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: 8556850
• 负责人: Yuri Sergeev
• 金额: $ 53.57万
• 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/8556850
• 关键词: 3-Dimensional; Active Sites; Adolescent; Affect; Affinity; Affinity Chromatography; Albinism; Amino Acid Sequence; Amino Acids; Binding; Binding Proteins; Binding Sites; Biological Assay; Bruch' s basal membrane structure; C-terminal; Calcium; Calcium Binding; Calcium ion; Catalytic Domain; Charge; Clinical; Clinical Research; Computational Biology; Computer Simulation; Consensus Sequence; Copper; Cysteine; DNA; Development; Diagnostic; Disease; Disease model; Elastic Fiber; Elasticity; Elastin; Elements; Engineering; Entropy; Enzymes; Epidermal Growth Factor; Escherichia coli; Extracellular Matrix; Eye; Eye diseases; Fiber; Functional disorder; Future; Gene Mutation; Generations; Genes; Genets; Genotype; Goals; Hair; Heat shock proteins; Hereditary Disease; Hereditary Eye Diseases; His-His-His-His-His-His; Homeostasis; Homology Modeling; Human; Hydrophobic Interactions; Hydrophobic Surfaces; Hydrophobicity; Inherited; Investigation; Ions; Knockout Mice; Larva; Lead; Length; Levodopa; Ligation; Link; Location; Macular degeneration; Medical; Medicine; Melanins; Membrane; Membrane Proteins; Metals; Microfibrils; Missense Mutation; Mixed Function Oxygenases; Molecular; Molecular Chaperones; Molecular Conformation; Molecular Models; Monophenol Monooxygenase; Mutagenesis; Mutation; National Human Genome Research Institute; Nature; Oculocutaneous Albinism; Pathogenicity; Patients; Peptide Signal Sequences; Peptides; Pharmaceutical Preparations; Phenotype; Physiological; Plant Resins; Positioning Attribute; Preventive; Process; Property; Proteins; Quinones; Reaction; Relative (related person); Reporting; Retinal Degeneration; Retinal Diseases; Role; Screening procedure; Sequence Homologs; Severities; Side; Simulate; Site; Skin; Solubility; Space Perception; Stress; Structure; Structure-Activity Relationship; Surface; TYRP1 gene; Temperature; Testing; Time; Transforming Growth Factors; Tyrosine; Variant; Visual system structure; Western Blotting; Work; X-Linked Retinoschisis; XLRS1 protein; Zinc; age group; age related; alpha-Crystallin A Chain; base; calcification; cohort; computer studies; design; disease phenotype; disease-causing mutation; disorder of macula of retina; fibrillin; fibrillin-2; flexibility; improved; insight; intermolecular interaction; male; molecular dynamics; molecular modeling; mutant; polypeptide; prevent; protein aggregation; protein expression; protein folding; protein protein interaction; protein structure; protein structure function; research study; three dimensional structure; 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, and provide a quantitative analysis of their impact on protein structure and stability. Here we use oculocutaneous albinism, autosomal dominant maculopathy, and X-linked retinoschisis (XLRS) as our disease models. 