Functional identification of HugZ, a heme oxygenase from Helicobacter pylori

Background

Iron is recognized as an important trace element, essential for most organisms including pathogenic bacteria. HugZ, a protein related to heme iron utilization, is involved in bacterial acquisition of iron from the host. We previously observed that a hugZ homologue is correlated with the adaptive colonization of Helicobacter pylori (H. pylori), a major gastro-enteric pathogen. However, its exact physiological role remains unclear.

Results

A gene homologous to hugZ, designated hp0318, identified in H. pylori ATCC 26695, exhibits 66% similarity to cj1613c of Campylobacter jejuni NCTC 11168. Soluble 6 × His fused-HugZ protein was expressed in vitro. Hemin-agrose affinity analysis indicated that the recombinant HugZ protein can bind to hemin. Absorption spectroscopy at 411 nm further revealed a heme:HugZ binding ratio of 1:1. Enzymatic assays showed that purified recombinant HugZ protein can degrade hemin into biliverdin and carbon monoxide in the presence of either ascorbic acid or NADPH and cytochrome P450 reductase. The biochemical and enzymatic characteristics agreed closely with those of Campylobacter jejuni Cj1613c protein, implying that hp0318 is a functional member of the HugZ family. A hugZ deletion mutant was obtained by homologous recombination. This mutant strain showed poor growth when hemoglobin was provided as the source of iron, partly because of its failure to utilize hemoglobin efficiently. Real-time quantitative PCR also confirmed that the expression of hugZ was regulated by iron levels.

Conclusion

These findings provide biochemical and genetic evidence that hugZ (hp0318) encodes a heme oxygenase involved in iron release/uptake in H. pylori.     

Crystal structure of HugZ, a novel heme oxygenase from Helicobacter pylori

The crystal structure of a heme oxygenase (HO) HugZ from Helicobacter pylori complexed with heme has been solved and refined at 1.8 Å resolution. HugZ is part of the iron acquisition mechanism of H. pylori, a major pathogen of human gastroenteric diseases. It is required for the adaptive colonization of H. pylori in hosts. Here, we report that HugZ is distinct from all other characterized HOs. It exists as a dimer in solution and in crystals, and the dimer adopts a split-barrel fold that is often found in FMN-binding proteins but has not been observed in hemoproteins. The heme is located at the intermonomer interface and is bound by both monomers. The heme iron is coordinated by the side chain of His²⁴⁵ and an azide molecule when it is present in crystallization conditions. Experiments show that Arg¹⁶⁶, which is involved in azide binding, is essential for HugZ enzymatic activity, whereas His²⁴⁵, surprisingly, is not, implying that HugZ has an enzymatic mechanism distinct from other HOs. The placement of the azide corroborates the observed γ-meso specificity for the heme degradation reaction, in contrast to most known HOs that have α-meso specificity. We demonstrate through sequence and structural comparisons that HugZ belongs to a new heme-binding protein family with a split-barrel fold. Members of this family are widespread in pathogenic bacteria and may play important roles in the iron acquisition of these bacteria.     

血红素氧合酶HugZ组氨酸-249对组氨酸-245侧链缺失补偿的结构基础

血红素氧合酶HugZ是幽门螺旋杆菌(Helicobacter pylori)利用宿主血红素作为铁源的关键蛋白．HugZ的His245残基侧链咪唑基与血红素中心铁配位结合,是酶活中心的重要组成部分．用定点突变的方法构建HugZ突变体H245A、H249A和H245A/H249A基因,并将突变体蛋白表达纯化．通过X射线晶体学途径解析了突变体H245A与血红素复合物的2.55Å分辨率晶体结构．结构解析表明,HugZ的His249残基侧链咪唑基团与血红素的铁原子结合,从而补偿了His245侧链缺失．这种结构特征在已知血红素氧合酶中未曾发现．Val238 ψ平面的可翻转和Gly239的柔性是His249能与血红素配位结合的关键原因,二者的共同作用改变了C端肽链的走向,使Val238与His249之间的柔性回折与α1螺旋的相互作用发生解离,并向远离血红素的方向伸展．HugZ蛋白与血红素结合的光谱实验证明HugZ柔性C端上的组氨酸残基有利于HugZ与血红素的结合．研究结果表明,含多个组氨酸残基柔性C端的存在有利于血红素氧合酶HugZ结合和分解血红素．     

Carbon monoxide affects electrical and contractile activity of rat myocardium

Background

Carbon monoxide (CO) is a toxic gas, which also acts in the organism as a neurotransmitter. It is generated as a by-product of heme breakdown catalyzed by heme oxygenase. We have investigated changes in electrical and contractile activity of isolated rat atrial and ventricular myocardium preparations under the influence of CO.

