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: 9362393
• 负责人: Yuri Sergeev
• 金额: $ 65.6万
• 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/9362393
• 关键词: 3-Dimensional; Adolescent; Affect; Alanine; Albinism; Area; Arginine; Binding; Biochemical; Biomass; Blindness; Catalytic Domain; Cattle; Charge; Childhood; Choroid; Choroideremia; Clinical; Clinical Data; Clinical Research; Collaborations; Collection; Communities; Computer Simulation; DNA; DNA Sequence Alteration; Disease; Disease model; Documentation; Elements; Enzymes; Eye; Eye diseases; Free Energy; Genes; Genetic; Genomic medicine; Genotype; Glutamic Acid; Goals; Hair; Hereditary Disease; Hereditary Eye Diseases; Heterogeneity; Histidine; Homology Modeling; Human; Hydrogen Bonding; In Vitro; Inbreeding; Individual; Inherited; Investigation; Knowledge; Leber' s amaurosis; Length; Libraries; Link; Macular degeneration; Measures; Medical Genetics; Melanins; Membrane; Membrane Proteins; Metabolic Pathway; Missense Mutation; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Molecular Genetics; Molecular Models; Monophenol Monooxygenase; Movement; Mutate; Mutation; Mutation Analysis; Natural Products; Nature; Night Blindness; Nucleotides; Oculocutaneous Albinism; Onset of illness; Patients; Pharmaceutical Preparations; Pharmacologic Substance; Phenotype; Phosphorylation; Phosphotransferases; Photoreceptors; Positioning Attribute; Preventive therapy; Process; Production; Proline; Protein Kinase; Proteins; Rab escort protein; Recombinants; Reporting; Retina; Retinal Diseases; Risk; Role; Saudi Arabia; Sequence Analysis; Sequence Homologs; Severities; Side; Skin; Sodium Chloride; Structure; Structure of retinal pigment epithelium; Symptoms; Technology; Testing; Threonine; Translational Research; Tribes; Valine; Variant; Visual; Visual Acuity; Visual Fields; Visual system structure; Water; Width; alpha helix; chaperonin CCT; clinical phenotype; computer studies; constriction; disease phenotype; disease-causing mutation; drug use screening; founder mutation; gene therapy; genetic variant; guanylate cyclase 1; high throughput screening; improved; inhibitor/antagonist; insight; interest; kindred; loss of function mutation; macular dystrophy; male; molecular dynamics; molecular modeling; mutant; next generation sequencing; novel; protein complex; protein expression; protein folding; protein purification; protein structure; protein structure function; research study; small molecule; tool

In order to understand how a pathogenic mutation causes inherited eye disease, it is necessary 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 perform molecular modeling to build protein structure, evaluate the severity of genetic missense changes from the atomic level of protein, and provide a quantitative analysis of the mutation impact on protein structure, stability and catalytic activity. We also do experimental in vitro studies for proteins of interest to measure the protein fold destabilization and changes in catalytic activity caused by the disease-related mutations. Finally we correlate these findings with clinical phenotypes from inherited eye disease. In addition, in collaboration with the National Center for Advancing Translational Sciences (NCATS) we search for drug activators of catalytic activity of mutant protein affected by genetic mutation. This year we were using oculocutaneous albinism, Stargardts macular degeneration, choroideremia (CHM), Leber congenital amaurosis (LCA), and others as our disease models. Oculocutaneous albinism 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 no or reduced TYR activity are classified as OCA1A or OCA1B forms, respectively. This year we performed large scale protein purifications for the human tyrosinase. In total 70 mg of catalytically active tyrosinase intra-melanosomal domain (truncated tyrosinase) were purified from the larval biomass and were a subject of high-throughput screens at the NCATS. To date 17,011 compounds from the Genesis Drug Collection, 2,108 compounds from the Natural Products Library, and 2,816 compounds from the NCATS Pharmaceutical Collection were successfully screened. First search has found new inhibitors (>100) and several activators of tyrosinase. Recently we also purified a recombinant full-length tyrosinase, which contains both, intra-melanosomal and trans-membrane domains. We demonstrated that full-length and truncated tyrosinases have similar enzymatic activities. This study validates our drug screening, where we are using the truncated protein. In addition, we performed biochemical, biophysical, and in silico studies of human recombinant tyrosinases to understand folding and stability of missense changes R402Q, P406L, R422Q, and R422W mimicking mutations in OCA1B form of