BackgroundMoyamoya disease is an idiopathic vascular disorder of intracranial arteries. Its susceptibility locus has been mapped to 17q25.3 in Japanese families, but the susceptibility gene is unknown.Methodology/Principal FindingsGenome-wide linkage analysis in eight three-generation families with moyamoya disease revealed linkage to 17q25.3 (P<10-4). Fine mapping demonstrated a 1.5-Mb disease locus bounded by D17S1806 and rs2280147. We conducted exome analysis of the eight index cases in these families, with results filtered through Ng criteria. There was a variant of p.N321S in PCMTD1 and p.R4810K in RNF213 in the 1.5-Mb locus of the eight index cases. The p.N321S variant in PCMTD1 could not be confirmed by the Sanger method. Sequencing RNF213 in 42 index cases confirmed p.R4810K and revealed it to be the only unregistered variant. Genotyping 39 SNPs around RNF213 revealed a founder haplotype transmitted in 42 families. Sequencing the 260-kb region covering the founder haplotype in one index case did not show any coding variants except p.R4810K. A case-control study demonstrated strong association of p.R4810K with moyamoya disease in East Asian populations (251 cases and 707 controls) with an odds ratio of 111.8 (P = 10−119). Sequencing of RNF213 in East Asian cases revealed additional novel variants: p.D4863N, p.E4950D, p.A5021V, p.D5160E, and p.E5176G. Among Caucasian cases, variants p.N3962D, p.D4013N, p.R4062Q and p.P4608S were identified. RNF213 encodes a 591-kDa cytosolic protein that possesses two functional domains: a Walker motif and a RING finger domain. These exhibit ATPase and ubiquitin ligase activities. Although the mutant alleles (p.R4810K or p.D4013N in the RING domain) did not affect transcription levels or ubiquitination activity, knockdown of RNF213 in zebrafish caused irregular wall formation in trunk arteries and abnormal sprouting vessels.Conclusions/SignificanceWe provide evidence suggesting, for the first time, the involvement of RNF213 in genetic susceptibility to moyamoya disease.

Misfolded or improperly assembled proteins in the endoplasmic reticulum (ER) are exported into the cytosol and degraded via the ubiquitin-proteasome pathway, a process termed ER-associated degradation (ERAD). Saccharomyces cerevisiae Hrd1p/Der3p is an ER membrane-spanning ubiquitin ligase that participates in ERAD of the cystic fibrosis transmembrane conductance regulator (CFTR) when CFTR is exogenously expressed in yeast cells. Two mammalian orthologues of yeast Hrd1p/Der3p, gp78 and HRD1, have been reported. Here, we demonstrate that gp78, but not HRD1, participates in ERAD of the CFTR mutant CFTR⌬F508, by specifically promoting ubiquitylation of CFTR⌬F508. Domain swapping experiments and deletion analysis revealed that gp78 binds to CFTR⌬F508 through its ubiquitin binding region, the so-called coupling of ubiquitin to ER degradation (CUE) domain. Gp78 polyubiquitylated in vitro an N-terminal ubiquitin-glutathione-S-transferase (GST)-fusion protein, but not GST alone. This suggests that gp78 recognizes the ubiquitin that is already conjugated to CFTR⌬F508 and catalyzes further polyubiquitylation of CFTR⌬F508 in a manner similar to that of a multiubiquitin chain assembly factor (E4). Furthermore, we revealed by small interfering RNA methods that the ubiquitin ligase RMA1 functioned as an E3 enzyme upstream of gp78. Our data demonstrates that gp78 cooperates with RMA1 with E4-like activity in the ERAD of CFTR⌬F508.

Quality control in the endoplasmic reticulum ensures that only properly folded proteins are retained in the cell through mechanisms that recognize and discard misfolded or unassembled proteins in a process called endoplasmic reticulum-associated degradation (ERAD). We previously cloned EDEM (ER degradation-enhancing ␣-mannosidase-like protein) and showed that it accelerates ERAD of misfolded glycoproteins. We now cloned mouse EDEM3, a soluble homolog of EDEM. EDEM3 consists of 931 amino acids and has all the signature motifs of Class I ␣-mannosidases (glycosyl hydrolase family 47) in its N-terminal domain and a protease-associated motif in its C-terminal region. EDEM3 accelerates glycoprotein ERAD in transfected HEK293 cells, as shown by increased degradation of misfolded ␣1-antitrypsin variant (null (Hong Kong)) and of TCR␣. Overexpression of EDEM3 also greatly stimulates mannose trimming not only from misfolded ␣1-AT null (Hong Kong) but also from total glycoproteins, in contrast to EDEM, which has no apparent ␣1,2-mannosidase activity. Furthermore, overexpression of the E147Q EDEM3 mutant, which has the mutation in one of the conserved acidic residues essential for enzyme activity of ␣1,2-mannosidases, abolishes the stimulation of mannose trimming and greatly decreases the stimulation of ERAD by EDEM3. These results show that EDEM3 has ␣1,2-mannosidase activity in vivo, suggesting that the mechanism whereby EDEM3 accelerates glycoprotein ERAD is different from that of EDEM.ER 3 quality control is an elaborate mechanism conserved from yeast to mammals, ensuring that newly synthesized proteins in the ER fold and assemble correctly and that only proteins that acquire their correct conformations are sorted further into the secretory pathway (1-4). During this process, proteins that fail to attain their native conformation due to mutations of the polypeptides or to ER stress conditions adverse for protein folding as well as orphan subunits are degraded in a process known as ER-associated degradation (ERAD) (3, 5-7). The recognition of misfolded proteins for ERAD is still poorly understood, but there is increasing evidence for a role of mannose trimming in the targeting of glycoproteins for ERAD (8, 9). In mammalian cells, overexpression of ER ␣-mannosidase I stimulates ERAD of misfolded glycoproteins (10, 11), whereas the ␣1,2-mannosidase inhibitors kifunensine and 1-deoxymannojirimycin stabilize misfolded glycoproteins (12-16). These observations suggested that Man 8 GlcNAc 2 isomer B, the major product of the ER ␣1,2-mannosidase, is a recognition marker for ERAD of glycoproteins, but this view is being challenged, since there is increasing evidence that trimming to smaller oligosaccharides occurs on ERAD substrates (10,(17)(18)(19). We previously cloned mouse EDEM (ER degradation enhancing ␣-mannosidase-like protein) as a cDNA whose expression is up-regulated by ER stress and showed that EDEM accelerates glycoprotein ERAD (20). EDEM is an integral ER membrane protein that has all the signature motifs of Class I ...

Moyamoya disease is an idiopathic human cerebrovascular disorder that is characterized by progressive stenosis and abnormal collateral vessels. We recently identified mysterin/RNF213 as its first susceptibility gene, which encodes a 591-kDa protein containing enzymatically active P-loop ATPase and ubiquitin ligase domains and is involved in proper vascular development in zebrafish. Here we demonstrate that mysterin further contains two tandem AAA+ ATPase modules and forms huge ring-shaped oligomeric complex. AAA+ ATPases are known to generally mediate various biophysical and mechanical processes with the characteristic ring-shaped structure. Fluorescence correlation spectroscopy and biochemical evaluation suggested that mysterin dynamically changes its oligomeric forms through ATP/ADP binding and hydrolysis cycles. Thus, the moyamoya disease-associated gene product is a unique protein that functions as ubiquitin ligase and AAA+ ATPase, which possibly contributes to vascular development through mechanical processes in the cell.