Structure-Function relation of Connexin disease mutations

连接蛋白疾病突变的结构-功能关系

• 批准号: 8536864
• 负责人: Thaddeus Andrew Bargiello
• 金额: $ 29.07万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8536864
• 关键词: Address; Amino Acid Substitution; Amino Acids; Anabolism; Antibodies; Biogenesis; Biological; Canis familiaris; Cell Line; Cell membrane; Cellular biology; Charcot-Marie-Tooth Disease; Collaborations; Computer Simulation; Computing Methodologies; Connexins; Crystallography; Cysteine; Defect; Development; Disease; Environment; Etiology; Failure; Fluorescence Spectrometry; Genes; Homology Modeling; Human; Immunoprecipitation; Inherited; Ions; Knowledge; Link; Maps; Measures; Membrane; Methods; Missense Mutation; Modeling; Modification; Molecular; Mono-S; Monoclonal Antibodies; Mutation; N-terminal; Pathway interactions; Peptides; Permeability; Phenotype; Plasma; Positioning Attribute; Probability; Process; Property; Research Personnel; Role; Second Messenger Systems; Solutions; Structural Models; Structure; Sulfhydryl Compounds; Testing; Validation; base; computer studies; deafness; disease-causing mutation; effective therapy; experience; flexibility; gain of function; insight; loss of function; loss of function mutation; molecular dynamics; mutant; prevent; research study; second messenger; skin disorder; therapeutic development; trafficking; voltage

DESCRIPTION (provided by applicant): The N-terminal domain (NT, residues 1-22) is an important determinant of perm-selectivity and voltage-dependent gating of connexin channels and a sensitive mutational target underlying two common inherited diseases: X-linked Charcot-Marie-Tooth (Cx32) and nonsyndromic and syndromic deafness (Cx26). This proposal will determine how disease causing mutations in the NT of Cx32 and Cx26 alter channel function and channel biosynthesis by applying synergistic computational and experimental approaches. Differences in function between wild type and disease causing NT mutations are hypothesized to arise from specific changes in channel structure. The study will examine 9 NT loci comprising mutations in both Cx32 and Cx26. In several cases, mutations of the same locus alter Cx26 and Cx32 channel function differently, suggesting that identical or homologous amino acid substitutions cause different structural defects in the two connexins. Studies will be guided by the crystal structure of a Cx26 hemichannel and a Cx32 homology model, both refined by all-atom molecular dynamics (MD) simulation and shown to closely correspond to the structure of the biological open channel. The study will solve the structure of mutant NT peptides by 2D NMR. Structural solutions of longer wild-type and mutant peptides (NT-CL domain, residues 1-114) in a membrane environment by 3D NMR, and assembled channels by x-ray crystallography have been initiated. Resulting atomic models of connexin channels will be refined by all-atom MD simulations, the permeabilities to ions and second messengers determined computationally and compared to experimental. This experimental strategy provides a sensitive test of the accuracy of atomic models, insights into molecular mechanisms of perm-selectivity and how these are changed by mutation, as well as testable hypotheses of structure-function relations. The study will investigate the role of the NT in channel biogenesis by determining the position and stability of the NT of connexin subunits inserted into canine microsomal membranes, the role of the NT in subunit oligomerization, and when and how the NT assumes its final position deep within the pore of assembled hemichannels prior to plasma membrane insertion. Parallel computational studies will provide a rigorous mechanistic framework that will guide these experimental studies. This new, fundamental knowledge will provide a framework for understanding the molecular defects of the class of disease causing NT mutations that are not plasma membrane inserted but trapped in cytosolic compartments and targeted for degradation. The project is highly collaborative, bringing together investigators with proven expertise in structural determination, computational methods and biophysical characterization of connexin channels. The results will provide new information fundamental to the elucidation of connexin disease etiology and to the development of effective treatments.

描述（由申请人提供）：N-末端结构域（NT，残基1-22）是连接蛋白通道的渗透选择性和电压依赖性门控的重要决定因素，也是两种常见遗传性疾病（X连锁腓骨肌萎缩症（Cx 32）和非综合征性和综合征性耳聋（Cx 26））的敏感突变靶点。该建议将确定如何致病的Cx 32和Cx 26的NT突变改变通道功能和通道生物合成，通过应用协同计算和实验方法。野生型和致病NT突变之间的功能差异被假设是由通道结构的特定变化引起的。该研究将检查包含Cx 32和Cx 26突变的9个NT基因座。在一些情况下，相同位点的突变改变Cx 26和Cx 32通道功能不同，这表明相同或同源的氨基酸取代导致两种连接蛋白的不同结构缺陷。研究将由Cx 26半通道和Cx 32同源模型的晶体结构指导，这两个模型都通过全原子分子动力学（MD）模拟进行了优化，并显示出与生物开放通道的结构密切对应。本研究将通过二维核磁共振技术解析突变NT肽的结构。较长的野生型和突变体肽（NT-CL结构域，残基1-114）在膜环境中通过3D NMR的结构解决方案，并通过X射线晶体学组装通道已开始。由此产生的连接蛋白通道的原子模型将通过全原子MD模拟进行改进，离子和第二信使的渗透性通过计算确定并与实验进行比较。这种实验策略提供了一个敏感的测试原子模型的准确性，深入了解分子机制的渗透选择性，以及如何改变突变，以及可测试的结构-功能关系的假设。该研究将通过确定插入犬微粒体膜的连接蛋白亚基的NT的位置和稳定性，NT在亚基寡聚化中的作用，以及NT何时以及如何在质膜插入之前在组装的半通道的孔深处假定其最终位置，来研究NT在通道生物发生中的作用。并行计算研究将提供一个严格的机制框架，将指导这些实验研究。这一新的基础知识将为理解导致NT突变的疾病的分子缺陷提供一个框架，这些NT突变不是质膜插入的，而是被困在胞质隔室中并被靶向降解。该项目是高度合作的，汇集了调查人员， 在连接蛋白通道的结构测定、计算方法和生物物理特性方面的专业知识。这些结果将为阐明连接蛋白疾病的病因和开发有效的治疗方法提供新的信息。