Paul Fox, PhD - US grants

The long term objective of this proposal is to understand the role of marine lipids on lesion formation in atherosclerosis. The proposal is based on four observations: (1) that smooth muscle cell (SMC) proliferation is an early and critical event in the formation of occlusive atherosclerotic lesions; (2) that ingestion of fish or fish oils containing n-3 polyunsaturated fatty acids (PUFAs) inhibits intimal thickening in experimental animals, and may decrease the incidence of coronary heart disease in man; (3) that cultured endothelial cells (EC) produce mitogens, especially a platelet-derived growth factor-like protein, that stimulate SMC proliferation in vitro; and (4) that growth factor production by cultured EC is regulatable and, as we have recently observed, is inhibited by fish oil emulsions and certain modified lipoproteins. To explain these observations, we propose that ingestion of fish oils results in an elevated level of specific marine lipids in plasma lipoproteins, that the altered lipoproteins inhibit EC production of growth factors, and that this regulatory mechanism is in part responsible for the observed decrease in SMC growth, and the proposed benefits of this diet. This hypothesis will be tested by pursuit of the following specific aims: 1. To characterize and identify the lipid(s) in marine oils that specifically inhibits EC production of growth factors. 2. To determine if cellular processing is required for the expression of the inhibitory activity of marine lipids. 3. To determine the biosynthetic step(s) regulated by marine lipids that causes the inhibition of growth factor production by EC. 4. To determine whether physiological lipoproteins enriched with marine lipids inhibit EC production of growth factors. 5. To determine if marine lipids inhibit the proliferation of cultured vascular SMC. Answers to these questions of vascular cell biology should contribute to our understanding of the cellular interactions of EC and SMC. The mechanistic insights resulting from this work may help to explain the recent epidemiological studies which suggest that diets rich in n-3 PUFAs are associated with reduced incidence of coronary heart disease, and may eventually lead to the development of pharmacologic inhibitors of atherogenesis.

The objective of this project is to test several ideas concerning the dynamic processes responsible for cresting spatial and temporal variation in ridge geometry on the slow- intermediate spreading rate ridge system in the South Atlantic from 31 to 36 South. Program involves two ship board legs to conduct geophysical studies including gravity, magnetics, and seismic refraction, along with Seabeam and dredging. Shipboard program is to be followed by lab based geochemical analysis and 3 dimensional numerical modeling of results. %%% This is an exciting project from a first rate team of investigators who are attempting a very thorough study of fundamental processes at ridge crests. The multi-disciplinary approach is a positive step forward, as is the idea of studying deep seated process such as mantle upwelling in conjunction with their surface expression in gravity, and geochemistry.

This is a project to map a section of the Mid-Atlantic Ridge in the South Atlantic and is designed to answer fundamental questions and to test hypothese central to understanding of a slow to intermediate spreading rate ridge system in the South Atlantic. The questions to be addreses include: 1) What is the segmentation geometry of the Mid-Atlantic Ridge in the South Atlantic and what is the relative abundance of first and second order discontinuities? 2) How does the segmentation geometry observed along a representative length of ridge compare with the early segmentation character of the South Atlantic in terms of the distribution of first and second order discontinuities? 3) What are the structual and kinematic characteristics associated with a change in plate boundary geometry and the creation of a second-order discontinuity? 4) What are the strutural and morphologic characteristics associated with the trnsfromation of a first-order discontinuity to a second-order discontinuity and what is the age of the offset when the boundary no longer behaves rigily? 5) What are the morphotectonic characteristics associated with evolution of a second-order discontinuity to a third-order discontinuity? 6) What is the distribution of rifted and non- rifted ridge segments along the MAR between 22S and 34S?

This research is designed to establish the morphological character, segmentation geometry, and structure of a significant length of the Mid-Atlantic Ridge in the central North Atlantic, and to test hypotheses on crustal accretion processes along a slowly spreading mid-ocean ridge. Questions to be addressed are: (1) What is the effect of the Azores hotspot on the morphology, segmentation geometry, and structure of the Mid-Atlantic Ridge? (2) Do major fracture zones in the central North Atlantic separate portions of the Mid-Atlantic Ridge with significantly different styles and wavelengths of segmentation? (3) How are differences in the pattern of mantle Bouguer anomalies at the Mid-Atlantic Ridge related to inter-segment morphotectonic variability? (4) What is the relative abundance of conventional transform faults and non-transform discontinuities along the Mid-Atlantic Ridge? (5) What controls the formation, stability, and evolution of non-transform offsets on the Mid-Atlantic Ridge? The principal investigators will participate in two cruises of the French ship R/V l'Atalante.

The regulation of oxidant and antioxidant activity has special significance in the development of cardiovascular disease in general, and atherosclerosis in particular. Recent evidence suggests that low density lipoprotein (LDL) that is modified by oxidation accumulates in atherosclerotic lesions, and in vitro studies suggest that oxidized LDL may be responsible for the dysfunctional behavior of lesion cells. The mechanisms of LDL oxidation are not known, but the cells present in lesions can oxidize LDL in vitro. The role of ceruloplasmin in any of these processes has not been investigated, but numerous reports of a potent antioxidant activity that can block the oxidation of lipids motivated us to begin studies of its role in LDL oxidation and lesion formation. Ceruloplasmin is an abundant 132 kDa acute-phase copper-protein carrying 95% of the copper in plasma. In contrast to previous reports, our preliminary studies show that ceruloplasmin is an extremely potent oxidant; at physiological levels it increases the oxidation of LDL by at least 25-fold. We have found that ceruloplasmin function is extremely sensitive to structural modification since oxidant activity (but not a distinct oxidase activity) is completely suppressed by (1) the removal of a single, specific bound copper or by (2) a single proteolytic event that cleaves ceruloplasmin into 116 and 19 kDa fragments. The latter finding may explain earlier reports of antioxidant activity. In other preliminary studies, we have observed that ceruloplasmin may be involved in cell- mediated oxidation. Ceruloplasmin can substitute for free metal ions in stimulating LDL oxidation by endothelial cells and smooth muscle cells. Ceruloplasmin may also regulate macrophage oxidation of LDL; in preliminary studies, we have found that agents that stimulate LDL oxidation by activated U937 cells (a monocytic line) also stimulate ceruloplasmin gene expression and protein production. Furthermore, anti- ceruloplasmin antibodies suppress much of the oxidant activity of U937 cells. Finally, a role for ceruloplasmin in oxidative processes in lesions is supported by our recent observation that ceruloplasmin is abundant in atherosclerotic lesions, but not in adjacent non-lesioned areas, of human carotid endarterectomy specimens. These new findings have led us to propose that the induced synthesis and secretion of ceruloplasmin by activated monocytes contributes to the oxidation of LDL by these and other vascular cells, and thus plays a critical role in pathological accumulation of oxidized lipoproteins in the vessel wall. The hypothesis will be tested in this proposal by examining the structural features of ceruloplasmin required for oxidant activity, by investigating its interaction with LDL, by determining the role of ceruloplasmin in vascular cell-mediated oxidation of LDL, and finally, by studies on the distribution and activity of ceruloplasmin in atherosclerotic lesions.

Oxidative modification of low density lipoprotein (LDL) is thought to be an important event during atherogenesis. The mechanism(s) by which lipoproteins are oxidized in vivo is largely unknown and a major-long term goal of this project. During the last several years we have explored the pro-oxidant activity of ceruloplasmin, a Cu-containing plasma protein, and have shown that it plays an important role in monocytic cell-mediated oxidative processes. Our evidence is that purified human ceruloplasmin induces LDL oxidation in vitro, that cultured monocytic cell-mediated oxidative processes. Our evidence is that purified human ceruloplasmin induces LDL oxidation in vitro, that cultured monocytic cells secrete ceruloplasmin is regulated at the mRNA and translational levels, and that immunohistochemical analysis shows that ceruloplasmin, in association with macrophages, is abundant in atherosclerotic lesions of human carotid endarterectomy specimens. These findings have led us to propose the following hypothesis: That lesion-derived cytokines stimulate macrophage production of ceruloplasmin, which as a Cu-containing, pro-oxidant molecule accelerates lipoprotein oxidation and atherosclerotic lesion formation. We will test this hypothesis by pursuing three specific aims. We will use biochemical and molecular approaches to determine the domains of ceruloplasmin involved in oxidant activity. We will investigate transcriptional and translational mechanisms that regulate ceruloplasmin synthesis by monocytic cells. Finally, we will determine the role of ceruloplasmin in vessel wall lipoprotein oxidation and atherosclerotic lesion formation in vivo. To accomplish this last aim, we will generate transgenic mice that over express ceruloplasmin and breed them with atherosclerosis-susceptible apo E-deficient mice. An understanding of the mechanisms underlying vessel wall oxidation processes is likely to have a major impact on the development of treatments that specifically reduce pathological oxidation while minimizing the effect of normal oxidative processes.

