Investigating the interplay of structural, molecular and spatial mechanisms that control SHP2 activity downstream of PD1

研究控制 PD1 下游 SHP2 活性的结构、分子和空间机制的相互作用

基本信息

批准号：
10002277
负责人：
Ye Zheng
金额：
$ 34.63万
依托单位：
SALK INSTITUTE FOR BIOLOGICAL STUDIES
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2021-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10002277
关键词：
Affect Affinity Animal Disease Models Antibodies Antibody Therapy Architecture Area Autoimmunity Binding Biochemical Biophysics Blocking Antibodies CD28 gene CTLA4 gene Cell Surface Receptors Cell membrane Cell physiology Complex Data Defect Deuterium Development Diabetes Mellitus Diabetic mouse Disease Disease Progression Drug Kinetics Ensure Extracellular Domain Fluorescence Microscopy Goals Hydrogen Imaging Techniques Imaging technology Immune response Immune system Immunomodulators Immunotherapy In Vitro Infection Kinetics Location Malignant Neoplasms Mass Spectrum Analysis Membrane Microscopy Molecular Molecular Conformation Movement Multiprotein Complexes Nature Onset of illness PD-1 pathway PTPN11 gene Pathway interactions Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Phosphotyrosine Plant Roots Post-Translational Protein Processing Process Research Resolution Signal Pathway Signal Transduction Signaling Molecule Specificity Structure Structure-Activity Relationship T Cell Receptor Signaling Pathway T cell response T-Lymphocyte Testing Time Tissues ZAP-70 Gene anti-CTLA-4 therapy anti-PD1 antibodies assault base biophysical techniques cancer immunotherapy cell type checkpoint therapy enzyme activity in vivo innovation inorganic phosphate insight interdisciplinary approach melanoma molecular imaging mouse model mutant novel novel drug class novel therapeutic intervention particle programmed cell death protein 1 receptor recruit response single molecule small molecule inhibitor spatial relationship spatiotemporal src Homology Region 2 Domain targeted treatment

项目摘要

ABSTRACT The immune system's ability to adjust the potency of its response to an external threat is exploited by most immunotherapies. Targeting the inhibitory receptors PD1 or CTLA4 to modulate the activity and function of T cells has been an extremely successful strategy for treating cancer. These checkpoint therapies block the extracellular domains of cell surface receptors with antibodies. However, antibodies often have inadequate pharmacokinetics and cannot penetrate relevant tissues. Alternative ways of inhibiting these trans-membrane receptors by targeting downstream effectors are currently not available, as the molecular mechanisms of signal transduction are not fully understood, and the identified intracellular signaling molecules are important in a wide range of cell types, signaling pathways, and cellular compartments. Thus, it is important to characterize signal transduction mechanisms that are specific to T cells. The recruitment and activation of downstream kinases and phosphatases by transmembrane receptors is one way that this specificity is achieved. This study investigates the mechanism by which the SHP2 phosphatase is recruited to and activated by the inhibitory receptor PD1 in T cells. To this end, a multidisciplinary approach will be employed that utilizes biochemical, structural, and biophysical approaches, as well as single molecule imaging, namely super resolution fluorescence microscopy and single particle tracking. First, the effects of SHP2 phosphorylation and PD1 binding on SHP2 conformation and phosphatase activity will be examined. Hydrogen-Deuterium Exchange – Mass spectrometry analyses of wild-type and mutant versions of SHP2 (in their phosphorylated and/or PD1 bound forms) will determine the nature and locations of conformational changes in SHP2. This information will be correlated to changes in activity to identify structure-function relationships. Second, interaction dynamics between SHP2 and PD1 will be analyzed in vivo and in vitro. Microscopy approaches will be used to determine recruitment kinetics of SHP2 to PD1 and correlate them to changes in SHP2 binding affinities. Mutant analyses will explore the molecular underpinning of these interactions and determine whether they can be altered to modulate T cell responses, as well as affect disease onset and progression in mouse models of melanoma and diabetes. Third, the spatio-temporal relations between SHP2, PD1, and components of the T cell receptor signaling pathway will be investigated using cutting edge single molecule imaging technologies. These approaches will also utilize wild-type and mutant versions of SHP2 to determine whether the membrane dynamics and distribution of SHP2 can be altered to change T cell immune responses. In conclusion, the suggested research will uncover mechanisms unique to the activation of SHP2 through the PD1 pathway in activated T cells. These mechanisms are potential targets for allosteric and small molecule inhibitors, thereby providing a viable alternative to current immunotherapies.

抽象的免疫系统能够调整其对外部威胁反应的效力的能力被大多数免疫疗法。靶向抑制受体PD1或CTLA4来调节T的活性和功能细胞一直是治疗癌症的极其成功的策略。这些检查点疗法阻止了具有抗体的细胞表面受体的细胞外结构域。但是，抗体通常不足药代动力学，无法穿透相关组织。抑制这些跨膜的替代方法目前尚无靶向下游效应器来接收器，因为信号的分子机制转导尚未完全了解，并且所鉴定的细胞内信号传导分子在A中很重要广泛的细胞类型，信号通路和细胞室。那很重要特定于T细胞的信号传递机制。下游的招募和激活跨膜接收器的激酶和磷酸酶是实现这种特异性的一种方法。这项研究研究SHP2磷酸酶被抑制和激活的机制 T细胞中的受体PD1。为此，将雇用一种使用生化的多学科方法结构和生物物理方法以及单分子成像，即超级分辨率荧光显微镜和单个颗粒跟踪。首先，SHP2磷酸化和PD1的影响将检查对SHP2构象和磷酸酶活性的结合。氢 - 居民交换 - SHP2的野生型和突变版本的质谱分析（在其磷酸化和/或PD1中结合形式）将确定SHP2中构象变化的性质和位置。这些信息将与活动的变化相关，以识别结构功能关系。其次，交互动力学 SHP2和PD1之间将在体内和体外进行分析。显微镜方法将用于确定 SHP2对PD1的募集动力学与SHP2结合亲和力的变化相关。突变分析将探索这些相互作用的分子基础，并确定是否可以将它们更改为调节T细胞反应，并影响黑色素瘤小鼠模型和糖尿病。第三，SHP2，PD1和T细胞接收器的组件之间的时空关系信号通路将使用尖端单分子成像技术研究。这些方法还将利用SHP2的野生型和突变版本来确定是否膜可以改变SHP2的动力学和分布以改变T细胞免疫反应。总之，建议的研究将发现SHP2通过PD1途径激活的机制活化的T细胞。这些机制是变构和小分子抑制剂的潜在目标，从而提供当前免疫疗法的可行替代方案。