Jerry Kaplan - US grants

DESCRIPTION (provided by applicant): Iron is an essential element but is toxic in excess. Malregulation of iron transport results in tissue injury, either from iron deprivation or overload. The concentration of free iron within cells is determined by regulated transmembrane iron import and export and by iron storage. We propose to identify and characterize systems that affect the transport of iron across the plasma membrane and into the vesicular apparatus. We will continue our approach of using yeast genetics to identify genes involved in iron metabolism. We have shown that the high affinity iron transport system of yeast involves a multicopper oxidase Fet3 and a transmembrane permease Ftr1. Transcription of this transport system is heme dependent and in the absence of heme transcription is repressed. We will determine how the repressor senses heme. Yeast and plants store iron in the vacuole. We identified the iron transporter Ccc1 that mediates iron export from cytosol to vacuole. We have shown that transcription of CCC1 is increased by iron. We will determine how the transcriptional activator of vacuolar iron import, Yap5, senses iron. Vertebrates store iron in ferritin and ferritin accumulation responds to increased cytosolic iron. We determined that expression of the iron exporter ferroportin or treatment with the permeable iron chelator desferasirox leads to ferritin iron release followed by ferritin degradation by the proteasome. We will determine biochemical changes in ferritin following iron release, how ferritin is marked for degradation and how ferritin is disassembled. The concentration of serum ferritin is a useful marker of iron overload disease, as there is a strong correlation with hepatic iron stores in hereditary hemochromatosis. We have determined the conditions that lead to ferritin secretion. Our data show that ferritin monomers can gain access to the secretory apparatus of cells. We propose to identify the sequences in ferritin that permit secretion. We will also test the hypothesis that secretion of ferritin is a mechanism that protects cells from ferritin induced decrease in free cytosolic iron. PUBLIC HEALTH RELEVANCE: Iron is an essential element but is toxic in excess. Malregulation of iron transport results in tissue injury, either from iron deprivation or overload. We will identify and characterize systems that affect the transport of iron across the plasma membrane and membranes of the vesicular system. We also propose to identify mechanisms that regulate iron storage. Our studies will provide information that may be used to manage and diagnose human diseases to altered iron metabolism.

alveolar macrophages; Chediak Higashi syndrome; receptor mediated endocytosis; intracellular transport; fungal proteins; G protein; protein sequence; membrane fusion; molecular cloning; vesicle /vacuole; lysosomes; alleles; potassium; protein structure function; phenotype; immunologic assay /test; laboratory rabbit; Chlamydophila psittaci; genetic strain; laboratory mouse; laboratory rat; flow cytometry;

Advances in molecular genetics have made it possible to correct genetic diseases by introducing a non-defective gene into the afflicted host via somatic cells. The objective of this proposal is to test the hypothesis that skin is the organ most amenable to correcting many genetic disorders via somatic cell gene therapy, especially those characterized by deficiency of a plasma protein. We propose to take advantage of a unique animal model to examine the possibility of curing diseases by use of genetically modified skin. Murine hypotransferrinemia is a recessive disorder resulting from a splicing defect in the plasma transferrin gene; transferrin being the major iron binding protein in plasma. Homozygotes for this disorder have drastically reduced levels of transferrin, but demonstrate iron overload disease of the liver, heart and pancreas. These mutants exhibit severe anemia (a faithful replica of the rare human disease atransferrinemia) and at the same time manifest the complications of the more common disease, hemochromatosis. We propose to evaluate the ability of skin implants, expressing transferrin, to provide the absent plasma protein and thereby alleviate the clinical manifestations of severe hypotransferrinemia. Fibroblasts taken from homozygote mice will be transduced in vitro with a retrovirus plasmid containing the cDNA for either human or murine transferrin. The genetically modified fibroblasts will be implanted subdermally and the concentration of plasma transferrin as well as objective indices of the disease state assayed. In a second project we propose to evaluate the use of skin cells expressing a truncated version transferrin to manage iron overload diseases. A number of diseases exist in which iron overload can not be treated by conventional phlebotomies, thalassemia being the most notable. We hypothesize that a shortened transferrin molecule, a truncated transferrin, would act as a perfect iron chelator. It wound bind iron with high affinity, not be recognized by transferrin receptors, and would be excreted in the urine. We propose to graft a dermal matrix of genetically modified fibroblasts expressing a truncated transferrin gene beneath the skin of homozygote hypotransferrinemic mice and assay these transfected mice for reduction in parenchymal tissue iron. We feel that our previous studies on iron metabolism, the existence of the mutant mouse and the fact that the relevant genes are in hand, offers a unique opportunity to examine the use of skin cells in somatic cell gene therapy.

