Folding of Proinsulin

胰岛素原的折叠

• 批准号: 7263990
• 负责人: MICHAEL Aaron WEISS
• 金额: $ 30.45万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7263990
• 关键词: Adopted; Affect; Affinity; Alanine; American; Amino Acid Sequence; Anabolism; Appendix; Atherosclerosis; Bacteriophage P22; Biochemical; Biological; Biophysics; Blindness; Boxing; C-Peptide; Cell Line; Cell Maintenance; Cell Nucleus; Cell physiology; Cells; Chronic Disease; Class; Collaborations; Color; Crying; Cultured Cells; Cystine; DKP-insulin; Diabetes Mellitus; Diabetic Angiopathies; Dipeptides; Disease; Disulfides; Endoplasmic Reticulum; Engineering; Epidemic; Escherichia coli; Evolution; Failure; Family suidae; Figs - dietary; Functional disorder; Genes; Guanidines; Heteronuclear NMR; High Pressure Liquid Chromatography; Homeostasis; Human; In Vitro; Insulin; Insulin Resistance; Investigation; Island; Kidney Failure; Kinetics; Knowledge; Label; Laboratories; Mammalian Cell; Medical; Metabolic; Methods; Methyl Green; Modeling; Molecular; Monitor; Motion; Mutation; Myocardial Ischemia; NMR Spectroscopy; Non-Insulin-Dependent Diabetes Mellitus; Numbers; Oranges; Oxidation-Reduction; Pancreas; Pathway interactions; Peptides; Personal Satisfaction; Pharmacologic Substance; Phase; Physiological; Physiology; Play; Positioning Attribute; Principal Investigator; Process; Proinsulin; Property; Proprotein Convertase 1; Proprotein Convertase 2; Protein Chemistry; Protein Dynamics; Proteins; Reaction; Relaxation; Residual state; Role; Scheme; Secretory Cell; Side; Site; Societies; Solutions; Staging; Stress; Structural Models; Structure; Structure of beta Cell of islet; Sulfhydryl Compounds; Tail; Techniques; Testing; Thermodynamics; Time; Tissues; Transfection; Variant; Vertebrates; analog; base; biosynthetic material; chemical synthesis; comparative; design; globular protein; guanidinium; in vivo; interest; member; millisecond; miniproinsulin; molecular modeling; monomer; mutant; nanosecond; novel; polypeptide; preference; preproinsulin; programs; protein folding; receptor binding; response; trafficking

DESCRIPTION (provided by applicant): This application focuses on a globular protein of central medical importance - human proinsulin - and investigates sequence determinants of its foldability. Proper folding of proinsulin is fundamental to the function of the pancreatic beta cell and maintenance of metabolic homeostasis in vertebrates. Little is known about the baseline structure of proinsulin due to long-standing difficulties in obtaining crystals and the intractability of its NMR spectrum due to aggregation. In Aims 1 and 2 we will overcome these limitations through heteronuclear NMR studies of an engineered monomer. We seek to compare the structure of the insulin moiety to that of insulin itself and to evaluate the extent of local order in the connecting peptide. Aim 3 investigates sequence determinants of foldability in the endoplasmic reticulum of mammalian secretory cell lines. These cell-biological studies will test the hypothesis that residues in a specific folding nucleus are required for proper folding independently of effects of mutations on the stability or receptor-binding affinity of the folded protein. We hypothesize that "folding mutations" will be found that block kinetic accessibility to the ground state. Aim 4 builds on these results to test whether putative in vivo folding mutations indeed impair oxidative folding of proinsulin in vitro. Because structural studies of nonfolding variants might be infeasible using biosynthetic material, we propose to employ total chemical synthesis to prepare the corresponding mutant insulins for functional, thermodynamic, and structural studies. Structures of "unfoldable" variants are sought in Aim 5. Can specific side chains in a protein serve as "kinetic guides" during folding - but be dispensible for the fuction of the protein once folded? Classical folding-pathway mutations have been identified in the trimeric tail-spike protein of phage P22. The proposed studies will test whether this paradigm generalizes to a monomeric folding reaction of central pharmaceutical and physiological importance. Of overarching interest would be the crystal structure of an "unfoldable" protein. Our results promise to have implications for the evolution of insulin-like sequences and the possible misfolding of human proinsulin in type II diabetes mellitus.

描述（由申请人提供）：本申请侧重于具有中心医学重要性的球形蛋白-人胰岛素原-并研究其可折叠性的序列决定因素。在脊椎动物中，胰岛素原的适当折叠是胰腺β细胞功能和维持代谢稳态的基础。