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Maleimide-peptide assay principle. (a) Catalytic cycle of the cysteine desulfurase NFS1. (1) formation of persulfide on NFS1 (NFS1-SSH) through PLP-catalyzed desulfurization of L-cysteine that generates L-alanine as a by-product. NFS1 persulfide can be reduced by thiols (RSH) by two different mechanisms: S-thiolation (2) or sulfur transfer (3). The S-thiolation pathway leads first to sulfide release and S-thiolation of NFS1 (NFS1-SSR) (2a) and then reduction of NFS1-SSR by a second thiol (2b). The sulfur transfer pathway leads first to sulfur transfer on the thiol that generates a persulfurated thiol (RSSH) (3a) and then hydrogen sulfide after reduction by a second thiol (3b). (b) Reaction scheme of alkylation assay principle by maleimide-peptide (MalP) for identification of persulfide. The reactions of formation and reduction of persulfide are quenched by adding MalP (dark blue) under denaturing conditions (SDS) that alkylates all the thiol groups including persulfurated and non-persulfurated cysteines. The presence of a persulfurated cysteine is revealed by the loss of the succinimide-peptide moiety (light blue) on reduction of the disulfide bond of the persulfide by DTT. The different species are then separated by SDS–PAGE. (c) Structure of the MalP16 maleimide-peptide. (d) Maleimide-peptide assay of NFS1 persulfide before and after reaction with L-cysteine. The alkylated forms of NFS1 are indicated as NFS1+7 and NFS1+6 corresponding to seven and six cysteine residues alkylated by MalP16, respectively.
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Friedreich's ataxia is a severe neurodegenerative disease caused by the decreased expression of frataxin, a mitochondrial protein that stimulates iron-sulfur (Fe-S) cluster biogenesis. In mammals, the primary steps of Fe-S cluster assembly are performed by the NFS1-ISD11-ISCU complex via the formation of a persulfide intermediate on NFS1. Here we s...
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... is not required for NFS1 persulfide formation. In mam- mals, the global rate of sulfide production is enhanced by FXN and decreased by ISCU when thiols are used as reductants 11,14 , but a step-by-step characterization of the effects of FXN and ISCU on the catalytic cycle of NFS1 is still lacking (Fig. 1a). To monitor the formation and reduction of NFS1 persulfide independently, we developed a new assay enabling the detection and quantification of persulfide. This assay is based on the chemical properties of the persulfide group, which contains both a terminal sulfur amenable to alkylation by maleimide compounds and a disulfide bond that ...
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... and reduction of NFS1 persulfide independently, we developed a new assay enabling the detection and quantification of persulfide. This assay is based on the chemical properties of the persulfide group, which contains both a terminal sulfur amenable to alkylation by maleimide compounds and a disulfide bond that can be cleaved by reduction (Fig. 1b). In brief, a persulfide is revealed by the loss of the succinimide-peptide moiety (the product of maleimide reaction with a sulfhydryl) upon cleavage of the sulfur-sulfur bond by dithiothreitol (DTT). To visualize the loss of the succinimide moiety, we engineered maleimide compounds with a peptide arm (maleimide-peptide, MalP) of a ...
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... of the sulfur-sulfur bond by dithiothreitol (DTT). To visualize the loss of the succinimide moiety, we engineered maleimide compounds with a peptide arm (maleimide-peptide, MalP) of a size sufficiently large to be detected by SDS-polyacrylamide gel electrophoresis (PAGE). The best results were obtained with a 16-mer MalP (MalP 16 : 1.95 kDa) (Fig. 1c). In the absence of L-cysteine (the substrate of NFS1), the seven cysteines of NFS1 were alkylated by MalP 16 (denoted NFS1 þ 7 species) and no loss of the succinimide-peptide moiety was observed upon reduction by DTT, indicating that none of NFS1 cysteines was initially persulfurated ( Fig. 1d and Supplementary Fig. 1a). Following ...
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... obtained with a 16-mer MalP (MalP 16 : 1.95 kDa) (Fig. 1c). In the absence of L-cysteine (the substrate of NFS1), the seven cysteines of NFS1 were alkylated by MalP 16 (denoted NFS1 þ 7 species) and no loss of the succinimide-peptide moiety was observed upon reduction by DTT, indicating that none of NFS1 cysteines was initially persulfurated ( Fig. 1d and Supplementary Fig. 1a). Following reaction with L-cysteine, the seven cysteines of NFS1 were still alkylated by MalP 16 but reduction by DTT led to the loss of one succinimide-peptide moiety from the NFS1 þ 7 species leading to formation of an NFS1 þ 6 species, thereby indicating the presence of a persulfide group on a single cysteine of NFS1 (Fig. ...
