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肽的产生在哺乳动物中发生在整个生命中，它们的进行性积累 在阿尔茨海默氏病的所有情况下，人脑都是不变且必要的特征。 Aβ的产生 需要β-分泌酶，然后是γ-分泌酶的蛋白水解，并了解这些过程 顺序切割是细胞生物学的基础。特别是识别holo-app的机制 依次裂解至Aβ肽对于设计安全有效的抑制剂和调节剂至关重要 这一过程是为了治疗并最终阻止与年龄相关的认知能力下降的主要部分。使用 生化和细胞生物学方法，我们最近发现α-和γ-的应用程序处理 分泌酶可能发生在大型多酸盐分数中，从而可以有效地分解 高分子重量（HMW）复合物中的底物稳定在四翼烷蛋白Web的成员（1）中。 关于协调α/γ处理的这种意外发现提出了一个问题，即是否类似机制 存在于从APP中产生Aβ的β-和γ-分泌酶切割，并且可能会产生类似的蛋白 来自许多其他β/γ底物的片段。在过去的几年中，在 解密PS/γ-分泌酶复合物的20-TMD结构（2）。但是，我们对 定义RIP的两步处理的细胞生物学机制。已经假定邮政 SHEDDASE CTF被运输到γ-分泌酶活性的膜位点（3），但是这种假定是如何进行的 膜内的运动发生，以便CTF正确找到并进入对接并活跃 γ-分泌酶的位点仍然是一个谜。我们在此R03中探测了RIP的强制性，两步的特征 早期调查员的应用。解决这些机制，并测试 针对β/γ络合物，我们提出以下两个特定目标。首先，我们将确认并描述 一种新型的催化活性β/γ-分泌酶复合物，我们最近从培养细胞中发现并分离 小鼠大脑和人脑使用1）蛋白质 - 蛋白质交互方法在内 FPLC，PLA，Nanobit（动力学可逆）和BIFC（不可逆），以及2）一种新型的实验范式 PI发明了通过新的新型酶酶表征对β/γ-分泌酶进行功能性酶促表征 自制AβELISA分析。其次，我们将搜索与此β/γ-相关的调节组件 通过1）蛋白质鉴定和定量蛋白质组学分析β/γ-分泌酶进行泌尿酶复合物 从培养细胞和人脑分离的复合物； 2）对潜在命中的基因操纵 蛋白质组学筛选以探索复杂的组装和稳定； 3）测试和设计小型化合物 基于我们发现的β/γ机制。拟议研究的完成将提供机械 通过单个蛋白酶复合物作为先前未识别的细胞对协调的蛋白水解的见解 生物事件，其参与AD发病机理，也是更特定的β/γ调节剂的靶标。