Identification of novel interacting partners of the TRIM29 E3-ubiquitin ligase in immortalized cells of normal prostate basal epithelium

Abstract

BACKGROUND: Based on the most recent findings, the E3 ubiquitin ligase TRIM29 participates in numerous cellular processes and holds significance in both the formation of normal basal epithelium and prostate cancer. Exploring these molecular mechanisms could be pivotal in developing novel cancer therapies. AIM: Identification of protein partners of TRIM29 and its truncated forms, followed by functional analysis of these proteins. Identification of the key molecular processes involving TRIM29. METHODS: Cell cultures from normal basal epithelium of the prostate with overexpression of the chimeric protein TRIM29-FLAG or its truncated forms lacking the B-Box domain or the Coiled-Coil domain were conducted. Protein partners of TRIM29 and its truncated forms were identified through protein immunoprecipitation followed by proteomic (HPLC-MS/MS) analysis. The findings were validated through Western blot analysis and immunocytochemistry. RESULTS: TRIM29 interacts with 288 proteins in normal prostate basal epithelium. Deletion of the B-Box has minimal impact on TRIM29's protein interactions, while deletion of the Coiled-Coil domain significantly reduces its protein partners and disrupts its dimerization. Our research indicates that TRIM29 is present in both the nucleus and cytoplasm, and deletion of functional domains does not affect its localization but alters binding to compartment-specific proteins. TRIM29 binds to cytoskeletal proteins, cellular stress response proteins, and RNA-binding proteins. Furthermore, TRIM29 enhances cell resistance to genotoxic agents and influences RNA splicing. CONCLUSION: Proteomic analysis reveals that in normal prostate basal epithelium, the E3-ubiquitin ligase TRIM29 binds to a broad spectrum of proteins with distinct functions in various cell compartments. Our findings align with other studies demonstrating involvement of TRIM29 in cytoskeletal rearrangement, cellular response to viral infections, and DNA damage. Additionally, our research demonstrates the interaction of TRIM29 with RNA-binding proteins and its potential role in regulating RNA splicing, particularly through the Coiled-Coil domain.