PRESENILIN ENDOPROTEASE & GAMMA-SECRETASE ACTIVITY

早老素内切蛋白酶

• 批准号: 6509704
• 负责人: Weiming Xia
• 金额: $ 26.13万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2004-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6509704
• 关键词: Alzheimer's disease; Escherichia coli; amyloid proteins; aspartate; endopeptidases; enzyme activity; gene expression; gene mutation; genetically modified animals; glutamates; laboratory mouse; membrane proteins; presenilin; protein binding; protein structure function; proteolysis

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.

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，在寻求破译AD的分子机制的主要目标是了解PS1和PS2的功能（和功能障碍）。与其他报告一致，我们已经表明，AD引起的突变PS1和2选择性地增强细胞生产和脑沉积的A β 42。我们还获得了少量APP和PS形成复合物的证据，并且我们证实了De Strooper等人的观察，即PS1的缺失显著降低APP的γ-分泌酶加工。最近，我们意外地发现，突变PS1中的2种不寻常和保守的跨膜（TM）腺苷酸酯中的任一种阻断了PS1的内蛋白水解，并急剧减少了Abeta的产生。 这些发现使我们推测早老素具有自蛋白水解和γ-分泌酶裂解活性。本研究将通过以下四个特定目的来验证这一假设：1）通过在PS1中引入Asp->Glu突变或轻微移动“活性位点”（2个对齐的Asp残基）来检测TM Asp残基在PS1内蛋白水解中的作用，并在E. 2）通过研究Asps的多个取代对细胞内和细胞外Abeta水平的影响以及在E. coli中共表达PS1 deltaE 9和C99来研究Asps残基在Abeta产生中的作用。3）分析PS2的蛋白水解活性，并检测野生型PS2是否能拯救Asp-PS1的生化表型（Abeta生成减少）和FAD突变PSI（增加的A β 42产生），以确定PS2是否具有与PS1非常相似的功能;和4）产生和表征Asp-mutant PS1 tg小鼠以证实PS在体内的γ-分泌酶活性。将APP tg小鼠与存活的Asp突变型PS1 tg小鼠杂交后，将检查后代的神经病理学效应。所有四个目标都是基于意外的，但有据可查的和可靠的初步数据。这些结果应该提供新的信息的跨膜蛋白（包括APP和PS1）的膜内蛋白水解的性质，并应阐明不寻常的机制Abeta的产生，一个新兴的治疗目标在AD。