Epiberberine: a potential rumen microbial urease inhibitor to reduce ammonia release screened by targeting UreG.

Rumen microbial urease inhibitors have been proposed for regulating nitrogen emission and improving nitrogen utilization efficiency in ruminant livestock industry. However, studies on plant-derived natural inhibitors of rumen microbial urease are limited. Urease accessory protein UreG, plays a crucial role in facilitating urease maturation, is a new target for design of urease inhibitor. The objective of this study was to select the potential effective inhibitor of rumen microbial urease from major protoberberine alkaloids in Rhizoma Coptidis by targeting UreG. Our results showed that berberine chloride and epiberberine exerted superior inhibition potential than other alkaloids based on GTPase activity study of UreG. Berberine chloride inhibition of UreG was mixed type, while inhibition kinetics type of epiberberine was uncompetitive. Furthermore, epiberberine was found to be more effective than berberine chloride in inhibiting the combination of nickel towards UreG and inducing changes in the second structure of UreG. Molecular modeling provided the rational structural basis for the higher inhibition potential of epiberberine, amino acid residues in G1 motif and G3 motif of UreG formed interactions with D ring of berberine chloride, while interacted with A ring and D ring of epiberberine. We further demonstrated the efficacy of epiberberine in the ruminal microbial fermentation with low ammonia release and urea degradation. In conclusion, our study clearly indicates that epiberberine is a promising candidate as a safe and effective inhibitor of rumen microbial urease and provides an optimal strategy and suitable feed additive for regulating nitrogen excretion in ruminants in the future. KEY POINTS: • Epiberberine is the most effective inhibitor of rumen urease from Rhizoma Coptidis. • Urease accessory protein UreG is an effective target for design of urease inhibitor. • Epiberberine may be used as natural feed additive to reducing NH3 release in ruminants.

Chelerythrine Chloride: A Potential Rumen Microbial Urease Inhibitor Screened by Targeting UreG.

Inhibition of ruminal microbial urease is of particular interest due to its crucial role in regulating urea-N utilization efficiency and nitrogen pollution in the livestock industry. Acetohydroxamic acid (AHA) is currently the only commercially available urease inhibitor, but it has adverse side effects. The urease accessory protein UreG, which facilitates the functional incorporation of the urease nickel metallocentre, has been proposed in developing urease inhibitor through disrupting urease maturation. The objective of this study was to screen natural compounds as potential urease inhibitors by targeting UreG in a predominant ruminal microbial urease. In silico screening and in vitro tests for potential inhibitors were performed using molecular docking and an assay for the GTPase activity of UreG. Chelerythrine chloride was selected as a potential urease inhibitor of UreG with an inhibition concentration IC50 value of 18.13 µM. It exhibited mixed inhibition, with the Ki value being 26.28 µM. We further explored its inhibition mechanism using isothermal titration calorimetry (ITC) and circular dichroism (CD) spectroscopy, and we found that chelerythrine chloride inhibited the binding of nickel to UreG and induced changes in the secondary structure, especially the α-helix and ß-sheet of UreG. Chelerythrine chloride formed a pi-anion interaction with the Asp41 residue of UreG, which is an important residue in initiating the conformational changes of UreG. In conclusion, chelerythrine chloride exhibited a potential inhibitory effect on urease, which provided new evidence for strategies to develop novel urease inhibitors targeting UreG to reduce nitrogen excretion from ruminants.

Substitution of residues in UreG to investigate UreE interactions and nickel binding in a predominant urease gene cluster from the ruminal metagenome.

Microbial ureases catalyze the hydrolysis of urea to ammonia, and inhibition of these enzymes in rumen has the potential to improve urea utilization efficiency and reduce urinary nitrogen excretion. Urease activity is catalyzed by a protein complex encoded by a gene cluster, and its accessory proteins (especially UreE and UreG) play important roles in transferring nickel to the active site for urease maturation. In this study, a predominant urease gene cluster (5290 bp) from the ruminal microbial metagenome was identified. Isothermal titration calorimetry (ITC) and analytical ultracentrifugation (AUC) analyses showed that the reaction of identified UreE with UreG was endothermic, and was dominated by a hydrophobic interaction, in which each UreE dimer bound 2 M equivalents of UreG monomer to form a UreE2-2UreG complex. Mutagenesis analyses showed that the UreG residues Glu-23, Asp-41, Glu-46, Glu-66, Cys-70, His-72, Asp-78, and Asp-118 were involved in the GTPase activity of UreG. Furthermore, variants of Cys-70 and His-72 involved in CPH motif of UreG, as well as the nearby Glu-66 and Asp-78, not only prevented interactions with UreE, but also prevented nickel binding. These data provide additional information regarding UreG residues that may be targeted for the design of new urease inhibitors.

[Subtotal petrosectomy - indications, surgical technique, experiences in Pécs]. / Subtotalis petrosectomia ­ javallatok, mutéti technika, pécsi tapasztalatok.

