Mitochondrial Rieske Iron-sulfur Protein Research Articles

Hypoxia-Induced Mitochondrial ROS and Function in Pulmonary Arterial Endothelial Cells.

Pulmonary artery endothelial cells (PAECs) are a major contributor to hypoxic pulmonary hypertension (PH) due to the possible roles of reactive oxygen species (ROS). However, the molecular mechanisms and functional roles of ROS in PAECs are not well established. In this study, we first used Amplex UltraRed reagent to assess hydrogen peroxide (H2O2) generation. The result indicated that hypoxic exposure resulted in a significant increase in Amplex UltraRed-derived fluorescence (i.e., H2O2 production) in human PAECs. To complement this result, we employed lucigenin as a probe to detect superoxide (O2-) production. Our assays showed that hypoxia largely increased O2- production. Hypoxia also enhanced H2O2 production in the mitochondria from PAECs. Using the genetically encoded H2O2 sensor HyPer, we further revealed the hypoxic ROS production in PAECs, which was fully blocked by the mitochondrial inhibitor rotenone or myxothiazol. Interestingly, hypoxia caused an increase in the migration of PAECs, determined by scratch wound assay. In contrast, nicotine, a major cigarette or e-cigarette component, had no effect. Moreover, hypoxia and nicotine co-exposure further increased migration. Transfection of lentiviral shRNAs specific for the mitochondrial Rieske iron-sulfur protein (RISP), which knocked down its expression and associated ROS generation, inhibited the hypoxic migration of PAECs. Hypoxia largely increased the proliferation of PAECs, determined using Ki67 staining and direct cell number accounting. Similarly, nicotine caused a large increase in proliferation. Moreover, hypoxia/nicotine co-exposure elicited a further increase in cell proliferation. RISP knockdown inhibited the proliferation of PAECs following hypoxia, nicotine exposure, and hypoxia/nicotine co-exposure. Taken together, our data demonstrate that hypoxia increases RISP-mediated mitochondrial ROS production, migration, and proliferation in human PAECs; nicotine has no effect on migration, increases proliferation, and promotes hypoxic proliferation; the effects of nicotine are largely mediated by RISP-dependent mitochondrial ROS signaling. Conceivably, PAECs may contribute to PH via the RISP-mediated mitochondrial ROS.

Mitochondrial Rieske iron-sulfur protein is a crucial molecule in COPD-associated pulmonary hypertension

Pulmonary hypertension (PH) often occurs in chronic obstructive pulmonary disease (COPD) and is the major cause of death in COPD patients. Molecular mechanisms for PH in COPD remain poorly understood, and clinical treatments are neither specific nor effective. Cigarette and e-cigarette smoking (CS) account for up to 90% of COPD cases, in which nicotine is the major active component and causes COPD-like cellular responses; moreover, hypoxia is a known key factor in the development of PH. In this study, we aim to develop a PH in COPD animal model and determine the underlying molecular mechanisms. Our findings revealed that co-exposure of nicotine (or CS) and hypoxia synergistically elevated PH (i.e., right ventricular pressure and weight) compared to nicotine, CS, or hypoxia alone. We also found that mitochondrial reactive oxygen species ([ROS] m ), assessed using mitochondrial-specific ROS indicator MitoSOX, was significantly increased in pulmonary artery smooth muscle cells (PASMCs) from CS patients with COPD compared to normal PASMCs from non-CS patients. The increased [ROS] m was blocked by lentiviral short hairpin RNA (shRNA)-mediated knockdown of Rieske iron-sulfur protein (RISP) in mitochondrial complex III. More importantly, SMC-specific RISP gene knockout blocked nicotine/hypoxia exposure-induced PA remodeling and hypertension, whereas SMC-specific RISP overexpression exacerbated PH in mice. We further discovered that RISP regulated DNA damage following nicotine/hypoxia co-exposure via ataxia telangiectasia-mutated (ATM) signaling. RISP also mediated nuclear factor (NF)-κB inflammatory signaling. Together, our studies for the first time demonstrate that nicotine/hypoxia co-exposure leads to RISP-dependent [ROS] m , ATM-mediated DNA damage-dependent NF-κB-associated inflammation, PA vasoremodeling and PH in COPD in a sex-dependent manner. Moreover, RISP, ATM, and NF-κB may serve as novel and effective molecular targets in treating COPD and its associated PH. NIH R01 HL122865, NIH R03AG070784, and NIH R01 HL108232 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Potential Important Role of Mitochondrial Rieske Iron-Sulfur Protein as a Novel Therapeutic Target for Pulmonary Hypertension in Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide, which is often due to pulmonary hypertension (PH). The underlying molecular mechanisms are poorly understood, and current medications are neither specific nor always effective. In this review, we highlight the recent findings on the roles of altered mitochondrial bioenergetics in PH in COPD. We also discuss the central role of mitochondrial reactive oxygen species (ROS) generation mediated by Rieske iron–sulfur protein (RISP) and review the contributions of RISP-dependent DNA damage and NF-κB-associated inflammatory signaling. Finally, the potential importance of mitochondrial RISP and its associated molecules as novel therapeutic targets for PH in COPD are meticulously discussed.

