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。为此，我们进行分子建模来构建蛋白质结构，从蛋白质原子水平评估遗传错义变化的严重性，并定量分析突变对蛋白质结构、稳定性和催化活性的影响。我们还对感兴趣的蛋白质进行体外实验研究，以测量由疾病相关突变引起的蛋白质折叠不稳定和催化活性的变化。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，我们与国家转化科学促进中心（NCATS）合作，寻找受基因突变影响的突变蛋白催化活性的药物激活剂。今年，我们使用眼皮肤白化病、Stargardts 黄斑变性、无脉络膜血症 (CHM)、莱伯先天性黑蒙 (LCA) 等作为我们的疾病模型。 眼皮肤白化病是一种罕见的黑色素合成遗传性疾病，会导致头发、皮肤和眼睛色素减退。酪氨酸酶 (TYR) 催化黑色素产生的限速第一步，其基因在许多眼皮肤白化病 (OCA1) 病例中发生突变，眼皮肤白化病是儿童失明的常染色体隐性遗传原因。 TYR 活性不存在或降低的患者分别被分类为 OCA1A 或 OCA1B 型。今年，我们对人类酪氨酸酶进行了大规模的蛋白质纯化。从幼虫生物质中纯化出总共 70 mg 具有催化活性的酪氨酸酶黑素体内结构域（截短的酪氨酸酶），并且是 NCATS 高通量筛选的对象。迄今为止，已成功筛选出 Genesis 药物保藏库中的 17,011 种化合物、天然产物库中的 2,108 种化合物以及 NCATS 药物保藏库中的 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，它包含黑素体内和跨膜结构域。我们证明全长酪氨酸酶和截短酪氨酸酶具有相似的酶活性。这项研究验证了我们的药物筛选，我们使用的是截短的蛋白质。此外，我们对人类重组酪氨酸酶进行了生物化学、生物物理和计算机研究，以了解模仿 OCA1B 形式白化病突变的 R402Q、P406L、R422Q 和 R422W 错义变化的折叠和稳定性。 我们还暗示利用分子模型来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相关的可能风险。 常染色体隐性遗传 Stargardts 病是青少年黄斑营养不良的最常见形式，由 ABCA4 基因突变引起。大约 50% 的致病性 ABCA4 错义突变发生在跨膜或核苷酸结合域中。使用分子建模获得这些域的原子结构。我们改进了引起突变的疾病的分子模型，并用临床数据测试了我们的预测，这些数据在今年得到了显着扩展。 CHM 是一种 X 连锁的视网膜色素上皮、光感受器和脉络膜变性，会导致夜盲症和视野进行性收缩，最终导致失明。 CHM 基因编码 Rab 护卫蛋白 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.、分子遗传学与基因组医学，2016）。 对 128 名受影响的男性进行了回顾性审查，分析了与 CHM 基因突变相关的症状发作、视力和视野。在 106 个 CHM 突变库中，发现了 4 个新的错义突变。 L80F、Q273H、M443V 和 L457P 突变预计将是影响蛋白质稳定性和折叠的严重变化，其效果类似于目前已知的 CHM 的其他类型突变。因此，所有 CHM 患者都有功能缺失突变，并且没有表现出功能性 REP-1。该结果证实了临床数据显示，夜盲症的发作、其他视觉症状的发作、视力或视野宽度没有显着的基因型-表型相关性。 我们还对 GUCY2 蛋白的结构进行了建模，以显示基因突变在 LCA 中的作用（Gradstein L. 等人，BMC Medical Genetics，2016）。本研究的目的是对患有 LCA 的大型近交贝都因以色列部落进行临床表征并确定其病因。 GUCY2D 的测序发现了一种新的错义突变（c.2129C>T；p.Ala710Val），导致 GUCY2D 编码的视网膜特异性酶鸟苷酸环化酶 1 (GC1) 的蛋白激酶结构域内的第 710 位丙氨酸被缬氨酸取代。分子模型表明，突变改变了 GC1 激酶 styk 结构域内调节片段的构象，并导致其螺旋结构丧失，可能抑制该片段内苏氨酸残基的磷酸化，而苏氨酸残基是激活蛋白质催化结构域所必需的。这是 GC1 中 p.Ala710Val 突变的第一份文献，也是有史以来第二个描述的其蛋白激酶结构域突变。这些发现扩大了 LCA 遗传变异的范围，突出了在具有相同 LCA 突变的个体中发现的表型异质性，并可能为这种先天性致盲疾病患者的基因治疗带来希望。由于研究的贝都因人起源于沙特阿拉伯，因此发现的突变可能是这个大社区的古代创始人突变。 LCA 基因突变的另一个目标是分子伴侣 CCT2（Minegishi 等人，Scientific Reports-Nature，2016）。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 个结构域中每一个的原子结构均通过同源性单独建模。使用牛 TRIC/CCT 分子伴侣作为结构模板构建了异八聚体的最终结构。使用水中 1ns 分子动力学以优化构象生成 CCT2 错义突变 T400P 和 R516H。 400 位脯氨酸残基的变化破坏了 α 螺旋构象，并使螺旋 14 的 C 帽区域中的几个氢键不稳定。此外，在 α 螺旋之间的界面中引入相对较大的脯氨酸残基可能会导致周围螺旋的向外运动。这种运动可能会减少这些螺旋维持的核苷酸结合。另一个突变体 R516H 位于 α 螺旋 18 的 C 帽中。在 CCT2 亚基中，带正电荷的精氨酸残基 (R516) 通过与带负电荷的谷氨酸 E509 形成盐桥来额外稳定 α 螺旋 18。组氨酸残基的突变破坏了预计会放松α螺旋结构的盐桥。已知产生这些突变的中间α螺旋对于CCT-伴侣蛋白环内形成很重要。已确定的无与伦比的候选 CCT2 及其突变体 T400P 和 R516H 蛋白预计会出现结构衰退，并且实际上在生化上不稳定。