Spin-Labeled Peptide Antibiotics Feix R01GM068829
Hemolysis, Lipid Oxidation and Vascular Dysfunction in Sickle Cell Disease Flewelen F31HL092773
Mechanisms of Metabolism of S-Nitrosothiols Hogg R01GM55792
Development of Biomedical EPR Instrumentation Hyde R01EB002052
HFSS Modeling in Aqueous Biological Samples for EPR Hyde R01EB001417
National Biomedical EPR Center Hyde P41EB001980
Advanced Instrumental Development Core: MCW. (Dartmouth College) Hyde U19AI091173
Advanced Research and Development of Chemical, Biological, Radiological, and Nuclear Medical Countermeasures Hyde HHSO100201100024C
Mitochondria-targeted Agents in Breast Cancer Kalyanaraman R01CA152810
Neuro-protection by Mitochondria-Targeted Antioxidants Kalyanaraman R01NS039958
Nitric Oxide Mediated Oxidation/Nitration in Membranes Kalyanaraman R01HL63119
Role of iNOS, Nitric Oxide & Arginase in Statin-Mediated Toxicity in Cancer Cells Kalyanaraman R01CA125112
Site-Directed Spin Labeling of ArnT Klug R01AI058024
Spin Labeling of MsbA Klug R01GM070642
Acquisition of Q-Band Pulsed EPR Capability Klug 1S10OD011937
Is Cholesterol Crystalline Domain a Barrier to Oxygen Transport in the Eye Lens? Subczynski R03TW008052
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, melittin. 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-melittin 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.
Sickle cell disease is one of the most commonly inherited blood disorders among Americans of Hispanic and African descent. Sickle red cells are more prone to hemolysis than normal red cells, resulting in increased levels of plasma hemoglobin, a known pro-oxidant. The vascular complications are likely due to this increase in oxidative stress. The long-term objective of this project is to understand the relationship between hemolysis, lipid oxidation and vascular dysfunction in sickle cell disease. This objective will be tested using the following Specific Aims: 1) Measure the level and oxidation state of plasma hemoglobin using novel techniques, a) Plasma hemoglobin levels and oxidation state will be determined using electron paramagnetic resonance spectroscopy. b) Hydroethidine will serve as a probe to detect plasma hemoglobin through a one-electron oxidation of HE in normal and sickle plasma and products resolving using HPLC techniques. 2) Measure oxidative products and the potential to support lipid oxidation in normal and sickle plasma, a) F2-isoprostanes, a vasoconstrictive lipid oxidation product, will be compared in both sickle cell and normal control plasma using liquid chromatography and mass spectrometry. b) The oxidation potential of sickle cell and normal plasma will be measured using an oxygen electrode-based method, c) The degree of correlation between these measurements and plasma hemoglobin levels will be examined. 3) Examine the relationship between hemolysis and vascular dysfunction using histological and vascular response assays in transgenic mouse models, a) Mouse plasma hemoglobin levels will be measured using EPR spectroscopy and an HE oxidation assay, b) Plasma F2-isoprostane levels and other oxidation potential will be measured using methods developed above; c) Measure vessel responsiveness using a facialis artery preparation; d) Analyze nitrotyrosine levels by western blot and immunohistochemistry; e) Examine histological changes using hematoxylin and eosin (H&E). The RELEVANCE to health in the proposed research project is gaining a more complete understanding of the role of hemoglobin and oxidative damage in the pathophysiology of sickle cell disease. Current treatments for an acute sickle cell crisis are mainly palliative, as patients seek medical care when complications present themselves. When completed, this research should lead to the discovery and exploitation of a new therapeutic target to aid in the treatment of sickle cell disease.
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.
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.
The goal of the proposed work is to improve sensitivity in 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.
