Grants:
NATIONAL BIOMEDICAL EPR CENTER, J. S. Hyde, P.I., P41-EB001980
The broad aim of the National Biomedical EPR Center is to create and maintain a comprehensive center with balance in all five categories of a Research Resource:
Site-directed spin labeling of ArnT, C.S. Klug, P.I., 5R01AI058024
Abstract: The ability of bacteria to resist host defense mechanisms is a major contributor to the virulence of bacterial infections. Bacterial resistance to antimicrobial peptides that play a key role in early stages of infection is especially significant. The proteins and substrates involved in the ability of bacteria such as Salmonella typhimurium and Escherichia coil to develop resistance to antimicrobial peptides have recently begun to be identified based on genetic analysis. The most recently identified protein involved in polymyxin resistance is the gene product for an inner membrane protein, termed ArnT, which is responsible for transferring an aminoarabinose moiety onto lipid A, conferring upon the bacteria resistance to the cationic antimicrobial peptide polymyxin. Obtaining a more thorough understanding of structure-function relationships in ArnT will be key to developing strategies to overcome resistance to polymyxin and other cationic peptides. Previous studies of ArnT have all involved in vivo enzymatic activity and genetic analyses to determine its role in polymyxin resistance; the ArnT protein has not previously been purified and studied by any methodology. The goal of this proposal is to study the structure of the purified inner membrane protein ArnT by site-directed spin labeling (SDSL) EPR spectroscopy in order to provide the first structural information on this newly identified transferase. A model is proposed in which the Salmonella typhimurium ArnT transferase is comprised of twelve transmembrane (-helices; this model will become the basis for the structural evaluation of the novel protein ArnT by SDSL EPR spectroscopy followed by the examination of structural changes in ArnT due to substrate recognition. In order to begin providing the first structural information on ArnT, a unique and new membrane protein, the following points will be addressed using SDSL EPR spectroscopy: 1) create and characterize a reactive-cysteine-free construct of ArnT; 2) evaluate the model predicting that ArnT is comprised of twelve transmembrane alpha-helices by nitroxide scanning through a putative transmembrane helical region; 3) explore the overall structural arrangement of ArnT by analyzing small sets of mutations placed within putative transmembrane, surface loop, and substrate binding regions; and 4) monitor local and global structural changes induced by substrate binding. It is anticipated that these studies will provide insights into the local and global structure of ArnT, a previously uncharacterized integral membrane protein, which is of fundamental importance in furthering our understanding of the structure and functional dynamics of membrane proteins.
Spin labeling of MsbA, C.S. Klug, P.I., 5R01GM070642
Abstract: The class of proteins termed ATP-binding cassette (ABC) transporters is one of the largest found in nature. Their ability, or lack thereof, to move a variety of ligands across a membrane bilayer using energy from ATP is fundamentally important to bacterial physiology and an array of human pathologies. ABC transporters mediate both the import and export of a wide variety of solutes including antibiotics, lipids, chemotherapy agents, sugars, amino acids, salts and metals. MsbA is the ABC transporter for lipid A that is found in the inner membranes of Gram-negative bacteria such as Escherichia coli. Without MsbA present, bacterial cells accumulate a toxic amount of lipid A, which is an essential component of the outer surface of the cell, within their inner membranes. A crystal structure of MsbA was recently obtained that provides an excellent starting point for structural and functional dynamics studies. Although a structure of MsbA is now available, many questions remain concerning its mechanism of transport. The goal of this proposal is to elucidate the conformational dynamics that occur in MsbA, a bacterial ABC transporter, upon binding ATP in its nucleotide binding domain and upon recognition and transport of lipid substrates in its helical core, utilizing site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy techniques. In order to address the proposal that the MsbA homodimer undergoes significant conformational rearrangements upon ATP and lipid binding that are essential to its function as a lipid exporter, the following points will be addressed: 1) evaluate the quaternary structure of MsbA reconstituted into lipid membranes; 2) investigate the conformational dynamics of the MsbA dimer upon ATP binding; and 3) investigate the conformational dynamics of the MsbA dimer upon lipid binding. It is anticipated that these studies will produce valuable insights into the local and global structural dynamics of MsbA as it functions in its role as a lipid transporter
Spin-Labeled Peptide Antibiotics , J. Feix, P.I., 1R01GM068829-01A2
