14 results on '"Shaunt Fereshetian"'
Search Results
2. Regulation of purine metabolism connects KCTD13 to a metabolic disorder with autistic features
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Jon M. Madison, Karen Duong, Ellen F. Vieux, Namrata D. Udeshi, Sumaiya Iqbal, Elise Requadt, Shaunt Fereshetian, Michael C. Lewis, Antonio S. Gomes, Kerry A. Pierce, Randall J. Platt, Feng Zhang, Arthur J. Campbell, Dennis Lal, Florence F. Wagner, Clary B. Clish, Steven A. Carr, Morgan Sheng, Edward M. Scolnick, and Jeffrey R. Cottrell
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Molecular Neuroscience ,Proteomics ,Metabolomics ,Science - Abstract
Summary: Genetic variation of the 16p11.2 deletion locus containing the KCTD13 gene and of CUL3 is linked with autism. This genetic connection suggested that substrates of a CUL3-KCTD13 ubiquitin ligase may be involved in disease pathogenesis. Comparison of Kctd13 mutant (Kctd13−/−) and wild-type neuronal ubiquitylomes identified adenylosuccinate synthetase (ADSS), an enzyme that catalyzes the first step in adenosine monophosphate (AMP) synthesis, as a KCTD13 ligase substrate. In Kctd13−/− neurons, there were increased levels of succinyl-adenosine (S-Ado), a metabolite downstream of ADSS. Notably, S-Ado levels are elevated in adenylosuccinate lyase deficiency, a metabolic disorder with autism and epilepsy phenotypes. The increased S-Ado levels in Kctd13−/− neurons were decreased by treatment with an ADSS inhibitor. Lastly, functional analysis of human KCTD13 variants suggests that KCTD13 variation may alter ubiquitination of ADSS. These data suggest that succinyl-AMP metabolites accumulate in Kctd13−/− neurons, and this observation may have implications for our understanding of 16p11.2 deletion syndrome.
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- 2021
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3. Multiomic characterization of oncogenic signaling mediated by wild-type and mutant RIT1
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Filip Mundt, Jacqueline Watson, Steven A. Carr, Sitapriya Moorthi, April Lo, Iris Fung, Shriya Kamlapurkar, Kristin D. Holmes, Alice H. Berger, Shaunt Fereshetian, and Philipp Mertins
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Mutant ,Guanosine ,GTPase ,Biology ,medicine.disease_cause ,Biochemistry ,Article ,chemistry.chemical_compound ,Germline mutation ,Oncogenic signaling ,medicine ,Humans ,Molecular Biology ,Wild type ,Oncogenes ,Cell Biology ,medicine.disease ,digestive system diseases ,respiratory tract diseases ,HEK293 Cells ,chemistry ,ras Proteins ,Cancer research ,Adenocarcinoma ,KRAS ,Signal Transduction - Abstract
Aberrant activation of the RAS family of guanosine triphosphatases (GTPases) is prevalent in lung adenocarcinoma, with somatic mutation of KRAS occurring in ~30% of tumors. We previously identified somatic mutations and amplifications of the gene encoding RAS family GTPase RIT1 in lung adenocarcinomas. To explore the biological pathways regulated by RIT1 and how they relate to the oncogenic KRAS network, we performed quantitative proteomic, phosphoproteomic, and transcriptomic profiling of isogenic lung epithelial cells in which we ectopically expressed wild-type or cancer-associated variants of RIT1 and KRAS. We found that both mutant KRAS and mutant RIT1 promoted canonical RAS signaling, and that overexpression of wild-type RIT1 partially phenocopied oncogenic RIT1 and KRAS, including induction of epithelial-to-mesenchymal transition. Our findings suggest that RIT1 protein abundance is a factor in its pathogenic function. Therefore, chromosomal amplification of wild-type RIT1 in lung and other cancers may be tumorigenic.
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- 2021
4. Protein and Imaging Biomarkers in the Eye for Early Detection of Alzheimer's Disease
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Manju L Subramanian, Steven Ness, Joshua S Agranat, Nicole Siegel, Shaunt Fereshetian, and Thor D. Stein
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retina ,lens ,Early detection ,Disease ,Review ,Bioinformatics ,vitreous ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Dementia ,crystalline ,business.industry ,General Neuroscience ,amyloid ,medicine.disease ,eye ,Psychiatry and Mental health ,Clinical Psychology ,cataract ,030221 ophthalmology & optometry ,Retinal imaging ,Geriatrics and Gerontology ,business ,Alzheimer’s disease ,030217 neurology & neurosurgery - Abstract
Alzheimer’s disease (AD) is one of the most common causes of dementia worldwide. Although no formal curative therapy exists for the treatment of AD, considerable research has been performed to identify biomarkers for early detection of this disease, and thus improved subsequent management. Given that the eye can be examined and imaged non-invasively with relative ease, it has emerged as an exciting area of research for evidence of biomarkers and to aid in the early diagnosis of AD. This review explores the current understanding of both protein and retinal imaging biomarkers in the eye. Herein, primary findings in the literature regarding AD biomarkers associated with the lens, retina, and other ocular structures are reviewed.
