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104. Enhancement of red blood cell transfusion compatibility using CRISPR‐mediated erythroblast gene editing

Author

Hawksworth, Joseph, primary, Satchwell, Timothy J, additional, Meinders, Marjolein, additional, Daniels, Deborah E, additional, Regan, Fiona, additional, Thornton, Nicole M, additional, Wilson, Marieangela C, additional, Dobbe, Johannes GG, additional, Streekstra, Geert J, additional, Trakarnsanga, Kongtana, additional, Heesom, Kate J, additional, Anstee, David J, additional, Frayne, Jan, additional, and Toye, Ashley M, additional

Published
2018
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106. Prediction of Fluoroquinolone Susceptibility Directly from Whole-Genome Sequence Data by Using Liquid Chromatography-Tandem Mass Spectrometry To Identify Mutant Genotypes

Author

Wan Nur Ismah, Wan Ahmad Kamil, primary, Takebayashi, Yuiko, additional, Findlay, Jacqueline, additional, Heesom, Kate J., additional, Jiménez-Castellanos, Juan-Carlos, additional, Zhang, Jay, additional, Graham, Lee, additional, Bowker, Karen, additional, Williams, O. Martin, additional, MacGowan, Alasdair P., additional, and Avison, Matthew B., additional

Published
2018
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111. Retromer- and WASH-dependent sorting of nutrient transporters requires a multivalent interaction network with ANKRD50

Author

Kvainickas, Arunas, Orgaz, Ana Jimenez, Nägele, Heike, Diedrich, Britta, Heesom, Kate J., Dengjel, Jörn, Cullen, Peter J., Steinberg, Florian, Kvainickas, Arunas, Orgaz, Ana Jimenez, Nägele, Heike, Diedrich, Britta, Heesom, Kate J., Dengjel, Jörn, Cullen, Peter J., and Steinberg, Florian

Abstract

Retromer and the associated actin-polymerizing WASH complex are essential for the endocytic recycling of a wide range of integral membrane proteins. A hereditary Parkinson's-disease-causing point mutation (D620N) in the retromer subunit VPS35 perturbs retromer's association with the WASH complex and also with the uncharacterized protein ankyrin-repeat-domain-containing protein 50 (ANKRD50). Here, we firmly establish ANKRD50 as a new and essential component of the SNX27– retromer–WASH super complex. Depletion of ANKRD50 in HeLa or U2OS cells phenocopied the loss of endosome-to-cell-surface recycling of multiple transmembrane proteins seen upon suppression of SNX27, retromer or WASH- complex components. Mass-spectrometry-based quantification of the cell surface proteome of ANKRD50-depleted cells identified amino acid transporters of the SLC1A family, among them SLC1A4, as additional cargo molecules that depend on ANKRD50 and retromer for their endocytic recycling. Mechanistically, we show that ANKRD50 simultaneously engages multiple parts of the SNX27–retromer–WASH complex machinery in a direct and co-operative interaction network that is needed to efficiently recycle the nutrient transporters GLUT1 (also known as SLC2A1) and SLC1A4, and potentially many other surface proteins.

Published
2017

113. Structural/mechanistic insights into the efficacy of nonclassical β-lactamase inhibitors against extensively drug resistantStenotrophomonas maltophiliaclinical isolates

Author

Calvopiña, Karina, primary, Hinchliffe, Philip, additional, Brem, Jürgen, additional, Heesom, Kate J., additional, Johnson, Samar, additional, Cain, Ricky, additional, Lohans, Christopher T., additional, Fishwick, Colin W. G., additional, Schofield, Christopher J., additional, Spencer, James, additional, and Avison, Matthew B., additional

Published
2017
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115. Retriever is a multiprotein complex for retromer-independent endosomal cargo recycling

Author

McNally, Kerrie E., primary, Faulkner, Rebecca, additional, Steinberg, Florian, additional, Gallon, Matthew, additional, Ghai, Rajesh, additional, Pim, David, additional, Langton, Paul, additional, Pearson, Neil, additional, Danson, Chris M., additional, Nägele, Heike, additional, Morris, Lindsey L., additional, Singla, Amika, additional, Overlee, Brittany L., additional, Heesom, Kate J., additional, Sessions, Richard, additional, Banks, Lawrence, additional, Collins, Brett M., additional, Berger, Imre, additional, Billadeau, Daniel D., additional, Burstein, Ezra, additional, and Cullen, Peter J., additional

