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: 10020005
• 负责人: Yuri Sergeev
• 金额: $ 101.35万
• 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/10020005
• 关键词: 3-Dimensional; Active Sites; Affect; Age related macular degeneration; Algorithms; Anabolism; Apoferritin; Binding; Biochemical; Biological Models; Biophysics; CDH23 gene; Cadherins; Calnexin; Clinical; Clinical Research; Complement Factor H; Complex; Computer Analysis; Computer Simulation; Computing Methodologies; Consensus; Consumption; Crowding; DNA Sequence Alteration; Data; Databases; Disease; Drug Screening; Elements; Energy-Generating Resources; Enzymes; Epidermal Growth Factor; Evaluation; Exclusion; Eye; Eye diseases; FBN1; Fatty acid glycerol esters; Fibronectins; Free Energy; Gel Chromatography; Genes; Genetic; Genetic Annotation; Genetic Diseases; Genotype; Glycoproteins; Goals; Guanosine Triphosphate; Hair; Hereditary Disease; Hereditary Eye Diseases; Homo; Homologous Gene; Homology Modeling; Human; Immunoglobulins; In Vitro; Individual; Inherited; Investigation; Kinetics; Knowledge; Laminin; Larva; Lectin; Length; Ligands; Measures; Melanins; Melanogenesis; Melanosomes; Membrane Proteins; Metabolic Pathway; Methods; Missense Mutation; Molecular; Molecular Chaperones; Molecular Weight; Monophenol Monooxygenase; Mutagenesis; Mutation; Oculocutaneous Albinism; Oxidases; PCDH15 gene; Pathogenicity; Patients; Pattern; Pharmaceutical Preparations; Phenotype; Preventive therapy; Process; Proteins; Proteome; Quality Control; Recombinant Proteins; Recombinants; Reporting; Retinitis Pigmentosa; Role; Sequence Alignment; Sequence Homologs; Severities; Skin Pigmentation; Structural Protein; Structure; System; TYRP1 gene; Technology; Tertiary Protein Structure; Time; Transforming Growth Factor beta; Tyrosinase related protein-1; Variant; Visual system structure; Water; Weight; Wood material; Work; calreticulin; disease phenotype; experimental study; fibrillin-2; genetic analysis; genetic variant; improved; in vivo; insight; interest; melanocyte; misfolded protein; models and simulation; molecular dynamics; molecular modeling; mutant; next generation sequencing; protective effect; protein expression; protein folding; protein protein interaction; protein structure; protein structure function; sedimentation equilibrium; small molecule; stereochemistry; tool; web site

To understand how a pathogenic mutation causes inherited eye disease, it is necessary to recognize how pathogenic mutations could affect protein structure-function, metabolic pathways, and how these perturbations could be associated clinical parameters describing the disease phenotype. For this purpose, we express and purifying proteins targeted by missense mutations in inherited eye disease with a purpose to mimic the effect of pathogenic mutations in vitro. Also, we are trying to develop computational methods, perform molecular modeling of atomic protein structures, and predict the effect of missense changes from the inherited disease. Results of this analysis are presented at the ocular proteome website. This year we successfully purified and analyzed the function of the full-length and truncated versions of human recombinant tyrosinase-related protein 1, TYRP1 and TYRP1tr. We also developed an effective method for the prediction of missense mutation severity in multidomain proteins from inherited disease. Tyrosinases are melanocyte specific enzymes involved in melanin biosynthesis. Mutations in their genes cause oculocutaneous albinism associated with reduced or altered pigmentation of skin, hair, and eyes. The recombinant human intra-melanosomal domains of tyrosinase, TYRtr(19-469), and TYRP1tr (25-472), and their full-length variants were studied in vitro to define their functional relationship (Dolinska et al, PCMR, 2019). Proteins were expressed or coexpressed in whole Trichoplusia ni larvae and purified. Their associations were studied using gel filtration and sedimentation equilibrium methods. Protection of TYRtr was demonstrated for the first time by measuring the kinetics of tyrosinase diphenol oxidase activity in the presence (1:1 and 1:20 molar ratios) or the absence of TYRP1tr for 10 hr under conditions mimicking melanosomal and ER pH values. Our data indicate that TYRtr incubation with excess TYRP1tr protects TYR, increasing its stability over time. However, this mechanism does not appear to involve the formation of stable heterooligomeric complexes to maintain the protective function of a protein. Our finding could be important for understanding the functions of tyrosinases in melanogenesis. To determine the specific molecular mechanism underlying tyrosinase interactions, we analyzed the potential interaction between the proteins using several different model systems in both in vitro and in vivo conditions. Using gel-filtration and sedimentation equilibrium methods, we illustrated that TYRP1tr is a monomeric glycoprotein with a weight-average molecular weight of 60 kDa and does not form stable homo-oligomers. Previous studies in vivo suggested that