Nongenomic effects of mineralocorticoid receptor activation in the cardiovascular system

Fifteen years ago Wehling and colleagues showed unequivocal rapid effects of aldosterone, neither mimicked by cortisol nor blocked by spironolactone, and postulated that these nongenomic effects are mediated via a membrane receptor distinct from the classical mineralocorticoid receptor (MR). Several recent studies have challenged this view. Alzamora et al. showed 11beta-hydroxysteroid denydrogenase 1 and 2 (11betaHSD1, 11betaHSD2) expression in human vascular smooth muscle cells, and that aldosterone rapidly raises intracellular pH via sodium-hydrogen exchange; cortisol is without effect and spironolactone does not block the aldosterone response. When, however, 11betaHSD activity is blocked by carbenoxolone, cortisol shows agonist effects indistinguishable from aldosterone; in addition, the effect of both aldosterone and cortisol is blocked by the open E-ring, water soluble MR antagonist RU28318. In rabbit cardiomyocytes, aldosterone increases intracellular [Na+] by activating Na+/K+/2Cl- cotransport, with secondary effects on Na+/K+ pump activity. Pump current rises approximately 10-fold within 15', is unaffected by actinomycin D or the MR antagonist canrenone, and not elevated by cortisol. Pump current is, however, completely blocked by the open E-ring, water soluble MR antagonist K+ canrenoate and stoichometrically by cortisol. PKCepsilon agonist peptides (but not PKCalpha, PKCdelta or scrambled PKCepsilon peptides) mimic the effect of aldosterone, and PKCepsilon antagonist peptides block the effect. Very recently, cortisol has been shown to mimic the effect of aldosterone when cardiomyocyte redox state is altered by the installation of oxidized glutathione (GSSG) via the pipet, paralleling the effect of carbenoxolone on vascular smooth cells and suggesting possible pathophysiologic roles for an always glucocorticoid occupied MR.     

Quantitative comparison of the hydrogen bond network of A-state and native ubiquitin by hydrogen bond scalar couplings

The backbone hydrogen bond (H-bond) network of the partially folded A-state of ubiquitin (60% methanol, 40% water, pH 2) has been characterized quantitatively by (h3)J(NC)(') H-bond scalar couplings between the (15)N nuclei of amino acid H-bond donors and the (13)C carbonyl nuclei of the acceptors. Results on (h3)J(NC)(') couplings and the amide proton ((1)H(N)) chemical shifts for the A-state are compared quantitatively to the native state. The (h3)J(NC)(') correlations of the A-state show intact, nativelike H-bonds of the first beta-hairpin beta1/beta2 and the alpha-helix, albeit at lower strength, whereas the H-bonds in the C-terminal part change from a pure beta-structure to an all alpha-helical H(N)(i)-->O(i-4) connectivity pattern. A residue-specific analysis reveals that the conformations within the conserved secondary structure segments are much more homogeneous in the A-state than in the native state. Thus, the strong asymmetry of (h3)J(NC)(') couplings and (1)H(N) chemical shifts between the interior and exterior sides of the native state alpha-helix vanishes in the A-state. This indicates that the bend of this helix around the native state hydrophobic core is released in the homogeneous solvent environment of the A-state. Similarly, an irregularity in the behavior of H-bond I3-->L15 in hairpin beta1/beta2, which results from strong contacts to strand beta5 in the native state, is absent in the A-state. These findings rationalize the behavior of the (1)H(N) chemical shifts in both states and indicate that the A-state is in many aspects similar to the onset of thermal denaturation of the native state.     

Muon disrupts AKT hydrogen bond network in cancer

A cancer microenvironment generates strong hydrogen bond network system by the positive feedback loops supporting cancer complexity and robustness. Such network functions through the AKT locus generating high entropic energy supporting cancer metastatic robustness. Charged lepton particle muon follows the rule of Bragg effect during a collision with hydrogen network in cancer cells. Muon beam dismantles hydrogen bond network in cancer by the muon-catalyzed fusion, leading to apoptosis of cancer cells. Muon induces cumulative energy appearance on the hydrogen bond network in a cancer cell with its fast decay to an electron and two neutrinos. Thus, muon beam, muonic atom, muon neutrino shower, and electrons simultaneously cause fast neutralization of the AKT hydrogen bond network by the conversion of hydrogen into deuterium or helium, inactivating the hydrogen bond networks and inducing failure of cancer complexity and robustness with the disappearance of a malignant phenotype.     