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... and Supplementary Fig. 1a). Following reaction with L-cysteine, the seven cysteines of NFS1 were still alkylated by MalP 16 but reduction by DTT led to the loss of one succinimide-peptide moiety from the NFS1 þ 7 species leading to formation of an NFS1 þ 6 species, thereby indicating the presence of a persulfide group on a single cysteine of NFS1 (Fig. ...
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... a first set of experiments, we monitored the influences of ISCU (U) and FXN (F) on the rates of persulfide formation (reaction 1, Fig. 1a) by the NFS1-ISD11 complex (NI). The NFS1-ISD11 complex (NI), ISCU (U) and FXN (F) have been shown to form homodimeric ternary and quaternary complexes with NI:U and NI:U:F stoichiometries of 1:1 and 1:1:1 for each subunit, respectively 11,14,23 , which will be denoted NIU and NIUF thereafter. We confirmed here by size exclusion ...
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... these conditions, NFS1 was almost entirely converted to its persulfurated form (B95%) 10 s after reaction with L-cysteine, and this irrespective of the presence of FXN and/or ISCU (Fig. 2a). Mammalian FXN is thus not required for persulfide formation on NFS1, in contrast to what was shown for the yeast homologue of FXN, Yfh1, even when using the concentrations of enzyme and L-cysteine reported in this study ( Supplementary Fig. 1b) 12 . ...
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... next investigated persulfide transfer from NFS1 to ISCU using the maleimide-peptide assay (Fig. 2d). Before reaction of the NIU complex with L-cysteine and upon reaction with MalP 16 , the majority of ISCU carried four succinimide-peptide moieties (denoted ISCU þ 4, see Supplementary Fig. 1c). A small amount of ISCU (o5%) was mono-persulfurated (ISCU þ 3) as a result of persulfuration of the overexpressed protein within bacteria. ...
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... it would regenerate the persulfide on NFS1 following its reduction, and DTT was added. We first noticed that NFS1 persulfide reduction was much slower than its formation (Fig. 4a, and see Fig. 2a), which indicated that reduction of NFS1 persulfide is the rate-limiting step of the cysteine desulfurase cycle under these conditions (reaction 2 or 3, Fig. 1a). Remarkably, ISCU and FXN both altered the rate of NFS1 persulfide reduction, but in opposite ways (Fig. 4a,b). While the binding of ISCU to the NI complex slowed down the reaction, the binding of FXN to the NIU complex enhanced it, but had no effect on the NI complex. The data were fitted assuming that reduction of NFS1 persulfide by ...
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... different mechanisms of reduction by thiols are feasible, through S-thiolation of NFS1 or sulfur transfer to the thiol (Fig. 1a). In the case of the S-thiolation pathway, a DTT adduct is expected that will appear as an NFS1 þ 6 species under non- reducing conditions in the alkylation assays. Under these conditions, no NFS1 þ 6 species was detected ( Supplementary Fig. 6c), indicating that NFS1 persulfide is reduced through sulfur transfer on DTT. We thus ...
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... L-cysteine was able to reduce NFS1 persulfide. Cystine (oxidized cysteine), which is the expected by-product of persulfide reduction by L-cysteine, was concomitantly generated in the reaction mixture, further supporting this conclusion ( Supplementary Fig. 7c-e). To determine whether L-cysteine reduces NFS1 through sulfur transfer or S-thiolation (Fig. 1a), we analysed the kinetics of persulfide formation under non-reducing conditions since an NFS1 þ 6 species is expected to appear in the case of the S-thiolation pathway. No L-cysteine adduct was detected under these conditions, which is consistent with the sulfur transfer mechanism of reduction ( Fig. 1d and Supplementary Fig. 1b). ...
