Mechanisms of Intercellular Heme Homeostasis in Liver

（请保持文字，不要PDF） 这项资助的长期目标是获得对维持有机体血红素稳态的细胞间机制的全面了解。在哺乳动物中，血红素是一种含铁的辅因子和信号分子，在所有有核细胞中合成，并且对于多种基本生物过程是必需的。作为一种疏水性和细胞毒性分子，血红素必须通过特定的细胞途径以高度受控的方式通过膜转运。目前的范式假定哺乳动物细胞血红素稳态仅由血红素生物合成（由5-氨基乙酰丙酸合酶（ALAS）驱动）和血红素降解之间的平衡来维持。虽然真核血红素转运蛋白的发现已经引起了新兴的概念，即细胞间和细胞内血红素运输途径也有助于维持血红素稳态，但在血红素生物合成和血红素降解的背景之外介导血红素稳态的途径和机制仍然知之甚少。我们最近未发表的研究使用新产生的肝细胞特异性ALAS敲除小鼠模型（Alas 1/2KO小鼠）强烈表明，肝细胞通过目前尚未探索的细胞间血红素稳态途径从网状内皮巨噬细胞获得“按需”血红素。在这个建议中，我们将测试我们的范式转变假说，肝细胞保持细胞血红素稳态收购的相当大一部分血红素从巨噬细胞使用Alas 1/2KO小鼠。为此，特定目标1将通过建立体外原代肝细胞共培养系统来鉴定直接参与血红素从巨噬细胞转移至肝细胞的细胞类型。具体目标2将采用在体内的方法，以确定参与肝细胞血红素收购途径的关键分子，并调查收购系统被激活的机制。具体目标3将确定额外的候选基因和途径，驱动肝细胞血红素收购使用单细胞RNA测序。这些研究将促进我们对肝脏如何在健康和疾病中维持血红素稳态的理解，并可能有助于提高我们对急性肝卟啉症中发生的危及生命的急性神经内脏攻击的发病机制的理解。

(PLEASE KEEP IN WORD, DO NOT PDF) The long-term goal of this grant is to obtain a comprehensive understanding of the intercellular mechanisms involved in the maintenance of organismal heme homeostasis. In mammals, heme, an iron-containing cofactor and signaling molecule, is synthesized in all nucleated cells and is essential for a diverse array of fundamental biological processes. As a hydrophobic and cytotoxic molecule, heme must be transported in a highly controlled manner through membranes via specific cellular pathways. The current paradigm posits that mammalian cellular heme homeostasis is maintained solely by the balance between heme biosynthesis, driven by 5-aminolevulinic acid synthase (ALAS), and heme degradation. While the discovery of eukaryotic heme transporters has given rise to the emerging notion that inter- and intra-cellular heme trafficking pathways also contribute to the maintenance of heme homeostasis, the pathways and mechanisms that mediate heme homeostasis outside of the context of heme biosynthesis and heme degradation remain poorly understood. Our recent unpublished studies using a newly generated hepatocyte-specific ALAS knockout mouse model (Alas1/2KO mice) strongly suggest that hepatocytes acquire “on demand” heme from reticuloendothelial macrophages through a currently unexplored intercellular heme homeostatic pathway. In this proposal, we will test our paradigm-shifting hypothesis that hepatocytes maintain cellular heme homeostasis by acquiring a substantial portion of their heme from macrophages using the Alas1/2KO mice. To this end, Specific Aim 1 will identify the cell types that are directly involved in heme transfer from macrophages to hepatocytes by establishing an in vitro primary liver cell co-culture system. Specific Aim 2 will employ in vivo approaches to identify key molecules involved in the hepatocyte heme acquisition pathway and to investigate the mechanisms by which the acquisition system is activated. Specific Aim 3 will identify additional candidate genes and pathways that drive hepatocyte heme acquisition using single cell RNA sequencing. These studies will advance our understanding of how liver maintains heme homeostasis in health and disease and may aid in improving our understanding of the pathogenesis of the life-threatening acute neurovisceral attacks that occur in the acute hepatic porphyrias.