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- 2021
5. Mapping the unique and shared functions of oncogenic KRAS and RIT1 with proteome and transcriptome profiling
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Steven A. Carr, Jacqueline Watson, Iris Fung, Sitapriya Moorthi, K. Holmes, April Lo, Philipp Mertins, Filip Mundt, Shaunt Fereshetian, and Alice H. Berger
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0303 health sciences ,GTPase ,Human leukocyte antigen ,Biology ,medicine.disease_cause ,digestive system diseases ,respiratory tract diseases ,Transcriptome ,03 medical and health sciences ,0302 clinical medicine ,Germline mutation ,030220 oncology & carcinogenesis ,Proteome ,medicine ,Cancer research ,Transcriptional regulation ,KRAS ,Technology Platforms ,Protein kinase B ,030304 developmental biology - Abstract
Aberrant activation of RAS oncogenes is prevalent in lung adenocarcinoma, with somatic mutation ofKRASoccurring in ∼30% of tumors. Recently, we identified somatic mutation of the RAS-family GTPaseRIT1in lung adenocarcinoma, but relatively little is known about the biological pathways regulated by RIT1 and how these relate to the oncogenic KRAS network. Here we present quantitative proteomic and transcriptomic profiles fromKRAS-mutant andRIT1-mutant isogenic lung epithelial cells and globally characterize the signaling networks regulated by each oncogene. We find that both mutant KRAS and mutant RIT1 promote S6 kinase, AKT, and RAF/MEK signaling, and promote epithelial-to-mesenchymal transition and immune evasion via HLA protein loss. However, KRAS and RIT1 diverge in regulation of phosphorylation sites on EGFR, USO1, and AHNAK proteins. The majority of the proteome changes are related to altered transcriptional regulation, but a small subset of proteins are differentially regulated by both oncoproteins at the post-transcriptional level, including intermediate filament proteins, metallothioneins, and MHC Class I proteins. These data provide the first global, unbiased characterization of oncogenic RIT1 network and identify the shared and divergent functions of oncogenic RIT1 and KRAS GTPases in lung cancer.
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- 2020
6. Rapid and deep-scale ubiquitylation profiling for biology and translational research
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Jessica A. Gasser, Shankha Satpathy, Tanya Svinkina, Shaunt Fereshetian, Steven A. Carr, Philipp Mertins, Benjamin L. Ebert, Deepak Mani, Namrata D. Udeshi, and Meagan E. Olive
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Proteomics ,0301 basic medicine ,Ubiquitylation ,Proteome ,Ion-mobility spectrometry ,Science ,Ubiquitin-Protein Ligases ,General Physics and Astronomy ,Breast Neoplasms ,Peptide ,Sensitivity and Specificity ,Quantitative accuracy ,Article ,General Biochemistry, Genetics and Molecular Biology ,Translational Research, Biomedical ,Ikaros Transcription Factor ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Protein ubiquitylation ,Ubiquitin ,Animals ,Humans ,lcsh:Science ,Casein Kinase Ialpha ,chemistry.chemical_classification ,Multidisciplinary ,Mass spectrometry ,Staining and Labeling ,biology ,Ubiquitination ,Proteins ,General Chemistry ,Ubiquitin ligase ,Cell biology ,030104 developmental biology ,chemistry ,030220 oncology & carcinogenesis ,biology.protein ,Female ,lcsh:Q ,Asymmetric waveform ,Multiple Myeloma ,Protein Processing, Post-Translational ,HeLa Cells - Abstract
Protein ubiquitylation is involved in a plethora of cellular processes. While antibodies directed at ubiquitin remnants (K-ɛ-GG) have improved the ability to monitor ubiquitylation using mass spectrometry, methods for highly multiplexed measurement of ubiquitylation in tissues and primary cells using sub-milligram amounts of sample remains a challenge. Here, we present a highly sensitive, rapid and multiplexed protocol termed UbiFast for quantifying ~10,000 ubiquitylation sites from as little as 500 μg peptide per sample from cells or tissue in a TMT10plex in ca. 5 h. High-field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) is used to improve quantitative accuracy for posttranslational modification analysis. We use the approach to rediscover substrates of the E3 ligase targeting drug lenalidomide and to identify proteins modulated by ubiquitylation in models of basal and luminal human breast cancer. The sensitivity and speed of the UbiFast method makes it suitable for large-scale studies in primary tissue samples., Comprehensive protein ubiquitylation profiling by mass spectrometry typically requires large sample amounts, limiting its applicability to tissue samples. Here, the authors present an optimized proteomics method that enables multiplexed ubiquitylome analysis of cells and tumor tissue samples.
