John D Hildebrandt
Medical University Of South Carolina
Project start date: 2009-04-01
Project end date: 2013-01-31
Sponsored Links Excellgen http://Excellgen.com
GENERATION AND FUNCTION OF VARIABLE PRENYL PROTEIN PROCESSING
John D Hildebrandt, Professor
Medical University Of South Carolina, 19 Hagood Ave., Suite 606, Charleston, Sc 29425
Grant 5R01GM082981-02 from National Institute Of General Medical Sciences
Abstract: About 2% of proteins in the human genome are predicted to be modified by prenylation. These include a broad range of substrates, but prominent among them are many key proteins involved in cell signaling, growth regulation, cell progression and differentiation; for example these include the ras proto-oncogene products and the 3 subunits of the heterotrimeric G proteins. The constituent reactions of this processing pathway are the targets of both currently used and investigational anticancer drugs. In addition, one class of target proteins for this modification, the 3 subunits of the heterotrimeric G proteins, are key constituents of the mediators of about half of all clinically useful drugs. Thus the physiological and pathological function of this processing pathway is closely related to the actions of a wide range of therapeutics from those involved in treatment of cardiovascular diseases to those involved in cancer chemotherapy. Prenylation involves multiple enzymatic steps. Following prenylation, the protein is typically proteolytically processed to remove the last 3 amino acids and the new C- terminus is carboxymethylated. We have characterized the heterogeneity of the prenylation of the heterotrimeric G protein 3 subunits and have found biologically significant variation in all three enzymatic reactions of this pathway. To characterize the functional significance of this variation we are concentrating on a specific subset of variably processed proteins (exemplified by G35 in humans) that are processed by prenylation but not by the subsequent reactions in this complex pathway. We are concentrating on this variant because its processing pattern is sequence dependent. About 10% of prenylated proteins have similar sequence determinants. Using G35 as a model protein we will elucidate the functional significance of this variable processing pattern by testing the hypothesis that G35 mediates unique signaling events in cells (Aim 1), by testing the hypothesis that differential proteolysis of prenylated G3 subunits determines their intracellular trafficking and functional site of action (Aim 2) and by identifying and validating protein-protein interactions related to the unique functions of G35 in cells (Aim 3). Finally, we will evaluate the general role of the variant prenyl processing signal in other predicted proteins from the human genome (Aim 4). These studies will extend the known diversity of the prenyl processing reactions and define the functional significance of this variation. The results of this work will have significance for the role of prenyl processing in cells, for how variation of prenyl processing is related to protein function, for the general mechanisms for signaling through heterotrimeric G proteins, for the actions of the large number of therapeutics that exert their effects through heterotrimeric G proteins, and for the mechanism, consequences and utility of anti-cancer drugs that specifically or coincidently target the protein prenylation reactions. The proposed project studies how a particular kind of lipid molecule referred to as a prenyl group when attached to proteins in cells alters their structure and function. The enzymes that add prenyl groups to proteins and the modified proteins themselves are targets for many different kinds of drugs including those used to treat heart diseases and cancer. The proposed work will increase our understanding of how these drugs work and will help us develop more effective drugs to treat these common illnesses
