Harry Ischiropoulos - US grants

DESCRIPTION Over the past ten years the molecular genetic basis of several rare mitochondrial DNA (mtDNA) disorders has been elucidated. However, the role of mtDNA damage and repair in common diseases remains largely unknown. Lung mtDNA may be at great risk as a result of the oxidative environment during oxygen metabolism and during application of therapeutic interventions such as breathing high oxygen concentrations. The partial reduction of oxygen to reactive species in combination with a newly described biochemistry involving nitrogen centered reactive species may occur in close proximity to mtDNA. Published data indicated that hyperoxia results in mitochondrial morphologic changes in lung endothelium and epithelium. The observed morphologic charges correlate with alterations in biochemical and metabolic function of the mitochondria. However, the effect of hyperoxia on lung mtDNA is virtually unexplored. Therefore, the major goal of this application is to determine the mechanisms of oxidative modification of lung mtDNA by reactive species. The proposed studies will establish the role of mtDNA damage and repair in lung cellular mitochondrial function and will be extended to examine the consequences on lung mtDNA after exposure to hyperoxia and hyperoxia with therapeutic levels of nitric oxide. Mitochondrial DNA damage will be determined and quantified by several end-point assays and will be correlated with mitochondrial function. Overall, the studies proposed in this application will establish mechanisms of oxidative mtDNA damage and determine whether mtDNA damage is part of the pathogenic mechanism of hyperoxic lung injury.

DESCRIPTION: Experiments evidenced have implicated oxidative stress as a contributing element in the neuropathology of degenerative disorders of late life such as Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis. Oxidative stress is the result of over production of reactive species which can overwhelm cellular neuroprotective mechanisms and inactivate critical processes for preserving cellular integrity in function. While much of the existing data has concentrated on the contribution of oxygen-derived reactive species, recent evidence has also implicated nitric oxide derived oxidants in the pathogenesis of neuronal injury. The toxicity of reactive oxidant species is coupled with the reactivity of nitric oxide. Since both nitric oxide and superoxide or free radicals they react in a near diffusion limited rate to form peroxynitrite. Recent data has revealed that peroxynitrite is a major oxidant generated in biological systems under pathologic conditions demonstrating that the proposed chemistry is possible in vivo. Furthermore, peroxynitrite reacts selectively with nitrogen residues of protein to form nitrotyrosine. Protein tyrosine nitration results in the inactivation of protein function and interferes with tyrosine phosphorylation a key event in cellular signal transduction. Previously this group of collaborative investigators has shown that exposure of PC12 cells to low levels of peroxynitrite resulted in significant and irreversible inhibition of DOPA synthesis. Preliminary data indicates that peroxynitrite is mediated nitration of tyrosine residues in the active site of tyrosine hydroxylase could account for the inhibition of DOPA synthesis. Moreover, tyrosine hydroxylase is detected after exposure of PC12 lysates to peroxynitrite or after induction of endogenous peroxynitrite production. The major goal of this proposed grant proposal is to elucidate the role of peroxynitrite-mediated tyrosine nitration in the inactivation of tyrosine hydroxylase in vitro as well as cellular and animal models of aging and Parkinson's disease. The investigators propose two major specific aims. 1) to determine if tyrosine nitration is responsible for the peroxynitrite inactivation of tyrosine hydroxylase. 2) characterize the peroxynitrite mediated modifications present in tyrosine hydroxylase in three models 1) PC12 cells stimulate to generate peroxynitrite, 2) brains of aged rats and 3) mouse brains following administration of MPTP.

