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. 2016 Apr 4;6(1):10-13.
doi: 10.4103/2045-9912.179341. eCollection 2016 Mar.

Cysteine-independent activation/inhibition of heme oxygenase-2

Dragic Vukomanovic  1 , Mona N Rahman  1 , Mahin D Maines  2 , Terence Rs Ozolinš  1 , Walter A Szarek  3 , Zongchao Jia  1 , Kanji Nakatsu  1
Affiliations

Cysteine-independent activation/inhibition of heme oxygenase-2

Dragic Vukomanovic et al. Med Gas Res. .

Abstract

Reactive thiols of cysteine (cys) residues in proteins play a key role in transforming chemical reactivity into a biological response. The heme oxygenase-2 (HO-2) isozyme contains two cys residues that have been implicated in binding of heme and also the regulation of its activity. In this paper, we address the question of a role for cys residues for the HO-2 inhibitors or activators designed in our laboratory. We tested the activity of full length recombinant human heme oxygenase-2 (FL-hHO-2) and its analog in which cys265 and cys282 were both replaced by alanine to determine the effect on activation by menadione (MD) and inhibition by QC-2350. Similar inhibition by QC-2350 and almost identical activation by MD was observed for both recombinant FL-hHO-2s. Our findings are interpreted to mean that thiols of FL-hHO-2s are not involved in HO-2 activation or inhibition by the compounds that have been designed and identified by us. Activation or inhibition of HO-2 by our compounds should be attributed to a mechanism other than altering binding affinity of HO-2 for heme through cys265 and cys282.

Keywords: HO-2 activator; HO-2 inhibitor; Heme degradation; QC-2350; heme oxygenase-2; in vitro; menadione; thiols.

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Conflict of interest statement

Conflicts of Interest: None declared.

Figures

Figure 1
Figure 1
Menadione activation of recombinant FL-hHO-2 (open circles) was similar to the same enzyme without thiols-FL-hHO-2 Cys265,282-Ala (closed triangles). Note: Heme oxygenase-2 (HO-2) activity was measured as described in Methods. The abscissa shows the log of the drug concentration (μM) and the ordinate shows HO-2 activity as pmol CO formed in 15 minutes by 0.7 μM FL-hHO-2s (the symbols represent the mean ± SD, n = 4). Where the SD bars are missing, the SD fell with the symbols. Inset shows the chemical structure of menadione. FL-hHO-2: Full length recombinant human heme oxygenase-2; FL-hHO-2 Cys265,282Ala: full length recombinant human heme oxygenase-2 in which cys265 and cys 282 were replaced with ala; CO: carbon monoxide.
Figure 2
Figure 2
QC-2350 inhibition of recombinant and microsomal HO-2. Note: (A) QC-2350 (HO-2 inhibitor) decreased the activity of both FL-hHO-2 (closed triangles) and FL-hHO-2 Cys265,282-Ala (open circles) almost identically when the data are plotted as % Control Activity. When the data are plotted as pmol CO formed in 15 minutes (B), a similar inhibition of the isozymes was observed. (C) QC-2350 inhibition of rat brain microsomal HO-2 was similar to both recombinant forms of HO-2, but was slightly more potent. HO-2 activities were measured as described in Methods. Preparation of microsomal HO-2 was described in our recent paper (Vlahakis et al., 2013). The abscissa shows the log of drug concentration (μM) and ordinate (B, C) shows HO-2 activity as pmol of CO formed in 15 minutes by 0.7 μM FL-hHO-2 (mean ± SD, n = 4). Inset in A shows the chemical structure of QC-2350. HO-2: Heme oxygenase-2; FL-hHO-2: full length recombinant human heme oxygenase-2; FL-hHO-2 Cys265,282Ala: full length recombinant human heme oxygenase-2 in which cys265 and cys 282 were replaced with ala; CO: carbon monoxide.
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