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:
(1) Resonator development
(2) W-band enhancement
(3) Broadband digital detection, signal analysis, and archiving in EPR spectroscopy
(4) Microwave oscillator development
(5) H1||H0 (parallel mode) EPR at Q- and W-band
(6) X-band/Q-band spin trapping methodology development: Structure/function aspects of NOS-generated radicals
Dissemination, including one workshop during the funded period
(1) Graduate and post-graduate training
(2) Training established investigators
(3) Training of young investigators
The functions of a Research Resource will be accomplished with expertise in the three main application areas of EPR spectroscopy: free radicals, transition metals, and spin labels and with competence in EPR instrumental development.
ADVANCED INSTRUMENTAL DEVELOPMENT CORE: MEDICAL COLLEGE OF WISCONSIN
Awardee Organization: DARTMOUTH COLLEGE
Project Number: 5U19AI091173-03 Sub-Project ID: 6786
Contact PI / Project Leader: HYDE, JAMES S
This core provides input from what is generally agreed to be the leading EPR instrumental development site in the world, the National Biomedical EPR Center at the Medical College of Wisconsin (MCW). The role of this core will be to provide leadership in the development of specific aspects of the technology needed to accomplish the goals of the CMCR. These developments will be carried out in collaboration with the projects and with the instrumental Core at Dartmouth, to facilitate the development of the best possible o prototype instruments for EPR dosimetry. The contributions of the core at MCW are described in four specific aims. Specific Aim 1 (50% of effort) is the Development of resonators for measurements at X-Band in vivo for nails and teeth; this will be the most extensive project and will focus on exploiting the breakthrough achieved in initial collaborative studies where for the first time, resonators that operate at the very sensitive X-Band frequency were successfully used to make measurements in vivo. This development is at the heart of project 3. During the course of the grant this core will attempt to make analogous resonators that can be used for in vivo tooth dosimetry, which could dramatically improve the already impressive sensitivity of this approach (Project 1). Specific Aim 2 (25% of effort) - Development of instrumentation to support measurements at X-Band, will especially focus on development of specifically designed microwave bridges that will facilitate both Projects 2 &3. These developments will be carried out in collaboration with a longstanding collaborative partner of both Dr. Hyde and Dr. Swartz, Dr. Froncisz of the Jagiellonian University in Krakow (Dr. Froncisz's support will come via a purchase agreement. Specific Aim 3 (20% of effort) - Improvements in resonators and bridges for measurements at L-Band. This core will similarly work with Dartmouth and Krakow in the development of improved L-Band bridges, in support of Project 1. It also will facilitate resonator development in project 1 by use of the very sophisticated modeling techniques that are highly developed at MCW, which enable different configurations of resonators to e=be rigorously evaluated so that construction can be focused on designs that are likely to be successful Specific Aim 4 -(5 % of effort) support of multifrequency studies will available an exceptional set of EPR spectrometers that can be of value to all three projects in helping to elucidate the characteristics of potentially overlapping components of EPR spectra.
MITOCHONDRIA-TARGETED AGENTS IN BREAST CANCER
5R01CA152810-03 Contact PI / Project Leader: KALYANARAMAN, BALARAMAN MEDICAL COLLEGE OF WISCONSIN
ABSTRACT We will develop mitochondria-targeted antioxidants (MTAs) and imaging probes that will mitigate cardiotoxicity and enhance antitumor efficacies of chemotherapeutic drugs. We will use doxorubicin (DOX), a front-line antitumor agent in breast cancer treatment. DOX causes delayed dose-dependent cardiotoxicity. Clinically, this side effect is managed with conventional antioxidants and iron chelators. This proposal provides a new adjuvant approach in breast cancer chemotherapy. Its genesis is based upon the following discoveries: 1) MTAs (e.g., Mito-Q, a synthetic drug analog of an endogenous antioxidant, Co-enzyme-Q, present in the mitochondrial respiratory chain) inhibit DOX-mediated cardiotoxicity in a preclinical animal model and in cardiomyocytes, and 2) MTAs (Mito-Q and Mito-CP, a nitroxide targeted to mitochondria) cause antiproliferative and cytotoxic effects in breast cancer cells (MCF-7 and MDA-MB-231) but not in non- transformed breast epithelial cells (MCF-10A) and significantly enhance DOX-induced breast cancer cell toxicity. We hypothesize that mitochondria-targeted antioxidants enhance DOX-mediated antitumor effects but