Publications by authors named "Steven P Gygi"

539 Publications

Proteomic analysis identifies the E3 ubiquitin ligase Pdzrn3 as a regulatory target of Wnt5a-Ror signaling.

Proc Natl Acad Sci U S A 2021 Jun;118(25)

Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis, CA 95616;

Wnt5a-Ror signaling is a conserved pathway that regulates morphogenetic processes during vertebrate development [R. T. Moon , 119, 97-111 (1993); I. Oishi , 8, 645-654 (2003)], but its downstream signaling events remain poorly understood. Through a large-scale proteomic screen in mouse embryonic fibroblasts, we identified the E3 ubiquitin ligase Pdzrn3 as a regulatory target of the Wnt5a-Ror pathway. Upon pathway activation, Pdzrn3 is degraded in a β-catenin-independent, ubiquitin-proteasome system-dependent manner. We developed a flow cytometry-based reporter to monitor Pdzrn3 abundance and delineated a signaling cascade involving Frizzled, Dishevelled, Casein kinase 1, and Glycogen synthase kinase 3 that regulates Pdzrn3 stability. Epistatically, Pdzrn3 is regulated independently of Kif26b, another Wnt5a-Ror effector. Wnt5a-dependent degradation of Pdzrn3 requires phosphorylation of three conserved amino acids within its C-terminal LNX3H domain [M. Flynn, O. Saha, P. Young, 11, 235 (2011)], which acts as a bona fide Wnt5a-responsive element. Importantly, this phospho-dependent degradation is essential for Wnt5a-Ror modulation of cell migration. Collectively, this work establishes a Wnt5a-Ror cell morphogenetic cascade involving Pdzrn3 phosphorylation and degradation.
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http://dx.doi.org/10.1073/pnas.2104944118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8237634PMC
June 2021

A Compendium of Murine (Phospho)Peptides Encompassing Different Isobaric Labeling and Data Acquisition Strategies.

J Proteome Res 2021 Jul 27;20(7):3678-3688. Epub 2021 May 27.

Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, United States.

Targeted mass spectrometry-based assays typically rely on previously acquired large data sets for peptide target selection. Such repositories are widely available for unlabeled peptides. However, they are less common for isobaric tagged peptides. Here we have assembled two series of six data sets originating from a mouse embryonic fibroblast cell line (NIH/3T3). One series is of peptides derived from a tryptic digest of a whole cell proteome and a second from enriched phosphopeptides. These data sets encompass three labeling approaches (unlabeled, TMT11-labeled, and TMTpro16-labeled) and two data acquisition strategies (ion trap MS2 with and without FAIMS-based gas phase separation). We identified a total of 1 509 526 peptide-spectrum matches which covered 11 482 proteins from the whole cell proteome tryptic digest, and 188 849 phosphopeptides from the phosphopeptide enrichment. The data sets were of similar depth, and while overlap across data sets was modest, protein overlap was high, thus reinforcing the comprehensiveness of these data sets. The data also supported FAIMS as a means to increase data set depth. These data sets provide a rich resource of peptides that may be used as starting points for targeted assays. Future data sets may be compiled for any genome-sequenced organism using the technologies and strategies highlighted herein. The data have been deposited in the ProteomeXchange Consortium with data set identifier PXD024298.
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http://dx.doi.org/10.1021/acs.jproteome.1c00247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8254770PMC
July 2021

UCP1 governs liver extracellular succinate and inflammatory pathogenesis.

Nat Metab 2021 05 17;3(5):604-617. Epub 2021 May 17.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA.

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.
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http://dx.doi.org/10.1038/s42255-021-00389-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8207988PMC
May 2021

Dual proteome-scale networks reveal cell-specific remodeling of the human interactome.

Cell 2021 May 6;184(11):3022-3040.e28. Epub 2021 May 6.

Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Thousands of interactions assemble proteins into modules that impart spatial and functional organization to the cellular proteome. Through affinity-purification mass spectrometry, we have created two proteome-scale, cell-line-specific interaction networks. The first, BioPlex 3.0, results from affinity purification of 10,128 human proteins-half the proteome-in 293T cells and includes 118,162 interactions among 14,586 proteins. The second results from 5,522 immunoprecipitations in HCT116 cells. These networks model the interactome whose structure encodes protein function, localization, and complex membership. Comparison across cell lines validates thousands of interactions and reveals extensive customization. Whereas shared interactions reside in core complexes and involve essential proteins, cell-specific interactions link these complexes, "rewiring" subnetworks within each cell's interactome. Interactions covary among proteins of shared function as the proteome remodels to produce each cell's phenotype. Viewable interactively online through BioPlexExplorer, these networks define principles of proteome organization and enable unknown protein characterization.
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http://dx.doi.org/10.1016/j.cell.2021.04.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8165030PMC
May 2021

Mapping Angiotensin II Type 1 Receptor-Biased Signaling Using Proximity Labeling and Proteomics Identifies Diverse Actions of Biased Agonists.

