Joseph L. Goldstein - US grants

[unreadable] DESCRIPTION (provided by applicant): [unreadable] This Program Project Grant began 30 years ago when we defined the LDL receptor (LDLR) pathway for control of cholesterol metabolism and showed that defects in LDLR produce Familial Hypercholesterolemia (FH) and its attendant atherosclerosis. After 30 years, our goals have broadened and the participants have increased, but the focus remains the same: to understand the genetic and molecular basis for regulation of lipid metabolism and to use this knowledge to prevent and treat lipid-related diseases, including atherosclerosis, diabetes, and neurologic diseases. During the last grant period, we published 183 papers. These papers report the following major advances: 1) discovery of Insig proteins as central to feedback control of cholesterol and fatty acid synthesis; 2) development of new methods to demonstrate direct cholesterol binding to the sterol-sensing protein Scap; 3) demonstration that cholesterol 24-hydroxylase mediates cholesterol turnover in adult brain; 4) discovery of new roles for LDLR family members in brain development and function (VLDL and apoE2 receptors) and in vascular integrity and atherogenesis (LRP1); 5) demonstration that cholesterol-rich caveolae regulate growth factor receptor tyrosine kinases; 6) identification of 4 new genes and their causative roles in 3 human genetic diseases: sitosterolemia (mutations in ABCG5 and ABCG8); autosomal recessive FH (adaptor protein ARH); and selective 25-hydroxyvitamin D deficiency (vitamin D 25-hydroxylase); 7) use of knockout mice to show that PCSK9 is a major regulator of LDLR protein levels in liver; and 8) development of the nonsynonymous codon approach to find causative genes in quantitative traits (e.g., loss-of-function mutations in PCSK9 lower LDL by 35% in 2% of African-Americans). [unreadable] [unreadable] We now apply for a 5-year renewal (Years 31-35) to further study these and related phenomena through an integrated and multidisciplinary approach. We propose to learn more about already-known molecules and discover new ones that regulate 3 processes: 1) cholesterol and fatty acid metabolism (SREBPs, Scap, Insigs, MOBP, HMG CoA reductase, PCSK9, LDLR, NPC1L1, ABCG5/G8, putative new insulin-sensitizing factor); 2) oxysterol and bile acid metabolism (cholesterol 24-hydroxylase, new sterol hydroxylases); and 3) caveolae membrane function, lipoprotein receptors, and cell signaling (caveolin-1, LRP1, LRP1B, PDGRF(, EGFR). We will continue to study these processes at all possible levels - the molecular (i.e., gene, mRNA, protein), the intact cell, the whole animal, and the human patient. In conducting these studies, we will use multiple techniques - biochemistry, molecular biology, genetics, cell biology, gene-manipulated mice, animal physiology, clinical genetics, and human genomics. This interdisciplinary approach is possible only through support of this Program Project Grant. [unreadable]

tissue /cell culture; biomedical facility; cell line; monoclonal antibody; fibroblasts;

transferase; membrane transport proteins; posttranslational modifications; enzyme mechanism; isoprenoid; blood lipoprotein metabolism; caveolas; geranyl compound; biological signal transduction; cell growth regulation; enzyme structure; oncoproteins; farnesyl compound; enzyme activity; immunoelectron microscopy; laboratory rabbit;

steroid metabolism; DNA binding protein; protein structure function; gene mutation; cholesterol; caveolas; blood lipoprotein metabolism; transcription factor; intracellular transport; homeostasis; lipid transport; proteolysis; hormone binding protein; CHO cells; molecular cloning;

