Characterization Of Human Glutaredoxin Binding Iron-sulfur Clusters And Their Functional Implications

Background:Glutaredoxin(GLRX),a member of the thioredoxin(Trx)family,plays an important role together with Trx system against oxidative stress and maintaining cellular redox balance.Four human glutaredoxins(GLRXs)have been found,including GLRX1,GLRX2,GLRX3,and GLRX5.GLRX1 and GLRX2 belong to the dithiol glutaredoxin group,proposed to serve as oxidative stress sensors,while GLRX3 and GLRX5 are monothiol forms,suggested to act as a scaffold for[2Fe-2S]cluster delivery and regulator for cellular iron homeostasis.Four GLRXs all have their own particular structures and function.Among them,GLRX2,GLRX3,and GLRX5 are capable of binding iron-sulfur clusters.GLRX2 may form a homodimer that binds a bridging[2Fe-2S]cluster with glutathione(GSH)as a ligand.Each iron of the iron-sulfur cluster is bound to the thiol of the cysteines,one of which is from N-terminal cysteine of the active site of GLRX2,another from GSH.GLRX3,whose crystal structure has not been revealed,was supposed to form a dimer that binds two bridging[2Fe-2S]clusters with GSH as a ligand,assembling a compound 2GLRX3-2[2Fe-2S]-4GSH.GLRX3 has two GRX domains,each containing one active site(-C-G-F-S-).Similar to GLRX2,the thiol of cystein of each active site and GSH bind to one iron of the iron-sulfur clusters,resulting in one domain dimer per cluster.GLRX3 is located in the cytosol and switches between the free monomer and the[2Fe-2S]cluster-bound dimer,which plays a role in cellular iron sensing and trafficking.The cytosolic iron responsive protein 1(IRP1)also serves as a cellular iron sensor.When the cellular iron increases,IRP1 is bound to the[4Fe-4S]cluster and turns into cytosolic aconitase.However,when the iron content decreases,IRP1 is dissociated from[4Fe-4S]cluster,and binds to the mRNA of untranslated regions(UTRs)of some iron related proteins,which facilitates cellular iron absorption and reduces iron storage.Objectives:The aims of this study are:(1)to determine whether isolated recombinant human GLRX3 is capable to transfer its iron-sulfur cluster to apo-IRP1 in vitro;(2)to investigate whether human GLRX3 may bind to other types of iron-sulfur clusters in addition to[2Fe-2S]cluster;(3)to reveal the roles that GLRXs and GSH play in iron-sulfur cluster transfer.Methods:Human GLRX3 was amplified by polymerase chain reaction(PCR)and ligated with pQE80L vector to construct a recombinant plasmid pQE80L-GLRX3-his6.The plasmid was transformed into the competent cells BL21(DE3).Soluble GLRX3-his6 fusion protein was overexpressed with the induction of isopropyl thio-?-D-galactoside(IPTG).After the bacteria were harvested by centrifugation and subsequently sonicated,GLRX3-his6 protein was purified with Ni-NTA column anaerobically.The protein was verified by sodium dodecyl sulfalte poly acrylamide gel electrophoresis(SDS-PAGE)and Coomassie stain.For the analysis of cluster types of GLRX3,UV-visible spectra were recorded.The freshly purified recombinant human GLRX3 was incubated with apo-IRP1 in an anaerobic glove box,followed by aconitase activity determination.Next,iron-sulfur cluster was assembled on GLRX3 by incubating the protein with sodium sulfide and ferric ammounium citrate in the presence or absence of GSH.UV-visible spectra were measured to estimate the iron-sulfur cluster composition.Aconitase activity was measured by incubating apo-IRP1 anaerobically with assembled GLRX3-[Fe-S]to confirm the possible type of iron-sulfur clusters bound to human GLRX3.GLRX2 was overexpressed in HEK293T cells,and mitochondrial and cytosolic aconitase activities were measured.BSO or diamide were added into HEK293T cells to inhibit the synthesis of GSH and oxidize sulfydry respectively,after which mitochondrial and cytosolic aconitase activities were determined.In the presence or absence of GLRX3,we transfered the iron-sulfur clusters from GLRX2 to apo-IRP1 to see the effect of GLRX3 on iron-sulfur cluster transfer from GLRX2 to IRP1.Results:(1)Isolated recombinant human GLRX3 prepared anaerobically from E.coli was brownish,and the UV-visible spectra of GLRX3 protein showed the presence of iron-sulfur cluster.The cluster could be transferred to the apo-protein IRP1 within 30 minutes to generate the aconitase activities.Two possible explanations were proposed:first,two[2Fe-2S]units converted to one[4Fe-4S]cluster,transferred to apo-IRP1 during the process;Second,some GLRX3 protein might directly bind[4Fe-4S]cluster in addtion to[2Fe-2S]reported previously and deliver[4Fe-4S]to apo-IRP1.(2)Isolated recombinant human GLRX2 showed a distinct UV-visible spectrum from that of GLRX3.The crystallographic structure has already comfirmed the presence of[2Fe-2S]cluster in GLRX2 dimer.GLRX2 was able to deliver its bound iron-sulfur cluster to apo-IRP1.However,the process was much slower than that of GLRX3,suggesting that recombinant GLRX3 may contain[4Fe-4S]cluster besides[2Fe-2S].(3)In the absence of GSH,anaerobically reconstituted GLRX3 was competent for activation of apo-IRP1 within 30 minutes.In the view of the UV-visible spectra of the reconstituted GLRX3,the iron-sulfur cluster was proposed to be[4Fe-4S]cluster.(4)In the presence of GSH,GLRX3 could bind assembled iron-sulfur cluster in vitro,and yet it was unable to be transferred to apo-IRP1 within 30 minutes.Taking into account the UV-visible spectrum,the cluster was supposed to be[2Fe-2S]cluster.(5)When mitochondrial GLRX2a was overexpressed in HEK293T cells,the activities of mitochondrial and cytosolic aconitase decreased obviously,possibly due to that GLRX2a binded more iron-sulfur clusters biosynthesized in mitochondria and prevented iron-sulfur cluster from transferring to mitochondrial and cytosolic aconitase.(6)Reduced synthesis of GSH in HEK293T and MEL cells resulted in decreased cellular aconitase activity,of which c-aco decreased more than m-aco.The results inferred that GSH played an important role in iron-sulfur clusters transfer from mitochondria to cytosol.(7)During the deliver of iron-sulfur clusters from GLRX2 to apo-IRP1 in vitro,GLRX3 prevented the cluster trafficking,indicating GLRX3 competed with IRP1 for acquiring iron-sulfur clusters from GLRX2.Conclusion:Our findings suggest that the monothiol human GLRX3 can incorporate[4Fe-4S]cluster in addition to[2Fe-2S]reported before in intro,and transfer them to the apo-protein IRP1 quickly,probably regulate cellular iron homeostasis together with IRP1.GLRX2,GSH and GLRX3 might play a role in iron-sulfur cluster trafficking from mitochondria to cytosol.