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

Area of Science:

• Biochemistry
• Molecular Biology
• Neuroscience

Background:

• Cerebral small vessel disease (SVD) is a common aging-related condition contributing to stroke and dementia.
• CADASIL, the most frequent inherited SVD, results from NOTCH3 gene mutations altering cysteine residues.
• The structural consequences of these NOTCH3 mutations on protein function are not fully understood.

Purpose of the Study:

• To investigate how CADASIL-associated NOTCH3 mutations affect protein oligomerization, thiol status, and stability.
• To determine the impact of a NOTCH3 N-terminal fragment (NTF) on wildtype and mutant NOTCH3 structure.

Main Methods:

• Preparation of wildtype and mutant recombinant NOTCH3 protein fragments.
• Analysis using gel electrophoresis, tandem mass spectrometry (MS/MS), and collision-induced unfolding.
• Assessment of disulfide bond formation, cysteine reduction, and protein structural stability.

Main Results:

• NOTCH3 mutants exhibited increased inappropriate disulfide bridges, reduced free cysteines, and structural instability.
• The NOTCH3 N-terminal fragment (NTF) further altered disulfide states in both wildtype and mutant proteins.
• NTF presence led to more reduced cysteines and enhanced destabilization of NOTCH3 structure.

Conclusions:

• CADASIL mutations induce specific cysteine alterations and affect NOTCH3 quaternary structure.
• Reductive factors, like NTF, significantly impact the structure and stability of the NOTCH3 protein relevant to SVD.
• These findings provide molecular insights into the pathogenesis of inherited cerebral small vessel disease.