Reinhard Kappl

Reversal of Mitochondrial Transhydrogenase Causes Oxidative Stress in Heart Failure

Mitochondrial reactive oxygen species (ROS) play a central role in most aging-related diseases. ROS are produced at the respiratory chain that demands NADH for electron transport and are eliminated by enzymes that require NADPH. The nicotinamide nucleotide transhydrogenase (Nnt) is considered a key antioxidative enzyme based on its ability to regenerate NADPH from NADH. Here, we show that pathological metabolic demand reverses the direction of the Nnt, consuming NADPH to support NADH and ATP production, but at the cost of NADPH-linked antioxidative capacity. In heart, reverse-mode Nnt is the dominant source for ROS during pressure overload. Due to a mutation of the Nnt gene, the inbred mouse strain C57BL/6J is protected from oxidative stress, heart failure, and death, making its use in cardiovascular research problematic. Targeting Nnt-mediated ROS with the tetrapeptide SS-31 rescued mortality in pressure overload-induced heart failure and could therefore have therapeutic potential in patients with this syndrome.

Redox regulation of calcium ion channels: chemical and physiological aspects.

Reactive oxygen species (ROS) are increasingly recognized as second messengers in many cellular processes. While high concentrations of oxidants damage proteins, lipids and DNA, ultimately resulting in cell death, selective and reversible oxidation of key residues in proteins is a physiological mechanism that can transiently alter their activity and function. Defects in ROS producing enzymes cause disturbed immune response and disease. Changes in the intracellular free Ca(2+) concentration are key triggers for diverse cellular functions. Ca(2+) homeostasis thus needs to be precisely tuned by channels, pumps, transporters and cellular buffering systems. Alterations of these key regulatory proteins by reversible or irreversible oxidation alter the physiological outcome following cell stimulation. It is therefore necessary to understand which proteins are regulated and if this regulation is relevant in a physiological- and/or pathophysiological context. Because ROS are inherently difficult to identify and to measure, we first review basic oxygen redox chemistry and methods of ROS detection with special emphasis on electron paramagnetic resonance (EPR) spectroscopy. We then focus on the present knowledge of redox regulation of Ca(2+) permeable ion channels such as voltage-gated (CaV) Ca(2+) channels, transient receptor potential (TRP) channels and Orai channels.

Heart-rate reduction by If-channel inhibition with ivabradine restores collateral artery growth in hypercholesterolemic atherosclerosis

AIMS Collateral arteries protect tissue from ischaemia. Heart rate correlates with vascular events in patients with arterial obstructive disease. Here, we tested the effect of heart-rate reduction (HRR) on collateral artery growth. METHODS AND RESULTS The I(f)-channel inhibitor ivabradine reduced heart rate by 11% in wild-type and 15% in apolipoprotein E (ApoE)(-/-) mice and restored endothelium-dependent relaxation in aortic rings of ApoE(-/-) mice. Microsphere perfusion and angiographies demonstrated that ivabradine did not change hindlimb perfusion in wild-type mice but improved perfusion in ApoE(-/-) mice from 40.5 ± 15.8-60.2 ± 18.5% ligated/unligated hindlimb. Heart rate reduction (13%) with metoprolol failed to improve endothelial function and perfusion. Protein expression of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS, and eNOS activity were increased in collateral tissue following ivabradine treatment of ApoE(-/-) mice. Co-treatment with nitric oxide-inhibitor N (G)-nitro-L-arginine methyl ester abolished the effects of ivabradine on arteriogenesis. Following ivabradine, classical inflammatory cytokine expression was lowered in ApoE(-/-) circulating mononuclear cells and in plasma, but unaltered in collateral-containing hindlimb tissue, where numbers of perivascular macrophages also remained unchanged. However, ivabradine reduced expression of anti-arteriogenic cytokines CXCL10and CXCL11 and of smooth muscle cell markers smoothelin and desmin in ApoE(-/-) hindlimb tissue. Endothelial nitric oxide synthase and inflammatory cytokine expression were unchanged in wild-type mice. Ivabradine did not affect cytokine production in HUVECs and THP1 mononuclear cells and had no effect on the membrane potential of HUVECs in patch-clamp experiments. CONCLUSION Ivabradine-induced HRR stimulates adaptive collateral artery growth. Important contributing mechanisms include improved endothelial function, eNOS activity, and modulation of inflammatory cytokine gene expression.

