Crystal structure of the Ate1 arginyl-tRNA-protein transferase and arginylation of N-degron substrates.

N-degron pathways are proteolytic systems that target proteins bearing N-terminal (Nt) degradation signals (degrons) called N-degrons. Nt-Arg of a protein is among Nt-residues that can be recognized as destabilizing ones by the Arg/N-degron pathway. A proteolytic cleavage of a protein can generate Arg at the N terminus of a resulting C-terminal (Ct) fragment either directly or after Nt-arginylation of that Ct-fragment by the Ate1 arginyl-tRNA-protein transferase (R-transferase), which uses Arg-tRNAArg as a cosubstrate. Ate1 can Nt-arginylate Nt-Asp, Nt-Glu, and oxidized Nt-Cys* (Cys-sulfinate or Cys-sulfonate) of proteins or short peptides. Ate1 genes of fungi, animals, and plants have been cloned decades ago, but a three-dimensional structure of Ate1 remained unknown. A detailed mechanism of arginylation is unknown as well. We describe here the crystal structure of the Ate1 R-transferase from the budding yeast Kluyveromyces lactis. The 58-kDa R-transferase comprises two domains that recognize, together, an acidic Nt-residue of an acceptor substrate, the Arg residue of Arg-tRNAArg, and a 3'-proximal segment of the tRNAArg moiety. The enzyme's active site is located, at least in part, between the two domains. In vitro and in vivo arginylation assays with site-directed Ate1 mutants that were suggested by structural results yielded inferences about specific binding sites of Ate1. We also analyzed the inhibition of Nt-arginylation activity of Ate1 by hemin (Fe3+-heme), and found that hemin induced the previously undescribed disulfide-mediated oligomerization of Ate1. Together, these results advance the understanding of R-transferase and the Arg/N-degron pathway.

Antimicrobial Resistance and Molecular Characterization of Extended-Spectrum ß-Lactamase-Producing Escherichia coli Isolated from Ducks in South Korea.

Ducks are potential carriers of pathogenic bacteria, which are capable of transmitting zoonotic diseases to humans. The global spread of Enterobacteriaceae carrying extended-spectrum ß-lactamase (ESBL) genes is a public health concern. This study investigated the prevalence of antimicrobial resistance in Escherichia coli isolated from ducks in Korea and described the molecular characteristics of the ESBLs they produced. A total of 146 E. coli isolates from 404 duck fecal and carcass samples in 85 duck farms were tested for antimicrobial resistance using the broth dilution method and were further characterized using molecular methods. We observed high resistance rates to tetracycline, trimethoprim/sulfamethoxazole, nalidixic acid, ampicillin, and ciprofloxacin. In total, six ceftiofur-resistant isolates (4.1%) were observed, which produced CTX-M-55 (n = 3) or CTX-M-65 ß-lactamase (n = 3). All CTX-M-producing E. coli isolates were also resistant to ciprofloxacin, with mutations in the quinolone resistance determining region of GyrA (S83L with or without D87N) and ParC (S80I), and three CTX-M-producing E. coli isolates carried plasmid-mediated quinolone resistance (PMQR) genes, qepA (n = 1), qnrS, and acc(6')-Ib-cr (n = 2). The transfer of blaCTX-M genes was observed in one isolate mediated by IncF-family plasmids but not in the co-resistant isolates carrying both blaCTX-M and PMQR genes. Pulsed-field gel electrophoresis and multilocus sequence typing demonstrated that CTX-M-producing isolates were heterogeneous; however, identical isolates were found in different farms and slaughterhouses. This study presents baseline data on antimicrobial resistance of E. coli derived from duck samples and is the first report of CTX-M-55 and CTX-M-65 ß-lactamase-producing E. coli isolated from ducks in Korea. The dissemination of ESBL-producing E. coli poses a potential risk to public health and therefore should be monitored.

Detection of oxazolidinone and phenicol resistant enterococcal isolates from duck feces and carcasses.

