. Tryptic phospho-peptides isolated by TiO(2) chromatography revealed only a single phosphosite in mitotic cells. Mitotic phosphorylation was abolished by roscovitine, suggesting the mitotic kinase is cyclin-dependent kinase 1. Cyclin-dependent kinase 1 phosphorylated full length dynamin II and Glutathione-S-Transferase-tagged-dynamin II-proline-rich domain in vitro, and mutation of Ser-764 to alanine reduced proline-rich domain phosphorylation by 80%, supporting that there is only a single major phosphosite. Ser-764 phosphorylation did not affect clathrin-mediated endocytosis or bulk endocytosis using penetratin-based phospho-deficient or phospho-mimetic peptides or following siRNA depletion/rescue experiments. Phospho-dynamin II was enriched at the mitotic centrosome, but this targeting was unaffected by the phospho-deficient or phospho-mimetic peptides. In contrast, the phospho-mimetic peptide displaced endogenous dynamin II, but not calcineurin, from the midbody and induced cytokinesis failure. Therefore, phosphorylation of dynamin II primarily occurs on a single site that regulates cytokinesis downstream of calcineurin, rather than regulating endocytosis or centrosome function.

HIV infection is influenced by dynamin at 3 independent points in the viral life cycle

Publisher: Wiley

Date: 11-04-2017

DOI: 10.1111/TRA.12481

Abstract: CD4 T cells are important cellular targets for HIV-1, yet the primary site of HIV fusion remains unresolved. Candidate fusion sites are either the plasma membrane or from within endosomes. One area of investigation compounding the controversy of this field, is the role of the protein dynamin in the HIV life cycle. To understand the role of dynamin in primary CD4 T cells we combined dynamin inhibition with a series of complementary assays based on single particle tracking, HIV fusion, detection of HIV DNA products and active viral transcription. We identify 3 levels of dynamin influence on the HIV life cycle. Firstly, dynamin influences productive infection by preventing cell cycle progression. Secondly, dynamin influences endocytosis rates and increases the probability of endosomal fusion. Finally, we provide evidence in resting CD4 T cells that dynamin directly regulates the HIV fusion reaction at the plasma membrane. We confirm this latter observation using 2 ergent dynamin modulating compounds, one that enhances dynamin conformations associated with dynamin ring formation (ryngo-1-23) and the other that preferentially targets dynamin conformations that appear in helices (dyngo-4a). This in-depth understanding of dynamin's roles in HIV infection clarifies recent controversies and furthermore provides evidence for dynamin regulation specifically in the HIV fusion reaction.

Targeting Glioma Stem Cells by Functional Inhibition of Dynamin 2: A Novel Treatment Strategy for Glioblastoma

Publisher: Informa UK Limited

Date: 16-03-2019

DOI: 10.1080/07357907.2019.1582060

Abstract: Glioma stem cells (GSCs) play major roles in drug resistance, tumour maintenance and recurrence of glioblastoma. We investigated inhibition of the GTPase dynamin 2 as a therapy for glioblastoma. Glioma cell lines and patient-derived GSCs were treated with dynamin inhibitors, Dynole 34-2 and CyDyn 4-36. We studied about cell viability, and GSC neurosphere formation in vitro and orthotopic tumour growth in vivo. Dynamin inhibition reduced glioblastoma cell line viability and suppressed neurosphere formation and migration of GSCs. Tumour growth was reduced by CyDyn 4-36 treatment. Dynamin 2 inhibition therefore represents a novel approach for stem cell-directed Glioblastoma therapy.

Identification of the 49-kDa Autoantigen Associated with Lymphocytic Hypophysitis as  -Enolase

Publisher: The Endocrine Society

Date: 02-2002

DOI: 10.1210/JC.87.2.752

PICK1 interacts with PACSIN to regulate AMPA receptor internalization and cerebellar long-term depression

Publisher: Proceedings of the National Academy of Sciences

Date: 05-08-2013

DOI: 10.1073/PNAS.1312467110

Abstract: The dynamic trafficking of AMPA receptors (AMPARs) into and out of synapses is crucial for synaptic transmission, plasticity, learning, and memory. The protein interacting with C-kinase 1 (PICK1) directly interacts with GluA2/3 subunits of the AMPARs. Although the role of PICK1 in regulating AMPAR trafficking and multiple forms of synaptic plasticity is known, the exact molecular mechanisms underlying this process remain unclear. Here, we report a unique interaction between PICK1 and all three members of the protein kinase C and casein kinase II substrate in neurons (PACSIN) family and show that they form a complex with AMPARs. Our results reveal that knockdown of the neuronal-specific protein, PACSIN1, leads to a significant reduction in AMPAR internalization following the activation of NMDA receptors in hippoc al neurons. The interaction between PICK1 and PACSIN1 is regulated by PACSIN1 phosphorylation within the variable region and is required for AMPAR endocytosis. Similarly, the binding of PICK1 to the ubiquitously expressed PACSIN2 is also regulated by the homologous phosphorylation sites within the PACSIN2-variable region. Genetic deletion of PACSIN2, which is highly expressed in Purkinje cells, eliminates cerebellar long-term depression. This deficit can be fully rescued by overexpressing wild-type PACSIN2, but not by a PACSIN2 phosphomimetic mutant, which does not bind PICK1 efficiently. Taken together, our data demonstrate that the interaction of PICK1 and PACSIN is required for the activity-dependent internalization of AMPARs and for the expression of long-term depression in the cerebellum.

Repurposing molecular mechanisms of transmitter release: a new job for syndapin at the fusion pore. Focus on “Syndapin 3 modulates fusion pore expansion in mouse neuroendocrine chromaffin cells

Publisher: American Physiological Society

Date: 05-2014

DOI: 10.1152/AJPCELL.00079.2014

Protein phosphorylation is required for endocytosis in nerve terminals: potential role for the dephosphins dynamin I and synaptojanin, but not AP180 or amphiphysin.

Publisher: Wiley

Date: 2001

DOI: 10.1046/J.1471-4159.2001.00049.X

Abstract: Dynamin I and at least five other nerve terminal proteins, hiphysins I and II, synaptojanin, epsin and eps15 (collectively called dephosphins), are coordinately dephosphorylated by calcineurin during endocytosis of synaptic vesicles. Here we have identified a new dephosphin, the essential endocytic protein AP180. Blocking dephosphorylation of the dephosphins is known to inhibit endocytosis, but the role of phosphorylation has not been determined. We show that the protein kinase C (PKC) antagonists Ro 31-8220 and Go 7874 block the rephosphorylation of dynamin I and synaptojanin that occurs during recovery from an initial depolarizing stimulus (S1). The rephosphorylation of AP180 and hiphysins 1 and 2, however, were unaffected by Ro 31-8220. Although these dephosphins share a single phosphatase, different protein kinases phosphorylated them after nerve terminal stimulation. The inhibitors were used to selectively examine the role of dynamin I and/or synaptojanin phosphorylation in endocytosis. Ro 31-8220 and Go 7874 did not block the initial S1 cycle of endocytosis, but strongly inhibited endocytosis following a second stimulus (S2). Therefore, phosphorylation of a subset of dephosphins, which includes dynamin I and synaptojanin, is required for the next round of stimulated synaptic vesicle retrieval.

The in Vivo Phosphorylation and Glycosylation of Human Insulin-like Growth Factor-binding Protein-5

Publisher: Elsevier BV

Date: 08-2007

DOI: 10.1074/MCP.M700027-MCP200

Dopamine and serotonin in two populations of synaptosomes isolated by percoll gradient centrifugation

Publisher: Elsevier BV

Date: 1986

DOI: 10.1016/0197-0186(86)90088-4

Abstract: A technique has recently been developed for the isolation of synaptosomes by centrifugation through percoll gradients. Utilizing this procedure, striatal synaptosomes were separated into two fractions, termed fractions 3 and 4, by their different sedimentation characteristics in percoll. The aim of this investigation was to determine whether there were any neurotransmitter differences between these fractions. The content of endogenous neurotransmitters dopamine (DA) and serotonin (5-HT) significantly differed between these fractions. Fraction 3 contained greater levels of 5-HT, while fraction 4 was enriched for DA. Both fractions were capable of releasing DA or 5-HT upon K(+) depolarization. The results raise the possibility that a relative enrichment of dopaminergic synaptosomes in fraction 4 and of serotonergic synaptosomes in fraction 3 has been achieved.

The Extracellular Domain of the Growth Hormone Receptor Interacts with Coactivator Activator to Promote Cell Proliferation

Publisher: The Endocrine Society

Date: 09-2008

DOI: 10.1210/ME.2008-0128

Abstract: The presence of GH receptor (GHR) in the cell nucleus correlates with cell ision, and targeting the GHR to the nucleus results in constitutive proliferation and transformation because of increased sensitivity to autocrine GH. Here we have sought additional mechanisms that might account for the enhanced proliferation seen with nuclear GHR, commencing with a yeast two-hybrid (Y2H) screen for interactors with the extracellular domain of the GHR [GH-binding protein (GHBP)]. We find that the GHBP is a transcriptional activator in yeast and mammalian cells, and this activity resides in the lower cytokine receptor module. Activity is dependent on S226, the conserved serine of the cytokine receptor consensus WSXWS box. By using parallel GHBP affinity columns and tandem mass spectrometry of tryptic digests of proteins bound to wild-type GHBP and S226A columns, we identified proteins that bind to the transcriptionally active GHBP. These include a nucleoporin and two transcriptional regulators, notably the coactivator activator (CoAA), which is also an RNA binding splicing protein. Binding of CoAA to the GHBP was confirmed by glutathione S-transferase pulldown and coimmunoprecipitation, and shown to be GH dependent in pro-B Ba/F3 cells. Importantly, stable expression of CoAA in Ba/F3 cells resulted in an increased maximum proliferation in response to GH, but not IL-3. Because CoAA overexpression has been identified in many cancers and its stable expression promotes cell proliferation and cell transformation in NIH-3T3 cells, we suggest CoAA contributes to the proliferative actions of nuclear GHR by the hormone-dependent recruitment of this powerful coactivator to the GHR.

Identification of a family of DNA-binding proteins with homology to RNA splicing factors

Publisher: Canadian Science Publishing

Date: 02-2006

DOI: 10.1139/O05-139

Abstract: We describe a unique family of human proteins that are capable of binding to the cAMP regulatory element (CRE) and that are homologous to RNA splicing proteins. A human cDNA was isolated that encodes a protein with a distinctive combination of modular domain structures: 2 leucine-zipper-like domains, a DNA-binding zinc-finger-like domain, an RNA-binding zinc-finger-like domain, and 2 coiled-coil protein–protein interaction domains. It also has a serine–arginine - rich domain, commonly found in proteins involved in RNA splicing. The protein was discovered using the CRE as bait in a yeast 1-hybrid assay. It was then shown to bind specifically to the CRE in vitro using gel shift assays. We have named the protein CRE-associated protein (CREAP). We show that it is widely expressed in human tissues but is highly expressed in several fetal tissues and in several regions of the adult brain. CREAP is closely related to 2 human proteins of unknown function. CREAP shows significant homology with a small nuclear ribonucleoprotein of yeast, Luc7p, involved in 5′ splice site recognition. The 3 human CREAP proteins form a unique family with the potential to act as transcription factors that link to RNA processing.Key words: multifunctional protein, zinc finger, bZIP, transcription factor, splicing factor, protein family, CRH, CRE.

Two mechanisms of synaptic vesicle recycling in rat brain nerve terminals.

