THE ROLE OF ALPHA HEMOGLOBIN STABILIZING PROTEIN IN HUMAN BETA THALASSEMIA
Mitchell J Weiss, Associate Professor Of Pediatrics
Children´s Hospital Of Philadelphia, Research Institute, Philadelphia, Pa 19104-4318
Grant 5R01HL087427-04 from National Heart, Lung, And Blood Institute
Abstract: We are working toward a new perspective in understanding and manipulating the pathophysiology of ? thalassemia, a common and debilitating inherited anemia. A hallmark of this disorder is excessive free ? hemoglobin (Hb), an unstable protein that generates reactive oxygen species (ROS) and forms cytotoxic precipitates. We identified alpha hemoglobin stabilizing protein (AHSP), an abundant erythroid protein that enhances the solubility of free ?Hb and limits its biochemical reactivity. Ahsp-/- mice exhibit hemolytic anemia with Hb precipitates and excessive ROS. Moreover, loss of AHSP exacerbates ? thalassemia in mice, raising the possibility that altered AHSP function or expression could modulate ? thalassemia phenotypes in humans. Preliminary data support both mechanisms. First, we discovered a naturally occurring missense mutation, AHSP N75I, which impairs protein function and is associated with unexpectedly severe p thalassemia in two pedigrees. Second, AHSP appears to be a quantitative trait locus (QTL) whose expression varies considerably between different individuals. Moreover, reduced AHSP expression associates with more severe clinical disease in several independent studies of small p thalassemia cohorts and pedigrees. Together, these findings lead to the hypothesis that AHSP is a genetic modifier of ? thalassemia. We will test this by analyzing thalassemic populations for AHSP gene mutations, including N75I, and determining their effects on gene expression and/or protein function. In addition, we will study how variations in erythroid AHSP expression affect nascent ?Hb pools, oxidative stress and clinical severity in p thalassemic patients. Our findings should provide new insights into the mechanisms of normal erythropoiesis and the pathophysiology of ? thalassemia. Ultimately, this information could provide a basis for developing novel therapeutic approaches to mitigate the toxicities of free ?Hb in ? thalassemia
Keywords: 2`, 7`-dichlorofluorescein diacetate; 2`, 7`-dichlorofluorescin diacetate; Active Oxygen; Affect; American; Anemia; Anemia, Erythroblastic; Anemia, Hemolytic; Anemia, Mediterranean; B-thalassemia; Binding; Binding (Molecular Function); Biochemical; Biology; Blood erythrocyte; Blood normocyte; Caring; Cells; Cellular injury; Clinic; Clinical; Clinical Research; Clinical Study; Code; Coding System; Cooley`s anemia; Crystallographies; Crystallography; DCFDA; DCFH-DA; DNA Alteration; DNA mutation; Data; Data Banks; Data Bases; Databank, Electronic; Databanks; Database, Electronic; Databases; Disease; Disorder; Dysfunction; Erythroblasts; Erythrocytes; Erythrocytes, Nucleated; Erythrocytic; Erythroid; Erythroid Cells; Erythropoiesis; Exhibits; Family; Frequencies (time pattern); Frequency; Functional disorder; Gene Alteration; Gene Expression; Gene Mutation; Gene Proteins; Genes; Genetic; Genetic Alteration; Genetic Change; Genetic Screening; Genetic defect; Genetic mutation; Genotype; Geographic Area; Geographic Locations; Geographic Region; Geographical Location; Globin; Haplotypes; Hemoglobin; Hemoglobinopathies; Hemoglobinopathies / Iron Metabolism; Hemolytic Anemia; Hereditary; Hospitals, Pediatric; Human; Human, General; Impairment; Individual; Inherited; Investigation; Investigators; Knowledge; Laboratories; Lead; Length of Life; Link; Longevity; Malaria; Mammals, Mice; Man (Taxonomy); Man, Modern; Marrow erythrocyte; MeSH Descriptors Class 4; Measures; Membrane; Mercaptans; Mercapto Compounds; Messenger RNA; Mice; Missense Mutation; Modification; Molecular Interaction; Murine; Mus; Mutation; Mutation, Missense; National Heart, Lung, and Blood Institute; Normoblasts; Nucleated red blood cell; Nucleated red cell; Oxidation-Reduction; Oxidative Stress; Oxygen Radicals; Paludism; Patients; Pb element; Pediatric Hospitals; Pedigree; Peptides; Phenotype; Philadelphia; Physiopathology; Plasmodium Infections; Polymorphism, Single Base; Population; Population Control; Population Study; Pro-Oxidants; Programs (PT); Programs [Publication Type]; Promoter Regions; Promoter Regions (Genetics); Promotor Regions; Promotor Regions (Genetics); Protein Gene Products; Proteins; QTL; Quantitative Trait Loci; RNA, Messenger; Reactive Oxygen Species; Recombinants; Red Blood Cells; Red Cell; Red blood corpuscule; Red cell of marrow; Redox; Research Personnel; Researchers; Resistance; Reticuloendothelial System, Erythrocytes; Role; SNP; SNPs; Sampling; Sequence Alteration; Severities; Single Nucleotide Polymorphism; Solubility; Structure; Sulfhydryl Compounds; Testing; Thalassemia; Thalassemia Major; Thalassemia intermedia; Therapeutic; Thiols; Toxic effect; Toxicities; Transferrin Receptor; Variant; Variation; Work; base; beta Thalassemia; blood corpuscles; cell damage; cell injury; clinical data repository; clinical data warehouse; clinical phenotype; cohort; cytotoxic; data repository; diacetyldichlorofluorescein; disease/disorder; gene product; genetic pedigree; genetic promoter element; genome mutation; geographic site; heavy metal Pb; heavy metal lead; insight; leuco-DCF diacetate; leukodiacetyl-2, 7-dichlorofluorescein; life span; lifespan; mRNA; mRNA Expression; membrane structure; mutant; novel; novel therapeutic intervention; nucleated RBCs; oxidation; oxidation reduction reaction; p-Globin; p-Thalassemia; pathophysiology; pedigree structure; programs; protein expression; protein function; protein structure; relational database; resistant; social role; sulfhydryl group; trait