attenuate DOX cardiotoxicity. Response to chemotherapy will be monitored by using the mitochondria-targeted technetium-labeled imaging agents (99mTc-Mito10-MAG3) in a chemically-induced breast carcinoma animal model. Specifically, we will: (i) Investigate the cytotoxic effects of MTAs alone and with DOX in breast cancer cells, (ii) Assess the cytotoxic effects of MTAs and DOX in breast cancer cells overexpressing multi-drug resistant protein, (iii) Evaluate the adjuvant chemotherapeutic effects of MTAs and DOX in an in vivo breast cancer model, and (iv) Assess the cardioprotective and oxy-radical scavenging effects of MTAs in DOX- treated cardiomyocytes and in DOX-treated rat cardiomyopathy model. These aims will be accomplished using HPLC-fluorescence and HPLC-electrochemical detection techniques, scintimammography and echocardiography. Abnormal generation of reactive oxygen species will be detected using novel species- and target-specific probes. We will develop innovative MTA-based adjuvant therapy that can be used to inhibit DOX-induced cardiotoxicity. This research may potentially lead to novel ways for improving the therapeutic efficacy of DOX and other antitumor agents used in breast cancer treatment.
The long-term goal of this multiple PI proposal is to develop neuroprotective strategies that involve synthesis and testing of mitochondria-targeted antioxidants in a preclinical mouse model of Parkinson's Disease (PD) that can be subsequently translated to patients with PD. In this proposal, we have combined the expertise and experience of individuals in synthetic organic chemistry, free radical biology, neuropharmacology, and neurotoxicology from two institutions, the Medical College of Wisconsin and Iowa State University. This proposal is based on the discovery that mitochondria- targeted antioxidants (MTAs) inhibit oxidative stress and neuronal damage in 1-methyl-4- phenylpyridinium (MPP+) treated neuronal cell culture models of PD as well as in 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP) animal models of PD. We hypothesize that MTAs provide an effective neuroprotective strategy for treatment of PD. As a corollary, we propose that MTAs attenuate mitochondria-derived reactive oxygen and nitrogen species (ROS/RNS), thereby protecting against inactivation of key redox sensors in response to mitochondrial neurotoxin exposure. Specifically, we will: (i) Design and synthesize MTAs and assess their cytoprotection in neuronal cell culture models of PD, (ii) Determine the molecular mechanisms of cytoprotection of MTAs in neuronal cell culture models of PD, (iii) Assess the neuroprotective effects of MTAs in a well-established preclinical MPTP mouse model of PD, (iv) Determine the activation/inactivation of key redox targets in mitochondria in response to MTA treatment in the preclinical MPTP mouse model, and (iv) Investigate the long-term tolerability of MTAs during chronic treatment in the mouse model. We will use several analytical techniques (low-temperature EPR, HPLC-fluorescence and electrochemical detection, HPLC/MS, proteomics) to detect and quantitate ROS/RNS, molecular biological approaches (apoptosis measurements, transcription factor translocation) to define the molecular mechanisms, and neurobehavioral and histopathological analyses to evaluate the neuroprotective effects. Abnormal generation of mitochondrial ROS/RNS in response to environmental toxins has been implicated in the pathogenesis of PD. Numerous antioxidants and iron chelators have been used with partial success in experimental animal models of PD. Emerging literature suggests that antioxidants specifically targeted to mitochondria might serve as promising neuroprotectants for treatment of PD. In this proposal, we will assess the neuroprotective efficacy of several novel MTAs in a cell culture and preclinical mouse model of PD. Systematic characterization of neuroprotective properties and long-term tolerability of novel MTAs in both cell culture and animal models will yield comprehensive preclinical data for the clinical development of efficacious mitochondria-targeted antioxidant therapies for PD. PUBLIC HEALTH RELEVANCE: Parkinson's Disease (PD) is a debilitating neurodegenerative disease. Effective treatment to intervene the progression of neurodegenerative processes in PD remains unavailable. Using a cell culture and a mouse model of PD, we propose to develop a "mitochondria-targeted" antioxidant - based neuroprotective strategy for treating PD. Proposed studies which bring together chemical and neuropharmacological expertise from two institutions (Medical College of Wisconsin and Iowa State University), will help us develop efficacious mitochondria-targeted antioxidants for treatment of PD as well as understand the possible neuroprotective mechanisms of these novel class of agents.
