The Roles Of Sulfide:quinone Oxidoreductase In Maintaining Mitochondrial Health And Cysteine Biosynthesis In Schizosaccharomyces Pombe

Sulfur is an essential element for all living organisms,and it participates in various metabolic processes in different forms.Sulfide(HS-and H2S),especially H2S,is often referred as the third gas signal molecules after nitric oxide(NO)and carbon monoxide(CO).H2S plays important physiological functions in the body,regulating a variety of processes,including vasodilation,anti-tumor,anti-inflammation,and antioxidation.H2S is produced enzymatically,and it is reversibly converted to reactive sulfane sulfur(RSS).The two sulfur species often coexist.H2S is believed to function via forming RSS.The sulfur atom in RSS is zero-valent sulfur,which has stronger nucleophilic properties and reducing activities than H2S.RSS,includes inorganic polysulfide(HSnH,n≥2),organic polysulfide(RSnH,n≥ 2),and polysulfane(RSSnR,n≥ 2).RSS plays critical functions in the cell,such as signaling,redox homeostasis,and metabolic regulation.Several RSS producing enzymes have been identified.Cystathionine beta-synthase(CBS)and cystathionine gamma-lyase(CSE)are mainly located in cytoplasm;3-mercaptopyruvate sulfurtransferase(3-MST)and cysteinyl-tRNA synthetase 2(CARS 2)are mainly in mitochondria.These four enzymes use cysteine or its oxidized form cystine as a precursor to generate RSS.In addition,sulfide:quinone oxidoreductase(SQR)oxidizes H2S to generate RSS in mitochondria.Recently,it is observed that impairing RSS biogenesis by knocking down CARS2 leads to obvious mitochondrial dysfunction,intimating that RSS metabolism is related with mitochondrial health.Mitochondria dysfunction is often associated with age-related disorders,including neurodegenerative diseases,metabolic syndrome,and carcinogenesis.Therefore,maintaining the functionality and integrity of mitochondria is important for human health.SQR is a specific RSS producer in the mitochondria of many eukaryotes including humans.As the first enzyme in the sulfide oxidation pathway,SQR catalyses the conversion of H2S to RSS and is critical for the whole sulfur metabolism.However,the role of SQR in maintaining mitochondrial health has not been reported,due to challenges in studying SQR directly in mammalian cells.First,knocking-out SQR is lethal to mammalian cell,and currently no specific SQR inhibiting compound is available.Second,even SQR is successfully knocked out/down,the possibility that cytoplasmic RSS transports into mitochondria for functional complementation cannot be excluded.Third,in the presence of persulfide dioxygenase(PDO),the effect of SQR overexpression maybe compromised.The commonly studied yeast,Schizosaccharomyces pombe,has been used as a good model for studying mitochondrial function in eukaryotic cells.Further,S.pombe does not contain genes encoding Cbs,Cse,Cars2,or Pdo for RSS metabolism;it has mitochondrial Sqr and 3-Mst,providing an ideal platform for studying the Sqr’s role in maintaining mitochondrial health.In this dissertation,S.pombe was used to investigate the main production pathway of RSS,the role of Sqr in maintaining mitochondrial health and in synthesizing cysteine.The details are as follows:1.The main production pathway of RSS in S.pombe was determined.The genes encoding Sqr and 3-Mst are denoted as hmt2 and tuml in S.pombe,For description convenience,we refer them as sqr and mst in this paper.We constructed a series of mutant strains,including Δsqr,Δmst,and ΔsqrΔmst.The intracellular RSS,growth,and H2S production of the wild-type and the three mutants were determined,and the results show that the Δsqr and ΔsqrΔmst strains were defective in RSS production and growth,but released increased H2S into the gas phase.The analysis of mRNA showed that Sqr and 3-Mst could not complement each other for RSS production.These results collectively support that the oxidation of H2S by Sqr is the main producer of intracellular RSS in S.prombe.2.Sqr also played a role in maintaining mitochondrial health.The sqr deletion led to various physiological changes including reduced viability of the mutant cells,changed cell morphology and early apoptosis.The Δsqr cells showed reduced growth in a non-fermentable medium,suggesting that mitochondria may be impaired.When a GFP was fused with a leading peptide to localize it into mitochondrial