ORCID Profile
0000-0002-6070-3774
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Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Biochemistry and Cell Biology | Protein Trafficking | Structural Biology (incl. Macromolecular Modelling) | Proteomics and Intermolecular Interactions (excl. Medical Proteomics) | Receptors and Membrane Biology | Biochemistry and cell biology | Biochemistry And Cell Biology Not Elsewhere Classified | Structural biology (incl. macromolecular modelling) | Protein Targeting And Signal Transduction | Inorganic materials (incl. nanomaterials) | Receptors and membrane biology | Biochemistry and Cell Biology not elsewhere classified | Nanoscale Characterisation | Biophysics | Nanomaterials | Other Physical Sciences | Theoretical And Computational Chemistry Not Elsewhere Classified | Nanotechnology | Cell Metabolism | Membrane Biology | Protein trafficking | Cell Neurochemistry
ANZSRC Socio-Economic Objective (SEO)
Expanding Knowledge in the Biological Sciences | Biological sciences | Neurodegenerative Disorders Related to Ageing | Expanding Knowledge in the Medical and Health Sciences | Other | Chemical sciences | Physical sciences | Expanding Knowledge in the Chemical Sciences | Expanding Knowledge in the Physical Sciences |
Related Links
Publications
Classification of the human phox homology (PX) domains based on their phosphoinositide binding specificities
Publisher: Springer Science and Business Media LLC
Date: 04-04-2019
DOI: 10.1038/S41467-019-09355-Y
Abstract: Phox homology (PX) domains are membrane interacting domains that bind to phosphatidylinositol phospholipids or phosphoinositides, markers of organelle identity in the endocytic system. Although many PX domains bind the canonical endosome-enriched lipid PtdIns3 P , others interact with alternative phosphoinositides, and a precise understanding of how these specificities arise has remained elusive. Here we systematically screen all human PX domains for their phospholipid preferences using liposome binding assays, biolayer interferometry and isothermal titration calorimetry. These analyses define four distinct classes of human PX domains that either bind specifically to PtdIns3 P , non-specifically to various di- and tri-phosphorylated phosphoinositides, bind both PtdIns3 P and other phosphoinositides, or associate with none of the lipids tested. A comprehensive evaluation of PX domain structures reveals two distinct binding sites that explain these specificities, providing a basis for defining and predicting the functional membrane interactions of the entire PX domain protein family.
Proteomic identification and structural basis for the interaction between sorting nexin SNX17 and PDLIM family proteins
Publisher: Cold Spring Harbor Laboratory
Date: 29-07-2021
DOI: 10.1101/2021.07.29.454387
Abstract: The sorting nexin SNX17 controls endosome-to-cell surface recycling of erse transmembrane cargo proteins including integrins, the amyloid precursor protein and lipoprotein receptors. This requires association with the multi-subunit Commander trafficking complex, which depends on the C-terminus of SNX17 through unknown mechanisms. Using affinity enrichment proteomics, we find that a C-terminal peptide of SNX17 is not only sufficient for Commander interaction but also associates with members of the actin-associated PDZ and LIM domain (PDLIM) family. We show that SNX17 contains a type III PSD95/Dlg/Zo1 (PDZ) binding motif (PDZbm) that binds specifically to the PDZ domains of PDLIM family proteins but not to other PDZ domains tested. The structure of the PDLIM7 PDZ domain bound to the SNX17 C-terminus was determined by NMR spectroscopy and reveals an unconventional perpendicular peptide interaction. Mutagenesis confirms the interaction is mediated by specific electrostatic contacts and a uniquely conserved proline-containing loop sequence in the PDLIM protein family. Our results define the mechanism of SNX17-PDLIM interaction and suggest that the PDLIM proteins may play a role in regulating the activity of SNX17 in conjunction with Commander and actin-rich endosomal trafficking domains.
Syntaxin 11 Binds Vti1b and Regulates Late Endosome to Lysosome Fusion in Macrophages
Publisher: Wiley
Date: 08-04-2011
DOI: 10.1111/J.1600-0854.2011.01189.X
Abstract: Syntaxin 11 (Stx11) is a SNARE protein enriched in cells of the immune system. Loss or mutation of Stx11 results in familial hemophagocytic lymphohistiocytosis type-4 (FHL-4), an autosomal recessive disorder of immune dysregulation characterized by high levels of inflammatory cytokines along with defects in T-cell and natural killer cell function. We show here Stx11 is located on endosomal membranes including late endosomes and lysosomes in macrophages. While Stx11 did not form a typical trans-SNARE complex, it did bind to the Q-SNARE Vti1b and was able to regulate the availability of Vti1b to form the Q-SNARE complexes Stx6/Stx7/Vtib and Stx7/Stx8/Vti1b. The mutant form of Stx11 sequestered Vti1b from forming the Q-SNARE complex that mediates late endosome to lysosome fusion. Depletion of Stx11 in activated macrophages leads to an accumulation of enlarged late endocytic compartments, increased trafficking to the cell surface and inhibition of late endosome to lysosome fusion. These phenotypes are rescued by the expression of an siRNA-resistant Stx11 construct in Stx11-depleted cells. Our results suggest that by regulating the availability of Vti1b, Stx11 regulates trafficking steps between late endosomes, lysosomes and the cell surface in macrophages.
De novo macrocyclic peptides for inhibiting, stabilizing, and probing the function of the retromer endosomal trafficking complex.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 03-12-2021
Abstract: Novel macrocyclic peptides are found that bind and modulate the function of the retromer membrane trafficking complex.
Towards a molecular understanding of endosomal trafficking by Retromer and Retriever
Publisher: Wiley
Date: 20-06-2019
DOI: 10.1111/TRA.12649
Abstract: Endosomes are dynamic intracellular compartments that control the sorting of a constant stream of different transmembrane cargos either for ESCRT-mediated degradation or for egress and recycling to compartments such as the Golgi and the plasma membrane. The recycling of cargos occurs within tubulovesicular membrane domains and is facilitated by peripheral membrane protein machineries that control both membrane remodelling and selection of specific transmembrane cargos. One of the primary sorting machineries is the Retromer complex, which controls the recycling of a large array of different cargo molecules in cooperation with various sorting nexin (SNX) adaptor proteins. Recently a Retromer-like complex was also identified that controls plasma membrane recycling of cargos including integrins and lipoprotein receptors. Termed "Retriever," this complex uses a different SNX family member SNX17 for cargo recognition, and cooperates with the COMMD/CCDC93/CCDC22 (CCC) complex to form a larger assembly called "Commander" to mediate endosomal trafficking. In this review we focus on recent advances that have begun to provide a molecular understanding of these two distantly related transport machineries.
Phosphoinositide binding by the SNX27 FERM domain regulates its localization at the immune synapse of activated T-cells
Publisher: The Company of Biologists
Date: 2015
DOI: 10.1242/JCS.158204
Abstract: Sorting nexin 27 (SNX27) controls the endosomal to cell-surface recycling of erse transmembrane protein cargos. Critical to this function is the recruitment of SNX27 to endosomes through the binding of phosphatidylinositol-3-phosphate (PtdIns3P) by the phox-homology (PX) domain. In T cells, SNX27 is polarized to the immunological synapse (IS) in an activation-dependent manner, but the molecular mechanisms underlying SNX27 translocation remain to be clarified. Here, we examined the phosphoinositide lipid-binding capabilities of full-length SNX27, and discovered a novel PtdInsP binding site within the C-terminal 4.1/ezrin/radixin/moesin (FERM) domain. This binding site showed a clear preference for di and tri-phosphorylated phophoinositides, and the interaction was confirmed through biophysical, mutagenesis and modeling approaches. At the IS of activated T-cells cell signaling regulates phosphoinositide dynamics, and we find that perturbing phosphoinositide binding by the SNX27 FERM domain alters its distribution in both endosomal recycling compartments and PtdIns(3,4,5)P3-enriched domains of the plasma membrane during synapse formation. Our results suggest that SNX27 undergoes dynamic partitioning between different membrane domains during IS assembly, and underscore the contribution of unique lipid interactions for SNX27 orchestration of cargo trafficking.
Structural predictions of the SNX-RGS proteins suggest they belong to a new class of lipid transfer proteins
Publisher: Cold Spring Harbor Laboratory
Date: 12-2021
DOI: 10.1101/2021.11.30.470681
Abstract: Recent advances in protein structure prediction using machine learning such as AlphaFold2 and RosettaFold presage a revolution in structural biology. Genome-wide predictions of protein structures are providing unprecedented insights into their architecture and intradomain interactions, and applications have already progressed towards assessing protein complex formation. Here we present detailed analyses of the sorting nexin proteins that contain regulator of G-protein signalling domains (SNX-RGS proteins), providing a key ex le of the ability of AlphaFold2 to reveal novel structures with previously unsuspected biological functions. These large proteins are conserved in most eukaryotes and are known to associate with lipid droplets (LDs) and sites of LD-membrane contacts, with key roles in regulating lipid metabolism. They possess five domains, including an N-terminal transmembrane domain that anchors them to the endoplasmic reticulum, an RGS domain, a lipid interacting phox homology (PX) domain and two additional domains named the PXA and PXC domains of unknown structure and function. Here we report the crystal structure of the RGS domain of sorting nexin 25 (SNX25) and show that the AlphaFold2 prediction closely matches the experimental structure. Analysing the full-length SNX-RGS proteins across multiple homologues and species we find that the distant PXA and PXC domains in fact fold into a single unique structure that notably features a large and conserved hydrophobic pocket. The nature of this pocket strongly suggests a role in lipid or fatty acid binding, and we propose that these molecules represent a new class of conserved lipid transfer proteins.
