A Generalizable Photo-Crosslinking Strategy to Identify Tyrosine Phosphatase Substrates

识别酪氨酸磷酸酶底物的通用光交联策略

基本信息

批准号：
10612641
负责人：
Neel H Shah
金额：
$ 19.36万
依托单位：
COLUMBIA UNIV NEW YORK MORNINGSIDE
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-01 至 2026-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10612641
关键词：
Active Sites Amber Amino Acids Animals Binding Binding Proteins Binding Sites Biochemical Bioinformatics Biological Assay Biological Models Cancer Etiology Catalysis Cell Line Cell physiology Cells Clinical Clinical Trials Complex Crosslinker Development Dominant-Negative Mutation Drug Targeting Electrophoretic Mobility Shift Assay Engineering Enzymes Event Exposure to Family Genetic Genetic Code Human Immune response Immunity Immunomodulators Immunoprecipitation In Vitro Individual Knowledge Light Malignant Neoplasms Mammalian Cell Mass Spectrum Analysis Measures Mediating Methods Modeling Modification Mutation Oncogenic PTPN1 gene PTPN11 gene PTPRC gene Peptides Phosphopeptides Phosphoric Monoester Hydrolases Phosphorylation Positioning Attribute Post-Translational Protein Processing Protein Conformation Protein Dephosphorylation Protein Engineering Protein Family Protein Tyrosine Kinase Protein Tyrosine Phosphatase Proteins Proteome Proteomics Protocols documentation Reporting Role Signal Pathway Signal Transduction Site Specificity Structure Substrate Specificity Techniques Testing Tumor Suppressor Proteins Tyrosine Phosphorylation Tyrosine Phosphorylation Site Ultraviolet Rays Work cancer cell cancer therapy design drug discovery enzyme activity enzyme substrate enzyme substrate complex experimental study interest member mutant overexpression personalized immunotherapy precision medicine protein function receptor targeted cancer therapy tool treatment response virtual

项目摘要

PROJECT SUMMARY Protein tyrosine phosphorylation is a common mechanism for relaying essential information in animal cells, and many cancers are driven by aberrant tyrosine phosphorylation events. This protein modification is mediated by two classes of enzymes: tyrosine kinases and tyrosine phosphatases, which phosphorylate and dephosphorylate, respectively, thousands of different proteins. Many tyrosine kinases are extremely well- characterized and are the targets of numerous clinically-approved cancer therapies. By contrast, most tyrosine phosphatases are not biochemically well-characterized, and in particular, we do not know the specific proteins that many of these enzymes dephosphorylate. Despite this dearth of knowledge, we known that a few tyrosine phosphatases directly contribute to cancer signaling, and genetic evidence suggests that many more are likely to be important cancer drivers, tumor suppressors, or modulators of the immune response against cancer cells. In order to elucidate the precise roles of individual tyrosine phosphatases in cellular processes, we need tools to rapidly and reliable identify their substrates. Here, we propose a strategy to identify direct substrates of tyrosine phosphatases in live cells, by combining protein engineering and mass spectrometry-based proteomics. A major challenge in the identification of tyrosine phosphatase substrates is that their interactions with their cognate enzymes are weak and transient, making them difficult to isolate from complex proteomic mixtures. To solve this problem, we will use genetic code expansion to introduce light-activatable crosslinkers into tyrosine phosphatases. In Aim 1, we will combine structural and evolutionary information to identify ideal positions on tyrosine phosphatases to place photo-crosslinkers, such that they do not perturb phosphatase function but can irreversibly capture a substrate when exposed to UV light. We will test out candidate positions on a model tyrosine phosphatase, PTP1B, with three different photo-crosslinkers. In Aim 2, we will examine the generality of our best designs by testing them on several tyrosine phosphatases and an array substrates. In Aim 3, we will establish methods to express the engineered tyrosine phosphatases in live mammalian cells, induce the capture of substrates with UV light, and identify those substrates using mass spectrometry. We will compare our strategy to the current state-of-the-art method, which relies on mutations in tyrosine phosphatases that significantly disrupt their function and only modestly stabilize their interactions with substrates. Successful development of our photo-crosslinking method will enable efficient identification of the substrates of any tyrosine phosphatases in virtually any cell line of interest. This technique could be used to delineate the roles of individual tyrosine phosphatases in cancer signaling and cancer-relevant immunity.

项目概要蛋白质酪氨酸磷酸化是动物细胞中传递基本信息的常见机制，并且许多癌症是由异常的酪氨酸磷酸化事件引起的。这种蛋白质修饰是由两类酶：酪氨酸激酶和酪氨酸磷酸酶，它们磷酸化和分别使数千种不同的蛋白质去磷酸化。许多酪氨酸激酶都非常好- 具有特征，并且是许多临床批准的癌症疗法的目标。相比之下，大多数酪氨酸磷酸酶的生化特性尚不明确，特别是我们不知道其具体作用许多这些酶使蛋白质去磷酸化。尽管缺乏知识，我们知道很少有酪氨酸磷酸酶直接参与癌症信号传导，遗传证据表明许多酪氨酸磷酸酶更多的可能是重要的癌症驱动因素、肿瘤抑制因子或免疫反应调节剂癌细胞。为了阐明单个酪氨酸磷酸酶在细胞过程中的精确作用，我们需要工具来快速可靠地识别其底物。在这里，我们提出了一个策略来识别直接通过结合蛋白质工程和质量研究活细胞中酪氨酸磷酸酶的底物基于光谱分析的蛋白质组学。鉴定酪氨酸磷酸酶底物的一个主要挑战是它们与底物的相互作用同源酶很弱且短暂，使得它们难以从复杂的蛋白质组混合物中分离出来。到为了解决这个问题，我们将使用遗传密码扩展将光活化交联剂引入到酪氨酸磷酸酶。在目标 1 中，我们将结合结构和进化信息来识别理想的在酪氨酸磷酸酶上放置光交联剂的位置，这样它们就不会干扰磷酸酶功能，但当暴露于紫外线时可以不可逆地捕获基材。我们将测试候选人职位模型酪氨酸磷酸酶 PTP1B，具有三种不同的光交联剂。在目标 2 中，我们将检查通过在几种酪氨酸磷酸酶和阵列基板上测试我们最好的设计的通用性。瞄准 3、我们将建立在活哺乳动物细胞中表达工程化酪氨酸磷酸酶的方法，诱导用紫外光捕获底物，并使用质谱法识别这些底物。我们会比较我们对当前最先进方法的策略，该方法依赖于酪氨酸磷酸酶的突变显着破坏它们的功能，并且只能适度稳定它们与底物的相互作用。我们的光交联方法的成功开发将能够有效识别几乎任何感兴趣的细胞系中任何酪氨酸磷酸酶的底物。这个技术可以用描述单个酪氨酸磷酸酶在癌症信号传导和癌症相关免疫中的作用。