Peptide name : FACT complex subunit SSRP1

Source/Organism : Human

Linear/Cyclic : Not found

Chirality : Not found

Peptide length: 709

C-terminal modification: Not found

N-terminal modification : Not found

Non-natural peptide information: None

Assay type : Antibody-based assay

Assay time : 48h

Activity : Not found

Cell line : HEK293

Cancer type : Not specified

Other activity : Not found

Amino acid composition bar chart :

Molecular mass : 81073.8677 Dalton

Aliphatic index : 0.606

Instability index : 46.9639

Hydrophobicity (GRAVY) : -0.921

Isoelectric point : 6.4466

Charge (pH 7) : -3.256

Aromaticity : 0.088

Molar extinction coefficient (cysteine, cystine): (65780, 66155)

Hydrophobic/hydrophilic ratio : 0.64801864

hydrophobic moment : -0.025

Missing amino acid : None

Most occurring amino acid : K

Most occurring amino acid frequency : 76

Least occurring amino acid : n

Least occurring amino acid frequency : 2

Secondary structure fraction (Helix, Turn, Sheet): (0.3, 0.3, 0.2)

SMILES Notation: 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Paper title : A Herpesviral Immediate Early Protein Promotes Transcription Elongation of Viral Transcripts.

Doi : https://doi.org/10.1128/mBio.00745-17

Abstract : Herpes simplex virus 1 (HSV-1) genes are transcribed by cellular RNA polymerase II (RNA Pol II). While four viral immediate early proteins (ICP4, ICP0, ICP27, and ICP22) function in some capacity in viral transcription, the mechanism by which ICP22 functions remains unclear. We observed that the FACT complex (comprised of SSRP1 and Spt16) was relocalized in infected cells as a function of ICP22. ICP22 was also required for the association of FACT and the transcription elongation factors SPT5 and SPT6 with viral genomes. We further demonstrated that the FACT complex interacts with ICP22 throughout infection. We therefore hypothesized that ICP22 recruits cellular transcription elongation factors to viral genomes for efficient transcription elongation of viral genes. We reevaluated the phenotype of an ICP22 mutant virus by determining the abundance of all viral mRNAs throughout infection by transcriptome sequencing (RNA-seq). The accumulation of almost all viral mRNAs late in infection was reduced compared to the wild type, regardless of kinetic class. Using chromatin immunoprecipitation sequencing (ChIP-seq), we mapped the location of RNA Pol II on viral genes and found that RNA Pol II levels on the bodies of viral genes were reduced in the ICP22 mutant compared to wild-type virus. In contrast, the association of RNA Pol II with transcription start sites in the mutant was not reduced. Taken together, our results indicate that ICP22 plays a role in recruiting elongation factors like the FACT complex to the HSV-1 genome to allow for efficient viral transcription elongation late in viral infection and ultimately infectious virion production.IMPORTANCE HSV-1 interacts with many cellular proteins throughout productive infection. Here, we demonstrate the interaction of a viral protein, ICP22, with a subset of cellular proteins known to be involved in transcription elongation. We determined that ICP22 is required to recruit the FACT complex and other transcription elongation factors to viral genomes and that in the absence of ICP22 viral transcription is globally reduced late in productive infection, due to an elongation defect. This insight defines a fundamental role of ICP22 in HSV-1 infection and elucidates the involvement of cellular factors in HSV-1 transcription.

Paper title : Lys-N and trypsin cover complementary parts of the phosphoproteome in a refined SCX-based approach.

Doi : https://doi.org/10.1021/ac9004309

Abstract : The analysis of proteome-wide phosphorylation events is still a major analytical challenge because of the enormous complexity of protein phosphorylation networks. In this work, we evaluate the complementarity of Lys-N, Lys-C, and trypsin with regard to their ability to contribute to the global analysis of the phosphoproteome. A refined version of low-pH strong cation exchange was used to efficiently separate N-terminally acetylated, phosphorylated, and nonmodified peptides. A total of 5036 nonredundant phosphopeptides could be identified with a false discovery rate of <1% from 1 mg of protein using a combination of the three enzymes. Our data revealed that the overlap between the phosphopeptide data sets generated with different proteases was marginal, whereas the overlap between two similarly generated tryptic data sets was found to be at least 4 times higher. In this way, the parallel use of Lys-N and trypsin enabled a 72% increase in the number of detected phosphopeptides as compared to trypsin alone, whereas a trypsin replicate experiment only led to a 25% increase. Thus, when focusing solely on the trypsin and Lys-N data, we identified 4671 nonredundant phosphopeptides. Further analysis of the detected sites showed that the Lys-N and trypsin data sets were enriched in significantly different phosphorylation motifs, further evidencing that multiprotease approaches are very valuable in phosphoproteome analyses.

Paper title : UIF, a New mRNA export adaptor that works together with REF/ALY, requires FACT for recruitment to mRNA.

Doi : https://doi.org/10.1016/j.cub.2009.09.041

Abstract : Messenger RNA (mRNA) export adaptors play an important role in the transport of mRNA from the nucleus to the cytoplasm. They couple early mRNA processing events such as 5' capping and 3' end formation with loading of the TAP/NXF1 export receptor onto mRNA. The canonical adaptor REF/ALY/Yra1 is recruited to mRNA via UAP56 and subsequently delivers the mRNA to NXF1 [1]. Knockdown of UAP56 [2, 3] and NXF1 [4-7] in higher eukaryotes efficiently blocks mRNA export, whereas knockdown of REF only causes a modest reduction, suggesting the existence of additional adaptors [8-10]. Here we identify a new UAP56-interacting factor, UIF, which functions as an export adaptor, binding NXF1 and delivering mRNA to the nuclear pore. REF and UIF are simultaneously found on the same mRNA molecules, and both proteins are required for efficient export of mRNA. We show that the histone chaperone FACT specifically binds UIF, but not REF, via the SSRP1 subunit, and this interaction is required for recruitment of UIF to mRNA. Together the results indicate that REF and UIF represent key human adaptors for the export of cellular mRNAs via the UAP56-NXF1 pathway.

