Significant evidence supports the hypothesis of amyloid beta (Aβ) as the cause of Alzheimer’s Disease (AD). Aβ is a peptide of variable length (38-43 amino acids) that is derived from proteolytic cleavage of the amyloid precursor protein (APP). Aβ peptides, particularly Aβ42, aggregate into plaques in the brains of individuals with AD and contribute to the neurodegeneration observed in this disease. Our lab previously developed an immunoprecipitation/mass spectrometry (IP/MS) protocol to study the metabolism of Aβ in the CNS, but this protocol was not sufficient for measurement of Aβ isoforms in the blood. Our present work aims to optimize the IP/MS protocol for measurement of 13C6-leucine labeled and unlabeled Aβ from human plasma in order to study Aβ metabolism in the blood. By labeling individuals with AD and healthy age-matched controls with 13C6-leucine, we can enable incorporation of 13C6-leucine into newly produced Aβ. Blood samples are taken from participants at regular intervals and processed using the IP/MS protocol. By measuring the C13:C12 ratio for various Aβ isoforms at each time point for an individual, labeling curves demonstrating AB production and clearance in the blood may be produced. If optimization of this protocol facilitates detection of differences in Aβ metabolism between clinical groups, this technology may be utilized as a blood test predictive of AD risk in the future.
In the field of medicine, intrinsically disordered proteins (IDPs) have been implicated in a number of diseases. For example, some IDPs aggregate readily and are implicated in debilitating brain diseases such as Alzheimer’s disease.
The purpose of the ongoing research is to develop a protocol to study IDPs and to understand the mechanisms of disorder-mediated protein interactions, which are key needs in structural biology and proteomics.Particularly in eukaryotic genomes, many proteins lack well-defined or ordered structures. Unlike structured proteins, IDPs have low sequence complexity. IDPs bind with high selectivity to their targets, as mediated by their disordered regions. IDPs are found in proteins that participate in cell signaling, transcription, and chromatin-remodeling functions. They undergo transitions to more ordered states upon binding to their targets or upon aggregation. This transition may be local or globular. For many IDPs, the disordered regions serve as “switches” in biological function. Switching to an ordered conformation upon interaction with their receptors (proteins or ligands) is an essential process in the protein function.
In this project, fast photochemical oxidation of proteins (FPOP), a new biophysical approach developed in the M. Gross lab, was used to show the change in structural conformation in CREB binding protein (CBP) and the activator of thyroid and retinoid receptors (ACTR) upon CBP binding ACTR. Our intention is to set forth a method for comparing structures of protein/protein complexes. With FPOP, we will monitor protein folding and binding on the time scale less than 1 µs, which may be essential for following the conformational switch in IDPs. By this example, a method for the detailed characterization of protein-protein interactions involving IDPs was demonstrated. Such an approach has higher throughput than NMR and X-ray crystallography. Application of this method for the rapid analysis of protein-ligand complexes may prove useful for studies of disease-related protein complexes, especially in drug discovery.