Steven G Clarke
University Of California Los Angeles
Project start date: 1978-12-01
Project end date: 2014-01-31
Sponsored Links Excellgen http://Excellgen.com
CONTROL OF EUCARYOTIC MEMBRANE FUNCTION BY METHYLATION
Steven G Clarke, Professor
University Of California Los Angeles Office Of Research Administration Los Angeles, Ca 90095
Grant 5R01GM026020-08 from National Institute Of General Medical Sciences IRG: PC
Abstract: We wish to define the role of reversible protein methylation in eucaryotic cells, particularly in human blood cells. We have shown that specific erythrocyte cytoskeletal and membrane proteins are methylated and demethylated in ester linkages at aspartyl residues. We are now concerned with the question of the physiological consequences of these reaction. Is the shape and deformability of the red cell, or its capacities to transporat anions and other metabolites across the plasma membrane, regulated by the level of methylation of these proteins? To pursue these questions we will ask whether the methylation of membrane proteins separated by dodecyl sufate gel electrophoresis from intact cells labeled with [3H-methyl] methionine is affected by the various conditions that a red cell is normally exposed to in the curculation. These conditions include plasma hormone levels as well as mechanical stresses on the membrane as erythrocytes pass through the capillary beds. We can also ask whether changes in protein methylation (induced for example by incubating cells with permeable inhibitors of the cytosolic protein methyltransferase) can affect any of the functional properties of red cells, or whether the in vitro activities of purified membrane proteins are modified by their degree of methylation. We will be interested in the factors that control the in vivo state of methylation and will pursue the identification and characterization of the protein carboxyl methyltransferase(s), protein methylestera(s), and endogenous small molecule substrates and inhibitors. We also propose to characterize protein methylation reactions in other types of cells. We will compare the methylation of membrane proteins in normal human erythrocytes with those of patients with disease expressed in these cells such as hereditary sperocytosis, sickle cell anemia, and muscular dystrophy. These experiments may not only provide evidence of the function of methylation in normal cells but may shed insight on the molecular nature of these disease. Because protein carboxyl methylation has already been shown to be involved in receptor function in bacterial chemotaxis, we will also investigate whether reversible methylation reactions play a role in human platelet functions such as clot retraction and colagen-and thrombin-induced shape changes and aggregation or in human neutrophil functions such as chemotaxis. Rats, rabbits, cats, dogs, chicken, sheep.
Keywords: ALKYL (GROUPS), METHYLATION-DEMETHYLATION, BIOLOGICAL TRANSPORT, MEMBRANE PERMEABILITY AND TRANSPORT, BLOOD CELLS, CARBOXYL GROUPS, CARBOXYMETHYLATION, CELLS (ORGANISMS), EUKARYOTIC (SEE ALSO SPECIFICS), PHYSIOLOGICAL CHEMISTRY STUDY SECTION, PROTEINS, MEMBRANE PROTEINS, BIOLOGICAL TRANSPORT, ION EXCHANGE AND TRANSPORT, BLOOD AND RE DISORDERS CONGENITAL (HEREDITARY), BLOOD CELLS, LEUKOCYTES, NEUTROPHILS, BLOOD PLATELETS, CELL COMPONENTS, CYTOSKELETON, DECARBOXYLASES, ENVIRONMENT, ORIENTATION, CHEMOTAXIS, ENZYME INHIBITORS, ENZYME MECHANISMS, ENZYMES, ISOENZYMES, ESTERASES, GENETICS, GENETIC REGULATION, GENETIC INDUCTION-REPRESSION-DEREPRESSION, TRANSLATION, MEMBRANE, ERYTHROCYTE GHOST AND MEMBRANE, PEPTIDES, POLYPEPTIDES, PROTEINS-PEPTIDES STRUCTURE, TRANSCARBOXYLASES, blood rheology, cell growth regulation, BIRDS, TURKEYS, BLOOD AND RE DISORDERS, ANEMIA HEMOLYTIC CONGENITAL, SPHEROCYTOSIS HEREDITARY (GENERAL), HEMOPROTEINS, HEMOGLOBINOPATHIES, SICKLE CELL ANEMIA, HUMAN, TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PROJECT, HYDROGEN, TRITIUM, MAMMALS, METABOLIC DISORDERS INBORN, MUSCULAR DYSTROPHIES, NUCLEOSIDYL AMINO ACIDS, S-ADENOSYLMETHIONINE, PHYSICAL SEPARATION, ELECTROPHORESIS, GEL, RADIOTRACERS, TISSUE (CELL) CULTURE
Project start date: 1978-12-01
Project end date: 1986-11-30
CONTROL OF EUKARYOTIC MEMBRANE FUNCTION BY METHYLATION
Steven G Clarke, Professor
University Of California Los Angeles Office Of Research Administration Los Angeles, Ca 90095
Grant 5R01GM026020-17 from National Institute Of General Medical Sciences IRG: PC
Abstract: The objective of this work is to understand the physiological role of enzymatic protein carboxyl methylation reactions in human tissues and other cells. We propose to continue our studies of an L-isoaspartyl/D-aspartyl methyltransferase (E.C. 2.1.1.77) that catalyzes the modification of damaged proteins containing these unusual residues. This enzyme is found in the cytosolic fraction of all mammalian tissues examined and recognizes the altered residues that result from spontaneous racemization, isomerization, and deamidation of normal L-aspartyl and L-asparaginyl residues in aged proteins. In model systems, the formation of methyl esters at L-isoaspartyl residues can lead to their conversion to L-aspartyl residues and suggests that this enzyme functions to repair certain types of covalent damage to intracellular proteins and limit their accumulation in aging cells. We will use a combination of biochemical and molecular biological techniques in in vitro and in intact cell systems, focusing particularly on human erythrocytes. Since the structure of this enzyme has been remarkably well conserved from bacteria to human cells, we will also utilize genetically-manipulatable microbial model systems. This methyltransferase may represent one of the first members of a class of enzymes that can check spontaneous damage to cellular proteins, and its disruption in pathological conditions may both decrease their useful lifetime and contribute to accelerated aging processes. our recent discovery of a new class of protein carboxyl methyltransferases suggests that these enzymes may also have roles in the regulation of cellular signalling reactions. Proteins that contain a C-terminal tetrapeptide sequence are modified by reactions that form C-terminal S-isoprenylated cysteine methyl esters. These proteins include the ras oncogene products as well as other small G-proteins, subunits of the large G-proteins, and nuclear lamin components. The isoprenylation and methylation of these proteins have been postulated to lead to both membrane association and to the control of their signalling activities. We will purify and characterize the enzymes involved in these reactions from both mammalian tissues and yeast. The ability to pharmacologically control these modification enzymes, and thus signalling proteins involved in cell division, may represent new therapies for cancer and other diseases.
