ABSTRACT
BACKGROUND: Bilophila wadsworthia, a strictly anaerobic, sulfite-reducing bacterium and common member of the human gut microbiota, has been associated with diseases such as appendicitis and colitis. It is specialized on organosulfonate respiration for energy conservation, i.e., utilization of dietary and host-derived organosulfonates, such as taurine (2-aminoethansulfonate), as sulfite donors for sulfite respiration, producing hydrogen sulfide (H2S), an important intestinal metabolite that may have beneficial as well as detrimental effects on the colonic environment. Its taurine desulfonation pathway involves the glycyl radical enzyme (GRE) isethionate sulfite-lyase (IslAB), which cleaves isethionate (2-hydroxyethanesulfonate) into acetaldehyde and sulfite. RESULTS: We demonstrate that taurine metabolism in B. wadsworthia 3.1.6 involves bacterial microcompartments (BMCs). First, we confirmed taurine-inducible production of BMCs by proteomic, transcriptomic and ultra-thin sectioning and electron-microscopical analyses. Then, we isolated BMCs from taurine-grown cells by density-gradient ultracentrifugation and analyzed their composition by proteomics as well as by enzyme assays, which suggested that the GRE IslAB and acetaldehyde dehydrogenase are located inside of the BMCs. Finally, we are discussing the recycling of cofactors in the IslAB-BMCs and a potential shuttling of electrons across the BMC shell by a potential iron-sulfur (FeS) cluster-containing shell protein identified by sequence analysis. CONCLUSIONS: We characterized a novel subclass of BMCs and broadened the spectrum of reactions known to take place enclosed in BMCs, which is of biotechnological interest. We also provided more details on the energy metabolism of the opportunistic pathobiont B. wadsworthia and on microbial H2S production in the human gut.
Subject(s)
Bilophila/metabolism , Bilophila/ultrastructure , Isethionic Acid/metabolism , Taurine/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bilophila/genetics , Cell Compartmentation , Gastrointestinal Microbiome , Gene Expression Profiling , Humans , Hydrogen Sulfide/metabolism , Proteomics , Sulfites/metabolismABSTRACT
Introduction: Thyroid hormones (THs) are known to have various effects on the cardiovascular system. However, the impact of TH levels on preexisting cardiac diseases is still unclear. Pressure overload due to arterial hypertension or aortic stenosis and aging are major risk factors for the development of structural and functional abnormalities and subsequent heart failure. Here, we assessed the sensitivity to altered TH levels in aged mice with maladaptive cardiac hypertrophy and cardiac dysfunction induced by transverse aortic constriction (TAC). Methods: Mice at the age of 12 months underwent TAC and received T4 or anti-thyroid medication in drinking water over the course of 4 weeks after induction of left ventricular pressure overload. Results: T4 excess or deprivation in older mice had no or only very little impact on cardiac function (fractional shortening), cardiac remodeling (cardiac wall thickness, heart weight, cardiomyocyte size, apoptosis, and interstitial fibrosis), and mortality. This is surprising because T4 excess or deprivation had significantly changed the outcome after TAC in young 8-week-old mice. Comparing the gene expression of deiodinases (Dio) 2 and 3 and TH receptor alpha (TRα) 1 and the dominant-negative acting isoform TRα2 between young and aged mice revealed that aged mice exhibited a higher expression of TRα2 and Dio3, while expression of Dio2 was reduced compared with young mice. These changes in Dio2 and 3 expressions might lead to reduced TH availability in the hearts of 12-month-old mice accompanied by reduced TRα action due to higher TRα2. Discussion: In summary, our study shows that low and high TH availability have little impact on cardiac function and remodeling in older mice with preexisting pressure-induced cardiac damage. This observation seems to be the result of an altered expression of deiodinases and TRα isoforms, thus suggesting that even though cardiovascular risk is increasing with age, the response to TH stress may be dampened in certain conditions.
Subject(s)
Heart Failure , Hypertension , Mice , Animals , Cardiomegaly/etiology , Cardiomegaly/metabolism , Heart Failure/etiology , Myocytes, Cardiac/metabolism , Thyroid Hormones/metabolism , Hypertension/complicationsABSTRACT
Background: Stimulation of ventricular hypertrophy and heart rate are two major cardiac effects of thyroid hormone (TH). The aim of this study was to determine in vivo which TH receptor (TR)-α or ß-and which mode of TR action-canonical gene expression or DNA-binding independent noncanonical action-mediate these effects. Methods: We compared global TRα and TRß knockout mice (TRαKO; TRßKO) with wild-type (WT) mice to determine the TR isoform responsible for T3 effects. The relevance of TR DNA binding was studied in mice with a mutation in the DNA-binding domain that selectively abrogates DNA binding and canonical TR action (TRαGS; TRßGS). Hearts were studied with echocardiography at baseline and after 7 weeks of T3 treatment. Gene expression was measured with real-time polymerase chain reaction. Heart rate was recorded with radiotelemetry transmitters for 7 weeks in untreated, hypothyroid, and T3-treated mice. Results: T3 induced ventricular hypertrophy in WT and TRßKO mice, but not in TRαKO mice. Hypertrophy was also induced in TRαGS mice. Thus, hypertrophy is mostly mediated by noncanonical TRα action. Similarly, repression of Mhy7 occurred in WT and TRαGS mice. Basal heart rate was largely dependent on canonical TRα action. But responsiveness to hypothyroidism and T3 treatment as well as expression of pacemaker gene Hcn2 were still preserved in TRαKO mice, demonstrating that TRß could compensate for absence of TRα. Conclusions: T3-induced cardiac hypertrophy could be attributed to noncanonical TRα action, whereas heart rate regulation was mediated by canonical TRα action. TRß could substitute for canonical but not noncanonical TRα action.
