Thomas Nebl

Structural basis for plasmepsin V inhibition that blocks export of malaria proteins to human erythrocytes

Publisher: Springer Science and Business Media LLC

Date: 27-07-2015

DOI: 10.1038/NSMB.3061

Abstract: Plasmepsin V, an essential aspartyl protease of malaria parasites, has a key role in the export of effector proteins to parasite-infected erythrocytes. Consequently, it is an important drug target for the two most virulent malaria parasites of humans, Plasmodium falciparum and Plasmodium vivax. We developed a potent inhibitor of plasmepsin V, called WEHI-842, which directly mimics the Plasmodium export element (PEXEL). WEHI-842 inhibits recombinant plasmepsin V with a half-maximal inhibitory concentration of 0.2 nM, efficiently blocks protein export and inhibits parasite growth. We obtained the structure of P. vivax plasmepsin V in complex with WEHI-842 to 2.4-Å resolution, which provides an explanation for the strict requirements for substrate and inhibitor binding. The structure characterizes both a plant-like fold and a malaria-specific helix-turn-helix motif that are likely to be important in cleavage of effector substrates for export.

Gastro-intestinal Digestibility of Processed Forms of Cow’s Milk Proteins under Simulated Infant and Adult Conditions Characterised by in vitro Methods

Publisher: Canadian Center of Science and Education

Date: 17-01-2019

DOI: 10.5539/JFR.V8N1P80

Abstract: Human digestion involves chemical, biochemical and fermentative processes integrated to achieve optimal nutrient absorption along the gastro-intestinal (GI) tract. In particular, efficient gastro-intestinal enzymatic digestion of proteins is critical for maximum upper intestinal protein absorption and may influence the distribution of dietary protein between the & lsquo host& rsquo and microbiota. However, the relative efficiency of infant versus adult conditions of GI digestion of cow& rsquo s milk products have not been systematically studied. Conditions for in vitro GI enzymatic digestion, optimised for either infant or adult conditions, were applied to test pasteurised, skimmed milk, skimmed milk powder (SMP) and whey and casein protein fractions of cow& rsquo s milk. Research methods included digestion monitoring by o-phthaldialdehyde (OPA) derivitisation of peptides and free amino acids, and digestate analysis to characterise low mass & lsquo absorbing& rsquo products (size exclusion chromatography) and larger non-absorbing products (700-3500 Da) by MALDI-TOF-MS. The results demonstrated significantly lower digestion efficiency for infant versus adult digestion conditions, particularly in the gastric phase. Digestibility of pasteurised milk and SMP were generally lower than either whey or casein protein fractions. Lower overall protein digestibility was associated with lower and higher proportions of & lsquo absorbing& rsquo and & lsquo non-absorbing& rsquo peptides, respectively. The higher yield of & lsquo non-absorbing& rsquo peptides for infant digestion may permit strategic release of peptides with receptor-antagonistic bioactivity along the gut, and/or may promote supply of nitrogen for the microbiota. The favouring of digestion-resistant peptides to the colon due to processing requires further research to also investigate potential negative relationships with health.

Purification of an insect juvenile hormone receptor complex enables insights into its post-translational phosphorylation

Publisher: Elsevier BV

Date: 12-2021

DOI: 10.1016/J.JBC.2021.101387

Menstrual fluid factors facilitate tissue repair: Identification and functional action in endometrial and skin repair

Publisher: Wiley

Date: 23-07-2019

DOI: 10.1096/FJ.201800086R

Supervillin modulation of focal adhesions involving TRIP6/ZRP-1

Publisher: Rockefeller University Press

Date: 31-07-2006

DOI: 10.1083/JCB.200512051

Abstract: Cell–substrate contacts, called focal adhesions (FAs), are dynamic in rapidly moving cells. We show that supervillin (SV)—a peripheral membrane protein that binds myosin II and F-actin in such cells—negatively regulates stress fibers, FAs, and cell–substrate adhesion. The major FA regulatory sequence within SV (SV342-571) binds to the LIM domains of two proteins in the zyxin family, thyroid receptor–interacting protein 6 (TRIP6) and lipoma-preferred partner (LPP), but not to zyxin itself. SV and TRIP6 colocalize within large FAs, where TRIP6 may help recruit SV. RNAi-mediated decreases in either protein increase cell adhesion to fibronectin. TRIP6 partially rescues SV effects on stress fibers and FAs, apparently by mislocating SV away from FAs. Thus, SV interactions with TRIP6 at FAs promote loss of FA structure and function. SV and TRIP6 binding partners suggest several specific mechanisms through which the SV–TRIP6 interaction may regulate FA maturation and/or disassembly.

Engineered enzymes that retain and regenerate their cofactors enable continuous-flow biocatalysis

Publisher: Springer Science and Business Media LLC

Date: 30-09-2019

DOI: 10.1038/S41929-019-0353-0

Dual Site-Specific Labeling of an Antibody Fragment through Sortase A and π-Clamp Conjugation

Publisher: American Chemical Society (ACS)

Date: 27-09-2019

DOI: 10.1021/ACS.BIOCONJCHEM.9B00639

Abstract: The functionalization of proteins with different cargo molecules is highly desirable for a broad range of applications. However, the reproducible production of defined conjugates with multiple functionalities is a significant challenge. Herein, we report the dual site-specific labeling of an antibody fragment, utilizing the orthogonal Sortase A and π-cl conjugation methods, and demonstrate that binding of the antibody fragment to its target receptor is retained after dual labeling.