[unreadable] DESCRIPTION (provided by applicant): [unreadable] This Program Project Grant began 30 years ago when we defined the LDL receptor (LDLR) pathway for control of cholesterol metabolism and showed that defects in LDLR produce Familial Hypercholesterolemia (FH) and its attendant atherosclerosis. After 30 years, our goals have broadened and the participants have increased, but the focus remains the same: to understand the genetic and molecular basis for regulation of lipid metabolism and to use this knowledge to prevent and treat lipid-related diseases, including atherosclerosis, diabetes, and neurologic diseases. During the last grant period, we published 183 papers. These papers report the following major advances: 1) discovery of Insig proteins as central to feedback control of cholesterol and fatty acid synthesis; 2) development of new methods to demonstrate direct cholesterol binding to the sterol-sensing protein Scap; 3) demonstration that cholesterol 24-hydroxylase mediates cholesterol turnover in adult brain; 4) discovery of new roles for LDLR family members in brain development and function (VLDL and apoE2 receptors) and in vascular integrity and atherogenesis (LRP1); 5) demonstration that cholesterol-rich caveolae regulate growth factor receptor tyrosine kinases; 6) identification of 4 new genes and their causative roles in 3 human genetic diseases: sitosterolemia (mutations in ABCG5 and ABCG8); autosomal recessive FH (adaptor protein ARH); and selective 25-hydroxyvitamin D deficiency (vitamin D 25-hydroxylase); 7) use of knockout mice to show that PCSK9 is a major regulator of LDLR protein levels in liver; and 8) development of the nonsynonymous codon approach to find causative genes in quantitative traits (e.g., loss-of-function mutations in PCSK9 lower LDL by 35% in 2% of African-Americans). [unreadable] [unreadable] We now apply for a 5-year renewal (Years 31-35) to further study these and related phenomena through an integrated and multidisciplinary approach. We propose to learn more about already-known molecules and discover new ones that regulate 3 processes: 1) cholesterol and fatty acid metabolism (SREBPs, Scap, Insigs, MOBP, HMG CoA reductase, PCSK9, LDLR, NPC1L1, ABCG5/G8, putative new insulin-sensitizing factor); 2) oxysterol and bile acid metabolism (cholesterol 24-hydroxylase, new sterol hydroxylases); and 3) caveolae membrane function, lipoprotein receptors, and cell signaling (caveolin-1, LRP1, LRP1B, PDGRF(, EGFR). We will continue to study these processes at all possible levels - the molecular (i.e., gene, mRNA, protein), the intact cell, the whole animal, and the human patient. In conducting these studies, we will use multiple techniques - biochemistry, molecular biology, genetics, cell biology, gene-manipulated mice, animal physiology, clinical genetics, and human genomics. This interdisciplinary approach is possible only through support of this Program Project Grant. [unreadable]

The Administrative Core coordinates the fiscal, scientific, and administrative aspects of the Program Project Grant. Although Drs. Goldstein and Brown share the overall administrative responsibility for this grant, their administrative duties are divided. Dr. Goldstein is in charge of budgetary and personnel matters. Dr. Brown is in charge of purchasing equipment and supervision of graduate students and postdoctoral fellows.

The Tissue Culture Core Laboratory will be responsible for providing investigators with cultured cells, antibodies, and facilities for routine microscopic analysis of biological samples. The core will be directed by Dr. Goldstein. He will be assisted by Drs. Y.K. Ho, Richard Anderson, Joachim Herz, and Rob Rawson. The technical work in the Tissue Culture Facility of this Core is carried out by four experienced technicians (Lisa Beatty, Angela Carroll, Jill Fairless, Marissa Hodgin) and one laboratory assistant (Rose Marshall). The Tissue Culture Facility of this Core is located in the Department of Molecular Genetics and consists of three suites of rooms that are used solely for tissue culture. One suite contains four work modules that open into a common work area;the second suite contains three work modules that open into a common work area;and the third suite contains two work modules, one for adenovirus work and the other for baculovirus. Each module is equipped with a sterile work area (Biological Safety Cabinet hood), one or more CO2 incubators, a refrigerator, and an inverted microscope. The common work area in each of the suites contains one or two sterile work areas. The entire facility contains a total of 12 inverted microscopes (6 of which are phase-contrast microscopes), 1 stereo microscope, 15 sterile work areas (hoods), 28 singlechamber CO2 incubators, 2 non-CO2 refrigerated incubators, 1 roller bottle incubator, 3 refrigerated incubator shakers, 3 table-top refrigerated centrifuges, and 9 refrigerators. Four liquid nitrogen freezers for storage of cell lines are located in the common work area adjacent to the work modules. In addition to this standard equipment, we have a Zeiss Axiovert 35 M inverted fluorescence microscope that allows us to visualize cells directly in the Petri dish (under sterile conditions) so as to determine whether or not they have taken up reconstituted fluorescent LDL. This method is the assay of choice for identifying and monitoring mutant cholesterol auxotrophic cell clones that are resistant to amphotericin and thus fail to take up fluorescent LDL (Research Project 1).

