PRESENILIN ENDOPROTEASE & GAMMA-SECRETASE ACTIVITY

早老素(PS)基因突变是主要遗传性阿尔茨海默病(AD)的主要原因。因此，研究阿尔茨海默病分子机制的一个主要目的是了解PS1和PS2的功能(和功能)。与其他报告一致，我们已经表明，导致AD的PS1和2的突变选择性地增强了Abeta42的细胞生产和脑沉积。我们还获得了少量APP和PS形成络合物的证据，我们证实了De Strooper等人的观察结果。PS1基因的缺失显著减少了APP的γ-分泌酶处理。最近，我们有了意想不到的发现，PS1中两个不寻常的和保守的跨膜(TM)天冬氨酸中的任何一个突变都阻止了PS1的内蛋白分解，并急剧减少了Abeta的产生。这些发现使我们假设早老素既具有自身蛋白分解活性，也具有伽马分泌酶切割活性。这一假说将通过四个特定的目的来验证：1)通过引入Asp-&Gt；Glu突变或稍微移动PS1中的“活性部位”(2个排列的Asp残基)来检测TM Asp残基在PS1内切蛋白中的作用，并在大肠杆菌中表达人PS1以寻找内切蛋白产物；2)通过研究Asps的多重取代对细胞内和细胞外Abeta水平的影响，以及通过在大肠杆菌中共表达PS1deltaE9和C99来证实PS1的内在伽马分泌酶活性，来检验Asp残基在Abeta生成中的作用；3)解剖PS2的蛋白分解活性，检测wt PS2能否挽救Asp突变体PS1(Abeta代减少)和Fad突变体PS1(Abeta42代增加)的生化表型，以确定PS2是否具有与PS L相似的功能；4)建立并鉴定Asp突变体PS1TG小鼠，以证实PS的体内伽玛分泌酶活性。将APP转基因小鼠与存活的天冬氨酸突变体PS1TG小鼠杂交后，将检测其后代的神经病理效应。所有四个目标都是基于意想不到的、但有充分证据和稳健的初步数据。这些结果将为跨膜蛋白(包括APP和PS1)膜内蛋白分解的性质提供新的信息，并应阐明Abeta产生的不寻常机制，Abeta是AD的一个新的治疗靶点。

Mutations in the presenilin (PS) genes are the major cause of dominantly inherited Alzheimer's disease (AD). Therefore, a major goal in the quest to decipher the molecular mechanisms of AD is to understand the function (and dysfunction) of PS1 and PS2. Consistent with other reports, we have shown that AD-causing mutations in PS1 and 2 selectively enhance the cellular production and cerebral deposition of Abeta42. We have also obtained evidence that small amounts of APP and PS form complexes, and we have confirmed the observation of De Strooper et al. that the deletion of PS1 markedly reduces gamma-secretase processing of APP. Recently, we made the unexpected discovery that mutating either of 2 unusual and conserved transmembrane (TM) aspartates in PS1 blocked PS1 endoproteolysis and sharply reduced Abeta production. These findings lead us to hypothesize that presenilins have both autoproteolysis and gamma-secretase cleavage activities. This hypothesis will be tested by carrying out four Specific Aims: 1) examine the role of the TM Asp residues in PS1 endoproteolysis by introducing an Asp->Glu mutation or slightly shifting the "active site" (the 2 aligned Asp residues) in PS1, and express human PS1 in E. coli to search for endoproteolytic products, 2) examine the role of the Asp residues in Abeta generation by studying the effect of multiple substitutions of the Asps on intracellular and extracellular Abeta levels, and by co-expressing PS1deltaE9 and C99 in E. coli to confirm the intrinsic gamma-secretase activity of PS1; 3) dissect the proteolytic activities of PS2 and examine if wt PS2 can rescue the biochemical phenotypes of Asp-mutant PS1 (reduced Abeta generation) and FAD mutant PSI (increased Abeta42 generation), in order to determine whether PS2 has a closely similar function to PS l; and 4) generate and characterize Asp-mutant PS1 tg mice to confirm the gamma-secretase activity of PS in vivo. After crossing APP tg mice with viable Asp-mutant PS1 tg mice, neuropathological effects in the offspring will be examined. All four Aims are based on unexpected but well documented and robust preliminary data. The results should provide novel information about the nature of the intramembranous proteolysis of transmembrane proteins (including APP and PS1) and should elucidate the unusual mechanism of Abeta generation, an emerging therapeutic target in AD.