Showing papers by "Steven P. Gygi published in 2007"
TL;DR: This work clarifies the preferred methodology by addressing four issues based on observed decoy hit frequencies: the major assumptions made with this database search strategy are reasonable, concatenated target-decoy database searches are preferable to separate target and decoydatabase searches, and the theoretical error associated with target-Decoy false positive (FP) rate measurements can be estimated.
Abstract: Liquid chromatography and tandem mass spectrometry (LC-MS/MS) has become the preferred method for conducting large-scale surveys of proteomes. Automated interpretation of tandem mass spectrometry (MS/MS) spectra can be problematic, however, for a variety of reasons. As most sequence search engines return results even for 'unmatchable' spectra, proteome researchers must devise ways to distinguish correct from incorrect peptide identifications. The target-decoy search strategy represents a straightforward and effective way to manage this effort. Despite the apparent simplicity of this method, some controversy surrounds its successful application. Here we clarify our preferred methodology by addressing four issues based on observed decoy hit frequencies: (i) the major assumptions made with this database search strategy are reasonable; (ii) concatenated target-decoy database searches are preferable to separate target and decoy database searches; (iii) the theoretical error associated with target-decoy false positive (FP) rate measurements can be estimated; and (iv) alternate methods for constructing decoy databases are similarly effective once certain considerations are taken into account.
3,602 citations
TL;DR: A large-scale proteomic analysis of proteins phosphorylated in response to DNA damage on consensus sites recognized by ATM and ATR is performed and more than 900 regulated phosphorylation sites encompassing over 700 proteins are identified.
Abstract: Cellular responses to DNA damage are mediated by a number of protein kinases, including ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related). The outlines of the signal transduction portion of this pathway are known, but little is known about the physiological scope of the DNA damage response (DDR). We performed a large-scale proteomic analysis of proteins phosphorylated in response to DNA damage on consensus sites recognized by ATM and ATR and identified more than 900 regulated phosphorylation sites encompassing over 700 proteins. Functional analysis of a subset of this data set indicated that this list is highly enriched for proteins involved in the DDR. This set of proteins is highly interconnected, and we identified a large number of protein modules and networks not previously linked to the DDR. This database paints a much broader landscape for the DDR than was previously appreciated and opens new avenues of investigation into the responses to DNA damage in mammals.
2,967 citations
TL;DR: By focusing on activated cell circuitry, the approach outlined here provides insight into cancer biology not available at the chromosomal and transcriptional levels and can be applied broadly across all human cancers.
2,177 citations
TL;DR: It is demonstrated that a combination of tandem phosphopeptide enrichment methods, high performance MS, and optimized database search/data filtering strategies is a powerful tool for surveying the phosphoproteome.
Abstract: Protein phosphorylation is a complex network of signaling and regulatory events that affects virtually every cellular process. Our understanding of the nature of this network as a whole remains limited, largely because of an array of technical challenges in the isolation and high-throughput sequencing of phosphorylated species. In the present work, we demonstrate that a combination of tandem phosphopeptide enrichment methods, high performance MS, and optimized database search/data filtering strategies is a powerful tool for surveying the phosphoproteome. Using our integrated analytical platform, we report the identification of 5,635 nonredundant phosphorylation sites from 2,328 proteins from mouse liver. From this list of sites, we extracted both novel and known motifs for specific Ser/Thr kinases including a "dipolar" motif. We also found that C-terminal phosphorylation was more frequent than at any other location and that the distribution of potential kinases for these sites was unique. Finally, we identified double phosphorylation motifs that may be involved in ordered phosphorylation.
747 citations
TL;DR: It is found that AMPK directly regulates mammalian FOXO3, a member of the FOXO family of Forkhead transcription factors known to promote resistance to oxidative stress, tumor suppression, and longevity, by phosphorylation by AMPK at six previously unidentified regulatory sites.
741 citations
TL;DR: An energy-sensing AMPK-FOXO pathway mediates the lifespan extension induced by a novel method of dietary restriction in C. elegans, suggesting a possible direct mechanism of regulation of FOXO/DAF-16 by AMPK.
717 citations
TL;DR: Phosphopeptide affinity proteomic analysis identified a protein, Abraxas, that directly binds the BRCA1 BRCT repeats through a phospho-Ser-X- X-Phe motif, forming a third type of B RCA1 complex.
