Novel Protein Sensors

描述(申请人提供)：蛋白质翻译后修饰是人类复杂性的核心，蛋白质翻译后修饰的变化与人类疾病密切相关。在这项提案中，我们将开发对翻译后修饰反应的蛋白质设计的一般方法，包括丝氨酸/苏氨酸和酪氨酸磷酸化；酪氨酸硫化；以及通过酪氨酸硝化、谷胱甘肽氧化和蛋白质S亚硝化的特定氧化。蛋白质磷酸化的失调在许多人类疾病中起着重要作用，包括癌症、心脏病、阿尔茨海默病和糖尿病。研究激酶和磷酸酶活性的新方法将在人类疾病中有潜在的不同应用。我们正在开发利用设计和合成来理解磷酸蛋白质组的方法，以便能够在不同的环境中以一种潜在的空间和时间可寻址的方式来确定激酶和磷酸酶的活性。这些方法对于分析特定蛋白激酶的活性是有用的，并且很容易适应于提供一种通用的方法来检测蛋白激酶的活性和不同的蛋白质翻译后修饰的活性。胞外酪氨酸的蛋白质硫化为细胞表面呈现和分子识别的改变提供了一个关键模式。酪氨酸硝化和蛋白质S亚硝化是与氧化应激相关的常见的翻译后修饰。细胞内氧化条件的变化会导致蛋白质和细胞功能的变化。具体目标1：开发对丝氨酸/苏氨酸和酪氨酸磷酸化反应的蛋白质模体。我们将开发新的、小的、设计的蛋白质序列(15-20个氨基酸)，其结构依赖于磷酸化，并且只有在磷酸化时才显示发光。该方法将被推广应用于一系列不同的激酶。将制备多个设计的基序，这些基序包含替代的激酶识别位点，将利用天然氨基酸作为可遗传编码的蛋白激酶活性传感器，并将被整合到蛋白质中，并在细胞模型中测试发光。具体目标2：设计对Tyr硫酸盐化反应的蛋白质基序。我们将开发一个分析蛋白质酪氨酸硫化的通用平台。具体目标3：设计对特定氧化反应的蛋白质。我们将开发可编码的蛋白质结构域，这些结构域对特定的氧化细胞条件做出反应，包括酪氨酸硝化、谷胱甘肽氧化和蛋白质S亚硝化。此外，这些方法将允许分析翻译后修饰的变化，这些变化发生在药物、其他小分子和其他改变激酶、磷酸酶、磺基转移酶和氧化活性的信号时。 公共卫生相关性：蛋白质修饰和细胞条件的变化在人类疾病中发挥核心作用，包括癌症、心脏病、阿尔茨海默病、艾滋病和糖尿病。我们将开发新的探针，以了解疾病中蛋白质和细胞条件发生的不同类型的变化。此外，这些方法将允许对药物、其他小分子和其他改变蛋白质状态的线索所发生的变化进行分析。了解疾病蛋白质与正常蛋白质的不同之处对于开发治疗这些疾病的新方法和确定新疗法的有效性非常重要。

DESCRIPTION (provided by applicant): Protein post-translational modifications are central to human complexity, and changes in post-translational modification of proteins are tightly associated with human disease. In this proposal, we will develop general approaches for the design of proteins responsive to post-translational modifications, including serine/threonine and tyrosine phosphorylation; tyrosine sulfation; and specific oxidation via tyrosine nitration, glutathione oxidation, and protein S-nitrosylation. Misregulation of protein phosphorylation plays an important role in numerous human diseases, including cancer, heart disease, Alzheimer's disease, and diabetes. New approaches to interrogate the activites of kinases and phosphatases will have potentially diverse applications in human disease. We are developing approaches employing design and synthesis to understand the phosphoproteome in order to allow the determination of kinase and phosphatase activities in diverse contexts and in a way that is potentially both spatially and temporally addressable. These approaches will be useful for the analysis of the activities of specific kinases and will be readily adaptable to provide a general approach to the detection of protein kinase activity and toward the activity of diverse protein postranslational modifications. Protein sulfation of extracellular tyrosines provides a critical mode for changes in cellular surface presentation and molecular recognition. Tyrosine nitration and protein S-nitrosylation are common post-translational modifications associated with oxidative stress. Changes in oxidative conditions in cells lead to changes in protein and cellular function. Specific aim 1: Development of protein motifs responsive to Ser/Thr and Tyr phosphorylation. We will develop novel, small, designed protein sequences (15-20 amino acids) whose structure is dependent on phosphorylation and which display luminescence only when phosphorylated. The approach will be generalized for application to a diverse series of kinases. Multiple designed motifs will be prepared which contain alternative kinase recognition sites, will utilize natural amino acids to enable their use as genetically encodable sensors of protein kinase activity, and will be incorporated into proteins and the luminescence tested in cellular models. Specific aim 2: Design of protein motifs responsive to Tyr sulfation. We will develop a general platform for the analysis of protein tyrosine sulfation. Specific aim 3: Design of proteins responsive to specific oxidation. We will develop encodable protein domains which are responsive to specific oxidative cellular conditions, including tyrosine nitration, glutathione oxidation, and protein S-nitrosylation. In addition, these approaches will allow the analysis of the changes in post-translational modification which occur in response to drugs, other small molecules, and other cues that change kinase, phosphatase, sulfotransferase, and oxidative activity. PUBLIC HEALTH RELEVANCE: Protein modifications and changes in cellular conditions play central role in human diseases, including cancer, heart disease, Alzheimer's disease, AIDS, and diabetes. We will develop new probes to understand the different types of changes that occur to proteins and in cellular conditions in disease. In addition, these approaches will allow the analysis of the changes which occur in response to drugs, other small molecules, and other cues that change protein state. Understanding how diseased proteins differ from normal proteins is important in developing new approaches to treat these diseases and to determine the effectiveness of new therapeutics.