Structural insight into poplar glutaredoxin C1 with a bridging iron-sulfur cluster at the active site

Glutaredoxins are glutathione-dependent enzymes that function to reduce disulfide bonds in vivo. Interestingly, a recent discovery indicates that some glutaredoxins can also exist in another form, an iron-sulfur protein [Lillig, C. H., et al. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 8168-8173]. This provides a direct connection between glutaredoxins and iron-sulfur proteins, suggesting a possible new regulatory role of iron-sulfur clusters along with the new functional switch of glutaredoxins. Biochemical studies have indicated that poplar glutaredoxin C1 (Grx-C1) is also such a biform protein. The apo form (monomer) of Grx-C1 is a regular glutaredoxin, and the holo form (dimer) is an iron-sulfur protein with a bridging [2Fe-2S] cluster. Here, we report the structural characterizations of poplar Grx-C1 in both the apo and holo forms by NMR spectroscopy. The solution structure of the reduced apo Grx-C1, which is the first plant Grx structure, shows a typical Grx fold. When poplar Grx-C1 forms a dimer with an iron-sulfur cluster, each subunit of the holo form still retains the overall fold of the apo form. The bridging iron-sulfur cluster in holo Grx-C1 is coordinated near the active site. In addition to the iron-sulfur cluster linker, helix alpha3 of each subunit is probably involved in the direct contact between the two subunits. Moreover, two glutathione molecules are identified in the vicinity of the iron-sulfur cluster and very likely participate in cluster coordination. Taken together, we propose that the bridging [2Fe-2S] cluster is coordinated by the first cysteine at the glutaredoxin active site from each subunit of holo Grx-C1, along with two cysteines from two glutathione molecules. Our studies reveal that holo Grx-C1 has a novel structural and iron-sulfur cluster coordination pattern for an iron-sulfur protein.     

MexAB-OprM-specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 1: discovery and early strategies for lead optimization

The identification of a series of compounds that specifically inhibit efflux by the MexAB-OprM pump system in Pseudomonas aeruginosa is described. Synthesis and in vitro structure-activity relationships (SARs) are outlined. Early leads lacked activity in animal models, and efforts to improve solubility and reduce serum protein binding by the introduction of polar groups are discussed.     

p38MAPK suppresses chronic pancreatitis by regulating HSP27 and BAD expression

Mitogen-activated protein kinases (MAPKs) are ubiquitous proteins that function in both normal and stress-related pathophysiological states of the cell. This study aimed to analyze the importance of p38MAPK in pancreatic injury using WBN/Kob rats with spontaneous chronic pancreatitis. Male WBN/Kob rats were injected with the p38MAPK inhibitor SB203580, starting at the age of 4 weeks, and sacrificed 6 weeks later. Compared with vehicle-treated rats, p38 inhibitor-treated rats exhibited a significant increase in pancreatic cell death and inflammation as assessed by histologic examination and myeloperoxidase activity, respectively. p38 inhibition decreased the expression of heat shock protein 27 (HSP27), an antioxidant protein, and enhanced accumulation of reactive oxygen species (ROS). In addition, the proapoptotic protein BAD was increased in the pancreas of rats treated with p38 inhibitor. In a pancreatic cell line (PANC-1), HSP27 knockdown augmented reactive oxygen species accumulation and cell death induced by tumor necrosis factor-α plus actinomycin D. In conclusion, p38MAPK suppresses chronic pancreatitis by upregulating HSP27 expression and downregulating BAD expression.     

Immune Suppression and Resistance Mediated by Constitutive Activation of Wnt/β-Catenin Signaling in Human Melanoma Cells

Cancer-induced immunosuppression is a major problem reducing antitumor effects of immunotherapies, but its molecular mechanism has not been well understood. We evaluated immunosuppressive roles of activated Wnt/β-catenin pathways in human melanoma for dendritic cells (DCs) and CTLs. IL-10 expression was associated with β-catenin accumulation in human melanoma cell lines and tissues and was induced by direct β-catenin/TCF binding to the IL-10 promoter. Culture supernatants from β-catenin-accumulated melanoma have activities to impair DC maturation and to induce possible regulatory DCs. Those immunosuppressive culture supernatant activities were reduced by knocking down β-catenin in melanoma cells, partly owing to downregulation of IL-10. Murine splenic and tumor-infiltrating DCs obtained from nude mice implanted with human mutant β-catenin-overexpressed melanoma cells had less ability to activate T cells than did DCs from mice with control melanoma cells, showing in vivo suppression of DCs by activated Wnt/β-catenin signaling in human melanoma. This in vivo DC suppression was restored by the administration of a β-catenin inhibitor, PKF115-584. β-catenin-overexpressed melanoma inhibited IFN-γ production by melanoma-specific CTLs in an IL-10-independent manner and is more resistant to CTL lysis in vitro and in vivo. These results indicate that Wnt/β-catenin pathways in human melanoma may be involved in immunosuppression and immunoresistance in both induction and effector phases of antitumor immunoresponses partly through IL-10 production, and they may be attractive targets for restoring immunocompetence in patients with Wnt/β-catenin-activated melanoma.     

