GAPDH regulates cellular heme insertion into inducible nitric oxide synthase

被引:108
作者
Chakravarti, Ritu [1 ]
Aulak, Kulwant S. [1 ]
Fox, Paul L. [2 ]
Stuehr, Dennis J. [1 ]
机构
[1] Cleveland Clin Fdn, Dept Pathobiol, Lerner Res Inst, Cleveland, OH 44195 USA
[2] Cleveland Clin Fdn, Dept Cell Biol, Lerner Res Inst, Cleveland, OH 44195 USA
关键词
GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE; BINDING; PROTEINS; ENZYME; DEATH; IDENTIFICATION; MITOCHONDRIAL; INHIBITOR; COMPLEXES; RECEPTOR;
D O I
10.1073/pnas.1008133107
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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.
引用
收藏
页码:18004 / 18009
页数:6
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