Project 1 consists of three subprojects. Subproject 1 will test the hypothesis that iron is transported across erythroid mitochondrial membranes as Fe(II). A ferroxidase is required on the inner mitochondrial membrane for effective iron uptake. Ferrochelatase is likely the required ferroxidase. This hypothesis will be tested with isolated mitochondria obtained from induced MEL cells. Genes responsible for erythroid-specific mitochondrial iron transport will be cloned and characterized and the effects of mutant ferrochelatase will be determined. Subproject 2 will define the properties of two iron regulatory element binding proteins, IRE-BP1 and IRE-BP2. The role of each of these proteins will be determined by targeted gene disruptions in ES cells and in transgenic animals. Subproject 3 will result in the creation of a model of human familial porphyria cutanea tarda (PCT) by targeted disruption of the uroporphyrinogen decarboxylase (URO-D) gene and the creation of transgenic animals heterozygous for the disruption. These animals will be used to test the hypothesis that phenotypic expression of PCT is dependent on a P450-mediated reaction which oxidizes the substrate of URO-D.

teacher; science education; health science research support; education evaluation /planning; secondary schools; pharmacy;

DESCRIPTION: (Adapted from investigator's abstract) The goal of this proposal is to define mitochondrial iron homeostasis in eukaryotes at the molecular and biochemical level by studying yeast genes that are involved in mitochondrial iron transport. Using different selection systems the applicants have identified yeast genes that result in alterations in cellular and mitochondrial iron metabolism. A yeast gene, YFH, was identified by its ability to complement the low-iron phenotype of a mutant bm-8 defective in intracellular iron metabolism. YFH is the homologue of the Frataxin gene, responsible for the human disease Friedreich's ataxia, and encodes a mitochondrial protein. A deletion in YFH results in mitochondrial iron accumulation, increased sensitivity to oxygen-mediated damage and a respiratory deficit. The applicants propose to determine how YFH affects mitochondrial iron accumulation and to also determine if the mammalian Frataxin encodes a mitochondrial protein. Analysis of the function of the yeast Frataxin homologue should clarify the etiology of Friedreich's ataxia. The BM8 gene is essential and the applicants will determine whether it is a mitochondrial protein and if so, will define its function. The applicants have already identified a family of genes that affect intracellular iron levels. These genes encode proteins that appear to be transporters; they have extensive hydrophobic domains and clusters of histidine residues in the putative cytosolic loops. One of the genes has been localized to the mitochondria. Based on this localization the applicants have called the family Mitochondrial Metal Transporters (MMT). They propose to determine the localization of the other two members of this family. Through the use of deletion and overexpression constructs the applicants propose to examine the function of these genes. They have also developed a selection system which is designed to identify genes that encode mitochondrial iron transporters, particularly those that deliver iron to ferrochelatase. Through the use of their mutant strains and genetic constructs the applicants propose to examine the relationship between mitochondrial iron accumulation and heme biosynthesis. They will determine if defective heme biosynthesis results, as it does in reticulocytes, in excessive iron deposition in mitochondria. These studies will help define the regulation of mitochondrial iron metabolism.

The objective of this project is to confirm the clinical diagnosis of Freidreich's ataxia in 49 patients attending the Muscular Dystrophy Association clinic who are thought to have Friedreich's ataxia. Diagnostic confirmation will be done by identifying the presence of a trinucleotide (GAA) expansion in intron 1 of the frataxin gene (the gene responsible for Freidreich's ataxia).