Keywords: 4-Amino-10-methylfolic Acid; 4-Amino-4-deoxy-10-methylpteroyl-L-glutamic Acid; Affect; Amino Acids; Anti-Cancer Agents; Anti-Tumor Agents; Anti-Tumor Drugs; Antineoplastic Agents; Antineoplastic Drugs; Antineoplastics; Antiproliferative Agents; Antiproliferative Drugs; Belief; Biochemical; Biochemical Reaction; Biochemistry; C-terminal; Cancer Drug; Cancers; Cardiac Diseases; Cardiac Disorders; Cardiovascular Diseases; Cell Communication and Signaling; Cell Signaling; Cells; Cellular Oncogene; Cellular Regulation; Chemistry, Biological; Chemotherapeutic Agents, Neoplastic Disease; Chemotherapy Protocol; Chemotherapy Regimen; Chemotherapy, Cancer, General; Chemotherapy-Oncologic Procedure; Classification; Combination Chemotherapy Regimen; Complex; Drug Delivery; Drug Delivery Systems; Drug Targeting; Drug Targetings; Drugs; EC 2; Enzymatic Biochemistry; Enzymatic Reaction; Enzymes; Enzymology; Esteroproteases; Event; Future; G-Proteins; GTP-Binding Proteins; GTP-Regulatory Proteins; Generalized Growth; Generations; Genome, Human; Growth; Guanine Nucleotide Coupling Protein; Guanine Nucleotide Regulatory Proteins; Heart Diseases; Heterogeneity; Heterotrimeric G Protein Subunit; Heterotrimeric G-Proteins; Heterotrimeric GTP-Binding Proteins; Human; Human Genome; Human, General; Intracellular Communication and Signaling; L-Glutamic acid, N-(4-(((2, 4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-; Lipids; Malignant Neoplasms; Malignant Tumor; Man (Taxonomy); Man, Modern; Mediating; Mediator; Mediator of Activation; Mediator of activation protein; Medication; Methotrexate; Methotrexate Methylaminopterin; Methotrexatum; Metotrexato; Modeling; Modification; Mutagenesis, Site-Directed; Oncogene Products; Oncogene Proteins; Oncoproteins; Pathway interactions; Pattern; Peptidases; Peptide Hydrolases; Pharmaceutic Preparations; Pharmaceutical Preparations; Physiologic; Physiological; Post-Translational Isoprenylation; Post-Translational Modifications; Post-Translational Protein Processing; Posttranslational Modifications; Process; Property; Property, LOINC Axis 2; Proteases; Protein Cleavage; Protein Isoprenylation; Protein Modification; Protein Modification, Post-Translational; Protein Prenylation; Protein Processing, Post-Translational; Protein Processing, Posttranslational; Protein/Amino Acid Biochemistry, Post-Translational Modification; Proteinases; Proteins; Proteolysis; Proteolytic Clipping; Proteolytic Enzymes; Proteolytic Processing; Proteomics; Proto-Oncogenes; Publishing; Quimioterapia; Reaction; Role; Series; Signal Transduction; Signal Transduction Systems; Signaling; Site; Site-Directed Mutagenesis; Site-Specific Mutagenesis; Structure; Systematics; Targeted DNA Modification; Targeted Modification; Testing; Therapeutic; Tissue Growth; Transferase; Treatment Efficacy; Tumor-Specific Treatment Agents; Variant; Variation; Work; aminoacid; anticancer agent; anticancer drug; biological signal transduction; c-ONC; cancer chemotherapy; carboxymethylation; cardiovascular disorder; cell growth regulation; computerized data processing; data processing; design; designing; drug development; drug/agent; gene product; heart disorder; inhibitor; inhibitor/antagonist; malignancy; neoplasm/cancer; ontogeny; pathway; prenyl; prenylation; protein function; protein protein interaction; protooncogene; rho; signal processing; social role; therapeutic efficacy; therapeutically effective; trafficking
Relevance: The proposed project studies how a particular kind of lipid molecule referred to as a prenyl group when attached to proteins in cells alters their structure and function. The enzymes that add prenyl groups to proteins and the modified proteins themselves are targets for many different kinds of drugs including those used to treat heart diseases and cancer. The proposed work will increase our understanding of how these drugs work and will help us develop more effective drugs to treat these common illnesses
Project start date: 2009-04-01
Project end date: 2013-01-31
Budget start date: 1-FEB-2010
Budget end date: 31-JAN-2011
PFA/PA: PA-07-070