Abstract: The BRCT repeats of the breast and ovarian cancer predisposition protein BRCA1 are essential for tumor suppression. Phosphopeptide affinity proteomic analysis identified a protein, Abraxas, that directly binds the BRCA1 BRCT repeats through a phospho-Ser-X-X-Phe motif. Abraxas binds BRCA1 to the mutual exclusion of BACH1 (BRCA1-associated C-terminal helicase) and CtIP (CtBP-interacting protein), forming a third type of BRCA1 complex. Abraxas recruits the ubiquitin-interacting motif (UIM)–containing protein RAP80 to BRCA1. Both Abraxas and RAP80 were required for DNA damage resistance, G2-M checkpoint control, and DNA repair. RAP80 was required for optimal accumulation of BRCA1 on damaged DNA (foci) in response to ionizing radiation, and the UIM domains alone were capable of foci formation. The RAP80-Abraxas complex may help recruit BRCA1 to DNA damage sites in part through recognition of ubiquitinated proteins.
677 citations
TL;DR: Mutation in FANCI is responsible for loss of a functional FA pathway in a patient with Fanconi anemia complementation group I, indicating the existence of a dual ubiquitin-locking mechanism required for ID complex function.
673 citations
TL;DR: The experiments suggest that the ESCRT pathway may be recruited to facilitate analogous membrane fission events during HIV budding, MVB vesicle formation, and the abscission stage of cytokinesis.
Abstract: TSG101 and ALIX both function in HIV budding and in vesicle formation at the multivesicular body (MVB), where they interact with other Endosomal Sorting Complex Required for Transport (ESCRT) pathway factors required for release of viruses and vesicles. Proteomic analyses revealed that ALIX and TSG101/ESCRT‐I also bind a series of proteins involved in cytokinesis, including CEP55, CD2AP, ROCK1, and IQGAP1. ALIX and TSG101 concentrate at centrosomes and are then recruited to the midbodies of dividing cells through direct interactions between the central CEP55 ‘hinge’ region and GPP‐based motifs within TSG101 and ALIX. ESCRT‐III and VPS4 proteins are also recruited, indicating that much of the ESCRT pathway localizes to the midbody. Depletion of ALIX and TSG101/ESCRT‐I inhibits the abscission step of HeLa cell cytokinesis, as does VPS4 overexpression, confirming a requirement for these proteins in cell division. Furthermore, ALIX point mutants that block CEP55 and CHMP4/ESCRT‐III binding also inhibit abscission, indicating that both interactions are essential. These experiments suggest that the ESCRT pathway may be recruited to facilitate analogous membrane fission events during HIV budding, MVB vesicle formation, and the abscission stage of cytokinesis.
651 citations
TL;DR: It is found that the U-box E3, CHIP, and Ring finger E3s, MuRF1 and Mdm2, with the E2, UbcH5, form a novel type of Ub chain that contains all seven possible linkages, but predominantly Lys48, Lys63, and Lys11 linkages.
472 citations
TL;DR: This work identifies a divergent E1 in vertebrates and sea urchin, Uba6, which specifically activates ubiquitin but not other UBLs in vitro and in vivo, and reveals unexpected complexity in the pathways that control the conjugation of Ubiquitin.
Abstract: Modification of proteins with ubiquitin or ubiquitin-like proteins (UBLs) by means of an E1-E2-E3 cascade controls many signalling networks. Ubiquitin conjugation involves adenylation and thioesterification of the carboxy-terminal carboxylate of ubiquitin by the E1-activating enzyme Ube1 (Uba1 in yeast), followed by ubiquitin transfer to an E2-conjugating enzyme through a transthiolation reaction. Charged E2s function with E3s to ubiquitinate substrates. It is currently thought that Ube1/Uba1 is the sole E1 for charging of E2s with ubiquitin in animals and fungi. Here we identify a divergent E1 in vertebrates and sea urchin, Uba6, which specifically activates ubiquitin but not other UBLs in vitro and in vivo. Human Uba6 and Ube1 have distinct preferences for E2 charging in vitro, and their specificity depends in part on their C-terminal ubiquitin-fold domains, which recruit E2s. In tissue culture cells, Uba6 is required for charging a previously uncharacterized Uba6-specific E2 (Use1), whereas Ube1 is required for charging the cell-cycle E2s Cdc34A and Cdc34B. Our data reveal unexpected complexity in the pathways that control the conjugation of ubiquitin, in which dual E1s orchestrate the charging of distinct cohorts of E2s.
