Identification of Cys385 in the isolated kinase insertion domain of heme-regulated eIF2α kinase (HRI) as the heme axial ligand by site-directed mutagenesis and spectral characterization

Hirai, Kyoko; Martínková, Markéta; Igarashi, Jun–ichi; Saiful, Islam S. M.; Yamauchi, Seigo; El-Mashtoly, Samir F.; Kitagawa, Teizo; Shimizu, Tôru

doi:10.1016/j.jinorgbio.2007.05.004

Cited by 16 publications

(15 citation statements)

References 14 publications

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“…Cys 409 of the kinase domain and a histidine (His 119 or His 120 ) of the N-terminal domain serve as ligands to this NO-binding heme. Characteristics of cysteine(thiolate) coordination are lost following reduction, suggesting that either a ligand switch or protonation to a cysteine(thiol) occurs to stabilize the more-electron-rich Fe(II) heme [54, 55]. HRI activity is specifically upregulated by formation of a five-coordinate NO adduct, which occurs owing to the strong trans influence of NO and the weakly coordinating nature of the histidine ligand.…”

Section: Discussionmentioning

confidence: 99%

Identification of Cys94 as the distal ligand to the Fe(III) heme in the transcriptional regulator RcoM-2 from Burkholderia xenovorans

et al. 2012

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The CO-responsive transcriptional regulator RcoM from Burkholderia xenovorans (BxRcoM) was recently identified as a Cys(thiolate)-ligated heme protein that undergoes a redox-mediated ligand switch; however, the Cys bound to the Fe(III) heme was not identified. To that end, we generated and purified three Cys-to-Ser variants of BxRcoM-2—C94S, C127S, and C130S—and examined their spectroscopic properties in order to identify the native Cys(thiolate) ligand. Electronic absorption, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopies demonstrate that the C127S and C130S variants, like wild-type BxRcoM-2, bind a six-coordinate low-spin Fe(III) heme using a Cys/His ligation motif. In contrast, electronic absorption and resonance Raman spectra of the C94S variant are most consistent with a mixture of five-coordinate high-spin and six-coordinate low-spin Fe(III) heme, neither of which are ligated by a Cys(thiolate) ligand. The EPR spectrum of C94S is dominated by a large, axial high-spin Fe(III) signal, confirming that the native ligation motif is not maintained in this variant. Together, these data reveal that Cys94 is the distal Fe(III) heme ligand in BxR-coM-2; by sequence alignment, Cys94 is also implicated as the distal Fe(III) heme ligand in BxRcoM-1, another homologue found in the same organism.

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Section: Discussionmentioning

confidence: 99%

Identification of Cys94 as the distal ligand to the Fe(III) heme in the transcriptional regulator RcoM-2 from Burkholderia xenovorans

et al. 2012

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“…Binding of heme to the N‐terminus is stable and it co‐purifies with HRI, whereas heme binding to the kinase insertion domain is reversible and inhibits HRI kinase activity . Heme inhibits HRI kinase activity in vitro and in vivo by promoting disulfide bond formation between HRI monomers, keeping them in an inactive dimer conformation . However, in the absence of heme, non‐covalent interactions between HRI molecules occurs, resulting in an active HRI dimer .…”

Section: Activation Of the Integrated Stress Responsementioning

confidence: 99%

“…Heme inhibits HRI kinase activity in vitro and in vivo by promoting disulfide bond formation between HRI monomers, keeping them in an inactive dimer conformation . However, in the absence of heme, non‐covalent interactions between HRI molecules occurs, resulting in an active HRI dimer . HRI can also be activated by other stresses including arsenite‐induced oxidative stress, heat shock, osmotic stress, 26S proteasome inhibition, and nitric oxide .…”

Section: Activation Of the Integrated Stress Responsementioning

confidence: 99%

The integrated stress response

et al. 2016

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In response to diverse stress stimuli, eukaryotic cells activate a common adaptive pathway, termed the integrated stress response (ISR), to restore cellular homeostasis. The core event in this pathway is the phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2a) by one of four members of the eIF2a kinase family, which leads to a decrease in global protein synthesis and the induction of selected genes, including the transcription factor ATF4, that together promote cellular recovery. The gene expression program activated by the ISR optimizes the cellular response to stress and is dependent on the cellular context, as well as on the nature and intensity of the stress stimuli. Although the ISR is primarily a pro-survival, homeostatic program, exposure to severe stress can drive signaling toward cell death. Here, we review current understanding of the ISR signaling and how it regulates cell fate under diverse types of stress.