J Proteome Res 2021 06 5;20(6):3256-3267. Epub 2021 May 5.

Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, United States.

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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http://dx.doi.org/10.1021/acs.jproteome.1c00080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8218870PMC
June 2021

A Semiautomated Paramagnetic Bead-Based Platform for Isobaric Tag Sample Preparation.

J Am Soc Mass Spectrom 2021 Jun 5;32(6):1519-1529. Epub 2021 May 5.

Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, United States.

The development of streamlined and high-throughput sample processing workflows is important for capitalizing on emerging advances and innovations in mass spectrometry-based applications. While the adaptation of new technologies and improved methodologies is fast paced, automation of upstream sample processing often lags. Here we have developed and implemented a semiautomated paramagnetic bead-based platform for isobaric tag sample preparation. We benchmarked the robot-assisted platform by comparing the protein abundance profiles of six common parental laboratory yeast strains in triplicate TMTpro16-plex experiments against an identical set of experiments in which the samples were manually processed. Both sets of experiments quantified similar numbers of proteins and peptides with good reproducibility. Using these data, we constructed an interactive website to explore the proteome profiles of six yeast strains. We also provide the community with open-source templates for automating routine proteomics workflows on an opentrons OT-2 liquid handler. The robot-assisted platform offers a versatile and affordable option for reproducible sample processing for a wide range of protein profiling applications.
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http://dx.doi.org/10.1021/jasms.1c00077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8210952PMC
June 2021

A human-airway-on-a-chip for the rapid identification of candidate antiviral therapeutics and prophylactics.

Nat Biomed Eng 2021 May 3. Epub 2021 May 3.

Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA.

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 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-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.
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http://dx.doi.org/10.1038/s41551-021-00718-9DOI Listing
May 2021

TMTpro-18plex: The Expanded and Complete Set of TMTpro Reagents for Sample Multiplexing.

J Proteome Res 2021 05 26;20(5):2964-2972. Epub 2021 Apr 26.

Department of Cell Biology, Harvard Medical School, Boston 02115, Massachusetts, United States.

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.
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http://dx.doi.org/10.1021/acs.jproteome.1c00168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8210943PMC
May 2021

Temporal proteomic changes induced by nicotine in human cells: A quantitative proteomics approach.

J Proteomics 2021 06 23;241:104244. Epub 2021 Apr 23.

Department of Cell Biology, Harvard Medical School, Boston, MA, USA. Electronic address:

Nicotine is a prominent active compound in tobacco and many smoking cessation products. Some of the biological effects of nicotine are well documented in in vitro and in vivo systems; however, data are scarce concerning the time-dependent changes on protein and phosphorylation events in response to nicotine. Here, we profiled the proteomes of SH-SY5Y and A549 cell lines subjected to acute (15 min, 1 h and 4 h) or chronic (24 h, 48 h) nicotine exposures. We used sample multiplexing (TMTpro16) and quantified more than 9000 proteins and over 7000 phosphorylation events per cell line. Among our findings, we determined a decrease in mitochondrial protein abundance for SH-SY5Y, while we detected alterations in several immune pathways, such as the complement system, for A549 following nicotine treatment. We also explored the proposed association between smoking (specifically nicotine) and SARS-CoV2. Here, we found several host proteins known to interact with viral proteins that were affected by nicotine in a time dependent manner. This dataset can be mined further to investigate the potential role of nicotine in different biological contexts. SIGNIFICANCE: Smoking is a major public health issue that is associated with several serious chronic, yet preventable diseases, including stroke, heart disease, type 2 diabetes, cancer, and susceptibility to infection. Tobacco smoke is a complex mixture of thousands of different compounds, among which nicotine is the main addictive compound. The biological effects of nicotine have been reported in several models, however very little data are available concerning the temporal proteomic and phosphoproteomic changes in response to nicotine. Here, we provide a dataset exploring the potential role of nicotine on different biological processes over time, including implications in the study of SARS-CoV2.
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http://dx.doi.org/10.1016/j.jprot.2021.104244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8199844PMC
June 2021

Time-resolved proteomics profiling of the ciliary Hedgehog response.

J Cell Biol 2021 May;220(5)

Center of Human and Molecular Biology, Saarland University School of Medicine, Homburg, Germany.

