Identification of residues in the heme domain of soluble guanylyl cyclase that are important for basal and stimulated catalytic activity

Nitric oxide signals through activation of soluble guanylyl cyclase (sGC), a heme-containing heterodimer. NO binds to the heme domain located in the N-terminal part of the β subunit of sGC resulting in increased production of cGMP in the catalytic domain located at the C-terminal part of sGC. Little is known about the mechanism by which the NO signaling is propagated from the receptor domain (heme domain) to the effector domain (catalytic domain), in particular events subsequent to the breakage of the bond between the heme iron and Histidine 105 (H105) of the β subunit. Our modeling of the heme-binding domain as well as previous homologous heme domain structures in different states point to two regions that could be critical for propagation of the NO activation signal. Structure-based mutational analysis of these regions revealed that residues T110 and R116 in the αF helix-β1 strand, and residues I41 and R40 in the αB-αC loop mediate propagation of activation between the heme domain and the catalytic domain. Biochemical analysis of these heme mutants allows refinement of the map of the residues that are critical for heme stability and propagation of the NO/YC-1 activation signal in sGC.     

Adenovirus serotype determines association and localization of the large E1B tumor antigen with cellular tumor antigen p53 in transformed cells.

The distribution and stability of the cellular tumor antigen p53 were studied in baby rat kidney cells transformed by region E1 sequences of nononcogenic adenovirus (Ad) type 5 (Ad5) or oncogenic type 12 (Ad12). In transformed cells expressing the large E1B T antigen of Ad5, p53 was associated with this T antigen. The complexed proteins were concentrated in a cytoplasmic body, which has been shown to consist of a cluster of 8-nm filaments (A. Zantema et al., Virology 142:44-58, 1985). In transformed cells expressing the E1B region of Ad12, however, no association between the viral large T antigen and p53 was detectable. In the latter case, both proteins were found almost exclusively in the nucleus. The stability of p53 in both Ad5- and Ad12-transformed cells was increased relative to that in primary cells or cells immortalized by the E1A region only. Thus, the increased stability of p53 in Ad-transformed cells is not caused by association with a viral T antigen, but it correlates with expression of E1B and with morphological transformation.     

Crystal structure of a non-toxic mutant of heat-labile enterotoxin, which is a potent mucosal adjuvant.

Two closely related bacterial toxins, heat-labile enterotoxin (LT-I) and cholera toxin (CT), not only invoke a toxic activity that affects many victims worldwide but also contain a beneficial mucosal adjuvant activity that significantly enhances the potency of vaccines in general. For the purpose of vaccine design it is most interesting that the undesirable toxic activity of these toxins can be eliminated by the single-site mutation Ser63Lys in the A subunit while the mucosal adjuvant activity is still present. The crystal structure of the Ser63Lys mutant of LT-I is determined at 2.0 A resolution. Its structure appears to be essentially the same as the wild-type LT-I structure. The substitution Ser63Lys was designed, based on the wild-type LT-I crystal structure, to decrease toxicity by interfering with NAD binding and/or catalysis. In the mutant crystal structure, the newly introduced lysine side chain is indeed positioned such that it could potentially obstruct the productive binding mode of the substrate NAD while at the same time its positive charge could possibly interfere with the critical function of nearby charged groups in the active site of LT-I. The fact that the Ser63Lys mutant of LT-I does not disrupt the wild-type LT-I structure makes the non-toxic mutant potentially suitable, from a structural point of view, to be used as a vaccine to prevent enterotoxigenic E. coli infections. The structural similarity of mutant and wild-type toxin might also be the reason why the inactive Ser63Lys variant retains its adjuvant activity.     

Crystal structure of cholera toxin B-pentamer bound to receptor GM1 pentasaccharide.

Cholera toxin (CT) is an AB5 hexameric protein responsible for the symptoms produced by Vibrio cholerae infection. In the first step of cell intoxication, the B-pentamer of the toxin binds specifically to the branched pentasaccharide moiety of ganglioside GM1 on the surface of target human intestinal epithelial cells. We present here the crystal structure of the cholera toxin B-pentamer complexed with the GM1 pentasaccharide. Each receptor binding site on the toxin is found to lie primarily within a single B-subunit, with a single solvent-mediated hydrogen bond from residue Gly 33 of an adjacent subunit. The large majority of interactions between the receptor and the toxin involve the 2 terminal sugars of GM1, galactose and sialic acid, with a smaller contribution from the N-acetyl galactosamine residue. The binding of GM1 to cholera toxin thus resembles a 2-fingered grip: the Gal(beta 1-3)GalNAc moiety representing the "forefinger" and the sialic acid representing the "thumb." The residues forming the binding site are conserved between cholera toxin and the homologous heat-labile enterotoxin from Escherichia coli, with the sole exception of His 13. Some reported differences in the binding affinity of the 2 toxins for gangliosides other than GM1 may be rationalized by sequence differences at this residue. The CTB5:GM1 pentasaccharide complex described here provides a detailed view of a protein:ganglioside specific binding interaction, and as such is of interest not only for understanding cholera pathogenesis and for the design of drugs and development of vaccines but also for modeling other protein:ganglioside interactions such as those involved in GM1-mediated signal transduction.     