Abstract: Infectious disease remains the leading cause of mortality worldwide. A significant aspect of this problem is the continuing rise of infections that are resistant to most, if not all, conventional antibiotics. To meet this challenge it is essential that new drug targets be identified, and new classes of antibiotics developed. Over the past two decades a large number of naturally-occurring antimicrobial peptides have been found in both vertebrate and invertebrate species that are capable of providing a rapid and broad-spectrum response against a wide variety of pathogens. Because the specificity of these peptides is based on recognition of general properties of the cell membrane the emergence of resistance is exceedingly rare, making them ideal starting points for the development of new antibiotics. A limiting factor in our ability to further enhance the efficacy of these peptides is the lack of detailed knowledge about their mechanism of action, and in particular the manner in which they interact with and disrupt the cell membrane. The goal of this proposal is to develop a clear understanding of peptide-membrane interactions and mechanism of action for a promising and well-defined class of antimicrobial peptides that are synthetic hybrids of the insect peptide cecropin A and the bee-venom peptide, mellitin. Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy provides a powerful and well-established approach for the analysis of peptide-membrane interactions that is uniquely suited to providing such a detailed understanding. Specifically, we will use both conventional and pulsed SDSL EPR to measure membrane binding affinities, determine structure, topology, degree of membrane insertion, and peptide-peptide interactions for cecropin-mellitin hybrid peptides in model membranes that mimic both eukaryotic and bacterial membranes and in intact cells. These properties will be related to antibiotic efficacy against a panel of drug-sensitive and drug-resistant bacteria. We will systematically modify peptide composition to define relationships between sequence, membrane interactions, and antibacterial efficacy. Finally, we will synthesize and evaluate the antibiotic efficacy and membrane interactions of peptidomimetic analogs composed of non-natural beta-amino acids. These studies will significantly advance our understanding of the mechanism of action of antimicrobial peptides, and contribute to the further development of peptide and peptidomimetic antibiotics.
Liquid Rafts in Eye Lens: Discrimination by Pulse EPR , SUBCZYNSKI, WITOLD K., P.I., 1R01EY015526-01A1
Abstract: DESCRIPTION (provided by applicant): The cell membrane has a 2-dimensional liquid-like structure containing domains that form and disperse continuously on various time and space scales. Rafts are membrane domains that require lipid interactions for their formation. The long-term objective of this proposal is to better understand the molecular mechanisms by which rafts form, are maintained and disintegrate in biological membranes, in particular in the plasma membrane of fiber cells of the eye lens. Detergent insolubility, which has been used to define rafts biochemically, does not reflect pre-existing structures and organization of the membrane. Furthermore, such an approach is not useful for understanding the size, lifetime and dynamics of the raft-constituent molecules and the raft itself. To address these issues, it is proposed to apply the pulse EPR spin labeling technique "discrimination by oxygen transport (DOT)" for in situ studies of rafts in both model and cell membranes. Since the spin-lattice relaxation time of spin labels is sufficiently long, membrane dynamics can be observed on the time scale 0.1 - 100 mu s. The DOT method permits discrimination of different membrane domains because the collision rate between O2 and the nitroxide moiety of spin labels (oxygen diffusion-concentration product) can be quite different in these domains. Additionally, membrane domains can be characterized by profiles of the oxygen diffusion concentration product in situ without the need for separation. This method is especially suitable for obtaining time-space characteristics of small/transient domains. It is hypothesized that rafts form liquid-ordered domains in the plasma membrane liquid-disordered environment. Membrane lipid composition as well as protein content is expected to modulate raft size and dynamics. The DOT method will be used to test the hypothesis on well-defined model systems in which domain size and the lipid exchange rate will be controlled by membrane lipid composition, selected protein and peptide content, and temperature. Furthermore, it will be used to study domain structure in cell membranes. These studies will include mature and aged fiber cell membranes in which the increased cholesterol/lipid ratio and elevated level of sphingomyelin create conditions favoring the formation of rafts. It is proposed: 1) to detect coexisting liquid-ordered and liquid-disordered domains in membranes containing cholesterol; 2) to evaluate the size and stability of the raft domains in model membranes made from raft-forming mixtures; 3) to examine how membrane anchored proteins and transmembrane alpha-helical peptides affect the organization and dynamics of these lipid raft domains; and 4) to apply the DOT method to look for raft domains in fiber cell plasma membranes of the eye lens during maturation and aging, as well as membrane models of mature, aged and cataractous lenses. Age-related nuclear cataract is a primary cause of blindness in the elderly in third world countries.
