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ABSTRACT All sulfur transfer pathways generally have in common an l-cysteine desulfurase as the initial sulfur-mobilizing enzyme, which serves as a sulfur donor for the biosynthesis of numerous sulfur-containing biomolecules in the cell. In Escherichia coli, the housekeeping l-cysteine desulfurase IscS functions as a hub for sulfur transfer through interactions with several partner proteins, which bind at different sites on IscS. So far, the interaction sites of IscU, Fdx, CyaY, and IscX involved in iron sulfur (Fe-S) cluster assembly, TusA, required for molybdenum cofactor biosynthesis and mnm5s2U34 transfer RNA (tRNA) modifications, and ThiI, involved in both the biosynthesis of thiamine and s4U8 tRNA modifications, have been mapped. Previous studies have suggested that IscS partner proteins bind only one at a time, with the exception of Fe-S cluster assembly, which involves the formation of a ternary complex involving IscS, IscU, and one of CyaY, Fdx, or IscX. Here, we show that the affinity of TusA for IscS is similar to but lower than that of IscU and that these proteins compete for binding to IscS. We show that heterocomplexes involving the IscS dimer and single IscU and TusA molecules are readily formed and that binding of both TusA and IscU to IscS affects its l-cysteine desulfurase activity. A model is proposed in which the delivery of sulfur to different sulfur-requiring pathways is controlled by sulfur acceptor protein levels, IscS-binding affinities, and acceptor protein-modulated IscS desulfurase activity.IMPORTANCEIron-sulfur clusters are evolutionarily ancient prosthetic groups. The housekeeping l-cysteine desulfurase IscS functions as a central core for sulfur transfer through interactions with several partner proteins, which bind at different sites on each IscS monomer with different affinities and partially overlapping binding sites. We show that heterocomplexes involving the IscS dimer and single IscU and TusA molecules at each site of the dimer are formed, thereby influencing the activity of IscS.

Abstract Background Malaria remains a public health concern globally. Resistance to anti-malarial drugs has consistently threatened the gains in controlling the malaria parasites. Currently, artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP) are the treatment regimens against Plasmodium falciparum infections in many African countries, including Kenya. Recurrent infections have been reported in patients treated with AL or DP, suggesting the possibility of reinfection or parasite recrudescence associated with the development of resistance against the two therapies. The Plasmodium falciparum cysteine desulfurase IscS (Pfnfs1) K65 selection marker has previously been associated with decreased lumefantrine susceptibility. This study evaluated the frequency of the Pfnfs1 K65 resistance marker and associated K65Q resistant allele in recurrent infections collected from P. falciparum-infected individuals living in Matayos, Busia County, in western Kenya. Methods Archived dried blood spots (DBS) of patients with recurrent malaria infection on clinical follow-up days after treatment with either AL or DP were used in the study. After extraction of genomic DNA, PCR amplification and sequencing analysis were employed to determine the frequencies of the Pfnfs1 K65 resistance marker and K65Q mutant allele in the recurrent infections. Plasmodium falciparum msp1 and P. falciparum msp2 genetic markers were used to distinguish recrudescent infections from new infections. Results The K65 wild-type allele was detected at a frequency of 41% while the K65Q mutant allele was detected at a frequency of 22% in the recurrent samples. 58% of the samples containing the K65 wild-type allele were AL treated samples and while 42% were DP treated samples. 79% of the samples with the K65Q mutation were AL treated samples and 21% were DP treated samples. The K65 wild-type allele was detected in three recrudescent infections (100%) identified from the AL treated samples. The K65 wild-type allele was detected in two recrudescent DP treated samples (67%) while the K65Q mutant allele was identified in one DP treated (33%) recrudescent sample. Conclusions The data demonstrate a higher frequency of the K65 resistance marker in patients with recurrent infection during the study period. The study underscores the need for consistent monitoring of molecular markers of resistance in regions of high malaria transmission.

As sulfur is part of many essential protein cofactors such as iron–sulfur clusters, molybdenum cofactors, or lipoic acid, its mobilization from cysteine represents a fundamental process. The abstraction of the sulfur atom from cysteine is catalysed by highly conserved pyridoxal 5ʹ-phosphate-dependent enzymes called cysteine desulfurases. The desulfuration of cysteine leads to the formation of a persulfide group on a conserved catalytic cysteine and the concomitant release of alanine. Sulfur is then transferred from cysteine desulfurases to different targets. Numerous studies have focused on cysteine desulfurases as sulfur-extracting enzymes for iron–sulfur cluster synthesis in mitochondria and chloroplasts but also for molybdenum cofactor sulfuration in the cytosol. Despite this, knowledge about the involvement of cysteine desulfurases in other pathways is quite rudimentary, particularly in photosynthetic organisms. In this review, we summarize current understanding of the different groups of cysteine desulfurases and their characteristics in terms of primary sequence, protein domain architecture, and subcellular localization. In addition, we review the roles of cysteine desulfurases in different fundamental pathways and highlight the gaps in our knowledge to encourage future work on unresolved issues especially in photosynthetic organisms. [ABSTRACT FROM AUTHOR]

Background: Malaria remains a public health concern globally. Resistance to anti-malarial drugs has consistently threatened the gains in controlling the malaria parasites. Currently, artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP) are the treatment regimens against Plasmodium falciparum infections in many African countries, including Kenya. Recurrent infections have been reported in patients treated with AL or DP, suggesting the possibility of reinfection or parasite recrudescence associated with the development of resistance against the two therapies. The Plasmodium falciparum cysteine desulfurase IscS (Pfnfs1) K65 selection marker has previously been associated with decreased lumefantrine susceptibility. This study evaluated the frequency of the Pfnfs1 K65 resistance marker and associated K65Q resistant allele in recurrent infections collected from P. falciparum-infected individuals living in Matayos, Busia County, in western Kenya. Methods: Archived dried blood spots (DBS) of patients with recurrent malaria infection on clinical follow-up days after treatment with either AL or DP were used in the study. After extraction of genomic DNA, PCR amplification and sequencing analysis were employed to determine the frequencies of the Pfnfs1 K65 resistance marker and K65Q mutant allele in the recurrent infections. Plasmodium falciparum msp1 and P. falciparum msp2 genetic markers were used to distinguish recrudescent infections from new infections. Results: The K65 wild-type allele was detected at a frequency of 41% while the K65Q mutant allele was detected at a frequency of 22% in the recurrent samples. 58% of the samples containing the K65 wild-type allele were AL treated samples and while 42% were DP treated samples. 79% of the samples with the K65Q mutation were AL treated samples and 21% were DP treated samples. The K65 wild-type allele was detected in three recrudescent infections (100%) identified from the AL treated samples. The K65 wild-type allele was detected in two recrudescent DP treated samples (67%) while the K65Q mutant allele was identified in one DP treated (33%) recrudescent sample. Conclusions: The data demonstrate a higher frequency of the K65 resistance marker in patients with recurrent infection during the study period. The study underscores the need for consistent monitoring of molecular markers of resistance in regions of high malaria transmission. [ABSTRACT FROM AUTHOR]

