Search Results (13)

All kingdoms of life have more than 150 different forms of RNA alterations, with tRNA accounting for around 80% of them. These chemical alterations include, among others, methylation, sulfuration, hydroxylation, and acetylation. These changes are necessary for the proper codon recognition and stability of tRNA. In Escherichia coli, sulfur modification at the wobble uridine (34) of lysine, glutamic acid, and glutamine is essential for codon and anticodon binding and prevents frameshifting during translation. Two important proteins that are involved in this thiolation modification are the L-cysteine desulfurase IscS, the initial sulfur donor, and tRNA-specific 2-thiouridylase MnmA, which adenylates and finally transfers the sulfur from IscS to the tRNA. tRNA-specific 2-thiouridylases are iron–sulfur clusters (Fe-S), either dependent or independent depending on the organism. Here, we dissect the controversy of whether the E. coli MnmA protein is an Fe-S cluster-dependent or independent protein. We show that when Fe-S clusters are bound to MnmA, tRNA thiolation is inhibited, making MnmA an Fe-S cluster-independent protein. We further show that 2-thiouridylase only binds to tRNA from its own organism. Full article

The Rhodanese-fold is a ubiquitous structural domain present in various protein subfamilies associated with different physiological functions or pathophysiological conditions in humans. Proteins harboring a Rhodanese domain are diverse in terms of domain architecture, with some representatives exhibiting one or several Rhodanese domains, fused or not to other structural domains. The most famous Rhodanese domains are catalytically active, thanks to an active-site loop containing an essential cysteine residue which allows for catalyzing sulfur transfer reactions involved in sulfur trafficking, hydrogen sulfide metabolism, biosynthesis of molybdenum cofactor, thio-modification of tRNAs or protein urmylation. In addition, they also catalyse phosphatase reactions linked to cell cycle regulation, and recent advances proposed a new role into tRNA hydroxylation, illustrating the catalytic versatility of Rhodanese domain. To date, no exhaustive analysis of Rhodanese containing protein equipment from humans is available. In this review, we focus on structural and biochemical properties of human-active Rhodanese-containing proteins, in order to provide a picture of their established or putative key roles in many essential biological functions. Full article

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. Full article

Iron–sulfur (Fe–S) clusters are essential cofactors for enzyme activity. These Fe–S clusters are present in structurally diverse forms, including [4Fe–4S] and [3Fe–4S]. Type-identification of the Fe–S cluster is indispensable in understanding the catalytic mechanism of enzymes. However, identifying [4Fe–4S] and [3Fe–4S] clusters in particular is challenging because of their rapid transformation in response to oxidation–reduction events. In this study, we focused on the relationship between the Fe–S cluster type and the catalytic activity of a tRNA-thiolation enzyme (TtuA). We reconstituted [4Fe–4S]-TtuA, prepared [3Fe–4S]-TtuA by oxidizing [4Fe–4S]-TtuA under strictly anaerobic conditions, and then observed changes in the Fe–S clusters in the samples and the enzymatic activity in the time-course experiments. Electron paramagnetic resonance analysis revealed that [3Fe–4S]-TtuA spontaneously transforms into [4Fe–4S]-TtuA in minutes to one hour without an additional free Fe source in the solution. Although the TtuA immediately after oxidation of [4Fe–4S]-TtuA was inactive [3Fe–4S]-TtuA, its activity recovered to a significant level compared to [4Fe–4S]-TtuA after one hour, corresponding to an increase of [4Fe–4S]-TtuA in the solution. Our findings reveal that [3Fe–4S]-TtuA is highly inactive and unstable. Moreover, time-course analysis of structural changes and activity under strictly anaerobic conditions further unraveled the Fe–S cluster type used by the tRNA-thiolation enzyme. Full article

Thrips palmi (Thysanoptera: Thripidae) is an important pest of vegetables, legumes, and ornamentals. In addition, it transmits several plant viruses. T. palmi genes associated with innate immunity, endocytosis-related pathways, and cuticular development are highly enriched in response to Groundnut bud necrosis orthotospovirus (GBNV, genus Orthotospovirus, family Tospoviridae) infection. As the previous transcriptomic study suggested the involvement of T. palmi UHRF1BP1 and PFAS in GBNV infection, these two genes were targeted for silencing using antisense oligonucleotides (ASOs), and the effects on thrips’ fitness and virus acquisition were observed. Phosphorothioate modification of ASOs was carried out by replacing the nonbridging oxygen atom with a sulfur atom at the 3′ position to increase nuclease stability. The modified ASOs were delivered orally through an artificial diet. Exposure to ASOs reduced the target mRNA expression up to 2.70-fold optimally. Silencing of T. palmi UHRF1BP1 and PFAS induced 93.33% mortality that further increased up to 100% with an increase in exposure. Silencing of T. palmi UHRF1BP1 and PFAS also produced morphological deformities in the treated T. palmi. GBNV titer in T. palmi significantly declined post-exposure to ASOs. This is the first-ever report of silencing T. palmi UHRF1BP1 and PFAS using modified ASO to induce mortality and impair virus transmission in T. palmi. T. palmi UHRF1BP1 and PFAS would be novel genetic targets to manage thrips and restrict the spread of tospovirus. Full article

