Bcl-2 protein Noxa in human T cell metabolism and differentiation

摘要 幼稚的淋巴细胞，一旦收到适当的共刺激信号，就会被激活，并经历 快速增殖爆裂，然后采取效应器功能，直接杀死目标‘入侵’生物或 癌细胞。这种快速增殖的爆发需要增加对合成代谢中葡萄糖的吸收和利用 为生物质生产积木的途径。为了支持增殖激活的T细胞切换到 谷氨酰胺作为氧化磷酸化(OXPHOS)的碳源。转变为有氧糖酵解和 激活的T细胞对谷氨酰胺的依赖增强是有文献记载的，但信号通路 促进这一转变的因素人们知之甚少。 我们小组最近的研究指出，人类BH3-2家族唯一的蛋白Noxa在控制BH3的表达中起着关键作用 这种激活诱导T细胞的代谢转换。根据定义，只有BH3-蛋白质是促凋亡的，但Noxa 在白血病细胞中也显示出促进生长和存活的功能。NOXA的这个非正则函数是 在人类原代T淋巴细胞中也很明显。TCR后T细胞中NOXA显著上调 参与，并在激活的细胞增殖时保持较高水平。此外，它还要求持续提供 谷氨酰胺将保持高表达。早期研究表明，NOXA在T细胞活化中起重要作用 以及与激活相关的新陈代谢重新编程。我们将检验一个中心假设，即-- NOXA是激活的人T细胞代谢转换为谷氨酰胺分解及其分化所必需的 到效应器的表型。 第一个目标是研究Noxa在T细胞激活和谷氨酰胺代谢中的作用。我们将决定 谷氨酰胺是否通过调节microRNAs介导T细胞NOXA的表达。这 AIM还将验证诺沙星是谷氨酰胺分解和活化T细胞增殖所必需的假设 使用CRISPR生成的Noxa KO初级T细胞、生化分析、代谢表型和 代谢组学。尽管Noxa的缺失会损害谷氨酰胺的分解，但并不影响线粒体的适合性。 受刺激的T细胞转变为氧化磷酸化的替代燃料来源。在目标2中，我们将使用多个 参数流式细胞术，以及代谢和功能分析，以验证诺沙星使T细胞 通过促进谷氨酰胺分解产生效应细胞，并阻止T细胞分化为调节因子(Treg) 或Memory(TM)表型，通过抑制脂肪酸氧化。 了解T细胞在扩增过程中的代谢需求将有助于发展 针对新陈代谢的治疗策略。这项研究可能会揭示利用串扰的途径 NOXA和谷氨酰胺代谢之间的关系，以更好地了解驱动代谢转换的细胞事件 在T细胞中。这项拟议的工作还将为了解诺沙星在推动T细胞分化为 这种蛋白质包括效应型、调节型或记忆亚型，并有助于评估其作为治疗工具的潜力。

SUMMARY Naïve lymphocytes, upon receiving appropriate co-stimulatory signals, become activated and go through a rapid proliferative burst before assuming effector functions directed at killing the target ‘invading’ organism or cancer cell. This rapid proliferative burst requires increased uptake and utilization of glucose in anabolic pathways that produce building blocks for biomass. To support proliferation activated T cells switch to glutamine as the carbon source for oxidative phosphorylation (OXPHOS). The shift to aerobic glycolysis and enhanced dependence on glutamine in activated T cells is well documented, but the signaling pathways that facilitate this shift are poorly understood. Recent studies from our group point to a key role for human Bcl-2 family BH3-only protein, Noxa, in controlling this activation-induced metabolic switch in T cells. BH3-only proteins are by definition pro-apoptotic, but Noxa exhibits a pro-growth and survival function as well, in leukemia cells. This non-canonical function of Noxa is also evident in primary human T lymphocytes. Noxa is significantly upregulated in T cells following TCR engagement, and remains high as the activated cells proliferate. Additionally, it requires constant availability of glutamine to remain highly expressed. Early studies suggest that Noxa plays an essential role in T cell activation and the metabolic reprogramming associated with activation. We will test a central hypothesis that states - Noxa is required for the metabolic switch to glutaminolysis in activated human T cells and for their differentiation to the effector phenotype. The first aim will investigate the role of Noxa in T cell activation and glutamine metabolism. We will determine whether glutamine mediates the induction of Noxa expression in T cells through regulation of microRNAs. This aim will also test the hypothesis that Noxa is required for glutaminolysis and for proliferation of activated T cells using novel CRISPR-generated Noxa KO primary T cells, biochemical assays, metabolic phenotyping, and metabolomics. Although loss of Noxa impairs glutaminolysis, it does not affect mitochondrial fitness suggesting stimulated T cells shift to an alternative fuel source for oxidative phosphorylation. In Aim 2 we will use multi- parameter flow cytometry, and metabolic and functional assays to test the hypothesis that Noxa enables T effector cell generation by promoting glutaminolysis, and prevents differentiation of T cells to a regulatory (Treg) or memory (TM) phenotype by suppressing fatty acid oxidation. An understanding of the metabolic needs of T cells as they undergo expansion will help to develop therapeutic strategies targeted at metabolism. This research could reveal avenues to exploit the crosstalk between Noxa and glutamine metabolism to better understand cellular events that drive the metabolic switch in T cells. The proposed work also will offer insights into Noxa’s role in driving differentiation of T cells into effector, regulatory or memory subtypes and help evaluate the protein’s potential as a therapeutic tool.