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: 10341499
• 负责人: Ku-Lung Hsu
• 金额: $ 34.37万
• 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/10341499
• 关键词: Active Sites; Address; Antibodies; Benchmarking; Binding; Biological Assay; Biological Process; Biology; CRISPR/Cas technology; Cancer Patient; Catalytic Domain; Cell Line; Cell Proliferation; Cell physiology; Cells; Chemicals; Chemistry; Clinic; Clinical; Clinical Trials; Communities; Complex; Development; Enzymes; Evaluation; Event; Exhibits; FDA approved; Felis catus; Genetic Models; Goals; Human; Individual; Knock-out; Knowledge; Label; Length; Malignant Neoplasms; Maps; Methodology; Methods; Modification; Molecular; Monitor; Noise; Oncogenes; Oncogenic; Outcome; PTPN11 gene; Peptides; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacology; Phenols; Phosphopeptides; Phosphoproteins; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Phosphotyrosine; Physiological; Post-Translational Protein Processing; Protein Dephosphorylation; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Protein phosphatase; Proteins; Proteome; Proteomics; Publications; Regulation; Research; Resources; Role; Sensitivity and Specificity; Signal Transduction; Site; Specificity; Substrate Specificity; Sulfur; System; Technology; Testing; Therapeutic; Transcriptional Activation; Triazoles; Tyrosine; Tyrosine Phosphorylation; Writing; anticancer treatment; bcr-abl Fusion Proteins; cell transformation; chemical reaction; human disease; improved; inhibitor; innovation; insight; interest; new therapeutic target; novel; pervanadate; phosphatase inhibitor; phosphoproteomics; precision medicine; programs; research clinical testing; small molecule; src Homology Region 2 Domain; 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）的酶和蛋白质。 结合结构域）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。异常磷酸酪氨酸（pTyr）信号传导 是许多人类疾病的基础，pTyr加工的分子组分的鉴定 揭示了信号转导的基本原理，为治疗提供了新的靶点。一个重要例子 是一种致癌的BCR-ABL融合蛋白，表现出组成型酪氨酸激酶活性，导致过度的 pTyr修饰以转化癌症细胞。小分子药物（格列卫TM）和抗体的开发 靶向pTyr信号传导的赫赛汀（HerceptinTM）疗法继续改变癌症治疗的选择， 诊所 尽管具有巨大的临床潜力，但生物医学领域仍然存在的一个关键障碍是， 将位点特异性磷酸化事件分配给功能变化，用于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 磷蛋白的需要，以解决功能磷酸化蛋白质组学分析的挑战。这是 尤其是Tyr的磷酸化。与磷酸化-Ser和-Thr相比，pTyr修饰代表了一种新的修饰方式。 人类磷酸化蛋白质组的罕见子集（~1%）。 拟议研究的目的是通过共价探针（称为SiteTraP）应用位点捕获 PTP癌基因酪氨酸蛋白磷酸酶非受体底物特异性的方法学 11型（SHP 2）。所提出的研究的意义是发展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-分配给直接在天然蛋白质上的单个PTP， 裂解物和细胞研究。 我们将测试2个独立但相关的针对基准的特定目标：（目标1） 在复杂蛋白质组中存在磷酸化-Ser和-Thr的情况下的SiteTraP，（目的1）SiteTraP对 捕获复杂蛋白质组中pTyr位点的SHP 2特异性去磷酸化，（目的2）SiteTraP的能力 用于捕获PTP破坏的活细胞中的全局pTyr活化，以及（目的2）SiteTraP的灵敏度和特异性 为SHP 2分配底物特异性，并确定SHP 2抑制剂如何破坏这些网络， 细胞 从广义上讲，我们提出的研究将是重要的生物医学界1）指导PTP抑制剂 开发靶向人类疾病中的特异性pTyr修饰，2）揭示活性位点的差异 和变构SHP 2抑制剂的作用模式，用于PTP药理学的基础和转化理解， 和3）获得对活细胞中PTP-底物网络和调节的更深入的理解。