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: 9155585
• 负责人: Yuri Sergeev
• 金额: $ 54.06万
• 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/9155585
• 关键词: 3-Dimensional; Adolescent; Affect; Age-Years; Alleles; Amino Acid Sequence; Atrophic; Binding; Binding Sites; Biochemical; Blindness; Brothers; C-terminal; Caliber; Catalytic Domain; Cations; Cell membrane; Cells; Charge; Childhood; Chimeric Proteins; Cilia; Clinical; Clinical Data; Clinical Research; Coiled-Coil Domain; Computer Simulation; Cone; Cyclic GMP; DNA; DNA Sequence Alteration; Data; Disease; Disease model; Electron Microscopy; Elements; Ependyma; Exclusion; Exhibits; Eye; Eye diseases; Female; Free Energy; Gated Ion Channel; Gene Mutation; Genes; Genetic; Genotype; Glutamic Acid; Goals; Hair; Hereditary Disease; Hereditary Eye Diseases; Human; In Vitro; Inherited; Insecta; Investigation; Knowledge; Length; Lysine; Macular degeneration; Measures; Melanins; Membrane; Membrane Proteins; Metabolic Pathway; Microtubules; Missense Mutation; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Molecular Genetics; Molecular Models; Monophenol Monooxygenase; Mus; Mutate; Mutation; Mutation Analysis; Nucleotides; Oculocutaneous Albinism; Parents; Patients; Peptide Sequence Determination; Peptides; Phenotype; Photoreceptors; Play; Positioning Attribute; Preventive; Process; Production; Proteins; RHOA gene; Recombinants; Rest; Retinal Cone; Retinal Degeneration; Retinal Diseases; Retinitis Pigmentosa; Risk; Role; Second Messenger Systems; Sequence Analysis; Sequence Homologs; Severities; Siblings; Side; Site; Skin; Stargardt' s disease; Structure; Technology; Testing; Tropomyosin; Variant; Ventricular; Vimentin; Visual system structure; Work; achromatopsia; base; cilium biogenesis; computer studies; disease phenotype; disease-causing mutation; flexibility; genetic analysis; genetic variant; glycosylation; improved; insight; macula; macular dystrophy; molecular dynamics; molecular modeling; mutant; next generation sequencing; novel; phosphoric diester hydrolase; protein expression; protein folding; protein structure; protein structure function; research study; retinal rods; second messenger; 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 to measure the protein folding destabilization and changes in catalytic activity caused by the disease-related mutations. Finally we correlate these findings with clinical data on inherited eye disease phenotype. Currently we were using oculocutaneous albinism,Stargardt macular degeneration, congenital achromatopsia,autosomal recessive retinitis pigmentosa 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 (TYR) 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 were doing large scale purifications, biochemical, biophysical, and in-silico studies of human recombinant tyrosinases including OCA1-causing mutant variants. We also studied a role of N-glycosylation in 5-, 6-, and 7-site deglycosylated recombinant variants. Surprisingly, OCA1A mutant variants, T373K and R77Q, and N-deglycosylated variants show low protein expression, protein purity, and very low or no catalytic activity. As well this year we performed experimental and in-silico study with a purpose to understand folding and stability of OCA1B causing missense variants R402Q, P406L, R422Q, and R422W. Autosomal recessive Stargardt disease is the most common form of juvenile macular dystrophy and results from mutations in the ABCA4 gene. Approximately 49% of pathogenic ABCA4 missense mutations occur in the trans-membrane or nucleotide binding domains.The atomic structure of these domains could be modeled using molecular modeling. This year we continued work on molecular modeling of disease causing mutations in Stargardts disease and testing our predictions with clinical and EyeGENE data. The large-scale mutational analysis and predictions of mutation severities from atomic structure could be useful tool for the functional annotation of genetic variants from next-generation sequencing data and establishing the genotype-to-phenotype relationships in genetic disease. In addition, we performed modeling of a CEP290 oligomeric structure and several mutant variants (Rachel R. et al., Human Molecular Genetics, 2015). CEP290 localized in the transition zone of connecting cilia, precisely to the