ABSTRACT
While cytoplasmic tRNA 2-thiolation protein 1 (Tuc1/Ncs6) and ubiquitin-related modifier-1 (Urm1) are important in the 2-thiolation of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at wobble uridines of tRNAs in eukaryotes, the biocatalytic roles and properties of Ncs6/Tuc1 and its homologs are poorly understood. Here we present the first report of an Ncs6 homolog of archaea (NcsA of Haloferax volcanii) that is essential for maintaining cellular pools of thiolated tRNA(Lys)UUU and for growth at high temperature. When purified from Hfx. volcanii, NcsA was found to be modified at Lys204 by isopeptide linkage to polymeric chains of the ubiquitin-fold protein SAMP2. The ubiquitin-activating E1 enzyme homolog of archaea (UbaA) was required for this covalent modification. Non-covalent protein partners that specifically associated with NcsA were also identified including UbaA, SAMP2, proteasome activating nucleotidase (PAN)-A/1, translation elongation factor aEF-1α and a ß-CASP ribonuclease homolog of the archaeal cleavage and polyadenylation specificity factor 1 family (aCPSF1). Together, our study reveals that NcsA is essential for growth at high temperature, required for formation of thiolated tRNA(Lys)UUU and intimately linked to homologs of ubiquitin-proteasome, translation and RNA processing systems.
Subject(s)
Archaeal Proteins , Haloferax volcanii , Proteasome Endopeptidase Complex , Protein Biosynthesis/physiology , RNA Processing, Post-Transcriptional/physiology , RNA, Archaeal , RNA, Transfer , Ubiquitins , Archaeal Proteins/genetics , Archaeal Proteins/metabolism , Haloferax volcanii/genetics , Haloferax volcanii/metabolism , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , RNA, Archaeal/genetics , RNA, Archaeal/metabolism , RNA, Transfer/genetics , RNA, Transfer/metabolism , Ubiquitins/genetics , Ubiquitins/metabolism , Uridine/genetics , Uridine/metabolismABSTRACT
Increasing resistance to every major class of antibiotics and a dearth of novel classes of antibacterial agents in development pipelines has created a dwindling reservoir of treatment options for serious bacterial infections. The bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV, are validated antibacterial drug targets with multiple prospective drug binding sites, including the catalytic site targeted by the fluoroquinolone antibiotics. However, growing resistance to fluoroquinolones, frequently mediated by mutations in the drug-binding site, is increasingly limiting the utility of this antibiotic class, prompting the search for other inhibitor classes that target different sites on the topoisomerase complexes. The highly conserved ATP-binding subunits of DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as excellent candidates for the development of dual-targeting antibacterial agents with broad-spectrum potential. However, to date, no natural product or small molecule inhibitors targeting these sites have succeeded in the clinic, and no inhibitors of these enzymes have yet been reported with broad-spectrum antibacterial activity encompassing the majority of Gram-negative pathogens. Using structure-based drug design (SBDD), we have created a novel dual-targeting pyrimidoindole inhibitor series with exquisite potency against GyrB and ParE enzymes from a broad range of clinically important pathogens. Inhibitors from this series demonstrate potent, broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens of clinical importance, including fluoroquinolone resistant and multidrug resistant strains. Lead compounds have been discovered with clinical potential; they are well tolerated in animals, and efficacious in Gram-negative infection models.
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , DNA Gyrase/metabolism , DNA Topoisomerase IV/antagonists & inhibitors , Drug Design , Topoisomerase II Inhibitors/chemistry , Topoisomerase II Inhibitors/pharmacology , Animals , Anti-Bacterial Agents/chemical synthesis , Bacteria/drug effects , Bacteria/enzymology , DNA Gyrase/chemistry , DNA Topoisomerase IV/chemistry , Drug Resistance, Bacterial/drug effects , Female , Indoles/chemical synthesis , Indoles/chemistry , Indoles/pharmacology , Mice , Microbial Sensitivity Tests , Models, Molecular , Protein Conformation , Topoisomerase II Inhibitors/chemical synthesisABSTRACT
BACKGROUND: The objective of this study was to identify factors motivating women to take part in endometriosis research and to determine if these factors differ for women participating in clinical versus basic science studies. METHODS: A consecutive series of 24 women volunteering for participation in endometriosis-related research were asked to indicate, in their own words, why they chose to volunteer. In addition, the women were asked to rate, on a scale of 0 to 10, sixteen potentially motivating factors. The information was gathered in the form of an anonymous self-administered questionnaire. RESULTS: Strong motivating factors (mean score > 8) included potential benefit to other women's health, improvement to one's own condition, and participation in scientific advancement. Weak motivating factors (mean score < 3) included financial compensation, making one's doctor happy, and use of 'natural' products. No difference was detected between clinical and basic science study participants. CONCLUSION: This study is the first study to specifically investigate the factors that motivate women to take part in endometriosis research. Understanding why women choose to take part in such research is important to the integrity of the informed consent process. The factors most strongly motivating women to participate in endometriosis research related to improving personal or public health; the weakest, to financial compensation and pleasing the doctor.