Our goal is to understand the machinery that maintains the constancy of cholesterol in cell membranes. We expect to discover new proteins and biochemical processes that control the production, intracellular transport, and storage of cholesterol. These discoveries will serve as a model that permits other scientists to expose related mechanisms that control the membrane content of other lipids that are essential for normal cell function. Defects in these control processes will likely be found to underlie diseases as diverse as atherosclerosis, neurodegeneration, and cancer. Cell membranes are fundamental to life, and defects in their function underlie many diseases. Membranes of different organelles have different lipid compositions. For example, cholesterol is markedly enriched in the plasma membrane compared with the ER. Scientists know the enzymes that synthesize and degrade the various lipids in different cell membranes. Yet, very little is known about how these enzymes are regulated so as to maintain differential lipid compositions. Our laboratory made the initial inroad into this problem through the discovery of the SREBP family of transcription factors that control synthesis and uptake of cholesterol and fatty acids. We discovered four proteins whose actions govern the regulated trafficking mechanism that dictates the activities of SREBPs. We are now in a unique position to decipher precisely how this system works at a molecular level. Our findings will drive the field of membrane biology at the level of fundamental understanding and at the level of disease. In the course of our work, we will continue to invent new methodologies, just as we have in the nearly 40 years since we first invented methods to discover receptor-mediated endocytosis and its specific application to LDL. We will uncover new concepts, just as we did when we defined the general processes of regulated-intramembrane proteolysis in 2000 and hydrophobic handoff in 2009. Our work will help scientists in many fields of biology to understand the membranes that play essential roles in all biologic functions.

The Tissue Culture Core Laboratory will be responsible for providing investigators with cultured cells, antibodies, and facilities for routine microscopy of biological samples. The core will be directed by Dr. Goldstein. He will be assisted by Drs. Y.K. Ho, Guosheng Liang, and Rob Rawson. The technical work in the Tissue Culture Facility of this Core is carried out by five experienced technicians (Lisa Beatty, Angela Carroll, Shomanike Head, Ijeoma Onwuneme, Muleya Kapaale) and one laboratory assistant (Dawn Rollins). The Tissue Culture Facility is located in the Department of Molecular Genetics and consists of three suites of rooms that are used solely for tissue culture. One suite contains four work modules that open into a common work area; the second suite contains three work modules that open into a common work area; and the third suite contains two work modules, one for adenovirus work and the other for baculovirus. Each module is equipped with a sterile work area (Biological Safety Cabinet hood), one or more CO2 incubators, a refrigerator, and an inverted microscope. The common work area in each of the suites contains one or two sterile work areas. The entire facility contains 14 inverted microscopes (6 of which are phase-contrast microscopes), 1 stereo microscope, 16 sterile work areas (hoods), 33 single-chamber CO2 incubators, 1 single-chamber multigas incubator, 2 non-C02 refrigerated incubators, 1 roller bottle incubator, 3 refrigerated incubator shakers, 4 table-top refrigerated centrifuges, and 11 refrigerators. Five liquid nitrogen freezers for storage of cell lines are located in the common work area adjacent to the work modules. In addition to this standard equipment, we have a Zeiss Axiovert 35 M inverted fluorescence microscope that allows us to visualize cells directly in the Petri dish (under sterile conditions) so as to determine whether or not they have taken up reconstituted fluorescent LDL or other fluorescent molecules.

C/PPG 2015 ? RP1 ? 30-line Summary A complex membrane protein called Scap is the central controller of lipid synthesis in animal cells. Located in the endoplasmic reticulum (ER), Scap binds and transports SREBPs (Sterol Regulatory-Element Binding Proteins) to the Golgi where their transcriptionally active portions are released from the membrane so that they can enter the nucleus. Nuclear SREBPs activate transcription of all genes necessary for synthesis of low density lipoprotein (LDL) receptors, cholesterol, and fatty acids. When cholesterol accumulates in ER membranes it binds to Scap, preventing its transport. Processing of SREBPs decreases and this decreases cholesterol synthesis and uptake from LDL. Scap is responsible for statin-induced up-regulation of LDL receptors and lowering of plasma LDL. In liver, insulin stimulates processing of one isoform of SREBP (SREBP- 1c) that activates synthesis of fatty acids and produces hypertriglyceridemia and fatty liver in Type 2 diabetes. Our laboratory discovered the SREBPs, Scap, and the SREBP-processing proteases. We have begun a molecular dissection of Scap, which has 8 transmembrane helices and several large structured hydrophilic loops. We showed that two large luminal loops (Loop1 and Loop7) bind to each other, and this binding is necessary for Scap to exit from the ER. Point mutations that disrupt this intramolecular binding prevent ER exit. When ER membrane cholesterol exceeds a sharp threshold, the cholesterol binds to Loop1 of Scap. We have hypothesized that cholesterol binding disrupts the Loop1-Loop7 complex and this dissociation prevents Scap from binding to COPII proteins that carry the Scap/SREBP complex to the Golgi. Aim 1 is designed to test the Loop1-Loop7 dissociation hypothesis. We recently produced a soluble fusion protein consisting of Loop1 and Loop7 joined by a linker that is cleavable by a protease. After cleavage, the two loops remain associated. So far, cholesterol addition in vitro has not dissociated the loops. We believe that dissociation may require cholesterol to be presented in a membrane. We propose experiments with liposomes to test this hypothesis. We also propose experiments to test the hypothesis that dissociation requires a conformational change in the 6 transmembrane helices that separate Loop1 and Loop7 in native Scap. Aim 2 is designed to identify drug-like molecules that bind to the sterol-binding site on Loop1 and inhibit SREBP processing. We propose a novel screen using a soluble cholesterol-binding bacterial hemolysin as a surrogate for Scap. We will also screen directly for inhibitors of [3H]cholesterol binding to Loop1. Our knockout studies in mice indicate that Scap inhibitors would be effective therapy for hypertriglyceridemia and fatty liver. Our laboratory has pioneered the molecular dissection of Scap and its functional domains, and we are prepared to extend this work to achieve a complete molecular understanding of this important protein machine.