This study is designed to determine if chelation therapy with desferrioxamine will reduce mitochondrial iron overload and arrest disease progression in patients with Friedreich ataxia. This study is based on findings in yeast that mutations of the frataxin gene (or in the yeast, the homolog of frataxin) result in mitochondrial iron overload and mitochondrial dysfunction. Seven patients have been entered on this study. No toxicity from desferrioxamine has been detected. All patients have had a decrease in total body iron stores, as indicated by a decrease in the serum ferritin concentration. No patient has developed iron-deficiency anemia. Abnormalities on cardiac biopsy have been noted in every patient with dense punctate deposits within mitochondria. Improvement in electroretinograms in several patients is the first indication that the therapy may be useful in arresting (or perhaps improving) neurologic function. The primary endpoint of this study is a reduction of cardiac mitochondrial iron overload as demonstrated by cardiac myocyte biopsies before and after one year of chelation. The first patients will reach the one-year study point in the autumn of 1999.

DESCRIPTION (provided by applicant) The University of Utah School of Medicine is applying for a five year grant to provide funds to support research expenses for medical students. It is our hope that a successful application will not only provide funds to support medical student research activities, but will increase the visibility, interest and quality of those opportunities. The University of Utah has had a strong commitment to biomedical research and to medical student research activities. These activities are supported by a standing committee which solicits students and sponsors and provides funds for research activities. This committee, through the P.l.'s on this application, report to the Dean's Office and the Sr. Vice President for Health Sciences. The Medical Student Research Program is a successful program that forms the cornerstone of academic medical research efforts. Over 100 faculty members from diverse departments participate in this program. This program has led to structured research experiences for over 100 students during the past five years. This program is directly responsible for students obtaining further research experience, and has led to the creation of an MD/PhD Program. Applicants for NRSA fellowships must be medical students in at least their second semester of study. MD/PhD candidates or holders of PhD degrees are not eligible. We request funding for 25 fellowships per year. This number is based on the past number of applicants and on further training for highly meritorious applicants.

[unreadable] DESCRIPTION (provided by applicant): Our goal is to define mitochondrial iron metabolism in eukaryotes at the molecular and biochemical level by studying yeast genes that are involved in mitochondrial iron transport and utilization. YFH1 is the yeast homologue of the mammalian Frataxin gene, which is responsible for Friedreich Ataxia. Defects in YFH1 result in excessive mitochondrial iron accumulation due to a defect in mitochondrial iron export and leads to respiratory deficit due to the generation of toxic oxygen radicals. We propose to determine how YFH1 affects mitochondrial iron export. We plan to test the hypothesis that defects in mitochondrial iron export result from defects in the mitochondrial iron-sulfur cluster synthetic pathway, and that Yfh1p is involved in iron-sulfur cluster syntheses. Genetic experiments are designed to identify proteins that interact with Yfh1p by generating dominant negative alleles of YFH1. Using biochemical assays to measure iron-sulfur cluster synthesis in isolated mitochondria, we plan to examine the affect of YFH1 mutant alleles. We also propose genetic approaches to identify genes that regulate the mitochondrial iron cycle. Different genes are implicated in mitochondrial metal transporter. We propose genetic and biochemical approaches to test the hypothesis that these genes are transition metal transporters which provide iron and other transition metals for mitochondrial processes. The genetic approaches involve identifying the phenotype of cells with deletions in multiple transporter genes. The biochemical approaches include studying metal transport in isolated mitochondria and in liposomes reconstituted with specific transporters. In mammals defective heme biosynthesis results in mitochondrial iron accumulation in reticulocytes but not in other cell types. We discovered that in yeast, defective heme synthesis inhibits high affinity iron transport, which prevents mitochondrial iron accumulation. We propose to determine the mechanism by which the absence of heme affects iron transport.