Erratum to “Mitochondrial Rieske iron-sulfur protein in pulmonary artery smooth muscle: A key primary signaling molecule in pulmonary hypertension. [Archives of Biochemistry and Biophysics 664 (2019) 68–75

Erratum to “Mitochondrial Rieske iron-sulfur protein in pulmonary artery smooth muscle: A key primary signaling molecule in pulmonary hypertension. [Archives of Biochemistry and Biophysics 664 (2019) 68–75

Rieske Iron-Sulfur Protein-Dependent Mitochondrial ROS-Mediated Dissociation of FKBP12.6/RyR2 Complex Plays an Essential Role in Pulmonary Hypertension

An abnormal increase in intracellular calcium concentration ([Ca2+]i, i.e., Ca2+ signaling) in pulmonary artery smooth muscle cells (PASMCs) has been generally believed to play an essential role in the development of pulmonary artery contraction and remodeling, thereby leading to devastating pulmonary hypertension. However, it is unclear which and how ion channels may mediate the abnormal increase in Ca2+ signaling in PASMCs and associated pulmonary hypertension. Herein we have for the first time found that RyR activity and Ca2+ release are significantly increased in PASMCs from mice with hypoxia-induced pulmonary hypertension, a most common type of pulmonary hypertension in clinic. Consistent with the increased RyR functions, SMC-specific RyR2 knockout (RyR2-/-) mice neither show the increased RyR activity and Ca2+ release nor develop pulmonary hypertension in mice following hypoxic exposure. Subcutaneous infusion of the RyR blocker tetracaine produces similar inhibitory effects. Our biochemical studies demonstrate that the endogenous RyR2 stabilizer (inhibitor) FK506 binding protein 12.6 (FKBP12.6) is dissociated from RyR2 in PASMCs from mice with pulmonary hypertension. FKBP12.6 knockout promotes, while subcutaneous infusion of the FKBP12.6 stabilizer S107 prevents, the development of pulmonary hypertension. Intravenous injection of lentiviral shRNAs specific for mitochondrial Rieske iron-sulfur protein (RISP) gene blocks the increased generation of PASMCs reactive oxygen species (ROS), dissociation of FKBP12.6/RyR2 complex, and pulmonary hypertension in mice. Taken together, we conclude that RISP-dependent mitochondrial ROS production may cause FKBP12.6/RyR2 complex dissociation, RyR2 hyperfunctions and Ca2+ release in PASMCs, which leads to pulmonary hypertension. Presumably, specific interventions targeted at RyR2, FKBP12.6 and RISP may become new and effective therapeutic strategies to treat pulmonary hypertension and other relevant vascular diseases.

Primary role of mitochondrial Rieske iron–sulfur protein in hypoxic ROS production in pulmonary artery myocytes

This study was designed to determine whether: (1) hypoxia could directly affect ROS production in isolated mitochondria and mitochondrial complex III from pulmonary artery smooth muscle cells (PASMCs) and (2) Rieske iron–sulfur protein in complex III might mediate hypoxic ROS production, leading to hypoxic pulmonary vasoconstriction (HPV). Our data, for the first time, demonstrate that hypoxia significantly enhances ROS production, measured by the standard ROS indicator dichlorodihydrofluorescein/diacetate, in isolated mitochondria from PASMCs. Studies using the newly developed, specific ROS biosensor pHyPer have found that hypoxia increases mitochondrial ROS generation in isolated PASMCs as well. Hypoxic ROS production has also been observed in isolated complex III. Rieske iron–sulfur protein silencing using siRNA abolishes the hypoxic ROS formation in isolated PASM complex III, mitochondria, and cells, whereas Rieske iron–sulfur protein overexpression produces the opposite effect. Rieske iron–sulfur protein silencing inhibits the hypoxic increase in [Ca 2+] i in PASMCs and hypoxic vasoconstriction in isolated PAs. These findings together provide novel evidence that mitochondria are the direct hypoxic targets in PASMCs, in which Rieske iron–sulfur protein in complex III may serve as an essential, primary molecule that mediates the hypoxic ROS generation, leading to an increase in intracellular Ca 2+ in PASMCs and HPV.

