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: 8737650
• 负责人: Yuri Sergeev
• 金额: $ 47.28万
• 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/8737650
• 关键词: 3-Dimensional; Affect; Age related macular degeneration; Albinism; Alternative Complement Pathway; Arginine; Behavior; Binding; Biomass; Blindness; Cells; Childhood; Clinical; Clinical Research; Clinical Trials; Complement 3b; Complement Factor H; Computer Simulation; Cysteine; DNA; Development; Disease; Disease model; Drug Design; Electroretinography; Elements; Endoplasmic Reticulum; Enzymatic Biochemistry; Enzyme Kinetics; Enzymes; Excision; Eye; Eye diseases; Free Energy; Functional disorder; Future; Gene Mutation; Genes; Genetic; Genotype; Glycoproteins; Goals; Grouping; Hair; Hereditary Disease; Hereditary Eye Diseases; Hospitals; Human; Individual; Inherited; Insecta; Investigation; Knowledge; Larva; Lead; Literature; London; Macular degeneration; Medical; Melanins; Membrane; Membrane Proteins; Methods; Missense Mutation; Modeling; Molecular; Molecular Chaperones; Molecular Models; Monophenol Monooxygenase; Mutate; Mutation; Nature; Oculocutaneous Albinism; Patients; Pharmaceutical Preparations; Phenotype; Pigment Epithelium of Eye; Preclinical Drug Evaluation; Preventive; Process; Production; Proline; Property; Proteins; Recombinants; Resolution; Retina; Retinal; Retinal Degeneration; Retinal Diseases; Risk; Roentgen Rays; Role; Sequence Homologs; Severities; Side; Simulate; Site; Skin; Solubility; Structure; Structure of retinal pigment epithelium; Structure-Activity Relationship; Temperature; Testing; Therapeutic; Therapeutic Intervention; Time; Variant; Visual system structure; Work; X-Linked Retinoschisis; XLRS1 protein; aged; base; beta pleated sheet; candidate selection; cohort; computer studies; disease phenotype; disorder risk; enzyme activity; gene therapy; glycosylation; in vivo; insight; molecular dynamics; molecular modeling; mutant; protein aggregation; protein folding; protein function; protein protein interaction; protein structure; protein structure function; research study; screening; small molecule; temperature sensitive mutant; tool

In order to understand how a pathogenic change in a gene causes disease, it is necessary to recognize how pathogenic mutations could affect a protein structure-function, protein-protein interactions in protein networks and how these changes could be associated with clinical parameters describing the disease phenotype. We imply molecular modeling to build protein structure, simulate the effect of pathogenic missense changes, provide a quantitative analysis of their impact on protein structure and stability, and corellate these findings with disease phenotype. Currently we use X-linked retinoschisis (XLRS), oculocutaneous albinism, and macular degeneration as our disease models. 1.XLRS is a vitreo-retinal degeneration caused by mutations in the protein retinoschisin (RS1), required for the structural and functional integrity of the retina. In our work the structural effect of mutational changes was analyzed in silico on the basis of atomic protein structure, molecular dynamics, and Gibbs free energy calculations. Previously, in 60 XLRS patients from NEI who share 27 missense mutations, the molecular models were correlated with retinal function as determined by the ERG and b-waves (Sergeev et al., 2010, HMG). To test the validity of this approach, we have now compared the molecular models with the electrophysiological features in a cohort of 38 XLRS patients from Moorfields Eye Hospital (London, UK) having one of 18 RS1 missense variants (Sergeev et al., 2013, HMG). Patients were grouped based on mutation severity predicted by molecular modeling: mild (class I), moderate (intermediate) and severe (class II). Most patients had an electronegative scotopic bright flash electroretinogram (ERG) (reduced