INOS AGGRESOME AS A PROTOTYPE OF A PHYSIOLOGIC AGGRESOME
Eissa Tony
Baylor College Of Medicinecity: Houston country: United States (us)
Grant 5R01HL080205-05 from National Heart, Lung, And Blood Institute
Abstract: Misfolding of proteins plays an important part in the pathogenesis of several lung diseases. Misfolded and aggregated proteins are handled in the cell through chaperone-mediated refolding, or destroyed by proteasomal degradation. Recent evidence suggests that cells have evolved a third pathway that involves sequestration of misfolded proteins into specialized "holding stations", close to the nucleus, called aggresomes. Recognizing the importance of this topic, the NHLBI convened a workshop to review protein processing related issues. The workshop emphasized the need for understanding the nature and the role of aggresome and the cellular mechanisms of its formation. We have discovered that cells regulate inducible nitric oxide synthase (iNOS) through aggresome formation. This newly discovered iNOS aggresome is the first described occurrence of an aggresome that is not associated with protein misfolding and which we termed "physiologic aggresome". This discovery sets the stage for a unique research opportunity. Study of the physiologic aggresome should reveal a wealth of information regarding how cells regulate proteins through aggresome formation. We hypothesize that A) iNOS physiologic aggresome shares certain features with what previously described as "pathologic" aggresome associated with misfolded proteins. Thus, pathologic aggresome may merely represent an acceleration of an established physiologic regulatory process. B) The regulation of aggresome formation in cells is linked to cell capacity to degrade proteins in a timely manner. Whenever cells sense that this capacity is likely to be exceeded due to the generation of either a misfolded protein or a large amount of a certain protein, they trigger aggresome formation. The decision of cells to resort to aggresome formation results in specific proteins migrating to participate in the formation and the regulation of the aggresome. To test these hypotheses we propose studies with the following specific aims 1) Characterization of mechanisms of formation and regulation of iNOS aggresome. 2) Identification of proteins forming iNOS aggresome. 3) Examination of the regulation of iNOS aggresome in cells harboring misfolded mutant proteins related to lung disease such as a1-antitrypsin mutants. The rationale for the proposed studies is that once these mechanisms are understood, they would greatly increase our understanding of cellular handing of misfolded proteins. Future therapeutic strategies can be designed to regulate these cellular responses in disease states
Keywords: 26 S proteasome complex; 26S ATP-Dependent Protease; 26S ATP-Dependent Proteasome; 26S protease; 26S proteasome; 26S Proteasome Complex; 26S Proteosome; aberrant protein folding; abnormal protein folding; Acceleration; Acrosomal Serine Protease Inhibitor; Air; Alpha-1 Antiproteinase; Antitrypsin; Area; Cell Culture Techniques; cell growth regulation; Cell Nucleus; Cells; Cellular Regulation; CFTR; CFTR Protein; Chaperone; Convulsive Therapy, Electric; Cultured Cells; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; cystic fibrosis transmembrane regulator; Data; Degradation Pathway; Degradative Pathway; design; designing; Disease; disease/disorder; Disorder; Educational workshop; electric shock treatment; electro-convulsive therapy; Electroconvulsive Psychotherapy; Electroconvulsive Shock Therapy; Electroconvulsive Therapy; Electroconvulsive treatment; electroplexy shock therapy; Electroshock Psychotherapy; Electroshock Therapy; Electroshock treatment; Epithelial Cells; fluid; Future; gene product; Generations; Genetic Alteration; Genetic Change; Genetic defect; genome mutation; Hand; Heat Shock; Heat-Shock Reaction; Heat-Shock Response; Hepatocyte Nitric Oxide