Angiotensin II type 1 receptor A1166C gene polymorphism and essential hypertension in Calabar and Uyo cities,Nigeria

OBJECTIVES:

The angiotensin II protein is a vasoconstrictor that exerts most of its influence through the angiotensin II type 1 receptor (AT₁R). Inconsistent association between the A1166C polymorphism of the AT₁R gene and hypertension has been reported among various populations but not among the peoples of Calabar and Uyo. This study was designed to determine the frequency of the A1166C polymorphism of the AT₁R gene and its association with hypertension in a sample population of Calabar and Uyo.

MATERIALS AND METHODS:

A population-based case control design consisting of total of 1224 participants, 612 each of patients and controls were randomly recruited from hypertension clinics and the general population. Genotyping of the A1166C allele of the AT₁R gene to identify variants was performed using polymerase chain reaction and restriction enzyme digestion. Multiple regressions were applied to test whether the A1166 genotypes were predictors of hypertension.

RESULTS:

99% of the study population had the wild type AA genotype, and 1% was AC heterozygous carriers of the A1166C polymorphism.

CONCLUSION:

The A1166C polymorphism was not a predictor of hypertension in the sample population of Calabar and Uyo.     

Anthraquinone-2-sulfonic acid (AQ2S) is A Novel Neurotherapeutic Agent

Anthraquinone derivatives such as emodin have recently been shown to protect in models of beta amyloid β (Aβ) and tau aggregation-induced cell death. The mechanisms of action possibly involve preconditioning effects, anti-aggregation properties, and/or enhancing the phosphatidylinositol-3-kinase (PI3K)/AKT survival mechanism. We studied several natural (emodin, rhein, and aloin) and synthetic (AQ2S) anthraquinones, to screen for post-treatment therapeutic benefit in two models of neuronal death, namely hydrogen peroxide (H₂O₂) and staurosporine (STS)-induced injury. Treatment with emodin, rhein, or aloin failed to reduce H₂O₂ injury. Moreover, consistent with emodin behaving like a mild toxin, it exacerbated oxidative injury at the highest concentration used (50 μM) in our post-treatment paradigm, and potently inhibited AKT. In contrast, AQ2S was neuroprotective. It reduced H₂O₂ injury at 50 and 75 μM. In addition, AQ2S potently inhibited staurosporine (STS)-induced injury. The mechanisms of action involve caspase inhibition and AKT activation. However, blockade of AKT signaling with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 failed to abolish AQ2S-mediated protection on the STS assay. This is the first study to report that AQ2S is a new neuroprotective compound and a novel caspase inhibitor.     

LncRNA H19 promotes the proliferation of pulmonary artery smooth muscle cells through AT<Subscript>1</Subscript>R via sponging let-7b in monocrotaline-induced pulmonary arterial hypertension

Background

Pulmonary arterial hypertension (PAH) is related to inflammation, and the lncRNA H19 is associated with inflammation. However, whether PDGF-BB-H19-let-7b-AT₁R axis contributes to the pathogenesis of PAH has not been thoroughly elucidated to date. This study investigated the role of H19 in PAH and its related mechanism.

Methods

In the present study, SD rats, C57/BL6 mice and H19?/? mice were injected with monocrotaline (MCT) to establish a PAH model. H19 was detected in the cytokine-stimulated pulmonary arterial smooth muscle cells (PASMCs), serum and lungs of rats/mice. H19 overexpression and knockdown experiments were also conducted. A dual luciferase reporter assay was used to explore whether let-7b is a sponge miRNA of H19, and AT₁R is a novel target of let-7b. A CCK-8 assay and flow cytometry were used to analyse cell proliferation.

Results

The results showed that H19 was highly expressed in the serum and lungs of MCT-induced rats/mice, and H19 was upregulated by PDGF-BB in vitro. H19 upregulated AT₁R expression via sponging miRNA let-7b following PDGF-BB stimulation. AT₁R is a novel target of let-7b. Moreover, the overexpression of H19 and AT₁R could facilitate PASMCs proliferation in vitro. H19 knockout protected mice from pulmonary artery remodeling and PAH following MCT treatment.