albinism. We also implied molecular modeling to investigate the potential structural and functional consequences as well as possible risks associated with genetic mutations causing inherited eye diseases. Autosomal recessive Stargardts disease is the most common form of juvenile macular dystrophy and results from mutations in the ABCA4 gene. Around 50% of pathogenic ABCA4 missense mutations occur in the trans-membrane or nucleotide binding domains. The atomic structure of these domains obtained using molecular modeling. We improved molecular modeling of disease causing mutations and testing our predictions with clinical data, which were significantly expanded on this year. CHM is an X-linked degeneration of the retinal pigment epithelium, photoreceptors, and choroid, which causes nyctalopia and progressive constriction of visual fields leading to blindness. The CHM gene encodes Rab escort protein 1 (REP-1). We performed molecular modeling of a REP-1 functional protein complex (Freund P.R., Sergeev Y.V., MacDonald I.M., Molecular Genetics & Genomics Medicine, 2016). A retrospective review of 128 affected males was performed analyzing the onset of symptoms, visual acuity, and visual fields with respect to their mutations in the CHM gene. In the pool of 106 CHM mutations, four novel missense mutations were discovered. The mutations, L80F, Q273H, M443V, and L457P, predicted to be severe changes affecting protein stability and folding with the effect similar to that of other types of mutations currently known for CHM. Therefore, all CHM patients have the Loss of Function mutations and show no functional REP-1. This result confirms the clinical data shown no significant genotype-phenotype correlation in respect to the onset of nyctalopia, the onset of other visual symptoms, visual acuity, or width of visual fields. We also modeled the structure for GUCY2 protein to show a role of genetic mutation in LCA (Gradstein L. et al., BMC Medical Genetics, 2016). The purpose of this study was to clinically characterize and identify the cause of disease in a large inbred Bedouin Israeli tribe with LCA. Sequencing of GUCY2D identified a novel missense mutation (c.2129C>T; p.Ala710Val) resulting in substitution of alanine by valine at position 710 within the protein kinase domain of the retina-specific enzyme guanylate cyclase 1 (GC1) encoded by GUCY2D. Molecular modeling implied that the mutation changes the conformation of the regulatory segment within the kinase styk-domain of GC1 and causes loss of its helical structure, likely inhibiting phosphorylation of threonine residue within this segment, which is needed to activate the catalytic domain of the protein. This is the first documentation of the p.Ala710Val mutation in GC1 and the second ever described mutation in its protein kinase domain. These findings enlarge the scope of genetic variability of LCA, highlight the phenotypic heterogeneity found amongst individuals harboring an identical LCA mutation, and possibly provide hope for gene therapy in patients with this congenital blinding disease. As the Bedouin kindred studied originates from Saudi Arabia, the mutation found might be an ancient founder mutation in that large community. Another target for genetic mutations in LCA is molecular chaperone CCT2 (Minegishi et al., Scientific Reports-Nature, 2016). To predict whether these mutants have actual impacts on disease onset, molecular modeling was conducted. Atomic structure of each of 8 domains was individually modeled by homology. The final structure of hetero-octamer was built using the bovine TRIC/CCT chaperone as a structural template. The CCT2 missense mutations T400P and R516H were generated in optimized conformation using the 1ns molecular dynamics in water. The change of the proline residue in position 400 disrupt alpha-helical conformation and destabilizes several hydrogen bonds in the area of C-cap of the helix 14. In addition, the introduction of relatively bulky proline residue in the interface between alpha-helices could cause outward movement of surrounding helices. This movement could potentially reduce the nucleotide binding maintained by these helices. Another mutant, R516H, is located in the C-cap of alpha-helix 18. In CCT2 subunit positively charged arginine residue (R516) additionally stabilize alpha-helix 18 by forming a salt bridge with negatively charged glutamic acid E509. Mutation to histidine residue breaks the salt bridge that predicted to loosen the alpha-helix structure. It is known that intermediate alpha-helices yielding these mutations are important for CCT-chaperonin intra-ring formation. Identified unrivalled candidate CCT2 and its mutants T400P and R516H proteins were predicted to have structural decays and indeed were biochemically instable.

