The Mechanism Of Sulfurtransferase To Maintain Mitochondrial Health In Eukaryotic Cells

Sulfur is one of the essential elements for living organisms.Important intracellular sulfurcontaining compounds include hydrogen sulfide(H2S),glutathione(GSH),sulfite,sulfate,and thiosulfate.H2S has been recognized as the third gasotransmitter after carbon monoxide(CO)and nitric oxide(NO).It exerts various functions including cytoprotection,anti-inflammation,angiogenesis,and vasodilation at low concentrations.The mechanism of H2S signaling has been controversial.It is widely believed that the signal is transmitted through persulfidation modification of the cysteine residues in proteins,but H2S cannot directly modify the proteins.Sulfane sulfur is a unique class of intracellular sulfur-containing compounds.This form of sulfur,also known as "S0",usually shows 0 or-1 valence.Compounds containing S0 with strong reactivity are called reactive sulfane sulfur,also known as reactive sulfur species(RSS).RSS is an efficient antioxidant and oxidant that reacts with both nucleophilic and electrophilic reagents.And it can directly modify cysteine residues in proteins.Since H2S and RSS can be interconverted intracellularly,RSS is considered as a direct signaling molecule.Although a variety of RSS-producing enzymes are found in cytoplasm,mitochondria are considered to be the main site of RSS production.In mitochondria,H2S is oxidized by sulfide:quinone oxidoreductase(SQR)to produce RSS,and excess RSS is oxidized to sulfite by peroxisulfide dioxygenase(PDO)or converted to thiosulfate by Rhod.Cysteinyl-tRNA synthase 2(CARS2),which converts cysteine to cysteine persulfide(CysSSH)in mitochondria,also has RSS-generating function.The altered RSS levels would lead to mitochondrial dysfunction,suggesting a close link between RSS biogenesis and mitochondrial health,but the underlying mechanisms remain elusive.Rhod belongs to sulfurtransferase family that is widely present in prokaryotes,eukaryotes and archaea.It catalyzes the reaction of glutathione persulfide(GSSH)with sulfite to produce thiosulfate.It can also catalyze the reaction of thiosulfate with GSH to produce GSSH.Rhod catalyzes sulfur transfer reactions with the ping-pong mechanism.First,the sulfane sulfur of thiosulfate or other sulfane sulfur donors is transferred to the thiol group of conserved Cys to form ES0,the enzyme-sulfur adduct.Second,ES0 transfers the sulfane sulfur to a nucleophilic acceptor and the occupation of Cys thiol is released.Thiosulfate is usually regarded as a stable sulfane sulfur carrier in biological systems.It is quite inert at neutral and alkaline pH.Its spontaneous oxidation to sulfate takes several months and decomposition only happens at very acidic pH.And it can’t react directly with Cys.How the stable thiosulfate reacts with Rhod to form the intermediates of ES0 has not been clearly described.Is there other enzyme(s)involving in RSS production in mitochondria besides SQR and C ARS2?Rhod is capable of producing GSSH using thiosulfate,indicating its potential for RSS production in mitochondria.To study this in mammalian cells is difficult due to the dominant roles of SQR and CARS2.As a classic eukaryotic microorganism,Saccharomyces cerevisiae contains no SQR or CARS2.As a single-domain Rhod,Rdl2 localized in mitochondria can also produce GSSH,which means that Rdl2 is likely to be involved in the production of mitochondrial RSS.The proliferation and migration of colon cancer cells were influenced by RSS levels.Will the deletion of TST,a tandem-domain of Rhod in human mitochondria,affects mitochondrial function and inhibits cell proliferation?In order to answer the above questions,this thesis focuses on the reaction mechanism and physiological functions of Rhod,as well as the relationship between RSS levels and mitochondrial homeostasis.(1)Enzymatic kinetics experiments with single-domain,multi-domain and tandemdomain Rhod revealed that the absence of either Arg or Thr at the active loop terminus resulted in loss of sulfutransferase activity.The absence of amino acids in the middle of the activity loop resulted in a decrease in sulfutransferase activity,but its activity was still retained.This may be due to the fact that the amino acid at the end of the active loop occupies a more important spatial position because of the proximity of its side chain to Cys.Arg and Thr were shown to promote the decomposition of thiosulfate through the formation of H2S2O3 or HS2O3-leading to the release of sulfane sulfur.Combining the above results,a reaction mechanism of Rhod was proposed:Thiosulfate shares hydrogen bonds with amino acids at the end of the active loop to form H2S2O3 or HS2O3-under physiological conditions,which leads to the release of sulfane sulfur.The released sulfane sulfur then reacts spontaneously with Cys to form the persulfidation intermediates.(2)Rdl2 is the main enzyme for RSS generation in S.cerevisiae mitochondria,in which no SQR is present.Rdl2 generated RSS by utilizing stable sulfur sources such as thiosulfate or dimethyl trisulfide(MeSSSMe)through sulfutransferase activity.The reaction rate between RSS and HO· was two magnitudes higher than that of HO· reacting with DNA.Thus Rdl2generated RSS can protect DNA from HO·attack.In vitro,H2S promoted the Fenton reaction to produce more HO·leading to increased DNA damage.Knocking out Rdl2 resulted in the change of mitochondrial morphology,loss of mitochondrial DNA(mit-DNA),reduction of oxidative phosphorylation efficiency,and change of iron metabolism.Rdl2-generated RSS protects mitochondria from ROS damage by scavenging HO·,revealing an elusive link between RSS biogenesis and mitochondrial health.(3)Knocking ou t TST resulted in a slight decrease of mitochondrial RSS in colon cancer cells HCT116,while affecting the health of mitochondria.The TST KO cell line becomed more sensitive to ROS-producing chemotherapeutic agents such as DMF.The DMF-treated TST KO cell line accumulated more ROS,which affected the morphology of mitochondria and reduced the membrane potential.In addition to causing necrosis in the TST KO cell line,DMF also increased the expression of caspase-3 which promoted cell apoptosis.Under hypoxic conditions,the expression levels of HIF1-α and VEGF were reduced in TST KO cell line.It indicates that the proliferation and migration of TST KO cell lines may be inhibited in vivo,which reveals the potential of TST as a therapeutic target for colon cancer.In summary,this work explained how the active loop-terminal amino acid helped stable thiosulfate to give S0 to form ES0.It was demonstrated that Rhod was involved in Litochondrial RSS production using a stable S0 carrier in S.cerevisiae.And Rhod-generated RSS maintained mitochondrial homeostasis by scavenging ROS and affected cancer cell proliferation and drug resistance.This thesis provides an important basis for further understanding the physiological function of Rhod and reveals the mechanism by which RSS maintains mitochondrial health in eukaryotic cells.It contributes to a better comprehension of how intracellular RSS is involved in regulating various biological activities.