Identifying the APP interactome influenced by phosphorylation of Thr668.

描述（由申请人提供）：本项目的总体目标是鉴定淀粉样前体蛋白（APP）胞质尾区（APPc）的生物结合伴侣，其依赖于残基T668（pT 668）的磷酸化和pT 668-P669肽键的异构体状态。磷酸化的APPc残基T668与阿尔茨海默氏病（AD）和增加生产的A？基于PI及其实验室对APPc的瞬时结构和T668磷酸化的局部结构后果的阐明，提出了两个探索性目的，包括将pT 668-P669肽键划分为不同的顺式（10%）和反式（90%）状态。假设这两种不同的结构将APP靶向至明显不同的结合伴侣，可能影响其蛋白水解命运并促进AD进展。在细胞中，与APPc的相互作用是竞争性的（由结合伴侣的亲和力和局部丰度决定），有时是合作性的（涉及多蛋白复合物的形成）。因此，虽然10%/90%是游离APPc的平衡顺式/反式平衡，但结合伴侣上异构体状态的分布将取决于结合蛋白的局部浓度和异构体特异性亲和力。我们的研究结果表明，脯氨酰异构酶Pin 1只作用于T668-磷酸化的APPc，加速重新平衡的自由顺式/反式人口通过异构化的pT 668-P669肽键，并防止致病加工的APP，支持这一假设，即异构体特异性结合伙伴的pT 668-P669基序参与的途径，影响生产的A？和AD的进展。APP通过两种不同的调节性膜内加工（RIP）途径被蛋白水解加工成特定片段，或者通过泛素介导的蛋白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，并且可以在该位点磷酸化，包括调节AD中涉及的特定基因转录的AICD片段。令人信服的文献表明，APP加工和降解，钙稳态，炎症和AD相关的基因调控都受到T668磷酸化的影响。在目的1中，提出了利用“诱饵”肽、精心选择的细胞系以及iTRAQ和GeLC-MS/MS蛋白质组学技术来确定磷酸化和非磷酸化T668 APPc的差异相互作用组的测定。在目标2中，将通过NMR或新开发的生化测定法（如果不适合NMR）研究目标1中鉴定的与AD相关途径相关的pT 668特异性的选定相互作用物的pT 668-P669肽键异构体特异性。这些研究应该产生关键的APP，CTF和AICD的相互作用打开或关闭T668磷酸化，以及它们的异构体特异性。该项目探索了新的领域，在其搜索异构体特异性结合合作伙伴的X-Pro基序。预期的结果是对APP的T668磷酸化信号的关键调控相互作用的详细基本理解，以及可能发现新的AD治疗药物开发的新靶点。

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.