DESCRIPTION (adapted from the application) The importance of iron is underscored by its participation in many cellular processes involving oxygen or redox reactions. Iron in excess of cellular needs is toxic; dietary overload or hereditary hemochromatosis leads to tissue iron deposition and injury, most likely due to redox activity of iron and consequent free radical reactions. The precise balance required to maintain appropriate cellular and tissue iron levels has led to mechanisms that regulate the synthesis of iron transport and storage proteins, e.g., transferrin receptor and ferritin. A role for copper in iron metabolism has been known for about 70 years. The important role for ceruloplasmin (Cp) in iron metabolism in vivo has been reinforced by the identification of "aceruloplasminemia" patients with Cp gene defects and massive iron deposits in many tissues, including the brain. This function for Cp has received support from studies showing that two Cp homologues, fet3p in yeast and hephaestin in mouse, play key roles in iron homeostasis. We have shown that the rate of Cp synthesis by HepG2 and Hep3B cells is tightly regulated by cellular iron status. Iron deficiency markedly increases Cp protein synthesis and gene expression. Nuclear "run-on" and mRNA stability studies indicate that regulation is by a transcriptional mechanism. We have new evidence that Cp transcription is regulated by hypoxia-inducible factor (HIF)-1 responsive elements since transcription is regulated by hypoxia and other HIF-1 activators. In addition, an enhancer element in the human Cp gene 5'-regulatory region contains HIF-1 responsive elements which increase reporter gene expression about 10-fold. In contrast to the stimulatory activity of iron deficiency, excess iron decreases Cp synthesis of HepG2 cells; surprisingly, a post-transcriptional mechanism has been observed in which Cp mRNA is destabilized by iron. These results support the important role of Cp in maintenance of cellular iron homeostasis. We hypothesize that the rate of hepatic Cp synthesis, like that of other proteins involved in iron transport, is regulated by cell iron status. In this application we will test the following specific aims: that a specific trans-activating factor(s) in iron-deficient HepG2 cells binds to a cis-acting element in the Cp 5'-regulatory region, thereby increasing Cp transcription. Furthermore, iron in excess alters the activity of a specific trans-acting factor(s) that binds to a region of the Cp 3'-UTR, thereby destabilizing the Cp mRNA. The long-term goal of this research is to understand the specific function of Cp in iron homeostasis, and especially its role in primary (genetic) and secondary (dietary) iron overload states.

This project is developing two new AAS degree programs in Manufacturing Engineering Technology and Plastics and Polymers Manufacturing. These programs are based on the development of new course materials and on the adaptation of curricula and courses from Sinclair Community College and the Pennsylvania College of Technology. The programs serve current manufacturing employees, employees who are newly entering the workforce or seeking a career change, and graduating high school students aspiring to develop essential technical career skills. Course content addresses critical workforce skills that have been identified by regional manufacturers. Articulation partnerships are established with Old Dominion University and Virginia Polytechnic Institute and State University. These partners offer third- and fourth-year-level manufacturing technology courses for program graduates.

Inflammation and macromolecule oxidation are inter-related processes that are major contributors to atherogenesis. An important role of Cp in inflammation is suggested by its increased plasma level during the acute phase reaction and by its synthesis by activated macrophages in sites of inflammation. Clinical studies have shown that elevated plasma Cp is a significant risk factor for atherosclerosis, restenosis after endarterectomy, and myocardial infarction. In view of our finding that Cp copper induces oxidative modification of LDL in vitro, Cp may represent an important molecular link connecting inflammation, oxidation, and atherosclerosis. In new preliminary studies, we show that Cp binds with high affinity to LDL, and that the interaction is required for LDL oxidation by Cp. Other preliminary evidence indicates that Cp binds LDL in human plasma, and that binding to LDL may be higher in patients with cardiovascular disease. Studies with epitope-specific antibodies to Cp localized the interaction site to the second major sequence domain of Cp. These structural studies may be relevant to human inflammatory diseases, e.g., atherosclerosis, since coding-altering, single nucleotide polymorphisms (SNPs) have been identified in the human Cp gene, and two are adjacent to the Cp/LDL binding site. Preliminary studies in mice with targeted deletion of the Cp gene suggests that Cp increases oxidative modification of proteins and lipids in inflammatory sites in vivo. From previous work and our new preliminary results we propose the following hypothesis: That in sites of inflammation, macrophage-derived Cp binds to LDL and causes specific copper ion-mediated oxidative modification of the protein and lipid components. We further propose that by defining the specific Cp domains and amino acids required for pro- and anti-inflammatory activities, recombinant, monofunctional Cp can be generated that can test the function of the individual activities in vivo. Finally, we propose that human Cp containing SNPs in these functional domains alter the inflammatory properties of Cp, and also the risk for atherosclerosis in humans. We will test this hypothesis by pursuing the following Specific Aims: Aim 1. Determine the structural requirements for pro- and anti-inflammatory Cp activities; we will analyze structural determinants of Cp required for LDL oxidase, ferroxidase, and nitrosation activities, and for Cp interaction with LDL. Aim 2. Investigate the role of Cp in oxidation during inflammatory processes in vivo. Cp-null mice will be subjected to sepsis and peritonitis models of inflammation, and specific lipid and protein oxidation products in peritoneal lavage and plasma will be measured by LC-mass spectrometry These studies will elucidate, at the molecular, animal, and patient level, the role of Cp activities in inflammation.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] Endothelial cell (EC) movement is initiated by angiogenic growth factors, which trigger a sequence of spatially polarized intracellular events including the activation of motility-regulating small GTPases and the assembly of actin-dependent, force-generating systems at the cell anterior. Our primary interest is the role of the plasma membrane in cell movement. We have shown that membrane microviscosity is a key determinant of motility, and that basic fibroblast growth factor increases EC plasma membrane microviscosity as measured by fluorescence recovery after photobleaching (FRAP). Spatial analysis shows a highly polarized gradient of microviscosity in plasma membranes of rapidly migrating EC, with a leading edge that is substantially more viscous than the trailing edge. An important role of cholesterol in generation of this membrane microviscosity gradient is suggested by an increase in cholesterol content of the membrane, by gradient reversal upon cholesterol removal, and by relocalization of a fluorescent cholesterol analog, NBD-cholesterol, to the front of moving ECs. In studies of the mechanism that drives membrane polarization we have observed that caveolin-1, an intracellular cholesterol transport protein, is also highly polarized and accumulates in the rear of migrating ECs. In studies of the mechanism by which membrane physical properties regulate motility, we have found that increased membrane microviscosity increases the binding of Racl to plasma membranes in the front of moving ECs. We have also found that the ability of actin to deform large unitamellar vesicles is decreased when microviscosity is high, i.e., at an elevated cholesterol-to-phospholipid ratio. From these data we propose as a hypothesis that angiogenic growth factors alter cholesterol synthesis and trafficking to increase the membrane microviscosity at the leading edge of the moving cell. We further propose that increased microviscosity increases cell movement by increasing Racl-binding to the plasma membrane and by altering the barrier properties to improve the efficiency by which actin filaments propel the cell forward. We will test this hypothesis in three Specific Aims: (1) Determine the molecular mechanism regulating polarization of membrane microviscosity during EC movement, (2) determine the mechanism by which microviscosity regulates Racl binding to membranes and (3) determine the role of membrane microviscosity in regulation of actin filament formation and function. The experiments will make use of cultured cells expressing GFP-tagged These studies will provide basic information on mechanisms regulating cell motility and may lead, in the long-term, to molecular strategies to inhibit or enhance cell migration. Pharmacological agents based on these results may be useful for inhibition of tumor angiogenesis or to enhance collateral blood vessel formation and the healing of synthetic vascular grafts. [unreadable] [unreadable]