Project start date: 2007-01-19
Project end date: 2010-12-31
Budget start date: 1-JAN-2010
Budget end date: 31-DEC-2010
5R01HL087427-04 (2010): $411250
Sponsored Links Excellgen http://Excellgen.com
The Role Of Alpha Hemoglobin Stabilizing Protein In Human Beta Thalassemia
Mitchell J Weiss
Children´s Hospital Of Philadelphia
Grant 5R01HL087427-03 from National Heart, Lung, And Blood Institute IRG: ELB
Abstract: We are working toward a new perspective in understanding and manipulating the pathophysiology of ? thalassemia, a common and debilitating inherited anemia. A hallmark of this disorder is excessive free ? hemoglobin (Hb), an unstable protein that generates reactive oxygen species (ROS) and forms cytotoxic precipitates. We identified alpha hemoglobin stabilizing protein (AHSP), an abundant erythroid protein that enhances the solubility of free ?Hb and limits its biochemical reactivity. Ahsp-/- mice exhibit hemolytic anemia with Hb precipitates and excessive ROS. Moreover, loss of AHSP exacerbates ? thalassemia in mice, raising the possibility that altered AHSP function or expression could modulate ? thalassemia phenotypes in humans. Preliminary data support both mechanisms. First, we discovered a naturally occurring missense mutation, AHSP N75I, which impairs protein function and is associated with unexpectedly severe p thalassemia in two pedigrees. Second, AHSP appears to be a quantitative trait locus (QTL) whose expression varies considerably between different individuals. Moreover, reduced AHSP expression associates with more severe clinical disease in several independent studies of small p thalassemia cohorts and pedigrees. Together, these findings lead to the hypothesis that AHSP is a genetic modifier of ? thalassemia. We will test this by analyzing thalassemic populations for AHSP gene mutations, including N75I, and determining their effects on gene expression and/or protein function. In addition, we will study how variations in erythroid AHSP expression affect nascent ?Hb pools, oxidative stress and clinical severity in p thalassemic patients. Our findings should provide new insights into the mechanisms of normal erythropoiesis and the pathophysiology of ? thalassemia. Ultimately, this information could provide a basis for developing novel therapeutic approaches to mitigate the toxicities of free ?Hb in ? thalassemia
Keywords: hemoglobin, protein structure function, thalassemia gene expression, gene mutation, nucleic acid sequence, oxidative stress, phenotype clinical research, histology, human genetic material tag, human subject, laboratory mouse, patient oriented research
Project start date: 2007-01-19
Project end date: 2010-12-31
Grants awarded to Mitchell J Weiss
The Role Of Alpha Hemoglobin Stabilizing Protein In Human Beta Thalassemia
Mitchell J Weiss
Children s Hospital Of Philadelphia Joseph Stokes, Jr. Research Institute Philadelphia, Pa 191044318
Grant 1R01HL087427-01A1 from National Heart, Lung, And Blood Institute IRG: ELB
Abstract: We are working toward a new perspective in understanding and manipulating the pathophysiology of ? thalassemia, a common and debilitating inherited anemia. A hallmark of this disorder is excessive free ? hemoglobin (Hb), an unstable protein that generates reactive oxygen species (ROS) and forms cytotoxic precipitates. We identified alpha hemoglobin stabilizing protein (AHSP), an abundant erythroid protein that enhances the solubility of free ?Hb and limits its biochemical reactivity. Ahsp-/- mice exhibit hemolytic anemia with Hb precipitates and excessive ROS. Moreover, loss of AHSP exacerbates ? thalassemia in mice, raising the possibility that altered AHSP function or expression could modulate ? thalassemia phenotypes in humans. Preliminary data support both mechanisms. First, we discovered a naturally occurring missense mutation, AHSP N75I, which impairs protein function and is associated with unexpectedly severe p thalassemia in two pedigrees. Second, AHSP appears to be a quantitative trait locus (QTL) whose expression varies considerably between different individuals. Moreover, reduced AHSP expression associates with more severe clinical disease in several independent studies of small p thalassemia cohorts and pedigrees. Together, these findings lead to the hypothesis that AHSP is a genetic modifier of ? thalassemia. We will test this by analyzing thalassemic populations for AHSP gene mutations, including N75I, and determining their effects on gene expression and/or protein function. In addition, we will study how variations in erythroid AHSP expression affect nascent ?Hb pools, oxidative stress and clinical severity in p thalassemic patients. Our findings should provide new insights into the mechanisms of normal erythropoiesis and the pathophysiology of ? thalassemia. Ultimately, this information could provide a basis for developing novel therapeutic approaches to mitigate the toxicities of free ?Hb in ? thalassemia.
