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.

细胞拥有能写(激酶)、能擦除(磷酸酶)和能读(磷酸专一性)的酶和蛋白质 结合结构域)跨蛋白质组的磷酸化修饰，以形成复杂的信号系统 调节增殖、分化和转录激活。异常磷酸酪氨酸(PTyr)信号转导 许多人类疾病和pTyr加工分子成分的鉴定 揭示了信号转导的基本原理，并为治疗提供了新的靶点。一个关键的例子 是致癌的bcr-abl融合蛋白，表现出结构性酪氨酸激酶活性，导致过量 PTyr修饰以转化癌细胞。小分子(GleevecTM)及其抗体的研究进展 针对pTyr信号转导的(HerceptinTM)疗法继续改变患者的抗癌治疗选择 诊所。 尽管有巨大的临床潜力，但生物医学领域仍然存在的一个关键障碍是 将特定部位的磷酸化事件分配给人类疾病的治疗靶向的功能变化。 能够克服低丰度和亚化学计量比磷酸化的技术--位置占有率 需要大量的磷蛋白来应对功能性磷蛋白组学研究的挑战。这是 尤其是对于Tyr的磷酸化。与磷酸丝氨酸和-苏氨酸相比，pTyr修饰代表了 人类磷蛋白质组的稀有亚群(~1%)。 拟议研究的目的是应用共价探针捕获位点(称为SiteTraP) PTP癌基因酪氨酸蛋白磷酸酶非受体底物专一性的确定方法 键入11(SHP2)。建议研究的意义在于开发一种化学蛋白质组学策略来 在蛋白质和pTyr位点水平上将底物特异性直接分配给天然蛋白质上的单个PTP 裂解物和细胞研究。 我们将针对基准测试两个独立但相关的特定目标：(目标1)pTyr特异性 SiteTraP在复杂蛋白质组中存在磷酸丝氨酸和-Thr时，(目标1)SiteTraP对 捕获复杂蛋白质组中SHP2特异性的pTyr位点去磷酸化，(目标2)SiteTraP的能力 用于捕获PTP破坏的活细胞中的全局pTyr激活，以及(目标2)SiteTraP的敏感性和特异性 将底物特异性分配给SHP2并确定SHP2抑制剂如何在活体中破坏这些网络 细胞。 总的来说，我们提出的研究将通过1)指导PTP抑制剂而对生物医学界具有重要意义 针对人类疾病中特定pTyr修饰的研究进展，2)揭示活性部位的差异 和变构SHP2抑制剂的作用模式，用于对PTP药理学的基本和翻译理解， 3)对PTP-底物网络及其在活细胞中的调控有更深入的了解。