Redox Modulation of Protein Phosphatases by Protein Carbonylation/De-Carbonylation Process in Arterial Neointimal Hyperplasia

Reactive oxygen species (ROS) production is an unavoidable outcome of aerobic metabolism. Traditionally, ROS had been considered to nonspecifically and indiscriminately react with most constituents of cells. In the past decade, numerous studies indicated that ROS can induce signaling cascades through ligand-receptor interactions. ROS influence many signaling events, mainly through oxidative modification of proteins. Pro-oxidative events are typically correlated with activation of signaling molecules. Paradoxically, many of the direct oxidation events are inhibitory, blocking activity of target molecules. Therefore, the oxidation of protein tyrosine phosphatases (PTPs) is particularly important in redox signaling, because the inactivation of protein phosphatases can induce a broad range of signaling pathways. PTPs possess a conserved domain with a reactive and redox-regulated thiol-containing cysteine, which when oxidized makes the protein inactive, and this oxidation can be relieved by thiol antioxidants. Recently, another oxidative modification- protein carbonylation attracted people’s attention. This metal-catalyzed carbonylation is an important mechanism of oxidative damage to proteins. Protein carbonylation was considered to be an irreversible, non-enzymeatic modification of proteins. Nevertheless, a pioneer study lately found an interesting event that carbonylated proteins can be decreased subsequent to the peak of carbonylation within short term, and this de-carbonylation process is proteasome-independent. Protein carbonylation/de-carbonylation may be another important mechanism to be involved in redox signaling. In this project, we will clarify this novel redox regulating pathway-protein carbonylation/de-carbonylation for regulating protein phosphatases including PTPs and Ser/Thr protein phosphatases (Ser/Thr PPases) in the pathology of arterial neointimal formation, a harmful disease strongly related to oxidative stress. Evaluating the redox regulation of “phosphatase tone” and the expression of protein carbonylation in protein phosphatases in arterial neointimal hyperplasia will be performed in the first year. We will then survey the carbonylated sites of these carbonylated protein phosphatases by mass spectrometry analysis, and determine the specificity of these carbonylated sites for regulating protein phosphatases in arterial neointimal hyperplasia. In the second year, we will determine the role of thioredoxin/glutaredoxin system and decarbonylases (aldo-keto reductases and short-chain dehydrogenases/reductases) on the de-carbonylation of carbonylated protein phosphatases in arterial neointimal hyperplasia. The advantages of overexpressing de-carbonylation process by lentiviral transduction will be assessed in vitro and in vivo. In the final year, we will survey the unknown carbonylated proteins by two-dimensional gel-based proteomics, determine their roles in arterial neointimal hyperplasia, and try to define the general de-carbonylation mechanisms. Based on our preliminary results, we have known the protein phosphatase activities of Ser/Thr PPases and PTPs were both down-regulated in H2O2-stimulated vascular smooth muscle cells (VSMCs). PP2A and SHP-1 were indeed carbonylated in H2O2-treated VSMCs, and the carbonyaltion/de-carbonylation process in VSMCs stimulated by H2O2 is proteasomal degradation-independent. These data collectively suggest that protein carbonylation also happened in protein phosphatases and this kind of oxidative modification is reversible. Furthermore, according to the data of 2D-gel electrophoresis, several carbonylated proteins clearly expressed in H2O2-treated VSMCs. With the accomplishment of this project, we can clarify a novel redox signaling pathway- the carbonylation/de-carbonylation of protein phosphatases, and determine its role in the progression of arterial neointimal formation. Overexpression of protein de-carbonylation and protection of these identified carbonylated proteins may present a promising therapy for the treatment of oxidative stress-related diseases.