Design of a methotrexate-controlled chemical dimerization system and its use in bio-electronic devices

Publisher: Springer Science and Business Media LLC

Date: 08-12-2021

DOI: 10.1038/S41467-021-27184-W

Abstract: Natural evolution produced polypeptides that selectively recognize chemical entities and their polymers, ranging from ions to proteins and nucleic acids. Such selective interactions serve as entry points to biological signaling and metabolic pathways. The ability to engineer artificial versions of such entry points is a key goal of synthetic biology, bioengineering and bioelectronics. We set out to map the optimal strategy for developing artificial small molecule:protein complexes that function as chemically induced dimerization (CID) systems. Using several starting points, we evolved CID systems controlled by a therapeutic drug methotrexate. Biophysical and structural analysis of methotrexate-controlled CID system reveals the critical role played by drug-induced conformational change in ligand-controlled protein complex assembly. We demonstrate utility of the developed CID by constructing electrochemical biosensors of methotrexate that enable quantification of methotrexate in human serum. Furthermore, using the methotrexate and functionally related biosensor of rapamycin we developed a multiplexed bioelectronic system that can perform repeated measurements of multiple analytes. The presented results open the door for construction of genetically encoded signaling systems for use in bioelectronics and diagnostics, as well as metabolic and signaling network engineering.

Proteomic analysis reveals novel proteins associated with the Plasmodium protein exporter PTEX and a loss of complex stability upon truncation of the core PTEX component, PTEX150

Publisher: Hindawi Limited

Date: 03-05-2016

DOI: 10.1111/CMI.12596

Abstract: The Plasmodium translocon for exported proteins (PTEX) has been established as the machinery responsible for the translocation of all classes of exported proteins beyond the parasitophorous vacuolar membrane of the intraerythrocytic malaria parasite. Protein export, particularly in the asexual blood stage, is crucial for parasite survival as exported proteins are involved in remodelling the host cell, an essential process for nutrient uptake, waste removal and immune evasion. Here, we have truncated the conserved C-terminus of one of the essential PTEX components, PTEX150, in Plasmodium falciparum in an attempt to create mutants of reduced functionality. Parasites tolerated C-terminal truncations of up to 125 amino acids with no reduction in growth, protein export or the establishment of new permeability pathways. Quantitative proteomic approaches however revealed a decrease in other PTEX subunits associating with PTEX150 in truncation mutants, suggesting a role for the C-terminus of PTEX150 in regulating PTEX stability. Our analyses also reveal three previously unreported PTEX-associated proteins, namely PV1, Pf113 and Hsp70-x (respective PlasmoDB numbers PF3D7_1129100, PF3D7_1420700 and PF3D7_0831700) and demonstrate that core PTEX proteins exist in various distinct multimeric forms outside the major complex.

Plasmodium falciparum exoerythrocytic forms require the PTEX translocon for development in human hepatocytes

Publisher: Research Square Platform LLC

Date: 03-2023

DOI: 10.21203/RS.3.RS-2632356/V1

Abstract: Plasmodium falciparum assembles a protein translocon (PTEX) at the parasitophorous vacuole membrane (PVM) of infected erythrocytes, through which several hundred proteins are exported. The preceding Plasmodium liver stage develops in hepatocytes within a PVM however, the importance of PTEX and identification of exported proteins in P. falciparum liver stages remains unexplored. Here, we apply the FlpL/ FRT system to P. falciparum NF54 to conditionally excise genes in sporozoites, enabling studies at the liver stage. Conditional disruption of PTEX components PTEX150 and EXP2 in sporozoites does not affect their development or infectivity but attenuates liver stage growth. While PTEX150-deficiency significantly reduces liver load in humanized mice, EXP2-deficiency conferred a severe fitness cost, demonstrating that PTEX is essential for P. falciparum liver stage development. We show that liver specific protein 2 (LISP2) and circumsporozoite protein (CSP) contain putative PEXEL sequences cleaved by plasmepsin V, yet they localize to the PVM of infected hepatocytes. The abundance of LISP2 is reduced in PTEX-deficient liver stages, suggesting this protein is degraded in the absence of a functioning PTEX complex. This study employs the FlpL/ FRT system for functional analysis of P. falciparum pre-erythrocytic biology, revealing that the protein export translocon required for growth in erythrocytes is essential for P. falciparum development in hepatocytes and normal LISP2 expression. It also describes two P. falciparum proteins that contain putative PEXEL motifs that are targeted to the PVM.

A Serpentine Receptor-Dependent, Gβ- and Ca2+ Influx-Independent Pathway Regulates Mitogen-Activated Protein Kinase ERK2 in Dictyostelium

Publisher: Elsevier BV

Date: 07-1999

DOI: 10.1006/BBRC.1999.0862

The X-ray crystal structure of the N-terminal domain of Ssr4, a Schizosaccharomyces pombe chromatin-remodelling protein

Publisher: International Union of Crystallography (IUCr)

Date: 25-11-2020

DOI: 10.1107/S2053230X20015216

Abstract: Ssr4 is a yeast protein from Schizosaccharomyces pombe and is an essential part of the chromatin-remodelling [SWI/SNF and RSC (remodelling the structure of chromatin)] complexes found in S. pombe . These complexes (or their homologues) regulate gene expression in eukaryotic organisms, affecting a large number of genes both positively and negatively. The downstream effects are seen in development, and in humans have implications for disease such as cancer. The chromatin structure is altered by modifying the DNA–histone contacts, thus opening up or closing down sections of DNA to specific transcription factors that regulate the transcription of genes. The Ssr4 sequence has little homology to other sequences in the Protein Data Bank, so the structure was solved using an iodine derivative with SAD phasing. The structure of the N-terminal domain is an antiparallel β-sheet of seven strands with α-helices on one side and random coil on the other. The structure is significantly different to deposited structures and was used as a target in the most recent Critical Assessment of Techniques for Protein Structure Prediction (CASP predictioncenter.org/) competition.

