Cholesterol-dependent enzyme activity of human TSPO1

Abstract

The amino acid sequence of the tryptophan-rich sensory proteins (TSPO) is substantially conserved throughout all kingdoms of life. Human mitochondrial TSPO1 (HsTSPO1) binds to porphyrins and steroids, although its interactions with these molecules remains unknown. HsTSPO1 is associated with numerous physiological and pathological disorders, but the underlying molecular mechanisms are unknown. Here, we disclose the finding of human mitochondrial TSPO as a cholesterol-dependent protoporphyrin IX oxygenase. The results of our biochemical characterization are consistent with structural data and evolutionary analysis. The dependence of HsTSPO1 activity on cholesterol may be the result of the coevolution of this membrane protein with the membrane system. Our study provides a molecular foundation for comprehending the various roles played by mitochondrial TSPO in normal physiological and pathological situations.

Keywords: NCMN; TSPO; bilindigin; cholesterol; protoporphyrin IX.

Fig. 1.

Enzyme activity and lipid dependency of HsTSPO1. (A) The general enzymatic reaction catalyzed by TSPO protein. TSPO catalyze the oxidative degradation of protoporphyrin IX into bilindigin. (B). The diagram shows the general procedure for preparation of HsTSPO1 NCMN particles. HsTSPO1 is a conserved mitochondrial membrane protein. Membrane-active polymers can extract HsTSPO1 directly from the mitochondrial membrane. After a single-step affinity purification, HsTSPO1 NCMN particles can be produced in the active state with associated native cell membrane lipids. (C). Fluorescence spectra showing HsTSPO1 enzyme activity. The enzyme activity was monitored by fluorescence decay of protoporphyrin IX-2 at 632 nm upon excitation at 405 nm with different doses of light pulses. (D) The light-pulse-dose-dependent fluorescence decay of protoporphyrin IX-2 upon exposure to UV 405 nm. (E) Lipidomic analysis of HsTSPO1 NCMN particles showing relative amount of different native lipid species that associated with HsTSPO1. Chol: cholesterol, Hex1Cer: Hex1 ceramide, PC: phosphatidylcholines. (F) Comparison of intrinsic tryptophan fluorescence spectrum HsTSPO1 in the presence of CHS (_A) and inactive HsTSPO1 in the absence of CHS (_B).

Fig. 2.

Comparative characterization of enzyme activities of HsTSPO1 and BcTSPO. Photoactivated fluorescence spectra are shown for various TSPO-PpIX preparations. (A–C) show the enzyme activity of HsTSPO1 and BcTSPO without additional inhibitors. (D–F) show the enzyme activity of HsTSPO1 and BcTSPO with PK11195 as inhibitors. (G–I) show the enzyme activity of HsTSPO1 and BcTSPO with diazepam as an inhibitor. (J–L) show the enzyme activity of HsTSPO1 and BcTSPO with hemin as an inhibitor. (A, D, G, and J) show DDM and LMNG-purified HsTSPO1 without CHS. (B, E, H, and K) show DDM and LMNG-purified HsTSPO1 with CHS. (C, F, I, and L) show BcTSPO purified without CHS. (J, K, and L) show the enzyme activity of HsTSPO1 and BcTSPO with hemin as an inhibitor. (A) shows that, in the absence of CHS, HsTSPO1 binds to PpIX but does not degrade PpIX into bilindigin as indicated by the decrease of peak 632 nm and appearance of the peak of 673 nm. (B and C) show that HsTSPO1 in the presence of CHS has similar enzyme activity as that of BcTSPO; (E, H, K and F, I, L) show that PK11195, Diazepam and Hemin inhibit both HsTSPO1 and BcTSPO; (D and G) show in the absence of CHS, PK11195, and Diazepam do not have obvious inhibition of PpIX binding to HsTSPO1; (J) shows that even in the absence of CHS, hemin inhibit binding of PpIX to HsTSPO1.

Fig. 3.