Paper title : FACT, a factor that facilitates transcript elongation through nucleosomes.

Doi : https://doi.org/10.1016/s0092-8674(00)80903-4

Abstract : The requirements for transcriptional activation by RNA polymerase II were examined using chromatin templates assembled in vitro and a transcription system composed of the human general transcription factors and RNA polymerase II. Activator-induced, energy-dependent chromatin remodeling promoted efficient preinitiation complex formation and transcription initiation, but was not sufficient for productive transcription. Polymerases that initiated transcription on remodeled chromatin templates encountered a block to transcription proximal to the promoter. Entry into productive transcription required an accessory factor present in HeLa cell nuclear extract, FACT (facilitates chromatin transcription), which we have purified. FACT acts subsequent to transcription initiation to release RNA polymerase II from a nucleosome-induced block to productive transcription. The biochemical properties and polypeptide composition of FACT suggest that it is a novel protein factor that facilitates transcript elongation through nucleosomes.

Paper title : FACT facilitates transcription-dependent nucleosome alteration.

Doi : https://doi.org/10.1126/science.1085703

Abstract : The FACT (facilitates chromatin transcription) complex is required for transcript elongation through nucleosomes by RNA polymerase II (Pol II) in vitro. Here, we show that FACT facilitates Pol II-driven transcription by destabilizing nucleosomal structure so that one histone H2A-H2B dimer is removed during enzyme passage. We also demonstrate that FACT possesses intrinsic histone chaperone activity and can deposit core histones onto DNA. Importantly, FACT activity requires both of its constituent subunits and is dependent on the highly acidic C terminus of its larger subunit, Spt16. These findings define the mechanism by which Pol II can transcribe through chromatin without disrupting its epigenetic status.

Paper title : The Flexible Ends of CENP-A Nucleosome Are Required for Mitotic Fidelity.

Doi : https://doi.org/10.1016/j.molcel.2016.06.023

Abstract : CENP-A is a histone variant, which replaces histone H3 at centromeres and confers unique properties to centromeric chromatin. The crystal structure of CENP-A nucleosome suggests flexible nucleosomal DNA ends, but their dynamics in solution remains elusive and their implication in centromere function is unknown. Using electron cryo-microscopy, we determined the dynamic solution properties of the CENP-A nucleosome. Our biochemical, proteomic, and genetic data reveal that higher flexibility of DNA ends impairs histone H1 binding to the CENP-A nucleosome. Substituting the 2-turn αN-helix of CENP-A with the 3-turn αN-helix of H3 results in compact particles with rigidified DNA ends, able to bind histone H1. In vivo replacement of CENP-A with H3-CENP-A hybrid nucleosomes leads to H1 recruitment, delocalization of kinetochore proteins, and significant mitotic and cytokinesis defects. Our data reveal that the evolutionarily conserved flexible ends of the CENP-A nucleosomes are essential to ensure the fidelity of the mitotic pathway.

Paper title : Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.

Doi : https://doi.org/10.1016/j.cell.2006.09.026

Abstract : Cell signaling mechanisms often transmit information via posttranslational protein modifications, most importantly reversible protein phosphorylation. Here we develop and apply a general mass spectrometric technology for identification and quantitation of phosphorylation sites as a function of stimulus, time, and subcellular location. We have detected 6,600 phosphorylation sites on 2,244 proteins and have determined their temporal dynamics after stimulating HeLa cells with epidermal growth factor (EGF) and recorded them in the Phosida database. Fourteen percent of phosphorylation sites are modulated at least 2-fold by EGF, and these were classified by their temporal profiles. Surprisingly, a majority of proteins contain multiple phosphorylation sites showing different kinetics, suggesting that they serve as platforms for integrating signals. In addition to protein kinase cascades, the targets of reversible phosphorylation include ubiquitin ligases, guanine nucleotide exchange factors, and at least 46 different transcriptional regulators. The dynamic phosphoproteome provides a missing link in a global, integrative view of cellular regulation.

Paper title : Nek9, a novel FACT-associated protein, modulates interphase progression.

Doi : https://doi.org/10.1074/jbc.M311477200

Abstract : The heterodimeric Spt16-Pob3/DUF/FACT complex is a class of chromatin structure modulators with important roles in replication and transcription. Although regarded as a transcription elongator for chromatin template, little is known about the mode of action and involvement in other molecular processes of the mammalian FACT. Here we report the identification of a novel interacting and functional partner of FACT, Nek9. Nek9 forms a stable, approximately 600-kDa complex with FACT in the interphase nuclei. Its active form is characterized by phosphorylation-dependent electrophoretic mobility shift and phosphorylation at a conserved residue within the activation loop (Thr(210)). When complexed with FACT, Nek9 exhibits markedly elevated phosphorylation on Thr(210). Cell cycle analysis on the Nek9(dsRNAi) cells directly implicated Nek9 in maintaining proper G(1) and S progression, a role temporally correlated to the formation of a phospho-Nek9-FACT complex. Collectively, these observations provide evidence that Nek9, potentially as an active enzymatic partner of FACT, mediates certain FACT-associated cellular processes, which are ultimately essential for interphase progression.

Paper title : SSRP1 functions as a co-activator of the transcriptional activator p63.

Doi : https://doi.org/10.1093/emboj/cdf540

Abstract : The p53 homolog p63 is a transcriptional activator. Here, we describe the identification of an HMG1-like protein SSRP1 as a co-activator of p63. Over expression of wild-type, but not deletion mutant, SSRP1 remarkably enhanced p63gamma-dependent luciferase activity, G1 arrest, apoptosis and expression of endogenous PIG3, p21(Waf1/cip1) and MDM2 in human p53-deficient lung carcinoma H1299 cells and mouse embryonic fibroblasts. Also, SSRP1 interacted to p63gamma in vitro and in cells, and resided with p63gamma at the p53-responsive DNA element sites of the cellular endogenous MDM2 and p21(Waf1/cip1) promoters. Moreover, N-terminus-deleted p63 (DeltaN-p63) bound to neither SSRP1 nor its central domain in vitro. Accordingly, SSRP1 was unable to stimulate DeltaN-p63-mediated residual luciferase activity and apoptosis in cells. Finally, the ectopic expression of the central p63-binding domain of SSRP1 inhibited p63-dependent transcription in cells. Thus, these results suggest that SSRP1 stimulates p63 activity by associating with this activator at the promoter.

