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

摘要 免疫系统的能力，以调整其效力的反应，以一个外部的威胁是利用大多数 免疫疗法靶向抑制性受体PD 1或CTLA 4调节T细胞的活性和功能 细胞是治疗癌症的一个非常成功的策略。这些检查点疗法阻断了 细胞表面受体的细胞外结构域与抗体。然而，抗体通常具有不足的 药代动力学，不能渗透相关组织。抑制这些跨膜的替代方法 通过靶向下游效应物的受体目前是不可用的，因为信号转导的分子机制 转导尚未完全理解，并且所鉴定的细胞内信号分子在细胞内信号转导中是重要的。 广泛的细胞类型、信号通路和细胞区室。因此，重要的是描述 信号转导机制是T细胞特有的。下游的招募和激活 激酶和磷酸酶是实现这种特异性的一种方式。本研究 研究了SHP 2磷酸酶被招募到抑制性细胞并被其激活的机制。 受体PD 1。为此，将采用多学科方法，利用生物化学， 结构和生物物理方法，以及单分子成像，即超分辨率 荧光显微镜和单粒子跟踪。首先，SHP 2磷酸化和PD 1 将检测对SHP 2构象和磷酸酶活性的结合。氢氘交换- SHP 2的野生型和突变体形式（以其磷酸化和/或PD 1形式）的质谱分析 结合形式）将决定SHP 2中构象变化的性质和位置。这些信息将 与活性变化相关，以确定结构-功能关系。第二，互动动态 将在体内和体外分析SHP 2和PD 1之间的差异。显微镜方法将用于确定 本发明的目的是研究SHP 2对PD 1的募集动力学，并将它们与SHP 2结合亲和力的变化相关联。突变体分析 将探索这些相互作用的分子基础，并确定它们是否可以改变， 调节T细胞应答，以及影响黑素瘤小鼠模型中的疾病发作和进展， 糖尿病第三，SHP 2、PD 1和T细胞受体组分之间的时空关系 将使用最先进的单分子成像技术研究信号通路。这些 方法也将利用野生型和突变型的SHP 2来确定膜是否 可以改变SHP 2的动力学和分布以改变T细胞免疫应答。最后 建议的研究将揭示通过PD 1通路激活SHP 2的独特机制， 活化的T细胞这些机制是变构抑制剂和小分子抑制剂的潜在靶点， 为目前的免疫疗法提供了一种可行的替代方案。

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.