We propose to examine the molecular mechanisms of vascular injury mediated by nitric oxide (NO), superoxide (O2) and peroxynitrite (ONOO). Endothelium is a major source of NO which has been shown to mediate important physiological functions such as vasodilatation, inhibition of platelet aggregation and neutrophil adherence. However, published reports have also implicated excessive production of NO as a contributing factor in cellular toxicity. Because NO has an unpaired electron, it reacts at a diffusion limited rate with superoxide to form ONOO. Peroxynitrite is a highly reactive molecule capable of oxidizing many biological molecules and inducing cellular and tissue injury. The reaction of ONOO with protein results in the addition of a nitro group in the ortho position of tyrosine residues to form nirotyrosine adducts. Nitrotyrosine has been detected in human and animal vascular endothelium in pathological disorders such as atherosclerosis, sepsis, inflammation and ischemia-reperfusion indicating that the formation of peroxynitrite is plausible in vivo. Moreover, the formation of nitrotyrosine may interfere with signal transduction events mediated by tyrosine-kinases. Peroxynitrite also exhibits selective reactivity with key cellular targets such as thiols, iron sulfur centers and zinc fingers. This selective reactivity of ONOO may also regulate important cellular functions such as respiration, signal transduction and transcriptional factors. We hypothesize that the toxicity of NO is mediated via the formation of ONOO which then acts as a selective modulator of cell signal transduction and transcriptional events. To evaluate the critical aspects of this hypothesis we propose to: 1) define the role of peroxynitrite in vascular endothelium injury, 2) examine the influence of peroxynitrite- mediated tyrosine nitration on tyrosine kinase-induced signal transduction events and 3) investigate the specific action of peoxynitrite in the activation of transcription factors as opposed to superoxide, hydrogen peroxide and nitric oxide. The proposed experiments will establish and characterize a cell model to examine important, new aspects of peroxynitrite-mediated pathology to vascular endothelium. The integration of our existing knowledge of peroxynitrite-mediated biochemical events with key cellular functions will rapidly advance understanding of pathogenic mechanisms and provide a basis for treatment of important problems in vascular medicine, including sepsis, atherosclerosis, inflammation and ischemia-reperfusion injury.

Limited number of studies that directly measure reactive species or oxidative stress in patients with ARDS are presently. A major limitation for measuring reactive species is their short half life in biological systems. Since reactive species modify biological molecules such as proteins, lipids and DNA, measurement of the modified targets provide the experimental tools for their detection and quantification. Protein carbonyls are derived by the direct oxidation of amino acid residues or conjugation of aldehydes that are formed by the oxidation of unsaturated lipids or sugars. Overall plasma protein carbonyls indicate the formation of oxidants. Nitration of protein tyrosine residues results in the formation of 3-nitrotyrosine. Previous we found that the reaction of peroxynitrate with C02 provides the necessary nitrating agent that explains the formation of plasm protein 3-nitrotyrosine. Peroxynitrate is formed by the nearly diffusion limited reaction of nitric oxide and superoxide. Urinary isoprostanes are generated by reactive species attack on arachidonate in lipid bilayers and is selective and sensitive indicator of lipid peroxidation. Therefore, the main function of the analytical chemistry core (Core C) is to measure the levels of modified plasma proteins (carbonyls and 3-nitrotyrosine) and urinary isoprostanes in the human samples for Project 3. Protein carbonyls will be also measured in cell lysates, lung tissue, perfusate and plasma in samples generated in Project 4. Core C will also function as a central processing, storage and distribution facility for the samples collected in Project 3. The analytical chemistry will be located at the Institute for Environmental Medicine. Strengths of Core C include the established protocols for clinical specimen collection, processing and storage, the experience of the investigators with measuring these biological markers and the understanding of the biochemical origin of these biological markers.

Oxidative stress is considered to be a significant component in the pathogenesis of bronchoplumonary dysplasia (BPD) and adult respiratory distress syndrome (ARDS). Oxidative stress can be defined as the pathogenic outcome created by the oxidation of critical tissue targets by reactive species which are generated at rates exceeding tissue antioxidant capacity. However, only few studies that directly measure reactive species or oxidative stress in patients with either BPD or ARDS are available. A major limitation for measuring reactive species is their short half life in biological systems. Since reactive species modify biological molecules such as proteins, lipids and DNA measurement of the modified targets provide the experimental tools for their detection and quantification. Preliminary data in this application indicate that serum protein modifications measured as 2, 4 dinitrophenylhydrazine reactive carbonyls and nitration of tyrosine residues may be indicate oxidative stress in BPD and ARDS. Protein carbonyls are derived by the direct oxidation of amino acid residues conjugation of aldehydes that formed by the oxidation of unsaturated lipids or sugars. Overall, plasma protein carbonyls indicate the formation of oxidants. Nitration of protein tyrosine residues results in the formation of 3-nitrotyrosine. Previously we found that the reaction of peroxynitrite with C02 provides the necessary nitrating agent that explains the formation of plasma protein 3-nitrotyrosine. Peroxynitrite is formed by the nearly diffusion limited reaction of nitric oxide and superoxide. Therefore this application will systematically examine the changes in these biological markers in BPD and ARDS. We propose that these protein modifications indicate generation of oxidants and nitrating species and correlate with the severity of patient's illness and fatal outcome. The critical aspects of our hypothesis will be tested by: 1) correlating the plasma protein levels of 3-nitrotyrosine in BPD and ARDS patients with the severity of clinical illness and outcome. 2) measuring the changes in plasma protein levels of 3-nitrotyrosine and carbonyls in ARDS and BPD patients after inhalation of therapeutic levels of nitric oxide. This application is strengthened by the established protocols for patient blood collection and processing, the experience with measuring these biological markers and the understanding of the biochemical origin of these biological markers.