Thesaurus Terms:
cell membrane, fiber cell, lens, membrane biogenesis, membrane structure
cataract, cholesterol, crosslink, lipid bilayer membrane, membrane model, membrane protein, organometallic compound, oxygen transport, phosphatidylcholine, sphingomyelin, thiosemicarbazone
electron spin resonance spectroscopy, human tissue, postmortem
Institution: MEDICAL COLLEGE OF WISCONSIN
PO BOX26509
MILWAUKEE, WI 532260509
Fiscal Year: 2005
Department: BIOPHYSICS
Project Start: 02-DEC-2004
Project End: 30-NOV-2009
ICD: NATIONAL EYE INSTITUTE
IRG: BBCB
VASCULAR SIGNALING BY FREE RADICALS, David R. Harter P.I., 5P01HL068769
Free Radical Core Facility, B. Kalyanaraman, P.I.
Abstract: Recently, numerous reports have defined actions of reactive oxygen species (ROS) or vascular biology, at many different levels. Many of these reports provide evidence that ROS mediate a variety of signaling events in the vascular wall including; ion channel activity, activation/inhibition of intracellular 2nd messengers, protein phosphorylation, activation of adhesion molecules, activation/inhibition of mitogenic pathways in endothelial and vascular muscle cells. The proposed Program Project Grant (PPG) will begin at the genetic; cellular, molecular, ionic, and whole animal level the mechanisms through which ROS mediate/modify vascular signaling events regulating vascular function, angiogenesis and apoptosis. Project 1 is directed by Dr. David Harder and will test the hypothesis that ROS act to initiate and/or modify signaling events regulating cerebral blood flow (CBF). His laboratory will study the action of ROS on cerebral vascular signaling through ion channel activity and activation of 2nd messengers in arteriolar muscle, define the role of radicals in autoregulation of 2nd messengers in arteriolar muscle, define the role of radicals in autoregulation of CBF, and functional hyperemia in response to neural activity. Finally, Project I will determine the action of ROS in modifying astrocyte mediated capillary angiogenesis in the brain. Project II directed by Dr. David Gutterman, will define the role of ROS in mediating flow (shear) induced dilation of human coronary arteries. Dr. Gutterman has demonstrated that in human coronary arterioles EDHF plays an important role in shear-induced dilation, and presents convincing data that ROS participate in this response. Project III, lead by Dr. William Chilian will define the action of ROS on endothelial ion channels responsible for setting and maintaining membrane potential. Dr. Chilian and colleagues will explore the novel hypothesis that O2-moves through endothelial CI-channels which may play a role in anti-oxidant defense mechanisms. Dr. Peter New man will direct Project IV and will test the hypothesis that PECAM-1 is a target for free radicals. Newman's group has shown that H2O2 induces phosphorylation, and OONO-nitration of tyrosine residues that activates and inhibits PECAM-1. PECAM-1 is an adhesion molecule with properties of an inhibitor receptor-regulation of PECAM-1 activity by free radicals is an important process that can effect cell-cell interaction and a variety of cellular signaling pathways. Project V is lead by Dr. Balaraman Kalyanaraman and will explore that hypothesis that ROS plays a pivotal role in endothelial and vascular mitotic activity. This project will study the actions of NO and ROS on apoptosis induced by oxidized LDL. Dr. Kalyanaraman and colleagues will define the paradoxical effect of Ros effect of ROS to both initiate and inhibit cellular proliferation in the vascular wall. These Projects will rely on a Free Radical and an Analytical Chemistry Core to measure and manipulate free radicals and to measure cellular signaling molecules. This Program brings together a critical measure cellular signaling molecules. This Program brings together a critical mass of recognized investigators and state-of-art techniques to define the biologic role of reactive oxygen and nitrogen species in vascular biology.
