811 results on '"Steven P. Gygi"'
Search Results
52. Supplementary Table S1 from Tissue-Specific Oncogenic Activity of KRASA146T
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Kevin M. Haigis, Kenneth D. Westover, Douglas A. Lauffenburger, Steven P. Gygi, Benjamin S. Braun, Pedro A. Perez-Mancera, John R. Engen, Yina Li, Jessica J. Gierut, Lana Bogdanova, Roxana E. Iacob, Thomas E. Wales, Sudershan Gondi, Phaedra C. Ghazi, Rebecca A. DeStefanis, Christian W. Johnson, Brian A. Joughin, Douglas K. Brubaker, Jonathan A. Nowak, Joshua Cook, Wei Yan, Carolina Morales, Tannie Q. Huang, Joao A. Paulo, Samantha Dale Strasser, Yi-Jang Lin, Jia Lu, Asim K. Bera, and Emily J. Poulin
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Data collection and refinement statistics
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- 2023
53. Data from Selective Alanine Transporter Utilization Creates a Targetable Metabolic Niche in Pancreatic Cancer
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Alec C. Kimmelman, Joseph D. Mancias, Dafna Bar-Sagi, Mark R. Philips, Huamin Wang, Drew R. Jones, Steven P. Gygi, Joao A. Paulo, Doug E. Biancur, Albert S.W. Sohn, Madeleine M. LaRue, Rebecca E. Rose, Joel Encarnación-Rosado, Keisuke Yamamoto, Qijia Yu, Kate E.R. Hollinshead, Caroline R. Amendola, and Seth J. Parker
- Abstract
Pancreatic ductal adenocarcinoma (PDAC) evolves a complex microenvironment comprised of multiple cell types, including pancreatic stellate cells (PSC). Previous studies have demonstrated that stromal supply of alanine, lipids, and nucleotides supports the metabolism, growth, and therapeutic resistance of PDAC. Here we demonstrate that alanine cross-talk between PSCs and PDAC is orchestrated by the utilization of specific transporters. PSCs utilize SLC1A4 and other transporters to rapidly exchange and maintain environmental alanine concentrations. Moreover, PDAC cells upregulate SLC38A2 to supply their increased alanine demand. Cells lacking SLC38A2 fail to concentrate intracellular alanine and undergo a profound metabolic crisis resulting in markedly impaired tumor growth. Our results demonstrate that stromal–cancer metabolic niches can form through differential transporter expression, creating unique therapeutic opportunities to target metabolic demands of cancer.Significance:This work identifies critical neutral amino acid transporters involved in channeling alanine between pancreatic stellate and PDAC cells. Targeting PDAC-specific alanine uptake results in a metabolic crisis impairing metabolism, proliferation, and tumor growth. PDAC cells specifically activate and require SLC38A2 to fuel their alanine demands that may be exploited therapeutically.This article is highlighted in the In This Issue feature, p. 890
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- 2023
54. Probing the missing mature β-cell proteomic landscape in differentiating patient iPSC-derived cells
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Heidrun Vethe, Yngvild Bjørlykke, Luiza M. Ghila, Joao A. Paulo, Hanne Scholz, Steven P. Gygi, Simona Chera, and Helge Ræder
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Medicine ,Science - Abstract
Abstract MODY1 is a maturity-onset monogenic diabetes, caused by heterozygous mutations of the HNF4A gene. To date the cellular and molecular mechanisms leading to disease onset remain largely unknown. In this study, we demonstrate that insulin-positive cells can be generated in vitro from human induced pluripotent stem cells (hiPSCs) derived from patients carrying a non-sense HNF4A mutation, proving for the first time, that a human HNF4A mutation is neither blocking the expression of the insulin genes nor the development of insulin-producing cells in vitro. However, regardless of the mutation or diabetes status, these insulin-producing cells are immature, a common downfall off most current β-cell differentiation protocols. To further address the immature state of the cells, in vitro differentiated cells and adult human islets were compared by global proteomic analysis. We report the predicted upstream regulators and signalling pathways characterizing the proteome landscape of each entity. Subsequently, we focused on the molecular components absent or misregulated in the in vitro differentiated cells, to probe the components involved in the deficient in vitro maturation towards fully functional β-cells. This analysis identified the modulation of key developmental signalling pathways representing potential targets for improving the efficiency of the current differentiation protocols.
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- 2017
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55. Compensatory metabolic networks in pancreatic cancers upon perturbation of glutamine metabolism
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Douglas E. Biancur, Joao A. Paulo, Beata Małachowska, Maria Quiles Del Rey, Cristovão M. Sousa, Xiaoxu Wang, Albert S. W. Sohn, Gerald C. Chu, Steven P. Gygi, J. Wade Harper, Wojciech Fendler, Joseph D. Mancias, and Alec C. Kimmelman
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Science - Abstract
Glutaminase inhibition (GLSi) has promising activity against certain cancers. Here, the authors show that GLSi has no effect on multiple mouse models of pancreatic cancer and characterize the metabolic pathways activated in response to GLSi whose concomitant inhibition may have therapeutic utility.
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- 2017
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56. Proteomic and Metabolomic Characterization of a Mammalian Cellular Transition from Quiescence to Proliferation
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Ho-Joon Lee, Mark P. Jedrychowski, Arunachalam Vinayagam, Ning Wu, Ng Shyh-Chang, Yanhui Hu, Chua Min-Wen, Jodene K. Moore, John M. Asara, Costas A. Lyssiotis, Norbert Perrimon, Steven P. Gygi, Lewis C. Cantley, and Marc W. Kirschner
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TMT proteomics ,metabolomics ,protein complexes ,IL-3 ,B cells ,quiescence ,cell growth ,cell cycle ,cancer metabolism ,methionine ,Biology (General) ,QH301-705.5 - Abstract
There exist similarities and differences in metabolism and physiology between normal proliferative cells and tumor cells. Once a cell enters the cell cycle, metabolic machinery is engaged to facilitate various processes. The kinetics and regulation of these metabolic changes have not been properly evaluated. To correlate the orchestration of these processes with the cell cycle, we analyzed the transition from quiescence to proliferation of a non-malignant murine pro-B lymphocyte cell line in response to IL-3. Using multiplex mass-spectrometry-based proteomics, we show that the transition to proliferation shares features generally attributed to cancer cells: upregulation of glycolysis, lipid metabolism, amino-acid synthesis, and nucleotide synthesis and downregulation of oxidative phosphorylation and the urea cycle. Furthermore, metabolomic profiling of this transition reveals similarities to cancer-related metabolic pathways. In particular, we find that methionine is consumed at a higher rate than that of other essential amino acids, with a potential link to maintenance of the epigenome.
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- 2017
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57. Aminoglycoside-driven biosynthesis of selenium-deficient Selenoprotein P
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Kostja Renko, Janine Martitz, Sandra Hybsier, Bjoern Heynisch, Linn Voss, Robert A. Everley, Steven P. Gygi, Mette Stoedter, Monika Wisniewska, Josef Köhrle, Vadim N. Gladyshev, and Lutz Schomburg
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Medicine ,Science - Abstract
Abstract Selenoprotein biosynthesis relies on the co-translational insertion of selenocysteine in response to UGA codons. Aminoglycoside antibiotics interfere with ribosomal function and may cause codon misreading. We hypothesized that biosynthesis of the selenium (Se) transporter selenoprotein P (SELENOP) is particularly sensitive to antibiotics due to its ten in frame UGA codons. As liver regulates Se metabolism, we tested the aminoglycosides G418 and gentamicin in hepatoma cell lines (HepG2, Hep3B and Hepa1-6) and in experimental mice. In vitro, SELENOP levels increased strongly in response to G418, whereas expression of the glutathione peroxidases GPX1 and GPX2 was marginally affected. Se content of G418-induced SELENOP was dependent on Se availability, and was completely suppressed by G418 under Se-poor conditions. Selenocysteine residues were replaced mainly by cysteine, tryptophan and arginine in a codon-specific manner. Interestingly, in young healthy mice, antibiotic treatment failed to affect Selenop biosynthesis to a detectable degree. These findings suggest that the interfering activity of aminoglycosides on selenoprotein biosynthesis can be severe, but depend on the Se status, and other parameters likely including age and general health. Focused analyses with aminoglycoside-treated patients are needed next to evaluate a possible interference of selenoprotein biosynthesis by the antibiotics and elucidate potential side effects.
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- 2017
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58. Evidence that C9ORF72 Dipeptide Repeat Proteins Associate with U2 snRNP to Cause Mis-splicing in ALS/FTD Patients
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Shanye Yin, Rodrigo Lopez-Gonzalez, Ryan C. Kunz, Jaya Gangopadhyay, Carl Borufka, Steven P. Gygi, Fen-Biao Gao, and Robin Reed
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pre-mRNA splicing ,U2 snRNP ,ALS ,FTD ,C9ORF72 ,toxic polydipeptide repeats ,DPRs ,poly-GR ,poly-PR ,iPSC-derived motor neurons ,Biology (General) ,QH301-705.5 - Abstract
Hexanucleotide repeat expansion in the C9ORF72 gene results in production of dipeptide repeat (DPR) proteins that may disrupt pre-mRNA splicing in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients. At present, the mechanisms underlying this mis-splicing are not understood. Here, we show that addition of proline-arginine (PR) and glycine-arginine (GR) toxic DPR peptides to nuclear extracts blocks spliceosome assembly and splicing, but not other types of RNA processing. Proteomic and biochemical analyses identified the U2 small nuclear ribonucleoprotein particle (snRNP) as a major interactor of PR and GR peptides. In addition, U2 snRNP, but not other splicing factors, mislocalizes from the nucleus to the cytoplasm both in C9ORF72 patient induced pluripotent stem cell (iPSC)-derived motor neurons and in HeLa cells treated with the toxic peptides. Bioinformatic studies support a specific role for U2-snRNP-dependent mis-splicing in C9ORF72 patient brains. Together, our data indicate that DPR-mediated dysfunction of U2 snRNP could account for as much as ∼44% of the mis-spliced cassette exons in C9ORF72 patient brains.