Conclusion

Our study showed that H19 is highly expressed in MCT-induced rodent lungs and upregulated by PDGF-BB. The H19-let-7b-AT₁R axis contributed to the pathogenesis of PAH by stimulating PASMCs proliferation. The H19 knockout had a protective role in the development of PAH. H19 may be a potential tar-get for the treatment of PAH.

     

Adenosine A2A receptor activation is determinant for BDNF actions upon GABA and glutamate release from rat hippocampal synaptosomes

Adenosine, through A_2A receptor (A_2AR) activation, can act as a metamodulator, controlling the actions of other modulators, as brain-derived neurotrophic factor (BDNF). Most of the metamodulatory actions of adenosine in the hippocampus have been evaluated in excitatory synapses. However, adenosine and BDNF can also influence GABAergic transmission. We thus evaluated the role of A_2AR on the modulatory effect of BDNF upon glutamate and GABA release from isolated hippocampal nerve terminals (synaptosomes). BDNF (30 ng/ml) enhanced K⁺-evoked [³H]glutamate release and inhibited the K⁺-evoked [³H]GABA release from synaptosomes. The effect of BDNF on both glutamate and GABA release requires tonic activation of adenosine A_2AR since for both neurotransmitters, the BDNF action was blocked by the A_2AR antagonist SCH 58261 (50 nM). In the presence of the A_2AR agonist, {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 (30 nM), the effect of BDNF on either glutamate or GABA release was, however, not potentiated. It is concluded that both the inhibitory actions of BDNF on GABA release as well as the facilitatory action of the neurotrophin on glutamate release are dependent on the activation of adenosine A_2AR by endogenous adenosine. However, these actions could not be further enhanced by exogenous activation of A_2AR.     

Themis2/ICB1 Is a Signaling Scaffold That Selectively Regulates Macrophage Toll-Like Receptor Signaling and Cytokine Production

Background

Thymocyte expressed molecule involved in selection 1 (Themis1, SwissProt accession number {"type":"entrez-protein","attrs":{"text":"Q8BGW0","term_id":"81896053","term_text":"Q8BGW0"}}Q8BGW0) is the recently characterised founder member of a novel family of proteins. A second member of this family, Themis2 ({"type":"entrez-protein","attrs":{"text":"Q91YX0","term_id":"73920022","term_text":"Q91YX0"}}Q91YX0), also known as ICB1 (Induced on contact with basement membrane 1), remains unreported at the protein level despite microarray and EST databases reporting Themis2 mRNA expression in B cells and macrophages.

Methodology/Principal Findings

Here we characterise Themis2 protein for the first time and show that it acts as a macrophage signalling scaffold, exerting a receptor-, mediator- and signalling pathway-specific effect on TLR responses in RAW 264.7 macrophages. Themis2 over-expression enhanced the LPS-induced production of TNF but not IL-6 or Cox-2, nor TNF production induced by ligands for TLR2 (PAM3) or TLR3 (poly I∶C). Moreover, LPS-induced activation of the MAP kinases ERK and p38 was enhanced in cells over-expressing Themis2 whereas the activation of JNK, IRF3 or NF-κB p65, was unaffected. Depletion of Themis2 protein by RNA inteference inhibited LPS-induced TNF production in primary human macrophages demonstrating a requirement for Themis2 in this event. Themis2 was inducibly tyrosine phosphorylated upon LPS challenge and interacted with Lyn kinase ({"type":"entrez-protein","attrs":{"text":"P25911","term_id":"2507208","term_text":"P25911"}}P25911), the Rho guanine nucleotide exchange factor, Vav ({"type":"entrez-protein","attrs":{"text":"P27870","term_id":"137483","term_text":"P27870"}}P27870), and the adaptor protein Grb2 ({"type":"entrez-protein","attrs":{"text":"Q60631","term_id":"2498425","term_text":"Q60631"}}Q60631). Mutation of either tyrosine 660 or a proline-rich sequence (PPPRPPK) simultaneously interrupted this complex and reduced by approximately 50% the capacity of Themis2 to promote LPS-induced TNF production. Finally, Themis2 protein expression was induced during macrophage development from murine bone marrow precursors and was regulated by inflammatory stimuli both in vitro and in vivo.