This is a second international conference, the first conference was held in May of 1997 in Ascona, Switzerland and attracted 70 scientists from all over the world. An indication of the success of the 1997 conference was exceptional quality of presentations, the length and extent of discussions and most of all the lively and animated evening discussions. During these discussions, which I vividly recall to date, controversial issues regarding peroxynitrite were critically discussed and evaluated. The resounding success of the first meeting and the overwhelming desire of all the participants to attend another conference resulted in the decision to put together a second meeting. The meeting is sponsored by the Nitric Oxide Society (see letters). We anticipate that the second meeting will bring together approximately 100 scientists; chemists, biochemists, biologists, physicians, neuroscientists and molecular biologists. The format will be similar to the first meeting and of Gordon conferences, with morning oral presentations followed by group discussions in the evening. The objectives will be to exchange information and engage in discussions regarding the chemistry, biology and medical applications of peroxynitrite. Peroxynitrite is the molecule formed by the reaction of nitric oxide and superoxide. A number of the chemical and biological functions of peroxynitrite have been described but almost monthly a new function or a possible role in human pathophysiology is reported. The number of laboratories contributing to investigations regarding peroxynitrite has grown over the years. A quick search in biomedical databases using peroxynitrite as the key word produced over 1,000 articles for the past eight years. However, the majority of the peer-reviewed articles (469) were published during the last two years following the Ascona meeting. Moreover, NIH currently funds a number of participants of the first meeting to work on peroxynitrite. Using the NIH search engine we found 86 projects that are currently funded by NIH to investigate the role of peroxynitrite in the pathogenic mechanism of neurodegenerative diseases, respiratory distress, cardiovascular disorders, cancer and a number of other disorders. Moreover, the most important advancements in this field are the recent evidence for the participation of peroxynitrite-mediated nitration of proteins in the pathogenesis of neurodegenerative diseases of aging.

