Molecular mechanism of membrane association of Bruton's Tyrosine Kinase

布鲁顿酪氨酸激酶膜缔合的分子机制

• 批准号: 10604872
• 负责人: Rachel McAllister
• 金额: $ 4.58万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10604872
• 关键词: Adaptive Immune System; Agammaglobulinaemia tyrosine kinase; Antibodies; Antigens; B cell differentiation; B lymphocyte immortalization; B lymphoid malignancy; B-Cell Activation; B-Cell Antigen Receptor; B-Lymphocytes; Binding; Binding Sites; Biochemical; Biological Assay; Cell Line; Cell Proliferation; Cell membrane; Chronic Lymphocytic Leukemia; Complement; Complex; Conserved Sequence; DNA; Data; Detection; Environment; Event; Generations; Genetic Transcription; Goals; Immunity; In Vitro; Informal Social Control; Learning; Length; Lipid Bilayers; Lipid Binding; Lipids; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Membrane; Membrane Proteins; Modeling; Molecular; Molecular Conformation; Mutation; NF-kappa B; Non-Hodgkin' s Lymphoma; PH Domain; Pathway interactions; Pharmacologic Substance; Phosphatidylinositols; Phosphatidylserine Synthase; Phosphatidylserines; Phosphotransferases; Predisposition; Proliferating; Protein Tyrosine Kinase; Proteins; Raji Cell; Receptor Activation; Receptor Signaling; Research; Resistance; Sequence Homology; Signal Pathway; Signal Transduction; Site; Specificity; Surface; Training; Tyrosine Kinase Inhibitor; United States Food and Drug Administration; Vesicle; Western Blotting; adaptive immune response; antigen binding; cancer cell; cancer therapy; chemotherapy; experimental study; in vivo; inhibitor; leukemia/lymphoma; mutant; novel; nuclear factors of activated T-cells; overexpression; pathogen; pharmacologic; phosphoproteomics; preservation; recruit; resistance mutation; response; side effect; stoichiometry; success; tripolyphosphate

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 适应性免疫系统由B细胞受体（BCR）途径驱动，其触发B细胞 分化和增殖。BCR的激活产生信号传导 脂磷脂酰肌醇-3，4，5-三磷酸（PIP 3）在质膜的内小叶上。的 非受体酪氨酸激酶布鲁顿酪氨酸激酶（Btk）的普列克底物蛋白同源（PH）结构域结合 这种PIP 3，触发了自抑制构象和反式自磷酸化的释放。自动- 磷酸化Btk激活下游途径，导致B细胞活化和增殖。过度活动 BCR信号可导致严重的恶性肿瘤，如慢性淋巴细胞白血病和非霍奇金淋巴瘤。 淋巴瘤第一个Btk抑制剂，伊曲替尼，于2013年被美国食品和药物管理局批准为 用于治疗B细胞恶性肿瘤的化疗的替代方案。成功的Btk抑制减缓了 通过减少转录调节因子NF-κB与DNA的活化和结合来抑制癌细胞增殖。的 最广泛使用的Btk抑制剂是伊曲替尼和第二代衍生物，其不可逆地结合Btk。 激酶结构域的ATP结合口袋。该口袋在酪氨酸激酶中高度保守， 因此，治疗导致显著的脱靶副作用和耐药性，这是由于在细胞中的突变引起的。 结合位点这些因素需要Btk内的替代抑制位点来促进B细胞增殖。 癌症治疗Btk活化的关键和初始步骤是其质膜缔合， PH域。PH结构域代表了一个有吸引力的抑制性靶标，因为存在低序列同源性 在班级中。然而，脂质特异性，PIP 3结合的化学计量以及它如何调节这些， 组装，全长Btk的功能性寡聚状态，所涉及的界面是未知的。目标 我的建议是确定脂质特异性，化学计量，膜的机制， 招募，和Btk的膜相关寡聚状态。通过定量 了解这些分子事件，我的目标是了解Btk在血浆中的功能， 膜是有规律的。我将使用古普塔实验室开发的工作流程直接检测蛋白质-蛋白质 和蛋白质-脂质相互作用。这将允许 我检测脂质与Btk相互作用的双层模拟血浆的脂质组成 膜以及膜结合Btk的低聚状态。我已经获得了初步的nativeMS 和囊泡缔合数据，其显示在体外与PS结合以及与双层缔合的能力 以PS依赖的方式。我将确定这种尚未定性的脂质的功能后果， 使用永生化B细胞系的相互作用。通过在PS清除剂存在下活化BCR， 我可以确定PS是否在BCR通路中起作用，使其成为抑制Btk的机制。