1. Oculocutaneous albinism (OCA) is a rare genetic disorder of melanin synthesis that results in hypopigmented hair, skin, and eyes. There are currently four types of OCA. For the first time, three full-length protein atomic structures, TYR (OCA1), TYRP1 (OCA3), and SLC45A2 (OCA4), were successfully modeled by homology and in silico analysis of missence changes from the NEI/NHGRI molecular diagnostic study has been performed. TYR is a type I trans-membrane monooxygenase. The 4-helix bundle is structurally conserved in different species to carry CuA and CuB ions essential for the catalytic reaction. The active site is formed by 6 His residues structurally coordinating the copper positions. Several missense changes from the collaborative NEI/NHGRI molecular diagnostic study were analyzed. S50L is found in the Cys-rich motif 1 of tyrosinase, whereas R298W is located within the Cys-rich motif 2. The mutations N364H, P384A, D394N, D437N and R403V disrupt the coordination of the copper ion center. A490D affects conformation of helix located in melanosomal membrane. TYRP1 is a type 1 membrane protein. This protein also has 2 Cys-rich motifs and an active site with 6 His residues coordinating 2 metal ions of unknown nature that could be either copper or zinc. A heterozygous missense mutation, A24T, was found in the border between a signal peptide and Cys-rich motif 1 of TYRP1. The structure of SLC45A2 is predicted as a multi-pass trans-membrane protein. The missense mutation L60R is predicted to be deleterious. To test our predictions on tyrosinase activity we engineered a construct of human C-terminal truncated tyrosinase, hTyrCtr. The expression of hTyrCtr in E. Coli was confirmed by Western blot analysis. Metal affinity chromatography shown poor binding to the column which suggests that C-terminal His-tag peptides have decreased binding capacity to the IMACS resin. In addition, L-Dopa enzymatic assay demonstrated that hTyrCtr is expressed as a non-active enzyme which might be due to either the loss of Cu2+ ions in a catalytic site or protein partial misfolding. In contrast, similar protein construct was implied for the protein expression in larvae. We shown that the hTyrCtr, and 2 mutant variants, R422Q and R422W, are active soluble proteins which catalyzes the rate-limiting conversions of tyrosine to DOPA and DOPA to DOPA-quinone. In perspective, a detailed understanding of protein structure and the mechanisms controlling tyrosine-modified tyrosinase interactions would allow to establish molecular chaperone screening for a future medical treatment of patients with the OCA-1B albinism. 2. Assembly of elastic fibers is critical for structural development as well as proper functioning of the extracellular matrix. Elastin and 10-nm fibrillin containing microfibrils form the major components of elastic fibers, which form integral part of extracellular matrices including Bruchs membrane. One of fibrillins, fibrillin-2 or FBN2, is a 2,912 amino acid polypeptide which consists of one amino-terminal trans-membrane domain, 4 epidermal growth factor-like (EGF) domains, 43 calcium-binding consensus sequences (Ca_EGF domains), and 9 transforming growth factor 1 binding protein-like (TB) domains. FBN-2 has 363 cysteine residues. The amino acid sequence of fibrillin-2 been