A New Method For The Research Of Disulfide In Mammalian Cells

Disulfide bonds play important roles in protein structure and stability. Disulfide bond formation is vital for cellular functionality. Disulfide can be formed in the ER and mitochondria, they exhibit different mechanistic properties. Protein oxidation has been implicated in many processes of human health such as apoptosis, aging, and regulation of the respiratory chain. Redox-sensitive green fluorescent proteins (GFP) allow real time visualization of the disulfide formation potential of the indicator. For better understanding of subcellular localization of protein disulfide, we stablely expressed redox sensitive green fluorescent proteins (roGFPs) in different subcellular compartments by tagging these proteins with organelle specific signal peptides and measured disulfide formation potential of these subcellular compartments. We found cytosol is the most reductive organelle and endoplasmic reticulum, golgi apparatus and mitochondria are the two organelles that have highest protein disulfide formation potential.As a result of the complexity inherent in cellular processes, multiple analytical methods are generally required to clearly elucidate their underlying mechanisms. For Multiple labeling of cell,we tag Halotag with organelle specific signal peptides, so they can be located in specific organelles. Halotag is a novel technology for cell imaging. The fluorophore might be interchanged among a variety of standard dyes. Covalent bond formation between the protein tag and the dyes is highly specific, occurs rapidly under physiological conditions, and is irreversible.To study disulfide protein-protein interactions,we describe a technology based on SNAP protein-fragment complementation assay. In this strategy, a reporter protein SNAP (derivated from hAGT) is fragmented into two inactive fragments to which interacting/binding proteins are fused. The interaction between fused proteins leads to the formation of a reassembled, active reporter. SNAP activity should be detected by fluorescein derivatives. At last, we identified a pair of SNAP fragments that complemented to produce detectable activity when fused to two interactive proteins. This have the advantagement of PCA and method for the covalent labeling of fusion proteins.