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

以前未被认识的 β/γ 分泌复合物作为 AD 的治疗靶点

• 批准号: 9902298
• 负责人: Lei Liu
• 金额: $ 17.9万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2021-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9902298
• 关键词: Abeta synthesis; Active Sites; Address; Affinity Chromatography; Age-associated memory impairment; Alzheimer' s Disease; Amyloid beta-Protein; Binding; Biochemical; Biological; Biological Assay; Brain; C-terminal; Cells; Cellular biology; Cleaved cell; Collection; Complement; Complex; Cultured Cells; Data; Docking; Enzyme-Linked Immunosorbent Assay; Event; Fluorescence; G-substrate; GTP-Binding Protein alpha Subunits, Gs; Generations; Human; In Situ; In Vitro; Integral Membrane Protein; Internet; Kinetics; Life; Ligation; Macromolecular Complexes; Mammals; Mediating; Membrane; Methods; Modeling; Molecular Weight; Movement; Mus; Neurons; Pathogenesis; Pathway interactions; Peptide Hydrolases; Peptides; Pharmacology; Physiological; Process; Property; Protein Fragment; Proteins; Proteolysis; Proteomics; Research Personnel; Route; Signal Transduction; Site; Structure; Synapses; Testing; Therapeutic; alpha secretase; amyloid precursor protein processing; base; beta secretase; beta-site APP cleaving enzyme 1; brain tissue; design; drug candidate; enzyme substrate; enzyme substrate complex; fast protein liquid chromatography; gamma secretase; genetic manipulation; inhibitor/antagonist; insight; member; novel; novel strategies; novel therapeutic intervention; presenilin; prevent; protein protein interaction; proteostasis; recruit; response; screening; secretase; targeted agent; therapeutic development; therapeutic target; thermostability

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.

Aβ肽的产生发生在哺乳动物的整个生命过程中，并且它们在哺乳动物中的逐渐积累。 人类大脑是所有阿尔茨海默病病例中不变和必要的特征。Aβ的产生 需要通过β-分泌酶和γ-分泌酶进行蛋白水解，并了解这些过程的基础 连续裂解是细胞生物学的基础。特别是，确定全息APP 被顺序切割为Aβ肽对于设计安全有效的A β抑制剂和调节剂至关重要。 这一过程，以治疗并最终预防年龄相关的认知能力下降的主要部分。使用 生物化学和细胞生物学方法，我们最近发现，APP加工的α-和γ- 分泌酶可以存在于大的多蛋白酶级分中，其允许有效的连续切割 在由四跨膜蛋白网（1）的成员稳定的高分子量（HMW）复合物内的底物。 这一关于协调α/γ加工的意外发现提出了一个问题，即是否存在类似的机制， 存在β-和γ-分泌酶裂解，其从APP产生Aβ，并可产生类似蛋白 许多其他β/γ底物的片段。在过去几年中，在以下方面取得了实质性进展： 解析PS/γ-分泌酶复合物的20-TMD结构（2）。然而，我们仍然知之甚少 定义RIP的两步处理的细胞生物学机制。据推测，后... sheddase CTFs被运输到γ-分泌酶活跃的膜位点（3），但这种推定的 膜内的运动发生，使得CTF正确地找到并进入对接并激活 γ-分泌酶的位点仍然是一个谜。我们在R 03中探讨的正是RIP的这一强制性的两步功能 早期研究者的应用。为了解决这些机制，并测试可行性， 针对β/γ复合物，我们提出了以下两个具体目标。首先，我们将确认和描述 我们最近发现并从培养细胞中分离出一种新的催化活性β/γ-分泌酶复合物， 小鼠脑和人脑，通过使用1）蛋白质-蛋白质相互作用方法，包括co-IP，非变性PAGE， FPLC，PLA，NanoBiT（可逆动力学）和BiFC（不可逆），以及2）一种新的实验范式 发明PI是为了通过一系列新的β/γ-分泌酶的功能性酶表征， 自制的Aβ ELISA测定法。其次，我们将寻找与这种β/γ- 通过1）β/γ-分泌酶的蛋白质鉴定和定量蛋白质组学分析 从培养的细胞和人脑中分离的复合物; 2）对来自 蛋白质组学筛选以探索复杂的组装和稳定性;以及3）测试和设计小化合物 基于我们发现的β/γ机制拟议研究的完成将提供 作为一种以前未被认识到的细胞，通过单一蛋白酶复合物对协调蛋白水解的深入了解 作为生物学事件，其参与AD发病机制，以及作为更特异性β/γ调节剂的靶标。