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: 8339794
• 负责人: Yuri Sergeev
• 金额: $ 68.83万
• 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/8339794
• 关键词: 3-Dimensional; Active Sites; Adolescent; Affect; Agaricales; Age; Agreement; Albinism; Binding; Catalytic Domain; Catechol Oxidase; Cells; Charge; Chinese Hamster Ovary Cell; Classification; Clinic; Clinical; Clinical Research; Computer Simulation; Copper; Cycloheximide; Cysteine; DNA; Data; Disease; Disease Progression; Disease model; EGF gene; Elements; Endoglycosidases; Enzymes; Excision; Eye; Eye diseases; Future; Gene Mutation; Genes; Genotype; Goals; Hair; Hemocyanin; Hereditary Eye Diseases; Human; Hydrophobic Surfaces; In Vitro; Individual; Inherited; Invertebrates; Investigation; Kinetics; Laminin; Larva; Lead; Link; Macular degeneration; Measures; Medical; Medicine; Melanins; Membrane; Membrane Proteins; Missense Mutation; Modeling; Molecular; Molecular Chaperones; Molecular Models; Molecular Sieve Chromatography; Monophenol Monooxygenase; Mus; Mutation; Oculocutaneous albinism type 1; Oligosaccharides; Pathway interactions; Patients; Peptide N-glycohydrolase F; Pharmaceutical Preparations; Phenotype; Pigmentation physiologic function; Pigments; Plants; Plasma; Preventive; Procedures; Process; Production; Property; Protein Biosynthesis; Proteins; Quinones; Residual state; Retinal Degeneration; Retinal Diseases; Role; Sequence Homologs; Severities; Side; Simulate; Site; Skin; Solubility; Structure; Structure-Activity Relationship; Testing; Tyrosine; Variant; Visual system structure; Work; X-Linked Retinoschisis; XLRS1 protein; albino mouse; analytical ultracentrifugation; base; cohort; computer studies; design; disease phenotype; enzyme activity; in vivo; insight; male; melanocyte; molecular dynamics; molecular modeling; mutant; older patient; protein aggregation; protein expression; protein folding; protein protein interaction; protein purification; protein structure; protein structure function; research study; three dimensional structure; tool; trend

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 X-linked retinoschisis (XLRS) and oculocutaneous albinism type 1 (OCA1) as our disease models. 1. Gene mutations that encode retinoschisin (RS1) cause X-linked retinoschisis (XLRS), a form of juvenile macular and retinal degeneration that affects males. Last year we suggested a severity score for the protein structure-based classification of the pathogenic missense mutations impact in human XLRS. Conclusions from this work were recently refined using the human XLRS affected cohort of patients from the NEI clinic. This analysis was applied independently to ERG b/a-wave ratios as a function of average patient age versus severity of XLRS phenotype. As result, phenotypic b/a-wave ratios declined for younger individuals accompanied by a corresponding rise of computed impact score, a measure of the structural severity of the mutation. Although this change is statistically less significant for older patients, a common trend showing a slight decrease with computed impact seems to be similar to that of described for younger individuals. Differences in phenotypes were considered for 3 groups of mutations, low- and high-impact missense changes and severe non-missense mutations. Low-impact missense changes show average b/a-wave ratio close to 1.0 for a maximum with 15 XLRS patients of different ages. This ratio corresponds to a maximum of 10 patients with an average value of approximately 0.6, which is consistent with more severe XLRS phenotype as expected for high-impact missense variants. In addition, the severity of high impact missense changes is confirmed by identical correspondence to 15 patients with the severe non-missense variants. This study confirmed that pathogenic missense mutations could be separated in mild and severe groups of XLRS phenotype by using the