Interrogation and validation of the interactome of neuronal Munc18-interacting Mint proteins with AlphaFold2
Publisher: Cold Spring Harbor Laboratory
Date: 21-02-2023
DOI: 10.1101/2023.02.20.529329
Abstract: Munc18-interacting proteins (Mints) are multi-domain adaptors that regulate neuronal membrane trafficking, signalling and neurotransmission. Mint1 and Mint2 are highly expressed in the brain and play overlapping roles in the regulation of synaptic vesicle fusion required for neurotransmitter release by interacting with the essential synaptic protein Munc18-1. The mechanism by which Mint1 mediates the interaction of Munc18-1 with Syntaxin1a (Sx1a) to control formation of the fusogenic SNARE complex and facilitate neurotransmission remains unclear. Here, we have studied both the specific interactions of the neuronal Mint proteins with Munc18-1 as well as their wider interactome. Using biochemical and biophysical binding approaches we identify a short acidic α -helical motif (AHM) within Mint1 and Mint2 that is necessary and sufficient for specific binding to Munc18-1. In silico modelling of this interaction with AlphaFold2 and extensive validation by structure-based mutagenesis showed that the AHM in Mint forms an α -helical structure that binds a conserved surface on Munc18-1 domain3b. In Munc18-1/2 double knockout cells we show that mutating the Mint-binding site significantly reduces the ability of Munc18-1 re-expression to rescue exocytosis. Although Munc18-1 can interact with Mint and Sx1a proteins simultaneously we find they have mutually reduced affinities, suggesting an allosteric coupling between the two proteins. Using AlphaFold2 we then examined the entire cellular network of putative interactions governed by the Mint scaffolds. Although this suggests many proposed binders do not associate directly with Mints, our analyses provide a structural model for their assembly with a variety of known and novel regulatory and cargo proteins including ARF3/ARF4 small GTPases, and the AP3 clathrin adaptor complex. Validation of Mint1 interaction with a new predicted binder TJAP1 provides experimental support that AlphaFold2 can correctly predict interactions across large-scale datasets. Overall, our data provides insights into the ersity of interactions mediated by the Mint family and shows that Mints may help facilitate a key trigger point in SNARE complex assembly and vesicle fusion.
STXBP1 encephalopathy
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 20-06-2019
DOI: 10.1212/WNL.0000000000007786
Abstract: De novo pathogenic variants in STXBP1 encoding syntaxin1-binding protein (STXBP1, also known as Munc18-1) lead to a range of early-onset neurocognitive conditions, most commonly early infantile epileptic encephalopathy type 4 (EIEE4, also called STXBP1 encephalopathy), a severe form of epilepsy associated with developmental delay/intellectual disability. Other neurologic features include autism spectrum disorder and movement disorders. The progression of neurologic symptoms has been reported in a few older affected in iduals, with the appearance of extrapyramidal features, reminiscent of early onset parkinsonism. Understanding the pathologic process is critical to improving therapies, as currently available antiepileptic drugs have shown limited success in controlling seizures in EIEE4 and there is no precision medication approach for the other neurologic features of the disorder. Basic research shows that genetic knockout of STXBP1 or other presynaptic proteins of the exocytic machinery leads to widespread perinatal neurodegeneration. The mechanism that regulates this effect is under scrutiny but shares intriguing hallmarks with classical neurodegenerative diseases, albeit appearing early during brain development. Most critically, recent evidence has revealed that STXBP1 controls the self-replicating aggregation of α-synuclein, a presynaptic protein involved in various neurodegenerative diseases that are collectively known as synucleinopathies, including Parkinson disease. In this review, we examine the tantalizing link among STXBP1 function, EIEE, and the neurodegenerative synucleinopathies, and suggest that neural development in EIEE could be further affected by concurrent synucleinopathic mechanisms.
Structure of the endosomal Commander complex linked to Ritscher-Schinzel syndrome
Publisher: Elsevier BV
Date: 05-2023
Copine-6 is a Ca2+sensor for activity-induced AMPA receptor exocytosis
Publisher: Cold Spring Harbor Laboratory
Date: 11-05-2023
DOI: 10.1101/2023.05.10.540298
Abstract: The recruitment of synaptic AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors underlies the strengthening of neuronal connectivity during learning and memory. This process is triggered by NMDA ( N -methyl- D -aspartate) receptor-dependent postsynaptic Ca 2+ influx. Synaptotagmin (Syt)-1 and −7 have been proposed as Ca 2+ -sensors for AMPA receptor exocytosis, but are functionally redundant. Here we identify a cytosolic C2 domain-containing Ca 2+ -binding protein Copine-6 that forms a complex with AMPA receptors. Loss of Copine-6 expression impairs activity-induced exocytosis of AMPA receptors in primary hippoc al neurons, which is rescued by wild-type Copine-6, but not Ca 2+ -binding mutants. In contrast, Copine-6 loss-of-function has no effects on steady-state expression or tetrodotoxin-induced synaptic upscaling of surface AMPA receptors. Loss of Syt-1/-7 significantly reduces Copine-6 protein expression. Interestingly, overexpression of wild-type Copine-6, but not the Ca 2+ -binding mutant, restores activity-dependent exocytosis of AMPA receptors in Syt-1/-7 double-knockdown neurons. We conclude that Copine-6 is a postsynaptic Ca 2+ -sensor that mediates AMPA receptor exocytosis during synaptic potentiation.
The Munc18-1 domain 3a loop is essential for neuroexocytosis but not for syntaxin-1A transport to the plasma membrane
Publisher: The Company of Biologists
Date: 06-2013
DOI: 10.1242/JCS.126813
Abstract: Munc18-1 plays a dual role in transporting syntaxin-1A (Sx1a) to the plasma membrane and regulating SNARE-mediated membrane fusion. As impairment of either function leads to a common exocytic defect, assigning specific roles for various Munc18-1 domains has proved difficult. Structural analyses predict that a loop region in Munc18-1 domain 3a could catalyse the conversion of Sx1a from a ‘closed’, fusion-incompetent to an ‘open’, fusion-competent conformation. As this conversion occurs at the plasma membrane, mutations in this loop could potentially separate the chaperone and exocytic functions of Munc18-1. Expression of a Munc18-1 deletion mutant lacking 17 residues of the domain 3a loop (Munc18-1Δ317–333) in PC12 cells deficient in endogenous Munc18 (DKD-PC12 cells) fully rescued transport of Sx1a to the plasma membrane, but not exocytic secretory granule fusion. In vitro binding of Munc18-1Δ317–333 to Sx1a was indistinguishable from that of full-length Munc18-1, consistent with the critical role of the closed conformation in Sx1a transport. However, in DKD-PC12 cells, Munc18-1Δ317–333 binding to Sx1a was greatly reduced compared to that of full-length Munc18-1, suggesting that closed conformation binding contributes little to the overall interaction at the cell surface. Furthermore, we found that Munc18-1Δ317–333 could bind SNARE complexes in vitro, suggesting that additional regulatory factors underpin the exocytic function of Munc18-1 in vivo. Together, these results point to a defined role for Munc18-1 in facilitating exocytosis linked to the loop region of domain 3a that is clearly distinct from its function in Sx1a transport.
Purification by reflux electrophoresis of whey proteins and of a recombinant protein expressed in Dictyostelium discoideum.
Publisher: Elsevier BV
Date: 06-1997
DOI: 10.1016/S0021-9673(97)00237-9
Abstract: Protein purification that combines the use of molecular mass exclusion membranes with electrophoresis is particularly powerful as it uses properties inherent to both techniques. The use of membranes allows efficient processing and is easily scaled up, while electrophoresis permits high resolution separation under mild conditions. The Gradiflow apparatus combines these two technologies as it uses polyacrylamide membranes to influence electrokinetic separations. The reflux electrophoresis process consists of a series of cycles incorporating a forward phase and a reverse phase. The forward phase involves collection of a target protein that passes through a separation membrane before trailing proteins in the same solution. The forward phase is repeated following clearance of the membrane in the reverse phase by reversing the current. We have devised a strategy to establish optimal reflux separation parameters, where membranes are chosen for a particular operating range and protein transfer is monitored at different pH values. In addition, forward and reverse phase times are determined during this process. Two ex les of the reflux method are described. In the first case, we described the purification strategy for proteins from a complex mixture which contains proteins of higher electrophoretic mobility than the target protein. This is a two-step procedure, where first proteins of higher mobility than the target protein are removed from the solution by a series of reflux cycles, so that the target protein remains as the leading fraction. In the second step the target protein is collected, as it has become the leading fraction of the remaining proteins. In the second ex le we report the development of a reflux strategy which allowed a rapid one-step preparative purification of a recombinant protein, expressed in Dictyostelium discoideum. These strategies demonstrate that the Gradiflow is amenable to a wide range of applications, as the protein of interest is not necessarily required to be the leading fraction in solution.
Crystal structure of a heptameric Sm-like protein complex from archaea: implications for the structure and evolution of snRNPs
Publisher: Elsevier BV
Date: 06-2001
Structural basis for the hijacking of endosomal sorting nexin proteins by Chlamydia trachomatis
Publisher: eLife Sciences Publications, Ltd
Date: 22-02-2017
DOI: 10.7554/ELIFE.22311
Abstract: During infection chlamydial pathogens form an intracellular membrane-bound replicative niche termed the inclusion, which is enriched with bacterial transmembrane proteins called Incs. Incs bind and manipulate host cell proteins to promote inclusion expansion and provide camouflage against innate immune responses. Sorting nexin (SNX) proteins that normally function in endosomal membrane trafficking are a major class of inclusion-associated host proteins, and are recruited by IncE/CT116. Crystal structures of the SNX5 phox-homology (PX) domain in complex with IncE define the precise molecular basis for these interactions. The binding site is unique to SNX5 and related family members SNX6 and SNX32. Intriguingly the site is also conserved in SNX5 homologues throughout evolution, suggesting that IncE captures SNX5-related proteins by mimicking a native host protein interaction. These findings thus provide the first mechanistic insights both into how chlamydial Incs hijack host proteins, and how SNX5-related PX domains function as scaffolds in protein complex assembly.
Cavin1 intrinsically disordered domains are essential for fuzzy electrostatic interactions and caveola formation
Publisher: Cold Spring Harbor Laboratory
Date: 12-11-2019
DOI: 10.1101/831149
Abstract: Caveolae are spherically shaped nanodomains of the plasma membrane, generated by cooperative assembly of caveolin and cavin proteins. Cavins are cytosolic peripheral membrane proteins with negatively charged intrinsically disordered regions (DR1-3) that flank positively charged α -helical regions (HR1 and HR2). Here we show that the three DR domains of Cavin1 are essential for caveola formation and dynamic trafficking of caveolae. Electrostatic interactions between DR and HR regions promote liquid-liquid phase separation behaviour of Cavin1 in vitro , assembly of Cavin1 oligomers in solution, generation of membrane curvature, association with caveolin-1 (CAV1), and Cavin1 recruitment to caveolae in cells. Removal of the first disordered region causes irreversible gel formation in vitro and results in aberrant caveola trafficking through the endosomal system. We propose a model for caveola assembly whereby fuzzy electrostatic interactions between Cavin1 and CAV1 proteins, combined with membrane lipid interactions, are required to generate membrane curvature and a metastable caveola coat.
