Gygi SP

References (11)

Title : DPP9 sequesters the C terminus of NLRP1 to repress inflammasome activation - Hollingsworth_2021_Nature_592_778
Author(s) : Hollingsworth LR , Sharif H , Griswold AR , Fontana P , Mintseris J , Dagbay KB , Paulo JA , Gygi SP , Bachovchin DA , Wu H
Ref : Nature , 592 :778 , 2021
Abstract : 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(1-4). Gain-of-function mutations of NLRP1 cause severe inflammatory diseases of the skin(4-6). NLRP1 contains a function-to-find domain that auto-proteolyses into noncovalently associated subdomains(7-9), and proteasomal degradation of the repressive N-terminal fragment of NLRP1 releases its inflammatory C-terminal fragment (NLRP1 CT)(10,11). 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(10,12-14). 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(10) 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.
ESTHER : Hollingsworth_2021_Nature_592_778
PubMedSearch : Hollingsworth_2021_Nature_592_778
PubMedID: 33731932
Gene_locus related to this paper: human-DPP9

Title : DPP9 directly sequesters the NLRP1 C-terminus to repress inflammasome activation - Hollingsworth_2020_Biorxiv__
Author(s) : Hollingsworth LR , Sharif H , Griswold AR , Fontana P , Mintseris J , Dagbay KB , Paulo JA , Gygi SP , Bachovchin DA , Wu
Ref : Biorxiv , : , 2020
Abstract : NLRP1 is a cytosolic inflammasome sensor that mediates activation of caspase-1, which in turn induces cytokine maturation and pyroptotic cell death1-6. Gain-of-function NLPR1 mutations cause skin inflammatory diseases including carcinoma, keratosis, and papillomatosis7-14. NLRP1 contains a unique function-to-find domain (FIIND) that autoproteolyzes into noncovalently associated subdomains15-18. Proteasomal degradation of the autoinhibitory N-terminal fragment (NT) activates NLRP1 by releasing the inflammatory C-terminal fragment (CT)19,20. Cytosolic dipeptidyl peptidases 8 and 9 (DPP8/9) interact with NLRP1, and small-molecule DPP8/9 inhibitors activate NLRP1 by poorly characterized mechanisms11,19,21. Here, we report cryo-EM structures of the human NLRP1-DPP9 complex, alone and in complex with the DPP8/9 inhibitor Val-boroPro (VbP). Surprisingly, the NLRP1-DPP9 complex is a ternary complex comprised of DPP9, one intact FIIND of a non-degraded full-length NLRP1 (NLRP1-FL) and one NLRP1-CT freed by NT degradation. The N-terminus of the NLRP1-CT unfolds and inserts into the DPP9 active site but is not cleaved by DPP9, and this binding is disrupted by VbP. Structure-based mutagenesis reveals that the binding of NLRP1-CT to DPP9 requires NLRP1-FL and vice versa, and inflammasome activation by ectopic NLRP1-CT expression is rescued by co-expressing autoproteolysis-deficient NLRP1-FL. Collectively, these data indicate that DPP9 functions as a 'bomb-diffuser' to prevent NLRP1-CTs from inducing inflammation during homeostatic protein turnover.
ESTHER : Hollingsworth_2020_Biorxiv__
PubMedSearch : Hollingsworth_2020_Biorxiv__
PubMedID:
Gene_locus related to this paper: human-DPP9

Title : A tissue-specific atlas of mouse protein phosphorylation and expression - Huttlin_2010_Cell_143_1174
Author(s) : Huttlin EL , Jedrychowski MP , Elias JE , Goswami T , Rad R , Beausoleil SA , Villen J , Haas W , Sowa ME , Gygi SP
Ref : Cell , 143 :1174 , 2010
Abstract : Although most tissues in an organism are genetically identical, the biochemistry of each is optimized to fulfill its unique physiological roles, with important consequences for human health and disease. Each tissue's unique physiology requires tightly regulated gene and protein expression coordinated by specialized, phosphorylation-dependent intracellular signaling. To better understand the role of phosphorylation in maintenance of physiological differences among tissues, we performed proteomic and phosphoproteomic characterizations of nine mouse tissues. We identified 12,039 proteins, including 6296 phosphoproteins harboring nearly 36,000 phosphorylation sites. Comparing protein abundances and phosphorylation levels revealed specialized, interconnected phosphorylation networks within each tissue while suggesting that many proteins are regulated by phosphorylation independently of their expression. Our data suggest that the "typical" phosphoprotein is widely expressed yet displays variable, often tissue-specific phosphorylation that tunes protein activity to the specific needs of each tissue. We offer this dataset as an online resource for the biological research community.
ESTHER : Huttlin_2010_Cell_143_1174
PubMedSearch : Huttlin_2010_Cell_143_1174
PubMedID: 21183079
Gene_locus related to this paper: mouse-abd12 , mouse-Ces2a , mouse-CMBL , mouse-ndr1 , mouse-ndr2 , mouse-ndr4 , mouse-f172a

