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Elucidating the Roles of CMPK2 in Mitochondrial Homeostasis and Antiviral Immunity
Microbial Pathogenesis And Immunology; TAMHSC; https://hdl.handle.net/20.500.14641/248; DHHS-NIH-National Heart, Lung, and Blood Institute
PROJECT SUMMARY: Cytidine/uridine monophosphate kinase 2 (CMPK2) is an interferon-regulated enzyme that was originally reported to catalyze the ATP-dependent phosphorylation of dCMP and dUMP to diphosphate forms in vitro. Due to a putative mitochondrial targeting sequence, CMPK2 was postulated to function in the mitochondrial deoxyribonucleotide salvage pathway necessary for the synthesis and maintenance of mitochondrial DNA (mtDNA). However, more recent data have revealed that CMPK2 prefers ribonucleotide diphosphate substrates in vitro and functions to restrict HIV and other RNA viruses in cell based assays. Beyond these seemingly contrasting findings, no other studies have addressed the cellular localization and tissue expression patterns of CMPK2 or utilized genetic knockouts to determine true biological activity. Therefore, the overall objective of this proposal is to close these knowledge gaps and mechanistically advance understanding of CMPK2 in mitochondrial function, tissue homeostasis, and antiviral innate immunity using a diverse toolkit of cell and animal models. The central hypothesis is that by maintaining mitochondrial homeostasis, CMPK2 boosts cell-intrinsic innate immunity and limits runaway inflammation triggered by mitochondrial stressors and viral infection. In support of this hypothesis, ongoing studies have revealed that CMPK2 localizes strongly to mitochondria and that ectopic overexpression of CMPK2 is sufficient to protect cells from RNA virus infection. Moreover, after systemic challenge with innate immune agonists, CMPK2 is markedly upregulated in the lungs and liver, and CMPK2 knockout mice exhibit elevated expression of proinflammatory cytokines and type I interferon (IFN-I) responses after Toll-like receptor (TLR) stimulation. To gain additional insight into how CMPK2 functions in mitochondria and antiviral immunity, two related, but independent aims are proposed. Aim 1 will elucidate the molecular mechanisms by which CMPK2 maintains mitochondrial homeostasis at rest and during stress. Here, CMPK2 knockout cells, novel lines reconstituted with mutant CMPK2 vectors lacking nucleotide kinase activity or mitochondrial targeting, and whole body CMPK2 knockout mice will be utilized. Aim 2 will determine that the mitochondrial activity of CMPK2 restricts coronavirus replication and maintains mitochondrial function during infection. Here, an intranasal mouse hepatitis virus challenge protocol that models acute respiratory distress syndrome and closely mirrors coronavirus pneumonia in humans will be employed. This research will fundamentally advance our understanding of how CMPK2 functions in mitochondrial homeostasis and antiviral innate immunity at both the cellular and organismal levels. Moreover, it may have a positive impact on public health by revealing novel CMPK2-centered strategies to maintain mitochondrial homeostasis, boost antiviral immunity, and limit damaging inflammation during coronavirus infection.
Elucidating the Roles of CMPK2 in Mitochondrial Homeostasis and Antiviral Immunity
Microbial Pathogenesis And Immunology; TAMHSC; https://hdl.handle.net/20.500.14641/248; DHHS-NIH-National Heart, Lung, and Blood Institute
PROJECT SUMMARY Cytidine/uridine monophosphate kinase 2 (CMPK2) is an interferon-regulated enzyme that was originally reported to catalyze the ATP-dependent phosphorylation of dCMP and dUMP to diphosphate forms in vitro. Due to a putative mitochondrial targeting sequence, CMPK2 was postulated to function in the mitochondrial deoxyribonucleotide salvage pathway necessary for the synthesis and maintenance of mitochondrial DNA (mtDNA). However, more recent data have revealed that CMPK2 prefers ribonucleotide diphosphate substrates in vitro and functions to restrict HIV and other RNA viruses in cell based assays. Beyond these seemingly contrasting findings, no other studies have addressed the cellular localization and tissue expression patterns of CMPK2 or utilized genetic knockouts to determine true biological activity. Therefore, the overall objective of this proposal is to close these knowledge gaps and mechanistically advance understanding of CMPK2 in mitochondrial function, tissue homeostasis, and antiviral innate immunity using a diverse toolkit of cell and animal models. The central hypothesis is that by maintaining mitochondrial homeostasis, CMPK2 boosts cell-intrinsic innate immunity