• Acp1 is involved in type II fatty acid synthesis and FeS cluster biogenesis. • Acp1 is a core component of the mitochondrial FeS cluster machinery. • Acp1 depletion leads to FeS cluster deficiency in both mitochondria and cytoplasm. • Cells unable to conjugate phosphopantetheine to Acp1 mimic Acp1-depletion effects. • Acp1 function requires phosphopantetheine attachment and associated fatty acid chains. Mitochondria perform vital biosynthetic processes, including fatty acid synthesis and iron-sulfur (FeS) cluster biogenesis. In Saccharomyces cerevisiae mitochondria, the acyl carrier protein Acp1 participates in type II fatty acid synthesis, requiring a 4-phosphopantetheine (PP) prosthetic group. Acp1 also interacts with the mitochondrial FeS cluster assembly complex that contains the cysteine desulfurase Nfs1. Here we investigated the role of Acp1 in FeS cluster biogenesis in mitochondria and cytoplasm. In the Acp1-depleted (Acp1↓) cells, biogenesis of mitochondrial FeS proteins was impaired, likely due to greatly reduced Nfs1 protein and/or its persulfide-forming activity. Formation of cytoplasmic FeS proteins was also deficient, suggesting a disruption in generating the (Fe-S) int intermediate, that is exported from mitochondria and is subsequently utilized for cytoplasmic FeS cluster assembly. Iron homeostasis was perturbed, with enhanced iron uptake into the cells and accumulation of iron in mitochondria. The Δppt2 strain, lacking the mitochondrial ability to add PP to Acp1, phenocopied the Acp1↓ cells. These data suggest that the holo form of Acp1 with the PP-conjugated acyl chain is required for stability of the Nfs1 protein and/or stimulation of its persulfide-forming activity. Thus, mitochondria lacking Acp1 (or Ppt2) cannot support FeS cluster biogenesis in mitochondria or cytoplasm, leading to disrupted iron homeostasis. [ABSTRACT FROM AUTHOR]

Escherichia coli cysteine desulfurase (CD), IscS, modifies basal metabolism by transferring sulphur (S) from L-cysteine to numerous cellular pathways, whereas NFS1, a human CD, is active only in the formation of the [Acp]2:[ISD11]2:[NFS1]2 complex. Despite the accumulation of red-coloured IscS in E. coli cells as a result of the deficiency of accessible iron, as revealed in our previous studies, the mechanism of the potential enzymatic reaction remains unclear. In this study, the N-terminus of IscS was fused with the C-terminus of NFS1, which was reported to be almost fully active as IscS and exhibits a pyridoxal 5′-phosphate (PLP) absorption peak at 395 nm. Moreover, SUMO-EH-IscS exhibited significant growth recovery and NADH-dehydrogenase I activity in the iscS mutant cells. Furthermore, through in vitro and in vivo experiments combined with high-performance liquid chromatography and ultra-performance liquid chromatography–tandem mass spectrometry, it was shown that the new absorption peaks of the IscS H104Q, IscS Q183E, IscS K206A, and IscS K206A&C328S variants at 340 and 350 nm may correspond to the enzyme reaction intermediates, Cys-ketimine and Cys-aldimine, respectively. However, after mutation of the conserved active-site residues, additional absorption peaks at 420 and 430 nm were associated with PLP migration in the active-site pocket. Additionally, the corresponding absorption peaks of Cys-quinonoid, Ala-ketimine, and Ala-aldimine intermediates in IscS were 510, 325, and 345 nm, respectively, as determined by site-directed mutagenesis and substrate/product-binding analyses during the CD reaction process. Notably, red IscS formed in vitro by incubating IscS variants (Q183E and K206A) with excess L-alanine and sulphide under aerobic conditions produced an absorption peak similar to the wild-type IscS, at 510 nm. Interestingly, site-directed mutation of IscS with hydrogen bonds to PLP at Asp180 and Gln183 resulted in a loss of enzymatic activity followed by an absorption peak consistent with NFS1 (420 nm). Furthermore, mutations at Asp180 or Lys206 inhibited the reaction of IscS in vitro with L-cysteine (substrate) and L-alanine (product). These results suggest that the conserved active site residues (His104, Asp180, and Gln183) and their hydrogen bond with PLP in the N-terminus of IscS play a key role in determining whether the L-cysteine substrate can enter the active-site pocket and regulate the enzymatic reaction process. Therefore, our findings provide a framework for evaluating the roles of conserved active-site residues, motifs, and domains in CDs.

The cysteine desulfurase SufS is a pyridoxal‐5′‐phosphate‐dependent enzyme and is essential for the SUF system, which participates in iron–sulfur cluster biosynthesis. Inhibition of SufS in the SUF system by D‐cycloserine (DCS) in Plasmodium falciparum apicoplast has recently been reported, indicating that SufS could be a target for malaria therapeutics. However, the mechanistic details underlying the inhibition of SufS by DCS have not yet been clarified. Moreover, inhibition of SufS by the other enantiomer, L‐cycloserine (LCS), has not been investigated. Herein, we investigated the structure‐based inhibition mechanisms of SufS by DCS and LCS using Bacillus subtilis SufS, whose catalytic mechanism has been well characterized in comparison to that of the P. falciparum SufS. Surprisingly, DCS‐ and LCS‐mediated inhibitions of SufS occur via distinct mechanisms resulting in pyridoxamine‐5′‐phosphate (PMP) in DCS‐mediated inhibition and PMP‐3‐hydroxyisoxazole adduct (PMP‐isoxazole) in LCS‐mediated inhibition. Biochemical and structural evaluation of SufS variants identified conserved His and Arg residues at the active site as the key determinants of the distinct inhibition mechanisms. The importance of structural elements involved in DCS and LCS‐mediated inhibitions of SufS provides valuable insights for the structure‐based design of new drugs targeting SufS. Database: Structural data are available in PDB database under the accession numbers 6KFY, 7CEO, 7CEP, 7CEQ, 7CER, 7CES, 7CET, 7CEU, 7E6A, 7E6B, 7E6C, 7E6D, 7E6E, and 7E6F. [ABSTRACT FROM AUTHOR]

Under conditions of oxidative stress or iron starvation, iron-sulfur cluster biogenesis in E. coli is initiated by the cysteine desulfurase, SufS, via the SUF pathway. SufS is a type II cysteine desulfurase that catalyzes the PLP-dependent breakage of an L-cysteine C-S bond to generate L-alanine and a covalent active site persulfide as products. The persulfide is transferred from SufS to SufE and then to the SufBC 2 D complex, which utilizes it in iron-sulfur cluster biogenesis. Several lines of evidence suggest two conserved arginine residues that line the solvent side of the SufS active site could be important for function. To investigate the mechanistic roles of R56 and R359, the residues were substituted using site-directed mutagenesis to obtain R56A/K and R359A/K SufS variants. Steady state kinetics indicated R56 and R359 have moderate defects in the desulfurase half reaction but major defects in the transpersulfurase step. Fluorescence polarization binding assays showed that the loss of activity was not due to a defect in forming the SufS/SufE complex. Structural characterization of R56A SufS shows loss of electron density for the α3-α4 loop at the R56/G57 positions, consistent with a requirement of R56 for proper loop conformation. The structure of R359A SufS exhibits a conformational change in the α3-α4 loop allowing R56 to enter the active site and mimics the residue's position in the PLP-cysteine aldimine structure. Taken together, the kinetic, binding, and structural data support a mechanism where R359 plays a role in linking SufS catalysis with modulation of the α3-α4 loop to promote a close-approach interaction of SufS and SufE conducive to persulfide transfer.

