Project description:BONCAT-MS analysis was performed against several HSV-1 infected and uninfected Hela cells.
PPI analysis of HSV-1 protein was performed against Flag-tagged HSV-1 protein expressed 293T cells.
Project description:CHD (chromodomain helicase DNA binding protein) family is composed of nine members of chromatin remodeling factors that regulate chromatin structure in an ATP-dependent manner. Among them, CHD4 contributes to many basic cellular functions during development mainly through multiple proteins interacting with CHD4 including NuRD (nucleosome remodeling and deacetylase activities) complex, which contains histone deacetylase HDAC1/2. However, functions of CHD4 that are not mediated by NuRD complexes have also been found, implying the existence of unknown proteins that are associated with CHD4. In the present study, we generated CHD4 FLAG-tag knock-in cells in HeLa-S3 and HEK293T cells and sought proteins bound to CHD4 with the use of immunoprecipitation and liquid chromatography and tandem MS (LC-MS/MS) analysis. We found that LCORL (ligand dependent nuclear receptor corepressor like) and NOL4L (nucleolar protein 4 like) were reproducibly identified as a novel CHD4 interactors. RNA-sequencing analysis of HEK293T cells depleted of CHD4, LCORL, and NOL4L revealed consistent upregulation of genes related to the Notch pathway. Our results thus suggest that both NOL4L and LCORL may cooperate with CHD4 to suppress the Notch pathway in mammalian cells.
Project description:Proteomic analysis of pervanadate-induced tyrosine-phosphorylated proteins in hepatocellular carcinoma WRL 68 cells Protein tyrosine phosphorylation plays critical roles in modulating biological processes such as cellular proliferation, differentiation, migration, apoptosis and metabolism. To profile tyrosine phosphorylated (pTyr) proteins as well as search novel pTyr proteins as cross-talk points among different cellular pathways, we developed a rapid and efficient approach to identify cellular pTyr proteins and their complexes by a combination of subcellular proteomics approach with signal transduction strategies. Human hepatocytic cells from WRL68 cell line were treated with pervanadate (POV), subfractionated into four fractions and then subjected to immunoaffinity purification with anti-pTyr antibody. The eluted mixtures of the anti-pTyr purification were identified by LC-MS/MS. Subcellular fractionation and affinity purification of tyrosine-phosphorylated proteins: WRL68 cells were first grown to 80% confluence in MEM complete medium and then the medium replaced with serum free media. After 15 h, the cells were either untreated or stimulated with 0.1 mM pervanadate (1 mM sodium orthovanadate, 3 mM H2O2) for 10 min. 150-mm cultures of WRL 68 cells were rinsed twice with 4? PBS and then scraped from the dish in 750?l of hypotonic buffer (10 mM Tris, 1 mM NaF, 10 mM IAA, pH 7.5) containing a cocktail of protein inhibitors. After a 20-min incubation on ice, the cells were passed about five times through a 25-g needle. The resulting lysate was subjected to a 15min centrifugation at 1000 rpm at 4?, after which the pellet was resuspended in 250?l of hypotonic buffer and re-extracted by a second round of the trituration and centrifugation. The supernatants of the first and second spins were combined, adjusted to 0.25 M NaCl, and separated into cytosolic (supernatant) and membrane (pellet) fractions bycentrifugation at 19,000 rpm (43,000 g) for 90 min at 4?. All the pellets and total cell lysate were resuspended in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholic acid sodium) containing 1mM pervanadate with a cocktail of protein inhibitors with sonication aid. Cleared cell lysates were incubated overnight at 4? with 30?l monoclonal anti-phosphotyrosine-agrose (Sigma). Precipitated immune complexes were washed three times with 1×HNTG (20 mM HEPES, 150 mM NaCl, 0.1% Triton X-100, 10% Glycerol, pH 7.5) and then eluted with 100 mM phenyl phosphate (Sigma) in lysis buffer at 4?. Enzyme digestion, mass spectrometry and protein identification: The sample was digested according to the published method. Chromatography was performed using a surveyor LC system (Thermo Finnigan,SanJose,CA) on C18 reverse phase column(RP, 180 µm x 150 mm, BioBasic® C18, 5 µm, Thermo Hypersil-Keystone). The pump flow rate is split 1:100 for a colum flow rate of 1.5 µL/min.The column effluent is directly electrosprayed using the orthogonal metal needle source without further splitting.Mobile phase A is 0.1% formic acid in water,and the B mobile phase is 0.1% formic acid in acetonitrile. The separation of peptides obtained by enzymatic digest of bile sample was achieved with a gradient of 2-80% B over 360 min.The column effluent from the reverse phase column was analyzed by LCQ Deca XP ion-trap mass spectrometer.The micro-electrospray interface uses a 30 µm metal needle that is orthogonal to the inlet of the LCQ.The mass spectrometer was set that one full MS scan was followed by three MS/MS scans on the three most intense ion from the MS spectrum with the following Dynamic Exclusion™ settings: repeat count, 1; repeat duration, 0.5 min; exclusion duration, 3.0 min. The acquired MS/MS spectra were automatically searched against protein database for human proteins (SWISS-PROT/TrEMBL) using the TurboSEQUEST program in the BioWorks™ 3.0 software suite. An accepted SEQUEST result had to have a ?Cn score of at least 0.1 (regardless of charge state) and Xcorr (one charge?1.5, two charges?2.0, three charges?2.5). Single peptides that alone identify a protein were manually validated after meeting the above criteria. Bioinformatics analysis: The pI and MW of the proteins were analyzed using ExPASy proteomics tools accessed from http://cn.expasy.org/tools/#proteome. The grand average hydropathicity (GRAVY) values were determined according to Kyte-Doolittle. The protein subcellular location annotation was from SwissProt &TrEMBL protein database (http://us.expasy.org/sprot/). The protein function family was categorized according to Gene Ontology (GO) annotation terms extracted by InterPro (http://www.ebi.ac.uk./interpro/). The annotation of protein phosphorylation was from SwissProt &TrEMBL protein database (http://us.expasy.org/sprot/) and PhosphoSite (http://www.phosphosite.org). The kinases were annotated according to human kinome. Keywords: other
