Auswahl der wissenschaftlichen Literatur zum Thema „Thiouridylase“

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.

ABSTRACTThe 2-thiouridine (s2U) modification of the wobble position in glutamate, glutamine, and lysine tRNA molecules serves to stabilize the anticodon structure, improving ribosomal binding and overall efficiency of the translational process. Biosynthesis of s2U inEscherichia colirequires a cysteine desulfurase (IscS), a thiouridylase (MnmA), and five intermediate sulfur-relay enzymes (TusABCDE). TheE. coliMnmA adenylates and subsequently thiolates tRNA to form the s2U modification.Bacillus subtilislacks IscS and the intermediate sulfur relay proteins, yet its genome contains a cysteine desulfurase gene,yrvO, directly adjacent tomnmA. The genomic synteny ofyrvOandmnmAcombined with the absence of the Tus proteins indicated a potential functionality of these proteins in s2U formation. Here, we provide evidence that theB. subtilisYrvO and MnmA are sufficient for s2U biosynthesis. A conditionalB. subtilisknockout strain showed that s2U abundance correlates with MnmA expression, andin vivocomplementation studies inE. coliIscS- or MnmA-deficient strains revealed the competency of these proteins in s2U biosynthesis.In vitroexperiments demonstrated s2U formation by YrvO and MnmA, and kinetic analysis established a partnership between theB. subtilisproteins that is contingent upon the presence of ATP. Furthermore, we observed that the slow-growth phenotype ofE. coliΔiscSand ΔmnmAstrains associated with s2U depletion is recovered byB. subtilis yrvOandmnmA. These results support the proposal that the involvement of a devoted cysteine desulfurase, YrvO, in s2U synthesis bypasses the need for a complex biosynthetic pathway by direct sulfur transfer to MnmA.IMPORTANCEThe 2-thiouridine (s2U) modification of the wobble position in glutamate, glutamine, and lysine tRNA is conserved in all three domains of life and stabilizes the anticodon structure, thus guaranteeing fidelity in translation. The biosynthesis of s2U inEscherichia colirequires seven proteins: the cysteine desulfurase IscS, the thiouridylase MnmA, and five intermediate sulfur-relay enzymes (TusABCDE).Bacillus subtilisand most Gram-positive bacteria lack a complete set of biosynthetic components. Interestingly, themnmAcoding sequence is located adjacent toyrvO, encoding a cysteine desulfurase. In this work, we provide evidence that theB. subtilisYrvO is able to transfer sulfur directly to MnmA. Both proteins are sufficient for s2U biosynthesis in a pathway independent of the one used inE. coli.

Мутации в гене TRMU, кодирующем одну из митохондриальных тРНК метилтрансфераз, были обнаружены при инфантильной гепатопатии, связанной с дефектом митохондриальной трансляции (OMIM#613070). Это заболевание является редким заболеванием с угрожающим жизни началом и во многих случаях с последующей спонтанной ремиссией. Своевременная диагностика и лечение таких больных имеют важное значение в клинической практике. В статье приводится описание пациента с печеночной недостаточностью, обусловленной мутациями в гене TRMU, и сравнение клинической картины с литературными данными. Mutations in the TRMU gene encoding the mitochondrial tRNA-specific 2-thiouridylase were found in infantile hepatopathy related to mitochondrial translation defect (OMIM# 613070). This condition is rare mitochondrial disorder with a life-threatening onset and with spontaneous remission, therefore a prompt diagnosis and treatment of these patients has importance in clinical practice. Here we describe a patient, with liver failure due to mutations in TRMU gene and compare with patients from literature.