TL;DR: In vitro reconstitution of USP1 deubiquitinating enzyme activity, using either ubiquitin-7-amido-4-methylcoumarin (Ub-AMC) or purified monoubiquitinated FANCD2 protein as substrates, demonstrates that UAF1 functions as an activator of USp1.
TL;DR: It is proposed that the exclusive association of U1 snRNP/SR proteins with RNAP II positions these splicing factors, which are known to function early in spliceosome assembly, close to the nascent pre-mRNA, so that these factors readily out-compete inhibitory hnRNP proteins, resulting in efficient splicesome assembly on nascentRNAP II transcripts.
TL;DR: Using immunoaffinity phosphopeptide isolation coupled with mass spectrometry to identify 570 sites phosphorylated in UV-damaged cells, 498 of which are previously undescribed, provide a rich resource for further deciphering ATM/ATR signaling and the pathways mediating the DNA damage response.
Abstract: To ensure survival in the face of genomic insult, cells have evolved complex mechanisms to respond to DNA damage, termed the DNA damage checkpoint. The serine/threonine kinases ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR) activate checkpoint signaling by phosphorylating substrate proteins at SQ/TQ motifs. Although some ATM/ATR substrates (Chk1, p53) have been identified, the lack of a more complete list of substrates limits current understanding of checkpoint pathways. Here, we use immunoaffinity phosphopeptide isolation coupled with mass spectrometry to identify 570 sites phosphorylated in UV-damaged cells, 498 of which are previously undescribed. Semiquantitative analysis yielded 24 known and 192 previously uncharacterized sites differentially phosphorylated upon UV damage, some of which were confirmed by SILAC, Western blotting, and immunoprecipitation/Western blotting. ATR-specific phosphorylation was investigated by using a Seckel syndrome (ATR mutant) cell line. Together, these results provide a rich resource for further deciphering ATM/ATR signaling and the pathways mediating the DNA damage response.
TL;DR: It is shown that transgene transcripts at centromeric repeats are processed into siRNAs and are therefore direct targets of RNAi, and that Cid14, a member of the Trf4/5 family of poly(A) polymerases, has poly( A) polymerase activity that is required for heterochromatic gene silencing.
TL;DR: The acquired enamel pellicle is a thin protein film forming upon exposure of tooth enamel surfaces to saliva and the structural analysis of this integument relies on efficient pellicles harvesting and protein identification procedures.
Abstract: The acquired enamel pellicle is a thin protein film forming upon exposure of tooth enamel surfaces to saliva. The structural analysis of this integument relies on efficient pellicle harvesting and protein identification procedures. Material from three individual subjects and two pooled samples yielded the identification by LC−ESI−MS/MS of 130 pellicle proteins of which 89 were found in three or more experiments. A high intersubject consistency in pellicle composition was observed. Keywords: acquired enamel pellicle • proteomics • oral • mass spectrometry • LC−ESI−MS/MS • linear ion trap • proteins • saliva
TL;DR: It is shown that Cdk1 has an unexpected role in controlling bud growth after bud emergence and that G1 cyclin–Cdk1 complexes specifically phosphorylate multiple proteins associated with Cdc24, the guanine nucleotide-exchange factor that activates the Cdc42 GTPase.
Abstract: The mechanisms that control cell growth during the cell cycle are poorly understood. In budding yeast, cyclin dependent kinase 1 (Cdk1) triggers polarization of the actin cytoskeleton and bud emergence in late G1 through activation of the Cdc42 GTPase. However, Cdk1 is not thought to be required for subsequent growth of the bud. Here, we show that Cdk1 has an unexpected role in controlling bud growth after bud emergence. Moreover, we show that G1 cyclin-Cdk1 complexes specifically phosphorylate multiple proteins associated with Cdc24, the guanine nucleotide-exchange factor (GEF) that activates the Cdc42 GTPase. A mutant form of a Cdc24-associated protein that fails to undergo Cdk1-dependent phosphorylation causes defects in bud growth. These results provide a direct link between Cdk1 activity and the control of polarized cell growth.
TL;DR: The first proteomic analysis of human e-CSF is reported and it is found that a large collection of protease inhibitors, extracellular matrix proteins, and transport proteins in CSF are identified, however, a surprising suite of signaling and intracellular proteins not predicted by previous proteomics analysis are found.