The evolving story of AtzT, a periplasmic binding protein

Publisher: International Union of Crystallography (IUCr)

Date: 31-10-2019

DOI: 10.1107/S2059798319013883

Abstract: Atrazine is an s -triazine-based herbicide that is used in many countries around the world in many millions of tons per year. A small number of organisms, such as Pseudomonas sp. strain ADP, have evolved to use this modified s -triazine as a food source, and the various genes required to metabolize atrazine can be found on a single plasmid. The atomic structures of seven of the eight proteins involved in the breakdown of atrazine by Pseudomonas sp. strain ADP have been determined by X-ray crystallography, but the structures of the proteins required by the cell to import atrazine for use as an energy source are still lacking. The structure of AtzT, a periplasmic binding protein that may be involved in the transport of a derivative of atrazine, 2-hydroxyatrazine, into the cell for mineralization, has now been determined. The structure was determined by SAD phasing using an ethylmercury phosphate derivative that diffracted X-rays to beyond 1.9 Å resolution. `Native' (guanine-bound) and 2-hydroxyatrazine-bound structures were also determined to high resolution (1.67 and 1.65 Å, respectively), showing that 2-hydroxyatrazine binds in a similar way to the purportedly native ligand. Structural similarities led to the belief that it may be possible to evolve AtzT from a purine-binding protein to a protein that can bind and detect atrazine in the environment.

Incorporation of hydroxyproline in bacterial collagen from Streptococcus pyogenes

Publisher: Elsevier BV

Date: 10-2018

DOI: 10.1016/J.ACTBIO.2018.09.012

Abstract: Bacterial collagen-like proteins differ from vertebrate collagens in that they do not contain hydroxyproline, which is seen as a characteristic of the vertebrate collagens, and which provides a significant contribution to the stability of the collagen triple-helix at body temperature. Despite this difference, the bacterial collagens are stable at around body temperature through inclusion of other stabilising sequence elements. Another difference is the lack of aggregation, and certain vertebrate collagen binding domains that can be introduced into the bacterial sequence lack full function when hydroxyproline is absent. In the present study we have demonstrated that a simple method utilising co-translational incorporation during fermentation can be used to incorporate hydroxyproline into the recombinant bacterial collagen. The presence and amount of hydroxyproline incorporation was shown by amino acid analysis and by mass spectrometry. A small increase in thermal stability was observed using circular dichroism spectroscopy. STATEMENT OF SIGNIFICANCE: Recombinant bacterial collagens provide a new opportunity for biomedical materials as they are readily produced in large quantity in E. coli. Unlike animal collagens, they are stable without the need for inclusion of a secondary modification system for hydroxyproline incorporation. In animal collagens, however, introduction of hydroxyproline is essential for stability and is also important for functional molecular interactions within the mammalian extracellular matrix. The present study has shown that hydroxyproline can be readily introduced into recombinant S. pyogenes bacterial collagen through direct co-translational incorporation of this modified imino acid during expression using the codons for proline in the introduced gene construct. This hydroxylation further improves the stability of the collagen and is available to enhance any introduced molecular functions.

Abstract

Publisher: eLife Sciences Publications, Ltd

Date: 2017

DOI: 10.7554/ELIFE.21083.001

An unexpected vestigial protein complex reveals the evolutionary origins of an s-triazine catabolic enzyme

Publisher: Elsevier BV

Date: 05-2018

DOI: 10.1074/JBC.RA118.001996

Membrane cytoskeleton: PIP2 pulls the strings

Publisher: Elsevier BV

Date: 05-2000

DOI: 10.1016/S0960-9822(00)00465-6

Abstract: A recent application of optical tweezers has shown that plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) levels control adhesion of the membrane bilayer to the underlying cytoskeleton, by regulated direct binding of PIP(2) to cytoskeletal proteins and/or indirect effects on cytoskeleton structure.

Effects of macro-nutrient, micro-nutrient composition and cooking conditions on in vitro digestibility of meat and aquatic dietary proteins

Publisher: Elsevier BV

Date: 07-2018

DOI: 10.1016/J.FOODCHEM.2018.01.164

Abstract: Animal and aquatic meats represent important sources of dietary protein and micro-nutrients. Although red and processed meats carry some risks for human health, sensory and nutritional advantages drive meat consumption. Therefore, it is important to understand how meat processing and cooking influence healthiness. The research aim was to investigate relationships of meat composition (proximates, amino acids and minerals) and cooking conditions (raw, 90 s microwave, 200 °C oven for 10 or 30 min) on protein digestibility, for a selection of four animal (beef, chicken, pork, kangaroo) and four aquatic meats (salmon, trout, prawn, oyster). Lean meats were minced before cooking followed by in vitro gastro-intestinal digestion and analysed for progress of hydrolysis, and size ranges of peptides using MALDI-TOF-MS. Correlation matrix analysis between compositional and functional parameters indicated that digestibility was significantly linked with protein and metal concentrations, likely reflecting moisture-dependent solubility and inter-mixing of sarcoplasmic metallo-proteins and insoluble myofibrillar proteins.

