{"database":"MassIVE","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Other":["ftp://massive-ftp.ucsd.edu/v06/MSV000096861/"]},"type":"primary"},"statusCode":"OK","statusCodeValue":200}],"scores":null,"additional":{"submitter":["Prof. S. Murty Srinivasula"],"full_dataset_link":["https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=78e536b8fe574ce7a87e77e81c26e1fa"],"submitter_email":["sms@iisertvm.ac.in"],"sample_protocol":[""],"repository":["MassIVE"],"file_size":["11"],"ptm_modification":["MS:1002864 - No post-translational-modifications are included in the identified peptides of this dataset"],"data_protocol":[""],"omics_type":["Proteomics"],"instrument_platform":["Q Exactive Plus"],"species":["Homo Sapiens (ncbitaxon:9606)"],"submitter_affiliation":["Indian Institute of Science Education and Research Thiruvananthapuram"],"pubmed_abstract":["The ubiquitin-proteasome system contributes to protein quality control, involving E3 ligases that ubiquitinate proteins and leading to their degradation. The dysregulation of protein degradation results in the abnormal accumulation of proteins and is implicated in the pathology of diverse diseases, making targeted protein degradation a promising therapeutic strategy. Here, we focus on RFFL, an endosome-associated RING E3 ligase involved in mitochondrial homeostasis and the clearance of misfolded cystic fibrosis transmembrane conductance regulator proteins. Using label-free quantitative mass spectrometry based proteomics for interactome and differential expression analyses, we systematically investigated and identified putative substrates of RFFL. For more confident identification, we performed these analyses on three cell lines that we generated: an RFFL knockout cell line generated using CRISPR/Cas9, another cell line rescuing RFFL expression when complemented with KO cells with stably expressing RFFL cDNA, and wild-type cells. We validated JMJD6 and DNAJB11 as substrates of endogenous RFFL, providing orthogonal validation and confidence in our screening approach. We demonstrated that RFFL ubiquitinates and degrades JMJD6 and DNAJB11 via the proteasomal pathway using in vivo assays. Interestingly, we also discovered a hitherto unknown role of RFFL in lipid metabolism. Collectively, this study provides the first comprehensive and unbiased analysis of RFFL substrates employing multiple complementary approaches."],"pubmed_title":["Quantitative Proteomic Analysis Reveals JMJD6 and DNAJB11 as Endogenous Substrates of E3 Ligase RFFL."],"pubmed_authors":["Narendradev Nikhil Dev ND, Marathe Soumitra S, Baboo Sabyasachi S, McClatchy Daniel B DB, Diedrich Jolene K JK, Jain Parul P, Purwar Rahul R, Yates John R JR, Srinivasula Srinivasa Murty SM"],"additional_accession":["PXD059848"]},"is_claimable":false,"name":"Quantitative proteomic analysis reveals JMJD6 and DNAJB11 as endogenous substrates of E3 ligase RFFL ","description":"A549 cells were washed with ice-cold PBS three times and harvested using mechanical scraping. Cell pellets were processed as described previously (Marathe et al., 2021). Briefly, the pellet was re-solubilized in Urea-Thiourea Buffer (6M Urea, 2M Thio-urea in Tris-Cl) containing protease inhibitor cocktail (#4693159001, Roche), phosphatase Inhibitor cocktail 2 (#P5726, Sigma) and phosphatase Inhibitor cocktail 3 (#P0044, Sigma). After protein concentration estimation using BCA (#23225, Pierce), 30ug of proteins from each sample were used for subsequent steps. Samples were reduced with 15mM DTT a 40oC for 60 minutes, followed by alkylation with 15 mM iodoacetamide for 30 minutes at room temperature in the dark. The digestion mixture was then diluted 4× with 50 mM ammonium bicarbonate, and the protein was digested with 1 ?g of MS-grade trypsin (#V5280, Promega) overnight at 37°C. The resulting peptide solutions were cleaned with C18 ZipTips, quantified using the Scopes method (Scopes, 1974), and 700 ng of peptides were injected into Thermo Q-Exactive plus instrument via EasyNLC through C18 PepMap EasySpray RSLC analytical column (#ES903, Thermo). The MS instrument was set to an MS1 resolution of 70,000 and an MS2 resolution of 17,500, with acquisition experiments optimized for 120 min LC gradients.\nThe MS spectra were analyzed using Proteome Discoverer version 2.2 using the standard LFQ workflows by Thermo Fisher as described previously (Marathe et al., 2021). Briefly, tandem mass spectra were searched against the UniProt human database and reversed sequences, using SEQUEST (Eng, McCormack and Yates, 1994). Full tryptic search with up to 2 missed cleavages and a minimum peptide length of 6 is considered. Carbamidomethylation on cystines (+57.021) was considered as a static modification, and oxidation on methionine (+15.995 Da) was considered as a dynamic modification. Precursor mass tolerance was set to 10 PPM, and fragment mass tolerance was set to 0.02 Da. Precursor ion-based quantification was performed using only unique peptides and the samples were normalized with respect to the total peptide amount. The false discovery rate (FDR) at the protein level was set to 0.01. Three biological replicates were used for analysis, and statistical analysis of identified proteins was performed using ANOVA (background-based).\n\nWT (AN1.raw,BN1.raw,CN1.raw)\nKO (AN2.raw,,BN2.raw,CN2.raw)\nRescue (AN3.raw,BN3.raw,CN3.raw)","dates":{"publication":"Wed Jan 15 21:33:00 GMT 2025"},"accession":"MSV000096861","cross_references":{"pubmed":["40568870"]}}