Abstract Evasion of apoptosis is a hallmark of cancer wherein overexpression and amplification of pro-survival BCL2-family genes like MCL1 is a common observation. MCL1 is frequently amplified in many hematological cancers like Multiple Myeloma (MM) that depend on it for survival. BH3 mimetic drugs, like the BCL2-specific inhibitor Venetoclax, have been successfully used in the clinic to treat certain cancers, and MCL1-selective inhibitors are currently in clinical development. While inhibition of MCL1 displays promising preclinical activity, many cancer models display acquired or intrinsic resistance to MCL1 inhibitors (MCL1i). As MCL1-targeted therapies progress clinically, understanding mechanisms that lead to resistance will be important to not only identify therapeutically-exploitable targets to combat resistance, but to also determine if these biomarkers could stratify patients most likely to respond to an MCL1i. Here, we used a genome-wide CRISPR knock-out screen to identify mechanisms of resistance to MCL1i AZD5991 in two MM cell lines, KMS11 and KMS34. We used a sgRNA library consisting of about 118,000 sgRNAs (~6 sgRNAs/gene), and treated the cells with DMSO or 1uM AZD5991 for 16 days (5 doublings). We identified 316 genes in KMS11 and 184 genes in KMS34 with >4-fold enrichment of sgRNAs; and 221 genes with >2-fold enrichment of sgRNAs in both cell lines. The sgRNAs targeting BAK and BAX were the most enriched overlapping hits. Using GSEA analysis of the 221 common genes with enriched sgRNAs, we discovered that the tRNA wobble uridine modification as the most enriched pathway. The tRNA U34 mcm5s2 modification is catalyzed by the elongator complex ELP1-6 and cytosolic thiouridylase CTU1/2. Each subunit of the elongator complex is essential for its function and loss of any subunit results in destabilization of the complex. By knocking out ELP4 in five MM cell lines (KMS11, KMS34, KMS12-PE, MM.1S, and RPMI-8226), we first validated the destabilization of the complex by showing a robust decrease in the protein levels of ELP1 and ELP3 via western blot. As the elongator complex has additional functions, we also knocked-out tRNA U34 modification pathway specific CTU1 in KMS11, KMS34, and KMS12-PE cells. We showed that genetic knock-out of ELP4 and CTU1 results in increased resistance to MCL1i in all cell lines tested. We observed the highest increase in MCL1i resistance upon ELP4-KO in KMS11 and RPMI-8226 cell lines. To understand the mechanism behind elongator complex mediated regulation of MCL1 dependency, we performed RNAseq and global proteomics in KMS11 cells (Parental, non-targeting control [NTC], ELP4-KO and CTU1-KO) and RPMI-8226 cells (Parental, NTC, and ELP4-KO). We show that the elongator complex is a regulator of IRE1-XBP1 axis of the ER stress response pathway; and knockout of IRE1 also results in MCL1i-resistance in KMS11 and RPMI8226 cell lines. Mechanistically, we show that loss of elongator complex-mediated downregulation of IRE1-XBP1 axis leads to stabilization of MCL1 and upregulation of BCL-XL and NOXA expression. We further show that upon treatment with MCL1i, KMS11-ELP4-KO cells have less disruption of MCL1:Bim complex and an increase in BCL-XL:Bim complex as compared with KMS11-NTC cells. The net increase in pro-survival MCL1 and BCL-XL proteins in ELP4-KO cells resulting in lower levels of unsequestered BIM upon AZD5991 treatment suggests a reduction in apoptotic priming. The mechanistic link between the elongator complex and ER stress response pathway led us to test ER stress inducing drugs in these cell lines. We observed that ELP4-KO results in increased resistance to proteasome inhibitor Bortezomib and other ER stress inducers like Tunicamycin, Thapsigargin, and BrefeldinA as a monotherapy or in combination with AZD5991. These data are consistent with our hypothesis that ELP4-KO cells have reduced apoptotic priming and are thus multi-drug resistant. As bortezomib is used in the clinic to treat MM patients, we asked if an elongator gene signature could be used to predict response to current therapies. We show that the elongator complex components could be used as a gene signature to stratify overall survival in MM patients (MMRF CoMMpass dataset). Moreover, ER stress response gene signature has been shown to be repressed in drug-resistant MM. Taken together, an integrated elongator and IRE-XBP1 gene signature could be a strong predictor of therapy response in MM . Disclosures Aryal: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Sundarrajan: AstraZeneca: Ended employment in the past 24 months. Boiko: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Jenkins: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Liu: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Ahdesmaki: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Bornot: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Jarnuczak: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Miele: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. McDermott: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Fawell: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Drew: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Boise: AbbVie/Genentech: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Research Funding. Cidado: AstraZeneca: Current Employment, Current equity holder in publicly-traded company.