A hydrogen bond in loop A is critical for the binding and function of the 5-HT3 receptor

The binding sites of Cys-loop receptors are formed from at least six loops (A-F). Here we have used mutagenesis, radioligand binding, voltage clamp electrophysiology, and homology modeling to probe the role of two residues in loop A of the 5-HT3 receptor: Asn128 and Glu129. The data show that substitution of Asn128, with a range of alternative natural and unnatural amino acids, changed the EC50 (from approximately 10-fold more potent to approximately 10-fold less potent than that of the wild type), increased the maximal peak current for mCPBG compared to 5-HT (R max) 2-19-fold, and decreased n H, indicating this residue is involved in receptor gating; we propose Asn128 faces away from the binding pocket and plays a role in facilitating transitions between conformational states. Substitutions of Glu129 resulted in functional receptors only when the residue could accept a hydrogen bond, but with both these and other substitutions, no [(3)H]granisetron binding could be detected, indicating a role in ligand binding. We propose that Glu129 faces into the binding pocket, where, through its ability to hydrogen bond, it plays a critical role in ligand binding. Thus, the data support a modified model of the 5-HT3 receptor binding site and show that loop A plays a critical role in both the ligand binding and function of this receptor.     

Identification of the erythropoietin receptor domain required for calcium channel activation.

Erythropoietin (Epo) activates a voltage-independent Ca2+ channel that is dependent on tyrosine phosphorylation. To identify the domain(s) of the Epo receptor (Epo-R) required for Epo-induced Ca2+ influx, Chinese hamster ovary (CHO) cells were transfected with wild-type or mutant Epo receptors subcloned into pTracer-cytomegalovirus vector. This vector contains an SV40 early promoter, which drives expression of the green fluorescent protein (GFP) gene, and a cytomegalovirus immediate-early promoter driving expression of the Epo-R. Successful transfection was verified in single cells by detection of GFP, and intracellular Ca2+ ([Ca]i) changes were simultaneously monitored with rhod-2. Transfection of CHO cells with pTracer encoding wild-type Epo-R, but not pTracer alone, resulted in an Epo-induced [Ca]i increase that was abolished in cells transfected with Epo-R F8 (all eight cytoplasmic tyrosines substituted). Transfection with carboxyl-terminal deletion mutants indicated that removal of the terminal four tyrosine phosphorylation sites, but not the tyrosine at position 479, abolished Epo-induced [Ca]i increase, suggesting that tyrosines at positions 443, 460, and/or 464 are important. In CHO cells transfected with mutant Epo-R in which phenylalanine was substituted for individual tyrosines, a significant increase in [Ca]i was observed with mutants Epo-R Y443F and Epo-R Y464F. The rise in [Ca]i was abolished in cells transfected with Epo-R Y460F. Results were confirmed with CHO cells transfected with plasmids expressing Epo-R mutants in which individual tyrosines were added back to Epo-R F8 and in stably transfected Ba/F3 cells. These results demonstrate a critical role for the Epo-R cytoplasmic tyrosine 460 in Epo-stimulated Ca2+ influx.     

