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

铁对于几乎所有形式的生命都是必需的，因为氧的结合和运输、ATP的产生以及DNA的复制和修复都需要铁。细胞必须维持足够的铁水平来支持这些过程；然而，考虑到铁在产生有毒活性氧中的作用，铁过量可能是有害的。为了达到这种平衡，细胞铁主要以一种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物。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是选择性货物受体，介导铁蛋白的自噬降解（“铁蛋白自噬”），从而释放铁供细胞使用。通过铁蛋白自噬途径的通量依赖于由细胞内铁水平调节的NCOA4蛋白水平。在充满铁的细胞条件下，NCOA4与E3泛素连接酶HERC2的结合增加，导致NCOA4的蛋白酶体降解。降低的NCOA4水平抑制铁蛋白自噬，从而增加铁蛋白铁储存。在缺铁条件下，HERC2与NCOA4的结合减少，导致NCOA4稳定并诱导铁蛋白自噬以释放铁。铁蛋白自噬参与铁依赖的生理过程，如红细胞生成，其中NCOA4介导铁蛋白铁释放，用于线粒体血红素合成。最近，在细胞培养模型中，铁蛋白自噬已被证明可调节铁凋亡，铁凋亡是一种由过量脂质过氧化介导的铁依赖性细胞死亡的新描述形式。铁质代谢失调和铁下垂已在血色素沉着症和急性缺血性肾损伤中被描述；然而，关于铁蛋白自噬在体内调节对铁下垂的敏感性方面的作用知之甚少。在本研究中，我们将研究NCOA4是细胞和全身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作用。Specific Aim 1将使用集成的定量蛋白质组学和细胞生物学实验工作流程定义细胞中调节NCOA4活性的生化机制。在具体目标2中，我们将利用NCOA4敲除和过表达的新型小鼠模型，确定体内铁蛋白自噬对调节全身铁稳态和红细胞生成的时空依赖性。最后，具体目标3将侧重于研究在病理生理条件下NCOA4在体内调节对铁下垂的敏感性中的作用。总之，拟议的体外和体内研究将确定NCOA4在感知和响应铁缺乏和过载中的基本作用，并确定追求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.