Effects And Mechanisms Ofendogenous Dimethyl Sulfide On Oxidative Stress-induced Damage

Part Ⅰ Effects of dimethyl sulfide on oxidative stress-induced damageAims:Dimethyl sulfide (DMS) is a volatile sulfide, which is the most important sulfur-containing substance in the atmosphere. DMS was found in human blood, urine, breath and skin tissue. However, the effects of DMS in higher animals remain largely unknown. This sectionintended to study the effect of DMS on oxidative stress injuries and explore its regulatory role in higher animals.Methods:Headspace solid-phase microextraction gas chromatography-mass spectrometry, reactive oxidative species (ROS) fluorescence assay, superoxide fluorescence assay,lipid oxide detection, cell survival assay, electron resonance spectrum, model animal lifespan assay.Results:(1)DMS exists in rat tissues. In lower organisms, such as nematodes and fruit flies, DMS concentration is lower. In brain and heart tissue, the DMS concentration in aged rats decreased.(2) In the rat brain tissue and heart tissue homogenates, physiological concentrations of DMS (1μM) significantly reduced the hydrogen peroxide-induced lipid peroxidation.(3) Physiological concentrations of DMS (0.2μM,1μM and5μM) significantly inhibited antimycin A (antimycin A, AA) induced ROS generation in PC12cells.(4)DMS (5μM) significantly inhibited AA-induced superoxide production. DMS (5μM) significantly reduced AA-inducedhydroxyl radicals generation.(5) DMS supplement extended the lifespan of C. elegans and Drosophila. The superoxide accumulation in aged nematodesdropped.Meanwhile,the MDA concentration in aged Drosophiladecreased. Conclusion:Our results suggest that DMS decreases ROS, protects cell from oxidative injuries, and extends lifespan. DMS may play an important role in the defense system to oxidative stress. Part IIThe mechanism underlying the protective effect of DMSonoxidativestress-induced damageAims:Methionine sulfoxide reductase A (MsrA) is an important member of the methionine sulfoxide reductase family.Because ofits importantrole in redox defense system, MsrA received extensive attention. MsrA expressed in most species, repairingdamaged protein. In recent years, several studies showed that MsrAnot only fixed protein damage, but also directly reduced reactive oxygen species. Ourprevious studies indicated that L-methionine may bean important antioxidant substrate of MsrA. Until now, there is few report found other endogenous small molecules as substrates involved in the MsrA antioxidant system. Thus, we explored the mechanisms underlined the MsrA catalytic antioxidant system. And we tried to find out the role of endogenous antioxidant DMSby chemical biological methods.Methods:Luminol enhanced chemiluminescence, electron spin resonance spectroscopy, adenoviral expressionshRNA interference,expression and purification of the fusion protein, Western blotting, liquid chromatography-mass spectrometry, fluorescence anisotropy measurement, homology modeling of protein structure, computer simulation of enzyme-substrate interactions, site-directed mutagenesis, headspace solid phase micro-extraction gas chromatography-mass spectrometry Results:(1)In vitro experiments showed that DMS decreased ROS at high concentration (1mM and10mM).The hydrogen peroxide signal decresedin chemiluminescenceassay and the hydroxyl radical signaldecreasedin ESR assay.(2) MsrA enhanced the DMS’s antioxidant capacity to hydrogen peroxide and hydroxyl radicals significantly.(3) When MsrA in PC12cell was knocked down by shRNA, the protective effects of DMS were blocked. DMS could significantly reduce the hydrogen peroxide-induced cell death and lipid peroxidation, while DMS could not elevate cell survival rate or reduce MDA production in MsrA shRNA virus group.(4) The lifespan effect of DMS was cancelled in a MsrA RNAi line of drosophila.(5) It is proved by fluorescence anisotropy test that DMS binded to MsrA directly. And DMS was oxidated to DMSO by MsrA. Homology modeling constructed the3D structure of MsrA. The binding patterns between MsrA and its substrates were stimulated to prove the priority of DMS to MsrA.(6) Site directed mutants were built to prove the key residues of MsrA. The catalytic activity of C72S, Y103A and El15A decreased.Conclusion:DMS is is an importantendogenous substrate of MsrA.Under oxidative stress, DMS binds to the key residues of MsrA to form an intermediateand transforms to DMSO by MsrA to reduce ROS.