Synthase; human NOS2A protein; ILD; Inducible Nitric Oxide Synthase; inhibitor; inhibitor/antagonist; INOS; iNOS enzyme; insoluble aggregate; Interphase; Interstitial Lung Diseases; Investigators; Link; liquid; Liquid substance; Lung diseases; Lung Diseases, Interstitial; lung disorder; Macrophage Nitric Oxide Synthase; Mediating; Molecular; Molecular Chaperones; Mucoviscidosis; mutant; Mutation; National Heart, Lung, and Blood Institute; Nature; Nitric Oxide Synthase 2A; nitric oxide synthase, Type II; NOS Type II; NOS2; NOS2A; NOS2A protein, human; Nucleus; PAI-3; Pathogenesis; Pathologic; pathologic protein folding; pathway; Pathway interactions; Physiologic; Physiological; Plasma Serine Protease Inhibitor; Plasminogen Activator Inhibitor 3; Plasminogen Activator Inhibitor III; Play; Process; programs; Programs (PT); Programs [Publication Type]; protein aggregate; Protein C Inhibitor; Protein C Inhibitor, Activated; protein mis-folding; protein misfolding; Protein Trafficking; protein transport; Proteins; prototype; Pulmonary Diseases; Pulmonary Disorder; Recommendation; Regulation; Research; Research Personnel; Researchers; Resort; Respiratory Disease; Respiratory Disorder; Respiratory System Disease; Respiratory System Disorder; response; Role; Serine or Cysteine Proteinase Inhibitor Clade A Member 5; Shock Therapy, Electric; shock treatment; social role; Staging; Testing; Therapeutic; Traffickings, Protein; Type 3 Plasminogen Activator Inhibitor; Workshop
Project start date: 2006-05-01
Project end date: 2012-02-28
Budget start date: 1-MAR-2010
Budget end date: 28-FEB-2012
5R01HL080205-05 (2010): $364125
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Eissa Tony
RESEARCH TRAINING IN LUNG DISEASE
Eissa Tony
Baylor College Of Medicinecity: Houston country: United States (us)
Grant 5T32HL007747-17 from National Heart, Lung, And Blood Institute
Abstract: This multidisciplinary T32 competitive renewal application will allow trainees at Baylor College of Medicine (BCM) to continue to successfully launch research careers in lung diseases. At this time, this Texas Medical Center based grant is the only T32 pulmonary research training program in eight contiguous southern states. Superb training opportunities exist at BCM in a number of areas, highly relevant to lung diseases. Trainees are recruited and selected from a large pool of outstanding applicants to BCM training programs in pulmonary as well as to individual mentors laboratories. Continuation of five postdoctoral trainee positions annually is requested, an increase from four in current cycle. Trainees are competitively selected for appointments to the T32 training program for two to three years of rigorous scientific training. Multi- disciplinary seventeen faculty members have been carefully selected from various departments to serve as mentors, based on their excellence in research and teaching. Most of the faculty are linked by collaborative research interests which have been strengthened during the previous period of this training program. Mentors in this program have strong record of federal funding and training experience. They provide training opportunities in allergic inflammation, innate and adaptive immunity, asthma and emphysema pathogenesis, cystic fibrosis, skeletal muscle biology, Nano-medicine, stem cell research, chemokine biology, adhesion molecules, signal transduction, molecular genetics, lung remodeling, cellular immunology, murine models of inflammation, infection and immunity, acute respiratory pathogens, tuberculosis, and clinical trial outcome analysis. Trainees enroll in graduate school courses and BCM NIH K30 supported Clinical Scientist Training Program, to acquire a sound fund of knowledge in basic science and pulmonary, the principles of conducting clinical research, and the ethical conduct of research. Trainees will be comprehensively mentored in hypothesis driven research, preparation and publication of manuscripts, presentations at national meetings, preparation of grant proposals, and skills for teaching and mentoring, and academic career development. The goal of this training program is to continue to develop pulmonary physician-scientists and researchers who will serve as future academic leaders