Archvillin, a muscle-specific isoform of supervillin, is an early expressed component of the costameric membrane skeleton

Publisher: The Company of Biologists

Date: 06-2003

DOI: 10.1242/JCS.00422

Abstract: The membrane skeleton protein supervillin binds tightly to both F-actin and membranes and can potentiate androgen receptor activity in non-muscle cells. We report that muscle, which constitutes the principal tissue source for supervillin sequences, contains a ∼250 kDa isoform of supervillin that localizes within nuclei and with dystrophin at costameres, regions of F-actin membrane attachment in skeletal muscle. The gene encoding this protein,`archvillin' (Latin, archi Greek, árchos `principal' or `chief'),contains an evolutionarily conserved, muscle-specific 5′ leader sequence. Archvillin cDNAs also contain four exons that encode ∼47 kDa of additional muscle-specific protein sequence in the form of two inserts within the function-rich N-terminus of supervillin. The first of these muscle-specific inserts contains two conserved nuclear targeting signals in addition to those found in sequences shared with supervillin. Archvillin, like supervillin, binds directly to radiolabeled F-actin and co-fractionates with plasma membranes. Colocalization of archvillin with membrane-associated actin filaments, non-muscle myosin II, and – to a lesser extent –vinculin was observed in myoblasts. Striking localizations of archvillin protein and mRNA were observed at the tips of differentiating myotubes. Transfected protein chimeras containing archvillin insert sequences inhibited myotube formation, consistent with a dominant-negative effect during early myogenesis. These data suggest that archvillin is among the first costameric proteins to assemble during myogenesis and that it contributes to myogenic membrane structure and differentiation.

Crystal structures of human ENPP1 in apo and bound forms

Publisher: International Union of Crystallography (IUCr)

Date: 17-08-2020

DOI: 10.1107/S2059798320010505

Abstract: Cancer is one of the leading causes of mortality in humans, and recent work has focused on the area of immuno-oncology, in which the immune system is used to specifically target cancerous cells. Ectonucleotide pyrophosphatase hosphodiesterase 1 (ENPP1) is an emerging therapeutic target in human cancers owing to its role in degrading cyclic GMP-AMP (cGAMP), an agonist of the stimulator of interferon genes (STING). The available structures of ENPP1 are of the mouse enzyme, and no structures are available with anything other than native nucleotides. Here, the first X-ray crystal structures of the human ENPP1 enzyme in an apo form, with bound nucleotides and with two known inhibitors are presented. The availability of these structures and a robust crystallization system will allow the development of structure-based drug-design c aigns against this attractive cancer therapeutic target.

A Mechanism for Actin Filament Severing by Malaria Parasite Actin Depolymerizing Factor 1 via a Low Affinity Binding Interface

Publisher: Elsevier BV

Date: 02-2014

DOI: 10.1074/JBC.M113.523365

Monosodium Urate Crystals Generate Nuclease-Resistant Neutrophil Extracellular Traps via a Distinct Molecular Pathway

Publisher: The American Association of Immunologists

Date: 03-2018

DOI: 10.4049/JIMMUNOL.1701382

Abstract: Neutrophil extracellular traps (NETs) and the cell death associated with it (NETosis) have been implicated in numerous diseases. Mechanistic studies of NETosis have typically relied on nonphysiological stimuli, such as PMA. The human disease of gout is caused by monosodium urate (MSU) crystals. We observed that DNA consistent with NETs is present in fluid from acutely inflamed joints of gout patients. NETs also coat the crystals found in uninflamed tophi of chronic gout patients. We developed a quantitative, live cell imaging assay, which measures the key features of NETosis, namely, cell death and chromatin decondensation. We show that MSU and other physiologically relevant crystals induce NETosis through a molecular pathway that is distinct from PMA and Candida hyphae. Crystals interact with lysosomes to induce NADPH oxidase-independent cell death, with postmortem chromatin decondensation mediated by neutrophil elastase. The resulting MSU-induced NETs are enriched for actin and are resistant to serum and DNase degradation. These findings demonstrate a distinct physiological NETosis pathway in response to MSU crystals, which coats MSU crystals in DNA that persists in tissues as gouty tophi.

The Glitazone Class of Drugs as Carbonic Anhydrase Inhibitors—A Spin-Off Discovery from Fragment Screening

Publisher: MDPI AG

Date: 18-05-2021

DOI: 10.3390/MOLECULES26103010

Abstract: The approved drugs that target carbonic anhydrases (CA, EC 4.2.1.1), a family of zinc metalloenzymes, comprise almost exclusively of primary sulfonamides (R-SO2NH2) as the zinc binding chemotype. New clinical applications for CA inhibitors, particularly for hard-to-treat cancers, has driven a growing interest in the development of novel CA inhibitors. We recently discovered that the thiazolidinedione heterocycle, where the ring nitrogen carries no substituent, is a new zinc binding group and an alternate CA inhibitor chemotype. This heterocycle is curiously also a substructure of the glitazone class of drugs used in the treatment options for type 2 diabetes. Herein, we investigate and characterise three glitazone drugs (troglitazone 11, rosiglitazone 12 and pioglitazone 13) for binding to CA using native mass spectrometry, protein X-ray crystallography and hydrogen–deuterium exchange (HDX) mass spectrometry, followed by CA enzyme inhibition studies. The glitazone drugs all displayed appreciable binding to and inhibition of CA isozymes. Given that thiazolidinediones are not credited as a zinc binding group nor known as CA inhibitors, our findings indicate that CA may be an off-target of these compounds when used clinically. Furthermore, thiazolidinediones may represent a new opportunity for the development of novel CA inhibitors as future drugs.