The heavy use or abuse of antimicrobials in food animals has caused an increase in antimicrobial resistance in enterococci of animal origin, which could get transmitted to those of human origin via the food chain. Since duck meat consumption has been on the rise in Korea, we conducted this study to provide information about the antimicrobial resistance of the enterococci obtained from healthy ducks and their carcasses. A total of 82 Enterococcus faecium and 174 E. faecalis isolated from duck fecal and carcass samples were investigated for antimicrobial resistance to 16 agents, using broth dilution method, and were further characterized using molecular methods. Most of E. faecium (84.1%) and E. faecalis (87.9%) isolates were resistant to one or more antimicrobials. Multi-drug resistant (MDR) isolates were observed in both E. faecium (40.2%) and E. faecalis (33.9%) with high frequencies. High rate of resistance was observed for tetracycline, ciprofloxacin, chloramphenicol, and erythromycin in both E. faecium and E. faecalis. Resistance to gentamicin, vancomycin, and daptomycin, in both E. faecium and E. faecalis, was, if at all, very rare. However, linezolid resistance was observed in nine E. faecium (11.0%) and one E. faecalis (0.6%). All, but one, Linezolid resistant (LR) isolates were also resistant to chloramphenicol and florfenicol. The novel transferable oxazolidinone and phenicol resistant gene, optrA, was found in six E. faecium isolates. All of them co-carried phenicol exporter gene fexA. None of the LR isolates had mutation in the 23S ribosomal RNA and in the ribosomal protein L3. Six LR E. faecium isolates had Asn130Lys mutation in the ribosomal protein L4, of which five also carried optrA gene. None of the isolates carried the multi-resistance gene cfr. Transfer of oxazolidinone and phenicol resistance was observed in five among the 10 LR isolates; two of them had optrA and fexA genes. Multi-drug resistant Enterococcus that also carried the resistance gene to a last-resort antimicrobial is a major concern for public health. Thus, to prevent the introduction of last-resort antimicrobial resistance into food chain, continuous surveillance of antimicrobial resistance in duck is imperative.

ACCORD: an assessment tool to determine the orientation of homodimeric coiled-coils.

The coiled-coil (CC) domain is a very important structural unit of proteins that plays critical roles in various biological functions. The major oligomeric state of CCs is a dimer, which can be either parallel or antiparallel. The orientation of each α-helix in a CC domain is critical for the molecular function of CC-containing proteins, but cannot be determined easily by sequence-based prediction. We developed a biochemical method for assessing differences between parallel and antiparallel CC homodimers and named it ACCORD (Assessment tool for homodimeric Coiled-Coil ORientation Decision). To validate this technique, we applied it to 15 different CC proteins with known structures, and the ACCORD results identified these proteins well, especially with long CCs. Furthermore, ACCORD was able to accurately determine the orientation of a CC domain of unknown directionality that was subsequently confirmed by X-ray crystallography and small angle X-ray scattering. Thus, ACCORD can be used as a tool to determine CC directionality to supplement the results of in silico prediction.

Structural basis for dual specificity of yeast N-terminal amidase in the N-end rule pathway.

The first step of the hierarchically organized Arg/N-end rule pathway of protein degradation is deamidation of the N-terminal glutamine and asparagine residues of substrate proteins to glutamate and aspartate, respectively. These reactions are catalyzed by the N-terminal amidase (Nt-amidase) Nta1 in fungi such as Saccharomyces cerevisiae, and by the glutamine-specific Ntaq1 and asparagine-specific Ntan1 Nt-amidases in mammals. To investigate the dual specificity of yeast Nta1 (yNta1) and the importance of second-position residues in Asn/Gln-bearing N-terminal degradation signals (N-degrons), we determined crystal structures of yNta1 in the apo state and in complex with various N-degron peptides. Both an Asn-peptide and a Gln-peptide fit well into the hollow active site pocket of yNta1, with the catalytic triad located deeper inside the active site. Specific hydrogen bonds stabilize interactions between N-degron peptides and hydrophobic peripheral regions of the active site pocket. Key determinants for substrate recognition were identified and thereafter confirmed by using structure-based mutagenesis. We also measured affinities between yNta1 (wild-type and its mutants) and specific peptides, and determined KM and kcat for peptides of each type. Together, these results elucidate, in structural and mechanistic detail, specific deamidation mechanisms in the first step of the N-end rule pathway.

Photoprotective properties of 20(S)-protopanaxatriol, an aglycone of ginseng saponins: Protection from ultraviolet-B radiation-induced oxidative stress in human epidermal keratinocytes.