DESCRIPTION (Adapted from the Applicant's Abstract): The activity of Tyrosine Hydroxylase (TH) is essential for the production of catecholamines. During the progression of Parkinson's disease (PD) distinct changes in TH activity and concentration have been described. A decrease in dopamine levels without a loss of either TH immunoreactivity or dopaminergic neurons has been described during the early phase of the disease. The middle stage of the disease is characterized by a loss in dopamine and immunoreactive TH without a loss of dopaminergic neurons. Loss of dopamine, TH and dopaminergic neurons characterize late phase of the disease. These distinct events in PD are faithfully reproduced in the 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) mouse model of PD. However, the biochemical basis to explain the changes in TH, activity and content prior to the death of dopaminergic neurons are not clearly understood. Our published data generated during the last two years of funding has provided a reasonable biochemical explanation for the changes in TH during the early phase of MPTP neurotoxicity. The data revealed that TH is a selective target for nitration. Nitration of tyrosine residues represents a post-translational protein modification that results from the reaction of nitrating agents with proteins. Nitrating agents such as peroxynitrite are formed during oxidative stress. Oxidative stress has been implicated in the pathogenesis of PD and in the MTPT neurotoxicity. The published data showed that for the first 6 hours post MPTP injection, nitration of a single tyrosine in TH results in the inactivation of the enzyme. The inactivation of TH paralleled the decline in dopamine levels in the mouse striatum whereas the levels of TH protein remain unchanged. However, 12 hours after the last MPTP injection, preliminary data indicated that an apparent non proteolytic, enzymatic process has repaired nitrated TH and this is reflected by an increase in the catalytic activity of the protein and in brain dopamine levels. At the same time the protein levels of TH have declined to nearly 50 percent of control. The loss of protein appears to be mediated by the ubiquitin-proteosome pathway. Based on these preliminary data we formulated the following working hypothesis: Protein nitration (specifically TH) represents a pathophysiology stimulus that is managed by two processes; non-proteolytic repair involving a unique denitrase, and/or protein degradation. Critical aspects of this working hypothesis will be examined by: 1) determining the kinetics of repair and degradation of TH in the mouse MPTP and in the PC12 cell models, 2) purifying and characterizing the brain denitrase activity, and 3) investigating the molecular mechanisms for the proteolytic degration of TH. This application is a natural extension of our previous work that elucidated the biochemical mechanism for the inactivation of tyrosine hydroxylase during the early stages of MPTP toxicity. The proposed experiments will elucidate biochemical, cellular and molecular changes in TH during the middle stages of MPTP toxicity and PD by integrating our experiences with protein nitration chemistry and biological chemistry of reactive species, with Dr. Horwitz's PC12 cell model and Dr. Przedborski's MPTP mouse model. The collaboration between the three different laboratories has been productive. Understanding the basic biochemical and molecular changes in TH during the progression of MPTP and PD will facilitate the development of approaches to correct the functional deficit in dopamine production in PD.

DESCRIPTION (provided by applicant): Experiments in this application will examine the molecular mechanisms responsible for the modulation of cellular metabolism and resistance to oxidants by endogenous nitric oxide (NO). Published data indicated that NO either directly mainly by reversible S-nitrosylation of critical cysteine residues or by elevating cGMP levels modulates the adaptive responses that render cells resistant to oxidative stress and apoptosis. However, the majority of the cellular models rely upon the deliver of NO by NO donors or by the induction of the inducible nitric oxide synthase (NOS). To study the contribution of NO generated by the low output endothelial NOS in the cellular protection against oxidants, we utilized ECV3O4 cells transfected with endothelial NOS. The transfected cells generated sufficient NO to induce elevation of cGMP in smooth muscle cells in an L-NAME inhabitable manner. Using this well-defined model preliminary data revealed that NO regulates the steady state of ATP, the flux of glucose by the glycolytic and pentose phosphate pathways and respiration. Moreover, this dynamic regulation of metabolism and mitochondrial bioenergetics was associated with an increased resistance to H2O2 exposure. Exposure to H2O2 at 50-100 pM induced a delayed cell death (18 hours after exposure) to nearly 50 percent of ECV3O4 but less than 20 percent in the ECV3O4-eNOS cells. Inhibition of NO production ameliorated the protective effect and restored the steady state levels of ATP and glucose fluxes. Preliminary data using human pulmonary artery endothelial cells confirmed the NO-dependent protection against H202 induced delayed cell death. These preliminary data together with scarce published data on the ability of NO to regulate metabolism suggest a previous unrecognized function of NO that may causally relate to adaptation against oxidative stress. We propose that the generation of low levels of NO by eNOS is sufficient to dynamically regulate cellular glucose metabolism and respiration providing a primary and previously unrecognized molecular mechanism for the NO-induced protection against oxidative stress. To examine these hypotheses we propose the following specific aims: (1) define the molecular mechanism(s) of nitric oxide-mediated regulation of cellular metabolism; (2) investigate the causal association between nitric oxide-dependent alterations in metabolism with the adaptation to oxidative stress; and (3) examine if endogenous nitric oxide regulation of mitochondrial respiration and mitochondrial function is responsible for the protection against oxidative stresses. Experiments in the first aim are focused on the allosteric, covalent and other regulatory functions of NO in critical enzymes that catalyze essential and irreversible steps in the glycolytic pathway and TCA cycle. The second aim will utilize biochemical, pharmacological and molecular approaches to provide evidence for the potential causal relationship between NO-mediated regulation of metabolism and resistance to oxidative stress. The third aim examines the importance of NO-regulated mitochondrial respiration and function in protecting cells from oxidant exposures and typical inducers of apoptosis. Overall the proposed experiments will evaluate in a systematic manner the critical role of endogenously generated NO as a mediator of cellular metabolism and respiration that enables cells to resist oxidative stress.

