title: Mutations in NOTCH3 cause the formation and retention of aggregates in the endoplasmic reticulum, leading to impaired cell proliferation.
abstract: Mutations in the human NOTCH3 gene cause cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), but the pathogenic mechanisms of the disorder remain unclear. We investigated the cytotoxic properties of mutant Notch3 using stable cell lines with inducible expression of either wild-type or two mutants p.R133C and p.C185R. We found that both mutants of Notch3 were prone to aggregation and retained in the endoplasmic reticulum (ER). The turnover rates of the mutated Notch3 proteins were strikingly slow, with half-lives greater than 6 days, whereas wild-type Notch3 was rapidly degraded, with a half-life of 0.7 days. The expression of mutant Notch3 also impaired cell proliferation compared with wild-type Notch3. In addition, cell lines expressing mutant Notch3 were more sensitive to proteasome inhibition resulting in cell death. These findings suggest that prolonged retention of mutant Notch3 aggregates in the ER decreases cell growth and increases sensitivity to other stresses. It is also possible that the aggregate-prone property of mutant Notch3 contributes to a pathogenic mechanism underlying CADASIL.
authors.affiliation: Department of Vascular Dementia Research, National Institute for Longevity Science, National Center for Geriatrics and Gerontology, Obu city, Aichi, Japan.
authors.forename: Keikichi
authors.initials: K
authors.lastname: Takahashi
authors.forename: Kayo
authors.initials: K
authors.lastname: Adachi
authors.forename: Kaichi
authors.initials: K
authors.lastname: Yoshizaki
authors.forename: Shohko
authors.initials: S
authors.lastname: Kunimoto
authors.forename: Raj N
authors.initials: RN
authors.lastname: Kalaria
authors.forename: Atsushi
authors.initials: A
authors.lastname: Watanabe
mesh_terms: D015153:Blotting, Western; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D018832:Molecular Chaperones / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D020836:Protein Structure, Quaternary; D021381:Protein Transport / Q000187:drug effects; D000071656:Receptor, Notch3; D051880:Receptors, Notch / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism
chemical_list: D007976:Leupeptins; D018832:Molecular Chaperones; D050505:Mutant Proteins; C499374:NOTCH3 protein, human; D000071656:Receptor, Notch3; D051880:Receptors, Notch; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde