Molecular basis for the bifunctional Uba4-Urm1 sulfur-relay system in tRNA thiolation and ubiquitin-like conjugation.

The chemical modification of tRNA bases by sulfur is crucial to tune translation and to optimize protein synthesis. In eukaryotes, the ubiquitin-related modifier 1 (Urm1) pathway is responsible for the synthesis of 2-thiolated wobble uridine (U34 ). During the key step of the modification cascade, the E1-like activating enzyme ubiquitin-like protein activator 4 (Uba4) first adenylates and thiocarboxylates the C-terminus of its substrate Urm1. Subsequently, activated thiocarboxylated Urm1 (Urm1-COSH) can serve as a sulfur donor for specific tRNA thiolases or participate in ubiquitin-like conjugation reactions. Structural and mechanistic details of Uba4 and Urm1 have remained elusive but are key to understand the evolutionary branch point between ubiquitin-like proteins (UBL) and sulfur-relay systems. Here, we report the crystal structures of full-length Uba4 and its heterodimeric complex with its substrate Urm1. We show how the two domains of Uba4 orchestrate recognition, binding, and thiocarboxylation of the C-terminus of Urm1. Finally, we uncover how the catalytic domains of Uba4 communicate efficiently during the reaction cycle and identify a mechanism that enables Uba4 to protect itself against self-conjugation with its own product, namely activated Urm1-COSH.

Fabrication and characterization of bovine serum albumin-phycocyanobilin conjugate: effect on antioxidant and ligand-binding properties.

BACKGROUND: Phycocyanobilin (PCB) is an open-chain blue tetrapyrrole chromophore of C-phycocyanin (C-PC), a major chromoprotein derived from the cyanobacterium Arthrospira platensis having numerous health-promoting effects. Relying on the ability of PCB to attach to the sulfhydryl group of proteins, we propose a new method for covalent attachment of PCB to bovine serum albumin (BSA) as a means of its functionalization. RESULTS: Traut's reagent (TR, 2-iminothiolane), modifying lysine residues, was used to optimize the introduction of sulfhydryl groups in BSA. A higher degree of BSA thiolation by TR induces more profound alterations of its structure, resulting in minor oligomerization and aggregation. A 50-fold molar excess of TR was found to be the optimal, balancing thiolation level and adverse effect on protein structure. PCB was covalently attached to newly introduced sulfhydryl groups at pH 9 at 20-fold PCB/BSA ratio. An increase in the TR/BSA molar ratio leads to increased efficiency of PCB conjugation with thiolated BSA. Compared to native BSA, BSA-PCB conjugate binds quercetin with similar affinity but has higher antioxidant activity and increased oxidative stability. CONCLUSIONS: PCB-modified BSA could serve as a stable, food-compatible carrier of bioactive PCB, but also bind other ligands that would be protected from oxidative damage due to the high antioxidant potential of covalently bound PCB. Thiolation by TR is, at the same time, a simple method for the covalent functionalization of virtually any protein by bioactive PCB or for obtaining PCB-based fluorescent probes. © 2024 Society of Chemical Industry.

Thiolation for Enhancing Photostability of Fluorophores at the Single-Molecule Level.

Fluorescent probes are essential for single-molecule imaging. However, their application in biological systems is often limited by the short photobleaching lifetime. To overcome this, we developed a novel thiolation strategy for squaraine dyes. By introducing thiolation of the central cyclobutene of squaraine (thio-squaraine), we observed a ≈5-fold increase in photobleaching lifetime. Our single-molecule data analysis attributes this improvement to improved photostability resulting from thiolation. Interestingly, bulk measurements show rapid oxidation of thio-squaraine to its oxo-analogue under irradiation, giving the perception of inferior photostability. This discrepancy between bulk and single-molecule environments can be ascribed to the factors in the latter, including larger intermolecular distances and restricted mobility, which reduce the interactions between a fluorophore and reactive oxygen species produced by other fluorophores, ultimately impacting photobleaching and photoconversion rate. We demonstrate the remarkable performance of thio-squaraine probes in various imaging buffers, such as glucose oxidase with catalase (GLOX) and GLOX+trolox. We successfully employed these photostable probes for single-molecule tracking of CD56 membrane protein and monitoring mitochondria movements in live neurons. CD56 tracking revealed distinct motion states and the corresponding protein fractions. This investigation is expected to propel the development of single-molecule imaging probes, particularly in scenarios where bulk measurements show suboptimal performance.

Ionic Liquid-Mediated Approach for the Synthesis of Site-Specific Thioether Conjugates.

