Novel role of endoplasmic reticulum-associated degradation in iron metabolism

我的实验室对内质网（ER）相关降解（ERAD）的（病理）生理重要性感兴趣，ERAD是一种主要的内质网质量控制机制，用于清除错误折叠的内质网蛋白以进行细胞质蛋白酶体降解。Sel1L-Hrd1蛋白复合物代表了从酵母到人类进化上最保守的ERAD机制。在过去的几年中，我们和其他人已经报道了Sel1L-Hrd1 ERAD以细胞类型和底物特异性的方式在健康和疾病中的生理意义；然而，我们对其生理作用的了解仍然有限。在本应用程序的初步数据中，我们进行了无偏倚的蛋白质组学筛选，确定了铜蓝蛋白（Cp）蛋白，一种调节铁稳态的氧化铁酶，作为肝脏中的ERAD底物。我们进一步发现，野生型和疾病突变型Cp都容易发生错误折叠，并被Sel1L- Hrd1 ERAD泛素化和降解。此外，肝细胞特异性sell缺陷小鼠在循环中表现出升高的Cp活性，并对缺铁引起的低色小细胞贫血具有抵抗力。这些数据表明肝细胞Sel1L-Hrd1 ERAD在Cp生物发生和全身铁稳态中的关键作用。这些发现是令人兴奋的，因为Cp是铁稳态的重要调节因子，因为人类的Cp错义突变会导致一种临床疾病，即急性纤溶酶血症，其特征是器官中铁的异常积累。然而，新生Cp在ER中的生物发生机制仍未被探索。因此，本应用的总体假设是，肝细胞中的Sel1L-Hrd1 ERAD分别通过调节野生型和疾病突变型Cp蛋白在生理和病理条件下的周转来控制全身铁稳态。我们将完成以下三个目标：(1)确定Sel1L-Hrd1 ERAD在铁代谢中的生理病理意义；(2)揭示ERAD调控Cp生物发生的分子机制；(3)阐明ERAD在急性纤溶酶血症发病机制中的病理意义。这些研究的完成不仅将揭示erd介导的铁代谢调控的意义和分子机制，还将为基础和病理条件下铁代谢的调控提供新的见解。与人类健康的相关性：铁体内平衡失调影响着全世界数百万人，导致缺铁性贫血，并在超载时增加患糖尿病、肝脏和肾脏疾病的风险。这一应用与平行的生理和生化研究将建立ERAD和铁代谢之间的直接联系，揭示ERAD和错误折叠相关的蛋白质毒性应激的新机制，并促进我们对蛋白质折叠缺陷相关疾病发病机制的理解。

My laboratory is interested in the (patho-)physiological importance of endoplasmic reticulum (ER)-associated degradation (ERAD), a principal ER quality-control machinery to clear misfolded ER proteins for cytosolic proteasomal degradation. The Sel1L-Hrd1 protein complex represents the most evolutionarily conserved ERAD machinery from yeast to humans. In the past several years, we and others have reported the physiological significance of Sel1L-Hrd1 ERAD in health and disease in a cell-type and substrate-specific manner; however, our understanding of its physiological role remains limited. In the preliminary data of this application, we performed an unbiased proteomics screen that led to the identification of ceruloplasmin (Cp) protein, a ferroxidase regulating iron homeostasis, as an ERAD substrate in the liver. We further showed that both wildtype and a disease mutant Cp are misfolding-prone and are ubiquitinated and degraded by Sel1L- Hrd1 ERAD. Moreover, hepatocyte-specific Sel1L-deficient mice exhibit elevated Cp activity in the circulation and are resistant to iron deficiency-induced hypochromic microcytic anemia. These data point to a critical role of hepatocyte Sel1L-Hrd1 ERAD in Cp biogenesis and systemic iron homeostasis. These findings are exciting because Cp is an essential regulator in iron homeostasis and because Cp missense mutations in humans cause a clinical condition known as aceruloplasminemia, characterized by abnormal iron accumulation in organs. However, the biogenesis of nascent Cp in the ER remains unexplored. Hence, the overarching hypothesis of this application is that Sel1L-Hrd1 ERAD in hepatocytes controls systemic iron homeostasis by regulating the turnover of both wildtype and disease mutant Cp proteins under physiological and pathological conditions, respectively. We will accomplish the following three Aims: (1) Determine the physiological and pathological significance of Sel1L-Hrd1 ERAD in iron metabolism; (2) Delineate the molecular mechanism underlying Cp biogenesis regulated by ERAD; and (3) Delineate the pathological importance of ERAD in the pathogenesis of aceruloplasminemia. Completion of these studies will not only delineate the significance and molecular mechanism underlying ERAD-mediated regulation of iron metabolism, but also provide novel insights into how iron metabolism is regulated under basal and pathological conditions. Relevance to human health: Disorders of iron homeostasis affect millions of individuals worldwide, which cause anemia in deficiency and increase the risk of diabetes, liver and kidney diseases upon overload. This application, with parallel physiological and biochemical studies, will establish a direct link between ERAD and iron metabolism, uncover novel mechanisms underlying ERAD and misfolding-associated proteotoxic stress, and advance our understanding of disease pathogenesis associated with protein folding defects in general.