Light-chain split luciferase assay implicates pathological NOTCH3 thiol reactivity in inherited cerebral small vessel...

Stereotyped mutations in NOTCH3 drive CADASIL, the leading inherited cause of stroke and vascular dementia. The vast majority of these mutations result in alterations in the number of cysteines in the gene product. However, non-cysteine-altering pathogenic mutations have also been identified, making it challenging to discriminate pathogenic from benign NOTCH3 sequence variants. Here, we present a method for quantitative assessment of NOTCH3 mutants, the light chain split luciferase (LSL) assay. In LSL, NOTCH3 mutant fragments, cloned between a split luciferase open reading frame, are transfected into cells, producing secreted luciferase activity that is dependent on the normal structure of NOTCH3. Insertion of point mutants that cause CADASIL results in significantly lower activity. Using a panel of 47 sequences, we determined the sensitivity and specificity of LSL for pathogenic NOTCH3 mutation discrimination to be 100% and 93%. LSL was also modestly successful in differentiating pathogenic proteins responsible for Marfan's disease and Stiff Skin Syndrome. Two additional parameters from the LSL analysis (TCEP rescue of activity and secretion index) were also shown to be useful in characterizing NOTCH3 mutants. We show that the spacing and primary sequence of the light chain module is an important component of the LSL assay, as a single light chain cysteine is critical for pathogenic sequence discrimination. Furthermore, we show that the activity of CADASIL mutant reporters is amplified by the application of cysteine-reactive iodoacetamide, suggesting that LSL may be deployed to screen for novel compounds that suppress pathogenic conformations of NOTCH3.