Paper title : Interaction of FACT, SSRP1, and the high mobility group (HMG) domain of SSRP1 with DNA damaged by the anticancer drug cisplatin.

Doi : https://doi.org/10.1074/jbc.M101208200

Abstract : The structure-specific recognition protein SSRP1, initially isolated from expression screening of a human B-cell cDNA library for proteins that bind to cisplatin (cis-diamminedichloroplatinum(II))-modified DNA, contains a single DNA-binding high mobility group (HMG) domain. Human SSRP1 purifies as a heterodimer of SSRP1 and Spt16 (FACT) that alleviates the nucleosomal block to transcription elongation by RNAPII in vitro. The affinity and specificity of FACT, SSRP1, and the isolated HMG domain of SSRP1 for cisplatin-damaged DNA were investigated by gel mobility shift assays. FACT exhibits both affinity and specificity for DNA damaged globally with cisplatin compared with unmodified DNA or DNA damaged globally with the clinically ineffective trans-DDP isomer. FACT binds the major 1,2-d(GpG) intrastrand cisplatin adduct, but its isolated SSRP1 subunit fails to form discrete, high affinity complexes with cisplatin-modified DNA under similar conditions. These results suggest that Spt16 primes SSRP1 for cisplatin-damaged DNA recognition by unveiling its HMG domain. As expected, the isolated HMG domain of SSRP1 is sufficient for specific binding to cisplatin-damaged DNA and binds the major cisplatin 1,2-d(GpG) intrastrand cross-link. The affinity and specificity of FACT for cisplatin-modified DNA, as well as its importance for transcription of chromatin, suggests that the interaction of FACT and cisplatin-damaged DNA may be crucial to the anticancer mechanism of cisplatin.

Paper title : Systematic analysis of protein pools, isoforms, and modifications affecting turnover and subcellular localization.

Doi : https://doi.org/10.1074/mcp.M111.013680

Abstract : In higher eukaryotes many genes encode protein isoforms whose properties and biological roles are often poorly characterized. Here we describe systematic approaches for detection of either distinct isoforms, or separate pools of the same isoform, with differential biological properties. Using information from ion intensities we have estimated protein abundance levels and using rates of change in stable isotope labeling with amino acids in cell culture isotope ratios we measured turnover rates and subcellular distribution for the HeLa cell proteome. Protein isoforms were detected using three data analysis strategies that evaluate differences between stable isotope labeling with amino acids in cell culture isotope ratios for specific groups of peptides within the total set of peptides assigned to a protein. The candidate approach compares stable isotope labeling with amino acids in cell culture isotope ratios for predicted isoform-specific peptides, with ratio values for peptides shared by all the isoforms. The rule of thirds approach compares the mean isotope ratio values for all peptides in each of three equal segments along the linear length of the protein, assessing differences between segment values. The three in a row approach compares mean isotope ratio values for each sequential group of three adjacent peptides, assessing differences with the mean value for all peptides assigned to the protein. Protein isoforms were also detected and their properties evaluated by fractionating cell extracts on one-dimensional SDS-PAGE prior to trypsin digestion and MS analysis and independently evaluating isotope ratio values for the same peptides isolated from different gel slices. The effect of protein phosphorylation on turnover rates was analyzed by comparing mean turnover values calculated for all peptides assigned to a protein, either including, or excluding, values for cognate phosphopeptides. Collectively, these experimental and analytical approaches provide a framework for expanding the functional annotation of the genome.

Paper title : Initial characterization of the human central proteome.

Doi : https://doi.org/10.1186/1752-0509-5-17

Abstract : BACKGROUND: On the basis of large proteomics datasets measured from seven human cell lines we consider their intersection as an approximation of the human central proteome, which is the set of proteins ubiquitously expressed in all human cells. Composition and properties of the central proteome are investigated through bioinformatics analyses. RESULTS: We experimentally identify a central proteome comprising 1,124 proteins that are ubiquitously and abundantly expressed in human cells using state of the art mass spectrometry and protein identification bioinformatics. The main represented functions are proteostasis, primary metabolism and proliferation. We further characterize the central proteome considering gene structures, conservation, interaction networks, pathways, drug targets, and coordination of biological processes. Among other new findings, we show that the central proteome is encoded by exon-rich genes, indicating an increased regulatory flexibility through alternative splicing to adapt to multiple environments, and that the protein interaction network linking the central proteome is very efficient for synchronizing translation with other biological processes. Surprisingly, at least 10% of the central proteome has no or very limited functional annotation. CONCLUSIONS: Our data and analysis provide a new and deeper description of the human central proteome compared to previous results thereby extending and complementing our knowledge of commonly expressed human proteins. All the data are made publicly available to help other researchers who, for instance, need to compare or link focused datasets to a common background.

Paper title : The HMG domain protein SSRP1/PREIIBF is involved in activation of the human embryonic beta-like globin gene.