Azotobacter vinelandii is a genetically tractable Gram-negative proteobacterium able to fix nitrogen (N2) under aerobic growth conditions. This narrative describes how biochemical-genetic approaches using A. vinelandii to study nitrogen fixation led to the formulation of the "scaffold hypothesis" for the assembly of both simple and complex [Fe-S] clusters associated with biological nitrogen fixation. These studies also led to the discovery of a parallel, but genetically distinct, pathway for maturation of [Fe-S] proteins that support central metabolic processes. • Azotobacter vinelandii is an obligately aerobic diazotroph. • Iron-sulfur proteins are essential to sustain all or most living organisms. • The catalytic components of biological nitrogen fixation are iron-sulfur proteins. • Simple and complex iron-sulfur clusters are assembled using molecular scaffolds. • Sulfur is supplied for assembly of iron-sulfur clusters by a cysteine desulfurase. • Iron-sulfur clusters are delivered to client proteins using carrier proteins. [ABSTRACT FROM AUTHOR]

Iron-sulfur (Fe-S) clusters are inorganic prosthetic groups in proteins composed exclusively of iron and inorganic sulfide. These cofactors are required in a wide range of critical cellular pathways. Iron-sulfur clusters do not form spontaneously in vivo; several proteins are required to mobilize sulfur and iron, assemble and traffic-nascent clusters. Bacteria have developed several Fe-S assembly systems, such as the ISC, NIF, and SUF systems. Interestingly, in Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), the SUF machinery is the primary Fe-S biogenesis system. This operon is essential for the viability of Mtb under normal growth conditions, and the genes it contains are known to be vulnerable, revealing the Mtb SUF system as an interesting target in the fight against tuberculosis. In the present study, two proteins of the Mtb SUF system were characterized for the first time: Rv1464(sufS) and Rv1465(sufU). The results presented reveal how these two proteins work together and thus provide insights into Fe-S biogenesis/metabolism by this pathogen. Combining biochemistry and structural approaches, we showed that Rv1464 is a type II cysteine-desulfurase enzyme and that Rv1465 is a zinc-dependent protein interacting with Rv1464. Endowed with a sulfurtransferase activity, Rv1465 significantly enhances the cysteine-desulfurase activity of Rv1464 by transferring the sulfur atom from persulfide on Rv1464 to its conserved Cys40 residue. The zinc ion is important for the sulfur transfer reaction between SufS and SufU, and His354 in SufS plays an essential role in this reaction. Finally, we showed that Mtb SufS-SufU is more resistant to oxidative stress than E. coli SufS-SufE and that the presence of zinc in SufU is likely responsible for this improved resistance. This study on Rv1464 and Rv1465 will help guide the design of future anti-tuberculosis agents.

Crohn’s disease (CD) is characterized by the chronic inflammation of the gastrointestinal tract. A dysbiotic microbiome and a defective immune system are linked to CD, where hydrogen sulfide (H2S) microbial producers positively correlate with the severity of the disease. Atopobium parvulum is a key H2S producer from the microbiome of CD patients. In this study, the biochemical characterization of two Atopobium parvulum cysteine desulfurases, ApSufS and ApCsdB, shows that the enzymes are allosterically regulated. Structural analyses reveal that ApSufS forms a dimer with conserved characteristics observed in type II cysteine desulfurases. Four residues surrounding the active site are essential to catalyse cysteine desulfurylation, and a segment of short-chain residues grant access for substrate binding. A better understanding of ApSufS will help future avenues for CD treatment. [ABSTRACT FROM AUTHOR]

Iron–sulfur clusters (Fe–S or ISC) are essential cofactors that function in a wide range of biological pathways. In mammalian cells, Fe–S biosynthesis primarily relies on mitochondria and involves a concerted group of evolutionary-conserved proteins forming the ISC pathway. In the early stage of the ISC pathway, the Fe–S core complex is required for de novo assembly of Fe–S. In humans, the Fe–S core complex comprises the cysteine desulfurase NFS1, the scaffold protein ISCU2, frataxin (FXN), the ferredoxin FDX2, and regulatory/accessory proteins ISD11 and Acyl Carrier Protein (ACP). In recent years, the field has made significant advances in unraveling the structure of the Fe–S core complex and the mechanism underlying its function. Herein, we review the key recent findings related to the Fe–S core complex and its components. We highlight some of the unanswered questions and provide a model of the Fe–S assembly within the complex. In addition, we briefly touch on the genetic diseases associated with mutations in the Fe–S core complex components. [ABSTRACT FROM AUTHOR]

Fe-S clusters are critical cofactors for redox chemistry in all organisms. The cysteine desulfurase, SufS, provides sulfur in the SUF Fe-S cluster bioassembly pathway. SufS is a dimeric, pyridoxal 5'-phosphate-dependent enzyme that uses cysteine as a substrate to generate alanine and a covalent persulfide on an active site cysteine residue. SufS enzymes are activated by an accessory transpersulfurase protein, either SufE or SufU depending on the organism, which accepts the persulfide product and delivers it to downstream partners for Fe-S assembly. Here, using Escherichia coli proteins, we present the first X-ray crystal structure of a SufS/SufE complex. There is a 1:1 stoichiometry with each monomeric unit of the EcSufS dimer bound to one EcSufE subunit, though one EcSufE is rotated ∼7° closer to the EcSufS active site. EcSufE makes clear interactions with the α16 helix of EcSufS and site-directed mutants of several α16 residues were deficient in EcSufE binding. Analysis of the EcSufE structure showed a loss of electron density at the EcSufS/EcSufE interface for a flexible loop containing the highly conserved residue R119. An R119A EcSufE variant binds EcSufS but is not active in cysteine desulfurase assays and fails to support Fe-S cluster bioassembly in vivo. 35 S-transfer assays suggest that R119A EcSufE can receive a persulfide, suggesting the residue may function in a release mechanism. The structure of the EcSufS/EcSufE complex allows for comparison with other cysteine desulfurases to understand mechanisms of protected persulfide transfer across protein interfaces., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