Keywords: hemoglobin, protein structure function, thalassemia, gene expression, gene mutation, nucleic acid sequence, oxidative stress, phenotype, clinical research, histology, human genetic material tag, human subject, laboratory mouse, patient oriented research
Project start date: 2007-01-19
Project end date: 2010-12-31
1R01HL087427-01A1 (2007): $412500
HEMATOPOIESIS FROM NORMAL AND PATIENT-DERIVED INDUCED PLURIPOTENT STEM CELLS
Mitchell J Weiss
Childrens Hospital Of Philadelphia, Research Institute, Philadelphia, Pa 19104-4318
Grant 5RC2HL101606-02 from National Heart, Lung, And Blood Institute
Abstract: This project addresses the NHLBI RC2 GO application entitled "Characterizing Differentiated Heart, Lung, and Blood Cells Derived by Reprogramming Human Embryonic and Induced Pluripotent Stem Cells." Emerging technologies to generate induced pluripotent stem cells (iPSCs) by reprogramming human somatic cells promises to revolutionize biomedical research and clinical medicine. Through in vitro culture methods, iPSCs can be differentiated into numerous cells types derived from all three germ layers. This raises the possibility that patient-derived iPSCs can be used to create relevant tissues for the study of many human disorders. In addition, iPSCs may provide starting material to manufacture transplantable cells for transfusion and regenerative therapies. However, the field is in its infancy and many core questions must be solved in order to realize these exciting long-term prospects. This proposal seeks to advance the use of iPSCs for the study of normal and pathological hematopoiesis. Multiple investigators with broad areas of expertise in hematopoiesis, embryonic stem cell/iPSC biology, chromatin biology, clinical hematology, bioinformatics, cell banking and bioethics/regulatory affairs will work together to pursue the following global issues 1) Mechanisms by which hematopoietic developmental potential might vary between different normal iPSC clones; 2) The extent to which iPSC-derived hematopoietic precursors resemble normal ones with respect to cellular phenotypes, gene expression and epigenetic signatures; 3) Whether hematopoietic disease phenotypes can be recapitulated by in vitro manipulation of patient-derived iPSCs. We will execute these studies using novel methods to create and culture iPSCs and state-of-the art tools to analyze and manipulate their resident genomes. Pursuit of these problems will serve as a framework in which to develop a facile infrastructure where investigators at our large pediatric institution can create, analyze, bank and distribute iPSCs from any patient of interest. If successful, this work will help to accelerate practical applications of iPSCs for the study and treatment of human diseases. This work will be based at Children´s Hospital of Philadelphia with subcontracts to The Pennsylvania State University (State College, PA) and The Coriell Institute for Medical Research (Camden, NJ). The project will create 6 new jobs, thereby stimulating the economy in three different regions of the Northeastern United States. Efforts to better understand blood production from patient-derived induced pluripotent stem cells (iPSCs) will enhance our understanding of blood disorders and generate new therapeutic approaches. Additionally, this work could create new general paradigms for studying the genesis of many normal tissues and their associated diseases
Keywords: 0-11 years old; Acute Megakaryoblastic Leukemia (FAB Type M7); Acute Megakaryocytic Leukemias; Address; Affect; Area; Arts; Bio-Informatics; Biochemical Genetics; Bioethics; Bioinformatics; Biology; Biomedical Research; Blood; Blood (Leukemia); Blood Cells; Blood Diseases; Blood, Cord; Body Tissues; CD34; CD34 gene; Cells; Cellular Morphology; Child; Child Youth; Childhood; Children (0-21); Chromatin; Clinic; Clinical; Clinical Medicine; Collaborations; Complement; Complement Proteins; Crossmatching, Tissue; Data; Development; Diamond-Blackfan anemia; Disease; Disorder; Down Syndrome; Down`s Syndrome; Downs Syndrome; Dysfunction; ERYF1; ERYF1 protein, human; ES cell; Embryo; Embryonic; Emergent Technologies; Emerging Technologies; Epigenetic; Epigenetic Change; Epigenetic Mechanism; Epigenetic Process; Erythro; Erythroblasts; Erythrocytes, Nucleated; Erythroid; Erythroid Transcription Factor; Erythropoiesis; Ethics, Biomedical; Fetal Liver; Foundations; Functional disorder; Future; GATA Binding Protein 1; GATA binding protein 1, human; GATA-1; GATA1; GATA1 gene; GATA1 protein, human; GF1; Gene Expression; Gene Transfer; Genes; Genetic Alteration; Genetic Change; Genetic Condition; Genetic Diseases; Genetic defect; Genetic, Biochemical; Genome; Germ Layers; Germ-Line Mutation; Germline Mutation; HPCA1; Heart; Hematologic Diseases; Hematological Disease; Hematological Disorder; Hematology; Hematopoiesis; Hematopoietic; Hematopoietic Cellular Control Mechanisms; Hereditary Disease; Hereditary Mutation; Histocompatibility Testing; Human; Human, Child; Human, General; Immunodeficient Mouse; In Vitro; Individual; Infrastructure; Institutes; Institution; Investigators; Jobs; Laboratories; Langdon Down syndrome; Leukemias, General; Longitudinal Studies; Lung; Man (Taxonomy); Man, Modern; Medical Research; Medical Sciences, Clinical; Megakaryoblastic Leukemia, Acute; Megakaryocytic Leukemia; Methods; Modeling; Modification; Molecular Disease; Mongolism; Mutation; Myeloid Leukemia, Acute, M7; NFE1; National Heart, Lung, and Blood Institute; New England; Normal Tissue; Normal tissue morphology; Normoblasts; Northeastern United States; Nucleated red blood cell; Nucleated red cell; Occupations; Patients; Pattern; Pediatric Hospitals; Pennsylvania; Peripheral Blood Cell; Phenotype; Philadelphia; Physiopathology; Predisposition; Preleukemia; Procedures; Process; Production; Professional Postions; Protocol; Protocols documentation; Regulatory Affairs; Research; Research Infrastructure; Research Personnel; Research Resources; Researchers; Resources; Respiratory System, Lung; Reticuloendothelial System, Blood; Ribosomal Proteins; Solid; Somatic Cell; Somatic Mutation; Source; Susceptibility; TRNSF; Technology; Tissue Crossmatchings; Tissue Typing; Tissues; Transcription Factor GATA1; Transfusion; Transplantation; Trisomy 21; Umbilical Cord Blood; Universities; Work; application in practice; base; blood disorder; cell bank; cell morphology; cell type; children; chromosome 21 trisomy syndrome; college; congenital acromicria syndrome; design; designing; disease phenotype; disease/disorder; embryonic stem cell; erythrold transcription factor 1; experience; experiment; experimental research; experimental study; fetal; fetal cord blood; genetic disorder; genetic manipulation; genome mutation; globin transcription factor 1; globin transcription factor 1, human; hematopoietic tissue; hereditary disorder; histocompatibility typing; homologous recombination; human GATA1 protein; human disease; induced pluripotent stem cell; infancy; infantile; interest; leukemia; long-term study; morbus Down; new technology; novel; novel therapeutic intervention; nucleated RBCs; pathophysiology; pediatric; practical application; progenitor; pseudohypertrophic progressive muscular dystrophy; public health relevance; pulmonary; regenerative therapy; repository; research study; stem cell of embryonic origin; tool; transfer of a gene; transient myeloproliferative disorder; transplant; trisomy 21 syndrome; youngster