L’atome de soufre est un élément abondant et indispensable à la vie. Il est présent dans une grande diversité de biomolécules contenant du soufre, tels que certains acides aminés essentiels - comme la cystéine et la méthionine, qui sont à la base de diverses voies métaboliques -, des thionucléosides des ARNs de transfert, et de certains cofacteurs indispensables à de nombreux processus biologiques, comme les centres fer-soufre [Fe-S]. Les centres [Fe-S] sont connus pour leur activité rédox et leur rôle dans des réactions de transferts d’électrons. Ils ont des fonctions cellulaires importantes dans la photosynthèse, la respiration et la régulation de la traduction génétique dans des conditions de stress. Ma thèse a consisté en l’étude de deux familles d’enzymes à centres [4Fe-4S] impliquées dans le métabolisme du soufre : plusieurs thiouridylases d'ARN de transfert (MnmA d’E. coli, ThiI de l’archée Methanococcus maripaludis), catalysant l’insertion d’un soufre dans les uridines d’ARNt, et une ThioUracile DéSulfidase (TudS) catalysant l’abstraction du soufre du thiouracile. En combinant diverses méthodes de caractérisation biochimique (tests d’activité in vitro, mutagénèse dirigée) et biophysiques (spectroscopies UV-visible, RPE, Mössbauer, cristallographie aux rayons X), nous avons pu démontrer la nature chimique et le rôle de cofacteur du centre [4Fe-4S] dans les réactions non rédox catalysées par ces métalloenzymes. L’identification d’un intermédiaire réactionnel [4Fe-5S] in crystallo, dans la structure de l’enzyme TudS, a permis de confirmer une nouvelle fonction des centres [4Fe-4S] dans la catalyse de réactions non-redox, précédemment proposée pour les thiouridylases d’ARN de transfert (TtuA) : le centre [4Fe-4S] étant ligandé par trois acides aminés seulement, le quatrième fer non coordonné jouerait le rôle d’acide Lewis en liant et activant l’atome de soufre du substrat (sulfure exogène ou thiouracile, respectivement) pour catalyser la réaction de thiolation (thiouridylases d’ARNt) ou déthiolation (TudS)
The sulfur atom is an abundant and essential element for life. It is present in a wide variety of sulfur-containing biomolecules, such as certain essential amino acids - the cysteine and the methionine, which are at the center of various metabolic pathways -, transfer RNA thionucleosides, and certain essential cofactors participating in many biological processes, such as iron-sulfur centers [Fe-S]. The [Fe-S] centers are known for their redox activity and their role in electron transfer reactions. They have important cellular functions in photosynthesis, respiration, and regulation of gene translation under stress conditions. My thesis consisted of the study of two families of enzymes with [4Fe-4S] centers involved in sulfur metabolism: several transfer RNA thiouridylases (MnmA from E. coli, ThiI from archaea Methanococcus maripaludis), catalyzing sulfur insertion into tRNA uridines, as well as a ThioUracil DeSulfidase (TudS) catalyzing sulfur abstraction from thiouracil. By combining various biochemical (in vitro activity tests, site-directed mutagenesis) and biophysical (UV-visible spectroscopy, EPR, Mössbauer, X-ray crystallography) characterization methods, we were able to demonstrate the chemical nature and the role of the [4Fe-4S] cluster in the non-redox reactions catalyzed by these metalloenzymes. Identifying a reaction intermediate [4Fe-5S] in crystal, in the structure of the enzyme TudS, has confirmed a new function of the [4Fe-4S] clusters in the catalysis of non-redox reactions, previously proposed for transfer RNA thiouridylases (TtuA): the [4Fe-4S] cluster being liganded by only three amino acids, the fourth uncoordinated iron would play the role of Lewis acid by binding and activating the sulfur atom of the substrate (exogenous sulfide or thiouracil, respectively) to catalyze the reaction of thiolation (tRNA thiouridylases) or dethiolation (TudS)