5R01GM082981-02 (2010): $292050
Grants awarded to John D Hildebrandt
TRAINING TO IMPROVE CARDIOVASCULAR DRUG THERAPY
John D Hildebrandt, Professor
Medical University Of South Carolina 171 Ashley Ave Charleston, Sc 29425
Grant 2T32HL007260-26 from National Heart, Lung, And Blood Institute IRG: ZHL1
Abstract: The program objective is to train promising new scientists in mechanisms of cardiovascular diseases, experimental strategies and technologies necessary for development of novel molecular therapeutics. The theme of the program is signal transduction within a multidisciplinary, integrated program that focuses on events involved in cardiac and vascular development, hypertension, genes/proteins involved in myocardial hypertrophy and failure, conduction abnormalities, structure and function of signaling molecules and signaling events in vascular smooth muscle, cardiac myocytes and conducting tissue. Training resources include molecular genetics with transgenic and knockout facilities, microscopy, image analysis, mass spectrometry and applied chemistry. Guidelines for entering predoctoral trainees include baccalaureate degree, GRE scores, letters of recommendation with prior research experience preferred. Guidelines for entering postdoctoral trainees include the Ph.D. and/or M.D. degrees; prior research experience and letters of recommendation. We value trainees with diverse backgrounds. Financial support is requested for 2, 4, 6, 6, 6, predoctoral trainees and 8 postdoctoral trainees/year for years 26-30. Training participants are faculty in Pharmacology, Medicine (Cardiology, Endocrinology, Rheumatology), Cell Biology and Anatomy, Biochemistry, Physiology and Surgery. Predoctoral trainees enter the Biomedical Sciences First Year Curriculum and then join the Cardiovascular Biology track in their second year as part of the Cell and Molecular Pharmacology and Experimental Therapeutics Ph.D program or the Cell and Molecular Biology and Pathobiology Ph.D. program. Thereafter, they take advanced courses in cardiovascular biology and those required by their individual Ph.D. program and carry out their dissertation research. Postdoctoral training emphasizes laboratory investigation with access to basic and advanced courses. All predoctoral and postdoctoral trainees meet with visiting scientists, present at local seminars and national meetings, and participate in joint journal clubs and seminars. Our aim is to produce outstanding investigators with a broad insight into cardiovascular dysfunction who will make significant contributions to the understanding of these disorders and development of therapies for cardiovascular diseases.
Project start date: 1977-07-01
Project end date: 2007-06-30
2T32HL007260-26 (2002): $334128
FUNCTION OF MODIFIED BRAIN SIGNALING PROTEINS
John D Hildebrandt, Professor
Pharmacologymedical University Of South Carolina
office Of Research And Sponsored Programs
charleston, Sc 29425
Grant 5R01NS038534-02 from National Institute Of Neurological Disorders And Stroke IRG: ZRG1
Abstract: The primary goal of this project is to determine the role of a novel G protein alpha/o subunit in signaling mechanisms in the brain. G proteins mediate the effects of extracellular signals on intracellular signaling pathways. They are particularly important in brain where the effects of many neurotransmitters are mediated by G protein-coupled receptors. G/o is the most abundant heterotrimeric G protein in brain, comprising about 0.2%-1% of total particulate protein. There is a single mammalian gene for the alpha/o protein, which gives rise to multiple mRNAs encoding at least two different Goalpha proteins. However, at least three major Goalpha proteins have been purified from brain. Preliminary Results reported here show that the third major isoform (alpha/oc) is a mixture of two proteins derived from the protein expressed from the major alpha/o mRNA (alpha/o1), but modified at one or the other of two specific asparagine residues. These two asparagine residues