Rapid mapping of interactions between human SNX-BAR proteins measured in vitro by alphascreen and single-molecule spectroscopy
Publisher: Elsevier BV
Date: 09-2014
The Munc18-1 domain 3a hinge-loop controls syntaxin-1A nanodomain assembly and engagement with the SNARE complex during secretory vesicle priming
Publisher: Rockefeller University Press
Date: 19-09-2016
Abstract: Munc18-1 and syntaxin-1A control SNARE-dependent neuroexocytosis and are organized in nanodomains on the plasma membrane of neurons and neurosecretory cells. Deciphering the intra- and intermolecular steps via which they prepare secretory vesicles (SVs) for fusion is key to understanding neuronal and hormonal communication. Here, we demonstrate that expression of a priming-deficient mutant lacking 17 residues of the domain 3a hinge-loop (Munc18-1Δ317-333) in PC12 cells engineered to knockdown Munc18-1/2 markedly prolonged SV docking. Single-molecule analysis revealed nonhomogeneous diffusion of Munc18-1 and syntaxin-1A in and out of partially overlapping nanodomains. Whereas Munc18-1WT mobility increased in response to stimulation, syntaxin-1A became less mobile. These Munc18-1 and syntaxin-1A diffusional switches were blocked by the expression of Munc18-1Δ317-333, suggesting that a conformational change in the Munc18-1 hinge-loop controls syntaxin-1A and subsequent SNARE complex assembly. Accordingly, syntaxin-1A confinement was prevented by expression of botulinum neurotoxin type E. The Munc18-1 domain 3a hinge-loop therefore controls syntaxin-1A engagement into SNARE complex formation during priming.
A variable undecad repeat domain in cavin1 regulates caveola formation and stability
Publisher: EMBO
Date: 18-07-2018
A phosphoinositide-binding cluster in cavin1 acts as a molecular sensor for cavin1 degradation.
Publisher: American Society for Cell Biology (ASCB)
Date: 15-10-2015
Abstract: Caveolae are abundant surface organelles implicated in a range of cellular processes. Two classes of proteins work together to generate caveolae: integral membrane proteins termed caveolins and cytoplasmic coat proteins called cavins. Caveolae respond to membrane stress by releasing cavins into the cytosol. A crucial aspect of this model is tight regulation of cytosolic pools of cavin under resting conditions. We now show that a recently identified region of cavin1 that can bind phosphoinositide (PI) lipids is also a major site of ubiquitylation. Ubiquitylation of lysines within this site leads to rapid proteasomal degradation. In cells that lack caveolins and caveolae, cavin1 is cytosolic and rapidly degraded as compared with cells in which cavin1 is associated with caveolae. Membrane stretching causes caveolar disassembly, release of cavin complexes into the cytosol, and increased proteasomal degradation of wild-type cavin1 but not mutant cavin1 lacking the major ubiquitylation site. Release of cavin1 from caveolae thus leads to exposure of key lysine residues in the PI-binding region, acting as a trigger for cavin1 ubiquitylation and down-regulation. This mutually exclusive PI-binding/ubiquitylation mechanism may help maintain low levels of cytosolic cavin1 in resting cells, a prerequisite for cavins acting as signaling modules following release from caveolae.
Milligram Quantities of Homogeneous Recombinant Full-Length Mouse Munc18c from Escherichia coli Cultures
Publisher: Public Library of Science (PLoS)
Date: 31-12-2013
Structural basis for the binding of the cancer targeting scorpion toxin, ClTx, to the vascular endothelia growth factor receptor neuropilin-1
Publisher: Elsevier BV
Date: 2021
Hepatocyte Growth Factor Acutely Perturbs Actin Filament Anchorage at the Epithelial Zonula Adherens
Publisher: Elsevier BV
Date: 03-2011
DOI: 10.1016/J.CUB.2011.02.018
Abstract: Cadherin adhesion molecules function in close cooperation with the actin cytoskeleton. At the zonula adherens (ZA) of polarized epithelial cells, E-cadherin adhesion induces the cortical recruitment of many key cytoskeletal regulators, which act in a dynamic integrated system to regulate junctional integrity and cell-cell interactions. This capacity for the cytoskeleton to support the ZA carries the implication that regulators of the junctional cytoskeleton might also be targeted to perturb junctional integrity. In this report, we now provide evidence for this hypothesis. We show that hepatocyte growth factor (HGF), which is well-known to disrupt cell-cell interactions, acutely perturbs ZA integrity much more rapidly than generally appreciated. This is accompanied by significant loss of junctional F-actin, a process that reflects loss of filament anchorage at the junctions. We demonstrate that this involves uncoupling of the unconventional motor myosin VI from junctional E-cadherin, a novel effect of HGF that is mediated by intracellular calcium. We conclude that regulators of the junctional cytoskeleton are likely to be major targets for cadherin junctions to be acutely modulated in development and perturbed in disease.
An Epilepsy-Associated GRIN2A Rare Variant Disrupts CaMKIIα Phosphorylation of GluN2A and NMDA Receptor Trafficking
Publisher: Elsevier BV
Date: 09-2020
Molecular identification of a BAR domain-containing coat complex for endosomal recycling of transmembrane proteins
Publisher: Springer Science and Business Media LLC
Date: 10-2019
Caveola mechanotransduction reinforces the cortical cytoskeleton to promote epithelial resilience
Publisher: Cold Spring Harbor Laboratory
Date: 29-03-2023
DOI: 10.1101/2023.03.29.534729
Abstract: As physical barriers, epithelia must preserve their integrity when challenged by mechanical stresses. Cell-cell junctions linked to the cortical cytoskeleton play key roles in this process, often with mechanotransduction mechanisms that reinforce tissues. Caveolae are mechanosensitive organelles that buffer tension via disassembly. Loss of caveolae, through caveolin-1 or cavin1 depletion, causes activation of PtdIns(4, 5)P 2 signalling, recruitment of FMNL2 formin, and enhanced cortical actin assembly. How this equates to physiological responses in epithelial cells containing endogenous caveolae is unknown. Here we examined the effect of mechanically-inducing acute disassembly of caveolae in epithelia. We show that perturbation of caveolae, through direct mechanical stress, reinforces the actin cortex at adherens junctions. Increasing interactions with membrane lipids by introducing multiple phosphatidylserine-binding undecad cavin1 (UC1) repeat domains into cavin1 rendered caveolae more stable to mechanical stimuli. This molecular stabilization blocked cortical reinforcement in response to mechanical stress. Cortical reinforcement elicited by the mechanically-induced disassembly of caveolae increased epithelial resilience against tensile stresses. These findings identify the actin cortex as a target of caveola mechanotransduction that contributes to epithelial integrity.
Low-resolution solution structures of Munc18:Syntaxin protein complexes indicate an open binding mode driven by the Syntaxin N-peptide
Publisher: Proceedings of the National Academy of Sciences
Date: 05-06-2012
Abstract: When nerve cells communicate, vesicles from one neuron fuse with the presynaptic membrane releasing chemicals that signal to the next. Similarly, when insulin binds its receptor on adipocytes or muscle, glucose transporter-4 vesicles fuse with the cell membrane, allowing glucose to be imported. These essential processes require the interaction of SNARE proteins on vesicle and cell membranes, as well as the enigmatic protein Munc18 that binds the SNARE protein Syntaxin. Here, we show that in solution the neuronal protein Syntaxin1a interacts with Munc18-1 whether or not the Syntaxin1a N-peptide is present. Conversely, the adipocyte protein Syntaxin4 does not bind its partner Munc18c unless the N-peptide is present. Solution-scattering data for the Munc18-1:Syntaxin1a complex in the absence of the N-peptide indicates that this complex adopts the inhibitory closed binding mode, exemplified by a crystal structure of the complex. However, when the N-peptide is present, the solution-scattering data indicate both Syntaxin1a and Syntaxin4 adopt extended conformations in complexes with their respective Munc18 partners. The low-resolution solution structure of the open Munc18:Syntaxin binding mode was modeled using data from cross-linking/mass spectrometry, small-angle X-ray scattering, and small-angle neutron scattering with contrast variation, indicating significant differences in Munc18:Syntaxin interactions compared with the closed binding mode. Overall, our results indicate that the neuronal Munc18-1:Syntaxin1a proteins can adopt two alternate and functionally distinct binding modes, closed and open, depending on the presence of the N-peptide, whereas Munc18c:Syntaxin4 adopts only the open binding mode.
Vps26B-retromer negatively regulates plasma membrane resensitization of PAR-2
Publisher: Wiley
Date: 15-07-2015
DOI: 10.1002/CBIN.10508
Abstract: Retromer is a trimeric complex composed of Vps26, Vps29, and Vps35 and has been shown to be involved in trafficking and sorting of transmembrane proteins within the endosome. The Vps26 paralog, Vps26B, defines a distinct retromer complex (Vps26B-retromer) in vivo and in vitro. Although endosomally associated, Vps26B-retromer does not bind the established retromer transmembrane cargo protein, cation-independent mannose 6-phosphate receptor (CI-M6PR), indicating it has a distinct role to retromer containing the Vps26A paralog. In the present study we use the previously established Vps26B-expressing HEK293 cell model to address the role of Vps26B-retromer in trafficking of the protease activated G-protein coupled receptor PAR-2 to the plasma membrane. In these cells there is no apparent defect in the initial activation of the receptor, as evidenced by release of intracellular calcium, ERK1/2 signaling and endocytosis of activated receptor PAR-2 into degradative organelles. However, we observe a significant delay in plasma membrane repopulation of the protease activated G protein-coupled receptor PAR-2 following stimulation, resulting in a defect in PAR-2 activation after resensitization. Here we propose that PAR-2 plasma membrane repopulation is regulated by Vps26B-retromer, describing a potential novel role for this complex.