Conclusions/Significance

We hypothesise that Themis2 may constitute a novel, physiological control point in macrophage inflammatory responses.     

Dopamine Receptors Modulate Cytotoxicity of Natural Killer Cells via cAMP-PKA-CREB Signaling Pathway

Dopamine (DA), a neurotransmitter in the nervous system, has been shown to modulate immune function. We have previously reported that five subtypes of DA receptors, including D1R, D2R, D3R, D4R and D5R, are expressed in T lymphocytes and they are involved in regulation of T cells. However, roles of these DA receptor subtypes and their coupled signal-transduction pathway in modulation of natural killer (NK) cells still remain to be clarified. The spleen of mice was harvested and NK cells were isolated and purified by negative selection using magnetic activated cell sorting. After NK cells were incubated with various drugs for 4 h, flow cytometry measured cytotoxicity of NK cells against YAC-1 lymphoma cells. NK cells expressed the five subtypes of DA receptors at mRNA and protein levels. Activation of D1-like receptors (including D1R and D5R) with agonist {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 enhanced NK cell cytotoxicity, but activation of D2-like receptors (including D2R, D3R and D4R) with agonist quinpirole attenuated NK cells. Simultaneously, {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 elevated D1R and D5R expression, cAMP content, and phosphorylated cAMP-response element-binding (CREB) level in NK cells, while quinpirole reduced D3R and D4R expression, cAMP content, and phosphorylated CREB level in NK cells. These effects of {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 were blocked by {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390, an antagonist of D1-like receptors, and quinpirole effects were abolished by haloperidol, an antagonist of D2-like receptors. In support these results, H89, an inhibitor of phosphokinase A (PKA), prevented the {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393-dependent enhancement of NK cells and forskolin, an activator of adenylyl cyclase (AC), counteracted the quinpirole-dependent suppression of NK cells. These findings show that DA receptor subtypes are involved in modulation of NK cells and suggest that D1-like receptors facilitate NK cells by stimulating D1R/D5R-cAMP-PKA-CREB signaling pathway and D2-like receptors suppress NK cells by inhibiting D3R/D4R-cAMP-PKA-CREB signaling pathway. The results may provide more targets of therapeutic strategy for neuroimmune diseases.     

Analysis of the Arabinose-5-Phosphate Isomerase of Bacteroides fragilis Provides Insight into Regulation of Single-Domain Arabinose Phosphate Isomerases

Arabinose-5-phosphate isomerases (APIs) catalyze the interconversion of d-ribulose-5-phosphate and d-arabinose-5-phosphate, the first step in the biosynthesis of 3-deoxy-d-manno-octulosonic acid (Kdo), an essential component of the lipopolysaccharide in Gram-negative bacteria. Classical APIs, such as Escherichia coli KdsD, contain a sugar isomerase domain and a tandem cystathionine beta-synthase domain. Despite substantial effort, little is known about structure-function relationships in these APIs. We recently reported an API containing only a sugar isomerase domain. This protein, c3406 from E. coli CFT073, has no known physiological function. In this study, we investigated a putative single-domain API from the anaerobic Gram-negative bacterium Bacteroides fragilis. This putative API (UniProt ID {"type":"entrez-protein","attrs":{"text":"Q5LIW1","term_id":"81317194","term_text":"Q5LIW1"}}Q5LIW1) is the only protein encoded by the B. fragilis genome with significant identity to any known API, suggesting that it is responsible for lipopolysaccharide biosynthesis in B. fragilis. We tested this hypothesis by preparing recombinant {"type":"entrez-protein","attrs":{"text":"Q5LIW1","term_id":"81317194","term_text":"Q5LIW1"}}Q5LIW1 protein (here referred to by the UniProt ID {"type":"entrez-protein","attrs":{"text":"Q5LIW1","term_id":"81317194","term_text":"Q5LIW1"}}Q5LIW1), characterizing its API activity in vitro, and demonstrating that the gene encoding {"type":"entrez-protein","attrs":{"text":"Q5LIW1","term_id":"81317194","term_text":"Q5LIW1"}}Q5LIW1 (GenBank ID {"type":"entrez-protein","attrs":{"text":"YP_209877.1","term_id":"60679733","term_text":"YP_209877.1"}}YP_209877.1) was able to complement an API-deficient E. coli strain. We demonstrated that {"type":"entrez-protein","attrs":{"text":"Q5LIW1","term_id":"81317194","term_text":"Q5LIW1"}}Q5LIW1 is inhibited by cytidine 5′-monophospho-3-deoxy-d-manno-2-octulosonic acid, the final product of the Kdo biosynthesis pathway, with a K_i of 1.91 μM. These results support the assertion that {"type":"entrez-protein","attrs":{"text":"Q5LIW1","term_id":"81317194","term_text":"Q5LIW1"}}Q5LIW1 is the API that supports lipopolysaccharide biosynthesis in B. fragilis and is subject to feedback regulation by CMP-Kdo. The sugar isomerase domain of E. coli KdsD, lacking the two cystathionine beta-synthase domains, demonstrated API activity and was further characterized. These results suggest that {"type":"entrez-protein","attrs":{"text":"Q5LIW1","term_id":"81317194","term_text":"Q5LIW1"}}Q5LIW1 may be a suitable system to study API structure-function relationships.     