Keywords: carboxymethylation, membrane protein, methylation, methyltransferase, protein metabolism, S adenosylmethionine, aging, aspartate, biological signal transduction, enzyme mechanism, enzyme substrate, erythrocyte, erythrocyte membrane, isozyme, membrane activity, protein sequence, Escherichia coli, RNA splicing, complementary DNA, gel electrophoresis, human tissue, molecular cloning, radiotracer, scintillation spectrometry, tissue /cell culture, yeast
Project start date: 1978-12-01
Project end date: 1995-12-06
5R01GM026020-17 (1995): $346576
5R01GM026020-16 (1994): $329034
CONTROL OF EUCARYOTIC MEMBRANE FUNCTION BY METHYLATION
Steven G Clarke, Professor
University Of California Los Angeles Office Of Research Administration Los Angeles, Ca 90095
Grant 5R01GM026020-15 from National Institute Of General Medical Sciences IRG: PC
Abstract: The objective of this work is to understand the physiological role of enzymatic protein carboxyl methylation reactions in human tissues and other cells. We propose to continue our studies of an L-isoaspartyl/D-aspartyl methyltransferase (E.C. 2.1.1.77) that catalyzes the modification of damaged proteins containing these unusual residues. This enzyme is found in the cytosolic fraction of all mammalian tissues examined and recognizes the altered residues that result from spontaneous racemization, isomerization, and deamidation of normal L-aspartyl and L-asparaginyl residues in aged proteins. In model systems, the formation of methyl esters at L-isoaspartyl residues can lead to their conversion to L-aspartyl residues and suggests that this enzyme functions to repair certain types of covalent damage to intracellular proteins and limit their accumulation in aging cells. We will use a combination of biochemical and molecular biological techniques in in vitro and in intact cell systems, focusing particularly on human erythrocytes. Since the structure of this enzyme has been remarkably well conserved from bacteria to human cells, we will also utilize genetically-manipulatable microbial model systems. This methyltransferase may represent one of the first members of a class of enzymes that can check spontaneous damage to cellular proteins, and its disruption in pathological conditions may both decrease their useful lifetime and contribute to accelerated aging processes. our recent discovery of a new class of protein carboxyl methyltransferases suggests that these enzymes may also have roles in the regulation of cellular signalling reactions. Proteins that contain a C-terminal tetrapeptide sequence are modified by reactions that form C-terminal S-isoprenylated cysteine methyl esters. These proteins include the ras oncogene products as well as other small G-proteins, subunits of the large G-proteins, and nuclear lamin components. The isoprenylation and methylation of these proteins have been postulated to lead to both membrane association and to the control of their signalling activities. We will purify and characterize the enzymes involved in these reactions from both mammalian tissues and yeast. The ability to pharmacologically control these modification enzymes, and thus signalling proteins involved in cell division, may represent new therapies for cancer and other diseases.
Keywords: carboxymethylation, membrane protein, methylation, methyltransferase, protein metabolism, S adenosylmethionine, aging, aspartate, biological signal transduction, enzyme mechanism, enzyme substrate, erythrocyte, erythrocyte membrane, isozyme, membrane activity, protein sequence, Escherichia coli, RNA splicing, complementary DNA, gel electrophoresis, human tissue, molecular cloning, radiotracer, scintillation spectrometry, tissue /cell culture, yeast
Project start date: 1978-12-01
Project end date: 1995-11-30
5R01GM026020-15 (1993): $323845
CONTROL OF EUKARYOTIC MEMBRANE FUNCTION BY METHYLATION
Steven G Clarke, Professor
University Of California Los Angeles Office Of Research Administration Los Angeles, Ca 90095
Grant 5R01GM026020-21 from National Institute Of General Medical Sciences IRG: PC
Abstract: The objective of this work is to understand the physiological role of enzymatic protein carboxyl methylation reactions in human tissues and other cells. We propose to continue our studies of an L-isoaspartyl/D-aspartyl methyltransferase (E.C. 2.1.1.77) that catalyzes the modification of damaged proteins containing these unusual residues. This enzyme is found in the cytosolic fraction of all mammalian tissues examined and recognizes the altered residues that result from spontaneous racemization, isomerization, and deamidation of normal L-aspartyl and L- asparaginyl residues in aged proteins. In model systems, the formation of methyl esters at L-isoaspartyl residues can lead to their conversion to L-aspartyl residues and suggests that this enzyme functions to repair certain types of covalent damage to intracellular proteins and limit their accumulation in aging cells. We will use a combination of biochemical and molecular biological techniques to characterize the human gene for this enzyme and its polymorphisms, to delineate the three- dimensional structure of the human enzyme, and to analyze residues responsible for binding and methylation of damaged protein substrates. We are also interested in examining the effects of enzyme overproduction and underproduction in transgenic mice. Since the structure of this enzyme has been remarkably well conserved from bacteria to human cells, we will also utilize genetically-manipulatable model systems, including bacteria and the nematode warm Caenorhabditis elegans. This methyltransferase may represent one of the first members of a class of enzymes that can check spontaneous damage to cellular proteins, and its disruption in pathological conditions may both decrease their useful lifetime and contribute to accelerated aging processes. Our recent discovery of a new class of protein carboxyl methyltransferase suggests that these enzymes may also have roles in the regulation of cellular metabolism including cell cycle control in tumor formation. In 1993, we found that one of the major cellular protein phosphatases (2A), which plays an essential role in modulating enzyme activity by reversing the action of various protein kinases, is a specific substrate for a novel C-terminal leucine methyltransferase. In this grant period, we will continue our investigation of this C-terminal leucine methylation reaction. We will purify and characterize the enzymes involved in these reactions from both mammalian tissues and yeast, and characterize potential demethylases as well. The ability to pharmacologically control these modification enzymes may represent new therapies for cancer and other diseases. Finally, we are interested in searching for additional new types of protein carboxyl methyltransferases in cells that may play unique roles in metabolism or signaling reactions.