Publisher: Wiley

Date: 10-2000

DOI: 10.1046/J.1471-4159.2000.0751645.X

Abstract: KCl and 4-aminopyridine (4-AP) evoke glutamate release from rat brain cortical nerve terminals by voltage cl ing or by Na(+) channel-generated repetitive action potentials, respectively. Stimulation by 4-AP but not KCl is largely mediated by protein kinase C (PKC). To determine whether KCl and 4-AP utilise the same mechanism to release glutamate, we correlated glutamate release with release of the hydrophobic synaptic vesicle (SV) marker FM2-10. A strong correlation was observed for increasing concentrations of KCl and after application of phorbol 12-myristate 13-acetate (PMA) or staurosporine. The parallel increase in exocytosis measured by two approaches suggested it occurred by a PKC-independent mechanism involving complete fusion of SVs with the plasma membrane. At low concentrations of 4-AP, alone or with staurosporine, glutamate and FM2-10 release also correlated. However, higher concentrations of 4-AP or of 4-AP plus PMA greatly increased glutamate release but did not further increase FM2-10 release. This ergence suggests that 4-AP recruits an additional mechanism of release during strong stimulation that is PKC dependent and is superimposed upon the first mechanism. This second mechanism is characteristic of kiss-and-run, which is not detectable by styryl dyes. Our data suggest that glutamate release in nerve terminals occurs via two mechanisms: (1) complete SV fusion, which is PKC independent and (2) a kiss-and-run-like mechanism, which is PKC dependent. Recruitment of a second release mechanism may be a widespread means to facilitate neurotransmitter release in central neurons.

Dynamin regulates the fusion pore of endo- and exocytotic vesicles as revealed by membrane capacitance measurements

Publisher: Elsevier BV

Date: 09-2017

DOI: 10.1016/J.BBAGEN.2017.06.022

Abstract: Dynamin is a multidomain GTPase exhibiting mechanochemical and catalytic properties involved in vesicle scission from the plasmalemma during endocytosis. New evidence indicates that dynamin is also involved in exocytotic release of catecholamines, suggesting the existence of a dynamin-regulated structure that couples endo- to exocytosis. Thus we here employed high-resolution cell-attached capacitance measurements and super-resolution structured illumination microscopy to directly examine single vesicle interactions with the plasmalemma in cultured rat astrocytes treated with distinct pharmacological modulators of dynamin activity. Fluorescent dextrans and the lipophilic plasmalemmal marker DiD were utilized to monitor uptake and distribution of vesicles in the peri-plasmalemmal space and in the cell cytosol. Dynamin inhibition with Dynole™-34-2 and Dyngo™-4a prevented vesicle internalization into the cytosol and decreased fusion pore conductance of vesicles that remained attached to the plasmalemma via a narrow fusion pore that lapsed into a state of repetitive opening and closing - flickering. In contrast, the dynamin activator Ryngo™-1-23 promoted vesicle internalization and favored fusion pore closure by prolonging closed and shortening open fusion pore dwell times. Immunocytochemical staining revealed dextran uptake into dynamin-positive vesicles and increased dextran uptake into Syt4- and VAMP2-positive vesicles after dynamin inhibition, indicating prolonged retention of these vesicles at the plasmalemma. Our results have provided direct evidence for a role of dynamin in regulation of fusion pore geometry and kinetics of endo- and exocytotic vesicles, indicating that both share a common dynamin-regulated structural intermediate, the fusion pore.

Identification and Characterisation of the RalA-ERp57 Interaction: Evidence for GDI Activity of ERp57

Publisher: Public Library of Science (PLoS)

Date: 30-11-2012

DOI: 10.1371/JOURNAL.PONE.0050879

Addressing the Challenges of High‐Throughput Cancer Tissue Proteomics for Clinical Application: ProCan

Publisher: Wiley

Date: 24-09-2019

DOI: 10.1002/PMIC.201900109

Abstract: The cancer tissue proteome has enormous potential as a source of novel predictive biomarkers in oncology. Progress in the development of mass spectrometry (MS)-based tissue proteomics now presents an opportunity to exploit this by applying the strategies of comprehensive molecular profiling and big-data analytics that are refined in other fields of 'omics research. ProCan (ProCan is a registered trademark) is a program aiming to generate high-quality tissue proteomic data across a broad spectrum of cancer types. It is based on data-independent acquisition-MS proteomic analysis of annotated tissue s les sourced through collaboration with expert clinical and cancer research groups. The practical requirements of a high-throughput translational research program have shaped the approach that ProCan is taking to address challenges in study design, s le preparation, raw data acquisition, and data analysis. The ultimate goal is to establish a large proteomics knowledge-base that, in combination with other cancer 'omics data, will accelerate cancer research.

Optimization of calmodulin-affinity chromatography for brain and organelles

Publisher: Elsevier BV

Date: 09-2015

DOI: 10.1016/J.EUPROT.2015.05.004

Phosphorylation of syndapin I F-BAR domain at two helix-capping motifs regulates membrane tubulation.

Publisher: Proceedings of the National Academy of Sciences

Date: 21-02-2012

DOI: 10.1073/PNAS.1108294109

Abstract: Syndapin I (PACSIN 1) is a synaptically enriched membrane tubulating protein that plays important roles in activity-dependent bulk endocytosis and neuronal morphogenesis. While syndapin I is an in vitro phosphoprotein, it is not known to be phosphorylated in neurons. Here, we report the identification of two phosphorylation sites, S76 and T181, of syndapin I from nerve terminals. Both residues are located at the N-terminal helix-capping motifs (N-Cap) of different α-helices in the F-BAR domain, important for F-BAR homodimer curvature and dimer-dimer filament assembly, respectively. Phospho-mimetic mutations of these residues regulate lipid-binding and tubulation both in vitro and in cells. Neither phosphosite regulated syndapin I function in activity-dependent bulk endocytosis. Rather, T181 phosphorylation was developmentally regulated and inhibited syndapin I function in neuronal morphogenesis. This suggests a novel mechanism for phosphorylation control of an F-BAR function through the regulation of α-helix interactions and stability within the folded F-BAR domain.

A comparative study of the role of adenylate cyclase in the release of adrenocorticotropin from the ovine and rat anterior pituitary

Publisher: Elsevier BV

Date: 05-1994

DOI: 10.1016/0303-7207(94)90232-1

Abstract: The interaction between corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) is important in the regulation of adrenocorticotropin (ACTH) release from the anterior pituitary (AP). CRF exerts its effect on the AP by activating the adenylate cyclase (AC) complex whereas AVP increases the turnover of phosphatidylinositol. In the rat and in man, CRF is the most potent ACTH secretagogue whereas AVP alone is only a weak agonist. Since recent studies in the sheep indicate a reversal of this order of potency, these studies were undertaken to test the hypothesis that a functional alteration of the AC in the ovine corticotrope might limit the ability of CRF to release ACTH from these cells. When rat AP cells were incubated with CRF, a dose-dependent increase in AC activity was observed. This effect was potentiated either by AVP or PMA, although neither agent alone altered AC activity. In contrast, CRF alone, or in combination with AVP or PMA, did not increase AC activity in ovine AP cells. Both cholera toxin (CT) and pertussis toxin (PT) caused a dose-dependent release of ACTH from rat and ovine AP cells, but the amount of ACTH released from the ovine AP cells by both agents was relatively reduced. In the ovine cells, however, AVP acted synergistically with CT or PT to markedly increase the release of ACTH to levels which approached those obtained when the rat AP cells were exposed to CT or PT alone. Forskolin increased AC activity in AP cells of both species, but to a much lower extent in ovine cells than in the rat cells. However, when the ovine cells were exposed to AVP, the AC response to forskolin became similar to the response observed in the rat cells when incubated with forskolin alone. Forskolin also released significantly less ACTH from the ovine AP cells, but AVP also acted synergistically with forskolin to greatly enhance the amount of ACTH released from these cells. Finally, 8-bromo-cyclic AMP produced a similar release of ACTH from both ovine and rat AP cells. We conclude that: (1) the decreased ability of CRF to increase ACTH release from the ovine AP reflects a net decrease in AC activity and cannot be ascribed to an ovine corticotropic resistance to cAMP (2) the decreased activity of the ovine corticotropic AC complex may in turn reflect functional alterations at the level of both the G proteins and the catalytic subunit (3) since AVP causes protein kinase C substrate phosphorylation in the ovine AP, AVP may increase AC activity in this tissue by phosphorylating the G proteins and/or the catalytic subunit.

The actin-binding and bundling protein, EPLIN, is required for cytokinesis

Publisher: Informa UK Limited

Date: 03-2009

DOI: 10.4161/CC.8.5.7878

Abstract: Cytokinesis involves two phases: (1) membrane ingression followed by (2) membrane abscission. The ingression phase generates a cleavage furrow and this requires co-operative function of the actin-myosin II contractile ring and septin filaments. We demonstrate that the actin-binding protein, EPLIN, locates to the cleavage furrow during cytokinesis and this is possibly via association with the contractile ring components, myosin II and the septin, Sept2. Depletion of EPLIN results in formation of multinucleated cells and this is associated with inefficient accumulation of active myosin II (MRLC(S19)) and Sept2 and their regulatory small GTPases, RhoA and Cdc42, respectively, to the cleavage furrow during the final stages of cytokinesis. We suggest that EPLIN may function during cytokinesis to maintain local accumulation of key cytokinesis proteins at the furrow.

The Brain Exocyst Complex Interacts with RalA in a GTP-dependent Manner

Publisher: Elsevier BV

Date: 08-2001

DOI: 10.1074/JBC.C100320200

A synthetic peptide analog of the putative substrate‐binding motif activates protein kinase C

Publisher: Wiley

Date: 05-06-1989

DOI: 10.1016/0014-5793(89)80632-5

Abstract: A 29-residue synthetic peptide, Leu530-Leu-Tyr-Glu-Met-Leu-Ala-Gly-Gln-Ala-Pro-Phe-Glu-Gly-Glu-Asp -Glu-Asp- Glu-Leu-Phe-Gln-Ser-Ile-Met-Glu-His-Asn-Val-NH2(558), corresponding to part of the catalytic domain of protein kinase C, is a potent activator of the enzyme, with a Ka of approx. 10 microM. Activation was 59 +/- 4% of that observed with phosphatidylserine, predominantly due to an increased Vmax, partially calcium-dependent, observed with all three isoenzymes (alpha, beta, gamma), and resulted in autophosphorylation. It is proposed that the region between Gly528 and Arg583 is part of the protein substrate binding region of protein kinase C and synthetic peptide analogs of this region activate the enzyme by blocking the action of the enzyme's basic pseudosubstrate autoregulatory region.

Dimerization of CtIP, a BRCA1- and CtBP-interacting protein, is mediated by an N-terminal coiled-coil motif

Publisher: Elsevier BV

Date: 06-2004

DOI: 10.1074/JBC.M313974200

Multisite phosphorylation of doublecortin by cyclin-dependent kinase 5

Publisher: Portland Press Ltd.

Date: 06-07-2004

DOI: 10.1042/BJ20040324

Abstract: Doublecortin (DCX) is a 40 kDa microtubule-associated protein required for normal neural migration and cortical layering during development. Mutations in the human DCX gene cause a disruption of cortical neuronal migration. Defects in cdk5 (cyclin-dependent kinase 5) also cause defects in neural migration and cortical layering. DCX is a substrate for cdk5 in vitro and in vivo and the major site of in vitro phosphorylation is Ser-297. We used a highly developed MS strategy to identify the cdk5 phosphorylation sites and determine the major and minor sites. Several phosphopeptides were identified from a tryptic digest of 32P-labelled, cdk5-phosphorylated DCX using a combination of off-line HPLC and matrix-assisted laser-desorption ionization-MS with alkaline phosphatase treatment. Tandem MS/MS enabled the identification of seven phosphorylation sites for cdk5. Monitoring of 32P label indicated that there was one major site, Ser-28, at the N-terminus, and a major site, Ser-339, in the serine roline-rich domain at the C-terminus. Five other sites, Ser-287, Thr-289, Ser-297, Thr-326 and Ser-332, were also found in the tail. Site-directed mutagenesis largely supported these findings. Single mutation of Ser-28 reduced but did not abolish phosphorylation. Double, rather than single, mutation for Ser-332 and Ser-339 was required to reduce overall phosphorylation, suggesting an interaction between these sites. Truncations of the tail produced a significant reduction in cdk5 phosphorylation of DCX. These results do not support Ser-297 as the major cdk5 phosphorylation site in DCX, but indicate that DCX is subject to complex multisite phosphorylation. This illustrates the importance of a well-developed MS strategy to identify phosphorylation sites.

