Interactions between the ADORA2b/Sphk1axis and the AE1-Hb switch in red blood cell aging in vivo and in vitro

ADORA2b/Sphk1axis 和 AE1-Hb 开关在体内和体外红细胞老化中的相互作用

• 批准号: 10369002
• 负责人: Angelo D'Alessandro
• 金额: $ 63.65万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-15 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10369002
• 关键词: Acclimatization; Adenosine; Adenosine A2B Receptor; Affect; Affinity; Age; Aging; Aldehyde-Lyases; Altitude; American; Animal Model; Anions; Antigens; Antioxidants; Binding; Biochemical; Blood; Blood Banks; Blood Circulation; Blood Transfusion; Blood donor; Cell Aging; Cell Nucleus; Cell Survival; Cells; Cellular Metabolic Process; Cellular biology; Chlorides; Complex; Cytolysis; Cytosol; Data; Disease; Docking; Energy Metabolism; Enzymes; Erythrocyte Transfusion; Erythrocytes; Exposure to; Face; Functional disorder; Gene Expression Regulation; Glucose; Glucosephosphate Dehydrogenase; Glucosephosphate Dehydrogenase Deficiency; Glyceraldehyde-3-Phosphate Dehydrogenases; Glycolysis; Health; Hemoglobin; Hospitals; Hour; Human; Human body; Hypoxia; In Vitro; Individual; Intervention; Iron; Life; Logistics; Longevity; Lung; Medical; Membrane; Metabolic; Metabolic stress; Metabolism; Modification; Morphology; Mus; N-terminal; NADH; NADP; Organelles; Oxidation-Reduction; Oxidative Stress; Oxygen; Pathologic; Pathology; Pathway interactions; Patients; Pentosephosphate Pathway; Peripheral; Persons; Plasma; Play; Predisposition; Procedures; Proteins; Proteomics; Reaction; Role; SPHK1 enzyme; Savings; Sea; Series; Sickle Cell Anemia; Signal Transduction; Site; Technology; Tissues; Translations; Vaccination; age related; base; cell age; cofactor; deoxyhemoglobin; diphosphoglycerate; gene product; in vivo; mouse model; normoxia; novel; oxidant stress; oxidative damage; repaired; response; senescence; sensor; tool

ABSTRACT Red Blood Cells (RBCs) represent ~83% of the total human cells in the body. RBC hemoglobin (Hb), which is critical for their function to carry and deliver oxygen to peripheral tissues, constitutes ~90% of the total protein content of a mature RBCs. During their lifespan of 120 days in the bloodstream, RBCs are constantly exposed to oxidant stress, which mostly arises from Fenton and Haber-Weiss reactions triggered by Hb-Iron in the presence of oxygen. However, RBCs lack nuclei and organelle and, as such, they cannot synthesize new proteins to replace oxidatively damaged components. Therefore, in order to cope with oxidant stress, RBCs have evolved unique mechanisms that rely on signaling axes and their capacity to trigger metabolic reprogramming to favor antioxidant defenses (the Pentose Phosphate Pathway – PPP) over energy metabolism (glycolysis). One such mechanism relies on the two most abundant proteins in RBC cytosols and membranes: Hb and anion exchanger 1 (AE1), respectively. Owing to its capacity to “sense” oxygen, in response to hypoxia, deoxygenated Hb migrates to the membrane, where it binds the N-terminus of AE1. This mechanism releases a series of glycolytic enzymes, which are inhibited under high-oxygen tensions owing to their binding to the same region of AE1 with high affinity for deoxygenated Hb. This phenomenon favors energy metabolism under low oxygen tensions (e.g., high-altitude hypoxia), while it creates a metabolic bottleneck in energy metabolism to promote a critical antioxidant pathway when oxidant stress is high: the Pentose Phosphate Pathway (PPP). This mechanism is referred to as the AE1-Hb switch in this proposal. Of note, glucose 6-phosphate dehydrogenase (G6PD) is not only the rate-limiting enzyme of the PPP, but also the target of the most common enzymopathy in humans, G6PD deficiency, which affects ~400 million people. While RBCs from G6PD-deficient subjects are perfectly healthy in the absence of oxidant stress, RBCs from these individuals are characterized by a shorter lifespan and susceptibility to lysis following oxidative insults. Oxidative stress to RBCs is not only relevant within the context of RBC senescence. Significant oxidant stress arises during RBC storage under blood bank conditions for blood transfusion purposes, a common in hospital medical procedure and a life-saving intervention for ~3-4 million Americans every year. However, little is known about the impact of the AE1-Hb switch and G6PD deficiency in the context of RBC aging in vitro (blood bank). In parallel, when studying human acclimatization to high-altitude hypoxia, we discovered a novel axis, the ADORA2b/Sphk1 axis, that interplays with the AE1-Hb switch to favor oxygen off-loading in healthy individuals as they climb to high-altitude, where oxygen is limited in comparison to sea level. By leveraging a combination of state-of-the art omics technologies (fluxomics and Xlinking proteomics) and a mix of well-established and novel animal models (exclusively developed for this proposal – e.g., G6PD-def mice) we will investigate the interplay of the AE1-Hb switch and ADORA2b/Sphk1 in the context of oxidant stress and hypoxia during RBC aging in vivo (senescence) and in vitro (blood bank).

