Biochemical Characterization and Cellular Effects of CADASIL Mutants of NOTCH3 He Meng 1 , Xiaojie Zhang 1 , Genggeng Yu1, Soo Jung Lee1, Y. Eugene Chen2, Igor Prudovsky4, Michael M. Wang1,3,5* 1 Department of Neurology, University of Michigan, Ann Arbor, Michigan, United States of America, 2 Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, United States of America, 3 Department of Molecular & Integrative Physiology, University of Michigan, Ann Arbor, Michigan, United States of America, 4 Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, Maine, United States of America, 5 Neurology Service, VA Ann Arbor Healthcare System, Ann Arbor, Michigan, United States of America Abstract Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is the best understood cause of dominantly inherited stroke and results from NOTCH3 mutations that lead to NOTCH3 protein accumulation and selective arterial smooth muscle degeneration. Previous studies show that NOTCH3 protein forms multimers. Here, we investigate protein interactions between NOTCH3 and other vascular Notch isoforms and characterize the effects of elevated NOTCH3 on smooth muscle gene regulation. We demonstrate that NOTCH3 forms heterodimers with NOTCH1, NOTCH3, and NOTCH4. R90C and C49Y mutant NOTCH3 form complexes which are more resistant to detergents than wild type NOTCH3 complexes. Using quantitative NOTCH3-luciferase clearance assays, we found significant inhibition of mutant NOTCH3 clearance. In coculture assays of NOTCH function, overexpressed wild type and mutant NOTCH3 significantly repressed NOTCH-regulated smooth muscle transcripts and potently impaired the activity of three independent smooth muscle promoters. Wildtype and R90C recombinant NOTCH3 proteins applied to cell cultures also blocked canonical Notch fuction. We conclude that CADASIL mutants of NOTCH3 complex with NOTCH1, 3, and 4, slow NOTCH3 clearance, and that overexpressed wild type and mutant NOTCH3 protein interfere with key NOTCH-mediated functions in smooth muscle cells. Citation: Meng H, Zhang X, Yu G, Lee SJ, Chen YE, et al. (2012) Biochemical Characterization and Cellular Effects of CADASIL Mutants of NOTCH3. PLoS ONE 7(9): e44964. doi:10.1371/journal.pone.0044964 Editor: Huaibin Cai, National Institute of Health, United States of America Received April 15, 2011; Accepted August 16, 2012; Published September 18, 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: These studies were supported by grants from the National Institutes of Health (NS041342 and NS054724 to MMW; HL068878 and HL89544 to YEC; HL035627 to IP), the American Heart Association (postdoctoral fellowship 0726004Z to HM), a Burroughs Wellcome Fund Career Award in Biomedical Sciences (MMW), and a Maine Cancer Foundation grant (IP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: micwang@umich.edu Introduction Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is an inherited arteriopathy marked by migraine headaches, premature stroke, and vascular dementia [1,2,3]. CADASIL is caused by mutations in NOTCH3 [4], which encodes an evolutionarily conserved transmembrane receptor [5,6,7,8,9,10,11]. Almost all mutations described to date result in a change in the number of cysteines in the extracellular domain of the protein, which has led to the hypothesis that abnormalities in NOTCH3 secondary structure cause the arterial pathology seen in CADASIL [12,13,14,15,16]. Tissues from patients with CADASIL display damage to smooth muscle cells of small arteries and arterioles, characterized by cell- cell separation and cell loss, accumulation of granular osmiophilic material (GOM) around cells, fibrosis, and marked accumulation of NOTCH3 protein in the media [17,18,19,20]. In spite of this detailed description of pathology, a comprehensive view of the molecular and cellular changes induced by NOTCH3 excess has yet to emerge. Notch signal transduction plays an important role in the development and homeostasis of blood vessels. Notch or Notch ligand knockout and activated Notch transgenic mice display marked vascular phenotypes, ranging from subtle arterio-venous specification disorders to severe angiogenic failure [21,22,23,24,25,26,27,28]. Notch also plays a role in homeostatic control and responses of the mature vasculature to disease [29,30,31,32]. Although a majority of work has focused on Notch in endothelial cells, smooth muscle Notch has also been shown to regulate cell growth, death, and differentiation [33,34,35]. The profound effect of the Notch system on blood vessels suggests that in CADASIL, interference with normal NOTCH signaling by mutant NOTCH3 could play a major mechanistic role. Recently, NOTCH3 protein has been shown to form multiprotein complexes [36,37]; CADASIL mutations in NOTCH3, moreover, have been shown to slow the degradation of NOTCH3 [38]. These studies suggest that smooth muscle cells in CADASIL accumulate NOTCH3 due to mutations in the protein and implicate protein accumulation as a component of the pathological process. However, to date, the cellular effects of NOTCH3 overexpression have not been addressed. The purpose of this study was to investigate whether excessive NOTCH3 is capable of forming complexes with other NOTCH proteins. We discovered that NOTCH3 could bind tightly to multiple vascular NOTCH PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e44964