Methods

Standard microelectrode technique was used for intracellular registration of electrical activity in isolated preparations of atrial and ventricular myocardium. Contractions of atrial myocardial stripes were registered via force transducer.

Results

CO (10^-4 - 10^-3 M) caused prominent decrease of action potential duration (APD) in working atrial myocardium as well as significant acceleration of sinus rhythm. In addition CO reduced force of contractions and other parameters of contractile activity. Inhibitor of heme oxygenase zinc protoporphyrin IX exerts opposite effects: prolongation of action potential, reduction of sinus rhythm rate and enhancement of contractile function. Therefore, endogenous CO, which may be generated in the heart due to the presence of active heme oxygenase, is likely to exert the same effects as exogenous CO applied to the perfusing medium. In ventricular myocardium preparations exogenous CO also induced shortening of action potential, while zinc protoporphyrin IX produced the opposite effect.

Conclusions

Thus, endogenous or exogenous carbon monoxide may act as an important regulator of electrical and contractile cardiac activity.     

Plesiomonas shigelloides hugZ encodes an iron-regulated heme binding protein required for heme iron utilization

Plesiomonas shigelloides is an intestinal pathogen that uses heme as an iron source. The P. shigelloides heme utilization system consists of 10 genes, 7 of which permit heme transport and 3 of which are associated with utilization of heme as an iron source once it is inside the cell. The goal of this study was to examine hugZ, 1 of the 3 genes associated with utilization of heme iron. DPH8, a hugZ mutant, failed to grow to full cell density in media containing heme as the iron source, indicating that hugZ is required for heme iron utilization. Western blots using antibodies against Vibrio cholerae HutZ to detect the P. shigelloides HugZ indicated that hugZ encodes an iron-regulated cytoplasmic protein, which is absent in DPH8. A heme affinity bead assay performed on soluble protein fractions from P. shigelloides DPH8/pHUG24.5 (pHUG24.5 encodes hugZ) indicated that HugZ binds heme. Heme utilization was restored in DPH8 by hox1, which encodes the alpha-heme oxygenase from Synechocystis sp. strain PCC6803. However, HugZ did not exhibit alpha-heme oxygenase activity in an assay that detects the conversion of heme to the bilin functional group present in phycobiliproteins. These results do not rule out that HugZ exhibits another type of heme oxygenase activity not detected in the assay.     

Heme-iron utilization by Leptospira interrogans requires a heme oxygenase and a plastidic-type ferredoxin-NADP reductase

Background

Heme oxygenase catalyzes the conversion of heme to iron, carbon monoxide and biliverdin employing oxygen and reducing equivalents. This enzyme is essential for heme-iron utilization and contributes to virulence in Leptospira interrogans.

Methods

A phylogenetic analysis was performed using heme oxygenases sequences from different organisms including saprophytic and pathogenic Leptospira species. L. interrogans heme oxygenase (LepHO) was cloned, overexpressed and purified. The structural and enzymatic properties of LepHO were analyzed by UV–vis spectrophotometry and ¹H NMR. Heme-degrading activity, ferrous iron release and biliverdin production were studied with different redox partners.

Results

A plastidic type, high efficiently ferredoxin-NADP⁺ reductase (LepFNR) provides the electrons for heme turnover by heme oxygenase in L. interrogans. This catalytic reaction does not require a ferredoxin. Moreover, LepFNR drives the heme degradation to completeness producing free iron and α-biliverdin as the final products. The phylogenetic divergence between heme oxygenases from saprophytic and pathogenic species supports the functional role of this enzyme in L. interrogans pathogenesis.

Conclusions

Heme-iron scavenging by LepHO in L. interrogans requires only LepFNR as redox partner. Thus, we report a new substrate of ferredoxin-NADP⁺ reductases different to ferredoxin and flavodoxin, the only recognized protein substrates of this flavoenzyme to date. The results presented here uncover a fundamental step of heme degradation in L. interrogans.

General significance

Our findings contribute to understand the heme-iron utilization pathway in Leptospira. Since iron is required for pathogen survival and infectivity, heme degradation pathway may be relevant for therapeutic applications.     