Keywords: Lung diseases; Research Training
Project start date: 1993-07-01
Project end date: 2015-06-30
Budget start date: 1-JUL-2011
Budget end date: 30-JUN-2012
PFA/PA: PA-08-226
5T32HL007747-17 (2011): $354330
2T32HL007747-16 (2010): $339880
CELLULAR REGULATION OF NITRIC OXIDE IN AIRWAY INFLAMMATION
Eissa Tony
Baylor College Of Medicinecity: Houston country: United States (us)
Grant 5R01HL084322-04 from National Heart, Lung, And Blood Institute
Abstract: Overproduction of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of airway inflammation of asthma. The long-term goal of this research is to understand the regulation of iNOS activity and to devise novel methods to regulate it. Although much is known about factors affecting the synthesis and catalytic activity of iNOS, little is known about its cellular regulation. We have recently shown that iNOS is degraded through the ubiquitin-proteasome pathway. The specificity of the ubiqutination system, is mainly provided by the specific ubiquitin ligase enzyme (E3) that recognizes and binds to the target protein. Therefore, identification and characterization of the E3 ubiquitin ligase for iNOS should be regarded as one of the most critical steps for understanding the mechanisms of iNOS cellular regulation. Our preliminary data identified an F-box-containing protein; we termed FBXinos that is a likely candidate to be the E3 ubiquitin ligase for iNOS. Additional preliminary data suggest that cells regulate NO synthesis by temporal and spatial regulation of iNOS. These mechanisms include a relatively rapid rate of iNOS turnover and sequestration of iNOS to a perinuclear location we termed the "physiologic aggresome". Interestingly, our preliminary data suggest that FBXinos is important not only for iNOS turnover but also for subcellular targeting of iNOS. We propose to test the following hypothesis An F-box-containing protein, FBXinos, is the E3 ligase for iNOS and it plays a central role in iNOS cellular regulation. The cellular regulation of this FBXinos further modulates iNOS and NO cellular levels. To test this hypothesis we propose studies with the following specific aims 1) Characterization of the regulation of iNOS by FBXinos. 2) Determine the mechanisms by which FBXinos ubiquitin ligase targets iNOS to the aggresome. 3) Characterization of the regulation of iNOS and FBXinos in airway inflammation. Studies will be conducted in cultured cells expressing iNOS and in primary bronchial epithelial cells cultured at the air/liquid interphase. The rationale for the proposed studies is that once these mechanisms are understood, they would greatly increase our understanding of cellular handling of iNOS. Future therapeutic strategies can be designed to regulate these cellular responses in disease states
Keywords: 20S Catalytic Proteasome; 20S Core Proteasome; 20S Proteasome; 20S Proteosome; 26 S proteasome complex; 26S ATP-Dependent Protease; 26S ATP-Dependent Proteasome; 26S protease; 26S proteasome; 26S Proteasome Complex; 26S Proteosome; Adaptor Protein; Adaptor Signaling Protein; Adrenal Cortex Hormones; Adverse effects; Affect; Air; airway epithelium infalmmation; airway inflammation; analog; APF-1; Arginine; Arginine, L-Isomer; Asthma; ATP-Dependent Proteolysis Factor 1; Binding; Binding (Molecular Function); Bronchial Asthma; cell biology; Cell Culture Techniques; cell growth regulation; Cells; Cellular biology; Cellular Regulation; Complex; Corticoids; Corticosteroids; Cultured Cells; Data; design; designing; Disease; disease/disorder; Disorder; E3 Ligase; E3 Ubiquitin Ligase; Endogenous Nitrate Vasodilator; endothelial cell derived relaxing