GAPDH regulates cellular heme insertion into inducible nitric oxide synthase
GAPDH regulates cellular heme insertion into inducible nitric oxide synthase
复制标题
DOI：
10.1073/pnas.1008133107
复制
发表时间：
2010-10-19
影响因子：
11.1
通讯作者：
Stuehr, Dennis J.
中科院分区：
文献类型：
--
作者：
Chakravarti, Ritu;Aulak, Kulwant S.;Stuehr, Dennis J.
血红素蛋白在生物学中起着重要的作用，但对血红素在哺乳动物细胞内的转运以及血红素是如何插入可溶性蛋白质中的知之甚少。我们最近发现，一氧化氮（NO）阻止细胞将血红素插入几种蛋白质，包括细胞色素P450，血红蛋白，NO脱氢酶和过氧化氢酶。这一发现使我们探索NO抑制的基础，并确定可能涉及的胞质蛋白，使用诱导型NO合酶（iNOS）作为模型靶点。令人惊讶的是，我们发现GAPDH起着关键作用。细胞内GAPDH与iNOS相关。纯GAPDH以NO敏感的方式与血红素或iNOS紧密结合。GAPDH敲低抑制了血红素插入iNOS，而血红素结合缺陷的GAPDH突变体充当了iNOS血红素插入的显性负性抑制剂。将细胞暴露于来自化学供体或通过iNOS诱导的NO导致GAPDH在Cys 152处变成S-亚硝基化。在细胞中表达GAPDH C152 S突变体或提供选择性阻断GAPDH S-亚硝基化的药物都使血红素插入iNOS抵抗NO抑制。我们认为GAPDH通过一个受其S-亚硝基化调节的过程将血红素传递给iNOS。我们的研究结果可能揭示了细胞内血红素运输的一个基本步骤，并揭示了NO可以控制这一过程的机制。
Heme proteins play essential roles in biology, but little is known about heme transport inside mammalian cells or how heme is inserted into soluble proteins. We recently found that nitric oxide (NO) blocks cells from inserting heme into several proteins, including cytochrome P450s, hemoglobin, NO synthases, and catalase. This finding led us to explore the basis for NO inhibition and to identify cytosolic proteins that may be involved, using inducible NO synthase (iNOS) as a model target. Surprisingly, we found that GAPDH plays a key role. GAPDH was associated with iNOS in cells. Pure GAPDH bound tightly to heme or to iNOS in an NO-sensitive manner. GAPDH knockdown inhibited heme insertion into iNOS and a GAPDH mutant with defective heme binding acted as a dominant negative inhibitor of iNOS heme insertion. Exposing cells to NO either from a chemical donor or by iNOS induction caused GAPDH to become S-nitrosylated at Cys152. Expressing a GAPDH C152S mutant in cells or providing a drug to selectively block GAPDH S-nitrosylation both made heme insertion into iNOS resistant to the NO inhibition. We propose that GAPDH delivers heme to iNOS through a process that is regulated by its S-nitrosylation. Our findings may uncover a fundamental step in intracellular heme trafficking, and reveal a mechanism whereby NO can govern the process.