Molecular mechanism of membrane association of Bruton's Tyrosine Kinase

项目总结 适应性免疫系统是由B细胞受体(BCR)途径驱动的，该途径触发B细胞 对抗原结合反应的分化和增殖。BCR的激活产生信令 脂质磷脂酰肌醇-3，4，5-三磷酸(PIP3)位于质膜内侧小叶上。这个 非受体酪氨酸激酶(BTK)的Pleckstrin同源(PH)结构域与 这个PIP3，触发了一种自动抑制的构象的释放和反式自动磷酸化。自动- 磷酸化的BTK激活下游通路，导致B细胞的激活和增殖。过度活跃 Bcr信号可导致严重的恶性肿瘤，如慢性淋巴细胞性白血病和非霍奇金氏病 淋巴瘤。第一种BTK抑制剂ibrutinib于2013年被美国食品和药物管理局批准为 治疗B细胞恶性肿瘤的化疗的替代方案。成功的BTK抑制速度放慢 通过减少转录调节因子NF-κB的激活和与dna的结合，实现癌细胞的增殖。这个 最广泛使用的BTK抑制剂是伊布鲁替尼和第二代衍生物，它们不可逆转地结合 激活域的ATP结合口袋。这个口袋在酪氨酸激酶和 因此，治疗会导致显著的非靶向副作用和耐药性，这是由于 结合部位。这些因素需要在BTK内有替代的抑制部位来促进B细胞的发展 癌症治疗。BTK激活的关键和起始步骤是其质膜结合。 PH域。由于具有较低的序列同源性，PH结构域是一个有吸引力的抑制靶点 在班上。然而，PIP3结合的脂特异性、化学计量比以及它是如何调节这些的 全长BTK的组装、功能低聚体状态以及所涉及的界面都是未知的。目标是 我的建议是确定膜脂的专一性、化学计量学、作用机制 募集，以及膜相关的BTK寡聚态。通过量化 了解这些分子事件，我的目标是了解BTK在血浆中的功能 膜是受调节的。我将使用古普塔实验室开发的工作流程来直接检测蛋白质-蛋白质 以及使用自然质谱学(NativeMS)从脂质双层获得的蛋白质-脂质相互作用。这将允许 在模拟血浆脂质成分的双层中检测与BTK相互作用的脂质 膜以及膜结合BTK的低聚态。我已获得初步的原生医学硕士学位 以及显示与PS体外结合以及与双分子层结合的能力的囊泡结合数据 以PS依赖的方式。我将确定这种尚未确定特征的脂质的功能后果 使用永生化的B细胞系进行相互作用。通过在PS清道夫存在的情况下激活BCR， 我可以确定PS是否在BCR途径中发挥作用，使其成为抑制BTK的机制。

PROJECT SUMMARY The adaptive immune system is driven by the B Cell receptor (BCR) pathway, which triggers B cell differentiation and proliferation in response to antigen binding. Activation of the BCR generates signaling lipid phosphatidylinositol – 3,4,5 – triphosphate (PIP3) on the inner leaflet of the plasma membrane. The pleckstrin homology (PH) domain of non-receptor tyrosine kinase Bruton's Tyrosine Kinase (Btk) binds to this PIP3, triggering release of an auto-inhibited conformation and trans auto-phosphorylation. Auto- phosphorylated Btk activates downstream pathways, leading to B cell activation and proliferation. Overactive BCR signaling can lead to severe malignancies, such as chronic lymphatic leukemia and non-Hodgkin's lymphoma. The first Btk inhibitor, ibrutinib, was approved in 2013 by the Food and Drug Administration as an alternative to chemotherapy for the treatment of B cell malignancies. Successful Btk inhibition slows cancer cell proliferation by reducing the activation and binding of transcription regulator NF-κB to DNA. The most widely used Btk inhibitors are ibrutinib and second-generation derivatives, which irreversibly bind the ATP-binding pocket of the kinase domain. This pocket is highly conserved among tyrosine kinases and consequently, treatment leads to significant off-target side effects and resistance due to mutations in the binding site. These factors necessitate alternative inhibitory sites within Btk for the advancement of B-cell cancer treatment. The critical and initial step in Btk activation is its plasma membrane association through the PH domain. The PH domain represents an attractive inhibitory target as there is low sequence homology among the class. However, the lipid specificity, stoichiometry of PIP3 binding and how it regulates these assemblies, functional oligomeric states of full-length Btk, the interfaces involved, are unknown. The goal of my proposal is to determine the lipid specificity, stoichiometry, mechanism of membrane recruitment, and the membrane-associated oligomeric states of Btk. Through a quantitative understanding of these molecular events, I aim to understand how the function of Btk at the plasma membrane is regulated. I will use a workflow developed by the Gupta lab to directly detect protein-protein and protein-lipid interactions from a lipid bilayer using native mass spectrometry (nativeMS). This will allow me to detect the lipids that interact with Btk in a bilayer mimicking the lipid composition of the plasma membrane as well as the oligomeric states of membrane bound Btk. I have obtained preliminary nativeMS and vesicle association data that shows in vitro binding to PS as well as the ability to associate with bilayers in a PS-dependent manner. I will determine the functional consequences of this yet uncharacterized lipid interaction using an immortalized B Cell line. By activating the BCR within the presence of a PS scavenger, I can determine whether PS plays a role in the BCR pathway, making it a mechanism for inhibition of Btk.