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- 2020
7. PPM1D-truncating mutations confer resistance to chemotherapy and sensitivity to PPM1D inhibition in hematopoietic cells
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Marie McConkey, Rob S. Sellar, Benjamin L. Ebert, John G. Doench, Siddhartha Jaiswal, Josephine Kahn, Karsten Krug, Shaunt Fereshetian, Dylan N. Adams, Shruti Bhatt, Peter Miller, Brenton G. Mar, Haoling Zhu, Christopher J. Gibson, Steven A. Carr, Alexander J. Silver, Anthony Letai, and Philipp Mertins
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0301 basic medicine ,Myeloid ,DNA damage ,Immunology ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Chemotherapy regimen ,Phenotype ,03 medical and health sciences ,Haematopoiesis ,Exon ,030104 developmental biology ,0302 clinical medicine ,medicine.anatomical_structure ,Apoptosis ,Cell culture ,030220 oncology & carcinogenesis ,Cancer research ,medicine - Abstract
Truncating mutations in the terminal exon of protein phosphatase Mg2+/Mn2+ 1D (PPM1D) have been identified in clonal hematopoiesis and myeloid neoplasms, with a striking enrichment in patients previously exposed to chemotherapy. In this study, we demonstrate that truncating PPM1D mutations confer a chemoresistance phenotype, resulting in the selective expansion of PPM1D-mutant hematopoietic cells in the presence of chemotherapy in vitro and in vivo. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease mutational profiling of PPM1D in the presence of chemotherapy selected for the same exon 6 mutations identified in patient samples. These exon 6 mutations encode for a truncated protein that displays elevated expression and activity due to loss of a C-terminal degradation domain. Global phosphoproteomic profiling revealed altered phosphorylation of target proteins in the presence of the mutation, highlighting multiple pathways including the DNA damage response (DDR). In the presence of chemotherapy, PPM1D-mutant cells have an abrogated DDR resulting in altered cell cycle progression, decreased apoptosis, and reduced mitochondrial priming. We demonstrate that treatment with an allosteric, small molecule inhibitor of PPM1D reverts the phosphoproteomic, DDR, apoptotic, and mitochondrial priming changes observed in PPM1D-mutant cells. Finally, we show that the inhibitor preferentially kills PPM1D-mutant cells, sensitizes the cells to chemotherapy, and reverses the chemoresistance phenotype. These results provide an explanation for the enrichment of truncating PPM1D mutations in the blood of patients exposed to chemotherapy and in therapy-related myeloid neoplasms, and demonstrate that PPM1D can be a targeted in the prevention of clonal expansion of PPM1D-mutant cells and the treatment of PPM1D-mutant disease.
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- 2018
8. Antibodies to biotin enable large-scale detection of biotinylation sites on proteins
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Vamsi K. Mootha, Dominic Ryan, Karsten Krug, Steven A. Carr, Karl R. Clauser, Tslil Ast, Namrata D. Udeshi, Kayvon Pedram, Tanya Svinkina, Alice Y. Ting, Samuel A. Myers, Shaunt Fereshetian, and Ozan Aygün
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0301 basic medicine ,Streptavidin ,Biotin ,Peptide ,Biology ,010402 general chemistry ,Tandem mass spectrometry ,Mass spectrometry ,01 natural sciences ,Biochemistry ,Antibodies ,Jurkat Cells ,03 medical and health sciences ,chemistry.chemical_compound ,Tandem Mass Spectrometry ,Humans ,Biotinylation ,Molecular Biology ,chemistry.chemical_classification ,Staining and Labeling ,Proteins ,Cell Biology ,0104 chemical sciences ,HEK293 Cells ,030104 developmental biology ,chemistry ,biology.protein ,Antibody ,Peptides ,Chromatography, Liquid ,Biotechnology ,Peroxidase - Abstract
Although purification of biotinylated molecules is highly efficient, identifying specific sites of biotinylation remains challenging. We show that anti-biotin antibodies enable unprecedented enrichment of biotinylated peptides from complex peptide mixtures. Live-cell proximity labeling using APEX peroxidase followed by anti-biotin enrichment and mass spectrometry yielded over 1,600 biotinylation sites on hundreds of proteins, an increase of more than 30-fold in the number of biotinylation sites identified compared to streptavidin-based enrichment of proteins.