Doi : https://doi.org/10.1128/MCB.18.5.2617

Abstract : The human embryonic beta-like globin (epsilon-globin) gene is expressed in primitive erythroid cells of the yolk sac during the first few weeks of development. We have previously shown that developmental stage-specific expression of the epsilon-globin gene is mediated by multiple positive and negative regulatory elements upstream of the start of transcription. Of particular interest is one positive regulatory element, PRE II, that works together with other elements (PRE I and PRE V) to confer developmental stage- and/or tissue-specific expression on a minimal promoter. An approximately 85- to 90-kDa PRE II binding factor (PREIIBF) was identified in the nuclei of erythroid cells and shown to bind specifically to a novel 19-bp region within PRE II; binding of this protein to PRE II resulted in bending of the target DNA and was required for promoter activation. In this report, we present the cDNA expression cloning of PREIIBF. The cDNA encodes a previously identified member of the HMG domain family of DNA binding proteins termed SSRP1. By a number of biochemical and immunological criteria, recombinant SSRP1 appears to be identical to the PREII binding factor from erythroid nuclei. A hallmark of HMG domain proteins is their ability to bend their target DNAs; therefore, as we speculated previously, DNA bending by SSRP1/PREIIBF may contribute to the mechanism by which PRE II synergizes with other regulatory elements located upstream and downstream. In contrast with reports from other investigators, we demonstrate that SSRP1 binds DNA with clear sequence specificity. Moreover, we show that SSRP1/PREIIBF lacks a classical activation domain but that binding by this protein to PRE II is required for activation of a minimal promoter in stable erythroid cell lines. These studies provide the first evidence that SSRP1 plays a role in transcriptional regulation. SSRP1/PREIIBF may serve an architectural function by helping to coordinate the assembly of a multiprotein complex required for stage-specific regulation of the human epsilon-globin gene.

Paper title : Comparative large scale characterization of plant versus mammal proteins reveals similar and idiosyncratic N-α-acetylation features.

Doi : https://doi.org/10.1074/mcp.M111.015131

Abstract : N-terminal modifications play a major role in the fate of proteins in terms of activity, stability, or subcellular compartmentalization. Such modifications remain poorly described and badly characterized in proteomic studies, and only a few comparison studies among organisms have been made available so far. Recent advances in the field now allow the enrichment and selection of N-terminal peptides in the course of proteome-wide mass spectrometry analyses. These targeted approaches unravel as a result the extent and nature of the protein N-terminal modifications. Here, we aimed at studying such modifications in the model plant Arabidopsis thaliana to compare these results with those obtained from a human sample analyzed in parallel. We applied large scale analysis to compile robust conclusions on both data sets. Our data show strong convergence of the characterized modifications especially for protein N-terminal methionine excision, co-translational N-α-acetylation, or N-myristoylation between animal and plant kingdoms. Because of the convergence of both the substrates and the N-α-acetylation machinery, it was possible to identify the N-acetyltransferases involved in such modifications for a small number of model plants. Finally, a high proportion of nuclear-encoded chloroplast proteins feature post-translational N-α-acetylation of the mature protein after removal of the transit peptide. Unlike animals, plants feature in a dedicated pathway for post-translational acetylation of organelle-targeted proteins. The corresponding machinery is yet to be discovered.

Paper title : Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis.

Doi : https://doi.org/10.1126/scisignal.2000475

Abstract : Eukaryotic cells replicate by a complex series of evolutionarily conserved events that are tightly regulated at defined stages of the cell division cycle. Progression through this cycle involves a large number of dedicated protein complexes and signaling pathways, and deregulation of this process is implicated in tumorigenesis. We applied high-resolution mass spectrometry-based proteomics to investigate the proteome and phosphoproteome of the human cell cycle on a global scale and quantified 6027 proteins and 20,443 unique phosphorylation sites and their dynamics. Co-regulated proteins and phosphorylation sites were grouped according to their cell cycle kinetics and compared to publicly available messenger RNA microarray data. Most detected phosphorylation sites and more than 20% of all quantified proteins showed substantial regulation, mainly in mitotic cells. Kinase-motif analysis revealed global activation during S phase of the DNA damage response network, which was mediated by phosphorylation by ATM or ATR or DNA-dependent protein kinases. We determined site-specific stoichiometry of more than 5000 sites and found that most of the up-regulated sites phosphorylated by cyclin-dependent kinase 1 (CDK1) or CDK2 were almost fully phosphorylated in mitotic cells. In particular, nuclear proteins and proteins involved in regulating metabolic processes have high phosphorylation site occupancy in mitosis. This suggests that these proteins may be inactivated by phosphorylation in mitotic cells.

Paper title : Histone H2B monoubiquitination functions cooperatively with FACT to regulate elongation by RNA polymerase II.

Doi : https://doi.org/10.1016/j.cell.2006.04.029

Abstract : Over the past years, a large number of histone posttranslational modifications have been described, some of which function to attain a repressed chromatin structure, while others facilitate activation by allowing access of regulators to DNA. Histone H2B monoubiquitination is a mark associated with transcriptional activity. Using a highly reconstituted chromatin-transcription system incorporating the inducible RARbeta2 promoter, we find that the establishment of H2B monoubiquitination by RNF20/40 and UbcH6 is dependent on the transcription elongation regulator complex PAF, the histone chaperone FACT, and transcription. H2B monoubiquitination facilitates FACT function, thereby stimulating transcript elongation and the generation of longer transcripts. These in vitro analyses and corroborating in vivo experiments demonstrate that elongation by RNA polymerase II through the nucleosomal barrier is minimally dependent upon (1) FACT and (2) the recruitment of PAF and the H2B monoubiquitination machinery.

Paper title : Requirement of RSF and FACT for transcription of chromatin templates in vitro.

Doi : https://doi.org/10.1126/science.282.5395.1900

Abstract : Transcription of naked DNA in vitro requires the general transcription factors and RNA polymerase II. However, this minimal set of factors is not sufficient for transcription when the DNA template is packaged into chromatin. Here, a factor that facilitates activator-dependent transcription initiation on chromatin templates was purified. This factor, remodeling and spacing factor (RSF), has adenosine triphosphate-dependent nucleosome-remodeling and spacing activities. Polymerases that initiate transcription with RSF can only extend their transcripts in the presence of FACT (facilitates chromatin transcription). Thus, the minimal factor requirements for activator-dependent transcription on chromatin templates in vitro have been defined.

Paper title : Systematic identification and analysis of mammalian small ubiquitin-like modifier substrates.

