Identifying the APP interactome influenced by phosphorylation of Thr668.

描述(申请人提供)：本项目的总体目标是确定依赖于残基T668(PT668)的磷酸化和pT668-P669肽键的异构体状态的淀粉样前体蛋白(APP)细胞质尾部(APPC)的生物结合伙伴。APPC残基T668的磷酸化与阿尔茨海默病(AD)和A？来自APP。在PI和她的实验室阐明APPC的瞬时结构和T668磷酸化的局部结构后果的基础上，提出了两个探索性目标，包括将pT668-P669肽键划分为不同的顺式(10%)和反式(90%)状态。这两种不同的结构被认为是将APP靶向于不同的结合伙伴，可能影响其蛋白分解命运并促进AD的进展。在细胞中，与APPC的相互作用是竞争性的(受结合伙伴的亲和力和局部丰度控制)，有时是合作的(涉及形成多蛋白复合体)。因此，虽然10%/90%是游离APPC的顺式/反式平衡，但异构态在结合伙伴上的分布将取决于结合蛋白的局部浓度和异构体特定亲和力。我们的研究发现，Pro异构酶Pin1仅作用于T668磷酸化的APPC，通过pT668-P669多肽键的异构化加速自由顺反种群的再平衡，并保护APP的致病过程，支持pT668-P669基序的异构体特异性结合伙伴参与影响A？和AD的进展。APP通过两条不同的调控膜内加工(RIP)途径被蛋白降解为特定的片段，或者通过泛素介导的蛋白酶体降解或自噬被完全降解。完整的APP及其RIP衍生的C末端片段(CTF)含有T668，并可在该位点被磷酸化，包括调节AD相关特定基因转录的AICD片段。令人信服的文献表明，APP的加工和降解、钙稳态、炎症和AD相关基因调控都受到T668磷酸化的影响。在目标1中，提出了利用“诱饵”肽、精心挑选的细胞系以及iTRAQ和GeLC-MS/MS蛋白质组学技术来确定磷酸和非磷酸-T668 APPC的差异相互作用的方法。在目标2中，将通过核磁共振或新开发的生化分析来研究在目标1中确定的与AD相关通路有关的pT668特异的相互作用体pT668-P669多肽键的异构体特异性，如果不符合核磁共振的话。这些研究应该产生关键的APP、CTF和AICD相互作用由T668磷酸化启动或关闭，以及它们的异构体特异性。该项目在寻找X-Pro基序的异构体特定结合伙伴方面探索了新的领域。预期的结果是对T668-APP磷酸化信号传递的关键调控相互作用有一个详细的基础了解，以及可能为开发新的AD疗法发现新的靶点。

DESCRIPTION (provided by applicant): The overall goal of this project is to identify biological binding partners of the amyloid precursor protein (APP) cytoplasmic tail (APPc) that depend on phosphorylation of residue T668 (pT668) and the isomer state of the pT668-P669 peptide bond. Phosphorylation of APPc residue T668 correlates with Alzheimer's disease (AD) and with increased production of A? from APP. Two exploratory Aims are proposed that build on the elucidation by the PI and her laboratory of the transient structure of APPc and the local structural consequences of T668 phosphorylation, including the partitioning of the pT668-P669 peptide bond into distinct cis (10%) and trans (90%) states. These two distinct structures are hypothesized to target APP to distinctly different binding partners, possibly influencing its proteolytic fate and contributing to AD progression. In the cell, interactions with APPc are competitive (governed by affinities and local abundances of binding partners) and sometimes cooperative (involve formation of multi-protein complexes). Thus, while 10%/90% is the equilibrium cis/trans balance of free APPc, the distribution of isomer states over bound partners will depend on the local concentrations and isomer-specific affinities of binding proteins. Our findings that prolyl isomerase Pin1 acts only on T668-phosphorylated APPc, accelerates re-equilibration of free cis/trans populations via isomerization of the pT668-P669 peptide bond, and protects against pathogenic processing of APP, support the hypothesis that isomer-specific binding partners of the pT668-P669 motif are involved in pathways that influence the production of A? and progression of AD. APP is proteolytically processed into specific fragments via two distinct regulated intramembrane processing (RIP) pathways, or is fully degraded via ubiquitin- mediated proteosomal degradation or autophagy. Intact APP and its RIP-derived C-terminal fragments (CTFs) contain T668 and can be phosphorylated at this site, including the AICD fragment that regulates transcription of specific genes implicated in AD. Convincing literature shows that APP processing and degradation, calcium homeostasis, inflammation, and AD-related gene regulation are all influenced by T668 phosphorylation. In Aim 1, assays are proposed that utilize "bait" peptides, carefully selected cell lines, and iTRAQ and GeLC-MS/MS proteomics technologies to determine the differential interactomes of phospho- and non-phospho-T668 APPc. In Aim 2, selected interactors specific for pT668 identified in Aim 1 that are implicated in AD-related pathways will be investigated for isomer specificity of the pT668-P669 peptide bond by NMR, or by newly developed biochemical assays if not amenable to NMR. These studies should yield key APP, CTF and AICD interactions switched on or off by T668 phosphorylation, as well as their isomer specificity. This project explores new territory in its search for isomer-specific binding partners of an X-Pro motif. The expected outcomes are a detailed fundamental understanding of key regulatory interactions signaled by T668-phosphorylation of APP, and the possible discovery of novel targets for the development of new AD therapeutics.