Influence of organic residues and soil incorporation on temporal measures of microbial biomass and plant available nitrogen

Aims

Despite our current understanding of plant nitrogen (N) uptake and soil N dynamics in arable systems, the supply and demand of N are infrequently matched as a result of variable seasonal and soil conditions. Consequently, inefficiencies in N utilisation often lead to constrained production and can contribute to potential environmental impacts. The aim of this study was to examine the influence of plant residue quality (C/N ratio) and extent of residue incorporation into soil on temporal changes in soil mineral N and the associated plant N uptake by wheat in the semi-arid agricultural production zone of Western Australia.

Methods

Oat (Avena sativa); lupin (Lupinus angustifolius) and field pea (Pisum sativum) were incorporated into a Red-Brown Earth using varying degrees of mechanical disturbance (0 to 100% residue incorporated). Soil samples for inorganic N (NO ₃ ^? and NH ₄ ⁺ ) profiles (0?C50?cm), microbial biomass-C (0?C50?cm) and plant N uptake were taken throughout the growing season of the subsequent wheat (Triticum aestivum) crop. Grain yield and yield components were determined at harvest.

Results

Despite observed treatment effects for plant residue type and soil disturbance, fluctuations in inorganic N were more readily influenced by seasonal variability associated with wet-dry cycles. Treatment effects resulting from residue management and extent of soil disturbance were also more readily distinguished in the NO ₃ ^? pool. The release of N from crop residues significantly increased (p?=?0.05) with greater soil-residue contact which related to the method of incorporation; the greater the extent of soil disturbance, the greater the net supply of inorganic N. Differences in microbial biomass-C were primarily associated with the type of plant residue incorporated, with higher microbial biomass generally associated with legume crops. No effect of residue incorporation method was noted for microbial biomass suggesting little effect of soil disturbance on the microbial population in this soil.

Conclusions

Despite differences in the magnitude of N release, neither crop type nor incorporation method significantly altered the timing or pattern of N release. As such asynchrony of N supply was not improved through residue or soil management, or through increased microbial biomass in this semi-arid environment. N fluxes were primarily controlled by abiotic factors (e.g. climate), which in this study dominated over imposed agricultural management practices associated with residue management.     

Membrane-bound progesterone receptors contain a cytochrome b5-like ligand-binding domain

Background

Membrane-associated progesterone receptors (MAPRs) are thought to mediate a number of rapid cellular effects not involving changes in gene expression. They do not show sequence similarity to any of the classical steroid receptors. We were interested in identifying distant homologs of MAPR better to understand their biological roles.

Results

We have identified MAPRs as distant homologs of cytochrome b ₅. We have also found regions homologous to cytochrome b ₅ in the mammalian HERC2 ubiquitin transferase proteins and a number of fungal chitin synthases.

Conclusions

In view of these findings, we propose that the heme-binding cytochrome b ₅ domain served as a template for the evolution of membrane-associated binding pockets for non-heme ligands.     

Crystal structure of CmlI,the arylamine oxygenase from the chloramphenicol biosynthetic pathway

The diiron cluster-containing oxygenase CmlI catalyzes the conversion of the aromatic amine precursor of chloramphenicol to the nitroaromatic moiety of the active antibiotic. The X-ray crystal structures of the fully active, N-terminally truncated CmlIΔ33 in the chemically reduced Fe²⁺/Fe²⁺ state and a cis μ-1,2(η ¹:η ¹)-peroxo complex are presented. These structures allow comparison with the homologous arylamine oxygenase AurF as well as other types of diiron cluster-containing oxygenases. The structural model of CmlIΔ33 crystallized at pH 6.8 lacks the oxo-bridge apparent from the enzyme optical spectrum in solution at higher pH. In its place, residue E236 forms a μ-1,3(η ¹:η ²) bridge between the irons in both models. This orientation of E236 stabilizes a helical region near the cluster which closes the active site to substrate binding in contrast to the open site found for AurF. A very similar closed structure was observed for the inactive dimanganese form of AurF. The observation of this same structure in different arylamine oxygenases may indicate that there are two structural states that are involved in regulation of the catalytic cycle. Both the structural studies and single crystal optical spectra indicate that the observed cis μ-1,2(η ¹:η ¹)-peroxo complex differs from the μ-η ¹:η ²-peroxo proposed from spectroscopic studies of a reactive intermediate formed in solution by addition of O₂ to diferrous CmlI. It is proposed that the structural changes required to open the active site also drive conversion of the µ-1,2-peroxo species to the reactive form.     

Estimating belowground nitrogen inputs of pea and canola and their contribution to soil inorganic N pools using 15N labeling

Background and aims

Crop species grown in a diversified crop rotation can influence soil N dynamics to varying degrees due to differences in the quantity and quality of the residues returned to the soil. The aim of this study was to quantify the contribution of N rhizodeposition by canola (Brassica napus L.) and pea (Pisum sativum L.) to the crop residue N balance and soil inorganic N pool.