DESCRIPTION (provided by applicant): The detection of oxidized proteins, lipids and DNA/RNA in human as well as animal and cellular models of neurodegenerative diseases has been documented. In the last decade additional chemistry based on the formation of reactive nitrogen oxides, byproducts of nitric oxide reactivity have been also documented by the detection of tyrosine-nitrated proteins mostly in the inclusions that characterize Parkinson's and other related diseases. Recent data has also indicated that reactive nitrogen oxides convert the free amino acid tyrosine to 3-nitrotyrosine. This unusual amino acid can be also formed in the CNS following the proteolytic degradation of nitrated proteins. Tyrosine is an important amino acid in the CNS since it serves as the building block for the formation of dopamine. We hypothesize that 3-nitrotyrosine interferes primarily with dopamine formation and tyrosine metabolism. Support for this hypothesis is derived from the sound documentation that 3-nitrotyrosine injected into rodent striatum selectively injures dopaminergic neurons. Another unrecognized feature of 3-nitrotyrosine may be the interference with mitochondrial respiration leading to the decline in ATP synthesis and additional production of reactive species. Preliminary data have also indicated a specific 3- nitrotyrosine-dependent disruption of microtubule assembly leading to the formation of soluble and insoluble protein aggregates. To test the critical aspects of these hypotheses we propose to evaluate the following: 1) determine if 3-nitrotyrosine interferes with the production, metabolism and biology of dopamine and tyrosine, 2) evaluate if 3-nitrotyroinse interferes with mitochondrial respiration and function and 3) examine if the formation of alpha/beta-tubulin aggregates resulting from the specific incorporation of 3-nitroyrosine into a-tubulin serves as a building block for the aggregation and/or fibrilization of alpha-synuclein. An array of biochemical, pharmacological, and molecular approaches that are already in place will be employed to perform the proposed experiments. The proposed experiments will evaluate the critical role of the endogenously generated unusual amino acid, 3-nitrotyrosine, as a central mediator responsible for alterations in fundamental regulatory pathways in dopamine metabolism, oxidative phosphorylation, and protein aggregation, which constitute well-recognized molecular targets responsible for neuronal injury and death in Parkinson's disease and related disorders. Overall a novel and previously unrecognized biological chemistry resulting from the formation of a modified amino acid, 3-nitrotyrosine, will be investigated in order to uncover multifaceted but potentially interrelated pathways that promote neuronal dysfunction in neurodegenerative disorders.

DESCRIPTION (provided by applicant): The purpose of this application is to acquire a hybrid triple quadrupole-ion trap mass spectrometer, specifically the QTRAP 5500 from Applied Biosystems, with unique capabilities to support actively ongoing studies that aim to uncover critical physiological pathways and causes of pediatric diseases. This instrument will be used exclusively for quantitative mass spectrometric applications by 9 NIH funded investigators at the Joseph Stokes, Jr. Research Institute of the Children's Hospital of Philadelphia. Ongoing projects initiated by these investigators have employed genomic discovery platforms, biochemical, molecular, different mass spectrometric and orthogonal experiments to discover proteins of interest, posttranslational modifications, proteolytic processing and networks of proteins that execute biological functions. Therefore all these projects are well advanced past the stages of discovery and specific and practical protein and peptide targets have been identified. The immediate challenge for these projects is to move beyond qualitative descriptions to explore these discoveries in depth by quantifying changes in proteins and peptides. This goal cannot be accomplished with typical antibody based western blot, ELISA or even with our current linear ion trap mass spectrometers since these approaches are often neither feasible nor practical due to lack of sensitivity, specificity, and/or throughput. Targeted peptide quantification is becoming the clear choice as a highly selective and sensitive methodological approach for quantitative evaluation of dynamic changes of proteins, protein networks, posttranslational modifications, validation and utilization of biological markers. Towards this goal the PI and co-Investigator in collaboration with the users of the proposed instrument are actively developing methodologies that employ stable isotope labeled peptides and multiple reaction monitoring (MRM) workflows for the quantification of proteins in human samples as well as in animal and cellular model systems. Multiple reaction monitoring is emerging as the standard technique for quantitative liquid chromatography tandem mass spectrometry (LC/MS/MS) experiments;however generation and application of these MRM workflows with our current ion trap platforms is limited. Therefore the installation and use of the proposed mass spectrometer is vital for enabling the development and implementation of methodologies for targeted peptide quantification to generate sizeable new knowledge and insights. Overall, the proposed projects although biologically diverse are united by the need to develop, validate and implement quantitative hypothesis-driven mass spectrometric approaches. Thus, while each research project stands alone in scientific value and integrity the added value of this combined proposal is substantial. PUBLIC HEALTH RELEVANCE: The instrument will have a major and immediate impact on the research programs of 9 NIH-funded investigators that are engaged in both basic and translational research aimed to uncover fundamental physiological processes and mechanisms of disease at the Children's Hospital of Philadelphia. The requested instrumentation will facilitate the quantification of potentially novel biological markers for the prognosis and diagnosis of pediatric diseases.