The primary cilium is a signaling compartment that interprets Hedgehog signals through changes of its protein, lipid, and second messenger compositions. Here, we combine proximity labeling of cilia with quantitative mass spectrometry to unbiasedly profile the time-dependent alterations of the ciliary proteome in response to Hedgehog. This approach correctly identifies the three factors known to undergo Hedgehog-regulated ciliary redistribution and reveals two such additional proteins. First, we find that a regulatory subunit of the cAMP-dependent protein kinase (PKA) rapidly exits cilia together with the G protein-coupled receptor GPR161 in response to Hedgehog, and we propose that the GPR161/PKA module senses and amplifies cAMP signals to modulate ciliary PKA activity. Second, we identify the phosphatase Paladin as a cell type-specific regulator of Hedgehog signaling that enters primary cilia upon pathway activation. The broad applicability of quantitative ciliary proteome profiling promises a rapid characterization of ciliopathies and their underlying signaling malfunctions.
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http://dx.doi.org/10.1083/jcb.202007207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8054476PMC
May 2021

Structures of chaperone-associated assembly intermediates reveal coordinated mechanisms of proteasome biogenesis.

Nat Struct Mol Biol 2021 05 12;28(5):418-425. Epub 2021 Apr 12.

Department of Pathology, Harvard Medical School and Brigham and Women's Hospital, Boston, MA, USA.

The proteasome mediates most selective protein degradation. Proteolysis occurs within the 20S core particle (CP), a barrel-shaped chamber with an αββα configuration. CP biogenesis proceeds through an ordered multistep pathway requiring five chaperones, Pba1-4 and Ump1. Using Saccharomyces cerevisiae, we report high-resolution structures of CP assembly intermediates by cryogenic-electron microscopy. The first structure corresponds to the 13S particle, which consists of a complete α-ring, partial β-ring (β2-4), Ump1 and Pba1/2. The second structure contains two additional subunits (β5-6) and represents a later pre-15S intermediate. These structures reveal the architecture and positions of Ump1 and β2/β5 propeptides, with important implications for their functions. Unexpectedly, Pba1's N terminus extends through an open CP pore, accessing the CP interior to contact Ump1 and the β5 propeptide. These results reveal how the coordinated activity of Ump1, Pba1 and the active site propeptides orchestrate key aspects of CP assembly.
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http://dx.doi.org/10.1038/s41594-021-00583-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8160580PMC
May 2021

ORF10-Cullin-2-ZYG11B complex is not required for SARS-CoV-2 infection.

Proc Natl Acad Sci U S A 2021 04;118(17)

Division of Genetics, Department of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115;

In order to understand the transmission and virulence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is necessary to understand the functions of each of the gene products encoded in the viral genome. One feature of the SARS-CoV-2 genome that is not present in related, common coronaviruses is ORF10, a putative 38-amino acid protein-coding gene. Proteomic studies found that ORF10 binds to an E3 ubiquitin ligase containing Cullin-2, Rbx1, Elongin B, Elongin C, and ZYG11B (CRL2). Since CRL2 mediates protein degradation, one possible role for ORF10 is to "hijack" CRL2 in order to target cellular, antiviral proteins for ubiquitylation and subsequent proteasomal degradation. Here, we investigated whether ORF10 hijacks CRL2 or functions in other ways, for example, as an inhibitor or substrate of CRL2 While we confirm the ORF10-ZYG11B interaction and show that the N terminus of ORF10 is critical for it, we find no evidence that ORF10 is functioning to inhibit or hijack CRL2 Furthermore, ZYG11B and its paralog ZER1 are dispensable for SARS-CoV-2 infection in cultured cells. We conclude that the interaction between ORF10 and CRL2 is not relevant for SARS-CoV-2 infection in vitro.
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http://dx.doi.org/10.1073/pnas.2023157118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092598PMC
April 2021

Iron Deficiency and Recovery in Yeast: A Quantitative Proteomics Approach.

J Proteome Res 2021 05 2;20(5):2751-2761. Epub 2021 Apr 2.

Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02155, United States.

