GENETIC ANALYSES OF GENES IN PRESENILIN RELATED PATHWAYS

早老素1和早老素2（PS1，PS2）都经历受调节的内切蛋白水解， 处理以产生两个稳定片段。我们最近发现， PS1和PS2将在远离正常细胞的位点交替切割。 细胞凋亡过程中的切割位点，以及当转染细胞中过表达时， 细胞系凋亡相关的PS1和PS2的内蛋白水解可以是 被凋亡半胱氨酸蛋白酶抑制剂zVAD阻断， 半胱天冬酶抑制剂，和zDEVD，一种选择性半胱天冬酶-3（CPP 32）抑制剂， 表明起作用的酶是半胱天冬酶-3家族蛋白酶。我们 已经将PS2中的凋亡切割位点鉴定为DSYDS（a.a.（第326-330段） 通过定点诱变。支持细胞凋亡的潜在作用， FAD中早老素的裂解，凋亡：正常裂解的比率 在表达FAD突变体的细胞中， PS2-N141 I与w.t. PS2表达细胞。由于FAD突变， PS1/PS2与A β 42：A β 40比率升高有关， 我们已经产生了初步的数据来测试是否抑制 caspase-3介导的突变型PS2的切割可以抑制突变型PS2的细胞增殖， Abeta 42：与FAD突变相关的Abeta 40比率。zVAD治疗 抑制了A β 42：总A β比率的增加， N141 I和M239 V FAD突变在PS2中减少44%。总的来说，这些发现 不仅表明早老素可能是细胞死亡底物， caspase 3家族蛋白酶对早老素的非凋亡裂解 可能改变A β 42：A β 40比值，并在发病机制中发挥作用 的FAD。鉴于这些新的发现，我们将测试以下内容 假设：1.早老素FAD突变导致细胞凋亡增加- caspase-3家族对早老素的相关内蛋白水解 蛋白酶; 2.早老素的凋亡相关裂解导致 增加的A β 42：A β 40比率;和4.细胞凋亡内蛋白水解 早老素改变亚细胞分布和/或分子水平， 早老素的相互作用。

Presenilin 1 and 2 (PS1, PS2) both undergo regulated endoproteolytic processing to yield two stable fragments. We have recently discovered that PS1 and PS2 will be alternatively cleaved at sites distal to the normal cleavage sites during apoptosis, and when over-expressed in transfected cell lines. Apoptosis-associated endoproteolysis of PS1 and PS2 can be blocked by the apoptotic cysteine protease inhibitors, zVAD, a general caspase inhibitor, and zDEVD, a selective caspase-3 (CPP32) inhibitor, indicating that the enzyme responsible is a caspase-3 family protease. We have identified the apoptotic cleavage site in PS2 as DSYDS (a.a. 326-330) by site directed mutagenesis. In support of a potential role for apoptotic cleavage of the presenilins in FAD, the ratio of apoptotic:normal cleavage fragments was elevated by over 3-fold in cells expressing the FAD mutant PS2-N141I as compared to w.t. PS2-expressing cells. Since FAD mutations in PS1/PS2 have been associated with an elevated ration of Abeta42:Abeta40, we have generated preliminary data to test whether inhibition of the caspase-3 mediated cleavage of mutant PS2 could repress the increased Abeta42:Abeta40 ration associated with FAD mutations. Treatment with zVAD repressed the increases in Abeta42:total Abeta ratio associated with the N141I and M239V FAD mutations in PS2 by 44%. Collectively, these findings not only suggest that the presenilins may be cell death substrates, but not apoptotic cleavage of the presenilins by a caspase 3 family protease may alter the Abeta42:Abeta40 ration and play a role in the pathogenesis of FAD. In view of these novel findings, we will test the following hypotheses: 1. Presenilin FAD mutations lead to increased apoptosis- associated endoproteolysis of the presenilins by a caspase-3 family protease; 2. Apoptosis-associated cleavage of the presenilins leads to an increased Abeta42:Abeta40 ratio; and 4. Apoptotic endoproteolysis of the presenilins alters the subcellular distribution and/or molecular interactions of the presenilins.