A transmembrane leucine zipper is required for activation of the dimeric receptor tyrosine kinase DDR1

Receptor tyrosine kinases of the discoidin domain family, DDR1 and DDR2, are activated by different types of collagen and play important roles in cell adhesion, migration, proliferation, and matrix remodeling. In a previous study, we found that collagen binding by the discoidin domain receptors (DDRs) requires dimerization of their extracellular domains (Leitinger, B. (2003) J. Biol. Chem. 278, 16761-16769), indicating that the paradigm of ligand-induced receptor dimerization may not apply to the DDRs. Using chemical cross-linking and co-immunoprecipitation of differently tagged DDRs, we now show that the DDRs form ligand-independent dimers in the biosynthetic pathway and on the cell surface. We further show that both the extracellular and the cytoplasmic domains are individually dispensable for DDR1 dimerization. The DDR1 transmembrane domain contains two putative dimerization motifs, a leucine zipper and a GXXXG motif. Mutations disrupting the leucine zipper strongly impaired collagen-induced transmembrane signaling, although the mutant DDR1 proteins were still able to dimerize, whereas mutation of the GXXXG motif had no effect. A bacterial reporter assay (named TOXCAT) showed that the DDR1 transmembrane domain has a strong potential for self-association in a biological membrane and that this interaction occurs via the leucine zipper and not the GXXXG motif. Our results demonstrate that the DDRs exist as stable dimers in the absence of ligand and that receptor activation requires specific interactions made by the transmembrane leucine zipper.     

Mu-opioid receptor and CREB activation are required for nicotine reward

Environmental cues associated with nicotine delivery are an important part of the stimulus that sustains smoking behavior and is often coupled with craving and relapse; however, the neuronal circuitry and molecular substrates underlying this process are still poorly understood. Exposure to an environment previously associated with rewarding properties of nicotine results in an increase of CREB phosphorylation similar to that seen following nicotine administration, and this response is absent in MOR(-/-) mice. Moreover, a single administration of an opioid receptor antagonist, naloxone, blocks both the conditioned molecular response (CREB phosphorylation) and the conditioned behavioral response (nicotine reward) in a place preference paradigm. Lastly, repeated nicotine administration results in increased expression of MORs. However, this effect, along with rewarding properties of nicotine, is blocked in mice with a targeted disruption in the CREB gene. Together, pharmacologic and genetic manipulations indicate that phosphorylation of CREB and upregulation of functional MORs are required for nicotine-conditioned reward.     

A novel halogen bond and a better-known hydrogen bond cooperation of neonicotinoid and insect nicotinic acetylcholine receptor recognition

Neonicotinoid insecticides target the insect nicotinic acetylcholine receptor (nAChR) and are highly effective against the piercing-sucking pests. To explore the molecular interaction mechanism between the neonicotinoids and the insect nAChR, some key neonicotinoid compounds were docked into Aplysia californica acetylcholine binding protein (Ac-AChBP), which serves as a suitable structural surrogate of the insect nAChR. The binding mode study showed that the hydrogen bond force between the electronegative pharmacophore of the neonicotinoids and Cys190NH of the target binding pocket is crucial to the high efficiency of the neonicotinoids. Increasing the coplanarity between the guanidine or amidine and the electronegative pharmacophore of the neonicotinoids could increase the Π-Π stacking effect with Tyr188 of the Ac-AChBP and thus enhance the insecticidal potency. The introduction of an azide group to the chloropyridine ring of the neonicotinoids would reduce its binding ability due to the disappearance of a novel halogen bonding interaction. A series of novel neonicotinoid molecules were designed based on the halogen bonding interaction and two compounds with 6-bromopyridine-3-yl and 6-(trifluoromethyl)-3-pyridinyl were found to be with potential insecticidal activities.     

Atomic dissection of the hydrogen bond network for transition-state analogue binding to purine nucleoside phosphorylase

