The Human Mercaptopyruvate Sulfurtransferase TUM1 Is Involved in Moco Biosynthesis, Cytosolic tRNA Thiolation and Cellular Bioenergetics in Human Embryonic Kidney Cells.

Sulfur is an important element that is incorporated into many biomolecules in humans. The incorporation and transfer of sulfur into biomolecules is, however, facilitated by a series of different sulfurtransferases. Among these sulfurtransferases is the human mercaptopyruvate sulfurtransferase (MPST) also designated as tRNA thiouridine modification protein (TUM1). The role of the human TUM1 protein has been suggested in a wide range of physiological processes in the cell among which are but not limited to involvement in Molybdenum cofactor (Moco) biosynthesis, cytosolic tRNA thiolation and generation of H2S as signaling molecule both in mitochondria and the cytosol. Previous interaction studies showed that TUM1 interacts with the L-cysteine desulfurase NFS1 and the Molybdenum cofactor biosynthesis protein 3 (MOCS3). Here, we show the roles of TUM1 in human cells using CRISPR/Cas9 genetically modified Human Embryonic Kidney cells. Here, we show that TUM1 is involved in the sulfur transfer for Molybdenum cofactor synthesis and tRNA thiomodification by spectrophotometric measurement of the activity of sulfite oxidase and liquid chromatography quantification of the level of sulfur-modified tRNA. Further, we show that TUM1 has a role in hydrogen sulfide production and cellular bioenergetics.

A-Type Carrier Proteins Are Involved in [4Fe-4S] Cluster Insertion into the Radical S-Adenosylmethionine Protein MoaA for the Synthesis of Active Molybdoenzymes.

Iron sulfur (Fe-S) clusters are important biological cofactors present in proteins with crucial biological functions, from photosynthesis to DNA repair, gene expression, and bioenergetic processes. For the insertion of Fe-S clusters into proteins, A-type carrier proteins have been identified. So far, three of them have been characterized in detail in Escherichia coli, namely, IscA, SufA, and ErpA, which were shown to partially replace each other in their roles in [4Fe-4S] cluster insertion into specific target proteins. To further expand the knowledge of [4Fe-4S] cluster insertion into proteins, we analyzed the complex Fe-S cluster-dependent network for the synthesis of the molybdenum cofactor (Moco) and the expression of genes encoding nitrate reductase in E. coli. Our studies include the identification of the A-type carrier proteins ErpA and IscA, involved in [4Fe-4S] cluster insertion into the radical S-adenosyl-methionine (SAM) enzyme MoaA. We show that ErpA and IscA can partially replace each other in their role to provide [4Fe-4S] clusters for MoaA. Since most genes expressing molybdoenzymes are regulated by the transcriptional regulator for fumarate and nitrate reduction (FNR) under anaerobic conditions, we also identified the proteins that are crucial to obtain an active FNR under conditions of nitrate respiration. We show that ErpA is essential for the FNR-dependent expression of the narGHJI operon, a role that cannot be compensated by IscA under the growth conditions tested. SufA does not appear to have a role in Fe-S cluster insertion into MoaA or FNR under anaerobic growth employing nitrate respiration, based on the low level of gene expression. IMPORTANCE Understanding the assembly of iron-sulfur (Fe-S) proteins is relevant to many fields, including nitrogen fixation, photosynthesis, bioenergetics, and gene regulation. Remaining critical gaps in our knowledge include how Fe-S clusters are transferred to their target proteins and how the specificity in this process is achieved, since different forms of Fe-S clusters need to be delivered to structurally highly diverse target proteins. Numerous Fe-S carrier proteins have been identified in prokaryotes like Escherichia coli, including ErpA, IscA, SufA, and NfuA. In addition, the diverse Fe-S cluster delivery proteins and their target proteins underlie a complex regulatory network of expression, to ensure that both proteins are synthesized under particular growth conditions.

The functional diversity of the prokaryotic sulfur carrier protein TusA.

Persulfide groups participate in a wide array of biochemical pathways and are chemically very versatile. The TusA protein has been identified as a central element supplying and transferring sulfur as persulfide to a number of important biosynthetic pathways, like molybdenum cofactor biosynthesis or thiomodifications in nucleosides of tRNAs. In recent years, it has furthermore become obvious that this protein is indispensable for the oxidation of sulfur compounds in the cytoplasm. Phylogenetic analyses revealed that different TusA protein variants exists in certain organisms, that have evolved to pursue specific roles in cellular pathways. The specific TusA-like proteins thereby cannot replace each other in their specific roles and are rather specific to one sulfur transfer pathway or shared between two pathways. While certain bacteria like Escherichia coli contain several copies of TusA-like proteins, in other bacteria like Allochromatium vinosum a single copy of TusA is present with an essential role for this organism. Here, we give an overview on the multiple roles of the various TusA-like proteins in sulfur transfer pathways in different organisms to shed light on the remaining mysteries of this versatile protein.

Identification of a protein-protein interaction network downstream of molybdenum cofactor biosynthesis in Arabidopsis thaliana.