Site-specific conjugation approaches are of great importance in drug discovery, notably for the synthesis of biochemical probes or molecular conjugates for targeted delivery. Herein, we report a mild ionic liquid (IL)-mediated thiolation technique that relies on the use of 1,3-ethyl-methyl imidazolium acetate, [C2 mim][OAc] as a solvent and precursor to generate activated IL, as well as a solvent for the conjugation reaction. First, a focused library of active ILs was prepared for functionalizing/conjugating cysteine-containing small molecules and unprotected peptides. Interestingly, a bifunctional active IL could also be successfully employed as a linker for the conjugation of peptides lacking Cys. This study sets the ground for further investigation of the use of active ILs for modifying, labeling or conjugating larger and more complex therapeutic modalities such as proteins and antibodies.

Effective Formation of New C(sp2 )-S Bonds via Photoactivation of Alkylamine-based Electron Donor-Acceptor Complexes.

A novel visible light promoted formation of CAryl- S bonds through electron donor-acceptor (EDA) complexes of alkylamines with 5- and 6-membered (hetero)arene halides is presented. This represents the first EDA-based thiolation method not relying on π-π or a thiolate-anion-π interactions and provides a facile access to heteroarene radicals, which can be suitably trapped by disulfide derivatives to form the corresponding versatile arylsulfides. Mechanistic investigations on the aspects of the whole process were conducted by spectroscopic measurements, demonstrating the hypothesized EDA complex formation. Moreover, the strength of this method has been proven by a gram-scale synthesis of thiolated products and the late-stage derivatization of an anticoagulant drug.

Methanogens-Driven Arsenic Methylation Preceding Formation of Methylated Thioarsenates in Sulfide-Rich Hot Springs.

Hot springs represent a major source of arsenic release into the environment. Speciation is typically reported to be dominated by arsenite, arsenate, and inorganic thiolated arsenates. Much less is known about the relevance and formation of methylated thioarsenates, a group with species of high mobility and toxicity. In hot spring samples taken from the Tengchong volcanic region in China, methylated thioarsenates contributed up to 13% to total arsenic. Enrichment cultures were obtained from the corresponding sediment samples and incubated to assess their capability to convert arsenite into methylated thioarsenates over time and in the presence of different microbial inhibitors. In contrast to observations in other environmental systems (e.g., paddy soils), there was no solid evidence, supporting that the sulfate-reducing bacteria contributed to the arsenic methylation. Methanosarcina, the sole genus of methanogens detected in the enrichment cultures, as well as Methanosarcina thermophila TM-1, a pure strain within the genus, did methylate arsenic. We propose that methylated thioarsenates in a typical sulfide-rich hot spring environment like Tengchong form via a combination of biotic arsenic methylation driven by thermophilic methanogens and arsenic thiolation with either geogenic sulfide or sulfide produced by sulfate-reducing bacteria.

Quick and Spontaneous Transformation between [3Fe-4S] and [4Fe-4S] Iron-Sulfur Clusters in the tRNA-Thiolation Enzyme TtuA.

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.

DBDMH-Promoted Methylthiolation in DMSO: A Metal-Free Protocol to Methyl Sulfur Compounds with Multifunctional Groups.

Organic thioethers play an important role in the discovery of drugs and natural products. However, the green synthesis of organic sulfide compounds remains a challenging task. The convenient and efficient synthesis of 5-alkoxy-3-halo-4-methylthio-2(5H)-furanones from DMSO is performed via the mediation of 1,3-dibromo-5,5-dimethylhydantoin (DBDMH), affording a facile route for the sulfur-functionalization of 3,4-dihalo-2(5H)-furanones under transition metal-free conditions. This new approach has demonstrated the functionalization of non-aromatic Csp2-X-type halides with unique structures containing C-X, C-O, C=O and C=C bonds. Compared with traditional synthesis methods using transition metal catalysts with ligands, this reaction has many advantages, such as the lower temperature, the shorter reaction time, the wide substrate range and good functional group tolerance. Notably, DMSO plays multiple roles, and is simultaneously used as an odorless methylthiolating reagent and safe solvent.

Manganese-Catalyzed Redox-Neutral Thiolation of Alkyl Halides with Thioformates.