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... through sulfur transfer or S-thiolation (Fig. 1a), we analysed the kinetics of persulfide formation under non-reducing conditions since an NFS1 þ 6 species is expected to appear in the case of the S-thiolation pathway. No L-cysteine adduct was detected under these conditions, which is consistent with the sulfur transfer mechanism of reduction ( Fig. 1d and Supplementary Fig. 1b). Moreover, the kinetics of Fe-S cluster reconstitution performed with L-cysteine as a reductant (without DTT), displayed a lag phase indicative of a delay in sulfide release ( Supplementary Fig. 8a-c). Such a lag phase is not expected in the case of the S-thiolation mechanism for which sulfide release should initiate immediately upon ...
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... is not expected in the case of the S-thiolation mechanism for which sulfide release should initiate immediately upon adding L-cysteine, whereas it is consistent with the sulfur transfer mechanism for which sulfide release is not directly correlated with NFS1 persulfide reduction but with the subsequent step of reduction of the persulfurated thiol (Fig. 1a, reaction (3b)). These data thus further support the sulfur transfer ...
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... or in the presence of FXN and/or ISCU, when extrapolated to t ¼ 0 did not intersect the y axis at y ¼ 0 and the amount of L-alanine generated was correlated to the amount of complex ( Supplementary Fig. 9c-g). This initial burst represents the amount of L-alanine generated during the first half-turnover of NFS1 persulfide formation (reaction 1, Fig. 1a) and the following part of the reaction (t40) reflects subsequent turnovers for which reduction is now required. The respective rates of these two different phases:420 mM s À 1 for persulfide formation and in the range of 0.1 to 1 mM s À 1 for the second part indicate that reduction is slowing down the reaction and thus that it is the ...
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... transferred to ISCU in a trans-persulfuration process leading to persulfuration of ISCU on one of its cysteine residues. Upon regeneration of NFS1 persulfide by L-cysteine, this persulfide is reduced by external thiols such as DTT, GSH and L-cysteine. The reduction of NFS1 persulfide likely proceeds by sulfur transfer rather than S-thiolation (Fig. 1a). ISCU persulfide is also reduced by external thiols but at a markedly slower rate than NFS1 persulfide. The analysis by mass spectrometry indicates that the persulfides generated on NFS1 and ISCU are mainly mono-persulfides. Although we cannot firmly establish which of the three conserved cysteine of ISCU (C35, C61 or C104) is the ...
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... which suggests that this might be a common feature of this class of enzymes 43,44,49 . Taken together, our results thus point to a unique molecular function of FXN as an enhancer of sulfur transfer to ISCU and small free thiols (Fig. 6). Sulfide release does occur subsequently, upon condensation of the persulfurated thiol with a second thiol (Fig. 1a, reaction (3b)), but is not directly catalysed by ...
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... these reactions correlated with physiologically relevant Fe-S cluster assembly on ISCU? We first determined that ISCU persulfide does not contribute to sulfide release and is not even reduced in Fe-S cluster reconstitution assays when thiols are used as reductants ( Supplementary Fig. 10a-d). Thus, under these conditions, sulfide production arises exclusively from reduction of NFS1 persulfide and FXN drives Fe-S cluster assembly by enhancing this reaction. ...
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... under these conditions, sulfide production arises exclusively from reduction of NFS1 persulfide and FXN drives Fe-S cluster assembly by enhancing this reaction. However, the rate of Fe-S cluster assembly is slow when DTT and/or L-cysteine are used as reductants (1 h to complete reconstitution on ISCU with L-cysteine) and the reconstitution is inefficient as at least 30 equivalents of sulfide ions are produced in 1 h with L-cysteine (according to the rate constants, Table 3) while only two equivalents would be required for building up a [2Fe2S] cluster (Supplementary Figs 8 and 10). These observa- tions suggest that L-cysteine (and also GSH which is an even less efficient reducer of NFS1 persulfide) are not the physiological reductants or that other accessory proteins might be required to secure Fe-S cluster assembly. ...
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... were used to determine the total number of cysteines available for alkylation by MalP 16 in NFS1, which contains seven cysteine residues, and ISCU, which contains four cysteine residues ( Supplementary Fig. 1a,c). MalP 16 (2 eq.) relative to total sulfhydryl content were added to NFS1 (20 mM) or to a mixture of NFS1 (20 mM) and ISCU (20 mM) in buffer C, and the alkylation reaction was stopped after 30 s using the TCA procedure. ...
