Proteomic analysis of Amyloid Precursor Protein signaling: mediators of toxicity

描述（由申请人提供）：阿尔茨海默病（AD）是痴呆症的最常见形式，也是美国最严重的医疗保健问题之一。虽然淀粉样蛋白-β（AB）通过AB-前体蛋白（APP）的裂解而积累已被确定为AD中潜在毒性的重要机制，但仍然没有有效的治疗方法。在体外数据的基础上，已经提出了介导AB毒性的各种机制，但在体内仍然很大程度上缺乏证据。我们最近发现，体内AB诱导的毒性的一个重要组成部分是依赖于APP在其细胞内结构域中Asp 664的裂解，因为动物在其他方面与明确定义的AD模型PDAPP小鼠相同，但携带Asp 664突变，继续产生和存款AB，但不发展AD样缺陷。A2结合APP并诱导APP多聚化，进而导致APP胞质尾部在Asp 664处裂解，随后导致突触和神经元损伤。使hAPP转基因动物抗AB积累的突变涉及APP的胞内信号结构域的稳定。我们的一般工作假设是，与APP的C-末端相关的蛋白质相互作用网络的信号复合物可能在体内介导AB毒性的信号通路中起关键作用。尽管与APP的C-末端相关的蛋白质相互作用已被广泛研究，但对APP的C-末端的信号传导知之甚少，并且尚未在人类相互作用组的更广泛背景下进行研究。此外，最近的研究采用传统的筛选方法未能检测到新的相互作用，这表明传统的方法可能已经用尽，不太可能产生新的信息。因此，映射出与APP的C-末端结构域相关的信号网络，我们建议应用一种新的方法来识别和分析支架蛋白在蛋白质组范围内的相互作用，基于目标辅助迭代筛选（TAIS）和功能关联指纹分析，三个主要的APP相关支架蛋白，JIP 1b/IB 1，X11/Mint和Fe 65。我们的具体目标是：1.定义JIP 1b/IB 1、X11/Mint 1和Fe 65的蛋白质-蛋白质相互作用结构域（PID）的识别共识。2.鉴定和验证人类蛋白质组中JIP 1b/IB 1、X11/Mint 1和Fe 65 PID的相互作用伙伴。3.对JIP 1b/IB 1、X11/Mint 1和Fe 65 PID的经验证的相互作用伴侣进行功能聚类，以确定通过这些支架蛋白整合的细胞功能和信号级联。拟议研究的长期目标是获得对体内介导AB毒性的细胞内信号传导途径的全面机制理解。确定AB毒性途径中的关键介质将允许合理设计旨在阻断AB毒性的新型治疗方法。 淀粉样蛋白-β（AB）通过AB-前体蛋白（APP）的裂解而积累被认为是阿尔茨海默病（AD）的毒性的基础，阿尔茨海默病是老年人中最常见的痴呆形式。我们建议应用一种新的蛋白质组学方法来阐明AB毒性的途径。旨在阻断AB毒性的治疗方法可以为AD的治疗或预防中旨在降低AB的治疗方法提供替代或补充。

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the most common form of dementia, and one of the most serious healthcare issues in the United States. Although the accumulation of amyloid-beta (AB) by cleavage of the AB-precursor protein (APP) has been identified as an important mechanism underlying toxicity in AD, effective treatments are still not available. Various mechanisms mediating AB toxicity have been proposed on the basis of in vitro data, but evidence for their relevance in vivo is still largely lacking. We recently showed that a significant component of AB-induced toxicity in vivo is dependent on the cleavage of APP at Asp664 in its intracellular domain, since animals otherwise identical to a well-defined model of AD, PDAPP mice, but carrying a mutation at Asp664 continue to produce and deposit AB but do not develop AD-like deficits. A2 binds APP and induces APP multimerization, leading in turn to cleavage of the APP cytosolic tail at Asp664, followed by synaptic and neuronal damage. The mutation that rendered hAPP transgenic animals resistant to AB accumulation involves the stabilization of the intracellular signaling domain of APP. Our general working hypothesis is that the signaling complexes of the protein interaction network associated with the C-terminus of APP may have a key role in signaling pathways mediating AB toxicity in vivo. Even though the protein interactions associated with the C-terminus of APP have been extensively researched, signaling from the C-terminus of APP is poorly understood and has not yet been studied in the wider context of the human interactome. Also, recent studies employing conventional screening methods failed to detect novel interactions, suggesting that conventional approaches may have been exhausted and are unlikely to yield new information. Therefore, to map out the signaling network associated with the C-terminal domain of APP we propose to apply a new methodology for the identification and analysis of interactions of scaffolding proteins on a proteome-wide scale, based on target-assisted iterative screening (TAIS) and functional association fingerprint analysis, to the three major APP-associated scaffolding proteins, JIP1b/IB1, X11/Mint and Fe65. Our specific aims are: 1. Define recognition consensuses for the protein-protein interaction domains (PIDs) of JIP1b/IB1, X11/Mint1 and Fe65. 2. Identify and validate interacting partners of JIP1b/IB1, X11/Mint1 and Fe65 PIDs in the human proteome. 3. Perform functional clustering of validated interacting partners of JIP1b/IB1, X11/Mint1 and Fe65 PIDs to determine cellular functionalities and signaling cascades integrated through these scaffolding proteins. The long-term goal of the proposed studies is to obtain a comprehensive mechanistic understanding of intracellular signaling pathways that mediate AB toxicity in vivo. The identification of key mediators in pathways of AB toxicity will allow for the rational design of novel therapeutic approaches aimed at blocking AB toxicity. The accumulation of amyloid-beta (AB) by cleavage of the AB-precursor protein (APP) is believed to underlie toxicity in Alzheimer's disease (AD), the most common form of dementia among older people. We propose to apply a new proteomic methodology to the elucidation of pathways of AB toxicity. Therapeutic approaches aimed at blocking AB toxicity may provide an alternative or a complement to those aimed at lowering AB in the treatment or prevention of AD.