Iron is an essential element for life, as it is critical for oxygen transport, cellular respiration, DNA synthesis, and metabolism. Disruptions in iron metabolism have been associated with several complex diseases like diabetes, cancer, infection susceptibility, neurodegeneration, and others; however, the molecular mechanisms linking iron metabolism with these diseases are not fully understood. A commonly used model to study iron deficiency (ID) is yeast, . Here, we used quantitative (phospho)proteomics to explore the early (4 and 6 h) and late (12 h) response to ID. We showed that metabolic pathways like the Krebs cycle, amino acid, and ergosterol biosynthesis were affected by ID. In addition, during the late response, several proteins related to the ubiquitin-proteasome system and autophagy were upregulated. We also explored the proteomic changes during a recovery period after 12 h of ID. Several proteins recovered their steady-state levels, but some others, such as cytochromes, did not recover during the time tested. Additionally, we showed that autophagy is active during ID, and some of the degraded proteins during ID can be rescued using KO strains for several key autophagy genes. Our results highlight the complex proteome changes occurring during ID and recovery. This study constitutes a valuable data set for researchers interested in iron biology, offering a temporal proteomic data set for ID, as well as a compendium the proteomic changes associated with episodes of iron recovery.
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http://dx.doi.org/10.1021/acs.jproteome.1c00035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8210944PMC
May 2021

Time-resolved phosphoproteomics reveals scaffolding and catalysis-responsive patterns of SHP2-dependent signaling.

Elife 2021 03 23;10. Epub 2021 Mar 23.

Department of Cancer Biology, Dana-Farber Cancer Institute Boston, Boston, United States.

SHP2 is a protein tyrosine phosphatase that normally potentiates intracellular signaling by growth factors, antigen receptors, and some cytokines, yet is frequently mutated in human cancer. Here, we examine the role of SHP2 in the responses of breast cancer cells to EGF by monitoring phosphoproteome dynamics when SHP2 is allosterically inhibited by SHP099. The dynamics of phosphotyrosine abundance at more than 400 tyrosine residues reveal six distinct response signatures following SHP099 treatment and washout. Remarkably, in addition to newly identified substrate sites on proteins such as occludin, ARHGAP35, and PLCγ2, another class of sites shows reduced phosphotyrosine abundance upon SHP2 inhibition. Sites of decreased phospho-abundance are enriched on proteins with two nearby phosphotyrosine residues, which can be directly protected from dephosphorylation by the paired SH2 domains of SHP2 itself. These findings highlight the distinct roles of the scaffolding and catalytic activities of SHP2 in effecting a transmembrane signaling response.
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http://dx.doi.org/10.7554/eLife.64251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8024022PMC
March 2021

Strain-Specific Peptide (SSP) Interference Reference Sample: A Genetically Encoded Quality Control for Isobaric Tagging Strategies.

Anal Chem 2021 03 18;93(12):5241-5247. Epub 2021 Mar 18.

Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, United States.

Isobaric tag-based sample multiplexing strategies are extensively used for global protein abundance profiling. However, such analyses are often confounded by ratio compression resulting from the co-isolation, co-fragmentation, and co-quantification of co-eluting peptides, termed "interference." Recent analytical strategies incorporating ion mobility and real-time database searching have helped to alleviate interference, yet further assessment is needed. Here, we present the strain-specific peptide (SSP) interference reference sample, a tandem mass tag (TMT)pro-labeled quality control that leverages the genetic variation in the proteomes of eight phylogenetically divergent mouse strains. Typically, a peptide with a missense mutation has a different mass and retention time than the reference or native peptide. TMT reporter ion signal for the native peptide in strains that encode the mutant peptide suggests interference which can be quantified and assessed using the interference-free index (IFI). We introduce the SSP by investigating interference in three common data acquisition methods and by showcasing improvements in the IFI when using ion mobility-based gas-phase fractionation. In addition, we provide a user-friendly, online viewer to visualize the data and streamline the calculation of the IFI. The SSP will aid in developing and optimizing isobaric tag-based experiments.
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http://dx.doi.org/10.1021/acs.analchem.0c05483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8210951PMC
March 2021

DPP9 sequesters the C terminus of NLRP1 to repress inflammasome activation.

Nature 2021 Apr 17;592(7856):778-783. Epub 2021 Mar 17.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.

