Molecular aspects of activation mechanisms of CF₁

The Ca2( +) -dependent ATPase activity of spinach chloroplast coupling factor 1 (CF?) is activated by treatment with dithiothreitol (DDT). If excess of this reagent is eliminated by gel filtration, an Eadie-Hofstee biphasic plot is obtained. These results are consistent with the existence of two active forms of the enzyme governed by the redox state. We have observed that SDS-polyacrylamide gel electrophoresis pattern is affected by the pretreatment of the samples under those two different conditions. Spontaneous activation of the samples, due to a limited proteolytic process, has also been detected. In this case the electrophoretic pattern was also affected. The protease implied in this process could be a cystein protease co-isolated with CF?. These observations suggest that limited proteolysis, as well as redox-induced changes, are involved in the physiological regulation of the enzyme.     

Antiarrhythmic properties of phenylpiperazine derivatives of phenytoin with α₁-adrenoceptor affinities

An association between α(1)-adrenoceptor affinities, hERG K(+)-antagonistic properties and antiarrhythmic activities for a series of phenylpiperazine derivatives of hydantoin (2a-21a) was investigated. New compounds were synthesized and tested for their affinity for α(1)-adrenoceptors in radioligand binding assay using [(3)H]-prazosin as a selective radioligand. Antiarrhythmic activities in adrenaline- and barium chloride-induced arrhythmia models, an influence of the phenylpiperazine derivatives on the ECG-components and blood pressure were tested in vivo in normotensive rats. The hERG K(+)-antagonistic properties of the most potent antiarrhythmic agents were investigated in silico by the use of program QikProp. The highest α(1)-adrenoceptor affinity (K(i)=4.7 nM) and the strongest antiarrhythmic activity in adrenaline induced arrhythmia (ED(50)=0.1 mg/kg) was found for 1-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-3-methyl-5,5-diphenylimidazolidine-2,4-dione hydrochloride (19a). The results indicated a significant correlation between α(1)-AR affinities (pK(i)) and antiarrhythmic activity (ED(50)) in adrenaline model (R(2)=0.92, p <0.005). Influence of the examined phenylpiperazine hydantoin derivatives on hERG K(+) channel, predicted by means of in silico methods, suggested their hERG K(+)-blocking properties.     

The binding interface of cytochrome c and cytochrome c₁ in the bc₁ complex: rationalizing the role of key residues

The interaction of cytochrome c with ubiquinol-cytochrome c oxidoreductase (bc₁ complex) has been studied for >30 years, yet many aspects remain unclear or controversial. We report the first molecular dynamic simulations of the cyt c-bc₁ complex interaction. Contrary to the results of crystallographic studies, our results show that there are multiple dynamic hydrogen bonds and salt bridges in the cyt c-c₁ interface. These include most of the basic cyt c residues previously implicated in chemical modification studies. We suggest that the static nature of x-ray structures can obscure the quantitative significance of electrostatic interactions between highly mobile residues. This provides a clear resolution of the discrepancy between the structural data and functional studies. It also suggests a general need to consider dynamic interactions of charged residues in protein-protein interfaces. In addition, a novel structural change in cyt c is reported, involving residues 21-25, which may be responsible for cyt c destabilization upon binding. We also propose a mechanism of interaction between cyt c₁ monomers responsible for limiting the binding of cyt c to only one molecule per bc₁ dimer by altering the affinity of the cytochrome c binding site on the second cyt c₁ monomer.     

Actions of a picomolar short-acting S1P₁ agonist in S1P₁-eGFP knock-in mice

Sphingosine 1-phosphate receptor 1 (S1P(1)) is critical for lymphocyte recirculation and is a clinical target for treatment of multiple sclerosis. By generating a short-duration S1P(1) agonist and mice in which fluorescently tagged S1P(1) replaces wild-type receptor, we elucidate physiological and agonist-perturbed changes in expression of S1P(1) at a subcellular level in vivo. We demonstrate differential downregulation of S1P(1) on lymphocytes and endothelia after agonist treatment.     

Increased expression of cannabinoid CB₁ receptors in Achilles tendinosis

Background

The endogenous cannabinoid system is involved in the control of pain. However, little is known as to the integrity of the cannabinoid system in human pain syndromes. Here we investigate the expression of the cannabinoid receptor 1 (CB₁) in human Achilles tendons from healthy volunteers and from patients with Achilles tendinosis.