Published
2017
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118. Prediction of fluoroquinolone susceptibility directly from whole genome sequence data using liquid chromatography-tandem mass spectrometry to identify mutant genotypes

Author

Wan Nur Ismah, Wan Ahmad Kamil, primary, Takebayashi, Yuiko, additional, Findlay, Jacqueline, additional, Heesom, Kate J., additional, Jiménez-Castellanos, Juan-Carlos, additional, Zhang, Jay, additional, Graham, Lee, additional, Bowker, Karen, additional, Williams, O. Martin, additional, MacGowan, Alasdair P., additional, and Avison, Matthew B., additional

Published
2017
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122. The Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) Binder Rasa3 Regulates Phosphoinositide 3-kinase (PI3K)-dependent Integrin αIIbβ3 Outside-in Signaling

Author

Battram, Anthony M., primary, Durrant, Tom N., additional, Agbani, Ejaife O., additional, Heesom, Kate J., additional, Paul, David S., additional, Piatt, Raymond, additional, Poole, Alastair W., additional, Cullen, Peter J., additional, Bergmeier, Wolfgang, additional, Moore, Samantha F., additional, and Hers, Ingeborg, additional

Published
2017
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124. Galaxy Integrated Omics:Web-based standards-compliant workflows for proteomics informed by transcriptomics

Author

Fan, Jun, Saha, Shyamasree, Barker, Gary, Heesom, Kate J, Ghali, Fawaz, Jones, Andrew Robert, Matthews, David A, and Bessant, Conrad

Subjects
ComputingMethodologies_PATTERNRECOGNITION
Abstract

With the recent advent of RNA-seq technology the proteomics community has begun to generate sample-specific protein databases for peptide and protein identification, an approach we call proteomics informed by transcriptomics (PIT). This approach has gained a lot of interest, particularly among researchers who work with non-model organisms or with particularly dynamic proteomes such as those observed in developmental biology and host-pathogen studies. PIT has been shown to improve coverage of known proteins, and to reveal potential novel gene products. However, many groups are impeded in their use of PIT by the complexity of the required data analysis. Necessarily, this analysis requires complex integration of a number of different software tools from at least two different communities, and because PIT has a range of biological applications a single software pipeline is not suitable for all use cases. To overcome these problems, we have created GIO, a software system that utilises the well-established Galaxy platform to make PIT analysis available to the typical bench scientist via a simple web interface. Within GIO we provide workflows for four common use cases: a standard search against a reference proteome; PIT protein identification without a reference genome; PIT protein identification using a genome guide; and PIT genome annotation. These workflows comprise individual tools that can be reconfigured and rearranged within the web interface to create new workflows to support additional use cases.

Published
2015

125. Misato Controls Mitotic Microtubule Generation by Stabilizing the Tubulin Chaperone Protein-1 Complex

Author

Palumbo, Valeria, Pellacani, Claudia, Heesom, Kate J., Rogala, Kacper B., Deane, Charlotte M., Mottier-Pavie, Violaine, Gatti, Maurizio, Bonaccorsi, Silvia, and Wakefield, James G.

Subjects
Genetics and Molecular Biology (all), Biological Sciences (all), Biochemistry, Genetics and Molecular Biology (all), Blotting, Western, Mitosis, Cell Cycle Proteins, macromolecular substances, Spindle Apparatus, Microtubules, Time-Lapse Imaging, Biochemistry, Polymerization, Cytoskeletal Proteins, Tubulin, Report, Animals, Drosophila Proteins, Immunoprecipitation, Drosophila, RNA Interference, human activities, Chaperonin Containing TCP-1, Molecular Chaperones
Abstract