The role of glycosylation in amyloid fibril formation of bovine κ-casein

Publisher: Elsevier BV

Date: 2023

DOI: 10.1016/J.CRFS.2023.100433

Role of Plasmepsin V in Export of Diverse Protein Families from the Plasmodium falciparum Exportome

Publisher: Wiley

Date: 27-02-2013

DOI: 10.1111/TRA.12053

Abstract: Plasmodium falciparum exports several hundred effector proteins that remodel the host erythrocyte and enable parasites to acquire nutrients, sequester in the circulation and evade immune responses. The majority of exported proteins contain the Plasmodium export element (PEXEL RxLxE/Q/D) in their N-terminus, which is proteolytically cleaved in the parasite endoplasmic reticulum by Plasmepsin V, and is necessary for export. Several exported proteins lack a PEXEL or contain noncanonical motifs. Here, we assessed whether Plasmepsin V could process the N-termini of erse protein families in P. falciparum. We show that Plasmepsin V cleaves N-terminal sequences from RIFIN, STEVOR and RESA multigene families, the latter of which contain a relaxed PEXEL (RxLxxE). However, Plasmepsin V does not cleave the N-terminal sequence of the major exported virulence factor erythrocyte membrane protein 1 (PfEMP1) or the PEXEL-negative exported proteins SBP-1 or REX-2. We probed the substrate specificity of Plasmepsin V and determined that lysine at the PEXEL P3 position, which is present in PfEMP1 and other putatively exported proteins, blocks Plasmepsin V activity. Furthermore, isoleucine at position P1 also blocked Plasmepsin V activity. The specificity of Plasmepsin V is therefore exquisitely confined and we have used this novel information to redefine the predicted P. falciparum PEXEL exportome.

Essential Role of the PfRh5/PfRipr/CyRPA Complex during Plasmodium falciparum Invasion of Erythrocytes

Publisher: Elsevier BV

Date: 07-2016

DOI: 10.1016/J.CHOM.2016.06.004

Abstract: Plasmodium falciparum parasites in the merozoite stage invade human erythrocytes and cause malaria. Invasion requires multiple interactions between merozoite ligands and erythrocyte receptors. P. falciparum reticulocyte binding homolog 5 (PfRh5) forms a complex with the PfRh5-interacting protein (PfRipr) and Cysteine-rich protective antigen (CyRPA) and binds erythrocytes via the host receptor basigin. However, the specific role that PfRipr and CyRPA play during invasion is unclear. Using P. falciparum lines conditionally expressing PfRipr and CyRPA, we show that loss of PfRipr or CyRPA function blocks growth due to the inability of merozoites to invade erythrocytes. Super-resolution microscopy revealed that PfRipr, CyRPA, and PfRh5 colocalize at the junction between merozoites and erythrocytes during invasion. PfRipr, CyRPA, and PfRipr/CyRPA/PfRh5-basigin complex is required for triggering the Ca(2+) release and establishing the tight junction. Together, these results establish that the PfRh5/PfRipr/CyRPA complex is essential in the sequential molecular events leading to parasite invasion of human erythrocytes.

Lipid Raft Membrane Skeletons

Publisher: Humana Press

Date: 2005

DOI: 10.1385/1-59259-803-X:047

High ploidy large cytoplasmic megakaryocytes are hematopoietic stem cells regulators and essential for platelet production

Publisher: Springer Science and Business Media LLC

Date: 13-04-2023

DOI: 10.1038/S41467-023-37780-7

Abstract: Megakaryocytes (MK) generate platelets. Recently, we and others, have reported MK also regulate hematopoietic stem cells (HSC). Here we show high ploidy large cytoplasmic megakaryocytes (LCM) are critical negative regulators of HSC and critical for platelet formation. Using a mouse knockout model ( Pf4-Srsf3 Δ/Δ ) with normal MK numbers, but essentially devoid of LCM, we demonstrate a pronounced increase in BM HSC concurrent with endogenous mobilization and extramedullary hematopoiesis. Severe thrombocytopenia is observed in animals with diminished LCM, although there is no change in MK ploidy distribution, uncoupling endoreduplication and platelet production. When HSC isolated from a microenvironment essentially devoid of LCM reconstitute hematopoiesis in lethally irradiated mice, the absence of LCM increases HSC in BM, blood and spleen, and the recapitulation of thrombocytopenia. In contrast, following a competitive transplant using minimal numbers of WT HSC together with HSC from a microenvironment with diminished LCM, sufficient WT HSC-generated LCM regulates a normal HSC pool and prevents thrombocytopenia. Importantly, LCM are conserved in humans.

