Abstract
AbstractCerebral small vessel disease (SVD) is a prevalent disease of aging and a major contributor to stroke and dementia. 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.
Funder
Foundation for the National Institutes of Health
American Heart Association
U.S. Department of Veterans Affairs
Publisher
Springer Science and Business Media LLC
Subject
General Agricultural and Biological Sciences,General Biochemistry, Genetics and Molecular Biology,Medicine (miscellaneous)
Reference30 articles.
1. Kim, H. W., Hong, J. & Jeon, J. C. Cerebral small vessel disease and Alzheimer’s disease: a review. Front. Neurol. 11, 927 (2020).
2. 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).
3. Dichgans, M., Ludwig, H., Müller-Höcker, 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. Genet. 8, 280–285 (2000).
4. Papakonstantinou, E. et al. NOTCH3 and CADASIL syndrome: a genetic and structural overview. EMBnet J. 24, e921 (2019).
5. Duering, M. et al. Co-aggregate formation of CADASIL-mutant NOTCH3: a single-particle analysis. Hum. Mol. Genet. 20, 3256–3265 (2011).
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