Nucleotide-binding domain and leucine-rich repeat pyrin-domain containing protein 1 (NLRP1) is an inflammasome sensor that mediates the activation of caspase-1 to induce cytokine maturation and pyroptosis. Gain-of-function mutations of NLRP1 cause severe inflammatory diseases of the skin. NLRP1 contains a function-to-find domain that auto-proteolyses into noncovalently associated subdomains, and proteasomal degradation of the repressive N-terminal fragment of NLRP1 releases its inflammatory C-terminal fragment (NLRP1 CT). Cytosolic dipeptidyl peptidases 8 and 9 (hereafter, DPP8/DPP9) both interact with NLRP1, and small-molecule inhibitors of DPP8/DPP9 activate NLRP1 by mechanisms that are currently unclear. Here we report cryo-electron microscopy structures of the human NLRP1-DPP9 complex alone and with Val-boroPro (VbP), an inhibitor of DPP8/DPP9. The structures reveal a ternary complex that comprises DPP9, full-length NLRP1 and the NLRPT CT. The binding of the NLRP1 CT to DPP9 requires full-length NLRP1, which suggests that NLRP1 activation is regulated by the ratio of NLRP1 CT to full-length NLRP1. Activation of the inflammasome by ectopic expression of the NLRP1 CT is consistently rescued by co-expression of autoproteolysis-deficient full-length NLRP1. The N terminus of the NLRP1 CT inserts into the DPP9 active site, and VbP disrupts this interaction. Thus, VbP weakens the NLRP1-DPP9 interaction and accelerates degradation of the N-terminal fragment to induce inflammasome activation. Overall, these data demonstrate that DPP9 quenches low levels of NLRP1 CT and thus serves as a checkpoint for activation of the NLRP1 inflammasome.
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http://dx.doi.org/10.1038/s41586-021-03350-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8299537PMC
April 2021

Peroxisomal-derived ether phospholipids link nucleotides to respirasome assembly.

Nat Chem Biol 2021 Jun 15;17(6):703-710. Epub 2021 Mar 15.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA.

The protein complexes of the mitochondrial electron transport chain exist in isolation and in higher order assemblies termed supercomplexes (SCs) or respirasomes (SC I+III+IV). The association of complexes I, III and IV into the respirasome is regulated by unknown mechanisms. Here, we designed a nanoluciferase complementation reporter for complex III and IV proximity to determine in vivo respirasome levels. In a chemical screen, we found that inhibitors of the de novo pyrimidine synthesis enzyme dihydroorotate dehydrogenase (DHODH) potently increased respirasome assembly and activity. By-passing DHODH inhibition via uridine supplementation decreases SC assembly by altering mitochondrial phospholipid composition, specifically elevated peroxisomal-derived ether phospholipids. Cell growth rates upon DHODH inhibition depend on ether lipid synthesis and SC assembly. These data reveal that nucleotide pools signal to peroxisomes to modulate synthesis and transport of ether phospholipids to mitochondria for SC assembly, which are necessary for optimal cell growth in conditions of nucleotide limitation.
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http://dx.doi.org/10.1038/s41589-021-00772-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8159895PMC
June 2021

Super Heavy TMTpro Labeling Reagent: An Alternative and Higher-Charge-State-Amenable Stable-Isotope-Labeled TMTpro Variant.

J Proteome Res 2021 05 10;20(5):3009-3013. Epub 2021 Mar 10.

Department of Cell Biology, Blavatnik Institute of Harvard Medical School, Boston, Massachusetts 02115, United States.

Stable isotope labeling is a leading strategy for mass-spectrometry-based peptide quantification. Whereas TMTpro isobaric tagging can quantify up to 16 multiplexed samples in a single experiment, nonisobaric, yet chromatographically indistinguishable, variants of TMTpro reagents can be used in conjunction with the isobaric tag series for various peptide-targeting applications. Here we test the performance of two nonisobaric TMTpro variants, a stable-isotope-free TMTproZero tag and a nearly fully isotope-labeled "super-heavy" variant, shTMTpro, in a targeted assay for peptides of charge state 4+. We label each peptide with TMTproZero or Super Heavy TMTpro reagents and separately spike each peptide into a TMTpro16-labeled background (equal amount of peptide across all 16 channels). We observe that the expected 1:1 reporter ion ratio is distorted when a TMTproZero-labeled peptide is used; however, we note no such interference when shTMTpro substitutes the TMTproZero tag. Our data suggest that using the Super Heavy TMTpro reagent is an improvement over the TMTproZero reagent for the accurate quantification of high-charge-state peptides for trigger-based multiplexed assays.
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http://dx.doi.org/10.1021/acs.jproteome.0c01056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8210946PMC
May 2021

Proteomic and transcriptomic profiling reveal different aspects of aging in the kidney.

Elife 2021 Mar 9;10. Epub 2021 Mar 9.

The Jackson Laboratory, Bar Harbor, United States.

Little is known about the molecular changes that take place in the kidney during the aging process. In order to better understand these changes, we measured mRNA and protein levels in genetically diverse mice at different ages. We observed distinctive change in mRNA and protein levels as a function of age. Changes in both mRNA and protein are associated with increased immune infiltration and decreases in mitochondrial function. Proteins show a greater extent of change and reveal changes in a wide array of biological processes including unique, organ-specific features of aging in kidney. Most importantly, we observed functionally important age-related changes in protein that occur in the absence of corresponding changes in mRNA. Our findings suggest that mRNA profiling alone provides an incomplete picture of molecular aging in the kidney and that examination of changes in proteins is essential to understand aging processes that are not transcriptionally regulated.
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http://dx.doi.org/10.7554/eLife.62585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8096428PMC
March 2021

Dual DNA and protein tagging of open chromatin unveils dynamics of epigenomic landscapes in leukemia.