Identification, cDNA Sequence and Deduced Amino Acid Sequence of the Mitochondrial Rieske Iron-Sulfur Protein from the Green Alga Chlamydomonas reinhardtii. Implications for Protein Targeting and Subunit Interaction

Specific oligonucleotide probes were used to isolate a cDNA clone for the mitochondrial Rieske iron-sulfur protein of the green alga Chlamydomonas reinhardtii. The protein is synthesized as a longer precursor with a cleavable N-terminal presequence of 54 amino acids but without a C-terminal extension. Comparison of the predicted secondary structure of this N-terminal sequence with that of the targeting signal of the chloroplast Rieske protein from C. reinhardtii [de Vitry (1994) J. Biol. Chem. 269, 7603-7609] indicates that, although they both have the potential to form amphiphilic alpha helices, the mito-chondrial presequence may form a more hydrophobic helix that could penetrate deeper into the membrane. The N-terminal part of the mature mitochondrial Rieske protein is characterized by a long, strongly hydrophilic N-terminal domain and by a positive charge in the middle of the hydrophobic stretch that is presumed to interact with the bc1 complex. Thus, the protein from C. reinhardtii differs from the Rieske proteins from mammals or fungi.

Functional analysis in yeast of cDNA coding for the mitochondrial Rieske iron-sulfur protein of higher plants.

cDNA clones coding for the nuclear-encoded mitochondrial Rieske iron-sulfur protein (RISP) have been isolated from maize and tobacco. Complementation analysis of hybrid proteins consisting of different domains of plant and yeast RISPs showed that the carboxyl two-thirds of the plant protein is functionally equivalent to that of the yeast protein. The amino terminus of the RISP, however, seems to be species specific because this region is not interchangeable between plant and yeast proteins. Complementation analysis of hybrid proteins also identified a structurally conserved domain probably essential for the function of bc1 complex RISPs. A specific domain from the plant RISP was found to cause temperature-sensitive respiratory growth in yeast. We have demonstrated that yeast can serve as a model system to study the structural and functional relationships of plant gene products that are enzymatic components of the mitochondrial respiratory chain.

Mutational analysis of the mitochondrial Rieske iron-sulfur protein of Saccharomyces cerevisiae. III. Import, protease processing, and assembly into the cytochrome bc1 complex of iron-sulfur protein lacking the iron-sulfur cluster.

We have used site-directed mutagenesis of the Saccharomyces cerevisiae Rieske iron-sulfur protein gene (RIP 1) to convert cysteines 159, 164, 178, and 180 to serines, and to convert histidines 161 and 181 to arginines. These 4 cysteines and 2 histidines are conserved in all Rieske proteins sequenced to date, and 4 of these 6 residues are thought to ligate the iron-sulfur cluster to the apoprotein. We have also converted histidine 184 to arginine. This histidine is conserved only in respiring organisms. The site-directed mutations of the six fully conserved putative iron-sulfur cluster ligands result in an inactive iron-sulfur protein, lacking iron-sulfur cluster, and failure of the yeast to grow on nonfermentable carbon sources. In contrast, when histidine 184 is replaced by arginine, the iron-sulfur cluster is assembled properly and the yeast grow on nonfermentable carbon sources. The site-directed mutations of the 6 fully conserved residues do not prevent post-translational import of iron-sulfur protein precursor into mitochondria, nor do the mutations prevent processing of iron-sulfur protein precursor to mature size protein by mitochondrial proteases. Optical spectra of mitochondria from the six mutants indicate that cytochrome b is normal, in contrast to the deranged spectrum of cytochrome b which results when the iron-sulfur protein gene is deleted. In addition, mature size iron-sulfur apoprotein is associated with cytochrome bc1 complex purified from a site-directed mutant in which iron-sulfur cluster is not inserted. These results indicate that mature size iron-sulfur apoprotein, lacking iron-sulfur cluster, is inserted into the cytochrome bc1 complex, where it interacts with and preserves the optical properties of cytochrome b. Insertion of the iron-sulfur cluster is not an obligatory prerequisite to processing of the protein to its final size. Either the processing protease cannot distinguish between iron-sulfur protein with or without the iron-sulfur cluster, or insertion of the iron-sulfur cluster occurs after the protein is processed to its mature size, possibly after it is assembled in the cytochrome bc1 complex.