Transition metal-catalyzed C-S cross-coupling has emerged as an important strategy to furnish thioethers; however, the dominant utilization of noble metal catalysts as well as the construction of challenging C(sp3 )-S bonds by transition metal-catalysis remain highly problematic. Earth-abundant manganese has gathered increasing interest as an attractive catalyst for new reaction development; nevertheless, C(sp3 )-S cross-coupling reaction by manganese catalysis has not been reported. Herein, we disclose a highly efficient manganese-catalyzed redox-neutral thiolation of a broad range of alkyl halides with thioformates as practical sulfuration agents. Strategically, employing easily synthesized thioformates as thiyl radical precursors allows access to various aryl and alkyl thioethers in good to excellent yields. Notably, this redox-neutral method avoids the utilization of strong bases, external ligands, forcing reaction conditions, and stoichiometric manganese, thus presenting apparent advantages, such as broad substrate scope, excellent functional group compatibility, and mild reaction conditions. Finally, the utilities of this method are also illustrated by downstream transformations and late-stage thiolation of structurally complex natural products and pharmaceuticals.

Widespread Occurrence of the Highly Toxic Dimethylated Monothioarsenate (DMMTA) in Rice Globally.

Arsenic (As) accumulation in rice is of global concern for human health and international trade. Rice is typically reported to contain inorganic As (iAs) and dimethylated arsenate (DMA), with current food guidelines limiting toxic iAs but not less-toxic DMA. Here, we show that the highly toxic dimethylated monothioarsenate (DMMTA) is also found in rice worldwide and has been unknowingly determined as less-toxic DMA by previous routine analytical methods. Using enzymatic extraction followed by high-performance liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS) analysis with a C18 column, DMMTA was detected in rice grains (n = 103) from a field survey from China and in polished rice grains (n = 140) from a global market-basket survey. Concentration ranged from <0.20 to 34.8 µg/kg (median 10.3 µg/kg), accounting for 0 to 21% of total As. A strong linear correlation was observed in all rice samples between DMA and DMMTA (being 30 ± 8% of DMA) concentrations. This robust relationship allows an estimation of DMMTA in rice grains from the DMA data reported in previous market-basket surveys, showing a general global geographical pattern with DMMTA concentration increasing from the equator toward high-latitude regions. Based on the global occurrence and potential high toxicity, DMMTA in rice should be considered in health risk assessments and for setting food regulations.

New Mobilization Pathway of Antimonite: Thiolation and Oxidation by Dissimilatory Metal-Reducing Bacteria via Elemental Sulfur Respiration.

Antimony (Sb) mobilization is widely explored with dissimilatory metal-reducing bacteria (DMRB) via microbial iron(III)-reduction. Here, our study found a previously unknown pathway whereby DMRB release adsorbed antimonite (SbIII-O) from goethite via elemental sulfur (S0) respiratory reduction under mild alkaline conditions. We incubated SbIII-O-loaded goethite with Shewanella oneidensis MR-1 in the presence of S0 at pH 8.5. The incubation results showed that MR-1 reduced S0 instead of goethite, and biogenic sulfide induced the formation of thioantimonite (SbIII-S). SbIII-S was then oxidized by S0 to mobile thioantimonate (SbV-S), resulting in over fourfold greater Sb release to water compared with the abiotic control. SbIV-S was identified as the intermediate during the oxidation process by Fourier transform ion cyclotron resonance mass spectrometry and electron spin resonance analysis. The existence of SbIV-S reveals that the oxidation of SbIII-S to SbV-S follows a two-step consecutive one-electron transfer from Sb to S atoms. SbV-S then links with SbIII-S by sharing S atoms and inhibits SbIII-S polymerization and SbIII2S3 precipitation like a "capping agent". This study clarifies the thiolation and oxidation pathway of SbIII-O to SbV-S by S0 respiration and expands the role of DMRB in the fate of Sb.

Mechanistic basis for impaired ferroptosis in cells expressing the African-centric S47 variant of p53.

A population-restricted single-nucleotide coding region polymorphism (SNP) at codon 47 exists in the human TP53 gene (P47S, hereafter P47 and S47). In studies aimed at identifying functional differences between these variants, we found that the African-specific S47 variant associates with an impaired response to agents that induce the oxidative stress-dependent, nonapoptotic cell death process of ferroptosis. This phenotype is manifested as a greater resistance to glutamate-induced cytotoxicity in cultured cells as well as increased carbon tetrachloride-mediated liver damage in a mouse model. The differential ferroptotic responses associate with intracellular antioxidant differences between P47 and S47 cells, including elevated abundance of the low molecular weight thiols coenzyme A (CoA) and glutathione in S47 cells. Importantly, the disparate ferroptosis phenotypes related to the P47S polymorphism are reversible. Exogenous administration of CoA provides protection against ferroptosis in cultured mouse and human cells, as well as in a mouse model. The combined data support a positive role for p53 in ferroptosis and identify CoA as a regulator of this cell death process. Together, these findings provide mechanistic insight linking redox regulation of p53 to small molecule antioxidants and stress signaling pathways. They also identify potential therapeutic approaches to redox-related pathologies.

