Electrophilic and Drug-Induced Stimulation of NOTCH3 N-terminal Fragment Oligomerization in Cerebrovascular Pathology

AbstractSmall vessel disease is a prevalent age-related condition linked to increased risk of dementia and stroke. We investigate the most commonly inherited form, CADASIL, caused by cysteine-involving mutations inNOTCH3. Recent studies highlight accumulation of NOTCH3 N-terminal fragmentation product (NTF) in disease. In vitro, NTF is capable of both spontaneous and catecholamine-enhanced cysteine-mediated oligomerization. Despite well-characterized genetic influence on CADASIL, environmental effects, including medication usage, on disease remain unclear. We studied effects of assorted electrophilic compounds and drugs on NTF oligomerization by SDS-PAGE and dynamic light scattering. We then examined direct proton pump inhibitor –NTF binding with antibodies designed against proton pump inhibitor–labeled proteins and mass spectrometry. Finally, we used monoclonal NTF antibodies with Proximity Ligation Assay to identify NTF oligomers in 3 CADASIL and 2 age-matched control brains. We identified enhancement of NTF oligomeri zation by two electrophilic cysteine-modifying compounds, N-ethylmaleimide and iodoacetamide, and an electrophilic compound capable of oxidizing cysteines, ferric chloride. Electrophilic clinical drugs (fenoldopam, omeprazole, tenatoprazole, lansoprazole, and rabeprazole) also promoted oligomerizati on, and we identified direct omeprazole-NTF and tenatoprazole-NTF complexes. Additionally, we provide novel evidence of NTF multimers in human CADASIL brains. ...
Source: Translational Stroke Research - Category: Neurology Source Type: research