factor; Endothelium-Derived Relaxing Factor; Enzymes; Epithelial Cells; experiment; experimental research; experimental study; F Box; F Box Domain; fluid; Future; gene product; Goals; Grant; Half-Life; Half-Lifes; Hepatocyte Nitric Oxide Synthase; High Mobility Protein 20; HMG-20; human NOS2A protein; In Vitro; Inducible Nitric Oxide Synthase; INOS; iNOS enzyme; Interphase; Investigators; Isoforms; L-Arginine; liquid; Liquid substance; Location; Macropain; Macrophage Nitric Oxide Synthase; Macroxyproteinase; Methods; Molecular; Molecular Interaction; Mononitrogen Monoxide; multicatalytic endopeptidase complex; Multicatalytic Proteinase; Nitric Oxide; Nitric Oxide Synthase 2A; nitric oxide synthase, Type II; Nitric Oxide, Endothelium-Derived; Nitrogen Monoxide; Nitrogen oxide; Nitrogen Protoxide; Normal Cell; NOS Type II; NOS2; NOS2A; NOS2A protein, human; novel; Pathogenesis; pathway; Pathway interactions; Physiologic; Physiological; Play; Process; programs; Programs (PT); Programs [Publication Type]; Prosome; Proteasome; Proteasome Endopeptidase Complex; protein degradation; Protein Degradation, Metabolic; Protein Degradation, Regulatory; Protein Isoforms; Protein Turnover; Proteins; Proteosome; Regulation; Research; Research Personnel; Research Proposals; research study; Researchers; response; Role; side effect; social role; Specificity; System; System, LOINC Axis 4; Testing; Therapeutic; Therapeutic Corticosteroid; therapy adverse effect; treatment adverse effect; Treatment Side Effects; ubiquination; Ubiquitilation; Ubiquitin; ubiquitin conjugation; ubiquitin ligase; ubiquitin-protein ligase; Ubiquitin-Protein Ligase E3; Ubiquitination; Ubiquitinoylation; Work
Project start date: 2007-02-01
Project end date: 2012-01-31
Budget start date: 1-FEB-2010
Budget end date: 31-JAN-2012
PFA/PA: PA-06-076
5R01HL084322-04 (2010): $375000
MOLECULAR MECHANISMS OF INOS DEGRADATION
Eissa Tony
Baylor College Of Medicinecity: Houston country: United States (us)
Grant 3R01HL069033-08S1 from National Heart, Lung, And Blood Institute
Abstract: Nitric oxide (NO), produced by the inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of inflammation associated with several pulmonary disorders including asthma, interstitial lung diseases, lung infection, and septic shock. The long term goal of this research is to understand the regulation of iNOS activity and devise novel methods to regulate it. Our recent data indicated that iNOS is primarily degraded by the ubiquitin-proteasome pathway. Further, we showed that cellular iNOS is regulated by sequestration into a perinuclear aggresome. In this proposal, we would like to study two complementary molecular mechanisms for iNOS degradation. Our preliminary data identify CHIP (carboxyl terminus of Hsp70- interacting protein) to play a major role in regulating iNOS degradation by the proteasome as well as for targeting iNOS to the aggresome. Our data further suggest that ADRM1 (adhesion regulating molecule 1) serves as a receptor for iNOS at the proteasome interface. For this grant period, we propose to test the following hypotheses a) iNOS is targeted to the proteasome by cooperative interaction between the ubiquitin pathway and the chaperone system, coordinated by CHIP and that the latter is critical for diverting excess iNOS to the aggresome. b). iNOS is handled at the proteasome by a specific receptor that deubiquitinates iNOS and delivers it to the proteasome. To test these hypotheses we propose studies with the following specific aims 1) Elucidate the mechanisms by which CHIP regulates iNOS proteasomal degradation. Our preliminary data suggest that CHIP interacts with and ubiquitinates iNOS. They further show that CHIP promotes iNOS degradation by the proteasome pathway. We will study the underlying mechanisms for CHIP regulation of iNOS degradation. Further, our preliminary data suggest that CHIP is expressed in primary airway epithelial cells. We will further examine CHIP regulation in normal primary cells as well as cells obtained from subjects with airway inflammation of asthma. 