Methodology

Cannabinoid CB₁ receptor immunoreactivity (CB₁IR) was evaluated in formalin-fixed biopsies from individuals suffering from painful Achilles tendinosis in comparison with healthy human Achilles tendons.

Principal Findings

CB₁IR was seen as a granular pattern in the tenocytes. CB₁IR was also observed in the blood vessel wall and in the perineurium of the nerve. Quantification of the immunoreactivity in tenocytes showed an increase of CB₁ receptor expression in tendinosis tissue compared to control tissue.

Conclusion

Expression of cannabinoid receptor 1 is increased in human Achilles tendinosis suggesting that the cannabinoid system may be dysregulated in this disorder.     

Role of AT₁ receptor-mediated salt retention in angiotensin II-dependent hypertension

Activation of type 1 angiotensin II (AT(1)) receptors in the kidney promotes blood pressure elevation and target organ damage, but whether renal AT(1) receptors influence the level of hypertension by stimulating sodium retention or by raising systemic vascular resistance has not been established. In the current studies, we used a kidney cross-transplantation strategy to determine whether increased sodium reabsorption by AT(1) receptors in the kidney mediates the chronic hypertensive response to angiotensin II. We found this to be true. In addition, we also identified a second, nontrivial component of blood pressure elevation induced by activation of renal AT(1) receptors that is sodium-independent. As the kidney has the capacity to limit the transmission of elevated systemic blood pressure into the renal microcirculation, prior studies struggled to clearly discriminate the relative contributions of blood pressure elevation vs. activation of AT(1) receptors to hypertensive kidney injury. In our model, we found that rapid surges in blood pressure, which may overcome the kidney's capacity to prevent perturbations in renal hemodynamics, correlate closely with kidney damage in hypertension. Moreover, maximal kidney injury in hypertension may require activation of a pool of nonrenal, systemic AT(1) receptors. These studies provide insight into precise mechanisms through which AT(1) receptor blockade influences the progression of hypertensive kidney disease.     

Assessment of dopamine D₁ receptor affinity and efficacy of three tetracyclic conformationally-restricted analogs of SKF38393

To assess the effect of conformational mobility on receptor activity, the β-phenyl substituent of dopamine D(1) agonist ligands of the phenylbenzazepine class, (±)-6,6a,7,8,9,13b-hexahydro-5H-benzo[d]naphtho[2,1-b]azepine-11,12-diol (8), and its oxygen and sulfur bioisosteres 9 and 10, respectively, were synthesized as conformationally-restricted analogs of SKF38393, a dopamine D(1)-selective partial agonist. Compounds trans-8b, 9, and 10 showed binding affinity comparable to that of SKF38393, but functionally, they displayed only very weak agonist activity. These results suggest that the conformationally-restricted structure of the analogs cannot adopt a binding orientation that is necessary for agonist activity.     

Identification of new antimalarial leads by use of virtual screening against cytochrome bc₁

Cytochrome bc(1) is a validated drug target in malaria parasites. The spread of Plasmodium falciparum strains resistant to multiple antimalarials emphasizes the urgent need for new drugs. We screened in silico the ZINC and MOE databases, using ligand- and structure-based approaches, to identify new leads for development. The most active compound presented an IC(50) value against cultured P. falciparum of 2 μM and a docking pose consistent with its activity.     

NirF is a periplasmic protein that binds d₁ heme as part of its essential role in d₁ heme biogenesis

The cytochrome cd? nitrite reductase from Paracoccus pantotrophus catalyses the one electron reduction of nitrite to nitric oxide using two heme cofactors. The site of nitrite reduction is the d? heme, which is synthesized under anaerobic conditions by using nirECFD-LGHJN gene products. In vivo studies with an unmarked deletion strain, ΔnirF, showed that this gene is essential for cd? assembly and consequently for denitrification, which was restored when the ΔnirF strain was complemented with wild-type, plasmid-borne, nirF. Removal of a signal sequence and deletion of a conserved N-terminal Gly-rich motif from the NirF coded on a plasmid resulted in loss of in vivo NirF activity. We demonstrate here that the product of the nirF gene is a periplasmic protein and, hence, must be involved in a late stage of the cofactor biosynthesis. In vitro studies with purified NirF established that it could bind d? heme. It is concluded that His41 of NirF, which aligns with His200 of the d? heme domain of cd?, is essential both for this binding and for the production of d? heme; replacement of His41 by Ala, Cys, Lys and Met all gave nonfunctional proteins. Potential functions of NirF are discussed.     