Distinct Protein Classes Including Novel Merozoite Surface Antigens in Raft-like Membranes of Plasmodium falciparum

Publisher: Elsevier BV

Date: 12-2005

DOI: 10.1074/JBC.M509631200

Identification of protein complexes in detergent-resistant membranes of Plasmodium falciparum schizonts

Publisher: Elsevier BV

Date: 08-2007

DOI: 10.1016/J.MOLBIOPARA.2007.04.013

Abstract: Merozoite surface proteins of the human malaria parasite Plasmodium falciparum are involved in initial contact with target erythrocytes, a process that begins a cascade of events required for successful invasion of these cells. In order to identify complexes that may play a role in invasion we purified detergent-resistant membranes (DRMs), known to be enriched in merozoite surface proteins, and used blue native-polyacrylamide gel electrophoresis (BN-PAGE) to isolate high molecular weight complexes for identification by mass spectrometry. Sixty-two proteins were detected and these mostly belonged to expected DRM proteins classes including GPI-anchored, multi-membrane spanning and rhoptry proteins. Proteins from seven known complexes were identified including MSP-1/7, the low (RAP1/2 and RAP1/3), and high (RhopH1/H2/H3) molecular weight rhoptry complexes, and the invasion motor complex (GAP45/GAP50/myosinA). Remarkably, a large proportion of identified spectra were derived from only 4 proteins: the GPI-anchored proteins MSP-1 and Pf92, the putative GPI-anchored protein Pf113 and RAP-1, the core component of the two RAP complexes. Each of these proteins predominated in high molecular weight species suggesting their aggregation in much larger complexes than anticipated. To demonstrate that the procedure had isolated novel complexes we focussed on MSP-1, which predominated as a distinct species at approximately 500 kDa by BN-PAGE, approximately twice its expected size. Chemical cross-linking supports the existence of a stable MSP-1 oligomer of approximately 500 kDa, probably comprising a highly stable homodimeric species. Our observations also suggests that oligomerization of MSP-1 is likely to occur outside the C-terminal epidermal growth factor (EGF)-like domains. Confirmation of MSP-1 oligomerization, together with the isolation of a number of known complexes by BN-PAGE, makes it highly likely that novel interactions occur amongst members of this proteome.

Identification and Stoichiometry of Glycosylphosphatidylinositol-anchored Membrane Proteins of the Human Malaria Parasite Plasmodium falciparum

Publisher: Elsevier BV

Date: 07-2006

DOI: 10.1074/MCP.M600035-MCP200

Protein Kinase A Dependent Phosphorylation of Apical Membrane Antigen 1 Plays an Important Role in Erythrocyte Invasion by the Malaria Parasite

Publisher: Public Library of Science (PLoS)

Date: 03-06-2010

DOI: 10.1371/JOURNAL.PPAT.1000941

Synergistic complexation of phenol functionalized polymer induced in situ microfiber formation for 3D printing of marine-based hydrogels

Publisher: Royal Society of Chemistry (RSC)

Date: 2022

DOI: 10.1039/D1GC04347A

Abstract: This study proposes a strategy to design tough and 3D printable marine-based hydrogels based on the synergistic complexation of a phenolated polyelectrolyte complex and enzyme-mediated crosslinking.

MSP1₁₉ miniproteins can serve as targets for invasion inhibitory antibodies in Plasmodium falciparum provided they contain the correct domains for cell surface trafficking

Publisher: Wiley

Date: 10-03-2008

DOI: 10.1111/J.1365-2958.2008.06140.X

Abstract: Antibodies from malaria-exposed in iduals can agglutinate merozoites released from Plasmodium schizonts, thereby preventing them from invading new erythrocytes. Merozoite coat proteins attached to the plasma membrane are major targets for host antibodies and are therefore considered important malaria vaccine candidates. Prominent among these is the abundant glycosylphosphatidylinositol (GPI)-anchored merozoite surface protein 1 (MSP1) and particularly its C-terminal fragment (MSP1(19)) comprised of two epidermal growth factor (EGF)-like modules. In this paper, we revisit the role of agglutination and immunity using transgenic fluorescent marker proteins. We describe expression of heterologous MSP1(19)'miniproteins' on the surface of Plasmodium falciparum merozoites. To correctly express these proteins, we determined that GPI-anchoring and the presence of a signal sequence do not allow default export of proteins from the endoplasmic reticulum to merozoite surface and that extra sequence elements are required. The EGFs are insufficient for correct trafficking unless they are fused to additional residues that normally reside upstream of this fragment. Antibodies specifically targeting the surface-expressed miniprotein can inhibit erythrocyte invasion in vitro despite the presence of endogenous MSP1. Using a line expressing a green fluorescent protein-MSP1 fusion protein, we demonstrate that one mode of inhibition by antibodies targeting the MSP1(19) domain is the rapid agglutinating of merozoites prior to erythrocyte attachment.

Export of malaria proteins requires co-translational processing of the PEXEL motif independent of phosphatidylinositol-3-phosphate binding

Publisher: Springer Science and Business Media LLC

Date: 02-2016

DOI: 10.1038/NCOMMS10470

Abstract: Plasmodium falciparum exports proteins into erythrocytes using the Plasmodium export element (PEXEL) motif, which is cleaved in the endoplasmic reticulum (ER) by plasmepsin V (PMV). A recent study reported that phosphatidylinositol-3-phosphate (PI(3)P) concentrated in the ER binds to PEXEL motifs and is required for export independent of PMV, and that PEXEL motifs are functionally interchangeable with RxLR motifs of oomycete effectors. Here we show that the PEXEL does not bind PI(3)P, and that this lipid is not concentrated in the ER. We find that RxLR motifs cannot mediate export in P. falciparum . Parasites expressing a mutated version of KAHRP, with the PEXEL motif repositioned near the signal sequence, prevented PMV cleavage. This mutant possessed the putative PI(3)P-binding residues but is not exported. Reinstatement of PEXEL to its original location restores processing by PMV and export. These results challenge the PI(3)P hypothesis and provide evidence that PEXEL position is conserved for co-translational processing and export.