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... the protein bands on SDS-PAGE were quantified using an Odyssey scanner (Li-Cor). The uncropped images of the gels used for the various figures are displayed in Supplementary Figs 11 and 12. The data obtained for persulfide reduction by DTT and GSH were fitted assuming a bimolecular mechanism as demonstrated in multiple-turnover kinetics for NFS1 persulfide reduction (see Fig. 5c,d) using equation (1) for second-order kinetic 42 , ...
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Friedreich’s ataxia (FRDA) is a progressive neuromuscular disorder caused by substantial decrease of mitochondrial protein frataxin responsible for biogenesis of iron-sulphur clusters and protection from oxidative damage. In this study, we investigated the antioxidant activities of a standardized aqueous extract from fruiting bodies of Hericium eri...
Citations
... De novo assembly begins with the desulfurase NFS1 converting cysteine to alanine. This releases sulfur to the scaffold protein ISCU, forming a [2Fe-2S] cluster through a conformational change, potentially activated by frataxin (FXN) [79]. The accessory protein ISD11 stabilizes the key enzyme NFS1 in the ISC core complex, ensuring efficient Fe-S cluster biogenesis. ...
Cancer stem cells (CSCs), with their ability of self-renewal, unlimited proliferation, and multi-directional differentiation, contribute to tumorigenesis, metastasis, recurrence, and resistance to conventional therapy and immunotherapy. Eliminating CSCs has long been thought to prevent tumorigenesis. Although known to negatively impact tumor prognosis, research revealed the unexpected role of iron metabolism as a key regulator of CSCs. This review explores recent advances in iron metabolism in CSCs, conventional cancer therapies targeting iron biochemistry, therapeutic resistance in these cells, and potential treatment options that could overcome them. These findings provide important insights into therapeutic modalities against intractable cancers.
... The (NIAU) 2 complex was shown to have increased affinity for FXN (X) 16,[27][28][29] . FXN binding to generate the intermediate (Fe-N S IAUX) 2 complex strongly increases the rate of persulfide transfer from NFS1 to ISCU2 (yielding (Fe-NIAU S X) 2 ) (Fig. 1a), implying a dedicated physiological role of FXN in this step, but FXN's precise mechanistic function remains unclear 17,25,26,30 . Studies performed aerobically suggested Cys69 ISCU2 and Cys138 ISCU2 as potential primary residues accepting the sulfur from NFS1, and Cys138 ISCU2 as the final acceptor 25 . ...
... FXN binding to generate the intermediate (Fe-N S IAUX) 2 complex strongly increases the rate of persulfide transfer from NFS1 to ISCU2 (yielding (Fe-NIAU S X) 2 ) (Fig. 1a), implying a dedicated physiological role of FXN in this step, but FXN's precise mechanistic function remains unclear 17,25,26,30 . Studies performed aerobically suggested Cys69 ISCU2 and Cys138 ISCU2 as potential primary residues accepting the sulfur from NFS1, and Cys138 ISCU2 as the final acceptor 25 . In the human (Zn-NIAU) 2 X-ray structure 15 , the latter residue is furthest from the sulfur-donating Cys381 NFS1 (distances for ligand-metal coordination and for Cys-Cys pairs in various ISC complex structures are compiled in Table 1). ...
... The Fe-containing conformation raised the important functional question of which Cys residue of ISCU2 may serve as the sulfur acceptor from the Cys loop Cys381 NFS1 . The latter is closest to the sulfur atoms of Cys95 ISCU2 (3.8 Å) and Cys69 ISCU2 (4.3 Å), yet rather distant 25 . Intriguingly, His137 ISCU2 , which is hydrogen-bonded to the water ligand of Fe between Cys381 NFS1 and Cys138 ISCU2 , appears to prevent a closer approach of the two Cys residues and to hinder direct sulfur transfer ( Supplementary Fig. 6). ...
