PDF(Pubmed)
Abstract：
Iron protoporphyrin IX (heme) is a redox-active cofactor that is bound in mammalian cells by GAPDH and allocated by a process influenced by physiologic levels of NO. This impacts the activity of many heme proteins including indoleamine dioxygenase-1 (IDO1), a redox enzyme involved in immune response and tumor growth. To gain further understanding we created a tetra-Cys human GAPDH reporter construct (TC-hGAPDH) which after labeling could indicate its heme binding by fluorescence quenching. When purified or expressed in a human cell line, TC-hGAPDH had properties like native GAPDH and heme binding quenched its fluorescence by 45-65%, allowing it to report on GAPDH binding of mitochondrially-generated heme in live cells in real time. In cells with active mitochondrial heme synthesis, low-level NO exposure increased heme allocation to IDO1 while keeping the TC-hGAPDH heme level constant due to replenishment by mitochondria. When mitochondrial heme synthesis was blocked, low NO caused a near complete transfer of the existing heme in TC-hGAPDH to IDO1 in a process that required IDO1 be able to bind the heme and have an active hsp90 present. Higher NO exposure had the opposite effect and caused IDO1 heme to transfer back to TC-hGAPDH. This demonstrated: (i) flow of mitochondrial heme through GAPDH is tightly coupled to target delivery, (ii) NO up- or down-regulates IDO1 activity by promoting a conserved heme exchange with GAPDH that goes in either direction according to the NO exposure level. The ability to drive a concentration-dependent, reversible protein heme exchange is unprecedented and reveals a new role for NO in biology.
摘要：
铁原卟啉IX（血红素）是一种氧化还原活性辅因子，在哺乳动物细胞中通过GAPDH结合，并通过受NO生理水平影响的过程分配。这会影响许多血红素蛋白的活性，包括吲哚胺双加氧酶-1（IDO1），一种参与免疫反应和肿瘤生长的氧化还原酶。为了获得进一步的理解,我们创建了四Cys人GAPDH报告构建体(TC-hGAPDH),其在标记后可以通过荧光猝灭指示其血红素结合。当在人细胞系中纯化或表达时，TC-hGAPDH具有天然GAPDH和血红素结合使其荧光猝灭45-65%的特性，允许它实时报告活细胞中线粒体生成血红素的GAPDH结合。在线粒体血红素合成活跃的细胞中，低水平的NO暴露会增加血红素对IDO1的分配，同时由于线粒体的补充而使TC-hGAPDH血红素水平保持恒定。当线粒体血红素合成被阻断时，在需要IDO1能够结合血红素并存在活性hsp90的过程中，低NO导致TC-hGAPDH中现有血红素几乎完全转移到IDO1。较高的NO暴露具有相反的作用，并导致IDO1血红素转移回TC-hGAPDH。这证明：（i）线粒体血红素通过GAPDH的流动与靶标递送紧密耦合，（ii）NO通过促进与GAPDH的保守血红素交换来上调或下调IDO1活性，该交换根据NO暴露水平朝任一方向发展。驱动浓度依赖性的能力，可逆蛋白血红素交换是前所未有的，揭示了NO在生物学中的新作用。