This "Produced in Virginia" implementation project is combining the efforts of Central Virginia Community College, Danville Community College, and the University of Virginia (with a third community college partner added during the second year of the project) to provide undergraduate connections in engineering education with the goal of increasing the number of students enrolling in and graduating from engineering associate and baccalaureate degree programs. The partnership is achieving this goal through the following strategies:
(1) Adapting and replicating the proven Priming the Pipeline model that encourages students to consider STEM careers and prepares them for success in college STEM courses with a new addition of delivering the university's introductory Explorations in Engineering course by distance education.
(2) Implementing new and upgraded curricula at all three community colleges, leading to an Associate of Science degree in engineering and guaranteeing students with a 3.4 average seamless transfer into the University of Virginia School of Engineering and Applied Science.
(3) Offering courses at the transfer and graduation level at the community colleges for engineering students from the University of Virginia's Bachelor of Science in Engineering Degree program as well as offering transfer courses at the University's main campus.
(4) Offering tuition assistance for students pursuing undergraduate engineering degrees and engaging those students in technical employment.
(5) Implementing a distance degree program pilot enabling students graduating from the Central Virginia A.S. degree program to earn undergraduate engineering degrees from the University of Virginia.
(6) Developing an academic mentoring program using Web 2.0 technology solutions.
The broader impact of this project is in serving as a viable and reproducible model for collaborations between community colleges and universities to facilitate student success in an era of rising costs. It is also contributing to the country's capacity in science and engineering in an increasingly competitive and changing global labor market.

DESCRIPTION (provided by applicant: Chronic inflammation is a major contributor to the onset and progression of atherosclerosis and other vascular disorders. Studies of inflammation have focused primarily on the initiating processes and less attention has been given to the mechanisms by which inflammation is terminated. During the last several years a new paradigm has been invoked in which endogenous mediators limit or reduce these responses as part of an active "resolution of chronic inflammation" process. In our own studies we have described the discovery of a novel translational control system that has the features of an endogenous regulator of the inflammatory response in myeloid cells. We have shown that interferon (IFN)-gamma, a classic activator of monocyte/macrophages, induces assembly of the IFN-Gamma-Activated Inhibitor of Translation (GAIT) complex that binds a specific RNA element in the 3'untranslated region (UTR) of target mRNAs, e.g., vascular endothelial growth factor (VEGF)-A, and inhibits their translation. Remarkably, the GAIT complex consists of four abundant, "house-keeping" proteins especially recruited for this function: Glu-Pro-tRNA synthetase (EPRS), ribosomal protein L13a, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and NS1-associated protein (NSAP1 or hnRNP Q). The complex assembles in two stages: An early, pre-GAIT complex of unknown function that contains EPRS and NSAP1, and a late, functional GAIT complex that contains all four proteins. We have elucidated the critical phosphorylation sites essential for GAIT complex activation, namely, Ser77 in L13a and Ser999 in EPRS. Also, we have reported that interaction of NSAP1 acts as an early negative regulator of EPRS binding to target RNAs, and that the inhibition is overcome by late joining of L13a and GAPDH to form an active GAIT complex. In new Preliminary Studies we show that the GAIT pathway is functional in mouse macrophages;however, the mouse GAIT complex is heterotrimeric, lacking NSAP1 and early negative regula- tion. We propose to take advantage of this new information to generate a comprehensive ensemble of genetically-altered mice with mutations in key phosphorylation sites of GAIT components that inactivate, constitutively activate, or modulate, GAIT function in vivo. We will investigate gene expression and the role of the GAIT pathway in atherosclerosis using the well-established apoE-null mouse model. Generation of these mouse models will permit us and others to investigate the role of post-transcriptional gene regulation in the resolution of inflammation, and also the pathological consequences resulting from its dysregulation. These experiments will reveal insights into post-transcriptional mechanisms that regulate the protein expression pro- file of inflammatory macrophages, and the models will provide a unique resource for rigorous analysis of the causes of inflammation-resolution and the consequences of its dysregulation. PUBLIC HEALTH RELEVANCE: Chronic inflammation is a major contributor to the onset and progression of atherosclerosis and other vascular disorders. Our laboratory has discovered a new pathway, the interferon-gamma-activated inhibitor of translation (GAIT) pathway, that functions to reduce or resolve inflammation by inhibiting the synthesis of pro-inflammatory proteins. We propose to develop genetically-modified mice to test the role of the GAIT pathway in preventing chronic inflammatory, cardiovascular disorders such as atherosclerosis. These models may reveal alternative targets for novel anti-inflammatory therapeutic strategies that, because of their specificity, may exhibit minimal adverse side-effects.

The scope of the Atherosclerosis and Lipoprotein Analysis Core facility is the quantitative assessment of atherosclerosis lesion areas at two locations in mouse models. For the aortic root assay, each individual investigator is only responsible for providing the excised saline-perfused heart in 10% phosphate buffered formalin. The Core personnel will then: 1) embed the heart in gelatin, 2) freeze the gelatin block in OCT, 3) prepare 5 slides containing four 12.5 micron thick sections each, that cover the first 500 microns of the aortic root, 4) stain the slides in oil red O, hematoxylin, and fast green, and 5) quantitate the lesion areas on one section per slide and calculate the mean lesion are per section. For the en face assay, the Core will provide a trained technologist to work under the project leader's approved protocol to: 1) anesthetize the mice, 2) perform saline perfusion, which euthanizes the mouse by exsanguination, 3) dissect the mouse to display the mouse aorta in situ, 4) remove the aorta and trim off adventitial tissue, 5) cut the aorta longitudinally and fix it flattened between two microscope slides, 6) stain the aorta with oil red O, 7) mount the aorta on a microscope slide, and 8) quantify the % surface lesion area in the entire aorta, as well as in the regions of the aortic arch, and thoracic and abdominal aortas.

DESCRIPTION (provided by applicant): Blood vessels are exposed to multiple stresses that can induce injury to the endothelium, which must undergo rapid repair to restore normal function of the vessel and the entire cardiovascular system. Endothelial cell (EC) migration has a critical role in this repair process, as well as in formation of new blood vessels. Polarization of multiple cell systems, including signaling, adhesion proteins, plasma membrane, secretion processes, cyto- skeletal arrangement, etc., is crucial for induction and control of cell migration. Recent studies have shown that 2-actin mRNA, and several other motility-related transcripts, accumulated in the front of moving cells, but little is known about the total ensemble of polarized mRNAs and their common localization elements. Our long-term goal is to understand the contribution of mRNA polarization to the development of asymmetry during EC movement, and the mechanisms by which mRNAs are polarized in planar moving cells. To accomplish this goal, we have initiated a comprehensive, global analysis of the mRNAs that become polarized during EC migration. We have applied the laser microdissection methodology to capture cell fragments from forward and rearward regions of planar-moving EC, a model representing the wound-healing process. We propose to couple this microdissection method with microarray technologies, followed by rigorous biochemical investigation to address the following hypothesis: cis-elements in mRNAs determine their localization in both the front and back of planar-migrating EC, and are responsible for targeting proteins to their site of function. We will test this hypothesis by pursuing the following specific aims: (1) identify novel mRNAs polarized in migrating EC, and (2) investigate RNA elements responsible for mRNA polarization during cell migration. Our studies will provide the first global analysis of mRNA localization during cell migration, and it will begin to fill important gaps in our understanding of cell polarization during migration. In particular, little is known about the RNA elements that induce mRNAs to accumulate in the cell front and virtually nothing is known about mRNAs (and their functional elements) that accumulate in the cell rear. In addition to the mechanistic contribution to our understanding of cell polarization, our work can reveal new insights into the regulation of cell movement, and has the potential of new interventions to alter motility. For example, identification of novel localization elements presents novel therapeutic targets for modulating mRNA localization and cell migration by RNA-based approaches, for example, by treatment with antisense RNA oligomers targeting the specific RNA element. Finally, our work is likely to provide and validate a technological advance that will permit global analysis of asymmetric mRNA in other systems. PUBLIC HEALTH RELEVANCE: Blood vessels are exposed to multiple stresses that can injure the endothelial cell (EC) lining of the vessel wall. Subsequent migration of EC is critical for vessel wall repair and restoration of normal blood vessel function. Polarization of the messenger RNAs (mRNA) encoding movement-related proteins is a newly discovered event that may contribute to cell directionality required for EC movement. Our studies will provide the first global analysis of mRNA polarization during planar cell migration, and it may reveal novel therapeutic targets for modulating localization of motility-related mRNAs and cell migration.