Parkinson's Disease (PD) is a debilitating neurodegenerative disease. Effective treatment to intervene the progression of neurodegenerative processes in PD remains unavailable. Using a cell culture and a mouse model of PD, we propose to develop a "mitochondria-targeted" antioxidant - based neuroprotective strategy for treating PD. Proposed studies which bring together chemical and neuropharmacological expertise from two institutions (Medical College of Wisconsin and Iowa State University), will help us develop efficacious mitochondria-targeted antioxidants for treatment of PD as well as understand the possible neuroprotective mechanisms of these novel class of agents.
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.
Long-term goal: Statins selectively inhibit the enzyme hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase leading to decreased cholesterol biosynthesis. Several natural and synthetic statins enhanced apoptosis in human lymphoblastoid, myeloma and breast cancer cells. This effect was directly related to their ability to inhibit HMG CoA reductase, which blocks the synthesis of isoprenylated small GTPases, and not by squalene, an immediate precursor of cholesterol. This proposal is based on the discovery that statins cause increased cytotoxicity to breast cancer cells through either increased expression of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) and/or decreased arginase expression. Statin-mediated cell death was partially reversed by 1400W, a more specific inhibitor of iNOS (NOS II), and by mevalonate, an immediate metabolic product of acetyl CoA/HMG-CoA reductase reaction. Mevalonate supplementation inhibited statin- induced iNOS and NO and restored arginase expression. Fluvastatin dose-dependently inhibited mammary tumor development in an in vivo animal model. Hypotheses to be tested are: (i) statins stimulate NO in breast cancer cells that is responsible for their proapoptotic, tumoricidal and antiproliferative effects, (ii) statins inhibit arginase expression and activity through inhibition of RhoA signaling in breast cancer cells, and (iii) supplementation with sepiapterin (iNOS co-factor) and tocotrienols potentiates statin-induced tumoricidal effects in breast cancer cells and in a rat model. Specific aims: (i) Assess the effects of various statins (lipophilic and hydrophilic) and tocotrienols on breast cancer cell proliferation, and apoptosis, (ii) Determine the induction of iNOS and NO formation in cells treated with statins alone and with sepiapterin and arginase inhibitors, (iii) Define the role of RhoA in statin-mediated NO generation, arginase expression, Nf:B inhibition and antiproliferative effects in breast cancer cells, (iv) Establish a chemopreventive rat model, and evaluate the effectiveness of statins alone and in combination with 3-tocotrienol or sepiapterin. Methods: We will use MCF- 7 and MDA-MB-231 cells and a chemically-induced breast cancer rat model. HPLC techniques will be used to detect and quantitate NO formation in cells treated with statins. Magnetic resonance imaging (MRI) will be used to assess the response to breast cancer therapy in a rat model. Significance: Recent research suggests that statins may prevent various types of cancers including breast cancer. However, the molecular mechanisms by which statins induce breast cancer cell death remain unknown. This proposal will advance our understanding of the chemopreventive and chemotherapeutic ability of statins, alone and in combination with naturally-occurring tocotrienols. Novelty: The overall goal is to elucidate the molecular mechanism by which statins exert antiproliferative/proapoptotic effects in breast cancer cells. The use of tocotrienols to synergistically enhance chemopreventive efficacy of statin in breast cancer cells and breast cancer animal model is innovative. MRI will be used to monitor chemopreventive effects of breast cancer in a rat model. PUBLIC HEARLTH RELEVANCE: Statins are one of the most widely prescribed group of drugs. Recent studies suggest that lipophilic statins may be beneficial for postmenopausal women. Studies also suggest that statins, when combined with other nutrients, become more potent as anticancer drugs. Breast cancer is the leading cause of death in women. Thus, it is both timely and important to understand the mechanism(s) by which statins kill breast cancer cells and to explore the possibility for clinical implementation of statins as chemopreventive drugs.
