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.


CONCLUSIONS
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.
Bingham
Brendan
B
Neuroscience Discovery Research, Wyeth Research, Princeton, New Jersey 08543-8000, USA. binghab@wyeth.com
Shen
Ru
R
Kotnis
Smita
S
Lo
C Frederick
CF
Ozenberger
Bradley A
BA
Ghosh
Nivedita
N
Kennedy
Jeffrey D
JD
Jacobsen
J Steven
JS
Grenier
Jill M
JM
DiStefano
Peter S
PS
Chiang
Lillian W
LW
Wood
Andrew
A
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D053148:Caspase 3 / Q000378:metabolism; D061945:Caspase Inhibitors; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000201:enzymology / Q000473:pathology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D047569:Laser Scanning Cytometry / Q000379:methods*; D009474:Neurons / Q000187:drug effects / Q000201:enzymology / Q000473:pathology*; D017354:Point Mutation; D000071449:Proprotein Convertase 9; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012694:Serine; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; C417985:butyloxycarbonyl-aspartyl-fluoromethyl ketone; D012694:Serine; C510804:PCSK9 protein, rat; D000071449:Proprotein Convertase 9; D012697:Serine Endopeptidases; D053148:Caspase 3