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- 2017
9. UbiFast, a rapid and deep-scale ubiquitylation profiling approach for biology and translational research
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Steven A. Carr, Shankha Satpathy, Philipp Mertins, Jessica A. Gasser, Deepak Mani, Namrata D. Udeshi, Benjamin L. Ebert, Tanya Svinkina, and Shaunt Fereshetian
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chemistry.chemical_classification ,0303 health sciences ,biology ,Peptide ,Quantitative accuracy ,3. Good health ,Ubiquitin ligase ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,Protein ubiquitylation ,Ubiquitin ,chemistry ,030220 oncology & carcinogenesis ,biology.protein ,Posttranslational modification ,Human breast ,030304 developmental biology - Abstract
Protein ubiquitylation is involved in a plethora of cellular processes. Defects in the ubiquitin system are at the root of many acquired and hereditary diseases. While antibodies directed at ubiquitin remnants (K-ε-GG) have improved the ability to monitor ubiquitylation using mass spectrometry, methods for highly-multiplexed measurement of ubiquitylation in tissues and primary cells using sub-milligram amounts of sample remains a challenge. Here we present a highly-sensitive, rapid and multiplexed protocol for quantifying ∼10,000 ubiquitylation sites from as little as 500 ug peptide per sample from cells or tissue in a TMT10 plex in ca. 5 hr. High-field Asymmetric Ion Mobility Spectrometry (FAIMS) is used to improve quantitative accuracy for posttranslational modification analysis. We use the approach to rediscover substrates of the E3 ligase targeting drug lenalidomide and to identify proteins modulated by ubiquitylation in models of basal and luminal human breast cancer. The sensitivity and speed of the UbiFast method makes it suitable for large-scale studies in primary tissue samples.
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- 2019
10. Combined Analysis of Metabolomes, Proteomes, and Transcriptomes of Hepatitis C Virus–Infected Cells and Liver to Identify Pathways Associated With Disease Development
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Ralf Bartenschlager, Kazuaki Chayama, Joachim Lupberger, Nabeel Bardeesy, Eloi R. Verrier, Nicolaas Van Renne, Tom Croonenborghs, Philipp Mertins, Armando Andres Roca Suarez, Steven A. Carr, Frank Jühling, Yujin Hoshida, Evelyn Ramberger, Mirjam B. Zeisel, Olivier Gevaert, Nourdine Hamdane, Marine A. Oudot, Gergö Meszaros, Alessia Virzì, Mohsen Nabian, Thomas F. Baumert, Daniel Brumaru, Romeo Ricci, Rileen Sinha, Naoto Fujiwara, Hussein El Saghire, Simonetta Bandiera, Marko Jovanovic, Carole Jamey, Celine Everaert, Shaunt Fereshetian, Nathalie Pochet, Laura Heydmann, Sarah C. Durand, Nassim Dali-Youcef, Atish Mukherji, Institut de Recherche sur les Maladies Virales et Hépatiques (IVH), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Harvard-MIT Division of Health Sciences and Technology [Cambridge], Massachusetts Institute of Technology (MIT), Evergrande Center for Immunologic Diseases [Boston, MA, USA] (Ann Romney Center for Neurologic Diseases), Brigham & Women's Hospital, Harvard Medical School, Université de Strasbourg (UNISTRA), Laboratoire de Biochimie et de Biologie Moléculaire, CHU Strasbourg-Hôpital de Hautepierre [Strasbourg], Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA), Department of Infectious Diseases [Heidelberg, Germany], Heidelberg University Hospital [Heidelberg], Division of Virus-Associated Carcinogenesis [Heidelberg, Germany], German Cancer Research Center - Deutsches Krebsforschungszentrum [Heidelberg] (DKFZ), The Broad Institute [Cambridge, MA, USA], Harvard University [Cambridge]-Massachusetts Institute of Technology (MIT), Proteomics Platform [Berlin, Germany], Max Delbrück Center for Molecular Medicine [Berlin] (MDC), Helmholtz-Gemeinschaft = Helmholtz Association-Helmholtz-Gemeinschaft = Helmholtz Association-Helmholtz Society [Berlin, Germany], Institute of