MECHANISMS OF METABOLISM OF S-NITROSOTHIOLS , Neil Hogg P.I., R01 GM55792
It has become apparent that S-nitrosothiols (RSNO), biological metabolites of nitric oxide and thiols, play divers roles in potentiating and modulating the effects of nitric oxide. Very little is known concerning the metabolism of RSNO. It is often assumed that these compounds spontaneously liberate nitric oxide and RSNO are commercially available as nitric oxide donors. However, this assumption is in error and RSNO are stable compounds that require an addition agent to promote decomposition. This project will investigate the molecular interactions of S-nitrosothiols with biological targets to understand more fully their role in biological systems. This will be done by addressing three hypotheses with associated specific aims: 1) The observations that protein thiols are the major intracellular targets of RSNO has led us to propose that the modification of protein thiol residues by RSNO is controlled by the local environment of the protein thiol. This specific aim will ask the crucial question of what makes a particular thiol susceptible to control by RSNO. II) Our observations that heme proteins can directly reduce S-nitrosothiols has led us to propose that ferrous heme groups are a major site of RSNO metabolism. In this specific aim we will explore the role played by heme moieties in the control of nitric oxide release from RSNO. III) Our observations concerning the metabolism of RSNO by endothelial cells has led us to propose that cells contain an active metabolic pathway for the metabolism of RSNO. These studies will investigate the mechanism of RSNO metabolism by various cell types. The biochemistry of RSNO has been linked to asthma, inflammation, hypertension, apoptosis and atherosclerosis. It is envisioned that this study will yield new insights into the roles played by these important compounds and aid pharmacological development of new and more selectively potent S-nitrosothiols.
DEVELOPMENT OF BIOMEDICAL ESR INSTRUMENTATION, J. S. Hyde, P.I., R01 EB002052
The proposal contains five projects that are concerned with pulse (or time-domain) saturation-recovery electron-spin resonance instrumentation and methodology. 1) Digital signal processors and improved time response. 2) 2D saturation recovery. 3) Multifrequency saturation recovery and FIDs. 4) Loop-gap resonators for saturation recovery. 5) Multiquantum readout of saturation recover.
HFSS MODELING IN AQUEOUS BIOLOGICAL SAMPLES FOR EPR, J. S. Hyde, P.I., R01 EB001417
The goal of the proposed work is to improve sensitivity in electron paramagnetic resonance (EPR) spectroscopy of aqueous fluid phase samples. There are two themes: (1) multifrequency enhancement from 1 to 35 GHz, and (2) optimization for samples of limited availability (ca., 1 microliter), intermediate availability (ca., 10-20 microliters) and unlimited availability (>100 microliters). This goal will be achieved by optimization of microwave resonator design and by optimization of aqueous sample cell design. The proposal is timely because of recent advances in software for finite element modeling of electromagnetic fields, coupled with greatly improved computing speeds. Computer aided design will be used for resonators, sample cells and field modulation coils, followed by experimental evaluation
EPR Sample-cell-Resonator Design and Construction, J.S. Hyde, 1R03EB007232-01A1
Abstract: DESCRIPTION (provided by applicant): This application is submitted in accordance with PA-02-057, Fogarty International Research Collaboration Award (FIRCA).The research will be done primarily in Poland, at the Jagiellonian University in Krakow in collaboration with Prof. Wojciech Froncisz, PhD, DSc, who is the Foreign Collaborator. It is an extension of NIH grant R01 EB001417, which is the Parent Grant. This proposal is device-design driven. The goal of the proposed work is to improve sensitivity in electron paramagnetic resonance (EPR) spectroscopy of aqueous fluid-phase samples. The proposal is timely because of recent advances in software for finite-element modeling of electromagnetic fields, computers with greatly improved computing speeds, and computer- controlled electric discharge machining (EDM). Computer-aided design will be used for resonators, sample cells and field modulation coils, followed by experimental evaluation. There are three Specific Aims, each of which involves the construction and evaluation in Poland of a sample-containing microwave resonator for EPR spectroscopy: (1) Loop-Gap Resonators (LGR) for Aqueous Samples. An LGR will be developed with ultra-high efficiency. (2) Uniform Field Cavity Resonators for Aqueous Samples. A Q-band uniform field cavity resonator will be developed for use with a novel sample-cell geometry and (3) Aqueous Sample Cell Clusters. A new Multipurpose cavity will be developed for use with layered flat cells. A theme of all three designs is use of EDM graphite, which has recently been proposed by us for EPR resonator construction. The P.I. serves as Director of The National Biomedical EPR Center, P41 EB00198Q, which is a Research Resource. Resonators developed under this FIRCA proposal will enhance the large number of Collaborative and Service Research projects carried out at the Resource. EPR spectroscopy is an important modality in biomedical research. Studies of short-lived radicals detected by spin trapping, of molecular structure using site-directed spin labeling, of biological or model membranes using spin probes, and of cell or tissue preparations are usually carried out in an aqueous environment. Work proposed here will improve the quality of data obtained in these experiments.