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- 2017
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59. A Temporal Proteomic Map of Epstein-Barr Virus Lytic Replication in B Cells
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Ina Ersing, Luis Nobre, Liang Wei Wang, Lior Soday, Yijie Ma, Joao A. Paulo, Yohei Narita, Camille W. Ashbaugh, Chang Jiang, Nicholas E. Grayson, Elliott Kieff, Steven P. Gygi, Michael P. Weekes, and Benjamin E. Gewurz
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Epstein-Barr virus ,herpesvirus ,lytic replication ,quantitative proteomics ,tandem mass tag ,host-pathogen interaction ,immune evasion ,B cell receptor ,complement ,viral evasion ,Biology (General) ,QH301-705.5 - Abstract
Epstein-Barr virus (EBV) replication contributes to multiple human diseases, including infectious mononucleosis, nasopharyngeal carcinoma, B cell lymphomas, and oral hairy leukoplakia. We performed systematic quantitative analyses of temporal changes in host and EBV proteins during lytic replication to gain insights into virus-host interactions, using conditional Burkitt lymphoma models of type I and II EBV infection. We quantified profiles of >8,000 cellular and 69 EBV proteins, including >500 plasma membrane proteins, providing temporal views of the lytic B cell proteome and EBV virome. Our approach revealed EBV-induced remodeling of cell cycle, innate and adaptive immune pathways, including upregulation of the complement cascade and proteasomal degradation of the B cell receptor complex, conserved between EBV types I and II. Cross-comparison with proteomic analyses of human cytomegalovirus infection and of a Kaposi-sarcoma-associated herpesvirus immunoevasin identified host factors targeted by multiple herpesviruses. Our results provide an important resource for studies of EBV replication.
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- 2017
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60. A peroxisomal ubiquitin ligase complex forms a retrotranslocation channel
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Peiqiang Feng, Xudong Wu, Satchal K. Erramilli, Joao A. Paulo, Pawel Knejski, Steven P. Gygi, Anthony A. Kossiakoff, and Tom A. Rapoport
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Multidisciplinary - Abstract
Peroxisomes are ubiquitous organelles that house various metabolic reactions and are essential for human health1–4. Luminal peroxisomal proteins are imported from the cytosol by mobile receptors, which then recycle back to the cytosol by a poorly understood process1–4. Recycling requires receptor modification by a membrane-embedded ubiquitin ligase complex comprising three RING finger domain-containing proteins (Pex2, Pex10 and Pex12)5,6. Here we report a cryo-electron microscopy structure of the ligase complex, which together with biochemical and in vivo experiments reveals its function as a retrotranslocation channel for peroxisomal import receptors. Each subunit of the complex contributes five transmembrane segments that co-assemble into an open channel. The three ring finger domains form a cytosolic tower, with ring finger 2 (RF2) positioned above the channel pore. We propose that the N terminus of a recycling receptor is inserted from the peroxisomal lumen into the pore and monoubiquitylated by RF2 to enable extraction into the cytosol. If recycling is compromised, receptors are polyubiquitylated by the concerted action of RF10 and RF12 and degraded. This polyubiquitylation pathway also maintains the homeostasis of other peroxisomal import factors. Our results clarify a crucial step during peroxisomal protein import and reveal why mutations in the ligase complex cause human disease.
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- 2022
61. Profiling Yeast Deletion Strains Using Sample Multiplexing and Network-Based Analyses
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Xinyue Liu, Jiaming Li, Steven P. Gygi, and Joao A. Paulo
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Fungal Proteins ,Saccharomyces cerevisiae Proteins ,Proteome ,Saccharomyces cerevisiae ,General Chemistry ,Biochemistry ,Article ,Gene Deletion - Abstract
The yeast, Saccharomyces cerevisiae, is a widely used model system for investigating conserved biological functions and pathways. Advancements in sample multiplexing have facilitated the study of the yeast proteome, yet many yeast proteins remain uncharacterized or only partially characterized. Yeast deletion strain collections are powerful resources for yeast proteome studies, uncovering the effects of gene function, genetic interactions, and cellular stresses. As complex biological systems cannot be understood by simply analyzing the individual components, a systems approach is often required in which a protein is represented as a component of large, interacting networks. Here, we evaluate the current state of yeast proteome analysis using isobaric tag-based sample multiplexing (TMTpro16) to profile the proteomes of 75 yeast deletion strains for which we measured the abundance of nearly 5000 proteins. Using statistical approaches, we highlighted covariance and regulation subnetworks and the enrichment of gene ontology classifications for covarying and coregulated proteins. This dataset presents a resource that is amenable to further data mining to study individual deletion strains, pathways, proteins, and/or interactions thereof while serving as a template for future network-based investigations using yeast deletion strain collections.
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- 2022
62. CDC7-independent G1/S transition revealed by targeted protein degradation
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Jan M. Suski, Nalin Ratnayeke, Marcin Braun, Tian Zhang, Vladislav Strmiska, Wojciech Michowski, Geylani Can, Antoine Simoneau, Konrad Snioch, Mikolaj Cup, Caitlin M. Sullivan, Xiaoji Wu, Joanna Nowacka, Timothy B. Branigan, Lindsey R. Pack, James A. DeCaprio, Yan Geng, Lee Zou, Steven P. Gygi, Johannes C. Walter, Tobias Meyer, and Piotr Sicinski
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DNA Replication ,Mice ,Multidisciplinary ,Proteolysis ,G1 Phase ,Animals ,Cell Cycle Proteins ,Protein Serine-Threonine Kinases ,Article ,S Phase - Abstract
Entry of mammalian cells into DNA synthesis (S-phase) represents a key event in cell division(1). According to the current cell-cycle models, the Cdc7 kinase constitutes an essential and rate-limiting trigger of DNA replication, acting together with cyclin-dependent kinase Cdk2. Here we show, using chemical-genetic systems which allow an acute shutdown of Cdc7 in in vitro cultured cells as well as in vivo in a living mouse, that Cdc7 is dispensable for cell division of many different cell types. We demonstrate that another cell-cycle kinase, Cdk1, is also active during G1/S transition both in cycling cells and in cells exiting quiescence. We show that Cdc7 and Cdk1 play functionally redundant roles during G1/S transition, and at least one of these kinases must be present to allow S-phase entry. These observations revise our understanding of cell-cycle progression by demonstrating that Cdk1 physiologically regulates two distinct transitions during cell division cycle, while Cdc7 plays a redundant function in DNA replication.
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- 2022
63. Author Correction: Sas-4 provides a scaffold for cytoplasmic complexes and tethers them in a centrosome
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Jayachandran Gopalakrishnan, Vito Mennella, Stephanie Blachon, Bo Zhai, Andrew H. Smith, Timothy L. Megraw, Daniela Nicastro, Steven P. Gygi, David A. Agard, and Tomer Avidor-Reiss
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Science - Abstract
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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- 2020
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64. Phosphorylation of FANCD2 Inhibits the FANCD2/FANCI Complex and Suppresses the Fanconi Anemia Pathway in the Absence of DNA Damage
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David Lopez-Martinez, Marian Kupculak, Di Yang, Yasunaga Yoshikawa, Chih-Chao Liang, Ronghu Wu, Steven P. Gygi, and Martin A. Cohn
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Biology (General) ,QH301-705.5 - Abstract
Summary: Interstrand crosslinks (ICLs) of the DNA helix are a deleterious form of DNA damage. ICLs can be repaired by the Fanconi anemia pathway. At the center of the pathway is the FANCD2/FANCI complex, recruitment of which to DNA is a critical step for repair. After recruitment, monoubiquitination of both FANCD2 and FANCI leads to their retention on chromatin, ensuring subsequent repair. However, regulation of recruitment is poorly understood. Here, we report a cluster of phosphosites on FANCD2 whose phosphorylation by CK2 inhibits both FANCD2 recruitment to ICLs and its monoubiquitination in vitro and in vivo. We have found that phosphorylated FANCD2 possesses reduced DNA binding activity, explaining the previous observations. Thus, we describe a regulatory mechanism operating as a molecular switch, where in the absence of DNA damage, the FANCD2/FANCI complex is prevented from loading onto DNA, effectively suppressing the FA pathway. : Lopez-Martinez et al. describe a regulatory switch in the Fanconi anemia pathway. Phosphorylation of FANCD2 reduces the DNA binding activity of the FANCD2/FANCI complex, thereby preventing spurious activation of the pathway in the absence of DNA damage. Keywords: Fanconi anemia, interstrand crosslink repair, ICL repair, FANCD2/FANCI, genome stability, DNA repair, CK2, casein kinase 2, phosphorylation, kinase
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- 2019
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65. Identification of UHRF2 as a novel DNA interstrand crosslink sensor protein.
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Anna Motnenko, Chih-Chao Liang, Di Yang, David Lopez-Martinez, Yasunaga Yoshikawa, Bao Zhan, Katherine E Ward, Jiayang Tian, Wilhelm Haas, Paolo Spingardi, Benedikt M Kessler, Skirmantas Kriaucionis, Steven P Gygi, and Martin A Cohn
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Genetics ,QH426-470 - Abstract
The Fanconi Anemia (FA) pathway is important for repairing interstrand crosslinks (ICLs) between the Watson-Crick strands of the DNA double helix. An initial and essential stage in the repair process is the detection of the ICL. Here, we report the identification of UHRF2, a paralogue of UHRF1, as an ICL sensor protein. UHRF2 is recruited to ICLs in the genome within seconds of their appearance. We show that UHRF2 cooperates with UHRF1, to ensure recruitment of FANCD2 to ICLs. A direct protein-protein interaction is formed between UHRF1 and UHRF2, and between either UHRF1 and UHRF2, and FANCD2. Importantly, we demonstrate that the essential monoubiquitination of FANCD2 is stimulated by UHRF1/UHRF2. The stimulation is mediating by a retention of FANCD2 on chromatin, allowing for its monoubiquitination by the FA core complex. Taken together, we uncover a mechanism of ICL sensing by UHRF2, leading to FANCD2 recruitment and retention at ICLs, in turn facilitating activation of FANCD2 by monoubiquitination.