Deceleration of the E1P-E2P transition and ion transport by mutation of potentially salt bridge-forming residues Lys-791 and Glu-820 in gastric H+/K+-ATPase

A lysine residue within the highly conserved center of the fifth transmembrane segment in P_IIC-type ATPase α-subunits is uniquely found in H,K-ATPases instead of a serine in all Na,K-ATPase isoforms. Because previous studies suggested a prominent role of this residue in determining the electrogenicity of non-gastric H,K-ATPase and in pK_a modulation of the proton-translocating residues in the gastric H,K-ATPases as well, we investigated its functional significance for ion transport by expressing several Lys-791 variants of the gastric H,K-ATPase in Xenopus oocytes. Although the mutant proteins were all detected at the cell surface, none of the investigated mutants displayed any measurable K⁺-induced stationary currents. In Rb⁺ uptake measurements, replacement of Lys-791 by Arg, Ala, Ser, and Glu substantially impaired transport activity and reduced the sensitivity toward the E₂-specific inhibitor {"type":"entrez-protein","attrs":{"text":"SCH28080","term_id":"1053015931","term_text":"SCH28080"}}SCH28080. Furthermore, voltage clamp fluorometry using a reporter site in the TM5/TM6 loop for labeling with tetra-methylrhodamine-6-maleimide revealed markedly changed fluorescence signals. All four investigated mutants exhibited a strong shift toward the E₁P state, in agreement with their reduced {"type":"entrez-protein","attrs":{"text":"SCH28080","term_id":"1053015931","term_text":"SCH28080"}}SCH28080 sensitivity, and an about 5–10-fold decreased forward rate constant of the E₁P ↔ E₂P conformational transition, thus explaining the E₁P shift and the reduced Rb⁺ transport activity. When Glu-820 in TM6 adjacent to Lys-791 was replaced by non-charged or positively charged amino acids, severe effects on fluorescence signals and Rb⁺ transport were also observed, whereas substitution by aspartate was less disturbing. These results suggest that formation of an E₂P-stabilizing interhelical salt bridge is essential to prevent futile proton exchange cycles of H⁺ pumping P-type ATPases.     

A role for cAMP in angiotensin II mediated inhibition of cell growth in AT1A receptor-transfected CHO-K1 cells