Summary Mitotic spindles are primarily composed of microtubules (MTs), generated by polymerization of α- and β-Tubulin hetero-dimers [1, 2]. Tubulins undergo a series of protein folding and post-translational modifications in order to fulfill their functions [3, 4]. Defects in Tubulin polymerization dramatically affect spindle formation and disrupt chromosome segregation. We recently described a role for the product of the conserved misato (mst) gene in regulating mitotic MT generation in flies [5], but the molecular function of Mst remains unknown. Here, we use affinity purification mass spectrometry (AP-MS) to identify interacting partners of Mst in the Drosophila embryo. We demonstrate that Mst associates stoichiometrically with the hetero-octameric Tubulin Chaperone Protein-1 (TCP-1) complex, with the hetero-hexameric Tubulin Prefoldin complex, and with proteins having conserved roles in generating MT-competent Tubulin. We show that RNAi-mediated in vivo depletion of any TCP-1 subunit phenocopies the effects of mutations in mst or the Prefoldin-encoding gene merry-go-round (mgr), leading to monopolar and disorganized mitotic spindles containing few MTs. Crucially, we demonstrate that Mst, but not Mgr, is required for TCP-1 complex stability and that both the efficiency of Tubulin polymerization and Tubulin stability are drastically compromised in mst mutants. Moreover, our structural bioinformatic analyses indicate that Mst resembles the three-dimensional structure of Tubulin monomers and might therefore occupy the TCP-1 complex central cavity. Collectively, our results suggest that Mst acts as a co-factor of the TCP-1 complex, playing an essential role in the Tubulin-folding processes required for proper assembly of spindle MTs., Highlights • Misato interacts biochemically with the Tubulin Chaperone Protein-1 (TCP-1) complex • Misato stabilizes the TCP-1 complex, possibly by filling its Tubulin-folding cavity • Loss of Misato or TCP-1 complex subunits leads to similar mitotic phenotypes • In the absence of Misato, Tubulin is unstable and unable to efficiently polymerize, Palumbo et al. show the conserved protein Misato interacts with the Tubulin Chaperone Protein-1 (TCP-1) complex in Drosophila. In the absence of Misato, TCP-1 subunit levels are reduced, functional Tubulin is unstable, and mitotic microtubule generation is compromised. The predicted structure of Misato suggests it acts as a molecular placeholder in the absence of Tubulin, stabilizing the TCP-1 complex.

Published
2015

126. Transduction with BBF2H7/CREB3L2 upregulates SEC23A protein in erythroblasts and partially corrects the hypo‐glycosylation phenotype associated with CDAII.

Author

Pellegrin, Stéphanie, Haydn‐Smith, Katy L., Hampton‐O'Neil, Lea A., Hawley, Bethan R., Heesom, Kate J., Fermo, Elisa, Bianchi, Paola, and Toye, Ashley M.

Abstract

The article offers a study on lentiviral transduction of BBF2H7/7/CREB3L2 into primary human erythroid cells which causes SEC23A upregulation and persistence during terminal erythroid differentiation in both normal and Congenital Dyserythropoietic Anaemia II (CDAII) cells, an autosomal recessive hereditary anaemia caused by mutations in SEC23B gene. Topics include cloning into pXLG3-GFP expression vector and concluded no detrimental effects on erythroblast proliferation or differentiation.

Published
2019
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127. Impact of OqxR loss of function on the envelope proteome of Klebsiella pneumoniae and susceptibility to antimicrobials.

Author

Ismah, Wan Ahmad Kamil Wan Nur, Takebayashi, Yuiko, Findlay, Jacqueline, Avison, Matthew B, Heesom, Kate J, and Wan Nur Ismah, Wan Ahmad Kamil

Subjects
KLEBSIELLA pneumoniae, PROTEOMICS, MICROBIAL sensitivity tests, BETA lactamases, GENETIC mutation, ANTIBIOTICS, BACTERIAL proteins, BETA lactam antibiotics, CELL physiology, COMPARATIVE studies, DRUG resistance in microorganisms, HYDROLASES, KLEBSIELLA, LIQUID chromatography, MASS spectrometry, RESEARCH methodology, MEDICAL cooperation, MEMBRANE proteins, RESEARCH, RESEARCH funding, EVALUATION research, KLEBSIELLA infections, MEMBRANE transport proteins, PHARMACODYNAMICS
Abstract