Doi : https://doi.org/10.1074/jbc.M411718200

Abstract : Small ubiquitin-like modifier (SUMO) regulates diverse cellular processes through its reversible, covalent attachment to target proteins. Many SUMO substrates are involved in transcription and chromatin structure. Sumoylation appears to regulate the functions of target proteins by changing their subcellular localization, increasing their stability, and/or mediating their binding to other proteins. Using an in vitro expression cloning approach, we have identified 40 human SUMO1 substrates. The spectrum of human SUMO1 substrates identified in our screen suggests general roles of sumoylation in transcription, chromosome structure, and RNA processing. We have validated the sumoylation of 24 substrates in living cells. Analysis of this panel of SUMO substrates leads to the following observations. 1) Sumoylation is more efficient in vitro than in living cells. Polysumoylation occurs on several substrates in vitro. 2) SUMO isopeptidases have little substrate specificity. 3) The SUMO ligases, PIAS1 and PIASxbeta, have broader substrate specificities than does PIASy. 4) Although SUMO1 and SUMO2 are equally efficiently conjugated to a given substrate in vitro, SUMO1 conjugation is more efficient in vivo. 5) Most SUMO substrates localize to the nucleus, and sumoylation does not generally affect their subcellular localization. Therefore, sumoylation appears to regulate the functions of its substrates through multiple, context-dependent mechanisms.

Paper title : System-wide temporal characterization of the proteome and phosphoproteome of human embryonic stem cell differentiation.

Doi : https://doi.org/10.1126/scisignal.2001570

Abstract : To elucidate cellular events underlying the pluripotency of human embryonic stem cells (hESCs), we performed parallel quantitative proteomic and phosphoproteomic analyses of hESCs during differentiation initiated by a diacylglycerol analog or transfer to media that had not been conditioned by feeder cells. We profiled 6521 proteins and 23,522 phosphorylation sites, of which almost 50% displayed dynamic changes in phosphorylation status during 24 hours of differentiation. These data are a resource for studies of the events associated with the maintenance of hESC pluripotency and those accompanying their differentiation. From these data, we identified a core hESC phosphoproteome of sites with similar robust changes in response to the two distinct treatments. These sites exhibited distinct dynamic phosphorylation patterns, which were linked to known or predicted kinases on the basis of the matching sequence motif. In addition to identifying previously unknown phosphorylation sites on factors associated with differentiation, such as kinases and transcription factors, we observed dynamic phosphorylation of DNA methyltransferases (DNMTs). We found a specific interaction of DNMTs during early differentiation with the PAF1 (polymerase-associated factor 1) transcriptional elongation complex, which binds to promoters of the pluripotency and known DNMT target genes encoding OCT4 and NANOG, thereby providing a possible molecular link for the silencing of these genes during differentiation.

Paper title : CK2 phosphorylates SSRP1 and inhibits its DNA-binding activity.

Doi : https://doi.org/10.1074/jbc.M413944200

Abstract : We have previously shown that CK2 associates with the human high-mobility group protein SSRP1 and that this association increases in response to UV irradiation. CK2 also phosphorylates SSRP1 in vitro. Here we extend this work by investigating CK2 regulation of SSRP1 function through phosphorylation. Phosphorylation of SSRP1 by CK2 inhibited the nonspecific DNA-binding activity of SSRP1 and FACT (facilitating chromatin-mediated transcription) complex in vitro. Using a serine/threonine-scanning Auto-spot peptide array coupled with a filter-based kinase assay with synthetic peptides as substrates, we identified serines 510, 657, and 688 as phosphorylation targets of CK2 in vitro. Mutagenesis of the three serines revealed that serine 510 was more important for the regulation of SSRP1 DNA-binding activity. Furthermore, we found that SSRP1 was phosphorylated in cells in response to UV (but not gamma) irradiation. These results suggest that CK2 regulates the DNA-binding ability of SSRP1 and that this regulation may be responsive to specific cell stresses.

Paper title : Combining protein-based IMAC, peptide-based IMAC, and MudPIT for efficient phosphoproteomic analysis.

Doi : https://doi.org/10.1021/pr0705441

Abstract : Immobilized metal affinity chromatography (IMAC) is a common strategy used for the enrichment of phosphopeptides from digested protein mixtures. However, this strategy by itself is inefficient when analyzing complex protein mixtures. Here, we assess the effectiveness of using protein-based IMAC as a pre-enrichment step prior to peptide-based IMAC. Ultimately, we couple the two IMAC-based enrichments and MudPIT in a quantitative phosphoproteomic analysis of the epidermal growth factor pathway in mammalian cells identifying 4470 unique phosphopeptides containing 4729 phosphorylation sites.

Paper title : An enzyme assisted RP-RPLC approach for in-depth analysis of human liver phosphoproteome.

Doi : https://doi.org/10.1016/j.jprot.2013.11.014

Abstract : UNLABELLED: Protein phosphorylation is one of the most common post-translational modifications. It plays key roles in regulating diverse biological processes of liver tissues. To better understand the role of protein phosphorylation in liver functions, it is essential to perform in-depth phosphoproteome analysis of human liver. Here, an enzyme assisted reversed-phase-reversed-phase liquid chromatography (RP-RPLC) approach with both RPLC separations operated with optimized acidic mobile phase was developed. High orthogonal separation was achieved by trypsin digestion of the Glu-C generated peptides in the fractions collected from the first RPLC separation. The phosphoproteome coverage was further improved by using two types of instruments, i.e. TripleTOF 5600 and LTQ Orbitrap Velos. A total of 22,446 phosphorylation sites, corresponding to 6526 nonredundant phosphoproteins were finally identified from normal human liver tissues. Of these sites, 15,229 sites were confidently localized with Ascore≥13. This dataset was the largest phosphoproteome dataset of human liver. It can be a public resource for the liver research community and holds promise for further biology studies. BIOLOGICAL SIGNIFICANCE: The enzyme assisted approach enabled the two RPLC separations operated both with optimized acidic mobile phases. The identifications from TripleTOF 5600 and Orbitrap Velos are highly complementary. The largest phosphoproteome dataset of human liver was generated.

Paper title : Toward a comprehensive characterization of a human cancer cell phosphoproteome.

