title: Comprehensive analysis of the ubiquitinome during oncogene-induced senescence in human fibroblasts.
abstract: Oncogene-induced senescence (OIS) is an important tumor suppression mechanism preventing uncontrolled proliferation in response to aberrant oncogenic signaling. The profound functional and morphological remodelling of the senescent cell involves extensive changes. In particular, alterations in protein ubiquitination during senescence have not been systematically analyzed previously. Here, we report the first global ubiquitination profile of primary human cells undergoing senescence. We employed a well-characterized in vitro model of OIS, primary human fibroblasts expressing oncogenic RAS. To compare the ubiquitinome of RAS-induced OIS and controls, ubiquitinated peptides were enriched by immune affinity purification and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS). We identified 4,472 ubiquitination sites, with 397 sites significantly changed (>3 standard deviations) in senescent cells. In addition, we performed mass spectrometry analysis of total proteins in OIS and control cells to account for parallel changes in both protein abundance and ubiquitin levels that did not affect the percentage of ubiquitination of a given protein. Pathway analysis revealed that the OIS-induced ubiquitinome alterations mainly affected 3 signaling networks: eIF2 signaling, eIF4/p70S6K signaling, and mTOR signaling. Interestingly, the majority of the changed ubiquitinated proteins in these pathways belong to the translation machinery. This includes several translation initiation factors (eIF2C2, eIF2B4, eIF3I, eIF3L, eIF4A1) and elongation factors (eEF1G, eEF1A) as well as 40S (RPS4X, RPS7, RPS11 and RPS20) and 60S ribosomal subunits (RPL10, RPL11, RPL18 and RPL35a). In addition, we observed enriched ubiquitination of aminoacyl-tRNA ligases (isoleucyl-, glutamine-, and tyrosine-tRNA ligase), which provide the amino acid-loaded tRNAs for protein synthesis. These results suggest that ubiquitination affects key components of the translation machinery to regulate protein synthesis during OIS. Our results thus point toward ubiquitination as a hitherto unappreciated regulatory mechanism during OIS.
authors.affiliation: a Gene Expression and Regulation Program ; The Wistar Institute Cancer Center; The Wistar Institute ; Philadelphia , PA USA.
authors.forename: Fee
authors.initials: F
authors.lastname: Bengsch
authors.forename: Zhigang
authors.initials: Z
authors.lastname: Tu
authors.forename: Hsin-Yao
authors.initials: HY
authors.lastname: Tang
authors.forename: Hengrui
authors.initials: H
authors.lastname: Zhu
authors.forename: David W
authors.initials: DW
authors.lastname: Speicher
authors.forename: Rugang
authors.initials: R
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D002478:Cells, Cultured; D016922:Cellular Senescence; D002851:Chromatography, High Pressure Liquid; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D039562:Eukaryotic Initiation Factor-4F / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling; D015689:Oncogene Protein p21(ras) / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000032:analysis / Q000378:metabolism; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D054875:Ubiquitination
chemical_list: D015852:Eukaryotic Initiation Factor-2; D039562:Eukaryotic Initiation Factor-4F; D010455:Peptides; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D058570:TOR Serine-Threonine Kinases; D015689:Oncogene Protein p21(ras)
keywords: mass spectrometry; oncogene-induced senescence; protein synthesis; protein translation; proteomics; ubiquitination