b/a-wave ratio) in keeping with predominant inner retinal dysfunction. An association between the type of structural RS1 alterations and the severity of b/a-wave reduction was found in all but the oldest group of patients, significant in patients aged 15V30 years. Severe RS1 missense changes were associated with a lower ERG b/a ratio than were mild changes, suggesting that the extent of inner retinal dysfunction is influenced by the effect of the mutations on protein structure. The majority of class I mutations showed no changes involving cysteine residues. Class II mutations caused severe perturbations due to the removal or insertion of cysteine residues or due to changes in the hydrophobic core. The ERG b/a ratio in intermediate cases was abnormal but showed significant variability, possibly related to the role of proline or arginine residues. The grouping in classes helps predict the severity of ERG abnormalities relating to global inner retinal dysfunction and may influence patient management and the selection of candidates for possible future therapeutic interventions. The knowledge of a functional phenotype (b/a-ratios) from the patient genotype potentially could be useful in clinical trials or other clinical studies to identify patient groups with severe and less severe phenotypes and might provide some rationale to choose medical treatment for each group. Indeed, the small molecule drug treatment might be more appropriate for patients with mild missense changes. In contrast, gene therapy could be suggested as a treatment to compensate the effect of the null protein caused by the severe missense change. Finally, this work confirms the findings in our previous study (Sergeev et al, 2010, HMG), makes additional observations with a finer grading scale, and contains a comprehensive statistical analysis of in silico predictions for the completely independent patient cohort. For the first time this work does clearly show that the specific mutation makes a difference in the XLRS clinical severity and quite likely in the clinical course. In general, the ability to establish genotype-to-phenotype relationships could be used to assess disease risk using atomic models of proteins for a broad spectrum of inherited eye disorders. 2.Oculocutaneous albinism (OCA) is a rare genetic disorder of melanin synthesis that results in hypopigmented hair, skin, and eyes. Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin production and its gene is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive (Simeonov et al., 2013, Human Mutation). Previous studies have shown that temperature sensitive mutants exit the endoplasmic reticulum and enter the endosomal compartment at 31oC, but not 37oC, suggesting that intracellular targeting is an important mechanism for loss of enzymatic activity. To test these predictions on tyrosinase activity the intra-melanosomal domain of human tyrosinase (residues 19 - 469) and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae (Dolinska et al., PLOS One, 2013, resubmitted). The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1mg per 10g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37oC and neutral pH. Glycosylation occurred at several Asn residues, and the enzymes kinetic and pharmacologic properties were similar to the authentic enzyme described in the literature. We furthermore characterized the enzymology of two temperature-sensitive enzymes. The intra-melanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure. Indeed, compared to WT enzyme, the R422Q mutant at 37oC had lowered helical and increased beta-sheet content. This conformational change is equivalent to partial (localized) protein unfolding with concomitant reduction in the enzymes function. Hence, temperature dependent changes in catalytic efficiency of R422Q appear to be associated with structural change. The temperature