are next to each other and near the C-terminus in a region of the protein involved in upstream receptor and downstream effector interactions. The proposed work will characterize the function, distribution and origin of these modified proteins based upon biochemical studies of purified and recombinant proteins. The Specific Aims are (1) To determine the functional differences between the Goalpha isoforms. (2) To determine the localization and distribution of the Goapha proteins in neural tissues, cell lines and subcellular fractions. (3) To determine the mechanism underlying the origin of the alpha/oc isoforms. These Specific Aims are designed to test the hypotheses that alpha/oa is a precursor of the alpha/oc proteins, and that the rate of production of alpha/oc is dependent upon the activity of the G protein signaling system, resulting in an altered signaling pathway with new properties as a result of the production of alpha/oc. Such a molecular mechanism affecting a major brain signaling protein would likely be an important component of memory, neural plasticity or other related neuroadaptive processes
Keywords: G protein, biological signal transduction, brain, protein isoform, protein structure /function RNA splicing, nerve /myelin protein, posttranslational modification, receptor coupling animal tissue, cell line, electron microscopy, laboratory rat, light microscopy
Project start date: 2000-03-10
Project end date: 2003-02-28
5R01NS038534-02 (2001): $214500
1R01NS038534-01A1 (2000): $214500
HORMONE-REGULATED GTP-BINDING PROTEINS
John D Hildebrandt, Professor
Medical University Of South Carolina 171 Ashley Ave Charleston, Sc 29425
Grant 5R01DK037219-09 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: PC
Abstract: The mechanisms for the interactions of a family of guanine nucleotide binding proteins (G proteins) which act as intermediaries between hormone receptors on the cell surface and their intracellular effector systems will be investigated. There are at least four distinct members of this family, most with multiple isoforms, that serve to coordinate the activities of at least six different intracellular regulatory pathways. These proteins mediate important hormone or hormone-like effects in essentially all mammalian cells. They all have a similar subunit composition and share one common subunit. This application proposes to investigate how this common subunit reversibly associates with the other subunits of the proteins and provides a mechanism by which they affect each others function, thereby coordinating intracellular regulatory pathways. There are five components of this project. (Specific Aim 1) The study of subunit interactions in intact membranes from s49 mouse lymphoma cells grown in culture. These studies will indicate the role of subunit dissociation in the behavior of proteins which have not been solubilized from membranes with detergents and subsequently purified away from other proteins which may modulate their function. (Specific Aim 2) The study of subunit dissociation of G proteins purified from bovine brain or human erythrocytes, or analogous recombinant proteins, in detergent solution; which will determine what regulates the interactions of the different proteins with their shared subunit. (Specific Aim 3) The study of the functional consequences of these reversible subunit interactions of purified G proteins in detergent solution, determined by examining the kinetics of GTP binding and hydrolysis to the purified proteins. (Specific Aim 4) The study of how these reversible subunit interactions affect a specific intracellular regulatory system in intact membranes from s49 cells, the CAMP-adenylyl cyclase system. (Specific Aim 5) The identification of the subunit binding sites on the proteins, determined primarily by isolating chemically-modified fragments of the proteins responsible for blocking subunit interactions. These studies will provide important structural information about these proteins and how their subunits interact.
