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The most commonly inherited SVD, CADASIL, is caused by dominantly acting cysteine-altering mutations in NOTCH3. These mutations change the number of cysteines from an even to an odd number, but the impact of these alterations on NOTCH3 protein structure remain unclear. Here, we prepared wildtype and four mutant recombinant NOTCH3 protein fragments to analyze the impact of CADASIL mutations on oligomerization, thiol status, and protein stability. Using gel electrophoresis, tandem MS\/MS, and collision-induced unfolding, we find that NOTCH3 mutant proteins feature increased amounts of inappropriate disulfide bridges, reduced cysteines, and structural instability. Presence of a second protein factor, an N-terminal fragment of NOTCH3 (NTF), is capable of further altering disulfide statuses of both wildtype and mutant proteins, leading to increased numbers of reduced cysteines and further destabilization of NOTCH3 structure. In sum, these studies identify specific cysteine residues alterations and quaternary structure induced by CADASIL mutations in NOTCH3; further, we validate that reductive factors alter the structure and stability of this small vessel disease protein.<\/jats:p>","DOI":"10.1038\/s42003-022-03259-2","type":"journal-article","created":{"date-parts":[[2022,4,7]],"date-time":"2022-04-07T10:20:54Z","timestamp":1649326854000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["Oligomerization, trans-reduction, and instability of mutant NOTCH3 in inherited vascular dementia"],"prefix":"10.1038","volume":"5","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6714-7563","authenticated-orcid":false,"given":"Kelly Z.","family":"Young","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2085-0577","authenticated-orcid":false,"given":"Carolina","family":"Rojas Ram\u00edrez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Simon G.","family":"Keep","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9759-2855","authenticated-orcid":false,"given":"John R.","family":"Gatti","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Soo Jung","family":"Lee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaojie","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Magdalena I.","family":"Ivanova","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6084-2328","authenticated-orcid":false,"given":"Brandon T.","family":"Ruotolo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5670-2496","authenticated-orcid":false,"given":"Michael M.","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2022,4,7]]},"reference":[{"key":"3259_CR1","doi-asserted-by":"publisher","first-page":"927","DOI":"10.3389\/fneur.2020.00927","volume":"11","author":"HW Kim","year":"2020","unstructured":"Kim, H. W., Hong, J. & Jeon, J. C. Cerebral small vessel disease and Alzheimer\u2019s disease: a review. Front. Neurol. 11, 927 (2020).","journal-title":"Front. Neurol."},{"key":"3259_CR2","first-page":"626","volume":"49","author":"KZ Young","year":"2020","unstructured":"Young, K. Z., Xu, G., Keep, S. G., Borjigin, J. & Wang, M. M. Small blood vessel disease in the brain theme issue overlapping protein accumulation profiles of CADASIL and CAA: is there a common mechanism driving cerebral small vessel disease? Am. J. Pathol. 49, 626 (2020).","journal-title":"Am. J. Pathol."},{"key":"3259_CR3","doi-asserted-by":"publisher","first-page":"280","DOI":"10.1038\/sj.ejhg.5200460","volume":"8","author":"M Dichgans","year":"2000","unstructured":"Dichgans, M., Ludwig, H., M\u00fcller-H\u00f6cker, J., Messerschmidt, A. & Gasser, T. Small in-frame deletions and missense mutations in CADASIL: 3D models predict misfolding of Notch3 EGF-like repeat domains. Eur. J. Hum. 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The effect of NOTCH3 pathogenic variant position on CADASIL disease severity: NOTCH3 EGFr 1-6 pathogenic variant are associated with a more severe phenotype and lower survival compared with EGFr 7-34 pathogenic variant. Genet. Med. 21, 676\u2013682 (2019).","journal-title":"Genet. Med."},{"key":"3259_CR18","doi-asserted-by":"publisher","first-page":"786","DOI":"10.1161\/STROKEAHA.114.007472","volume":"46","author":"FA Wollenweber","year":"2015","unstructured":"Wollenweber, F. A. et al. Cysteine-sparing CADASIL mutations in NOTCH3 show proaggregatory properties in vitro. Stroke 46, 786\u2013792 (2015).","journal-title":"Stroke"},{"key":"3259_CR19","doi-asserted-by":"publisher","first-page":"e0234797","DOI":"10.1371\/journal.pone.0234797","volume":"15","author":"H Kim","year":"2020","unstructured":"Kim, H., Lim, Y.-M., Lee, E.-J., Oh, Y. J. & Kim, K.-K. 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The use of de-identified human samples obtained at autopsy was not considered regulated human subjects research by the University of Michigan IRB (HUM00026522) or the VA Ann Arbor Healthcare System Research and Development Committee.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"The authors declare no competing interests.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"331"}}