Maturation of iron-sulfur proteins in eukaryotes is initiated in mitochondria by the core iron-sulfur cluster assembly (ISC) complex, consisting of the cysteine desulfurase sub-complex NFS1-ISD11-ACP1, the scaffold protein ISCU2, the electron donor ferredoxin FDX2, and frataxin, a protein dysfunctional in Friedreich’s ataxia. The core ISC complex synthesizes [2Fe-2S] clusters de novo from Fe and a persulfide (SSH) bound at conserved cluster assembly site residues. Here, we elucidate the poorly understood Fe-dependent mechanism of persulfide transfer from cysteine desulfurase NFS1 to ISCU2. High-resolution cryo-EM structures obtained from anaerobically prepared samples provide snapshots that both visualize different stages of persulfide transfer from Cys381NFS1 to Cys138ISCU2 and clarify the molecular role of frataxin in optimally positioning assembly site residues for fast sulfur transfer. Biochemical analyses assign ISCU2 residues essential for sulfur transfer, and reveal that Cys138ISCU2 rapidly receives the persulfide without a detectable intermediate. Mössbauer spectroscopy assessing the Fe coordination of various sulfur transfer intermediates shows a dynamic equilibrium between pre- and post-sulfur-transfer states shifted by frataxin. Collectively, our study defines crucial mechanistic stages of physiological [2Fe-2S] cluster assembly and clarifies frataxin’s molecular role in this fundamental process.
... Thus, during the alternative approach to tackle this metabolic imbalance in mitochondrial cancer bioenergetics, the observations of the authors that IM downregulates remarkably the expression of both SCO2 and FXN genes were expanded. The former is involved in the biogenesis of COX, while the latter in the assembly of Fe/S clusters of the mitochondrial respiratory chain (66,67), both associated with COX deficiency (24). As proposed by Michelakis et al (31), DCA was utilized as a potential metabolic-targeting therapy for cancer capable of blocking the lactate production at the last steps of glycolysis. ...
Tumor malignant cells are characterized by dysregulation of mitochondrial bioenergetics due to the 'Warburg effect'. In the present study, this metabolic imbalance was explored as a potential target for novel cancer chemotherapy. Imatinib (IM) downregulates the expression levels of SCO2 and FRATAXIN (FXN) genes involved in the heme-dependent cytochrome c oxidase biosynthesis and assembly pathway in human erythroleukemic IM-sensitive K-562 chronic myeloid leukemia cells (K-562). In the present study, it was investigated whether the treatment of cancer cells with IM (an inhibitor of oxidative phosphorylation) separately, or together with dichloroacetate (DCA) (an inhibitor of glycolysis), can inhibit cell proliferation or cause death. Human K-562 and IM-chemoresistant K-562 chronic myeloid leukemia cells (K-562R), as well as human colorectal carcinoma cells HCT-116 (+/+p53) and (−/−p53, with double TP53 knock-in disruptions), were employed. Treatments of these cells with either IM (1 or 2 μM) and/or DCA (4 mM) were also assessed for the levels of several process biomarkers including SCO2, FXN, lactate dehydrogenase A, glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase M2, hypoxia inducing factor-1a, heme oxygenase-1, NF-κB, stem cell factor and vascular endothelial growth factor via western blot analysis. Computational network biology models were also applied to reveal the connections between the ten proteins examined. Combination treatment of IM with DCA caused extensive cell death (>75%) in K-562 and considerable (>45%) in HCT-116 (+/+p53) cultures, but less in K-562R and HCT-116 (−/−p53), with the latter deficient in full length p53 protein. Such treatment, markedly reduced reactive oxygen species levels, as measured by flow-cytometry, in K-562 cells and affected the oxidative phosphorylation and glycolytic biomarkers in all lines examined. These findings indicated, that targeting of cancer mitochondrial bioenergetics with such a combination treatment was very effective, although chemoresistance to IM in leukemia and the absence of a full length p53 in colorectal cells affected its impact.
... Frataxin was shown to interact in vitro with the mitochondrial FeS cluster assembly machinery [21][22][23][24]. The contribution of frataxin to the synthesis of the FeS clusters is still under debate, however a consensus exists pointing to a function as sulfide production stimulator or accelerator of sulfur transfer from cysteine [25][26][27][28][29][30]. It is worth noting that FRDA models and patients' cells show impaired cellular respiration and reduced ATP production [31][32][33][34][35][36][37][38][39], which is particularly relevant in energyintensive cells, as neurons and cardiomyocytes. ...
