PRESENILIN DOMAINS AND RECONSTITUTION OF CATALYSIS

描述（由申请人提供）：γ-分泌酶具有蛋白水解活性，可催化从跨膜阿尔茨海默淀粉样前体蛋白（APP）生成淀粉样β-肽（AP）的最后一步。越来越多的证据表明，早老素蛋白（PS1和PS2）是γ-分泌酶裂解所必需的。PS蛋白被内切蛋白水解切割，产生N-末端片段（NTF）和C-末端片段（CTF）。NTF和CTF作为异二聚体保持稳定结合，并与称为nicastrin、aph-1和pen-2蛋白的伴侣蛋白组装在一起，以形成高分子量（约10/6 kDa）复合物，该复合物被认为构成功能性γ分泌酶。γ-分泌酶裂解是不寻常的，因为它显然发生在膜双层的无水环境中。许多跨膜蛋白以类似的方式加工的认识激发了一个一般概念的形成：调节性膜内蛋白水解，或RIP。该提案的总体目标是推进我们对PS蛋白的结构和功能的理解。在目的1中，将设计突变PS分子，其允许定位对于（i）PS的内切蛋白水解加工和（ii）APP的膜内加工以释放A β 40、A β 42和APP胞内结构域（AICD）重要的结构域。将使用连续截短和点突变策略。在目标2中，我们将阐明从重组PS1及其伙伴nicastrin、aph-1和pen-2在昆虫细胞中表达后产生乙酰基蛋白酶活性所需的条件。这项调查将允许γ-分泌酶活性的最小功能单元的识别和潜在的支持的概念，PS1确实是γ-分泌酶复合物的催化组分。

DESCRIPTION (provided by applicant): gamma-Secretase is the proteolytic activity that catalyzes the final step in the generation of the amyloid beta-peptide (AP) from the transmembrane Alzheimer amyloid precursor protein (APP). Growing evidence suggests that the presenilin proteins (PS1 and PS2) are required in order for gamma-secretase cleavage to occur. The PS proteins are endoproteolytically cleaved, generating an N-terminal fragment (NTF) and a C-terminal fragment (CTF). The NTF and CTF remain stably associated as heterodimers and assemble together with the partner proteins known as nicastrin, aph-1, and pen-2 proteins in order to form the high molecular weight (approximately 10/6 kDa) complex that is believed to constitute the functional gamma secretase. Gamma-Secretase cleavage is unusual in that it apparently occurs within the anhydrous environment of the membrane bilayer. The realization that numerous transmembrane proteins are processed in a similar fashion has inspired the formulation of a general concept: regulated intramembrane proteolysis, or RIP. The general goal of this proposal is to advance our understanding of the structure and function of PS proteins. In Aim 1 mutant PS molecules will be designed that permit the localization of domains that are important for (i) endoproteolytic processing of PS, and for (ii) intramembranous processing of APP to liberate Abeta40, Abeta42, and the APP intracellular domain (AICD). Successive truncation and point mutagenesis strategies will be utilized. In Aim 2, we will elucidate the conditions required for generating aspartyl proteinase activity from recombinant PS1 and its partners nicastrin, aph-1 and pen-2, following their expression in insect cells. This investigation will permit the identification of the minimal functional unit for gamma-secretase activity and potentially support the notion that PS1 is indeed the catalytic component of the gamma-secretase complex.