A previously unrecognized β/γ-secretases complex as a therapeutic target for AD

Aβ多肽的产生贯穿哺乳动物的一生，它们在体内的逐渐积累 在所有阿尔茨海默病病例中，人脑是一个不变的和必要的特征。一种β的生产 要求先用β-分泌酶再用γ-分泌酶进行蛋白质分解，并了解其背后的过程 顺序分裂是细胞生物学的基础。特别是，识别holo-app的机制 被顺序切割成Aβ多肽是设计安全有效的抑制和调节剂的关键 这一过程才能治疗并最终预防很大一部分与年龄相关的认知能力下降。vbl.使用 生化和细胞生物学方法，我们最近发现由α和γ处理APP- 分泌酶可以出现在大的、多酶组分中，从而允许有效的顺序切割 高分子量(HMW)复合体中的底物，该复合体由Tetraspanin网的成员稳定(1)。 这一关于协调α/γ处理的意外发现提出了一个问题，即类似的机制是否 存在于β-和γ-分泌酶切割，它从APP产生β，并可以产生类似的蛋白质 许多其他β/γ底物的碎片。在过去的几年里，在以下方面取得了重大进展 破译PS/γ-分泌酶复合体的20-TMD结构(2)。然而，我们仍然对此知之甚少 定义RIP的两步处理的细胞生物学机制。据推测，邮局- 脱落酶CTF被运输到γ分泌酶活跃的膜部位(3)，但这种假定如何 在膜内发生运动，以便CTF正确地找到并进入对接和活动 γ分泌酶的位置仍然是一个谜。我们在本R03中探讨的正是RIP的这一强制性的两步特性 由早期调查人员提出的申请。为了解决这些机制并测试以下各项的可行性 针对β/γ综合体，我们提出了以下两个具体目标。首先，我们将确认和描述 我们最近从培养细胞中发现并分离出了一种新的具有催化活性的β/γ-分泌酶复合体， 利用1)蛋白质-蛋白质相互作用的方法，包括共IP，自然PAGE， FPLC、PLA、Nanobit(动力学可逆)和BIFC(不可逆)；2)新的实验范式 发明PI是为了通过收集一系列新的β/γ-分泌酶来进行功能性酶特性的 自制A-β酶联免疫吸附试验。第二，我们将搜索与此β/γ相关的监管组件- 分泌酶复合体：1)β/γ-分泌酶的蛋白质鉴定和定量蛋白质组学分析 从培养细胞和人脑中分离出的复合体；2)对来自 蛋白质组筛选以探索复杂的组装和稳定性；3)测试和设计小分子化合物 基于我们发现的β/γ机制。拟议研究的完成将提供机械性的 对单一蛋白酶复合体作为以前未被识别的细胞的协调蛋白分解的洞察 生物学事件，其在AD发病机制中的参与，以及作为更特定的β/γ调节剂的靶标。

The production of Aβ peptides occurs throughout life in mammals, and their progressive accumulation in human brain is an invariant and necessary feature in all cases of Alzheimer’s disease. The production of Aβ requires proteolysis by β-secretase followed by γ-secretase, and understanding the process underlying these sequential cleavages is fundamental to cell biology. In particular, identifying the mechanism by which holo-APP is cleaved sequentially to Aβ peptides is critical for designing safe and effective inhibitors and modulators of this process in order to treat and ultimately prevent a major portion of age-related cognitive decline. Using biochemical and cell biological approaches, we recently discovered that APP processing by α- and γ- secretases can occur in a large, multi-protease fraction that allows for efficient sequential cleavages of substrates within a high molecular weight (HMW) complex stabilized by members of the tetraspanin web(1). This unexpected finding about coordinated α/γ processing raised the question of whether a similar mechanism exists for the β- and γ-secretase cleavages which generates Aβ from APP and could create analogous protein fragments from many other β/γ substrates. In the last few years, there has been substantial progress in deciphering the 20-TMD structure of the PS/γ-secretase complex(2). However, we still know very little about the cell biological mechanism of the two-step processing that defines RIP. It has been assumed that the post- sheddase CTFs are trafficked to a membrane site where γ-secretase is active(3), but how such presumptive movement within the membrane occurs so that the CTFs correctly finds and enters the docking and active sites of γ-secretase remains a mystery. It is this obligatory, 2-step feature of RIP that we probe in this R03 application by an early stage investigator. To address these mechanisms and also test the feasibility of targeting the β/γ complex, we propose the following two Specific Aims. First, we will confirm and characterize a novel, catalytically active β/γ-secretase complex we recently discovered and isolated from cultured cells, mouse brain and human brain by using 1) protein-protein-interaction approaches including co-IP, native PAGE, FPLC, PLA, NanoBiT (reversible with kinetics) and BiFC (irreversible), and 2) a novel experimental paradigm the PI invented to perform functional enzymatic characterization of β/γ-secretases through a collection of new homemade Aβ ELISA assays. Second, we will search for regulatory components associated with this β/γ- secretase complex through 1) protein identification and quantitative proteomics analysis of β/γ-secretases complexes isolated from cultured cells and human brain; 2) genetic manipulation of potential hits from proteomic screening to explore complex assembly and stabilization; and 3) test and design small compounds based on the β/γ-mechanism we’ve discovered. The completion of the proposed study will provide mechanistic insights into the coordinated proteolysis by a single protease complex as a previously unrecognized cell biological event, its involvement in AD pathogenesis, and as a target for more specific β/γ-modulators.