Cloning and sequencing of a cDNA encoding the Rieske iron-sulfur protein of bovine heart mitochondrial ubiquinol-cytochrome c reductase

Two cDNA clones encoding bovine heart mitochondrial Rieske iron-sulfur protein were obtained by immunological screening of a bovine heart cDNA expression library in λgt11 with antiserum directed against Rieske iron-sulfur protein isolated from bovine heart mitochondrial ubiquinol-cytochrome c reductase. The cDNA inserts were 1005 and 1100 base pairs with an open reading frame of 807 base pairs which encoded a 196-amino acid mature Rieske iron-sulfur protein and a 73-amino acid presequence. The amino acid sequence of Rieske iron-sulfur protein deduced from nucleotide sequencing is the same as that obtained from protein sequencing except at residues #73 and #191 which are Ser and Asp instead of Ala and Gly, respectively.

Mutational Analysis of the Mitochondrial Rieske Iron-Sulfur Protein of Saccharomyces cerevisiae: I. Construction of a RIP1 deletion strain and isolation of temperature-sensitive mutants

Abstract A protocol has been devised to permit mutational analysis of the Rieske iron-sulfur protein of the mitochondrial cytochrome bc1 complex of Saccharomyces cerevisiae. The gene for this iron-sulfur protein (RIP1) has recently been cloned and sequenced (Beckmann, J. D., Ljungdahl, P. O., Lopez, J. L., and Trumpower, B. L. (1987) J. Biol. Chem. 262, 8901-8909). We have constructed a stable yeast deletion strain, JPJ1, in which the chromosomal copy of RIP1 was displaced by the yeast LEU2 gene by homologous recombination. A linear DNA fragment containing the LEU2 gene was inserted at the breakpoints of an 800-base pair deletion of the iron-sulfur protein gene and used to transform a leu- yeast strain. Leu+ transformants were obtained which were unable to grow on nonfermentable carbon sources. Southern analysis of the transformant, JPJ1, confirmed that the chromosomal copy of the RIP1 gene was deleted and replaced by the LEU2 gene. The genotype of JPJ1 was confirmed by genetic crosses. JPJ1 cannot grow on nonfermentable carbon sources but can be complemented to respiratory competence and transformed by yeast vectors containing the wild type RIP1 gene. The ability to complement strain JPJ1 with episomally encoded iron-sulfur protein provided the basis of a selection protocol by which mutagenized plasmids containing the RIP1 gene were assayed for mutations affecting respiratory growth. Five mutants of RIP1 were identified by their ability to complement JPJ1 to temperature-sensitive respiratory growth. DNA sequence analysis demonstrated that temperature-sensitive respiratory growth resulted from single point mutations within the protein coding region of RIP1. These mutations altered a single amino acid residue in each case. Mutations were dispersed throughout the terminal two-thirds of the protein. Each mutation was recessive and did not affect fermentative growth on dextrose. However, each mutation exerted unique temperature-sensitive growth characteristics on media containing the nonfermentable carbon source glycerol.

2,3-Dimercaptopropan-1-ol (BAL). An aerobic electron-transport inhibitor, but an anaerobic photosynthetic electron donor

The effect of 2,3-dimercaptopropan-1-ol (BAL) on photosynthetic electron transport was studied in Oscillatoria limnetica cells as well as in spinach chloroplasts. Preincubation of the cyanobacterial cells with BAL under aerobic conditions caused inhibition of the sulfide-dependent H 2 evolution. The inhibited site was bypassed by N, N, N′, N′-tetramethyl- p-phenylene diamine. Similarly, NADP photoreduction by spinach chloroplasts was inhibited by BAL pretreatment and restored by the addition of reduced 2,6-dichlorophenol-indophenol. These results accord previous data demonstrating damage of mitochondrial Rieske iron-sulfur protein after BAL treatment (Slater, E.C. and De Vries, S. (1980) Nature 288, 717–718). However, in chloroplasts there was no change in the Rieske center ESR spectrum after BAL treatment. When added anaerobically, BAL served as an electron donor for light-dependent, H 2 evolution in cyanobacteria or for NADP reduction in spinach chloroplasts. These reactions were insensitive to 3-(3′,4′-dichlorophenyl)-1,1dimethylurea (DCMU), but sensitive to 2,5-dibromothymoquinone or to aerobic pretreatment with BAL. The Rieske protein ESR signal of chloroplasts ( g = 1.89) which disappeared upon illumination in the presence of DCMU and methyl viologen, was restored by BAL. It is suggested that as opposed to its aerobic inhibitory effect, BAL donates electrons to the Rieske protein under anaerobic conditions.