G-protein coupled Angiotensin II receptors (AT_1A), mediate cellular responses through multiple signal transduction pathways. In AT_1A receptor-transfected CHO-K1 cells (T3CHO/AT_1A), angiotensin II (AII) stimulated a dose-dependent (EC₅₀=3.3 nM) increase in cAMP accumulation, which was inhibited by the selective AT₁, nonpeptide receptor antagonist EXP3174. Activation of protein kinase C, or increasing intracellular Ca²⁺ with ATP, the calcium ionophore A23187 or ionomycin failed to stimulate cAMP accumulation. Thus, AII-induced cAMP accumulation was not secondary to activation of a protein kinase C- or Ca²⁺/calmodulin-dependent pathway. Since cAMP has an established role in cellular growth responses, we investigated the effect of the AII-mediated increase in cAMP on cell number and [³H]thymidine incorporation in T3CHOA/AT_1A cells. AII (1 M) significantly inhibited cell number (51% at 96 h) and [³H]thymidine incorporation (68% at 24 h) compared to vehicle controls. These effects were blocked by EXP3174, confirming that these responses were mediated through the AT₁ receptor. Forskolin (10 M) and the cAMP analog dibutyryl-cAMP (1 mM) also inhibited [³H]thymidine incorporation by 55 and 25% respectively. We extended our investigation on the effect of AII-stimulated increases in cAMP, to determine the role for established growth related signaling events, i.e., mitogen-activated protein kinase activity and tyrosine phosphorylation of cellular proteins. AII-stimulated mitogen-activated protein kinase activity and phosphorylation of the 42 and 44 kD forms. These events were unaffected by forskolin stimulated increases in cAMP, thus the AII-stimulated mitogen-activated protein kinase activity was independent of cAMP in these cells. AII also stimulated tyrosine phosphorylation of a number of cellular proteins in T3CHO/AT_1A cells, in particular a 127 kD protein. The phosphorylation of the 127 kD protein was transient, reaching a maximum at 1 min, and returning to basal levels within 10 min. The dephosphorylation of this protein was blocked by a selective inhibitor of cAMP dependent protein kinase A, H89-dihydrochloride and preexposure to forskolin prevented the AII-induced transient tyrosine phosphorylation of the 127 kD protein. These data suggest that cAMP, and therefore protein kinase A can contribute to AII-mediated growth inhibition by stimulating the dephosphorylation of substrates that are tyrosine phosphorylated in response to AII.     

ANG II-induced cell proliferation is dually mediated by c-Src/Yes/Fyn-regulated ERK1/2 activation in the cytoplasm and PKCzeta-controlled ERK1/2 activity within the nucleus

High-affinity binding of angiotensin II (ANG II) to the ANG II type 1 receptor (AT₁R) results in the activation of ERK1/2 mitogen-activated protein kinases (MAPK). However, the precise mechanism of ANG II-induced ERK1/2 activation has not been fully characterized. Here, we investigated the signaling events leading to ANG II-induced ERK1/2 activation using a c-Src/Yes/Fyn tyrosine kinase-deficient mouse embryonic fibroblast (MEF) cell line stably transfected with the AT₁R (SYF/AT₁). ERK1/2 activation was reduced by 50% within these cells compared with wild-type controls (WT/AT₁). The remaining 50% of intracellular ERK1/2 activation was dependent upon heterotrimeric G protein and protein kinase C zeta (PKC) activation. Therefore, ANG II-induced ERK1/2 activation occurs via two independent mechanisms. We next investigated whether a loss of either c-Src/Yes/Fyn or PKC signaling affected ERK1/2 nuclear translocation and cell proliferation in response to ANG II. ANG II-induced cell proliferation was markedly reduced in SYF/AT₁ cells compared with WT/AT₁ cells (P < 0.01), but interestingly, ERK2 nuclear translocation was normal. ANG II-induced nuclear translocation of ERK2 was blocked via pretreatment of WT/AT₁ cells with a PKC pseudosubstrate. ANG II-induced cell proliferation was significantly reduced in PKC pseudosubstrate-treated WT/AT₁ cells (P < 0.01) and was completely blocked in SYF/AT₁ cells treated with this same compound. Thus ANG II-induced cell proliferation appears to be regulated by both ERK1/2-driven nuclear and cytoplasmic events. In response to ANG II, the ability of ERK1/2 to remain within the cytoplasm or translocate into the nucleus is controlled by c-Src/Yes/Fyn or heterotrimeric G protein/PKC signaling, respectively. Src family tyrosine kinases; angiotensin II     

Diacylglycerol regulates acute hypoxic pulmonary vasoconstriction via TRPC6

Background

Hypoxic pulmonary vasoconstriction (HPV) is an essential mechanism of the lung that matches blood perfusion to alveolar ventilation to optimize gas exchange. Recently we have demonstrated that acute but not sustained HPV is critically dependent on the classical transient receptor potential 6 (TRPC6) channel. However, the mechanism of TRPC6 activation during acute HPV remains elusive. We hypothesize that a diacylglycerol (DAG)-dependent activation of TRPC6 regulates acute HPV.