Title : A quantitative atlas of mitotic phosphorylation - Dephoure_2008_Proc.Natl.Acad.Sci.U.S.A_105_10762
Author(s) : Dephoure N , Zhou C , Villen J , Beausoleil SA , Bakalarski CE , Elledge SJ , Gygi SP
Ref : Proc Natl Acad Sci U S A , 105 :10762 , 2008
Abstract : The eukaryotic cell division cycle is characterized by a sequence of orderly and highly regulated events resulting in the duplication and separation of all cellular material into two newly formed daughter cells. Protein phosphorylation by cyclin-dependent kinases (CDKs) drives this cycle. To gain further insight into how phosphorylation regulates the cell cycle, we sought to identify proteins whose phosphorylation is cell cycle regulated. Using stable isotope labeling along with a two-step strategy for phosphopeptide enrichment and high mass accuracy mass spectrometry, we examined protein phosphorylation in a human cell line arrested in the G(1) and mitotic phases of the cell cycle. We report the identification of >14,000 different phosphorylation events, more than half of which, to our knowledge, have not been described in the literature, along with relative quantitative data for the majority of these sites. We observed >1,000 proteins with increased phosphorylation in mitosis including many known cell cycle regulators. The majority of sites on regulated phosphopeptides lie in [S/T]P motifs, the minimum required sequence for CDKs, suggesting that many of the proteins may be CDK substrates. Analysis of non-proline site-containing phosphopeptides identified two unique motifs that suggest there are at least two undiscovered mitotic kinases.
ESTHER : Dephoure_2008_Proc.Natl.Acad.Sci.U.S.A_105_10762
PubMedSearch : Dephoure_2008_Proc.Natl.Acad.Sci.U.S.A_105_10762
PubMedID: 18669648
Gene_locus related to this paper: human-KANSL3

Title : Phosphoproteome analysis of Drosophila melanogaster embryos - Zhai_2008_J.Proteome.Res_7_1675
Author(s) : Zhai B , Villen J , Beausoleil SA , Mintseris J , Gygi SP
Ref : J Proteome Res , 7 :1675 , 2008
Abstract : Protein phosphorylation is a key regulatory event in most cellular processes and development. Mass spectrometry-based proteomics provides a framework for the large-scale identification and characterization of phosphorylation sites. Here, we used a well-established phosphopeptide enrichment and identification strategy including the combination of strong cation exchange chromatography, immobilized metal affinity chromatography, and high-accuracy mass spectrometry instrumentation to study phosphorylation in developing Drosophila embryos. In total, 13,720 different phosphorylation sites were discovered from 2702 proteins with an estimated false-discovery rate (FDR) of 0.63% at the peptide level. Because of the large size of the data set, both novel and known phosphorylation motifs were extracted using the Motif-X algorithm, including those representative of potential ordered phosphorylation events.
ESTHER : Zhai_2008_J.Proteome.Res_7_1675
PubMedSearch : Zhai_2008_J.Proteome.Res_7_1675
PubMedID: 18327897