The molybdenum cofactor (Moco) is ubiquitously present in all kingdoms of life and vitally important for survival. Among animals, loss of the Moco-containing enzyme (Mo-enzyme) sulphite oxidase is lethal, while for plants the loss of nitrate reductase prohibits nitrogen assimilation. Moco is highly oxygen-sensitive, which obviates a freely diffusible pool and necessitates protein-mediated distribution. During the highly conserved Moco biosynthesis pathway, intermediates are channelled through a multi-protein complex facilitating protected transport. However, the mechanism by which Moco is subsequently transferred to apo-enzymes is still unclear. Moco user enzymes can be divided into two families: the sulphite oxidase (SO) and the xanthine oxidoreductase (XOR) family. The latter requires a final sulphurisation of Moco catalysed via ABA3. To examine Moco transfer towards apo-Mo-enzymes, two different and independent protein-protein interaction assays were performed in vivo: bimolecular fluorescence complementation and split luciferase. The results revealed a direct contact between Moco producer molybdenum insertase CNX1, which represents the last biosynthesis step, and members of the SO family. However, no protein contact was observed between Moco producer CNX1 and apo-enzymes of the XOR family or between CNX1 and the Moco sulphurase ABA3. Instead, the Moco-binding protein MOBP2 was identified as a mediator between CNX1 and ABA3. This interaction was followed by contact between ABA3 and enzymes of the XOR family. These results allow to describe an interaction matrix of proteins beyond Moco biosynthesis and to demonstrate the complexity of transferring a prosthetic group after biosynthesis.

Molecular characterization of novel immunodominant molybdenum cofactor biosynthesis protein C1 (Rv3111) from Mycobacterium tuberculosis H37Rv.

BACKGROUND: In the molybdenum cofactor biosynthesis pathway, MoaA and MoaC catalyze the first step of transformation of GTP to cPMP. In M. tuberculosis H37Rv, three different genes (Rv3111, Rv0864 and Rv3324c) encode for MoaC homologs. Out of these three only MoaC1 (Rv3111) is secretory in nature. METHODS: We have characterized MoaC1 protein through biophysical, in-silico, and immunological techniques. RESULTS: We have characterized the conformation and thermodynamic stability of MoaC1, and have established its secretory nature by demonstrating the presence of anti-MoaC1 antibodies in human tuberculosis patients' sera. Further, MoaC1 elicited a dominant Th1 immune response in mice characterized by increased induction of IL-2 and IFN-γ. CONCLUSION: Integrating these results, we conclude that MoaC1 is a structured secretory protein capable of binding with GTP and eliciting induced immune response. GENERAL SIGNIFICANCE: This study would be useful for the development of vaccines against tuberculosis and to improve methods used for diagnosis of tuberculosis.

Moco biosynthesis and the ATAC acetyltransferase engage translation initiation by inhibiting latent PKR activity.

Molybdenum cofactor (Moco) biosynthesis is linked to c-Jun N-terminal kinase (JNK) signaling in Drosophila through MoaE, a molybdopterin (MPT) synthase subunit that is also a component of the Ada Two A containing (ATAC) acetyltransferase complex. Here, we show that human MPT synthase and ATAC inhibited PKR, a double-stranded RNA-dependent protein kinase, to facilitate translation initiation of iron-responsive mRNA. MPT synthase and ATAC directly interacted with PKR and suppressed latent autophosphorylation of PKR and its downstream phosphorylation of JNK and eukaryotic initiation factor 2α (eIF2α). The suppression of eIF2α phosphorylation via MPT synthase and ATAC prevented sequestration of the guanine nucleotide exchange factor eIF2B, which recycles eIF2-GDP to eIF2-GTP, resulting in the promotion of translation initiation. Indeed, translation of the iron storage protein, ferritin, was reduced in the absence of MPT synthase or ATAC subunits. Thus, MPT synthase and ATAC regulate latent PKR signaling and link transcription and translation initiation.

Structural characterization of gephyrin by AFM and SAXS reveals a mixture of compact and extended states.

Gephyrin is a trimeric protein involved in the final steps of molybdenum-cofactor (Moco) biosynthesis and in the clustering of inhibitory glycine and GABAA receptors at postsynaptic specializations. Each protomer consists of stably folded domains (referred to as the G and E domains) located at either terminus and connected by a proteolytically sensitive linker of ∼150 residues. Both terminal domains can oligomerize in their isolated forms; however, in the context of the full-length protein only the G-domain trimer is permanently present, whereas E-domain dimerization is prevented. Atomic force microscopy (AFM) and small-angle X-ray scattering (SAXS) reveal a high degree of flexibility in the structure of gephyrin. The results imply an equilibrium between compact and extended conformational states in solution, with a preference for compact states. CD spectroscopy suggests that a partial compaction is achieved by interactions of the linker with the G and E domains. Taken together, the data provide a rationale for the role of the linker in the overall structure and the conformational dynamics of gephyrin.

TusA (YhhP) and IscS are required for molybdenum cofactor-dependent base-analog detoxification.

Lack of molybdenum cofactor (Moco) in Escherichia coli leads to hypersensitivity to the mutagenic and toxic effects of N-hydroxylated base analogs, such as 6-N-hydroxylaminopurine (HAP). This phenotype is due to the loss of two Moco-dependent activities, YcbX and YiiM, that are capable of reducing HAP to adenine. Here, we describe two novel HAP-sensitive mutants containing a defect in iscS or tusA (yhhP) gene. IscS is a major L-cysteine desulfurase involved in iron-sulfur cluster synthesis, thiamine synthesis, and tRNA thiomodification. TusA is a small sulfur-carrier protein that interacts with IscS. We show that both IscS and TusA operate within the Moco-dependent pathway. Like other Moco-deficient strains, tusA and iscS mutants are HAP sensitive and resistant to chlorate under anaerobic conditions. The base-analog sensitivity of iscS or tusA strains could be suppressed by supplying exogenous L-cysteine or sulfide or by an increase in endogenous sulfur donors (cysB constitutive mutant). The data suggest that iscS and tusA mutants have a defect in the mobilization of sulfur required for active YcbX/YiiM proteins as well as nitrate reductase, presumably due to lack of functional Moco. Overall, our data imply a novel and indispensable role of the IscS/TusA complex in the activity of several molybdoenzymes.