eNOS and the RADICAL MECHANISM OF ANTITUMOR ANTHRACYCLINES, B. Kalyanaraman, P.I., R01 CA77822
Abstract: Long-term goal: The long-term goal of this renewal is to unravel the free radical mechanisms by which doxorubicin (DOX), a cancer chemotherapeutic drug that is currently used in the clinic, induces cardiotoxicity in cancer patients.Hypothesis: The general hypothesis to be tested is that DOX-induced apoptosis in cardiomyocytes and endothelial cells is predominantly mediated by hydrogen peroxide (H202) and that antioxidants or antioxidant enzymes that detoxify H202 are antiapoptotic. Specific Aims: First, we will investigate the effect of glutathione peroxidase (GPxI) overexpression on DOX-induced apoptosis in myocytes and endothelial cells. Next, we will demonstrate the role of endothelial nitric oxide synthase (eNOS) in DOX-induced apoptosis. The objective here is to determine whether the DOX/eNOS generated H202 is responsible for apoptosis. DOX-induced generation of reactive oxygen species (ROS) and expression of pro- and antiapoptotic proteins in myocytes and endothelial cells will be assessed using antisense eNOS. Finally, we will determine the mechanism of DOX-induced iron uptake and its implications in apoptosis. After establishing the role of ROS and eNOS in DOX-induced apoptosis, we will investigate the mechanism of antiapoptotic action of selected metalloporphyrins in this system. Methods: We will use cardiomyocytes isolated from adult rat hearts, cultured bovine aortic endothelial cells, and adenovirally transfected GPx1 overexpressing cells. Apoptosis will be detected by several methods including TUNEL analysis, caspase activity, mitochondrial cytochrome c release, Bcl-2 and Bax activity. Superoxide levels will be determined by fluorescence and spin-trapping. We will use the state-of-the-art ESR technique to detect the various redox states of metalloporphyrins. Significance: Drug toxicity is a serious side effect of cancer chemotherapy and severely limits the clinical usefulness of most widely used drugs such as doxorubicin. Children treated with DOX for leukemia develop cardiomyopathy years after cessation of DOX chemotherapy. Further understanding of the oxidative mechanisms may lead to an effective antioxidant/antiapoptotic therapy for minimizing cardiotoxicity. Novelty: Emerging literature indicate that cardiomyocyte apoptosis contributes to heart failure. Understanding the relationship between DOX-induced apoptosis and ROS formation may help discover novel antioxidant/antiapoptotic therapy in oxidant-induced disease
PEROXIDE, NO AND IRON SIGNALING IN ENDOTHELIAL DAMAGE, B. Kalyanaraman, P.I., 1R01 HL073056
Abstract: Long-term goal: The long-term goal of this proposal is to unravel the role of oxidant-induced iron signaling mechanism in endothelial cell apoptosis. Emerging studies indicate that the cellular oxidative damage caused by reactive oxygen and nitrogen species (ROS/RNS) is critically controlled by cellular iron homeostasis. Hypothesis: The general hypotheses to be tested are that (i) peroxide (e.g., H2O2, lipid hydroperoxide)-induced endothelial oxidative damage and apoptosis are mediated by transferrin receptor (TfR)-dependent uptake of transferrin (Tf)-iron and that the TfR is an effective "gatekeeper" and modulator of oxidant-induced apoptosis in endothelial cells, and (ii) nitric oxide (NO) and antioxidants mitigate peroxide-induced endothelial damage by inhibiting iron signaling mechanism. Specific aims: First, we will investigate the effect of extracellular and intracellular hydroperoxides on endothelial iron signaling and apoptosis. Next, we will investigate the effect of cell-permeable esterase-specific NO donors and NO synthase inhibitors on peroxide-induced iron signaling and apoptosis. The objective here will be to establish the link between iron, oxidative/nitrosative stress, and peroxide-induced endothelial toxicity. Finally, we will investigate the effect of antioxidant (enzyme) supplementation on peroxide-induced intracellular oxidative stress, iron signaling, and apoptosis. Methods: We will use the bovine aortic endothelial cells (BAEC) and human aortic endothelial cells (HAEC). The following redox- parameters will be measured: GSH and lipid peroxides; aconitase and iron-regulatory protein activities; TfR expression, 55Fe uptake; caspase signaling, and apoptosis. Superoxide and hydroxyl radicals will be determined by fluorescence and novel spin-trapping (immunoassay) techniques. Significance: Endothelial cell injury is an early oxidative insult in many cardiovascular diseases. Several clinical and basic research studies implicated a role for oxidant-induced iron signaling in vascular oxidative damage. Novelty: This proposal presents a new insight on peroxide-induced iron signaling in endothelial apoptosis and on the role of NO and antioxidants in mitigating these effects.
