The role of NCOA4-mediated ferritinophagy in iron homeostasis and ferroptosis

NCOA4介导的铁蛋白吞噬作用在铁稳态和铁下垂中的作用铁是几乎所有形式的生命所必需的，因为铁在氧结合和运输、ATP生产以及DNA复制和修复中都需要铁。细胞必须维持足够的铁水平来支持这些过程；然而，鉴于铁在产生有毒的活性氧物种中的作用，过量的铁可能是有害的。为了达到这种平衡，细胞铁主要以无毒的形式储存在铁蛋白中，铁蛋白是一种由24个铁蛋白轻链和重链亚基组成的蛋白质复合体。然而，直到最近，铁蛋白在细胞需求时释放铁的机制还不清楚。我们确定NCOA4是一种选择性的货物受体，它介导铁蛋白的自噬降解(“噬铁蛋白”)，从而释放铁供细胞使用。通过吞铁蛋白途径的通量依赖于NCOA4蛋白水平，而NCOA4蛋白水平受细胞内铁水平的调节。在铁充足的细胞条件下，NCOA4与E3泛素连接酶HERC2的结合增加，导致NCOA4的蛋白酶体降解。降低NCOA4水平可抑制铁蛋白吞噬，从而增加铁蛋白铁的储存。在缺铁条件下，HERC2与NCOA4的结合减少，导致NCOA4稳定并诱导吞铁释放铁。吞铁蛋白参与了铁依赖的生理过程，如红细胞生成，其中NCOA4介导铁蛋白铁的释放，以合成线粒体血红素。最近，在细胞培养模型中，噬铁蛋白被证明可以调节铁下垂，这是一种新描述的铁依赖的细胞死亡形式，由过度的脂质过氧化介导。铁代谢紊乱和铁下垂在血色素沉着症和急性缺血性肾损伤中已被描述；然而，对于吞铁蛋白在体内调节铁下垂敏感性的作用知之甚少。在这项拟议的工作中，我们将检验这一假说，即NCOA4是细胞和系统铁稳态的关键调节因子，在生理和病理生理中具有特定的时间和空间调节铁稳态的作用。具体目标1将使用一个整合的定量蛋白质组和细胞生物学实验工作流程来定义调节细胞中NCOA4活性的生化机制。在特定的目标2中，我们将使用新的NCOA4基因敲除和过度表达的小鼠模型来确定体内噬铁蛋白在调节全身铁稳态和红细胞生成方面的时间和空间依赖性。最后，具体目标3将重点研究NCOA4在体内调节在病理生理条件下对铁性下垂的敏感性的作用。总之，拟议的体外和体内研究将确定NCOA4在感知和应对铁缺乏和过载方面的基本作用，并确定将NCOA4作为多种铁相关疾病的治疗靶点的效用。

The role of NCOA4-mediated ferritinophagy in iron homeostasis and ferroptosis Iron is essential for nearly all forms of life given the requirement for iron in oxygen binding and transport, ATP production, and DNA replication and repair. Cells must maintain sufficient iron levels to support these processes; however, iron excess can be detrimental given the role of iron in generation of toxic reactive oxygen species. To achieve this balance, cellular iron is primarily stored in a non-toxic form in ferritin, a protein complex composed of 24 subunits of ferritin light and heavy chain subunits. However, the mechanism of iron release from ferritin in times of cellular demand was unclear until recently. We identified NCOA4 as the selective cargo receptor that mediates the autophagic degradation of ferritin (“ferritinophagy”) thereby releasing iron for use by the cell. Flux through the ferritinophagy pathway is dependent on NCOA4 protein levels which are regulated by intracellular iron levels. Under iron-replete cellular conditions, NCOA4 binding to HERC2, an E3 ubiquitin ligase, is increased, leading to proteasomal degradation of NCOA4. Decreased NCOA4 levels inhibit ferritinophagy thereby increasing ferritin iron storage. Under iron-deficient conditions, HERC2 binding to NCOA4 is decreased, leading to NCOA4 stabilization and induction of ferritinophagy for iron release. Ferritinophagy is involved in iron- dependent physiological processes such as erythropoiesis, where NCOA4 mediates ferritin iron release for mitochondrial heme synthesis. Recently, in cell culture models, ferritinophagy has been shown to regulate ferroptosis, a newly described form of iron-dependent cell death mediated by excess lipid peroxidation. Dysregulation of iron metabolism and ferroptosis have been described in hemochromatosis and acute ischemic kidney injury; however, little is known about the in vivo role of ferritinophagy in regulating sensitivity to ferroptosis. In the proposed work, we will examine the hypothesis that NCOA4 is a key regulator of cellular and systemic iron homeostasis with specific temporal and spatial roles in regulating iron homeostasis in physiology and pathophysiology. Specific Aim 1 will define the biochemical mechanisms regulating NCOA4 activity in cells using an integrated quantitative proteomic and cell biologic experimentation workflow. In specific aim 2, we will determine the temporal and spatial dependency of ferritinophagy in vivo for regulating systemic iron homeostasis and erythropoiesis using novel mouse models of NCOA4 knockout and over-expression. Finally, specific aim 3 will focus on examining a role for NCOA4 in regulating sensitivity to ferroptosis in vivo under pathophysiologic conditions. Together, the proposed in vitro and in vivo studies will identify fundamental roles of NCOA4 in sensing and responding to iron deficiency and overload and determine the utility of pursuing NCOA4 as a therapeutic target for multiple iron-related disorders.