Thiolated Hydroxypropyl-ß-cyclodextrin: A Potential Multifunctional Excipient for Ocular Drug Delivery.

The goal of this study was the design and evaluation of a thiolated cyclodextrin providing high drug solubilizing and mucoadhesive properties for ocular drug delivery. Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) was thiolated via a microwave-assisted method, resulting in a degree of thiolation of 33%. Mucoadhesive properties of thiolated HP-ß-CD (HP-ß-CD-SH) were determined via rheological measurements and ex vivo studies on isolated porcine cornea. Due to thiolation of HP-ß-CD, a 2-fold increase of mucus viscosity and a 1.4-fold increase in residence time on isolated corneal tissue were achieved. After instillation, the mean precorneal residence time and AUC of dexamethasone (DMS) eye drops were 4-fold and 11.7-fold enhanced by HP-ß-CD-SH, respectively. Furthermore, in the presence of HP-ß-CD-SH, a constant high level of DMS in aqueous humour between 30 and 150 min after administration was observed. These results suggest that HP-ß-CD-SH is an excellent excipient for ocular formulations of poorly soluble drugs in order to prolong their ocular residence time and bioavailability.

Blood Thiol Redox State in Chronic Kidney Disease.

Thiols (sulfhydryl groups) are effective antioxidants that can preserve the correct structure of proteins, and can protect cells and tissues from damage induced by oxidative stress. Abnormal levels of thiols have been measured in the blood of patients with moderate-to-severe chronic kidney disease (CKD) compared to healthy subjects, as well as in end-stage renal disease (ESRD) patients on haemodialysis or peritoneal dialysis. The levels of protein thiols (a measure of the endogenous antioxidant capacity inversely related to protein oxidation) and S-thiolated proteins (mixed disulphides of protein thiols and low molecular mass thiols), and the protein thiolation index (the molar ratio of the S-thiolated proteins to free protein thiols in plasma) have been investigated in the plasma or red blood cells of CKD and ESRD patients as possible biomarkers of oxidative stress. This type of minimally invasive analysis provides valuable information on the redox status of the less-easily accessible tissues and organs, and of the whole organism. This review provides an overview of reversible modifications in protein thiols in the setting of CKD and renal replacement therapy. The evidence suggests that protein thiols, S-thiolated proteins, and the protein thiolation index are promising biomarkers of reversible oxidative stress that could be included in the routine monitoring of CKD and ESRD patients.

Electroreductive Nickel-Catalyzed Thiolation: Efficient Cross-Electrophile Coupling for C-S Formation.

Sulfur-containing molecules are of utmost topical importance towards the effective development of pharmaceuticals and functional materials. Herein, we present an efficient and mild electrochemical thiolation by cross-electrophile coupling of alkyl bromides with functionalized bench-stable thiosulfonates to access alkyl sulfides with excellent efficacy and broad functional group tolerance. Cyclic voltammetry and potentiostatic analysis were performed to elucidate mechanistic insights into this electrocatalytic thiolation reaction.

Protein thiolation index in microvolumes of plasma.

Protein Thiolation Index (PTI) has been recently proposed as a new biomarker of oxidative stress. It is calculated by measuring both free thiols and S-thiolated proteins in plasma with the assumption that this redox ratio is altered by a pro-oxidant stimulus. Here the original protocol was modified and adapted to the use of microvolumes of blood collected by finger prick and down to 3 µl blood was shown to be the lowest volume suitable for this kind of analysis. The new procedure was used to evaluate both the circadian rhythm and the annual fluctuations of PTI in healthy humans.

Dynamics of Dimethylated Monothioarsenate (DMMTA) in Paddy Soils and Its Accumulation in Rice Grains.

Arsenic species transformation in paddy soils has important implications for arsenic accumulation in rice grains and its safety to the consumers. Methylated thioarsenates including highly toxic dimethylated monothioarsenate (DMMTA) have been detected in paddy soils, but their production and dynamics remain poorly understood. In the present study, we first optimized a HPLC-ICP-MS method to quantify methylated thioarsenate species. Using this method together with 10 mM diethylenetriamine pentaacetate (DTPA) to preserve As speciation, we investigated methylated thioarsenate species in porewaters of seven As-contaminated soils incubated under flooded conditions and of two paddy fields. DMMTA was the main methylated thioarsenate species in the porewaters in both incubated soils and paddy fields, with concentrations ranging from 0.2 to 36.2 µg/L and representing ca. 58% of its precursor dimethylarsenate (DMA). The temporal production and dynamics of DMMTA were linked with the DMA concentrations. When soils were drained, DMMTA was converted to DMA. In the two paddy fields, DMMTA concentrations in rice grains were 0.4-10.1 µg/kg. Addition of sulfur fertilizer and rice straw incorporation increased grain DMMTA by 9-28%. These results suggest that DMMTA is an important As species in paddy soils and can accumulate in rice grains, presenting a risk to food safety and human health.