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- 2018
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66. Proteostasis and lysosomal quality control deficits in Alzheimer’s disease neurons
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Ching-Chieh Chou, Ryan Vest, Miguel A. Prado, Joshua Wilson-Grady, Joao A. Paulo, Yohei Shibuya, Patricia Moran-Losada, Ting-Ting Lee, Jian Luo, Steven P. Gygi, Jeffery W. Kelly, Daniel Finley, Marius Wernig, Tony Wyss-Coray, and Judith Frydman
- Subjects
Article - Abstract
SummaryThe role of proteostasis and organelle homeostasis dysfunction in human aging and Alzheimer’s disease (AD) remains unclear. Analyzing proteome-wide changes in human donor fibroblasts and their corresponding transdifferentiated neurons (tNeurons), we find aging and AD synergistically impair multiple proteostasis pathways, most notably lysosomal quality control (LQC). In particular, we show that ESCRT-mediated lysosomal repair defects are associated with both sporadic and PSEN1 familial AD. Aging- and AD-linked defects are detected in fibroblasts but highly exacerbated in tNeurons, leading to enhanced neuronal vulnerability, unrepaired lysosomal damage, inflammatory factor secretion and cytotoxicity. Surprisingly, tNeurons from aged and AD donors spontaneously develop amyloid-β inclusions co-localizing with LQC markers, LAMP1/2-positive lysosomes and proteostasis factors; we observe similar inclusions in brain tissue from AD patients and APP-transgenic mice. Importantly, compounds enhancing lysosomal function broadly ameliorate these AD-associated pathologies. Our findings establish cell-autonomous LQC dysfunction in neurons as a central vulnerability in aging and AD pathogenesis.
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- 2023
67. Genetic dissection of the pluripotent proteome through multi-omics data integration
- Author
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Selcan Aydin, Duy T. Pham, Tian Zhang, Gregory R. Keele, Daniel A. Skelly, Joao A. Paulo, Matthew Pankratz, Ted Choi, Steven P. Gygi, Laura G. Reinholdt, Christopher L. Baker, Gary A. Churchill, and Steven C. Munger
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Genetics ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Article - Abstract
Genetic background drives phenotypic variability in pluripotent stem cells (PSCs). Most studies to date have used transcript abundance as the primary molecular readout of cell state in PSCs. We performed a comprehensive proteogenomics analysis of 190 genetically diverse mouse embryonic stem cell (mESC) lines. The quantitative proteome is highly variable across lines, and we identified pluripotency-associated pathways that were differentially activated in the proteomics data that were not evident in transcriptome data from the same lines. Integration of protein abundance to transcript levels and chromatin accessibility revealed broad co-variation across molecular layers as well as shared and unique drivers of quantitative variation in pluripotency-associated pathways. Quantitative trait locus (QTL) mapping localized the drivers of these multi-omic signatures to genomic hotspots. This study reveals post-transcriptional mechanisms and genetic interactions that underlie quantitative variability in the pluripotent proteome and provides a regulatory map for mESCs that can provide a basis for future mechanistic studies.
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- 2023
68. Author Reply to Peer Reviews of UBXN1 maintains ER proteostasis and represses UPR activation by modulating translation independently of the p97 ATPase
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Malavika Raman, Brittany Ann Ahlstedt, Rakesh Ganji, Sirisha Mukkavalli, Joao Paulo, and Steven P Gygi
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- 2023
69. Frontotemporal Dementia Patient Neurons With Progranulin Deficiency Display Protein Dyshomeostasis
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Lisa Elia, Bianca Herting, Amela Alijagic, Christina Buselli, Leela Wong, Grace Morrison, Miguel A. Prado, Joao A. Paulo, Steven P. Gygi, Daniel Finley, and Steven Finkbeiner
- Subjects
Article - Abstract
Haploinsufficiency of progranulin (PGRN) causes frontotemporal dementia (FTD), a devastating neurodegenerative disease with no effective treatment. PGRN is required for efficient proteostasis, as loss of neuronal PGRN results in dysfunctional lysosomes and impaired clearance and cytoplasmic aggregation of TDP-43, a protein involved in neurodegeneration in FTD. These and other events lead to neurodegeneration and neuroinflammation. However, the detailed mechanisms leading to protein dyshomeostasis in PGRN-deficient cells remain unclear. We report here the development of human cell models of FTD with PGRN-deficiency to explore the molecular mechanisms underlying proteostasis breakdown and TDP-43 aggregation in FTD. Neurons differentiated from FTD patient induced pluripotent stem cells (iPSCs) have reduced PGRN levels, and the neurons recapitulate key disease features, including impaired lysosomal function, defective TDP-43 turnover and accumulation, neurodegeneration, and death. Proteomic analysis revealed altered levels of proteins linked to the autophagy-lysosome pathway (ALP) and the ubiquitin-proteasome system (UPS) in FTD patient neurons, providing new mechanistic insights into the link between PGRN-deficiency and disease pathobiology.
- Published
- 2023
70. RBM43 links adipose inflammation and energy expenditure through translational regulation of PGC1α
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Phillip A. Dumesic, Sarah E. Wilensky, Symanthika Bose, Jonathan G. Van Vranken, Steven P. Gygi, and Bruce M. Spiegelman
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Article - Abstract
Adipose thermogenesis involves specialized mitochondrial function that counteracts metabolic disease through dissipation of chemical energy as heat. However, inflammation present in obese adipose tissue can impair oxidative metabolism. Here, we show that PGC1α, a key governor of mitochondrial biogenesis and thermogenesis, is negatively regulated at the level of mRNA translation by the little-known RNA-binding protein RBM43.Rbm43is expressed selectively in white adipose depots that have low thermogenic potential, and is induced by inflammatory cytokines. RBM43 suppresses mitochondrial and thermogenic gene expression in a PGC1α-dependent manner and its loss protects cells from cytokine-induced mitochondrial impairment. In mice, adipocyte-selectiveRbm43disruption increases PGC1α translation, resulting in mitochondrial biogenesis and adipose thermogenesis. These changes are accompanied by improvements in glucose homeostasis during diet-induced obesity that are independent of body weight. The action of RBM43 suggests a translational mechanism by which inflammatory signals associated with metabolic disease dampen mitochondrial function and thermogenesis.
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- 2023
71. A proteome-wide atlas of drug mechanism of action
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Dylan C. Mitchell, Miljan Kuljanin, Jiaming Li, Jonathan G. Van Vranken, Nathan Bulloch, Devin K. Schweppe, Edward L. Huttlin, and Steven P. Gygi
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Biomedical Engineering ,Molecular Medicine ,Bioengineering ,Applied Microbiology and Biotechnology ,Biotechnology - Published
- 2023
72. Rixosomal RNA degradation contributes to silencing of Polycomb target genes
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Haining Zhou, Chad B. Stein, Tiasha A. Shafiq, Gergana Shipkovenska, Marian Kalocsay, Joao A. Paulo, Jiuchun Zhang, Zhenhua Luo, Steven P. Gygi, Karen Adelman, and Danesh Moazed
- Subjects
Polycomb Repressive Complex 1 ,Multidisciplinary ,Heterochromatin ,RNA Stability ,Exoribonucleases ,Schizosaccharomyces ,Polycomb Repressive Complex 2 ,Humans ,Polycomb-Group Proteins ,Gene Silencing ,macromolecular substances - Abstract
Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) are histone-modifying and -binding complexes that mediate the formation of facultative heterochromatin and are required for silencing of developmental genes and maintenance of cell fate1–3. Multiple pathways of RNA decay work together to establish and maintain heterochromatin in fission yeast, including a recently identified role for a conserved RNA-degradation complex known as the rixosome or RIX1 complex4–6. Whether RNA degradation also has a role in the stability of mammalian heterochromatin remains unknown. Here we show that the rixosome contributes to silencing of many Polycomb targets in human cells. The rixosome associates with human PRC complexes and is enriched at promoters of Polycomb target genes. Depletion of either the rixosome or Polycomb results in accumulation of paused and elongating RNA polymerase at Polycomb target genes. We identify point mutations in the RING1B subunit of PRC1 that disrupt the interaction between PRC1 and the rixosome and result in diminished silencing, suggesting that direct recruitment of the rixosome to chromatin is required for silencing. Finally, we show that the RNA endonuclease and kinase activities of the rixosome and the downstream XRN2 exoribonuclease, which degrades RNAs with 5′ monophosphate groups generated by the rixosome, are required for silencing. Our findings suggest that rixosomal degradation of nascent RNA is conserved from fission yeast to human, with a primary role in RNA degradation at facultative heterochromatin in human cells.
- Published
- 2022
73. Adiporedoxin, an upstream regulator of ER oxidative folding and protein secretion in adipocytes
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Mark P. Jedrychowski, Libin Liu, Collette J. Laflamme, Kalypso Karastergiou, Tova Meshulam, Shi-Ying Ding, Yuanyuan Wu, Mi-Jeong Lee, Steven P. Gygi, Susan K. Fried, and Paul F. Pilch
- Subjects
Adipocyte ,Adipokine ,Protein secretion ,Endoplasmic reticulum ,Oxidoreductase ,Disulfide bond formation ,Internal medicine ,RC31-1245 - Abstract
Objective: Adipocytes are robust protein secretors, most notably of adipokines, hormone-like polypeptides, which act in an endocrine and paracrine fashion to affect numerous physiological processes such as energy balance and insulin sensitivity. To understand how such proteins are assembled for secretion we describe the function of a novel endoplasmic reticulum oxidoreductase, adiporedoxin (Adrx). Methods: Adrx knockdown and overexpressing 3T3-L1 murine adipocyte cell lines and a knockout mouse model were used to assess the influence of Adrx on secreted proteins as well as the redox state of ER resident chaperones. The metabolic phenotypes of Adrx null mice were characterized and compared to WT mice. The correlation of Adrx levels BMI, adiponectin levels, and other inflammatory markers from adipose tissue of human subjects was also studied. Results: Adiporedoxin functions via a CXXC active site, and is upstream of protein disulfide isomerase whose direct function is disulfide bond formation, and ultimately protein secretion. Over and under expression of Adrx in vitro enhances and reduces, respectively, the secretion of the disulfide-bonded proteins including adiponectin and collagen isoforms. On a chow diet, Adrx null mice have normal body weights, and glucose tolerance, are moderately hyperinsulinemic, have reduced levels of circulating adiponectin and are virtually free of adipocyte fibrosis resulting in a complex phenotype tending towards insulin resistance. Adrx protein levels in human adipose tissue correlate positively with adiponectin levels and negatively with the inflammatory marker phospho-Jun kinase. Conclusion: These data support the notion that Adrx plays a critical role in adipocyte biology and in the regulation of mouse and human metabolism via its modulation of adipocyte protein secretion.
