Functional roles of lipids in early B cell receptor signaling.

描述(申请人提供)：质膜脂类的物理性质在B细胞受体(BCR)激活中起着至关重要的作用，但关于脂类如何调节这一信号通路中蛋白质的组织和活性却知之甚少。克服这一基本知识差距对于阐明这一基本生物学功能的潜在机制是必要的，并将导致开发针对免疫相关疾病的新干预措施。这项建议中概述的实验将确定质膜脂在调节BCR和免疫反应的下游介质之间的相互作用中所起的作用，并将开发调节细胞内脂质介导的相互作用的实验方法。工作假说是，健康的B细胞利用其膜脂的关键成分维持的异质性，平衡BCR、激活激酶和下调磷酸酶之间的相互作用，以促进静止细胞的早期激活步骤和抑制受体活性。这项拟议的研究通过量化BCR和质膜蛋白之间的脂类介导的相互作用来检验这一工作假说，这些BCR在正常的B细胞中和在有针对性的脂类异质性扰动的细胞中的相互作用。 在广泛的初步数据的指导下，将追求两个特定的目标：1)量化和调节BCR和最小的膜锚之间的有效相互作用；2)确定脂类在早期B细胞受体激活中的作用。这两个目标都将利用申请人实验室开发的一种新的和经过测试的方法，使用超分辨率成像技术直接测量质膜蛋白之间的相互作用势。这两个目标还将应用一类新的膜扰动，以改变细胞中分离的质膜囊泡的相行为和交联型BCR受体簇的组成。在第一个目标中，将测量BCR与在B细胞中表达的膜蛋白锚定基序之间的相互作用能量，并且相互作用将受到生化扰动的调节。在第二个目标下，将测量参与BCR信号的全长蛋白质的活性，并将在一系列实验条件下量化它们与其他信号成分的有效相互作用。将开发一个包括蛋白质和脂类相互作用的预测模型，并将研究具有已知bcr信号缺陷的淋巴瘤细胞的质膜异质性。虽然这项拟议的研究将在B细胞中进行，但它对研究脂类在其他生物过程和细胞类型中的作用具有重要意义。这种方法是创新的，因为它借鉴了尖端的实验方法，以及脂类通过调节嵌入蛋白质之间的有效相互作用来影响功能过程的独特视角。这项拟议的工作意义重大，因为它将建立一种通过脂类调节免疫信号过程的机制，从而能够通过操纵质膜物理性质来治疗免疫相关疾病的新策略。

DESCRIPTION (provided by applicant): The physical properties of plasma membrane lipids play vital roles in B cell receptor (BCR) activation, yet remarkably little is known regarding how lipids regulate the organization and activity of proteins within this signaling pathway. Overcoming this basic knowledge gap is necessary to elucidate the mechanisms underlying this essential biological function and will lead to the development of novel interventions for immune- related diseases. The experiments outlined in this proposal will identify the role plasma membrane lipids in regulating interactions between the BCR and down-stream mediators of the immune response, and will develop experimental methods to modulate lipid-mediated interactions in cells. The working hypothesis is that healthy B cells exploit heterogeneity maintained by a critical composition of its membrane lipids, to balance interactions between the BCR, activating kinases, and down-regulating phosphatases in order to facilitate early activation steps and suppress receptor activity in resting cells. The proposed research tests this working hypothesis by quantifying lipid-mediated interactions between the BCR and plasma membrane proteins in normal B cells and in cells treated with targeted perturbations of lipid heterogeneity. Guided by extensive preliminary data, two specific aims will be pursued: 1) Quantify and modulate effective interactions between the BCR and minimal membrane anchors and 2) Identify the roles of lipids in early B cell receptor activation. Both aims will utilize a new and tested method developed in the applicant's laboratory to directly measure interaction potentials between plasma membrane proteins using super-resolution imaging techniques. Both aims will also apply a novel class of membrane perturbations shown to modify the phase behavior of isolated plasma membrane vesicles and the composition of cross-linked BCR receptor clusters in cells. In the first aim, interaction energies will be measured between the BCR and membrane protein anchor motifs expressed in B cells and interactions will be modulated with biochemical perturbations. Under the second aim, the activity of full length proteins involved in BCR signaling will be measured and their effective interactions with other signaling components will be quantified over a range of experimental conditions. A predictive model will be developed that includes protein and lipid interactions, and plasma membrane heterogeneity will be investigated in lymphoma cells with known defects in BCR signaling. Although the proposed research will take place in B cells, it has implications for studying of role of lipids in other biological proceses and cell types. This approach is innovative because it draws on cutting edge experimental methodologies as well as the unique perspective that lipids impact functional processes by modulating effective interactions between embedded proteins. The proposed work is significant because it will establish a mechanism for lipid-mediated control of immune signaling processes enabling new strategies for the treatment of immune related disease through manipulation of plasma membrane physical properties.