Relevance: RELEVANCE Efforts to better understand blood production from patient-derived induced pluripotent stem cells (iPSCs) will enhance our understanding of blood disorders and generate new therapeutic approaches. Additionally, this work could create new general paradigms for studying the genesis of many normal tissues and their associated diseases
Project start date: 2009-09-30
Project end date: 2011-08-31
Budget start date: 1-SEP-2010
Budget end date: 31-AUG-2011
PFA/PA: RFA-OD-09-004
5RC2HL101606-02 (2010): $964802
1RC2HL101606-01 (2009): $997217
MOLECULAR AND BIOLOGICAL ACTIVITIES OF ALPHA HEMOGLOBIN STABILIZING PROTEIN
Mitchell J Weiss, Associate Professor Of Pediatrics
Children´s Hospital Of Philadelphia, Research Institute, Philadelphia, Pa 19104-4318
Grant 3R01DK061692-07S1 from National Institute Of Diabetes And Digestive And Kidney Diseases
Abstract: This award is issued in response to Notice OD-09-060, Recovery Act Administrative Supplements Providing Summer Research Experiences for Students and Science Educators. Our studies address a critical problem in hemoglobin (Hb) biology how inherently unstable globin protein subunits are folded and maintained during normal and pathological erythropoiesis. We discovered alpha hemoglobin stabilizing protein (AHSP), an erythroid protein that specifically binds free ? globin subunit, stabilizes its structure and limits its pro-oxidant activities. Our preliminary studies suggest two distinct functions for AHSP. First, to detoxify excess ? globin that accumulates during normal erythropoiesis and in various anemias, particularly ? thalassemia. Second, to fold and stabilize newly formed ? globin subunits en route to HbA (?2??2) synthesis. Of potential importance to both functions, we discovered that degradation of AHSP mRNA is accelerated by iron, an essential component of HbA and determinant of nascent globin protein stability. Our overall view is that AHSP facilitates normal HbA synthesis and also buffers against imbalances that arise from genetic or environmental stresses, such as thalassemias and iron deficiency. Now we seek to better understand AHSP activities and their relevance to human health. Aim 1 uses mouse genetics to investigate AHSP functions in vivo. We will examine the consequences of manipulated AHSP expression in thalassemias and create Ahsp gene missense mutations in mice to probe mechanisms of AHSP protein function. Aim 2 studies the biochemical properties of AHSP. We will test in vitro if AHSP promotes reconstitution of HbA from its purified apo-globin and heme components and search for new erythroid proteins that interact with ? globin-AHSP complexes. Aim 3 examines the mechanisms by which iron regulates AHSP expression and the physiological implications of this pathway during altered iron homeostasis. If successful, our work will establish new basic principles of Hb biology and erythropoiesis. In addition, there are potential practical long-term benefits. For example, understanding how AHSP detoxifies excess ?Hb should illustrate novel therapeutic approaches for human ? thalassemias. Elucidating the role of AHSP in HbA synthesis may provide tools to optimize the manufacture of recombinant Hb-based blood substitutes. Finally, defining functional interactions between iron and AHSP could provide insights into the pathophysiology and management of iron overload and deficiency states. Our work examines how the blood oxygen carrier hemoglobin is stabilized and assembled during red blood cell formation. If successful, our experiments will enhance general knowledge about how blood is formed. In addition, we will provide new insights toward understanding and treating common and debilitating anemias such as thalassemia and iron deficiency
Keywords: 3` Untranslated Regions; 3`UTR; Active Oxygen; Address; Affinity; Anemia; Artificial Erythrocytes; Assay; Autoregulation; Binding; Binding (Molecular Function); Binding Proteins; Bioassay; Biochemical; Biologic Assays; Biological; Biological Assay; Biology; Blood; Blood Substitutes; Blood erythrocyte; Blood normocyte; Blood, Artificial; Breeding; Buffers; Capital; Carrier State; Chaperone; Clinical; Collaborations; Complex; Crystallography, X-Ray; Crystallography, X-Ray Diffraction; Crystallography, X-Ray/Neutron; Crystallography, Xray; Data; Defect; Degradation, mRNA; Disease; Disorder; Dysfunction; Erythrocyte Substitutes; Erythrocytes; Erythrocytic; Erythroid; Erythropoiesis; Fe element; Ferrate(2-), (7, 12-diethenyl-3, 8, 13, 17-tetramethyl-21H, 23H-porphine-2, 18-dipropanoato(4-)-N21, N22, N23, N24)-, dihydrogen, (SP-4-2)-; Ferroprotoporphyrin; Functional disorder; Gene Expression; Gene Proteins; Gene Transfer; Genes; Genetic; Genetic Alteration; Genetic Change; Genetic defect; Globin; Goals; Health; Hematopoietic; Heme; Heme Iron; Heme b; Hemoglobin; Hemolysis; Histidine; Histidine, L-isomer; Homeostasis; Human; Human Biology; Human, General; Hybrids; In Vitro; Individual; Iron; Iron Overload; Kinetic; Kinetics; Knowledge; L-Histidine; Letters; Ligand Binding Protein; Link; Mammals, Mice; Man (Taxonomy); Man, Modern; Maps; Marrow erythrocyte; Measures; Messenger RNA; Metals; Mice; Mice, Mutant Strains; Mice, Transgenic; Missense Mutation; Molecular; Molecular Chaperones; Molecular Configuration; Molecular Conformation; Molecular Interaction; Molecular Stereochemistry; Mouse Protein; Murine; Mus; Mutant Strains Mice; Mutation; Mutation, Missense; Nomenclature; O element; O2 element; Organ; Oxygen; Oxygen Radicals; Pathway interactions; Physiologic; Physiological; Physiological Homeostasis; Physiopathology; Pro-Oxidants; Process; Production; Property; Property, LOINC Axis 2; Protein Binding; Protein Gene Products; Protein Subunits; Proteins; Protoheme; Protoheme IX; RNA, Messenger; Reactive Oxygen Species; Recombinants; Red Blood Cells; Red Cell; Red Cell Substitutes; Red blood corpuscule; Red cell of marrow; Regulation; Response Elements; Reticuloendothelial System, Blood; Reticuloendothelial System, Erythrocytes; Rice; Role; Route; Single Crystal Diffraction; Solubility; Stress; Structure; Testing; Thalassemia; Thalassemia intermedia; Toxic effect; Toxicities; Transcript; Transgenic Mice; Universities; Work; X Ray Crystallographies; X-Ray Crystallography; Yeasts; alpha Globin; base; blood corpuscles; conformation; conformational state; cytotoxic; disease carrier state; disease/disorder; experiment; experimental research; experimental study; ferroheme; gene product; genome mutation; in vitro testing; in vivo; insight; mRNA; mRNA Transcript Degradation; mouse model; mouse mutant; mutant; novel therapeutic intervention; overexpression; p-Globin; pathophysiology; pathway; protein complex; protein expression; protein function; public health relevance; reconstitute; reconstitution; research study; social role; tissue culture; tool; transfer of a gene
Project start date: 2009-08-01
