Structure of the bacteriophage T4 DNA adenine methyltransferase

DNA-adenine methylation at certain GATC sites plays a pivotal role in bacterial and phage gene expression as well as bacterial virulence. We report here the crystal structures of the bacteriophage T4Dam DNA adenine methyltransferase (MTase) in a binary complex with the methyl-donor product S-adenosyl-L-homocysteine (AdoHcy) and in a ternary complex with a synthetic 12-bp DNA duplex and AdoHcy. T4Dam contains two domains: a seven-stranded catalytic domain that harbors the binding site for AdoHcy and a DNA binding domain consisting of a five-helix bundle and a beta-hairpin that is conserved in the family of GATC-related MTase orthologs. Unexpectedly, the sequence-specific T4Dam bound to DNA in a nonspecific mode that contained two Dam monomers per synthetic duplex, even though the DNA contains a single GATC site. The ternary structure provides a rare snapshot of an enzyme poised for linear diffusion along the DNA.     

Crystal structure of the BAFF-BAFF-R complex and its implications for receptor activation

B-cell activating factor (BAFF) is a key regulator of B-lymphocyte development. Its biological role is mediated by the specific receptors BCMA, TACI and BAFF-R. We have determined the crystal structure of the extracellular domain of BAFF-R bound to BAFF at a resolution of 3.3 A. The cysteine-rich domain (CRD) of the BAFF-R extracellular domain adopts a beta-hairpin structure and binds to the virus-like BAFF cage in a 1:1 molar ratio. The conserved DxL motif of BAFF-R is located on the tip of the beta-turn and is indispensable in the binding of BAFF. The crystal structure shows that a unique dimeric contact occurs between the BAFF-R monomers in the virus-like cage complex. The extracellular domain of TACI contains two CRDs, both of which contain the DxL motif. Modeling of TACI-BAFF complex suggests that both CDRs simultaneously interact with the BAFF dimer in the virus-like cage.     

Myosin heavy chain and physiological adaptation of the rat diaphragm in elastase-induced emphysema

Background

Platelets are thought to play a role in a variety of inflammatory conditions in the lung, some of which may lead to fibrosis. In the current study we tested the hypothesis that whole platelets and platelet lysate can mediate remodelling of extracellular matrix in vitro by affecting fibroblast-mediated contraction of a collagen gel. We also sought to determine to what extent platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β) contribute to this effect.

Methods

Washed platelets, isolated from healthy blood donors, and platelet lysate (freezing and thawing), were cast together with human lung fibroblasts in three-dimensional collagen gels. The gels were then released and cultured for four days. PDGF and TGF-β₁ concentrations were measured in culture supernatants by ELISA.

Results

Both platelets and platelet lysate augmented fibroblast-mediated gel contraction in a time and concentration dependent manner (19.9% ± 0.1 (mean ± SEM) of initial area vs. 48.0% ± 0.4 at 48 hours; P < 0.001 and 41.5% ± 0.6 vs. 60.6% ± 0.3 at 48 hours; P < 0.001, respectively). Fixed platelets had no effect in the system. Both TGF-β₁ and PDGF-AA/AB were released in co-culture. PDGF-AA/AB had a maximum release at 24 hours whereas TGF-β₁ release increased with longer culture periods. Neutralising antibodies to these mediators partially inhibited platelet-induced gel contraction.

Conclusion

We conclude that platelets may promote remodelling of extracellular matrix in vitro and that PDGF and TGF-β partially mediate this effect, also indicating a role for other mediators. The findings may be an important mechanism in regulating repair processes after injury.     

TRPC5 as a candidate for the nonselective cation channel activated by muscarinic stimulation in murine stomach

We investigated which transient receptor potential (TRP) channel is responsible for the nonselective cation channel (NSCC) activated by carbachol (CCh) in murine stomach with RT-PCR and the electrophysiological method. All seven types of TRP mRNA were detected in murine stomach with RT-PCR. When each TRP channel was expressed, the current-voltage relationship of mTRP5 was most similar to that recorded in murine gastric myocytes. mTRP5 showed a conductance order of Cs(+) > K(+) > Na(+), similar to that in the murine stomach. With 0.2 mM GTPgammaS in the pipette solution, the current was activated transiently in both NSCC in the murine stomach and the expressed mTRP5. Both NSCC activated by CCh in murine stomach and mTRP5 were inhibited by intracellularly applied anti-G(q/11) antibody, PLC inhibitor U-73122, IICR inhibitor 2-aminoethoxydiphenylborate, and nonspecific cation channel blockers La(3+) and flufenamate. There were two other unique properties. Both the native NSCC and mTRP5 were activated by 1-oleoyl-2-acetyl-sn-glycerol. Without the activation of NSCC by CCh, the NSCC in murine stomach was constitutively active like mTRP5. From the above results, we suggest that mTRP5 might be a candidate for the NSCC activated by ACh or CCh in murine stomach.     

