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Biochemical Characterization and Cellular Effects of CADASIL Mutants of NOTCH3

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 stud...

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Autores principales: Meng, He, Zhang, Xiaojie, Yu, Genggeng, Lee, Soo Jung, Chen, Y. Eugene, Prudovsky, Igor, Wang, Michael M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3445613/
https://www.ncbi.nlm.nih.gov/pubmed/23028706
http://dx.doi.org/10.1371/journal.pone.0044964
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author Meng, He
Zhang, Xiaojie
Yu, Genggeng
Lee, Soo Jung
Chen, Y. Eugene
Prudovsky, Igor
Wang, Michael M.
author_facet Meng, He
Zhang, Xiaojie
Yu, Genggeng
Lee, Soo Jung
Chen, Y. Eugene
Prudovsky, Igor
Wang, Michael M.
author_sort Meng, He
collection PubMed
description 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.
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spelling pubmed-34456132012-10-01 Biochemical Characterization and Cellular Effects of CADASIL Mutants of NOTCH3 Meng, He Zhang, Xiaojie Yu, Genggeng Lee, Soo Jung Chen, Y. Eugene Prudovsky, Igor Wang, Michael M. PLoS One Research Article 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. Public Library of Science 2012-09-18 /pmc/articles/PMC3445613/ /pubmed/23028706 http://dx.doi.org/10.1371/journal.pone.0044964 Text en https://creativecommons.org/publicdomain/zero/1.0/ This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
spellingShingle Research Article
Meng, He
Zhang, Xiaojie
Yu, Genggeng
Lee, Soo Jung
Chen, Y. Eugene
Prudovsky, Igor
Wang, Michael M.
Biochemical Characterization and Cellular Effects of CADASIL Mutants of NOTCH3
title Biochemical Characterization and Cellular Effects of CADASIL Mutants of NOTCH3
title_full Biochemical Characterization and Cellular Effects of CADASIL Mutants of NOTCH3
title_fullStr Biochemical Characterization and Cellular Effects of CADASIL Mutants of NOTCH3
title_full_unstemmed Biochemical Characterization and Cellular Effects of CADASIL Mutants of NOTCH3
title_short Biochemical Characterization and Cellular Effects of CADASIL Mutants of NOTCH3
title_sort biochemical characterization and cellular effects of cadasil mutants of notch3
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3445613/
https://www.ncbi.nlm.nih.gov/pubmed/23028706
http://dx.doi.org/10.1371/journal.pone.0044964
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