Moyamoya Disease-AssociatedRNF213Alleles Encode Dominant Negative Alleles That Globally Impair Ubiquitylation

Abstract

Single nucleotide polymorphisms (SNPs) in RNF213, which encodes a 591kDa protein with AAA+ ATPase and RING E3 domains, are associated with a rare, autosomal dominant cerebrovascular disorder, Moyamoya disease (MMD). MMD-associated SNPs primarily localize to the C-terminal region of RNF213, and some affect conserved residues in the RING domain.Although the autosomal dominant inheritance of MMD could most easily be explained by RNF213 gain-of-function, the type of ubiquitylation catalyzed by RNF213 and the effects of MMDassociated SNPs on its E3-ligase activity have remained unclear. We found that the RING domain of RNF213 uses the E2-conjugating enzyme UBE2D2 to catalyze predominantly K6-dependent poly-ubiquitination events comprising a mixture of typical and atypical ubiquitin linkages. MMDassociated SNPs encode proteins with decreased E3-ligase activity and the most frequent MMD allele, RNF213 R4810K , is a dominant negative mutant that decreases ubiquitylation globally. By contrast, MMD-associated RNF213 SNPs do not affect ATPase activity. We propose that decreased RNF213 E3-ligase activity is central to MMD pathogenesis.the RNF213 R4810K allele (Cecchi et al., 2014;Moteki et al., 2015). Several other rare RNF213 variants are found in MMD patients of diverse ethnicities (Cecchi et al., 2014;Kobayashi et al., 2016;Moteki et al., 2015). Although ~2% of Japanese individuals have RNF213 R4810K , the prevalence of MMD is low (~0.006%), indicating that additional genetic and/or environmental modifiers are required for pathogenesis (Ran et al., 2013).RNF213 encodes an ~591kDa protein containing tandem AAA+ ATPase domains and a RING E3 domain (Fig. 1a). The AAA+ ATPase domains mediate RNF213 oligomerization into a homo-hexamer (Morito et al., 2014). Nucleotide (ATP/ADP) binding to the first ATPase domain stabilizes the oligomer, and destabilization occurs upon ATP hydrolysis by the second domain.Less, however, is known about the RNF213 E3 domain. RING E3 domains catalyze UB (ubiquitin) transfer from (an) E2 ubiquitin-conjugating enzyme(s) to lysine residues of E3-bound substrate(s), resulting in isopeptide bond formation (Deshaies and Joazeiro, 2009). Mono-ubiquitylation joins a single UB molecule to a substrate, although multiple sites on the same substrate can be mono-ubiquitylated ("multi-monoubiquitylation"). Poly-ubiquitylation occurs when UB residues are added sequentially to a specific lysine residue in a previously conjugated UB, forming a UB chain. The N-terminal methionine and seven lysine residues (K6, K11, K27, K29, K33, K48 and K63) in UB can participate in polyubiquitylation, leading to linear and/or branched UB chains (Yau and Rape, 2016). Some E2s appear to promote specific types of UB linkages; others are more promiscuous. Most E3 ubiquitin ligases can interact with several E2s, thereby catalyzing production of different types of UB chains.Depending on the specific UB linkage, ubiquitylation regulates diverse cellular processes, including protein degradation, endocytic trafficking, inflammation, transl...

E3 ubiquitin ligase RNF213 employs a non-canonical zinc finger active site and is allosterically regulated by ATP

“…A number of publications reported that the isolated RING domain from RNF213 has E3 activity (1) that works with either the E2 UBE2D2/UbcH5b (21), or the E2 UBE2N/Ubc13 (14,22). However, our recent report (16) reveals that the RING domain is not required for basal E3 autoubiquitination activity of full-length RNF213.…”