AlphaFold and RoseTTAFold are deep learning-based approaches that predict the structure of proteins from their amino acid sequences. Remarkable success has recently been achieved in the prediction accuracy of not only the fold of the target protein but also the position of its amino acid side chains. In this article, I question the accuracy of these methods to predict iron–sulfur binding sites. I analyze three-dimensional models calculated by AlphaFold and RoseTTAFold of Fe–S–dependent enzymes, for which no structure of a homologous protein has been solved experimentally. In all cases, the amino acids that presumably coordinate the cluster were gathered together and facing each other, which led to a quite accurate model of the Fe–S cluster binding site. Yet, cysteine candidates were often involved in intramolecular disulfide bonds, and the number and identity of the protein amino acids that should ligate the cluster were not always clear. The experimental structure determination of the protein with its Fe–S cluster and in complex with substrate/inhibitor/product is still needed to unambiguously visualize the coordination state of the cluster and understand the conformational changes occurring during catalysis. Full article

Various sulfur-containing biomolecules include iron–sulfur clusters that act as cofactors for enzymes, sulfur-containing vitamins such as thiamin, and sulfur-modified nucleosides in RNA, in addition to methionine and cysteine in proteins. Sulfur-containing nucleosides are post-transcriptionally introduced into tRNA molecules, where they ensure precise codon recognition or stabilization of tRNA structure, thereby maintaining cellular proteome integrity. Modulating sulfur modification controls the translation efficiency of specific groups of genes, allowing organisms to adapt to specific environments. The biosynthesis of tRNA sulfur nucleosides involves elaborate ‘sulfur trafficking systems’ within cellular sulfur metabolism and ‘modification enzymes’ that incorporate sulfur atoms into tRNA. This review provides an up-to-date overview of advances in our knowledge of the mechanisms involved. It covers the functions, biosynthesis, and biodegradation of sulfur-containing nucleosides as well as the reaction mechanisms of biosynthetic enzymes catalyzed by the iron–sulfur clusters, and identification of enzymes involved in the de-modification of sulfur atoms of RNA. The mechanistic similarity of these opposite reactions is discussed. Mutations in genes related to these pathways can cause human diseases (e.g., cancer, diabetes, and mitochondrial diseases), emphasizing the importance of these pathways. Full article

Urm1 (ubiquitin related modifier 1) is a molecular fossil in the class of ubiquitin-like proteins (UBLs). It encompasses characteristics of classical UBLs, such as ubiquitin or SUMO (small ubiquitin-related modifier), but also of bacterial sulfur-carrier proteins (SCP). Since its main function is to modify tRNA, Urm1 acts in a non-canonical manner. Uba4, the activating enzyme of Urm1, contains two domains: a classical E1-like domain (AD), which activates Urm1, and a rhodanese homology domain (RHD). This sulfurtransferase domain catalyzes the formation of a C-terminal thiocarboxylate on Urm1. Thiocarboxylated Urm1 is the sulfur donor for 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U), a chemical nucleotide modification at the wobble position in tRNA. This thio-modification is conserved in all domains of life and optimizes translation. The absence of Urm1 increases stress sensitivity in yeast triggered by defects in protein homeostasis, a hallmark of neurological defects in higher organisms. In contrast, elevated levels of tRNA modifying enzymes promote the appearance of certain types of cancer and the formation of metastasis. Here, we summarize recent findings on the unique features that place Urm1 at the intersection of UBL and SCP and make Urm1 an excellent model for studying the evolution of protein conjugation and sulfur-carrier systems. Full article

Sulfur is an essential element required for plant growth. It can be found as a thiol group of proteins or non-protein molecules, and as various sulfur-containing small biomolecules, including iron-sulfur (Fe/S) clusters, molybdenum cofactor (Moco), and sulfur-modified nucleotides. Thiol-mediated redox regulation has been well investigated, whereas biosynthesis pathways of the sulfur-containing small biomolecules have not yet been clearly described. In order to understand overall sulfur transfer processes in plant cells, it is important to elucidate the relationships among various sulfur delivery pathways as well as to investigate their interactions. In this review, we summarize the information from recent studies on the biosynthesis pathways of several sulfur-containing small biomolecules and the proteins participating in these processes. In addition, we show characteristic features of gene expression in Arabidopsis at the early stage of sulfate depletion from the medium, and we provide insights into sulfur transfer processes in plant cells. Full article