Subject(s)
Cardiomegaly , Heart Rate , Mice, Knockout , Thyroid Hormone Receptors alpha , Thyroid Hormone Receptors beta , Triiodothyronine , Animals , Male , Mice , Cardiomegaly/metabolism , Cardiomegaly/genetics , Hypothyroidism/metabolism , Hypothyroidism/genetics , Protein Isoforms/metabolism , Thyroid Hormone Receptors alpha/genetics , Thyroid Hormone Receptors alpha/metabolism , Thyroid Hormone Receptors beta/genetics , Thyroid Hormone Receptors beta/metabolismABSTRACT
In tRNAAsp, tRNAAsn, tRNATyr, and tRNAHis of most bacteria and eukaryotes, the anticodon wobble position may be occupied by the modified nucleoside queuosine, which affects the speed and the accuracy of translation. Since eukaryotes are not able to synthesize queuosine de novo, they have to salvage queuine (the queuosine base) as a micronutrient from food and/or the gut microbiome. The heterodimeric Zn2+ containing enzyme tRNA-guanine transglycosylase (TGT) catalyzes the insertion of queuine into the above-named tRNAs in exchange for the genetically encoded guanine. This enzyme has attracted medical interest since it was shown to be potentially useful for the treatment of multiple sclerosis. In addition, TGT inactivation via gene knockout leads to the suppressed cell proliferation and migration of certain breast cancer cells, which may render this enzyme a potential target for the design of compounds supporting breast cancer therapy. As a prerequisite to fully exploit the medical potential of eukaryotic TGT, we have determined and analyzed a number of crystal structures of the functional murine TGT with and without bound queuine. In addition, we have investigated the importance of two residues of its non-catalytic subunit on dimer stability and determined the Michaelis-Menten parameters of murine TGT with respect to tRNA and several natural and artificial nucleobase substrates. Ultimately, on the basis of available TGT crystal structures, we provide an entirely conclusive reaction mechanism for this enzyme, which in detail explains why the TGT-catalyzed insertion of some nucleobases into tRNA occurs reversibly while that of others is irreversible.
Subject(s)
Pentosyltransferases/chemistry , Animals , Eukaryotic Cells/metabolism , Female , Guanine/metabolism , Humans , Mice , Nucleoside Q , RNA, Transfer/chemistryABSTRACT
Peroxisome proliferator-activated receptor ß/δ (PPARß/δ) is a ligand-regulated nuclear receptor with essential functions in metabolism and inflammation. We have synthesized a new derivative [methyl 3-(N-(4-(hexylamino)-2-methoxyphenyl)sulfamoyl)thiophene-2-carboxylate (ST247) structurally related to the published PPARß/δ inhibitory ligand methyl 3-(N-(2-methoxy-4-(phenylamino)phenyl)sulfamoyl)thiophene-2-carboxylate (GSK0660). ST247 has a higher affinity to PPARß/δ than GSK0660, and at equimolar concentrations, it more efficiently 1) induces the interaction with corepressors both in vitro and in vivo, 2) inhibits the agonist-induced transcriptional activity of PPARß/δ, and 3) down-regulates basal level expression of the peroxisome proliferator responsive element-driven PPARß/δ target gene ANGPTL4. Methyl 3-(N-(4-(tert-butylamino)-2-methoxyphenyl)sulfamoyl)thiophene-2-carboxylate (PT-S58), another high-affinity derivative from our series, also efficiently inhibits agonist-induced transcriptional activation, but in contrast to ST247, it does not enhance the interaction of PPARß/δ with corepressors. PT-S58 rather prevents corepressor recruitment triggered by the inverse agonist ST247. These findings classify ST247 as an inverse agonist, whereas PT-S58 is the first pure PPARß/δ antagonist described to date. It is noteworthy that ST247 and PT-S58 are also effective on PPRE-independent functions of PPARß/δ: in monocytic cells, both ligands modulate expression of the activation marker CCL2 in the opposite direction as an established PPARß/δ agonist. The possibility to differentially modulate specific functions of PPARß/δ makes these novel compounds invaluable tools to advance our understanding of PPARß/δ biology.
