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

基本信息

批准号：
8938334
负责人：
Yuri Sergeev
金额：
$ 53.34万
依托单位：
NATIONAL EYE INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
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/8938334
关键词：
3-Dimensional Affect Amino Acids Autoimmune Diseases Autoimmunity Autophagocytosis B-Lymphocytes Binding Proteins Biochemical Biomass Blindness Calcium Binding Calorimetry Cells Childhood Chinese People Clinical Research Complex Computer Analysis Computer Simulation Computing Methodologies Consensus Sequence Cysteine DNA Data Analyses Dissociation Ectopia Lentis Elements Entropy Enzyme Kinetics Enzymes Epidermal Growth Factor Equilibrium Eye Eye diseases FBN1 Family Fiber Free Energy Gene Mutation Genes Genetic Genotype Glycoproteins Goals Hair Hereditary Disease Hereditary Eye Diseases Human In Vitro Inherited Insecta Interleukins Investigation Knowledge Larva Literature Macular degeneration Manuscripts Mass Spectrum Analysis Measures Medicine Melanins Membrane Membrane Proteins Methods Molecular Molecular Chaperones Molecular Genetics Molecular Models Monophenol Monooxygenase Mutate Mutation Nature Oculocutaneous Albinism Paper Pathogenesis Pathogenicity Patients Pattern Pharmaceutical Preparations Phenotype Preventive Process Production Property Proteins Protocols documentation Publishing Recombinants Retinal Diseases Risk Role Sampling Saphenous Vein Science Sequence Analysis Sequence Homologs Severities Side Simulate Skin Structure Technology Temperature Tertiary Protein Structure Therapeutic Human Experimentation Thermodynamics Transforming Growth Factors Variant Vision Visual system structure Work analytical ultracentrifugation base computer studies dimer enthalpy enzyme activity fibrillin-2 flexibility genetic variant glycosylation improved in vivo insight interest molecular dynamics molecular modeling monomer mutant next generation sequencing novel polypeptide protein expression protein protein interaction protein structure protein structure function research study sedimentation equilibrium small molecule temperature sensitive mutant tool

项目摘要

This year we continue biochemical and biophysical studies on human tyrosinase and mutant variants involved in oculocutaneous albinism OCA1B. We also published our method on thermodynamic analysis of weak protein interactions using sedimentation equilibrium and use molecular modeling to build structures of multi-domain proteins, simulate the effect of pathogenic missense changes, and provide a quantitative analysis of their impact on protein structure and stability for proteins involved in inherited eye disease. 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 reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hindered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes. Currently there is no biologically active human recombinant tyrosinase available in preparative quantities. Therefore we were interested to have an active human protein and OCA1B-related mutant variants expressed and purified for biochemical and biophysical studies to study their enzymatic properties, thermal sensitivity, and structural properties. The intra-melanosomal domain of human tyrosinase (residues 19469) and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae (Dolinska et al., PlosOne, 2014). The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of .1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37oC and neutral pH. According to mass-spectroscopy analysis glycosylation occurred at five Asn residues, and the enzymes kinetic and pharmacologic properties were similar to the authentic enzyme described in the literature. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure. The intra-melanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in-vivo function. This advance allows for the structure function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1B. Recently we were invited to submit a manuscript on a method on thermodynamic analysis of weak protein interactions using sedimentation equilibrium. This paper was recently published in Current Protocols in Protein Science (Sergeev et al, 2014). Thermodynamics experiments are necessary to find Gibbs free energy, enthalpy, and entropy and other thermodynamics parameters. However traditional calorimetry is not a sensitive enough method to measure weak interactions between protein molecules and require large protein samples. Our method is novel and based on analytical ultracentrifugation and require a direct determination of the dissociation constant (Kd) for protein-protein interactions as a function of temperature. The dissociation constant, which characterizes the strength of protein association in oligomeric complexes formed by weak forces, can be precisely measured by sedimentation equilibrium. The Kd is dependent on local protein interactions and the temperature. Proteins self-associate to form dimers and tetramers. Purified proteins are used to study the thermodynamics of protein interactions using the analytical ultracentrifuge. In this approach, monomer-dimer equilibrium constants are directly measured at various temperatures. Data analysis is used to derive thermodynamic parameters, such as Gibbs free energy, enthalpy, and entropy, which can predict which weak forces are involved in protein association. Lately we were using molecular modeling to investigate the potential structural and functional consequences as well as possible risks associated with genetic mutations affecting proteins composed of numerous repetitive domains. Mutations in such proteins often cause the inherited eye disease. However computational methods for the analysis of such structures are not establishes yet. These proteins are very large molecules difficult for the structural and computational analysis. However such an analysis could be performed using isolated structural domains. In first case, the analysis of genetic mutations has been implied to the multi-domain FBN-2 protein associated with macular degeneration and AMD phenotypes (Ratnapriya et al., Human Molecular Genetics, 2014). The fibrillin-2 protein, FBN2, is a 2,912 amino acid polypeptide which has one amino-terminal trans-membrane domain, 4 epidermal growth factor-like (EGF) domains, 43 calcium-binding consensus sequences (Ca_EGF domains), and 9 transforming growth factor β1 binding protein-like (TB) domains. FBN-2 has 363 cysteine residues. We have analyzed several mutations in the computationally isolated Ca_EGF domains and structural consequences of these changes. Mutations with severe phenotype are likely to cause a change in the fiber flexibility, packaging, glycosylation pattern, and pathogenicity. Similar approach in the analysis of mutant variants have been applied to novel mutation in fibrillin-1 protein, FBN1, causes ectopia lentis and varicose great saphenous vein in one Chinese autosomal dominant family (Fu et al., Molecular Vision, 2014). We have also participated in collaborative work on the role of chaperone-like molecules in the pathogenesis of inherited eye disease (Valapala M. et al., Autophagy, 2014), the role of IL27 p28 in autoimmunity (Chong WP et al., J. Autoimmunity, 2014), and the biochemical characterization of Interleukin-35 which induces regulatory B cells suppressing autoimmune disease (Wang R.X. et al., Nature Medicine, 2014).

