Dissection and Manipulation of the Cellular Response to Iron Restriction

项目总结/摘要 红系铁限制反应是两种最常见的贫血类型的基础：慢性贫血， 疾病和炎症（ACDI）和缺铁性贫血（IDA）。这些贫血症导致了全球性的 发病率和死亡率的负担，ACDI尚无最佳治疗方法。这种血统特异性 祖细胞对营养缺乏的反应涉及异柠檬酸产生的损失， 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制剂， 使用临床上可行的给药方案在鼠ACDI中持续有效。上次融资的结果 期间确定了与铁限制相关的促红细胞生成素（Epo）抵抗的分子基础， 临床问题（J. Exp. Med. 2018）。从本质上讲，缺铁导致细胞表面传递铁的失败。 Epo受体（EpoR）和相关因子（Scrib和TfR 2）。这种异常可被异柠檬酸逆转 恢复Epo反应性的治疗。最近，我们发现铁和异柠檬酸 以红系特异性方式调节高尔基体的完整性。此外，红系铁限制诱导 微管细胞骨架的早期和持续破坏，已知微管细胞骨架是高尔基体的关键结构 上维护异柠檬酸盐处理不能阻止最初的微管破坏，但能促进其 在稍后的时间点重新组装。我们对患者标本的研究以及先前发表的报告支持 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， 分阶段的人红系祖细胞，以确定可能有助于微管不稳定性的特征。在 在所有阶段，红系细胞表现出稳定微管相关蛋白的显著缺乏 （MAPs），如普遍存在的MAP 4，并大量表达微管去稳定剂Stathmin 1 （STMN 1）。因此，我们假设存在一个非经典的，铁调节的，稳定的MAP， 检查铁蛋白重链（FTH 1），已知具有微管捆绑活性，并受 铁.红系铁限制导致蛋白水解和非蛋白水解FTH 1迅速和有效的下降， 机制，后者可能涉及IRP翻译抑制。异柠檬酸盐挽救了FTH 1水平，但没有 防止早期蛋白水解;其拯救的可能靶点是IRP 1，已知受异柠檬酸调节， 参与红细胞铁限制反应。重要的是，FTH 1的慢病毒敲低被破坏， 微管和受损分化的方式类似于铁限制。囊泡和蛋白质转运 可能通过微管依赖性或非依赖性机制发生。值得注意的是，我们发现了 在红系细胞中内源性FTH 1与EpoR的强而特异的相互作用，暗示FTH 1在 受体囊泡的微管募集。拟议的实验将测试FTH 1 参与红系铁限制反应的两个关键组成部分：1）铁的专门途径， 敏感的EpoR囊泡转运和2）铁和异柠檬酸调节的微管稳定性维持。

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.