Ginsenosides are responsible for diverse pharmacological properties ascribed to ginseng, a plant used in traditional medicine. Ginsenosides are classified into three categories: Protopanaxadiol, protopanaxatriol (PPT) and oleanolic acid. As an aglycone of PPT-type ginsenosides, PPT exists in two stereoisomeric forms, 20(S)-PPT and 20(R)­PPT. The 20(S)­PPT stereoisomer is a major metabolic product of PPT­type ginsenosides produced in the gastrointestinal tract. In the present study, 20(S)-PPT suppressed the elevation of reactive oxygen species in HaCaT cells following irradiation with ultraviolet (UV)­B. In addition, 20(S)­PPT inhibited UV­B­induced gelatinase activities of matrix metalloproteinase-2 and -9 in HaCaT cells, and suppressed UV­B­induced expression and secretion of these proteins. Accordingly, 20(S)­PPT restored the total glutathione levels in UV­B­irradiated keratinocytes. Taken together, these data indicated that 20(S)­PPT may possess photoprotective properties that combat the effects of UV­B radiation.

Protective properties of ginsenoside Rb3 against UV-B radiation-induced oxidative stress in HaCaT keratinocytes.

This work aimed to evaluate the skin anti-photoaging properties of ginsenoside Rb3 (Rb3), one of the main protopanaxdiol-type ginsenosides from ginseng, in HaCaT keratinocytes. The skin anti-photoaging activity was assessed by analyzing the levels of reactive oxygen species (ROS), pro-matrix metalloproteinase-2 (proMMP-2), pro-matrix metalloproteinase-9 (proMMP-9), total glutathione (GSH), and superoxide dismutase (SOD) activity as well as cell viability in HaCaT keratinocytes under UV-B irradiation. When HaCaT keratinocytes were exposed to Rb3 prior to UV-B irradiation, Rb3 exhibited suppressive activities on UV-B-induced ROS, proMMP-2, and proMMP-9 enhancements. On the contrary, Rb3 displayed enhancing activities on UV-B-reduced total GSH and SOD activity levels. Rb3 could not interfere with cell viabilities in UV-B-irradiated HaCaT keratinocytes. Rb3 plays a protective role against UV-B-induced oxidative stress in human HaCaT keratinocytes, proposing its potential skin anti-photoaging properties.

Protective properties of ginsenoside Rb1 against UV-B radiation-induced oxidative stress in human dermal keratinocytes.

Ginsenosides, also known as ginseng saponins, are responsible for most pharmacological effect of ginseng. Ginsenoside Rb1 (Rb1) exerts a variety of pharmacological properties, including anti-inflammatory, antistress, anti-aging and anti-neurodegenerative activities. The aim of the present work was to assess the skin anti-photoaging properties of Rb1 in human dermal keratinocyte HaCaT cells. The anti-photoaging activity was evaluated by analyzing the levels of reactive oxygen species (ROS) and matrix metalloproteinases (MMPs) as well as cell viability for HaCaT cells under UV-B irradiation. Rb1 was able to suppress the ROS levels which were elevated under UV-B irradiation, and unable to influence cellular survival in UV-B-irradiated HaCaT cells. Rb1 diminished the enhancement of MMP-2 gelatinolytic activity in conditioned medium, which corresponded with the decreased MMP-2 protein levels in both conditioned medium and cellular lysate prepared from UV-B-irradiated HaCaT cultures. Rb1 could restore the total glutathione (GSH) and superoxide dismutase (SOD) activity diminished in UV-B-irradiated HaCaT cells. Ginsenoside Rb1 possesses skin anti-photoaging properties through scavenging ROS and decreasing MMP-2 levels possibly by enhancing antioxidant activity in keratinocytes under UV-B irradiation.

Ginsenoside Rb2 Attenuates UV-B Radiation-Induced Reactive Oxygen Species and Matrix Metalloproteinase-2 through Upregulation of Antioxidant Components in Human Dermal Fibroblasts.