Enzyme characteristics of aminotransferase FumI of Sphingopyxis sp. MTA144 for deamination of hydrolyzed fumonisin B₁

Fumonisins are carcinogenic mycotoxins that are frequently found as natural contaminants in maize from warm climate regions around the world. The aminotransferase FumI is encoded as part of a gene cluster of Sphingopyxis sp. MTA144, which enables this bacterial strain to degrade fumonisin B₁ and related fumonisins. FumI catalyzes the deamination of the first intermediate of the catabolic pathway, hydrolyzed fumonisin B₁. We used a preparation of purified, His-tagged FumI, produced recombinantly in Escherichia coli in soluble form, for enzyme characterization. The structure of the reaction product was studied by NMR and identified as 2-keto hydrolyzed fumonisin B₁. Pyruvate was found to be the preferred co-substrate and amino group receptor (K _M = 490 μM at 10 μM hydrolyzed fumonisin B₁) of FumI, but other α-keto acids were also accepted as co-substrates. Addition of the co-enzyme pyridoxal phosphate to the enzyme preparation enhanced activity, and saturation was already reached at the lowest tested concentration of 10 μM. The enzyme showed activity in the range of pH 6 to 10 with an optimum at pH 8.5, and in the range of 6°C to 50°C with an optimum at 35°C. The aminotransferase worked best at low salt concentration. FumI activity could be recovered after preincubation at pH 4.0 or higher, but not lower. The aminotransferase was denatured after preincubation at 60°C for 1 h, and the residual activity was also reduced after preincubation at lower temperatures. At optimum conditions, the kinetic parameters K _M = 1.1 μM and k _cat = 104/min were determined with 5 mM pyruvate as co-substrate. Based on the enzyme characteristics, a technological application of FumI, in combination with the fumonisin carboxylesterase FumD for hydrolysis of fumonisins, for deamination and detoxification of hydrolyzed fumonisins seems possible, if the enzyme properties are considered.     

FTIR 2D correlation spectroscopy of α₁ and α₂ fractions of an alkali-pretreated gelatin

An alkali-pretreated gelatin (pI~4.9) was fractionated by means of alcohol coacervation and semi-preparative gel chromatography. The thermal responses of the isolated α fractions, the coacervate and the total gelatin were investigated by 2D-correlation FTIR spectroscopy in the amide I band region (1600-1700 cm?1). The gelation temperature was the same for all examined samples (24.5°C) while the melting temperature of the α? fraction was lower (30°C) than that of the other samples (32.5°C). The 2D COS plots indicate that on cooling (gelation) the core sequence of conformational changes is the same for all samples. On heating, however, the α? fraction deviates from the α?-containing samples and shows an earlier disappearance of the triple helix signal in the event sequence. The lower melting temperature (less thermostable gelatin gel) of the α? fraction thus results from a different conformational cascade of the α? chains upon melting. In all samples the initial conformational changes take place in the β-turns, providing further evidence for the models proposed previously.     

Quantitative genomics of 30 complex phenotypes in Wagyu x Angus F₁ progeny

In the present study, a total of 91 genes involved in various pathways were investigated for their associations with six carcass traits and twenty-four fatty acid composition phenotypes in a Wagyu×Angus reference population, including 43 Wagyu bulls and their potential 791 F(1) progeny. Of the 182 SNPs evaluated, 102 SNPs that were in Hardy-Weinberg equilibrium with minor allele frequencies (MAF>0.15) were selected for parentage assignment and association studies with these quantitative traits. The parentage assignment revealed that 40 of 43 Wagyu sires produced over 96.71% of the calves in the population. Linkage disequilibrium analysis identified 75 of 102 SNPs derived from 54 genes as tagged SNPs. After Bonferroni correction, single-marker analysis revealed a total of 113 significant associations between 44 genes and 29 phenotypes (adjusted P<0.05). Multiple-marker analysis confirmed single-gene associations for 10 traits, but revealed two-gene networks for 9 traits and three-gene networks for 8 traits. Particularly, we observed that TNF (tumor necrosis factor) gene is significantly associated with both beef marbling score (P=0.0016) and palmitic acid (C16:0) (P=0.0043), RCAN1 (regulator of calcineurin 1) with rib-eye area (P=0.0103), ASB3 (ankyrin repeat and SOCS box-containing 3) with backfat (P=0.0392), ABCA1 (ATP-binding cassette A1) with both palmitic acid (C16:0) (P=0.0025) and oleic acid (C18:1n9) (P=0.0114), SLC27A1(solute carrier family 27 A1) with oleic acid (C18:1n9) (P=0.0155), CRH (corticotropin releasing hormone) with both linolenic acid (OMEGA-3) (P=0.0200) and OMEGA 6:3 RATIO (P=0.0054), SLC27A2 (solute carrier family 27 A2) with both linoleic acid (OMEGA-6) (P=0.0121) and FAT (P=0.0333), GNG3 (guanine nucleotide binding protein gamma 3 with desaturase 9 (P=0.0115), and EFEMP1 (EGF containing fibulin-like extracellular matrix protein 1), PLTP (phospholipid transfer protein) and DSEL (dermatan sulfate epimerase-like) with conjugated linoleic acid (P=0.0042-0.0044), respectively, in the Wagyu x Angus F(1) population. In addition, we observed an interesting phenomenon that crossbreeding of different breeds might change gene actions to dominant and overdominant modes, thus explaining the origin of heterosis. The present study confirmed that these important families or pathway-based genes are useful targets for improving meat quality traits and healthful beef products in cattle.     

