| Summary: | 碩士 === 國立陽明大學 === 生物藥學研究所 === 99 === PICK1 (Protein interacting with C-kinase 1) is a scaffold protein that predominantly locates to mitochondria in NIH3T3 cells. Our previous study showed that PICK1 stabilized mitochondrial membrane potential by recruiting protein kinase Cα (PKCα) . Mitochondria are highly mobile organelles that continuously undergo fusion and fission. Disturbance in mitochondrial dynamic has been shown to be associated with several physiological and pathological conditions. Our previous studies showed that down-regulation of PICK1 by shRNA in NIH3T3 cells significantly increased dependency on glucose for survival. In this study, neither the activation of caspase-3 nor the apoptotic nuclei was observed after cells were cultured under low glucose conditions, suggesting that apoptosis was not the predominant form of cell death under these conditions. Interestingly, we observed formation of autolysosomes as demonstrated by MDC (monodancyl-cadaverin) staining and increased LC3-II (light chain 3-II) levels under low glucose conditions in NIH3T3 parental cells. These results suggested that glucose starvation induced autophagy instead of apoptosis. Furthermore, knockdown of PICK1 promoted mitochondrial fragmentation under both normal and low glucose conditions, suggesting a role of PICK1 in regulating mitochondria morphology. Glucose starvation also induced a rapid processing of L-OPA1, a mitochondrial protein known to regulate mitochondria fusion, to S-OPA1 in PICK1 knockdown cells, which has been shown to be related to the loss of mitochondrial membrane potential. This result suggested that knockdown of PICK1 may promote mitochondrial dysfunction under low glucose conditions. The expression level of mitochondrial fusion protein, Mfn2, was not decreased in low glucose-triggered mitochondrial fragmentation. Overexpression of Mfn2 in PICK1-knockdown NIH3T3 cells increased mitochondrial fusion, but did not reverse the low glucose-induced cell death. Taken together, this study suggests that under low glucose conditions, knockdown of PICK1 promotes mitochondrial dysfunction which leads to autophagic cell death.
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