DESCRIPTION (provided by applicant): This application tests the hypothesis that biochemically distinct oligomers of a-synuclein induce neuron degeneration in part by inhibiting the function of chaperone-mediated autophagy (CMA). a-Synuclein (a- syn) has been identified as one of the major components of the protein inclusions in the brains of subjects with sporadic and familial forms of Parkinson's disease (PD) and related neurodegenerative disorders. Deregulation in dopamine metabolism resulting in increased cytosolic dopamine that undergoes oxidation has been also considered as contributing pathogenic mechanism for the selective neuron dysfunction and death in PD. We have provided evidence that oxidized dopamine interacts with a-syn and propose that this interaction unifies two potential neurotoxic mechanisms responsible for PD and disorders characterized by a-syn inclusions. Despite the profound implications in the pathogenesis of disease the interaction of a-syn with dopamine in vivo has not been evaluated. Therefore we will elevate the levels of dopamine in the substantia nigra of the mice expressing human a-syn with the pathogenic A53T mutation driven by the mouse PrP promoter by injecting lentiviral vectors that deliver cDNA of human tyrosine hydroxylase (TH) with N-terminus R37E, R38E mutation (TH-RREE), which is not feed-back inhibited by dopamine. In these mice we will then: 1) Compare and contrast the effects of dopamine on regional formation and biochemical properties of a-syn oligomers. Experiments will define the regional distribution, biochemical and biophysical properties of the soluble a-syn oligomers. Experiments will also test the ex vivo effects of oligomers on neuron dysfunction, vesicular association and a-syn aggregation. 2) Evaluate the regional association of a-syn with CMA and determine the in vivo effects of increasing dopamine levels. Experiments will test the novel hypothesis that compromised function of CMA resulting from the interaction with oxidized dopamine-induced a-syn oligomers leads to neuron dysfunction by the accumulation of S-nitrosylated glyceraldehyde phosphate dehydrogenase. This hypothesis unites the effect of a-syn oligomers in blocking CMA with the previously established effects of nitric oxide-induced dopaminergic neuron death. 3) Investigate the effect of increasing the levels of dopamine on the phenotype of A53T a-syn expressing mice. Onset of phenotype will be determined followed by a comprehensive immunohistochemical and biochemical analysis of the brains for dopamine neuron viability, inclusion formation, and astrogliosis. PUBLIC HEALTH RELEVANCE: The conversion of soluble proteins into insoluble aggregates is a common pathological hallmark of age-related neurodegenerative disorders. In Parkinson's disease and related disorders the presence of intracellular inclusions composed mostly of aggregated a-synuclein represents an indispensable neuropathological diagnostic tool and suggests that the formation of insoluble a-synuclein is an important factor in pathogenesis. Parkinson's disease and related disorders are also characterized by the loss of neurons that synthesize the neurotransmitter dopamine. It has been postulated but not tested in a living organism that the aggregation and potential toxicity of a-synuclein is intimately influenced by dopamine. Experiments in this application offer a unique opportunity to explore the significance of the dopamine a-synuclein interaction in vivo and generated mice that may recapitulate additional features of the disease such as degeneration of dopamine-producing neurons improving our most basic understanding of the molecular mechanism of these neurodegenerative disorders.

