Cleavage of 14-3-3 protein during programmed cell death = 세포예정사에서 14-3-3 단백질의 절단

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Caspase-mediated proteolysis is a critical and central element in the apoptotic process. Although it is important to identify the downstream molecular targets of capases, systematic and broadly applicable strategies to identify their substrates are not commonly available. This study established a systematic method for screening the substrate proteins for caspases by using a modified yeast two-hybrid system. The B42, the acidic domain of a transcriptional activator and a model substrate, poly(ADP-ribose) polymerase (PARP) were fused to the Lex A DNA-binding domain. The fusion protein was expressed in the yeast strain, EGY48, while caspase-3 was expressed under GAL1 inducible promoter in the yeast strain, YM4271. These two yeast cells expressing the substrate protein and the caspase were mated and the cleavage of the substrate was monitored by measuring the ß-galactosidase activity. It was demonstrated that the cleavage of a model substrate (PARP), was effectively detected in the system. The results indicate that this method is of useful for screening the substrate proteins of caspases and possibly of other proteases. 14-3-3ε protein was identified as one of the caspase-3 substrates by the modified yeast two-hybrid system. The cellular 14-3-3ε protein was also cleaved in response to the treatment of apoptosis inducers in cultured mammalian cells. Asp238 of 14-3-3ε protein was determined as the site of cleavage by caspase-3. The affinity of the cleaved 14-3-3 mutant protein (D238) to Bad, a death promoting Bcl-2 family protein, was lower than that of wild type or the uncleavable mutant 14-3-3ε protein (D238A). However, Bad associated with the cellular Bcl-x(L) more effectively in human 293T cells co-expressing Bad with the truncated form of 14-3-3ε protein (D238) than in control cells co-expressing Bad with wild type or the uncleavable mutant 14-3-3ε protein (D238A). The present study suggests that the cleavage of 14-3-3 protein during apoptosis promotes cell deat...
Joe, Cheol-O.researcher조철오researcher
한국과학기술원 : 생물과학과,
Issue Date
231024/325007  / 000995232

학위논문(박사) - 한국과학기술원 : 생물과학과, 2003.8, [ vii, 112 p. ]


14-3-3 protein; apoptosis; caspase; 캐스페이즈; 14-3-3 단백질; 세포예정사

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