Public Health [Berlin, Germany], Charité - UniversitätsMedizin = Charité - University Hospital [Berlin], Hiroshima University, Liver Research Project Center [Hiroshima, Japan], Massachusetts General Hospital [Boston], Harold C. Simmons Comprehensive Cancer Center [Dallas, TX, États-Unis], University of Texas Southwestern Medical Center [Dallas], Cell Circuits Program [Cambridge, MA, USA], Broad Institute of MIT and Harvard (BROAD INSTITUTE), Harvard Medical School [Boston] (HMS)-Massachusetts Institute of Technology (MIT)-Massachusetts General Hospital [Boston]-Harvard Medical School [Boston] (HMS)-Massachusetts Institute of Technology (MIT)-Massachusetts General Hospital [Boston], Stanford Center for BioMedical Informatics Research (BMIR), Stanford University, Department of Neurology [Cambridge, MA, USA] (Ann Romney Center for Neurologic Diseases), Harvard Medical School [Boston] (HMS)-Brigham and Women's Hospital [Boston], Department of Neurology [Boston], Harvard Medical School [Boston] (HMS)-Massachusetts General Hospital [Boston], Pôle hépato-digestif [Strasbourg], Nouvel Hôpital Civil, Hospices Civils de Strasbourg-Institut Hospitalo-Universitaire de strasbourg, This work was supported by the European Union (ERC-AdG-2014 HEPCIR to Thomas F. Baumert and Yujin Hoshida and EU H2020 HEPCAR 667273 to Thomas F. Baumert and Joachim Lupberger), the Agence nationale de recherche sur le sida et les hépatites virales (ECTZ4236 to Joachim Lupberger and ECTZ4446 to Armando Andres Roca Suarez), the French Cancer Agency (ARC IHU201301187 to Thomas F. Baumert), the US Department of Defense (W81XWH-16-1-0363 to Thomas F. Baumert and Yujin Hoshida), the National Institutes of Health (National Institute of Allergy and Infectious Diseases R03AI131066 to Nathalie Pochet and Thomas F. Baumert, National Cancer Institute 1R21CA209940 to Nathalie Pochet, Thomas F. Baumert, and Olivier Gevaert, National Institute of Allergy and Infectious Diseases 5U19AI123862-02 to Thomas F. Baumert, National Cancer Institute/Informatics Technology for Cancer Research (ITCR) U01 CA214846 to Nathalie Pochet and Olivier Gevaert), the Fondation de l’Université de Strasbourg (HEPKIN) (TBA-DON-0002) and the INSERM Plan Cancer 2019-2023 to Thomas F. Baumert. This work has benefitted from support by the Initiative of Excellence IDEX-Unistra (ANR-10-IDEX-0002-02 to Joachim Lupberger) and has been published under the framework of the LABEX ANR-10-LAB-28 (HEPSYS). INSERM Plan Cancer, IDEX, and LABEX are initiatives from the French program 'Investments for the Future.' The work of Ralf Bartenschlager was supported by the Deutsche Forschungsgemeinschaft (TRR179, TP9). Kazuaki Chayama was supported by the Research Program on Hepatitis from the Japanese Agency for Medical Research and Development (AMED) Japan (JP18fk0210020h0002). The work of Romeo Ricci and Gergö Meszaros was supported by a European Research Council (ERC) starting grant (ERC-2011-StG, 281271-STRESS METABOL), by the European Foundation for the Study of Diabetes (EFSD)/Lilly European Diabetes Research Program grant, and by the ANR-10-LABX-0030-INRT grant, a French State fund managed by the ANR under the frame program Investissements d’Avenir ANR-10-IDEX0002-02., daulny, anne, and Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
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0301 basic medicine ,Proteomics ,Cirrhosis ,Peroxisome proliferator-activated receptor ,Datasets as Topic ,Hepacivirus ,medicine.disease_cause ,Sciences du Vivant [q-bio]/Cancer ,Transcriptome ,Liver disease ,Mice ,0302 clinical medicine ,Sciences du Vivant [q-bio]/Immunologie ,HCC ,chemistry.chemical_classification ,[SDV.MHEP] Life Sciences [q-bio]/Human health and pathology ,Fatty liver ,Gastroenterology ,Sciences du Vivant [q-bio]/Biotechnologies ,metabolic disease ,3. Good health ,Liver ,Hepatocellular carcinoma ,030211 gastroenterology & hepatology ,signal transduction ,STAT3 Transcription Factor ,Hepatitis C virus ,Biology ,Sciences du Vivant [q-bio]/Médecine humaine et pathologie ,03 medical and health sciences ,Cell Line, Tumor ,medicine ,Peroxisomes ,Animals ,Humans ,Metabolomics ,Transplantation Chimera ,Hepatology ,Gene Expression Profiling ,immune regulation ,[SDV.MHEP.HEG]Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology ,Hepatitis C, Chronic ,medicine.disease ,[SDV.MHEP.HEG] Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology ,digestive system diseases ,Disease Models, Animal ,030104 developmental biology ,Glucose ,chemistry ,Cancer research ,Hepatocytes ,Steatohepatitis ,[SDV.MHEP]Life Sciences [q-bio]/Human health and pathology - Abstract
Comment inMulti-omic Analyses Reveal Complex Interactions Between HCV and Hepatocytes Demonstrating That the Red Queen Is Up and Running. [Gastroenterology. 2019]; International audience; BACKGROUND & AIMS:The mechanisms of hepatitis C virus (HCV) infection, liver disease progression, and hepatocarcinogenesis are only partially understood. We performed genomic, proteomic, and metabolomic analyses of HCV-infected cells and chimeric mice to learn more about these processes.METHODS:Huh7.5.1dif (hepatocyte-like cells) were infected with culture-derived HCV and used in RNA sequencing, proteomic, metabolomic, and integrative genomic analyses. uPA/SCID (urokinase-type plasminogen activator/severe combined immunodeficiency) mice were injected with serum from HCV-infected patients; 8 weeks later, liver tissues were collected and analyzed by RNA sequencing and proteomics. Using differential expression, gene set enrichment analyses, and protein interaction mapping, we identified pathways that changed in response to HCV infection. We validated our findings in studies of liver tissues from 216 patients with HCV infection and early-stage cirrhosis and paired biopsy specimens from 99 patients with hepatocellular carcinoma, including 17 patients with histologic features of steatohepatitis. Cirrhotic liver tissues from patients with HCV infection were classified into 2 groups based on relative peroxisome function; outcomes assessed included Child-Pugh class, development of hepatocellular carcinoma, survival, and steatohepatitis. Hepatocellular carcinomas were classified according to steatohepatitis; the outcome was relative peroxisomal function.RESULTS:We quantified 21,950 messenger RNAs (mRNAs) and 8297 proteins in HCV-infected cells. Upon HCV infection of hepatocyte-like cells and chimeric mice, we observed significant changes in levels of mRNAs and proteins involved in metabolism and hepatocarcinogenesis. HCV infection of hepatocyte-like cells significantly increased levels of the mRNAs, but not proteins, that regulate the innate immune response; we believe this was due to the inhibition of translation in these cells. HCV infection of hepatocyte-like cells increased glucose consumption and metabolism and the STAT3 signaling pathway and reduced peroxisome function. Peroxisomes mediate β-oxidation of very long-chain fatty acids; we found intracellular accumulation of very long-chain fatty acids in HCV-infected cells, which is also observed in patients with fatty liver disease. Cells in livers from HCV-infected mice had significant reductions in levels of the mRNAs and proteins associated with peroxisome function, indicating perturbation of peroxisomes. We found that defects in peroxisome function were associated with outcomes and features of HCV-associated cirrhosis, fatty liver disease, and hepatocellular carcinoma in patients.CONCLUSIONS:We performed combined transcriptome, proteome, and metabolome analyses of liver tissues from HCV-infected hepatocyte-like cells and HCV-infected mice. We found that HCV infection increases glucose metabolism and the STAT3 signaling pathway and thereby reduces peroxisome function; alterations in the expression levels of peroxisome genes were associated with outcomes of patients with liver diseases. These findings provide insights into liver disease pathogenesis and might be used to identify new therapeutic targets.