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.
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.
ACQUISITION OF Q-BAND PULSED EPR CAPABILITY (Klug, PI, 1S10OD011937-01 )
This application is for an upgrade to Q-band for our current Bruker X-band E580 pulse spectrometer capable of running DEER (double electron-electron resonance), DQC (double quantum coherence), and ENDOR (electron nuclear double resonance) experiments at cryogenic temperatures. The primary use of both the current and upgraded instrument will be to quantitate distance measurements between paramagnetic probes on or within biomedically relevant proteins. The major advantages of upgrading to Q-band (35 GHz) DEER from X-band (9 GHz) DEER are a >10-fold increase in sensitivity and overall higher quality distance data. The improvement in resolution, accuracy, identification, signal intensity and the collection of longer distances will be of considerable benefit to an array of biological projects. Additional advantages of pulsed Q- band over X-band include smaller sample volumes, lower overall sample concentration requirements, and significantly decreased data collection times. Pulsed Q-band ENDOR also provides technological and fundamental benefits over X-band ENDOR. The Q-band resonator allows for significantly higher radio frequency (RF) fields for a given supplied power, which is often the limiting factor in ENDOR sensitivity, and the microwave field is increased at Q-band; these allow for the study of fast-relaxing systems that are not amenable to pulsed X-band or continuous-wave ENDOR. In addition, 14N and 1H ENDOR resonances typically overlap at X-band, yet are readily assigned at Q-band. Each one of these is a remarkable advantage to biological research projects and coupled with the considerable improvement in data accuracy will have a substantial impact on current and future structural biology studies of soluble and membrane proteins and protein complexes. Frequencies higher than Q-band provide little additional benefit to the proposed experiments, thus this upgrade request is for the optimum instrument for long-term pulse EPR spectroscopy use. This type of state-of-the-art Q-band pulsed EPR instrumentation is lacking not only at the Medical College of Wisconsin but in entire the state of Wisconsin. Five major users are identified who will measure long-range distances within proteins and protein complexes. In addition, numerous minor users are identified, each with a need for advanced pulse instrumentation not available in the region. Specifically, the research benefiting from the requested upgrade will contribute to a better understanding of the physiology of disease processes such as epilepsy, arrhythmia, neurological disorders, and cancer, and to the development of novel antibiotics and cancer therapeutic agents. Additional opportunities are expected to be uncovered once the success of the initially proposed projects is evident, opening up further avenues of interdisciplinary science. This upgraded instrumentation capability will immediately and significantly advance the productivity of the projects outlined in this proposal as well as enhance the research environment for the surrounding community.