All sulfur transfer pathways generally have in common an l-cysteine desulfurase as the initial sulfur-mobilizing enzyme, which serves as a sulfur donor for the biosynthesis of numerous sulfur-containing biomolecules in the cell. In Escherichia coli , the housekeeping l-cysteine desulfurase IscS functions as a hub for sulfur transfer through interactions with several partner proteins, which bind at different sites on IscS. So far, the interaction sites of IscU, Fdx, CyaY, and IscX involved in iron sulfur (Fe-S) cluster assembly, TusA, required for molybdenum cofactor biosynthesis and mnm 5 s 2 U34 transfer RNA (tRNA) modifications, and ThiI, involved in both the biosynthesis of thiamine and s 4 U8 tRNA modifications, have been mapped. Previous studies have suggested that IscS partner proteins bind only one at a time, with the exception of Fe-S cluster assembly, which involves the formation of a ternary complex involving IscS, IscU, and one of CyaY, Fdx, or IscX. Here, we show that the affinity of TusA for IscS is similar to but lower than that of IscU and that these proteins compete for binding to IscS. We show that heterocomplexes involving the IscS dimer and single IscU and TusA molecules are readily formed and that binding of both TusA and IscU to IscS affects its l-cysteine desulfurase activity. A model is proposed in which the delivery of sulfur to different sulfur-requiring pathways is controlled by sulfur acceptor protein levels, IscS-binding affinities, and acceptor protein-modulated IscS desulfurase activity.IMPORTANCEIron-sulfur clusters are evolutionarily ancient prosthetic groups. The housekeeping l-cysteine desulfurase IscS functions as a central core for sulfur transfer through interactions with several partner proteins, which bind at different sites on each IscS monomer with different affinities and partially overlapping binding sites. We show that heterocomplexes involving the IscS dimer and single IscU and TusA molecules at each site of the dimer are formed, thereby influencing the activity of IscS.

Arthrospira platensis (Ap) has several advantageous adaptive mechanisms including its efficient response to elevated oxidative stress (OS). This study intends to identify the antioxidant effect, in vitro and in vivo, of a novel peptide (GP13), Ap-derived cysteine-desulfurase (CDS) peptide, for antioxidant role and the associated mechanism(s) by which the GP13 elicits cytoprotection under elevated OS.Using bioinformatics tools, a short sequence of amino acids GP13 with a predicted antioxidant effect has been identified from the cysteine sulfurase domain of ApCDS between 243 and 616 amino acids. The synthesized GP13 peptide expressed significant antioxidant activity against free-radicals at various concentrations (10–80 µM), as established through the DPPH, ABTS, SARS, and HRS assays.Besides, GP13 demonstrated no cytotoxicity on Vero cells and human leucocytes. In H2O2-induced human leucocytes, the peptide exhibited maximum ROS scavenging activity at a concentration of 80 µM. Reduced apoptotic body formation was identified using Hoechst 33,342 staining in GP13-exposed Vero cells. In vivo results revealed that pre-treated zebrafish larvae with GP13 (10–80 µM) ameliorated the H2O2-induced oxidative damage. Further, GP13 inhibited the H2O2-induced caspase-3-dependent apoptotic response at 96 h post fertilized (hpf) zebrafish larvae.Together, results demonstrated that in GP13 treated (80 µM) zebrafish there was a reduction in the expression of lipid peroxidation level; and an increase in antioxidant-enzymes. Therefore, it is possible that GP13 peptide derived from ApCDS has in vitro and in vivo antioxidant activity and that GP13 should be further investigated for potential therapeutic utility in OS-mediated complications.

Selenium is a vital micronutrient in many organisms. While traces are required for microbial utilization, excess amounts are toxic; thus, selenium can be regarded as a biological double-edged sword. Selenium is chemically similar to the essential element sulfur, but curiously, evolution has selected the former over the latter for a subset of oxidoreductases. Enzymes involved in sulfur metabolism are less discriminate in terms of preventing selenium incorporation; however, its specific incorporation into selenoproteins reveals a highly discriminate process that is not completely understood. We have identified SclA, a NifS-like protein in the nosocomial pathogen, Enterococcus faecalis , and characterized its enzymatic activity and specificity for l -selenocysteine over l -cysteine. It is known that Asp-146 is required for selenocysteine specificity in the human selenocysteine lyase. Thus, using computational biology, we compared the bacterial and mammalian enzymes and identified His-100, an Asp-146 ortholog in SclA, and generated site-directed mutants in order to study the residue's potential role in the l -selenocysteine discrimination mechanism. The proteins were overexpressed, purified, and characterized for their biochemical properties. All mutants exhibited varying Michaelis-Menten behavior towards l -selenocysteine, but His-100 was not found to be essential for this activity. Additionally, l -cysteine acted as a competitive inhibitor of all enzymes with higher affinity than l -selenocysteine. Finally, we discovered that SclA exhibited low activity with l -cysteine as a poor substrate regardless of mutations. We conclude that His-100 is not required for l -selenocysteine specificity, underscoring the inherent differences in discriminatory mechanisms between bacterial NifS-like proteins and mammalian selenocysteine lyases. • SclA, a NifS-like protein in Enterococcus faecalis , degrades l -selenocysteine. • SclA is similar to human selenocysteine lyase based on computational biology. • His-100 of SclA is not required for discrimination of l -selenocysteine. • l -Cysteine inhibits SclA in a dose-dependent manner. • SclA can utilize l -cysteine as a poor alternative substrate. [ABSTRACT FROM AUTHOR]

Due to the inherent low immunogenicity and immunosuppressive tumor microenvironment (TME) of malignant cancers, the clinical efficacy and application of tumor immunotherapy have been limited. Herein, a bimetallic drug-gene co-loading network (Cu/ZIF-8@U-104@siNFS1-HA) is developed that increased the intracellular labile iron pool (LIP) and enhanced the weakly acidic TME by co-suppressing the dual enzymatic activities of carbonic anhydrase IX (CA IX) and cysteine desulfurylase (NFS1), inducing a safe and efficient initial tumor immunogenic ferroptosis. During this process, Cu 2+ is responsively released to deplete glutathione (GSH) and reduce the enzyme activity of glutathione peroxidase 4 (GPX4), achieving the co-inhibition of the three enzymes and further inducing lipid peroxidation (LPO). Additionally, the reactive oxygen species (ROS) storm in target cells promoted the generation of large numbers of double-stranded DNA breaks. The presence of Zn 2+ substantially increased the expression of cGAS/STING, which cooperated with ferroptosis to strengthen the immunogenic cell death (ICD) response and remodel the immunosuppressive TME. In brief, Cu/ZIF-8@U-104@siNFS1-HA linked ferroptosis with immunotherapy through multiple pathways, including the increase in LIP, regulation of pH, depletion of GSH/GPX4, and activation of STING, effectively inhibiting cancer growth and metastasis., (© 2023 Wiley‐VCH GmbH.)

