Heme delivery to heme oxygenase-2 involves glyceraldehyde-3-phosphate dehydrogenase

Abstract

Heme regulatory motifs (HRMs) are found in a variety of proteins with diverse biological functions. In heme oxygenase-2 (HO2), heme binds to the HRMs and is readily transferred to the catalytic site in the core of the protein. To further define this heme transfer mechanism, we evaluated the ability of GAPDH, a known heme chaperone, to transfer heme to the HRMs and/or the catalytic core of HO2. Our results indicate GAPDH and HO2 form a complex in vitro. We have followed heme insertion at both sites by fluorescence quenching in HEK293 cells with HO2 reporter constructs. Upon mutation of residues essential for heme binding at each site in our reporter construct, we found that HO2 binds heme at the core and the HRMs in live cells and that heme delivery to HO2 is dependent on the presence of GAPDH that is competent for heme binding. In sum, GAPDH is involved in heme delivery to HO2 but, surprisingly, not to a specific site on HO2. Our results thus emphasize the importance of heme binding to both the core and the HRMs and the interplay of HO2 with the heme pool via GAPDH to maintain cellular heme homeostasis.

Keywords: GAPDH; chaperone; glyceraldehyde-3-phosphate dehydrogenase; heme oxygenase-2; heme regulatory motifs; heme trafficking.

Figure 1:

Heme delivery to and heme transfer within HO2. GAPDH is proposed to deliver heme to apo-HO2 (top). Once heme is bound to HO2, it can be transferred between the catalytic core region (with ligation via His45, red) and the C-terminal HRMs (centered at Cys265 and Cys282, blue) of HO2. A linear representation of HO2 (bottom) indicates residues involved in heme binding.

Figure 2:

Binding interaction of HO2 and its fragments with GAPDH. The panels show the change in residual polarized fluorescence of FITC-labeled HO2 protein constructs during titration with GAPDH. (A) FITC-labeled full-length HO2 (1–288). (B) FITC-labeled HO2 (1–248). (C) FITC-labeled HO2 (213–288). Points are the mean ± SD for three samples and are representative of three independent experiments

Figure 3:

Effect of the TC motif on heme binding to the HO2 catalytic core, HO2 (1–248), and HRM-containing tail, HO2 (213–288). (A) The loop region (K176-G181, teal) adjacent to the distal helix was substituted with a TC motif in TCcore-HO2, as indicated on the structure of the catalytic core of heme-bound HO2 (PDB code 2RGZ). Two other regions, indicated in yellow, were also changed to a TC motif, but those variants were not investigated further. (B) Overlay of the CD spectra of HO2 (1–248) (blue) and TCcore-HO2 (1–248) (red). (C) Spectra of the heme-bound forms of HO2 (1–248) (blue), TCcore-HO2 (1–248) (red), and FlAsH bound TCcore-HO2 (1–248) (green). (D) Difference titrations of HO2 (1–248) (blue), TCcore-HO2 (1–248) (red), and FlAsH-bound TCcore-HO2 (1–248) (green) with heme. The percent of the total difference in absorbance at 406 nm was plotted and fit to a K_D of 3.6 nM. (E) The difference in absorbance of FlAsH bound TCcore-HO2 (1–248) upon titration with heme (green) is plotted against the percent of total fluorescence intensity at 532 nm after excitation at 485 nm as heme was added to the protein (orange). (F–H) Same as panels (A–C) except with C282A HO2 (213–288) (blue), TCtail-HO2 (213–288) (red), and FlAsH-bound TCtail-HO2 (213–288) (green). The change in fluorescence of FlAsH bound TCtail-HO2 (213–288) is plotted in orange in H.

Figure 4:

Characterization of TC motif-containing HO2 (1–288). (A) Spectra of the heme-bound forms of HO2 (1–288) (blue), TCcore-HO2 (1–288) (red), and TCtail-HO2 (1–288) (green). (B) Difference titrations of HO2 (1–288) (blue), TCcore-HO2 (1–288) (red), and TCtail-HO2 (1–288) (green) with heme. The percent of the total difference in absorbance at 406 nm was plotted and fit to a K_D of 3.6 nM. (C) Steady state activity of HO2 (1–288) as compared to the activity of TCcore-HO2 (1–288) and TCtail-HO2 (1–288) with and without FlAsH bound. (D) The change in the percent of total fluorescence intensity at 532 nm after excitation at 485 nm as heme was added to TCcore-G163D/C265A/C282A-HO2 (1–288) (blue) and TCtail-G163D/C265A-HO2 (1–288) (red). Data was fit to a K_D of 3.6 nM. (E) The change in the percent of total fluorescence intensity at 532 nm after excitation at 485 nm as heme was added to TCcore-H45W/G159W-HO2 (1–288) (blue) and TCtail-H45W/G159W-HO2 (1–288) (red). Both curves were fit to a K_D of 55 nM. (F) The change in the percent of total fluorescence intensity at 532 nm after excitation at 485 nm as heme was added to TCcore-H45W/G159W/C265A/C282A-HO2 (1–288) (blue) and TCtail-H45W/G159W/C265A-HO2 (1–288) (red).

Figure 5:

Heme delivery to apo-TCcore-HO2 proteins in living cells and the importance of GAPDH. The apo-TCcore-HO2 protein (A–C) or the G163D/C265A/C282A (D–F) or the H45W/G159W (G–I) variants of the TCcore-HO2 protein was expressed in heme-deficient HEK293 cells and FlAsH-labeled. The change in fluorescence intensity versus time was monitored after adding vehiclHO2 ( buffer or 5 µM heme to the cell cultures, and a decrease in FlAsH fluorescence intensity indicates heme incorporation. In this Figure, “siGAPDH” indicates heme was added to cells transfected with siGAPDH RNA; “scramble RNA” indicates heme was added to cells transfected with scramble RNA; “HA-GAPDH H53A” indicates heme was added to cells transfected with siGAPDH RNA and siRNA resistant HA-GAPDH H53A; and “HA-GAPDH WT” indicates heme was added to cells transfected with siGAPDH RNA and siRNA resistant HA-GAPDH. Kinetic traces are the mean ± SD of three wells and are representative of two independent experiments.

Figure 6:

Heme delivery to apo-TCtail-HO2 proteins in living cells and the importance of GAPDH. The apo-TCtail-HO2 protein (A–C) or the G163D/C265A (D–F) or the H45W/G159W (G–I) variants of the TCtail-HO2 protein was expressed in heme-deficient HEK293 cells and FlAsH-labeled. The change in fluorescence intensity versus time was monitored after adding vehicle buffer or 5 µM heme to the cell cultures, and a decrease in FlAsH fluorescence intensity indicates heme incorporation. In this Figure, “siGAPDH” indicates heme was added to cells transfected with siGAPDH RNA; “scramble RNA” indicates heme was added to cells transfected with scramble RNA; “HA-GAPDH H53A” indicates heme was added to cells transfected with siGAPDH RNA and siRNA resistant HA-GAPDH H53A; and “HA-GAPDH WT” indicates heme was added to cells transfected with siGAPDH RNA and siRNA resistant HA-GAPDH. Kinetic traces are the mean ± SD of three wells and are representative of two independent experiments.