Keywords: carboxymethylation, membrane protein, methylation, methyltransferase, protein metabolism, aging, aspartate, enzyme mechanism, enzyme substrate, genetic polymorphism, phosphoprotein phosphatase, protein sequence, protein structure /function, Caenorhabditis elegans, X ray crystallography, gel electrophoresis, human genetic material tag, human tissue, laboratory mouse, laboratory rat, molecular cloning, radiotracer, tissue /cell culture, transgenic animal
Project start date: 1978-12-01
Project end date: 1999-12-09
5R01GM026020-21 (1999): $376122
5R01GM026020-20 (1998): $361964
5R01GM026020-19 (1997): $348043
Sponsored Links Excellgen http://Excellgen.com
CONTROL OF EUCARYOTIC MEMBRANE FUNCTION BY METHYLATION
Steven G Clarke, Professor
University Of California Los Angeles Office Of Research Administration Los Angeles, Ca 90095
Grant 5R01GM026020-13 from National Institute Of General Medical Sciences IRG: PC
Abstract: The objective of this proposal is to understand the physiological significance of an enzymatic methylation reaction which appears to be a step in the metabolism of altered intracellular proteins. This ubiquitous enzymatic activity catalyzes the S- adenosylmethionine-dependent methyl esterification of isomerized and racemized aspartic acid residues in a wide variety of membrane and cytosolic proteins. Normally functional L- aspartyl and L-asparaginyl residues can be altered by spontaneous aging and/or damage reactions to form D-aspartyl residues and L- isoaspartyl transpeptidation products. Proteins containing these latter residues may not be expected to be fully functional, and we are interested in pursuing the possibility that the enzymatic methylation of these proteins may lead to their repair and/or degradation. Since the chemical deterioration, replacement, or repair of various macromolecules is likely to be a major determinant in the aging process of an organism, understanding the role of these reactions may contribute significantly to our concepts of the stability of cellular physiological systems over extended periods of time. We propose to concentrate our efforts on the methylation of abnormal proteins in the human erythrocyte system, where the absence of protein synthesis simplifies the experimental design. Although previous work has shown that similar methylation reactions occur in other mammalian tissues, differences in the metabolism of the methylated proteins may exist and we will be interested in comparing these reactions in non-erythroid tissues. Our specific goals in this project period include furthering our understanding of the enzymology of protein carboxyl methyltransferases, characterizing the specific sites of methylation on membrane and cytosolic proteins in the cell, and delineating the various possible pathways for the metabolism of methylated proteins. We will continue to use synthetic peptides as models for the methylated and demethylation reactions. Our long term goal will be to understand the functional role of protein covalent modification reactions that are involved in the repair and/or specific degradation of cellular proteins.