Evidence that the stimulation by arginine vasopressin of the release of adrenocorticotropin from the ovine anterior pituitary involves the activation of protein kinase C

Publisher: Elsevier BV

Date: 09-1992

DOI: 10.1016/0303-7207(92)90231-T

Abstract: These studies were undertaken to evaluate the role of protein kinase C (PKC) in the regulation by arginine vasopressin (AVP) of adrenocorticotropin (ACTH) secretion from the ovine anterior pituitary. AVP caused the rapid translocation of PKC from the cytosol to the cell membrane in ovine anterior pituitary cells that was maximal at 5 min. This phenomenon, which is a known concomitant of C-kinase activation, was produced to a greater extent by phorbol 12-myristate 13-acetate (PMA) but not by corticotropin-releasing factor (CRF). To determine whether AVP activated corticotrope PKC, we assessed the ability of three different PKC inhibitors (H-7, sphingosine, and retinal) to modify basal, AVP-, PMA-, and CRF-stimulated ACTH release. In addition to inhibiting the in vitro activity of purified PKC, each compound also caused in vitro inhibition of the protein kinase A (PKA) catalytic subunit, indicating that none could be considered to be a specific inhibitor of PKC and the PKA catalytic subunit. As determined by the mean IC50 values required for the in vitro inhibition of PKC and the PKA catalytic subunit, sphingosine was judged to be the most selective and H-7 the least selective PKC inhibitor. A 4 h exposure to each inhibitor caused a dose-dependent increase in basal ACTH release and attenuation of both AVP- and PMA-stimulated ACTH release. H-7 and retinal, in concentrations that caused a 20-50% inhibition of PKA, also attenuated CRF-stimulated ACTH release however, this effect was not observed with sphingosine in concentrations that caused only a 10-20% inhibition of PKA. We conclude that: (1) AVP causes the direct activation of PKC in the ovine anterior pituitary and that C kinase activation is important in mediating the effect of AVP on ACTH release (2) the finding that inhibition of PKC elevates ACTH suggests that basal ACTH secretion is also partly regulated by PKC (3) since CRF does not cause PKC translocation in ovine anterior pituitary cells, it is unlikely that PKC plays a physiological role in the action of CRF on the corticotrope (4) the finding that H-7 and retinal attenuate CRF-stimulated ACTH secretion suggests that CRF activates PKA in corticotropes.

How to Fill a Synapse

Publisher: American Association for the Advancement of Science (AAAS)

Date: 27-04-2007

DOI: 10.1126/SCIENCE.1142705

Phosphorylation regulates copper-responsive trafficking of the Menkes copper transporting P-type ATPase

Publisher: Elsevier BV

Date: 12-2009

DOI: 10.1016/J.BIOCEL.2009.06.008

The phosphorylation of p25/TPPP by LIM kinase 1 inhibits its ability to assemble microtubules

Publisher: Elsevier BV

Date: 12-2007

DOI: 10.1016/J.YEXCR.2007.08.012

Abstract: LIM kinase 1 (LIMK1) is a key regulator of actin dynamics as it phosphorylates and inactivates cofilin, an actin-depolymerizing factor. LIMK1 activity is also required for microtubule disassembly in endothelial cells. A search for LIMK1-interacting proteins identified p25alpha, a phosphoprotein that promotes tubulin polymerization. We found that p25 is phosphorylated by LIMK1 on serine residues in vitro and in cells. Immunoblotting analysis revealed that p25 is not a brain specific protein as previously reported, but is expressed in all mouse tissues. Immunofluorescence analysis demonstrated that endogenous p25 is co-localized with microtubules and is also found in the nucleus. Down-regulation of p25 by siRNA decreased microtubule levels while its overexpression in stable NIH-3T3 cell lines increased cell size and levels of stable tubulin. Bacterially expressed unphosphorylated p25 promotes microtubule assembly in vitro however, when phosphorylated in cells, p25 lost its ability to assemble microtubule. Our results represent a surprising connection between the tubulin and the actin cytoskeleton mediated by LIMK1. We propose that the LIMK1 phosphorylation of p25 blocks p25 activity, thus promoting microtubule disassembly.

Myristyl Trimethyl Ammonium Bromide and Octadecyl Trimethyl Ammonium Bromide Are Surface-Active Small Molecule Dynamin Inhibitors that Block Endocytosis Mediated by Dynamin I or Dynamin II

Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)

Date: 16-08-2007

DOI: 10.1124/MOL.107.034207

Abstract: Dynamin is a GTPase enzyme involved in membrane constriction and fission during endocytosis. Phospholipid binding via its pleckstrin homology domain maximally stimulates dynamin activity. We developed a series of surface-active small-molecule inhibitors, such as myristyl trimethyl ammonium bromide (MiTMAB) and octadecyltrimethyl ammonium bromide (OcTMAB), and we now show MiTMAB targets the dynamin-phospholipid interaction. MiTMAB inhibited dynamin GTPase activity, with a Ki of 940 +/- 25 nM. It potently inhibited receptor-mediated endocytosis (RME) of transferrin or epidermal growth factor (EGF) in a range of cells without blocking EGF binding, receptor number, or autophosphorylation. RME inhibition was rapidly reversed after washout. The rank order of potency for a variety of MiTMAB analogs on RME matched the rank order for dynamin inhibition, suggesting dynamin recruitment to the membrane is a primary cellular target. MiTMAB also inhibited synaptic vesicle endocytosis in rat brain nerve terminals (synaptosomes) without inducing depolarization or morphological defects. Therefore, the drug rapidly and reversibly blocks multiple forms of endocytosis with no acute cellular damage. The unique mechanism of action of MiTMAB provides an important tool to better understand dynamin-mediated membrane trafficking events in a variety of cells.

Inhibition of clathrin by pitstop 2 activates the spindle assembly checkpoint and induces cell death in dividing HeLa cancer cells

Publisher: Springer Science and Business Media LLC

Date: 17-01-2013

DOI: 10.1186/1476-4598-12-4

Abstract: During metaphase clathrin stabilises the mitotic spindle kinetochore(K)-fibres. Many anti-mitotic compounds target microtubule dynamics. Pitstop 2™ is the first small molecule inhibitor of clathrin terminal domain and inhibits clathrin-mediated endocytosis. We investigated its effects on a second function for clathrin in mitosis. Pitstop 2 did not impair clathrin recruitment to the spindle but disrupted its function once stationed there. Pitstop 2 trapped HeLa cells in metaphase through loss of mitotic spindle integrity and activation of the spindle assembly checkpoint, phenocopying clathrin depletion and aurora A kinase inhibition. Pitstop 2 is therefore a new tool for investigating clathrin spindle dynamics. Pitstop 2 reduced viability in iding HeLa cells, without affecting iding non-cancerous NIH3T3 cells, suggesting that clathrin is a possible novel anti-mitotic drug target.

A tachykinin peptide receptor joins an elite club

Publisher: Elsevier BV

Date: 1988

DOI: 10.1016/0165-6147(88)90228-3

Abstract: To examine mediators of mifepristone treatment on improvements in health-related quality of life (HRQOL) among women with symptomatic fibroids. The study s le included women with symptomatic uterine fibroids who were treated with 5mg or 2.5mg of mifepristone or placebo. Assessments of uterine size (ultrasound), pain (McGill pain questionnaire), bleeding (diary), anemia (gm/dL), and HRQOL measured using the uterine fibroid symptom quality of life scale were done at baseline, 3 months, and 6 months. The improvements in HRQOL that could be explained by changes in these clinical factors were assessed. The final s le included 62 women. Treatment with mifepristone was associated with significant improvement in HRQOL, which was explained in part by reduction in pain (28%, P<0.001) and bleeding (18%, P<0.001). Reduction in uterine volume was of marginal significance (P=0.05) and was associated with a decrease in HRQOL (7%). Much of the impact of treatment on HRQOL (61%) remained unexplained in this model. Improvements in HRQOL after treatment with mifepristone are partly explained by improvements in pain and bleeding, but not uterine size. However, most of the improvement in HRQOL is not explained by improvements in these clinical parameters.

Pyrimidyn Compounds: Dual-Action Small Molecule Pyrimidine-Based Dynamin Inhibitors

Publisher: American Chemical Society (ACS)

Date: 30-05-2013

DOI: 10.1021/CB400137P

Mutations in the SPTLC1 protein cause mitochondrial structural abnormalities and endoplasmic reticulum stress in lymphoblasts

Publisher: Mary Ann Liebert Inc

Date: 07-2014

DOI: 10.1089/DNA.2013.2182

Abstract: Mutations in serine palmitoyltransferase long chain subunit 1 (SPTLC1) cause the typical length-dependent axonal degeneration hereditary sensory neuropathy type 1 (HSN1). Transmission electron microscopy studies on SPTLC1 mutant lymphoblasts derived from patients revealed specific structural abnormalities of mitochondria. Swollen mitochondria with abnormal cristae were clustered around the nucleus, with some mitochondria being wrapped in rough endoplasmic reticulum (ER) membranes. Total mitochondrial counts revealed a significant change in mitochondrial numbers between healthy and diseased lymphocytes but did not reveal any change in length to width ratios nor were there any changes to cellular function. However, there was a notable change in ER homeostasis, as assessed using key ER stress markers, BiP and ERO1-Lα, displaying reduced protein expression. The observations suggest that SPTLC1 mutations cause mitochondrial abnormalities and ER stress in HSN1 cells.

Pituitary autoantibodies

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 08-2006

DOI: 10.1097/01.MED.0000235324.46788.47

Dynamin II function is required for EGF-mediated Stat3 activation but not Erk1/2 phosphorylation

Publisher: Informa UK Limited

Date: 11-05-2012

DOI: 10.3109/08977194.2012.683189

Abstract: Signalling from receptor tyrosine kinases is elicited by ligand binding which initiates the activation of many downstream signalling cascades. Endocytosis has been widely accepted as one mechanism in which cells inactivate signalling by internalising and subsequently degrading activated receptors. However, it is now evident that endocytosis of signalling receptors is important in initiation and sustaining downstream signalling. We and others have previously shown that epidermal growth factor receptor (EGFR) overexpression and activation of signal transducer and activator of transcription 3 (Stat3) are associated with tumourigenicity. Here, we examine the role of endocytosis in EGFR signal attenuation and differential signalling. Inhibition of dynamin II (Dyn II), a GTPase required for endocytosis, with a small molecular weight inhibitor, led to reduced EGF-mediated Stat3 phosphorylation and transcriptional activity in the A431 and HN5 human tumour cell lines. However, Dyn II inhibition had minimal effect on EGF-mediated EGFR and Erk1/2 phosphorylation, which is often regarded responsible for the tumourigenic function of the EGFR. Interestingly, this effect on Stat3 activation was not due to reduced EGFR/Stat3 association. Likewise, cells transfected with Dyn II siRNA or stably transfected with Dyn II shRNA had reduced EGF-mediated phospho-Stat3 levels but similar EGF-mediated phospho-EGFR and phospho-Erk1/2 levels compared with controls. Dyn II siRNA also reduced Stat3 transcriptional reporter activity and inhibits Stat3 accumulating into the nucleus. Taken together, our data suggest that the activation status of Stat3 and Erk1/2 and the sustainability of these signals are potentially due to the spatial and temporal control of the EGFR within the cell. This notion may have implications on therapeutic targeting and efficacy when using inhibitors to proteins either regulating endocytosis and/or signalling.

The phospho-dependent dynamin-syndapin interaction triggers activity-dependent bulk endocytosis of synaptic vesicles.

Publisher: Society for Neuroscience

Date: 17-06-2009

DOI: 10.1523/JNEUROSCI.1976-09.2009

Basal and KCl-stimulated corticotropin-releasing hormone release from human placental syncytiotrophoblasts is inhibited by sodium nitroprusside

Publisher: The Endocrine Society

Date: 08-1994

DOI: 10.1210/JC.79.2.519

Calcineurin activity is required for the completion of cytokinesis

Publisher: Springer Science and Business Media LLC

Date: 23-05-2010

DOI: 10.1007/S00018-010-0401-Z

Abstract: Successful completion of cytokinesis requires the spatio-temporal regulation of protein phosphorylation and the coordinated activity of protein kinases and phosphatases. Many mitotic protein kinases are well characterized while mitotic phosphatases are largely unknown. Here, we show that the Ca(2+)- and calmodulin-dependent phosphatase, calcineurin (CaN), is required for cytokinesis in mammalian cells, functioning specifically at the abscission stage. CaN inhibitors induce multinucleation in HeLa cells and prolong the time cells spend connected via an extended intracellular bridge. Upon Ca(2+) influx during cytokinesis, CaN is activated, targeting a set of proteins for dephosphorylation, including dynamin II (dynII). At the intracellular bridge, phospho-dynII and CaN are co-localized to dual flanking midbody rings (FMRs) that reside on either side of the central midbody ring. CaN activity and disassembly of the FMRs coincide with abscission. Thus, CaN activity at the midbody plays a key role in regulating the completion of cytokinesis in mammalian cells.