TYRP1 functions as a molecular chaperone for TYR in the ER, which suggests that interactions of TYRP1 with TYR occur in the ER and that homo-oligomerization of TYR is a step-in proper protein maturation within the ER. However, this idea could be easily criticized. First, there is no direct observation that TYRP1 helps tyrosinase maintain a native protein fold in vitro. However, the lectin chaperones calnexin and calreticulin, which are components of the ER quality control system associate with TYR to help the protein folding. Second, there is no observation of TYRP1 consuming energy to perform the protective function, in contrast to chaperone-like molecules requiring ATP or GTP as an energy source to perform their function. Third, according to our in vitro data, TYRP1 is monomeric and does not form a large oligomeric homo-complex to provide a protein surface for the binding of misfolded proteins, as suggested previously. From this view, the mechanism of the TYRP1 protection could be different from that of classical chaperone function. In addition, we observed an improvement in catalytic activity and a threefold increase in TYRtr efficiency with a 20fold excess of TYRP1tr at the pH of melanosomes (pH 5.5). The significant excess of TYRP1tr led us to the possible explanation that its protective effect could be related to molecular crowding (Minton, 2001, 2006), which is a mechanism that has been frequently reported for other proteins. Molecular crowding has been shown to enhance the protein stability, intrinsic catalytic efficiency, and structure of proteins. Crowding is characterized by molecular volume exclusion due to the excess of a highly concentrated protecting protein with decreases the available space ligands may occupy, bringing them in closer proximity to a corresponding active site, and complex pattern of interatomic interactions at protein surfaces of apoferritin (Sergeev et al., 2018). Therefore, molecular crowding might provide an explanation for the improvement in TYRtr function with an excess of protecting protein, TYRP1tr. One of the challenging tasks in the analysis of genetic alterations is related to the absence structure of multi-domain proteins, some of which contained up to several hundred structural domains and atomic structure are not available. These proteins account for 70% of the eukaryotic proteome and in the majority are very difficult for computational analysis. In genetic disease, multi-domain proteins are affected by numerous missense mutations. The mutation effects on protein stability and their roles in genetic disease are unspoken. With a purpose to understand how to analyze such proteins, we selected proteins targeted by genetic alterations in inherited eye disease (Wood Ortiz & Sergeev, Scientific Reports,2019). The domain stability evaluation was performed for nine proteins, Eyes Shut homolog, Fibrillin-1, Fibrillin-2, Complement Factor H, Protocadherin-15, Protocadherin Fat 1, Protocadherin Fat 4, Roundabout homolog 3, and Cadherin-23. Alterations in these proteins cause genetic eye diseases such as retinitis pigmentosa, age-related macular degeneration, and others. To simplify the analysis, the proteins were split into individual domains. Each domain structure was built using homology modeling and then divided into 7 groups using protein fold similarities. These domain groups were epidermal growth factor-like, laminin globular, sushi, immunoglobulin-like C2-type, fibronectin type-III, cadherin, and transforming growth factor-beta. In total, the 291 protein domain structures were individually homology-modeled, equilibrated using 2 ns molecular dynamics in water to achieve better domain stereochemistry, and subjected to global computational mutagenesis to evaluate the effect of mutations on the protein stability. Mutation propensities within each group of domains were then averaged to find residues critical for protein stability of domain fold. The consensus derived from the sequence alignment shows that the critical residues determined by global mutagenesis are conserved within each group. From the global mutagenesis, we concluded that 80% of known disease-related genetic variants are associated with the residues critical for proper maintaining of protein fold and are expected to have significant destabilizing effects on domain structure. Our work provides an in-silico quantification of protein stability and could help to analyze the complex relationship among missense mutations, multi-domain protein stability, and disease phenotypes in inherited eye disease. Results of analysis of multi-domain proteins were incorporated in the ocular proteome web-site at the NEI Commons (https://neicommons.nei.nih.gov/#/proteomeData). The latest version of the ocular proteome web site contains in-silico predictions for 1,407,120 missense mutations associated with 111 protein structures from 163 inherited eye diseases. However, further progress in a building of the ocular proteome database is limited due to the computational complexity of the algorithm and a large volume of data.