DESCRIPTION (provided by applicant): The long-term goal of our research program is to elucidate the post-transcriptional mechanisms that modulate gene expression in inflammation of the vasculature. Interferon (IFN)-3 is the classic activator of monocyte/macrophages, and it induces rapid transcription of inflammatory growth factors, proteases, chemokines, and generators of radical species. If unregulated, this process becomes chronic and monocyte/macrophage products accumulate, damage host tissue, and contribute to chronic disorders of blood vessels, e.g., atherosclerosis. The termination of inflammation is not a passive process that begins after elimination of the initial insult;in contrast, intrinsic mechanisms actively limit expression of potentially injurious proteins. Recently, investigators have recognized the important role of post-transcriptional processes in limiting or resolving inflammation. We have discovered a novel translational control pathway that acts as an endogenous regulator of the inflammatory response. In myeloid cells, IFN-3 induces assembly of the heterotetrameric, IFN-Gamma-Activated Inhibitor of Translation (GAIT) complex, which binds an RNA element in the 3'untranslated region of certain pro-inflammatory target mRNAs, e.g., vascular endothelial growth factor-A, and inhibits their translation. In Preliminary Studies we show that one GAIT protein, glutamyl-prolyl-tRNA synthetase (EPRS), is central to the GAIT system because it is responsible for target mRNA recognition, and its function is regulated by phosphorylation and binding of the other 3 GAIT proteins. We suggest EPRS is not an inert, protein-binding scaffold, but rather a dynamic system subject to stimulus-inducible modifications that regulate GAIT complex assembly and function. Based on these results, we propose the following hypothesis: Phosphorylation of EPRS by IFN-3- dependent kinases causes conformational changes in EPRS that regulate assembly of the GAIT complex, which silences translation of inflammatory mRNA targets and contributes to the resolution of chronic inflammation. We will test this hypothesis by pursuit of three Specific Aims. In Aim 1 we will determine the EPRS domains required for GAIT complex assembly and GAIT element-binding. In Aim 2 we will determine the role of EPRS phosphorylation in GAIT complex assembly and function. In Aim 3 we will investigate the anti- inflammatory function of EPRS and the GAIT complex in vivo. Dysregulation of anti-inflammatory mechanisms in vascular disease is an important, under-investigated area. Our studies will elucidate the molecular mechanism of a post-transcriptional pathway that regulates the protein expression profile of inflammatory macrophages, and will improve our understanding of mechanisms in the limitation and resolution of chronic inflammation. Understanding endogenous, anti-inflammatory "off" mechanisms is crucial because defects can contribute to inflammatory disorders, and because the pathway itself may present alternative targets for development of novel anti-inflammatory therapeutics to reduce vascular disease. PUBLIC HEALTH RELEVANCE: Our studies will elucidate a new pathway that regulates the synthesis of inflammatory proteins by macrophages, an important process in the development of vascular diseases such as atherosclerosis. The pathway under investigation contributes to the limitation and resolution of chronic inflammation, an important causative factor in disease progression. A deeper understanding of inflammatory "stop" pathways is important because defects in these pathways can contribute to vascular disorders, and because the pathway itself may present alternative targets for development of novel anti-inflammatory therapeutics.

DESCRIPTION (provided by applicant): Our long-term goal is to understand the interactions between elements in noncoding regions of vertebrate mRNAs, and their cognate binding proteins, and how they integrate signals from disparate stimuli to control translation. Transcript-selective translational control is mediated by interactions of RNA-binding proteins to sequence/structural elements in non-coding regions, most often the 5'- or 3'-untranslated region (UTR) of the target transcript. In addition to protein-RNA interactions, RNA-RNA interactions also regulate gene expression, e.g., riboswitches in the UTR of bacterial mRNAs contain proximate structural elements that undergo conformational change in response to specific metabolites, and control translation. Recent experiments in our laboratory suggest that human vascular endothelial growth factor (VEGF)-A mRNA contains adjoining elements that function as a novel stimulus-dependent, protein-directed riboswitch that exists in two metastable conformations: a translation-silencing and a translation-permissive conformer. The binary switch is controlled by integration of two signals, interferon (IFN)-? and hypoxia, that regulate the amount or activity of the binding factors. Upon cell stimulation by IFN-?, Glu-Pro tRNA synthetase (EPRS) is released from its residence from the tRNA multisynthetase complex and joins the GAIT (IFN-Gamma-Activated Inhibitor of Translation) complex. EPRS binds a defined, 29-nt GAIT element in the VEGF-A mRNA 3'UTR, stabilizing the translation-silencing conformer and inhibiting translation. However, superimposition of hypoxia on IFN-? stimulation increases the level of heterogeneous nuclear ribonucleoprotein (hnRNP) L that binds a CA-rich element directly upstream of the GAIT element, stabilizing the translation-permissive conformer and allowing VEGF-A expression. We propose the following specific hypothesis: The myeloid cell integrates signals from IFN-? and hypoxia by regulating the relative amounts of hnRNP L and GAIT complex, which in turn dictate the conformation of the VEGF-A 3'UTR to either permit or suppress VEGF-A mRNA translation. We will test this hypothesis by pursuing the following Specific Aims: Aim 1: Determine sequences and secondary structures in the VEGF-A mRNA 3'UTR required for binary switch function. Aim 2: Determine the role of VEGF-A 3'UTR binding proteins in switch function. Aim 3: Investigate regulation of the VEGF-A 3'UTR binary switch by IFN-? and hypoxia. We hypothesize that the switch evolved to maintain VEGF-A expression and angiogenesis in hypoxic, inflammatory tissues. Tumors, also residing in hypoxic, inflammatory sites, may take advantage of the VEGF-A switch to stimulate inward blood vessel growth to provide nourishment and permit tumor growth. Thus, the VEGF-A switch represents a novel therapeutic target to specifically inhibit tumor macrophage expression of VEGF-A. We also speculate that the VEGF-A switch may represent the founding member of a family of protein-directed riboswitches in vertebrates that integrate other physiological or pathological stimuli to control gene expression. PUBLIC HEALTH RELEVANCE Certain messenger RNAs respond to changes in their environment by altering their folding structure and their rate of expression of protein products. Although these "riboswitches" are found primarily in bacteria, we have found a similar switch in the mRNA encoding human vascular endothelial growth factor (VEGF), a protein critical for blood vessel formation. The VEGF riboswitch is sensitive to inflammation and hypoxia, two conditions found in the tumor environment, and an understanding of its mechanism may reveal insights into tumor growth and potential therapies to inhibit the process.

DESCRIPTION (provided by applicant): Chronic kidney disease (CKD) is characterized by mild to severe anemia with severe pathological consequences. Treatment of anemia in CKD patients by intravenous injection of iron and erythropoietin (Epo) in combination with dialysis, significantly improves results. Optimal treatment is hampered by the lack of quantitative information on iron and Epo dosage, and by the high cost of recombinant Epo. Moreover, excess iron exposes the patient to cardiac injury, presumably by iron-induced, free radical oxidative damage. The long- term goal of our research program is to develop a predictive model of whole-body iron metabolism that will take advantage of recent and continuing advances in understanding the genetics and cell biology of iron transport mechanisms. The model is expected to provide quantitative, mechanism-based guidance for personalized treatment of patients with CKD to maximize efficacy and minimize injurious side-effects of iron supplements. We will take a systems biology approach in which mathematical modeling and simulation is used in the design and analysis of experiments. Our approach spans diverse research areas and necessitates an experienced research team with expertise in experimental biology, computational modeling, and medicine. We have assembled a unique multidisciplinary team of recognized experts in all major aspects of this application. The team is led by a cell and molecular biologist (Paul Fox, Ph.D., P.I.) and by a biomedical engineer with specific expertise in mathematical modeling of complex biological systems (Gerald Saidel, Ph.D. Co-I). They share long-standing interest in experimental and computational/modeling aspects of iron metabolism. The other essential members of our team are: Saul Nurko, M.D., a nephrologist with expertise in treatment of CKD anemia, Linda Graham, M.D., a vascular surgeon who has improved the surgical model of CKD in the mouse, Alan Lichtin, M.D. and Roy Silverstein, M.D., hematologists with expertise in treatment of hematopoietic disease and cell biology, respectively, and Marc Penn, M.D., Ph.D., a cardiologist with expertise in heart physiology and failure. Our team is highly integrated with multiple interactions among the participants. Our model will uniquely take advantage of the kinetic characteristics of iron transport proteins as opposed to previous diffusion-based models. The whole-body model will be used to develop optimal treatment regimens for the anemia of CKD. The regimens will be tested in vivo in a surgical model of CKD in mice. Adverse effects on the heart will be measured as cardiac iron accumulation and cardiac function and hypertrophy. The potential impact of this work is very high. A predictive, mechanism-based mathematical model of whole-body iron metabolism will provide testable insights into the interactions between the key iron homeostatic systems - the small intestine, bone marrow, liver, and marrow. Moreover, this model can provide future clinicians with a tool for personalized treatment of CKD (and potentially other anemias) that can optimize erythropoiesis, minimize cost, and most importantly, reduce adverse side-effects such as cardiac hypertrophy and failure. PUBLIC HEALTH RELEVANCE: Chronic kidney disease (CKD) patients are generally afflicted with mild to severe anemia, which can lead to heart failure and other cardiovascular disorders. The anemia-related symptoms of kidney disease are improved by treatment with the hormone erythropoietin and iron, but their beneficial effect on heart disease is controversial, and recent studies show increased risk of heart disease upon over-treatment. We have assemble a unique multidisciplinary research team of scientists and clinicians, with expertise in kidney and heart disease, cell biology, iron metabolism, systems biology, and mathematical modeling. Our goal is to investigate the mechanisms regulating iron homeostasis during of CKD, and devise regimens for personalized treatment that will improve health and minimize risk.