The redox state of transglutaminase 2 controls arterial remodeling

While inward remodeling of small arteries in response to low blood flow, hypertension, and chronic vasoconstriction depends on type 2 transglutaminase (TG2), the mechanisms of action have remained unresolved. We studied the regulation of TG2 activity, its (sub) cellular localization, substrates, and its specific mode of action during small artery inward remodeling. We found that inward remodeling of isolated mouse mesenteric arteries by exogenous TG2 required the presence of a reducing agent. The effect of TG2 depended on its cross-linking activity, as indicated by the lack of effect of mutant TG2. The cell-permeable reducing agent DTT, but not the cell-impermeable reducing agent TCEP, induced translocation of endogenous TG2 and high membrane-bound transglutaminase activity. This coincided with inward remodeling, characterized by a stiffening of the artery. The remodeling could be inhibited by a TG2 inhibitor and by the nitric oxide donor, SNAP. Using a pull-down assay and mass spectrometry, 21 proteins were identified as TG2 cross-linking substrates, including fibronectin, collagen and nidogen. Inward remodeling induced by low blood flow was associated with the upregulation of several anti-oxidant proteins, notably glutathione-S-transferase, and selenoprotein P. In conclusion, these results show that a reduced state induces smooth muscle membrane-bound TG2 activity. Inward remodeling results from the cross-linking of vicinal matrix proteins, causing a stiffening of the arterial wall.     

Tazobactam forms a stoichiometric trans-enamine intermediate in the E166A variant of SHV-1 beta-lactamase: 1.63 A crystal structure

Many pathogenic bacteria develop antibiotic resistance by utilizing beta-lactamases to degrade penicillin-like antibiotics. A commonly prescribed mechanism-based inhibitor of beta-lactamases is tazobactam, which can function either irreversibly or in a transient manner. We have demonstrated previously that the reaction between tazobactam and a deacylation deficient variant of SHV-1 beta-lactamase, E166A, could be followed in single crystals using Raman microscopy [Helfand, M. S., et al. (2003) Biochemistry 42, 13386-13392]. The Raman data show that maximal populations of an enamine-like intermediate occur 20-30 min after "soaking in" has commenced. By flash-freezing crystals in this time frame, we were able to trap the enamine species. The resulting 1.63 A resolution crystal structure revealed tazobactam covalently bound in the trans-enamine intermediate state with close to 100% occupancy in the active site. The Raman data also indicated that tazobactam forms a larger population of enamine than sulbactam or clavulanic acid does and that tazobactam's intermediate is also the most long-lived. The crystal structure provides a rationale for this finding since only tazobactam is able to form favorable intra- and intermolecular interactions in the active site that stabilize this trans-enamine intermediate. These interactions involve both the sulfone and triazolyl groups that distinguish tazobactam from clavulanic acid and sulbactam, respectively. The observed stabilization of the transient intermediate of tazobactam is thought to contribute to tazobactam's superior in vitro and in vivo clinical efficacy. Understanding the structural details of differing inhibitor effectiveness can aid the design of improved mechanism-based beta-lactamase inhibitors.     

5-Methyltetrahydrofolic acid and folic acid measured in plasma with liquid chromatography tandem mass spectrometry: applications to folate absorption and metabolism

