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 1 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 scanning 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 1 proapoptotic effect. Results Wild‐type NARC 1 was found to have substantial proapoptotic effects that were only partially reversible by BAF. Mutation of the active site serine or deletion of the catalytic domain resulted in a reduced level of cell death, consistent with loss of the BAF‐sensitive component of cell death. NH 2 ‐terminal deletion constructs of NARC 1 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 BAF. 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 1 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 1 protein. © 2006 International Society for Analytical Cytology