Proteins From Hereditary Eye Diseases: In-silico and Experimental Studies

遗传性眼病的蛋白质：计算机模拟和实验研究

基本信息

批准号：
7968424
负责人：
Yuri Sergeev
金额：
$ 68.91万
依托单位：
NATIONAL EYE INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7968424
关键词：
Affect Age related macular degeneration Aging Astrocytes Biochemistry Boxing Cells Choroideremia Collaborations Complex Computational Biology Computer Simulation Computing Methodologies Crystallins Degenerative Disorder Diagnosis Disease Electroretinography Elements Eye Eye diseases Fibroblasts Gene Mutation Genes Genotype Goals Hereditary Eye Diseases Homo Inherited Investigation Laboratories Lead Manuscripts Measures Mediating Membrane Proteins Missense Mutation Molecular Molecular Chaperones Molecular Genetics Molecular Models Mononuclear Mutation National Institute of Arthritis and Musculoskeletal and Skin Diseases Pathology Patients Phenotype Positioning Attribute Preventive Process Protein Analysis Proteins Proteolysis Publications Rattus Recombinant Proteins Retina Role Severities Site Site-Directed Mutagenesis Structural Protein Structure of retinal pigment epithelium Structure-Activity Relationship Syndrome System Time Transcription factor genes Visual system structure Western Blotting Work X-Linked Retinoschisis XLRS1 protein base beta-Crystallins disease phenotype functional genomics lens meetings molecular dynamics molecular modeling mutant novel protein aggregation protein expression protein folding protein protein interaction protein structure protein structure function research study tool

项目摘要

In order to understand how a pathogenic change in a gene causes disease, it is necessary to recognize a protein structure-function and a role in protein networks. We are looking for a general approach in which computational methods are used to probe the severity of protein structural perturbations caused by pathogenic mutation and to provide a basis for associating these changes with disease phenotype. The implications of such an approach to the kingdom of proteins affected by inherited and degenerative eye disease could create a universal scale to compare effects of mutations at the atomic level, thus, guiding better diagnosis of inherited disease. We successfully applied this approach in molecular modeling of retinoschisin with functional analysis of 27 mutations from 61 X-linked retinoschisis (XLRS) patients and demonstrated that mutational change in RS1 protein structure affects the phenotype of XLRS measured from electroretinogram (manuscript is ready for submission). In 10 pathogenic mutations causing choroideremia missense L550P, truncation and deletion mutants were predicted using computational modeling to be associated with a partial or total loss of the Rab excort protein 1 (REP-1). The presumptive loss of protein was confirmed by Western Blot analysis of protein from mononuclear cells and fibroblasts from CHM patients. Recently, I have applied molecular modeling to explain a functional role of three mutations in the paried box transcription factor gene, PAX2, which may cause papillorenal syndrome. In collaboration with Dr. Brian Brooks I performed molecular modeling of the DNA-PAX2 complex to show a structural effect of these missense mutations. The manuscript describing this work and in which I am a co-author, has received a good review in the PLOS Molecular Genetics and is expected to be accepted. In the experimental part of our work, we have used recombinant protein expression and purification to generate wild type and modified beta-crystallin proteins. Protein folding/misfolding, protein aggregation, subunits exchange, stability and formation of oligomeric homo- and hetero-associates in protein-protein interactions were studied. These characterizations, lead to a better understanding of the different elements of protein structure defining native interactions such as terminal extensions, globular domains and their structural parts. Recently, we demonstrated using site directed mutagenesis, protein expression/purification, limited proteolysis and molecular modeling, that the amino terminal of betaB1 extension contains structural features which position a mobile loop in the vicinity of these processing sites. The loop is derived from residues 48-56 which appear critical for mediating protein interactions with betaA3-crystallin. This work is done in collaboration with Dr. Paul Wingfield (NIAMS) and his laboratory. These results were presented at ARVO meeting 2009 and the manuscript is accepted for publication in Biochemistry. In collaboration with Dr. Sinha laboratory, I used molecular dynamic simulations to show that the 10-residue insertion in betaA3/A1-crystallin creates a loop at the C-terminus that sterically affects protein-protein interactions. This was confirmed by my collaborators who showed aggregation of the mutant betaA3/A1 in the lens. In addition, this study showed for the first time that betaA3/A1 is expressed in the astrocytes, has a novel function in the remodeling of the retina and is also expressed in retinal pigment epithelium cells where it shows age-related macular degeneration (AMD)-like pathology in the aging Nuc1 spontaneous mutant rats.

为了了解基因的致病性变化如何导致疾病，有必要认识蛋白质的结构-功能和蛋白质网络中的作用。我们正在寻找一种通用方法，其中使用计算方法来探测致病突变引起的蛋白质结构扰动的严重性，并为将这些变化与疾病表型相关联提供基础。这种方法对受遗传性和退行性眼病影响的蛋白质王国的影响可以创建一个通用的尺度来比较原子水平上突变的影响，从而指导更好地诊断遗传性疾病。我们成功地将这种方法应用于视网膜劈裂素的分子建模，对 61 名 X 连锁视网膜劈裂症 (XLRS) 患者的 27 个突变进行了功能分析，并证明 RS1 蛋白结构的突变变化影响视网膜电图测量的 XLRS 表型（手稿已准备好提交）。在导致无脉络膜血症错义 L550P 的 10 种致病性突变中，使用计算模型预测截短和缺失突变体与 Rab 排泄蛋白 1 (REP-1) 的部分或全部丢失相关。对 CHM 患者的单核细胞和成纤维细胞的蛋白质进行蛋白质印迹分析，证实了推定的蛋白质损失。最近，我应用分子模型来解释配对盒转录因子基因 PAX2 中三种突变的功能作用，这可能导致乳头肾综合征。我与 Brian Brooks 博士合作，对 DNA-PAX2 复合物进行了分子建模，以显示这些错义突变的结构效应。我作为合著者描述这项工作的手稿已在《公共科学图书馆分子遗传学》上获得了良好的评论，预计将被接受。在我们工作的实验部分，我们使用重组蛋白表达和纯化来生成野生型和修饰的 β-晶状体蛋白。研究了蛋白质-蛋白质相互作用中蛋白质折叠/错误折叠、蛋白质聚集、亚基交换、稳定性和寡聚同源和异源缔合体的形成。这些表征有助于更好地理解定义天然相互作用的蛋白质结构的不同元素，例如末端延伸、球状结构域及其结构部分。最近，我们使用定点诱变、蛋白质表达/纯化、有限蛋白水解和分子建模证明，βB1 延伸的氨基末端包含将移动环定位在这些加工位点附近的结构特征。该环源自残基 48-56，这些残基对于介导蛋白质与 betaA3-晶状体蛋白的相互作用至关重要。这项工作是与 Paul Wingfield 博士 (NIAMS) 及其实验室合作完成的。这些结果已在 2009 年 ARVO 会议上发表，手稿已被《生物化学》接受发表。我与 Sinha 博士实验室合作，使用分子动力学模拟表明，βA3/A1-晶状体蛋白中的 10 个残基插入在 C 末端形成了一个环，在空间上影响蛋白质-蛋白质相互作用。我的合作者证实了这一点，他们展示了突变体 betaA3/A1 在晶状体中的聚集。此外，这项研究首次表明，βA3/A1在星形胶质细胞中表达，在视网膜重塑中具有新功能，并且也在视网膜色素上皮细胞中表达，在视网膜色素上皮细胞中表现出年龄相关性黄斑变性（AMD）衰老的 Nuc1 自发突变大鼠的类似病理学。