MAPK mediates PKC-dependent contraction of cat esophageal and lower esophageal sphincter circular smooth muscle

Esophageal (ESO) circular muscle contraction and lower esophageal sphincter (LES) tone are PKC dependent. Because MAPKs may be involved in PKC-dependent contraction, we examined ERK1/ERK2 and p38 MAPKs in ESO and LES. In permeabilized LES muscle cells, ERK1/2 antibodies reduced 1,2-dioctanoylglycerol (DG)- and threshold ACh-induced contraction, which are PKC dependent, but not maximal ACh, which is calmodulin dependent. LES tone was reduced by the ERK1/2 kinase inhibitor PD-98059 and by the p38 MAPK inhibitor SB-203580. In permeable ESO cells, ACh contraction was reduced by ERK1/ERK2 and p38 MAPK antibodies and by PD-98059 and SB-203580. ACh increased MAPK activity and phosphorylation of MAPK and of p38 MAPK. The 27-kDa heat shock protein (HSP27) antibodies reduced ACh contraction. HSP27 and p38 MAPK antibodies together caused no greater inhibition than either one alone. p38 MAPK and HSP27 coprecipitated after ACh stimulation, suggesting that HSP27 is linked to p38 MAPK. These data suggest that PKC-dependent contraction in ESO and LES is mediated by the following two distinct MAPK pathways: ERK1/2 and HSP27-linked p38 MAPK.     

Roles of different peptide fragments derived from proadrenomedullin in the regulation of vascular tone in isolated rat aorta

The effects of proadrenomedullin N-terminal 20 peptide (PAMP) and adrenotensin (ADT) on adrenomedullin (ADM)-induced vasodilation were investigated in aortic rings from rat. ADM (10(-9) to 10(-7)M) relaxed the aorta preconstricted with phenylephrine in a concentration-dependent manner. Denudation of endothelium or pretreatment with nitric oxide synthase (NOS) inhibitor, L-NAME, attenuated the vasodilatory action of ADM. ADM-induced vasorelaxation in the aortic rings with endothelium was converted to contraction by PAMP, but not by ADT. The ADM-induced vasodilation was not affected by PAMP in aorta rings without endothelium or in intact aortic rings pretreated with L-NAME. ADM-stimulated nitrite production and NOS activity of the aortas, which was inhibited by PAMP, ADT or PAMP plus ADT. ADM, PAMP, and ADT increased the cyclic adenosine monophosphate (cAMP) contents in vascular tissue. The combination of ADM with PAMP or ADT caused a smaller increase in cAMP level as compared with that of PAMP or ADT alone. These results show that ADM-induced endothelium-dependent vasodilation could be converted to vasoconstriction in the presence of PAMP, probably through a NO-dependent pathway. There was no indication that cAMP was involved in the converting effect of PAMP on ADM vasodilator action.     

Substrate binding and catalysis in trichosanthin occur in different sites as revealed by the complex structures of several E85 mutants

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) which possesses rRNA N-glycosidase activity. In recent years, its immunomodulatory, anti-tumor and anti-HIV properties have been revealed. Here we report the crystal structures of several E85 mutant TCS complexes with adenosine-5'-monophosphate (AMP) and adenine. In E85Q TCS/AMP and E85A TCS/AMP, near the active site of the molecule and parallel to the aromatic ring of Tyr70, an AMP molecule is bound to the mutant without being hydrolyzed. In the E85R TCS/adenine complex, the hydrolyzed product adenine is located in the active pocket where it occupies a position similar to that in the TCS/NADPH complex. Significantly, AMP is bound in a position different to that of adenine. In comparison with these structures, we suggest that there are at least two subsites in the active site of TCS, one for initial substrate recognition as revealed by the AMP site and another for catalysis as represented by the NADPH site. Based on these complex structures, the function of residue 85 and the mechanism of catalysis are proposed.     

PROSPECT II: protein structure prediction program for genome-scale applications

A new method for fold recognition is developed and added to the general protein structure prediction package PROSPECT (http://compbio.ornl.gov/PROSPECT/). The new method (PROSPECT II) has four key features. (i) We have developed an efficient way to utilize the evolutionary information for evaluating the threading potentials including singleton and pairwise energies. (ii) We have developed a two-stage threading strategy: (a) threading using dynamic programming without considering the pairwise energy and (b) fold recognition considering all the energy terms, including the pairwise energy calculated from the dynamic programming threading alignments. (iii) We have developed a combined z-score scheme for fold recognition, which takes into consideration the z-scores of each energy term. (iv) Based on the z-scores, we have developed a confidence index, which measures the reliability of a prediction and a possible structure-function relationship based on a statistical analysis of a large data set consisting of threadings of 600 query proteins against the entire FSSP templates. Tests on several benchmark sets indicate that the evolutionary information and other new features of PROSPECT II greatly improve the alignment accuracy. We also demonstrate that the performance of PROSPECT II on fold recognition is significantly better than any other method available at all levels of similarity. Improvement in the sensitivity of the fold recognition, especially at the superfamily and fold levels, makes PROSPECT II a reliable and fully automated protein structure and function prediction program for genome-scale applications.