Doi : https://doi.org/10.1021/pr300630k

Abstract : Mass spectrometry (MS)-based phosphoproteomics has achieved extraordinary success in qualitative and quantitative analysis of cellular protein phosphorylation. Considering that an estimated level of phosphorylation in a cell is placed at well above 100,000 sites, there is still much room for improvement. Here, we attempt to extend the depth of phosphoproteome coverage while maintaining realistic aspirations in terms of available material, robustness, and instrument running time. We developed three strategies, where each provided a different balance between these three key parameters. The first strategy simply used enrichment by Ti(4+)-IMAC followed by reversed chromatography LC-MS (termed 1D). The second strategy incorporated an additional fractionation step through the use of HILIC (2D). Finally, a third strategy was designed employing first an SCX fractionation, followed by Ti(4+)-IMAC enrichment and additional fractionation by HILIC (3D). A preliminary evaluation was performed on the HeLa cell line. Detecting 3700 phosphopeptides in about 2 h, the 1D strategy was found to be the most sensitive but limited in comprehensivity, mainly due to issues with complexity and dynamic range. Overall, the best balance was achieved using the 2D based strategy, identifying close to 17,000 phosphopeptides with less than 1 mg of material in about 48 h. Subsequently, we confirmed the findings with the K562 cell sample. When sufficient material was available, the 3D strategy increased phosphoproteome allowing over 22,000 unique phosphopeptides to be identified. Unfortunately, the 3D strategy required more time and over 1 mg of material before it started to outperform 2D. Ultimately, combining all strategies, we were able to identify over 16,000 and nearly 24,000 unique phosphorylation sites from the cancer cell lines HeLa and K562, respectively. In summary, we demonstrate the need to carry out extensive fractionation for deep mining of the phosphoproteome and provide a guide for appropriate strategies depending on sample amount and/or analysis time.

Paper title : A quantitative atlas of mitotic phosphorylation.

Doi : https://doi.org/10.1073/pnas.0805139105

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.

Paper title : Lysine acetylation targets protein complexes and co-regulates major cellular functions.

Doi : https://doi.org/10.1126/science.1175371

Abstract : Lysine acetylation is a reversible posttranslational modification of proteins and plays a key role in regulating gene expression. Technological limitations have so far prevented a global analysis of lysine acetylation's cellular roles. We used high-resolution mass spectrometry to identify 3600 lysine acetylation sites on 1750 proteins and quantified acetylation changes in response to the deacetylase inhibitors suberoylanilide hydroxamic acid and MS-275. Lysine acetylation preferentially targets large macromolecular complexes involved in diverse cellular processes, such as chromatin remodeling, cell cycle, splicing, nuclear transport, and actin nucleation. Acetylation impaired phosphorylation-dependent interactions of 14-3-3 and regulated the yeast cyclin-dependent kinase Cdc28. Our data demonstrate that the regulatory scope of lysine acetylation is broad and comparable with that of other major posttranslational modifications.

Paper title : The chromatin-specific transcription elongation factor FACT comprises human SPT16 and SSRP1 proteins.

Doi : https://doi.org/10.1038/22350

Abstract : The regulation of gene expression depends critically upon chromatin structure. Transcription of protein-coding genes can be reconstituted on naked DNA with only the general transcription factors and RNA polymerase II. This minimal system cannot transcribe DNA packaged into chromatin, indicating that accessory factors may facilitate access to DNA. Two classes of accessory factor, ATP-dependent chromatin-remodelling enzymes and histone acetyltransferases, facilitate transcription initiation from chromatin templates. FACT (for facilitates chromatin transcription) is a chromatin-specific elongation factor required for transcription of chromatin templates in vitro. Here we show that FACT comprises a new human homologue of the Saccharomyces cerevisiae Spt16/Cdc68 protein and the high-mobility group-1-like protein structure-specific recognition protein-1. Yeast SPT16/CDC68 is an essential gene that has been implicated in transcription and cell-cycle regulation. Consistent with our biochemical analysis of FACT, we provide evidence that Spt16/Cdc68 is involved in transcript elongation in vivo. Moreover, FACT specifically interacts with nucleosomes and histone H2A/H2B dimers, indicating that it may work by promoting nucleosome disassembly upon transcription. In support of this model, we show that FACT activity is abrogated by covalently crosslinking nucleosomal histones.

Paper title : The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

Doi : https://doi.org/10.1101/gr.2596504

Abstract : The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

Paper title : Isolation and characterization of human cDNA clones encoding a high mobility group box protein that recognizes structural distortions to DNA caused by binding of the anticancer agent cisplatin.

Doi : https://doi.org/10.1073/pnas.89.6.2307

Abstract : Human cDNA clones encoding a structure-specific recognition protein, SSRP1, that binds specifically to DNA modified with cisplatin have been isolated and characterized. The SSRP1 gene maps to human chromosome 11q12. The cDNA clones, obtained by using partial-length cDNAs described previously, predict an 81-kDa protein containing several highly charged domains and a stretch of 75 amino acids 47% identical to a portion of the high mobility group (HMG) protein HMG1. This HMG box most likely constitutes the structure recognition element for cisplatin-modified DNA, with the probable recognition motif being the local duplex unwinding and bending toward the major groove that occurs upon formation of intrastrand cis-[Pt(NH3)2]2+ d(GpG) and d(ApG) cross-links. Although the DNA recognition properties of members of the HMG-box family of proteins have been characterized with respect to their sequence specificity, the present work demonstrates that proteins with this domain can recognize particular DNA structures as well. The Pt-DNA SSRP described here is the human homolog of a recently identified mouse protein that binds to recombination signal sequences [Shirakata, M., Hüppi, K., Usuda, S., Okazaki, K., Yoshida, K. & Sakano, H. (1991) Mol. Cell. Biol. 11, 4528-4536]. These sequences have been postulated to form stem-loop structures, further implicating local bends and unwinding in DNA as a recognition target for HMG-box proteins. Expression analysis in a variety of tissues and cisplatin-resistant cell lines and the inability of cisplatin to induce the message in HeLa cells argue against a direct link between SSRP1 mRNA levels and the response of cells to the drug.