为了了解致病性突变如何导致遗传性眼病，有必要认识到致病性突变如何影响蛋白质结构功能、代谢途径，以及这些扰动如何与描述疾病表型的临床参数相关联。为此，我们表达并纯化了遗传性眼病中错义突变所针对的蛋白质，目的是在体外模拟致病突变的影响。此外，我们正在尝试开发计算方法，对原子蛋白质结构进行分子建模，并预测遗传性疾病错义变化的影响。该分析的结果发布在眼部蛋白质组网站上。今年我们成功纯化并分析了人重组酪氨酸酶相关蛋白1、TYRP1和TYRP1tr的全长和截短版本的功能。我们还开发了一种有效的方法来预测遗传性疾病多域蛋白错义突变的严重程度。 酪氨酸酶是参与黑色素生物合成的黑素细胞特异性酶。它们的基因突变会导致与皮肤、头发和眼睛色素沉着减少或改变相关的眼皮肤白化病。对酪氨酸酶 TYRtr(19-469) 和 TYRP1tr (25-472) 的重组人黑素体内结构域及其全长变体进行了体外研究，以确定它们的功能关系 (Dolinska 等人，PCMR，2019)。在整个粉纹夜蛾幼虫中表达或共表达蛋白质并纯化。使用凝胶过滤和沉降平衡方法研究了它们的关联。通过在模拟黑素体和 ER pH 值的条件下测量存在（1:1 和 1:20 摩尔比）或不存在 TYRP1tr 的情况下酪氨酸酶二酚氧化酶活性 10 小时的动力学，首次证明了 TYRtr 的保护作用。我们的数据表明，TYRtr 与过量 TYRP1tr 一起孵育可以保护 TYR，随着时间的推移提高其稳定性。然而，这种机制似乎并不涉及形成稳定的异寡聚复合物以维持蛋白质的保护功能。 我们的发现对于理解酪氨酸酶在黑色素生成中的功能可能很重要。 为了确定酪氨酸酶相互作用的具体分子机制，我们在体外和体内条件下使用几种不同的模型系统分析了蛋白质之间的潜在相互作用。使用凝胶过滤和沉降平衡方法，我们证明TYRP1tr是一种重均分子量为60 kDa的单体糖蛋白，并且不形成稳定的同源寡聚物。 先前的体内研究表明，TYRP1 在 ER 中充当 TYR 的分子伴侣，这表明 TYRP1 与 TYR 的相互作用发生在 ER 中，并且 TYR 的同源寡聚化是 ER 内蛋白质正确成熟的一个步骤。 然而，这个想法很容易受到批评。首先，没有直接观察到 TYRP1 有助于酪氨酸酶在体外维持天然蛋白质折叠。 然而，凝集素伴侣钙联蛋白和钙网蛋白是 ER 质量控制系统的组成部分，与 TYR 结合以帮助蛋白质折叠。其次，没有观察到 TYRP1 消耗能量来执行保护功能，而类伴侣分子则需要 ATP 或 GTP 作为能源来执行其功能。第三，根据我们的体外数据，TYRP1是单体的，并且不会形成大的寡聚同源复合物来为错误折叠蛋白的结合提供蛋白表面，如之前所建议的。从这个角度来看，TYRP1 的保护机制可能不同于经典的伴侣功能。 此外，我们观察到在黑素体的 pH 值（pH 5.5）下，当 TYRP1tr 过量 20 倍时，催化活性得到改善，并且 TYRtr 效率提高了三倍。 