Methods

Canola and pea were grown in a soil-sand mixture and were subject to cotton-wick ¹⁵N labeling in a greenhouse experiment. Nitrogen-15 recovered in the soil and roots were used to estimate N rhizodeposition.

Results

Belowground N, including root N and N rhizodeposits, comprised 70 % and 61 % of total crop residue N for canola and pea, respectively. Canola released the greatest amount of total root-derived N to the soil, which was related to greater root biomass production by canola. However, root-derived N in the soil inorganic N pool was greater under pea (13 %) than canola (4 %).

Conclusions

Our results show a significant belowground N contribution to total crop residue from pea and canola. Further investigation is required to determine whether input of the more labile N rhizodeposits of pea improves soil N supply to succeeding crops or increases the potential for N loss from the soil system relative to canola.     

Neuropeptide-inducible upregulation of proteasome activity precedes nuclear factor kappa B activation in androgen-independent prostate cancer cells

Background

Upregulation of nuclear factor kappa B (NF??B) activity and neuroendocrine differentiation are two mechanisms known to be involved in prostate cancer (PC) progression to castration resistance. We have observed that major components of these pathways, including NF??B, proteasome, neutral endopeptidase (NEP) and endothelin 1 (ET-1), exhibit an inverse and mirror image pattern in androgen-dependent (AD) and -independent (AI) states in vitro.

Methods

We have now investigated for evidence of a direct mechanistic connection between these pathways with the use of immunocytochemistry (ICC), western blot analysis, electrophoretic mobility shift assay (EMSA) and proteasome activity assessment.

Results

Neuropeptide (NP) stimulation induced nuclear translocation of NF??B in a dose-dependent manner in AI cells, also evident as reduced total inhibitor ??B (I??B) levels and increased DNA binding in EMSA. These effects were preceded by increased 20?S proteasome activity at lower doses and at earlier times and were at least partially reversed under conditions of NP deprivation induced by specific NP receptor inhibitors, as well as NF??B, I??B kinase (IKK) and proteasome inhibitors. AD cells showed no appreciable nuclear translocation upon NP stimulation, with less intense DNA binding signal on EMSA.

Conclusions

Our results support evidence for a direct mechanistic connection between the NPs and NF??B/proteasome signaling pathways, with a distinct NP-induced profile in the more aggressive AI cancer state.     

Insights into mechanism and functional consequences of heme binding to hemolysin-activating lysine acyltransferase HlyC from Escherichia coli

Background

Tight regulation of heme homeostasis is a critical mechanism in pathogenic bacteria since heme functions as iron source and prosthetic group, but is also toxic at elevated concentrations. Hemolysin-activating lysine-acyltransferase (HlyC) from Escherichia coli is crucial for maturation of hemolysin A, which lyses several mammalian cells including erythrocytes liberating large amounts of heme for bacterial uptake. A possible impact and functional consequences of the released heme on events employing bacterial HlyC have remained unexplored.

Anti-MJ/NXP-2 autoantibody specificity in a cohort of adult Italian patients with polymyositis/dermatomyositis

Introduction

Increased frequencies of hyperuricemia and gout have been associated with primary hyperparathyroidism, and recent clinical trials of parathyroid hormone (PTH) have reported hyperuricemic adverse events. We evaluated the potential population impact of PTH on serum uric acid (SUA) levels by using a nationally representative sample of United States adults.

Methods

By using data from 8,316 participants aged 18 years and older in the National Health and Nutrition Examination Survey 2003 to 2006, we examined the relation between serum PTH and SUA levels with weighted linear regression. Additionally, we examined the relation with hyperuricemia by using weighted logistic regression.

Results

SUA levels increased with increasing serum PTH concentration. After adjusting for age, sex, dietary factors, glomerular filtration rate (GFR), and other potentially related biomarkers (calcium, phosphorus, alkaline-phosphatase, 25-hydroxyvitamin D), the SUA level differences from the bottom (referent) to top quintiles of serum PTH levels were 0, 8, 13, 14, and 19 ??M (95% CI, 12 to 26; P for trend, < 0.001). These estimates were larger among renally impaired individuals (multivariate SUA difference between the extreme quintiles of PTH, 26 versus 15 ??M among those with GFR ?? 60 versus < 60 ml/min per 1.73 m², respectively) (P for interaction = 0.004). The odds of hyperuricemia by various definitions increased with increasing PTH levels as well (multivariate P values for trend, < 0.05).

