A study of TRIM32 self-ubiquitination

Abstract

TRIMs (tripartite motif proteins) constitute a family of ubiquitin E3 ligases that are involved in a variety of cellular processes including autophagy and carcinogenesis. TRIM32 contains an N-terminal RING finger domain, B-box region, a coiled-coil domain and six NHL repeats in the C-terminal region, which are involved in protein dimerization and substrate recognitions. Various mutations in the NHL domains are associated with limb-girdle muscular dystrophy 2H (LGMD2H). TRIM32 is shown to have the capability to ubiquitinate actin and thus possibly participates in myofibrillar protein turnover, especially during the period of muscle transformation and adaptation. TRIM32 is activated by self-ubiquitination induced upon dimerization. Mass spectrometry analysis of EGFP-TRIM32 expressed in HEK293 FlpIn cells suggested three lysine residues, K50, K247 and K401, as putative target sites for the self-ubiquitination activity. The aim of this work was to investigate the importance of these lysine residues for TRIM32 mediated self-ubiquitination. Expression plasmids of EGFP-TRIM32 single, double and triple mutants were established by site–directed mutagenesis. These mutants were transfected into various cell lines and TRIM32 self-ubiquitination analyzed by Western blotting. Introduction of single or double K to R mutations did not seem to abolish self-ubiquitination. However, the double mutant EGFP-TRIM32-K247R/K401R and the triple mutant EGFP-TRIM32-K49R/K247R/K401 displayed a faster migration band on the blot, suggesting that these proteins were cleaved. Inhibition of the lysosome or the proteasome did not inhibit cleavage of EGFP-TRIM32 K49R/K247R/K401R. Localization studies using confocal microscopy showed that both EGFP-TRIM32 WT and the K to R double and triple mutants had a tendency to aggregate in cells, clustering with the Golgi protein GM130 and the autophagy receptor p62/SQSTM1. Since mutations in TRIM32 leads to muscle dystrophy, the second aim of this project was to establish myoblast TRIM32 KO cell lines. These cells would represent good model systems for functional analysis of TRIM32 mutations. Guide RNAs (gRNA) sequences for mouse and rat TRIM32 were selected by bioinformatics tools and cloned into the CRISPR/Cas9 pX458 plasmid. Successfully cloned plasmids were transfected into the mouse myoblast cell line C2C12 and the rat myoblast cell line H9c2. Transfected cells were selected by cell sorting, and expanded clones analyzed by Western blotting. Two promising myoblast C2C12 TRIM32 KO cell lines were successfully established.