The purpose of Computational Chemistry sub-Core is to provide computational/modeling support for the investigators within the Program Projects. Computational chemistry and molecular modeling techniques will be used to gain structural/functional insight into specific molecular interactions present in the biomolecular complexes studied within different projects of the PPG. This sub-Core will integrate experimental data produced by Projects and other Cores in the Program with theoretical methods in order to produce structural information needed to elucidate the nature of interactions in these biosystems and the relationship between their structure and function. For example, the sub-Core will provide atomistic models for biomolecular complexes like protein L13A-RNA complex, eNOS complex with HSP-90 and caveolin, and HDL-PON1-MPO complex, which are investigated in Projects 3, 2 and 1, respectively, using molecular visualization/building programs (Pymol, SwissPDBViewer, Autodock4 and Modeller), and hydrogen-deuterium exchange and small angle neutron and X-ray scattering calculations. The interaction interface between different components of the complexes will be constructed using docking (Autodock4). The docking experiments will identify specific interactions between amino acid residues for protein-protein complexes, or between RNA nucleotides with amino acid residues for RNA-protein complexes, or between amino acid residues and lipids for lipoproteins. All solvated systems will be subjected to molecular dynamics simulations. The trajectory resulted from the simulation will be analyzed to determine the change in the conformation during simulation, the change in the pattern of H-bonds and salt-bridges, the change in the secondary structure and so forth. To investigate conformational changes that occur on a microsecond scale and are important for the functionality of the biomolecular system, coarse-grained simulations will be performed in which atoms are grouped together in beads and a bead-to-bead simplified force field is used. The theoretical understanding resulted from the computational/modeling investigation will be further used by the Projects to design new experiments.

The long-term goal of Project 2 is to elucidate the role of ribosomal protein L13a in post-transcriptional regulation of inflammatory gene expression in monocyte/macrophages. Interferon (IFN)-gamma is the classic activator of monocyte/macrophages; it induces rapid transcription of inflammatory growth factors, proteases, chemokines, and generators of radical species. If unregulated, this process becomes chronic and monocyte/macrophage products accumulate, damage host tissue, and contribute to chronic disorders of blood vessels, e.g., atherosclerosis. We have discovered a novel translational control pathway that acts as an endogenous regulator of the inflammatory response. In myeloid cells, IFN-gamma induces assembly of the IFN-Gamma-Activated Inhibitor of Translation (GAIT) complex, which binds an RNA element in the 3¿untranslated region of pro-inflammatory target mRNAs, and inhibits their translation. In Preliminary Studies we show that one GAIT protein, L13a, has a critical role in the GAIT system: its function is regulated by phosphorylation, it induces conformational changes in other GAIT proteins to regulate target mRNA recognition, and by interaction with eIF4G it is responsible for the observed translational silencing. Recently, we have shown that stress can alter GAIT system activity and influence inflammatory gene expression. Based on these results, we propose the following hypothesis: IFN-gamma-dependent phosphorylation of L13a induces a conformational change that facilitates its release from the 60S ribosomal subunit, formation of the GAIT complex, and binding to eIF4G to cause translational silencing of inflammatory transcripts; moreover, physiological stress can alter GAIT system function and inflammatory gene expression. We will test this hypothesis by pursuit of three Specific Aims. In Aim 1 we will determine the mechanism of inducible release of L13a from ribosome in the response to inflammatory stimulus. In Aim 2 we will determine L13a interactions required for GAIT complex assembly and silencing of inflammatory gene expression. In Aim 3 we will determine the mechanisms by which stress alters GAIT complex function and inflammatory gene expression.

The purpose of Computational Chemistry sub-Core is to provide computational/modeling support for the investigators within the Program Projects. Computational chemistry and molecular modeling techniques will be used to gain structural/functional insight into specific molecular interactions present in the biomolecular complexes studied within different projects of the PPG. This sub-Core will integrate experimental data produced by Projects and other Cores in the Program with theoretical methods in order to produce structural information needed to elucidate the nature of interactions in these biosystems and the relationship between their structure and function. For example, the sub-Core will provide atomistic models for biomolecular complexes like protein L13A-RNA complex, eNOS complex with HSP-90 and caveolin, and HDL-PON1-MPO complex, which are investigated in Projects 3, 2 and 1, respectively, using molecular visualization/building programs (Pymol, SwissPDBViewer, Autodock4 and Modeller), and hydrogen-deuterium exchange and small angle neutron and X-ray scattering calculations. The interaction interface between different components of the complexes will be constructed using docking (Autodock4). The docking experiments will identify specific interactions between amino acid residues for protein-protein complexes, or between RNA nucleotides with amino acid residues for RNA-protein complexes, or between amino acid residues and lipids for lipoproteins. All solvated systems will be subjected to molecular dynamics simulations. The trajectory resulted from the simulation will be analyzed to determine the change in the conformation during simulation, the change in the pattern of H-bonds and salt-bridges, the change in the secondary structure and so forth. To investigate conformational changes that occur on a microsecond scale and are important for the functionality of the biomolecular system, coarse-grained simulations will be performed in which atoms are grouped together in beads and a bead-to-bead simplified force field is used. The theoretical understanding resulted from the computational/modeling investigation will be further used by the Projects to design new experiments.

DESCRIPTION (provided by applicant): Project Summary/Abstract Our long-term goal is to understand how interactions between elements in noncoding regions of vertebrate mRNAs and their cognate binding proteins integrate signals from disparate stimuli to control translation. Tran- script-selective translationl control is mediated by interactions of RNA-binding proteins to sequence/structural elements in the 5'- or 3'-untranslated region (UTR) of target transcripts. Recently, an additional layer of com plexity has been recognized in which element pairs act as condition-dependent RNA switches. For example, riboswitches are proximate structural elements in the UTR of multiple bacterial mRNAs that undergo conforma- tional changes in response to specific metabolites. We have reported an analogous, stimulus-dependent switch in the 3'UTR of human vascular endothelial growth factor (VEGF)-A mRNA. VEGF-A mRNA contains adjoining elements that function as a novel stimulus-dependent, protein-directed RNA switch that exists in two metastable conformations: a translation-silencing and a translation-permissive conformer. The binary switch is controlled by integration of two signals, interferon (IFN)-¿ and hypoxia, that regulate the amount or activity of the binding factors. Upon cell stimulation by IFN-¿, phosphorylation of Glu-Pro tRNA synthetase (EPRS) initiates formation of the GAIT (IFN-Gamma-Activated Inhibitor of Translation) complex. EPRS binds a defined, GAIT element in the VEGF-A mRNA 3'UTR, stabilizing the translation-silencing conformer and inhibiting translation. However, superimposition of hypoxia on IFN-¿ stimulation induces phosphorylation of hnRNP L at Tyr359 that initiates assembly of a newly discovered 3-component HILDA complex that binds a CA-rich element directly upstream of the GAIT element, stabilizing the translation-permissive conformer and allowing VEGF-A expression. We propose the following specific hypothesis: Myeloid cells integrate signals from IFN-¿ and hypoxia by inducing Tyr359 phosphorylation of hnRNP L and assembly of the HILDA complex, which in turn directs an RNA switch in the 3'-UTR of VEGF-A and other inflammation-related mRNAs to regulate translation. We will test this hypothesis by pursuing the following Specific Aims: Aim 1: Investigate molecular mechanisms regulating hnRNP L expression and localization; Aim 2: Determine the functions of HILDA components in regulating the RNA switch; Aim 3: Identify novel transcripts controlled by protein-directed RNA switches. We suggest that the switch evolved to maintain VEGF-A expression and angiogenesis in hypoxic, inflammatory tissues. Tumors, also residing in hypoxic, inflammatory sites, may take advantage of the VEGF-A switch to stimulate inward blood vessel growth to provide nourishment and permit tumor growth. Thus, the VEGF-A switch represents a novel therapeutic target to specifically inhibit tumor macrophage expression of VEGF-A. We also speculate that the VEGF-A switch may represent the founding member of a family of protein-directed RNA switches in vertebrates that integrate physiological or pathological stimuli to control gene expression.