- Published
- 2015
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74. Clonal populations of a human TNBC model display significant functional heterogeneity and divergent growth dynamics in distinct contexts
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Timour Baslan, Ron C. J. Schackmann, Maurizio Callari, G. Kenneth Gray, Laura M. Selfors, Chandler M Friend, Tian Zhang, Hendrik J. Kuiken, Carlos Caldas, Frank Stegmeier, Jett Crowdis, Steven P. Gygi, Joan S. Brugge, Hyo-eun C. Bhang, and Sabin Dhakal
- Subjects
Cancer Research ,Copy number analysis ,RNA ,Triple Negative Breast Neoplasms ,Biology ,Article ,In vitro ,law.invention ,Genetic Heterogeneity ,Mice ,In vivo ,Tumor progression ,Interferon ,law ,Mutation ,Proteome ,Tumor Microenvironment ,Genetics ,Cancer research ,medicine ,Animals ,Humans ,Suppressor ,Molecular Biology ,medicine.drug - Abstract
Intratumoral heterogeneity has been described for various tumor types and models of human cancer, and can have profound effects on tumor progression and drug resistance. This study describes an in-depth analysis of molecular and functional heterogeneity among subclonal populations (SCPs) derived from a single triple-negative breast cancer cell line, including copy number analysis, whole-exome and RNA sequencing, proteome analysis, and barcode analysis of clonal dynamics, as well as functional assays. The SCPs were found to have multiple unique genetic alterations and displayed significant variation in anchorage independent growth and tumor forming ability. Analyses of clonal dynamics in SCP mixtures using DNA barcode technology revealed selection for distinct clonal populations in different in vitro and in vivo environmental contexts, demonstrating that in vitro propagation of cancer cell lines using different culture conditions can contribute to the establishment of unique strains. These analyses also revealed strong enrichment of a single SCP during the development of xenograft tumors in immune-compromised mice. This SCP displayed attenuated interferon signaling in vivo and reduced sensitivity to the antiproliferative effects of type I interferons. Reduction in interferon signaling was found to provide a selective advantage within the xenograft microenvironment specifically. In concordance with the previously described role of interferon signaling as tumor suppressor, these findings suggest that similar selective pressures may be operative in human cancer and patient-derived xenograft models.
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- 2021
75. Liver RBFOX2 regulates cholesterol homeostasis via Scarb1 alternative splicing in mice
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Helen A. B. Paterson, Sijia Yu, Natalia Artigas, Miguel A. Prado, Nejc Haberman, Yi-Fang Wang, Andrew M. Jobbins, Elena Pahita, Joao Mokochinski, Zoe Hall, Maryse Guerin, Joao A. Paulo, Soon Seng Ng, Francesc Villarroya, Sheikh Tamir Rashid, Wilfried Le Goff, Boris Lenhard, Inês Cebola, Daniel Finley, Steven P. Gygi, Christopher R. Sibley, and Santiago Vernia
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Scarb1 (SR-BI/II) ,pre-mRNA alternative splicing ,Endocrinology, Diabetes and Metabolism ,RNA-Binding Proteins ,MAFLD ,cholesterol ,Cell Biology ,Scavenger Receptors, Class B ,Mice ,Alternative Splicing ,Cholesterol ,splice-switching oligonucleotide (SSO) ,Liver ,Physiology (medical) ,Internal Medicine ,Animals ,Protein Isoforms ,RNA ,Homeostasis ,RNA Splicing Factors ,triglyceride ,RBFOX2 - Abstract
RNA alternative splicing (AS) expands the regulatory potential of eukaryotic genomes. The mechanisms regulating liver-specific AS profiles and their contribution to liver function are poorly understood. Here, we identify a key role for the splicing factor RNA-binding Fox protein 2 (RBFOX2) in maintaining cholesterol homeostasis in a lipogenic environment in the liver. Using enhanced individual-nucleotide-resolution ultra-violet cross-linking and immunoprecipitation, we identify physiologically relevant targets of RBFOX2 in mouse liver, including the scavenger receptor class B type I (Scarb1). RBFOX2 function is decreased in the liver in diet-induced obesity, causing a Scarb1 isoform switch and alteration of hepatocyte lipid homeostasis. Our findings demonstrate that specific AS programmes actively maintain liver physiology, and underlie the lipotoxic effects of obesogenic diets when dysregulated. Splice-switching oligonucleotides targeting this network alleviate obesity-induced inflammation in the liver and promote an anti-atherogenic lipoprotein profile in the blood, underscoring the potential of isoform-specific RNA therapeutics for treating metabolism-associated diseases.
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- 2022
76. Phosphate Starvation Signaling Increases Mitochondrial Membrane Potential through Respiration-independent Mechanisms
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Yeyun Ouyang, Corey N. Cunningham, Jordan A. Berg, Ashish G. Toshniwal, Casey E. Hughes, Jonathan G. Van Vranken, Mi-Young Jeong, Ahmad A. Cluntun, Geanette Lam, Jacob M. Winter, Emel Akdoǧan, Katja K. Dove, Steven P. Gygi, Cory D Dunn, Dennis R Winge, and Jared Rutter
- Abstract
Mitochondrial membrane potential directly powers many critical functions of mitochondria, including ATP production, mitochondrial protein import, and metabolite transport. Its loss is a cardinal feature of aging and mitochondrial diseases, and cells closely monitor membrane potential as an indicator of mitochondrial health. Given its central importance, it is logical that cells would modulate mitochondrial membrane potential in response to demand and environmental cues, but there has been little exploration of this question. We report that loss of the Sit4 protein phosphatase in yeast increases mitochondrial membrane potential, both through inducing the electron transport chain and the phosphate starvation response. Indeed, a similarly elevated mitochondrial membrane potential is also elicited simply by phosphate starvation or by abrogation of the Pho85-dependent phosphate sensing pathway. This enhanced membrane potential is primarily driven by an unexpected activity of the ADP/ATP carrier. We also demonstrate that this connection between phosphate limitation and enhancement of the mitochondrial membrane potential is also observed in primary and immortalized mammalian cells as well as inDrosophila. These data suggest that mitochondrial membrane potential is subject to environmental stimuli and intracellular signaling regulation and raise the possibility for therapeutic enhancement of mitochondrial functions even with defective mitochondria.
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- 2022
77. Membrane curvature-generating proteins crucial for autophagosome formation
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Ning Wang, Yoko Shibata, Joao A. Paulo, Steven P. Gygi, and Tom A. Rapoport
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Autophagy is essential for cellular homeostasis and begins with the formation of a phagophore, a cup-like membrane sheet consisting of two closely apposed lipid bilayers connected by a highly curved rim. How the membrane sheet forms, bends, and eventually generates an autophagosome that enwraps cargo remains enigmatic. Specifically, it is unclear how the high membrane curvature of the phagophore rim, an energetically unfavorable state, is stabilized. Here, we demonstrate that phagophore formation requires the conserved, membrane curvature-generating REEP1 proteins. The REEP1 family proteins (REEP1-4 in vertebrates) differ from the related endoplasmic reticulum-shaping REEPs in abundance and membrane topology. In fission yeast, the single REEP1 ortholog is involved in both bulk and selective autophagy. It is recruited at early stages of phagophore formation and required for their maturation into autophagosomes. The function of REEP1 relies on its ability to generate high membrane curvature and its localization to the phagophore rim. Mammalian REEP1 proteins also associate with phagophores upon induction of autophagy and colocalize with early autophagic markers. We propose that REEP1 proteins stabilize the phagophore’s highly curved rim so that the two membrane sheets are kept in close proximity to form the autophagosome. Defective autophagy may underlie the effect of curvature-compromising mutations in human REEP1 proteins linked to neurological disease.
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- 2022
78. Phosphorylation of RXRα mediates the effect of JNK to suppress hepatic FGF21 expression and promote metabolic syndrome
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Santiago Vernia, Alexandra Lee, Norman J. Kennedy, Myoung Sook Han, Marta Isasa, Julie Cavanagh-Kyros, Armanda Roy, Aafreen Syed, Shanzah Chaudhry, Yvonne J. K. Edwards, Steven P. Gygi, Guangping Gao, and Roger J. Davis
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Fibroblast Growth Factors ,Metabolic Syndrome ,Mice, Knockout ,Mice ,Multidisciplinary ,Retinoid X Receptor alpha ,Liver ,MAP Kinase Kinase 4 ,Hepatocytes ,Animals ,PPAR alpha ,Phosphorylation ,Carrier Proteins - Abstract
The cJun NH2-terminal kinase (JNK) signaling pathway in the liver promotes systemic changes in metabolism by regulating peroxisome proliferator-activated receptor α (PPARα)-dependent expression of the hepatokine fibroblast growth factor 21 (FGF21). Hepatocyte-specific gene ablation studies demonstrated that theMapk9gene (encoding JNK2) plays a key mechanistic role. Mutually exclusive inclusion of exons 7a and 7b yields expression of the isoforms JNK2α and JNK2β. Here we demonstrate thatFgf21gene expression and metabolic regulation are primarily regulated by the JNK2α isoform. To identify relevant substrates of JNK2α, we performed a quantitative phosphoproteomic study of livers isolated from control mice, mice with JNK deficiency in hepatocytes, and mice that express only JNK2α or JNK2β in hepatocytes. We identified the JNK substrate retinoid X receptor α (RXRα) as a protein that exhibited JNK2α-promoted phosphorylation in vivo. RXRα functions as a heterodimeric partner of PPARα and may therefore mediate the effects of JNK2α signaling onFgf21expression. To test this hypothesis, we established mice with hepatocyte-specific expression of wild-type or mutated RXRα proteins. We found that the RXRα phosphorylation site Ser260was required for suppression ofFgf21gene expression. Collectively, these data establish a JNK-mediated signaling pathway that regulates hepaticFgf21expression.
