Choi E K, Zaidi N F, Miller J S, Crowley A C, Merriam D E, Lilliehook C, Buxbaum J D, Wasco W
Genetics and Aging Unit, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129, USA.
J Biol Chem. 2001 Jun 1;276(22):19197-204. doi: 10.1074/jbc.M008597200. Epub 2001 Mar 9.
Calsenilin is a member of the recoverin family of neuronal calcium-binding proteins that we have previously shown to interact with presenilin 1 (PS1) and presenilin 2 (PS2) holoproteins. The expression of calsenilin can regulate the levels of a proteolytic product of PS2 (Buxbaum, J. D., Choi, E. K., Luo, Y., Lilliehook, C., Crowley, A. C., Merriam, D. E., and Wasco, W. (1998) Nat. Med. 4, 1177-1181) and reverse the presenilin-mediated enhancement of calcium signaling (Leissring, M. A., Yamasaki, T. R., Wasco, W., Buxbaum, J. D., Parker, I., and LaFerla, F. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8590-8593). Here, we have used cultured mammalian cells that transiently or stably express calsenilin to extend the characterization of calsenilin and of the calsenilin-PS2 interaction. We have found that calsenilin has the ability to interact with endogenous 25-kDa C-terminal fragment (CTF) that is a product of regulated endoproteolytic cleavage of PS2 and that the presence of the N141I PS2 mutation does not significantly alter the interaction of calsenilin with PS2. Interestingly, when the 25-kDa PS2 CTF and the 20-kDa PS2 CTF are both present, calsenilin preferentially interacts with the 20-kDa CTF. Increases in the 20-kDa fragment are associated with the presence of familial Alzheimer's disease-associated mutations (Kim, T., Pettingell, W. H., Jung, Y., Kovacs, D. M., and Tanzi, R. E. (1997) Science 277, 373-376). However, the finding that the production of the 20-kDa fragment is regulated by the phosphorylation of PS2 (Walter, J., Schindzielorz, A., Grunberg, J., and Haass, C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1391-1396) suggests that it is a regulated physiological event that also occurs in the absence of the familial Alzheimer's disease-associated mutations in PS2. Finally, we have demonstrated that calsenilin is a substrate for caspase-3, and we have used site-directed mutagenesis to map the caspase-3 cleavage site to a region that is proximal to the calcium binding domain of calsenilin.
钙结合蛋白（Calsenilin）是神经元钙结合蛋白恢复蛋白家族的成员，我们之前已证明它能与早老素1（PS1）和早老素2（PS2）全蛋白相互作用。钙结合蛋白的表达可调节PS2蛋白水解产物的水平（Buxbaum, J. D., Choi, E. K., Luo, Y., Lilliehook, C., Crowley, A. C., Merriam, D. E., and Wasco, W. (1998) 《自然医学》4, 1177 - 1181），并逆转早老素介导的钙信号增强（Leissring, M. A., Yamasaki, T. R., Wasco, W., Buxbaum, J. D., Parker, I., and LaFerla, F. M. (2000) 《美国国家科学院院刊》97, 8590 - 8593）。在此，我们使用了瞬时或稳定表达钙结合蛋白的培养哺乳动物细胞，以进一步表征钙结合蛋白及其与PS2的相互作用。我们发现钙结合蛋白能够与内源性25 kDa的C末端片段（CTF）相互作用，该片段是PS2经调控的内蛋白水解切割产物，并且N141I PS2突变的存在并不会显著改变钙结合蛋白与PS2的相互作用。有趣的是，当25 kDa的PS2 CTF和20 kDa的PS2 CTF同时存在时，钙结合蛋白优先与20 kDa的CTF相互作用。20 kDa片段增加与家族性阿尔茨海默病相关突变的存在有关（Kim, T., Pettingell, W. H., Jung, Y., Kovacs, D. M., and Tanzi, R. E. (1997) 《科学》277, 373 - 376）。然而，20 kDa片段的产生受PS2磷酸化调节这一发现（Walter, J., Schindzielorz, A., Grunberg, J., and Haass, C. (1999) 《美国国家科学院院刊》96, 1391 - 1396）表明，这是一个受调控的生理事件，在PS2不存在家族性阿尔茨海默病相关突变时也会发生。最后，我们证明钙结合蛋白是半胱天冬酶-3的底物，并使用定点诱变将半胱天冬酶-3切割位点定位到钙结合蛋白钙结合结构域近端的一个区域。