Phox homology band 4.1/ezrin/radixin/moesin-like proteins function as molecular scaffolds that interact with cargo receptors and Ras GTPases
Publisher: Proceedings of the National Academy of Sciences
Date: 21-04-2011
Abstract: Following endocytosis, the fates of receptors, channels, and other transmembrane proteins are decided via specific endosomal sorting pathways, including recycling to the cell surface for continued activity. Two distinct phox-homology (PX)-domain-containing proteins, sorting nexin (SNX) 17 and SNX27, are critical regulators of recycling from endosomes to the cell surface. In this study we demonstrate that SNX17, SNX27, and SNX31 all possess a novel 4.1/ezrin/radixin/moesin (FERM)-like domain. SNX17 has been shown to bind to Asn-Pro-Xaa-Tyr (NPxY) sequences in the cytoplasmic tails of cargo such as LDL receptors and the amyloid precursor protein, and we find that both SNX17 and SNX27 display similar affinities for NPxY sorting motifs, suggesting conserved functions in endosomal recycling. Furthermore, we show for the first time that all three proteins are able to bind the Ras GTPase through their FERM-like domains. These interactions place the PX-FERM-like proteins at a hub of endosomal sorting and signaling processes. Studies of the SNX17 PX domain coupled with cellular localization experiments reveal the mechanistic basis for endosomal localization of the PX-FERM-like proteins, and structures of SNX17 and SNX27 determined by small angle X-ray scattering show that they adopt non-self-assembling, modular structures in solution. In summary, this work defines a novel family of proteins that participate in a network of interactions that will impact on both endosomal protein trafficking and compartment specific Ras signaling cascades.
Cavin1 intrinsically disordered domains are essential for fuzzy electrostatic interactions and caveola formation
Publisher: Springer Science and Business Media LLC
Date: 10-02-2021
DOI: 10.1038/S41467-021-21035-4
Abstract: Caveolae are spherically shaped nanodomains of the plasma membrane, generated by cooperative assembly of caveolin and cavin proteins. Cavins are cytosolic peripheral membrane proteins with negatively charged intrinsically disordered regions that flank positively charged α-helical regions. Here, we show that the three disordered domains of Cavin1 are essential for caveola formation and dynamic trafficking of caveolae. Electrostatic interactions between disordered regions and α-helical regions promote liquid-liquid phase separation behaviour of Cavin1 in vitro, assembly of Cavin1 oligomers in solution, generation of membrane curvature, association with caveolin-1, and Cavin1 recruitment to caveolae in cells. Removal of the first disordered region causes irreversible gel formation in vitro and results in aberrant caveola trafficking through the endosomal system. We propose a model for caveola assembly whereby fuzzy electrostatic interactions between Cavin1 and caveolin-1 proteins, combined with membrane lipid interactions, are required to generate membrane curvature and a metastable caveola coat.
Recognising the signals for endosomal trafficking
Publisher: Elsevier BV
Date: 08-2020
Author response: Structural insights into the architecture and membrane interactions of the conserved COMMD proteins
Publisher: eLife Sciences Publications, Ltd
Date: 27-06-2018
Introduction to special issue on endosome dynamics
Publisher: Elsevier BV
Date: 07-2014
Structure of Vps26B and mapping of its interaction with the retromer protein complex
Publisher: Wiley
Date: 11-12-2008
DOI: 10.1111/J.1600-0854.2007.00688.X
Abstract: Retromer is a heteromeric protein complex with important roles in endosomal membrane trafficking, most notably in the retrograde transport of lysosomal hydrolase receptors from endosomes to the Golgi. The core of retromer is composed of three subunits vacuolar protein sorting (Vps)35, Vps26 and Vps29, and in mammals, there are two paralogues of the medium subunit Vps26A and Vps26B. We find that both Vps26A and Vps26B bind to Vps35/Vps29 with nanomolar affinity and compete for a single-binding site to define distinct retromer complexes in vitro and in vivo. We have determined the crystal structure of mouse Vps26B and compare this structure with that of Vps26A. Vps26 proteins have a striking similarity to the arrestin family of proteins that regulate the signalling and endocytosis of G-protein-coupled receptors, although we observe that surface residues involved in arrestin function are not conserved in Vps26. Using structure-based mutagenesis, we show that both Vps26A and Vps26B are incorporated into retromer complexes through binding of Vps35 to a highly conserved surface patch within the C-terminal subdomain and that this interaction is required for endosomal recruitment of the proteins.
Presynaptic targeting of botulinum neurotoxin type A requires a tripartite PSG‐Syt1 ‐SV2 plasma membrane nanocluster for synaptic vesicle entry
Publisher: EMBO
Date: 25-05-2023
Abstract: The unique nerve terminal targeting of botulinum neurotoxin type A (BoNT/A) is due to its capacity to bind two receptors on the neuronal plasma membrane: polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Whether and how PSGs and SV2 may coordinate other proteins for BoNT/A recruitment and internalization remains unknown. Here, we demonstrate that the targeted endocytosis of BoNT/A into synaptic vesicles (SVs) requires a tripartite surface nanocluster. Live‐cell super‐resolution imaging and electron microscopy of catalytically inactivated BoNT/A wildtype and receptor‐binding‐deficient mutants in cultured hippoc al neurons demonstrated that BoNT/A must bind coincidentally to a PSG and SV2 to target synaptic vesicles. We reveal that BoNT/A simultaneously interacts with a preassembled PSG‐synaptotagmin‐1 (Syt1) complex and SV2 on the neuronal plasma membrane, facilitating Syt1‐SV2 nanoclustering that controls endocytic sorting of the toxin into synaptic vesicles. Syt1 CRISPRi knockdown suppressed BoNT/A‐ and BoNT/E‐induced neurointoxication as quantified by SNAP‐25 cleavage, suggesting that this tripartite nanocluster may be a unifying entry point for selected botulinum neurotoxins that hijack this for synaptic vesicle targeting.
Molecular Basis for Membrane Recruitment by the PX and C2 Domains of Class II Phosphoinositide 3-Kinase-C2α
Publisher: Elsevier BV
Date: 12-2018
DOI: 10.1016/J.STR.2018.08.010
Abstract: Phosphorylation of phosphoinositides by the class II phosphatidylinositol 3-kinase (PI3K) PI3K-C2α is essential for many processes, including neuroexocytosis and formation of clathrin-coated vesicles. A defining feature of the class II PI3Ks is a C-terminal module composed of phox-homology (PX) and C2 membrane interacting domains however, the mechanisms that control their specific cellular localization remain poorly understood. Here we report the crystal structure of the C2 domain of PI3K-C2α in complex with the phosphoinositide head-group mimic inositol hexaphosphate, revealing two distinct pockets for membrane binding. The C2 domain preferentially binds to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol (3,4,5)-trisphosphate, and low-resolution structures of the combined PX-C2 module by small-angle X-ray scattering reveal a compact conformation in which cooperative lipid binding by each domain binding can occur. Finally, we demonstrate an unexpected role for calcium in perturbing the membrane interactions of the PX-C2 module, which we speculate may be important for regulating the activity of PI3K-C2α.
Class IIa Histone Deacetylases Drive Toll-like Receptor-Inducible Glycolysis and Macrophage Inflammatory Responses via Pyruvate Kinase M2.
Publisher: Elsevier BV
Date: 02-2020
Neuropilin-1 is a host factor for SARS-CoV-2 infection
Publisher: American Association for the Advancement of Science (AAAS)
Date: 13-11-2020
Abstract: Virus-host interactions determine cellular entry and spreading in tissues. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the earlier SARS-CoV use angiotensin-converting enzyme 2 (ACE2) as a receptor however, their tissue tropism differs, raising the possibility that additional host factors are involved. The spike protein of SARS-CoV-2 contains a cleavage site for the protease furin that is absent from SARS-CoV (see the Perspective by Kielian). Cantuti-Castelvetri et al. now show that neuropilin-1 (NRP1), which is known to bind furin-cleaved substrates, potentiates SARS-CoV-2 infectivity. NRP1 is abundantly expressed in the respiratory and olfactory epithelium, with highest expression in endothelial and epithelial cells. Daly et al. found that the furin-cleaved S1 fragment of the spike protein binds directly to cell surface NRP1 and blocking this interaction with a small-molecule inhibitor or monoclonal antibodies reduced viral infection in cell culture. Understanding the role of NRP1 in SARS-CoV-2 infection may suggest potential targets for future antiviral therapeutics. Science , this issue p. 856 , p. 861 see also p. 765
Sorting nexin 27 (SNX27) regulates the trafficking and activity of the glutamine transporter ASCT2
Publisher: Elsevier BV
Date: 05-2018
Caveolae Control Contractile Tension for Epithelia to Eliminate Tumor Cells
Publisher: Elsevier BV
Date: 07-2020
Structural and thermodynamic analysis of the GFP:GFP‐nanobody complex
Publisher: Wiley
Date: 18-11-2010
DOI: 10.1002/PRO.519
Towards a generic prototyping approach for therapeutically-relevant peptides and proteins in a cell-free translation system
Publisher: Springer Science and Business Media LLC
Date: 11-01-2022
DOI: 10.1038/S41467-021-27854-9
Abstract: Advances in peptide and protein therapeutics increased the need for rapid and cost-effective polypeptide prototyping. While in vitro translation systems are well suited for fast and multiplexed polypeptide prototyping, they suffer from misfolding, aggregation and disulfide-bond scrambling of the translated products. Here we propose that efficient folding of in vitro produced disulfide-rich peptides and proteins can be achieved if performed in an aggregation-free and thermodynamically controlled folding environment. To this end, we modify an E. coli -based in vitro translation system to allow co-translational capture of translated products by affinity matrix. This process reduces protein aggregation and enables productive oxidative folding and recycling of misfolded states under thermodynamic control. In this study we show that the developed approach is likely to be generally applicable for prototyping of a wide variety of disulfide-constrained peptides, macrocyclic peptides with non-native bonds and antibody fragments in amounts sufficient for interaction analysis and biological activity assessment.
Elucidating the Lipid Binding Properties of Membrane-Active Peptides Using Cyclised Nanodiscs
Publisher: Frontiers Media SA
Date: 16-04-2019
Proteomic identification and structural basis for the interaction between sorting nexin SNX17 and PDLIM family proteins
Publisher: Elsevier BV
Date: 12-2022
DOI: 10.1016/J.STR.2022.10.001
Abstract: The sorting nexin SNX17 controls endosomal recycling of transmembrane cargo proteins including integrins, the amyloid precursor protein, and lipoprotein receptors. This requires association with the Commander trafficking complex and depends on the C terminus of SNX17 through unknown mechanisms. Using proteomics, we find that the SNX17 C terminus is sufficient for Commander interaction and also associates with members of the PDZ and LIM domain (PDLIM) family. SNX17 contains a type III PDZ binding motif that binds specifically to the PDLIM proteins. The structure of the PDLIM7 PDZ domain bound to the SNX17 C terminus reveals an unconventional perpendicular peptide interaction mediated by electrostatic contacts and a uniquely conserved proline-containing loop sequence in the PDLIM protein family. Our results define the mechanism of SNX17-PDLIM interaction and suggest that the PDLIM proteins may play a role in regulating the activity of SNX17 in conjunction with Commander and actin-rich endosomal trafficking domains.