Background: In Klebsiella pneumoniae, loss-of-function mutations in the transcriptional repressors RamR and OqxR both have an impact on the production of efflux pumps and porins relevant to antimicrobial efflux/entry.Objectives: To define, in an otherwise isogenic background, the relative effects of OqxR and RamR loss-of-function mutations on envelope protein production, envelope permeability and antimicrobial susceptibility. We also investigated the clinical relevance of an OqxR loss-of-function mutation, particularly in the context of β-lactam susceptibility.Methods: Envelope permeability was estimated using a fluorescent dye accumulation assay. Antimicrobial susceptibility was measured using disc testing. Total envelope protein production was quantified using LC-MS/MS proteomics and quantitative RT-PCR was used to measure transcript levels.Results: Loss of RamR or OqxR reduced envelope permeability in K. pneumoniae by 45%-55% relative to the WT. RamR loss activated AcrAB efflux pump production ∼5-fold and this reduced β-lactam susceptibility, conferring ertapenem non-susceptibility even in the absence of a carbapenemase. In contrast, OqxR loss specifically activated OqxAB efflux pump production >10 000-fold. This reduced fluoroquinolone susceptibility but had little impact on β-lactam susceptibility even in the presence of a β-lactamase.Conclusions: Whilst OqxR loss and RamR loss are both seen in K. pneumoniae clinical isolates, only RamR loss significantly stimulates AcrAB efflux pump production. This means that only RamR mutants have significantly reduced β-lactamase-mediated β-lactam susceptibility and therefore represent a greater clinical threat. [ABSTRACT FROM AUTHOR]

Published
2018
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128. Integrative Transcriptome and Proteome Profiling of Insulin-Resistant Kidney Cell Models and Patient Biopsies Reveals Common and Cell Type-Specific Mechanisms Underpinning Diabetic Kidney Disease

Author

Coward, Richard, Lay, Abigail C., Tran, Van Du T., Nair, Viji, Betin, Virginie M., Burdet, Frédéric, Mehl, Florence, Kryvokhyzha, Dmytro, Ahmad, Abrar, Lewis, Philip A., Wilson, Marieangela C., Menon, Rajasree, Otto, Edgar A., Heesom, Kate J., Looker, Helen C., Nelson, Robert G., Ju, Wenjun, Kretzler, Matthias, Satchell, Simon C., and Gomez, Maria F.

Published
2023
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129. TFG Promotes Organization of Transitional ER and Efficient Collagen Secretion

Author

McCaughey, Janine, Miller, Victoria J., Stevenson, Nicola L., Brown, Anna K., Budnik, Annika, Heesom, Kate J., Alibhai, Dominic, Stephens, David J., McCaughey, Janine, Miller, Victoria J., Stevenson, Nicola L., Brown, Anna K., Budnik, Annika, Heesom, Kate J., Alibhai, Dominic, and Stephens, David J.

Abstract

Collagen is the most abundant protein in the animal kingdom. It is of fundamental importance during development for cell differentiation and tissue morphogenesis as well as in pathological processes such as fibrosis and cancer cell migration. However, our understanding of the mechanisms of procollagen secretion remains limited. Here, we show that TFG organizes transitional ER (tER) and ER exit sites (ERESs) into larger structures. Depletion of TFG results in dispersion of tER elements that remain associated with individual ER-Golgi intermediate compartments (ERGICs) as largely functional ERESs. We show that TFG is not required for the transport and packaging of small soluble cargoes but is necessary for the export of procollagen from the ER. Our work therefore suggests a key relationship between the structure and function of ERESs and a central role for TFG in optimizing COPII assembly for procollagen export.

Published
2016

130. Protein arginine methyltransferase 5 is a key regulator of the MYCN oncoprotein in neuroblastoma cells

Author

Park, Ji Hyun, Szemes, Marianna, Vieira, Gabriella Cunha C, Melegh, Zsombor B, Malik, Sally, Heesom, Kate J, Von Wallwitz-Freitas, Laura, Greenhough, Alexander, Brown, Keith W, Zheng, Y George, Catchpoole, Daniel, Deery, Michael J, and Malik, Karim T A

Subjects
Oncogene Proteins, N-Myc Proto-Oncogene Protein, Protein-Arginine N-Methyltransferases, Brain Neoplasms, Protein Stability, Cell Cycle, Molecular Sequence Data, Nuclear Proteins, Apoptosis, Neuroblastoma, Cell Line, Tumor, Gene Knockdown Techniques, Humans, 1112 Oncology and Carcinogenesis, Oncology & Carcinogenesis, Amino Acid Sequence, neoplasms, Protein Binding
Abstract

