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Updated: Jun 8, 2026
08:32
Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells
Published on: March 16, 2017
GAPDH regulates cellular heme insertion into inducible nitric oxide synthase
Ritu Chakravarti1, Kulwant S Aulak, Paul L Fox
1Department of Pathobiology,Lerner Research institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
Summary
Nitric oxide (NO) inhibits heme insertion into proteins by affecting Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This suggests GAPDH is crucial for intracellular heme transport and NO regulation.
Area of Science:
- Cellular Biology
- Biochemistry
- Molecular Biology
Background:
- Heme proteins are vital, yet intracellular heme transport and insertion remain poorly understood.
- Nitric oxide (NO) was found to inhibit heme insertion into key proteins like cytochrome P450s and hemoglobin.
Purpose of the Study:
- To investigate the mechanism of NO inhibition on heme insertion.
- To identify cytosolic proteins involved in heme transport, using inducible NO synthase (iNOS) as a model.
- To elucidate the role of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in this process.
Main Methods:
- Investigated the association between GAPDH and iNOS in cells.
- Assessed the binding of pure GAPDH to heme and iNOS under varying NO conditions.
- Utilized GAPDH knockdown and mutant strains to study heme insertion.
- Examined the S-nitrosylation of GAPDH at Cys152 upon NO exposure.
- Tested the effect of a GAPDH C152S mutant and a selective S-nitrosylation blocking drug on heme insertion.
Main Results:
- GAPDH was found to associate with iNOS and bind heme and iNOS in an NO-sensitive manner.
- GAPDH knockdown inhibited heme insertion into iNOS.
- NO exposure led to S-nitrosylation of GAPDH at Cys152.
- Mutating Cys152 or blocking GAPDH S-nitrosylation rendered iNOS heme insertion resistant to NO inhibition.
Conclusions:
- GAPDH acts as a heme delivery protein to iNOS, with its function regulated by S-nitrosylation.
- This uncovers a fundamental mechanism in intracellular heme trafficking.
- Nitric oxide (NO) governs heme insertion into proteins via GAPDH S-nitrosylation.
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