GAPDH regulates cellular heme insertion into inducible nitric oxide synthase

Abstract

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.

Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors

“…Our findings that GAPDH and NO regulate heme availability and mobilization are highly reminiscent of and supported by previous studies demonstrating that GAPDH and NO cooperate to control heme insertion into nitric oxide synthase (32). As such, it is tempting to speculate that Tdh3p is the source of NO-mobilized heme.…”

“…Given the HS1-M7A K d II of 25 nM, we estimate that heme is buffered at an upper limit of 2.5 nM in the mitochondria and nucleus. This concentration corresponds to fewer than six molecules in the nucleus [V nuc ∼ 5 fL (30) (32). Could NO also mobilize LH pools?…”

“…Although previous studies have indicated that H 2 O 2 and NO can regulate heme transfer between specific protein pairs (32, 36), our results suggest that NO can mobilize cell-wide LH pools to regulate heme-dependent processes in multiple compartments. The mechanism of NOmediated heme mobilization is unknown, but likely involves S-nitrosation of certain hemoproteins and heme dissociation (32,37).…”

Regulation of heme biosynthesis and transport in metazoa

“…GAPDH is known to be an enzyme catalyzing the conversion of glyceraldehyde-3-phosphate into 1,3-bisphosphoglycerate. Chakravarti et al [76] found that GAPDH interacts with both heme and iNOS. Knockdown of GAPDH or mutation of the heme-binding site in GAPDH significantly reduced heme incorporation into iNOS.…”

“…Knockdown of GAPDH or mutation of the heme-binding site in GAPDH significantly reduced heme incorporation into iNOS. The product of iNOS, nitric oxide (NO), also inhibited heme insertion into iNOS through S-nitrosylation of GAPDH [76]. Heme insertion into sGC was found to be mediated by HSP90, which preferentially interacts with apo-sGC [77].…”