Dissection and Manipulation of the Cellular Response to Iron Restriction

项目摘要/摘要 红细胞性铁限制反应是两种最常见的贫血类型的基础：慢性 疾病和炎症(ACDI)和缺铁性贫血(IDA)。这些贫血使一个重大的全球 发病率和死亡率的负担，ACDI目前还没有最佳的治疗方法。这种特定于世系的 祖细胞对营养缺乏的反应涉及异柠檬酸产生的损失，并可通过以下方式逆转 在体外和体内提供外源性异柠檬酸。我们最近开发了一种口服配方，显示了 使用临床上可行的剂量方案对小鼠ACDI的持续疗效。上一次资助的结果 时期确定了与铁限制有关的促红细胞生成素(EPO)抵抗的分子基础，铁限制是主要的 临床问题(J.Exp.地中海医院。2018年)。从本质上讲，缺铁导致细胞表面转运失败。 促红细胞生成素受体(EPOR)及其相关因子(Scrib和TfR2)。这种异常被异柠檬酸逆转。 恢复促红细胞生成素反应性的治疗。最近，我们发现铁和异柠檬酸 以红系特有的方式调节高尔基体的完整性。此外，红细胞铁限制诱导 微管细胞骨架的早期和持续破坏，这是一种已知对高尔基体至关重要的结构 维修。异柠檬酸治疗不能阻止最初的微管破裂，但促进了微管破裂 在以后的时间点重新组装。我们对患者样本的研究，以及之前发表的报告，支持 这种微管反应的临床意义。然后，我们挖掘了一个全面的蛋白质组数据集 分期人类红系祖细胞，以确定可能导致微管不稳定性的特征。在… 在所有阶段，红系细胞都表现出稳定微管相关蛋白的显著不足。 (MAP)，如普遍存在的MAP4，并大量表达微管失稳剂stathmin1 (STMN1)。因此，我们假设存在一个非规范的、铁调节的、稳定的映射和 检测了铁蛋白重链(FTH1)，已知具有微管结合活性，并受 铁制的。由于蛋白水解性和非蛋白水解性，红细胞铁限制导致FTH1迅速而有力地下降 机制，后者可能涉及IRP翻译抑制。异柠檬酸挽救了FTH1水平，但没有 防止早期蛋白分解；其拯救的一个可能的目标是Irp1，已知受异柠檬酸和 参与红细胞铁限制反应。重要的是，慢病毒对FTH1的敲除被中断 微管和分化受损，类似于铁限制的方式。囊泡和蛋白质转运 可能通过微管依赖或非独立机制发生。值得注意的是，我们发现了一个 红系细胞内源性FTH1与EPOR强而特异的相互作用 受体囊泡的微管重新聚集。拟议的实验将检验FTH1的假设 参与了红细胞铁限制反应的两个关键成分：1)铁的一种特殊途径-- 敏感的EPOR囊泡运输和2)铁和异柠檬酸调节的微管稳定性的维持。

Project Summary/Abstract The erythroid iron restriction response underlies two of the most common types of anemia: anemia of chronic disease and inflammation (ACDI) and iron deficiency anemia (IDA). These anemias confer a major global burden of morbidity and mortality, with no optimal therapies yet available for ACDI. This lineage-specific progenitor response to nutrient deprivation involves loss of isocitrate production and can be reversed by providing exogenous isocitrate in vitro and in vivo. We have recently developed an oral formulation showing sustained efficacy in murine ACDI using a clinically feasible dosing regimen. Results from the last funding period identified the molecular basis for erythropoietin (Epo) resistance associated with iron restriction, a major clinical problem (J. Exp. Med. 2018). In essence, iron deprivation caused a failure in cell surface delivery of the Epo receptor (EpoR) and associated factors (Scrib and TfR2). This abnormality was reversed by isocitrate treatment which restored Epo responsiveness. More recently, we have discovered that iron and isocitrate modulate Golgi integrity in an erythroid lineage-specific manner. Furthermore, erythroid iron restriction induced an early and sustained disruption of the microtubule cytoskeleton, a structure known to be critical for Golgi maintenance. Isocitrate treatment did not prevent the initial microtubule disruption but promoted its reassembly at later time points. Our studies of patient specimens, as well as prior published reports, support the clinical relevance of this microtubule response. We then mined a comprehensive proteomic dataset on staged human erythroid progenitors to identify features that might contribute to the microtubule instability. At all stages, erythroid cells manifested a striking deficiency of stabilizing microtubule-associated proteins (MAPs), such as the ubiquitous MAP4, and abundantly expressed the microtubule destabilizer, Stathmin 1 (STMN1). We therefore postulated the existence of a non-canonical, iron-regulated, stabilizing MAP and examined ferritin heavy chain (FTH1), known to possess microtubule bundling activity and be controlled by iron. Erythroid iron restriction caused a prompt and potent FTH1 decline due to proteolytic and non-proteolytic mechanisms, the latter likely involving IRP translational repression. Isocitrate rescued FTH1 levels but did not prevent early proteolysis; a likely target in its rescue is IRP1, known to be regulated by isocitrate and to participate in the erythroid iron restriction response. Importantly, lentiviral knockdown of FTH1 disrupted microtubules and impaired differentiation in a manner similar to iron restriction. Vesicular and protein transport may occur through either microtubule-dependent or –independent mechanisms. Notably, we discovered a strong and specific interaction of endogenous FTH1 with EpoR in erythroid cells, implicating FTH1 in microtubule recruitment of receptor vesicles. The proposed experiments will test the hypothesis that FTH1 participates in two key components of the erythroid iron restriction response: 1) a specialized pathway of iron- sensitive EpoR vesicular transport and 2) iron- and isocitrate-regulated maintenance of microtubule stability.