Quantitative in vivo Analyses Reveal Calcium-dependent Phosphorylation Sites and Identifies a Novel Component of the Toxoplasma Invasion Motor Complex

Publisher: Public Library of Science (PLoS)

Date: 29-09-2011

DOI: 10.1371/JOURNAL.PPAT.1002222

Multiple signalling pathways connect chemoattractant receptors and calcium channels in Dictyostelium

Publisher: Springer Science and Business Media LLC

Date: 2002

DOI: 10.1023/A:1024496232604

Abstract: Dictyostelium mutants expressing aequorin were used to study and compare the roles of heterotrimeric G-proteins and the second messengers IP3 and cGMP in regulating folate- and cAMP receptor-activated [Ca2+]i signals. The calcium responses of vegetative cells to folate were dramatically impaired in Gbeta and Galpha4 null mutants but were restored with altered kinetics and temperature-sensitivity in Gbeta null mutants overexpressing wild type and temperature-sensitive Gbeta isoforms. Folic acid receptors thus mediate changes in [Ca2+]i via a Galpha4betagamma-dependent pathway. Neither folate nor cAMP-induced [Ca2+]i signals were significantly altered in PLC null transformants, but [Ca2+]i changes elicited by both attractants were significantly prolonged in two stmF mutants lacking cGMP-specific phosphodiesterase activity. This confirms an important role of cGMP in regulating receptor-activated Ca2+ uptake and/or extrusion systems. This cGMP-dependent part of the Ca2+ response to cAMP stimuli was developmentally down-regulated and all but disappeared by the time the cells reached full aggregation competence after 8 h of starvation. The results suggest that folate and cAMP receptor-activated [Ca2+]i signals are regulated in a complex manner via multiple signalling pathways, one that is G-protein- and cGMP-dependent (present at the vegetative and early poststarvation stage) and another that is G-protein-independent (dominant in fully aggregation-competent cells at approximately 8 h poststarvation).

Alternative splicing of Bim and Erk-mediated Bim EL phosphorylation are dispensable for hematopoietic homeostasis in vivo

Publisher: Springer Science and Business Media LLC

Date: 13-01-2012

DOI: 10.1038/CDD.2011.198

Effects of chemical composition and baking on in vitro digestibility of proteins in breads made from selected gluten-containing and gluten-free flours

Publisher: Elsevier BV

Date: 10-2017

DOI: 10.1016/J.FOODCHEM.2017.04.158

Abstract: Breads prepared from cereal grains are a dietary staple, providing a significant proportion of daily energy, but not necessarily of dietary protein. However, good digestibility of proteins in bread is important to avoid potential immunogenic effects of undigested peptides, including for those gluten-intolerant. Four gluten-containing (white wheat, wholemeal wheat, spelt and rye) and four gluten-free (chick pea, lupin, buckwheat, amaranth) flours were used to make yeast-leavened breads standardized for protein. In vitro gastro-intestinal digestion of pre-mixes, doughs and breads baked for 20 and 35min was conducted followed by correlation analysis between fitted parameters of digestion profiles, chemical composition (protein, non-fibre carbohydrates, fibre, ash and total polyphenolics) and amino acid profiles. The results indicated that digestibility generally increased during proofing and decreased during baking. Relatively higher protein digestibility was correlated with ratio of non-fibre carbohydrate to protein and lower digestibility with increasing contents of fibre and total polyphenolics in pre-mixes.

The exported chaperone Hsp70-x supports virulence functions for Plasmodium falciparum blood stage parasites

Publisher: Public Library of Science (PLoS)

Date: 21-07-2017

DOI: 10.1371/JOURNAL.PONE.0181656

Plasmepsin V cleaves malaria effector proteins in a distinct endoplasmic reticulum translocation interactome for export to the erythrocyte

Publisher: Springer Science and Business Media LLC

Date: 20-08-2018

DOI: 10.1038/S41564-018-0219-2

Abstract: Plasmodium falciparum exports hundreds of virulence proteins within infected erythrocytes, a process that requires cleavage of a pentameric motif called Plasmodium export element or vacuolar transport signal by the endoplasmic reticulum (ER)-resident protease plasmepsin V. We identified plasmepsin V-binding proteins that form a unique interactome required for the translocation of effector cargo into the parasite ER. These interactions are functionally distinct from the Sec61-signal peptidase complex required for the translocation of proteins destined for the classical secretory pathway. This interactome does not involve the signal peptidase (SPC21) and consists of PfSec61, PfSPC25, plasmepsin V and PfSec62, which is an essential component of the post-translational ER translocon. Together, they form a distinct portal for the recognition and translocation of a large subset of Plasmodium export element effector proteins into the ER, thereby remodelling the infected erythrocyte that is required for parasite survival and pathogenesis.