The Administrative Core (Core C) coordinates the fiscal, scientific, and administrative aspects of the Program Project Grant (PPG). Dr. Joseph L. Goldstein will serve as the Director for the overall PPG. He has been the Director on this Program Project Grant since its beginning in 1977. Dr. Goldstein, who will be responsible for the PPG's overall scientific and financial matters, will be assisted in his administrative duties by Drs. Michael Brown and Jay Horton. Dr. Brown will be in charge of students and postdoctoral researchers. Dr. Horton will handle business matters concerning animals, human research protocols, and laboratory equipment. Drs. Horton and Goldstein will be assisted by a Financial Affairs Manager (Kay Pokladnik) who has worked on the PPG for 32 years; by an Administrative Coordinator (Mrs. Karen Schiller) who worked for 4 years as a Senior Grants and Contracts Specialist in the Office of Grants Management at UT Southwestern prior to working on this PPG for the last 7 years; and by a Laboratory Manager (Linda Donnelly), who has been associated with this PPG for 15 years. The Administrative Core will be responsible for personnel matters, for placing all purchase orders, for handling all protocols involving animals and human subjects, and for preparing weekly accounts of expenditures of each project for review by Drs. Horton and Goldstein. In addition to these administrative responsibilities, the Core will also be responsible for the typing and preparation of all our manuscripts, grant applications, and research progress reports. C/PPG 2015 ? Core C ? 30-line Summary

Core A?Tissue Culture Core will be responsible for providing investigators with cultured cells, monoclonal antibodies, recombinant proteins, and recombinant adeno-associated viruses. The Core will be directed by Dr. Joseph Goldstein, who has served as its Principal Investigator for the past 40 years. The technical work in the Core is carried out by five experienced technicians, one of whom (Lisa Beatty) has been in charge of this facility for the past 28 years. The physical facilities of the Core consists of three suites of rooms that are used solely for tissue culture. The facility is equipped with 40 incubators, 14 inverted microscopes, 1 stereo microscope, 16 sterile work areas (hoods), 2 roller bottle apparatuses, 5 refrigerated incubator shakers, 3 table-top refrigerated centrifuges, 11 refrigerators, and 7 liquid nitrogen freezers for storage of cell lines. The success of the entire Program Project Grant depends on the smooth operation of this Core. Over the past 40 years, we have developed considerable experience in maintaining quality control and in growing multiple cell lines, including over 1100 different primary human fibroblast cell strains, derived from skin biopsies from normal subjects as well as from patients with metabolic disorders. Since 1984, tens of thousands of transfection experiments have been carried out in which we have introduced into various cell lines (e.g., HEK- 293, CHO, and SV589 cells) multiple plasmid constructs containing either the protein-coding region or the promoter/enhancer region of multiple genes. From these transfections, more than 3000 stable and permanent cell lines have been clonally established and frozen away in multiple aliquots. In addition to maintenance of stock cell lines and preparation of cultured cells for experiments, the Core is involved in the following activities: 1) Generation and maintenance of mouse and rabbit hybridoma cell lines and production of monoclonal antibodies from culture medium; 2) Purification by Protein A-Sepharose chromatography of more than 25 different polyclonal rabbit antibodies directed against multiple proteins; 3) Growth of large volumes of suspension-culture cells that allow efficient transfection of cDNAs and production of their encoded proteins; 4) Isolating, maintaining, and freezing away cloned cell lines that have been transfected with mutated versions of various cDNA and promoter/enhancer constructs; 5) Maintenance of insect cells in suspension culture for production of recombinant proteins by infecting these cells with recombinant baculoviruses encoding cloned cDNAs; 6) Production of recombinant adeno-associated viruses and lentiviruses, which are used for evaluating the function of genes in cultured cells and in the liver after in vivo injection; 7) Sending aliquots of monoclonal and polyclonal antibodies and cell lines to hundreds of investigators who request them. C/PPG 2015 ? Core A ? 30-line Summary