THE STUDY OF PROTEIN DAMAGE IN AGING AND AGE-RELATED DISEASES IN BRAIN USING BI

Louisiana State Univ A&M Col Baton Rouge, Baton Rouge LA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Oxidative stress such as protein carbonylation, nitrosylation and glutathionylation formation, induced by reactive oxygen species has been implicated as a contributing factor to Alzheimer Disease (AD). They are considered markers of oxidative stress in aging and AD. The overall goal of this research is to identify the major carbonylated and nitorsylated proteins in the aging brain that contribute to the pathogenesis of AD and determine which neurological pathways are affected by the presence of these oxidative damaged proteins. Our first specific aim is to develop a "micro-fluidic chip" to enrich a minute amount of damaged proteins prior to proteomics. The microchip is replicated from a nickel mold master using hot embossing. This surface of microchip is chemically modified to specifically bind to carbonyls. The capture of the protein carbonyls is further validated with in-chip protein labeling and fluorescence microscopy. This microchip based protein carbonyl enrichment proves to be a very sensitive and specific affinity device to enrich protein carbonyls from minuscule samples. It can detect and capture 40 femtomole of carbonlys. Our second specific aim is to develop a proteomic reactor to simplify the processing of complex proteomic samples by combining multiple proteomic steps and allow for the identification of carbonylated, nitrosylated and glutathionylated proteins. This will help to understand the longitudinal change in the identities and relative ratios of specific protein targets of oxidative damage. Liver mitochondrial proteins from Sprague Dawley rats subjected to hydrogen peroxide oxidation are used to confirm the feasibility of quantitative identification of carbonylated proteins using the proteomic reactor and iTRAQ labeling. Additionally, we identify mitochondrial proteins susceptible to carbonylation and nitrosylation in a dose-dependent manner from In vitro oxidative stress model-HT29 human colon adenocarcinoma cell with menodione treatment using quantitative proteomics. In the future study, to understand the early and progressive cellular changes in AD development and progression, using quantitative proteomic profiling, this laboratory will investigate protein carbonylation in the AD transgenic mouse model at three stages of disease progression: long before (2-months old), immediately before (6-months old) and after (12-months old), the appearance of amyloid pathology and cognitive impairment.