PRESENILIN ENDOPROTEASE & GAMMA-SECRETASE ACTIVITY

早老素（PS）基因突变是显性遗传性阿尔茨海默病（AD）的主要原因。因此，了解PS1和PS2的功能（和功能障碍）是破解AD分子机制的一个主要目标。与其他报道一致，我们已经表明，ad引起的PS1和2突变选择性地增强了Abeta42的细胞产生和脑沉积。我们也获得了少量APP和PS形成复合物的证据，我们证实了De Strooper等人的观察，即PS1的缺失显著降低了APP的γ -分泌酶加工。最近，我们意外发现，突变PS1中2种不寻常且保守的跨膜（TM）天冬氨酸中的任何一种都会阻断PS1的内源性蛋白水解，并急剧减少β的产生。这些发现使我们假设早老素具有自身蛋白水解和γ -分泌酶裂解活性。这一假设将通过实施四个具体目标来检验：1)通过引入Asp->Glu突变或轻微移动PS1的“活性位点”（2个排列的Asp残基）来检测TM Asp残基在PS1内源性蛋白水解中的作用，并在大肠杆菌中表达人类PS1以寻找内源性蛋白水解产物；2)通过研究Asp的多次取代对细胞内和细胞外β水平的影响来检测Asp残基在β生成中的作用。并通过在大肠杆菌中共表达PS1deltaE9和C99来证实PS1的内在γ -分泌酶活性；3)解剖PS2的蛋白水解活性，检测wt PS2是否能挽救asp突变体PS1 （Abeta代减少）和FAD突变体PSI （Abeta42代增加）的生化表型，以确定PS2是否具有与PS1非常相似的功能；4)生成并鉴定asp突变体PS1 tg小鼠，以证实PS在体内的γ分泌酶活性。将APP tg小鼠与活的asp突变体PS1 tg小鼠杂交后，检测其对后代的神经病理学影响。所有四个目标都是基于意想不到但有充分记录和可靠的初步数据。这些结果应该为跨膜蛋白（包括APP和PS1）的膜内蛋白水解的性质提供新的信息，并应该阐明Abeta生成的不寻常机制，这是阿尔茨海默病的一个新兴治疗靶点。

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.