Methods

We investigated the effect of the DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) on normoxic vascular tone in isolated perfused and ventilated mouse lungs from TRPC6-deficient and wild-type mice. Moreover, the effects of OAG, the DAG kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"R59949","term_id":"830644","term_text":"R59949"}}R59949 and the phospholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075"}}U73122 on the strength of HPV were investigated compared to those on non-hypoxia-induced vasoconstriction elicited by the thromboxane mimeticum U46619.

Results

OAG increased normoxic vascular tone in lungs from wild-type mice, but not in lungs from TRPC6-deficient mice. Under conditions of repetitive hypoxic ventilation, OAG as well as {"type":"entrez-nucleotide","attrs":{"text":"R59949","term_id":"830644","term_text":"R59949"}}R59949 dose-dependently attenuated the strength of acute HPV whereas U46619-induced vasoconstrictions were not reduced. Like OAG, {"type":"entrez-nucleotide","attrs":{"text":"R59949","term_id":"830644","term_text":"R59949"}}R59949 mimicked HPV, since it induced a dose-dependent vasoconstriction during normoxic ventilation. In contrast, {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075"}}U73122, a blocker of DAG synthesis, inhibited acute HPV whereas {"type":"entrez-nucleotide","attrs":{"text":"U73343","term_id":"1688125"}}U73343, the inactive form of {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075"}}U73122, had no effect on HPV.

Conclusion

These findings support the conclusion that the TRPC6-dependency of acute HPV is induced via DAG.     

The 1.5-? Structure of XplA-heme,an Unusual Cytochrome P450 Heme Domain That Catalyzes Reductive Biotransformation of Royal Demolition Explosive