Immucillin-H (ImmH) and immucillin-G (ImmG) were previously reported as transition-state analogues for bovine purine nucleoside phosphorylase (PNP) and are the most powerful inhibitors reported for the enzyme (K(i) = 23 and 30 pM). Sixteen new immucillins are used to probe the atomic interactions that cause tight binding for bovine PNP. Eight analogues of ImmH are identified with equilibrium dissociation constants of 1 nM or below. A novel crystal structure of bovine PNP-ImmG-PO(4) is described. Crystal structures of ImmH and ImmG bound to bovine PNP indicate that nearly every H-bond donor/acceptor site on the inhibitor is fully engaged in favorable H-bond partners. Chemical modification of the immucillins is used to quantitate the energetics for each contact at the catalytic site. Conversion of the 6-carbonyl oxygen to a 6-amino group (ImmH to ImmA) increases the dissociation constant from 23 pM to 2.6 million pM. Conversion of the 4'-imino group to a 4'-oxygen (ImmH to 9-deazainosine) increases the dissociation constant from 23 pM to 2.0 million pM. Substituents that induce small pK(a) changes at N-7 demonstrate modest loss of affinity. Thus, 8-F or 8-CH(3)-substitutions decrease affinity less than 10-fold. But a change in the deazapurine ring to convert N-7 from a H-bond donor to a H-bond acceptor (ImmH to 4-aza-3-deaza-ImmH) decreases affinity by >10(7). Introduction of a methylene bridge between 9-deazahypoxanthine and the iminoribitol (9-(1'-CH(2))-ImmH) increased the distance between leaving and oxacarbenium groups and increased K(i) to 91 000 pM. Catalytic site energetics for 20 substitutions in the transition-state analogue are analyzed in this approach. Disruption of the H-bond pattern that defines the transition-state ensemble leads to a large decrease in binding affinity. Changes in a single H-bond contact site cause up to 10.1 kcal/mol loss of binding energy, requiring a cooperative H-bond pattern in binding the transition-state analogues. Groups involved in leaving group activation and ribooxacarbenium ion stabilization are central to the H-bond network that provides transition-state stabilization and tight binding of the immucillins.     

Proposal for a hydrogen bond network in the active site of the prototypic gamma-class carbonic anhydrase

The crystal structure of Cam, the prototypic gamma-class carbonic anhydrase, reveals active site residues Gln75, Asn73, and Asn 202 previously hypothesized to participate in catalysis. These potential roles were investigated for the first time by kinetic analyses of site-specific replacement variants of the zinc and cobalt forms of Cam. Gln75 replacement variants showed large decreases in k(cat)/K(m) relative to wild-type. Further, the Gln75 variants showed a loss of the pK(a) in pH versus k(cat)/K(m) profiles previously attributed to ionization of the metal-bound water yielding the hydroxyl group attacking CO(2). These results support the previously proposed role for Gln75 in hydrogen bonding with the catalytic hydroxyl orienting it for attack on CO(2). Kinetic analyses of Asn73 variants were consistent with a role in hydrogen bonding with Gln75 to position it for optimal interaction with the catalytic hydroxyl. Kinetic analyses of Asn202 variants showed substantial decreases in k(cat)/K(m) relative to the wild-type enzyme supporting the previously hypothesized role in polarizing CO(2) and facilitating attack from the metal-bound hydroxyl. On the basis of results presented here, and previously reported structural analyses, we present a catalytic mechanism involving Gln75, Asn73, and Asn202 that also suggests a role for Glu62 not previously recognized. Finally, the results suggest that the gamma-, beta-, and alpha-class carbonic anhydrases each independently evolved variations of a fundamental hydrogen bond network essential for catalysis.     

Evidence for an extended hydrogen bond network in the binding site of the nicotinic receptor: role of the vicinal disulfide of the alpha1 subunit

The defining feature of the α subunits of the family of nicotinic acetylcholine receptors is a vicinal disulfide between Cys-192 and Cys-193. Although this structure has played a pivotal role in a number of pioneering studies of nicotinic receptors, its functional role in native receptors remains uncertain. Using mutant cycle analysis and unnatural residue mutagenesis, including backbone mutagenesis of the peptide bond of the vicinal disulfide, we have established the presence of a network of hydrogen bonds that extends from that peptide NH, across a β turn to another backbone hydrogen bond, and then across the subunit interface to the side chain of a functionally important Asp residue in the non-α subunit. We propose that the role of the vicinal disulfide is to distort the β turn and thereby properly position a backbone NH for intersubunit hydrogen bonding to the key Asp.     

A simple hydrogen bond potential function for conformational studies

Since hydrogen bonding plays an important role in determining energetically favourable conformations of biological molecules, a computationally simple and convenient semiempirical function has been evaluated by constraining a function to conform to the experimentally observed data for isolated simple systems of hydrogen bonded dimers. The function is found to be satisfactory for use in conformational energy calculations.     