SNT-1 Functions as the Ca2+ Sensor for Tonic and Evoked Neurotransmitter Release in Caenorhabditis Elegans
Publisher: Society for Neuroscience
Date: 14-05-2018
Homomeric Ring Assemblies of Eukaryotic Sm Proteins Have Affinity for Both RNA and DNA
Publisher: Elsevier BV
Date: 05-2003
A molecular code for endosomal recycling of phosphorylated cargos by the SNX27–retromer complex
Publisher: Springer Science and Business Media LLC
Date: 05-09-2016
DOI: 10.1038/NSMB.3290
Abstract: Recycling of internalized receptors from endosomal compartments is essential for the receptors' cell-surface homeostasis. Sorting nexin 27 (SNX27) cooperates with the retromer complex in the recycling of proteins containing type I PSD95-Dlg-ZO1 (PDZ)-binding motifs. Here we define specific acidic amino acid sequences upstream of the PDZ-binding motif required for high-affinity engagement of the human SNX27 PDZ domain. However, a subset of SNX27 ligands, such as the β
Caveolae
Publisher: Elsevier BV
Date: 04-2018
DOI: 10.1016/J.CUB.2017.11.075
Abstract: Caveolae are one of the most abundant and striking features of the plasma membrane of many mammalian cell types. These surface pits have fascinated biologists since their discovery by the pioneers of electron microscopy in the middle of the last century, but we are only just starting to understand their multiple functions. Molecular understanding of caveolar formation is advancing rapidly and we now know that sculpting the membrane to generate the characteristic bulb-shaped caveolar pit involves the coordinated action of integral membrane proteins and peripheral membrane coat proteins in a process dependent on their multiple interactions with membrane lipids. The resulting structure is further stabilised by protein complexes at the caveolar neck. Caveolae can bud to generate an endocytic carrier but can also be disassembled in response to specific stimuli to function as a mechanoprotective device. These structures have also been linked to numerous signalling pathways. Here, we will briefly summarise the current molecular and structural understanding of caveolar formation and dynamics, discuss how the crucial structural components of caveolae work together to generate a dynamic sensing domain, and discuss the implications of recent studies on the erse roles proposed for caveolae in different cells and tissues.
FBXL4 suppresses mitophagy by restricting the accumulation of NIX and BNIP3 mitophagy receptors
Publisher: Cold Spring Harbor Laboratory
Date: 12-10-2022
DOI: 10.1101/2022.10.12.511867
Abstract: Cells selectively remove damaged or excessive mitochondria through mitophagy, a specialized form of autophagy, to maintain mitochondrial quality and quantity. Mitophagy is induced in response to erse conditions, including hypoxia, cellular differentiation, and mitochondrial damage. However, the mechanisms by which cells remove specific dysfunctional mitochondria under steady-state conditions to fine-tune mitochondrial content are not well understood. Here, we report that SCF FBXL4 , an SKP1/CUL1/F-box protein ubiquitin ligase complex, localizes to the mitochondrial outer membrane in unstressed cells and mediates the constitutive ubiquitylation and degradation of the mitophagy receptors NIX and BNIP3 to suppress basal levels of mitophagy. We demonstrate that, unlike wild-type FBXL4, pathogenic variants of FBXL4 that cause encephalopathic mtDNA depletion syndrome (MTDPS13), do not efficiently interact with the core SCF ubiquitin ligase machinery or mediate the degradation of NIX and BNIP3. Thus, we reveal a molecular mechanism that actively suppresses mitophagy via preventing NIX and BNIP3 accumulation and propose that excessive basal mitophagy in the FBXL4-associated mtDNA depletion syndrome is caused by dysregulation of NIX and BNIP3 turnover.
SNX27 links DGK zeta to the control of transcriptional and metabolic programs in T lymphocytes
Publisher: Springer Science and Business Media LLC
Date: 27-11-2017
DOI: 10.1038/S41598-017-16370-W
Abstract: Sorting nexin 27 (SNX27) recycles PSD-95, Dlg1, ZO-1 (PDZ) domain-interacting membrane proteins and is essential to sustain adequate brain functions. Here we define a fundamental SNX27 function in T lymphocytes controlling antigen-induced transcriptional activation and metabolic reprogramming. SNX27 limits the activation of diacylglycerol (DAG)-based signals through its high affinity PDZ-interacting cargo DAG kinase ζ (DGKζ). SNX27 silencing in human T cells enhanced T cell receptor (TCR)-stimulated activator protein 1 (AP-1)- and nuclear factor κB (NF-κB)-mediated transcription. Transcription did not increase upon DGKζ silencing, suggesting that DGKζ function is dependent on SNX27. The enhanced transcriptional activation in SNX27-silenced cells contrasted with defective activation of the mammalian target of rapamycin (mTOR) pathway. The analysis of Snx27 −/− mice supported a role for SNX27 in the control of T cell growth. This study broadens our understanding of SNX27 as an integrator of lipid-based signals with the control of transcription and metabolic pathways.
Structural insights into the architecture and membrane interactions of the conserved COMMD proteins
Publisher: eLife Sciences Publications, Ltd
Date: 08-2018
DOI: 10.7554/ELIFE.35898
Abstract: The COMMD proteins are a conserved family of proteins with central roles in intracellular membrane trafficking and transcription. They form oligomeric complexes with each other and act as components of a larger assembly called the CCC complex, which is localized to endosomal compartments and mediates the transport of several transmembrane cargos. How these complexes are formed however is completely unknown. Here, we have systematically characterised the interactions between human COMMD proteins, and determined structures of COMMD proteins using X-ray crystallography and X-ray scattering to provide insights into the underlying mechanisms of homo- and heteromeric assembly. All COMMD proteins possess an α-helical N-terminal domain, and a highly conserved C-terminal domain that forms a tightly interlocked dimeric structure responsible for COMMD-COMMD interactions. The COMM domains also bind directly to components of CCC and mediate non-specific membrane association. Overall these studies show that COMMD proteins function as obligatory dimers with conserved domain architectures.
Retromer's Role in Endosomal Trafficking and Impaired Function in Neurodegenerative Diseases
Publisher: Bentham Science Publishers Ltd.
Date: 08-05-2017
Assembly and solution structure of the core retromer protein complex
Publisher: Wiley
Date: 15-10-2011
DOI: 10.1111/J.1600-0854.2010.01124.X
Abstract: Retromer is a peripheral membrane protein complex that has pleiotropic roles in endosomal membrane trafficking. The core of retromer possesses three subunits, VPS35, VPS29 and VPS26, that play different roles in binding to cargo, regulatory proteins and complex stabilization. We have performed an investigation of the thermodynamics of core retromer assembly using isothermal titration calorimetry (ITC) demonstrating that VPS35 acts as the central subunit to which VPS29 and VPS26 bind independently. Furthermore, we confirm that the conserved PRLYL motif of the large VPS35 subunit is critical for direct VPS26 interaction. Heat capacity measurements of VPS29 and VPS26 binding to VPS35 indicate extensive binding interfaces and suggest conformational alterations in VPS29 or VPS35 upon complex formation. Solution studies of the retromer core using small-angle X-ray scattering allow us to propose a model whereby VPS35 forms an extended platform with VPS29 and VPS26 bound at distal ends, with the potential for forming dimeric assemblies.
Insights into the PX (phox-homology) domain and SNX (sorting nexin) protein families: Structures, functions and roles in disease
Publisher: Portland Press Ltd.
Date: 14-12-2012
DOI: 10.1042/BJ20111226
Abstract: The mammalian genome encodes 49 proteins that possess a PX (phox-homology) domain, responsible for membrane attachment to organelles of the secretory and endocytic system via binding of phosphoinositide lipids. The PX domain proteins, most of which are classified as SNXs (sorting nexins), constitute an extremely erse family of molecules that play varied roles in membrane trafficking, cell signalling, membrane remodelling and organelle motility. In the present review, we present an overview of the family, incorporating recent functional and structural insights, and propose an updated classification of the proteins into distinct subfamilies on the basis of these insights. Almost all PX domain proteins bind PtdIns3P and are recruited to early endosomal membranes. Although other specificities and localizations have been reported for a select few family members, the molecular basis for binding to other lipids is still not clear. The PX domain is also emerging as an important protein–protein interaction domain, binding endocytic and exocytic machinery, transmembrane proteins and many other molecules. A comprehensive survey of the molecular interactions governed by PX proteins highlights the functional ersity of the family as trafficking cargo adaptors and membrane-associated scaffolds regulating cell signalling. Finally, we examine the mounting evidence linking PX proteins to different disorders, in particular focusing on their emerging importance in both pathogen invasion and amyloid production in Alzheimer's disease.
Cavin4 interacts with Bin1 to promote T-tubule formation and stability in developing skeletal muscle
Publisher: Rockefeller University Press
Date: 11-10-2021
Abstract: The cavin proteins are essential for caveola biogenesis and function. Here, we identify a role for the muscle-specific component, Cavin4, in skeletal muscle T-tubule development by analyzing two vertebrate systems, mouse and zebrafish. In both models, Cavin4 localized to T-tubules, and loss of Cavin4 resulted in aberrant T-tubule maturation. In zebrafish, which possess duplicated cavin4 paralogs, Cavin4b was shown to directly interact with the T-tubule–associated BAR domain protein Bin1. Loss of both Cavin4a and Cavin4b caused aberrant accumulation of interconnected caveolae within the T-tubules, a fragmented T-tubule network enriched in Caveolin-3, and an impaired Ca2+ response upon mechanical stimulation. We propose a role for Cavin4 in remodeling the T-tubule membrane early in development by recycling caveolar components from the T-tubule to the sarcolemma. This generates a stable T-tubule domain lacking caveolae that is essential for T-tubule function.