region of Y-linkers between central microtubules and plasma membrane. Based on the requirement of CEP290 in photoreceptor and ventricular ependyma ciliogenesis, we sought to predict CEP290 tertiary structure to gain further insight into its possible mechanism of action at the transition zone. Protein sequence information on centrosomal protein of 290 kDa (CEP290) from human and mice was used to predict structurally disordered regions and structural coiled-coil domains. Domain prediction of both mouse and human CEP290 identified extensive coiled-coils throughout the protein, almost to the exclusion of other domains. Only a few other cilia proteins share such a domain structure. To explore the tertiary structure of CEP290, we identified six coiled coil domains covering the full length of the protein, interspersed with five small peptide regions exhibiting structural disorder, which indicate five nodes that might maintain molecular flexibility of an otherwise rigid, rod-like molecule. We demonstrated that CEP290 has the propensity to form oligomeric structures. The entire protein is predicted to have a maximum theoretical length of about 348 nm, thus forming a long, thin rod-like molecule with approximate diameter 1.4 nm. In contrast, the truncated CEP290 protein of 116 kDa had a relatively high propensity to form a dimeric coiled-coil structure compared with that of the full-length trimeric protein. The structures of full-length and truncated CEP290 are interrupted by the coiled-coil vimentin-like structure (residues 696 to 752). The rest of the truncated CEP290 protein revealed 13% sequence identity to tropomyosin with 12 coiled-coil heptad motifs (HxxHCxC) confirming the coiled-coil conformation. The 116 kDa protein is shortened by 58% and predicted to have a maximum length of 145 nm. This study resulted in a model of photoreceptor connecting cilium which agrees with electron microscopy data. Our work clarifies possible positions for rod-like coiled-coil domain proteins such as Cep290, which localize to the region of the Y-linkers between the plasma membrane and the microtubule ring. 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 disease. Such an examples are proteins, PDE6C and CNGA1, involved in congenital achromatopsia, macular atrophy, and autosomal recessive retinitis pigmentosa, respectively (Katagari S et al, Ophthalmic Genetics, 2015; PLOS One, 2014). In the first case, the ophthalmic examination revealed achromatopsia and severe macular atrophy in the older female sibling at 30 years of age and mild macular atrophy in the brother at 26 years of age. The genetic analysis identified a novel homozygous PDE6C mutation E591K as the disease-causing allele in the siblings. Each parent was heterozygous for the mutation. Molecular modeling showed that the mutation could cause a conformational change in the PDE6C protein and result in reduced phosphodiesterase activity. The PDEase catalytic domain has a binding site for divalent Zn2+ cations. The E591K mutation replaced a negatively-charged glutamic acid with a positively-charged lysine residue. This alteration dramatically changes the interaction between two helices, H5 and H12, located within the catalytic domain. The results of this modeling analysis were consistent with results of a structural study of phosphodiesterase inhibition by the C-terminal region of the g-subunit. In our PDE6C model, the H5 and H12 helices were positioned close to the H- and M-loops, residues 610-632 and 748-770, respectively. These loops formed a distinct interface that contributed to the g-subunit binding site. The disruption of this interface causes retinal degeneration in atrd3 mice. Our findings indicated that the H5 and H12 helices might be involved in the stabilization of the g-subunit binding site. PDE6C plays an important role in cone photoreceptors by rapidly decreasing intracellular levels of the second messenger cGMP. Reportedly, known PDE6C gene mutations reduce PDE activity, based on data from a PDE5/PDE6 chimeric protein expressed in Sf9 insect cells. Therefore, the E591K mutation might reduce PDE activity and thereby disturb the closure of the cGMP-gated ion channel in the cone outer segment membrane, resulting in the loss of hyperpolarization in the cone photoreceptors and leading to achromatopsia.