Sulfur-containing transfer ribonucleic acids (tRNAs) are ubiquitous biomolecules found in all organisms that possess a variety of functions. For decades, their roles in processes such as translation, structural stability, and cellular protection have been elucidated and appreciated. These thionucleosides are found in all types of bacteria; however, their biosynthetic pathways are distinct among different groups of bacteria. Considering that many of the thio-tRNA biosynthetic enzymes are absent in Gram-positive bacteria, recent studies have addressed how sulfur trafficking is regulated in these prokaryotic species. Interestingly, a novel proposal has been given for interplay among thionucleosides and the biosynthesis of other thiocofactors, through participation of shared-enzyme intermediates, the functions of which are impacted by the availability of substrate as well as metabolic demand of thiocofactors. This review describes the occurrence of thio-modifications in bacterial tRNA and current methods for detection of these modifications that have enabled studies on the biosynthesis and functions of S-containing tRNA across bacteria. It provides insight into potential modes of regulation and potential evolutionary events responsible for divergence in sulfur metabolism among prokaryotes. Full article

Post-translational tRNA modifications have very broad diversity and are present in all domains of life. They are important for proper tRNA functions. In this review, we emphasize the recent advances on the biosynthesis of sulfur-containing tRNA nucleosides including the 2-thiouridine (s²U) derivatives, 4-thiouridine (s⁴U), 2-thiocytidine (s²C), and 2-methylthioadenosine (ms²A). Their biosynthetic pathways have two major types depending on the requirement of iron–sulfur (Fe–S) clusters. In all cases, the first step in bacteria and eukaryotes is to activate the sulfur atom of free l-cysteine by cysteine desulfurases, generating a persulfide (R-S-SH) group. In some archaea, a cysteine desulfurase is missing. The following steps of the bacterial s²U and s⁴U formation are Fe–S cluster independent, and the activated sulfur is transferred by persulfide-carrier proteins. By contrast, the biosynthesis of bacterial s²C and ms²A require Fe–S cluster dependent enzymes. A recent study shows that the archaeal s⁴U synthetase (ThiI) and the eukaryotic cytosolic 2-thiouridine synthetase (Ncs6) are Fe–S enzymes; this expands the role of Fe–S enzymes in tRNA thiolation to the Archaea and Eukarya domains. The detailed reaction mechanisms of Fe–S cluster depend s²U and s⁴U formation await further investigations. Full article

The wobble uridine (U₃₄) of transfer RNAs (tRNAs) for two-box codon recognition, i.e., tRNA^Lys_UUU, tRNA^Glu_UUC, and tRNA^Gln_UUG, harbor a sulfur- (thio-) and a methyl-derivative structure at the second and fifth positions of U₃₄, respectively. Both modifications are necessary to construct the proper anticodon loop structure and to enable them to exert their functions in translation. Thio-modification of U₃₄ (s²U₃₄) is found in both cytosolic tRNAs (cy-tRNAs) and mitochondrial tRNAs (mt-tRNAs). Although l-cysteine desulfurase is required in both cases, subsequent sulfur transfer pathways to cy-tRNAs and mt-tRNAs are different due to their distinct intracellular locations. The s²U₃₄ formation in cy-tRNAs involves a sulfur delivery system required for the biosynthesis of iron-sulfur (Fe/S) clusters and certain resultant Fe/S proteins. This review addresses presumed sulfur delivery pathways for the s²U₃₄ formation in distinct intracellular locations, especially that for cy-tRNAs in comparison with that for mt-tRNAs. Full article

Modifications of transfer RNA (tRNA) have been shown to play critical roles in the biogenesis, metabolism, structural stability and function of RNA molecules, and the specific modifications of nucleobases with sulfur atoms in tRNA are present in pro- and eukaryotes. Here, especially the thiomodifications xm⁵s²U at the wobble position 34 in tRNAs for Lys, Gln and Glu, were suggested to have an important role during the translation process by ensuring accurate deciphering of the genetic code and by stabilization of the tRNA structure. The trafficking and delivery of sulfur nucleosides is a complex process carried out by sulfur relay systems involving numerous proteins, which not only deliver sulfur to the specific tRNAs but also to other sulfur-containing molecules including iron–sulfur clusters, thiamin, biotin, lipoic acid and molybdopterin (MPT). Among the biosynthesis of these sulfur-containing molecules, the biosynthesis of the molybdenum cofactor (Moco) and the synthesis of thio-modified tRNAs in particular show a surprising link by sharing protein components for sulfur mobilization in pro- and eukaryotes. Full article