ABSTRACT DNA phosphorothioate (PT) modification, in which the nonbridging oxygen in the sugar-phosphate backbone is substituted by sulfur, is catalyzed by DndABCDE or SspABCD in a double-stranded or single-stranded manner, respectively. In Dnd and Ssp systems, mobilization of sulfur in PT formation starts with the activation of the sulfur atom of cysteine catalyzed by the DndA and SspA cysteine desulfurases, respectively. Despite playing the same biochemical role, SspA cannot be functionally replaced by DndA, indicating its unique physiological properties. In this study, we solved the crystal structure of Vibrio cyclitrophicus SspA in complex with its natural substrate, cysteine, and cofactor, pyridoxal phosphate (PLP), at a resolution of 1.80 Å. Our solved structure revealed the molecular mechanism that SspA employs to recognize its cysteine substrate and PLP cofactor, suggesting a common binding mode shared by cysteine desulfurases. In addition, although the distance between the catalytic Cys314 and the substrate cysteine is 8.9 Å, which is too far for direct interaction, our structural modeling and biochemical analysis revealed a conformational change in the active site region toward the cysteine substrate to move them close to each other to facilitate the nucleophilic attack. Finally, the pulldown analysis showed that SspA could form a complex with SspD, an ATP pyrophosphatase, suggesting that SspD might potentially accept the activated sulfur atom directly from SspA, providing further insights into the biochemical pathway of Ssp-mediated PT modification. IMPORTANCE Apart from its roles in Fe-S cluster assembly, tRNA thiolation, and sulfur-containing cofactor biosynthesis, cysteine desulfurase serves as a sulfur donor in the DNA PT modification, in which a sulfur atom substitutes a nonbridging oxygen in the DNA phosphodiester backbone. The initial sulfur mobilization from l-cysteine is catalyzed by the SspA cysteine desulfurase in the SspABCD-mediated DNA PT modification system. By determining the crystal structure of SspA, the study presents the molecular mechanism that SspA employs to recognize its cysteine substrate and PLP cofactor. To overcome the long distance (8.9 Å) between the catalytic Cys314 and the cysteine substrate, a conformational change occurs to bring Cys314 to the vicinity of the substrate, allowing for nucleophilic attack.

Iron–sulfur clusters (Fe–S or ISC) are essential cofactors that function in a wide range of biological pathways. In mammalian cells, Fe–S biosynthesis primarily relies on mitochondria and involves a concerted group of evolutionary-conserved proteins forming the ISC pathway. In the early stage of the ISC pathway, the Fe–S core complex is required for de novo assembly of Fe–S. In humans, the Fe–S core complex comprises the cysteine desulfurase NFS1, the scaffold protein ISCU2, frataxin (FXN), the ferredoxin FDX2, and regulatory/accessory proteins ISD11 and Acyl Carrier Protein (ACP). In recent years, the field has made significant advances in unraveling the structure of the Fe–S core complex and the mechanism underlying its function. Herein, we review the key recent findings related to the Fe–S core complex and its components. We highlight some of the unanswered questions and provide a model of the Fe–S assembly within the complex. In addition, we briefly touch on the genetic diseases associated with mutations in the Fe–S core complex components.

Mitochondria are essential in most eukaryotes and are involved in numerous biological functions including ATP production, cofactor biosyntheses, apoptosis, lipid synthesis, and steroid metabolism. Work over the past two decades has uncovered the biogenesis of cellular iron-sulfur (Fe/S) proteins as the essential and minimal function of mitochondria. This process is catalyzed by the bacteria-derived iron-sulfur cluster assembly (ISC) machinery and has been dissected into three major steps: de novo synthesis of a [2Fe-2S] cluster on a scaffold protein; Hsp70 chaperone–mediated trafficking of the cluster and insertion into [2Fe-2S] target apoproteins; and catalytic conversion of the [2Fe-2S] into a [4Fe-4S] cluster and subsequent insertion into recipient apoproteins. ISC components of the first two steps are also required for biogenesis of numerous essential cytosolic and nuclear Fe/S proteins, explaining the essentiality of mitochondria. This review summarizes the molecular mechanisms underlying the ISC protein–mediated maturation of mitochondrial Fe/S proteins and the importance for human disease. [ABSTRACT FROM AUTHOR]

Cysteine desulfurase enzymes catalyze sulfur mobilization from l‐cysteine to sulfur‐containing biomolecules such as iron‐sulfur (Fe‐S) clusters and thio‐tRNAs. The enzymes utilize the cofactor pyridoxal‐5'‐phosphate (PLP), which forms the external substrate‐ and product‐aldimines and ketimines during catalysis and are grouped into two types (I and II) based on their different catalytic loops. To clarify the structure‐based catalytic mechanisms for each group, we determined the structures of the external substrate‐ and product‐aldimines as catalytic intermediates of NifS (type I) and SufS (type II) that are involved in Fe‐S cluster biosynthesis using X‐ray crystallographic snapshot analysis. As a common intermediate structure, the thiol group of the PLP‐l‐cysteine external aldimine is stabilized by the conserved histidine adjacent to PLP through a polar interaction. This interaction makes the thiol group orientated for subsequent nucleophilic attack by a conserved cysteine residue on the catalytic loop in the state of PLP‐l‐cysteine ketimine, which is formed from the PLP‐l‐cysteine aldimine. Unlike the intermediates, structural changes of the loops were different between the type I and II enzymes. In the type I enzyme, conformational and topological change of the loop is necessary for nucleophilic attack by the cysteine. In contrast, the loop in type II cysteine desulfurase enzymes showed no large conformational change; rather, it might possibly orient the thiol group of the catalytic cysteine for nucleophilic attack toward PLP‐l‐cysteine. The present structures allow a revision of the catalytic mechanism and may provide a clue for consideration of enzyme function, structural diversity, and evolution of cysteine desulfurase enzymes. Database: Structural data are available in PDB database under the accession numbers 5WT2, 5WT4, 5ZSP, 5ZST, 5ZS9, 5ZSK, 5ZSO, 6KFZ, 6KG0, and 6KG1. [ABSTRACT FROM AUTHOR]

Cysteine serves as the sulfur source for the biosynthesis of Fe–S clusters and thio‐cofactors, molecules that are required for core metabolic processes in all organisms. Therefore, cysteine desulfurases, which mobilize sulfur for its incorporation into thio‐cofactors by cleaving the Cα—S bond of cysteine, are ubiquitous in nature. SufS, a type 2 cysteine desulfurase that is present in plants and microorganisms, mobilizes sulfur from cysteine to the transpersulfurase SufE to initiate Fe–S biosynthesis. Here, a 1.5 Å resolution X‐ray crystal structure of the Escherichia coli SufS homodimer is reported which adopts a state in which the two monomers are rotated relative to their resting state, displacing a β‐hairpin from its typical position blocking transpersulfurase access to the SufS active site. A global structure and sequence analysis of SufS family members indicates that the active‐site β‐hairpin is likely to require adjacent structural elements to function as a β‐latch regulating access to the SufS active site. [ABSTRACT FROM AUTHOR]

