Abstract
TRIM E3 ubiquitin ligases regulate multiple cellular processes, and their dysfunction is linked to disease. They are characterised by a conserved N-terminal tripartite motif comprising a RING, B-box domains, and a coiled-coil region, with C-terminal domains often mediating substrate recruitment. TRIM proteins are grouped into 11 classes based on C-terminal domain identity. Class VI TRIMs, TRIM24, TRIM33, and TRIM28, have been described as transcriptional regulators, a function linked to their C-terminal plant homeodomain and bromodomain, and independent of their ubiquitination activity. It is unclear whether E3 ligase activity is regulated in family members where the C-terminal domains function independently. Here, we provide a detailed biochemical characterisation of the RING domains of class VI TRIMs and describe the solution structure of the TRIM28 RING. Our study reveals a lack of activity of the isolated RING domains, which may be linked to the absence of self-association. We propose that class VI TRIMs exist in an inactive state and require additional regulatory events to stimulate E3 ligase activity, ensuring that associated chromatin-remodelling factors are not injudiciously degraded.
Highlights
Modification of proteins with either a single ubiquitin molecule or poly-ubiquitin chains is a regulatory signalling mechanism integral to many cellular functions
To assess the basic E3 ubiquitin ligase activity of class VI TRIM proteins, we produced the RING domains of TRIM24, TRIM33, and TRIM28 in isolation to rule out any autoinhibitory effects from adjacent domains present in the full-length proteins
TRIM24 has been reported to catalyse p53 ubiquitination in conjunction with UBE2E2 [30], whereas TRIM28 activity has been linked with UBE2D [36], and TRIM33 has been proposed to function with both UBE2E1 and UBE2D [34, 46]
Summary
Modification of proteins with either a single ubiquitin molecule or poly-ubiquitin chains is a regulatory signalling mechanism integral to many cellular functions. Ubiquitin becomes covalently attached to a substrate lysine residue in a process catalysed by a multienzyme cascade, involving E1 activating enzymes, E2 conjugating enzymes, and E3 ligase enzymes. The largest family of E3s, RING-type E3 ligases, act as adaptors and catalyse the direct transfer of ubiquitin from the E2–Ub conjugate to the substrate [2, 3]. The TRIM protein family constitutes the largest subfamily of RING E3 ligases, with more than 70 family members in humans that regulate many cellular processes. The precise function of B-box domains is not fully understood, but they are often considered to mediate protein–protein interactions; in this context, they are involved in autoinhibition of TRIM21 [8] and homo-oligomerisation of TRIM5α [9]. The coiled-coil domain drives homo-dimerisation, which occurs in an antiparallel fashion [10, 11] and may facilitate hetero-oligomerisation of TRIM proteins [12]
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