为了了解致病性突变如何引起遗传性眼病，有必要认识到致病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。为此，我们执行分子建模以建立蛋白质结构，评估遗传错过的严重程度与蛋白质原子水平的变化，并对突变对蛋白质结构，稳定性和催化活性的影响进行定量分析。我们还对感兴趣的蛋白质进行实验性研究，以测量蛋白质折叠的不稳定和由疾病相关突变引起的催化活性的变化。最后，我们将这些发现与遗传性眼病的临床表型相关联。此外，与国家前进的转化科学中心（NCAT）合作，我们搜索受遗传突变影响的突变蛋白催化活性的药物激活剂。今年，我们使用眼皮白化病，Stargardts黄斑变性，绒毛膜血症（CHM），Leber先天性症（LCA）和其他人作为我们的疾病模型。 眼皮白化症是一种罕见的黑色素合成遗传疾病，导致头发，皮肤和眼睛不足。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。没有或降低的TYR活性的患者分别归类为OCA1A或OCA1B形式。今年，我们为人酪氨酸酶进行了大规模蛋白质净化。从幼体生物量中纯化了总共70毫克催化活性酪氨酸酶内球体内结构域（截短的酪氨酸酶），并且是NCATS上的高通量筛选的主题。迄今为止，来自Genesis药物收集的17,011种化合物，2,108种来自天然产品库的化合物以及NCATS Pharmaceutical Collection的2,816种化合物。首次搜索发现了新的抑制剂（> 100）和几种酪氨酸酶激活剂。最近，我们还纯化了一种重组全长酪氨酸酶，该酪氨酸酶既包含叶lan体内和跨膜结构域。我们证明了全长和截短的酪氨酸酶具有相似的酶促活性。这项研究验证了我们使用截短蛋白质的药物筛查。此外，我们在对人重组酪氨酸酶的生物化学，生物物理和硅研究中进行了了解，以了解错义变化的折叠和稳定性R402Q，P406L，R422Q和R422W，模仿OCA1B形式的模仿突变。 我们还暗示了分子建模，以研究潜在的结构和功能后果，以及与导致遗传性眼病的基因突变有关的可能风险。 常染色体隐性星巴特疾病是少年黄斑营养不良的最常见形式，是ABCA4基因突变的结果。大约50％的病原ABCA4错义突变发生在反膜或核苷酸结合结构域中。这些结构域的原子结构使用分子建模获得。我们改善了引起突变的疾病分子建模，并通过临床数据测试了我们的预测，今年有显着扩展。 CHM是视网膜色素上皮，光感受器和脉络膜的X连锁变性，会导致Nyctalopia和视野的逐渐限制导致失明。 CHM基因编码RAB伴随蛋白1（REP-1）。我们对REP-1功能蛋白复合物进行了分子建模（Freund P.R.，Sergeev Y.V.，MacDonald I.M.，分子遗传学和基因组医学，2016年）。 对128名受影响的男性进行了回顾性综述，分析了CHM基因突变的症状，视力和视野的发作。在106个CHM突变的池中，发现了四个新型的错义突变。突变，L80F，Q273H，M443V和L457P，预计会发生严重的变化，影响蛋白质稳定性和折叠，其效果与CHM当前已知的其他类型突变相似。因此，所有CHM患者都有功能突变的丧失，并且没有功能性REP-1。该结果证实了临床数据显示，与Nyctalopia的发作，其他视觉症状，视力或视野的宽度有关，没有明显的基因型 - 表型相关性。 我们还对GUCY2蛋白的结构进行了建模，以显示LCA遗传突变的作用（Gradstein L.等，BMC Medical Genetics，2016）。这项研究的目的是在临床上表征和确定具有LCA的大近交贝都因人部落中的疾病原因。 GUCY2D的测序确定了一种新型的错义突变（C.2129C> t; p.Ala710Val），从而导致valine在VARINE蛋白激酶结构域内的位置7​​10在视网膜特异性酶环鸟苷酸环化酶1（GC1）中用GucCy2d替代了丙氨酸的蛋白激酶结构域（GC1）。分子建模暗示突变改变了GC1激酶造型域内调节段的构象，并导致其螺旋结构的丧失，可能会抑制该段内苏氨酸残基的磷酸化，这是激活蛋白质催化域所需的。这是GC1中P.Ala710VAL突变的第一个文献，也是其蛋白激酶结构域中的第二个描述的突变。这些发现扩大了LCA遗传变异的范围，突出了具有相同LCA突变的个体中发现的表型异质性，并可能为这种先天性盲目疾病的患者提供了基因治疗的希望。正如贝都因（Bedouin）所研究的那样起源于沙特阿拉伯，发现该突变可能是该大型社区中的古老创始人突变。 LCA遗传突变的另一个靶标是分子伴侣CCT2（Minegishi等人，科学报告 - 基因，2016年）。为了预测这些突变体是否对疾病发作有实际影响，进行了分子建模。 8个域中的每个域的原子结构都是通过同源性模型的。杂糖剂的最终结构是使用牛/CCT伴侣伴侣作为结构模板构建的。 CCT2错义突变T400P和R516H是使用水中的1NS分子动力学在优化构象中生成的。位置上脯氨酸残基的变化400破坏了α-螺旋构象，并破坏了螺旋螺旋14的C-CAP区域中的几个氢键。此外，在α-螺旋中引入相对较大的脯氨酸残基在α-螺旋中的引入可能会导致周围螺旋的外部运动。这种运动可能有可能减少这些螺旋螺旋的核苷酸结合。另一个突变体R516H位于Alpha-helix 18的C-CAP中。在CCT2亚基阳性电荷的精氨酸残基（R516）中，还通过形成带负电荷的谷氨酸E509的盐桥，稳定α-螺旋18。对组氨酸残基的突变破坏了预测会松开α-螺旋结构的盐桥。众所周知，产生这些突变的中间α-螺旋对于CCT-链球菌素内部的形成很重要。鉴定出无与伦比的候选CCT2及其突变体T400P和R516H蛋白被预测具有结构性衰减，实际上在生化上是不稳定的。