In vitro oxidative inactivation of human presequence protease (hPreP)

Abstract

The mitochondrial peptidasome called presequence protease (PreP) is responsible for the degradation of presequences and other unstructured peptides including the amyloid-β peptide, whose accumulation may have deleterious effects on mitochondrial function. Recent studies showed that PreP activity is reduced in Alzheimer disease (AD) patients and AD mouse models compared to controls, which correlated with an enhanced reactive oxygen species production in mitochondria. In this study, we have investigated the effects of a biologically relevant oxidant, hydrogen peroxide (H(2)O(2)), on the activity of recombinant human PreP (hPreP). H(2)O(2) inhibited hPreP activity in a concentration-dependent manner, resulting in oxidation of amino acid residues (detected by carbonylation) and lowered protein stability. Substitution of the evolutionarily conserved methionine 206 for leucine resulted in increased sensitivity of hPreP to oxidation, indicating a possible protective role of M206 as internal antioxidant. The activity of hPreP oxidized at low concentrations of H(2)O(2) could be restored by methionine sulfoxide reductase A (MsrA), an enzyme that localizes to the mitochondrial matrix, suggesting that hPreP constitutes a substrate for MsrA. In summary, our in vitro results suggest a possible redox control of hPreP in the mitochondrial matrix and support the protective role of the conserved methionine 206 residue as an internal antioxidant.

Fig. 1

Effect of exposure to hydrogen peroxide on hPreP wild-type activity. After 4 h incubation with H₂O₂, the hydrogen peroxide was removed and hPreP activity assayed as the cleavage of four substrates. (A) Representative gel showing cleavage of C1 by hPreP, resulting in a change in migration on agarose gel due to the charge profile of the peptide. (B) Effect of oxidation on the rate of substrate V degradation by hPreP (average of three experiments). Time course analysis of (C) pF₁β and (D) Aβ degradation by hPreP incubated in the absence or presence (0.5 or 5 mM) of H₂O₂. Shown are representative gels (corresponding to one of the three experiments) and an estimation of the degradation rate (in the first 10 min for pF₁β and in the first 45 min for Aβ).

Fig. 2

Consequences of hPreP wild-type oxidation. Upon incubation with H₂O₂ for 4 h, hPreP was analyzed by (A) SDS–PAGE (0.7 µg per lane) and (B) carbonyl immunoblot (4 µg per lane), as described under Materials and methods. Ponceau S staining is shown as a loading control. (C) hPreP samples were incubated in the presence or absence of 5 mM H₂O₂ for 4 h; subsequently treated with 10 ng trypsin for 1, 10, and 30 min; and then analyzed by SDS–PAGE (2 µg per lane).

Fig. 3

Localization of the analyzed methionine residues in the hPreP structural model, constructed based on the structure of AtPreP1 (PDB ID: 2FGE) [51]. Methionines are shown in red, the active-site histidines are blue, the substrate peptide is green, and the zinc ion is magenta. The detailed location of M206 within the hPreP active site is highlighted. Inset shows an alignment of PreP sequences from A. thaliana (AtPreP1 and AtPreP2), Plasmodium falciparum (falcilysin, Fln), S. cerevisiae (Mop112), Bos taurus (PreP_Bt), Canis familiaris (PreP_Cf), Mus musculus (PreP_Mm), Rattus norvergicus (PreP_Rn), Schizosaccharomyces pombe (PreP_Sp), and Homo sapiens (hPreP). The alignment is restricted to the region around M206 (full alignment is shown in Supplementary Fig. S2).

Fig. 4

(A) Effect of H₂O₂ exposure (4 h) on the activity of hPreP^M206L compared to hPreP wild type (wt), as assayed by the degradation of the fluorogenic substrate V. (B) Time course inactivation of hPreP wt and hPreP^M206L upon exposure to 0.5 mM H₂O₂ analyzed by the substrate V assay (results shown are averages of three experiments). The regression lines (inset) were fit for a first-order reaction for time points from 30 to 240 min and gave R² of 0.96 and 0.99, for the hPreP wt and hPreP^M206L data sets, respectively. The ratio between the inactivation rates of hPreP^M206L and hPreP wt showed a threefold difference. 100% activity corresponds to a specific activity of 491.6 ± 54.2 ng substrate V degraded min⁻¹ µg protein⁻¹ for wt hPreP and 656.2 ± 32.7 ng substrate V degraded min⁻¹ µg protein⁻¹ for hPreP^M206L.

Fig. 5

Recovery of hPreP wt activity by MsrA. hPreP was exposed to H₂O₂ for 4 h at the concentrations of 0.5 or 5 mM. The H₂O₂ was then removed by filtration and hPreP further incubated for 2 h at 37 °C with DTT and MsrA as indicated. After this incubation the hPreP activity was assayed using substrate V. (A) MsrA effect on hPreP oxidized with 0.5 mM H₂O₂. (B) MsrA effect on hPreP oxidized with 5 mM H₂O₂. Statistical analysis was made using Student’s t test. Inset in (A) shows that MsrA itself has no degradation activity against substrate V.