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Reham Mahmoud Moh’d Milhem
Department of Pathology
College of Medicine and Health Sciences
Dissertation
Title
ELUCIDATION OF THE CELLULAR AND MOLECULAR MECHANISMS OF MISSENSE MUTATIONS ASSOCIATED WITH FA-
MILIAL EXUDATIVE VITREORETINOPATHY AND CONGENITAL MYASTHEN-IC SYNDROME
Faculty Advisor
Prof. Bassam Ali
Defense Date
22 February 2015
Abstract
The endoplasmic reticulum (ER), within eukaryotic cells, is a hub for protein folding and assembly. Mis-folded 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 cyste-ine-rich domain (Fz-CRD) required for dimerization in the
ER. Mutations in FZD4 and MuSK genes are known to cause familial exudative vitreoretinopathy (FEVR, an autosomal
dominant disease) and congeni-tal 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. 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 by confocal microscopy imaging and glycosidase
treatments. Abnormal trafficking was demonstrated in 10 of 21 stud-ied mutants; nine mutants were within Fz-CRD and
one was distant from Fz-CRD. These ER-retained mu-tants were improperly N-glycosylated confirming ER-localization.
They were tagged with polyubiquitin chains confirming targeting for proteasomal degradation. The half-lives of wild-type
MuSK and P344R-MuSK were 90 and 37 min, respectively; the latter half-life improved on incubation with the proteasomal
inhibitor MG132. The P344R-MuSK kinase mutant showed around 50% of its in vivo autophosphoryla-tion 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% dime-thyl sulfoxide, 10 μM thapsigargin, or 1 μM curcumin). Trafficking of
wild-type FZD4 was not affected by co-expression with any of the nine ER-retained mutants, suggesting haploinsufficiency
as the mechanism of disease. Thus, all nine studied Fz-CRD mutants of FEVR and CMS resulted in misfolded proteins. In
contrast, only one of the 12 mutants outside Fz-CRD resulted in ER-retention. These findings demon-strate a common
mechanism for diseases associated with Fz-CRD missense mutations. Disorders of Fz-CRD could be amenable to novel
therapies that alleviate protein misfolding.
Research Relevance and Potential Impact
Reham Milhem’s PhD research resulted in the elucidation of the cellular mechanisms underlying many mutations causing two ge-netic disorders,
namely congenital myasthenic syndrome and familial exudative vitreoretinopathy. These findings are original and are likely to have impact on our
understanding of the parthenogenesis of these incurable disorders and possibly help in develop-ing novel therapies for them. The results have been
published in journals that are in the top 5-10% of their respective fields.