Nat Methods 2021 03 1;18(3):293-302. Epub 2021 Mar 1.

Cancer Research Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.

The architecture of chromatin regulates eukaryotic cell states by controlling transcription factor access to sites of gene regulation. Here we describe a dual transposase-peroxidase approach, integrative DNA and protein tagging (iDAPT), which detects both DNA (iDAPT-seq) and protein (iDAPT-MS) associated with accessible regions of chromatin. In addition to direct identification of bound transcription factors, iDAPT enables the inference of their gene regulatory networks, protein interactors and regulation of chromatin accessibility. We applied iDAPT to profile the epigenomic consequences of granulocytic differentiation of acute promyelocytic leukemia, yielding previously undescribed mechanistic insights. Our findings demonstrate the power of iDAPT as a platform for studying the dynamic epigenomic landscapes and their transcription factor components associated with biological phenomena and disease.
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http://dx.doi.org/10.1038/s41592-021-01077-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8272231PMC
March 2021

Categorization of Phosphorylation Site Behavior during the Diauxic Shift in .

J Proteome Res 2021 05 25;20(5):2487-2496. Epub 2021 Feb 25.

Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, United States.

Protein phosphorylation has long been recognized as an essential regulator of protein activity, structure, complex formation, and subcellular localization among other cellular mechanisms. However, interpretation of the changes in protein phosphorylation is difficult. To address this difficulty, we measured protein and phosphorylation site changes across 11 points of a time course and developed a method for categorizing phosphorylation site behavior relative to protein level changes using the diauxic shift in yeast as a model and TMT11 sample multiplexing. We classified quantified proteins into behavioral categories that reflected differences in kinase activity, protein complex structure, and growth and metabolic pathway regulation across different phases of the diauxic shift. These data also provide a valuable resource for the study of fermentative versus respiratory growth and set a new benchmark for temporal quantitative proteomics and phosphoproteomics for the diauxic shift in . Data are available via ProteomeXchange with identifier PXD022741.
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http://dx.doi.org/10.1021/acs.jproteome.0c00943DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8210948PMC
May 2021

A cold-stress-inducible PERK/OGT axis controls TOM70-assisted mitochondrial protein import and cristae formation.

Cell Metab 2021 Mar 15;33(3):598-614.e7. Epub 2021 Feb 15.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Department of Cell Biology, Harvard Medical School, Boston, MA 02215, USA. Electronic address:

The architecture of cristae provides a spatial mitochondrial organization that contains functional respiratory complexes. Several protein components including OPA1 and MICOS complex subunits organize cristae structure, but upstream regulatory mechanisms are largely unknown. Here, in vivo and in vitro reconstitution experiments show that the endoplasmic reticulum (ER) kinase PERK promotes cristae formation by increasing TOM70-assisted mitochondrial import of MIC19, a critical subunit of the MICOS complex. Cold stress or β-adrenergic stimulation activates PERK that phosphorylates O-linked N-acetylglucosamine transferase (OGT). Phosphorylated OGT glycosylates TOM70 on Ser94, enhancing MIC19 protein import into mitochondria and promoting cristae formation and respiration. In addition, PERK-activated OGT O-GlcNAcylates and attenuates CK2α activity, which mediates TOM70 Ser94 phosphorylation and decreases MIC19 mitochondrial protein import. We have identified a cold-stress inter-organelle PERK-OGT-TOM70 axis that increases cell respiration through mitochondrial protein import and subsequent cristae formation. These studies have significant implications in cellular bioenergetics and adaptations to stress conditions.
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http://dx.doi.org/10.1016/j.cmet.2021.01.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7962155PMC
March 2021

Targeting oncoproteins with a positive selection assay for protein degraders.

Sci Adv 2021 Feb 5;7(6). Epub 2021 Feb 5.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA.