Introduction: Subtotal petrosectomy (STP) has been known for decades and it is considered being on the borderline between ear and skull-base surgery. Due to its excessive radicalism and adverse effects on hearing, it has been a forgotten surgical technique until recently. Over the last decade, due to its many benefits and the ability to combine it with modern hearing rehabilitation methods, STP has reappeared, providing a definitive solution to a number of problematic middl-ear disorders. Aim: In our retrospective clinical study, we wanted to highlight the effectiveness of STP, analyzing the efficiency of this surgery for the first time in domestic patient records, and to report our own experiences to date. Method: We processed data from 45 surgeries on our 44 patients. In addition to presenting the various surgical indications, we review the steps of the operation, analyze the audiological results, including the various methods of hearing rehabilitation, and also analyze the complications that have occurred. Our results are also summarized in the light of international professional literature. Results: Of our 44 patients, 23 were female and 21 were male. Patients had a mean age of 44.6 ± 20.5 years and a mean follow-up of 23 ± 16 months. Of our patients, 25 (57%) had undergone at least two previous unsuccessful ear operations, and 6 (14%) had deafness before surgery. The most common indication was chronic otitis media with cholesteatoma. 13 patients underwent 14 cochlear implantations (23%), in addition 6 patients received bone anchored hearing aids (BAHA) (14%), and 2 patients (5%) underwent VSB (Vibrant Soundbridge) placement against the round window membrane. Complications occurred in 11 of our patients, the most common of which was cerebrospinal fluid leak (5 cases, 11%) and wound dehiscence (3 cases, 7%). Data from 3 patients were available for processing the audiological results. Conclusion: STP is an extremely favorable surgical solution for a number of middle-ear pathologies that have previously caused major problems. The main reason for its growing popularity is that it can be combined with a number of modern, effective methods of hearing rehabilitation. Orv Hetil. 2020; 161(14): 544-553.

Coptisine-induced inhibition of Helicobacter pylori: elucidation of specific mechanisms by probing urease active site and its maturation process.

In this study, we examined the anti-Helicobactor pylori effects of the main protoberberine-type alkaloids in Rhizoma Coptidis. Coptisine exerted varying antibacterial and bactericidal effects against three standard H. pylori strains and eleven clinical isolates, including four drug-resistant strains, with minimum inhibitory concentrations ranging from 25 to 50 µg/mL and minimal bactericidal concentrations ranging from 37.5 to 125 µg/mL. Coptisine's anti-H. pylori effects derived from specific inhibition of urease in vivo. In vitro, coptisine inactivated urease in a concentration-dependent manner through slow-binding inhibition and involved binding to the urease active site sulfhydryl group. Coptisine inhibition of H. pylori urease (HPU) was mixed type, while inhibition of jack bean urease was non-competitive. Importantly, coptisine also inhibited HPU by binding to its nickel metallocentre. Besides, coptisine interfered with urease maturation by inhibiting activity of prototypical urease accessory protein UreG and formation of UreG dimers and by promoting dissociation of nickel from UreG dimers. These findings demonstrate that coptisine inhibits urease activity by targeting its active site and inhibiting its maturation, thereby effectively inhibiting H. pylori. Coptisine may thus be an effective anti-H. pylori agent.

UreE-UreG complex facilitates nickel transfer and preactivates GTPase of UreG in Helicobacter pylori.

The pathogenicity of Helicobacter pylori relies heavily on urease, which converts urea to ammonia to neutralize the stomach acid. Incorporation of Ni(2+) into the active site of urease requires a battery of chaperones. Both metallochaperones UreE and UreG play important roles in the urease activation. In this study, we demonstrate that, in the presence of GTP and Mg(2+), UreG binds Ni(2+) with an affinity (Kd) of ∼0.36 µm. The GTPase activity of Ni(2+)-UreG is stimulated by both K(+) (or NH4 (+)) and HCO3 (-) to a biologically relevant level, suggesting that K(+)/NH4 (+) and HCO3 (-) might serve as GTPase elements of UreG. We show that complexation of UreE and UreG results in two protein complexes, i.e. 2E-2G and 2E-G, with the former being formed only in the presence of both GTP and Mg(2+). Mutagenesis studies reveal that Arg-101 on UreE and Cys-66 on UreG are critical for stabilization of 2E-2G complex. Combined biophysical and bioassay studies show that the formation of 2E-2G complex not only facilitates nickel transfer from UreE to UreG, but also enhances the binding of GTP. This suggests that UreE might also serve as a structural scaffold for recruitment of GTP to UreG. Importantly, we demonstrate for the first time that UreE serves as a bridge to grasp Ni(2+) from HypA, subsequently donating it to UreG. The study expands our horizons on the molecular details of nickel translocation among metallochaperones UreE, UreG, and HypA, which further extends our knowledge on the urease maturation process.

Molecular landscape of the interaction between the urease accessory proteins UreE and UreG.

Urease, the most efficient enzyme so far discovered, depends on the presence of nickel ions in the catalytic site for its activity. The transformation of inactive apo-urease into active holo-urease requires the insertion of two Ni(II) ions in the substrate binding site, a process that involves the interaction of four accessory proteins named UreD, UreF, UreG and UreE. This study, carried out using calorimetric and NMR-based structural analysis, is focused on the interaction between UreE and UreG from Sporosarcina pasteurii, a highly ureolytic bacterium. Isothermal calorimetric protein-protein titrations revealed the occurrence of a binding event between SpUreE and SpUreG, entailing two independent steps with positive cooperativity (Kd1=42±9µM; Kd2=1.7±0.3µM). This was interpreted as indicating the formation of the (UreE)2(UreG)2 hetero-oligomer upon binding of two UreG monomers onto the pre-formed UreE dimer. The molecular details of this interaction were elucidated using high-resolution NMR spectroscopy. The occurrence of SpUreE chemical shift perturbations upon addition of SpUreG was investigated and analyzed to establish the protein-protein interaction site. The latter appears to involve the Ni(II) binding site as well as mobile portions on the C-terminal and the N-terminal domains. Docking calculations based on the information obtained from NMR provided a structural basis for the protein-protein contact site. The high sequence and structural similarity within these protein classes suggests a generality of the interaction mode among homologous proteins. The implications of these results on the molecular details of the urease activation process are considered and analyzed.