Approximately half of poor prognosis neuroblastomas (NBs) are characterized by pathognomonic MYCN gene amplification and MYCN over-expression. Here we present data showing that short-interfering RNA mediated depletion of the protein arginine methyltransferase 5 (PRMT5) in cell-lines representative of NBs with MYCN gene amplification leads to greatly impaired growth and apoptosis. Growth suppression is not apparent in the MYCN-negative SH-SY5Y NB cell-line, or in two immortalized human fibroblast cell-lines. Immunoblotting of NB cell-lines shows that high PRMT5 expression is strongly associated with MYCN-amplification (P < 0.004, Mann-Whitney U-test) and immunohistochemical analysis of primary NBs reveals that whilst PRMT5 protein is ubiquitously expressed in the cytoplasm of most cells, MYCN-amplified tumours exhibit pronounced nuclear PRMT5 staining. PRMT5 knockdown in MYCN-overexpressing cells, including the SHEP-21N cell-line with inducible MYCN expression leads to a dramatic decrease in MYCN protein and MYCN-associated cell-death in SHEP-21N cells. Quantitative gene expression analysis and cycloheximide chase experiments suggest that PRMT5 regulates MYCN at a post-transcriptional level. Reciprocal co-immunoprecipitation experiments demonstrated that endogenous PRMT5 and MYCN interact in both SK-N-BE(2)C and NGP cell lines. By using liquid chromatography - tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated MYCN protein, we identified several potential sites of arginine dimethylation on the MYCN protein. Together our studies implicate PRMT5 in a novel mode of MYCN post-translational regulation and suggest PRMT5 plays a major role in NB tumorigenesis. Small-molecule inhibitors of PRMT5 may therefore represent a novel therapeutic strategy for neuroblastoma and other cancers driven by the MYCN oncogene.

Published
2014

131. High-throughput quantitative proteomic analysis of dengue virus type 2 infected A549 cells

Author

Chiu, Han-Chen, Hannemann, Holger, Heesom, Kate J., Matthews, David A., and Davidson, Andrew D.

Subjects
Proteomics, Viral Diseases, Cytoplasm, Proteome, lcsh:Medicine, Nerve Tissue Proteins, Pathogenesis, Pathology and Laboratory Medicine, Biochemistry, Microbiology, Dengue Fever, Dengue, Viral Proteins, Tandem Mass Spectrometry, Virology, Cell Line, Tumor, Medicine and Health Sciences, Humans, lcsh:Science, Adaptor Proteins, Signal Transducing, Cell Nucleus, lcsh:R, Biology and Life Sciences, Membrane Proteins, Dengue Virus, Tropical Diseases, Infectious Diseases, HEK293 Cells, Host-Pathogen Interactions, lcsh:Q, Carrier Proteins, Protein Abundance, Research Article, Neglected Tropical Diseases
Abstract

Disease caused by dengue virus is a global health concern with up to 390 million individuals infected annually worldwide. There are no vaccines or antiviral compounds available to either prevent or treat dengue disease which may be fatal. To increase our understanding of the interaction of dengue virus with the host cell, we analyzed changes in the proteome of human A549 cells in response to dengue virus type 2 infection using stable isotope labelling in cell culture (SILAC) in combination with high-throughput mass spectrometry (MS). Mock and infected A549 cells were fractionated into nuclear and cytoplasmic extracts before analysis to identify proteins that redistribute between cellular compartments during infection and reduce the complexity of the analysis. We identified and quantified 3098 and 2115 proteins in the cytoplasmic and nuclear fractions respectively. Proteins that showed a significant alteration in amount during infection were examined using gene enrichment, pathway and network analysis tools. The analyses revealed that dengue virus infection modulated the amounts of proteins involved in the interferon and unfolded protein responses, lipid metabolism and the cell cycle. The SILAC-MS results were validated for a select number of proteins over a time course of infection by Western blotting and immunofluorescence microscopy. Our study demonstrates for the first time the power of SILAC-MS for identifying and quantifying novel changes in cellular protein amounts in response to dengue virus infection.

