A Generalizable Photo-Crosslinking Strategy to Identify Tyrosine Phosphatase Substrates

项目总结 蛋白质酪氨酸磷酸化是动物细胞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驱动的。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 分别对数千种不同的蛋白质进行去磷酸化。很多酪氨酸酶都非常好- 是许多临床批准的癌症疗法的特点和靶点。相比之下，大多数酪氨酸 磷酸酶的生物化学特性不是很好，尤其是，我们不知道具体的 这些酶中的许多酶能使蛋白质去磷酸化。尽管知识匮乏，但我们知道一个 很少有酪氨酸磷酸酶直接参与癌症信号传递，而遗传证据表明，许多 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的调节器。 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，我们 需要工具来快速、可靠地识别它们的底物。在这里，我们提出了一种策略，以确定直接 结合蛋白质工程和质量的活细胞酪氨酸磷酸酶底物 以光谱为基础的蛋白质组学。 鉴定酪氨酸磷酸酶底物的一个主要挑战是它们与它们的 同源酶是弱的和瞬时的，使得它们很难从复杂的蛋白质组混合物中分离出来。至 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，我们将结合结构和进化信息来识别理想 酪氨酸磷酸酶上放置光交联剂的位置，以便它们不扰乱磷酸酶 功能，但在暴露在紫外线下时可以不可逆转地捕获基材。我们将对候选人的职位进行测试 在具有三种不同光交联剂的模型酪氨酸磷酸酶PTP1B上。在目标2中，我们将研究 通过在几种酪氨酸磷酸酶和阵列基板上测试我们最佳设计的通用性。在AIM 3，我们将建立在哺乳动物活细胞中表达工程酪氨酸磷酸酶的方法，诱导 用紫外光捕捉底物，并用质谱仪鉴定这些底物。我们会比较一下 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 严重扰乱它们的功能，仅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使我们能够有效地识别 任何酪氨酸磷酸酶的底物，几乎在任何感兴趣的细胞系中。这项技术可以用来 描述单个酪氨酸磷酸酶在癌症信号转导和癌症相关免疫中的作用。

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.