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

总结 幼稚淋巴细胞在接收到适当的共刺激信号后，被激活并经历一个周期。 快速增殖爆发，然后承担效应子功能，直接杀死目标“入侵”生物体，或 癌细胞这种快速的增殖爆发需要增加葡萄糖的摄取和利用， 产生生物质的基本单元的途径。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。向有氧糖酵解的转变， 活化T细胞对谷氨酰胺的依赖性增强已得到充分证明，但信号通路 促进这一转变的因素却知之甚少。 我们小组最近的研究指出，人类Bcl-2家族BH 3蛋白Noxa在控制 这种激活诱导的T细胞代谢转换。BH 3-only蛋白是促凋亡蛋白，但Noxa 在白血病细胞中也表现出促生长和存活功能。Noxa的这个非正则函数是 在原代人T淋巴细胞中也是明显的。Noxa在TCR后T细胞中显著上调 参与，并保持高的活化细胞增殖。此外，它还需要持续提供 谷氨酰胺保持高表达。早期的研究表明，Noxa在T细胞活化中起着重要作用 以及与激活相关的代谢重编程。我们将检验一个中心假设，即- Noxa是活化的人T细胞代谢转换为γ-氨基裂解及其分化所必需的 到效应子表型。 第一个目标是研究Noxa在T细胞活化和谷氨酰胺代谢中的作用。我们将确定 谷氨酰胺是否通过调节microRNA介导T细胞中Noxa表达的诱导。这 本研究还将检验Noxa是γ-氨基裂解和活化T细胞增殖所必需的假说 使用新的CRISPR产生的Noxa KO原代T细胞，生物化学测定，代谢表型， 代谢组学尽管Noxa的缺失会损害线粒体氨解，但它并不影响线粒体的适应性，这表明 受刺激的T细胞转变为氧化磷酸化的替代燃料源。在Aim 2中，我们将使用多- 参数流式细胞术，以及代谢和功能测定，以检验Noxa使T 通过促进γ-氨基裂解产生效应细胞，并阻止T细胞分化为调节性T细胞（Treg） 或记忆（TM）表型。 了解T细胞在扩增过程中的代谢需求将有助于发展 针对代谢的治疗策略。这项研究可以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深入了解Noxa在驱动T细胞分化为T细胞中的作用。 效应子，调节或记忆亚型，并帮助评估蛋白质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.