This research will be done primarily in Krakow, Poland, at the Jagiellonian University in collaboration with Dr. Marta Pasenkiewicz-Gierula as an extension of NIH Grant No. R01-EY015526. Cholesterol, the most prominent sterol of mammalian cells, is located predominantly in the plasma membrane, where it comprises 40-45 mol% of the total lipids. However, in membranes of fiber cells, the cholesterol level is extremely high, showing cholesterol-to-phospholipids mole ratios from 1 to 2 in the cortex of the lens to as high as 3 to 4 in the lens nucleus, which leads to the formation of immiscible cholesterol crystalline domains within this membrane. The long-term objective of this proposal is to achieve a greater understanding of the function of cholesterol in membranes of the eye lens, including the effect on the transport of metabolites, particularly oxygen, within the lens. It is proposed to (1) build computer models of selected domains of the fiber cell membrane, with special attention paid to its cholesterol crystalline domain, which can occupy as much as 50% of the cell surface in the lens nucleus, and (2) calculate the oxygen permeability coefficient across membrane domains arising from an oxygen concentration gradient across the membrane. This will allow testing of the hypothesis that the rigid cholesterol crystalline domain can be a barrier to oxygen transport, which, if true, should help to maintain low oxygen concentration in the eye-lens interior. Finally, it is proposed to (3) calculate profiles of both oxygen concentration and the oxygen translational diffusion coefficient across membrane domains and, based on these profiles, determine their oxygen permeability coefficients. Two methods of evaluation of the oxygen permeability coefficient will be compared, namely one that is based on oxygen flux in an oxygen concentration gradient, and one that depends on the details of oxygen self-diffusion in a membrane at equilibrium. Computer experiments will be performed using molecular dynamics (MD) membrane simulations to provide information that is not available from experimental studies about oxygen permeability, structure, and the dynamics of the cholesterol crystalline domain. When possible, the results of MD simulations will be compared with experimental data acquired by the PI under support of the Parent Grant. PUBLIC HEALTH RELEVANCE: Age-related cataracts are a major cause of blindness in developing countries. The reason for the onset of cataracts is unknown, but a great deal of evidence suggests that an increase in oxygen concentration in the lens interior can lead to the development of cataracts. The proposed studies will generate important fundamental information about the contribution of cholesterol to the process of oxygen transport within the eye lens, which should increase our understanding of the role cholesterol plays and, in turn, help contribute to the prevention of age-related nuclear cataracts.
Cholesterol Crystalline Domain Function in Eye Lens: EPR Spin-Labeling Studies (Subczynski, PI, R01 EY015526)
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.
Endothelial dysfunction (ED) is a critical event in the pathophysiology of atherosclerosis and other cardiovascular diseases (CVD). The underlying causes of ED have not been fully established, although unbalanced production of nitric oxide (NO) and reactive oxygen production (ROS) are causally involved. Enzymatic production of NO is dependent on optimal basal tetrahydrobiopterin (BH4) levels in the endothelium. Increased availability of the cofactor by genetic manipulation or pharmacological supplementation has been shown to be beneficial, although the mechanisms controlling BH4 in the endothelium are poorly understood. The broad objectives of this renewal is designed to bridge the gap in knowledge and is based upon the hypothesis that altering BH4 metabolism by lipid peroxidation products and ROS has important consequences in normal NO/ROS fluxes and endothelial physiology favoring phenotypical changes associated with atherogenesis. The present proposal is built on three findings: (i) BH4-free endothelial nitric oxide synthase (eNOS) generates ~100 nmoles superoxide/min/mg protein which is inhibited by BH4 (micromolar range); (ii) BH4 depletion in endothelial cells increases superoxide production by calcium-dependent mechanisms; (iii) 4-hydroxy-2-nonenal (HNE) is a potent inhibitor of BH4 synthesis, depletes BH4 and NO, and increases superoxide production in endothelial cells. The evaluation of the consequences of increased superoxide production from eNOS is critical to the implications of NO and ROS oxidant signaling in disease. Specifically we will: 1) establish the mechanisms increasing uncoupled eNOS-dependent superoxide by HNE and peroxides; 2) investigate the influence of BH4 depletion by HNE and peroxides on increased mitochondrial-superoxide release and dysfunction; 3) elucidate the role of BH4 in limiting oxidative damage, mitochondrial dysfunction and changes in cell phenotype. In the execution of this proposal we will use established endothelial cell cultures and also cells isolated from GTPCH-transgenic mice to examine the influence of endogenous variation in BH4 in the endothelial responses which have been linked to protection, "re-coupling" of eNOS and/or additional antioxidant activity. Generally this proposal is aimed at understanding these fundamental mechanisms that should provide the basis for developing new and improved strategies in the prevention and treatment of atherosclerosis and CVD.