Project description:In order to understand the underline mechanism of SHMT2 (serine hydroxymethyltransferase 2) effect on tumor growth, proteome and metabolome analysis were carried on an engineered HeLa cell line (HeLa-SHMT2-shSHMT2, short as HeLa-Ss), which has inducible SHMT2 over-expression or suppression by treating cell with tetracycline or IPTG, respectively. SHMT2 over-expression in HeLa-ss cell increased cell proliferation in vitro and in vivo, deceased expression of several mitochondrial complex I and III proteins, and increased glycine and glutathione levels in cells. BioID method identified more than 20 SHMT2 associated proteins that are involved in oxidation-reduction process. These results indicate SHMT2 involves in the regulation of cellular redox balance. SHMT2 suppression only reduced growth of cells under glycine depletion condition in cell culture. It increased expression of several proteins involved in glutaminolysis and amino acid transporters, and elevated metabolites related to glutamine metabolism. These results indicate tumor cells have a compensatory reaction after SHMT2 suppression. Further reducing glycine levels in cells by sodium benzoate caused cell death in cultured cell and slightly reduced tumor growth in vivo. Benzoate treatment induces more changes in protein expressions and metabolite levels, and it may provide a new addition for tumor treatment.
Project description:Analysis of HeLa cells at 24 hours after transfection with wild type miR-1, miR-124, miR-181 versus control transfected HeLa cells. Results were compared to protein down-regulation at 48 hours measured by SILAC-MS. Analysis of HeLa cells at 24 hours after transfection with wild type miR-1, miR-124, miR-181 versus control transfected HeLa cells. Results were compared to protein down-regulation at 48 hours measured by SILAC-MS.
Project description:Analysis of HeLa cells at 24 hours after transfection with wild type miR-1, miR-124, miR-181 versus control transfected HeLa cells. Results were compared to protein down-regulation at 48 hours measured by SILAC-MS.
Project description:Our understanding of the functions of DNA elements is limited by the paucity of information about the spectrum of proteins that occupy these genomic regions. Here we describe an approach to identify proteins associated with genomic regions whose chromatin is marked by specific modified histones, which we term chromatin profiling. We used chromatin immunoprecipitation followed by mass spectrometry (ChIP-MS) to identify proteins associated with genomic regions marked by histone H3K27Ac, H3K4me3, H3K79me2 and H3K36me3 in mouse embryonic stem (mES) cells. We identified 385 known and 224 novel candidate proteins associated with these histone-marked genomic segments and confirmed that several of the novel candidates are indeed associated with histone-marked segments of the genome. Future study of the novel candidates, many of which have been implicated in various diseases, should lead to an improved understanding of gene control and its dysregulation in disease. ChIP-seq for nucleosomes with modified histones and DNA-binding proteins in mouse embryonic stem cells, and DNA-binding proteins in Jurkat cells
Project description:It is becoming increasingly clear that cells infected with pathogens can signal to bystander cells. Infected cells have the ability to alert and instruct bystander cells to mount pro-inflammatory cytokine response, thus contributing to clearing infections. Here we analyse secretome of HeLa cells infected with Salmonella enterica serovar Typhimurium strain SL1344. Cells were infected with a MOI of 100 for 14 hours. Secretome from mock- and Salmonella-infected cells was collected for mass-spectrometry analysis.
Project description:Toxoplasma gondii is a ubiquitous apicomplexan parasite of mammals and birds and an important pathogen of humans. IFN-g is the major mediator of host resistance against T. gondii but intriguingly, parasite-infected host cells including macrophages are severely impaired to respond to IFN-g due to defective transcriptional activation of target genes. Here, we tested the possibility that the impaired responsiveness of T. gondii-infected macrophages to IFN-g can be restored by inhibiting histone deacetylases (HDACs) using the class I-specific inhibitor MS-275. Treatment of RAW264.7 cells with MS-275 indeed increased MHC class II surface expression in infected and non-infected cells and largely abolished the inhibition of IFN-g-regulated MHC class II expression exerted by T. gondii. Genome-wide transcriptome profiling revealed that MS-275 increased mean mRNA levels of IFN-g-regulated genes particularly in non-infected macrophages. Transcript levels of 33% of IFN-g secondary response genes but only those of a few primary response genes were also increased by MS-275 in T. gondii-infected cells. Importantly, the unresponsiveness of parasite-infected cells to IFN-g was however not abolished by MS-275. Furthermore, MS-275 also up-regulated several anti-inflammatory cytokines or signaling molecules in T. gondii-infected macrophages. It additionally regulated expression of more than 2500 genes in non-infected macrophages expression of which was surprisingly counteracted by prior infection with T. gondii. FACS analysis and immunofluorescence microscopy revealed that MS-275 did not considerably diminish the number of parasite-positive cells or the intracellular replication in macrophages stimulated or not with IFN-g. Thus, a supportive therapy using MS-275 appears inappropriate for treatment of toxoplasmosis.