Most intracellular proteins lack hydrophobic pockets suitable for altering their function with drug-like small molecules. Recent studies indicate that some undruggable proteins can be targeted by compounds that can degrade them. For example, thalidomide-like drugs (IMiDs) degrade the critical multiple myeloma transcription factors IKZF1 and IKZF3 by recruiting them to the cereblon E3 ubiquitin ligase. Current loss of signal ("down") assays for identifying degraders often exhibit poor signal-to-noise ratios, narrow dynamic ranges, and false positives from compounds that nonspecifically suppress transcription or translation. Here, we describe a gain of signal ("up") assay for degraders. In arrayed chemical screens, we identified novel IMiD-like IKZF1 degraders and Spautin-1, which, unlike the IMiDs, degrades IKZF1 in a cereblon-independent manner. In a pooled CRISPR-Cas9-based screen, we found that CDK2 regulates the abundance of the ASCL1 oncogenic transcription factor. This methodology should facilitate the identification of drugs that directly or indirectly degrade undruggable proteins.
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http://dx.doi.org/10.1126/sciadv.abd6263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864573PMC
February 2021

Enrichment of Neurodegenerative Microglia Signature in Brain-Derived Extracellular Vesicles Isolated from Alzheimer's Disease Mouse Models.

J Proteome Res 2021 03 3;20(3):1733-1743. Epub 2021 Feb 3.

Department of Pharmacology & Experimental Therapeutics, Boston University School of Medicine, Boston, Massachusetts 02118, United States.

Extracellular vesicles (EVs) are secreted by any neural cells in the central nervous system for molecular clearance, cellular communications, and disease spread in multiple neurodegenerative diseases, including Alzheimer's disease (AD), although their exact molecular mechanism is poorly understood. We hypothesize that high-resolution proteomic profiling of EVs separated from animal models of AD would determine the composition of EV contents and their cellular origin. Here, we examined recently developed transgenic mice (CAST.), which express familial AD-linked mutations of amyloid precursor protein () and presenilin-1 () in the CAST/EiJ mouse strain and develop hippocampal neurodegeneration. Quantitative proteomics analysis of EVs separated from CAST. and age-matched control mice by tandem mass tag-mass spectrometry identified a total of 3444 unique proteins, which are enriched in neuron-, astrocyte-, oligodendrocyte-, and microglia-specific molecules. CAST.-derived EVs show significant enrichment of Psen1, APP, and Itgax and reduction of Wdr61, Pmpca, Aldh1a2, Calu, Anp32b, Actn4, and Ndufv2 compared to WT-derived EVs, suggesting the involvement of Aβ-processing complex and disease-associated/neurodegenerative microglia (DAM/MGnD) in EV secretion. In addition, Itgax and Apoe, DAM/MGnD markers, in EVs show a positive correlation with and mRNA expression from brain tissue in CAST. mice. These datasets indicate the significant contribution of Aβ plaque and neurodegeneration-induced DAM/MGnD microglia for EV secretion in CAST. mice and shed light on understanding AD pathogenesis.
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http://dx.doi.org/10.1021/acs.jproteome.0c00934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7944570PMC
March 2021

Tetracyclines promote survival and fitness in mitochondrial disease models.

Nat Metab 2021 01 18;3(1):33-42. Epub 2021 Jan 18.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA.

Mitochondrial diseases (MDs) are a heterogeneous group of disorders resulting from mutations in nuclear or mitochondrial DNA genes encoding mitochondrial proteins. MDs cause pathologies with severe tissue damage and ultimately death. There are no cures for MDs and current treatments are only palliative. Here we show that tetracyclines improve fitness of cultured MD cells and ameliorate disease in a mouse model of Leigh syndrome. To identify small molecules that prevent cellular damage and death under nutrient stress conditions, we conduct a chemical high-throughput screen with cells carrying human MD mutations and discover a series of antibiotics that maintain survival of various MD cells. We subsequently show that a sub-library of tetracycline analogues, including doxycycline, rescues cell death and inflammatory signatures in mutant cells through partial and selective inhibition of mitochondrial translation, resulting in an ATF4-independent mitohormetic response. Doxycycline treatment strongly promotes fitness and survival of Ndufs4 mice, a preclinical Leigh syndrome mouse model. A proteomic analysis of brain tissue reveals that doxycycline treatment largely prevents neuronal death and the accumulation of neuroimmune and inflammatory proteins in Ndufs4 mice, indicating a potential causal role for these proteins in the brain pathology. Our findings suggest that tetracyclines deserve further evaluation as potential drugs for the treatment of MDs.
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http://dx.doi.org/10.1038/s42255-020-00334-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7856165PMC
January 2021

Proteomics of broad deubiquitylase inhibition unmasks redundant enzyme function to reveal substrates and assess enzyme specificity.

Cell Chem Biol 2021 04 7;28(4):487-502.e5. Epub 2021 Jan 7.