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。我们还进行实验性体外研究，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表型的临床数据相关联。目前，我们正在使用眼皮白化病，Stargardt黄斑变性，先天性高瘤，常染色体隐性视网膜色素炎等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）催化速率限制，黑色素产生的第一步及其基因（Tyr）在许多眼皮白化病（OCA1）的情况下被突变（OCA1），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，我们对人类重组酪氨酸酶（包括引起OCA1的突变体变体）进行了大规模纯化，生化，生物物理和硅内研究。我们还研究了N-糖基化在5-，6-和7位退化重组变体中的作用。令人惊讶的是，OCA1A突变体变体T373K和R77Q和N-糖基化变体显示出低蛋白质表达，蛋白质纯度，并且非常低或无催化活性。同样，今年，我们进行了实验性和silico研究，目的是了解oca1b的折叠和稳定性，导致错义变体R402Q，P406L，R422Q和R422W。 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基因突变引起的。大约49％的致病ABCA4错义突变发生在跨膜或核苷酸结合结构域中。这些结构域的原子结构可以使用分子建模进行建模。今年，我们继续研究疾病的分子建模，导致Star​​gardts疾病中的突变，并通过临床和眼睛的数据测试我们的预测。来自原子结构的大规模突变分析和突变严重程度的预测可能是从下一代测序数据中对遗传变异的功能注释的有用工具，并建立了遗传疾病中基因型至上型关系的有用工具。 此外，我们对CEP290低聚结构和几种突变体变体进行了建模（Rachel R.等，人类分子遗传学，2015年）。 CEP290位于连接纤毛的过渡区域，正好与中央微管和质膜之间的Y链链区域。根据CEP290在光感受器和心室c骨纤毛发生中的需求，我们试图预测CEP290三级结构，以进一步深入了解其在过渡区的可能作用机理。人和小鼠的290 kDa（CEP290）中心体蛋白的蛋白质序列信息用于预测结构无序的区域和结构性盘绕螺旋结构域。小鼠和人类CEP290的域预测都鉴定了整个蛋白质中的广泛盘绕螺旋，几乎排除了其他结构域。只有其他几个纤毛蛋白共享这种域结构。为了探索CEP290的三级结构，我们鉴定了六个盘绕的线圈结构域，涵盖了蛋白质的全长，并散布着五个表现出结构性疾病的小肽区域，这表明五个可能维持原本刚性刚性的杆状分子的分子柔韧性。我们证明了CEP290具有形成寡聚结构的倾向。预计整个蛋白质的最大理论长度约为348 nm，因此形成一个长，薄的杆状分子，直径近似于直径1.4 nm。相比之下，与全长三聚蛋白相比，截短的116 kDa的CEP290蛋白具有相对较高的形成二聚体盘绕结构的倾向。全长和截短的CEP290的结构被盘绕的螺旋蛋白样结构（残基696至752）中断。其余的截短的CEP290蛋白显示出具有12个盘绕螺旋型含量基序（HXXHCXC）的tropomyosin的13％序列同一性，从而确认了盘绕螺旋构象的构象。 116 kDa蛋白缩短了58％，预计最大长度为145 nm。这项研究导致了连接纤毛的感光器模型，该模型与电子显微镜数据一致。我们的工作阐明了类似杆状的盘绕螺旋型域蛋白（例如CEP290）的位置，该蛋白位于质膜和微管环之间的Y-LINCER区域。 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。这样的例子是蛋白质，PDE6C和CNGA1，分别参与先天性乙酰基瘤，黄斑萎缩和常染色体隐性视网膜炎色素（Katagari S等人，Ophthalmic Genetics，2015; Plos One，2014年）。在第一种情况下，眼科检查显示30岁的老年雌性兄弟姐妹的赤乳症和严重的黄斑萎缩，而兄弟则为26岁的兄弟中的轻度黄斑萎缩。遗传分析确定了一种新型的纯合PDE6C突变E591K是兄弟姐妹中引起疾病​​的等位基因。每个父母都是突变的杂合子。分子建模表明，该突变可能导致PDE6C蛋白的构象变化，并导致磷酸二酯酶活性降低。 PDESE催化域具有用于二价Zn2+阳离子的结合位点。 E591K突变用呈阳性的赖氨酸残基代替了带负荷利亚的谷氨酸。这种改变极大地改变了位于催化域内的两个螺旋H5和H12之间的相互作用。该建模分析的结果与G-Subunit的C末端区域对磷酸二酯酶抑制的结构研究结果一致。在我们的PDE6C模型中，H5和H12螺旋分别位于H-和M-LOOPS附近，残基610-632和748-770。这些环形成了有助于G-Subunit结合位点的独特界面。该界面的破坏会导致ATRD3小鼠的视网膜变性。我们的发现表明，H5和H12螺旋可能参与G-Subunit结合位点的稳定。 PDE6C通过迅速降低第二信使CGMP的细胞内水平来在锥形感光体中起重要作用。据报道，已知的PDE6C基因突变基于在SF9昆虫细胞中表达的PDE5/PDE6嵌合蛋白的数据降低了PDE活性。因此，E591K突变可能会降低PDE活性，从而干扰锥外段膜中CGMP门控的离子通道的闭合，从而导致锥形感受器中的超极化损失并导致丘脑瘤。