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.

From Ancient Pathways to Aging Cells—Connecting Metabolism and Cellular Senescence

“…MDM2 also antagonizes glycolysis and promotes senescence by targeting PGM for degradation (Mikawa et al, 2014). Protein ubiquitination states change notably with senescence, most notably in proteins responsible for regulating translation, including several eukaryotic initiation factors (eIFs), elongation factors, ribosomal subunits, and mTOR signaling complexes (Bengsch et al, 2015). The mitochondrial ubiquitin ligase MARCH5 antagonizes senescence (presumably MiDAS) by promoting degradation of mitofusin 1 and dynamin-regulated protein 1 (DRP1) (Park et al, 2010).…”

Translational identification of transcriptional signatures of major depression and antidepressant response

“…Thus, our candidate gene list contained two players involved in the translational machinery: the immunoglobulin mu-binding protein 2 (IGHMBP2), encoding a DNA/RNA helicase (De Planell-Saguer et al, 2009), and RPL35A, encoding a component of the large subunit of the ribosome. RPL35A was found to be among the most ubiquitinated proteins during senescence (Bengsch et al, 2015). In addition, we noticed that among our FIGURE 7 | Validation of candidate gene expression in human blood.…”

The Immortal Senescence

“…The changes described above are just a fraction of a larger scale metabolic alterations occurring in cells undergoing OIS, and the global metabolic changes occurring during oncogene-induced senescence have been the focus of study of several groups (102–106). Some of the other pathways altered during OIS include the oxidation of fatty acids (103), glucose metabolism (6), mitochondrial oxygen consumption (103), as well as protein ubiquitination (106).…”

“…Some of the other pathways altered during OIS include the oxidation of fatty acids (103), glucose metabolism (6), mitochondrial oxygen consumption (103), as well as protein ubiquitination (106). …”

“…The process of ubiquitination is highly dynamic, being regulated by both ubiquitin ligases (E1, E2, and E3 enzymes) which add ubiquitin moieties to proteins, and deubiquitinating enzymes (DUBs) which instead remove the tag (109). A recent paper profiled the changes in protein ubiquitination patterns occurring during OIS and identified most of the alterations being clustered within the mammalian target of rapamycin (mTOR) downstream effectors pathways: 4EBP-EIF4E, p70S6K and EEF2K/EIF2 (106). These pathway plays a prominent role in the translational control of cell growth and proliferation (110).…”