molecular grading scale. Thus, low- and high-impact missense changes in protein structure are associated with mild and severe changes in the XLRS phenotype. The results for the expected XLRS phenotypes currently predicted for 115 missense changes. From our analysis, half of the mild mutations could be considered as mutations which were partially or fully exposed at the protein surface; another half are the mutations predicted to be buried in the hydrophobic core. Mutations from this latter group were variants related to insertion, exchange, or removal of charged residues or replacement with a homologous hydrophobic or polar residue. In contrast, the majority of missense variants were predicted to cause severe changes, related to insertion or removal of cysteine residues (23 mutations) and/or buried in the hydrophobic core. Finally, this study confirms results of our previous work that severe phenotype maximum structure perturbations are related to dramatic changes in a protein hydrophobic core or to the deletion or insertion of cysteine residues affecting in general the stability of protein fold. The kinetics of the disease progression might depend on the degree of the mutation impact on protein stability caused by the pathogenic missense change. In the future, we have to incorporate age, genotype, and molecular modeling into ERG analysis to understand a functional role of pathogenic mutations. 2. Oculocutaneous albinism type 1 (OCA1) is an autosomal recessive disorder characterized by absence of pigment (melanin) in eyes, hair and skin. Pathogenic mutations causing OCA1 decrease or abolish the melanogenic pathway by affecting tyrosinase enzymatic activity which is the rate limiting step in pigment production. An atomic structure of mammalian tyrosinase currently is not available. We imply molecular modeling to generate the mouse tyrosinase atomic structure by homology to the available crystal structures of prokaryotic and mushroom tyrosinase, invertebrate hemocyanin, and plant catechol oxidase. Molecular modeling has demonstrated that the copper-binding domain is conserved between bacterial, mushroom and mammalian tyrosinases. In addition, mammalian tyrosinase most likely contain EGF-like structural motif at the N-terminus. The effect of the c-2J (R77L) and c-h (H420R) tyrosinase mutants was modeled in silico. The c-2J mutation, R77L, occurs in an EGF/laminin-like motif. Although the precise function of this domain is not known, the R77L mutation is expected to have a severe effect on tyrosine binding, which is take place at the hydrophobic surface of the catalytic site. Thus, we would expect that elevating tyrosine concentrations would have little to no effect on mutant tyrosinase function, in agreement with in vivo results. The analysis of the Himalayan (c-h) mutation, H420R, proposes a weaker structural change. The model predicts a role in the coordination of copper near the active site where elevated tyrosine concentrations binding might stabilize the enzyme enough to help for less efficient copper coordination and residual enzymatic activity. These predictions are consistent with in vivo data. In silico analysis suggested that H420R but not R77L tyrosinase can effectively bind tyrosine. The ambient tyrosine might act as a small molecular chaperone and selectively stabilize the protein in Hymalayan mice. We tested this idea in vitro by expressing either wild-type, R77L, or H420R mutant tyrosinase proteins in Chinese-hamster ovary (CHO) cells and measuring tyrosinase protein stability using cycloheximide to inhibit new protein synthesis. The elevated tyrosine stabilizes H240R, but not R77L, tyrosinase. These results agree with the in vivo observations that pharmacological elevation of plasma tyrosine increases pigmentation in the Himalayan model of OCA-1B, but not in the Tyrc-2J/c-2J model of OCA-1A. We also investigated the enzymatic