The pathogenesis of ACTA1‐related congenital fiber type disproportion

Publisher: Wiley

Date: 23-03-2007

DOI: 10.1002/ANA.21112

Abstract: Mutations in ACTA1 have been associated with a variety of changes in muscle histology that likely result from fundamental differences in the way that ACTA1 mutations disrupt muscle function. Recently, we reported three patients with congenital fiber type disproportion (CFTD) caused by novel heterozygous missense mutations in ACTA1 (D292V, L221P, P332S) with marked type 1 fiber hypotrophy as the only pathological finding on muscle biopsy. We have investigated the basis for the histological differences between these CFTD patients and patients with ACTA1 nemaline myopathy (NM). Mass spectrometry and two-dimensional gel electrophoresis demonstrate that mutant actin accounts for 25 and 50% of alpha-skeletal actin in the skeletal muscle of patients with the P332S and D292V mutations, respectively, consistent with a dominant-negative disease mechanism. In vitro motility studies indicate that abnormal interactions between actin and tropomyosin are the likely principal cause of muscle weakness for D292V, with tropomyosin stabilized in the "switched off" position. Both the D292V and P322S CFTD mutations are associated with normal sarcomeric structure on electron microscopy, which is atypical for severe NM. In contrast, we found no clear difference between ACTA1 mutations associated with NM and CFTD in tendency to polymerize or aggregate in C2C12 expression models. These data suggest that ACTA1 CFTD mutations cause weakness by disrupting sarcomere function rather than structure. We raise the possibility that the presence or absence of structural disorganization when mutant actin incorporates into sarcomeres may be an important determinant of whether the histological patterns of CFTD or NM develop in ACTA1 myopathy.

The in vitro phosphorylation of actin from rat cerebral cortex

Publisher: Springer Science and Business Media LLC

Date: 07-1983

DOI: 10.1007/BF00964547

Abstract: KSHV is endemic in Uganda and the HIV epidemic has dramatically increased the incidence of Kaposi sarcoma (KS). To investigate the role of KSHV in the development of KS, we obtained KS biopsies from ART-naïve, HIV-positive in iduals in Uganda and analyzed the tumors using RNAseq to globally characterize the KSHV transcriptome. Phylogenetic analysis of ORF75 sequences from 23 tumors revealed 6 distinct genetic clusters with KSHV strains exhibiting M, N or P alleles. RNA reads mapping to specific unique coding sequence (UCDS) features were quantitated using a gene feature file previously developed to globally analyze and quantitate KSHV transcription in infected endothelial cells. A pattern of high level expression was detected in the KSHV latency region that was common to all KS tumors. The clear majority of transcription was derived from the downstream latency transcript promoter P3(LTd) flanking ORF72, with little evidence of transcription from the P1(LTc) latency promoter, which is constitutive in KSHV-infected lymphomas and tissue-culture cells. RNAseq data provided evidence of alternate P3(LTd) transcript editing, splicing and termination resulting in multiple gene products, with 90% of the P3(LTd) transcripts spliced to release the intronic source of the microRNAs K1-9 and 11. The spliced transcripts encode a regulatory uORF upstream of Kaposin A with alterations in intervening repeat sequences yielding novel or deleted Kaposin B/C-like sequences. Hierarchical clustering and PCA analysis of KSHV transcripts revealed three clusters of tumors with different latent and lytic gene expression profiles. Paradoxically, tumors with a latent phenotype had high levels of total KSHV transcription, while tumors with a lytic phenotype had low levels of total KSHV transcription. Morphologically distinct KS tumors from the same in idual showed similar KSHV gene expression profiles suggesting that the tumor microenvironment and host response play important roles in the activation level of KSHV within the infected tumor cells.

Role of dynamin in elongated cell migration in a 3D matrix

Publisher: Elsevier BV

Date: 03-2015

DOI: 10.1016/J.BBAMCR.2014.12.008

Abstract: The use of 3-dimensional (3D) collagen gels has yielded new insights into the migratory behaviour of cancer cells. While the large GTPase dynamin has emerged as an important regulator of cancer cell migration and invasion under 2D conditions, its role in 3D migration is unclear. We have used a potent dynamin modulator, a bis-tyrphostin derivative, Ryngo® 1-23, to investigate the role of dynamin in 3D migration in 3 different cell lines. The compound specifically inhibits persistent, elongated 3D migration in U87MG and SMA-560 cells. Treated U87MG cells adopt a rounded morphology that is not due to apoptosis, loss of matrix metalloprotease activity or inhibition of clathrin-mediated endocytosis. Given that Ryngo 1-23 is known to regulate dynamin oligomerisation and actin dynamics at the leading edge, we analysed actin filament distribution. Ryngo 1-23 induced a switch in actin filament organization in 3D cultures resulting in the generation of multiple short actin-rich microspikes. Correlated with the change in actin filament distribution, cells displayed reduced collagen gel contraction. Since acto-myosin force transmission to the extra-cellular matrix underpins persistent, elongated migration, our results suggest that Ryngo 1-23 modulates this process in 3D migration via dynamin-mediated regulation of acto-myosin force transmission to the extra-cellular matrix.

Analysis of synaptic vesicle endocytosis in synaptosomes by high-content screening

Publisher: Springer Science and Business Media LLC

Date: 05-07-2012

DOI: 10.1038/NPROT.2012.070

Abstract: Small molecules modulating synaptic vesicle endocytosis (SVE) may ultimately be useful for diseases where pathological neurotransmission is implicated. Only a small number of specific SVE modulators have been identified to date. Slow progress is due to the laborious nature of traditional approaches to study SVE, in which nerve terminals are identified and studied in cultured neurons, typically yielding data from 10-20 synapses per experiment. We provide a protocol for a quantitative, high-throughput method for studying SVE in thousands of nerve terminals. Rat forebrain synaptosomes are attached to 96-well microplates and depolarized SVE is then quantified by uptake of the dye FM4-64, which is imaged by high-content screening. Synaptosomes that have been frozen and stored can be used in place of fresh synaptosomes, reducing the experimental time and animal numbers required. With a supply of frozen synaptosomes, the assay can be performed within a day, including data analysis.

Dynamin inhibitors induce caspase-mediated apoptosis following cytokinesis failure in human cancer cells and this is blocked by Bcl-2 overexpression

Publisher: Springer Science and Business Media LLC

Date: 28-06-2011

DOI: 10.1186/1476-4598-10-78

Abstract: The aim of both classical (e.g. taxol) and targeted anti-mitotic agents (e.g. Aurora kinase inhibitors) is to disrupt the mitotic spindle. Such compounds are currently used in the clinic and/or are being tested in clinical trials for cancer treatment. We recently reported a new class of targeted anti-mitotic compounds that do not disrupt the mitotic spindle, but exclusively block completion of cytokinesis. This new class includes MiTMAB and OcTMAB (MiTMABs), which are potent inhibitors of the endocytic protein, dynamin. Like other anti-mitotics, MiTMABs are highly cytotoxic and possess anti-proliferative properties, which appear to be selective for cancer cells. The cellular response following cytokinesis failure and the mechanistic pathway involved is unknown. We show that MiTMABs induce cell death specifically following cytokinesis failure via the intrinsic apoptotic pathway. This involves cleavage of caspase-8, -9, -3 and PARP, DNA fragmentation and membrane blebbing. Apoptosis was blocked by the pan-caspase inhibitor, ZVAD, and in HeLa cells stably expressing the anti-apoptotic protein, Bcl-2. This resulted in an accumulation of polyploid cells. Caspases were not cleaved in MiTMAB-treated cells that did not enter mitosis. This is consistent with the model that apoptosis induced by MiTMABs occurs exclusively following cytokinesis failure. Cytokinesis failure induced by cytochalasin B also resulted in apoptosis, suggesting that disruption of this process is generally toxic to cells. Collectively, these data indicate that MiTMAB-induced apoptosis is dependent on both polyploidization and specific intracellular signalling components. This suggests that dynamin and potentially other cytokinesis factors are novel targets for development of cancer therapeutics.

Actin- and Dynamin-Dependent Maturation of Bulk Endocytosis Restores Neurotransmission following Synaptic Depletion

Publisher: Public Library of Science (PLoS)

Date: 22-05-2012

DOI: 10.1371/JOURNAL.PONE.0036913

A Case Study and Methodology for OpenSWATH Parameter Optimization Using the ProCan90 Data Set and 45 810 Computational Analysis Runs

Publisher: American Chemical Society (ACS)

Date: 17-01-2019

DOI: 10.1021/ACS.JPROTEOME.8B00709

Abstract: In the current study, we show how ProCan90, a curated data set of HEK293 technical replicates, can be used to optimize the configuration options for algorithms in the OpenSWATH pipeline. Furthermore, we use this case study as a proof of concept for horizontal scaling of such a pipeline to allow 45 810 computational analysis runs of OpenSWATH to be completed within four and a half days on a budget of US $10 000. Through the use of Amazon Web Services (AWS), we have successfully processed each of the ProCan 90 files with 506 combinations of input parameters. In total, the project consumed more than 340 000 core hours of compute and generated in excess of 26 TB of data. Using the resulting data and a set of quantitative metrics, we show an analysis pathway that allows the calculation of two optimal parameter sets, one for a compute rich environment (where run time is not a constraint), and another for a compute poor environment (where run time is optimized). For the same input files and the compute rich parameter set, we show a 29.8% improvement in the number of quality protein (>2 peptide) identifications found compared to the current OpenSWATH defaults, with negligible adverse effects on quantification reproducibility or drop in identification confidence, and a median run time of 75 min (103% increase). For the compute poor parameter set, we find a 55% improvement in the run time from the default parameter set, at the expense of a 3.4% decrease in the number of quality protein identifications, and an intensity CV decrease from 14.0% to 13.7%.

Author Correction: Small molecule inhibition of Dynamin-dependent endocytosis targets multiple niche signals and impairs leukemia stem cells

Publisher: Springer Science and Business Media LLC

Date: 19-02-2021

DOI: 10.1038/S41467-021-21688-1

Abstract: A Correction to this paper has been published: 0.1038/s41467-021-21688-1

Mechanisms of synaptic vesicle recycling illuminated by fluorescent dyes.

Publisher: Wiley

Date: 12-1999

DOI: 10.1046/J.1471-4159.1999.0732227.X

Abstract: The recycling of synaptic vesicles in nerve terminals involves multiple steps, underlies all aspects of synaptic transmission, and is a key to understanding the basis of synaptic plasticity. The development of styryl dyes as fluorescent molecules that label recycling synaptic vesicles has revolutionized the way in which synaptic vesicle recycling can be investigated, by allowing an examination of processes in neurons that have long been inaccessible. In this review, we evaluate the major aspects of synaptic vesicle recycling that have been revealed and advanced by studies with styryl dyes, focussing upon synaptic vesicle fusion, retrieval, and trafficking. The greatest impact of styryl dyes has been to allow the routine visualization of endocytosis in central nerve terminals for the first time. This has revealed the kinetics of endocytosis, its underlying sequential steps, and its regulation by Ca2+. In studies of exocytosis, styryl dyes have helped distinguish between different modes of vesicle fusion, provided direct support for the quantal nature of exocytosis and endocytosis, and revealed how the probability of exocytosis varies enormously from one nerve terminal to another. Synaptic vesicle labelling with styryl dyes has helped our understanding of vesicle trafficking by allowing better understanding of different synaptic vesicle pools within the nerve terminal, vesicle intermixing, and vesicle clustering at release sites. Finally, the dyes are now being used in innovative ways to reveal further insights into synaptic plasticity.