Friedreich ataxia (FRDA) is a rare, inherited neurodegenerative disease caused by an expanded GAA repeat in the first intron of the FXN gene, leading to transcriptional silencing and reduced expression of frataxin. Frataxin participates in the mitochondrial assembly of FeS clusters, redox cofactors of the respiratory complexes I, II and III. To date it is still unclear how frataxin deficiency culminates in the decrease of bioenergetics efficiency in FRDA patients’ cells. We previously demonstrated that in healthy cells frataxin is closely attached to the mitochondrial cristae, which contain both the FeS cluster assembly machinery and the respiratory chain complexes, whereas in FRDA patients’ cells with impaired respiration the residual frataxin is largely displaced in the matrix. To gain novel insights into the function of frataxin in the mitochondrial pathophysiology, and in the upstream metabolic defects leading to FRDA disease onset and progression, here we explored the potential interaction of frataxin with the FeS cluster-containing respiratory complexes I, II and III. Using healthy cells and different FRDA cellular models we found that frataxin interacts with these three respiratory complexes. Furthermore, by EPR spectroscopy, we observed that in mitochondria from FRDA patients’ cells the decreased level of frataxin specifically affects the FeS cluster content of complex I. Remarkably, we also found that the frataxin-like protein Nqo15 from T. thermophilus complex I ameliorates the mitochondrial respiratory phenotype when expressed in FRDA patient’s cells. Our data point to a structural and functional interaction of frataxin with complex I and open a perspective to explore therapeutic rationales for FRDA targeted to this respiratory complex.
... Full-length hFXN protein (1-210, Fig. 1) expressed in the cytosol, translocates to the mitochondria where it undergoes sequential mitochondrial processing peptidase (MPP) cleavage at G 41 -L 42 and K 80 -S 81 to produce hFXN-M protein ( Fig. 1) 5,6 . Mitochondrial hFXN-M protein plays an important role in the biogenesis and maintenance of Fe-S clusters and in persulfide processing [7][8][9][10] . Epigenetic silencing of the FXN gene due to the presence of GAA triplet repeats in intron 1 of both alleles of the FXN gene (homozygous patients) results in reduced transcription of FXN mRNA, reduced expression of full-length hFXN in the cytosol, and reduced amounts of hFXN-M produced in the mitochondria 1,11 . ...
Deficiency in human mature frataxin (hFXN-M) protein is responsible for the devastating neurodegenerative and cardiodegenerative disease of Friedreich’s ataxia (FRDA). It results primarily through epigenetic silencing of the FXN gene by GAA triplet repeats on intron 1 of both alleles. GAA repeat lengths are most commonly between 600 and 1200 but can reach 1700. A subset of approximately 3% of FRDA patients have GAA repeats on one allele and a mutation on the other. FRDA patients die most commonly in their 30s from heart disease. Therefore, increasing expression of heart hFXN-M using gene therapy offers a way to prevent early mortality in FRDA. We used rhesus macaque monkeys to test the pharmacology of an adeno-associated virus (AAV)hu68.CB7.hFXN therapy. The advantage of using non-human primates for hFXN-M gene therapy studies is that hFXN-M and monkey FXN-M (mFXN-M) are 98.5% identical, which limits potential immunologic side-effects. However, this presented a formidable bioanalytical challenge in quantification of proteins with almost identical sequences. This could be overcome by the development of a species-specific quantitative mass spectrometry-based method, which has revealed for the first time, robust transgene-specific human protein expression in monkey heart tissue. The dose response is non-linear resulting in a ten-fold increase in monkey heart hFXN-M protein expression with only a three-fold increase in dose of the vector.
... The process starts with cysteine desulfurase, NFS, a PLP-dependent enzyme, which converts cysteine to alanine, generating protein persulfide (Fig. 8) (Mueller, 2006;Zhang et al., 2010). On the way to the iron-sulfur cluster assembly, this persulfide is then transferred further to other protein targets, as well as LMW thiols (Parent et al., 2015). In addition, the cysteine desulfurase persulfide shuttles the sulfhydryl sulfur to a rhodanese-like sulfur transferase, ThiI, forming ThiI persulfide. ...
Significance:
Protein persulfidation (the formation of RSSH), an evolutionarily conserved oxidative posttranslational modification in which thiol groups in cysteine residues are converted into persulfides, has emerged as one of the main mechanisms through which hydrogen sulfide (H2S) conveys its signaling.
Recent advances:
New methodological advances in persulfide labeling started unraveling the chemical biology of this modification and its role in (patho)physiology. Some of the key metabolic enzymes are regulated by persulfidation. RSSH levels are important for the cellular defense against oxidative injury, and they decrease with aging, leaving proteins vulnerable to oxidative damage. Persulfidation is dysregulated in many diseases.
