Folding of Proinsulin

胰岛素原的折叠

基本信息

批准号：
7474694
负责人：
MICHAEL Aaron WEISS
金额：
$ 29.85万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-09-30 至 2010-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7474694
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

项目摘要

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）：本申请重点关注具有重要医学意义的球状蛋白——人胰岛素原——并研究其可折叠性的序列决定因素。胰岛素原的正确折叠对于胰腺β细胞的功能和脊椎动物代谢稳态的维持至关重要。由于长期以来获得晶体的困难以及由于聚集导致的核磁共振谱的难以处理，人们对胰岛素原的基线结构知之甚少。在目标 1 和 2 中，我们将通过工程单体的异核核磁共振研究克服这些限制。我们试图将胰岛素部分的结构与胰岛素本身的结构进行比较，并评估连接肽中局部有序的程度。目标 3 研究哺乳动物分泌细胞系内质网折叠性的序列决定因素。这些细胞生物学研究将检验这样的假设：特定折叠核中的残基是正确折叠所必需的，与突变对折叠蛋白的稳定性或受体结合亲和力的影响无关。我们假设将发现“折叠突变”，阻止动力学可达基态。目标 4 以这些结果为基础，测试推定的体内折叠突变是否确实会损害体外胰岛素原的氧化折叠。由于使用生物合成材料对非折叠变体进行结构研究可能不可行，因此我们建议采用全化学合成来制备相应的突变型胰岛素，用于功能、热力学和结构研究。目标 5 中寻找“可折叠”变体的结构。蛋白质中的特定侧链能否在折叠过程中充当“动力学引导”，但对于蛋白质折叠后的功能来说是可有可无的？噬菌体 P22 的三聚体尾尖蛋白中已鉴定出经典折叠途径突变。拟议的研究将测试这种范式是否可以推广到具有重要药学和生理学重要性的单体折叠反应。最令人感兴趣的是“不可折叠”蛋白质的晶体结构。我们的结果有望对胰岛素样序列的进化以及 II 型糖尿病中人胰岛素原可能的错误折叠产生影响。