Keywords: carboxymethylation, membrane protein, methylation, methyltransferase, protein metabolism, S adenosylmethionine, aspartate, carboxyltransferase, cell growth regulation, cytoskeleton, enzyme mechanism, enzyme substrate, erythrocyte, erythrocyte membrane, membrane permeability, molecular site, peptide chemical synthesis, protein biosynthesis, protein structure, Escherichia coli, gel electrophoresis, human tissue from nonrelated source, radiotracer, sickle cell anemia, tissue /cell culture
Project start date: 1978-12-01
Project end date: 1991-11-30
Grants awarded to Steven G Clarke
CONTROL OF EUCARYOTIC FUNCTION BY METHYLATION
Steven G Clarke, Professor
University Of California Los Angeles, Office Of Research Administration, Los Angeles, Ca 90095
Grant 2R01GM026020-32 from National Institute Of General Medical Sciences
Abstract: We will investigate how physiological functions are controlled by methylation reactions, focusing on the enzymes that catalyze the modification of protein arginine, lysine, and isoaspartyl residues. We are especially interested in methyltransferases that are involved in signal transduction and metabolic regulatory pathways where the presence or absence of the methyl group can modulate the function of the methyl-accepting species. These reactions are important in protecting organisms from environmental stresses and from the accumulation of spontaneous damage in aging cells. We will also develop methods to identify new types of methyltransferases that may catalyze previously unrecognized reactions in these pathways. We will determine the enzymology and functional roles of new members of the eucaryotic family of protein arginine methyltransferases (PRMTs). These enzymes alter the ability of the arginine residue to interact with RNA, DNA, and protein partners and have been shown to have roles in gene regulation, DNA repair, and intracellular signaling pathways. We will focus our work on mammalian systems, but will also use yeast and trypanosomes as model systems. We will pay special attention to the human PRMT7 protein that has been implicated in tumor formation and stem cell survival. Our overall goal is to establish the complete cast of characters of the enzymes that catalyze these modifications in nature so that their functions, especially in signaling and gene regulation in health and disease, can be fully understood. We will characterize new roles in intracellular signaling for the protein repair L-isoaspartyl/D-aspartyl methyltransferase. Here, we will utilize both mouse and worm (Caenorhabditis elegans) systems to explore the relationships between the accumulation of age-damaged proteins, their recognition by the protein repair methyltransferase, and the responses of the insulin/insulin-like signaling system to increase stress resistance and longevity. We will test several hypotheses to explain the physiological role of the linkage, including direct recognition of damaged proteins or methylated proteins by either the signaling pathway itself or a transcriptional system that upregulates one or more crucial members of the signaling pathway. We will determine the role of lysine protein methylation reactions in the translational apparatus, including ribosomal proteins and elongation factors. We will work in both yeast and mammalian cells to understand how the modifications of ribosomal proteins and eEF1A contribute to translational control and resistance to environmental toxins. Finally, we will use bioinformatic and biochemical approaches to search for and to characterize new types of methyltransferases in both yeast and humans. We are especially interested in identifying potential novel sites of methyltransferase inhibition associated with elevated plasma homocysteine levels in humans that have been linked to cardiovascular and neurological diseases. Our work focuses on how the modification of proteins by the addition of methyl groups can control body function, including pathways of signal transduction in metabolism and macromolecular assembly. We are also interested in how these pathways are altered in cancer and diseases associated with aging
Keywords: 0-11 years old; 2-amino-4-mercaptobutyric acid; 4q31; Ademetionine; Adenosine, 5`-((3-amino-3-carboxypropyl)methylsulfonio)-5`-deoxy-, inner salt, (3S)-; AdoMet; Age; Aging; Amino Acyl T RNA; Area; Arg-Lys; Arginine; Arginine Methylase; Arginine, L-Isomer; Attention; Bacteria; Bio-Informatics; Biochemical; Bioinformatics; Biological Models; Biology; Blood Plasma; Buffaloes; Butanoic acid, 4, 4`-dithiobis(2-amino)-; C elegans; C.elegans; Caenorhabditis elegans; Cancers; Cardiovascular; Cardiovascular Body System; Cardiovascular system; Cardiovascular system (all sites); Cell Aging; Cell Communication and Signaling; Cell Senescence; Cell Signaling; Cell Survival; Cell Viability; Cells; Cellular Aging; Child; Child Youth; Children (0-21); Collaborations; Complex; Coupled; D-Aspartyl-L-Isoaspartyl Methyltransferase; DNA; DNA Damage Repair; DNA Repair; Deoxyribonucleic Acid; Disease; Disorder; EC 2.1.1; Elongation Factor; Environmental Toxin; Enzymatic Biochemistry; Enzymes; Enzymology; Esterification; Eukaryote; Eukaryotic Cell; Event; Factor, Elongation; Family; Gene Action Regulation; Gene Expression; Gene Expression Regulation; Gene Regulation; Gene Regulation Process; Goals; Grant; Health; Histone (Arginine) Methyltransferase; Histones; Homocysteine; Homocysteine, L-Isomer; Homocystine; Human; Human, Child; Human, General; Humulin R; Insulin; Insulin (ox), 8A-L-threonine-10A-L-isoleucine-30B-L-threonine-; Insulin, Regular; Intermediary Metabolism; Intracellular Communication and Signaling; Intracellular Signaling Proteins; Isoaspartyl-Aspartyl Protein Methyltransferase; Knock-out; Knockout; L-Arginine; L-Isoaspartyl Protein Carboxymethyltransferase; L-Lysine; Laboratories; Length of Life; Linguistic; Linguistics; Link; Longevity; Lysine; METBL; Malignant Neoplasms; Malignant Tumor; Mammalia; Mammalian Cell; Mammals; Mammals, General; Mammals, Mice; Man (Taxonomy); Man, Modern; Maps; Mass Spectrum; Mass Spectrum Analysis; Metabolic; Metabolic