Endogenous hydrogen sulfide (H2S), which is primarily generated by 3-mercaptopyruvate sulfurtransferase (3-MST) in Escherichia coli (E. coli) under aerobic conditions, renders bacteria highly resistant to oxidative stress. However, the biosynthetic pathway and physiological role of this gas under anaerobic conditions remains largely unknown. In the present study, we demonstrate that cysteine desulfurase (IscS), not 3-MST, is the primary source of endogenous H2S in E. coli under anaerobic conditions. A significant decrease in H2S production under anaerobic conditions was observed in E. coli upon deletion of IscS, but not in 3-MST-deficient bacteria (ΔmstA). Furthermore, the H2S-producing activity of recombinant IscS using L-cysteine as a substrate exhibited an approximately 2.6-fold increase in the presence of dithiothreitol (DTT), indicating that H2S production catalyzed by IscS was greatly increased under reducing conditions. The activity of IscS was regulated under the different redox conditions and the midpoint redox potential was determined to be −329 ± 1.6 mV. Moreover, in E. coli cells H2S production from IscS is regulated under oxidative and reductive stress. A mutant E. coli (ΔiscS) strain lacking a chromosomal copy of the IscS-encoding gene iscS showed significant growth defects and low levels of ATP under both aerobic and anaerobic conditions. The growth defects could be fully restored after addition of 500 μM Na2S (an H2S donor) under anaerobic conditions, but not by the addition of cysteine, sodium sulfite or sodium sulfate. We also showed that the addition of 500 μM Na2S to culture medium stimulates ATP synthesis in the mutant E. coli (ΔiscS) strain in the logarithmic growth phase but suppresses ATP synthesis in wild-type E. coli. Our results reveal a new H2S-producing pathway in E. coli under anaerobic conditions and show that hydrogen sulfide from IscS contributes to sustaining cell growth and bioenergetics under oxygen-deficient conditions.

Endogenous hydrogen sulfide (H2S), which is primarily generated by 3-mercaptopyruvate sulfurtransferase (3-MST) in Escherichia coli (E. coli) under aerobic conditions, renders bacteria highly resistant to oxidative stress. However, the biosynthetic pathway and physiological role of this gas under anaerobic conditions remains largely unknown. In the present study, we demonstrate that cysteine desulfurase (IscS), not 3-MST, is the primary source of endogenous H2S in E. coli under anaerobic conditions. A significant decrease in H2S production under anaerobic conditions was observed in E. coli upon deletion of IscS, but not in 3-MST-deficient bacteria (Δ mstA). Furthermore, the H2S-producing activity of recombinant IscS using L -cysteine as a substrate exhibited an approximately 2.6-fold increase in the presence of dithiothreitol (DTT), indicating that H2S production catalyzed by IscS was greatly increased under reducing conditions. The activity of IscS was regulated under the different redox conditions and the midpoint redox potential was determined to be −329 ± 1.6 mV. Moreover, in E. coli cells H2S production from IscS is regulated under oxidative and reductive stress. A mutant E. coli (Δ iscS) strain lacking a chromosomal copy of the IscS-encoding gene iscS showed significant growth defects and low levels of ATP under both aerobic and anaerobic conditions. The growth defects could be fully restored after addition of 500 μM Na2S (an H2S donor) under anaerobic conditions, but not by the addition of cysteine, sodium sulfite or sodium sulfate. We also showed that the addition of 500 μM Na2S to culture medium stimulates ATP synthesis in the mutant E. coli (Δ iscS) strain in the logarithmic growth phase but suppresses ATP synthesis in wild-type E. coli. Our results reveal a new H2S-producing pathway in E. coli under anaerobic conditions and show that hydrogen sulfide from IscS contributes to sustaining cell growth and bioenergetics under oxygen-deficient conditions. [ABSTRACT FROM AUTHOR]

Key message: The ISC Fe–S cluster biosynthetic pathway would play a key role in the regulation of iron and sulfur homeostasis in plants. The Arabidopsis thaliana mitochondrial cysteine desulfurase AtNFS1 has an essential role in cellular ISC Fe–S cluster assembly, and this pathway is one of the main sinks for iron (Fe) and sulfur (S) in the plant. In different plant species it has been reported a close relationship between Fe and S metabolisms; however, the regulation of both nutrient homeostasis is not fully understood. In this study, we have characterized AtNFS1 overexpressing and knockdown mutant Arabidopsis plants. Plants showed alterations in the ISC Fe–S biosynthetic pathway genes and in the activity of Fe–S enzymes. Genes involved in Fe and S uptakes, assimilation, and regulation were up-regulated in overexpressing plants and down-regulated in knockdown plants. Furthermore, the plant nutritional status in different tissues was in accordance with those gene activities: overexpressing lines accumulated increased amounts of Fe and S and mutant plant had lower contents of S. In summary, our results suggest that the ISC Fe–S cluster biosynthetic pathway plays a crucial role in the homeostasis of Fe and S in plants, and that it may be important in their regulation. [ABSTRACT FROM AUTHOR]

Abstract Iron-sulfur (Fe-S) clusters are critical metallo-cofactors required for cell function. Assembly of these cofactors is a carefully controlled process in cells to avoid toxicity from free iron and sulfide. In Plasmodium , two pathways for these Fe-S cluster biogenesis have been reported; ISC pathway in the mitochondria and SUF pathway functional in the apicoplast. Amongst these, SUF pathway is reported essential for the apicoplast maintenance and parasite survival. Many of its components have been studied from P. falciparum and P. berghei in recent years, still few queries remain to be addressed; one of them being the assembly and transfer of Fe-S clusters. In this study, using P. vivax clinical isolates, we have shown the in vitro interaction of SUF pathway proteins SufS and SufE responsible for sulfur mobilization in the apicoplast. The sulfur mobilized by the SufSE complex assembles on the scaffold protein Pv SufA along with iron provided by the external source. Here, we demonstrate in vitro transfer of these labile Fe-S clusters from the scaffold protein on to an apo-protein, Pv IspG (a protein involved in penultimate step of Isoprenoids biosynthesis pathway) in order to provide an insight into the interaction of different components for the biosynthesis and transfer of Fe-S clusters. Our analysis indicate that inspite of the presence of variations in pathway proteins, the overall pathway remains well conserved in the clinical isolates when compared to that reported in lab strains. Graphical abstract Image 1 Highlights • Elucidation of Fe-S clusters assembly & transfer in the SUF pathway of P. vivax. • Sulfur mobilized by Pv SufSE complex is acquired by Pv SufA to form Fe-S clusters. • In vitro transfer of Fe-S clusters from Pv SufA to apo-protein Pv IspG. [ABSTRACT FROM AUTHOR]

Iron-Sulfur (Fe-S) clusters and proteins are essential to many growth and developmental processes. In plants, they exist in the plastids, mitochondria, cytosol, and nucleus. Six types of Fe-S clusters are found in the plastid: classic 2Fe-2S, NEET-type 2Fe-2S, Rieske-type 2Fe-2S, 3Fe-4S, 4Fe-4S, and siroheme 4Fe-4S. Classic, NEET-type, and Rieske-type 2Fe-2S clusters have the same 2Fe-2S core; similarly, common and siroheme 4Fe-4S clusters have the same 4Fe-4S core. Plastidial Fe-S clusters are assembled by the sulfur mobilization (SUF) pathway, which contains cysteine desulfurase (EC 2.8.1.7), sulfur transferase (EC 2.8.1.3), Fe-S scaffold complex, and Fe-S carrier proteins. The plastidial cysteine desulfurase-sulfur transferase-Fe-S-scaffold complex system is responsible for de novo assembly of all plastidial Fe-S clusters. However, different types of Fe-S clusters are transferred to recipient proteins via respective Fe-S carrier proteins. This review focuses on recent discoveries on the molecular functions of different assembly and transfer factors involved in the plastidial SUF pathway. It also discusses potential points for regulation of the SUF pathway, relationships among the plastidial, mitochondrial, and cytosolic Fe-S assembly and transfer pathways, as well as several open questions about the carrier proteins for Rieske-type 2Fe-2S, NEET-type 2Fe-2S, and 3F-4S clusters.