De novomacrocyclic peptides for inhibiting, stabilising and probing the function of the Retromer endosomal trafficking complex
Publisher: Cold Spring Harbor Laboratory
Date: 04-12-2020
DOI: 10.1101/2020.12.03.410779
Abstract: The Retromer complex (Vps35-Vps26-Vps29) is essential for endosomal membrane trafficking and signalling. Mutations in Retromer cause late-onset Parkinson’s disease, while viral and bacterial pathogens can hijack the complex during cellular infection. To modulate and probe its function we have created a novel series of macrocyclic peptides that bind Retromer with high affinity and specificity. Crystal structures show the majority of cyclic peptides bind to Vps29 via a Pro-Leu-containing sequence, structurally mimicking known interactors such as TBC1D5, and blocking their interaction with Retromer in vitro and in cells. By contrast, macrocyclic peptide RT-L4 binds Retromer at the Vps35-Vps26 interface and is a more effective molecular chaperone than reported small molecules, suggesting a new therapeutic avenue for targeting Retromer. Finally, tagged peptides can be used to probe the cellular localisation of Retromer and its functional interactions in cells, providing novel tools for studying Retromer function.
ESCPE-1 Mediates Retrograde Endosomal Sorting of the SARS-CoV-2 Host Factor Neuropilin-1
Publisher: Cold Spring Harbor Laboratory
Date: 22-01-2022
DOI: 10.1101/2022.01.20.477115
Abstract: Endosomal sorting maintains cellular homeostasis by recycling transmembrane proteins and associated proteins and lipids (termed ‘cargoes’) from the endosomal network to multiple subcellular destinations, including retrograde traffic to the trans -Golgi network (TGN). Viral and bacterial pathogens subvert retrograde trafficking machinery to facilitate infectivity. Here, we develop a proteomic screen to identify novel retrograde cargo proteins of the Endosomal SNX-BAR Sorting Complex Promoting Exit-1 (ESCPE-1). Using this methodology, we identify Neuropilin-1 (NRP1), a recently characterised host factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as a cargo directly bound and trafficked by ESCPE-1. ESCPE-1 mediates retrograde trafficking of engineered nanoparticles functionalised with the NRP1-interacting peptide of the SARS-CoV-2 Spike protein. ESCPE-1 sorting of NRP1 may therefore play a role in the intracellular membrane trafficking of NRP1-interacting viruses such as SARS-CoV-2.
Origins of PDZ Binding Specificity. A Computational and Experimental Study Using NHERF1 and the Parathyroid Hormone Receptor
Publisher: American Chemical Society (ACS)
Date: 14-04-2017
Cavin family proteins and the assembly of caveolae
Publisher: The Company of Biologists
Date: 04-2015
DOI: 10.1242/JCS.167866
Abstract: Caveolae are an abundant feature of the plasma membrane in many cells. Until recently, they were generally considered to be membrane invaginations whose formation primarily driven by integral membrane proteins called caveolins. However, the past decade has seen the emergence of the cavin family of peripheral membrane proteins as essential coat components and regulators of caveola biogenesis. In this Commentary, we summarise recent data on the role of cavins in caveola formation, highlighting structural studies that provide new insights into cavin coat assembly. In mammals, there are four cavin family members that associate through homo- and hetero-oligomerisation to form distinct subcomplexes on caveolae, which can be released into the cell in response to stimuli. Studies from several labs have provided a better understanding of cavin stoichiometry and the molecular basis for their oligomerisation, as well as identifying interactions with membrane phospholipids that may be important for caveola function. We propose a model in which coincident, low-affinity electrostatically controlled protein–protein and protein–lipid interactions allow the formation of caveolae, generating a meta-stable structure that can respond to plasma membrane stress by release of cavins.
Structural basis for endosomal trafficking of diverse transmembrane cargos by PX-FERM proteins
Publisher: Proceedings of the National Academy of Sciences
Date: 04-02-2013
Abstract: Transit of proteins through the endosomal organelle following endocytosis is critical for regulating the homeostasis of cell-surface proteins and controlling signal transduction pathways. However, the mechanisms that control these membrane-transport processes are poorly understood. The Phox-homology (PX) domain-containing proteins sorting nexin (SNX) 17, SNX27, and SNX31 have emerged recently as key regulators of endosomal recycling and bind conserved Asn-Pro-Xaa-Tyr–sorting signals in transmembrane cargos via an atypical band, 4.1/ezrin/radixin/moesin (FERM) domain. Here we present the crystal structure of the SNX17 FERM domain bound to the sorting motif of the P-selectin adhesion protein, revealing both the architecture of the atypical FERM domain and the molecular basis for recognition of these essential sorting sequences. We further show that the PX-FERM proteins share a promiscuous ability to bind a wide array of putative cargo molecules, including receptor tyrosine kinases, and propose a model for their coordinated molecular interactions with membrane, cargo, and regulatory proteins.
Little evidence that FAM65B belongs to the family of phox homology (PX) and bin/amphiphysin/rvs (BAR) domain-containing proteins
Publisher: Proceedings of the National Academy of Sciences
Date: 19-09-2014
Reconciling the regulatory role of Munc18 proteins in SNARE-complex assembly
Publisher: International Union of Crystallography (IUCr)
Date: 21-10-2014
DOI: 10.1107/S2052252514020727
Abstract: Membrane fusion is essential for human health, playing a vital role in processes as erse as neurotransmission and blood glucose control. Two protein families are key: (1) the Sec1p/Munc18 (SM) and (2) the soluble N -ethylmaleimide-sensitive attachment protein receptor (SNARE) proteins. Whilst the essential nature of these proteins is irrefutable, their exact regulatory roles in membrane fusion remain controversial. In particular, whether SM proteins promote and/or inhibit the SNARE-complex formation required for membrane fusion is not resolved. Crystal structures of SM proteins alone and in complex with their cognate SNARE proteins have provided some insight, however, these structures lack the transmembrane spanning regions of the SNARE proteins and may not accurately reflect the native state. Here, we review the literature surrounding the regulatory role of mammalian Munc18 SM proteins required for exocytosis in eukaryotes. Our analysis suggests that the conflicting roles reported for these SM proteins may reflect differences in experimental design. SNARE proteins appear to require C-terminal immobilization or anchoring, for ex le through a transmembrane domain, to form a functional fusion complex in the presence of Munc18 proteins.
Downregulation of SNX27 expression does not exacerbate amyloidogenesis in the APP/PS1 Alzheimer's disease mouse model.
Publisher: Elsevier BV
Date: 05-2019
DOI: 10.1016/J.NEUROBIOLAGING.2019.01.011
Abstract: There is in vitro evidence that sorting nexin family member 27 (SNX27), a member of the retromer complex, changes the distribution of the amyloid-beta (Aβ) precursor protein (APP) to promote its recycling and thereby prevent the production of Aβ, the toxic protein associated with Alzheimer's disease (AD). In this study, we analyzed the phenotype of the familial AD APP/PS mouse strain lacking one copy of the SNX27 gene. The reduction in SNX27 expression had no significant effect on the in vivo accumulation of soluble, total, or plaque-deposited Aβ, which is overproduced by the familial APP/PS transgenes. Hippoc al structure and cholinergic basal forebrain neuronal health were also unaffected. Nonetheless, mild positive and negative effects of age and/or genotype on spatial navigation performance were observed in SNX27
Structure-Based Reassessment of the Caveolin Signaling Model: Do Caveolae Regulate Signaling through Caveolin-Protein Interactions?
Publisher: Elsevier BV
Date: 07-2012
Vps26A and Vps26B Subunits Define Distinct Retromer Complexes
Publisher: Wiley
Date: 17-10-2011
DOI: 10.1111/J.1600-0854.2011.01284.X
Abstract: The trimeric Vps29-Vps35-Vps26 sub-complex of retromer mediates retrograde transport of transmembrane proteins from endosomes to the trans-Golgi network. Our group has recently identified a Vps26 paralogue, Vps26B, which is able to suppress the expression of Vps26A when exogenously expressed in mammalian cells and defines a distinct retromer complex (Vps26B-retromer) in vivo and in vitro. In this study, we use HEK293 cells stably expressing either Vps26A-myc or Vps26B-myc to address the role of retromer cargo transport and subcellular localization of the two core retromer complexes as defined by the two mammalian-specific Vps26 paralogues. Vps26B-retromer, like Vps26A-retromer, associates with TBC1D5 and GOLPH3. In contrast, no interaction between Vps26B-retromer and cation-independent mannose 6-phosphate receptor (CI-M6PR) was detected, leading to a degradation of this receptor and an increase in cathepsin D secretion. Colocalization of Vps26 paralogues with different endosomally located Rab proteins shows prolonged association of Vps26B-retromer with maturing endosomes relative to Vps26A-retromer. Interestingly, the cycling of CI-M6PR is restored upon deletion of the variable Vps26B C-terminal region indicating that this region is directly responsible for the differential function of the two paralogues. In summary, we show that the two distinct retromer complexes defined by different Vps26 paralogues are not functionally equivalent and that the Vps26B C-terminal region can control cargo selection of the Vps26B-retromer.
FBXL4 suppresses mitophagy by restricting the accumulation of NIX and BNIP3 mitophagy receptors
Publisher: EMBO
Date: 10-05-2023
Abstract: To maintain both mitochondrial quality and quantity, cells selectively remove damaged or excessive mitochondria through mitophagy, which is a specialised form of autophagy. Mitophagy is induced in response to erse conditions, including hypoxia, cellular differentiation and mitochondrial damage. However, the mechanisms that govern the removal of specific dysfunctional mitochondria under steady‐state conditions to fine‐tune mitochondrial content are not well understood. Here, we report that SCF FBXL4 , an SKP1/CUL1/F‐box protein ubiquitin ligase complex, localises to the mitochondrial outer membrane in unstressed cells and mediates the constitutive ubiquitylation and degradation of the mitophagy receptors NIX and BNIP3 to suppress basal levels of mitophagy. We demonstrate that the pathogenic variants of FBXL4 that cause encephalopathic mtDNA depletion syndrome (MTDPS13) do not efficiently interact with the core SCF ubiquitin ligase machinery or mediate the degradation of NIX and BNIP3. Thus, we reveal a molecular mechanism whereby FBXL4 actively suppresses mitophagy by preventing NIX and BNIP3 accumulation. We propose that the dysregulation of NIX and BNIP3 turnover causes excessive basal mitophagy in FBXL4‐associated mtDNA depletion syndrome.