Critical issues:
A relatively new field of signaling by protein persulfidation still has many unanswered questions: the mechanism(s) of persulfide formation and transpersulfidation and the identification of "protein persulfidases", the improvement of methods to monitor RSSH changes and identify protein targets, and understanding the mechanisms through which this modification controls important (patho)physiological functions.
Future directions:
Deep mechanistic studies using more selective and sensitive RSSH labeling techniques will provide high-resolution structural, functional, quantitative, and spatiotemporal information on RSSH dynamics and help with better understanding how H2S-derived protein persulfidation affects protein structure and function in health and disease. This knowledge could pave the way for targeted drug design for a wide variety of pathologies.
... Iron delivery is a matter of debate, so far it is likely that locally available free ferrous (Fe(II)) ions are used since no dedicated donor protein was identified [59]. Frataxin (FXN) was considered as an iron donor protein [66,75], however, this activity was questioned and it has been shown that FXN rather has a rate enhancement role in cluster biogenesis (see below) [73,76]. On the scaffold protein, simpler [2Fe-2S] structures are assembled, which are transported from the mitochondria if necessary and delivered to target apoproteins by dedicated chaperones creating the Fe-S holoproteins or undergo further maturation to generate [4Fe-4S] and more complex clusters [63]. ...
... They concluded that the rate-limiting step of the system is the sulfide release from NFS1-persulfide. It appeared that ISCU-SSH did not contribute to sulfide production by physiological reductants like Cys-SH or GSH, therefore it was postulated that a dedicated reductase system should be operative to release sulfide for cluster production and that the sulfane sulfur transfer to biological thiols might serve an alternative purpose, i.e. contribution to cellular sulfur trafficking [76]. The same group later revealed that the binding of zinc (Zn 2+ ) ion to ISCU hindered iron binding in earlier reconstitution attempts in vitro and reshuffled persulfide reduction to NFS1-SSH. ...
Reactive sulfur species (RSS) entail a diverse family of sulfur derivatives that have emerged as important effector molecules in H2S-mediated biological events. RSS (including H2S) can exert their biological roles via widespread interactions with metalloproteins. Metalloproteins are essential components along the metabolic route of oxygen in the body, from the transport and storage of O2, through cellular respiration, to the maintenance of redox homeostasis by elimination of reactive oxygen species (ROS). Moreover, heme peroxidases contribute to immune defense by killing pathogens using oxygen-derived H2O2 as a precursor for stronger oxidants. Coordination and redox reactions with metal centers are primary means of RSS to alter fundamental cellular functions. In addition to RSS-mediated metalloprotein functions, the reduction of high-valent metal centers by RSS results in radical formation and opens the way for subsequent per- and polysulfide formation, which may have implications in cellular protection against oxidative stress and in redox signaling. Furthermore, recent findings pointed out the potential role of RSS as substrates for mitochondrial energy production and their cytoprotective capacity, with the involvement of metalloproteins. The current review summarizes the interactions of RSS with protein metal centers and their biological implications with special emphasis on mechanistic aspects, sulfide-mediated signaling, and pathophysiological consequences. A deeper understanding of the biological actions of reactive sulfur species on a molecular level is primordial in H2S-related drug development and the advancement of redox medicine.
... Later, another thiol label-based probe, a maleimide compound containing a peptide arm, designated maleimide peptide (MalP, 1.95 kDa), was used to study the persulfidation of the iron-sulfur cluster machinery in mammalian proteins (Parent et al., 2015). As with the red maleimide fluorescent probe, both -SH and -SSH are labeled with MalP, and only the labeled -SSHs are further reduced by the subsequent DTT incubation and release the succinimide-peptide moiety (the product of the maleimide reaction with a sulfhydryl). ...
Significance:
Hydrogen sulfide (H2S) is a multitasking potent regulator that facilitates plant growth, development, and responses to environmental stimuli.
Recent advances:
The important beneficial effects of H2S in various aspects of plant physiology aroused the interest of this chemical for agriculture. Protein cysteine persulfidation has been recognized as the main redox regulatory mechanism of H2S signaling. An increasing number of studies, including large-scale proteomic analyses and function characterizations, have revealed that H2S-mediated persulfidations directly regulate protein functions, altering downstream signaling in plants. To date, the importance of H2S-mediated persufidation in several abscisic acid signaling-controlling key proteins has been assessed as well as their role in stomatal movements, largely contributing to the understanding of the plant H2S-regulatory mechanism.