XplA is a cytochrome P450 of unique structural organization, consisting of a heme- domain that is C-terminally fused to its native flavodoxin redox partner. XplA, along with flavodoxin reductase XplB, has been shown to catalyze the breakdown of the nitramine explosive and pollutant hexahydro-1,3,5-trinitro-1,3,5-triazine (royal demolition explosive) by reductive denitration. The structure of the heme domain of XplA (XplA-heme) has been solved in two crystal forms: as a dimer in space group P2₁ to a resolution of 1.9 Å and as a monomer in space group P2₁2₁2 to a resolution of 1.5 Å, with the ligand imidazole bound at the heme iron. Although it shares the overall fold of cytochromes P450 of known structure, XplA-heme is unusual in that the kinked I-helix that traverses the distal face of the heme is broken by Met-394 and Ala-395 in place of the well conserved Asp/Glu plus Thr/Ser, important in oxidative P450s for the scission of the dioxygen bond prior to substrate oxygenation. The heme environment of XplA-heme is hydrophobic, featuring a cluster of three methionines above the heme, including Met-394. Imidazole was observed bound to the heme iron and is in close proximity to the side chain of Gln-438, which is situated over the distal face of the heme. Imidazole is also hydrogen-bonded to a water molecule that sits in place of the threonine side-chain hydroxyl exemplified by Thr-252 in Cyt-P450cam. Both Gln-438 → Ala and Ala-395 → Thr mutants of XplA-heme displayed markedly reduced activity compared with the wild type for royal demolition explosive degradation when combined with surrogate electron donors.Royal demolition explosive (RDX)² or cyclotrimethylenetrinitramine 1 (see Fig. 1) is a widely used explosive compound with both military and civil applications. The extensive global usage of RDX has resulted in concerns over environmental contamination, because it is both recalcitrant to degradation, leading to contamination in soil and ground water, and a potent convulsant and possible carcinogen. The bioremediation of RDX has thus been the focus of increasing research in recent years, with a number of bacterial strains reported to catalyze its degradation (1–3). Among these, Bruce and co-workers, using a selective enrichment technique, isolated Rhodococcus rhodochrous strain 11Y from an RDX-contaminated site, which was able to grow on RDX as the sole nitrogen source (4). The products of biotransformation of RDX by this bacterium were shown to be nitrite and formaldehyde. A gene cluster in strain 11Y essential for RDX degradation was identified and shown to contain a novel cytochrome P450, termed XplA, and a redox partner, XplB, which were shown together to be capable of catalyzing the biotransformation of RDX in vitro (5). Near identical xplA and xplB genes have now been identified in strains within the Actinomycetales isolated from geographically distinct sites (6). Interestingly, these genes have been found to be plasmid encoded providing compelling evidence for recent lateral gene transfer. In the interests of developing an efficient technology for the targeted phytoremediation of RDX, the XplA-XplB system was expressed in transgenic strains of Arabidopsis thaliana and shown to successfully remediate RDX from contaminated soil (5, 7).Open in a separate windowFIGURE 1.Biocatalyzed routes proposed for the degradation of the nitramine exposive RDX 1 by cytochrome P450 XplA from Rhodococcus rhodochrous 11Y. Pathway A corresponds to the route proposed under anaerobic conditions to give the cleavage product metabolite methylene dinitramine 6; pathway B is that postulated to occur under aerobic conditions to give cleavage product 4-nitro-2,4-diazabutanal 7.RDX has an unusual chemical structure, featuring three nitramine (N-NO₂) bonds on a saturated six-membered ring, a functionality that has few precedents among natural products (8). The products isolated from XplA-XplB-catalyzed degradation of RDX in vitro were shown to be different under aerobic and anaerobic conditions after initial denitration to imine intermediate 2 (Fig. 1) (5). Under anaerobic conditions A, 2 would undergo hydration to give intermediate 4. Ring cleavage would yield the isolable metabolite methylene dinitramine 6. The aerobic process B was thought to proceed via successive denitration of two nitramine groups, to give the di-imine 3, which would then be twice hydrated to give the unstable intermediate 5, which would undergo spontaneous ring cleavage to give the isolable product 4-nitro-2,4-diazabutanal 7, formaldehyde, and two equivalents of nitrite.Although the reductive denitration of, for example, glycerol trinitrate (9) and other nitroheterocylic drugs (10), has previously been described for mammalian cytochromes P450, the best known reactions catalyzed by this family of enzymes include a wide range of oxidative chemical reactions, including hydroxylation, heteroatom dealkylation, and C–C bond cleavage reactions most commonly using molecular oxygen and involving cleavage of the O–O bond after dioxygen is bound by the heme-iron (11). A sequence alignment of some cyt-P450 for which the structures have been determined (Fig. 2) shows that the well conserved residues Glu/Asp-Thr/Ser that are thought to be a requirement for oxidative chemistry (12, 13) and to aid cleavage of the scissile O–O bond are not conserved in XplA, being replaced by methionine-394 and alanine-395. In the interests of illuminating the molecular mechanism and substrate specificity in XplA, we were thus interested in obtaining a structure of the heme domain of the enzyme. In this report, the structure of XplA-heme domain in two crystal forms is presented: the first with two molecules in the asymmetric unit, derived from the attempted crystallization of full-length XplA and the other derived from the subcloned, isolated XplA-heme domain, with one molecule in the asymmetric unit and the heme iron ligated to the substrate analogue imidazole. The results reveal a possible access channel for ligand transport and, in combination with mutational studies, demonstrate a highly unusual active site environment for substrate binding, suggesting substrate binding or catalytic roles for Gln-438 and Ala-395.Open in a separate windowFIGURE 2.Amino acid sequence alignment for portions of selected CYP heme domains for which the structures have been solved. P450_pikC (from Streptomyces venezuelae; Uniprot accession no. {"type":"entrez-protein","attrs":{"text":"O87605","term_id":"75539043","term_text":"O87605"}}O87605); P450_cin (from Citrobacter braakii; {"type":"entrez-protein","attrs":{"text":"Q8VQ86","term_id":"75529099","term_text":"Q8VQ86"}}Q8VQ86); P450_eryF (Saccharopolyspora erythrea; {"type":"entrez-protein","attrs":{"text":"Q00441","term_id":"729202","term_text":"Q00441"}}Q00441); P450_moxa (Nonomuraea recticatena; {"type":"entrez-protein","attrs":{"text":"Q2L6S8","term_id":"123293472","term_text":"Q2L6S8"}}Q2L6S8); P450_staP (Streptomyces sp. TP-A0274; {"type":"entrez-protein","attrs":{"text":"Q83WG3","term_id":"81321418","term_text":"Q83WG3"}}Q83WG3); P450_BioI (B. subtilis; {"type":"entrez-protein","attrs":{"text":"P53554","term_id":"1705469","term_text":"P53554"}}P53554); P450_epok (Sorangium cellulosum; {"type":"entrez-protein","attrs":{"text":"Q9KIZ4","term_id":"41688519","term_text":"Q9KIZ4"}}Q9KIZ4); P450_terp (Pseudomonas sp.; {"type":"entrez-protein","attrs":{"text":"P33006","term_id":"416837","term_text":"P33006"}}P33006); P450_xpla (Rhodococcus sp. 11Y; {"type":"entrez-protein","attrs":{"text":"Q8GPH7","term_id":"81322300","term_text":"Q8GPH7"}}Q8GPH7); P450_cam (Pseudomonas putida; {"type":"entrez-protein","attrs":{"text":"P00183","term_id":"117297","term_text":"P00183"}}P00183); and P450_BM3 (Bacillus megaterium; {"type":"entrez-protein","attrs":{"text":"P14779","term_id":"117298","term_text":"P14779"}}P14779). Each catalyzes oxidative chemistry and most apart from P450_cin contain the (E/D)(S/T) dyad (shown in red) demonstrated to be important in the scission of the dioxygen bond as a prerequisite of substrate mono-oxygenation. XplA possesses Met-394 and Ala-395 (shown in green).     