Published
2014

136. Comparative effects of overproducing the AraC-type transcriptional regulators MarA, SoxS, RarA and RamA on antimicrobial drug susceptibility inKlebsiella pneumoniae

Author

Jiménez-Castellanos, Juan-Carlos, primary, Wan Ahmad Kamil, Wan Nur Ismah, additional, Cheung, Ching Hei Phoebe, additional, Tobin, Maryann S., additional, Brown, James, additional, Isaac, Sophie G., additional, Heesom, Kate J., additional, Schneiders, Thamarai, additional, and Avison, Matthew B., additional

Published
2016
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141. Envelope proteome changes driven by RamA overproduction in Klebsiella pneumoniae that enhance acquired β-lactam resistance.

Author

Jiménez-Castellanos, Juan-Carlos, Wan Nur Ismah, Wan Ahmad Kamil, Yuiko Takebayashi, Findlay, Jacqueline, Schneiders, Thamarai, Heesom, Kate J., Avison, Matthew B., and Takebayashi, Yuiko

Subjects
KLEBSIELLA pneumoniae, PROTEOMICS, BETA lactam antibiotics, DRUG resistance in bacteria, MICROBIAL sensitivity tests, LIQUID chromatography-mass spectrometry, PORINS (Proteins), THERAPEUTICS
Abstract

Objectives: In Klebsiella pneumoniae, overproduction of RamA results in reduced envelope permeability and reduced antimicrobial susceptibility but clinically relevant resistance is rarely observed. Here we have tested whether RamA overproduction can enhance acquired β-lactam resistance mechanisms in K. pneumoniae and have defined the envelope protein abundance changes upon RamA overproduction during growth in low and high osmolarity media.Methods: Envelope permeability was estimated using a fluorescent dye accumulation assay. β-Lactam susceptibility was measured using disc testing. Total envelope protein production was quantified using LC-MS/MS proteomics and transcript levels were quantified using real-time RT-PCR.Results: RamA overproduction enhanced β-lactamase-mediated β-lactam resistance, in some cases dramatically, without altering β-lactamase production. It increased production of efflux pumps and decreased OmpK35 porin production, though micF overexpression showed that OmpK35 reduction has little impact on envelope permeability. A survey of K. pneumoniae bloodstream isolates revealed ramA hyperexpression in 3 of 4 carbapenemase producers, 1 of 21 CTX-M producers and 2 of 19 strains not carrying CTX-M or carbapenemases.Conclusions: Whilst RamA is not a key mediator of antibiotic resistance in K. pneumoniae on its own, it is potentially important for enhancing the spectrum of acquired β-lactamase-mediated β-lactam resistance. LC-MS/MS proteomics analysis has revealed that this enhancement is achieved predominantly through activation of efflux pump production. [ABSTRACT FROM AUTHOR]

Published
2018
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143. Cytoskeletal proteins bound to heat-shock protein 70 may elicit resistance to simian immunodeficiency virus infection of CD4+ T cells

Author

Bergmeier, Lesley A, Babaahmady, Kaboutar, Pido-Lopez, Jeffrey, Heesom, Kate J, Kelly, Charles G, and Lehner, Thomas

Subjects
CD4-Positive T-Lymphocytes, Binding Sites, viruses, Original Articles, APOBEC-3G Deaminase, Virus Replication, Actins, Mass Spectrometry, Profilins, Actin Depolymerizing Factors, Neutralization Tests, Cytidine Deaminase, Animals, Humans, Macaca, Electrophoresis, Gel, Two-Dimensional, HSP70 Heat-Shock Proteins, Simian Immunodeficiency Virus
Abstract

This study is based on the evidence that immunization of macaques with human CD4(+) T cells elicits prevention of simian immunodeficiency virus (SIV) infection. We hypothesized that heat-shock protein 70 (HSP70) isolated from CD4(+) T cells may act as a chaperone and carry the protective host proteins. Two moieties of HSP70 were affinity-purified from human CD4(+) T cells; an ADP preparation with HSP70-bound proteins (ADP-HSP) and an ATP control preparation. Immunization of rhesus macaques with these preparations showed significant inhibition of SIVmac251 infectivity ex vivo in CD4(+) T cells only with the ADP-HSP (P = 0.01). Proteomic analysis identified three cytoskeletal elements, cofilin, profilin and gamma-actin, exclusively in the ADP-HSP preparation. Investigation of the mechanism of prevention of SIV replication suggests that antibodies to the cytoskeletal proteins may inhibit actin depolymerization and facilitate viral degradation by the innate antiviral APOBEC3G. As cytoskeletal proteins are critical in the formation of virological and immunological synapses, finding specific antibodies and anti-SIV/human immunodeficiency virus (HIV) factors suggests a novel insight into HIV-1 immunopathogenesis.