NITRIC OXIDE MEDIATED OXIDATION/NITRATION IN MEMBRANES, B. Kalyanaraman, P.I., R01 HL63119
Increased levels of nitrotyrosine and nitrated proteins have been detected in a variety of pulmonary and cardiovascular diseases, and in neurodegenerative and chronic inflammatory disorders. The overall objective of this R01 application is to obtain new mechanistic insight into how the hydrophobic interior of biological membranes facilitates oxidation and nitration reactions of reactive nitrogen species (RNS), such as peroxynitrite (ONOO or ONOOH) or nitrogen dioxide radical (NO2). This proposal is based on the following recent discoveries: 1) peroxynitrite can cross lipid membranes through anion transport channels or passive diffusion at rates significantly faster than their reaction with any other target molecule in the aqueous phase. 2) The reaction between NO and O2 is significantly faster in the membrane interior. 3) Peroxynitrite and NO2 cause extensive nitration of alpha-tocopherol in membranes under conditions where tyrosine nitration in the aqueous phase was negligible. The investigators hypothesize that nitration of phenols and nitrosation of thiols by RNS in biological systems is increased in a hydrophobic environment. To investigate the nitration and nitrosation reactions in membranes, they will synthesize tyrosylated lipid and tyrosine- or cysteine-containing peptides that are anchored at defined locations in the lipid bilayer. The investigators will use HPLC, stop-flow spectrophotometry, mass spectrometry, and spin trapping to investigate nitration and nitrosation reactions in membranes. Specifically, the PI will: 1) compare the yields of formation of nitro-gama-tocopherol in membranes and nitrotyrosine in the aqueous phase; 2) detect and characterize nitration products of tyrosylated lipid; 3) determine the mechanism of nitration and nitrosation of tyrosine- and cysteine-containing peptides in membranes; and 4) use nitro-gama-tocopherol or nitrated transmembrane peptide as a marker product to detect peroxynitrite formation from nitric oxide synthase enzymes. This comprehensive study of RNS reactions in simple well-defined model membrane system may provide new mechanistic insight for understanding oxidative and nitrosative stress in pulmonary cardiovascular, neurodegenerative, and inflammatory diseases.
ROLE OF NEURONAL NOS AND SUPEROXIDE IN NEURODEGENERATION, B. Kalyanaraman, P.I., R01 NS39958
The long-term goal of this proposed research is to understand the role of ROS and RNS in the onset of age-related neurodegenerative diseases. The general hypothesis to be tested is that nNOS can generate both superoxide and nitric oxide in a ratio that is regulated by flavin cofactors, redox active compounds, L-arginine and tetrahydrobiopterin (BH4). The specific hypothesis to be tested is that nNOS plays a crucial role in the neurotoxicity of MPP+, a mitochondrial neurotoxin that induces Parkinsonian symptoms. Specific aims: 1) the kinetic of superoxide and NO formation from purified nNOS in the presence and absence of MPP+ will be determined. 2) The role of nNOS in the cytotoxic and apoptotic effects of MPP+ in neuronal cells will be determined. Similar experiments will be determined in BH4-deficient neuronal cells that do not synthesize NO. In these systems, ROS and RNS formation will be measured. 3) The effect of MPP+ toxicity and apoptosis in neuronal cells isolated from nNOS knockout mice will be determined. Methods: Extracellular generation of superoxide will be measured by spin-trapping and intracellular formation of superoxide will be determined by monitoring mitochondrial aconitase activity. Aconitase carbonyls and nitrated protein will be determined by immunochemical methods. significance results from this study should provide new mechanistic insight into ROS and RNS formation from nNOS. Neuronal NOS has been implicated in the onset of several age-related neurodegeneration disorder including Parkinson's, Huntington's, and Lou Gehrig's diseases. Inhibition of nNOS and scavenger of ROS have been shown to exert neuroprotection in animal model mimicking these pathologies. New therapeutic strategies for treating age-associated neurodegenerative diseases may emerge from these studies. Novelty: Novel aspect of this proposal is the ability to measure simultaneously both NO and superoxide from purified nNOS in response to MPP+ and other co-factors. The superoxide-mediated oxidation of 4Fe-4S clusters in aconitase inactivates the enzyme and increases oxidative stress. The availability of novel superoxide dismutase mimetics has made it possible to prove more thoroughly regarding the nature of ROS causing the inactivation of aconitase in the intracellular milieu.