Project end date: 2010-07-31
Budget start date: 1-AUG-2009
Budget end date: 31-JUL-2010
PFA/PA: PA-07-070
3R01DK061692-07S1 (2009): $8111
Characterization Of A Alpha-globin Chaperone Protein
Mitchell J Weiss
Children s Hospital Of Philadelphia Joseph Stokes, Jr. Research Institute Philadelphia, Pa 191044318
Grant 5R01DK061692-05 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: HEM
Abstract: Recent discoveries in our laboratory offer new insights into normal erythroid biology and beta-thalassemia. The high- level production of hemoglobin that occurs during erythroid maturation is tightly coordinated so as to minimize toxicities caused by accumulation of individual alpha- and beta- globin subunits, which tend to precipitate in cells. Prior studies of normal and beta-thalassemic erythroid precursors predict that compensatory mechanisms exist to neutralize free alpha-globin. To learn more about the control of hemoglobin production, we isolated RNA transcripts that are induced by the essential transcription factor GATA-1, a global regulator of erythropoiesis. We identified Erythroid Differentiation Related Factor (EDRF), a small, abundant highly erythroid-specific protein that is strongly upregulated during terminal erythroid maturation and appears to be a direct GATA-1 target gene. We determined that alpha-globin is a specific EDRF binding partner in two independent protein interaction screens. EDRF interacts with free alpha-globin but not with beta-globin or hemoglobin A (alpha2beta2). Moreover, EDRF markedly inhibits precipitation of free alpha-globin in solution and in mammalian cells. Our findings raise the possibility that EDRF acts as a chaperone protein to prevent precipitation and subsequent toxicity of free alpha-globin in erythroid cells. Now that we have established a physical and functional connection between EDRF and alpha-globin in vitro and in heterologous cells, we will study the significance of this association in normal erythropoiesis. Structure-function analyses in Aim 1 will define the domains that are required for physical and functional interactions between EDRF and alpha-globin. In Aim 2, we will assess the biological role of EDRF and its association with alpha-globin in established cell lines and in primary erythroid cells derived from in vitro culture of EDRF gene-targeted embryonic stem (ES) cells. To this end, we have developed EDRF heterozygous and homozygous-null ES cells. In Aim 3, we will determine the hematopoietic consequences of altered EDRF expression in mice. By genetically manipulating EDRF and free alpha-globin levels, we will determine how their relative stoichiometry affects viability and differentiation of erythroid cells. Specifically, we will establish whether EDRF-null animals exhibit excessive alpha- globin precipitation in erythroid precursors, and whether altered EDRF gene expression affects the severity of beta-thalassemia, a disorder that is distinguished by alpha-globin precipitation. Our studies to characterize a highly expressed erythroid specific protein that prevents aggregation of free alpha-globin are important for understanding how hemoglobin chain balance is modulated by non-globin proteins during normal erythropoiesis and might provide a novel approach to alleviate the deleterious effects of excessive alpha-globin in beta-thalassemia.
Keywords: erythropoiesis, globin, molecular chaperone, protein structure function, embryonic stem cell, erythroid stem cell, gene interaction, hematopoietic stem cell, hemoglobin, messenger RNA, mutant, phenotype, protein metabolism, protein protein interaction, thalassemia, transcription factor, gene targeting, genetic manipulation, genetically modified animal, laboratory mouse, stoichiometry, tissue /cell culture, transfection /expression vector
Project start date: 2002-04-01
Project end date: 2008-01-31
5R01DK061692-05 (2006): $346899
5R01DK061692-04 (2005): $355247
5R01DK061692-03 (2004): $355247
Characterization Of A Alpha-globin Chaperone Protein
Mitchell J Weiss
Children s Hospital Of Philadelphia Joseph Stokes, Jr. Research Institute Philadelphia, Pa 191044318
Grant 3R01DK061692-02S1 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: HEM
Abstract: Recent discoveries in our laboratory offer new insights into normal erythroid biology and beta-thalassemia. The high- level production of hemoglobin that occurs during erythroid maturation is tightly coordinated so as to minimize toxicities caused by accumulation of individual alpha- and beta- globin subunits, which tend to precipitate in cells. Prior studies of normal and beta-thalassemic erythroid precursors predict that compensatory mechanisms exist to neutralize free alpha-globin. To learn more about the control of hemoglobin production, we isolated RNA transcripts that are induced by the essential transcription factor GATA-1, a global regulator of erythropoiesis. We identified Erythroid Differentiation Related Factor (EDRF), a small, abundant highly erythroid-specific protein that is strongly upregulated during terminal erythroid maturation and appears to be a direct GATA-1 target gene. We determined that alpha-globin is a specific EDRF binding partner in two independent protein interaction screens. EDRF interacts with free alpha-globin but not with beta-globin or hemoglobin A (alpha2beta2). Moreover, EDRF markedly inhibits precipitation of free alpha-globin in solution and in mammalian cells. Our findings raise the possibility that EDRF acts as a chaperone protein to prevent precipitation and subsequent toxicity of free alpha-globin in erythroid cells. Now that we have established a physical and functional connection between EDRF and alpha-globin in vitro and in heterologous cells, we will study the significance of this association in normal erythropoiesis. Structure-function analyses in Aim 1 will define the domains that are required for physical and functional interactions between EDRF and alpha-globin. In Aim 2, we will assess the biological role of EDRF and its association with alpha-globin in established cell lines and in primary erythroid cells derived from in vitro culture of EDRF gene-targeted embryonic stem (ES) cells. To this end, we have developed EDRF heterozygous and homozygous-null ES cells. In Aim 3, we will determine the hematopoietic consequences of altered EDRF expression in mice. By genetically manipulating EDRF and free alpha-globin levels, we will determine how their relative stoichiometry affects viability and differentiation of erythroid cells. Specifically, we will establish whether EDRF-null animals exhibit excessive alpha- globin precipitation in erythroid precursors, and whether altered EDRF gene expression affects the severity of beta-thalassemia, a disorder that is distinguished by alpha-globin precipitation. Our studies to characterize a highly expressed erythroid specific protein that prevents aggregation of free alpha-globin are important for understanding how hemoglobin chain balance is modulated by non-globin proteins during normal erythropoiesis and might provide a novel approach to alleviate the deleterious effects of excessive alpha-globin in beta-thalassemia.