Identification of TPIT and other novel autoantigens in lymphocytic hypophysitis; immunoscreening of a pituitary cDNA library and development of immunoprecipitation assays

Publisher: Oxford University Press (OUP)

Date: 03-2012

DOI: 10.1530/EJE-11-1015

Abstract: Lymphocytic hypophysitis is an organ-specific autoimmune disease of the pituitary gland. A specific and sensitive serological test currently does not exist to aid in the diagnosis. To identify target autoantigens in lymphocytic hypophysitis and develop a diagnostic assay for these proteins. A pituitary cDNA expression library was immunoscreened using sera from four patients with lymphocytic hypophysitis. Relevant cDNA clones from screening, along with previously identified autoantigens pituitary gland-specific factor 1a and 2 (PGSF1a and PGSF2) and neuron-specific enolase (NSE) were tested in an in vitro transcription and translation immunoprecipitation assay. The corticotroph-specific transcription factor, TPIT, was investigated separately as a candidate autoantigen. Significantly positive autoantibody reactivity against TPIT was found in 9/86 hypophysitis patients vs 1/90 controls ( P =0.018). The reactivity against TPIT was not specific for lymphocytic hypophysitis with autoantibodies detectable in the sera from patients with other autoimmune endocrine diseases. Autoantibodies were also detected against chromodomain-helicase-DNA binding protein 8, presynaptic cytomatrix protein (piccolo), Ca 2 + -dependent secretion activator, PGSF2 and NSE in serum s les from patients with lymphocytic hypophysitis, but at a frequency that did not differ from healthy controls. Importantly, 8/86 patients with lymphocytic hypophysitis had autoantibodies against any two autoantigens in comparison with 0/90 controls ( P =0.0093). TPIT, a corticotroph-specific transcription factor, was identified as a target autoantigen in 10.5% of patients with lymphocytic hypophysitis. Further autoantigens related to vesicle processing were also identified as potential autoantigens with different immunoreactivity patterns in patients and controls.

Membrane fission by dynamin: what we know and what we need to know

Publisher: EMBO

Date: 26-09-2016

DOI: 10.15252/EMBJ.201694613

Inhibition of Dynamin Mediated Endocytosis by the Dynoles—Synthesis and Functional Activity of a Family of Indoles

Publisher: American Chemical Society (ACS)

Date: 21-05-2009

DOI: 10.1021/JM900036M

Abstract: Screening identified two bisindolylmaleimides as 100 microM inhibitors of the GTPase activity of dynamin I. Focused library approaches allowed development of indole-based dynamin inhibitors called dynoles. 100-Fold in vitro enhancement of potency was noted with the best inhibitor, 2-cyano-3-(1-(2-(dimethylamino)ethyl)-1H-indol-3-yl)-N-octylacrylamide (dynole 34-2), a 1.3 +/- 0.3 microM dynamin I inhibitor. Dynole 34-2 potently inhibited receptor mediated endocytosis (RME) internalization of Texas red-transferrin. The rank order of potency for a variety of dynole analogues on RME in U2OS cells matched their rank order for dynamin inhibition, suggesting that the mechanism of inhibition is via dynamin. Dynoles are the most active dynamin I inhibitors reported for in vitro or RME evaluations. Dynole 34-2 is 15-fold more active than dynasore against dynamin I and 6-fold more active against dynamin mediated RME (IC(50) approximately 15 microM RME IC(50) approximately 80 microM). The dynoles represent a new series of tools to better probe endocytosis and dynamin-mediated trafficking events in a variety of cells.

Ral: Mediator of membrane trafficking

Publisher: Elsevier BV

Date: 2006

DOI: 10.1016/J.BIOCEL.2006.04.006

Abstract: Ral is a multifunctional small GTPase involved in tumorigenesis and in controlling intracellular membrane trafficking. It is mainly activated by factors downstream of Ras, or independently of these factors and operates by protein-protein interactions with an expanding repertoire of partners. RalA is a positive regulator of calcium-evoked exocytosis via binding phospholipase D and is involved in G protein coupled receptor signalling by binding phospholipase C-delta1. The binding of Ral to calmodulin links to intracellular trafficking events. Another link is direct binding of activated Ral (Ral-GTP) to the endocytic and exocytic machineries. Ral-GTP binds RalBP1, which connects to receptor-mediated endocytosis via AP-2. Alternatively, Ral-GTP binds the exocyst complex, which controls secretory vesicle trafficking in regulated secretion and filopodia formation. Thus, Ral-GTP "chooses" between different membrane trafficking pathways. Other Ral partners are still being uncovered that may provide further mechanistic insights into how Ral controls erse membrane trafficking pathways.

Engineering plasticization resistant gas separation membranes using metal–organic nanocapsules

Publisher: Royal Society of Chemistry (RSC)

Date: 2020

DOI: 10.1039/D0SC01498B

Abstract: Ultra-small metal–organic nanocapsules (MONCs) with open metal sites (OMSs) are used as multi-dentate nodes to form coordinative crosslinking networks with polymers.

From Spanish fly to room-temperature ionic liquids (RTILs): synthesis, thermal stability and inhibition of dynamin 1 GTPase by a novel class of RTILs

Publisher: Royal Society of Chemistry (RSC)

Date: 2008

DOI: 10.1039/B707092F

Phosphorylation of septin 3 on Ser-91 by cGMP-dependent protein kinase-I in nerve terminals

Publisher: Portland Press Ltd.

Date: 27-07-2004

DOI: 10.1042/BJ20040455

Abstract: The septins are a family of GTPase enzymes required for cytokinesis and play a role in exocytosis. Among the ten vertebrate septins, Sept5 (CDCrel-1) and Sept3 (G-septin) are primarily concentrated in the brain, wherein Sept3 is a substrate for PKG-I (cGMP-dependent protein kinase-I) in nerve terminals. There are two motifs for potential PKG-I phosphorylation in Sept3, Thr-55 and Ser-91, but phosphoamino acid analysis revealed that the primary site is a serine. Derivatization of phosphoserine to S-propylcysteine followed by N-terminal sequence analysis revealed Ser-91 as a major phosphorylation site. Tandem MS revealed a single phosphorylation site at Ser-91. Substitution of Ser-91 with Ala in a synthetic peptide abolished phosphorylation. Mutation of Ser-91 to Ala in recombinant Sept3 also abolished PKG phosphorylation, confirming that Ser-91 is the major site in vitro. Antibodies raised against a peptide containing phospho-Ser-91 detected phospho-Sept3 only in the cytosol of nerve terminals, whereas Sept3 was located in a peripheral membrane extract. Therefore Sept3 is phosphorylated on Ser-91 in nerve terminals and its phosphorylation may contribute to the regulation of its subcellular localization in neurons.

High-Content Drug Discovery Screening of Endocytosis Pathways

Publisher: Springer US

Date: 22-11-2020

DOI: 10.1007/978-1-0716-1044-2_5

Dynamin1 long- and short-tail isoforms exploit distinct recruitment and spatial patterns to form endocytic nanoclusters

Publisher: Research Square Platform LLC

Date: 14-03-2023

DOI: 10.21203/RS.3.RS-2641489/V1

Abstract: Endocytosis requires a coordinated framework of molecular interactions that ultimately lead to the fission of nascent endosomes. How cytosolic proteins, such as dynamin, timely concentrate at discrete sites that are sparsely distributed across the plasma membrane remains poorly understood. Two dynamin 1 (Dyn1) major splice variants differ by the length of their C-terminal proline-rich region (short-tail and long-tail). Using sptPALM in PC12 cells, neurons and MEF cells, we demonstrate that short-tail Dyn1 isoforms Dyn1ab and Dyn1bb display an activity-dependent recruitment to the membrane, promptly followed by concentration into nanoclusters. These nanoclusters were sensitive to both Calcineurin and Dyn1 GTPase inhibitors, and were larger, denser, and more numerous than that of long-tail isoform Dyn1aa. Spatiotemporal modelling confirmed that Dyn1 isoforms perform distinct search patterns and undergo dimensional reduction to generate endocytic nanoclusters, with short-tail isoforms more robustly exploiting lateral trapping in the generation of nanoclusters compared to long-tail isoform Dyn1aa.

Dephosphin/dynamin is a neuronal phosphoprotein concentrated in nerve terminals: Evidence from rat cerebellum

Publisher: Elsevier BV

Date: 02-1995

DOI: 10.1016/0306-4522(94)00337-5

Abstract: Dephosphin/dynamin is a 94,000/96,000 mol. wt protein kinase C substrate from rat brain that is phosphorylated in nerve terminals and dephosphorylated upon stimulation of exocytosis and synaptic vesicle recycling. Phosphorylation activates an intrinsic GTPase activity and dephosphin may play a role in endocytosis [Robinson P. J. et al. (1993) Nature 365, 163-166]. In this study a specific polyclonal antiserum to dephosphin was used to investigate its distribution in rat brain by immunoblotting and immunocytochemistry. Immunoblots of various organs of the rat showed that dephosphin was detectable only in the whole brain and not in the testes, lung, kidney, adrenals, heart, liver or skeletal muscle. Immunoblotting of various regions of the brain revealed high levels of dephosphin, particularly in the hippoc us, cerebellum and cerebral cortex, but its absence from the anterior pituitary. Synaptosomes were prepared from these three regions and labelled with 32Pi for 60 min, followed by incubation in control or 41 mM K+ depolarizing buffer. Dephosphin was present in each region and was stoichiometrically dephosphorylated by depolarization, indicating the presence and regulation of dephosphin in intact cerebellar nerve terminals. The cerebellum was selected for detailed study, using conventional light and confocal microscopy, owing to its ordered and well-characterized structure. Immunostaining was abundant within the cerebellar cortex and deep cerebellar nuclei, but almost entirely absent from the medulla. In the cortex many neuronal cells contained dephosphin-like immunoreactivity which was also evident in perikarya, axons, and nerve terminals. Dephosphin-like immunoreactivity was not detected in the radial Bergman glial cells. The greatest concentrations were observed in synaptic terminals, particularly in granular layer glomeruli and basket cell terminals surrounding Purkinje cell bodies and dendrites. Dephosphin therefore appears to be exclusive to neuronal tissue, but is distributed widely throughout the brain. It is located in many neuronal cell types of the cerebellum and may be particularly enriched in synaptic terminals, where it is regulated by phosphorylation and dephosphorylation. This distribution suggests a role for dephosphin in synaptic vesicle cycling in nerve terminals.

Phosphorylation of phospholamban in aortic smooth muscle cells and heart by calcium/calmodulin-dependent protein kinase II

Publisher: Elsevier BV

Date: 08-1994

DOI: 10.1016/0898-6568(94)90045-0

Abstract: Phospholamban is a negative regulator of the sarcoplasmic reticulum Ca(2+)-pumping ATPase. Phosphorylation of phospholamban activates the ATPase and decreases the level of cytosolic calcium. Phospholamban is phosphorylated in heart by cAMP-dependent protein kinase, cGMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase II (CM-kinase-II) and in smooth muscle cells by cGMP-dependent protein kinase. In contrast to heart muscle, phospholamban is poorly phosphorylated by CM-kinase-II in extracts of rat aortic smooth muscle cells. Rat aorta phospholamban amino acid sequence was identical to dog heart. The peptide substrate specificity of CM-kinase-II from rat aorta was the same as that from rat heart. The lack of phosphorylation of rat aorta phospholamban by the CM-kinase-II appears to result from the relatively low abundance of phospholamban in smooth muscle.

