Molecular mechanism of membrane association of Bruton's Tyrosine Kinase

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.

项目摘要 自适应免疫系统由B细胞接收器（BCR）途径驱动，该途径触发B细胞 响应抗原结合的分化和增殖。 BCR的激活产生信号传导 脂质磷脂酰肌醇 - 3,4,5 - 质膜内部小叶上的三磷酸（PIP3）。这 非受体酪氨酸激酶Bruton的酪氨酸激酶（BTK）的Pleckstrin同源性（pH）结构域结合 该PIP3触发自动抑制构象和反式自动磷酸化的释放。汽车- 磷酸化的BTK激活下游途径，导致B细胞激活和增殖。过度活跃 BCR信号传导可能导致严重的恶性肿瘤，例如慢性淋巴白血病和非霍奇金 淋巴瘤。第一个BTK抑制剂Ibrutinib于2013年被食品药品监督管理局批准 用于治疗B细胞恶性肿瘤的化学疗法的替代方法。成功的BTK抑制作用速度 通过减少转录调节剂NF-κB与DNA的激活和结合，癌细胞增殖。这 最广泛使用的BTK抑制剂是ibrutinib和第二代衍生物，它们不可逆转地结合了 激酶域的ATP结合口袋。这个口袋在酪氨酸激酶中是高度保守的， 因此，由于在 绑定位点。这些因素需要BTK内的替代抑制位点以提高B细胞 癌症治疗。 BTK激活的关键和初始步骤是通过其质膜关联 pH域。 pH结构域代表一个有吸引力的抑制靶标，因为序列同源性较低 在班级中。但是，脂质特异性，PIP3结合的化学计量及其如何调节这些 组件，全长BTK的功能性寡聚状态，所涉及的接口，尚不清楚。目标 我的建议是确定脂质特异性，化学计量，膜的机理 招募，以及与膜相关的BTK的寡聚状态。通过定量 了解这些分子事件，我的目标是了解BTK的功能如何在等离子体中 膜受调节。我将使用Gupta Lab开发的工作流程直接检测蛋白质蛋白质 使用天然质谱法（Nativems）来自脂质双层的蛋白质脂质相互作用。这将允许 我检测在模仿血浆脂质组成的双层中与BTK相互作用的脂质 膜以及膜结合BTK的低聚状态。我已经获得了初步的本质 和囊泡关联数据，显示在体外结合PS以及与BiLayers关联的能力 以PS依赖性方式。我将确定这种但未表征的脂质的功能后果 使用永生的B细胞系相互作用。通过在PS清真寺的存在下激活BCR， 我可以确定PS是否在BCR途径中起作用，使其成为抑制BTK的机制。