Electron spin and electron nuclear double resonance of the [FeO2]− centre from irradiated oxyhemo- and oxymyoglobin

Abstract Exposure of glassy and icy solutions of oxyhemoglobin (HbO 2 ) and oxymyoglobin (MbO 2 ) to X- or ultraviolet irradiation at 77 K yields ESR- and ENDOR-active [FeO 2 ] − centres due to addition of an electron to the dioxygen. The centres are shown to be primary ones within the temperature range 4.2 K to 77 K. The rhombic ESR spectra exhibit distinct spectral changes due to various centres formed in α- and β-chains of HbO 2 and MbO 2 upon annealing. Depending on the solvent and pH, up to seven different centres can be distinguished in the ESR spectra, via shifts in g -elements in HbO 2 , and up to three in MbO 2 , all of which decay into ferric signals at about 200 K. Their spectral parameters, including 17 O-hyperfine splitting for the primary species, are given. ENDOR spectra reveal the presence of an exchangeable 1 H-coupling (approx. 10 MHz) in Mb and in both α- and β-chains of Hb for the primary centres which is attributable to an H-bond of the O 2 ligand to nitrogen NE2 of distal His-E7. Tentative assignments for several 1 H-couplings to amino acid residues (Val-E11, Phe-CD1, His-E7, His-F8) on the proximal and distal side of the heme are given for Mb. Differences between the various primary and the annealed centres mainly involve the loss of the exchangeable proton in Hb, Mb and isolated β-chains. The results are discussed in terms of an initial electron trap in the Fe-O-O unit stabilized by the hydrogen bond to the distal histidine proton, the latter being possibly transferred upon annealing.

Quercetinase QueD of Streptomyces sp. FLA, a monocupin dioxygenase with a preference for nickel and cobalt

Quercetinase (QueD) of Streptomyces sp. FLA is an enzyme of the monocupin family and catalyzes the 2,4-dioxygenolytic cleavage of the flavonol quercetin. After expression of the queD gene in Escherichia coli, high specific QueD activity was found in crude cell extracts when the growth medium was supplemented with NiCl 2 or CoCl 2, but not when Mn (2+), Fe (2+), Cu (2+), or Zn (2+) was added. The metal occupancy of Ni- and Co-QueD purified from these cells was </=50%, presumably due to strong overproduction of QueD in E. coli. Circular dichroism spectroscopy indicated the same folded structure with a high content of beta-sheet for the Ni and Co protein. The apparent kinetic constants for quercetin of Ni-QueD ( k cat = 40.1 s (-1), and K m = 5.75 microM) and Co-QueD ( k cat = 7.6 s (-1), and K m = 0.96 muM) indicate similar catalytic efficiencies; however, the approximately 5-fold lower apparent K m value of Ni-QueD for dioxygen suggests that the nickel enzyme performs better under physiological conditions. The pH dependence of k cat,app indicates that an ionizable group with a p K a near 6.8 has to be deprotonated for catalysis. Electron paramagnetic resonance spectra of resting Co-QueD are indicative of a high-spin ( S = (3)/ 2) Co (2+) species in a tetrahedral or trigonal-bipyramidal coordination geometry. Anoxic binding of quercetin to QueD drastically altered the hyperfine pattern at g approximately 6 without changing the valence state of the Co(II) center and elicited a hypsochromic shift of UV-vis absorption band I of quercetin. On the basis of spectroscopic data, and considering the organic chemistry of flavonols, a nonredox role of the metal center in catalysis is discussed.

Bacterial Degradation of Quinoline and Derivatives—Pathways and Their Biocatalysts

A series of interesting enzymes were discovered during investigations on the degradation of quinoline by microorganisms. These include the molybdenum-containing hydroxylases that catalyze the transformation 1→2 and the unusual 2,4-dioxygenases that catalyze the reaction 3→4. The application of the hydroxylases may even be interesting in industry, because several quinoline derivatives are used as pharmaceuticals or agrochemicals.

Inverse regulation of melanoma growth and migration by Orai1/STIM2‐dependent calcium entry

Spontaneous melanoma phenotype switching is controlled by unknown environmental factors and may determine melanoma outcome and responsiveness to anticancer therapy. We show that Orai1 and STIM2 are highly expressed and control store‐operated Ca2+ entry in human melanoma. Lower extracellular Ca2+ or silencing of Orai1/STIM2 caused a decrease in intracellular Ca2+, which correlated with enhanced proliferation and increased expression of microphthalmia‐associated transcription factor, a marker for proliferative melanoma phenotype. In contrast, the invasive and migratory potential of melanoma cells was reduced upon silencing of Orai1 and/or STIM2. Accordingly, markers for a non‐proliferative, tumor‐maintaining phenotype such as JARID1B and Brn2 decreased. Immunohistochemical staining of primary melanomas and lymph node metastases revealed a heterogeneous distribution of Orai1 and STIM2 with elevated expression in the invasive rim of the tumor. In summary, our results support a dynamic model in which Orai1 and STIM2 inversely control melanoma growth and invasion. Pharmacological tuning of Orai1 and particularly STIM2 might thus prevent metastatic spread and render melanomas more susceptible to conventional therapy.

