TRIAD3/RNF216 E3 ligase specifically synthesises K63-linked ubiquitin chains and is inactivated by mutations associated with Gordon Holmes syndrome

Abstract

TRIAD3/RNF216 is a ubiquitin ligase of the RING-in-between-RING family. Recent publications identified TRIAD3 mutations in patients with neurological diseases, including Gordon Holmes syndrome and Huntington-like disorder. To understand the functional relevance of these disease-associated mutations, we have tested the ubiquitin ligase activity of mutated TRIAD3 in vitro. Several of these point mutations completely abrogated TRIAD3’s catalytic activity. Using mass spectrometry, we identified new TRIAD3-interacting proteins/substrates from mouse brain lysate, which provide a new link between TRIAD3 and processes involving clathrin-mediated endocytosis. Strikingly, we found that TRIAD3 synthesises specifically lysine-63 (K63)-linked poly-ubiquitin chains in vitro, a chain type that usually plays a role in mediating signalling events rather than triggering proteasomal degradation. Therefore, this finding is of great importance to further understand TRIAD3’s cellular role and loss-of-function in disease.

Chain reactions: molecular mechanisms of RBR ubiquitin ligases

“…Notably, the catalytic triad residues are not completely conserved across all RBR ligases ( Figure 3B). RNF216, for example, is an active E3 ligase despite lacking the active site histidine ( Figure 3B) [25,26]. Together, these observations suggest that alternate active site configurations and catalytic mechanisms exist within the RBR family.…”

“…Unlike the previously described UBA and CUE domains, these motifs consist of a single helix only, but similarly interact with the canonical Ile44 ubiquitin surface. Curiously, the MIU downstream of the RNF216 RING2 appears to be critical for E3 ligase activity, although its molecular basis remains unclear [25,26]. The unstructured N-terminus of RNF216 also houses three SUMO-interaction motifs (SIMs), which are able to bind SUMO2 chains in vitro [25].…”

“…Recently, RNF216 (TRIAD3) has been shown to form free K63-linked ubiquitin chains in vitro , making it only the second RBR to exhibit preference for a single chain type [ 25 , 26 ]. This contrasts with previous studies which had implicated RNF216 in the proteasomal degradation of substrates via K48-linked polyubiquitination [ 28–34 ].…”

New roles for the de-ubiquitylating enzyme OTUD4 in an RNA-protein network and RNA granules

“…Mouse brain lysate from cortex or cerebellum of 6–8-week-old C57BL/6 mice was prepared as described in Schwintzer et al (2019). All animal experiments were performed according to approved guidelines.…”

“…Mass spectrometry and data analysis was performed as in Schwintzer et al (2019). Proteins that were identified in at least two out of three experiments per tissue and were enriched ≥30 fold over control samples (HA-affinity purification from HA-vector-transfected cells; pulldown from mouse brain lysate) (based on peak area) were considered putative interactors.…”