Phosphorylation of splicing factor SF1 on Ser20 by cGMP-dependent protein kinase regulates spliceosome assembly

Publisher: Wiley

Date: 16-08-1999

DOI: 10.1093/EMBOJ/18.16.4549

Telomerase Recognizes G-Quadruplex and Linear DNA as Distinct Substrates

Publisher: American Chemical Society (ACS)

Date: 18-09-2007

DOI: 10.1021/BI700993Q

Abstract: Telomeric DNA can assemble into a nonlinear, higher-order conformation known as a G-quadruplex. Here, we demonstrate by electrospray ionization mass spectrometry that the two repeat telomeric sequence d(TGGGGTTGGGGT) from Tetrahymena thermophila gives rise to a novel parallel four-stranded G-quadruplex in the presence of sodium. The G-quadruplex directly interacts with the catalytic subunit of Tetrahymena telomerase (TERT) with micromolar affinity, and the presence of telomerase RNA is not obligatory for this interaction. Both N- and C-terminal halves of TERT bind the G-quadruplex independently. This G-quadruplex is a robust substrate for both recombinant and cell extract-derived telomerase in vitro. Furthermore, the G-quadruplex weakens the affinity of wild-type telomerase for the incoming nucleotide (dTTP) and likely perturbs the nucleotide binding pocket of the enzyme. In agreement with this, a lysine to alanine substitution at amino acid 538 (K538A) within motif 1 of TERT dramatically reduces the ability of telomerase to extend G-quadruplex but not linear DNA. The K538A mutant retains binding affinity for the quadruplex. This suggests that telomerase undergoes changes in conformation in its active site to specifically accommodate binding and subsequent extension of G-quadruplex DNA. We propose that telomerase recognizes G-quadruplex DNA as a substrate that is distinct from linear DNA.

Green chemistry approaches to the Knoevenagel condensation: comparison of ethanol, water and solvent free (dry grind) approaches

Publisher: Elsevier BV

Date: 04-2002

DOI: 10.1016/S0040-4039(02)00480-X

Endocytosis Inhibition in Humans to Improve Responses to ADCC-Mediating Antibodies

Publisher: Elsevier BV

Date: 03-2020

DOI: 10.1016/J.CELL.2020.02.019

Small molecule inhibition of Dynamin-dependent endocytosis targets multiple niche signals and impairs leukemia stem cells

Publisher: Springer Science and Business Media LLC

Date: 04-12-2020

DOI: 10.1038/S41467-020-20091-6

Abstract: Intensive chemotherapy for acute leukemia can usually induce complete remission, but fails in many patients to eradicate the leukemia stem cells responsible for relapse. There is accumulating evidence that these relapse-inducing cells are maintained and protected by signals provided by the microenvironment. Thus, inhibition of niche signals is a proposed strategy to target leukemia stem cells but this requires knowledge of the critical signals and may be subject to compensatory mechanisms. Signals from the niche require receptor-mediated endocytosis, a generic process dependent on the Dynamin family of large GTPases. Here, we show that Dynole 34-2, a potent inhibitor of Dynamin GTPase activity, can block transduction of key signalling pathways and overcome chemoresistance of leukemia stem cells. Our results provide a significant conceptual advance in therapeutic strategies for acute leukemia that may be applicable to other malignancies in which signals from the niche are involved in disease progression and chemoresistance.

Pyrimidine-Based Inhibitors of Dynamin I GTPase Activity: Competitive Inhibition at the Pleckstrin Homology Domain

Publisher: American Chemical Society (ACS)

Date: 20-12-2016

DOI: 10.1021/ACS.JMEDCHEM.6B01422

Abstract: The large GTPase dynamin mediates membrane fission during clathrin-mediated endocytosis (CME). The aminopyrimidine compounds were reported to disrupt dynamin localization to the plasma membrane via the PH domain and implicate this mechanism in the inhibition of CME. We have used a computational approach of binding site identification, docking, and interaction energy calculations to design and synthesize a new library of aminopyrimidine analogues targeting site-2 of the pleckstrin homology (PH) domain. The optimized analogues showed low micromolar inhibition against both dynamin I (IC

Long chain amines and long chain ammonium salts as novel inhibitors of dynamin GTPase activity

Publisher: Elsevier BV

Date: 06-2004

DOI: 10.1016/J.BMCL.2004.03.096

Chapter 12 Phosphoproteomics

Publisher: Elsevier

Date: 2008

DOI: 10.1016/S0166-526X(08)00212-2

Pituitary autoantibodies in lymphocytic hypophysitis target both gamma- and alpha-Enolase - a link with pregnancy?

Publisher: Informa UK Limited

Date: 2002

DOI: 10.1076/APAB.110.1.94.897

Abstract: The first target autoantigen to have been identified in lymphocytic hypophysitis is a 49 kDa protein, identified as alpha-enolase. Pituitary autoimmunity is strongly associated with pregnancy and we have shown that pituitary autoantibodies from patients with peripartum lymphocytic hypophysitis also recognise enolase in the placenta. Enolase exists in different forms as a number of isoenzymes, which are homo- or heterodimers of three subunits, alpha, beta and gamma. alphaalpha-enolase is ubiquitous, betabeta-enolase is muscle-specific and gammagamma-enolase, which is restricted to neuronal tissue and neuroendocrine cells, is known as neuron-specific enolase (NSE). NSE is expressed in normal human pituitary and pituitary neoplasms. The current study investigated which isoforms of enolase in pituitary and placenta reacted with the sera of patients with lymphocytic hypophysitis. Immunoblotting of two-dimensional gels of human pituitary cytosolic proteins showed that autoantibodies in patient sera react with both an acidic form, and more neutral forms of enolase. Immunoblotting with a monoclonal antibody to NSE confirmed the identity of the acidic enolase isoform as the gammagamma-isoform in both pituitary and placental s les. Gamma-enolase, i.e. NSE, was detected by immunohistochemistry in term placenta in decidua, syncytiotrophoblasts, anchoring villi and terminal villi. Our study is the first to describe the cellular localisation of NSE in normal human placenta, thus establishing a direct link between pituitary and placental autoantigens. This link provides a theoretical basis for the strong prediliction of lymphocytic hypophysitis to occur during or after pregnancy.

A rapid Percoll gradient procedure for preparation of synaptosomes

Publisher: Springer Science and Business Media LLC

Date: 16-10-2008

DOI: 10.1038/NPROT.2008.171

Abstract: Homogenization of fresh brain tissue in isotonic medium shears plasma membranes causing nerve terminals to become separated from their axons and postsynaptic connections. The nerve terminal membranes then reseal to form synaptosomes. The discontinuous Percoll gradient procedure described here is designed to isolate synaptosomes from brain homogenates in the minimum time to allow functional experiments to be performed. Synaptosomes are isolated using a medium-speed centrifuge, while maintaining isotonic conditions and minimizing mechanically damaging resuspension steps. This protocol has advantages over other procedures in terms of speed and by producing relatively homogeneous synaptosomes, minimizing the presence of synaptic and glial plasma membranes and extrasynaptosomal mitochondria. The purified synaptosomes are viable and take up and release neurotransmitters very efficiently. A typical yield of synaptosomes is between 2.5 and 4 mg of synaptosomal protein per gram rat brain. The procedure takes approximately 1 h from homogenization of the brain until collection of the synaptosomal suspension from the Percoll gradient.

Calcium-stimulated protein kinases from rat cerebral cortex are inactivated by preincubation

Publisher: Elsevier BV

Date: 10-1981

DOI: 10.1016/S0006-291X(81)80138-6

Abstract: Controversy exists about the optimal treatment for patients with a traumatic acute subdural haematoma (ASDH) and an intracerebral haematoma/contusion (t-ICH). Treatment varies largely between different regions. The effect of this practice variation on patient outcome is unknown. Here, we present the protocol for a prospective multicentre observational study aimed at comparing the effectiveness of different treatment strategies in patients with ASDH and/or t-ICH. Specifically, the aims are to compare (1) an acute surgical approach to an expectant approach and (2) craniotomy to decompressive craniectomy when evacuating the haematoma. Patients presenting to the emergency room with an ASDH and/or an t-ICH are eligible for inclusion. Standardised prospective data on patient and injury characteristics, treatment and outcome will be collected on 1000 ASDH and 750 t-ICH patients in 60-70 centres within two multicentre prospective observational cohort studies: the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) and Neurotraumatology Quality Registry (Net-QuRe). The interventions of interest are acute surgery, defined as surgery directly after the first CT at presentation versus late or no surgery and craniotomy versus decompressive craniectomy. The primary outcome measure is the Glasgow Outcome Score-Extended at 6 months. Secondary outcome measures include in-hospital mortality, quality of life and neuropsychological tests. In the primary analysis, the effect of treatment preference (eg, proportion of patients in which the intervention under study is preferred) per hospital will be analysed with random effects ordinal regression models, adjusted for casemix and stratified by study. Such a hospital-level approach reduces confounding by the indication. Sensitivity analyses will include propensity score matching, with treatment defined on patient level. This study is designed to determine the best acute management strategy for ASDH and t-ICH by exploiting the existing between-hospital variability in surgical management. Ethics approval was obtained in all participating countries. Results of surgical management of ASDH and t-ICH/contusion will separately be submitted for publication in a peer-reviewed journal. NCT02210221 and NL 5761.

Phosphorylation of synaptosomal cytoplasmic proteins: Inhibition of calcium-activated, phospholipid-dependent protein kinase (protein kinase c) by bay k 8644

Publisher: Elsevier BV

Date: 1988

DOI: 10.1016/0197-0186(88)90122-2

Abstract: The phosphorylation of specific substrates of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was examined in striatal synaptosomal cytoplasm. The phosphoprotein substrata were termed group C phosphoprotems and were ided into two subgroups: group C(1) phosphoproteins (P83, P45A, P21 and P18) were found in both cytoplasm and synaptosomal membranes and, although stimulated by phosphatidylserine, only required exogamous calcium for their labeling group C(2) phosphoproteins (P120, P96, P21.5, P18.5 and P16) were found predominantly in the cytoplasm and were absolutely dependent upon exogenous calcium and phosphatidylserme for their labeling. Several criteria were used to identify these proteins as specific protein kinase C substrates: (a) their phosphorylation was stimulated to a greater extent by Ca(2+) hosphatidylserine/diolein than by Ca(2+) alone or Cal(2+) /calmodulin (group C(1)) or was completely dependent upon Ca(2+) hosphatdylserine/diolein (group C(2)) (b) supermaximal concentrations of the cAMP-dependent protein kinase inhibitor were without effect (c) their phosphorylation was stimulated by oleic acid, which selectively activates protein kinase C in the absence of Ca(2+) (d) NaCl, which inhibited cAMP- and Ca(2+)/calmodulindependent phosphorylation, slightly increased phosphorylation of group C(1) and slightly decreased phosphorylation of group C(2) phosphoproteins. Maximal phosphorylation of P96 and other group C phosphoproteins occurred within 60 s and was followed by a slow decay rate while substrata of calmodulin-dependent protein kinase were maximally labeled within 20-30 s and rapidly dephosphorylated. The phosphorylation of all group C phosphoproteins was inhibited by the calcium channel agomst BAY K 8644, however, group C(2) phosphoproteins were considerably more sensitive. The IC(50) for inhibition of P96 labeling was 19 ?M. but for P83 was 190 ?M. Group B phosphoproteins were also slightly inhibited, and the IC(50) for P63 was 290 ?M. No inhibitory effects of another dihydropyridine, nifedipine, or of verapamil were detected in this concentration range. BAY K 8644 did not displace [(3)H]phorbol-12,13-dibutyrate binding, nor was the inhibition decreased by increasing phosphatidylserine concentrations. BAY K 8644 had no effect on the rate of dephosphorylation of any phosphoprotein, indicating that it is unlikely to inhibit a protein phosphatase. BAY K 8644 may, therefore, prove to be a valuable tool for discriminating protein kinase C activity from the activity of other protein kinases. We conclude that BAY K 8644 interacts either with a specific subgroup of protein kinase C substrata or with one of two putative forms of protein kinase C.

Opportunities for pharmacoproteomics in biomarker discovery

Publisher: Wiley

Date: 22-09-2022

DOI: 10.1002/PMIC.202200031

Abstract: Proteomic data are a uniquely valuable resource for drug response prediction and biomarker discovery because most drugs interact directly with proteins in target cells rather than with DNA or RNA. Recent advances in mass spectrometry and associated processing methods have enabled the generation of large‐scale proteomic datasets. Here we review the significant opportunities that currently exist to combine large‐scale proteomic data with drug‐related research, a field termed pharmacoproteomics. We describe successful applications of drug response prediction using molecular data, with an emphasis on oncology. We focus on technical advances in data‐independent acquisition mass spectrometry (DIA‐MS) that can facilitate the discovery of protein biomarkers for drug responses, alongside the increased availability of big biomedical data. We spotlight new opportunities for machine learning in pharmacoproteomics, driven by the combination of these large datasets and improved high‐performance computing. Finally, we explore the value of pre‐clinical models for pharmacoproteomic studies and the accompanying challenges of clinical validation. We propose that pharmacoproteomics offers the potential for novel discovery and innovation within the cancer landscape.

