Proapoptotic effects of NARC 1 (=PCSK9), the gene encoding a novel serine proteinase

Background: NARC 1/PCSK9 encodes a novel serine proteinase known to play a role in cholesterol homeostasis. NARC 1 mRNA expression in cerebellar granule neurons (CGNs) was discovered to be induced following an apoptotic injury. Coregulation of known apoptotic mediators (caspase-3 and death receptor 6) raises the possibility that NARC I might be involved in the propagation of apoptotic signaling in neurons. Methods: CGNs were transfected with EGFP-fusion constructs of wild-type and mutant NARC 1, and a laser scan. p ning cytometry-based method of scoring cell death in transfectants was applied. Use of the poly-caspase inhibitor BAF allowed assessment of the caspase-dependence of the NARC I proapoptotic effect. Results: Wild-type NARC I was found to have substantial proapoptotic effects that were only partially reversible by BAF. Mutation of the active site serine or deletion of the cataIN,tic domain resulted in a reduced level of cell death, consistent With loss of the BAF-sensitive component of cell death. NH2-terminal deletion constructs of NARC I had effects similar to wild-type, both in the absence and presence of BAF, whereas expression of COOH-terminal deletion mutants produced a rate of cell death similar to wild-type in the absence of BAF treatment, but which lacked the capacity to be reduced by treatment with BAE. Conclusion: The mechanism by which NARC 1-EGFP over-expression induces cell death in cultured CGNs remains unclear. Mutation analysis established a positive correlation between the presence of the Narc I active site serine in the transiently expressed protein and induction of the BAF-sensitive component of the cell death phenotype. A caspase-independent component proved sufficiently complex to map discretely within the Narc I protein. (c) 2006 International Society for Analytical Cytology.