Adrenomedullin disulfide bond mimetics uncover structural requirements for AM1 receptor activation

Adrenomedullin (ADM) is a vasoactive peptide hormone of 52 amino acids and belongs to the calcitonin peptide superfamily. Its vasodilative effects are mediated by the interaction with the calcitonin receptor‐like receptor (CLR), a class B G protein‐coupled receptor (GPCR), associated with the receptor activity modifying protein 2 (RAMP2) and functionally described as AM‐1 receptor (AM₁R). A disulfide‐bonded ring structure consisting of six amino acids between Cys¹⁶ and Cys²¹ has been shown to be a key motif for receptor activation. However, the specific structural requirements remain to be elucidated. To investigate the influence of ring size and position of additional functional groups that replace the native disulfide bond, we generated ADM analogs containing thioether, thioacetal, alkane, and lactam bonds between amino acids 16 and 21 by Fmoc/t‐Bu solid phase peptide synthesis. Activity studies of the ADM disulfide bond mimetics (DSBM) revealed a strong impact of structural parameters. Interestingly, an increased ring size was tolerated but the activity of lactam‐based mimetics depended on its position within the bridging structure. Furthermore, we found the thioacetal as well as the thioether‐based mimetics to be well accepted with full AM₁R activity. While a reduced selectivity over the calcitonin gene‐related peptide receptor (CGRPR) was observed for the thioethers, the thioacetal was able to retain a wild–type‐like selectivity profile. The carbon analog in contrast displayed weak antagonistic properties. These results provide insight into the structural requirements for AM₁R activation as well as new possibilities for the development of metabolically stabilized analogs for therapeutic applications of ADM.     

A hydrogen bond network in the active site of Anabaena ferredoxin-NADP+ reductase modulates its catalytic efficiency

Ferredoxin-nicotinamide–adenine dinucleotide phosphate (NADP⁺) reductase (FNR) catalyses the production of reduced nicotinamide–adenine dinucleotide phosphate (NADPH) in photosynthetic organisms, where its flavin adenine dinucleotide (FAD) cofactor takes two electrons from two reduced ferredoxin (Fd) molecules in two sequential steps, and transfers them to NADP⁺ in a single hydride transfer (HT) step. Despite the good knowledge of this catalytic machinery, additional roles can still be envisaged for already reported key residues, and new features are added to residues not previously identified as having a particular role in the mechanism. Here, we analyse for the first time the role of Ser59 in Anabaena FNR, a residue suggested by recent theoretical simulations as putatively involved in competent binding of the coenzyme in the active site by cooperating with Ser80. We show that Ser59 indirectly modulates the geometry of the active site, the interaction with substrates and the electronic properties of the isoalloxazine ring, and in consequence the electron transfer (ET) and HT processes. Additionally, we revise the role of Tyr79 and Ser80, previously investigated in homologous enzymes from plants. Our results probe that the active site of FNR is tuned by a H-bond network that involves the side-chains of these residues and that results to critical optimal substrate binding, exchange of electrons and, particularly, competent disposition of the C4n (hydride acceptor/donor) of the nicotinamide moiety of the coenzyme during the reversible HT event.     

Rat lung possesses the mineralocorticoid receptor.

Lung cytosol from male, adrenalectomized rats was screened for the mineralocorticoid receptor (MCR) by a polyclonal antiserum raised in the rabbit against rat renal antigen. Western blot analysis revealed a single 98 kDa band, like the MCR purified biochemically. The MCR could also be photolabelled for the first time by 3H-R 5020 in this very 98 kDa region that was displaced by RU 26752 specific to MCR. Immune IgG was able to precipitate the MCR-3H-RU 26752 complex, and to displace the same to high molecular weight regions during gel permeation chromatography on Sephacryl columns. Thus, MCR mediated actions need to be redefined. Furthermore, the technique of photochemical labelling forms a novel tool to assess MCR specificity, and to dissect its structure and function.