DESCRIPTION (provided by applicant): This application requests support for travel expenses to enable young U.S. investigators to attend and participate in the biennial Bioiron World Congress on Iron Metabolism (Bioiron 2005). The application also requests support provide travel expenses for senior investigators whose expertise may not be in iron but whose participation in the meeting would be of benefit to the field. A special emphasis will be given to the support of women, members of minority subpopulations, persons with disabilities and other individuals who have been traditionally underrepresented in science. Bioiron 2005 is recognized as the premier international conference on iron in physiology and medicine. The Bioiron conferences bring together clinicians and researchers in a meeting that combines state-of-the-art sessions and symposia given by the world's leading investigators with peer-reviewed presentations and posters of the latest research results. Recent progress in this field, especially the discovery of genes whose products are involved in the regulation of cellular iron uptake and distribution in humans, has made participation in this meeting essential for the development of young U.S. investigators working in this area. Conference abstracts are published in full and distributed to all participants. Major topics to be considered in Bioiron 2005 include, animal models of disorders of iron metabolism, mechanisms and regulation of iron transport and storage, analysis of genes that lead to iron overload disease including both HFE and non-HFE hereditary hemochromatosis. Sessions will focus on the role of iron in the cardiovascular and pulmonary systems. Special attention will be given to clinical issues such as treatment of iron overload disorders and anemia, and the potential role of iron-chelator therapy in disease prevention and management. Bioiron 2005 will be held in Prague Czech Republic from May 22 to May 28 2005. The overall goal of the Bioiron (2005) World Congress is the international dissemination of important new research results to investigators working in the field of iron metabolism.