Rab8a interacts directly with PI3Kγ to modulate TLR4-driven PI3K and mTOR signalling
Publisher: Springer Science and Business Media LLC
Date: 15-07-2014
DOI: 10.1038/NCOMMS5407
Abstract: Toll-like receptor 4 (TLR4) is activated by bacterial lipopolysaccharide (LPS) to mount innate immune responses. The TLR4-induced release of pro- and anti-inflammatory cytokines generates robust inflammatory responses, which must then be restrained to avoid disease. New mechanisms for the critical regulation of TLR-induced cytokine responses are still emerging. Here we find TLR4 complexes localized in LPS-induced dorsal ruffles on the surface of macrophages. We discover that the small GTPase Rab8a is enriched in these ruffles and recruits phosphatidylinositol 3-kinase (PI3Kγ) as an effector by interacting directly through its Ras-binding domain. Rab8a and PI3Kγ function to regulate Akt signalling generated by surface TLR4. Rab8a and PI3Kγ do not affect TLR4 endocytosis, but instead regulate mammalian target of rapamycin signalling as a mechanism for biasing the cytokine profile to constrain inflammation in innate immunity.
Structure and Membrane Binding Properties of the Endosomal Tetratricopeptide Repeat (TPR) Domain-containing Sorting Nexins SNX20 and SNX21
Publisher: Elsevier BV
Date: 06-2015
Increased Polyubiquitination and Proteasomal Degradation of a Munc18-1 Disease-Linked Mutant Causes Temperature-Sensitive Defect in Exocytosis
Publisher: Elsevier BV
Date: 10-2014
DOI: 10.1016/J.CELREP.2014.08.059
Abstract: Munc18-1 is a critical component of the core machinery controlling neuroexocytosis. Recently, mutations in Munc18-1 leading to the development of early infantile epileptic encephalopathy have been discovered. However, which degradative pathway controls Munc18-1 levels and how it impacts on neuroexocytosis in this pathology is unknown. Using neurosecretory cells deficient in Munc18, we show that a disease-linked mutation, C180Y, renders the protein unstable at 37°C. Although the mutated protein retains its function as t-SNARE chaperone, neuroexocytosis is impaired, a defect that can be rescued at a lower permissive temperature. We reveal that Munc18-1 undergoes K48-linked polyubiquitination, which is highly increased by the mutation, leading to proteasomal, but not lysosomal, degradation. Our data demonstrate that functional Munc18-1 levels are controlled through polyubiquitination and proteasomal degradation. The C180Y disease-causing mutation greatly potentiates this degradative pathway, rendering Munc18-1 unable to facilitate neuroexocytosis, a phenotype that is reversed at a permissive temperature.
EhC2B, a C2 domain-containing protein, promotes erythrophagocytosis in Entamoeba histolytica via actin nucleation
Publisher: Public Library of Science (PLoS)
Date: 04-05-2020
VAMP2/Munc18-1 domain 3a interaction controls the nanoscale reorganization underpinning vesicular priming
Publisher: Cold Spring Harbor Laboratory
Date: 11-09-2023
Revisiting interaction specificity reveals neuronal and adipocyte Munc18 membrane fusion regulatory proteins differ in their binding interactions with partner SNARE Syntaxins
Publisher: Public Library of Science (PLoS)
Date: 31-10-2017
Munc18-1 is a molecular chaperone for α-synuclein, controlling its self-replicating aggregation
Publisher: Rockefeller University Press
Date: 05-09-2016
Abstract: Munc18-1 is a key component of the exocytic machinery that controls neurotransmitter release. Munc18-1 heterozygous mutations cause developmental defects and epileptic phenotypes, including infantile epileptic encephalopathy (EIEE), suggestive of a gain of pathological function. Here, we used single-molecule analysis, gene-edited cells, and neurons to demonstrate that Munc18-1 EIEE-causing mutants form large polymers that coaggregate wild-type Munc18-1 in vitro and in cells. Surprisingly, Munc18-1 EIEE mutants also form Lewy body–like structures that contain α-synuclein (α-Syn). We reveal that Munc18-1 binds α-Syn, and its EIEE mutants coaggregate α-Syn. Likewise, removal of endogenous Munc18-1 increases the aggregative propensity of α-SynWT and that of the Parkinson’s disease–causing α-SynA30P mutant, an effect rescued by Munc18-1WT expression, indicative of chaperone activity. Coexpression of the α-SynA30P mutant with Munc18-1 reduced the number of α-SynA30P aggregates. Munc18-1 mutations and haploinsufficiency may therefore trigger a pathogenic gain of function through both the corruption of native Munc18-1 and a perturbed chaperone activity for α-Syn leading to aggregation-induced neurodegeneration.
The Phox Homology (PX) Domain
Publisher: Springer International Publishing
Date: 2018
Abstract: The phox-homology (PX) domain is a phosphoinositide-binding domain conserved in all eukaryotes and present in 49 human proteins. Proteins containing PX domains, many of which are also known as sorting nexins (SNXs), have a large variety of functions in membrane trafficking, cell signaling, and lipid metabolism in association with membranes of the secretory and endocytic system. In this review we discuss the structural basis for both canonical lipid interactions with the endosome-enriched lipid phosphatidylinositol-3-phosphate (PtdIns3P) as well as non-canonical lipids that promote membrane association. We also describe recent advances in defining the erse mechanisms by which PX domains interact with other proteins including the retromer trafficking complex and proteins secreted by bacterial pathogens. Like other membrane interacting domains, the attachment of PX domain proteins to specific membranes is often facilitated by additional interactions that contribute to binding avidity, and we discuss this coincidence detection for several known ex les.
Structure of the membrane-assembled retromer coat determined by cryo-electron tomography.
Publisher: Springer Science and Business Media LLC
Date: 09-2018
Parkinson Disease-linked Vps35 R524W Mutation Impairs the Endosomal Association of Retromer and Induces -Synuclein Aggregation
Publisher: Elsevier BV
Date: 08-2016
Structural Basis for Different Phosphoinositide Specificities of the PX Domains of Sorting Nexins Regulating G-protein Signaling
Publisher: Elsevier BV
Date: 10-2014
Sorting nexin 27 couples PTHR trafficking to retromer for signal regulation in osteoblasts during bone growth
Publisher: American Society for Cell Biology (ASCB)
Date: 15-04-2016
Abstract: The parathyroid hormone 1 receptor (PTHR) is central to the process of bone formation and remodeling. PTHR signaling requires receptor internalization into endosomes, which is then terminated by recycling or degradation. Here we show that sorting nexin 27 (SNX27) functions as an adaptor that couples PTHR to the retromer trafficking complex. SNX27 binds directly to the C-terminal PDZ-binding motif of PTHR, wiring it to retromer for endosomal sorting. The structure of SNX27 bound to the PTHR motif reveals a high-affinity interface involving conserved electrostatic interactions. Mechanistically, depletion of SNX27 or retromer augments intracellular PTHR signaling in endosomes. Osteoblasts genetically lacking SNX27 show similar disruptions in PTHR signaling and greatly reduced capacity for bone mineralization, contributing to profound skeletal deficits in SNX27-knockout mice. Taken together, our data support a critical role for SNX27-retromer mediated transport of PTHR in normal bone development.
Regulation of the divalent metal ion transporter via membrane budding.
Publisher: Springer Science and Business Media LLC
Date: 21-06-2016
Abstract: The release of extracellular vesicles (EVs) is important for both normal physiology and disease. However, a basic understanding of the targeting of EV cargoes, composition and mechanism of release is lacking. Here we present evidence that the alent metal ion transporter (DMT1) is unexpectedly regulated through release in EVs. This process involves the Nedd4-2 ubiquitin ligase, and the adaptor proteins Arrdc1 and Arrdc4 via different budding mechanisms. We show that mouse gut explants release endogenous DMT1 in EVs. Although we observed no change in the relative amount of DMT1 released in EVs from gut explants in Arrdc1 or Arrdc4 deficient mice, the extent of EVs released was significantly reduced indicating an adaptor role in biogenesis. Furthermore, using Arrdc1 or Arrdc4 knockout mouse embryonic fibroblasts, we show that both Arrdc1 and Arrdc4 are non-redundant positive regulators of EV release. Our results suggest that DMT1 release from the plasma membrane into EVs may represent a novel mechanism for the maintenance of iron homeostasis, which may also be important for the regulation of other membrane proteins.
Molecular Characterization of Caveolin-induced Membrane Curvature
Publisher: Elsevier BV
Date: 10-2015
A unique PDZ domain and arrestin-like fold interaction reveals mechanistic details of endocytic recycling by SNX27-retromer
Publisher: Proceedings of the National Academy of Sciences
Date: 18-08-2014
Abstract: Cell surface proteins are regulated by a constant cycle of internalization and recycling from intracellular compartments called endosomes. From these organelles, two protein sorting platforms, sorting nexin 27 (SNX27) and the retromer complex, play a critical role in the retrieval of various proteins responsible for ion transport, glucose metabolism, neurotransmission, and other cell functions. Based on the three-dimensional structure of SNX27 in complex with the retromer subunit VPS26, we define the mechanism by which these proteins cooperate to drive endosomal cargo sorting. Retromer and SNX27 dysfunction is implicated in various disorders, including diabetes, Down syndrome, Parkinson disease, and Alzheimer’s disease, and this work provides important insights into the assembly of this essential endosomal sorting machinery.
Drosophila Snazarus Regulates a Lipid Droplet Population at Plasma Membrane-Droplet Contacts in Adipocytes
Publisher: Elsevier BV
Date: 09-2019
The Vps35 D620N mutation linked to Parkinson's disease disrupts the cargo sorting function of retromer.
Publisher: Wiley
Date: 14-11-2014
DOI: 10.1111/TRA.12136
Abstract: The retromer is a trimeric cargo-recognition protein complex composed of Vps26, Vps29 and Vps35 associated with protein trafficking within endosomes. Recently, a pathogenic point mutation within the Vps35 subunit (D620N) was linked to the manifestation of Parkinson's disease (PD). Here, we investigated details underlying the molecular mechanism by which the D620N mutation in Vps35 modulates retromer function, including examination of retromer's subcellular localization and its capacity to sort cargo. We show that expression of the PD-linked Vps35 D620N mutant redistributes retromer-positive endosomes to a perinuclear subcellular localization and that these endosomes are enlarged in both model cell lines and fibroblasts isolated from a PD patient. Vps35 D620N is correctly folded and binds Vps29 and Vps26A with the same affinity as wild-type Vps35. While PD-linked point mutant Vps35 D620N interacts with the cation-independent mannose-6-phosphate receptor (CI-M6PR), a known retromer cargo, we find that its expression disrupts the trafficking of cathepsin D, a CI-M6PR ligand and protease responsible for degradation of α-synuclein, a causative agent of PD. In summary, we find that the expression of Vps35 D620N leads to endosomal alterations and trafficking defects that may partly explain its action in PD.