inner membrane,laser confocal microscopy revealed that mitochondria in Δsqr were mostly fragmentized and scattered,suggesting that sqr knock-out disrupts mitochondrial morphological integrity.A series of physicochemical indicators that can characterize mitochondrial function,including mtDNA copy number,mitochondrial membrane potential,oxygen consumption,and ROS,were also tested,and the results demonstrated that mitochondria health was impaired in the Δsqr cells.Combined transcriptome(RNA-seq)and targeted metabolome analyses of wt and Δsqr strains showed that sqr knock-out led to wide changes in transcriptome and metabolome profiles,including dramatically changes in the TCA cycle,the electron transport chain,cysteine biosynthesis,etc.,which deserve further study.In conclusion,we reveal for the first time that mitochondria health is heavily dependent on Sqr.3.Mitochondria are the key organelles where sulfide is utilized to generate cysteine and oxidized to RSS by enzymes in S.pombe.We observed that the Δsqr strain totally lost the growth in basic medium and exhibited defects in cysteine synthesis.Further investigation revealed that the growth of Δsqr cells was arrested in the G2 phase of its cell cycle,and cysteine supplementation could restore the growth.The complementary effect of cysteine was also manifested in that the Δsqr strain significantly improved cell viability and restored mitochondrial morphological integrity and function to certain extents.The conversion of cysteine to RSS compensates for the loss of sqr,as we detected a significant increase in the content of RSS and H2S accumulation in both wt and Δsqr cells following cysteine supplementation to the medium.Cysteine synthase(Cys11)is also located in mitochondria,and its mRNA and protein levels were not reduced in the Δsqr strain.A cysteine synthase deletion strain Acys11 was contructed.Interestingly,Δcys11 andΔsqr strains were observed to exhibit similar phenotypes and physiological changes,suggesting that both SQR and Cysll may be involved in producing cysteine.How dose SQR affect cysteine synthesisin mitochondria?It is an interesting question worthy of further study.4.The sqr knock-out blocked cysteine biosynthesis was confirmed.Several lines of evidence supported that RSS was the intracellular substrate for cysteine biosynthesis in S.pombe.First,Cysll was shown to catalyze GSSH and OAS to produce Cys-SSG,which was reduced by GSH to produce Cys-SH and GSSG.When GSSH,H2S and S2O32-were used to determine kinetic parameters including Kcat,kcat/Km,and Vmax,Cysll preferred to use GSSH and H2S over S2O32-.Second,theΔsqr strain produced more H2S and less RSS than wt strain,butit displayed a cysteine-deficient phenotype,suggesting that H2S was not effectively used by Cys11.Third,the intracellular RSS was around 100 μM-400 μM;whereas,H2S was 15μM-50 μM.Other studies reported that intracellular H2S is even low to 10 nM-30 nM quantified with different methods.Therefore,RSS provides a relatively constant substrate reservoir for Cys11.Cysteine synthases from different microorganisms were tested,and they all used RSS as a substrate for cysteine synthesis.The finding that RSS instead of H2S was used by cysteine synthases to synthesize cysteine may rewrite the cysteine biosynthesis route in S.pombe and other eukaryotes and prokaryotes.In summary,we found that Sqr,mitochondrial health,and cysteine biosynthesis are interwound in S.pombe.First,the Sqr-catalyzed H2S oxidation to RSS and 3-Mst(and other rhodaneses)-catalyzed conversion of cysteine to RSS are the two resources of intracellular RSS.When exogenous cysteine nutrient is lacking,the former becomes the main pathway of RSS production.Second,RSS is the de facto substrate for cysteine biosynthesis other than H2S.Sqr knock-out results in RSS shortage,thereby leading to cysteine deficiency.Third,both RSS and cysteine are important for maintaining mitochondrial health.However,cysteine functions at least partially via forming RSS.Herein,we revealed that Sqr is the main RSS generating enzyme in S.pombe mitochondria.Fourth,the mitochondrial integrity and functionality were impaired in the Δsqr strain.These findings suggest that Sqr might be a potential new target for inhibiting cell proliferation and provide a new perspective for understanding of mitochondrial diseases under physiological or pathological conditions.