Cooperation between a T†Domain and a Minimal C-Terminal Docking Domain to Enable Specific Assembly in a Multiprotein NRPS.

Non-ribosomal peptide synthetases (NRPS) produce natural products from amino acid building blocks. They often consist of multiple polypeptide chains which assemble in a specific linear order via specialized N- and C-terminal docking domains (N/C DDs). Typically, docking domains function independently from other domains in NRPS assembly. Thus, docking domain replacements enable the assembly of "designer" NRPS from proteins that normally do not interact. The multiprotein "peptide-antimicrobial-Xenorhabdus" (PAX) peptide-producing PaxS NRPS is assembled from the three proteins PaxA, PaxB and PaxC. Herein, we show that the small C DD of PaxA cooperates with its preceding thiolation (T1 ) domain to bind the N DD of PaxB with very high affinity, establishing a structural and thermodynamical basis for this unprecedented docking interaction, and we test its functional importance in vivo in a truncated PaxS assembly line. Similar docking interactions are apparently present in other NRPS systems.

Direct Allylic C(sp3 )-H and Vinylic C(sp2 )-H Thiolation with Hydrogen Evolution by Quantum Dots and Visible Light.

Direct allylic C-H thiolation is straightforward for allylic C(sp3 )-S bond formation. However, strong interactions between thiol and transition metal catalysts lead to deactivation of the catalytic cycle or oxidation of sulfur atom under oxidative condition. Thus, direct allylic C(sp3 )-H thiolation has proved difficult. Represented herein is an exceptional for direct, efficient, atom- and step-economic thiolation of allylic C(sp3 )-H and thiol S-H under visible light irradiation. Radical trapping experiments and electron paramagnetic resonance (EPR) spectroscopy identified the allylic radical and thiyl radical generated on the surface of photocatalyst quantum dots (QDs). The C-S bond formation does not require external oxidants and radical initiators, and hydrogen (H2 ) is produced as byproduct. When vinylic C(sp2 )-H was used instead of allylic C(sp3 )-H bond, the radical-radical cross-coupling of C(sp2 )-H and S-H was achieved with liberation of H2 . Such a unique transformation opens up a door toward direct C-H and S-H coupling for valuable organosulfur chemistry.

Mitochondria export iron-sulfur and sulfur intermediates to the cytoplasm for iron-sulfur cluster assembly and tRNA thiolation in yeast.

Iron-sulfur clusters are essential cofactors of proteins. In eukaryotes, iron-sulfur cluster biogenesis requires a mitochondrial iron-sulfur cluster machinery (ISC) and a cytoplasmic iron-sulfur protein assembly machinery (CIA). Here we used mitochondria and cytoplasm isolated from yeast cells, and [35S]cysteine to detect cytoplasmic Fe-35S cluster assembly on a purified apoprotein substrate. We showed that mitochondria generate an intermediate, called (Fe-S)int, needed for cytoplasmic iron-sulfur cluster assembly. The mitochondrial biosynthesis of (Fe-S)int required ISC components such as Nfs1 cysteine desulfurase, Isu1/2 scaffold, and Ssq1 chaperone. Mitochondria then exported (Fe-S)int via the Atm1 transporter in the inner membrane, and we detected (Fe-S)int in active form. When (Fe-S)int was added to cytoplasm, CIA utilized it for iron-sulfur cluster assembly without any further help from the mitochondria. We found that both iron and sulfur for cytoplasmic iron-sulfur cluster assembly originate from the mitochondria, revealing a surprising and novel mitochondrial role. Mitochondrial (Fe-S)int export was most efficient in the presence of cytoplasm containing an apoprotein substrate, suggesting that mitochondria respond to the cytoplasmic demand for iron-sulfur cluster synthesis. Of note, the (Fe-S)int is distinct from the sulfur intermediate called Sint, which is also made and exported by mitochondria but is instead used for cytoplasmic tRNA thiolation. In summary, our findings establish a direct and vital role of mitochondria in cytoplasmic iron-sulfur cluster assembly in yeast cells.