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

• 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
• 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

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引起疾病，有必要识别蛋白质结构功能和在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了基础。这种方法对受遗传和退化性眼病影响的蛋白质王国的含义可能会产生普遍的规模，以比较原子水平的突变的影响，从而指导更好地诊断遗传性疾病。我们通过对61个X连锁视网膜（XLRS）患者的27个突变进行功能分析，成功地将这种方法应用于视网膜的分子建模中，并证明RS1蛋白结构中的突变变化会影响从电子图中测量的XLRS的表型（手稿已准备好出现）。在10个导致脉络膜血症错义L550p的致病突变中，使用计算模型预测截断和缺失突变体与RAB Excort蛋白1（REP-1）的部分或总损失相关。通过对单核细胞的蛋白质和CHM患者的成纤维细胞的蛋白质分析，证实了蛋白质的假定丧失。最近，我应用了分子建模来解释三个突变在培养箱转录因子基因Pax2中的功能作用，这可能会导致乳头状综合征。我与Brian Brooks博士合作进行了DNA-PAX2复合物的分子建模，以显示这些错义突变的结构效应。描述这项工作的手稿和我是合着者，在PLOS分子遗传学方面得到了良好的综述，并有望被接受。 在我们工作的实验部分中，我们使用重组蛋白表达和纯化来产生野生型和改良的β-晶状蛋白蛋白。研究了蛋白质折叠/错误折叠，蛋白质聚集，亚基交换，稳定性以及蛋白质 - 蛋白质相互作用中寡聚同型和异方酶的形成。这些特征可以更好地理解定义天然相互作用的蛋白质结构的不同元素，例如末端扩展，球状结构域及其结构部分。最近，我们使用位置定向诱变，蛋白质表达/纯化，有限的蛋白水解和分子建模证明，BETAB1扩展的氨基末端包含结构特征，这些特征将移动循环定位在这些加工位点附近。循环源自残基48-56，对于介导蛋白质与β3-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。这些结果在2009年的Arvo会议上提出，并接受了手稿以发表生物化学。我与Sinha博士实验室合作，我使用分子动力学模拟表明，β3/A1-晶状体中的10个残留插入在C末端会产生一个循环，从而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，他在镜头中显示了突变体β3/A1的聚合。此外，这项研究首次表明，β3/A1在星形胶质细胞中表达，在视网膜的重塑中具有新功能，并且在视网膜色素上皮细胞中也表达，其中显示了与年龄相关的黄斑变性（AMD） - 类似于年龄相关的黄斑变性（AMD） - 类似于衰老的nuc1 nuc1 nuc1 nuc1 aptontane spontaneos突变突变体。