activity of R77L and H420R mutant proteins in vitro, compared to wild-type protein in albino mouse melanocytes, so-called Melan-c cells. A saturating concentration of tyrosine increased enzyme activity in Melan-c cells expressing the H420R mutant tyrosinase, but not R77L mutant tyrosinase, regardless of comparable levels of protein expression. This suggests that elevated tyrosine results in increased enzymatic activity of tyrosinase in Melan-c cells. The results are consistent with previous in silico analysis and in vivo data. Tyrosinase is a Type I membrane protein with a single trans-membrane fragment located at the C-terminus. We designed, expressed in larvae, developed a high-yield protein purification procedure, and purified the human tyrosinase with the truncated trans-membrane fragment. The modified human tyrosinase is an enzymaticaly active soluble protein which catalyzes the rate-limiting conversions of tyrosine to DOPA and DOPA to DOPA-quinone. We used the PNGase F endoglycosidase to show that the modified tyrosinase contains N-linked oligosaccharides. The tyrosinase is a monomeric protein (Mr 56 kDa) as demonstrated by size-exclusion chromatography and analytical ultracentrifugation. In perspective, a detailed understanding of protein structure and the mechanisms controlling tyrosine-modified tyrosinase interactions would allow in silico and large-scale drug scrinning for a future medical treatment of patients with the OCA-1B albinism.

为了了解基因的致病性变化如何导致疾病，有必要认识到致病性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的临床参数相关联。表型。我们利用分子建模来构建蛋白质结构，模拟致病性错义变化的影响，并对其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 X 连锁视网膜劈裂症 (XLRS) 和 1 型眼皮肤白化病 (OCA1) 作为我们的疾病模型。 1. 编码视网膜劈裂素 (RS1) 的基因突变会导致 X 连锁视网膜劈裂症 (XLRS)，这是一种影响男性的青少年黄斑和视网膜变性。去年，我们建议对人类 XLRS 中致病性错义突变影响的基于蛋白质结构的分类进行严重性评分。最近使用 NEI 诊所受 XLRS 影响的人类患者队列完善了这项工作的结论。该分析独立应用于 ERG b/a 波比率，作为患者平均年龄与 XLRS 表型严重程度的函数。结果，年轻个体的表型 b/a 波比率下降，同时计算出的影响评分（突变结构严重程度的衡量标准）相应上升。尽管这种变化对于老年患者来说在统计上不太显着，但显示出计算影响略有下降的常见趋势似乎与针对年轻个体所描述的相似。 考虑了 3 组突变、低影响和高影响的错义变化以及严重的非错义突变的表型差异。低影响错义变化显示 15 名不同年龄的 XLRS 患者的最大平均 b/a 波比接近 1.0。该比率对应于最多 10 名患者，平均值约为 0.6，这与高影响错义变异所预期的更严重的 XLRS 表型一致。此外，高影响错义变化的严重性通过与 15 名具有严重非错义变异的患者的相同对应关系得到证实。这项研究证实，通过使用分子分级量表，可以将致病性错义突变分为轻度和重度 XLRS 表型组。因此，蛋白质结构的低影响和高影响错义变化与 XLRS 表型的轻度和严重变化相关。 预期 XLRS 表型的结果目前预测有 115 个错义变化。根据我们的分析，一半的轻度突变可以被认为是部分或完全暴露在蛋白质表面的突变；另一半是预计埋藏在疏水核心中的突变。后一组的突变是与带电残基的插入、交换或去除或用同源疏水或极性残基替换相关的变体。相比之下，大多数错义变体预计会引起严重的变化，与半胱氨酸残基的插入或去除（23 个突变）和/或埋藏在疏水核心中有关。最后，这项研究证实了我们之前的工作结果，即严重的表型最大结构扰动与蛋白质疏水核心的显着变化有关，或者与影响蛋白质折叠稳定性的半胱氨酸残基的删除或插入有关。疾病进展的动力学可能取决于致病性错义变化引起的突变对蛋白质稳定性的影响程度。未来，我们必须将年龄、基因型和分子模型纳入 ERG 分析中，以了解致病突变的功能作用。 2. 1 型眼皮肤白化病 (OCA1) 是一种常染色体隐性遗传疾病，其特征是眼睛、头发和皮肤中缺乏色素（黑色素）。导致 OCA1 的致病突变通过影响酪氨酸酶活性来减少或消除黑色素生成途径，酪氨酸酶活性是色素产生的限速步骤。目前哺乳动物酪氨酸酶的原子结构尚不清楚。我们意味着通过与原核和蘑菇酪氨酸酶、无脊椎动物血蓝蛋白和植物儿茶酚氧化酶的可用晶体结构同源来生成小鼠酪氨酸酶原子结构的分子建模。 分子模型表明，铜结合域在细菌、蘑菇和哺乳动物酪氨酸酶之间是保守的。此外，哺乳动物酪氨酸酶很可能在 N 末端含有 EGF 样结构基序。 c-2J (R77L) 和 c-h (H420R) 酪氨酸酶突变体的作用在计算机中建模。 c-2J 突变 R77L 发生在 EGF/层粘连蛋白样基序中。尽管该结构域的确切功能尚不清楚，但预计 R77L 突变会对酪氨酸结合产生严重影响，酪氨酸结合发生在催化位点的疏水表面。因此，我们预计提高酪氨酸浓度对突变酪氨酸酶功能几乎没有影响，这与体内结果一致。对喜马拉雅 (c-h) 突变 H420R 的分析表明结构变化较弱。该模型预测了铜在活性位点附近的配位中的作用，其中结合的酪氨酸浓度升高可能足以稳定酶，从而有助于降低铜配位效率和残留酶活性。 这些预测与体内数据一致。 计算机分析表明，H420R 而不是 R77L 酪氨酸酶可以有效结合酪氨酸。周围的酪氨酸可能充当小分子伴侣并选择性地稳定喜马拉雅小鼠中的蛋白质。 我们通过在中国仓鼠卵巢 (CHO) 细胞中表达野生型、R77L 或 H420R 突变酪氨酸酶蛋白并使用放线菌酮抑制新蛋白质合成来测量酪氨酸酶蛋白稳定性，在体外测试了这一想法。 升高的酪氨酸可以稳定 H240R，但不能稳定 R77L（酪氨酸酶）。这些结果与体内观察结果一致，即血浆酪氨酸的药理升高会增加 OCA-1B 喜马拉雅模型中的色素沉着，但在 OCA-1A 的 Tyrc-2J/c-2J 模型中则不然。 我们还研究了 R77L 和 H420R 突变蛋白的体外酶活性，并与白化小鼠黑素细胞（即所谓的 Melan-c 细胞）中的野生型蛋白进行比较。 无论蛋白质表达水平如何，饱和浓度的酪氨酸都会增加表达 H420R 突变型酪氨酸酶的 Melan-c 细胞中的酶活性，但不会增加 R77L 突变型酪氨酸酶。这表明酪氨酸升高导致 Melan-c 细胞中酪氨酸酶的酶活性增加。结果与之前的计算机分析和体内数据一致。 酪氨酸酶是一种 I 型膜蛋白，在 C 末端有一个跨膜片段。我们设计并在幼虫中表达，开发了高产蛋白纯化程序，并用截短的跨膜片段纯化了人酪氨酸酶。修饰的人酪氨酸酶是一种具有酶活性的可溶性蛋白质，可催化酪氨酸限速转化为多巴以及多巴转化为多巴醌。我们使用 PNGase F 糖苷内切酶证明修饰后的酪氨酸酶含有 N-连接寡糖。尺寸排阻色谱和分析超速离心证明酪氨酸酶是一种单体蛋白 (Mr 56 kDa)。从长远来看，对蛋白质结构和控制酪氨酸修饰酪氨酸酶相互作用的机制的详细了解将允许对 OCA-1B 白化病患者的未来医学治疗进行计算机模拟和大规模药物筛选。