Imiquimod Suppresses Propagation of Herpes Simplex Virus 1 by Upregulation of Cystatin A via the Adenosine Receptor A1 Pathway

Imiquimod is recognized as an agonist for Toll-like receptor 7 (TLR7) in immunocompetent cells. TLR7, as well as TLR3 and TLR8, triggers the immune responses, such as the production of type I interferons (IFNs) and proinflammatory cytokines via recognition of viral nucleic acids in the infected cells. In this study, we proposed that imiquimod has an IFN-independent antiviral effect in nonimmune cells. Imiquimod, but not resiquimod, suppressed replication of human herpes simplex virus 1 (HSV-1) in FL cells. We analyzed alternation of gene expression by treatment with imiquimod using microarray analysis. Neither type I IFNs, nor TLRs, nor IFN-inducible antiviral genes were induced in imiquimod-treated FL cells. Cystatin A, a host cysteine protease inhibitor, was strongly upregulated by imiquimod and took a major part in the anti-HSV-1 activity deduced by the suppression experiment using its small interfering RNA. Upregulation of cystatin A was suggested to be mediated by antagonizing adenosine receptor A(1) and activating the protein kinase A pathway. Imiquimod, but not resiquimod, was shown to interact with adenosine receptor A(1). Imiquimod-induced anti-HSV-1 activity was observed in other cells, such as HeLa, SiHa, and CaSki cells, in a manner consistent with the cystatin A induction by imiquimod. These results indicated that imiquimod acted as an antagonist for adenosine receptor A(1) and induced a host antiviral protein, cystatin A. The process occurred independently of TLR7 and type I IFNs.     

CXCL14-like immunoreactivity in growth hormone-containing cells of urodele pituitaries

Immunohistochemical techniques were employed to investigate the distribution of a chemokine, namely, CXCL14-like immunoreactivity in the axolotl (Ambystoma mexicanum) and Japanese black salamander (Hynobius nigrescens) pituitaries. CXCL14-immunoreactive cells concentrated at an area of the pars distalis adjacent to the pars intermedia. We found that these cells correspond to the cells immunoreactive to an antibody against rat growth hormone (GH). Immunoelectron microscopy indicated that the CXCL14-like substance and GH coexisted on the secretory granules in the axolotl pituitary. Western blot analysis of axolotl pituitary extracts revealed the anti-human CXCL14 antibody labeled an approximately 16.6-kDa band that was not labeled by the anti-GH antibody. The CXCL14-like substance in the pars distalis may participate in GH functions in these species.     

High-temperature sorbose fermentation with thermotolerant Gluconobacter frateurii CHM43 and its mutant strain adapted to higher temperature

We succeeded in obtaining a strain adapted to higher temperature from a thermotolerant strain, Gluconobacter frateurii CHM43, for sorbose fermentation. The adapted strain showed higher growth and L: -sorbose production than original CHM43 strain at higher temperature around 38.5-40?°C. It was also shown to be useful even with the fermentation without temperature control. To understand the sorbose fermentation ability of the adapted strain at higher temperature, D: -sorbitol-oxidizing respiratory chain was compared with the CHM43 strain and the adapted strain. We found that the activity of pyrroloquinoline quinone (PQQ)-dependent glycerol dehydrogenase (GLDH), which is a primary dehydrogenase of the respiratory chain and responsible for L: -sorbose production, was decreased when the temperature increased, but the decreased activity of GLDH was recovered by the addition of PQQ. Since the adapted strain was found to produce more PQQ than the CHM43 strain, it was suggested that the adapted strain keeps GLDH as holoenzyme with the increased PQQ production, and thus produces more L: -sorbose and grows better under higher temperature.