Subject(s)
PPAR delta/metabolism , PPAR-beta/metabolism , Sulfonamides/metabolism , Thiophenes/metabolism , Animals , Base Sequence , Binding Sites , Cell Line , Chromatin Immunoprecipitation , DNA Primers , Fluorescence Resonance Energy Transfer , Humans , Ligands , Mice , PPAR delta/antagonists & inhibitors , PPAR-beta/antagonists & inhibitors , Real-Time Polymerase Chain ReactionABSTRACT
Bacterial tRNA-guanine transglycosylase (Tgt) is involved in the biosynthesis of the modified tRNA nucleoside queuosine present in the anticodon wobble position of tRNAs specific for aspartate, asparagine, histidine, and tyrosine. Inactivation of the tgt gene leads to decreased pathogenicity of Shigella bacteria. Therefore, Tgt constitutes a putative target for Shigellosis drug therapy. Since it is only active as homodimer, interference with dimer-interface formation may, in addition to active-site inhibition, provide further means to disable this protein. A cluster of four aromatic residues seems important to stabilize the homodimer. We mutated residues of this aromatic cluster and analyzed each mutated variant with respect to the dimer and thermal stability or enzyme activity by applying native mass spectrometry, a thermal shift assay, enzyme kinetics, and X-ray crystallography. Our structural studies indicate a strong influence of pH on the homodimer stability. Apparently, protonation of a histidine within the aromatic cluster supports the collapse of an essential structural motif within the dimer interface at slightly acidic pH.
Subject(s)
Pentosyltransferases/chemistry , Zymomonas/enzymology , Catalytic Domain , Crystallography, X-Ray , Enzyme Stability , Models, Molecular , Mutation , Pentosyltransferases/genetics , Protein Conformation , Protein Multimerization , Zymomonas/chemistry , Zymomonas/geneticsABSTRACT
GSK0660 (1) is the first peroxisome proliferator-activated receptor (PPAR) ß/δ-selective inhibitory ligand described in the literature. Based on its structure, we designed and synthesized a series of modified compounds to establish preliminary structure-activity relationships. Most beneficial for increased binding affinity towards the PPARß/δ ligand binding domain was the replacement of the 4'-aminophenyl substituent by medium-length n-alkyl chains, such as n-butyl or iso-pentyl. These compounds show activity down to the one-digit nanomolar range, thus possessing up to a tenfold higher binding affinity compared with GSK0660. Additionally, the subtype-specific inhibition of PPARß/δ was confirmed in a cell-based assay making these compounds invaluable tools for the further exploration of the functions of PPARß/δ.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , PPAR delta/antagonists & inhibitors , PPAR-beta/antagonists & inhibitors , Sulfones/chemistry , Sulfones/pharmacology , Thiophenes/chemistry , Thiophenes/pharmacology , Animals , Cell Line , Humans , PPAR delta/metabolism , PPAR-beta/metabolism , Structure-Activity RelationshipABSTRACT
Structure-based drug design is an integral part of industrial and academic drug discovery projects. Initial lead structures are, in general, optimized in terms of affinity using iterative cycles comprising synthesis, biological evaluation, computational methods, and structural analysis. X-ray crystallography commonly suggests the existence of a single well-defined state, termed binding mode, which is generally assumed to be consistent in a series of similar ligands and therefore used for the following optimization process. During the further development of symmetrically disubstituted 3,4-amino-pyrrolidines as human immunodeficiency virus type 1 protease inhibitors, we discovered that, by modification of the P1/P1' moieties of our lead structure, the activity of the inhibitors towards the active-site mutation Ile84Val was altered, however, not being explainable with the initial underlying structure-activity relationship. The cocrystallization of the most potent derivative in complex with the human immunodeficiency virus type 1 protease surprisingly led to two different crystal forms (P2(1)2(1)2(1) and P6(1)22). Structural analysis revealed two completely different binding modes; the interaction of the pyrrolidine nitrogen atom with the catalytic aspartates remains as the only similarity. The study presented clearly demonstrates that structural biology has to escort the entire lead optimization process not to fail by an initially observed binding orientation.
Subject(s)
HIV Protease Inhibitors/metabolism , HIV Protease Inhibitors/pharmacology , HIV Protease/metabolism , HIV-1/enzymology , Pyrrolidines/metabolism , Pyrrolidines/pharmacology , Crystallography, X-Ray , HIV Protease/chemistry , HIV Protease Inhibitors/chemistry , HIV-1/drug effects , Humans , Hydrogen Bonding/drug effects , Kinetics , Models, Molecular , Protein Binding/drug effects , Static ElectricityABSTRACT
HIV protease is a well-established drug target in antiviral chemotherapy. Immense research efforts have been made to discover effective inhibitors, thus making the enzyme one of the most studied and best characterized proteins. Although the protease exhibits high flexibility, all approved drugs target virtually the same protein conformation. The development of viral cross-resistance demands the generation of inhibitors with novel scaffolds and deviating modes of binding. Herein we report the design and the short, high-yielding stereoselective synthesis of a series of chiral, symmetric pyrrolidine-based inhibitors targeting the open-flap conformation of the protease. The obtained co-crystal structure with one derivative provides a valuable starting point for further inhibitor design.