Keywords: erythropoiesis, globin, molecular chaperone, protein structure function, embryonic stem cell, erythroid stem cell, gene interaction, hematopoietic stem cell, hemoglobin, messenger RNA, mutant, phenotype, protein metabolism, protein protein interaction, thalassemia, transcription factor, gene targeting, genetic manipulation, laboratory mouse, stoichiometry, tissue /cell culture, transfection /expression vector, transgenic animal
Project start date: 2002-04-01
Project end date: 2007-01-31
3R01DK061692-02S1 (2003): $34000
3R01DK061692-02S2 (2003): $104747
Characterization Of A Alpha-globin Chaperone Protein
Mitchell J Weiss
Children s Hospital Of Philadelphia Joseph Stokes, Jr. Research Institute Philadelphia, Pa 191044318
Grant 1R01DK061692-01 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: HEM
Abstract: Recent discoveries in our laboratory offer new insights into normal erythroid biology and beta-thalassemia. The high- level production of hemoglobin that occurs during erythroid maturation is tightly coordinated so as to minimize toxicities caused by accumulation of individual alpha- and beta- globin subunits, which tend to precipitate in cells. Prior studies of normal and beta-thalassemic erythroid precursors predict that compensatory mechanisms exist to neutralize free alpha-globin. To learn more about the control of hemoglobin production, we isolated RNA transcripts that are induced by the essential transcription factor GATA-1, a global regulator of erythropoiesis. We identified Erythroid Differentiation Related Factor (EDRF), a small, abundant highly erythroid-specific protein that is strongly upregulated during terminal erythroid maturation and appears to be a direct GATA-1 target gene. We determined that alpha-globin is a specific EDRF binding partner in two independent protein interaction screens. EDRF interacts with free alpha-globin but not with beta-globin or hemoglobin A (alpha2beta2). Moreover, EDRF markedly inhibits precipitation of free alpha-globin in solution and in mammalian cells. Our findings raise the possibility that EDRF acts as a chaperone protein to prevent precipitation and subsequent toxicity of free alpha-globin in erythroid cells. Now that we have established a physical and functional connection between EDRF and alpha-globin in vitro and in heterologous cells, we will study the significance of this association in normal erythropoiesis. Structure-function analyses in Aim 1 will define the domains that are required for physical and functional interactions between EDRF and alpha-globin. In Aim 2, we will assess the biological role of EDRF and its association with alpha-globin in established cell lines and in primary erythroid cells derived from in vitro culture of EDRF gene-targeted embryonic stem (ES) cells. To this end, we have developed EDRF heterozygous and homozygous-null ES cells. In Aim 3, we will determine the hematopoietic consequences of altered EDRF expression in mice. By genetically manipulating EDRF and free alpha-globin levels, we will determine how their relative stoichiometry affects viability and differentiation of erythroid cells. Specifically, we will establish whether EDRF-null animals exhibit excessive alpha- globin precipitation in erythroid precursors, and whether altered EDRF gene expression affects the severity of beta-thalassemia, a disorder that is distinguished by alpha-globin precipitation. Our studies to characterize a highly expressed erythroid specific protein that prevents aggregation of free alpha-globin are important for understanding how hemoglobin chain balance is modulated by non-globin proteins during normal erythropoiesis and might provide a novel approach to alleviate the deleterious effects of excessive alpha-globin in beta-thalassemia.