Title : Phosphoproteome analysis of fission yeast - Wilson-Grady_2008_J.Proteome.Res_7_1088
Author(s) : Wilson-Grady JT , Villen J , Gygi SP
Ref : J Proteome Res , 7 :1088 , 2008
Abstract : Phosphorylation is a key regulator of many events in eukaryotic cells. The acquisition of large-scale phosphorylation data sets from model organisms can pinpoint conserved regulatory inputs and reveal kinase-substrate relationships. Here, we provide the first large-scale phosphorylation analysis of the fission yeast, Schizosaccharomyces pombe. Protein from thiabendazole-treated cells was separated by preparative SDS-PAGE and digested with trypsin. The resulting peptides were subjected to either IMAC or TiO2 phosphopeptide enrichment methods and then analyzed by LC-MS/MS using an LTQ-Orbitrap mass spectrometer. In total, 2887 distinct phosphorylation sites were identified from 1194 proteins with an estimated false-discovery rate of <0.5% at the peptide level. A comparison of the two different enrichment methods is presented, supporting the finding that they are complementary. Finally, phosphorylation sites were examined for phosphorylation-specific motifs and evolutionary conservation. These analyses revealed both motifs and specific phosphorylation events identified in S. pombe were conserved and predicted novel phosphorylation in mammals.
ESTHER : Wilson-Grady_2008_J.Proteome.Res_7_1088
PubMedSearch : Wilson-Grady_2008_J.Proteome.Res_7_1088
PubMedID: 18257517
Gene_locus related to this paper: schpo-SPCC5E4.05C

Title : Large-scale phosphorylation analysis of alpha-factor-arrested Saccharomyces cerevisiae - Li_2007_J.Proteome.Res_6_1190
Author(s) : Li X , Gerber SA , Rudner AD , Beausoleil SA , Haas W , Villen J , Elias JE , Gygi SP
Ref : J Proteome Res , 6 :1190 , 2007
Abstract : Protein phosphorylation is essential for numerous cellular processes. Large-scale profiling of phosphoproteins continues to enhance the depth and speed at which we understand these processes. The development of effective phosphoprotein and peptide enrichment techniques and improvements to mass spectrometric instrumentation have intensified phosphoproteomic research in recent years, leading to unprecedented achievements. Here, we describe a large-scale phosphorylation analysis of alpha-factor-arrested yeast. Using a multidimensional separation strategy involving preparative SDS-PAGE for prefractionation, in-gel digestion with trypsin, and immobilized metal affinity chromatography (IMAC) enrichment of phosphopeptides, followed by LC-MS/MS analysis employing a hybrid LTQ-Orbitrap mass spectrometer, we were able to catalog a substantial portion of the phosphoproteins present in yeast whole-cell lysate. This analysis yielded the confident identification of 2288 nonredundant phosphorylation sites from 985 proteins. The ambiguity score (Ascore) algorithm was utilized to determine the certainty of site localization for the entire data set. In addition, the size of the data set permitted extraction of known and novel kinase motifs using the Motif-X algorithm. Finally, a large number of members of the pheromone signaling pathway were found as phosphoproteins and are discussed.
ESTHER : Li_2007_J.Proteome.Res_6_1190
PubMedSearch : Li_2007_J.Proteome.Res_6_1190
PubMedID: 17330950
Gene_locus related to this paper: yeast-YDR428C

Title : Two different Argonaute complexes are required for siRNA generation and heterochromatin assembly in fission yeast - Buker_2007_Nat.Struct.Mol.Biol_14_200
Author(s) : Buker SM , Iida T , Buhler M , Villen J , Gygi SP , Nakayama J , Moazed D
Ref : Nat Struct Mol Biol , 14 :200 , 2007
Abstract : The RNA-induced transcriptional silencing (RITS) complex, containing Ago1, Chp1, Tas3 and centromeric small interfering RNAs (siRNAs), is required for heterochromatic gene silencing at centromeres. Here, we identify a second fission yeast Argonaute complex (Argonaute siRNA chaperone, ARC), which contains, in addition to Ago1, two previously uncharacterized proteins, Arb1 and Arb2, both of which are required for histone H3 Lys9 (H3-K9) methylation, heterochromatin assembly and siRNA generation. Furthermore, whereas siRNAs in the RITS complex are mostly single-stranded, siRNAs associated with ARC are mostly double-stranded, indicating that Arb1 and Arb2 inhibit the release of the siRNA passenger strand from Ago1. Consistent with this observation, purified Arb1 inhibits the slicer activity of Ago1 in vitro, and purified catalytically inactive Ago1 contains only double-stranded siRNA. Finally, we show that slicer activity is required for the siRNA-dependent association of Ago1 with chromatin and for the spreading of histone H3-K9 methylation.
ESTHER : Buker_2007_Nat.Struct.Mol.Biol_14_200
PubMedSearch : Buker_2007_Nat.Struct.Mol.Biol_14_200
PubMedID: 17310250

