Oxidatively-modified proteins: I. Proteomic identification of specifically-oxidized proteins in Alzheimer's disease brain. II. Modulation of synaptosomal membrane lipid bilayer asymmetry by 4-hydroxy-2-trans-nonenal.

摘要

The studies described is this dissertation were performed with the intent of investigating the role of oxidative stress as a relevant event in Alzheimer's disease (AD). AD is a neurodegenerative disorder associated with age, which results in loss of memory and cognitive function. Extensive experimental evidence indicates that oxidative stress might play an important role in modulating the degenerative events characteristic of this disorder. Reaction of radical species might occur close to the site of formation or in different locations, and results in attack on proteins, lipids and DNA. The subsequent oxidative damage to these biomolecules is particularly pronounced in the brain, due to its high oxygen consumption and lower antioxidant defense.; It has been demonstrated that protein oxidation often results in loss in protein function. Introduction of protein carbonyls and nitration of tyrosine, markers of protein oxidation, even on few key amino acids, inevitably change the protein conformation, which is often a cause for its dysfunction. Identification of these targets of protein oxidation would represent very valuable evidence toward the understanding of how oxidative stress modulates cellular life at a protein level. Discovery of which protein is selectively oxidized in AD brain may provide deep insights into the oxidation-related mechanism of neurodegeneration, in which a few key oxidized proteins are not capable of expleting their function and thus impair the metabolic, signaling pathway, structural maintenance or antioxidant function they modulate.; The introduction of proteomics techniques has allowed a relatively easy approach to investigate these targets. It couples 2-dimensional polyacrilamide electrophoresis (2D-PAGE) and mass spectrometry (MS) analysis, which help in elucidating the dynamism of cellular systems by studying the products of genes, proteins, and their structure, function, localization and thus modification in condition of disease. Identification of oxidized protein in AD is a proteomic achievement toward understanding of the role of oxidative stress in AD brain and shows the potentiality of proteomics in providing important information in condition of disease. Collection of this information offers the opportunity to study mechanisms of disease, conditions in which proteins are often involved in pathological events, and the identification of new potential therapeutic targets. In our studies, proteomics is also applied to study the consequences of a dysfunctional protein that has been identified as target of protein oxidation in AD brain, in an animal model of disease. This has been achieved by proteomic analysis of the GAD mouse.; Oxidative damage occurs also in brain lipids, that relates to apoptosis in AD, and leads to production of reactive aldehydes, among which 4-hydroxynonenal (HNE) and acrolein. This dissertation also investigates the effects of HNE and acrolein, lipid peroxidation products, in the maintenance of phospholipid asymmetry in synaptic membranes.; The data collected for this dissertation provide additional evidence sustaining the oxidative stress hypothesis in AD.