Keywords: G protein, guanine nucleotide binding protein, hormone regulation /control mechanism, protein structure function, active site, adenylate cyclase, cyclic AMP, erythrocyte membrane, hormone receptor, intracellular transport, membrane protein, phospholipid, platelet, protein isoform, protein sequence, recombinant protein, second messenger, detergent, disc gel electrophoresis, guanosine triphosphate, high performance liquid chromatography, human tissue, laboratory rabbit, neoplastic cell culture for noncancer research, protein purification, solution
Project start date: 1991-07-01
Project end date: 1995-11-30
5R01DK037219-09 (1995): $223256
5R01DK037219-08 (1994): $214669
HORMONE REGULATED GTP-BINDING PROTEINS
John D Hildebrandt, Professor
Medical University Of South Carolina 171 Ashley Ave Charleston, Sc 29425
Grant 5R01DK037219-07 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: PC
Abstract: The mechanisms for the interactions of a family of guanine nucleotide binding proteins (G proteins) which act as intermediaries between hormone receptors on the cell surface and their intracellular effector systems will be investigated. There are at least four distinct members of this family, most with multiple isoforms, that serve to coordinate the activities of at least six different intracellular regulatory pathways. These proteins mediate important hormone or hormone-like effects in essentially all mammalian cells. They all have a similar subunit composition and share one common subunit. This application proposes to investigate how this common subunit reversibly associates with the other subunits of the proteins and provides a mechanism by which they affect each others function, thereby coordinating intracellular regulatory pathways. There are five components of this project. (Specific Aim 1) The study of subunit interactions in intact membranes from s49 mouse lymphoma cells grown in culture. These studies will indicate the role of subunit dissociation in the behavior of proteins which have not been solubilized from membranes with detergents and subsequently purified away from other proteins which may modulate their function. (Specific Aim 2) The study of subunit dissociation of G proteins purified from bovine brain or human erythrocytes, or analogous recombinant proteins, in detergent solution; which will determine what regulates the interactions of the different proteins with their shared subunit. (Specific Aim 3) The study of the functional consequences of these reversible subunit interactions of purified G proteins in detergent solution, determined by examining the kinetics of GTP binding and hydrolysis to the purified proteins. (Specific Aim 4) The study of how these reversible subunit interactions affect a specific intracellular regulatory system in intact membranes from s49 cells, the CAMP-adenylyl cyclase system. (Specific Aim 5) The identification of the subunit binding sites on the proteins, determined primarily by isolating chemically-modified fragments of the proteins responsible for blocking subunit interactions. These studies will provide important structural information about these proteins and how their subunits interact.
Keywords: G protein, guanine nucleotide binding protein, hormone regulation /control mechanism, protein structure function, active site, adenylate cyclase, cyclic AMP, erythrocyte membrane, hormone receptor, intracellular transport, membrane protein, phospholipid, platelet, protein isoform, protein sequence, recombinant protein, second messenger, detergent, disc gel electrophoresis, guanosine triphosphate, high performance liquid chromatography, human tissue, laboratory rabbit, neoplastic cell culture for noncancer research, protein purification, solution
Project start date: 1991-07-01
Project end date: 1995-11-30
5R01DK037219-07 (1993): $203546
5R01DK037219-06 (1992): $194729
HORMONE-REGULATED GTP-BINDING PROTEINS
John D Hildebrandt, Professor
Worcester Foundation For Biomedical Res Shrewsbury, Ma 01545
Grant 5R01DK037219-03 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: PC
Abstract: The consequences of the structural and functional homology of a family of guanine nucleotide binding proteins for their activation by hormone receptors will be investigated. These GTP binding proteins act as intermediaries between hormones receptor on the cell surface and intracellular second messenger systems. At least three different members of this family have been identified, and there are indications that there are additional such proteins. These proteins have a similar subunit composition and a number of common functional properties. There are at least four different second messenger functions affected by these proteins. (Specific Aim 1) Experiments will attempt to identify additional members of this family in various rat and bovine tissues based upon the common properties of the already identified proteins. Small quantities of new proteins will be purified and compared structurally and functionally with the other members of this family. (Specific Aim 2) The interactions of newly discovered and/or previously identified proteins with hormone receptors will be studied. Receptor interactions will be detected by hormone-induced subunit dissociation of the proteins and hormone induced guanine nucleotide binding. Initial studies will use human platelet membranes which contain a well characterized second messenger system for the synthesis of cAMP. This system is regulated by two of these proteins, one mediating stimulation by PGE1 and another inhibition by epinephrine. These functional studies will then be extended to various rat and bovine tissues containing different patterns of hormone receptors and GTP binding proteins. (Specific Aim 3) The tissue-specific distribution of those members of this family which are substrates for bacterial toxins in rat and bovine tissues will be characterized. Novel forms identified, or forms not previously amenable to isolation, will be purified and characterized. Preliminary studies indicate that testis may be an advantageous source of one such form of the GTP binding protein regulating stimulation of adenylate cyclase. There are indications that this form of the protein may preferentially mediate hormonal stimulation of adenylate cyclase.