Dynole 34-2 and Acrylo-Dyn 2-30, Novel Dynamin GTPase Chemical Biology Probes

Publisher: Springer US

Date: 2022

DOI: 10.1007/978-1-0716-1916-2_17

Abstract: This protocol describes the chemical synthesis of the dynamin inhibitors Dynole 34-2 and Acrylo-Dyn 2-30, and their chemical scaffold matched partner inactive compounds. The chosen active and inactive paired compounds represent potent dynamin inhibitors and very closely related dynamin-inactive compounds, with the synthesis of three of the four compounds readily possible via a common intermediate. Combined with the assay data provided, this allows the interrogation of dynamin in vitro and potentially in vivo.

Synthesis of Phthaladyn-29 and Naphthalimide-10, GTP Site Directed Dynamin GTPase Inhibitors

Publisher: Springer US

Date: 2022

DOI: 10.1007/978-1-0716-1916-2_18

Abstract: Herein we describe the detailed synthesis of the dynamin inhibitors Phthaladyn-29 and Napthaladyn-10, and their chemical scaffold matched partner inactive compounds. Combined with the assay data provided, this allows the interrogation of dynamin in vitro and potentially in vivo.

Clathrin-independent carriers form a high capacity endocytic sorting system at the leading edge of migrating cells

Publisher: Rockefeller University Press

Date: 16-08-2010

DOI: 10.1083/JCB.201002119

Abstract: Although the importance of clathrin- and caveolin-independent endocytic pathways has recently emerged, key aspects of these routes remain unknown. Using quantitative ultrastructural approaches, we show that clathrin-independent carriers (CLICs) account for approximately three times the volume internalized by the clathrin-mediated endocytic pathway, forming the major pathway involved in uptake of fluid and bulk membrane in fibroblasts. Electron tomographic analysis of the 3D morphology of the earliest carriers shows that they are multidomain organelles that form a complex sorting station as they mature. Proteomic analysis provides direct links between CLICs, cellular adhesion turnover, and migration. Consistent with this, CLIC-mediated endocytosis of key cargo proteins, CD44 and Thy-1, is polarized at the leading edge of migrating fibroblasts, while transient ablation of CLICs impairs their ability to migrate. These studies provide the first quantitative ultrastructural analysis and molecular characterization of the major endocytic pathway in fibroblasts, a pathway that provides rapid membrane turnover at the leading edge of migrating cells.

Superfluous Role of Mammalian Septins 3 and 5 in Neuronal Development and Synaptic Transmission

Publisher: Informa UK Limited

Date: 12-2008

DOI: 10.1128/MCB.00035-08

Targeting membrane trafficking in infection prophylaxis: dynamin inhibitors

Publisher: Elsevier BV

Date: 02-2013

DOI: 10.1016/J.TCB.2012.10.007

Abstract: Many pathogens hijack existing endocytic trafficking pathways to exert toxic effects in cells. Dynamin controls various steps of the intoxication process used by numerous pathogenic bacteria, viruses, and toxins. Targeting dynamin with pharmaceutical compounds may therefore have prophylactic potential. Here we review the growing number of pathogens requiring dynamin-dependent trafficking to intoxicate cells, outline the mode of internalization that leads to their pathogenicity, and highlight the protective effect of pharmacological and genetic approaches targeting dynamin function. We also assess the methodologies used to investigate the role of dynamin in the intoxication process and discuss the validity and potential pitfalls of using dynamin inhibitors (DIs) as therapeutics.

Phosphorylation of proteins in chick ciliary ganglion under conditions that induce long-lasting changes in synaptic transmission: Phosphoprotein targets for nitric oxide action

Publisher: Elsevier BV

Date: 02-1999

DOI: 10.1016/S0306-4522(98)00452-7

Abstract: Production of nitric oxide and the activation of protein kinases are required for long-term potentiation of synaptic transmission at the giant synapses in chicken ciliary ganglion. In the present study, we investigated the ability of nitric oxide to regulate the phosphorylation of endogenous proteins under conditions that induced long-term potentiation in intact ciliary ganglion and the protein kinases responsible for the phosphorylation of these proteins in lysed ciliary ganglion. Using Calcium Green-1 we showed that the nitric oxide donor sodium nitroprusside did not change the intraterminal Ca2+ dynamics in ciliary ganglion. Two dimensional phosphopeptide analysis of 32Pi-labelled intact ciliary ganglion showed that the sodium nitroprusside (300 microM) increased the phosphorylation of several phosphopeptides (P50a, P50b and P41) derived from proteins at 50,000 and 41,000 mol. wts which we have called nitric oxide-responsive phosphoproteins. A similar stimulation of phosphorylation was achieved by 8-bromo-cyclic AMP (100 microM), which also induced long-term potentiation, but not by phorbol dibutyrate (2 microM) that does not induce long-term potentiation in ciliary ganglion. When subcellular fractions from lysed ciliary ganglion were labelled in vitro by [gamma-32P]ATP in the presence of purified cGMP-dependent, cAMP-dependent or Ca2+-phospholipid-dependent protein kinases, we identified cyclic GMP-dependent protein kinase substrates that gave rise to phosphopeptides co-migrating with P50a, P50b and P41 from 32Pi-labelled intact ciliary ganglion. P50a and P41 were derived from soluble proteins while P50b was derived from a membrane-associated protein. The proteins giving rise to P50a, P50b and P41 were also substrates for cyclic AMP-dependent protein kinase, but not for calcium and phospholipid-dependent protein kinase in vitro, suggesting that nitric oxide-responsive phosphoproteins are convergence points in information processing in vivo and their phosphorylation might represent an important mechanism in nitric oxide-mediated synaptic plasticity in ciliary ganglion.

Iminochromene Inhibitors of Dynamins I and II GTPase Activity and Endocytosis

Publisher: American Chemical Society (ACS)

Date: 27-05-2010

DOI: 10.1021/JM100119C

Abstract: Herein we report the synthesis of discrete iminochromene ("iminodyn") libraries (14-38) as potential inhibitors of dynamin GTPase. Thirteen iminodyns were active (IC(50) values of 260 nM to 100 microM), with N,N-(ethane-1,2-diyl)bis(7,8-dihydroxy-2-iminochromene-3-carboxamide) (17), N,N-(ethane-1,2-diyl)bis(7,8-dihydroxy-2-iminochromene-3-carboxamide) (22), and N,N-(ethane-1,2-diyl)bis(7,8-dihydroxy-2-iminochromene-3-carboxamide) (23) (IC(50) values of 330 +/- 70, 450 +/- 50, and 260 +/- 80 nM, respectively) being the most potent. Five of the most potent iminodyns all inhibited dynamins I and II approximately equally. Iminodyn-22 displayed uncompetitive inhibition with respect to GTP. Selected iminodyns were evaluated for their ability to block receptor mediated endocytosis (RME, mediated by dynamin II) and synaptic vesicle endocytosis (SVE, mediated by dynamin I), with 17 showing no activity while 22 returned RME and SVE IC(50) values of 10.7 +/- 4.5 and 99.5 +/- 1.7 microM, respectively. The iminodyns reported herein represent a new chemical class of the first nanomolar potent dynamin inhibitors that are also effective endocytosis inhibitors.

Prognostic Association of YB-1 Expression in Breast Cancers: A Matter of Antibody

Publisher: Public Library of Science (PLoS)

Date: 10-06-2011

DOI: 10.1371/JOURNAL.PONE.0020603

Prodrugs of the Archetypal Dynamin Inhibitor Bis‐T‐22

Publisher: Wiley

Date: 09-11-2022

DOI: 10.1002/CMDC.202200400

Abstract: The Bis‐T series of compounds comprise some of the most potent inhibitors of dynamin GTPase activity yet reported, e. g., (2 E ,2′ E )‐ N , N ′‐(propane‐1,3‐diyl)bis(2‐cyano‐3‐(3,4‐dihydroxyphenyl)acrylamide) ( 2 ), Bis‐T‐22. The catechol moieties are believed to limit cell permeability, rendering these compounds largely inactive in cells. To solve this problem, a prodrug strategy was envisaged and eight ester analogues were synthesised. The shortest and bulkiest esters (acetate and butyl/ tert ‐butyl) were found to be insoluble under physiological conditions, whilst the remaining five were soluble and stable under these conditions. These five were analysed for plasma stability and half‐lives ranged from ∼2.3 min (propionic ester 4 ), increasing with size and bulk, to greater than 24 hr (dimethyl carbamate 10 ). Similar profiles where observed with the rate of formation of Bis‐T‐22 with half‐lives ranging from ∼25 mins (propionic ester 4 ). Propionic ester 4 was chosen to undergo further testing and was found to inhibit endocytosis in a dose‐dependent manner with IC 50 ∼8 μM, suggesting this compound is able to effectively cross the cell membrane where it is rapidly hydrolysed to the desired Bis‐T‐22 parent compound.

Synthesis of Dynole 34-2, Dynole 2-24 and Dyngo 4a for investigating dynamin GTPase

Publisher: Springer Science and Business Media LLC

Date: 20-03-2014

DOI: 10.1038/NPROT.2014.046

Abstract: Dynamin is a large GTPase with roles in membrane fission during clathrin-mediated endocytosis, in actin dynamics and in cytokinesis. Defects in dynamin have been linked to human diseases. The synthesis of a dynamin modulator toolkit comprising two different inhibitor classes is described. The first series comprises Dynole 34-2, Dynole 2-24 and the inactive control Dynole 31-2. The Dynole compounds act on the dynamin G domain, are not GTP competitive and can be synthesized in 2-3 d. Knoevenagel condensation of 1-(3-(dimethylamino)propyl)-1H-indole-3-carbaldehyde (1) with cyanoamides (2 and 3) affords Dynole 31-2 and Dynole 34-2, respectively. Reductive amination of 1 with decylamine gives Dynole 2-24. The second series acts at an allosteric site in the G domain of dynamin and comprises Dyngo 4a and Dyngo Ø (inactive control). Both are synthesized in an overnight reaction via condensation of 3-hydroxy-2-naphthoic hydrazide with 2,4,5-trihydroxybenzaldehyde to afford Dyngo 4a, or with benzaldehyde to afford Dyngo Ø.

Styryl dye-based synaptic vesicle recycling assay in cultured cerebellar granule neurons.

Publisher: Humana Press

Date: 2008

DOI: 10.1007/978-1-59745-261-8_25

Abstract: Neurons transmit information by exocytosis of synaptic vesicles (SV), which contain neurotransmitter. Exocytosis is followed by efficient retrieval of the plasma membrane by endocytosis to generate a new SV. SV retrieval supports multiple cycles of synaptic transmission. Over the years, styryl dyes have been widely used to probe the mechanism of SV recycling in the processes of cultured neurons. The styryl dye method is complementary to electrophysiological measurements or genetic reporter approaches. Owing to their ease to culture, cerebellar granule neurons provide a robust neuronal model system for the assay. These cells are readily transfected with various DNA constructs to study the function of exocytic or endocytic proteins in SV recycling.

The Impact of Commonly Used Alkylating Agents on Artifactual Peptide Modification

Publisher: American Chemical Society (ACS)

Date: 23-08-2017

DOI: 10.1021/ACS.JPROTEOME.7B00022

Abstract: Iodoacetamide is by far the most commonly used agent for alkylation of cysteine during s le preparation for proteomics. An alternative, 2-chloroacetamide, has recently been suggested to reduce the alkylation of residues other than cysteine, such as the N-terminus, Asp, Glu, Lys, Ser, Thr, and Tyr. Here we show that although 2-chloroacetamide reduces the level of off-target alkylation, it exhibits a range of adverse effects. The most significant of these is methionine oxidation, which increases to a maximum of 40% of all Met-containing peptides, compared with 2-5% with iodoacetamide. Increases were also observed for mono- and dioxidized tryptophan. No additional differences between the alkylating reagents were observed for a range of other post-translational modifications and digestion parameters. The deleterious effects were observed for 2-chloroacetamide from three separate suppliers. The adverse impact of 2-chloroacetamide on methionine oxidation suggests that it is not the ideal alkylating reagent for proteomics.