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- 2019
11. Regulation of purine metabolism connects KCTD13 to a metabolic disorder with autistic features
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Clary B. Clish, Sumaiya Iqbal, Dennis Lal, Karen Duong, Namrata D. Udeshi, Arthur J. Campbell, Edward M. Scolnick, Kerry A. Pierce, Feng Zhang, Michael C. Lewis, Jon M. Madison, Antonio S. Gomes, Florence F. Wagner, Randall Jeffrey Platt, Jeffrey R. Cottrell, Morgan Sheng, Ellen F. Vieux, Shaunt Fereshetian, Steven A. Carr, and Elise Requadt
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Proteomics ,0301 basic medicine ,Adenosine monophosphate ,Mutant ,02 engineering and technology ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Ubiquitin ,medicine ,Metabolomics ,lcsh:Science ,Purine metabolism ,Adenylosuccinate lyase deficiency ,chemistry.chemical_classification ,DNA ligase ,Multidisciplinary ,biology ,Adenylosuccinate synthase ,021001 nanoscience & nanotechnology ,medicine.disease ,Ubiquitin ligase ,Cell biology ,030104 developmental biology ,chemistry ,biology.protein ,lcsh:Q ,Molecular Neuroscience ,0210 nano-technology - Abstract
Summary Genetic variation of the 16p11.2 deletion locus containing the KCTD13 gene and of CUL3 is linked with autism. This genetic connection suggested that substrates of a CUL3-KCTD13 ubiquitin ligase may be involved in disease pathogenesis. Comparison of Kctd13 mutant (Kctd13−/−) and wild-type neuronal ubiquitylomes identified adenylosuccinate synthetase (ADSS), an enzyme that catalyzes the first step in adenosine monophosphate (AMP) synthesis, as a KCTD13 ligase substrate. In Kctd13−/− neurons, there were increased levels of succinyl-adenosine (S-Ado), a metabolite downstream of ADSS. Notably, S-Ado levels are elevated in adenylosuccinate lyase deficiency, a metabolic disorder with autism and epilepsy phenotypes. The increased S-Ado levels in Kctd13−/− neurons were decreased by treatment with an ADSS inhibitor. Lastly, functional analysis of human KCTD13 variants suggests that KCTD13 variation may alter ubiquitination of ADSS. These data suggest that succinyl-AMP metabolites accumulate in Kctd13−/− neurons, and this observation may have implications for our understanding of 16p11.2 deletion syndrome., Graphical abstract, Highlights • KCTD13 deletion leads to decreases in ubiquitination and increases in levels of ADSS • KCTD13 deletion increases S-Ado levels, a metabolite observed in ADSL deficiency • Treatment of KCTD13 deletion neurons with an ADSS inhibitor reduces S-Ado levels • Human KCTD13 variants can alter ubiquitination of ADSS, Molecular Neuroscience; Proteomics; Metabolomics
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- 2021
12. Targeting wild-type KRAS-amplified gastroesophageal cancer through combined MEK and SHP2 inhibition
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Francesco Graziano, J. Alan Diehl, Yu Imamura, Les Henderson, Rachel Rendak, Peng Xu, Jens Puschhof, Charles Zou, Sarit Schwartz, Steven A. Carr, Jin Zhou, Philipp Mertins, Xinsen Xu, Masayuki Watanabe, Wei-Li Liao, Eiji Oki, Annamaria Ruzzo, Gabrielle S. Wong, Tianxia Li, Daniel V.T. Catenacci, Jie Bin Liu, Adam J. Bass, Kwok-Kin Wong, Anil K. Rustgi, Steven E. Schumacher, Rameen Beroukhim, Emily O'Day, Shaunt Fereshetian, Kenichi Nakamura, Hideo Baba, David Xu, Zhong Wu, Karin Jensen, Fabiola Cecchi, Austin M. Dulak, Christopher Szeto, James M. McFarland, and Todd Hembrough
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0301 basic medicine ,MAPK/ERK pathway ,Esophageal Neoplasms ,endocrine system diseases ,Pyridones ,Protein Tyrosine Phosphatase, Non-Receptor Type 11 ,Pyrimidinones ,Protein tyrosine phosphatase ,Biology ,medicine.disease_cause ,General Biochemistry, Genetics and Molecular Biology ,Proto-Oncogene Proteins p21(ras) ,Mice ,03 medical and health sciences ,Piperidines ,Stomach Neoplasms ,In vivo ,Cell Line, Tumor ,Gene duplication ,medicine ,Animals ,Humans ,Protein Kinase Inhibitors ,neoplasms ,Mitogen-Activated Protein Kinase Kinases ,Gene Amplification ,Wild type ,Cancer ,General Medicine ,medicine.disease ,digestive system diseases ,respiratory tract diseases ,3. Good health ,Disease Models, Animal ,Pyrimidines ,030104 developmental biology ,SOS1 ,Cancer research ,KRAS - Abstract
The role of KRAS, when activated through canonical mutations, has been well established in cancer1. Here we explore a secondary means of KRAS activation in cancer: focal high-level amplification of the KRAS gene in the absence of coding mutations. These amplifications occur most commonly in esophageal, gastric and ovarian adenocarcinomas2-4. KRAS-amplified gastric cancer models show marked overexpression of the KRAS protein and are insensitive to MAPK blockade owing to their capacity to adaptively respond by rapidly increasing KRAS-GTP levels. Here we demonstrate that inhibition of the guanine-exchange factors SOS1 and SOS2 or the protein tyrosine phosphatase SHP2 can attenuate this adaptive process and that targeting these factors, both genetically and pharmacologically, can enhance the sensitivity of KRAS-amplified models to MEK inhibition in both in vitro and in vivo settings. These data demonstrate the relevance of copy-number amplification as a mechanism of KRAS activation, and uncover the therapeutic potential for targeting of these tumors through combined SHP2 and MEK inhibition.
