National Biomedical EPR Center (Klug, PI, 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:
- Technological research:
- (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.
LptA-Mediated Transport of LPS (Klug, PI, R01 GM108817)
Lipopolysaccharide (LPS) is the major component of the outer leaflet of the outer membrane (OM) of Gram-negative bacteria such as Escherichia coli, Salmonella typhimurium and many other important pathogens. LPS, also referred to as endotoxin, is essential for survival in this large class of bacteria and serves as a first line of defense against hostile environments encountered during host infection. Given the essential role of LPS in the survival of Gram-negative bacteria - i.e., the bacterial cells die if any step o LPS transport does not occur - and the unique cell surface it creates, a detailed understanding of the proteins and mechanisms involved in LPS synthesis and transport will be the foundation on which to develop novel antibiotics against these promising new drug targets. Many of the proteins involved in LPS transport have been identified through recent genetics studies, suggesting that a set of seven inner membrane (IM), periplasmic, and OM proteins (named LptA, LptB, LptC, LptD, LptE, LptF, and LptG) are directly involved in moving LPS from the IM to the OM. However, the mechanism of how this group of proteins transports LPS to the OM is yet unknown. One of the most striking questions about this process is how the hydrophobic domain of LPS crosses the periplasm. Therefore, the proposed studies will focus on how the periplasmic protein LptA receives LPS from the IM-associated protein LptC, how LptA protects the hydrophobic acyl chains of LPS as it crosses the periplasm, and how LptA delivers LPS to LptDE at the OM. The successful completion of the proposed studies will include the development of a novel functional assessment tool for LptA, the creation of a comprehensive library of in vivo growth assay results to identify LptA amino acids critical for its structure or function, the identification of the specific LptA sites and conformational changes involved in LPS binding, and the characterization of the interactions between LptA and its binding partners LptC, LptDE, and LPS. The results of the novel genetic screenings, the laser light scattering analyses, the innovative electron paramagnetic resonance (EPR) spectroscopy studies, and the isothermal titration calorimetry measurements will provide detailed insights into the mechanism of LPS transport across the periplasm of Gram-negative bacteria. This unique knowledge will greatly enhance our growing understanding of LPS transport in bacteria and set the stage for future studies on the other Lpt proteins of unknown structure and function.
Cholesterol Crystalline Domain Function in Eye Lens: EPR Spin-Labeling Study (Subczynski, PI, R01 EY015526)
This competitive renewal grant is a request to support further analysis of age-related changes in membranes of human eye lens fiber-cells in order to elucidate major differences occurring in transparent and cataractous lenses. This proposal seeks to improve the methodology of membrane studies to the single lens level, which will allow us to consider donor health history information provided by the Eye Bank. The long-range goal is to understand the role of eye lens membranes in maintaining lens transparency. The lens membranes have unique lipid compositions and structures thought to maintain low oxygen concentration in the lens interior, and thus, protect against cataract formation. Our research will provide a basis to develop alternative strategies to prevent the onset or slow the progression of lens opacification. The emphasis will be on the role of the lipid bilayer portion of fiber-cell membranes in maintaining fiber-cell and lens homeostasis. Important progress in the previous grant period includes the identification of the role of cholesterol and the crucial role of cholesterol bilayer domains (CBDs) in particular. The presence of the CBD ensures that the surrounding phospholipid bilayer is saturated with cholesterol. The saturating cholesterol content in fiber-cell membranes keeps the bulk physical properties of lens-lipid membranes consistent and independent of changes in phospholipid composition. Thus, the CBD helps to maintain lens-membrane homeostasis while the membrane phospholipid composition changes significantly with age. We will (i) continue to adapt, refine, and improve our recently developed methods for the quantification of lipid domains in intact fiber-cell membranes to membranes derived from a single lens, and (ii) based on single lens measurements, examine changes in fiber-cell membranes occurring with age and cataract formation. Special attention will be paid to determine major differences in the organization of lipids in lens membranes of people with cataracts and age-matched clear lenses, as well as to the structure of lens membranes of people who retain clear lenses into their eighth and ninth decades. Finally (iii), we will test the hypothesis that an increase in oxygen tension in the lens (one of the causes of cataract formation) initiates lipid peroxidation and drastically changes the organization of lipids in fiber cell membranes, including the formation of CBDs and cholesterol crystals. Our studies will be based on the use of state-of-the-art EPR techniques and methods available and developed at the National Biomedical EPR Center at the Medical College of Wisconsin. EPR spin-labeling methods permit identification of membrane domains, give information about structure and molecular dynamics as a function of the membrane depth in coexisting domains, and allow quantification of lipids in these domains. They also are capable of measuring oxygen transport within and across membrane domains.
Mechanism of Activation and Membrane Interactions of Pseudomonas Toxin ExoU (Feix, PI, R01 GM114234)
Multi-drug resistant (MDR) bacterial infections represent one of the most serious challenges facing health care today. Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is a leading cause of hospital-acquired infections, and is particularly problematic for patients who are immunosuppressed or require mechanical ventilation. P. aeruginosa persists chronically in cystic fibrosis patients, resulting in irreversible lung damage and mortality. Both natural and acquired characteristics, including the expression of several multidrug efflux systems, make P. aeruginosa infections especially difficult to treat. In addition to its intrinsic drug resistance, the infectivity of P. aeruginosa is enhanced by expression of a Type III secretion system (T3SS). This needle-type apparatus directly injects effector proteins into eukaryotic host cells to facilitate bacterial surival and dissemination. The most cytotoxic T3SS effector produced by P. aeruginosa is the phospholipase, ExoU. This application builds upon our previous discovery that ubiquitin and ubiquitinated proteins act as essential cofactors for the activation of ExoU. The interaction of ExoU with ubiquitin is novel in that it does not involve the covalent attachment or removal of ubiquitin subunits, leading us to hypothesize that ubiquitin acts as a scaffold to induce the folding of ExoU into a catalytically-active conformation. We will employ innovative site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) methods in conjunction with genetic and biochemical approaches to characterize the structure of ExoU in solution, elucidate the structure of the ExoU-ubiquitin complex, and investigate ExoU-membrane interactions. These studies will advance the application of new methods to investigate protein-protein interactions, facilitate the development of novel inhibitors of ExoU virulence, and promote our understanding of bacterial toxins that target the membrane interface.
Advanced Research and Development of Chemical, Biological, Radiological, and Nuclear Medical Countermeasures (Hyde HHSO100201100024C)
Mitochondria-Targeted Agents in Breast Cancer (Kalyanaraman, PI, R01 CA152810)
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.
Neuroprotection by Mitochondria-Targeted Antioxidants (Kalyanaraman, PI, R01 NS039958)
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.