Mammalian Septins Nomenclature

Publisher: American Society for Cell Biology (ASCB)

Date: 12-2002

DOI: 10.1091/MBC.E02-07-0438

Abstract: There are 10 known mammalian septin genes, some of which produce multiple splice variants. The current nomenclature for the genes and gene products is very confusing, with several different names having been given to the same gene product and distinct names given to splice variants of the same gene. Moreover, some names are based on those of yeast or Drosophilaseptins that are not the closest homologues. Therefore, we suggest that the mammalian septin field adopt a common nomenclature system, based on that adopted by the Mouse Genomic Nomenclature Committee and accepted by the Human Genome Organization Gene Nomenclature Committee. The human and mouse septin genes will be namedSEPT1–SEPT10 and Sept1–Sept10, respectively. Splice variants will be designated by an underscore followed by a lowercase “v” and a number, e.g., SEPT4_v1.

Regulation of protein kinases by pseudosubstrate prototopes

Publisher: No publisher found

Date: 1989

DOI: 10.1016/0898-6568(89)90049-1

Identification of cGMP-dependent protein kinase and its specific substrates in the anterior pituitary

Publisher: Elsevier BV

Date: 09-1996

DOI: 10.1016/0303-7207(96)03881-6

Abstract: In the anterior pituitary, cGMP is produced in response to a number of stimuli, but intracellular events distal to cGMP production are obscure. Since cGMP-dependent protein kinase (PKG) is a major effector of cGMP actions in other tissues we have determined whether PKG and its specific substrates might be present and responsive to external signals in the ovine anterior pituitary. Photoaffinity labelling with [32P]cGMP revealed a specific 78 kDa protein in ovine anterior pituitary that comigrated with purified bovine lung PKG-I. PKG in protein extracts from anterior pituitary or cultured anterior pituitary cells was enriched by DEAE ion-exchange chromatography and assayed for activity. Both tissue and cultured cells had a relatively high PKG activity by comparison with aortic smooth muscle (known high activity) and brain (known low activity). Subcellular distribution studies showed that in anterior pituitary, aortic and brain, PKG activity was present in both cytosol and triton-extracted membrane fractions, while in platelets the activity was associated with only the membrane fraction. To determine if this PKG might be responsive to extracellular signals an activity ratio assay was used. Incubation of cultured cells with atrial natriuretic peptide (ANP) and sodium nitroprusside, activators of membrane and cytosolic guanylate cyclases respectively, increased the activity of PKG. To determine events distal to PKG activation, a search for potential substrates of PKG was performed. Few substrates were detectable upon addition of purified PKG to tissue lysates due to the high background activity of endogenous protein kinases in the anterior pituitary. However, 19 substrates of PKG were detected in heat-stable and 14 in acid-soluble protein extracts of the anterior pituitary, in which background phosphorylation was almost abolished. After partial purification through Q-Sepharose ion-exchange chromatography some of these proteins were preferentially phosphorylated by addition of PKG-I, while the others were additionally substrates of exogenous cAMP-dependent protein kinase (PKA) or Ca2+ and phospholipid-dependent protein kinase (PKC). A 132-kDa substrate showed an identical phosphopeptide map to a PKG substrate previously described in vascular smooth muscle and platelets. These data demonstrate for the first time the presence of functional PKG activity and multiple PKG substrates in the anterior pituitary where they may play a role in mediating the intracellular actions of cGMP.

Transformer-based deep learning integrates multi-omic data with cancer pathways

Publisher: Cold Spring Harbor Laboratory

Date: 31-10-2022

DOI: 10.1101/2022.10.27.514141

Abstract: Multi-omic data analysis incorporating machine learning has the potential to significantly improve cancer diagnosis and prognosis. Traditional machine learning methods are usually limited to omic measurements, omitting existing domain knowledge such as the biological networks that link molecular entities in various omic data types. We develop a Transformer-based explainable deep learning model, DeePathNet, which integrates cancer-specific pathway information into multi-omic data analysis. Using a variety of big datasets, including ProCan-DepMapSanger, CCLE and TCGA, we show that DeePathNet outperforms traditional methods for the prediction of drug response and classification of cancer type and subtype. Combining biomedical knowledge and state-of-the-art deep learning methods, DeePathNet enables biomarker discovery at the pathway level, maximising the power of data-driven approaches to cancer research. DeePathNet is available on GitHub at github.com/CMRI-ProCan/DeePathNet .

Two fractions enriched for striatal synaptosomes isolated by percoll gradient centrifugation: synaptosome morphology, dopamine and serotonin receptor distribution, and adenylate cyclase activity

Publisher: Elsevier BV

Date: 1989

DOI: 10.1016/0197-0186(89)90142-3

Abstract: The present report describes the morphology and biochemical characteristics of two fractions containing rat striatal synaptosomes prepared by percoll density gradient centrifugation. The two fractions, designated fraction 3 and fraction 4, contained the same absolute number of synaptosomal profiles, however, fraction 4 contained a population of significantly larger synaptosomes. The mean diameter of synaptosomes in fraction 4 was 0.91 ?m, while synaptosomes in fraction 3 had a mean diameter of 0.59 ?m. Fraction 3 contained considerably fewer mitochondria, either within or external to the synaptosomes, and contained a greater number of synaptic plasma membranes than fraction 4. The larger fraction 4 synaptosomes contained less plasma membrane, but contained a larger percentage of mitochondria (10%). The distribution of binding sites for spiroperidol, SCH-23390, serotonin, muscimol, and nitrendipine, as well as adenylate cyclase and cAMP-dependent protein kinase activities were compared in membranes from these two fractions. In fraction 3 the number of recognition sites (B(max)) for D-1, 5-HT, GABA, nitrendipine, and forskolin receptors were 2- to 4-fold larger, while those for D-2 5-HT, adenylate cyclase and cAMP-dependent protein kinase activities were 1.5- to 2.0-fold greater than in fraction 4. In no case was there difference in K(d), suggesting that the number of binding sites was different. Since the radioligands that are preferential markers of receptor sites localized in postsynaptic membranes were concentrated in fraction 3, we conclude that this fraction may contain a large amount of postsynaptic membranes as well as the synaptosome component and may be suited to a variety of receptor binding studies. By contrast, fraction 4 synaptosomes contain presynaptic dopamine terminals [Robinson P.J. and Lovenberg W. (1986) Neurochem. Int.9, 455-458] and less nonsynaptosomal membrane contamination and should be valuable for functional studies on presynaptic mechanisms.

Arginine vasopressin (AVP) causes the reversible phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein in the ovine anterior pituitary: evidence that MARCKS phosphoryla

Publisher: Elsevier BV

Date: 05-1994

DOI: 10.1016/0303-7207(94)90241-0

Abstract: We have recently shown that AVP causes a protein kinase C (PKC)-dependent increase in ACTH release and biosynthesis in ovine anterior pituitary cells. In these cells, AVP also causes the translocation of PKC from the cytosol to the cell membrane which is maximal at 5 min, but the intracellular events distal to protein kinase C activation that underlie ACTH secretion have not been well characterized to date. Since the MARCKS protein has been implicated in neurosecretion and is phosphorylated by PKC in synaptosomes, studies were carried out to determine whether AVP might cause MARCKS phosphorylation in the ovine anterior pituitary, and to determine whether this phenomenon might be temporally correlated with PKC translocation and the release of ACTH. When cytosolic fractions of rat brain, ovine anterior pituitary, and cultured ovine anterior pituitary cells were incubated with purified PKC, several proteins were phosphorylated including those in the region of 83-85 kDa. After precipitation of the proteins with 40% acetic acid, the 83-85 kDa phosphoproteins were selectively recovered in the acid soluble phase. Phosphopeptide maps of either the 83 or 85 kDa proteins were generated with Staphylococcus aureus V8 protease and revealed 13 and 9 kDa phosphopeptides, which are characteristic of the authentic MARCKS protein. An identical phosphopeptide map was also obtained when the MARCKS protein was selectively extracted from intact 32P-labeled anterior pituitary cells. MARCKS phosphorylation was markedly increased when ovine anterior pituitary cells were exposed to 1 microM phorbol 12-myristate 13-acetate (PMA). When the cells were exposed to 1 microM AVP, MARCKS phosphorylation increased at 15 s and reached the maximal plateau value at 30 s. MARCKS phosphorylation then started to diminish at 2 min, and baseline levels were attained by 10 min. In the same cells, AVP stimulated ACTH release in a biphasic manner - during the first 30 s, there resulted a rapid burst of ACTH secretion that was followed by a slower, but sustained rate of secretion. We conclude that: (1) AVP causes a rapid, and reversible, phosphorylation of the MARCKS protein in the ovine anterior pituitary (2) since the AVP-induced increase in MARCKS phosphorylation occurs much earlier in these cells than does PKC trans-location, MARCKS phosphorylation may provide a more sensitive index of the onset of PKC activation than the translocation assay (3) the close temporal association between MARCKS phosphorylation and the rapid early release of ACTH suggests that MARCKS phosphorylation may be involved in the initial intracellular events that underly exocytosis of the hormone.

The molecular pathology of amyloid deposition in Alzheimer's disease

Publisher: Springer Science and Business Media LLC

Date: 06-1991

DOI: 10.1007/BF02935560

Sorting Nexin 9 Recruits Clathrin Heavy Chain to the Mitotic Spindle for Chromosome Alignment and Segregation

Publisher: Public Library of Science (PLoS)

Date: 05-07-2013

DOI: 10.1371/JOURNAL.PONE.0068387

The interaction of assembly protein AP180 and clathrin is inhibited by multi-site phospho-mimetics

Publisher: Elsevier BV

Date: 10-2019

DOI: 10.1016/J.NEUINT.2019.104474

Abstract: Clathrin-mediated endocytosis at the nerve terminal is dependent on assembly protein 180 (AP180) and adapter protein complex 2 (AP2). Both membrane adapter proteins bind to each other and to clathrin, to drive assembly of the clathrin coat over nascent synaptic vesicles. Using knowledge of in vivo phosphorylation sites, AP180 was mutated to determine the effect on binding. N-terminally truncated AP180 exhibited phospho-mimetic (Ser/Thr to Glu)-dependent interaction with AP2, but not clathrin. C-terminally truncated and full length phospho-mutant AP180 bound less AP2 than wild type. However, there was no difference in AP2 binding for the phospho-mimetic or phospho-deficient (Ser/Thr to Ala) AP180 mutants. Thus, the phospho-mutant approach did not provide clarity for the role of phosphorylation in AP180-AP2 binding. Clathrin exhibited a phospho-mimetic-dependent interaction with full-length AP180. Furthermore, phospho-mimetic AP180 was deficient at assembling clathrin cages. These latter discoveries support a model where AP180 phosphorylation inhibits clathrin binding and assembly.

Inha University

Location: No location found

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École Polytechnique Fédérale De Lausanne

Location: Switzerland

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Seoul National University

Location: Korea, Republic of

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Inha University

Location: Korea, Republic of

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University Of Sydney

Location: Australia

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The University Of Texas At Austin

Location: United States of America

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ShanghaiTech University

Location: China

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The University Of Newcastle

Location: Australia

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Children's Medical Research Institute

Location: Australia

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Discovery Projects - Grant ID: DP0987706

Start Date: 03-2009

End Date: 03-2013

Amount: $300,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP180101781

Start Date: 02-2018

End Date: 12-2022

Amount: $802,912.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP1095780

Start Date: 06-2010

End Date: 06-2013

Amount: $438,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP120100754

Start Date: 07-2012

End Date: 12-2014

Amount: $300,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE100100142

Start Date: 2010

End Date: 07-2011

Amount: $500,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0666572

Start Date: 2006

End Date: 09-2009

Amount: $265,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE100100150

Start Date: 2010

End Date: 12-2012

Amount: $500,000.00

Funder: Australian Research Council