We describe a liquid chromatography (LC) tandem mass spectrometry (MS-MS) method for the determination of 5-methyltetrahydrofolic acid (5-methylTHF) and folic acid concentrations and enrichments in human plasma. It was used to study absorption and initial metabolism in five volunteers with two simultaneously administered oral test doses ([(13)C(6)]folic acid in capsules and [(2)H(2)]folic acid in a drink). [(13)C(5)]5-methylTHF and [(2)H(4)]folic acid were used as internal standards. Plasma samples (2 ml) were purified using folate binding protein affinity columns, followed by a concentration step. After LC separation, folates were detected using positive electrospray ionization MS-MS under multiple reaction monitoring conditions. Calibrations were linear for 5-methylTHF over the range 1.2 x 10(-11) (=limit of detection) to 3.2 x 10(-7)mol/L and for folic acid over the range 5 x 10(-10) (=limit of detection) to 4.5 x 10(-8)mol/L. For 5-methylTHF concentration in plasma, intraassay coefficient of variation was within 8.6% (and for unlabeled 5-methylTHF it was within 2.8%) and interassay coefficient of variation was within 9.0%. For folic acid concentrations these coefficient of variations were within 7.5% and within 6.5%, respectively. The [(13)C(6)] and [(2)H(2)] isotopomers of folic acid and 5-methylTHF were measured in the plasma of each volunteer for 8h. After accounting for the time delay due to capsule opening, the modeling results showed no significant differences in absorption time, first pass effect, and elimination rate in the folic acid test doses in capsule or drink. We conclude that LC-MS-MS offers increased sensitivity for quantification of plasma concentrations and enrichments of 5-methylTHF and folic acid and is applicable to stable-isotope studies in humans.     

STING Millennium Suite: integrated software for extensive analyses of 3d structures of proteins and their complexes

Background  

The integration of many aspects of protein/DNA structure analysis is an important requirement for software products in general area of structural bioinformatics. In fact, there are too few software packages on the internet which can be described as successful in this respect. We might say that what is still missing is publicly available, web based software for interactive analysis of the sequence/structure/function of proteins and their complexes with DNA and ligands. Some of existing software packages do have certain level of integration and do offer analysis of several structure related parameters, however not to the extent generally demanded by a user.     

Potassium channels regulated by inositol 1,3,4,5-tetrakisphosphate and internal calcium in DDT1 MF-2 smooth muscle cells

This study was carried out to determine the intracellular components responsible for the transmembrane current evoked by stimulation of H1-histaminergic receptors in DDT1 MF-2 smooth muscle cells. Histamine elicited an outward current that was reversed below the K+ equilibrium potential and passed voltage-independent K+ channels. A histamine concentration-dependent rise in outward current and in cytoplasmic-free Ca2+ with similar time courses was observed. The histamine-induced current was not found after depletion of internal Ca2+ stores, suggesting a coupling between internal Ca2+ and K+ current. The time course of the initial increase in inositol (1,4,5)-trisphosphate (Ins (1,4,5)P3) caused by histamine differs from that of the internal Ca2+ response. However, a significant concentration-dependent increase in inositol (1,3,4,5)-tetrakisphosphate (Ins (1,3,4,5)P4) was seen during the whole stimulating period. The role of internal Ca2+, Ins (1,4,5)P3, and Ins (1,3,4,5)P4 on the outward current was also examined by the addition of these substances directly to the cytoplasm. Internal application of Ca2+ increased the amplitude and duration of the histamine-induced current whereas internal EGTA suppressed the outward current. Internal Ins (1,4,5)P3 did not affect the histamine-induced K+ current, Ins (1,3,4,5)P4 inhibited the outward current, and the combination of Ins (1,3,4,5)P4 and Ca2+ abolished this response. The noradrenaline response evoked under normal conditions is not reflected by a change in transmembrane current or a change in Ins (1,3,4,5)P4 but is associated with an increase in Ins (1,4,5)P3 and internal Ca2+. Stimulation of alpha 1-adrenoceptors, however, also evoked an outward current after the addition of Ins (1,3,4,5)P4 intracellularly. It is concluded that K+ channels, carrying the histamine outward current, are activated from the combined action of internal Ca2+ and Ins (1,3,4,5)P4.     

Heart Failure in Chronic Myocarditis: A Role for microRNAs?

Myocarditis is an inflammatory disease of the heart, which can persist over a long time. During this time, known as the chronic phase of myocarditis, ongoing inflammation damages the cardiomyocytes. The loss of cardiac cells culminates in the development of dilated cardiomyopathy, often followed by non-ischemic heart failure due to diminished cardiac function. During the course of the disease, expression levels of non-coding small RNAs, called microRNAs (miRNAs), change. Although mainly studied in the acute setting, some of these changes in expression level appear to persist in the chronic phase. In addition to being a much-needed diagnostic tool, these miRNA could provide new treatment options. miRNA-based intervention strategies already showed promising results in the treatment of ischemic cardiovascular diseases in preclinical animal models. By implementing more knowledge on the role of miRNAs in the progression towards heart failure, this can potentially be used in the development of miRNA-based therapeutic interventions in the treatment of myocarditis and thereby preventing the progression towards heart failure. The first part of this review will focus on the natural course of myocarditis and the progression towards heart failure. Secondly, we will discuss the current knowledge on alterations of miRNA expression patterns, and suggest some possible future interventions.