Keywords: erythropoiesis, globin, molecular chaperone, protein structure function, embryonic stem cell, erythroid stem cell, gene interaction, hematopoietic stem cell, hemoglobin, messenger RNA, mutant, phenotype, protein metabolism, protein protein interaction, thalassemia, transcription factor, gene targeting, genetic manipulation, laboratory mouse, stoichiometry, tissue /cell culture, transfection /expression vector, transgenic animal
Project start date: 2002-04-01
Project end date: 2007-03-31
1R01DK061692-01 (2002): $376763
Sponsored Links Excellgen http://Excellgen.com
MOLECULAR AND BIOLOGICAL ACTIVITIES OF ALPHA HEMOGLOBIN STABILIZING PROTEIN
Mitchell J Weiss, Associate Professor Of Pediatrics
Children´s Hospital Of Philadelphia, Research Institute, Philadelphia, Pa 19104-4318
Grant 5R01DK061692-08 from National Institute Of Diabetes And Digestive And Kidney Diseases
Abstract: Our studies address a critical problem in hemoglobin (Hb) biology how inherently unstable globin protein subunits are folded and maintained during normal and pathological erythropoiesis. We discovered alpha hemoglobin stabilizing protein (AHSP), an erythroid protein that specifically binds free ? globin subunit, stabilizes its structure and limits its pro-oxidant activities. Our preliminary studies suggest two distinct functions for AHSP. First, to detoxify excess ? globin that accumulates during normal erythropoiesis and in various anemias, particularly ? thalassemia. Second, to fold and stabilize newly formed ? globin subunits en route to HbA (?2??2) synthesis. Of potential importance to both functions, we discovered that degradation of AHSP mRNA is accelerated by iron, an essential component of HbA and determinant of nascent globin protein stability. Our overall view is that AHSP facilitates normal HbA synthesis and also buffers against imbalances that arise from genetic or environmental stresses, such as thalassemias and iron deficiency. Now we seek to better understand AHSP activities and their relevance to human health. Aim 1 uses mouse genetics to investigate AHSP functions in vivo. We will examine the consequences of manipulated AHSP expression in thalassemias and create Ahsp gene missense mutations in mice to probe mechanisms of AHSP protein function. Aim 2 studies the biochemical properties of AHSP. We will test in vitro if AHSP promotes reconstitution of HbA from its purified apo-globin and heme components and search for new erythroid proteins that interact with ? globin-AHSP complexes. Aim 3 examines the mechanisms by which iron regulates AHSP expression and the physiological implications of this pathway during altered iron homeostasis. If successful, our work will establish new basic principles of Hb biology and erythropoiesis. In addition, there are potential practical long-term benefits. For example, understanding how AHSP detoxifies excess ?Hb should illustrate novel therapeutic approaches for human ? thalassemias. Elucidating the role of AHSP in HbA synthesis may provide tools to optimize the manufacture of recombinant Hb-based blood substitutes. Finally, defining functional interactions between iron and AHSP could provide insights into the pathophysiology and management of iron overload and deficiency states. Our work examines how the blood oxygen carrier hemoglobin is stabilized and assembled during red blood cell formation. If successful, our experiments will enhance general knowledge about how blood is formed. In addition, we will provide new insights toward understanding and treating common and debilitating anemias such as thalassemia and iron deficiency
Keywords: 3` Untranslated Regions; 3`UTR; Active Oxygen; Address; Affinity; Anemia; Artificial Erythrocytes; Assay; Autoregulation; Binding; Binding (Molecular Function); Binding Proteins; Bioassay; Biochemical; Biologic Assays; Biological; Biological Assay; Biology; Blood; Blood Substitutes; Blood erythrocyte; Blood normocyte; Blood, Artificial; Breeding; Buffers; Capital; Carrier State; Chaperone; Clinical; Collaborations; Complex; Crystallography, X-Ray; Crystallography, X-Ray Diffraction; Crystallography, X-Ray/Neutron; Crystallography, Xray; Data; Defect; Degradation, mRNA; Disease; Disorder; Dysfunction; Erythrocyte Substitutes; Erythrocytes; Erythrocytic; Erythroid; Erythropoiesis; Fe element; Ferrate(2-), (7, 12-diethenyl-3, 8, 13, 17-tetramethyl-21H, 23H-porphine-2, 18-dipropanoato(4-)-N21, N22, N23, N24)-, dihydrogen, (SP-4-2)-; Ferroprotoporphyrin; Functional disorder; Gene Expression; Gene Proteins; Gene Transfer; Genes; Genetic; Genetic Alteration; Genetic Change; Genetic defect; Globin; Goals; Health; Hematopoietic; Heme; Heme Iron; Heme b; Hemoglobin; Hemolysis; Histidine; Histidine, L-isomer; Homeostasis; Human; Human Biology; Human, General; Hybrids; In Vitro; Individual; Iron; Iron Overload; Kinetic; Kinetics; Knowledge; L-Histidine; Letters; Ligand Binding Protein; Link; Mammals, Mice; Man (Taxonomy); Man, Modern; Maps; Marrow erythrocyte; Measures; Messenger RNA; Metals; Mice; Mice, Mutant Strains; Mice, Transgenic; Missense Mutation; Molecular; Molecular Chaperones; Molecular Configuration; Molecular Conformation; Molecular Interaction; Molecular Stereochemistry; Mouse Protein; Murine; Mus; Mutant Strains Mice; Mutation; Mutation, Missense; Nomenclature; O element; O2 element; Organ; Oxygen; Oxygen Radicals; Pathway interactions; Physiologic; Physiological; Physiological Homeostasis; Physiopathology; Pro-Oxidants; Process; Production; Property; Property, LOINC Axis 2; Protein Binding; Protein Gene Products; Protein Subunits; Proteins; Protoheme; Protoheme IX; RNA, Messenger; Reactive Oxygen Species; Recombinants; Red Blood Cells; Red Cell; Red Cell Substitutes; Red blood corpuscule; Red cell of marrow; Regulation; Response Elements; Reticuloendothelial System, Blood; Reticuloendothelial System, Erythrocytes; Rice; Role; Route; Single Crystal Diffraction; Solubility; Stress; Structure; Testing; Thalassemia; Thalassemia intermedia; Toxic effect; Toxicities; Transcript; Transgenic Mice; Universities; Work; X Ray Crystallographies; X-Ray Crystallography; Yeasts; alpha Globin; base; blood corpuscles; conformation; conformational state; cytotoxic; disease carrier state; disease/disorder; experiment; experimental research; experimental study; ferroheme; gene product; genome mutation; in vitro testing; in vivo; insight; mRNA; mRNA Transcript Degradation; mouse model; mouse mutant; mutant; novel therapeutic intervention; overexpression; p-Globin; pathophysiology; pathway; protein complex; protein expression; protein function; public health relevance; reconstitute; reconstitution; research study; social role; tissue culture; tool; transfer of a gene
Project start date: 2002-04-21
Project end date: 2013-01-31
Budget start date: 1-FEB-2010
Budget end date: 31-JAN-2011
PFA/PA: PA-07-070
5R01DK061692-08 (2010): $341861
Transcription Factor GATA-1 In Erythro-Megakaryocytic De
Mitchell J Weiss
Children s Hospital Of Philadelphia Joseph Stokes, Jr. Research Institute Philadelphia, Pa 191044318
Grant 5R21DK075966-02 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: HP
Abstract: Most leukemias are caused by somatic mutations that disrupt transcription factor function. One example is GATA-1, a nuclear protein required for erythroid and megakaryocytic maturation. Nearly all patients with Down s Syndrome-associated transient myeloproliferative disorder (TMD) or acute megakaryoblastic leukemia (AMKL) exhibit somatic GATA1 gene mutations that result in the exclusive production of an abnormal amino-truncated protein, termed GATA-1 short. We showed that in murine embryonic stem cells and embryos, loss of GATA-1 causes a previously unappreciated block at the bipotential megakaryocytic-erythroid progenitor (MEP) stage of hematopoiesis, where development also appears to be perturbed in AMKL. The leukemia-associated GATA-1 short protein fails to relieve this block and actually drives proliferation in a subset of arrested MEPs. Hence, we hypothesize that GATA-1 promotes normal MEP maturation and that derangements in this function contribute to Down s syndrome-associated TMD and AMKL. In particular, we believe that loss of specific GATA-1 functions activate an aberrant self-renewal program in MEPs, which contributes to development of the leukemic stem cell. Now, we will define the genetic program through which normal GATA-1 controls MEP development and investigate how this program becomes deranged by GATA-1 short in murine ES cells and in human fetal hematopoietic progenitors from both normal and trisomy 21 individuals.
