Mechanisms restraining the accumulation of antibody secreting cells

B淋巴细胞是唯一能够发育成抗体分泌细胞(ASC)的免疫细胞类型，这种细胞类型通常被称为浆细胞。定义调控ASC发育的基因和细胞过程、寿命以及ASC分泌的抗体数量是基本问题--这些问题的答案可能与体内许多其他动态细胞系统相关。这一知识的实际应用直接关系到疫苗的开发、对自身免疫/过敏状况的干预、反常增加的ASC数量对健康衰老的影响以及哺乳动物细胞生产合成生物疗法。B淋巴细胞的激活和ASC发育的途径是由基因转录的动态变化以及转录后对mRNA的定位、翻译和稳定性的调节所支撑的。这些共同作用决定了蛋白质的产生、地点、时间和数量。ASC功能和存活的翻译控制机制是前沿生物科学新兴和重要领域中一个成熟的进一步发现的领域，这些领域包括将应激反应的分子生物学与细胞分化联系起来；避免蛋白毒性；以及细胞寿命的决定因素。这些知识可能与几乎所有的生物系统和在生物技术或医学环境中的实际应用有关。该项目建立在调节ASC发育和生存的RNA结合蛋白(RBP)的新的遗传筛查基础上。筛查确定了ASC积累所需的基因，以及当缺失时导致ASC形成和存活增加的基因。这一筛选的结果已经发表在预印服务器上，这项拨款提案试图了解两个多蛋白复合体[CCR4NOT和eIF3]中模块的体内免疫学、细胞生物学和分子机制，这些模块对转录、RNA衰变和翻译是基本的。这些模块是限制ASC的发育或存活，还是影响体内的两者？在ASC中，CCR4NOT和eIF3模块是否在物理上和功能上相互耦合，以形成连接转录、mRNA稳定性和翻译的连续体？它们是否调节内质网和氧化应激等动态平衡应激反应，而氧化应激是ASC分泌输出的基础？它们是否调节了蛋白质平衡与营养感应和氧化感应通路之间的联系？模块成分是由高等真核生物中发现的基因编码的，因此对独特的多细胞生物学很可能是重要的。我们建议使用定量评估体内ASC形成和功能的方法来研究这些基因，因为这是一种强大的生理系统选择。我们建议将其与细胞生物学的体外研究结合起来，通过综合数据驱动的转录组学方法，考虑到mRNA的丰度和周转及其翻译效率。此外，我们将确定限制ASC功能的基因编码的蛋白质所在的多蛋白复合体的组成和位置。这种方法的结合可以提供对控制基因表达的不同过程是如何耦合来调节细胞功能和动态平衡的机械性洞察。

The B lymphocyte is the only immune cell type capable of development into the antibody secreting cell (ASC) a cell type commonly referred to as the plasma cell. Defining the genes and cellular processes that regulate ASC development, lifespan and what regulates the amount of antibody secreted by an ASC are fundamental questions -with answers that are likely to be relevant to many other dynamic cell systems in the body. The practical application of this knowledge is of direct relevance to the development of vaccines, interventions for autoimmune/allergic conditions, the impact on healthy ageing of abnormally increased numbers of ASC and the production of synthetic biological therapeutics by mammalian cells. B lymphocyte activation and the pathway of ASC development is underpinned by dynamic changes in gene transcription coupled to post-transcriptional regulation of mRNA localisation, translation and stability. These act together to determine, where, when and in what amounts proteins are produced. The mechanisms of translational control of ASC function and survival is an area ripe for further discoveries in the emerging and important areas of frontier bioscience: these include linking the molecular biology of stress responses to cell differentiation; the avoidance of proteotoxicity; and the determinants of cellular longevity. Knowledge of these may have relevance to almost all biological systems and practical application in biotechnology or medical contexts.This project builds on a new genetic screen for RNA binding proteins (RBP) that regulate the development and survival of ASC. The screen identified genes that were required for the accumulation of ASC and also genes which, when deleted, led to increased ASC formation and survival. The findings of this screen have been published on a preprint server, and this grant proposal is seeking to understand the in vivo immunology, the cell biology and molecular mechanism of modules within two multiprotein complexes [CCR4NOT and EIF3] known to be fundamental for transcription, RNA decay and translation.Do these modules limit ASC development or survival, or do they affect both in vivo? Are the CCR4NOT and EIF3 modules coupled physically and functionally to each other in ASC to form a continuum that connects transcription mRNA stability and translation? Are they regulating homeostatic stress responses such as ER and oxidative stress that is fundamental for the secretory output of ASC? Do they mediate a mechanism to interlink proteostasis with nutrient sensing and oxidative sensing pathways? The module components are encoded by genes only found in higher eukaryotes and thus likely to be important for the unique biology of multicellularity.We propose to study these genes using methods that evaluate quantitatively ASC formation and function in vivo as this is the powerful physiological system of choice. We propose to combine this with in vitro studies of cell biology informed by integrative data-driven transcriptomics methods that take into account the abundance and turnover of mRNA and its translation efficiency. Furthermore, we will determine the composition and location of the multiprotein complexes in which the protein encoded by the genes that limit ASC function reside. This combination of approaches can provide mechanistic insight into how the different processes which control gene expression are coupled to regulate cell function and homeostasis.