Elucidation of the Cellular and Molecular Mechanisms of Missense Mutations Associated With Familial Exudative Vitreoretinopathy and Congenital Myasthenic Syndrome

Reham Mahmoud Mohammed Milhem

Abstract

The endoplasmic reticulum (ER), within eukaryotic cells, is a hub for protein folding and assembly. Misfolded proteins and unassembled subunits of protein complexes are retained in the ER and degraded by a process termed endoplasmic reticulum associated degradation (ERAD). Frizzled class receptor 4 (FZD4) and muscle, skeletal, receptor tyrosine kinase (MuSK) are Wnt receptors. These proteins contain the frizzled cysteine-rich domain (Fz-CRD) required for dimerization in the ER. Mutations in FZD4 and MuSK genes are known to cause familial exudative vitroretinopathy (FEVR, an autosomal dominant disease) and congenital myasthenic syndrome (CMS, an autosomal recessive disease), respectively. It was hypothesized that missense mutations within Fz-CRD lead to misfolding of FZD4 and MuSK proteins and consequent ER-retention. Investigating the molecular mechanism of these mutations is important since misfolded protein and ER-targeted therapies are in development being developed. Wild-type and mutants of FZD4 and MuSK were expressed at 37 °C in Hela, COS-7, and HEK293 cells and their subcellular localizations were investigated using confocal microscopy imaging and glycosidase treatments. Abnormal trafficking was demonstrated in 10 of 21 mutants studied; nine mutants were within Fz-CRD and one was distant from Fz-CRD. These ER-retained mutants were improperly N-glycosylated confirming ER-localization. They were tagged with polyubiqutin chains confirming targeting for proteasomal degradation. The half-lives of wild-type MuSK and P344R-MuSK were 45 and 37 minutes, respectively; the latter half-life improved on incubation with proteasomal inhibitor MG132. The P344R-MuSK kinase mutant showed around 50% of its in vivo autophosphorylation activity. Trafficking defects in three of the 10 mutants (M105T-FZD4, C204Y-FZD4, and P344R-MuSK) were rescued by expression at 27 °C and by chemical chaperones (2.5-7.5% glycerol, 0.1-1% dimethyl sulfoxide, 10 μM curcumin). Trafficking of wild-type FZD4 was not affected by c0-expression with any of the nine ER-retained mutants, suggesting haploinsufficiency as the mechanism of disease. Thus, all nine Fz-CRD mutants of FEVR and CMS studied resulted in misfolded proteins. In contrast, only one of the 12 mutants outside Fz-CRD resulted in ER-retention. These findings demonstrated a common mechanism for diseases associated with Fz-CRD missense mutations. Disorders of Fz-CRD may be receptive to novel therapies that alleviate protein misfolding.