Critical issues:
The molecular mechanisms of the H2S sensing and transduction in plants remain elusive. The correlation between H2S-mediated persulfidation with other oxidative posttranslational modifications of cysteines are still to be explored.
Future directions:
Implementation of advanced detection approaches for the spatiotemporal monitoring of H2S levels in cells and the current proteomic profiling strategies for the identification and quantification of the cysteine site-specific persulfidation will provide insight into the H2S signaling in plants.
... This positive effect on AtNFS1 activity indicates that both proteins act as AtNFS1 regulators (Fig. 2). For human frataxin, it was shown that the protein is not required for the formation of the persulfide on Homo sapiens HsNFS1 itself, but that it accelerates the sulfane sulfur (S 0 ) transfer from HsNFS1 to HsISCU (Parent et al., 2015;Gervason et al., 2019). Furthermore, yeast ScNFS1 is prone to aggregation in the absence of ScISD11, implying a stabilizing effect of ScISD11 (Adam et al., 2006). ...
Since the discovery of an autonomous iron-sulfur cluster (Fe-S) assembly machinery in mitochondria, significant efforts to examine the nature of this process were made. The assembly of Fe-S clusters occurs in two distinct steps with the initial synthesis of [2Fe-2S] clusters by a first machinery followed by a subsequent assembly into [4Fe-4S] clusters by a second machinery. Despite this knowledge, we still have only a rudimentary understanding of how Fe-S clusters are transferred and distributed among their respective apoproteins. Especially considering that continuous protein turnover and particularly sacrificial destruction of clusters for synthesis of biotin and lipoic acid reveals possible bottlenecks in the supply chain of Fe-S clusters. Considering available information from other species, this review explores the mitochondrial assembly machinery of Arabidopsis and provides the current knowledge about the respective transfer steps to apoproteins. Furthermore, this review highlights biotin synthase and lipoyl synthase, which both utilize Fe-S clusters as sulfur source. After extraction of sulfur atoms from these clusters, the remains of the clusters likely fall apart releasing sulfide as a highly toxic by-product. Immediate refixation through local cysteine biosynthesis is therefore an essential salvage pathway and emphasises the physiological need for cysteine biosynthesis in plant mitochondria.
... Considering a previous result that cysteine persulfide is converted to sulfide in an ETC-dependent manner [21], persulfides are likely to serve as electron acceptors for electrons from the ETC and may prevent aberrant electron acceptance by dioxygen and consequently avoid generation of excessive reactive oxygen species. Considering their possible roles in the synthesis of iron-sulfur clusters [42], persulfides may serve as a decoy to protect persulfide-based sulfur transfer for the incorporation of sulfur into iron-sulfur clusters, which is essential for ETC function. The necessity of sulfide oxidation by SQOR for persulfide-mediated mitochondrial activation implies three aspects of sulfide metabolism: protection from sulfide toxicity [23,24], recovery of electrons leaked from the ETC via persulfides and regeneration of persulfides from sulfide. ...
NF-E2-related factor 2 (NRF2) plays a crucial role in the maintenance of cellular homeostasis by regulating various enzymes and proteins that are involved in the redox reactions utilizing sulfur. While substantial impacts of NRF2 on mitochondrial activity have been described, the precise mechanism by which NRF2 regulates mitochondrial function is still not fully understood. Here, we demonstrated that NRF2 increased intracellular persulfides by upregulating the cystine transporter xCT encoded by Slc7a11, a well-known NRF2 target gene. Persulfides have been shown to play an important role in mitochondrial function. Supplementation with glutathione trisulfide (GSSSG), which is a form of persulfide, elevated the mitochondrial membrane potential (MMP), increased the oxygen consumption rate (OCR) and promoted ATP production. Persulfide-mediated mitochondrial activation was shown to require the mitochondrial sulfur oxidation pathway, especially sulfide quinone oxidoreductase (SQOR). Consistently, NRF2-mediated mitochondrial activation was also dependent on SQOR activity. This study clarified that the facilitation of persulfide production and sulfur metabolism in mitochondria by increasing cysteine availability is one of the mechanisms for NRF2-dependent mitochondrial activation.