Intermonomer electron transfer between the low-potential b hemes of cytochrome bc₁

Cytochrome (cyt) bc(1) is a structural dimer with its monomers consisting of the Fe-S protein, cyt b, and cyt c(1) subunits. Its three-dimensional architecture depicts it as a symmetrical homodimer, but the mobility of the head domain of the Fe-S protein indicates that the functional enzyme exists in asymmetrical heterodimeric conformations. Here, we report a new genetic system for studying intra- and intermonomer interactions within the cyt bc(1) using the facultative phototrophic bacterium Rhodobacter capsulatus. The system involves two different sets of independently expressed cyt bc(1) structural genes carried by two plasmids that are coharbored by a cell without its endogenous enzyme. Our results indicate that coexpressed cyt bc(1) subunits were matured, assorted, and assembled in vivo into homo- and heterodimeric enzymes that can bear different mutations in each monomer. Using the system, the occurrence of intermonomer electron transfer between the low-potential b hemes of cyt bc(1) was probed by choosing mutations that perturb electron transfer at the hydroquinone oxidation (Q(o)) and quinone reduction (Q(i)) sites of the enzyme. The data demonstrate that active heterodimeric variants, formed of monomers carrying mutations that abolish only one of the two (Q(o) or Q(i)) active sites of each monomer, are produced, and they support photosynthetic growth of R. capsulatus. Detailed analyses of the physicochemical properties of membranes of these mutants, as well as purified homo- and heterodimeric cyt bc(1) preparations, demonstrated that efficient and productive electron transfer occurs between the low-potential b(L) hemes of the monomers in a heterodimeric enzyme. Overall findings are discussed with respect to intra- and intermonomer interactions that take place during the catalytic turnover of cyt bc(1).     

Synthesis and in vitro evaluation of derivatives of the β₁-adrenergic receptor antagonist HX-CH 44

Isopropyl- and fluoroisopropyl-amino derivatives of the β(1)-adrenergic receptor antagonist 2-[4-[3-(tert-butyl-amino)-2-hydroxypropoxy]phenyl]-3-methyl-6-methoxy-4(3H)-quinazolinone ((±)HX-CH 44) were synthesized, including a concise and efficient preparation of the core, 2-(4-hydroxyphenyl)-6-methoxy-3-methylquinazolin-4(3H)-one. In vitro binding assays showed that the fluorinated analog was selective towards β(1)-adrenergic receptors over β(2)-adrenergic and 5-HT(1A) receptors. An X-ray crystallographic characterization of the fluorinated analog is also reported.     

The Na-K-ATPase α₁β₁ heterodimer as a cell adhesion molecule in epithelia

The ion gradients generated by the Na-K-ATPase play a critical role in epithelia by driving transepithelial transport of various solutes. The efficiency of this Na-K-ATPase-driven vectorial transport depends on the integrity of epithelial junctions that maintain polar distribution of membrane transporters, including the basolateral sodium pump, and restrict paracellular diffusion of solutes. The review summarizes the data showing that, in addition to pumping ions, the Na-K-ATPase located at the sites of cell-cell junction acts as a cell adhesion molecule by interacting with the Na-K-ATPase of the adjacent cell in the intercellular space accompanied by anchoring to the cytoskeleton in the cytoplasm. The review also discusses the experimental evidence on the importance of a specific amino acid region in the extracellular domain of the Na-K-ATPase β(1) subunit for the Na-K-ATPase trans-dimerization and intercellular adhesion. Furthermore, a possible role of N-glycans linked to the Na-K-ATPase β(1) subunit in regulation of epithelial junctions by modulating β(1)-β(1) interactions is discussed.     

