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.
The long-term goal of this project is to discover critical binding event(s) that influence the amyloidogenic processing fate of the amyloid precursor protein (APP). The proposed studies take a novel approach toward this goal by determining the cis vs. trans isomer specificity of binding partners that interact with the phospho- Thr668-Pro669 peptide bond in the APP cytoplasmic domain (APPc), hypothesized to be a molecular switch governing the APP processing fate. 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.