Regulation of PP2AC Carboxylmethylation and Cellular Localisation by Inhibitory Class G-Protein Coupled Receptors in Cardiomyocytes

The enzymatic activity of the type 2A protein phosphatase (PP2A) holoenzyme, a major serine/threonine phosphatase in the heart, is conferred by its catalytic subunit (PP2A_C). PP2A_C activity and subcellular localisation can be regulated by reversible carboxylmethylation of its C-terminal leucine309 (leu309) residue. Previous studies have shown that the stimulation of adenosine type 1 receptors (A1.Rs) induces PP2A_C carboxylmethylation and altered subcellular distribution in adult rat ventricular myocytes (ARVM). In the current study, we show that the enzymatic components that regulate the carboxylmethylation status of PP2A_C, leucine carboxylmethyltransferase-1 (LCMT-1) and phosphatase methylesterase-1 (PME-1) are abundantly expressed in, and almost entirely localised in the cytoplasm of ARVM. The stimulation of G_i-coupled A1.Rs with N⁶-cyclopentyladenosine (CPA), and of other G_i-coupled receptors such as muscarinic M₂ receptors (stimulated with carbachol) and angiotensin II AT₂ receptors (stimulated with {"type":"entrez-protein","attrs":{"text":"CGP42112","term_id":"874777115"}}CGP42112) in ARVM, induced PP2A_C carboxylmethylation at leu309 in a concentration-dependent manner. Exposure of ARVM to 10 µM CPA increased the cellular association between PP2A_C and its methyltransferase LCMT-1, but not its esterase PME-1. Stimulation of A1.Rs with 10 µM CPA increased the phosphorylation of protein kinase B at ser473, which was abolished by the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (20 µM), thereby confirming that PI3K activity is upregulated in response to A1.R stimulation by CPA in ARVM. A1.R-induced PP2A_C translocation to the particulate fraction was abrogated by adenoviral expression of the alpha subunit (Gα_t1) coupled to the transducin G-protein coupled receptor. A similar inhibitory effect on A1.R-induced PP2A_C translocation was also seen with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (20 µM). These data suggest that in ARVM, A1.R-induced PP2A_C translocation to the particulate fraction occurs through a G_iPCR-Gβγ-PI3K mediated intracellular signalling pathway, which may involve elevated PP2A_C carboxylmethylation at leu309.