used to generate a native and mutant variant structures for the Ca-EGF motifs 12-19 by homology modeling. Protein fold of Ca_EGF domain is maintained by 6 conserved cysteines which form 3 SS-bridges. In addition, negatively charged conserved residues are either involved in direct ligation to calcium or involved in stabilizing the calcium-binding site. Calcium ion improves the fold stability, help to fix a relative orientation of two neighbor Ca_EGF domains, and stabilize a spatial orientation of FBN-2. Disease-causing mutation E1144K introduces a positive charge into the negatively charged cavity and decreases the Ca-binding affinity. The interaction of K1144 and E1178 change a relative orientation of a neighbor domain. E1438K mutation is expected to have a similar structural effect. Both mutations are associated with a severe phenotype of disease and could change of microfiber packing and elasticity. The M1247T change is affecting the hydrophobic surface loop. The SS-bridge C1246-C1257 stabilizes the native fold of a protein by lowering entropy of the polypeptide chain and by condensing hydrophobic residues from the surface loop into local hydrophobic core using hydrophobic interactions. Thus, the mutation M1247T might affect the SS-bond stability and/or intermolecular interactions. Other mutations are mild changes. Mutations with severe phenotype are likely to cause a change in the fiber flexibility, packaging, and pathogenicity. This might cause the loss of elastic fibers, thickening and calcification of Bruchs membrane which are associated with dominant maculopathy and AMD pathophysiology. 3. Gene mutations that encode retinoschisin (RS1) cause X-linked retinoschisis (XLRS), a form of juvenile macular and retinal degeneration that affects males. Molecular modeling predicted an association between the type of structural RS1 alterations and the severity of full-field ERG phenotype in all but the oldest group of patients. This is now a second study (Hum Mol Genet 19:1302, 2010) that indicates a genotype-ERGphenotype correlation, and it was done with a totally separate and independent cohort. There was a significant association between the predicted severity of RS1 perturbation and both photopic and scotopic ERG b/a-ratios, but only for one age group (15-30 years). Severe RS1 missense changes were associated with a lower ERG b/a ratio than for mild and moderate missense changes, suggesting a quantitatively distinct ERG phenotype. Age-related differences in dark-adapted ERG parameters are consistent with those reported previously in the RS1 knockout mouse. 4. One of possible clinical implications in a human eye disease is using chaperones for the stabilization of native protein structure in mutant variants affected by genetic mutations. This stabilization could be performed in controllable fashion by using small heat shock proteins (sHSPs) with genetically engineered structure. Our recent study have indicated a role for changing of protein hydrophobicity in the thermal adaptation of alpha-crystallin A and suggested ways to produce sHSP variants with altered chaperone-like activity. In this