Trans-Reduction of Cerebral Small Vessel Disease Proteins by Notch-Derived EGF-like Sequences

Area of Science:

• Biochemistry
• Molecular Biology
• Neuroscience

Background:

• Cysteine oxidation states regulate extracellular protein function and disease.
• Mutations in NOTCH3 are linked to cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), affecting vascular smooth muscle cells.
• Altered NOTCH3 cysteine states are implicated in CADASIL pathophysiology.

Purpose of the Study:

• To investigate the novel redox-modulating property of the NOTCH3 second EGF-like domain.
• To determine the effect of this property on NOTCH3 and other extracellular proteins.
• To explore the role of this mechanism in CADASIL.

Main Methods:

• Synthesis of NOTCH3 N-terminal fragment 2 (NTF2) peptides.
• In vitro reduction assays using NTF2 and NOTCH3 ectodomain polypeptides.
• Analysis of NTF2 interaction with NOTCH3 and other proteins (TSP2, CTSH).
• Examination of NOTCH3 and cleaved NOTCH3 in CADASIL patient cerebral arteries.

Main Results:

• NTF2 peptides reduce NOTCH3 ectodomain polypeptides in a dose- and time-dependent manner.
• NTF2 preferentially reduces EGF-like domains 12-15 of NOTCH3.
• NTF2 also reduces TSP2 and CTSH and forms complexes with the NOTCH3 ectodomain.
• CADASIL mutations enhance NOTCH3 susceptibility to NTF2-mediated reduction and trans-reduction.
• Cleaved NOTCH3 and NTF2 co-localize in cerebral arteries of CADASIL patients.

Conclusions:

• The NOTCH3 N-terminal fragment 2 possesses intrinsic cysteine redox-modulating activity.
• This activity, termed protein trans-reduction, targets NOTCH3 and other vascular proteins.
• Enhanced trans-reduction of mutant NOTCH3 suggests a novel mechanism contributing to CADASIL cerebrovascular pathology.