Proteomics

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Oligomerization, trans-reduction, and instability of mutant NOTCH3 in inherited vascular dementia


ABSTRACT: Cerebral 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.

INSTRUMENT(S): Orbitrap Fusion, Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

DISEASE(S): Cadasil

SUBMITTER: Kelly Young  

LAB HEAD: Michael M. Wang

PROVIDER: PXD031097 | Pride | 2022-05-20

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
PRF_Q_2019_M_WANG_89_47567.msf Msf
PRF_Q_2019_M_WANG_89_47567.raw Raw
PRF_Q_2019_M_WANG_89_47568.msf Msf
PRF_Q_2019_M_WANG_89_47568.raw Raw
PRF_Q_2019_M_WANG_89_47569.msf Msf
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Publications

Oligomerization, trans-reduction, and instability of mutant NOTCH3 in inherited vascular dementia.

Young Kelly Z KZ   Rojas Ramírez Carolina C   Keep Simon G SG   Gatti John R JR   Lee Soo Jung SJ   Zhang Xiaojie X   Ivanova Magdalena I MI   Ruotolo Brandon T BT   Wang Michael M MM  

Communications biology 20220407 1


Cerebral 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 m  ...[more]

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