2) Determine the role of CHIP in targeting of iNOS to the aggresome. Our preliminary data suggest a role for CHIP in triaging iNOS between the proteasome and the aggresome pathways. We will examine the molecular mechanisms governing these interactions. 3) Characterize the regulation of iNOS by ADRM1, a novel proteasome associated protein. Our preliminary data suggest that ADRM1 plays a role in iNOS degradation. We will determine if ADRM1 is a specific receptor for iNOS at the proteasome interface. The modulatory effects of ADRM1 on iNOS will be elucidated. The rationale for the proposed studies is that once these mechanisms are understood, they would greatly increase our understanding of cellular handling of iNOS and how NO levels are regulated. Future therapeutic strategies can be designed to regulate these cellular responses in disease states. PUBLIC HEALTH RELEVANCE. Nitric oxide, produced by the inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of inflammation associated with several pulmonary disorders including asthma, interstitial lung diseases, lung infection, and septic shock. The long-term goal of this research is to understand the regulation of iNOS activity and devise novel methods to regulate it. The rationale for the proposed studies is that once these mechanisms are understood, future therapeutic strategies can be designed to regulate these cellular responses in disease states
Keywords: 20S Catalytic Proteasome; 20S Core Proteasome; 20S Proteasome; 20S Proteosome; Adhesions; Affect; Air; airway epithelium infalmmation; airway inflammation; APF-1; Asthma; ATP-Dependent Proteolysis Factor 1; Binding; Binding (Molecular Function); Boxing; Bronchial Asthma; C-terminal; cell biology; Cell Culture Techniques; Cells; Cellular biology; cellular targeting; Chaperone; Complex; Cultured Cells; Data; Degradation Pathway; Degradative Pathway; design; designing; Deubiquitination; Disease; disease/disorder; Disorder; Dissociation; Endogenous Nitrate Vasodilator; endothelial cell derived relaxing factor; Endothelium-Derived Relaxing Factor; enzyme activity; Enzymes; Epithelial Cells; fluid; Future; gene product; Goals; Grant; Health; Hepatocyte Nitric Oxide Synthase; High Mobility Protein 20; HMG-20; Host Defense; human NOS2A protein; ILD; Inducible Nitric Oxide Synthase; Infection; Inflammation; INFLM; INOS; iNOS enzyme; Interstitial Lung Diseases; liquid; Liquid substance; Lung; Lung diseases; Lung Diseases, Interstitial; lung disorder; Macropain; Macrophage Nitric Oxide Synthase; Macroxyproteinase; Methods; microbial; Molecular; Molecular Chaperones; Molecular Interaction; Mononitrogen Monoxide; multicatalytic endopeptidase complex; Multicatalytic Proteinase; Nitric Oxide; Nitric Oxide Synthase 2A; nitric oxide synthase, Type II; Nitric Oxide, Endothelium-Derived; Nitrogen Monoxide; Nitrogen oxide; Nitrogen Protoxide; NOS Type II; NOS2; NOS2A; NOS2A protein, human; novel; pathogen; Pathogenesis; pathway; Pathway interactions; Play; Polyubiquitin Gene Product; Process; Production; Prosome; Proteasome; Proteasome Endopeptidase Complex; protein complex; Proteins; Proteosome; pulmonary; Pulmonary Diseases; Pulmonary Disorder; receptor; Receptor Protein; recruit; Recruitment Activity; Regulation; Research; Research Proposals; Respiratory Disease; Respiratory Disorder; Respiratory System Disease; Respiratory System Disorder; Respiratory System, Lung; response; Role; Septic Shock; social role; Specificity; Staging; System; System, LOINC Axis 4; Testing; Therapeutic; Triage; ubiquination; Ubiquitilation; Ubiquitin; Ubiquitin C; ubiquitin conjugation; Ubiquitination; Ubiquitinoylation; Work
Project start date: 2001-12-01
Project end date: 2012-04-30
Budget start date: 1-JAN-2011
Budget end date: 30-APR-2011
PFA/PA: PA-07-070
3R01HL069033-08S1 (2011): $22295
5R01HL069033-09 (2011): $426002