Conclusions

These nationally representative data indicate that serum PTH levels are independently associated with serum uric acid levels and the frequency of hyperuricemia at the population level.     

Evidence of shift in C4 species range in central Argentina during the late Holocene

Aim

Millennial-scale biogeographic changes are well understood in many parts of the world, but little is known about long-term vegetation dynamics in subtropical regions. Here we investigate shifts in C₃/C₄ plant abundance occurred in central Argentina during the past few millennia

Methods

We determined present day soil organic matter ??¹³C signatures of grasslands, shrublands and woodlands, containing different mixtures of C₃ and C₄ plants. We measured past changes in the relative cover of C₃/C₄ plants by comparing ??¹³C values in soil profiles with present day ??¹³C signatures. We analyzed ¹⁴C activity in soil depths that showed major changes in vegetation.

Results

Present day relative cover of C₃/C₄ plants determines whole ecosystem ??¹³C signatures integrated as litter and superficial soil organic matter (R²?=?0.78; p?<?0.01). Deeper soils show a consistent shift in ??¹³C, indicating a continuous replacement of C₄ by C₃ plants since 3,870 (±210) YBP. During this period, the relative abundance of C₃ plants increased 32% (average across sites) with significant changes being observed in all studied ecosystems.

Conclusions

Our results show that C₄ species were more abundant in the past, but C₃ species became dominant during the late Holocene. We identified increases in the relative C₃/C₄ cover in grasslands, shrublands and woodlands, suggesting a physiological basis for changes in vegetation. The replacement of C₄ by C₃ plants coincided with changes in climate towards colder and wetter conditions and could represent a climatically driven shift in the C₄ species optimum range.     

Heme oxygenase is involved in H2O2-induced lateral root formation in apocynin-treated rice

Key message

Apocynin is a natural organic compound structurally related to vanillin. We demonstrated that hydrogen peroxide and heme oxygenase participated in apocynin-induced lateral root formation in rice.

Abstract

Apocynin, also known as acetovanillone, is a natural organic compound structurally related to vanillin. Information concerning the effect of apocynin on plants is limited. In this study, we examined the effect of apocynin on lateral root (LR) formation in rice. Treatment with apocynin induced LR formation and increased H₂O₂ production, but had no effect on nitric oxide production. Diphenyleneiodonium chloride, an inhibitor of H₂O₂ generating NADPH oxidase, was effective in reducing apocynin-induced H₂O₂ production and LR formation. Apocynin treatment also increased superoxide dismutase activity and decreased catalase activity. H₂O₂ application was able to increase the number of LRs. Moreover, H₂O₂ production caused by H₂O₂ and apocynin was localized in the root area corresponding to the LR emergence. Treatment with H₂O₂ and apocynin also increased heme oxygenase (HO) activity and induced OsHO1 mRNA expression. Lateral root formation and HO activity induced by H₂O₂ and apocynin were reduced by Zn protoporphyrin IX (the specific inhibitor of HO). Our data suggest that both H₂O₂ and HO are required for apocynin-induced LR formation in rice.     

Genetic testing in diffuse parenchymal lung disease

Background

Mutations in SCO2 cause cytochrome c oxidase deficiency (COX) and a fatal infantile cardioencephalomyopathy. SCO2 encodes a protein involved in COX copper metabolism; supplementation with copper salts rescues the defect in patients?? cells. Bezafibrate (BZF), an approved hypolipidemic agent, ameliorates the COX deficiency in mice with mutations in COX10, another COX-assembly gene.

Methods

We have investigated the effect of BZF and copper in cells with SCO2 mutations using spectrophotometric methods to analyse respiratory chain activities and a luciferase assay to measure ATP production..

Results

Individual mitochondrial enzymes displayed different responses to BZF. COX activity increased by about 40% above basal levels (both in controls and patients), with SCO2 cells reaching 75-80% COX activity compared to untreated controls. The increase in COX was paralleled by an increase in ATP production. The effect was dose-dependent: it was negligible with 100 ??M BZF, and peaked at 400 ??M BZF. Higher BZF concentrations were associated with a relative decline of COX activity, indicating that the therapeutic range of this drug is very narrow. Combined treatment with 100 ??M CuCl₂ and 200 ??M BZF (which are only marginally effective when administered individually) achieved complete rescue of COX activity in SCO2 cells.

Conclusions

These data are crucial to design therapeutic trials for this otherwise fatal disorder. The additive effect of copper and BZF will allow to employ lower doses of each drug and to reduce their potential toxic effects. The exact mechanism of action of BZF remains to be determined.