TYRP1tr 的显着过量使我们得出了可能的解释，即其保护作用可能与分子拥挤有关（Minton，2001，2006），这是其他蛋白质经常报道的一种机制。分子拥挤已被证明可以增强蛋白质稳定性、内在催化效率和蛋白质结构。拥挤的特点是由于高度浓缩的保护蛋白过量而导致分子体积排斥，配体可能占据的可用空间减少，使它们更接近相应的活性位点，以及脱铁铁蛋白表面原子间相互作用的复杂模式（Sergeev等人，2018）。因此，分子拥挤可能为过量保护蛋白 TYRP1tr 改善 TYRtr 功能提供了解释。 遗传改变分析中具有挑战性的任务之一与多结构域蛋白质的缺乏结构有关，其中一些蛋白质包含多达数百个结构域，并且原子结构不可用。这些蛋白质占真核蛋白质组的 70%，其中大多数很难进行计算分析。在遗传疾病中，多结构域蛋白受到许多错义突变的影响。突变对蛋白质稳定性的影响及其在遗传疾病中的作用是不言而喻的。为了了解如何分析此类蛋白质，我们选择了遗传性眼病遗传改变的目标蛋白质（Wood Ortiz & Sergeev，科学报告，2019）。对九种蛋白质进行了结构域稳定性评估：Eyes Shut 同源物、Fibrillin-1、Fibrillin-2、补体因子 H、原钙粘蛋白-15、原钙粘蛋白脂肪 1、原钙粘蛋白脂肪 4、Roundabout 同源物 3 和钙粘蛋白-23。这些蛋白质的改变会导致遗传性眼部疾病，例如色素性视网膜炎、年龄相关性黄斑变性等。为了简化分析，蛋白质被分成单独的结构域。使用同源建模构建每个结构域结构，然后使用蛋白质折叠相似性将其分为 7 组。这些结构域组包括表皮生长因子样、层粘连蛋白球状、寿司状、免疫球蛋白样 C2 型、纤连蛋白 III 型、钙粘蛋白和转化生长因子-β。总共，对 291 个蛋白质结构域结构进行了单独同源建模，使用水中 2 ns 分子动力学进行平衡，以实现更好的结构域立体化学，并进行全局计算诱变以评估突变对蛋白质稳定性的影响。然后对每组结构域内的突变倾向进行平均，以找到对结构域折叠的蛋白质稳定性至关重要的残基。从序列比对得出的共识表明，通过全局诱变确定的关键残基在每组内都是保守的。从整体诱变中，我们得出的结论是，80% 的已知疾病相关遗传变异与对正确维持蛋白质折叠至关重要的残基相关，并且预计会对结构域结构产生显着的不稳定影响。我们的工作提供了蛋白质稳定性的计算机量化，有助于分析错义突变、多结构域蛋白质稳定性和遗传性眼病的疾病表型之间的复杂关系。 多结构域蛋白质的分析结果已纳入 NEI Commons 的眼部蛋白质组网站 (https://neicommons.nei.nih.gov/#/proteomeData)。眼部蛋白质组网站的最新版本包含 1,407,120 个错义突变的计算机预测，这些突变与 163 种遗传性眼病的 111 种蛋白质结构相关。然而，由于算法的计算复杂性和数据量大，眼部蛋白质组数据库建设的进一步进展受到限制。