Related Experiment Video
Updated: Apr 16, 2026
Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations
Published on: November 7, 2019
Comprehensive analysis of the ubiquitinome during oncogene-induced senescence in human fibroblasts
Fee Bengsch1, Zhigang Tu, Hsin-Yao Tang
1a Gene Expression and Regulation Program ; The Wistar Institute Cancer Center; The Wistar Institute ; Philadelphia , PA USA.
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.
Insights
Oncogene-induced senescence (OIS) involves significant protein ubiquitination changes, particularly affecting translation machinery. This study reveals ubiquitination as a key regulator of protein synthesis during OIS, offering new insights into tumor suppression.
Area of Science:
- Cellular senescence
- Cancer biology
- Proteomics
Background:
- Oncogene-induced senescence (OIS) is a critical tumor suppression mechanism.
- Senescent cells undergo substantial functional and morphological changes.
- Protein ubiquitination alterations during senescence remain largely uncharacterized.
Purpose of the Study:
- To perform the first global ubiquitination profiling of primary human cells undergoing OIS.
- To identify specific ubiquitination changes and affected signaling pathways in OIS.
- To elucidate the role of ubiquitination in regulating protein synthesis during OIS.
Main Methods:
- Utilized a well-established in vitro model of OIS using primary human fibroblasts expressing oncogenic RAS.
- Employed immune affinity purification of ubiquitinated peptides followed by liquid chromatography tandem mass spectrometry (LC-MS/MS).
- Performed mass spectrometry analysis of total proteins to differentiate ubiquitination changes from protein abundance variations.
Main Results:
- Identified 4,472 ubiquitination sites, with 397 significantly altered in senescent cells.
- Discovered that OIS-induced ubiquitination changes predominantly impact eIF2, eIF4/p70S6K, and mTOR signaling networks.
- Found that the majority of altered ubiquitinated proteins are components of the translation machinery, including initiation/elongation factors and ribosomal subunits.
Conclusions:
- Ubiquitination significantly affects key components of the translation machinery during OIS.
- These ubiquitination changes regulate protein synthesis in senescent cells.
- Ubiquitination emerges as a previously unappreciated regulatory mechanism in OIS and tumor suppression.
More Related Videos
13:59A Quantitative Measurement of Reactive Oxygen Species and Senescence-associated Secretory Phenotype in Normal Human Fibroblasts During Oncogene-induced Senescence
Published on: August 12, 2018
08:18Induction and Validation of Cellular Senescence in Primary Human Cells
Published on: June 20, 2018
Related Concept Videos
02:04Covalently Linked Protein Regulators
These groups modify specific amino acids in a protein....
02:15Replicative Cell Senescence
02:15Replicative Cell Senescence
01:13The Proteasome
In this pathway, the target proteins are first tagged with small proteins called ubiquitin. This involves participation of a series of enzymes including— E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3...
01:20The Retinoblastoma Gene
The first-ever tumor suppressor gene called Rb was identified in retinoblastoma - a rare eye tumor in children. In inherited forms of the disease, a child inherits one defective copy of the Rb gene, which predisposes them to retinoblastoma. However,...