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表型的临床数据相关联。目前我们使用眼皮肤白化病、Stargardt黄斑变性、先天性全色盲、常染色体隐性遗传色素性视网膜炎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 (TYR) 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，我们对人类重组酪氨酸酶（包括引起 OCA1 的突变变体）进行大规模纯化、生物化学、生物物理和计算机模拟研究。我们还研究了 N-糖基化在 5、6 和 7 位点去糖基化重组变体中的作用。令人惊讶的是，OCA1A 突变变体、T373K 和 R77Q 以及 N-去糖基化变体显示出低蛋白表达、蛋白纯度以及非常低或没有催化活性。今年，我们也进行了实验和计算机模拟研究，目的是了解导致错义变异 R402Q、P406L、R422Q 和 R422W 的 OCA1B 的折叠和稳定性。 常染色体隐性遗传 Stargardt 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 49% 的致病性 ABCA4 错义突变发生在跨膜或核苷酸结合域中。这些域的原子结构可以使用分子建模来建模。今年，我们继续研究引起斯塔加特病突变的分子模型，并用临床和 EyeGENE 数据测试我们的预测。大规模突变分析和原子结构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进行功能注释并建立遗传疾病中基因型与表型关系的有用工具。 此外，我们对 CEP290 寡聚结构和几种突变变体进行了建模（Rachel R. 等人，人类分子遗传学，2015）。 CEP290 定位于连接纤毛的过渡区，精确地定位于中央微管和质膜之间的 Y 接头区域。基于CEP290在光感受器和室管膜纤毛发生中的要求，我们试图预测CEP290的三级结构，以进一步了解其在过渡区的可能作用机制。来自人和小鼠的 290 kDa 中心体蛋白 (CEP290) 的蛋白质序列信息用于预测结构无序区域和结构卷曲螺旋结构域。小鼠和人类 CEP290 的结构域预测在整个蛋白质中发现了广泛的卷曲螺旋，几乎排除了其他结构域。只有少数其他纤毛蛋白具有这样的结构域。为了探索 CEP290 的三级结构，我们确定了覆盖蛋白质全长的 6 个卷曲螺旋结构域，其中散布着 5 个表现出结构无序的小肽区域，这表明五个节点可能保持刚性棒状分子的分子灵活性。我们证明了 CEP290 具有形成寡聚结构的倾向。整个蛋白质预计最大理论长度约为348 nm，从而形成一个长而细的棒状分子，直径约为1.4 nm。相反，与全长三聚体蛋白相比，116 kDa的截短CEP290蛋白具有相对较高的形成二聚体卷曲螺旋结构的倾向。全长和截短的 CEP290 的结构被卷曲螺旋波形蛋白样结构（残基 696 至 752）打断。截短的 CEP290 蛋白的其余部分显示与原肌球蛋白有 13% 的序列同一性，具有 12 个卷曲螺旋七联体基序 (HxxHCxC)，证实了卷曲螺旋构象。 116 kDa 的蛋白质缩短了 58%，预计最大长度为 145 nm。这项研究建立了一个连接纤毛的光感受器模型，与电子显微镜数据一致。我们的工作阐明了杆状卷曲螺旋结构域蛋白（例如 Cep290）的可能位置，该蛋白定位于质膜和微管环之间的 Y 连接子区域。 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。这样的例子是蛋白质 PDE6C 和 CNGA1，分别与先天性全色盲、黄斑萎缩和常染色体隐性遗传色素性视网膜炎有关（Katagari S 等人，眼科遗传学，2015；PLOS One，2014）。在第一个病例中，眼科检查显示，30 岁的姐姐患有色盲和严重黄斑萎缩，26 岁的哥哥患有轻度黄斑萎缩。遗传分析确定了一种新的纯合 PDE6C 突变 E591K 作为这对兄弟姐妹的致病等位基因。每个父母的突变都是杂合的。分子模型表明，该突变可能导致 PDE6C 蛋白构象发生变化，导致磷酸二酯酶活性降低。 PDEase 催化结构域具有二价 Zn2+ 阳离子的结合位点。 E591K 突变用带正电的赖氨酸残基取代了带负电的谷氨酸。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。该模型分析的结果与 g 亚基 C 末端区域抑制磷酸二酯酶的结构研究结果一致。在我们的 PDE6C 模型中，H5 和 H12 螺旋的位置分别靠近 H 环和 M 环，即残基 610-632 和 748-770。这些环形成了一个独特的界面，有助于 g 亚基结合位点。该界面的破坏会导致 atrd3 小鼠的视网膜变性。我们的研究结果表明，H5 和 H12 螺旋可能参与 g 亚基结合位点的稳定。 PDE6C 通过快速降低第二信使 cGMP 的细胞内水平在视锥光感受器中发挥重要作用。据报道，根据 Sf9 昆虫细胞中表达的 PDE5/PDE6 嵌合蛋白的数据，已知的 PDE6C 基因突变会降低 PDE 活性。因此，E591K突变可能会降低PDE活性，从而扰乱视锥细胞外节膜中cGMP门控离子通道的关闭，导致视锥细胞光感受器超极化的丧失并导致全色盲。