and limits runaway inflammation triggered by mitochondrial stressors and viral infection. In support of this hypothesis, ongoing studies have revealed that CMPK2 localizes strongly to mitochondria and that ectopic overexpression of CMPK2 is sufficient to protect cells from RNA virus infection. Moreover, after systemic challenge with innate immune agonists, CMPK2 is markedly upregulated in the lungs and liver, and CMPK2 knockout mice exhibit elevated expression of proinflammatory cytokines and type I interferon (IFN-I) responses after Toll-like receptor (TLR) stimulation. To gain additional insight into how CMPK2 functions in mitochondria and antiviral immunity, two related, but independent aims are proposed. Aim 1 will elucidate the molecular mechanisms by which CMPK2 maintains mitochondrial homeostasis at rest and during stress. Here, CMPK2 knockout cells, novel lines reconstituted with mutant CMPK2 vectors lacking nucleotide kinase activity or mitochondrial targeting, and whole body CMPK2 knockout mice will be utilized. Aim 2 will determine that the mitochondrial activity of CMPK2 restricts coronavirus replication and maintains mitochondrial function during infection. Here, an intranasal mouse hepatitis virus challenge protocol that models acute respiratory distress syndrome and closely mirrors coronavirus pneumonia in humans will be employed. This research will fundamentally advance our understanding of how CMPK2 functions in mitochondrial homeostasis and antiviral innate immunity at both the cellular and organismal levels. Moreover, it may have a positive impact on public health by revealing novel CMPK2-centered strategies to maintain mitochondrial homeostasis, boost antiviral immunity, and limit damaging inflammation during coronavirus infection.
Exercise Training-Enhanced Reactive Oxygen Species as Protective Mechanisms in the Coronary Microcirculation
Veterinary - Physiology & Pharmacology; TAMU; https://hdl.handle.net/20.500.14641/207; DHHS-NIH-National Heart, Lung, and Blood Institute
Project Summary Regular exercise is a proven, powerful and cost-effective intervention for the treatment and secondary prevention of coronary artery disease. However, a detailed understanding of the fundamental cellular and molecular mechanisms that underlie exercise-induced cardioprotection are lacking, limiting the development of effective new therapeutic strategies for diseased patients. Despite recent advances in the appreciation of reactive oxygen species (ROS) as critical regulators of cell signaling, the details of the specific contributions of these molecules to physiologic signaling and functional adaptions in the vascular system remain to be elucidated. This is particularly true in the coronary microcirculation where studies determining the contributions of ROS in the control of blood flow are sparse. The proposed studies will utilize a combination of in vitro and in vivo approaches to determine how exercise-induced adaptations in ROS signaling affect vascular reactivity and coronary blood flow into both control and ischemic myocardium, an area that has been largely unexplored in the coronary circulation. The overarching hypothesis is that ROS play a critical and protective role in the exercise training-induced restoration of vasodilation responses in the coronary microcirculation and thereby enhances perfusion and contractile function of the at-risk myocardium. Aim 1 will determine exercise training- induced adaptations in ROS production in hearts subjected to chronic coronary artery occlusion. Aim 2 will determine the effects of exercise training on the expression and subcellular localization of candidate sources of ROS production and associated regulatory subunit proteins in microvascular endothelium of hearts subjected to chronic coronary artery occlusion. Aim 3 will identify the adaptations by which exercise training promotes downstream signaling pathway(s) for ROS-mediated dilation in arterioles isolated from hearts subjected to chronic coronary artery occlusion. Aim 4 will identify the signaling mechanisms by which exercise training enhances regional perfusion and myocardial contractile function at rest and during dobutamine-induced myocardial stress in hearts subjected to chronic coronary occlusion. These studies are of high impact since the knowledge gained will provide novel insight into the protective role of ROS in the cardiovascular system. The proposed studies will provide important new information with significant mechanistic insight into human ischemic heart disease and identify the role of ROS signaling in the control of coronary blood flow in health, disease, and exercise adaptation.