Department of Cell Biology, Blavatnik Institute at Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Deubiquitylating enzymes (DUBs) counteract ubiquitylation to control stability or activity of substrates. Identification of DUB substrates is challenging because multiple DUBs can act on the same substrate, thwarting genetic approaches. Here, we circumvent redundancy by chemically inhibiting multiple DUBs simultaneously in Xenopus egg extract. We used quantitative mass spectrometry to identify proteins whose ubiquitylation or stability is altered by broad DUB inhibition, and confirmed their DUB-dependent regulation with human orthologs, demonstrating evolutionary conservation. We next extended this method to profile DUB specificity. By adding recombinant DUBs to extract where DUB activity was broadly inhibited, but ubiquitylation and degradation were active at physiological rates, we profiled the ability of DUBs to rescue degradation of these substrates. We found that USP7 has a unique ability to broadly antagonize degradation. Together, we present an approach to identify DUB substrates and characterize DUB specificity that overcomes challenges posed by DUB redundancy.
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http://dx.doi.org/10.1016/j.chembiol.2020.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8052291PMC
April 2021

Rapid toxin sequestration modifies poison frog physiology.

J Exp Biol 2021 02 9;224(Pt 3). Epub 2021 Feb 9.

Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.

Poison frogs sequester chemical defenses from their diet of leaf litter arthropods for defense against predation. Little is known about the physiological adaptations that confer this unusual bioaccumulation ability. We conducted an alkaloid-feeding experiment with the Diablito poison frog () to determine how quickly alkaloids are accumulated and how toxins modify frog physiology using quantitative proteomics. Diablito frogs rapidly accumulated the alkaloid decahydroquinoline within 4 days, and dietary alkaloid exposure altered protein abundance in the intestines, liver and skin. Many proteins that increased in abundance with decahydroquinoline accumulation are plasma glycoproteins, including the complement system and the toxin-binding protein saxiphilin. Other protein classes that change in abundance with decahydroquinoline accumulation are membrane proteins involved in small molecule transport and metabolism. Overall, this work shows that poison frogs can rapidly accumulate alkaloids, which alter carrier protein abundance, initiate an immune response, and alter small molecule transport and metabolism dynamics across tissues.
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http://dx.doi.org/10.1242/jeb.230342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7888741PMC
February 2021

Reimagining high-throughput profiling of reactive cysteines for cell-based screening of large electrophile libraries.

Nat Biotechnol 2021 05 4;39(5):630-641. Epub 2021 Jan 4.

Department of Cell Biology, Harvard Medical School, Boston, MA, USA.

Current methods used for measuring amino acid side-chain reactivity lack the throughput needed to screen large chemical libraries for interactions across the proteome. Here we redesigned the workflow for activity-based protein profiling of reactive cysteine residues by using a smaller desthiobiotin-based probe, sample multiplexing, reduced protein starting amounts and software to boost data acquisition in real time on the mass spectrometer. Our method, streamlined cysteine activity-based protein profiling (SLC-ABPP), achieved a 42-fold improvement in sample throughput, corresponding to profiling library members at a depth of >8,000 reactive cysteine sites at 18 min per compound. We applied it to identify proteome-wide targets of covalent inhibitors to mutant Kirsten rat sarcoma (KRAS) and Bruton's tyrosine kinase (BTK). In addition, we created a resource of cysteine reactivity to 285 electrophiles in three human cell lines, which includes >20,000 cysteines from >6,000 proteins per line. The goal of proteome-wide profiling of cysteine reactivity across thousand-member libraries under several cellular contexts is now within reach.
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http://dx.doi.org/10.1038/s41587-020-00778-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8316984PMC
May 2021

Obesity Shapes Metabolism in the Tumor Microenvironment to Suppress Anti-Tumor Immunity.

Cell 2020 12 9;183(7):1848-1866.e26. Epub 2020 Dec 9.

Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Obesity is a major cancer risk factor, but how differences in systemic metabolism change the tumor microenvironment (TME) and impact anti-tumor immunity is not understood. Here, we demonstrate that high-fat diet (HFD)-induced obesity impairs CD8 T cell function in the murine TME, accelerating tumor growth. We generate a single-cell resolution atlas of cellular metabolism in the TME, detailing how it changes with diet-induced obesity. We find that tumor and CD8 T cells display distinct metabolic adaptations to obesity. Tumor cells increase fat uptake with HFD, whereas tumor-infiltrating CD8 T cells do not. These differential adaptations lead to altered fatty acid partitioning in HFD tumors, impairing CD8 T cell infiltration and function. Blocking metabolic reprogramming by tumor cells in obese mice improves anti-tumor immunity. Analysis of human cancers reveals similar transcriptional changes in CD8 T cell markers, suggesting interventions that exploit metabolism to improve cancer immunotherapy.
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http://dx.doi.org/10.1016/j.cell.2020.11.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8064125PMC
December 2020
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