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“…Binding inhibits Bach1/MafK association with the HO-1 promoter, inducing subcellular relocalization of Bach1 and degradation (37,44). In addition to Bach1, various heme-mediated protein functions require HRMs: the yeast transcriptional activator Hap1 that transcribes genes encoding various cytochromes, catalase, and Rox1, which represses anaerobic genes under high heme concentration (see reference 12 and references within); the hemeregulated inhibitor kinase that controls the activity of the translation initiator factor eIF-2␣ in stressed erythroid cells (4,11); the erythroid 5-aminolevulinic acid synthase precursors whose transport to the mitochondria is mediated by heme binding to HRMs (20); the heme lyase found in both Saccharomyces cerevisiae and Neurospora crassa (35); the mammalian nuclear factor erythroid 2 that plays a critical role in erythroid differentiation (22); the HO-2 that metabolizes heme (21); the iron regulatory protein 2 (IRP2), a regulator of iron metabolism in mammals (13), and the iron response regulator (Irr) in bacteria whose turnover depends on the cellular iron availability (28,42).…”

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confidence: 99%

A Novel Heme-Regulatory Motif Mediates Heme-Dependent Degradation of the Circadian Factor Period 2

Yang

Kim²,

Lucas³

et al. 2008

Molecular and Cellular Biology

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Although efforts have been made to identify circadian-controlled genes regulating cell cycle progression and cell death, little is known about the metabolic signals modulating circadian regulation of gene expression. We identify heme, an iron-containing prosthetic group, as a regulatory ligand controlling human Period-2 (hPer2) stability. Furthermore, we define a novel heme-regulatory motif within the C terminus of hPer2 (SC 841 PA) as necessary for heme binding and protein destabilization. Spectroscopy reveals that whereas the PAS domain binds to both the ferric and ferrous forms of heme, SC 841 PA binds exclusively to ferric heme, thus acting as a redox sensor. Consequently, binding prevents hPer2 from interacting with its stabilizing counterpart cryptochrome. In vivo, hPer2 downregulation is suppressed by inhibitors of heme synthesis or proteasome activity, while SA 841 PA is sufficient to stabilize hPer2 in transfected cells. Moreover, heme binding to the SC 841 PA motif directly impacts circadian gene expression, resulting in altered period length. Overall, the data support a model where heme-mediated oxidation triggers hPer2 degradation, thus controlling heterodimerization and ultimately gene transcription.Cellular homeostasis depends on a delicate balance between metabolic activity and gene expression. Heme is a prosthetic group essential for transport and storage of oxygen that is involved in the generation of cellular energy by respiration and synthesis and degradation of lipids and in oxidative damage. Heme-based sensor proteins detect and respond to variations in oxygen, carbon monoxide, and nitric oxide levels and cellular redox state by acting on transcription, translation, protein translocation, and protein assembly (8,25).Heme binding to transcription factors is found in both prokaryotes and lower eukaryotes; however, only three cases have been identified in higher eukaryotes: the basic leucine zipper transcription factor Bach1, the circadian transcription factor NPAS2, and the nuclear orphan receptor 23,30,

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Identification of Cys385 in the isolated kinase insertion domain of heme-regulated eIF2α kinase (HRI) as the heme axial ligand by site-directed mutagenesis and spectral characterization

Cited by 16 publications

References 14 publications

Identification of Cys94 as the distal ligand to the Fe(III) heme in the transcriptional regulator RcoM-2 from Burkholderia xenovorans

Identification of Cys94 as the distal ligand to the Fe(III) heme in the transcriptional regulator RcoM-2 from Burkholderia xenovorans

The integrated stress response

A Novel Heme-Regulatory Motif Mediates Heme-Dependent Degradation of the Circadian Factor Period 2

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