Defining and targeting substrate specificity of protein tyrosine phosphatases

蛋白质酪氨酸磷酸酶的底物特异性的定义和靶向

• 批准号: 10580475
• 负责人: Ku-Lung Hsu
• 金额: $ 24.99万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2025-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10580475
• 关键词: Active Sites; Address; Antibodies; Benchmarking; Binding; Biological Process; Cells; Chemicals; Clinic; Clinical; Communities; Complex; Development; Enzymes; Event; Exhibits; FDA approved; Goals; Human; Individual; Knowledge; Malignant Neoplasms; Methodology; Modification; Molecular; Oncogenes; Oncogenic; PTPN11 gene; Pharmaceutical Preparations; Pharmacology; Phosphoproteins; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Phosphotyrosine; Post-Translational Protein Processing; Protein Dephosphorylation; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Protein phosphatase; Proteins; Proteome; Proteomics; Regulation; Research; Role; Sensitivity and Specificity; Signal Transduction; Site; Specificity; Substrate Specificity; System; Technology; Testing; Therapeutic; Transcriptional Activation; Tyrosine; Writing; anticancer treatment; bcr-abl Fusion Proteins; cell transformation; human disease; inhibitor; interest; new therapeutic target; phosphatase inhibitor; phosphoproteomics; programs; research clinical testing; small molecule; therapeutic target

Cells possess enzymes and proteins that can write (kinase), erase (phosphatase), and read (phospho-specific binding domains) phosphorylation modifications across the proteome to form a complex signaling system for regulating proliferation, differentiation, and transcriptional activation. Aberrant phosphotyrosine (pTyr) signaling underlie many human diseases and the identification of molecular components of pTyr processing has uncovered fundamental principles of signal transduction and furnished new targets for therapy. A key example is the oncogenic BCR-ABL fusion protein that exhibits constitutive tyrosine kinase activity resulting in excessive pTyr modifications to transform cells in cancer. The development of small molecule (GleevecTM) and antibody (HerceptinTM) therapeutics that target pTyr signaling continues to transform anti-cancer treatment options in the clinic. Despite enormous clinical potential, a critical barrier that remains in the biomedical field is the ability to assign site specific phosphorylation events to functional changes for therapeutic targeting in human disease. Technologies capable of overcoming the low abundance and substoichiometric phosphorylation-site occupancy of phosphoproteins are needed to address the challenge of functional phosphoproteomic profiling. This is especially true for phosphorylation of Tyr. Compared with phospho-Ser and -Thr, pTyr modifications represent a rare subset (~1%) of the human phosphoproteome. The objective of the proposed studies is to apply a site trapping by covalent probes (dubbed SiteTraP) methodology to assign substrate specificity to the PTP oncogene, tyrosine-protein phosphatase non-receptor type 11 (SHP2). The significance of the proposed studies is development of a chemical proteomics strategy to assign substrate specificity – at the protein and pTyr site level – to individual PTPs directly on native proteins in lysate and cellular studies. We will test 2 independent, yet related specific aims directed at benchmarking: (Aim 1) pTyr specificity of SiteTraP in the presence of phospho-Ser and -Thr in complex proteomes, (Aim 1) Sensitivity of SiteTraP for capturing SHP2-specific dephosphorylation of pTyr sites in complex proteomes, (Aim 2) Capability of SiteTraP for capturing global pTyr activation in PTP-disrupted live cells, and (Aim 2) Sensitivity and specificity of SiteTraP for assigning substrate specificity to SHP2 and determining how SHP2 inhibitors disrupt these networks in live cells. Broadly, our proposed studies will be important for the biomedical community by 1) guiding PTP inhibitor development for targeting specific pTyr modifications in human disease, 2) revealing differences in active site and allosteric SHP2 inhibitor mode of action for basic and translational understanding of PTP pharmacology, and 3) gain a deeper understanding of PTP-substrate networks and regulation in live cells.

细胞拥有可以写入（激酶）、擦除（磷酸酶）和读取（磷酸特异性酶）的酶和蛋白质。 结合域）跨蛋白质组的磷酸化修饰，形成复杂的信号系统 调节增殖、分化和转录激活异常的磷酸酪氨酸 (pTyr) 信号。 pTyr 加工的分子成分的鉴定是许多人类疾病的根源 揭示了信号转导的基本原理并为治疗提供了新的目标。 是致癌的 BCR-ABL 融合蛋白，具有组成型酪氨酸激酶活性，导致过量 pTyr 修饰可转化癌症细胞。小分子 (GleevecTM) 和抗体的开发。 (HerceptinTM) 靶向 pTyr 信号传导的疗法继续改变抗癌治疗选择 诊所。 尽管具有巨大的临床潜力，但生物医学领域仍然存在一个关键障碍： 将位点特异性磷酸化事件分配给功能变化，以实现人类疾病的治疗靶向。 能够克服低丰度和亚化学计量磷酸化位点占用的技术 需要大量的磷蛋白来应对功能性磷酸蛋白质组分析的挑战。 与磷酸化-Ser 和-Thr 相比，pTyr 修饰尤其适用于 Tyr 的磷酸化。 人类磷酸化蛋白质组的罕见子集（~1%）。 拟议研究的目的是应用共价探针的位点捕获（称为 SiteTraP） 将底物特异性分配给 PTP 癌基因、酪氨酸蛋白磷酸酶非受体的方法 11 型（SHP2）的研究意义在于开发化学蛋白质组学策略。 将底物特异性（在蛋白质和 pTyr 位点水平）直接分配给蛋白质中天然蛋白质上的各个 PTP 裂解物和细胞研究。 我们将测试 2 个独立但相关的具体目标，旨在进行基准测试：（目标 1）pTyr 特异性 SiteTraP 在复杂蛋白质组中存在磷酸-Ser 和 -Thr 的情况下，（目标 1）SiteTraP 对 捕获复杂蛋白质组中 pTyr 位点的 SHP2 特异性去磷酸化，（目标 2）SiteTraP 的功能 用于捕获 PTP 破坏的活细胞中的全局 pTyr 激活，以及（目标 2）SiteTraP 的灵敏度和特异性 用于分配 SHP2 的底物特异性并确定 SHP2 抑制剂如何在体内破坏这些网络 细胞。 总的来说，我们提出的研究对于生物医学界来说很重要：1）指导 PTP 抑制剂 针对人类疾病中特定 pTyr 修饰的开发，2) 揭示活性位点的差异 和变构 SHP2 抑制剂作用模式，用于 PTP 药理学的基本和转化理解， 3) 更深入地了解活细胞中的 PTP 底物网络和调节。