Title : A probability-based approach for high-throughput protein phosphorylation analysis and site localization - Beausoleil_2006_Nat.Biotechnol_24_1285
Author(s) : Beausoleil SA , Villen J , Gerber SA , Rush J , Gygi SP
Ref : Nat Biotechnol , 24 :1285 , 2006
Abstract : Data analysis and interpretation remain major logistical challenges when attempting to identify large numbers of protein phosphorylation sites by nanoscale reverse-phase liquid chromatography/tandem mass spectrometry (LC-MS/MS) (Supplementary Figure 1 online). In this report we address challenges that are often only addressable by laborious manual validation, including data set error, data set sensitivity and phosphorylation site localization. We provide a large-scale phosphorylation data set with a measured error rate as determined by the target-decoy approach, we demonstrate an approach to maximize data set sensitivity by efficiently distracting incorrect peptide spectral matches (PSMs), and we present a probability-based score, the Ascore, that measures the probability of correct phosphorylation site localization based on the presence and intensity of site-determining ions in MS/MS spectra. We applied our methods in a fully automated fashion to nocodazole-arrested HeLa cell lysate where we identified 1,761 nonredundant phosphorylation sites from 491 proteins with a peptide false-positive rate of 1.3%.
ESTHER : Beausoleil_2006_Nat.Biotechnol_24_1285
PubMedSearch : Beausoleil_2006_Nat.Biotechnol_24_1285
PubMedID: 16964243

Title : Large-scale characterization of HeLa cell nuclear phosphoproteins - Beausoleil_2004_Proc.Natl.Acad.Sci.U.S.A_101_12130
Author(s) : Beausoleil SA , Jedrychowski M , Schwartz D , Elias JE , Villen J , Li J , Cohn MA , Cantley LC , Gygi SP
Ref : Proc Natl Acad Sci U S A , 101 :12130 , 2004
Abstract : Determining the site of a regulatory phosphorylation event is often essential for elucidating specific kinase-substrate relationships, providing a handle for understanding essential signaling pathways and ultimately allowing insights into numerous disease pathologies. Despite intense research efforts to elucidate mechanisms of protein phosphorylation regulation, efficient, large-scale identification and characterization of phosphorylation sites remains an unsolved problem. In this report we describe an application of existing technology for the isolation and identification of phosphorylation sites. By using a strategy based on strong cation exchange chromatography, phosphopeptides were enriched from the nuclear fraction of HeLa cell lysate. From 967 proteins, 2,002 phosphorylation sites were determined by tandem MS. This unprecedented large collection of sites permitted a detailed accounting of known and unknown kinase motifs and substrates.
ESTHER : Beausoleil_2004_Proc.Natl.Acad.Sci.U.S.A_101_12130
PubMedSearch : Beausoleil_2004_Proc.Natl.Acad.Sci.U.S.A_101_12130
PubMedID: 15302935
Gene_locus related to this paper: human-KANSL3

Title : Identification of RFC(Ctf18p, Ctf8p, Dcc1p): an alternative RFC complex required for sister chromatid cohesion in S. cerevisiae - Mayer_2001_Mol.Cell_7_959
Author(s) : Mayer ML , Gygi SP , Aebersold R , Hieter P
Ref : Mol Cell , 7 :959 , 2001
Abstract : We have identified and characterized an alternative RFC complex RFC(Ctf18p, Ctf8p, Dcc1p) that is required for sister chromatid cohesion and faithful chromosome transmission. Ctf18p, Ctf8p, and Dcc1p interact physically in a complex with Rfc2p, Rfc3p, Rfc4p, and Rfc5p but not with Rfc1p or Rad24p. Deletion of CTF18, CTF8, or DCC1 singly or in combination (ctf18Deltactf8Deltadcc1Delta) leads to sensitivity to microtubule depolymerizing drugs and a severe sister chromatid cohesion defect. Furthermore, temperature-sensitive mutations in RFC4 result in precocious sister chromatid separation. Our results highlight a novel function of the RFC proteins and support a model in which sister chromatid cohesion is established at the replication fork via a polymerase switching mechanism and a replication-coupled remodeling of chromatin.
ESTHER : Mayer_2001_Mol.Cell_7_959
PubMedSearch : Mayer_2001_Mol.Cell_7_959
PubMedID: 11389843