Synaptotagmin 1 mediates toxicity of botulinum neurotoxin type A
Publisher: Cold Spring Harbor Laboratory
Date: 03-2022
DOI: 10.1101/2022.02.28.482398
Abstract: The extreme neurotoxicity of botulinum neurotoxins (BoNTs), broadly used as a therapeutics, is mediated by their direct binding to two plasma membrane receptors: polysialogangliosides (PSGs) and serotype-dependently either synaptotagmin 1/2 (Syt1/2) or synaptic vesicle glycoprotein 2 (SV2). Here, we demonstrate that although BoNT/A serotype binds directly to PSG and SV2, its neurotoxicity depends on Syt1. Using single-molecule superresolution microscopy of pathological concentrations of BoNT/A holotoxins, we demonstrate that the toxin binds to a preassembled PSG-Syt1 complex that forms nanoclusters with SV2 on the plasma membrane of hippoc al neurons. This coincidental interaction controls the selective endocytic targeting of BoNT/A into synaptic vesicles, leading to incapacitation of neuronal communication. Our results suggest that Syt1-SV2 plasma membrane nanoclusters may acts as a common target for various BoNT serotypes. BoNT/A hijacks an intrinsic tripartite PSG-Syt1-SV2 endocytic sorting mechanism to incapacitate neuronal communication
Possible roles for Munc18-1 domain 3a and Syntaxin1 N-peptide and C-terminal anchor in SNARE complex formation
Publisher: Proceedings of the National Academy of Sciences
Date: 30-12-2011
Abstract: Munc18-1 and Syntaxin1 are essential proteins for SNARE-mediated neurotransmission. Munc18-1 participates in synaptic vesicle fusion via dual roles: as a docking/chaperone protein by binding closed Syntaxin1, and as a fusion protein that binds SNARE complexes in a Syntaxin1 N-peptide dependent manner. The two roles are associated with a closed–open Syntaxin1 conformational transition. Here, we show that Syntaxin N-peptide binding to Munc18-1 is not highly selective, suggesting that other parts of the SNARE complex are involved in binding to Munc18-1. We also find that Syntaxin1, with an N peptide and a physically anchored C terminus, binds to Munc18-1 and that this complex can participate in SNARE complex formation. We report a Munc18-1–N-peptide crystal structure that, together with other data, reveals how Munc18-1 might transit from a conformation that binds closed Syntaxin1 to one that may be compatible with binding open Syntaxin1 and SNARE complexes. Our results suggest the possibility that structural transitions occur in both Munc18-1 and Syntaxin1 during their binary interaction. We hypothesize that Munc18-1 domain 3a undergoes a conformational change that may allow coiled-coil interactions with SNARE complexes.
The recognition of membrane-bound PtdIns3P by PX domains
Publisher: Wiley
Date: 12-05-2014
DOI: 10.1002/PROT.24593
Abstract: Phox-homology (PX) domains target proteins to the organelles of the secretary and endocytic systems by binding to phosphatidylinositol phospholipids (PIPs). Among all the structures of PX domains that have been solved, only three have been solved in a complex with the main physiological ligand: PtdIns3P. In this work, molecular dynamic simulations have been used to explore the structure and dynamics of the p40(phox) -PX domain and the SNX17-PX domain and their interaction with membrane-bound PtdIns3P. In the simulations, both PX domains associated spontaneously with the membrane-bound PtdIns3P and formed stable complexes. The interaction between the p40(phox) -PX domain and PtdIns3P in the membrane was found to be similar to the crystal structure of the p40(phox) -PX-PtdIns3P complex that is available. The interaction between the SNX17-PX domain and PtdIns3P was similar to that observed in the p40(phox) -PX-PtdIns3P complex however, some residues adopted different orientations. The simulations also showed that nonspecific interactions between the β1-β2 loop and the membrane play an important role in the interaction of membrane bound PtdIns3P and different PX domains. The behaviour of unbound PtdIns3P within a 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) membrane environment was also examined and compared to the available experimental data and simulation studies of related molecules.
A novel dual Ca2+ sensor system regulates Ca2+-dependent neurotransmitter release
Publisher: Rockefeller University Press
Date: 11-02-2021
Abstract: Ca2+-dependent neurotransmitter release requires synaptotagmins as Ca2+ sensors to trigger synaptic vesicle (SV) exocytosis via binding of their tandem C2 domains—C2A and C2B—to Ca2+. We have previously demonstrated that SNT-1, a mouse synaptotagmin-1 (Syt1) homologue, functions as the fast Ca2+ sensor in Caenorhabditis elegans. Here, we report a new Ca2+ sensor, SNT-3, which triggers delayed Ca2+-dependent neurotransmitter release. snt-1 snt-3 double mutants abolish evoked synaptic transmission, demonstrating that C. elegans NMJs use a dual Ca2+ sensor system. SNT-3 possesses canonical aspartate residues in both C2 domains, but lacks an N-terminal transmembrane (TM) domain. Biochemical evidence demonstrates that SNT-3 binds both Ca2+ and the plasma membrane. Functional analysis shows that SNT-3 is activated when SNT-1 function is impaired, triggering SV release that is loosely coupled to Ca2+ entry. Compared with SNT-1, which is tethered to SVs, SNT-3 is not associated with SV. Eliminating the SV tethering of SNT-1 by removing the TM domain or the whole N terminus rescues fast release kinetics, demonstrating that cytoplasmic SNT-1 is still functional and triggers fast neurotransmitter release, but also exhibits decreased evoked litude and release probability. These results suggest that the fast and slow properties of SV release are determined by the intrinsically different C2 domains in SNT-1 and SNT-3, rather than their N-termini–mediated membrane tethering. Our findings therefore reveal a novel dual Ca2+ sensor system in C. elegans and provide significant insights into Ca2+-regulated exocytosis.
Fragment-based screening by X-ray crystallography, MS and isothermal titration calorimetry to identify PNMT (phenylethanolamine N-methyltransferase) inhibitors
Publisher: Portland Press Ltd.
Date: 14-09-2010
DOI: 10.1042/BJ20100651
Abstract: CNS (central nervous system) adrenaline (epinephrine) is implicated in a wide range of physiological and pathological conditions. PNMT (phenylethanolamine N-methyltransferase) catalyses the final step in the biosynthesis of adrenaline, the conversion of noradrenaline (norepinephrine) to adrenaline by methylation. To help elucidate the role of CNS adrenaline, and to develop potential drug leads, potent, selective and CNS-active inhibitors are required. The fragment screening approach has advantages over other lead discovery methods including high hit rates, more efficient hits and the ability to s le chemical ersity more easily. In the present study we applied fragment-based screening approaches to the enzyme PNMT. We used crystallography as the primary screen and identified 12 hits from a small commercial library of 384 drug-like fragments. The hits include nine chemicals with two fused rings and three single-ring chemical systems. Eight of the hits come from three chemical classes: benzimidazoles (a known class of PNMT inhibitor), purines and quinolines. Nine of the hits have measurable binding affinities (~5–700 μM) as determined by isothermal titration calorimetry and all nine have ligand efficiencies of 0.39 kcal/mol per heavy atom or better (1 kcal≈4.184 kJ). We synthesized five elaborated benzimidazole compounds and characterized their binding to PNMT, showing for the first time how this class of inhibitors interact with the noradrenaline-binding site. Finally, we performed a pilot study with PNMT for fragment-based screening by MS showing that this approach could be used as a fast and efficient first-pass screening method prior to characterization of binding mode and affinity of hits.
SNX27–Retromer directly binds ESCPE-1 to transfer cargo proteins during endosomal recycling
Publisher: Public Library of Science (PLoS)
Date: 13-04-2022
DOI: 10.1371/JOURNAL.PBIO.3001601
Abstract: Coat complexes coordinate cargo recognition through cargo adaptors with biogenesis of transport carriers during integral membrane protein trafficking. Here, we combine biochemical, structural, and cellular analyses to establish the mechanistic basis through which SNX27–Retromer, a major endosomal cargo adaptor, couples to the membrane remodeling endosomal SNX-BAR sorting complex for promoting exit 1 (ESCPE-1). In showing that the SNX27 FERM (4.1/ezrin/radixin/moesin) domain directly binds acidic-Asp-Leu-Phe (aDLF) motifs in the SNX1/SNX2 subunits of ESCPE-1, we propose a handover model where SNX27–Retromer captured cargo proteins are transferred into ESCPE-1 transport carriers to promote endosome-to-plasma membrane recycling. By revealing that assembly of the SNX27:Retromer:ESCPE-1 coat evolved in a stepwise manner during early metazoan evolution, likely reflecting the increasing complexity of endosome-to-plasma membrane recycling from the ancestral opisthokont to modern animals, we provide further evidence of the functional ersification of yeast pentameric Retromer in the recycling of hundreds of integral membrane proteins in metazoans.
Functional characterization of retromer in GLUT4 storage vesicle formation and adipocyte differentiation
Publisher: Wiley
Date: 18-11-2016
DOI: 10.1096/FJ.15-274704
Abstract: Insulin-stimulated translocation of glucose transporter 4 (GLUT4) storage vesicles (GSVs), the specialized intracellular compartments within mature adipocytes, to the plasma membrane (PM) is a fundamental cellular process for maintaining glucose homeostasis. Using 2 independent adipocyte cell line models, human primary Simpson-Golabi-Behmel syndrome and mouse 3T3-L1 fibroblast cell lines, we demonstrate that the endosome-associated protein-sorting complex retromer colocalizes with GLUT4 on the GSVs by confocal microscopy in mature adipocytes. By use of both confocal microscopy and differential ultracentrifugation techniques, retromer is redistributed to the PM of mature adipocytes upon insulin stimulation. Furthermore, stable knockdown of the retromer subunit-vacuolar protein-sorting 35, or the retromer-associated protein sorting nexin 27, by lentivirus-delivered small hairpin RNA impaired the adipogenesis process when compared to nonsilence control. The knockdown of retromer decreased peroxisome proliferator activated receptor γ expression during differentiation, generating adipocytes with decreased levels of GSVs, lipid droplet accumulation, and insulin-stimulated glucose uptake. In conclusion, our study demonstrates a role for retromer in the GSV formation and adipogenesis.
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