Keywords: FATTY ACIDS, EICOSANOIDS, PROSTAGLANDINS, PROSTAGLANDIN E, PHENYLALKYLAMINES, CATECHOLAMINES, EPINEPHRINE, PROTEINS, BINDING PROTEINS, PROTEINS, BINDING PROTEINS, GUANINE NUCLEOTIDE BINDING PROTEINS, BIOLOGICAL SIGNAL TRANSDUCTION, SECOND MESSENGERS, BLOOD PLATELETS, CELL COMPONENTS, CELL MEMBRANE, IMMUNOLOGY, ANTIGENS BACTERIAL, BACTERIAL TOXINS, NUCLEOTIDYL-CYCLASES, ADENYLATE CYCLASE, PURINE NUCLEOTIDES, ADENINE NUCLEOTIDES, AMP CYCLIC, RECEPTORS, HORMONE RECEPTORS, ANIMALS, CHORDATES, MAMMALS, LAGOMORPHS, ANIMALS, CHORDATES, MAMMALS, RODENTS, MYOMORPHA, RATS (LABORATORY), ANIMALS, CHORDATES, MAMMALS, UNGULATES, CATTLE, HUMAN, HUMAN TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PR OJECT, PHYSICAL SEPARATION, CHROMATOGRAPHY, AFFINITY, PHYSICAL SEPARATION, ELECTROPHORESIS, DISC, PURINE NUCLEOTIDES, GUANINE NUCLEOTIDES, GTP, TISSUE (CELL) CULTURE
Project start date: 1986-04-01
Project end date: 1990-03-31
HORMONE REGULATED GTP-BINDING PROTEINS
John D Hildebrandt, Professor
Pharmacologymedical University Of South Carolina
office Of Research And Sponsored Programs
charleston, Sc 29425
Grant 3R01DK037219-13S1 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: PC
Abstract: Reversible G protein subunit interactions will be studied to determine the properties of these proteins important for mediating hormone action. G proteins are heterotrimeric GTP binding proteins composed of alpha, beta and gamma subunits. They mediate signalling across the plasma membranes of cells for many hormones and neurotransmitters, as well as for numerous other regulatory substances. External signals activate G proteins inside cells by stimulating GTP-dependent dissociation of their alpha subunit from their associated betagamma dimer. These parts of the G protein then independently control signalling pathways inside cells so that the cells respond appropriately to signals in their environment. Errors in such signalling systems are likely contributors to disease processes such as diabetes, essential hypertension and cancer. Based upon the fact that some hormones require very specific G protein isoform combinations for their effects, it is hypothesized that G protein subunit dissociation and reassociation during hormone signalling provides a key mechanism for coordinating cell responses. For example, if G protein subunits form transient or dynamic heterotrimeric complexes, hormones will only be able to stimulate cells when G proteins containing the correct subunits are present. To test this hypothesis the regions of betagamma dimers that bind G protein alpha subunits will be identified and this information will be used to develop sensitive and general assays for G protein activation by receptors in intact membranes and cells. The Specific Aims of this application are 1). To determine the structure and diversity of naturally occurring gamma subunits in G protein heterotrimers. (2). To determine the post-translational modifications of the beta subunits of the G proteins. 3). To use the information from 1) and 2) to define regions in G protein subunits involved in the binding of betagamma dimers to alpha subunits. 4). To determine if G protein heterotrimers are formed of specific subunit combinations in cells and whether reversible subunit dissociation produces heterotrimers of changing subunit composition. 5). To determine the occurrence, regulation and function of G protein subunit dissociation in intact membranes using fluorescently-labeled peptides based upon the work in Specific Aims 1-3. These studies will test the hypothesis that changing G protein subunit composition alters hormonal responses of cells. Regardless of the validity of this hypothesis, these studies will define the properties of the G proteins important for the action of the hormones that regulate them