Calcium binding and transport by coenzyme Q.

Coenzyme Q10 (CoQ10) is one of the essential components of the mitochondrial electron-transport chain (ETC) with the primary function to transfer electrons along and protons across the inner mitochondrial membrane (IMM). The concomitant proton gradient across the IMM is essential for the process of oxidative phosphorylation and consequently ATP production. Cytochrome P450 (CYP450) monoxygenase enzymes are known to induce structural changes in a variety of compounds and are expressed in the IMM. However, it is unknown if CYP450 interacts with CoQ10 and how such an interaction would affect mitochondrial function. Using voltammetry, UV-vis spectrometry, electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), fluorescence microscopy and high performance liquid chromatography-mass spectrometry (HPLC-MS), we show that both CoQ10 and its analogue CoQ1, when exposed to CYP450 or alkaline media, undergo structural changes through a complex reaction pathway and form quinone structures with distinct properties. Hereby, one or both methoxy groups at positions 2 and 3 on the quinone ring are replaced by hydroxyl groups in a time-dependent manner. In comparison with the native forms, the electrochemically reduced forms of the new hydroxylated CoQs have higher antioxidative potential and are also now able to bind and transport Ca(2+) across artificial biomimetic membranes. Our results open new perspectives on the physiological importance of CoQ10 and its analogues, not only as electron and proton transporters, but also as potential regulators of mitochondrial Ca(2+) and redox homeostasis.

Recognition of bacterial signal peptides by mammalian formyl peptide receptors: a new mechanism for sensing pathogens.

Background: The function of formyl peptide receptors (FPRs) is incompletely understood. Results: We report the identification of bacterial signal peptides as potent activators of mammalian FPRs and innate immune responses and define critical features underlying FPR peptide recognition. Conclusion: These findings identify a molecular signature for FPR activation. Significance: Our results define a novel mechanism for sensing bacteria. Formyl peptide receptors (FPRs) are G-protein-coupled receptors that function as chemoattractant receptors in innate immune responses. Here we perform systematic structure-function analyses of FPRs from six mammalian species using structurally diverse FPR peptide agonists and identify a common set of conserved agonist properties with typical features of pathogen-associated molecular patterns. Guided by these results, we discover that bacterial signal peptides, normally used to translocate proteins across cytoplasmic membranes, are a vast family of natural FPR agonists. N-terminally formylated signal peptide fragments with variable sequence and length activate human and mouse FPR1 and FPR2 at low nanomolar concentrations, thus establishing FPR1 and FPR2 as sensitive and broad signal peptide receptors. The vomeronasal receptor mFpr-rs1 and its sequence orthologue hFPR3 also react to signal peptides but are much more narrowly tuned in signal peptide recognition. Furthermore, all signal peptides examined here function as potent activators of the innate immune system. They elicit robust, FPR-dependent calcium mobilization in human and mouse leukocytes and trigger a range of classical innate defense mechanisms, such as the production of reactive oxygen species, metalloprotease release, and chemotaxis. Thus, bacterial signal peptides constitute a novel class of immune activators that are likely to contribute to mammalian immune defense against bacteria. This evolutionarily conserved detection mechanism combines structural promiscuity with high specificity and enables discrimination between bacterial and eukaryotic signal sequences. With at least 175,542 predicted sequences, bacterial signal peptides represent the largest and structurally most heterogeneous class of G-protein-coupled receptor agonists currently known for the innate immune system.

Genome Mining in Sorangium cellulosum So ce56: IDENTIFICATION AND CHARACTERIZATION OF THE HOMOLOGOUS ELECTRON TRANSFER PROTEINS OF A MYXOBACTERIAL CYTOCHROME P450*

Myxobacteria, especially members of the genus Sorangium, are known for their biotechnological potential as producers of pharmaceutically valuable secondary metabolites. The biosynthesis of several of those myxobacterial compounds includes cytochrome P450 activity. Although class I cytochrome P450 enzymes occur wide-spread in bacteria and rely on ferredoxins and ferredoxin reductases as essential electron mediators, the study of these proteins is often neglected. Therefore, we decided to search in the Sorangium cellulosum So ce56 genome for putative interaction partners of cytochromes P450. In this work we report the investigation of eight myxobacterial ferredoxins and two ferredoxin reductases with respect to their activity in cytochrome P450 systems. Intriguingly, we found not only one, but two ferredoxins whose ability to sustain an endogenous So ce56 cytochrome P450 was demonstrated by CYP260A1-dependent conversion of nootkatone. Moreover, we could demonstrate that the two ferredoxins were able to receive electrons from both ferredoxin reductases. These findings indicate that S. cellulosum can alternate between different electron transport pathways to sustain cytochrome P450 activity.