Iron-sulfur (Fe-S) clusters are ubiquit1ous cofactors present in all domains of life. The chemistries catalyzed by these inorganic cofactors are diverse and their associated enzymes are involved in many cellular processes. Despite the wide range of structures reported for Fe-S clusters inserted into proteins, the biological synthesis of all Fe-S clusters starts with the assembly of simple units of 2Fe-2S and 4Fe-4S clusters. Several systems have been associated with the formation of Fe-S clusters in bacteria with varying phylogenetic origins and number of biosynthetic and regulatory components. All systems, however, construct Fe-S clusters through a similar biosynthetic scheme involving three main steps: (1) sulfur activation by a cysteine desulfurase, (2) cluster assembly by a scaffold protein, and (3) guided delivery of Fe-S units to either final acceptors or biosynthetic enzymes involved in the formation of complex metalloclusters. Another unifying feature on the biological formation of Fe-S clusters in bacteria is that these systems are tightly regulated by a network of protein interactions. Thus, the formation of transient protein complexes among biosynthetic components allows for the direct transfer of reactive sulfur and Fe-S intermediates preventing oxygen damage and reactions with nonphysiological targets. Recent studies revealed the importance of reciprocal signature sequence motifs that enable specific protein-protein interactions and consequently guide the transactions between physiological donors and acceptors. Such findings provide insights into strategies used by bacteria to regulate the flow of reactive intermediates and provide protein barcodes to uncover yet-unidentified cellular components involved in Fe-S metabolism. [ABSTRACT FROM AUTHOR]

Cysteine desulfhydrase (CDes) can catalyze the degradation of cysteine producing hydrogen sulfide. In this study, D-cysteine desulfhydrase from wheat (TaD-CDes) was cloned and overexpressed in Arabidopsis thaliana. The physiological effects of TaD-CDes were determined by investigating seed germination, root growth, stomatal closure, and drought resistance in the TaD-CDes plants. Results showed that, compared with wild-type plants (WT), seed germination, root growth, and stomatal closure of the TaD-CDes plants were more sensitive to ABA, resulting from up-regulation of ABA-responsive genes (such as PYR1, ABI1, ABI2, HAB1, HAB2, SnRK2, ABF2, and ABF4). Moreover, although TaD-CDes mediated ABA-induced stomatal closure, TaD-CDes-overexpressing plants did not show higher drought resistance than WT, which might be attributed to their increased stomatal densities. [ABSTRACT FROM AUTHOR]

Highlights • A novel predictor is develop to predict S-sulfenylation sites. • The combined features is used to predict and analyze S-sulfenylation sites. • mRMR feature selection method is adopted to remove the redundant features. • A free online service is available for prediction. Abstract Cysteine S-sulfenylation is an important protein post-translational modification, which plays a crucial role in transcriptional regulation, cell signaling, and protein functions. To better elucidate the molecular mechanism of S-sulfenylation, it is important to identify S-sulfenylated substrates and their corresponding S-sulfenylation sites accurately. In this study, a novel bioinformatics tool named Sulf_FSVM is proposed to predict S-sulfenylation sites by using multiple feature extraction and fuzzy support vector machine algorithm. On the one hand, amino acid factors, binary encoding, and the composition of k-spaced amino acid pairs features are incorporated to encode S-sulfenylation sites. And the maximum relevance minimum redundancy method are adopted to remove the redundant features. On the other hand, a fuzzy support vector machine algorithm is used to handle the class imbalance and noise problem in S-sulfenylation sites training dataset. As illustrated by 10-fold cross-validation, the performance of Sulf_FSVM achieves a satisfactory performance with a Sensitivity of 73.26%, a Specificity of 70.78%, an Accuracy of 71.07% and a Matthew's correlation coefficient of 0.2971. Independent tests also show that Sulf_FSVM significantly outperforms existing S-sulfenylation sites predictors. Therefore, Sulf_FSVM can be a useful tool for accurate prediction of protein S-sulfenylation sites. Graphical abstract Image, graphical abstract [ABSTRACT FROM AUTHOR]

Background Selenophosphate, the key selenium donor for the synthesis of selenoprotein and selenium-modified tRNA, is produced by selenophosphate synthetase (SPS) from ATP, selenide, and H 2 O. Although free selenide can be used as the in vitro selenium substrate for selenophosphate synthesis, the precise physiological system that donates in vivo selenium substrate to SPS has not yet been characterized completely. Scope of review In this review, we discuss selenium metabolism with respect to the delivery of selenium to SPS in selenoprotein biosynthesis. Major conclusions Glutathione, selenocysteine lyase, cysteine desulfurase, and selenium-binding proteins are the candidates of selenium delivery system to SPS. The thioredoxin system is also implicated in the selenium delivery to SPS in Escherichia coli . General significance Selenium delivered via a protein-bound selenopersulfide intermediate emerges as a central element not only in achieving specific selenoprotein biosynthesis but also in preventing the occurrence of toxic free selenide in the cell. This article is part of a Special Issue entitled “Selenium research in biochemistry and biophysics – 200 year anniversary”. [ABSTRACT FROM AUTHOR]

A nonheme Fe(ii) complex (1) that models substrate-bound cysteine dioxygenase (CDO) reacts with O2 at −80 °C to yield a purple intermediate (2). Analysis with spectroscopic and computational methods determined that 2 features a thiolate-ligated Fe(iii) center bound to a superoxide radical, mimicking the putative structure of a key CDO intermediate. [ABSTRACT FROM AUTHOR]