今年，我们继续对涉及眼皮白化病OCA1B的人类酪氨酸酶和突变体变体进行生化和生物物理研究。我们还使用沉积平衡发表了有关弱蛋白质相互作用的热力学分析的方法，并使用分子建模来构建多域蛋白质的结构，模拟致病性错义变化的影响，并对其对蛋白质结构的影响和稳定性进行定量分析，使其对蛋白质结构的影响以及涉及遗传性眼病的蛋白质的稳定性。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。 TyR活性降低的患者被归类为OCA1B；一些OCA1B突变对温度敏感。 OCA1的治疗研究受到部分阻碍，部分原因是没有纯化，活跃的，重组的野生型和突变的人类酶。当前，没有制备量的生物活性人类重组酪氨酸酶可用。因此，我们有兴趣具有活性的人蛋白和与OCA1b相关的突变体变体，并纯化用于生化和生物物理研究，以研究其酶特性，热敏感性和结构特性。人酪氨酸酶（残基19469）和两个相关温度敏感的突变体R422Q和R422W的绿素体内结构域在昆虫细胞中表达，并在Ni幼虫中产生（Dolinska等人，Plosone，Plosone，2014）。删除了短的跨膜片段，以避免潜在的蛋白质不溶性，同时保留酶的所有其他功能特征。获得纯化的酪氨酸酶，每10 g幼虫生物量的产率为0.1 mg。该蛋白是一种单体糖酶，在37oC和中性pH下具有最大酶活性。根据质谱分析，糖基化发生在五个ASN残基处，动力学和药理学特性与文献中描述的真实酶相似。与野生型蛋白相比，两个纯化的突变体的活性较低，温度敏感。这些差异与二级结构中的构象扰动有关。重组野生型和突变的人酪氨酸酶的螺旋内粒域是可溶性单体糖蛋白，其活性反映了它们的体内功能。这种进步允许对不同突变蛋白的结构函数分析，并与它们相应的人类表型相关。它还提供了一种重要的工具，可以发现可以改善酪氨酸酶活性并治疗OCA1B的药物。最近，我们被邀请提交有关使用沉积平衡对弱蛋白质相互作用的热力学分析方法提交手稿。本文最近发表在当前蛋白质科学方案中（Sergeev等，2014）。热力学实验对于找到Gibbs自由能，焓和熵以及其他热力学参数是必需的。但是，传统的量热法不是一种足够敏感的方法来测量蛋白质分子之间的弱相互作用，并且需要大型蛋白质样品。我们的方法是新颖的，并且基于分析性超速离心，需要直接确定蛋白质 - 蛋白质相互作用的分离常数（KD）作为温度的函数。解离常数可以通过沉积平衡来精确测量由弱力形成的寡聚复合物中蛋白质缔合的强度。 KD取决于局部蛋白质相互作用和温度。蛋白质自相关以形成二聚体和四聚体。纯化的蛋白质用于研究蛋白质相互作用的热力学使用分析性超级离心分离。在这种方法中，单体二聚体平衡常数直接在各种温度下测量。数据分析用于得出热力学参数，例如Gibbs自由能，焓和熵，这些参数可以预测哪些弱力与蛋白质缔合有关。最近，我们使用分子建模来研究潜在的结构和功能后果，以及与影响由许多重复域组成的蛋白质相关的可能风险。这种蛋白质中的突变通常会引起遗传性眼病。但是，尚未建立用于分析此类结构的计算方法。这些蛋白质很难进行结构和计算分析。但是，可以使用孤立的结构域进行这种分析。在第一种情况下，对与黄斑变性和AMD表型相关的多域FBN-2蛋白的遗传突变分析已暗示（Ratnapriya等，人类分子遗传学，2014年）。 Fibrillin-2蛋白FBN2是一个2,912个氨基酸多肽，具有一个氨基末端跨膜结构域，4个表皮生长因子样（EGF）域，43个钙结合共识序列（CA_EGF域）和9转化生长因子β1粘合因子β1型蛋白质样子（TB）样子（TB）样子（TB）样子（TB）样子。 FBN-2具有363个半胱氨酸残基。我们已经分析了这些变化的计算分离的CA_EGF域和结构性后果中的几个突变。严重表型的突变可能会导致纤维柔韧性，包装，糖基化模式和致病性的变化。在对突变变体分析中的类似方法已应用于原纤维蛋白-1蛋白FBN1中的新突变，在一个中国常染色体显性himal氏家族中引起了宽易梁的异端和质量大隐静脉（Fu等人，Molecular Vision，2014年）。我们还参与了有关伴侣蛋白样分子在遗传性眼病发病机理中的作用（Valapala M.等人，自噬，2014年），IL27 p28在自身免疫中的作用（Chong WP等人，J。AutoMummunity，2014）的作用（chong wp et al。抑制自身免疫性疾病（Wang R.X.等，自然医学，2014年）。