work we use molecular modeling, computational biology, and side-directed mutagenesis to evaluate the effect of mutations and to establish a link between sHSPs hydrophobicity and physiological temperatures. sHSPs maintain cellular homeostasis by preventing stress and disease-induced protein aggregation. In addition, our work provided an evidence for an evolutionary mechanism that has adapted chaperone activity to different environmental temperatures though the alteration of hydrophobicity at crucial locations in the protein structure. This combination of experimental and computational design potentially could be used to create a new generation of artificial chaperones with a purpose to improve stability of mutant variants in inherited eye disease.

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，并对它们对蛋白质结构和稳定性的影响进行定量分析。在这里，我们使用眼皮白化病，常染色体显性baculopathy和X连锁视网膜（XLR）作为我们的疾病模型。 1。眼皮白化病（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。目前有四种类型的OCA。首次通过同源性和硅基分析，对NEI/NHGRI分子诊断研究的失误变化进行了成功建模的三个全长蛋白质原子结构Tyr（OCA1），Tyrp1（OCA3）和SLC45A2（OCA4）。 Tyr是I型跨膜单加氧酶。 4-螺旋束在不同物种上在结构上保守，可携带Cua和Cub离子，对催化反应必不可少。活性位点由6个残留物在结构上协调铜位置形成。分析了协作NEI/NHGRI分子诊断研究的几种错义变化。 S50L在酪氨酸酶的Cys富基序1中发现，而R298W位于富含Cys的基序2中。 A490D会影响位于黑色素体膜中的螺旋的构象。 Tyrp1是1型膜蛋白。该蛋白质还具有2个富含Cys的基序和一个活性位点，其残留物与可能是铜或锌的2个未知性质的残基配位。在信号肽和Tyrp1的Cys富基序之间发现了杂合的错义突变A24T。 SLC45A2的结构被预测为多通跨膜蛋白。预计错义突变L60R是有害的。 为了测试我们对酪氨酸酶活性的预测，我们设计了人类C末端截短的酪氨酸酶Htyrctr的构建体。通过蛋白质印迹分析证实了大肠杆菌中htyrctr的表达。金属亲和力色谱显示与色谱柱结合较差，这表明C末端HIS-TAG肽的结合能力降低了与iMacs树脂的结合能力。此外，L-DOPA酶试验表明，HTYRCTR表示为一种非活性酶，这可能是由于催化位点中Cu2+离子的丢失或蛋白质部分错误折叠所致。相反，对于幼虫的蛋白质表达暗示了相似的蛋白质构建体。我们表明，HTYRCTR和2个突变体变体R422Q和R422W是活跃的可溶性蛋白，可催化酪氨酸到DOPA和DOPA到Dopa-喹酮的速率限制转换。从角度来看，对蛋白质结构和控制酪氨酸改性酪氨酸酶相互作用的机制的详细理解将允许建立分子伴侣筛选，以对OCA-1B白化病患者的未来医疗治疗进行筛查。 2。弹性纤维的组装对于结构发育以及细胞外基质的正常功能至关重要。 含有微纤维的弹性蛋白和10-nm纤维蛋白形成了弹性纤维的主要成分，弹性纤维形成了包括Bruchs膜在内的细胞外基质的组成部分。 Fibrillins之一Fibrillin-2或FBN2是一种2,912个氨基酸多肽，由一个氨基末端的跨膜结构域组成，4个表皮生长因子（EGF）结构域，43个钙结合结合序列（CA_EGF结构域），以及9个转化的生长因子（CA_EGF结构域）。 FBN-2具有363个半胱氨酸残基。 Fibrillin-2的氨基酸序列用于通过同源性建模为12-19的CA-EGF基序生成天然和突变变体结构。 CA_EGF结构域的蛋白质折叠由6个保守的半胱氨酸保持，形成3个SS-Bridges。此外，带负电荷的保守残基与钙直接连接或稳定钙结合位点有关。 钙离子可以提高折叠稳定性，有助于固定两个邻居CA_EGF域的相对方向，并稳定FBN-2的空间取向。引起疾病的突变E1144K向带负电荷的腔引入正电荷，并降低了CA结合亲和力。 K1144和E1178的相互作用改变了邻居域的相对方向。 E1438K突变有望具有相似的结构效应。这两种突变都与严重的疾病表型有关，并且可能会改变超细纤维堆积和弹性。 M1247T变化正在影响疏水表面环。 SS桥C1246-C1257通过降低多肽链的熵并使用疏水相互作用将疏水性残基从表面环凝结到局部疏水性核心，从而稳定蛋白质的天然褶皱。因此，突变M1247T可能会影响SS键的稳定性和/或分子间相互作用。其他突变是轻微的变化。严重表型的突变可能会导致纤维柔韧性，包装和致病性发生变化。这可能会导致弹性纤维的丧失，与主导肿瘤和AMD病理生理学相关的BRUCHS膜的增厚和钙化。 3。编码视网膜气概（RS1）的基因突变引起X连接的视网膜静脉（XLRS），这是一种影响男性的幼年黄斑和视网膜变性的形式。分子建模预测结构RS1改变的类型与除最古老的患者以外的所有型号ERG表型的严重程度之间存在关联。现在，这是一项第二项研究（Hum Mol Genet 19：1302，2010），它表明基因型 - ergenotype相关性，它是通过完全独立且独立的队列进行的。 RS1扰动的预测严重程度与光波器和SCOTOPIC ERG B/A-RATIOS之间存在显着关联，但仅适用于一个年龄段（15-30岁）。严重的RS1错义变化与比轻度和中等错义变化的ERG B/A比低有关，这表明具有定量的ERG表型。黑暗适应性ERG参数的年龄相关差异与先前在RS1敲除小鼠中报道的参数一致。 4。人眼病中可能的临床意义之一是使用伴侣来稳定受基因突变影响的突变体变异体中天然蛋白质结构。可以通过使用具有基因工程结构的小热激蛋白（SHSP）来以可控的方式进行这种稳定。我们最近的研究表明，蛋白质疏水性在α-晶状体A的热适应中的作用，并建议产生具有伴侣样活性改变的SHSP变体的方法。在这项工作中，我们使用分子建模，计算生物学和侧向诱变来评估突变的效果，并在SHSP疏水性和生理温度之间建立联系。 SHSP通过预防应激和疾病诱导的蛋白质聚集来维持细胞稳态。此外，我们的工作为进化机制提供了证据，该机制使伴侣活性适应了不同的环境温度，尽管蛋白质结构中关键位置的疏水性改变了。实验设计和计算设计的这种结合可能可用于创建新一代的人造伴侣，目的是提高遗传性眼病中突变变体的稳定性。