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- 2022
79. A multi-purpose, regenerable, proteome-scale, human phosphoserine resource for phosphoproteomics
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Brandon M. Gassaway, Jiaming Li, Ramin Rad, Julian Mintseris, Kyle Mohler, Tyler Levy, Mike Aguiar, Sean A. Beausoleil, Joao A. Paulo, Jesse Rinehart, Edward L. Huttlin, and Steven P. Gygi
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Phosphopeptides ,Proteomics ,Phosphoserine ,Proteome ,Humans ,Cell Biology ,Phosphorylation ,Molecular Biology ,Biochemistry ,Article ,Mass Spectrometry ,Biotechnology - Abstract
Mass-spectrometry-based phosphoproteomics has become indispensable for understanding cellular signaling in complex biological systems. Despite the central role of protein phosphorylation, the field still lacks inexpensive, regenerable, and diverse phosphopeptides with ground-truth phosphorylation positions. Here, we present Iterative Synthetically Phosphorylated Isomers (iSPI), a proteome-scale library of human-derived phosphoserine-containing phosphopeptides that is inexpensive, regenerable, and diverse, with precisely known positions of phosphorylation. We demonstrate possible uses of iSPI, including use as a phosphopeptide standard, a tool to evaluate and optimize phosphorylation-site localization algorithms, and a benchmark to compare performance across data analysis pipelines. We also present AScorePro, an updated version of the AScore algorithm specifically optimized for phosphorylation-site localization in higher energy fragmentation spectra, and the FLR viewer, a web tool for phosphorylation-site localization, to enable community use of the iSPI resource. iSPI and its associated data constitute a useful, multi-purpose resource for the phosphoproteomics community.
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- 2022
80. APC/CCdc20-mediated degradation of Clb4 prompts astral microtubule stabilization at anaphase onset
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Federico Zucca, Clara Visintin, Jiaming Li, Steven P. Gygi, and Rosella Visintin
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Cdc20 Proteins ,Cyclins ,Animals ,Spindle Apparatus ,Cell Biology ,Cyclin B ,Anaphase ,Microtubules ,Anaphase-Promoting Complex-Cyclosome - Abstract
Key for accurate chromosome partitioning to the offspring is the ability of mitotic spindle microtubules to respond to different molecular signals and remodel their dynamics accordingly. Spindle microtubules are conventionally divided into three classes: kinetochore, interpolar, and astral microtubules (kMTs, iMTs, and aMTs, respectively). Among all, aMT regulation remains elusive. Here, we show that aMT dynamics are tightly regulated. aMTs remain unstable up to metaphase and are stabilized at anaphase onset. This switch in aMT dynamics, important for proper spindle orientation, specifically requires the degradation of the mitotic cyclin Clb4 by the Anaphase Promoting Complex bound to its activator subunit Cdc20 (APC/CCdc20). These data highlight a unique role for mitotic cyclin Clb4 in controlling aMT regulating factors, of which Kip2 is a prime candidate, provide a framework to understand aMT regulation in vertebrates, and uncover mechanistic principles of how the APC/CCdc20 choreographs the timing of late mitotic events by sequentially impacting on the three classes of spindle microtubules.
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- 2022
81. Regulation of protein abundance in genetically diverse mouse populations
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Gary A. Churchill, Steven C. Munger, Tian Zhang, Steven P. Gygi, Matthew Vincent, Ginger D. Shaw, Duy Pham, Gregory R. Keele, Fernando Pardo-Manuel de Villena, Timothy A. Bell, Joao A. Paulo, Martin T. Ferris, and Pablo Hock
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Inbred strain ,Evolutionary biology ,Abundance (ecology) ,Genetic variation ,Proteome ,Heritability ,Quantitative trait locus ,Biology ,Proteomics ,Phenotype - Abstract
Summary Genetically diverse mouse populations are powerful tools for characterizing the regulation of the proteome and its relationship to whole-organism phenotypes. We used mass spectrometry to profile and quantify the abundance of 6,798 proteins in liver tissue from mice of both sexes across 58 Collaborative Cross (CC) inbred strains. We previously collected liver proteomics data from the related Diversity Outbred (DO) mice and their founder strains. We show concordance across the proteomics datasets despite being generated from separate experiments, allowing comparative analysis. We map protein abundance quantitative trait loci (pQTLs), identifying 1,087 local and 285 distal in the CC mice and 1,706 local and 414 distal in the DO mice. We find that regulatory effects on individual proteins are conserved across the mouse populations, in particular for local genetic variation and sex differences. In comparison, proteins that form complexes are often co-regulated, displaying varying genetic architectures, and overall show lower heritability and map fewer pQTLs. We have made this resource publicly available to enable quantitative analyses of the regulation of the proteome.
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- 2022
82. Proteomic Profiling of Extracellular Vesicles Derived from Cerebrospinal Fluid of Alzheimer’s Disease Patients: A Pilot Study
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Satoshi Muraoka, Mark P. Jedrychowski, Kiran Yanamandra, Seiko Ikezu, Steven P. Gygi, and Tsuneya Ikezu
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Alzheimer’s disease ,biomarker ,cerebrospinal fluid ,extracellular vesicles ,proteome ,Cytology ,QH573-671 - Abstract
Pathological hallmarks of Alzheimer’s disease (AD) are deposits of amyloid beta (Aβ) and hyper-phosphorylated tau aggregates in brain plaques. Recent studies have highlighted the importance of Aβ and tau-containing extracellular vesicles (EVs) in AD. We therefore examined EVs separated from cerebrospinal fluid (CSF) of AD, mild cognitive impairment (MCI), and control (CTRL) patient samples to profile the protein composition of CSF EV. EV fractions were separated from AD (n = 13), MCI (n = 10), and CTRL (n = 10) CSF samples using MagCapture Exosome Isolation kit. The CSF-derived EV proteins were identified and quantified by label-free and tandem mass tag (TMT)-labeled mass spectrometry. Label-free proteomics analysis identified 2546 proteins that were significantly enriched for extracellular exosome ontology by Gene Ontology analysis. Canonical Pathway Analysis revealed glia-related signaling. Quantitative proteomics analysis, moreover, showed that EVs expressed 1284 unique proteins in AD, MCI and CTRL groups. Statistical analysis identified three proteins—HSPA1A, NPEPPS, and PTGFRN—involved in AD progression. In addition, the PTGFRN showed a moderate correlation with amyloid plaque (rho = 0.404, p = 0.027) and tangle scores (rho = 0.500, p = 0.005) in AD, MCI and CTRL. Based on the CSF EV proteomics, these data indicate that three proteins, HSPA1A, NPEPPS and PTGFRN, may be used to monitor the progression of MCI to AD.
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- 2020
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83. Apolipoprotein E is a pancreatic extracellular factor that maintains mature β-cell gene expression.
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Ahmed I Mahmoud, Francisco X Galdos, Katherine A Dinan, Mark P Jedrychowski, Jeffrey C Davis, Ana Vujic, Inbal Rachmin, Christian Shigley, James R Pancoast, Samuel Lee, Jennifer Hollister-Lock, Catherine M MacGillivray, Steven P Gygi, Douglas A Melton, Gordon C Weir, and Richard T Lee
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Medicine ,Science - Abstract
The in vivo microenvironment of tissues provides myriad unique signals to cells. Thus, following isolation, many cell types change in culture, often preserving some but not all of their in vivo characteristics in culture. At least some of the in vivo microenvironment may be mimicked by providing specific cues to cultured cells. Here, we show that after isolation and during maintenance in culture, adherent rat islets reduce expression of key β-cell transcription factors necessary for β-cell function and that soluble pancreatic decellularized matrix (DCM) can enhance β-cell gene expression. Following chromatographic fractionation of pancreatic DCM, we performed proteomics to identify soluble factors that can maintain β-cell stability and function. We identified Apolipoprotein E (ApoE) as an extracellular protein that significantly increased the expression of key β-cell genes. The ApoE effect on beta cells was mediated at least in part through the JAK/STAT signaling pathway. Together, these results reveal a role for ApoE as an extracellular factor that can maintain the mature β-cell gene expression profile.
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- 2018
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84. Filamin C Cardiomyopathy Variants Cause Protein and Lysosome Accumulation
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Joshua M. Gorham, Christine E. Seidman, Christopher S. Chen, Jonathan G. Seidman, Subramanian Sundaram, Christopher N. Toepfer, Radhika Agarwal, Jourdan K. Ewoldt, Steven P. Gygi, Joao A. Paulo, Steven R. DePalma, Qi Zhang, and Anant Chopra
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Sarcomeres ,Physiology ,Filamins ,Induced Pluripotent Stem Cells ,Autophagy ,Biology ,Filamin ,Myocardial Contraction ,Sarcomere ,Article ,Protein–protein interaction ,Cell biology ,medicine.anatomical_structure ,Lysosome ,medicine ,Humans ,Myocytes, Cardiac ,FLNC ,Sarcomere organization ,Cardiomyopathies ,Lysosomes ,Cardiology and Cardiovascular Medicine ,Haploinsufficiency ,Cells, Cultured - Abstract
Rationale: Dominant heterozygous variants in filamin C ( FLNC ) cause diverse cardiomyopathies, although the underlying molecular mechanisms remain poorly understood. Objective: We aimed to define the molecular mechanisms by which FLNC variants altered human cardiomyocyte gene and protein expression, sarcomere structure, and contractile performance. Methods and Results: Using CRISPR/Cas9, we introduced FLNC variants into human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs). We compared isogenic hiPSC-CMs with normal (wild-type), ablated expression ( FLNC −/− ), or haploinsufficiency ( FLNC +/− ) that causes dilated cardiomyopathy. We also studied a heterozygous in-frame deletion ( FLNC +/Δ7aa ) which did not affect FLNC expression but caused aggregate formation, similar to FLNC variants associated with hypertrophic cardiomyopathy. FLNC −/− hiPSC-CMs demonstrated profound sarcomere misassembly and reduced contractility. Although sarcomere formation and function were unaffected in FLNC +/ − and FLNC +/Δ7aa hiPSC-CMs, these heterozygous variants caused increases in lysosome content, enhancement of autophagic flux, and accumulation of FLNC-binding partners and Z-disc proteins. Conclusions: FLNC expression is required for sarcomere organization and physiological function. Variants that produce misfolded FLNC proteins cause the accumulation of FLNC and FLNC-binding partners which leads to increased lysosome expression and activation of autophagic pathways. Surprisingly, similar pathways were activated in FLNC haploinsufficient hiPSC-CMs, likely initiated by the loss of stoichiometric FLNC protein interactions and impaired turnover of proteins at the Z-disc. These results indicate that both FLNC haploinsufficient variants and variants that produce misfolded FLNC protein cause disease by similar proteotoxic mechanisms and indicate the therapeutic potential for augmenting protein degradative pathways to treat a wide range of FLNC -related cardiomyopathies.