Allosteric property of the (Na⁺+K⁺)-ATPase β₁ subunit

(Na(+)+K(+))-ATPase (NKA) comprises two basic α and β subunits: The larger α subunit catalyzes the hydrolysis of ATP for active transport of Na(+) and K(+) ions across the plasma membrane; the smaller β subunit does not take part in the catalytic process of the enzyme. Little is known about allosteric regulation of the NKA β subunit. Here, we report a surprising finding that extracellular stimuli on the native β(1) subunit can generate a significant impact on the catalytic function of NKA. By using a β(1) subunit-specific monoclonal antibody JY2948, we found that the JY2948-β(1) subunit interaction markedly enhances the catalytic activity of the enzyme and increases the apparent affinity of Na(+) and K(+) ions for both ouabain-resistant rat NKA and ouabain-sensitive dog NKA. This study provides the first evidence to identify an allosteric binding site residing on the NKA β(1) subunit and uncovers the latent allosteric property of the β(1) subunit, which remotely controls the NKA catalytic function.     

Synthesis of a quinazoline derivative: a new α₁-adrenoceptor ligand for conjugation to quantum dots to study α₁-adrenoceptors in living cells

Quantum dots (QDs) that are conjugated to small molecule derivatives of drugs and endogenous ligands may be useful tools to study the distribution and dynamic of membrane bound receptors, ion channels and transporters in live cells. In order to use these tools, it is necessary to functionalize QDs with bioactive ligands. In this paper, we successfully synthesized a ligand of α(1)-adrenoceptor that could be conjugated to QDs. In addition, the conjugation of the ligands to QDs and their biological activity were evaluated through binding assay with 30 nM QD conjugates in living human embryonic kidney 293 cells.     

Lysosomal accumulation of Trk protein in brain of GM₁ -gangliosidosis mouse and its restoration by chemical chaperone

G(M1) -gangliosidosis is a fatal neurodegenerative disorder caused by deficiency of lysosomal acid β-galactosidase (β-gal). Accumulation of its substrate ganglioside G(M1) (G(M1) ) in lysosomes and other parts of the cell leads to progressive neurodegeneration, but underlying mechanisms remain unclear. Previous studies demonstrated an essential role for interaction of G(M1) with tropomyosin receptor kinase (Trk) receptors in neuronal growth, survival and differentiation. In this study we demonstrate accumulation of G(M1) in the cell-surface rafts and lysosomes of the β-gal knockout (β-gal-/-) mouse brain association with accumulation of Trk receptors and enhancement of its downstream signaling. Immunofluorescence and subcellular fractionation analysis revealed accumulation of Trk receptors in the late endosomes/lysosomes of the β-gal-/- mouse brain and their association with ubiquitin and p62. Administration of a chemical chaperone to β-gal-/- mouse expressing human mutant R201C protein resulted in a marked reduction of intracellular storage of G(M1) and phosphorylated Trk. These findings indicate that G(M1) accumulation in rafts causes activation of Trk signaling, which may participate in the pathogenesis of G(M1) -gangliosidosis.     

The CB₁ receptor-mediated endocannabinoid signaling and NGF: the novel targets of curcumin

Increasing interest has recently been attracted towards the identification of natural compounds including those with antidepressant properties. Curcumin has shown promising antidepressant effect, however, its molecular target(s) have not been well defined. Based on the interaction between the neurotrophins and endocannabinoid system as well as their contribution to the emotional reactivity and antidepressant action, here we show that 4-week treatment with curcumin, similar to the classical antidepressant amitriptyline, results in the sustained elevation of brain nerve growth factor (NGF) and endocannabinoids in dose-dependent and brain region-specific fashion. Pretreatment with cannabinoid CB₁ receptor neutral antagonist AM4113, but not the CB₂ antagonist SR144528, prevents the enhancement of brain NGF contents. AM4113 exerts no effect by itself. Our findings by presenting the CB₁ receptor-mediated endocannabinoid signaling and NGF as novel targets for curcumin, suggest that more attention should be focused on the therapeutic potential of herbal medicines including curcumin.