AIMS: The antiphotoaging activities of ginsenoside Rb2 on the skin, one of the predominant protopanaxadiol-type ginsenosides, were evaluated in cultured human dermal fibroblasts. METHODS: The antiphotoaging activity was examined by analyzing the levels of reactive oxygen species (ROS), matrix metalloproteinase-2 (MMP-2), total glutathione (GSH) and superoxide dismutase (SOD) activity as well as cell viability for fibroblasts under UV-B irradiation. RESULTS: When cultured fibroblasts were exposed to Rb2 prior to UV-B irradiation, Rb2 displayed suppressive activities on UV-B-induced ROS elevation and MMP-2 on both activity and protein levels, while it exhibited an enhancing activity on total GSH level and SOD activity diminished by UV-B irradiation. However, Rb2 could not interfere with cell viabilities in UV-B-irradiated fibroblasts. CONCLUSION: Ginsenoside Rb2 plays a photoprotective role against UV-B-induced oxidative stress in human dermal fibroblasts, which implies its skin antiphotoaging potential.

Suppressive properties of ginsenoside Rb2, a protopanaxadiol-type ginseng saponin, on reactive oxygen species and matrix metalloproteinase-2 in UV-B-irradiated human dermal keratinocytes.

Ginsenosides, also known as ginseng saponins, are the principal bioactive ingredients of ginseng, which are responsible for its diverse pharmacological activities. The present work aimed to assess skin anti-photoaging properties of ginsenoside Rb2 (Rb2), one of the predominant protopanaxadiol-type ginsenosides, in human epidermal keratinocyte HaCaT cells under UV-B irradiation. When the cultured keratinocytes were subjected to Rb2 prior to UV-B irradiation, Rb2 displayed suppressive activities on UV-B-induced reactive oxygen species elevation and matrix metalloproteinase-2 expression and secretion. However, Rb2 at the used concentrations was unable to modulate cellular survivals in the UV-B-irradiated keratinocytes. In brief, Rb2 possesses a protective role against the photoaging of human keratinocyte cells under UV-B irradiation.

Stereoselective suppressive effects of protopanaxadiol epimers on UV-B-induced reactive oxygen species and matrix metalloproteinase-2 in human dermal keratinocytes.

This study aimed to assess the skin-related anti-photoaging activities of the 2 epimeric forms of protopanaxadiol (PPD), 20(S)-PPD and 20(R)-PPD, in cultured human keratinocytes (HaCaT cells). The anti-photoaging activity was evaluated by analyzing the levels of reactive oxygen species (ROS) and matrix metalloproteinases (MMPs), as well as cell viability for HaCaT cells under UV-B irradiation. The activities for MMP-2 and -1 in conditioned medium were determined using gelatin zymography, and MMP-2 protein in the conditioned medium was detected using Western blot analysis. 20(S)-PPD, but not 20(R)-PPD, suppressed UV-B-induced ROS elevation. Neither of the epimers, at the concentrations used, exhibited cytotoxicity, irrespective of UV-B irradiation. 20(S)-PPD, but not 20(R)-PPD, exhibited an inhibitory effect on UV-B-induced MMP-2 activity and expression in HaCaT cells. In brief, only 20(S)-PPD, a major metabolic product of PPD-type ginsenosides, inhibits UV-B-induced ROS and MMP-2 elevation, implying its stereospecific anti-photoaging activity on the skin.

Skin anti-photoaging properties of ginsenoside Rh2 epimers in UV-B-irradiated human keratinocyte cells.

Ginseng, one of the most widely used herbal medicines, has a wide range of therapeutic and pharmacological applications. Ginsenosides are the major bioactive ingredients of ginseng, which are responsible for various pharmacological activities of ginseng. Ginsenoside Rh2, known as an antitumour ginsenoside, exists as two different stereoisomeric forms, 20(S)-ginsenoside Rh2 [20(S)-Rh2] and 20(R)-ginsenoside Rh2 [20(R)-Rh2]. This work aimed to assess and compare skin anti-photoaging activities of 20(S)-Rh2 and 20(R)-Rh2 in UV-B-irradiated HaCat cells. 20(S)-Rh2, but not 20(R)-Rh2, was able to suppress UV-B-induced ROS production in HaCat cells. Both stereoisomeric forms could not modulate cellular survival and NO level in UV-B-irradiated HaCat cells. Both 20(S)-Rh2 and 20(R)-Rh2 exhibited suppressive effects on UV-B-induced MMP-2 activity and expression in HaCat cells. In brief, the two stereoisomers of ginsenoside Rh2, 20(S)-Rh2 and 20(R)-Rh2, possess skin anti-photoaging effects but possibly in different fashions.