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- 2021
85. The Proteasome Distinguishes between Heterotypic and Homotypic Lysine-11-Linked Polyubiquitin Chains
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Guinevere L. Grice, Ian T. Lobb, Michael P. Weekes, Steven P. Gygi, Robin Antrobus, and James A. Nathan
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Biology (General) ,QH301-705.5 - Abstract
Proteasome-mediated degradation occurs with proteins principally modified with lysine-48 polyubiquitin chains. Whether the proteasome also can bind atypical ubiquitin chains, including those linked by lysine-11, has not been well established. This is critically important, as lysine-11 polyubiquitination has been implicated in both proteasome-mediated degradation and non-degradative outcomes. Here we demonstrate that pure homotypic lysine-11-linked chains do not bind strongly to the mammalian proteasome. By contrast, heterotypic polyubiquitin chains, containing lysine-11 and lysine-48 linkages, not only bind to the proteasome but also stimulate the proteasomal degradation of the cell-cycle regulator cyclin B1. Thus, while heterotypic lysine-11-linked chains facilitate proteasomal degradation, homotypic lysine-11 linkages adopt conformations that prevent association with the proteasome. Our data demonstrate the capacity of the proteasome to bind ubiquitin chains of distinct topology, with implications for the recognition and diverse biological functions of mixed ubiquitin chains.
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- 2015
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86. A Critical Role for PKR Complexes with TRBP in Immunometabolic Regulation and eIF2α Phosphorylation in Obesity
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Takahisa Nakamura, Ryan C. Kunz, Cai Zhang, Taishi Kimura, Celvie L. Yuan, Brenna Baccaro, Yuka Namiki, Steven P. Gygi, and Gökhan S. Hotamisligil
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Biology (General) ,QH301-705.5 - Abstract
Aberrant stress and inflammatory responses are key factors in the pathogenesis of obesity and metabolic dysfunction, and the double-stranded RNA-dependent kinase (PKR) has been proposed to play an important role in integrating these pathways. Here, we report the formation of a complex between PKR and TAR RNA-binding protein (TRBP) during metabolic and obesity-induced stress, which is critical for the regulation of eukaryotic translation initiation factor 2 alpha (eIF2α) phosphorylation and c-Jun N-terminal kinase (JNK) activation. We show that TRBP phosphorylation is induced in the setting of metabolic stress, leading to PKR activation. Suppression of hepatic TRBP reduced inflammation, JNK activity, and eIF2α phosphorylation and improved systemic insulin resistance and glucose metabolism, while TRBP overexpression exacerbated the impairment in glucose homeostasis in obese mice. These data indicate that the association between PKR and TRBP integrates metabolism with translational control and inflammatory signaling and plays important roles in metabolic homeostasis and disease.
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- 2015
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87. UHRF1 Is a Sensor for DNA Interstrand Crosslinks and Recruits FANCD2 to Initiate the Fanconi Anemia Pathway
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Chih-Chao Liang, Bao Zhan, Yasunaga Yoshikawa, Wilhelm Haas, Steven P. Gygi, and Martin A. Cohn
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Biology (General) ,QH301-705.5 - Abstract
The Fanconi anemia (FA) pathway is critical for the cellular response to toxic DNA interstrand crosslinks (ICLs). Using a biochemical purification strategy, we identified UHRF1 as a protein that specifically interacts with ICLs in vitro and in vivo. Reduction of cellular levels of UHRF1 by RNAi attenuates the FA pathway and sensitizes cells to mitomycin C. Knockdown cells display a drastic reduction in FANCD2 foci formation. Using live-cell imaging, we observe that UHRF1 is rapidly recruited to chromatin in response to DNA crosslinking agents and that this recruitment both precedes and is required for the recruitment of FANCD2 to ICLs. Based on these results, we describe a mechanism of ICL sensing and propose that UHRF1 is a critical factor that binds to ICLs. In turn, this binding is necessary for the subsequent recruitment of FANCD2, which allows the DNA repair process to initiate.
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- 2015
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88. Global proteome metastability response in isogenic animals to missense mutations and polyglutamine expansions in aging
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Xiaojing Sui, Miguel A. Prado, Joao A. Paulo, Steven P. Gygi, Daniel Finley, and Richard I. Morimoto
- Abstract
The conformational stability of the proteome has tremendous implications for the health of the cell and its capacity to determine longevity or susceptibility to age-associated degenerative diseases. For humans, this question of proteome conformational stability has the additional complexity from non-synonymous mutations in thousands of protein coding genes challenging the capacity of the proteostasis network to properly fold, transport, assemble and degrade proteins. Here, we quantify the proteome-wide capacity to such challenges using the isogenic organism Caenorhabditis elegans by examining the dynamics of global proteome conformational stability in animals expressing different temperature-sensitive (ts) proteins or short polyglutamine (polyQ) expansions in the context of biological aging. Using limited proteolysis of native extracts together with tandem mass tag-based quantitative proteomics, we identify proteins that become metastable under these conditions and monitor the effects on proteome solubility and abundance. Expression of different mutant proteins in the same tissue identifies hundreds to a thousand proteins that become metastable affecting multiple compartments and processes in a cell autonomous and non-autonomous manner. Comparison of the network of metastable proteins, however, reveals only a small number of common proteins. The most dramatic effects on global proteome dynamics occur in aging with one-third of the proteome undergoing conformational changes in early adulthood. These age-dependent metastable proteins overlap substantially with ts proteins and polyQ; moreover, expression of polyQ accelerates the aging phenotype. Together, these results reveal that the proteome responds to misfolding one-at-a-time to generate a metastable sub-proteome network with features of a fingerprint for which aging is the dominant determinant of proteome metastability.
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- 2022
89. The ancestral ESCRT protein TOM1L2 selects ubiquitinated cargoes for retrieval from cilia
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Swapnil Rohidas Shinde, David U. Mick, Erika Aoki, Rachel B. Rodrigues, Steven P. Gygi, and Maxence V. Nachury
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Cell Biology ,Molecular Biology ,General Biochemistry, Genetics and Molecular Biology ,Developmental Biology - Abstract
Many G protein-coupled receptors (GPCRs) reside within cilia of mammalian cells and must undergo regulated exit from cilia for the appropriate transduction of signals such as Hedgehog morphogens. Lysine 63-linked ubiquitin (K63Ub) chains mark GPCRs for regulated removal from cilia, but the molecular basis of K63Ub recognition inside cilia remains elusive. Here we show that the BBSome –the trafficking complex in charge of retrieving GPCRs from cilia– engages the ancestral endosomal sorting factor TOM1L2 (Target of Myb1-Like 2) to recognize UbK63 chains within cilia. TOM1L2 directly binds to UbK63 chains and to the BBSome and targeted disruption of the TOM1L2/BBSome interaction results in the accumulation of TOM1L2, ubiquitin and the GPCRs SSTR3, Smoothened and GPR161 inside cilia. Strikingly, the single cell algaChlamydomonasalso requires its TOM1L2 orthologue to clear ubiquitinated proteins from cilia. We conclude that TOM1L2 broadly enables the retrieval of UbK63-tagged proteins by the ciliary trafficking machinery.
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- 2022
90. Mapping the GALNT1 substrate landscape with versatile proteomics tools
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Amir Ata Saei, Susanna L. Lundström, Hezheng Lyu, Hassan Gharibi, Weiqi Lu, Pan Fang, Xuepei Zhang, Zhaowei Meng, Jijing Wang, Massimiliano Gaetani, Ákos Végvári, Steven P. Gygi, and Roman A. Zubarev
- Abstract
O-GalNAc type glycosylation is a common post-translational modification (PTM) of proteins catalyzed by polypeptide GalNAc transferases, but the substrate specificity of these transferases is poorly understood. Here we develop a strategy based on integral thermal proteome solubility profiling to identify and prioritize the protein substrates of polypeptide N-acetylgalactosaminyltransferase 1 (GALNT1). Combined with glycoprotein enrichment followed by HCD and soft EThcD gas-phase fragmentation technique, we uncover hundreds of novel GALNT1 substrates in two model human cell lines. GALNT1-mediated O-glycosylation is more common on Thr than Ser residues, with a strong preference for Pro at positions +3 and +4 in respect to O-glycosylation. These results implicate GALNT1 in potentially regulating proteins in several diverse pathways, including some unexpected processes, such as TCA cycle and DNA transcription. This study depicts a roadmap for identification of functional substrates for glycosyltransferases, facilitating fundamental insight into the role of glycosylation in homeostasis and disease.
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- 2022
91. The Sestrins Interact with GATOR2 to Negatively Regulate the Amino-Acid-Sensing Pathway Upstream of mTORC1
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Lynne Chantranupong, Rachel L. Wolfson, Jose M. Orozco, Robert A. Saxton, Sonia M. Scaria, Liron Bar-Peled, Eric Spooner, Marta Isasa, Steven P. Gygi, and David M. Sabatini
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Biology (General) ,QH301-705.5 - Abstract
Summary: The mechanistic target of rapamycin complex 1 (mTORC1) kinase is a major regulator of cell growth that responds to numerous environmental cues. A key input is amino acids, which act through the heterodimeric Rag GTPases (RagA or RagB bound to RagC or RagD) in order to promote the translocation of mTORC1 to the lysosomal surface, its site of activation. GATOR2 is a complex of unknown function that positively regulates mTORC1 signaling by acting upstream of or in parallel to GATOR1, which is a GTPase-activating protein (GAP) for RagA or RagB and an inhibitor of the amino-acid-sensing pathway. Here, we find that the Sestrins, a family of poorly understood growth regulators (Sestrin1–Sestrin3), interact with GATOR2 in an amino-acid-sensitive fashion. Sestrin2-mediated inhibition of mTORC1 signaling requires GATOR1 and the Rag GTPases, and the Sestrins regulate the localization of mTORC1 in response to amino acids. Thus, we identify the Sestrins as GATOR2-interacting proteins that regulate the amino-acid-sensing branch of the mTORC1 pathway. : The mTORC1 kinase is a major growth regulator that responds to numerous environmental inputs, including amino acids. Chantranupong et al. now identify the Sestrins as components of the mTORC1 amino-acid-sensing pathway. The Sestrins interact with GATOR2 in an amino-acid-sensitive manner and function as negative regulators of the pathway by preventing proper mTORC1 localization to the lysosome in response to amino acids.