Processes; Metabolism; Methods; Methylation; Methyltransferase; Methyltransferase PIMT; Mice; Mice, Transgenic; Model System; Models, Biologic; Modification; Mother Cells; Murine; Mus; Myelin Basic Protein (Arginine) Methyltransferase; Nature; Nerve Cells; Nerve Unit; Nervous System Diseases; Neural Cell; Neurocyte; Neurologic Disorders; Neurological Disorders; Neurons; Novolin R; Organ System, Cardiovascular; Organism; PCMT1 Gene Product; Pathology; Pathway interactions; Peptide Biosynthesis, Ribosomal; Photometry/Spectrum Analysis, Mass; Physiologic; Physiological; Plasma; Post-Translational Modifications; Post-Translational Protein Processing; Posttranslational Modifications; Process; Progenitor Cells; Protein Arginine Methyltransferase; Protein Biosynthesis; Protein Biosynthesis, Ribosomal; Protein D-Aspartate-L-Isoaspartate Methyltransferase; Protein L-Isoaspartate O-Methyltransferase; Protein L-Isoaspartyl Methyltransferase; Protein Methylase I; Protein Methylation; Protein Methyltransferase I; Protein Modification; Protein Modification, Post-Translational; Protein Processing, Post-Translational; Protein Processing, Posttranslational; Protein Synthesis, Ribosomal; Protein-Arginine N-Methyltransferase; Protein-D-Asp Methyltransferase; Protein-D-Aspartate Methyltransferase; Protein-L-Isoaspartate Methyltransferase; Protein-L-Isoaspartate-D-Aspartate-O-Methyltransferase; Protein-beta-Aspartate Methyltransferase; Protein/Amino Acid Biochemistry, Post-Translational Modification; Proteins; RNA, Transfer, Amino Acyl; RNA-Protein Interaction; Reaction; Regulation; Regulatory Pathway; Research; Resistance; Reticuloendothelial System, Serum, Plasma; Ribonucleic acids, transfer; Ribosomal Proteins; Ribosomes; Role; S-Adenosyl-L-methionine[{..}]protein-L-arginine N-methyltransferase; S-Adenosylmethionine; SAMe; Senescence; Senescence, Cellular; Senescence, Replicative; Serum, Plasma; Side; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Signal Transduction Systems; Signaling; Signaling Proteins, Intracellular; Site; Specificity; Spectrometry, Mass; Spectroscopy, Mass; Spectrum Analyses, Mass; Spectrum Analysis, Mass; Stem cells; Stress; Sum; System; System, LOINC Axis 4; Testing; Toxic Environmental Agents; Toxic Environmental Substances; Transfer RNA; Transgenic Mice; Translations; Triplet Codon-Amino Acid Adaptor; Trypanosoma; Trypanosome; Unscheduled DNA Synthesis; Vascular, Heart; Work; Yeasts; aminoacyl tRNA; arginine-lysine; arginyllysine; biological signal transduction; cardiovascular risk; cardiovascular risk factor; cell age; children; circulatory system; college; disease/disorder; environmental toxicant; eukaryotida; gene product; interest; life span; lifespan; living system; macromolecular assembly; malignancy; man; man`s; member; methyl group; methylase; neoplasm/cancer; nervous system disorder; neurological disease; neuronal; novel; overexpression; pathway; pcm Gene Product; protein synthesis; public health relevance; repair; repaired; resistant; response; s-adenosyl-l-methionine; senescent; social role; tRNA; tRNA-Amino Acyl; transmethylase; tumor; yeast protein; youngster
Relevance: Our work focuses on how the modification of proteins by the addition of methyl groups can control body function, including pathways of signal transduction in metabolism and macromolecular assembly. We are also interested in how these pathways are altered in cancer and diseases associated with aging
Project start date: 1978-12-01
Project end date: 2014-01-31
Budget start date: 1-APR-2010
Budget end date: 31-JAN-2011
PFA/PA: PA-07-070
2R01GM026020-32 (2010): $531468
Research Training In Cellular And Molecular Biology
Steven G Clarke, Professor
University Of California Los Angeles Office Of Research Administration Los Angeles, Ca 90095
Grant 5T32GM007185-33 from National Institute Of General Medical Sciences IRG: BRT
Abstract: This proposal seeks renewal for UCLA s predoctoral Cellular and Molecular Biology Training Program. Now in its 30th year, the program has trained 414 Ph.D. candidates for careers in biomedical research and education. These students have gone on to make discoveries that have improved human health and we expect that future trainees will also follow in their footsteps with the training received here. The program is interdepartmental and includes faculty and students in both the UCLA College and the School of Medicine, including the Departments of Chemistry and Biochemistry; Molecular, Cell and Developmental Biology; Microbiology, Immunology and Molecular Genetics; Biological Chemistry; and Neurobiology, as well as, the Interdepartmental Program in Molecular Biology. There are currently 40 training faculty with primary appointments in nine departments, including those listed above and Pathology and Laboratory Medicine; Physiological Sciences; Medicine; and Urology. Major research areas include biochemistry, cell biology, developmental biology, gene expression, macromolecular structure, molecular and cellular immunology, neurobiology, virology and microbial pathology. Trainees are self-nominated from each of the six major Ph.D. programs involved. Students are selected on a highly competitive basis using the criteria of their academic and research achievements. Appointments are made for a maximum period of three years. A small number of students enter the Training Program as first year Ph.D. students; the majority, however, come in after a year of graduate work when they have chosen their dissertation research director. In addition to meeting the University and Departmental or Program requirements for their degrees, trainees participate in a research ethics course, as well as in seminars and conferences organized by the Training Program. This proposal requests stipends for 45 trainees. The facilities are those of a major undergraduate and graduate campus of approximately 33,000 students that includes a medical school faculty. All of the departments involved in this program are in close proximity to each other and to the interdepartmental Molecular Biology Institute, which facilitates interactions of both students and faculty.