抽象的 红细胞（RBC）占体内人类总细胞的约83％。 RBC血红蛋白（HB）， 它们的功能至关重要的至关重要 成熟的RBC的内容。在血液中的120天的寿命中，RBC不断暴露 氧化物应激，主要是由HB铁触发的Fenton和Haber-Weiss反应引起的 氧的存在。但是，RBC缺乏核和细胞器，因此，它们无法合成新的 蛋白质代替氧化损坏的成分。因此，为了应对氧化应激，RBC具有 进化的独特机制依赖信号轴及其触发代谢重编程的能力 有利于抗氧化剂防御（戊糖磷酸途径 - PPP），而不是能量代谢（糖酵解）。 这样一种机制依赖于RBC细胞质和膜中的两种最丰富的蛋白质：HB和阴离子 交换器1（AE1）。由于其具有“感官”氧气的能力，响应缺氧，脱氧 HB迁移到膜，该膜结合了AE1的N端。这种机制释放了一系列 糖酵解酶，由于它们与同一区域的结合，它们在高氧张力下抑制 AE1对脱氧Hb具有高亲和力。这种现象有利于低氧下的能量代谢 紧张局势（例如高空缺氧），而它会在能量代谢中产生代谢瓶颈以促进 当氧化应激较高时，临界抗氧化剂途径：戊糖磷酸盐途径（PPP）。这 该提案中的机制称为AE1-HB开关。值得注意的是，葡萄糖6-磷酸脱氢酶 （G6PD）不仅是PPP的限速酶，而且是最常见的酶病的靶标 人类，G6PD缺乏症，影响约4亿人。虽然来自G6PD缺陷受试者的RBC是 在没有氧化物应力的情况下，完全健康，来自这些个体的RBC的特征是较短 氧化损伤后的寿命和裂解的易感性。对RBC的氧化应激不仅与 RBC感应的上下文。在血库下的RBC存储期间，出现了明显的氧化应激 输血目的的条件，在医院医疗程序中常见和挽救生命的干预措施 每年约3-400万美国人。但是，对AE1-HB开关和G6PD的影响知之甚少 在RBC体外衰老的背景下（血库）不足。同时，研究人类适应于 高空缺氧，我们发现了一个新型轴，Adora2b/sphk1轴，与AE1-HB相互作用 切换到健康个体爬到高空时的氧气下载，其中氧气受到限制 与海平面进行比较。通过利用最先进的艺术技术技术（通量学和 Xlinking蛋白质组学）以及建立了良好和新颖的动物模型的混合（专为此而开发 提案 - 例如，G6PD-DEF小鼠）我们将研究AE1-HB开关和ADORA2B/SPHK1的相互作用 RBC衰老在体内（感应）和体外（血液库）期间的氧化应激和缺氧的背景。