Chronic inflammation is a principal cause of atherosclerosis and other vascular diseases. Inflammation is an orchestrated response to trauma incited by tissue injury or microbial invasion of the host. The inflammatory response is initiated by cytokines that induce cascades of signaling events culminating in the expression of new gene products, some of them toxic to invading organisms. Uncontrolled production or accumulation of inflammatory products can be injurious to the host organism. Mechanisms have evolved that limit the production of these products and permit resolution of the inflammatory response. The central hypothesis of this continuing Program Project is that pro- and anti-inflammatory processes in vascular cells are tightly regulated by endogenous signaling pathways, and their dysregulation contributes to vascular diseases such as atherosclerosis. We will investigate this hypothesis through three highly focused, but well-integrated projects led by a team of accomplished experts in diverse areas of vascular inflammation. In Project 1, Dr. Xiaoxia Li investigates the macrophage signaling pathways initiated by the interleukin-1R (IL-1 receptor)/TLR (Toll-like receptor) superfamily which can lead to either a pro-inflammatory, transcriptional program of gene expression, or an anti-inflammatory, post-transcriptional program. The theme of pro- and anti-inflammation, and also post-transcriptional regulation, continues in Project 2, led by Dr. Paul Fox, which focuses on a distinct post-transcriptional mechanism in monocyte/macrophages in which interferon-??induces phosphorylation- dependent formation of a complex that binds select inflammatory transcripts and inhibits their translation. The goal of Project 3, led by Dr. Paul DiCorleto, is to understand the role of the tumor necrosis factor receptor p75 and the transcription factor HOXA9 in the transcriptional regulation of pro-inflammatory genes in endothelial cells. Three scientific cores (Cell Culture, Atherosclerosis and Lipoprotein Analysis, and Macromolecular Interaction) and an Administration Core will provide multi-project support, expertise, and service in a cost-effective manner, which will significantly strengthen each investigator's research effort.

Leaders meet on a bi-weekly basis to discuss any scientific, administrative or fiscal matters pertaining to the program. Weekly scientific (research-in-progress) meetings are also held which are attended by

Project Summary/Abstract Protein S-nitrosylation is a ubiquitous, post-translational effector mechanism in which a Cys residue is modified by NO. Dysregulated S-nitrosylation is associated with diverse inflammatory diseases and pathological condi- tions. A concept is emerging in which site-specific modification of disease-related proteins, rather than a global nitrosative activity, contributes to disease initiation and progression. However, molecular mechanisms directing site selectivity are not understood. Our long-term goal is to elucidate mechanisms underlying selective S- nitrosylation, and its pathophysiological role in cardiovascular disease (CVD). We recently observed a remark- able S-nitrosylation-mediated inactivation of the GAIT (IFN-Gamma-Activated Inhibitor of Translation) transla- tional control system, discovered in our laboratory. Interferon (IFN)-?, a prototypic activator of myeloid cells, induces assembly of the GAIT complex that binds RNA elements in the 3?UTR of select target mRNAs, e.g., vascular endothelial growth factor-A, and inhibits their translation. The GAIT complex consists of four ?house- keeping? proteins including ribosomal protein L13a and GAPDH. We recently reported that low density lipopro- tein (LDL) oxidized by the physiological myeloperoxidase (MPO)-H2O2-NO2- system (LDLox) inactivates the GAIT system, thereby increasing expression of VEGF-A and other GAIT targets. LDLox induces S-nitrosylation of GAPDH, inactivating the chaperone-like activity by which GAPDH protects L13a, resulting in degradation of nearly the entire cell complement of L13a. An unprecedented site-specific S-nitrosylation of GAPDH at Cys247 is essential for loss of shielding activity. Recently, we found that LDLox plus IFN-? markedly induce iNOS (inducible nitric oxide synthase) in human monocytes and is required for S-nitrosylation of GAPDH at Cys247. We also showed that a heterotrimeric complex of S100A8, S100A9, and iNOS is cotranslationally assembled, and specifically S-nitrosylates GAPDH at Cys247. Serum and LDL from CVD subjects induce Cys247 GAPDH S- nitrosylation, L13a degradation, and VEGF-A expression in monocytes, supporting the physiological signifi- cance of these events. To further investigate the function of GAPDH S-nitrosylation in vivo, we have engineered Cys245 (mouse analog of human Cys247)-to-Ala knock-in mice. We have begun to investigate the role of S100A8/9 on global S-nitrosylation of macrophage proteins, and on macrophage function. The iNOS- S100A8/9 complex induces S-nitrosylation of a cohort of ~100 proteins, with likely involvement in macrophage gene expression, lipid metabolism, and foam cell formation. In this Project we will test the following hypothesis: LDLox, in the presence of IFN-? induces iNOS and subsequent cotranslational assembly of a ternary iNOS- S100A8/A9 nitrosylase complex that directs nitrosylation of Cys247 on GAPDH and other selected targets, causing dysregulation of the GAIT system and contributing to accelerated atherosclerotic lesion progression. Our results can lead to new biomarkers with strong predictive power, and potentially to new therapeutic targets that disrupt site-selective S-nitrosylation.

Project 3 (P3). Noncanonical activities of a tRNA synthetase in metabolism and atherosclerosis Paul L. Fox, Ph.D., Project Leader Project Summary/Abstract The long-term goal of Project 3 is to understand the noncanonical function of an extraordinary tRNA synthetase, the Glu-Pro tRNA synthetase or EPRS, in diet-induced obesity and consequent cardiovascular disease, particularly atherosclerosis. The recent explosive epidemic of obesity, insulin resistance, and cardiovascular disease has begun an unprecedented decline in the health-span of adults in the U.S., and likewise threatens an equally unprecedented economic burden. In 1942, James Neel suggested a possible rationale for the genetic selection of genes causing these pathologies. He posited that metabolic pathways were naturally selected to efficiently store fat and carbohydrate during periods of food scarcity; however, the same genes and pathways are detrimental during periods of plentiful, calorie-rich food supply as in the Western world today. A kinase cascade involving the mammalian target of rapamycin (mTORC1) and ribosomal protein S6 kinase-1 (S6K1) is implicated as a key metabolic pathway regulating food utilization, and is conserved from Drosophila to humans. Despite intense study, the key downstream effector(s) of mTORC1-S6K1, and consequent cell mechanisms that regulate metabolism remains unknown. During the previous Project period, we made fundamental in vitro and in vivo discoveries that revealed phosphorylated EPRS as a key downstream effector of mTORC1-S6K1, regulating post-transcriptional, inflammation-related pathways in macrophages and metabolic pathways in adipocytes. Inflammatory macrophages permeate the more abundant adipocytes in adipose tissue of obese subjects, and EPRS phosphorylation in both cell types by S6K1 might functionally couple these cells, and contribute importantly to obesity-associated cardiovascular disease. To test the role of phospho-EPRS in these processes we have generated genetically-modified EPRS phospho-deficient and phospho-mimetic knock-in mice. Preliminary studies show that phospho-deficient EPRS mice phenocopy S6K1-null mice, e.g., small size and low fat mass, and will permit rigorous investigation of the role of EPRS in mTORC1-S6K1-driven mechanisms in vivo. We propose to test the following hypothesis: Phospho-EPRS is a critical effector of the mTORC1-S6K1 signaling pathway in both adipocytes and macrophages, and contributes importantly to diet- induced obesity and atherosclerosis. Our discovery that EPRS is a key mTORC1-S6K1 effector activated by agonists of both inflammation and metabolism provides a molecular link between these processes. We anticipate our studies will reveal new mechanisms underlying obesity and atherosclerosis, and can provide novel therapeutic targets for treatment of these related disorders.

