title: Improved plasma membrane expression of the trafficking defective P344R mutant of muscle, skeletal, receptor tyrosine kinase (MuSK) causing congenital myasthenic syndrome.
abstract: Muscle, skeletal, receptor tyrosine kinase (MuSK) is a key organizer at the postsynaptic membrane and critical for proper development and maintenance of the neuromuscular junction. Mutations in MUSK result in congenital myasthenic syndrome (CMS). We hypothesized that the CMS-causing missense mutation (P344R), found within the cysteine-rich domain of the protein, will affect its conformational tertiary structure. Consequently, the protein will misfold, get retained in the endoplasmic reticulum (ER) and lose its biological function through degradation by the highly conserved ER associated degradation (ERAD) machinery. We report that P344R-MuSK mutant is trafficking-deficient when expressed at 37°C in HeLa, COS-7 and HEK293 cell lines. It colocalized with the ER marker calnexin in contrast to wild-type MuSK which localized to the plasma membrane. The N-glycosylation status of P344R-MuSK is that of an immature and not properly post-translationally modified protein. Inhibition of protein synthesis showed that the P344R mutant's half-life is shorter than wild-type MuSK protein. Proteasomal inhibition resulted in the stabilization of the mutant protein. The mutant protein is highly ubiquitinated compared to wild-type confirming targeting for proteasomal degradation. The mutant showed around 50% of its in vivo autophosphorylation activity. P344R-MuSK mutant's trafficking defect is correctable by culturing the expressing cells at 27°C. Moreover, chemical compounds namely 2.5% glycerol, 1% dimethyl sulfoxide, 10 μM thapsigargin and 1 μM curcumin improved the maturation and exit of the mutant protein from the ER. These findings open perspectives for potential therapeutic intervention for patients with CMS harboring the P344R-MuSK mutation.
authors.affiliation: Department of Pathology, College of Medicine and Health Sciences, United Arab Emirates University, P.O. Box 17666, Al-Ain, United Arab Emirates.
authors.forename: Reham M
authors.initials: RM
authors.lastname: Milhem
authors.affiliation: Department of Pediatrics, College of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates.
authors.forename: Lihadh
authors.initials: L
authors.lastname: Al-Gazali
authors.affiliation: Department of Pathology, College of Medicine and Health Sciences, United Arab Emirates University, P.O. Box 17666, Al-Ain, United Arab Emirates. Electronic address: bassam.ali@uaeu.ac.ae.
authors.forename: Bassam R
authors.initials: BR
authors.lastname: Ali
mesh_terms: D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism; D002522:Chlorocebus aethiops; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D009132:Muscles / Q000201:enzymology* / Q000378:metabolism; D009154:Mutation; D020125:Mutation, Missense / Q000235:genetics; D020294:Myasthenic Syndromes, Congenital / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011950:Receptors, Cholinergic / Q000235:genetics / Q000378:metabolism*
chemical_list: D011950:Receptors, Cholinergic; C095714:MUSK protein, human; D020794:Receptor Protein-Tyrosine Kinases
keywords: Congenital Myasthenic Syndromes (CMSs); Endoplasmic Reticulum Associated Degradation (ERAD); Muscle, skeletal, receptor tyrosine kinase (MuSK); Neuromuscular junction; Proteasome; Protein misfolding; Trafficking; Ubiquitination