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- 2018
13. Deep, Quantitative Coverage of the Lysine Acetylome Using Novel Anti-acetyl-lysine Antibodies and an Optimized Proteomic Workflow
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Jana Qiao, Eric Kuhn, Tanya Svinkina, Shaunt Fereshetian, Jacob D. Jaffe, Steven A. Carr, Hongbo Gu, Philipp Mertins, Namrata D. Udeshi, and Jeffrey C. Silva
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Proteomics ,Special Issue Articles ,Lysine ,Peptide ,Mass spectrometry ,Biochemistry ,Mass Spectrometry ,Analytical Chemistry ,Workflow ,Jurkat Cells ,Mice ,Ubiquitin ,Stable isotope labeling by amino acids in cell culture ,Animals ,Humans ,Molecular Biology ,chemistry.chemical_classification ,Chromatography ,biology ,Chemistry ,Antibodies, Monoclonal ,Mammary Neoplasms, Experimental ,Acetylation ,Liver ,biology.protein ,Phosphorylation ,Female ,Protein Processing, Post-Translational - Abstract
Introduction of antibodies specific for acetylated lysine has significantly improved the detection of endogenous acetylation sites by mass spectrometry. Here, we describe a new, commercially available mixture of anti-lysine acetylation (Kac) antibodies and show its utility for in-depth profiling of the acetylome. Specifically, seven complementary monoclones with high specificity for Kac were combined into a final anti-Kac reagent which results in at least a twofold increase in identification of Kac peptides over a commonly used Kac antibody. We outline optimal antibody usage conditions, effective offline basic reversed phase separation, and use of state-of-the-art LC-MS technology for achieving unprecedented coverage of the acetylome. The methods were applied to quantify acetylation sites in suberoylanilide hydroxamic acid-treated Jurkat cells. Over 10,000 Kac peptides from over 3000 Kac proteins were quantified from a single stable isotope labeling by amino acids in cell culture labeled sample using 7.5 mg of peptide input per state. This constitutes the deepest coverage of acetylation sites in quantitative experiments obtained to-date. The approach was also applied to breast tumor xenograft samples using isobaric mass tag labeling of peptides (iTRAQ4, TMT6 and TMT10-plex reagents) for quantification. Greater than 6700 Kac peptides from over 2300 Kac proteins were quantified using 1 mg of tumor protein per iTRAQ 4-plex channel. The novel reagents and methods we describe here enable quantitative, global acetylome analyses with depth and sensitivity approaching that obtained for other well-studied post-translational modifications such as phosphorylation and ubiquitylation, and should have widespread application in biological and clinical studies employing mass spectrometry-based proteomics.
- Published
- 2015
14. Author Correction: Targeting wild-type KRAS-amplified gastroesophageal cancer through combined MEK and SHP2 inhibition
- Author
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Tianxia Li, Jens Puschhof, Daniel V.T. Catenacci, Austin M. Dulak, Charles Zou, Steven E. Schumacher, Francesco Graziano, David Xu, Adam J. Bass, Kwok-Kin Wong, Rameen Beroukhim, Philipp Mertins, Les Henderson, Karin Jensen, Fabiola Cecchi, Kenichi Nakamura, Hideo Baba, Rachel Rendak, Christopher Szeto, Sarit Schwartz, Steven A. Carr, Peng Xu, James M. McFarland, Anil K. Rustgi, Todd Hembrough, Masayuki Watanabe, Annamaria Ruzzo, Zhong Wu, J. Alan Diehl, Xinsen Xu, Gabrielle S. Wong, Wei-Li Liao, Eiji Oki, Jin Zhou, Jie Bin Liu, Emily O'Day, Shaunt Fereshetian, and Yu Imamura
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0301 basic medicine ,business.industry ,Wild type ,MEDLINE ,General Medicine ,medicine.disease_cause ,Molecular medicine ,General Biochemistry, Genetics and Molecular Biology ,Blot ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Gastroesophageal cancer ,030220 oncology & carcinogenesis ,Cancer research ,medicine ,KRAS ,business - Abstract
In the Supplementary Information originally published with this article, a lane was missing in the β-actin blot in Supplementary Fig. 2. The lane has been added. The error has been corrected in the Supplementary Information associated with this article.
- Published
- 2018
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