DESCRIPTION (provided by applicant): Ferroportin (Fpn, IREG1, MTP1) is the only identified transmembrane iron exporter in vertebrates. Our preliminary data indicate that hepcidin inhibits cellular iron export by inducing the internalization and degradation of Fpn. The goals of this proposal are to understand the mechanism of Fpn-mediated iron export and the mechanism of hepcidin-mediated Fpn internalization. The entry of iron into plasma is highly regulated and is affected by iron stores, inflammation and hypoxia. Chronic inflammation results in decreased plasma iron and an iron-limited anemia, referred to as the Anemia of Chronic Disease. Increased iron stores or inflammation result in high levels of hepcidin, a peptide secreted by liver that regulates iron metabolism. We hypothesize that this interaction is a major controlling factor in mammalian iron homeostasis. We will determine if metal export by Fpn is specific for iron through the use of metal sensitive fluorescent dyes. We will define the structure of Fpn and determine the number of transmembrane domains and the location of intracellular and extracellular domains. Biochemical and genetic approaches will be used to determine how hepcidin binds to Fpn and how Fpn is internalized. Human hepcidin will be modified to generate radiolabeled and photoaffinity ligands, which will then be used to characterize the hepcidin/Fpn interaction. By selectively changing amino acid residues in human and fish hepcidins, we will define residues critical for Fpn binding. Residues in Fpn that are required for internalization and metal transport will be identified through genetic screens and biochemical examination of hepcidin treated Fpn. RNAi silencing will be used to determine if Fpn is the only mammalian iron exporter or if are there are other iron export systems. These studies will probe the mechanism that underlines the regulation of iron export by hepcidin and will define the roles of hepcidin and Fpn in iron physiology.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] This application requests support for travel expenses to enable young U.S. investigators to attend and [unreadable] participate in the biennial BioIron World Congress on Iron Metabolism (BioIron 2007). The application also requests support to provide travel expenses for senior investigators whose expertise may not be in iron but whose participation in the meeting would be of benefit to the field. A special emphasis will be given to the support of women, members of minority subpopulations, persons with disabilities and other individuals who have been traditionally underrepresented in science. BioIron 2007 is recognized as the premier international conference on iron in physiology and medicine. The BioIron conferences bring together clinicians and researchers in a meeting that combines state-of-the-art sessions and symposia given by the world's leading investigators with peer-reviewed presentations and posters of the latest research results. Recent progress in this field, especially the discovery of genes whose products are involved in the regulation of cellular iron uptake and distribution in humans, has made participation in this meeting essential for the development of young U.S. investigators working in this area. Conference abstracts are published in full and distributed to all participants. Major topics to be considered in BioIron 2007 include, animal models of disorders of iron metabolism, mechanisms and regulation of iron transport and storage, analysis of genes that lead to iron overload disease and anemia. Sessions will focus on the role of iron in the cardiovascular and pulmonary systems. Special attention will be given to clinical issues such as treatment of iron overload disorders and anemia, and the potential role of iron-chelator therapy in disease prevention and management. BioIron 2007 will be held in Kyoto Japan from April 1 to April 6 2007. The overall goal of the BioIron 2007 World Congress is the international dissemination of important new research results to investigators working in the field of iron metabolism. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): We seek to acquire a Becton Dickinson Pathway Bioimager system, designed for image-based high- Throughput Screens (HTS). This is an automated microscope capable of imaging a 96-well plate in less than 20 minutes. This instrument can generate confocal z-stacks, and has automated liquid and sample handling functions-all essential requirements for the applications proposed herein. No other instrument currently available to university researchers meets these needs. As the research proposals in our application demonstrate, there is an overwhelming interest in performing high-throughput imaging screens at the University of Utah. Of the 18 research proposals contained herein, 15 are from NIH-funded investigators. Our pilot studies demonstrate we have the necessary technical expertise to manage and analyze the large volumes of data that these screens will produce; moreover analyzing these data will provide a forum for collaboration with members of the Department of Computer Science, and with Myriad Pharmaceuticals, Inc. Thus, the instrument will also foster interdisciplinary academic and industrial collaborations. Finally, the fact that the BD Pathway will be part of a university core facility will further enhance its benefits to users- instrument maintenance and staff salaries will be paid through university funds and existing oversight procedures will ensure fair and equitable access to the instrument. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The University of Utah School of Medicine is applying for a five year grant to provide funds to support research expenses for medical students. The goals of this proposal are to enable interested students to evaluate a potential career as a physician and to enhance the qualifications of those students interested in academic medicine. We believe that a research experience will increase critical thinking, which is important to the diagnosis and treatment of patients. It is our hope that a successful application will not only provide funds to support medical student research activities, but will increase the visibility, interest and quality of those opportunities. The University of Utah has had a strong commitment to biomedical research and to medical student research activities. These activities are supported by an oversight committee which solicits students and sponsors and provides funds for research activities. This committee, through the P.I. on this application, reports to the Dean's Office and the Sr. Vice President for Health Sciences. The Medical Student Research Program is a successful program that forms the cornerstone of academic medical research efforts. Over 100 faculty members from diverse departments participate in this program. This program has led to structured research experiences for over 125 students during the past five years. This program is directly responsible for students obtaining further research experience, and has led to the creation of an MD/PhD Program and a Medical Student Research Interest Group. Applicants for NRSA fellowships must be medical students in at least their second semester of study. MD/PhD Candidates or holders of PhD degrees are not eligible. We request funding for 30 fellowships per year. This number is based on the past number of applicants, an increase in medical student class size and on further training for highly meritorious applicants. (End of Abstract)

DESCRIPTION, OVERALL (provided by applicant): We propose a Center of Exellence in Molecular Hematology (CEHM) with the theme of metal metabolism and heme biosynthesis. The CEMH will support the activities of 17 NIH-supported investigators from multiple departments at the University of Utah and five other universities. These investigators focus on iron, copper, and zinc and on the biosynthesis of porphyrins and heme in normal and disease states. Research in metal metabolism emphasizes the genetic and biochemical basis of metals, including homeostasis uptake, transport, and cellular distribution. Genes of interest are studied in yeast, zebrafish, worms, and mouse models. Results from model systems are used to test clinical hypotheses. These studies are facilitated by the availability of large pedigrees with inherited iron overload disorders. Research in porphyria and heme biosynthesis extends from structural studies of porphyrin biosynthetic enzymes to animal models of human porphyrins. Again, large pedigrees with inherited disorders of porphyrin metabolism are available for translational studies. The CEMH will support four Cores that will facilitate research on metals and heme. These Cores include X-Ray Crystallography, Protein Purification, and Metabolomics. We expect the Metabolomics Core to be national reference centers for studies on metals and heme. The CEMH will also support pilot projects directed both at translational research and at identification of genes that affect metal uptake transport and distribution. The pilot projects take advantage of Utah's strengths in genetic analysis, metal physiology, and a large accessible patient population.