Red-shifting impact of urea, CHAPS, and DPC on the intrinsic HsTSPO1 tryptophan fluorescence in the presence of CHS. (A) I, Control, active HsTSPO1 in the presence of 0.02% CHS and 0.05%v LMNG; II, HsTSPO1 in the presence of 0.02% CHS and 0.05%v LMNG and 6 M of Urea; (B) I, Control, active HsTSPO1 in the presence of 0.02% CHS, 0.05%v LMNG, and 0% CHAPS; II, HsTSPO1 in the presence of 0.02% CHS and 0.05%v LMNG and 1% CHAPS; III, HsTSPO1 in the presence of 0.02% CHS and 0.05%v LMNG and 0.02% CHAPS; IV, HsTSPO1 in the presence of 0.05%v LMNG and absence of CHS; (C) I, Control, active HsTSPO1 in the presence of 0.02% CHS and 0.05%v LMNG; II, HsTSPO1 in the presence of 0.02% CHS and 0.05%v LMNG and 2% DPC without heating; III, HsTSPO1 in the presence of 0.02% CHS and 0.05%v LMNG and 2% DPC and heated at 40 °C for 30 min.

Fig. 4.

Intrinsic tryptophan fluorescence analysis of HsTSPO1 in the presence and absence of CHS. (A) Intrinsic tryptophan fluorescence quenching spectra of HsTSPO1 in the presence of 0.02% CHS and various concentrations of hemin. I, 0 μL of 1 mg/mL hemin; II, 1μL of 1 mg/mL hemin; III, 2 μL of 1 mg/mL hemin; (B) Intrinsic tryptophan fluorescence quenching spectra of HsTSPO1 in the absence of CHS and various concentrations of hemin. I, 0μL of 1 mg/mL hemin; II, 1μL of 1 mg/mL hemin; III, 2 μL of 1 mg/mL hemin; (C) Intrinsic tryptophan fluorescence quenching spectra of HsTSPO1 in the presence of 0.02% CHS and various concentrations of PpIX. I, 0μL of 1 mg/mL PpIX; II, 1 μL of 1 mg/mL PpIX; III, 2 µL of 1 mg/mL PpIX; (D) Intrinsic tryptophan fluorescence quenching spectra of HsTSPO1 in the absence of 0.02% CHS and various concentrations of PpIX. I, 0 μL of 1 mg/mL PpIX; II, 1 μL of 1 mg/mL PpIX; III, 2 μL of 1 mg/mL PpIX; (E) Intrinsic tryptophan fluorescence quenching spectra of HsTSPO1 in the presence of 0.02% CHS and various concentrations of diazepam. I, 0 μL of 1 mg/mL Diazepam; II, 1 μ L of 1 mg/mL Diazepam; III, 2 μ L of 1 mg/mL Diazepam; (F) Intrinsic tryptophan fluorescence quenching spectra of HsTSPO1 in the absence of CHS and various concentrations of diazepam. I, 0 μL of 1 mg/mL PK11195; II, 1 μL of 1 mg/mL PK11195; III, 2 μL of 1 mg/mL PK11195; (G) Intrinsic tryptophan fluorescence quenching spectra of HsTSPO1 in the presence of 0.02% CHS and various concentrations of PK11195. I, 0 μL of 1 mg/mL PK11195; II, 1 μ L of 1 mg/mL PK11195; III, 2 μ L of 1 mg/mL PK11195; (H) Intrinsic tryptophan fluorescence quenching spectra of HsTSPO1 in the absence of CHS and various concentrations of PK11195. I, 0 μL of 1 mg/mL PK11195; II, 1 μL of 1 mg/mL PK11195; III, 2 μL of 1 mg/mL PK11195.

Fig. 5.

Crystal structure of the BcTSPO/hematin complex (PDB ID: 8VGU, chain (A) A single hemin molecule is located in the active site of BcTSPO. (B) Interaction between hemin and BcTSPO and relative positions of the four conserved tryptophan residues to the porphyrin molecule. (C) Conservativeness of the active site and potential cholesterol binding site on the HsTSPO1 structural model. (D) Conserved potential cholesterol binding site between TM2 and TM5 on the surface of the HsTSPO1 structural model. 60° anticlockwise rotation (viewed from Top to Bottom) relative to the orientation displayed in panel (D and E) Conservativeness of the active site of HsTSPO1 structural model. The active site is displayed as an open-book view, i.e., 180° relative to each half of the active site. (F) 2D view of the relative positions of conserved tryptophan’s and hemin. (G) 2D chemical structure of proposed bilindigin molecule based on mass spectrometric analysis.