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引起疾病，有必要认识到致病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表型的临床参数相关。我们暗示分子建模以建立蛋白质结构，模拟致病错义变化的影响，并对它们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连锁的视网膜（XLR）和眼皮白化病（OCA1）作为我们的疾病模型。 1。编码视网膜气概（RS1）的基因突变引起X连接的视网膜静脉（XLRS），这是一种影响男性的幼年黄斑和视网膜变性的形式。去年，我们提出了基于蛋白质结构的致病错义突变影响人类XLR的严重程度评分。最近，使用人类XLRS影响了NEI诊所的患者队列的人群，从这项工作中得出了结论。该分析是独立地应用于ERG B/A波的比率，这是平均患者年龄与XLRS表型严重程度的函数。结果，年轻人的表型B/A波比下降，伴随着相应的计算影响评分的上升，这是突变的结构严重程度的度量。尽管对于老年患者而言，这种变化在统计学上的显着意义较小，但与年轻人所描述的相似的常见趋势似乎与所描述的影响相似。 考虑了3组突变，低影响和高影响的错义变化和严重的非松散突变的表型差异。低影响的错义变化显示，平均B/A波比率接近1.0，最大为15个XLRS患者，年龄不同。该比率对应于10例平均值约为0.6的患者，这与高影响力错义变体所期望的更严重的XLRS表型一致。此外，与15例严重非错觉变体患者相同的对应证实了高影响力变化的严重程度。这项研究证实，通过使用分子渐变量表，可以在轻度和严重的XLR表型中分离致病性错义突变。因此，蛋白质结构的低影响和高影响的错义变化与XLRS表型的轻度和严重变化有关。 目前预期的XLR表型的结果预测，预计115个错义变化。从我们的分析中，一半的温和突变可以被视为在蛋白质表面部分或完全暴露的突变。另一半是预计将埋在疏水核心中的突变。后一组的突变是与插入，交换或去除带电残基或用同源疏水或极性残基替换有关的变体。相反，预计大多数错义变体会导致严重的变化，这与插入或去除半胱氨酸残基（23个突变）和/或埋在疏水性核心有关。最后，这项研究证实了我们先前的工作的结果，即严重的表型最大结构扰动与蛋白质疏水性核心的急剧变化有关，或与一般影响蛋白质折叠稳定性的半胱氨酸残基的缺失或插入。疾病进展的动力学可能取决于致病错义变化引起的突变对蛋白质稳定性的影响。将来，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。眼皮白化病（OCA1）是一种常染色体隐性疾病，其特征是眼睛，头发和皮肤中没有色素（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，这是色素产生的速率限制步骤。哺乳动物酪氨酸酶的原子结构目前尚不可用。我们暗示分子建模是通过与原核生物和蘑菇酪氨酸酶，无脊椎动物血胶蛋白和植物儿茶酚氧化酶的可用晶体结构同源性生成小鼠酪氨酸酶原子结构。 分子建模表明，铜结合结构域在细菌，蘑菇和哺乳动物酪氨酸酶之间保守。此外，哺乳动物酪氨酸酶很可能在N末端包含EGF样结构基序。在计算机中对C-2J（R77L）和C-H（H420R）酪氨酸酶突变体的作用进行了建模。 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，我们预计与体内结果一致，升高酪氨酸浓度对突变酪氨酸酶功能几乎没有影响。喜马拉雅（C-H）突变H420R的分析提出了较弱的结构变化。该模型可以预测在铜的配位中的作用，在活性位点附近的铜浓度升高可能会稳定酶，以帮助较低效率的铜配位和残留酶活性。 这些预测与体内数据一致。 在硅分析中，H420R而不是R77L酪氨酸酶可以有效地结合酪氨酸。环境酪氨酸可能充当小分子伴侣，并有选择地稳定蛋白质的蛋白质。 我们通过在中汉斯特卵巢（CHO）细胞中表达野生型，R77L或H420R突变酪氨酸酶蛋白并使用环己酰亚胺抑制新蛋白质合成的酪氨酸酶蛋白稳定性来在体外测试这一想法。 升高的酪氨酸可以稳定H240R，而不是R77L酪氨酸酶。这些结果与体内观察结果一致，即在OCA-1B的喜马拉雅模型中，血浆酪氨酸的药理升高会增加色素沉着，而在OCA-1A的Tyrc-2J/C-2J模型中则不增加。 我们还研究了与白化小鼠黑素细胞中的野生型蛋白相比，在体外研究了R77L和H420R突变蛋白的酶活性，所谓的Melan-C细胞。 在表达H420R突变酪氨酸酶的MELAN-C细胞中，酪氨酸的饱和浓度增加了酶活性，而不论蛋白质表达水平可比，而R77L突变酪氨酸酶都不会。这表明抑制酪氨酸的升高会导致酪氨酸酶在Melan-C细胞中的酶促活性增加。结果与以前的硅分析和体内数据一致。 酪氨酸酶是一种I型膜蛋白，其单个跨膜片段位于C末端。我们在幼虫中设计，开发了一种高收益蛋白纯化程序，并用截短的跨膜片段纯化了人类酪氨酸酶。改良的人酪氨酸酶是一种酶促的可溶性蛋白质，可催化酪氨酸对DOPA和DOPA与DOPA- Quinone的速率限制转化。我们使用PNGase F内糖苷酶表明修饰的酪氨酸酶含有N连接的寡糖。酪氨酸酶是一种单体蛋白（MR 56 kDa），如尺寸排斥色谱和分析性超速离心所证明。从角度来看，对蛋白质结构和控制酪氨酸改性酪氨酸酶相互作用的机制的详细理解将允许在硅和大规模药物中刮擦，以便对OCA-1B白化症患者进行未来的医学治疗。