DESCRIPTION (provided by applicant): This is a competing continuation application for the University of Utah's Research Training in Hematology Program, a program established in 1943 by Dr. M.M. Wintrobe. Twenty-four faculty members serve as research preceptors for trainees, and the faculty consists of both physician-scientists and basic scientists from the Departments of Medicine, Biochemistry, Chemistry, Human Genetics, Oncological Sciences, Pathology, and Pediatrics. Research groups participating in the Training Program include the Molecular Regulation of Metal and Heme Metabolism and the Hematopoiesis-Cell Differentiation Group. The special attribute of this multidisciplinary training program is the faculty with which trainees can interact, with both basic and clinical investigators. The unifying element is the common objective to train post-doctoral fellows and graduate students who can conduct studies at the cutting edge of hematologic research. In our program, physician-trainees interact with basic science post-doctoral trainees and graduate students every day, and this interaction promotes an expanded view of hematologic research for both groups of post-doctoral fellows and the graduate students working in the laboratories of the training faculty. A plan is presented to expand the pre-doctoral Training Program by incorporating a newly created University of Utah Med-into-Grad Program. This program is designed to transform basic science graduate education by integrating intensive, clinically-relevant education into pre-doctoral training. The program will permit graduate students to obtain dual degrees, a department-specific Ph.D. degree and a medical school-wide Master of Science in clinical investigation. The application requests support for six pre-doctoral trainees and six post-doctoral fellows (a mixture of physician-trainees and Ph.D. post-doctoral trainees). PUBLIC HEALTH RELEVANCE: Hematology has led medical sciences into an era of enormous advances in the cellular and molecular basis of many diseases. The leading role of hematology is explained, in part, by the ease of access to blood and bone marrow cells. An expanded core of investigators trained in molecular biology and cell physiology is required to exploit new opportunities afforded by advances in technology. The Hematology Research Training Program at the University of Utah is designed to train these investigators.

DESCRIPTION (provided by applicant): Our goal is to define mitochondrial iron metabolism in eukaryotes at the molecular and biochemical level by studying proteins required for mitochondrial iron transport and utilization. We have identified mitochondrial iron importers in yeast, Mrs3/Mrs4, and vertebrates, Mitoferrin1 and Mitoferrin2. We demonstrated that the mitoferrins show tissue specific expression patterns and tissue specific functions. We have generated mice with floxed alleles of both Mitoferrin1 and Mitoferrin2. We will utilize specific expression of Cre recombinase to identify the tissue specific function of the mitoferrins. We will examine the mechanism of mitochondrial dysfunction by deleting mitoferrins in vitro in specific cell types through the use of recombinant Tat-Cre. Through genetic screens we identified genes that are required for hemoglobinization in developing erythrocytes. We will determine the function of these genes. We will determine if Abcb10 has roles other than stabilizing Mfrn1, if deletion of Atpif1 affects ferrochelatase activity through altered mitochondrial pH and if reductio of SLC25A39, the Mtm1 yeast homologue, affects mitochondrial superoxide dismutase activity. We determined that the mitochondrial iron pool is tightly regulated, but can be increased by overexpression of mitochondrial iron importers. We will determine the physiological consequences of increased levels of mitochondrial iron in both yeast and mammalian cells. We had identified Mmt1 and Mmt2 as mitochondrial iron exporters. We discovered that MMT1 and MMT2 are transcriptionally upregulated by low iron and by oxidant stress. We have identified Yap1 as the oxidant stress activated transcription factor for MMT1. We will identify the transcriptional factor responsible for the low iron induction of MMT1/MMT2 and the physiological function of mitochondrial iron export. We will determine if mammalian mitochondria can act as a iron reservoir and if there are mammalian mitochondrial iron exporters.