Improvement of heme oxygenase-1-based heme sensor for quantifying free heme in biological samples

Taira, Junichi; Nakashima, Y.; Yoshihara, Shun; Koga, Shinya; Sueda, Shinji; Komatsu, Hideyuki; Higashimoto, Yuichiro; Takahashi, Toru; Tanioka, Nohito; Shimizu, Hiroko; Morimatsu, Hiroshi; Sakamoto, Hiroshi

doi:10.1016/j.ab.2015.08.004

Cited by 5 publications

(7 citation statements)

References 13 publications

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“…The sensor comprised an EGFP-cytochrome b 562 fusion protein, 176 and it was subsequently optimized for enhanced RET. 177 Fluorescently labeled variants of heme oxygenase-1 (K18C 178 and D140H 179 variants) have also been used to detect heme in vitro. These sensors were a step forward in terms of heme quantification, but were not deployed in cells for real-time monitoring of heme concentrations.…”

Section: Quantifying Heme Concentrations In Cellsmentioning

confidence: 99%

Understanding the Logistics for the Distribution of Heme in Cells

Gallio

Fung

Cammack-Najera

et al. 2021

JACS Au

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Heme is essential for the survival of virtually all living systems—from bacteria, fungi, and yeast, through plants to animals. No eukaryote has been identified that can survive without heme. There are thousands of different proteins that require heme in order to function properly, and these are responsible for processes such as oxygen transport, electron transfer, oxidative stress response, respiration, and catalysis. Further to this, in the past few years, heme has been shown to have an important regulatory role in cells, in processes such as transcription, regulation of the circadian clock, and the gating of ion channels. To act in a regulatory capacity, heme needs to move from its place of synthesis (in mitochondria) to other locations in cells. But while there is detailed information on how the heme lifecycle begins (heme synthesis), and how it ends (heme degradation), what happens in between is largely a mystery. Here we summarize recent information on the quantification of heme in cells, and we present a discussion of a mechanistic framework that could meet the logistical challenge of heme distribution.

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Section: Quantifying Heme Concentrations In Cellsmentioning

confidence: 99%

Understanding the Logistics for the Distribution of Heme in Cells

Gallio

Fung

Cammack-Najera

et al. 2021

JACS Au

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“…Third, the free heme concentration in the renal tissue of our rat RM-AKI model was not directly measured. The usual levels of free heme in normal cells is too low (<10 −9 M) to be assessed using conventional methods, although we have developed a novel heme sensor using fluorescently labeled HO-1 that enables us to measure free heme concentration in the rat liver [ 37 ]. Moreover, it has been reported that the microsomal heme concentration in the kidney is almost one-third of that in the liver [ 10 , 15 ], suggesting that free heme concentration is significantly lower in the kidney than in the liver.…”

Section: Discussionmentioning

confidence: 99%

Protective effect of tin chloride on rhabdomyolysis-induced acute kidney injury in rats

et al. 2022

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The heme component of myoglobin plays a crucial role in the pathogenesis of rhabdomyolysis-associated acute kidney injury (RM-AKI). Heme oxiganenase-1 (HO-1) is the rate-limiting enzyme of heme catabolism, and its metabolites, iron, biliverdin, and carbon monoxide, have antioxidant properties. Tin chloride (SnCl2) is a kidney specific HO-1 inducer. In this study, we examined whether the induction of HO-1 in the kidney by SnCl₂ pretreatment ameliorates RM-AKI in rats and if the effect is due to the degradation of excess renal free heme. We developed an RM-AKI rat (male Sprague-Dawley rats) model by injecting glycerol (Gly) in the hind limbs. RM-AKI rats were pretreated with saline or SnCl₂ or additional SnMP (tin mesoporphyrin, a specific HO inhibitor) followed by Gly treatment. Serum blood urea nitrogen (BUN) and creatinine (Crea) were measured as indicators of renal function. Renal free heme level was assessed based on the levels of δ-aminolevulinate synthase (ALAS1), a heme biosynthetic enzyme, and nuclear BTB and CNC homology 1 (Bach1), an inhibitory transcription factor of HO-1. Elevated free heme levels lead to decreases in ALAS1 and nuclear Bach1. After 24 h of Gly injection, serum BUN and Crea levels in saline-pretreated rats were significantly higher than those in untreated control rats. In contrast, SnCl₂-pretreated rats showed no significant increase in the indices. However, additional treatment of SnMP abolished the beneficial effect of SnCl₂. Renal ALAS1 mRNA levels and renal nuclear Bach1 protein levels in the saline pretreated rats were significantly lower than those in control rats 3 h after Gly injection. In contrast, the levels in SnCl₂-pretreated rats were not altered. The findings indicate that SnCl2 pretreatment confers protection against RM-AKI by virtue of HO-1 induction in the renal system, at least in part through excess free heme degradation.

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“…Single-turnover reactions of recombinant EGFP-Δ19 and EGFP-Δ19/ D159H, in complex with heme, were performed using a system utilizing ascorbate as an electron donor. 41 The system comprised 2 μM heme-complexed proteins, 20 mM phosphate buffer (pH 7.4), and 50 mM ascorbate. UV−vis spectra of the complexes in the wavelength region from 350 to 450 nm were recorded before and after the addition of ascorbate at 25 °C.…”

Section: A Rmentioning

confidence: 99%

Complex Formation of Heme Oxygenase-2 with Heme Is Competitively Inhibited by the Cytosolic Domain of Caveolin-1

et al. 2021

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The mechanism and physiological functions of heme oxygenase-2 (HO-2)-mediated carbon monoxide (CO) production, accompanied by heme metabolism, have been studied intensively in recent years. The enzymatic activity of constitutively expressed HO-2 must be strictly controlled in terms of the toxicity and chemical stability of CO. In this study, the molecular interaction between HO-2 and caveolin-1 and its effect on HO action were evaluated. An enzyme kinetics assay with residues 82–101 of caveolin-1, also called the caveolin scaffold domain, inhibited HO-2 activity in a competitive manner. Analytical ultracentrifugation and a hemin titration assay suggested that the inhibitory effect was generated by direct binding of caveolin-1 to aromatic residues, which were defined as components of the caveolin-binding motif in the HO-2 heme pocket. Herein, we developed a HO-2-based fluorescence bioprobe, namely EGFP-Δ19/D159H, which was capable of quantifying heme binding by HO-2 as the initial step in the CO production. The fluorescence of EGFP-Δ19/D159H decreased in accordance with 5-aminolevulinic acid-facilitated heme biosynthesis in COS-7 cells. In contrast, expression of the N-terminal cytosolic domain of caveolin-1 (residues 1–101) increased the probe fluorescence, suggesting that the cytosolic domain of caveolin-1 potently inhibits the binding of heme to the heme pocket of EGFP-Δ19/D159H. Taken together, our results suggest that caveolin-1 is a negative regulator of HO-2 enzymatic action. Moreover, our bioprobe EGFP-Δ19/D159H represents a powerful tool for use in future studies addressing HO-2-mediated CO production.

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Improvement of heme oxygenase-1-based heme sensor for quantifying free heme in biological samples

Cited by 5 publications

References 13 publications

Understanding the Logistics for the Distribution of Heme in Cells

Understanding the Logistics for the Distribution of Heme in Cells

Protective effect of tin chloride on rhabdomyolysis-induced acute kidney injury in rats

Complex Formation of Heme Oxygenase-2 with Heme Is Competitively Inhibited by the Cytosolic Domain of Caveolin-1

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