Exercise Training-Enhanced Reactive Oxygen Species as Protective Mechanisms in the Coronary Microcirculation
Veterinary - Physiology & Pharmacology; TAMU; https://hdl.handle.net/20.500.14641/207; DHHS-NIH-National Heart, Lung, and Blood Institute
Project Summary Regular exercise is a proven, powerful and cost-effective intervention for the treatment and secondary prevention of coronary artery disease. However, a detailed understanding of the fundamental cellular and molecular mechanisms that underlie exercise-induced cardioprotection are lacking, limiting the development of effective new therapeutic strategies for diseased patients. Despite recent advances in the appreciation of reactive oxygen species (ROS) as critical regulators of cell signaling, the details of the specific contributions of these molecules to physiologic signaling and functional adaptions in the vascular system remain to be elucidated. This is particularly true in the coronary microcirculation where studies determining the contributions of ROS in the control of blood flow are sparse. The proposed studies will utilize a combination of in vitro and in vivo approaches to determine how exercise-induced adaptations in ROS signaling affect vascular reactivity and coronary blood flow into both control and ischemic myocardium, an area that has been largely unexplored in the coronary circulation. The overarching hypothesis is that ROS play a critical and protective role in the exercise training-induced restoration of vasodilation responses in the coronary microcirculation and thereby enhances perfusion and contractile function of the at-risk myocardium. Aim 1 will determine exercise training- induced adaptations in ROS production in hearts subjected to chronic coronary artery occlusion. Aim 2 will determine the effects of exercise training on the expression and subcellular localization of candidate sources of ROS production and associated regulatory subunit proteins in microvascular endothelium of hearts subjected to chronic coronary artery occlusion. Aim 3 will identify the adaptations by which exercise training promotes downstream signaling pathway(s) for ROS-mediated dilation in arterioles isolated from hearts subjected to chronic coronary artery occlusion. Aim 4 will identify the signaling mechanisms by which exercise training enhances regional perfusion and myocardial contractile function at rest and during dobutamine-induced myocardial stress in hearts subjected to chronic coronary occlusion. These studies are of high impact since the knowledge gained will provide novel insight into the protective role of ROS in the cardiovascular system. The proposed studies will provide important new information with significant mechanistic insight into human ischemic heart disease and identify the role of ROS signaling in the control of coronary blood flow in health, disease, and exercise adaptation.
Role of TET dioxygenase associated immune mechanisms in cardiac injury and repair
Ibt-Ctr For Epigenetics & Disease Prev; TAMHSC; https://hdl.handle.net/20.500.14641/220; DHHS-NIH-National Heart, Lung, and Blood Institute
Project Summary/ Abstract: Clonal hematopoiesis of indeterminate potential (CHIP) is defined as an expansion of somatic hematopoietic blood cell clone in individual without hematological disorders. Recent exome sequencing identified hematopoietic stem and progenitor cells (HSPCs) with frequent mutations of epigenetic regulators (e.g., the DNA methylcytosine dioxygenase TET2) that exhibited growth advantage with clonal expansion during aging. Interestingly, CHIP individuals with somatic TET2 mutations tend to have high risk of coronary cardiovascular diseases (CVD). This discovery heralds the advent of a molecular era in the dissection of novel pathogenic mechanisms underlying CHIP-CVD convergence. In animal studies that mimic clonal hematopoiesis, Tet2 LOF has been found to accelerate atherosclerosis and heart failure. While these studies provided detailed phenotypic characterizations, the underlying molecular mechanisms and the causal relations between TET2 LOF in CHIP and increased CVD risk remain largely unresolved. The PI’s laboratory has developed a set of unique tools to address this critical clinically-relevant knowledge gap, including (i) tissue specific Tet2-deficient mouse models (specific ablation of Tet2 in the myeloid lineage or in HSPCs) with reporter genes to enable real-time lineage tracing in vivo during cardiac injury; and (ii) dCas9 based epigenome editing tools that allow the interrogation of causal effects between epigenotypes and phenotypes. The team proposes to test the hypothesis that Tet2 controls the activity of enhancers that regulate the expression of key genes required for maintaining the proper function of monocytes/ macrophages in the reparative response to ischemic injury (e.g., myocardial infarction or MI). Aim 1 will address how Tet2 loss impairs myeloid cells and HSPCs that actively participate in the post-MI cardiac repair process. Aim 2 will address how Tet2 deficiency disrupts enhancer activities in key genes that are essential for proinflammatory to reparative monocyte conversion, thereby perturbing the biphasic post-MI response of monocyte to compromise timely resolution of inflammation and cardiac repair. The idea of restoring Tet2/5hmC function will be further tested to intervene post-MI tissue repair. This study introduces a new dimension to dissect CVD pathogenesis by focusing on the interplay between the cardiovascular system and the immune-hematopoietic system. Completion of this project is anticipated to yield novel insights on how somatic TET2 mutations-associated clonal hematopoiesis increases the risk of cardiovascular disease (CVD) and impairs cardiac function under stress. More clinically relevant, discoveries made in this study are also expected to establish the preclinical rationale for targeting defective epigenetic regulators to prevent and treat CVD.