Abstract: During vertebrate central nervous system development, the apical neuroepithelium is bathed with embryonic Cerebrospinal Fluid (e-CSF) which plays regulatory roles in cortical cell proliferation and maintenance. Here, we report the first proteomic analysis of human e-CSF and compare it to an extensive proteomic analysis of rat e-CSF. As expected, we identified a large collection of protease inhibitors, extracellular matrix proteins, and transport proteins in CSF. However, we also found a surprising suite of signaling and intracellular proteins not predicted by previous proteomic analysis. Some of the intracellular proteins are likely to represent the contents of microvesicles recently described within the CSF (Marzesco, A. M., et al. J. Cell Sci. 2005, 118 (Pt. 13), 2849-2858). Defining the rich composition of e-CSF will enable a greater understanding of its concerted actions during critical stages of brain development.
TL;DR: It is shown that CYLD is also required for timely entry into mitosis, and it is proposed that this additional function of CYLD could provide an explanation for the benign nature of most cylindroma lesions.
Abstract: Mutations in the cylindromatosis (CYLD) gene cause benign tumors of skin appendages, referred to as cylindromas. The CYLD gene encodes a deubiquitinating enzyme that removes Lys-63-linked ubiquitin chains from I kappa B kinase signaling components and thereby inhibits NF-kappaB pathway activation. The dysregulation of NF-kappaB activity has been proposed to promote cell transformation in part by increasing apoptosis resistance, but it is not clear whether this is CYLD's only or predominant tumor-suppressing function. Here, we show that CYLD is also required for timely entry into mitosis. Consistent with a cell-cycle regulatory function, CYLD localizes to microtubules in interphase and the midbody during telophase, and its protein levels decrease as cells exit from mitosis. We identified the protein kinase Plk1 as a potential target of CYLD in the regulation of mitotic entry, based on their physical interaction and similar loss-of-function and overexpression phenotypes. Our findings raise the possibility that, as with other genes regulating tumorigenesis, CYLD has not only tumor-suppressing (apoptosis regulation) but also tumor-promoting activities (enhancer of mitotic entry). We propose that this additional function of CYLD could provide an explanation for the benign nature of most cylindroma lesions.
TL;DR: It is shown that slicer activity is required for the siRNA-dependent association of Ago1 with chromatin and for the spreading of histone H3-K9 methylation.
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.
TL;DR: It is reported here that MVP is rapidly recruited to lipid rafts when human lung epithelial cells are infected with Pseudomonas aeruginosa, and maximal recruitment is dependent on bacterial binding to the cystic fibrosis transmembrane conductance regulator.
Abstract: The airway epithelium plays an essential role in innate immunity to lung pathogens. Ribonucleoprotein particles primarily composed of major vault protein (MVP) are highly expressed in cells that encounter xenobiotics. However, a clear biologic function for MVP is not established. We report here that MVP is rapidly recruited to lipid rafts when human lung epithelial cells are infected with Pseudomonas aeruginosa, and maximal recruitment is dependent on bacterial binding to the cystic fibrosis transmembrane conductance regulator. MVP was also essential for optimal epithelial cell internalization and clearance of P. aeruginosa. These results suggest that MVP makes a substantial contribution to epithelial cell-mediated resistance to infection.
TL;DR: This work demonstrates that Mek1 is activated in response to DSBs by autophosphorylation of two conserved threonines, T327 and T331, in the Mek1 activation loop.
Abstract: Mek1 is a meiosis-specific kinase in budding yeast which promotes recombination between homologous chromosomes by suppressing double-strand break (DSB) repair between sister chromatids. Previous work has shown that in the absence of the meiosis-specific recombinase gene, DMC1, cells arrest in prophase due to unrepaired DSBs and that Mek1 kinase activity is required in this situation to prevent repair of the breaks using sister chromatids. This work demonstrates that Mek1 is activated in response to DSBs by autophosphorylation of two conserved threonines, T327 and T331, in the Mek1 activation loop. Using a version of Mek1 that can be conditionally dimerized during meiosis, Mek1 function was shown to be promoted by dimerization, perhaps as a way of enabling autophosphorylation of the activation loop in trans. A putative HOP1-dependent dimerization domain within the C terminus of Mek1 has been identified. Dimerization alone, however, is insufficient for activation, as DSBs and Mek1 recruitment to the meiosis-specific chromosomal core protein Red1 are also necessary. Phosphorylation of S320 in the activation loop inhibits sister chromatid repair specifically in dmc1-arrested cells. Ectopic dimerization of Mek1 bypasses the requirement for S320 phosphorylation, suggesting this phosphorylation is necessary for maintenance of Mek1 dimers during checkpointinduced arrest.