Keywords: Downs syndrome, acute myelogenous leukemia, cell differentiation, cell growth regulation, erythroid stem cell, megakaryocyte, myeloproliferative neoplasm, nuclear protein, protein structure function, transcription factor, cord blood, embryonic stem cell, neoplasm /cancer genetics, neoplastic process, NOD mouse, RNA interference, SCID mouse, clinical research, human subject, newborn human (0-6 weeks), patient oriented research, small interfering RNA, stem cell transplantation, tissue /cell culture
Project start date: 2006-08-01
Project end date: 2008-07-31
5R21DK075966-02 (2007): $200269
1R21DK075966-01 (2006): $247500
Antiproliferative Actions Of GATA-1 During Hematopoiesis
Mitchell J Weiss
Children s Hospital Of Philadelphia Joseph Stokes, Jr. Research Institute Philadelphia, Pa 191044318
Grant 5R01DK064037-03 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: HP
Abstract: GATA-1 is an essential hematopoietic transcription factor that coordinates proliferation arrest with cellular maturation, a function of relevance to normal tissue formation and cancer. Germline mutations in GATA-1 cause inherited dyserythropoietic anemia and thrombocytopenia, while somatic mutations are associated with acute megakaryocytic leukemia (AMKL). The extent of GATA-1 actions and its critical targets are not fully defined; here we propose to investigate its antiproliferative functions. Preliminary findings support the hypothesis that GATA-1 inhibits cell cycle progression by repressing mitogenic genes and activating antiproliferative ones. Remarkably, at least two oncogenes, Kit and Myc, are inhibited directly by GATA-1 in erythroid cells, highlighting a significant role for gene repression in the GATA-1 antiproliferative program. GATA-1 also triggers proliferation arrest and maturation of cultured GATA-1- megakaryocytes through pathways that are currently unexplored. Our current goals are to investigate the mechanisms by which GATA-1 regulates Myc, Kit (Aim 1), and four other potentially key cell cycle effector genes whose expression are controlled by GATA-1 (Aim 2), as identified in our preliminary studies. In Aim 2, we will also examine the functions of these genes by manipulating their expression in erythroid cells and determining the effects on the GATA-1 antiproliferative program. These experiments address the poorly understood role of GATA-1 in gene repression and characterize new molecular targets, several of which were not previously implicated in hematopoiesis. To complement our work on erythroid cells, we will investigate the effects of GATA-1 on megakaryocyte proliferation (Aim 3). Specifically, we will define the transcriptional program associated with proliferation arrest caused by normal GATA-1 and examine mutations previously associated with hyperproliferation and/or maturation defects. These findings will provide further insights into how altered GATA function causes AMKL. Moreover, comparative analysis between erythroblasts and megakaryocytes should reveal aspects of GATA-1-mediated cell cycle that are either common or unique to these related lineages. Our major long-term goals are to define the regulatory hierarchies through which GATA-1 orchestrates cell cycle arrest during normal hematopoietic differentiation and to apply this knowledge to the study of cytopenias and leukemia(s) associated with altered GATA-1 function
Keywords: cell growth regulation, cell proliferation, hematopoiesis, hemorrhagic thrombocythemia, transcription factor, binding site, cell cycle, dyserythropoietic anemia, gene expression, gene mutation, genetic mapping, thrombocytopenia, SCID mouse, flow cytometry, polymerase chain reaction, tissue /cell culture
Project start date: 2004-09-15
Project end date: 2007-06-30
5R01DK064037-03 (2006): $243149
5R01DK064037-02 (2005): $249000
1R01DK064037-01A1 (2004): $249000
Mitchell J Weiss
Childrens Hospital Of Philadelphia
Project start date: 2002-04-21
Project end date: 2013-01-31
The Function And Regulation Of Alpha Hemoglobin Stabilizing Protein (AHSP) In Hem
Mitchell J Weiss
Children s Hospital Of Philadelphia Joseph Stokes, Jr. Research Institute Philadelphia, Pa 191044318
Grant 2R56DK061692-06 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: ELB
Keywords: hemoglobin, protein
Project start date: 2002-04-01
Project end date: 2008-01-31
2R56DK061692-06 (2007): $82500
UNIQUE ROLES FOR GATA 1 AND GATA 2 IN ERYTHROPOIESIS
Mitchell J Weiss
Children s Hospital Boston 300 Longwood Ave Boston, Ma 021155737
Grant 5K08HL003364-02 from National Heart, Lung, And Blood Institute IRG: RTR
Project start date: 1995-08-15
Project end date: 1997-06-30
5K08HL003364-02 (1996): $82620
Sponsored Links Excellgen http://Excellgen.com