- Published
- 2014
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92. Abstract 3485: Intrinsically disordered regions of the ARID1A/B tumor suppressors encode an interaction network within biomolecular condensates that directs mSWI/SNF chromatin remodeler complex activity
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Ajinkya Patil, Amy R. Strom, Clayton K. Collings, Joao A. Paulo, Tobias Wauer, Akshay Sankar, Jessica D. St.Laurent, Kasey S. Cervantes, Steven P. Gygi, Clifford P. Brangwynne, and Cigall Kadoch
- Subjects
Cancer Research ,Oncology - Abstract
The mammalian SWI/SNF (mSWI/SNF or BAF) ATP-dependent chromatin remodeling complexes collectively represent one of the most frequently mutated cellular entities in cancer, second only to TP53. Mutations across the 29 human genes that encode mSWI/SNF complex subunits occur in over 20% of human cancers, with mutations affecting ARID1A and ARID1B, the largest BAF subunits, being the most frequent. However, the functional contributions of these subunits, particularly their commonly mutated N-terminal intrinsically disordered regions (IDRs) and a highly conserved ARID DNA-binding domain, to BAF function remain poorly understood. Here, we demonstrate that the IDRs of ARID1A/B, coupled with the ARID domain, drive biomolecular condensate formation and BAF chromatin localization in cells. We define ARID1A/B IDRs as two-part systems, facilitating homotypic BAF complex interactions (i.e., valence generated by localized condensation) and heterotypic complex interactions, that together establish a highly specific, sequence-encoded protein interaction network within condensates. Both types of interactions are required for appropriate genome-wide targeting of BAF complexes, DNA accessibility generation, and appropriate gene expression. Replacement of the ARID1A N-terminal IDR with IDRs derived from two unrelated proteins FUS and DDX4, rescues generic condensation of BAF but not chromatin occupancy, DNA accessibility, and heterotypic interactions, highlighting the sequence-specificity embedded in the IDR of ARID1A. Taken together, these data establish a role for the largest and most frequently perturbed IDRs within a major chromatin remodeler and explain how biomolecular condensate formation enables both genomic localization and functional partner recruitment. Furthermore, these findings lay the groundwork for mapping IDR sequence specificity or “grammar”, that dictates the co-condensation network formation, and suggests that targeted disruption of these mechanisms may represent new targeted therapeutic opportunities across multiple cancers. Citation Format: Ajinkya Patil, Amy R. Strom, Clayton K. Collings, Joao A. Paulo, Tobias Wauer, Akshay Sankar, Jessica D. St.Laurent, Kasey S. Cervantes, Steven P. Gygi, Clifford P. Brangwynne, Cigall Kadoch. Intrinsically disordered regions of the ARID1A/B tumor suppressors encode an interaction network within biomolecular condensates that directs mSWI/SNF chromatin remodeler complex activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3485.
- Published
- 2023
93. UCP1 governs liver extracellular succinate and inflammatory pathogenesis
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Hannah Prendeville, Cathal Harmon, Nika N. Danial, Anita Reddy, Edward T. Chouchani, Accalia Fu, Nhien V. Tran, Ryan Garrity, Lydia Lynch, Steven P. Gygi, Evanna L. Mills, John Szpyt, Haopeng Xiao, Mark P. Jedrychowski, and Gary A. Bradshaw
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medicine.medical_specialty ,Adipose Tissue, White ,Endocrinology, Diabetes and Metabolism ,Succinic Acid ,Adipose tissue ,Inflammation ,Biology ,Article ,Hepatitis ,Receptors, G-Protein-Coupled ,Pathogenesis ,Mice ,Non-alcoholic Fatty Liver Disease ,Stress, Physiological ,Physiology (medical) ,Internal medicine ,Glucose Intolerance ,Internal Medicine ,Succinate receptor 1 ,medicine ,Humans ,Animals ,Uncoupling Protein 1 ,Fatty liver ,Cell Biology ,Adipose Tissue, Beige ,medicine.disease ,Fibrosis ,Thermogenin ,Glucose ,Endocrinology ,Hepatic stellate cell ,Disease Susceptibility ,medicine.symptom ,Extracellular Space ,Thermogenesis ,Metabolic Networks and Pathways ,Signal Transduction - Abstract
Non-alcoholic fatty liver disease (NAFLD), the most prevalent liver pathology worldwide, is intimately linked with obesity and type 2 diabetes. Liver inflammation is a hallmark of NAFLD and is thought to contribute to tissue fibrosis and disease pathogenesis. Uncoupling protein 1 (UCP1) is exclusively expressed in brown and beige adipocytes, and has been extensively studied for its capacity to elevate thermogenesis and reverse obesity. Here we identify an endocrine pathway regulated by UCP1 that antagonizes liver inflammation and pathology, independent of effects on obesity. We show that, without UCP1, brown and beige fat exhibit a diminished capacity to clear succinate from the circulation. Moreover, UCP1KO mice exhibit elevated extracellular succinate in liver tissue that drives inflammation through ligation of its cognate receptor succinate receptor 1 (SUCNR1) in liver-resident stellate cell and macrophage populations. Conversely, increasing brown and beige adipocyte content in mice antagonizes SUCNR1-dependent inflammatory signalling in the liver. We show that this UCP1-succinate–SUCNR1 axis is necessary to regulate liver immune cell infiltration and pathology, and systemic glucose intolerance in an obesogenic environment. As such, the therapeutic use of brown and beige adipocytes and UCP1 extends beyond thermogenesis and may be leveraged to antagonize NAFLD and SUCNR1-dependent liver inflammation. UCP1 is exclusively expressed in brown and beige adipocytes, where it drives thermogenesis through futile substrate cycling. Mills et al. identify a endocrine pathway mediated by the UCP1 catabolic circuit that antagonizes liver inflammation by lowering the concentration of succinate in the liver extracellular fluid.
- Published
- 2021
94. Mapping Angiotensin II Type 1 Receptor-Biased Signaling Using Proximity Labeling and Proteomics Identifies Diverse Actions of Biased Agonists
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Steven P. Gygi, Howard A. Rockman, Joao A. Paulo, Jialu Wang, Conrad T Pfeiffer, and Xue Jiang
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Proteomics ,0301 basic medicine ,Agonist ,Cell signaling ,030102 biochemistry & molecular biology ,Chemistry ,medicine.drug_class ,Signal transducing adaptor protein ,General Chemistry ,Ligands ,Biochemistry ,Angiotensin II ,Partial agonist ,Article ,Receptor, Angiotensin, Type 1 ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,Functional selectivity ,medicine ,Receptor ,beta-Arrestins ,Signal Transduction - Abstract
Angiotensin II type 1 receptors (AT1Rs) are one of the most widely studied G-protein-coupled receptors. To fully appreciate the diversity in cellular signaling profiles activated by AT1R transducer-biased ligands, we utilized peroxidase-catalyzed proximity labeling to capture proteins in close proximity to AT1Rs in response to six different ligands: angiotensin II (full agonist), S1I8 (partial agonist), TRV055 and TRV056 (G-protein-biased agonists), and TRV026 and TRV027 (β-arrestin-biased agonists) at 90 s, 10 min, and 60 min after stimulation (ProteomeXchange Identifier PXD023814). We systematically analyzed the kinetics of AT1R trafficking and determined that distinct ligands lead AT1R to different cellular compartments for downstream signaling activation and receptor degradation/recycling. Distinct proximity labeling of proteins from a number of functional classes, including GTPases, adaptor proteins, and kinases, was activated by different ligands suggesting unique signaling and physiological roles of the AT1R. Ligands within the same class, that is, either G-protein-biased or β-arrestin-biased, shared high similarity in their labeling profiles. A comparison between ligand classes revealed distinct signaling activation such as greater labeling by G-protein-biased ligands on ESCRT-0 complex proteins that act as the sorting machinery for ubiquitinated proteins. Our study provides a comprehensive analysis of AT1R receptor-trafficking kinetics and signaling activation profiles induced by distinct classes of ligands.
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- 2021
95. A human-airway-on-a-chip for the rapid identification of candidate antiviral therapeutics and prophylactics
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Crystal Yuri Oh, Ilona Golynker, Sarah E. Gilpin, Haiqing Bai, Rachelle Prantil-Baun, Amanda Jiang, Danni Y. Zhu, Steven P. Gygi, Mercy Soong, Melissa Rodas, Marisa McGrath, Longlong Si, Robert Haupt, Seongmin Kim, James Logue, Daniel Blanco-Melo, Tristan X. Jordan, Randy A. Albrecht, Rasmus Møller, Tian Zhang, Donald E. Ingber, Kohei Oishi, Justin J. Frere, Kambez H. Benam, Daisy A. Hoagland, Girija Goyal, Kenneth E. Carlson, Wen-Chun Liu, Rani K. Powers, Shu Horiuchi, Benjamin E. Nilsson-Payant, Skyler Uhl, Roberto Plebani, Matthew B. Frieman, Benjamin R. tenOever, Stuart Weston, Atiq Nurani, and Wuji Cao
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Male ,0301 basic medicine ,Drug ,Oseltamivir ,media_common.quotation_subject ,medicine.medical_treatment ,Green Fluorescent Proteins ,Biomedical Engineering ,Medicine (miscellaneous) ,Bioengineering ,Amodiaquine ,Antiviral Agents ,Article ,Cell Line ,03 medical and health sciences ,chemistry.chemical_compound ,COVID-19 Testing ,0302 clinical medicine ,Immune system ,Cricetinae ,Lab-On-A-Chip Devices ,medicine ,Animals ,Humans ,media_common ,SARS-CoV-2 ,business.industry ,COVID-19 ,Hydroxychloroquine ,Virus Internalization ,respiratory system ,Virology ,COVID-19 Drug Treatment ,respiratory tract diseases ,Computer Science Applications ,Nafamostat ,030104 developmental biology ,Cytokine ,chemistry ,Cell culture ,Female ,business ,030217 neurology & neurosurgery ,Biotechnology ,medicine.drug - Abstract
The rapid repurposing of antivirals is particularly pressing during pandemics. However, rapid assays for assessing candidate drugs typically involve in vitro screens and cell lines that do not recapitulate human physiology at the tissue and organ levels. Here, we show that a microfluidic bronchial-airway-on-a-chip lined by highly differentiated human bronchial-airway epithelium and pulmonary endothelium can model viral infection, strain-dependent virulence, cytokine production, and the recruitment of circulating immune cells. In airway chips infected with influenza A, the co-administration of nafamostat with oseltamivir doubled the treatment-time window for oseltamivir. In chips infected with pseudotyped SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), clinically relevant doses of the antimalarial drug amodiaquine inhibited infection, but clinical doses of hydroxychloroquine and other antiviral drugs that inhibit the entry of pseudotyped SARS-CoV-2 in cell lines under static conditions did not. We also show that amodiaquine showed substantial prophylactic and therapeutic activities in hamsters challenged with native SARS-CoV-2. The human airway-on-a-chip may accelerate the identification of therapeutics and prophylactics with repurposing potential., One-sentence editorial summary: A microfluidic bronchial-airway-on-a-chip lined by human bronchial-airway epithelium and pulmonary endothelium can be used to rapidly identify antiviral therapeutics and prophylactics with repurposing potential.
