Abstract
TRIM32 is an E3 ligase implicated in diverse biological pathways and pathologies such as muscular dystrophy and cancer. TRIM32 are expressed both as full-length proteins, and as a truncated protein. The mechanisms for regulating these isoforms are poorly understood. Here we identify a PEST sequence in TRIM32 located in the unstructured region between the RING-BBox-CoiledCoil domains and the NHL repeats. The PEST sequence directs cleavage of TRIM32, generating a truncated protein similarly to the short isoform. We map three lysine residues that regulate PEST mediated cleavage and auto-ubiquitylation activity of TRIM32. Mimicking acetylation of lysine K247 completely inhibits TRIM32 cleavage, while the lysines K50 and K401 are implicated in auto-ubiquitylation activity. We show that the short isoform of TRIM32 is catalytic inactive, suggesting a dominant negative role. These findings uncover that TRIM32 is regulated by post-translational modifications of three lysine residues, and a conserved PEST sequence.
Highlights
Tripartite motif (TRIM) proteins have emerged as a large family of ubiquitin E3 ligases that are involved in a wide range of cellular processes such as development, differentiation, immunity and carcinogenesis [1]
Auto-ubiquitylation seems to be a prerequisite for the catalytic activity of TRIM32, as the LGMD2H mutant is unable to ubiquitylate the substrate protein p62/SQSTM1 [7]
All proteins were stably expressed in the HEK293 FlpIn TRIM32 knock out (KO) cells described previously [7]
Summary
Tripartite motif (TRIM) proteins have emerged as a large family of ubiquitin E3 ligases that are involved in a wide range of cellular processes such as development, differentiation, immunity and carcinogenesis [1]. As a member of the TRIM family, TRIM32 shows the common RBCC domain organization in its N-terminal, consisting of a “Really interesting New gene” (RING) domain, followed by one B-box domain, and a Coiled Coil (CC) region [2]. TRIM32 is characterized by six NHL repeats in its C-terminus. The RING domain is essential for the E3 ligase activity, while the B-box domain is necessary to modulate chain assembly rate of ubiquitin units. The NHL repeats are found to be involved in dimerization and cargo recognition [4]. TRIM32 oligomerization is a pre-requisite for its catalytic activity
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