Keywords: G protein, guanine nucleotide binding protein, hormone regulation /control mechanism, protein structure /function active site, biological polymorphism, biological signal transduction, chemical association, dimer, hormone receptor, membrane protein, posttranslational modification, protein biosynthesis, protein isoform, protein sequence, protein structure, receptor coupling, recombinant protein SDS polyacrylamide gel electrophoresis, high performance liquid chromatography, immunoprecipitation, laboratory rabbit, molecular cloning, protein purification, site directed mutagenesis
Project start date: 1991-07-01
Project end date: 2000-07-14
3R01DK037219-13S1 (2000): $85591
Sponsored Links Excellgen http://Excellgen.com
5R01DK037219-13 (1999): $261183
5R01DK037219-12 (1998): $251137
5R01DK037219-11 (1997): $241476
HORMONE REGULATED GTP BINDING PROTEINS
John D Hildebrandt, Professor
Medical University Of South Carolina 171 Ashley Ave Charleston, Sc 29425
Grant 5R01DK037219-17 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: PC
Abstract: Heterotrimeric G proteins mediate signaling across the plasma membranes of cells for many hormones and neurotransmitters, as well as for a wide variety of other regulatory agents. They are known or likely sites for disease processes such as cholera and pertussis, and their role in cellular regulation makes them likely contributors to the pathology, if not etiology, of complex diseases such as diabetes, essential hypertension and cancer. Understanding how these proteins function in normal signaling events is essential to describing their role in disease states and to providing information about the future possible development of these proteins as targets for pharmacologic intervention. One of the two main hypotheses upon which this research is based is that the widespread involvement of these proteins in cell signaling depends upon the immense structural and functional diversity of the G protein subunits. A primary objective of the proposed studies is to define the role of subunit diversity in signaling by heterotrimeric G proteins. Thus, the first two Specific Aims are (1) To determine the origin and nature of the structural diversity of the beta and gamma subunits of G proteins. These studies take advantage of protein chemistry and mass spectrometry expertise associated with this project. (2) To determine the functional differences between Gbetagamma dimers containing (a) different beta or gamma isoforms or (b) differently modified gamma subunit isoforms. These studies use primarily molecular biology approaches to apply the information generated under Specific Aim 1 to the biochemical characterization of the G protein subunits. An important functional step in the action of G proteins is the GTP-dependent dissociation of their a subunit from their associated betagamma dimer. This reaction has diverse implications for the cellular signaling processes mediated by G proteins, in large part due to their structural and functional diversity. We hypothesize that G protein subunit dissociation is a key step in signal transduction that can determine to what signals cells respond, as well as the nature of that response. To test this hypothesis the third Specific Aim is (3) To determine the physiological role of subunit dissociation in intact membranes and intact cells. These studies will specifically test whether the G protein subunit diversity characterized under Specific Aims 1 and 2 is utilized by cells to generate changing patterns of G protein heterotrimers with varying signaling properties. The results of this project will define the potential range of functional diversity associated with these proteins, and in so doing define their diversity as potential drug targets.