DESCRIPTION (Provided by applicant): Despite the advent of effective statin therapies, atherosclerosis remains the leading cause of cardiovascular disease in the United States and worldwide. In addition to the well-known role of cholesterol as a major risk factor contributing to cardiovascular disease, evidence from many laboratories points to two other critical contributory factors, namely, dysregulated inflammation and metabolism. The latter two factors are inter- dependent, and metabolically active cells, in response to excess nutrient input, can generate a low-grade, chronic inflammation, referred to as metaflammation. This concept has become increasingly important as an epidemic of obesity in the Western world is driving the incidence of insulin resistance, diabetes, and athero- sclerosis, and consequent morbidity and mortality. The long-term goal of our Program Project is to gain a deep and mechanistic understanding of the genetic events and cellular and physiological processes underlying the connections between inflammation, metabolism, and atherogenesis. A central hypothesis is that macrophages are critically important early responders to metabolic stress, and that they in turn, by paracrine mechanisms, influence the responses of other cells crucial in metaflammation, particularly adipocytes and endothelial cells. The specific objectives of this Program are (i) to understand the molecular basis of interleukin-1 receptor/Toll- like receptor-mediated inflammatory responses in macrophages and EC, and delineate their roles in the initiation and pathogenesis of obesity-associated inflammatory disease (Project 1, Dr. Xiaoxia Li), (ii) to utilize mouse genetic, genomic, and proteomic approaches to identify atherosclerosis modifier genes and genetic modifiers of macrophage foam cell lipid droplet metabolism (Project 2, Dr. Jonathan Smith), and (iii) to determine the noncanonical function of glutamyl-prolyl tRNA synthetase (EPRS) as a critical effector of the mTORC1-S6K1 signaling pathway in adipocytes and macrophages, and its contribution to diet-induced obesity and atherosclerosis (Project 3, Dr. Paul Fox). The Project Leaders are a highly integrated and synergistic group employing distinct but complementary approaches, i.e., cell signaling (Li), mouse genetics/genomics/proteomics (Smith), and biochemistry (Fox). Three scientific Cores - Primary Cell and Mouse Metabolism, Atherosclerosis and Lipoprotein Analysis, and Macromolecular Interaction - and an Administration Core provide multi-project support, expertise, and service in a cost-effective manner, significantly strengthening each of the Projects. We anticipate our collaborative effort will lead to the discovery of novel genes, signaling pathways, and macro-molecular interactions that promote or restrict atherogenesis, and that can be leveraged to reduce disease initiation and progression, and support health and longevity.

Macromolecular Interaction Core (Core D). Paul L. Fox, Ph.D., Core Leader Project Summary/Abstract The Macromolecular Interaction Core has served this Program Project for the last 5 years. The Core serves the common need of all three Projects for investigation of protein-protein and protein-RNA interactions. The principal objective of Core D is to make available robust, state-of-the-art techniques for accurate and reproducible measurement of macromolecular interactions. The Core provides not only access to instrumentation and reagents, but more importantly provides experience and expertise in setting up the methods, and analyzing and interpreting the results. As noted in the Personnel section, Dr. Fox, Core Leader has about 15 years of experience investigating (and publishing on) macromolecular interactions (protein- protein, protein-RNA, protein-DNA) and their role in macrophage inflammatory gene expression and endothelial cell polarization and migration1-18. Dr. Jia, Core Manager, has expertise and proven experience in all of the physical and biochemical methods to be provided by the Core4,6,8-10,12,13. The Macromolecular Interaction Core will provide a diversity of services including provision of reagents, assistance in probe design and construction, design and performance of experiments, and data analysis and interpretation of results. An educational component is a major strength of this Core. All of the methods provided by Core D require technical expertise and experience not generally available in a modern cell or molecular biology laboratory; additionally, some methods require unfamiliar computational analysis methods.

Administrative Core (Core A) Paul L. Fox, Ph.D., Core Leader Project Summary/Abstract Our continuing Program Project aims to develop a better understanding of the mechanisms linking metabolism, obesity, inflammation, and atherosclerosis. The PPG is comprise of 3 interrelated Projects, 3 supporting Scientific Cores, and an Administrative Core. The Administrative Core is responsible for all NIH interactions, including assembly and preparation of this application, coordination of annual non-competitive renewal-related submissions, and all other NIH-related interactions. In addition, the Administrative core is responsible for fostering and coordinating collaborative research activities within the PPG. The Core is also responsible for coordinating communication and meetings with Internal and External Advisory Committees. The Core will provide clerical support for all Projects and Scientific Cores, such as for manuscript preparation and submission, art and photography assistance, and assistance with manuscript deposition to PubMed Central.

? DESCRIPTION (provided by applicant): Translation of mRNAs normally terminates at the first in-frame stop codon encountered by the ribosome; however, in some circumstances, translation can continue beyond the stop codon to a downstream stop codon, generating a novel protein with a C-terminal polypeptide extension. Such translational readthrough (TR) events are best understood in viruses, and evidence for TR of vertebrate mRNAs has been accumulating in the last two years. However, essentially nothing is known about the molecular mechanisms that regulate TR in vertebrate mRNAs. We have recently reported that protein-driven, programmed TR of human VEGFA (vascular endothelial growth factor-A) mRNA in endothelial cells (EC) generates a novel isoform of VEGF-A, a secreted angiogenic mitogen that regulates blood vessel formation. Readthrough generates a protein termed VEGF-Ax (x for extended) containing a 22-amino acid C-terminus extension. Remarkably, the RNA sequence that encodes the 22-amino acid extension also functions as a recoding cis-acting element (Ax element) essential for efficient TR. Readthrough is stimulated by interaction of heterogeneous nuclear ribonucleoprotein (hnRNP) A2B1 with the Ax element. Recent preliminary studies show an ensemble of proteins, including hnRNP L, DDX5, and HuR, also bind the Ax element. VEGF-Ax expression is down-regulated during C2C12 myoblast differentiation, but is remarkably efficient in human platelets. Based on these initial studies, we propose the following specific hypothesis: Assembly of a multiprotein complex on the structured VEGFA mRNA Ax element interacts with the ribosome or release factors to recode the canonical stop codon, regulating cell- and context-dependent TR and VEGF-Ax generation. We will test this hypothesis by pursuing three Specific Aims: Aim 1. Identify Ax element-binding trans-acting factors (AxE-TAF) and their role in TR of VEGFA mRNA in EC. We will identify and validate AxE-TAFs, and determine their interactions with each other, their affinities for the Ax element, and their influence on TR efficiency. We will also investigate the role of post-translational modifications (PTM) of these factors on their interactions and cell localization. Aim 2. Investigate determinants of cell- and context-dependent TR efficiency of VEGFA mRNA. We will investigate the mechanisms underlying suppressed VEGF-Ax production during C2C12 myoblast differentiation, and extraordinarily efficient VEGF-Ax expression in human platelets. We will determine the influence of localization, expression, and PTM of regulatory trans-acting factors on assembly of the readthrough complex, binding to the Ax element, and readthrough efficiency. Aim 3. Determine molecular mechanisms regulating translational readthrough of VEGFA mRNA. We will determine the folding structure of the Ax RNA element and its functional regions and residues. We will test the hypothesis that readthrough is promoted by the interaction of AxE-TAFs with ribosomes or ribosomal release factors, and identify the tRNA responsible for Ser insertion. TR has recently emerged as a mechanism of expansion of the human proteome. Successful completion of the proposed studies will not only elucidate mechanisms of readthrough of VEGFA mRNA, but also will provide a foundation for investigation of mechanisms regulating TR of other mRNAs and systems.

