MMD-associated RNF213 SNPs encode dominant-negative alleles that globally impair ubiquitylation

Abhishek Bhardwaj,Wei Zhang,Sachdev S Sidhu,Robert S Banh,Benjamin G Neel

doi:10.26508/lsa.202000807

Abhishek Bhardwaj, Wei Zhang + Show 3 more

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https://doi.org/10.26508/lsa.202000807

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Abstract

Single-nucleotide polymorphisms (SNPs) in RNF213, which encodes a 591-kD 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 explained by RNF213 gain-of-function, the type of ubiquitylation catalyzed by RNF213 and the effects of MMD-associated SNPs on its E3 ligase activity have remained unclear. We found that RNF213 uses the E2-conjugating enzymes UBE2D2 and UBE2L3 to catalyze distinct ubiquitylation events. RNF213-UBED2 catalyzes K6 and, to a lesser extent, K48-dependent poly-ubiquitylation in vitro, whereas RNF213-UBE2L3 catalyzes K6-, K11-, and K48-dependent poly-ubiquitylation events. MMD-associated SNPs encode proteins with decreased E3 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. Our results suggest that decreased RNF213 E3 ligase activity is central to MMD pathogenesis.

Highlights

Moyamoya disease (MMD) is a rare, autosomal dominant disorder characterized by stenotic/occlusive lesions in the circle of Willis with surrounding abnormal blood vessels in the brain (Nanba et al, 2006)
We report that RNF213 has K6- and K48-ubiquitin–dependent E3 ligase activity mediated by the E2 UBE2D2 and K6, K11, and K48-ubiquitin– dependent E3 ligase activity mediated by UBE2L3
An unbiased screen showed that RNF213 interacts well with UBE2D2 and UBE2L3 (Pao et al, 2018), whereas other work suggests that RNF213 predominantly uses UBE2L3 (Ahel et al, 2020)

Summary

Introduction

Moyamoya disease (MMD) is a rare, autosomal dominant disorder characterized by stenotic/occlusive lesions in the circle of Willis with surrounding abnormal blood vessels in the brain (Nanba et al, 2006). These vessels have a “puff of smoke” appearance in imaging studies ( the Japanese term, “moyamoya”) and are believed to develop around the occlusive lesions to compensate for lack of blood flow. On average, ~50% of Asian (and ~80% of Japanese) MMD families carry the RNF213R4810K allele (Cecchi et al, 2014; Moteki et al, 2015). ~2% of Japanese individuals have RNF213R4810K, the prevalence of MMD is low (~0.006%), indicating that additional genetic and/or environmental modifiers are required for pathogenesis (Ran et al, 2013)

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