Keywords: G protein, guanine nucleotide binding protein, hormone regulation /control mechanism, protein structure function, chemical association, dimer, membrane protein, protein isoform, recombinant protein, high performance liquid chromatography, laboratory rabbit, matrix assisted laser desorption ionization
Project start date: 1991-07-01
Project end date: 2005-06-30
5R01DK037219-17 (2003): $312519
5R01DK037219-16 (2002): $303417
5R01DK037219-15 (2001): $294580
2R01DK037219-14A1 (2000): $283695
Generation And Function Of Variable Prenyl Protein Processing
John D Hildebrandt, Professor
Cell And Molecular Pharmacologymedical University Of South Carolina
Grant 1R01GM082981-01A2 from National Institute Of General Medical Sciences IRG: MIST
Abstract: About 2% of proteins in the human genome are predicted to be modified by prenylation. These include a broad range of substrates, but prominent among them are many key proteins involved in cell signaling, growth regulation, cell progression and differentiation; for example these include the ras proto-oncogene products and the 3 subunits of the heterotrimeric G proteins. The constituent reactions of this processing pathway are the targets of both currently used and investigational anticancer drugs. In addition, one class of target proteins for this modification, the 3 subunits of the heterotrimeric G proteins, are key constituents of the mediators of about half of all clinically useful drugs. Thus the physiological and pathological function of this processing pathway is closely related to the actions of a wide range of therapeutics from those involved in treatment of cardiovascular diseases to those involved in cancer chemotherapy. Prenylation involves multiple enzymatic steps. Following prenylation, the protein is typically proteolytically processed to remove the last 3 amino acids and the new C- terminus is carboxymethylated. We have characterized the heterogeneity of the prenylation of the heterotrimeric G protein 3 subunits and have found biologically significant variation in all three enzymatic reactions of this pathway. To characterize the functional significance of this variation we are concentrating on a specific subset of variably processed proteins (exemplified by G35 in humans) that are processed by prenylation but not by the subsequent reactions in this complex pathway. We are concentrating on this variant because its processing pattern is sequence dependent. About 10% of prenylated proteins have similar sequence determinants. Using G35 as a model protein we will elucidate the functional significance of this variable processing pattern by testing the hypothesis that G35 mediates unique signaling events in cells (Aim 1), by testing the hypothesis that differential proteolysis of prenylated G3 subunits determines their intracellular trafficking and functional site of action (Aim 2) and by identifying and validating protein-protein interactions related to the unique functions of G35 in cells (Aim 3). Finally, we will evaluate the general role of the variant prenyl processing signal in other predicted proteins from the human genome (Aim 4). These studies will extend the known diversity of the prenyl processing reactions and define the functional significance of this variation. The results of this work will have significance for the role of prenyl processing in cells, for how variation of prenyl processing is related to protein function, for the general mechanisms for signaling through heterotrimeric G proteins, for the actions of the large number of therapeutics that exert their effects through heterotrimeric G proteins, and for the mechanism, consequences and utility of anti-cancer drugs that specifically or coincidently target the protein prenylation reactions. The proposed project studies how a particular kind of lipid molecule referred to as a prenyl group when attached to proteins in cells alters their structure and function. The enzymes that add prenyl groups to proteins and the modified proteins themselves are targets for many different kinds of drugs including those used to treat heart diseases and cancer. The proposed work will increase our understanding of how these drugs work and will help us develop more effective drugs to treat these common illnesses
Project start date: 2009-04-01
Project end date: 2013-01-31
3R01GM082981-01A2S1 (2009): $273992
TRAINING TO IMPROVE CARDIOVASCULAR DRUG THERAPY
John D Hildebrandt, Professor
Medicinemedical University Of South Carolina
office Of Research And Sponsored Programs
charleston, Sc 29425
Grant 5T32HL007260-25 from National Heart, Lung, And Blood Institute IRG: ZHL1
Project start date: 1977-07-01
Project end date: 2002-06-30
5T32HL007260-25 (2001): $278524
Sponsored Links Excellgen http://Excellgen.com
HORMONE REGULATED GTP-BINDING PROTEINS
John D Hildebrandt, Professor
Pharmacologymedical University Of South Carolina
office Of Research And Sponsored Programs
charleston, Sc 29425
Grant 2R01DK037219-10 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: PC
Project start date: 1991-07-01
Project end date: 1999-11-30
2R01DK037219-10 (1996): $262464
HORMONE-REGULATED GTP-BINGING PROTEINS
John D Hildebrandt, Professor
Worcester Foundation For Biomedical Res Shrewsbury, Ma 01545
Grant 2R01DK037219-04A2 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: PC
Project start date: 1986-04-01
Project end date: 1991-06-30