DESCRIPTION (provided by applicant): Activation of the coagulation cascade converts soluble fibrinogen to insoluble fibrin, which polymerizes to produce, along with platelets, the haemostatic clot. Whereas the normal activation of the coagulation cascade is essential for life, inappropriate activation or failure to dissolve deposited fibrin in a timely manner may result in fibrin-dependent organ pathology. Indeed pathologically induced thrombogenesis produces venous thromboembolism (VTE), a major cause of morbidity and mortality in the USA. The pulmonary vascular compartment is a major target for VTE and other clinical syndromes characterized by fibrin deposition. However, it is unclear if the structure and mechanical properties of fibrin influences the severity of VTE and pulmonary complications. Recent studies have provided provocative new insights regarding the presence of fibrin structures with altered architecture and mechanical properties in subjects with coronary artery disease. These new findings provide mechanistic associations with the previously documented epidemiological findings between fibrinogen/fibrin and risk of cardiovascular complications. Despite similar epidemiological associations, the functional consequences and contributions of fibrin networks for the risk of developing VTE and pulmonary dysfunction remain untested. Therefore we propose that abnormal fibrin structures that are resistant to fibrinolysis induce pulmonary vascular abnormalities confer a high risk for VTE and acute lung injury. This hypothesis will be tested by: 1) Quantifying fibrin-clot turbidity, fibrin structure, fibrinolytic resistance and levels of oxidatively-modified fibrinogen in patients with clinically documented deep venous thrombosis. 2) Testing the functional consequences of thromboemboli generated from fibrinogen isolated from clinically documented DVT subjects in a mouse model of pulmonary thromboemboli challenge. 3) Ascertaining the functional consequences of fibrin generated in vivo from fibrinogen isolated from clinically documented DVT subjects in a mouse model of acute thromboembolism. Collectively these mouse models aim to evaluate for the first time the influence of variant fibrin structures in deriving pulmonary vascular complications. Successful completion of these specific aims will provide a systematic study of the biochemical and biophysical properties of fibrin clots in subjects at risk for acute thromboembolism and explore the potential biological consequences of altered fibrin assemblies in vivo.

DESCRIPTION (provided by applicant): Nitric oxide (NO) is a versatile free radical that mediates numerous biological functions within every major organ system. An emerging molecular pathway by which NO accomplish functional diversity is the specific modification of protein cysteine residues to form S-nitrosocysteine. This post-translational modification, S-nitrosylation, impacts protein function and location. Despite considerable advances with individual proteins, the biological chemistry, the dependency on specific nitric oxide synthases (NOS) and the structural elements that govern the modification of specific cysteine residues in vivo are vastly unknown. Moreover comprehensive studies exploring protein signaling pathways or interrelated protein clusters that are regulated by S-nitrosylation have not been performed. To provide insights for these important biological questions, sensitive, validated and quantitative proteomic approaches are needed but are not currently available. To this end, during the last funding period we developed and implemented a novel mass spectrometry-based proteomic approach. The new method achieved specific, efficient, complementary and selective identification of the modified cysteine residue. Currently implementation of the method has precisely pinpointed the site of S-nitrosylation in 741 peptides, which were independently matched to 521 proteins in mouse liver, heart, lung brain and thymus. These proteins constitute the largest datasets of endogenous S-nitrosylated proteins to date. Using this robust new methodology we propose to: (1) Define the structural elements that govern the specificity of S-nitrosylation, (2) Elucidate the functional networks and signaling pathways that are influenced by S-nitrosylation and (3) Determine if S-nitrosylation represents the molecular link between eNOS and leptin in the regulation of liver lipid metabolism. By uncovering the endogenous S-nitrosocysteine proteomes of mouse liver, brain, lung and heart and applying multiple analytical and computational tools, the structural properties that govern the specificity and selectivity of S-nitrosylation in vivo will be defined. By identifying the S-nitrosocysteine proteome of mice which do not express S-nitrosoglutathione reductase (GSNOR), the enzyme that metabolizes S-nitrosoglutathione (GSNO), we will elucidate the structural elements governing the in vivo GSNO-mediated S-nitrosylation. Functional pathway and network analyses in conjunction with quantitative assessment of S-nitrosoproteomes derived from endothelial NOS (eNOS), neuronal NOS (nNOS) and GSNOR null mice will test NOS specific functional regulation in signaling cascades within and across the four different organs. A novel hypothesis linking eNOS-mediated S-nitrosylation with leptin in the regulation of liver lipid metabolism will be explored. Overall the comprehensive large-scale study of protein structures and functional pathways will significantly improve our appreciation of S-nitrosylation in nitric oxide biology.