Paper title : FACT relieves DSIF/NELF-mediated inhibition of transcriptional elongation and reveals functional differences between P-TEFb and TFIIH.

Doi : https://doi.org/10.1016/s1097-2765(00)80272-5

Abstract : We report that the chromatin-specific transcription elongation factor FACT functions in conjunction with the RNA polymerase II CTD kinase P-TEFb to alleviate transcription inhibition by DSIF (DRB sensitivity-inducing factor) and NELF (negative elongation factor). We find that the kinase activity of TFIIH is dispensable for this activity, demonstrating that TFIIH-mediated CTD phosphorylation is not involved in the regulation of FACT and DSIF/NELF activities. Thus, we propose a novel transcriptional regulatory network in which DSIF/NELF inhibition of transcription is prevented by P-TEFb in cooperation with FACT. This study uncovers a novel role for FACT in the regulation of transcription on naked DNA that is independent of its activities on chromatin templates. In addition, this study reveals functional differences between P-TEFb and TFIIH in the regulation of transcription.

Paper title : Coupling caspase cleavage and ubiquitin-proteasome-dependent degradation of SSRP1 during apoptosis.

Doi : https://doi.org/10.1038/sj.cdd.4401878

Abstract : Structure-specific recognition protein (SSRP1) is an 87 kDa protein that heterodimerizes with Spt16 to form FACT, a complex initially shown to facilitate chromatin transcription. Despite its crucial roles in transcription and replication, little is known about the dynamics of FACT turnover in vivo. Here, we show that SSRP1 is cleaved during apoptosis by caspase 3 and/or 7 at the DQHD(450) site. Analysis of the resulting fragments suggests that cleavage of SSRP1 generates a truncated, chromatin-associated form of FACT. Furthermore, the N-terminal product is stabilized by proteasome inhibitors and ubiquitylated in cells, suggesting degradation through the ubiquitin-proteasome pathway. These results demonstrate that SSRP1 degradation during apoptosis is a two-step process coupling caspase cleavage and ubiquitin-dependent proteolysis.

Paper title : Quantitative phosphoproteomic analysis of T cell receptor signaling reveals system-wide modulation of protein-protein interactions.

Doi : https://doi.org/10.1126/scisignal.2000007

Abstract : Protein phosphorylation events during T cell receptor (TCR) signaling control the formation of complexes among proteins proximal to the TCR, the activation of kinase cascades, and the activation of transcription factors; however, the mode and extent of the influence of phosphorylation in coordinating the diverse phenomena associated with T cell activation are unclear. Therefore, we used the human Jurkat T cell leukemia cell line as a model system and performed large-scale quantitative phosphoproteomic analyses of TCR signaling. We identified 10,665 unique phosphorylation sites, of which 696 showed TCR-responsive changes. In addition, we analyzed broad trends in phosphorylation data sets to uncover underlying mechanisms associated with T cell activation. We found that, upon stimulation of the TCR, phosphorylation events extensively targeted protein modules involved in all of the salient phenomena associated with T cell activation: patterning of surface proteins, endocytosis of the TCR, formation of the F-actin cup, inside-out activation of integrins, polarization of microtubules, production of cytokines, and alternative splicing of messenger RNA. Further, case-by-case analysis of TCR-responsive phosphorylation sites on proteins belonging to relevant functional modules together with network analysis allowed us to deduce that serine-threonine (S-T) phosphorylation modulated protein-protein interactions (PPIs) in a system-wide fashion. We also provide experimental support for this inference by showing that phosphorylation of tubulin on six distinct serine residues abrogated PPIs during the assembly of microtubules. We propose that modulation of PPIs by stimulus-dependent changes in S-T phosphorylation state is a widespread phenomenon applicable to many other signaling systems.

Paper title : p53 serine 392 phosphorylation increases after UV through induction of the assembly of the CK2.hSPT16.SSRP1 complex.

Doi : https://doi.org/10.1074/jbc.M209820200

Abstract : Previously, we purified a UV-responsive p53 serine 392 kinase from F9 and HeLa cells and found that its activity is attributed to a high molecular weight protein complex containing the protein kinase CK2, along with the chromatin-associated factors hSPT16 and SSRP1. Here we determine that these proteins interact in vitro and in cells via non-overlapping domains and provide evidence consistent with the idea that hSPT16 and SSRP1 change the conformation of CK2 upon binding such that it specifically targets p53 over other substrates. Also, UV irradiation apparently induces the association of the complex, thereby increasing the specificity of CK2 for p53 at the expense of other cellular CK2 substrates and leading to an overall increase in p53 serine 392 phosphorylation.

Paper title : High prevalence of autoantibodies against the nuclear high mobility group (HMG) protein SSRP1 in sera from patients with systemic lupus erythematosus, but not other rheumatic diseases.

Doi : https://doi.org/Not available

Abstract : OBJECTIVE: To evaluate the presence of autoantibodies to the high mobility group (HMG) structure specific recognition protein I (SSRP1) in sera from patients with systemic lupus erythematosus (SLE) or other rheumatic diseases. METHODS: Antibodies to SSRP1(anti-SSRP1) were measured in sera from patients with SLE, Sjogren's syndrome (SS), ulcerative colitis (UC), systemic sclerosis (SSc), rheumatoid arthritis (RA), and sera from healthy individuals by both an enzyme-linked immunoassay (ELISA) and Western blotting (WB) using the recombinant SSRP1 N-terminus as antigen. RESULTS: We found 28.8% of the sera from patients with SLE contained anti-SSRPI by both ELISA and WB assay, compared to 8.3% of the sera from healthy individuals. When the 40 sera from patients with other autoimmune diseases were tested, only 2 sera (5%) from individuals with SS showed a moderate reactivity to SSRPI in both ELISA and WB assays. CONCLUSION: The results show that anti-SSRPI can be identified in sera from patients with SLE, but not with other rheumatic diseases and may thus help the diagnosis of SLE in the presence of appropriate clinical findings.