Project start date: 1975-07-01
Project end date: 2011-06-30
5T32GM007185-33 (2007): $1126704
2T32GM007185-32 (2006): $1018632
5T32GM007185-31 (2005): $1015072
5T32GM007185-30 (2004): $1048592
5T32GM007185-29 (2003): $1018200
5T32GM007185-28 (2002): $917769
5T32GM007185-26 (2000): $782946
5T32GM007185-25 (1999): $762546
5T32GM007185-24 (1998): $630438
Sponsored Links Excellgen http://Excellgen.com
5T32GM007185-36 (2010): $1120272
5T32GM007185-35 (2009): $1134209
5T32GM007185-34 (2008): $1126718
2T32GM007185-27 (2001): $879550
CONTROL OF EUCARYOTIC FUNCTION BY METHYLATION
Steven G Clarke, Professor
University Of California Los Angeles Office Of Research Administration Los Angeles, Ca 90095
Grant 5R37GM026020-26 from National Institute Of General Medical Sciences IRG: PC
Abstract: The objective of this work is to understand the physiological role of several related members of a family of S-adenosylmethionine-dependent methyltransferases in aging, metabolic control, and signal transduction. We will continue our work to characterize the protein L-isoaspartate (D- aspartate) O-methyltransferase that recognizes age-damaged proteins and catalyzes the initial step of a protein repair reaction. Seizures occur as a result of the loss of function of this enzyme in transgenic knockout mice. We will investigate the factors leading to the onset of seizures to help understand the mechanisms involved in human epilepsy and its potential control. We will compare the role of this protein repair enzyme to an enzyme that we have recently discovered (trans-aconitate methyltransferase) that recognizes a spontaneously formed inhibitor of the citric acid cycle in a potential detoxification reaction. We also propose to characterize members of an expanding family of protein arginine methyltransferases. These enzymes interact with signaling molecules such as the interferon receptor, the TIS21 protein and SH3-domain-containing proteins. We will now characterize these gene products to better understand the role of these enzymes in metabolic control, including a novel enzyme we have recently discovered that methylates the delta, or internal guanidino nitrogen atom, or arginine residues. Finally, we will examine the enzymes that catalyze the carboxyl methylation of an elongation factor in protein synthesis that may be regulated by a methylation/demethylation cycle. These enzymes all appear to be members of one revolutionarily related family with a probably common three dimensional structure. While one group of enzymes appears to function in reducing the accumulation of the potentially toxic products generated spontaneously during the aging process, the other group appears to regulate the cell s metabolism and its signal transduction pathways.
Keywords: carboxymethylation, enzyme activity, methylation, methyltransferase, protein metabolism, Krebs cycle, RNA binding protein, Saccharomyces cerevisiae, aging, aspartate, biological signal transduction, brain metabolism, detoxification, eukaryote, open reading frame, protein structure function, translation factor, gene targeting, human genetic material tag, human tissue, laboratory mouse, tissue /cell culture, transgenic animal
Project start date: 1978-12-01
Project end date: 2004-11-30
5R37GM026020-26 (2004): $451868
5R37GM026020-25 (2003): $445256
5R37GM026020-24 (2002): $438622
5R37GM026020-23 (2001): $431628
2R37GM026020-22 (2000): $438672
ENYZMES AFFECTING THE ACCUMULATION OF ALTERED PROTEINS
Steven G Clarke, Professor
Chemistry And Biochemistryuniversity Of California Los Angeles
office Of Research Administration
los Angeles, Ca 90095
Grant 5R01AG018000-03 from National Institute On Aging IRG: ZAG1
Abstract: Adapted ). The manifestations of aging result in part from cells becoming less efficient at self-repair with time. These reactions represent one part of the battle of organisms to maintain the structural integrity of essential macromolecules in the face of the molecules intrinsic instabilities. Defects in these mechanisms may underlie pathologies where the aging process can be accelerated. The objective of this work is to understand how aging organisms prevent the accumulation of covalently altered proteins that can compromise cellular functions. These investigators will characterize the role of the protein L-isoaspartate (D-aspartate) O-methyltransferase that recognizes spontaneously-damaged proteins and catalyzes the initial step of a protein repair reaction. The discovery of this pathway reveals that macromolecular repair may not be just for DNA, but for proteins as well. These investigators propose to ask how the potential accumulation of damaged proteins in aging is reduced by methylation and other pathways in viva. They will utilize model organisms including bacteria, yeast, worms, and plants. Specifically, we will characterize protein damage in the bacterium Escherichia coli. They will study mutant phenotypes of both the methyltransferase pcm gene and the sure gene shown to be present in an operon with pcm. They will also study the role of associated enzymes that are involved in the metabolism of isoaspartyl-containing proteins and peptides, including isoaspartyl dipeptidases. They will analyze mutants of the protein repair methyltransferase in the nematode worm Casnorhabditis elegant. They will ask how the yeast Saccharomyces cerevisiae can avoid the accumulation of proteins containing altered aspartyl residues in spite of the fact that it naturally lacks the methyltransferase. Finally, they will examine the role of the methylation reaction in controlling protein damage in higher plants, including corn and Arabidopsis. These studies will hopefully not only provide a new window to view protein life but may also suggest that the biological aging process may be closely linked to how well cells can keep polypeptides free of spontaneous covalent damage