DESCRIPTION (provided by applicant): GRC NO 2013 The Gordon Research Conference on Nitric Oxide (NO) is designed to provide biologists, chemists, clinicians and other scientists with state-of-the-art knowledge on the basic structure/function relationships of NO generating systems, and the biology of NO as a signaling and effector molecule in physiology and pathophysiology. A long-term goal is to use this knowledge in development of novel NO based therapeutics in particular for treatment and prevention of cardiovascular disease. This research conference is held every two years and the next conference will take place at the Ventura Beach Marriott in Ventura, CA on Feb 17-22, 2013. Approximately 160 participants from academia, government and industry are expected to attend. Funds are requested to support conference registration and travel costs fees for participants (i.e. speakers/discussion leaders and for exceptional graduate students/post-doctoral candidates and other junior scientists working in the field of NO biology). The speakers have been selected to address unresolved questions and cover emerging new areas in the field and to balance the program with senior and junior investigators. In a specific effort to promote and showcase the work of younger scientists, 12 highly talented junior researchers (5 females) have been invited to give full talks. Senior leaders in the field will chair these sessions. Sessions will focus on novel insights on the regulation of NO formation and signaling. Chemically related nitrogen oxide species with signaling properties including S-nitrosothiols, nitrite and nitro fatty acids will be discussed in depth. In addition, therapeutic avenues will be covered with an emphasis on how enhancement of NO bioavailability via newly described principles might be used to treat and prevent cardiovascular and metabolic disease. 1998 Nobel laureate Louis J Ignarro has kindly agreed to present the Keynote Lecture. In addition, there are two prominently featured and moderated poster sessions selected from abstracts submitted to the meeting, a special session on Current Controversies and Late-Breaking Findings and a Hot Topics session that encourages last-minute submissions of exciting findings from new or established investigators culminating in awards given to several of the junior investigators. The main strength of this meeting is the opportunity for cross-disciplinary interactions in a highly focused, yet informal intellectually stimulating atmosphere. The Gordon Conference on NO plays an essential role in providing guidance and exploring new vistas in this important field of basic and translational research.

Project Summary/Abstract While the molecular mechanisms for the regulation of vascular tone by nitric oxide are well appreciated, the targets of nitric oxide (NO) signaling at the proteome level are incomplete. Decline in synthesis and bioavailability of NO is central to the pathogenesis of cardiovascular diseases and in age-dependent deterioration of vascular function and cardiac muscle performance. However, the signaling pathways affected by the decline in bioavailable NO during ageing remain unclear. Furthermore, several current clinical trials aim to restore the levels of NO in humans with hopes to improve cardiovascular and skeletal muscle physiological function and prevent vascular disease and disability in aged populations. Presently, the signaling pathways reestablished by pharmacological restoration of NO remain unknown. Therefore, we propose to use chemoselective, high- resolution, mass spectrometry-based proteomic technologies to identify and quantify for the first time at the organ and cellular level the two post-translational modifications protein phosphorylation and cysteine S-nitrosation, that constitute the two principle NO signaling pathways. We will resolve changes in the canonical signaling cascade, activation of soluble guanylate cyclase, production of cGMP and Ser/Thr phosphorylation of proteins and the complimentary selective S-nitrosylation of cysteine residues as a function of gender, ageing and in the setting of NO deficiency before and after restoration of bioavailable NO. Guided by preliminary data we will also investigate a novel mechanism for the regulation of the NAD-dependent protein deacetylase sirtuin 2 by NO. This regulatory function may have important cardioprotective functions. Completion of the proposed aims will provide new mechanistic insights and a framework for system-level appreciation of NO signalling in the cardiovascular system.