sensitivity of the R422W mutant is not clearly detected by far-UV CD. This suggests that, at least in part, the temperature sensitive behavior is intrinsic to the tyrosinase structure. In conclusion, we have described methods which result in the production of pure wild-type and mutant tyrosinase proteins in quantities sufficient for targeted or panel drug screening. Pure recombinant enzyme would also have the potential for direct therapeutic treatment, if delivered to the retinal pigment epithelium of the eye prior to foveal development. Purified proteins can also be used for crystal screening and X-ray structure determination. High resolution structural information could be used, for example, in rational drug designs. Also, structural determinations could help understand at a molecular level how individual mutations contribute to the diverse phenotype in patients with albinism. 3.Age-related macular degeneration. Recently we have implied molecular modeling to investigate possibly risks of genetic mutations associated with macular degeneration and the potential functional consequences of the K155Q variant (Zhan X. et al., 2013, Nature Genetics, accepted).Our analysis identified K155Q as a rare C3 variant associated with a 2.91-fold increased risk of macular degeneration. Molecular modeling suggests that together with rare CFH variant R1210C and previously described common C3 variant R102G, K155Q may reduce binding of CFH to C3b, inhibiting the ability of Factor H to inactivate the alternative complement pathway.

为了了解基因的致病性变化如何引起疾病，有必要认识到致病突变如何影响蛋白质结构功能，蛋白质网络中的蛋白质 - 蛋白质相互作用以及这些变化如何与描述疾病表型的临床参数相关。我们暗示分子建模以建立蛋白质结构，模拟致病错义变化的影响，对其对蛋白质结构和稳定性的影响进行定量分析，并与疾病表型相结合。目前，我们将X连锁的视网膜（XLR），眼皮白化病和黄斑变性作为我们的疾病模型。 1.XLRS是由视网膜视网膜气概（RS1）突变引起的玻璃体 - 视网膜变性，是视网膜的结构和功能完整性所必需的。在我们的工作中，根据原子蛋白结构，分子动力学和Gibbs自由能计算，在计算机中分析了突变变化的结构效应。以前，在具有27个错义突变的NEI的60名XLRS患者中，分子模型与由ERG和B波的确定的视网膜功能相关（Sergeev等，2010，HMG）。为了测试这种方法的有效性，我们现在将分子模型与来自Moorfields眼科医院（英国伦敦）的38例XLRS患者组成的分子模型与具有18 RS1 rs1误差变体中的一个（Sergeev等，2013，HMG）中的人进行了比较。根据通过分子建模预测的突变严重程度进行分组：轻度（I类），中度（中级）和严重（II类）。大多数患者的电负性Scotopic明亮闪光电子图（ERG）（降低了B/A波比），这与主要视网膜功能障碍一致。在最古老的患者中，发现结构性RS1改变的类型与B/A波降低的严重程度之间存在关联，这对于15V30岁的患者很重要。与轻度变化相比，严重的RS1错义变化与ERG B/A的比率低有关，这表明内部视网膜功能障碍的程度受突变对蛋白质结构的影响的影响。 I类突变的大多数没有显示涉及半胱氨酸残基的变化。 II类突变由于去除或插入半胱氨酸残基而导致严重的扰动或由于疏水核心的变化。中间病例中的ERG B/A比异常，但显示出显着的变异性，可能与脯氨酸或精氨酸残基的作用有关。课程中的分组有助于预测与全球内部视网膜功能障碍有关的ERG异常的严重性，并可能影响患者的管理以及选择候选人以进行未来的治疗干预措施。患者基因型的功能表型（B/A-RATIOS）的知识可能在临床试验或其他临床研究中有用，以鉴定患有严重和较差表型的患者群体，并可能提供一些基本原理来为每个组选择医疗治疗。实际上，小分子药物治疗可能更适合于轻度错义变化的患者。相比之下，可以建议将基因治疗作为一种治疗方法来补偿因严重的错义变化而导致的无效蛋白的作用。 最后，这项工作证实了我们先前的研究中的发现（Sergeev等，2010，HMG），以更细的评分量表进行了其他观察结果，并包含对完全独立的患者队列的计算机预测中的全面统计分析。这项工作首次清楚地表明，特定的突变会在XLRS临床严重程度上有所不同，并且很可能在临床过程中。通常，建立基因型到表型关系的能力可用于使用蛋白质的原子模型来评估疾病的风险，以用于广泛的遗传性眼部疾病。 2.胶质性白化病（OCA）是一种罕见的黑色素合成遗传疾病，导致头发，皮肤和眼睛不形成。酪氨酸酶（Tyr）催化了限制速率，黑色素产生的第一步及其基因在许多眼皮白化病（OCA1）的情况下被突变，这是童年失明的常染色体隐性原因。 TyR活性降低的患者被归类为OCA1B；某些OCA1b突变对温度敏感（Simeonov等，2013，人类突变）。先前的研究表明，温度敏感的突变体退出内质网，并在31oC处进入内体室，但没有37oC，这表明细胞内靶向是损失酶活性的重要机制。为了测试酪氨酸酶活性的这些预测，人酪氨酸酶内螺体内结构域（残基19-469）和两个相关温度敏感的突变体，R422Q和R422W在昆虫细胞中表达，并在T. ni幼虫中产生，并在T. ni幼虫中产生（Dolinska等人（Dolinska等）（Dolinska et al。 删除了短的跨膜片段，以避免潜在的蛋白质不溶性，同时保留酶的所有其他功能特征。纯化的酪氨酸酶以每10克幼虫生物量> 1mg的屈服获得。该蛋白是一种单体糖酶，在37oC和中性pH下具有最大酶活性。糖基化发生在几个ASN残基上，并且动力学和药理学特性与文献中描述的正宗酶相似。我们进一步表征了两种温度敏感酶的酶学。重组野生型和突变的人酪氨酸酶的螺旋内域域是可溶性单体糖蛋白，其活性反映了它们的体内功能。与野生型蛋白相比，两个纯化的突变体的活性较低，温度敏感。这些差异与二级结构中的构象扰动有关。实际上，与WT酶相比，37oC处的R422Q突变体降低了螺旋型和增加的β表含量。这种构象变化等同于部分（局部）蛋白随着酶功能的降低而展开。因此，R422Q催化效率的温度依赖性变化似乎与结构变化有关。 Far-UV CD无法清楚地检测到R422W突变体的温度灵敏度。这表明至少部分地，温度敏感的行为是酪氨酸酶结构的固有的。总之，我们描述了导致纯野生型和突变酪氨酸酶蛋白的产生的方法，足以用于靶向或面板药物筛查。 如果在动脉凹发育前递送至眼睛的视网膜色素上皮，则纯重组酶也有可能直接治疗。纯化的蛋白质也可以用于晶体筛选和X射线结构测定。高分辨率的结构信息可以在理性的药物设计中使用。同样，结构测定可以帮助您了解分子水平的单个突变如何促进白化病患者的多种表型。 3.与年龄相关的黄斑变性。最近，我们隐含了分子建模，以研究与黄斑变性有关的遗传突变的风险以及K155Q变体的潜在功能后果（Zhan X.等，2013，自然遗传学，被接受）。您的分析确定了K155Q是罕见的C3变体，与2.91-毛细血管的风险相关。分子建模表明，与稀有的CFH变体R1210C一起，并先前描述的常见C3变体R102G，K155Q可以降低CFH与C3B的结合，从而抑制因子H灭活替代补体途径的能力。