Keywords: aging, aspartate, enzyme mechanism, methyltransferase, protein metabolism gene mutation Arabidopsis, Caenorhabditis elegans, Escherichia coli, Saccharomyces cerevisiae, grain
Project start date: 2000-06-15
Project end date: 2004-05-31
5R01AG018000-03 (2002): $225909
Sponsored Links Excellgen http://Excellgen.com
5R01AG018000-02 (2001): $225909
1R01AG018000-01 (2000): $225909
5R01AG018000-04 (2003): $225909
Linked Protein Repair, Proteolysis, And Oxidation In Aging
Steven G Clarke, Professor
Chemistry And Biochemistryuniversity Of California Los Angeles
office Of Research Administration
los Angeles, Ca 90095
Grant 1R21AG032303-01 from National Institute On Aging IRG: ZAG1
Abstract: A significant part of the loss of human function in aging may be due to the build-up of damaged proteins. Proteins, responsible for most of the catalytic and structural operations of the body, can spontaneously break down with time. As organisms age, proteins can accumulate enough chemical damage to become inactivated, or even toxic. The success of aging organisms may depend upon their ability to first recognize which proteins are damaged, and then to either repair or remove these species. In this proposal, we want to understand how organisms integrate protein repair and proteolytic pathways to stem the accumulation of damaged proteins. We are particularly interested in how a major type of spontaneous damage, the isomerization and racemization of protein aspartyl and asparaginyl residues, is minimized by a combination of molecular repair initiated by the L-isoaspartyl-(D-aspartyl) protein O- methyltransferase enzyme and specific proteolytic degradation reactions. We propose to use mice, yeast (Saccharomyces cerevisiae), and nematode worms (Caenorhabditis elegans) as model systems. Each of these systems has advantages to aid us in deciphering the pathways that may also be used in humans. We will first examine the links between protein repair and proteolysis pathways in mice. We will focus on pathways used in animals lacking the protein repair methyltransferase. We have previously established that the accumulation of damaged aspartyl residues in repair deficient mice levels off after about 60 days of age. At the same time, the levels of damaged peptides in the urine of the mice increases, suggesting that a proteolytic system to remove the unrepaired proteins is activated. We propose to characterize this back-up system and to find its role in the normal aging process. We will then examine the metabolism of proteins containing damaged aspartyl residues in the yeast S. cerevisiae that lacks the protein repair methyltransferase. We have shown that proteins containing damaged aspartyl residues do not accumulate in yeast, although they appear to be formed at the same rate as in other organisms. We thus propose that yeast have specific proteolytic systems to prevent the accumulation of these altered proteins and will characterize them by a combination of biochemical and genetic approaches. Finally, we will compare the repair/proteolysis responses of mice to those that occur in aging worms. Previous work in our laboratory has suggested that proteolysis may be coupled to protein repair in the nematode C. elegans. We will characterize worms deficient in the L-isoaspartyl methyltransferase, focusing on two larval stages of worms that are specialized for survival and that appear to be most affected in the absence of the repair enzyme. Similarities in repair, signaling, and proteolysis systems in worms and mice suggest that what we learn here will be important for human health. 7. PROJECT NARRATIVE We want to understand how human cells can perform molecular repair and replacement processes that contribute to healthy aging and how defects in these pathways lead to disease. Protein molecules essential for body functions are continuously being degraded by spontaneous chemical processes. Unless damaged molecules are repaired or replaced, their accumulation can slow or stop normal physiological functions
Project start date: 2008-08-15
Project end date: 2010-07-31
1R21AG032303-01 (2008): $210875
CONTROL OF EUCARYOTIC FUNCTION BY METHYLATION
Steven G Clarke
Department/ Educational Institution Type:
Grant 3R01GM026020-32S1 from National Institute Of General Medical Sciences
Keywords: 0-11 years old; 2-amino-4-mercaptobutyric acid; 4q31; Ademetionine; Adenosine, 5`-((3-amino-3-carboxypropyl)methylsulfonio)-5`-deoxy-, inner salt, (3S)-; AdoMet; Age; Aging; Amino Acyl T RNA; aminoacyl tRNA; Area; Arg-Lys; Arginine; Arginine Methylase; Arginine, L-Isomer; arginine-lysine; arginyllysine; Attention; Bacteria; Bio-Informatics; Biochemical; Bioinformatics; Biological Models; biological signal transduction; Biology; Blood Plasma; Buffaloes; Butanoic acid, 4, 4`-dithiobis(2-amino)-; C elegans; C.elegans; Caenorhabditis elegans; Cancers; Cardiovascular; Cardiovascular Body System; cardiovascular risk; cardiovascular risk factor; Cardiovascular system; Cardiovascular system (all sites); cell age; Cell Aging; Cell Communication and Signaling; Cell Senescence; Cell Signaling; Cell Survival; Cell Viability; Cells; Cellular Aging; Child; Child Youth; children; Children (0-21); circulatory system; Collaborations; college; Complex; Coupled; D-Aspartyl-L-Isoaspartyl Methyltransferase; Deoxyribonucleic Acid; Disease; disease/disorder; Disorder; DNA; DNA Damage Repair; DNA Repair; EC 2.1.1; Elongation Factor; environmental toxicant; Environmental Toxin; Enzymatic Biochemistry; Enzymes; Enzymology; Esterification; Eukaryote; Eukaryotic Cell; eukaryotida; Event; Family; Gene Action Regulation; Gene Expression; Gene Expression Regulation; gene product; Gene Regulation; Gene Regulation Process; Goals; Grant; Health; Histone (Arginine) Methyltransferase; Histones; Homocysteine; Homocysteine, L-Isomer; Homocystine; Human; Human, Child; Human, General; Humulin R; Insulin; Insulin (ox), 8A-L-threonine-10A-L-isoleucine-30B-L-threonine-; Insulin, Regular; interest; Intermediary Metabolism; Intracellular Communication and Signaling; Intracellular Signaling Proteins; Isoaspartyl-Aspartyl Protein Methyltransferase; Knock-out; Knockout; L-Arginine; L-Isoaspartyl Protein Carboxymethyltransferase; L-Lysine; Laboratories; Length of Life; life span; lifespan; Linguistic; Linguistics; Link; living system; Longevity; Lysine; macromolecular assembly; malignancy; Malignant Neoplasms; Malignant Tumor; Mammalia; Mammalian Cell; Mammals; Mammals, General; Mammals, Mice; man; Man (Taxonomy); man`s; Man, Modern; Maps; Mass Spectrum; Mass Spectrum Analysis; member; Metabolic; Metabolic Processes; Metabolism; METBL; Methods; methyl group; methylase; Methylation; Methyltransferase; Methyltransferase PIMT; Mice; Model System; Models, Biologic; Modification; Mother Cells; Murine; Mus; Myelin Basic Protein (Arginine) Methyltransferase; Nature; neoplasm/cancer; Nerve Cells; Nerve Unit; Nervous System Diseases; nervous system disorder; Neural Cell; Neurocyte; Neurologic Disorders; neurological disease; Neurological Disorders; neuronal; Neurons; novel; Novolin R; Organ System, Cardiovascular; Organism; overexpression; Pathology; pathway; Pathway interactions; pcm Gene Product; PCMT1 Gene Product; Peptide Biosynthesis, Ribosomal; Photometry/Spectrum Analysis, Mass; Physiologic; Physiological; Plasma; Post-Translational Modifications; Post-Translational Protein Processing; Posttranslational Modifications; Process; Progenitor Cells; Protein Arginine Methyltransferase; Protein Biosynthesis; Protein Biosynthesis, Ribosomal; Protein D-Aspartate-L-Isoaspartate Methyltransferase; Protein L-Isoaspartate O-Methyltransferase; Protein L-Isoaspartyl Methyltransferase; Protein Methylase I; Protein Methylation; Protein Methyltransferase I; Protein Modification; Protein Modification, Post-Translational; Protein Processing, Post-Translational; Protein Processing, Posttranslational; protein synthesis; Protein Synthesis, Ribosomal; Protein-Arginine N-Methyltransferase; Protein-beta-Aspartate Methyltransferase; Protein-D-Asp Methyltransferase; Protein-D-Aspartate Methyltransferase; Protein-L-Isoaspartate Methyltransferase; Protein-L-Isoaspartate-D-Aspartate-O-Methyltransferase; Protein/Amino Acid Biochemistry, Post-Translational Modification; Proteins; public health relevance; Reaction; Regulation; Regulatory Pathway; repair; repaired; Research; Resistance; resistant; response; Reticuloendothelial System, Serum, Plasma; Ribonucleic acids, transfer; Ribosomal Proteins; Ribosomes; RNA, Transfer, Amino Acyl; RNA-Protein Interaction; Role; s-adenosyl-l-methionine; S-Adenosyl-L-methionine[{..}]protein-L-arginine N-methyltransferase; S-Adenosylmethionine; SAMe; Senescence; Senescence, Cellular; Senescence, Replicative; senescent; Serum, Plasma; Side; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Signal Transduction Systems; Signaling; Site; social role; Specificity; Spectrometry, Mass; Spectroscopy, Mass; Spectrum Analyses, Mass; Spectrum Analysis, Mass; Stem cells; Stress; Sum; System; System, LOINC Axis 4; Testing; Toxic Environmental Agents; Toxic Environmental Substances; Transfer RNA; Transgenic Mice; Translations; transmethylase; Triplet Codon-Amino Acid Adaptor; tRNA; tRNA-Amino Acyl; Trypanosoma; Trypanosome; tumor; Unscheduled DNA Synthesis; Vascular, Heart; Work; yeast protein; Yeasts; youngster
Relevance: Our work focuses on how the modification of proteins by the addition of methyl groups can control body function, including pathways of signal transduction in metabolism and macromolecular assembly. We are also interested in how these pathways are altered in cancer and diseases associated with aging
Project start date: 1978-12-01
Project end date: 2014-01-31
Budget start date: 1-APR-2010
Budget end date: 31-JAN-2011
PFA/PA: PA-07-070
3R01GM026020-32S1 (2011): $18910
5R37GM026020-31 (2009): $569432
5R37GM026020-29 (2007): $557757
5R37GM026020-28 (2006): $581907
3R37GM026020-27S1 (2005): $17099
4R37GM026020-27 (2005): $543973
Sponsored Links Excellgen http://Excellgen.com
RESEARCH TRAINING IN CELLULAR AND MOLECULAR BIOLOGY
Steven G Clarke, Professor
Molecular Biology Instituteuniversity Of California Los Angeles
office Of Research Administration
los Angeles, Ca 90095
Grant 5T32GM007185-23 from National Institute Of General Medical Sciences IRG: ZGM1
Project start date: 1975-07-01
Project end date: 2001-06-30
5T32GM007185-23 (1997): $623549
CONTROL OF EUKARYOTIC MEMBRANE FUNCTION BY METHYLATION
Steven G Clarke, Professor
University Of California Los Angeles Office Of Research Administration Los Angeles, Ca 90095
Grant 2R01GM026020-18 from National Institute Of General Medical Sciences IRG: PC
Project start date: 1978-12-01
Project end date: 1999-11-30
2R01GM026020-18 (1996): $334658
RESEARCH TRAINING IN CELLULAR AND MOLECULAR BIOLOGY
Steven G Clarke, Professor
University Of California Los Angeles Office Of Research Administration Los Angeles, Ca 90095
Grant 2T32GM007185-22 from National Institute Of General Medical Sciences IRG: ZGM1
Project start date: 1975-07-01
Project end date: 2001-06-30
2T32GM007185-22 (1996): $585642
5T32GM007185-21 (1995): $506733
5T32GM007185-20 (1994): $534590
5T32GM007185-19 (1993): $491127
CELLULAR AND MOLECULAR BIOLOGY
Steven G Clarke, Professor
University Of California Los Angeles Office Of Research Administration Los Angeles, Ca 90095
Grant 5T32GM007185-18 from National Institute Of General Medical Sciences IRG: CMBD
Project start date: 1975-07-01
Project end date: 1996-06-30
5T32GM007185-18 (1992): $502848
Sponsored Links Excellgen http://Excellgen.com