Human de novo iron-sulfur (Fe-S) assembly complex consists of cysteine desulfurase NFS1, accessory protein ISD11, acyl carrier protein ACP, scaffold protein ISCU, and allosteric activator frataxin (FXN). FXN binds the NFS1-ISD11-ACP-ISCU complex (SDAU), to activate the desulfurase activity and Fe-S cluster biosynthesis. In the absence of FXN, the NFS1-ISD11-ACP (SDA) complex was reportedly inhibited by binding of recombinant ISCU. Recent studies also reported a substitution at position Met141 on the yeast ISCU orthologue Isu, to Ile, Leu, Val, or Cys, could bypass the requirement of FXN for Fe-S cluster biosynthesis and cell viability. Here, we show that recombinant human ISCU binds zinc(II) ion, as previously demonstrated with the E. coli orthologue IscU. Surprisingly, the relative proportion between zinc-bound and zinc-depleted forms varies among purification batches. Importantly the presence of zinc in ISCU impacts SDAU desulfurase activity. Indeed, removal of zinc(II) ion from ISCU causes a moderate but significant increase in activity compared to SDA alone, and FXN can activate both zinc-depleted and zinc-bound forms of ISCU complexed to SDA. Taking into consideration the inhibition of desulfurase activity by zinc-bound ISCU, we characterized wild type ISCU and the M140I, M140L, and M140V variants under both zinc-bound and zinc-depleted conditions, and did not observe significant differences in the biochemical and biophysical properties between wild-type and variants. Importantly, in the absence of FXN, ISCU variants behaved like wild-type and did not stimulate the desulfurase activity of the SDA complex. This study therefore identifies an important regulatory role for zinc-bound ISCU in modulation of the human Fe-S assembly system in vitro and reports no ‘FXN bypass’ effect on mutations at position Met140 in human ISCU. Furthermore, this study also calls for caution in interpreting studies involving recombinant ISCU by taking into consideration the influence of the bound zinc(II) ion on SDAU complex activity. [ABSTRACT FROM AUTHOR]

Abstract: RNA interference is widely used to analyze gene functions via phenotypic knockdown of target transcripts in mosquitoes, which transmit numerous mosquito‐borne diseases. Functional analysis of mosquito genes is indispensable to understand and reduce transmission of mosquito‐borne diseases in mosquitoes. Intrathoracic injection of double‐stranded RNA (dsRNA) remains the simplest and most customizable method in mosquitoes for functional analysis of the genes of interest. However, achieving consistent and effective knockdown by dsRNAi is often elusive and may require extensive optimization. We tested the effectiveness of gene silencing by intrathoracic injection of four different quantities of dsRNA targeting two Aedes aegypti genes, cysteine desulfurylase (Nfs1) and short‐chain dehydrogenase (SDH). We found that Nfs1 gene has a lower expression level upon silencing than SDH gene. In the case of the gene that is easier to silence, Nfs1 gene expression was significantly silenced by all four tested quantities of dsRNA up to 21 days post infection (d.p.i.), but silencing of SDH, the gene that is difficult to silence, was less effective, with knockdown lasting up to 9 d.p.i. only when 1,000 ng of dsRNA was used. Based on our observation, intrathoracic injection of 500 ng of dsRNAs per mosquito is recommended to achieve effective knockdown for well‐silenced transcripts such as Nfs1 for up to 3 weeks. This includes most in vivo bioassays involving arboviral infections in Ae. aegypti. The estimated quantities of dsRNA described in this study should be applicable to most Ae. aegypti dsRNAi studies and thus provide a guideline to develop efficient dsRNAi in other experimental investigations. [ABSTRACT FROM AUTHOR]

Frataxin (FXN) is involved in mitochondrial iron‑sulfur (Fe-S) cluster biogenesis and serves to accelerate Fe-S cluster formation. FXN deficiency is associated with Friedreich ataxia, a neurodegenerative disease. We have used a combination of isothermal titration calorimetry and multinuclear NMR spectroscopy to investigate interactions among the components of the biological machine that carries out the assembly of iron‑sulfur clusters in human mitochondria. Our results show that FXN tightly binds a single Fe 2+ but not Fe 3+ . While FXN (with or without bound Fe 2+ ) does not bind the scaffold protein ISCU directly, the two proteins interact mutually when each is bound to the cysteine desulfurase complex ([NFS1] 2 :[ISD11] 2 :[Acp] 2 ), abbreviated as (NIA) 2 , where “N” represents the cysteine desulfurase (NFS1), “I” represents the accessory protein (ISD11), and “A” represents acyl carrier protein (Acp). FXN binds (NIA) 2 weakly in the absence of ISCU but more strongly in its presence. Fe 2+ -FXN binds to the (NIA) 2 -ISCU 2 complex without release of iron. However, upon the addition of both l -cysteine and a reductant (either reduced FDX2 or DTT), Fe 2+ is released from FXN as consistent with Fe 2+ -FXN being the proximal source of iron for Fe-S cluster assembly. [ABSTRACT FROM AUTHOR]

The cysteine desulfurase Nfs1/Isd11 uses the amino acid cysteine as the substrate and its activity is absolutely required for contributing persulfide sulfur to the essential process of iron-sulfur (Fe–S) cluster assembly in mitochondria. Here we describe a novel regulatory process involving phosphorylation of Nfs1 in mitochondria. Phosphorylation enhanced cysteine desulfurase activity, while dephosphorylation decreased its activity. Nfs1 phosphopeptides were identified, and the corresponding phosphosite mutants showed impaired persulfide formation. Nfs1 pull down from mitochondria recovered an associated kinase activity, and Yck2, a kinase present in the pull down, was able to phosphorylate Nfs1 in vitro and stimulate cysteine desulfurase activity. Yck2 exhibited an eclipsed distribution in the mitochondrial matrix, although other cellular localizations have been previously described. Mitochondria lacking the Yck2 protein kinase (∆ yck2 ) showed less phosphorylating activity for Nfs1. Compared with wild-type mitochondria, ∆ yck2 mitochondria revealed slower persulfide formation on Nfs1 consistent with a role of Yck2 in regulating mitochondrial cysteine desulfurase activity. We propose that Nfs1 phosphorylation may provide a means of rapid adaptation to increased metabolic demand for sulfur and Fe–S clusters within mitochondria. [ABSTRACT FROM AUTHOR]

Iron-Sulfur (Fe-S) clusters and proteins are essential to many growth and developmental processes. In plants, they exist in the plastids, mitochondria, cytosol and nucleus. Six types of Fe-S clusters are found in the plastid: classic 2Fe-2S, NEET-type 2Fe-2S, Rieske-type 2Fe-2S, 3Fe-4S, 4Fe-4S and siroheme 4Fe-4S. Classic, NEET-type, and Rieske-type 2Fe-2S clusters have the same 2Fe-2S core; similarly, common and siroheme 4Fe-4S clusters have the same 4Fe-4S core. Plastidial Fe-S clusters are assembled by the sulfur mobilization (SUF) pathway, which contains cysteine desulfurase (EC 2.8.1.7), sulfur transferase (EC 2.8.1.3), Fe-S scaffold complex and Fe-S carrier proteins. The plastidial cysteine desulfurase-sulfur transferase-Fe-S-scaffold complex system is responsible for de novo assembly of all plastidial Fe-S clusters. However, different types of Fe-S clusters are transferred to recipient proteins via respective Fe-S carrier proteins. This review focuses on recent discoveries on the molecular functions of different assembly and transfer factors involved in the plastidial SUF pathway. It also discusses potential points for regulation of the SUF pathway, relationships among the plastidial, mitochondrial and cytosolic Fe-S assembly and transfer pathways, as well as several open questions about the carrier proteins for Rieske-type 2Fe-2S, NEET-type 2Fe-2S, and 3F-4S clusters. [ABSTRACT FROM AUTHOR]