Proprotein convertase 5/6 cleaves platelet-derived growth factor A in the human endometrium in preparation for embryo implantation

Publisher: Oxford University Press (OUP)

Date: 27-11-2014

DOI: 10.1093/MOLEHR/GAU109

Abstract: Establishment of endometrial receptivity is vital for successful embryo implantation. Proprotein convertase 5/6 (referred to as PC6) is up-regulated in the human endometrium specifically at the time of epithelial receptivity. PC6, a serine protease of the proprotein convertase family, plays an important role in converting precursor proteins into their active forms through specific proteolysis. The proform of platelet-derived growth factor A (pro-PDGFA) requires PC cleavage to convert to the active-PDGFA. We investigated the PC6-mediated activation of PDGFA in the human endometrium during the establishment of receptivity. Proteomic analysis identified that the pro-PDGFA was increased in the conditioned medium of HEC1A cells in which PC6 was stably knocked down by small interfering RNA (PC6-siRNA). Western blot analysis demonstrated an accumulation of the pro-PDGFA but a reduction in the active-PDGFA in PC6-siRNA cell lysates and medium compared with control. PC6 cleavage of pro-PDGFA was further confirmed in vitro by incubation of recombinant pro-PDGFA with PC6. Immunohistochemistry revealed cycle-stage-specific localization of the active-PDGFA in the human endometrium. During the non-receptive phase, the active-PDGFA was barely detectable. In contrast, it was localized specifically to the apical surface of the luminal and glandular epithelium in the receptive phase. Furthermore, the active-PDGFA was detected in uterine lavage with levels being significantly higher in the receptive than the non-receptive phase. We thus identified that the secreted PDGFA may serve as a biomarker for endometrial receptivity. This is also the first study demonstrating that the active-PDGFA localizes to the apical surface of the endometrium during receptivity.

Neutralizing inhibitors in the airways of naïve ferrets do not play a major role in modulating the virulence of H3 subtype influenza A viruses.

Publisher: Elsevier BV

Date: 07-2016

DOI: 10.1016/J.VIROL.2016.01.024

Abstract: Many insights regarding the pathogenesis of human influenza A virus (IAV) infections have come from studies in mice and ferrets. Surfactant protein (SP)-D is the major neutralizing inhibitor of IAV in mouse airway fluids and SP-D-resistant IAV mutants show enhanced virus replication and virulence in mice. Herein, we demonstrate that sialylated glycoproteins, rather than SP-D, represent the major neutralizing inhibitors against H3 subtype viruses in airway fluids from naïve ferrets. Moreover, while resistance to neutralizing inhibitors is a critical factor in modulating virus replication and disease in the mouse model, it does not appear to be so in the ferret model, as H3 mutants resistant to either SP-D or sialylated glycoproteins in ferret airway fluids did not show enhanced virulence in ferrets. These data have important implications for our understanding of pathogenesis and immunity to human IAV infections in these two widely used animal models of infection.

Stimulation of innate immune responses by malarial glycosylphosphatidylinositol via pattern recognition receptors

Publisher: Cambridge University Press (CUP)

Date: 03-2005

DOI: 10.1017/S0031182005008152

Abstract: The glycosylphosphatidylinositol (GPI) anchor of Plasmodium falciparum is thought to function as a critical toxin that contributes to severe malarial pathogenesis by eliciting the production of proinflammatory responses by the innate immune system of mammalian hosts. Analysis of the fine structure of P. falciparum GPI suggests a requirement for the presence of both core glycan and lipid moieties in the recognition and signalling of parasite glycolipids by host immune cells. It has been demonstrated that GPI anchors of various parasitic protozoa can mediate cellular immune responses via members of the Toll-like family of pattern recognition receptors (TLRs). Recent studies indicate that GPI anchors of P. falciparum and other protozoa are preferentially recognized by TLR-2, involving the MyD88-dependent activation of specific signalling pathways that mediate the production of proinflammatory cytokines and nitric oxide from host macrophages in vitro . However, the contribution of malaria GPI toxin to severe disease syndromes and the role of specific TLRs or other pattern recognition receptors in innate immunity in vivo is only just beginning to be characterized. A better understanding of the molecular mechanisms underlying severe malarial pathogenesis may yet lead to substantial new insights with important implications for the development of novel therapeutics for malaria treatment.

Structures of the transcriptional regulator BgaR, a lactose sensor

Publisher: International Union of Crystallography (IUCr)

Date: 26-06-2019

DOI: 10.1107/S2059798319008131

Abstract: The structure of BgaR, a transcriptional regulator of the lactose operon in Clostridium perfringens , has been solved by SAD phasing using a mercury derivative. BgaR is an exquisite sensor of lactose, with a binding affinity in the low-micromolar range. This sensor and regulator has been captured bound to lactose and to lactulose as well as in a nominal apo form, and was compared with AraC, another saccharide-binding transcriptional regulator. It is shown that the saccharides bind in the N-terminal region of a jelly-roll fold, but that part of the saccharide is exposed to bulk solvent. This differs from the classical AraC saccharide-binding site, which is mostly sequestered from the bulk solvent. The structures of BgaR bound to lactose and to lactulose highlight how specific and nonspecific interactions lead to a higher binding affinity of BgaR for lactose compared with lactulose. Moreover, solving multiple structures of BgaR in different space groups, both bound to saccharides and unbound, verified that the dimer interface along a C-terminal helix is similar to the dimer interface observed in AraC.

Université Libre De Bruxelles (ULB)

Location: Belgium

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Université Libre De Bruxelles

Location: Belgium

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Commonwealth Scientific And Industrial Research Organisation

Location: Australia

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CSIRO

Location: Australia

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CSIRO Biomedical Manufacturing

Location: Australia

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Linkage Infrastructure, Equipment And Facilities - Grant ID: LE210100122

Start Date: 2021

End Date: 12-2021

Amount: $725,000.00

Funder: Australian Research Council