TL;DR: It is demonstrated that two related human proteins (MVB12A and MVB12B) constitute the fourth class of metazoan ESCRT-I subunits, despite lacking identifiable sequence homology to Mvb12p, and indicate that the MVB 12 subunits play a unique role in regulating ESC RT-mediated virus budding.
TL;DR: These findings suggest the importance of both enzymatic and nonenzymatic roles of spLsd1 in regulating heterochromatin propagation and euchromatic transcription and also suggest that misregulation of sp lsd1/2 is likely to impact the epigenetic state of the cell.
01 Jan 2007
TL;DR: The SecY complex associates with the ribosome to form a protein translocation channel in the bacterial plasma membrane to capture and translocates the nascent chain.
Abstract: The SecY complex associates with the ribosome to form a protein translocation channel in the bacterial plasma membrane. We have used cryo-electron microscopy and quantitative mass spectrometry to show that a nontranslating E. coli ribosome binds to a single SecY complex. The crystal structure of an archaeal SecY complex was then docked into the electron density maps. In the resulting model, two cytoplasmic loops of SecY extend into the exit tunnel near proteins L23, L29, and L24. The loop between transmembrane helices 8 and 9 interacts with helices H59 and H50 in the large subunit RNA, while the 6/7 loop interacts with H7. We also show that point mutations of basic residues within either loop abolish ribosome binding. We suggest that SecY binds to this primary site on the ribosome and subsequently captures and translocates the nascent chain.
TL;DR: In this paper, it was shown that the SecY complex associates with the ribosome to form a protein translocation channel in the bacterial plasma membrane, and point mutations of basic residues within either loop abolish ribosomal binding.
TL;DR: A dominant-negative version of CHMP3, which specifically prevents targeting of AMSH to endosomes, inhibits degradation but not internalization of EGFR, suggesting that endosomal AMSH is a functional component of the multivesicular body pathway.
TL;DR: It is suggested that caveolae may have a function of particular use for the fat cell, namely the modulation of fatty acid flux across the plasma membrane.
TL;DR: It is found that the combination of highly accurate precursor masses generated from one survey scan in the FT-ICR cell, coupled with ten data-dependent tandem MS scans in a lower-resolution linear ion trap, provides more identifications in both mixtures than the other examined methods.
Abstract: Proteomic analyses via tandem mass spectrometry have been greatly enhanced by the recent development of fast, highly accurate instrumentation. However, successful application of these developments to high-throughput experiments requires careful optimization of many variables which adversely affect each other, such as mass accuracy and data collection speed. We examined the performance of three shotgun-style acquisition methods ranging in their data collection speed and use of mass accuracy in identifying proteins from yeast-derived complex peptide and phosphopeptide-enriched mixtures. We find that the combination of highly accurate precursor masses generated from one survey scan in the FT-ICR cell, coupled with ten data-dependent tandem MS scans in a lower-resolution linear ion trap, provides more identifications in both mixtures than the other examined methods. For phosphopeptide identifications in particular, this method identified over twice as many unique phosphopeptides as the second-ranked, lower-resolution method from triplicate 90-min analyses (744 ± 50 vs. 308 ± 50, respectively). We also examined the performance of four popular peptide assignment algorithms (Mascot, Sequest, OMSSA, and Tandem) in analyzing the results from both high-and low-resolution data. When compared in the context of a false positive rate of approximately 1%, the performance differences between algorithms were much larger for phosphopeptide analyses than for an unenriched, complex mixture. Based upon these findings, acquisition speed, mass accuracy, and the choice of assignment algorithm all largely affect the number of peptides and proteins identified in high-throughput studies.
TL;DR: This chapter discusses the development of an AQUA method and demonstrates its usefulness in the measurement of endogenous levels of the human protein separase at a functionally relevant phosphorylation site, serine 1126.
Abstract: The absolute quantification (AQUA) strategy provides a means to determine the precise protein or modified protein levels directly from cells or tissues The technique is based on two major principles: stable isotope dilution theory and the use of synthetic peptides containing such stable isotopes to exactly mimic native counterparts after proteolysis These peptides can be synthesized with modifications such as phosphorylation methylation, and acetylation to allow for the direct, quantitative analysis of posttranslationally modified proteins In this chapter, we discuss the development of an AQUA method and demonstrate its usefulness in the measurement of endogenous levels of the human protein separase at a functionally relevant phosphorylation site, serine 1126