DECIPHERING THE FUNCTIONAL BIOLOGY OF THE E3 UBIQUITIN LIGASE TRIP12 USING SILAC-BASED QUANTITATIVE PROTEOMICS APPROACH

Abstract

Thyroid hormone receptor interacting protein (TRIP12) is an E3 ubiquitin ligase of the HECT domain family, involved in the cell cycle control, DNA damage response, and TGF-b signaling. TRIP12 de novo mutations are common in ~10% of intellectual disability disorders. Moreover, there are ~4–10% of somatic mutations of TRIP12 in cancer patients. The biological significance and the molecular pathways governed by TRIP12 are not well established. The overall goal of my PhD dissertation is to understand the molecular biology of TRIP12 with an aim to delineate the contribution of TRIP12 in physiology and disease. To understand TRIP12-mediated pathways, I performed the stable isotope labeling of amino acids in cell culture (SILAC) to detect changes in protein abundance in the absence of TRIP12. We used quantitative proteomics in sgTRIP12 versus wildtype HCT116 colorectal cancer cells and found up to 80% of differentially expressed proteins to be significantly downregulated in sgTRIP12 cells. I performed pathway enrichment analysis using the SRplot online tool on the SILAC dataset and discovered that protein translation pathways were the topmost enriched pathways in sgTRIP12 cells. Subsequently, I used protein translation coupled with OPP-labeling of nascent protein cells and found impaired protein translation in TRIP12 knockdown cells. Consistent with that, phosphorylation of EIF2S1 at Serine 51, a surrogate marker of impaired protein translation, was upregulated in TRIP12 knockdown cells. Further, I discovered that the mTOR pathway, a major regulator of protein translation, is downregulated in sgTRIP12 cells. Mechanistically TRIP12 positively regulates mTOR signaling via ubiquitylation-mediated inhibition of G3BP1 – a negative regulator of mTOR. Co-deletion of G3BP1/TRIP12 restored nearly all cellular and molecular phenotypes of TRIP12 knockdown cells. My thesis highlights the unusual propensity of mTOR signaling regulation by TRIP12 E3 ubiquitin ligase, which may explain some of TRIP12 biology in health and disease.

Date of Award	2024
Original language	American English
Awarding Institution	HBKU College of Health & Life Sciences