Role of TET dioxygenase associated immune mechanisms in cardiac injury and repair
Ibt-Ctr For Epigenetics & Disease Prev; TAMHSC; https://hdl.handle.net/20.500.14641/220; DHHS-NIH-National Heart, Lung, and Blood Institute
Project Summary/ Abstract Clonal hematopoiesis of indeterminate potential (CHIP) is defined as an expansion of somatic hematopoietic blood cell clone in individual without hematological disorders. Recent exome sequencing identified hematopoietic stem and progenitor cells (HSPCs) with frequent mutations of epigenetic regulators (e.g., the DNA methylcytosine dioxygenase TET2) that exhibited growth advantage with clonal expansion during aging. Interestingly, CHIP individuals with somatic TET2 mutations tend to have high risk of coronary cardiovascular diseases (CVD). This discovery heralds the advent of a molecular era in the dissection of novel pathogenic mechanisms underlying CHIP-CVD convergence. In animal studies that mimic clonal hematopoiesis, Tet2 LOF has been found to accelerate atherosclerosis and heart failure. While these studies provided detailed phenotypic characterizations, the underlying molecular mechanisms and the causal relations between TET2 LOF in CHIP and increased CVD risk remain largely unresolved. The PI’s laboratory has developed a set of unique tools to address this critical clinically-relevant knowledge gap, including (i) tissue specific Tet2-deficient mouse models (specific ablation of Tet2 in the myeloid lineage or in HSPCs) with reporter genes to enable real-time lineage tracing in vivo during cardiac injury; and (ii) dCas9 based epigenome editing tools that allow the interrogation of causal effects between epigenotypes and phenotypes. The team proposes to test the hypothesis that Tet2 controls the activity of enhancers that regulate the expression of key genes required for maintaining the proper function of monocytes/ macrophages in the reparative response to ischemic injury (e.g., myocardial infarction or MI). Aim 1 will address how Tet2 loss impairs myeloid cells and HSPCs that actively participate in the post-MI cardiac repair process. Aim 2 will address how Tet2 deficiency disrupts enhancer activities in key genes that are essential for proinflammatory to reparative monocyte conversion, thereby perturbing the biphasic post-MI response of monocyte to compromise timely resolution of inflammation and cardiac repair. The idea of restoring Tet2/5hmC function will be further tested to intervene post-MI tissue repair. This study introduces a new dimension to dissect CVD pathogenesis by focusing on the interplay between the cardiovascular system and the immune-hematopoietic system. Completion of this project is anticipated to yield novel insights on how somatic TET2 mutations-associated clonal hematopoiesis increases the risk of cardiovascular disease (CVD) and impairs cardiac function under stress. More clinically relevant, discoveries made in this study are also expected to establish the preclinical rationale for targeting defective epigenetic regulators to prevent and treat CVD.
The Role of Hemoglobin Alpha in Diabetes-Related Vascular Dysfunction
Medical Physiology; TAMHSC; https://hdl.handle.net/20.500.14641/551; DHHS-NIH-National Heart, Lung, and Blood Institute
Scientific Abstract Hemoglobin, the oxygen carrying protein expressed in erythrocytes, can be glycated following elevations in blood glucose. Some amino acids, such as the N-terminal valine of the hemoglobin beta chain are highly susceptible to glycation in diabetic patients. This specific glycated isoform, termed HbA1c, has been used by clinicians as an overall picture of a diabetic patient’s ability to control their glucose over a 3-month period and as an indicator for future cardiovascular risks. Recently, it was observed that the alpha chain of hemoglobin, but not the beta chain, is expressed in endothelial cells lining arteries where it interacts with endothelial nitric oxide synthase (eNOS) to modulate nitric oxide (NO) release. Hemoglobin alpha is known to be glycated at a number of sites, including one in the putative eNOS interaction domain. Since it is well recognized that vascular dysfunction underlies many of the pathologies in diabetic patients, it was hypothesized that the hemoglobin alpha expressed in the endothelium will have aberrant function in diabetes mellitus, likely due to a glycation event. The aim of the current proposal is to examine the role of hemoglobin alpha and any possible glycated forms of hemoglobin alpha in the endothelium of a murine model of diabetes. Using pharmacological and genetic approaches, the interaction between hemoglobin alpha and eNOS will be disrupted and the influence on the development of vascular dysfunction will be explored. This work has the potential to identify both a novel biomarker of vascular risk and also a potential therapeutic target for pharmacological treatments.

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