Activation of Heme-regulated Eukaryotic Initiation Factor 2α Kinase by Nitric Oxide Is Induced by the Formation of a Five-coordinate NO-Heme Complex

Abstract

Heme-regulated eukaryotic initiation factor 2␣ kinase (HRI) regulates the synthesis of hemoglobin in reticulocytes in response to heme availability. HRI contains a tightly bound heme at the N-terminal domain. Earlier reports show that nitric oxide (NO) regulates HRI catalysis. However, the mechanism of this process remains unclear. In the present study, we utilize in vitro kinase assays, optical absorption, electron spin resonance (ESR), and resonance Raman spectra of purified full-length HRI for the first time to elucidate the regulation mechanism of NO. HRI was activated via heme upon NO binding, and the

Eukaryotic initiation factor 2α kinase is a nitric oxide‐responsive mercury sensor enzyme: Potent inhibition of catalysis by the mercury cation and reversal by nitric oxide

“…A plasmid encoding (His) 6 -tagged wild-type HRI comprising residues 1-619 (His 6 HRI) from mouse liver cDNA was originally constructed using the pET28 vector (Novagen, Madison, WI, USA), as described previously [6,11]. We introduced a PreScissione protease (Amersham Biosciences, Piscataway, NJ, USA) recognition site in the expression vector to remove the (His) 6 -tag.…”

“…We introduced a PreScissione protease (Amersham Biosciences, Piscataway, NJ, USA) recognition site in the expression vector to remove the (His) 6 -tag. The resulting protein contains extra Gly-Pro-His residues upstream of the Met residue.…”

“…(His) 6 -tagged HRI was expressed in Escherichia coli BL21 (DE3) Codon Plus RIL (Stratagene, La Jolla, CA, USA) harboring pET28a-PreScission/HRI, and purified using a previous protocol [6,11]. The (His) 6 tag was cut with PreScissione protease, according to the manufacturer's protocol.…”

“…Primary mouse anti-(His) 6 monoclonal IgM (H-3), goat anti-HRI IgG (S-16), rabbit anti-eIF2a IgG (FL-315), and goat anti-phosphorylated eIF2a IgG (Ser52) antibodies were purchased from Santa Cruz Biotechnology, Inc. For immunoblotting, the membrane was blocked for 1 h with 5% bovine serum albumin in Tris-buffered saline (20 mM Tris-HCl, pH 7.7, 137 mM NaCl) containing 1% Tween 20 (TBST), and incubated overnight at room temperature with primary antibody diluted in TBST. After washing with TBST, the membrane was treated with horseradish peroxidase-conjugated sheep anti-mouse IgG, donkey anti-rabbit IgG (Amersham Biosciences) or donkey antigoat IgG (Santa Cruz Biotechnology) for 1 h. Immunoreactive protein bands were visualized with ECL reagents (Amersham Biosciences), and detected using a Fuji Film Chemiluminescence Reader LAS-3000 IDX6 (Tokyo, Japan).…”

“…However, the sensing systems of these eIF2a kinases in response to cell stress or emergency are different. For example, GCN2 mediates the termination of translation under conditions of amino acid shortage [1] and UV exposure [2], PERK is activated during accumulation of denatured proteins [2], PKR activity is stimulated upon viral infection [3], and HRI acts when cells face a shortage of heme [4][5][6].…”