BICARBONATE ENHANCES PEROXIDATION OF SOD/ALS MUTANTS, B. Kalyanaraman, P.I., R01 NS40494
Proposed research has a long-term goal to unravel the molecular mechanisms by which mutations of Sod1 gene encoding Cu, Zn superoxide dismutase (SOD1) produce selective motor neuron degeneration in amyotrophic lateral sclerosis (ALS) or Lou Gehrig's disease. The central hypothesis of this proposal is that bicarbonate anion enhances the peroxidative potential of SOD1 and its mutant variants via generation of a highly potent carbonate anion radical (CO3.-). Specific Aims include demonstration of CO3.- radical generation using purified enzymes; studies on the role of carbonate radical in hydroxylation, oxidation and nitration reactions; characterization of bicarbonate effects on oxidation/nitration reaction catalyzed by spinal cord extracts of transgenic ALS mutant mice and wild type animals. Four major analytical techniques to be used include electron spin resonance spectroscopy (ESR) and spin trapping; HPLC; UV-visible spectroscopy and chemiluminescence
THE FORMATION AND METABOLISM OF S-NITROSOTHIOLS IN VASCULAR CELLS, Y. Zhang, P.I. AHA 0310032Z
S-nitrosothiols (RSNOs), biologically active metabolites of nitric oxide (NO) play an important role in a variety of physiological events, especially in the vasculature. RSNOs are potent vasodilator and antiplatelet agents. S-nitrosation of proteins has been suggested as a regulatory reaction comparable to phosphorylation. RSNOs are also thought to be NO reservoirs to transport and store NO. However, little is known about the mechanisms of RSNO formation and metabolism in cells. We hypothesize that RSNOs are formed and actively metabolized in vascular cells.
We test our hypotheses by specific aims: 1. Determine the mechanisms of cellular RSNO formation upon RSNO addition by comparing the cellular RSNO formation under different treatments -NO released from NO donors, RSNOs, or increase of endogenous NO by stimulating enodothelial nitric oxide synthase (eNOS) and by inducing inducible NOS, examining the role of nitrogen oxides, the intermediates generated during NO autoxidation using an NO scavenger and by modulating oxygen level in cell culture, and studying the influence of GSH depletion on cellular RSNO formation; 2. Determine the mechanisms of RSNO metabolism in cells by introducing S-nitroso probes, including low-molecular-mass S-nitrosothiols, S-nitroso peptides and protein S-nitrosothiols into cells, monitoring their decay in the intracellular milieu and examining the influence of intracellular thiol depletion and extracellular ascorbate addition on the metabolism of these probes. Bovine aortic endothelial cells and murine macrophage Raw 264.7 cells will be used in this study. Various methods will be used to introduce S-nitroso probes into cells, including a lipid – mediated delivery system and the synthesis of cell-permeable peptides. The cellular RSNO formation and decomposition will be detected using ozone-based chemiluminescent method, fluorescence microscope and high performance liquid chromatography.
The goal of this project is to elucidate the mechanisms of RSNO formation and metabolism in cells. It has fundamental significance to understand the biological roles of RSNO. The development of RSNOs as a potential medicine for vasodilation and antiplatelet requires a better understanding about the metabolism of these compounds in biological system. From this point of view this project is also important for the clinical use of RSNOs.