- Published
- 2021
96. TMTpro-18plex: The Expanded and Complete Set of TMTpro Reagents for Sample Multiplexing
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Ian Pike, Jiaming Li, Thomas M. Roberts, Steven P. Gygi, Karsten Kuhn, John C. Rogers, Joao A. Paulo, Zhenying Cai, and Ryan Bomgarden
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Proteomics ,0301 basic medicine ,Profiling (computer programming) ,Proteome ,030102 biochemistry & molecular biology ,Chemistry ,General Chemistry ,Missing data ,Biochemistry ,Isogenic human disease models ,Multiplexing ,Article ,Cell Line ,Set (abstract data type) ,Data set ,03 medical and health sciences ,030104 developmental biology ,Reagent ,Indicators and Reagents ,Peptides ,Biological system - Abstract
The development of the TMTpro-16plex series expanded the breadth of commercial isobaric tagging reagents by nearly 50% over classic TMT-11plex. In addition to the described 16plex reagents, the proline-based TMTpro molecule can accommodate two additional combinations of heavy carbon and nitrogen isotopes. Here, we introduce the final two labeling reagents, TMTpro-134C and TMTpro-135N, which permit the simultaneous global protein profiling of 18 samples with essentially no missing values. For example, six conditions with three biological replicates can now be perfectly accommodated. We showcase the 18plex reagent set by profiling the proteome and phosphoproteome of a pair of isogenic mammary epithelial cell lines under three conditions in triplicate. We compare the depth and quantitative performance of this data set with a TMTpro-16plex experiment in which two samples were omitted. Our analysis revealed similar numbers of quantified peptides and proteins, with high quantitative correlation. We interrogated further the TMTpro-18plex data set by highlighting changes in protein abundance profiles under different conditions in the isogenic cell lines. We conclude that TMTpro-18plex further expands the sample multiplexing landscape, allowing for complex and innovative experimental designs.
- Published
- 2021
97. Quantitative multiplexed proteomics of Taenia solium cysts obtained from the skeletal muscle and central nervous system of pigs.
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José Navarrete-Perea, Marta Isasa, Joao A Paulo, Ricardo Corral-Corral, Jeanette Flores-Bautista, Beatriz Hernández-Téllez, Raúl J Bobes, Gladis Fragoso, Edda Sciutto, Xavier Soberón, Steven P Gygi, and Juan P Laclette
- Subjects
Arctic medicine. Tropical medicine ,RC955-962 ,Public aspects of medicine ,RA1-1270 - Abstract
In human and porcine cysticercosis caused by the tapeworm Taenia solium, the larval stage (cysts) can infest several tissues including the central nervous system (CNS) and the skeletal muscles (SM). The cyst's proteomics changes associated with the tissue localization in the host tissues have been poorly studied. Quantitative multiplexed proteomics has the power to evaluate global proteome changes in response to different conditions. Here, using a TMT-multiplexed strategy we identified and quantified over 4,200 proteins in cysts obtained from the SM and CNS of pigs, of which 891 were host proteins. To our knowledge, this is the most extensive intermixing of host and parasite proteins reported for tapeworm infections.Several antigens in cysticercosis, i.e., GP50, paramyosin and a calcium-binding protein were enriched in skeletal muscle cysts. Our results suggested the occurrence of tissue-enriched antigen that could be useful in the improvement of the immunodiagnosis for cysticercosis. Using several algorithms for epitope detection, we selected 42 highly antigenic proteins enriched for each tissue localization of the cysts. Taking into account the fold changes and the antigen/epitope contents, we selected 10 proteins and produced synthetic peptides from the best epitopes. Nine peptides were recognized by serum antibodies of cysticercotic pigs, suggesting that those peptides are antigens. Mixtures of peptides derived from SM and CNS cysts yielded better results than mixtures of peptides derived from a single tissue location, however the identification of the 'optimal' tissue-enriched antigens remains to be discovered. Through machine learning technologies, we determined that a reliable immunodiagnostic test for porcine cysticercosis required at least five different antigenic determinants.
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- 2017
- Full Text
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98. Kinesin superfamily protein Kif26b links Wnt5a-Ror signaling to the control of cell and tissue behaviors in vertebrates
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Michael W Susman, Edith P Karuna, Ryan C Kunz, Taranjit S Gujral, Andrea V Cantú, Shannon S Choi, Brigette Y Jong, Kyoko Okada, Michael K Scales, Jennie Hum, Linda S Hu, Marc W Kirschner, Ryuichi Nishinakamura, Soichiro Yamada, Diana J Laird, Li-En Jao, Steven P Gygi, Michael E Greenberg, and Hsin-Yi Henry Ho
- Subjects
noncanonical Wnt signaling ,Ror ,signal transduction ,tissue morphogenesis ,regulated proteolysis ,Kif26b ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
Wnt5a-Ror signaling constitutes a developmental pathway crucial for embryonic tissue morphogenesis, reproduction and adult tissue regeneration, yet the molecular mechanisms by which the Wnt5a-Ror pathway mediates these processes are largely unknown. Using a proteomic screen, we identify the kinesin superfamily protein Kif26b as a downstream target of the Wnt5a-Ror pathway. Wnt5a-Ror, through a process independent of the canonical Wnt/β-catenin-dependent pathway, regulates the cellular stability of Kif26b by inducing its degradation via the ubiquitin-proteasome system. Through this mechanism, Kif26b modulates the migratory behavior of cultured mesenchymal cells in a Wnt5a-dependent manner. Genetic perturbation of Kif26b function in vivo caused embryonic axis malformations and depletion of primordial germ cells in the developing gonad, two phenotypes characteristic of disrupted Wnt5a-Ror signaling. These findings indicate that Kif26b links Wnt5a-Ror signaling to the control of morphogenetic cell and tissue behaviors in vertebrates and reveal a new role for regulated proteolysis in noncanonical Wnt5a-Ror signal transduction.
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- 2017
- Full Text
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99. Ubiquilin1 promotes antigen-receptor mediated proliferation by eliminating mislocalized mitochondrial proteins
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Alexandra M Whiteley, Miguel A Prado, Ivan Peng, Alexander R Abbas, Benjamin Haley, Joao A Paulo, Mike Reichelt, Anand Katakam, Meredith Sagolla, Zora Modrusan, Dong Yun Lee, Merone Roose-Girma, Donald S Kirkpatrick, Brent S McKenzie, Steven P Gygi, Daniel Finley, and Eric J Brown
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B cell ,mitochondria ,ubiquilin ,cell cycle ,protein synthesis ,BCR ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
Ubiquilins (Ubqlns) are a family of ubiquitin receptors that promote the delivery of hydrophobic and aggregated ubiquitinated proteins to the proteasome for degradation. We carried out a proteomic analysis of a B cell lymphoma-derived cell line, BJAB, that requires UBQLN1 for survival to identify UBQLN1 client proteins. When UBQLN1 expression was acutely inhibited, 120 mitochondrial proteins were enriched in the cytoplasm, suggesting that the accumulation of mitochondrial client proteins in the absence of UBQLN1 is cytostatic. Using a Ubqln1−/− mouse strain, we found that B cell receptor (BCR) ligation of Ubqln1−/− B cells led to a defect in cell cycle entry. As in BJAB cells, mitochondrial proteins accumulated in BCR-stimulated cells, leading to protein synthesis inhibition and cell cycle block. Thus, UBQLN1 plays an important role in clearing mislocalized mitochondrial proteins upon cell stimulation, and its absence leads to suppression of protein synthesis and cell cycle arrest.
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- 2017
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100. Scaffold-mediated gating of Cdc42 signalling flux
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Péter Rapali, Romain Mitteau, Craig Braun, Aurèlie Massoni-Laporte, Caner Ünlü, Laure Bataille, Floriane Saint Arramon, Steven P Gygi, and Derek McCusker
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cell polarity ,Rho GTPase ,signaling ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
Scaffold proteins modulate signalling pathway activity spatially and temporally. In budding yeast, the scaffold Bem1 contributes to polarity axis establishment by regulating the GTPase Cdc42. Although different models have been proposed for Bem1 function, there is little direct evidence for an underlying mechanism. Here, we find that Bem1 directly augments the guanine exchange factor (GEF) activity of Cdc24. Bem1 also increases GEF phosphorylation by the p21-activated kinase (PAK), Cla4. Phosphorylation abrogates the scaffold-dependent stimulation of GEF activity, rendering Cdc24 insensitive to additional Bem1. Thus, Bem1 stimulates GEF activity in a reversible fashion, contributing to signalling flux through Cdc42. The contribution of Bem1 to GTPase dynamics was borne-out by in vivo imaging: active Cdc42 was enriched at the cell pole in hypophosphorylated cdc24 mutants, while hyperphosphorylated cdc24 mutants that were resistant to scaffold stimulation displayed a deficit in active Cdc42 at the pole. These findings illustrate the self-regulatory properties that scaffold proteins confer on signalling pathways.
- Published
- 2017
- Full Text
- View/download PDF
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