GENETIC ANALYSES OF GENES IN PRESENILIN RELATED PATHWAYS

早老素1和2(PS1、PS2)均经历调节的内蛋白分解 处理以产生两个稳定的碎片。我们最近发现， PS1和PS2将在正常的远端被交替切割 在细胞凋亡过程中的切割位点，以及在转染后过表达的情况 细胞系。PS1和PS2的凋亡相关内蛋白降解可以是 被凋亡的半胱氨酸蛋白酶抑制剂zVAD阻断，一般 Caspase抑制剂和选择性caspase-3(CPP32)抑制剂zDEVD， 这表明起作用的酶是caspase-3家族的蛋白酶。我们 已确定PS2中的细胞凋亡裂解位点为DSYDS(A.A.326-330) 通过定点突变。支持细胞凋亡的潜在作用 FAD中早老素的切割、细胞凋亡与正常切割的比率 在表达Fad突变体的细胞中，片段增加了3倍以上 PS2-N141I与W.T.表达PS2的细胞。由于FAD基因突变在 PS1/PS2与Abeta42：Abeta40的比率升高有关， 我们已经生成了初步数据来测试是否抑制了 Caspase-3介导的突变体PS2的切割可抑制增加的 Abeta42：与FAD突变相关的Abeta40基因。ZVAD治疗 抑制了Abeta42：与Abeta42有关的总Abeta比率的增加 PS2基因N141I和M239V FAD突变44%。总而言之，这些发现 不仅表明早老素可能是细胞死亡的底物，而且 半胱氨酸蛋白酶3家族对早老素的非凋亡性切割 可能改变Abeta42：Abeta40比值，在发病机制中发挥作用 时尚。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切作用 2.与细胞凋亡相关的早老素的切割导致 Abeta42：Abeta40比值增加；以及4.细胞凋亡性内蛋白分解 早老素改变亚细胞分布和/或分子 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.