Published
2010

144. Retromer Binding to FAM21 and the WASH Complex Is Perturbed by the Parkinson Disease-Linked VPS35(D620N) Mutation.

Author

McGough, Ian J., Steinberg, Florian, Jia, Da, Barbuti, Peter A., McMillan, Kirsty J., Heesom, Kate J., Caldwell, Maeve A., Billadeau, Daniel D., Rosen, Michael K., Cullen, Peter J., McGough, Ian J., Steinberg, Florian, Jia, Da, Barbuti, Peter A., McMillan, Kirsty J., Heesom, Kate J., Caldwell, Maeve A., Billadeau, Daniel D., Rosen, Michael K., and Cullen, Peter J.

Abstract

Retromer is a protein assembly that plays a central role in orchestrating export of transmembrane-spanning cargo proteins from endosomes into retrieval pathways destined for the Golgi apparatus and the plasma membrane [1]. Recently, a specific mutation in the retromer component VPS35, VPS35(D620N), has linked retromer dysfunction to familial autosomal dominant and sporadic Parkinson disease [2, 3]. However, the effect of this mutation on retromer function remains poorly characterized. Here we established that in cells expressing VPS35(D620N) there is a perturbation in endosome-to-TGN transport but not endosome-to-plasma membrane recycling, which we confirm in patient cells harboring the VPS35(D620N) mutation. Through comparative stable isotope labeling by amino acids in cell culture (SILAC)-based analysis of wild-type VPS35 versus the VPS35(D620N) mutant interactomes, we establish that the major defect of the D620N mutation lies in the association to the actin-nucleating Wiskott-Aldrich syndrome and SCAR homolog (WASH) complex. Moreover, using isothermal calorimetry, we establish that the primary defect of the VPS35(D620N) mutant is a 2.2 +/- 0.5-fold decrease in affinity for the WASH complex component FAM21. These data define the primary molecular defect in retromer assembly that arises from the VPS35(D620N) mutation and, by revealing functional effects on retromer-mediated endosome-to-TGN transport, provide new insight into retromer deregulation in Parkinson disease.

Published
2014
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145. Protein arginine methyltransferase 5 is a key regulator of the MYCN oncoprotein in neuroblastoma cells

Author

Park, Ji Hyun, primary, Szemes, Marianna, additional, Vieira, Gabriella Cunha, additional, Melegh, Zsombor, additional, Malik, Sally, additional, Heesom, Kate J., additional, Von Wallwitz-Freitas, Laura, additional, Greenhough, Alexander, additional, Brown, Keith W., additional, Zheng, Y. George, additional, Catchpoole, Daniel, additional, Deery, Michael J., additional, and Malik, Karim, additional

Published
2014
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147. Qualitative and Quantitative Comparison of the Proteome of Erythroid Cells Differentiated from Human iPSCs and Adult Erythroid Cells by Multiplex TMT Labelling and NanoLC-MS/MS

Author

Trakarnsanga, Kongtana, primary, Wilson, Marieangela C., additional, Griffiths, Rebecca E., additional, Toye, Ashley M., additional, Carpenter, Lee, additional, Heesom, Kate J., additional, Parsons, Steve F., additional, Anstee, David J., additional, and Frayne, Jan, additional

Published
2014
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148. Retromer Binding to FAM21 and the WASH Complex Is Perturbed by the Parkinson Disease-Linked VPS35(D620N) Mutation

Author

McGough, Ian J., primary, Steinberg, Florian, additional, Jia, Da, additional, Barbuti, Peter A., additional, McMillan, Kirsty J., additional, Heesom, Kate J., additional, Whone, Alan L., additional, Caldwell, Maeve A., additional, Billadeau, Daniel D., additional, Rosen, Michael K., additional, and Cullen, Peter J., additional

Published
2014
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