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

• 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
• 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

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.

项目摘要 蛋白质酪氨酸磷酸化是动物细胞中传递重要信息的常见机制， 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 分别去磷酸化成千上万种不同的蛋白质。许多酪氨酸激酶都非常好- 其特征在于，并且是许多临床批准的癌症疗法的靶点。相比之下，大多数酪氨酸 磷酸酶的生物化学特性不好，特别是，我们不知道具体的 这些酶去磷酸化的蛋白质。尽管知识匮乏，我们知道， 很少有酪氨酸磷酸酶直接参与癌症信号传导，遗传学证据表明， 更多的可能是重要的癌症驱动因子、肿瘤抑制因子或免疫反应的调节剂， 癌细胞为了阐明单个酪氨酸磷酸酶在细胞过程中的确切作用， 需要工具来快速和可靠地识别它们的基底。在这里，我们提出了一个战略，以确定直接 活细胞中酪氨酸磷酸酶的底物，通过结合蛋白质工程和质量 基于光谱的蛋白质组学 鉴定酪氨酸磷酸酶底物的一个主要挑战是它们与它们的底物的相互作用。 同源酶是弱的和瞬时的，使得它们难以从复杂的蛋白质组混合物中分离。到 为了解决这个问题，我们将使用遗传密码扩展将光激活交联剂引入到 酪氨酸磷酸酶在目标1中，我们将结合联合收割机的结构和进化信息来确定理想的 酪氨酸磷酸酶上的位置，以放置光交联剂，使得它们不干扰磷酸酶 但是当暴露于UV光时可以不可逆地捕获底物。我们将测试候选人的位置 在模型酪氨酸磷酸酶，PTP 1B上，用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一个阵列底物上测试我们的最佳设计的一般性。在Aim中 3、建立在活的哺乳动物细胞中表达工程酪氨酸磷酸酶的方法， 用UV光捕获底物，并使用质谱法鉴定这些底物。我们将比较 我们的策略是目前最先进的方法，它依赖于酪氨酸磷酸酶的突变， 显著地破坏它们功能且仅适度地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地识别 在几乎任何感兴趣的细胞系中的任何酪氨酸磷酸酶的底物。这项技术可以用于 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。