Paper title : A DNA damage-induced p53 serine 392 kinase complex contains CK2, hSpt16, and SSRP1.

Doi : https://doi.org/10.1016/s1097-2765(01)00176-9

Abstract : Phosphorylation of the human p53 protein at Ser-392 has been shown to be responsive to UV but not gamma irradiation. Here we describe identification and purification of a mammalian UV-activated protein kinase complex that phosphorylates Ser-392 of p53 in vitro. This kinase complex contains casein kinase 2 (CK2) and the chromatin transcriptional elongation factor FACT (a heterodimer of hSpt16 and SSRP1). In vitro studies show that FACT alters the specificity of CK2 in the complex such that it selectively phosphorylates p53 over other substrates including casein. In addition, phosphorylation by the kinase complex enhances p53 activity. These results thus provide a potential mechanism for p53 activation by UV irradiation.

Paper title : N-terminal acetylome analyses and functional insights of the N-terminal acetyltransferase NatB.

Doi : https://doi.org/10.1073/pnas.1210303109

Abstract : Protein N-terminal acetylation (Nt-acetylation) is an important mediator of protein function, stability, sorting, and localization. Although the responsible enzymes are thought to be fairly well characterized, the lack of identified in vivo substrates, the occurrence of Nt-acetylation substrates displaying yet uncharacterized N-terminal acetyltransferase (NAT) specificities, and emerging evidence of posttranslational Nt-acetylation, necessitate the use of genetic models and quantitative proteomics. NatB, which targets Met-Glu-, Met-Asp-, and Met-Asn-starting protein N termini, is presumed to Nt-acetylate 15% of all yeast and 18% of all human proteins. We here report on the evolutionary traits of NatB from yeast to human and demonstrate that ectopically expressed hNatB in a yNatB-Δ yeast strain partially complements the natB-Δ phenotypes and partially restores the yNatB Nt-acetylome. Overall, combining quantitative N-terminomics with yeast studies and knockdown of hNatB in human cell lines, led to the unambiguous identification of 180 human and 110 yeast NatB substrates. Interestingly, these substrates included Met-Gln- N-termini, which are thus now classified as in vivo NatB substrates. We also demonstrate the requirement of hNatB activity for maintaining the structure and function of actomyosin fibers and for proper cellular migration. In addition, expression of tropomyosin-1 restored the altered focal adhesions and cellular migration defects observed in hNatB-depleted HeLa cells, indicative for the conserved link between NatB, tropomyosin, and actin cable function from yeast to human.

Paper title : Large-scale phosphoproteome analysis of human liver tissue by enrichment and fractionation of phosphopeptides with strong anion exchange chromatography.

Doi : https://doi.org/10.1002/pmic.200700884

Abstract : The mixture of phosphopeptides enriched from proteome samples are very complex. To reduce the complexity it is necessary to fractionate the phosphopeptides. However, conventional enrichment methods typically only enrich phosphopeptides but not fractionate phosphopeptides. In this study, the application of strong anion exchange (SAX) chromatography for enrichment and fractionation of phosphopeptides was presented. It was found that phosphopeptides were highly enriched by SAX and majority of unmodified peptides did not bind onto SAX. Compared with Fe(3+) immobilized metal affinity chromatography (Fe(3+)-IMAC), almost double phosphopeptides were identified from the same sample when only one fraction was generated by SAX. SAX and Fe(3+)-IMAC showed the complementarity in enrichment and identification of phosphopeptides. It was also demonstrated that SAX have the ability to fractionate phosphopeptides under gradient elution based on their different interaction with SAX adsorbent. SAX was further applied to enrich and fractionate phosphopeptides in tryptic digest of proteins extracted from human liver tissue adjacent to tumorous region for phosphoproteome profiling. This resulted in the highly confident identification of 274 phosphorylation sites from 305 unique phosphopeptides corresponding to 168 proteins at false discovery rate (FDR) of 0.96%.

Paper title : Maturation of the infant microbiome community structure and function across multiple body sites and in relation to mode of delivery.

Doi : https://doi.org/10.1038/nm.4272

Abstract : Human microbial communities are characterized by their taxonomic, metagenomic and metabolic diversity, which varies by distinct body sites and influences human physiology. However, when and how microbial communities within each body niche acquire unique taxonomical and functional signatures in early life remains underexplored. We thus sought to determine the taxonomic composition and potential metabolic function of the neonatal and early infant microbiota across multiple body sites and assess the effect of the mode of delivery and its potential confounders or modifiers. A cohort of pregnant women in their early third trimester (n = 81) were prospectively enrolled for longitudinal sampling through 6 weeks after delivery, and a second matched cross-sectional cohort (n = 81) was additionally recruited for sampling once at the time of delivery. Samples across multiple body sites, including stool, oral gingiva, nares, skin and vagina were collected for each maternal-infant dyad. Whole-genome shotgun sequencing and sequencing analysis of the gene encoding the 16S rRNA were performed to interrogate the composition and function of the neonatal and maternal microbiota. We found that the neonatal microbiota and its associated functional pathways were relatively homogeneous across all body sites at delivery, with the notable exception of the neonatal meconium. However, by 6 weeks after delivery, the infant microbiota structure and function had substantially expanded and diversified, with the body site serving as the primary determinant of the composition of the bacterial community and its functional capacity. Although minor variations in the neonatal (immediately at birth) microbiota community structure were associated with the cesarean mode of delivery in some body sites (oral gingiva, nares and skin; R2 = 0.038), this was not true for neonatal stool (meconium; Mann-Whitney P &gt; 0.05), and there was no observable difference in community function regardless of delivery mode. For infants at 6 weeks of age, the microbiota structure and function had expanded and diversified with demonstrable body site specificity (P &lt; 0.001, R2 = 0.189) but without discernable differences in community structure or function between infants delivered vaginally or by cesarean surgery (P = 0.057, R2 = 0.007). We conclude that within the first 6 weeks of life, the infant microbiota undergoes substantial reorganization, which is primarily driven by body site and not by mode of delivery.