Project Summary/Abstract Obesity is an epidemic-scale problem in the U.S. and worldwide with enormous health and economic costs. The mTORC1-S6 kinase 1 (S6K1) axis drives anabolic pathways determining obesity. We recently identified glutamyl-prolyl tRNA synthetase (EPRS) as an mTORC1-S6K1 target that contributes to mouse adiposity. Insulin-stimulated EPRS phosphorylation at Ser999 by S6K1 in adipocytes induces its binding to fatty acid transport protein 1 (FATP1) and translocation to the plasma membrane to increase long-chain fatty acid (LCFA) uptake. Recent studies reveal that phosphorylation of S6K1 by cyclin-dependent kinase 5 (Cdk5) at Ser424 and Ser429 in the S6K1 C-terminus are required for phosphorylation of EPRS, but not for canonical substrates such as RPS6. This unexpected finding indicates that embedded in S6K1 is a target-selective phospho-code in which combinatorial phospho-site phosphorylation determines kinase targets. To identify additional targets of multi-phosphorylated S6K1 (termed S6K1*) but not mTORC1-activated S6K1, we transfected HEK cells with S6K1 bearing phospho-mimetic mutations at the 3 phospho-sites, or wild-type S6K1 cDNA. Three new S6K1* targets were identified by mass spectrometry and validated in adipocytes ? coenzyme A synthase (COASY), cortactin, and lipocalin 2. Importantly, all are implicated in adipocyte lipid metabolism: P-EPRS transports FATP1 to the plasma membrane for increased LCFA uptake; COASY catalyzes the final two steps of synthesis of coenzyme A, required for LCFA activation; lipocalin 2 increases LCFA ?-oxidation and insulin resistance; and cortactin is required for insulin-stimulated transport of Glut4-containing vesicles to plasma membranes. We propose that S6K1* directs an adipocyte lipid metabolon, and is a major contributor to obesity-related phenotypes driven by the mTORC1-S6K1 axis. We will test this hypothesis by pursuit of 3 Specific Aims: In Aim 1 we determine S6K1*/target docking domains. By mass spectrometry and site-directed mutation analysis, we will determine specific S6K1*-directed phosphorylation sites in the targets. In Aim 2 we determine the function of phosphorylated S6K1* targets in adipocyte lipid metabolism. We will determine the mechanism of insulin-stimulated transport and binding of P-EPRS to the adipocyte plasma membrane; the role of phosphorylation in COASY catalytic activity and localization; whether P-cortactin transports P-EPRS/FATP1-containing vesicles to the plasma membrane; and extracellular secretion and intracellular localization of P-lipocalin 2, and its role in LCFA oxidation. In Aim 3 we elucidate In vivo role of S6K1* in lipid metabolism and obesity. We will determine the effect of diet-induced obesity on the S6K1* activation pathway and on target phosphorylation in mice. Taking advantage of our new mouse model (generated by Crispr-Cas9 technology) bearing a Ser429-to-Ala mutation in Rps6kb1 (mouse gene encoding S6K1) that lack S6K1* activity, while retaining canonical S6K1 activity, we will test the role of S6K1* in target phosphorylation in vivo, in lipid metabolism, and in diet-induced obesity.

Project Summary/Abstract Obesity is an epidemic-scale problem in the U.S. affecting about 35% of the adult population and 20% of children under 19 years of age. Elevated body mass index is associated with hypertension, dyslipidemia, and hyperinsulinemia ? all risk factors for multiple pathologies including diabetes, cardiovascular disease, cancer, shortened lifespan, and even depression. The long-term goal of our research program is to develop a low- molecular weight inhibitor (LMWI) of a newly discovered adiposity-driving metabolic pathway elucidated by our laboratory. The immediate goal of this application is to develop assays and necessary reagents to permit pilot screens and prepare for a future high-throughput screen (HTS) of small-molecule inhibitors to discover therapeutic agents to prevent or reduce obesity and its pathological consequences. We recently reported a new target of the mTORC1-S6K1 axis, namely, glutamyl-prolyl tRNA synthetase (EPRS). S6K1 directly phosphorylates EPRS at Ser999 in the linker domain that joins the catalytic synthetase domains. Remarkably, genetically-modified mice with a phospho-deficient Ser999-to-Ala mutation exhibit marked reduction in weight and white adipose tissue. They are metabolically healthy as indicated by improved glucose tolerance and extended lifespan, and mice remain lean when fed a high-fat diet. These results strongly implicate EPRS as a critical downstream target of mTORC1-S6K1 that determines adiposity. We propose to use AlphaScreen technology to seek LMWIs of S6K1-mediated phosphorylation of EPRS. We will take advantage of recent findings in our laboratory that show strong binding between S6K1 and EPRS. Inhibition of this binding specifically blocks EPRS phosphorylation without inhibiting the catalytic activity of S6K1 or phosphorylation of its canonical targets such as ribosomal protein S6. Thus, we anticipate our approach will reveal small-molecule inhibitors that prevent fat accumulation without disrupting the principal functions of the mTORC1-S6K1 axis, such as global protein synthesis. We further expect that such LMWIs will exhibit markedly reduced adverse side effects compared to known inhibitors of mTORC1, such as rapamycin. As a specific hypothesis, we propose that an effective LMWI of the interaction of S6K1* with EPRS will safely and efficiently reduce fat accumulation in adipocytes and whole body adiposity. Here we will develop in vitro and cellular assays to facilitate discovery and validation of such inhibitors. In Aim 1 we will develop an AlphaScreen-based assay to interrogate S6K1*/EPRS interaction, and its inhibition. In Aim 2 we will develop orthogonal assays for validation, determination of selectivity and structure-activity relationship, and assessment of cell function and toxicity. In Aim 3 we will use these newly-developed assays to conduct pilot screens, and validate and triage candidates. Completion of these studies will provide the reagents and assays necessary for a future HTS of large, diverse compound libraries, validation and prioritization of candidates, testing of structurally-related compounds, and chemical modification to maximize efficacy to permit subsequent testing in mouse models of dietary obesity.

Project Summary/Abstract Advanced age is the leading risk factor for Alzheimer's disease (AD). Our program is based on a central tenet of geroscience that select pathways and mechanisms are shared between advanced age and chronic disease, and knowledge of one can inform the other. The mTORC1-S6K1 kinase axis is an obesity-activated pathway that restricts longevity, and targeting this pathway extends lifespan in animal models. This pathway has been implicated in AD pathogenesis, and pre-clinical intervention studies are promising. However, pharmacological inhibition causes harmful side-effects, deterring therapeutic application. We have discovered a Cdk5-driven bifurcation of the mTORC1-S6K1 pathway that contributes to aging and adiposity. We identified a novel, triply- phosphorylated form of S6K1 (we term S6K1*) phosphorylated at two sites in the C-terminus, as well as at Thr389, the classical mTORC1 activation site. Multi-site phosphorylated S6K1 directs phosphorylation of novel targets, including the dual function tRNA synthetase, Glu-Pro tRNA synthetase (EPRS), coenzyme A synthase (CoASY), and lipocalin-2 (Lcn2). Importantly, mice bearing a phospho-deficient mutation of EPRS at the critical Ser999 residue are lean and exhibit ~120-day lifespan extension. Ser999 EPRS phosphorylation is required for elevated adipocyte expression of key longevity-related adipokines, including monocyte chemoattractant protein- 1 (MCP1) and plasminogen-activator-1 (PAI-1). MCP1 is a pro-inflammatory protein predominant in the senescence-associated secretory phenotype (SASP); PAI-1 is a marker and mediator of cell senescence and aging, and a null mutation in SERPINE1, the gene encoding PAI-1, protects against biological aging. Importantly, these novel, age-related targets of S6K1* also are implicated in AD progression. For example, serum MCP1 level is associated with cognitive decline in mouse AD models and AD patients, and PAI-1 knockout, or pharmacologic inhibition, reduces AD in mice. We hypothesize that the extended mTORC1-S6K1 pathway and its effectors contribute to AD onset and progression, and that genetic inhibition of the pathway will retard AD onset and reduce its severity with minimal adverse side effects. Also, mice fed a high-fat diet will show that obesity influences aging and AD progression by a common pathway. We will test these hypotheses in two Specific Aims. In the first Aim, we will elucidate the role of the S6K1* pathway in AD progression. AD- susceptible mice will be bred with two genetic mouse models of S6K1* pathway inhibition developed in our laboratory, namely, EPRS phospho-deficient knock-in mice bearing a Ser999-to-Ala mutation, and our newly developed S6K1 Ser429-to-Ala mouse model, that lacks the extended S6K1* substrate selection, but exhibits unaltered canonical S6K1 kinase activity. We will determine effects of S6K1* pathway inhibition on AD pathology and adverse cognitive side-effects. In the second Aim we will determine the influence of obesity on S6K1* pathway-mediated AD progression. Our program will establish new mechanisms and molecular targets for intervention in the aging process and AD.