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. 2021 Dec 31;23(1):443.
doi: 10.3390/ijms23010443.

Oxidized Substrates of APEH as a Tool to Study the Endoprotease Activity of the Enzyme

Annamaria Sandomenico  1 , Marta Gogliettino  2 , Emanuela Iaccarino  1 , Carmela Fusco  2   3 , Andrea Caporale  1 , Menotti Ruvo  1 , Gianna Palmieri  2 , Ennio Cocca  2
Affiliations

Oxidized Substrates of APEH as a Tool to Study the Endoprotease Activity of the Enzyme

Annamaria Sandomenico et al. Int J Mol Sci. .

Abstract

APEH is a ubiquitous and cytosolic serine protease belonging to the prolyl oligopeptidase (POP) family, playing a critical role in the processes of degradation of proteins through both exo- and endopeptidase events. Endopeptidase activity has been associated with protein oxidation; however, the actual mechanisms have yet to be elucidated. We show that a synthetic fragment of GDF11 spanning the region 48-64 acquires sensitivity to the endopeptidase activity of APEH only when the methionines are transformed into the corresponding sulphoxide derivatives. The data suggest that the presence of sulphoxide-modified methionines is an important prerequisite for the substrates to be processed by APEH and that the residue is crucial for switching the enzyme activity from exo- to endoprotease. The cleavage occurs on residues placed on the C-terminal side of Met(O), with an efficiency depending on the methionine adjacent residues, which thereby may play a crucial role in driving and modulating APEH endoprotease activity.

Keywords: APEH; endoproteolytic activity; oxidative stress; oxidized methionine; oxidized substrates.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Scheme of the hydrolysis of GDF11[48-64]ox by trypsin and identification of the oxidation sites on methionines.
Figure 2
Figure 2
LC-MS analysis of GDF11[48-64]ox following tryptic digestion. Base peaks profiles of the peptide after oxidation and treatment with trypsin (A). EIC analysis of Fragment 1 (Ac-EYMFMQK), (B). EIC analysis of Fragment 2 (YPHTHLVQQA-NH2), (C). Mass spectrum of Fragment 1, (D). Mass spectrum of Fragment 2, (E).
Figure 3
Figure 3
LC-MS analysis of the GDF11[48-64]ox before and after treatment with APEH for 48 h. Base Peak profiles of GDF11[48-64]ox (A) before and (B) after treatment with APEH where the 3 peaks generated are indicated by arrows. (C–F) EIC profiles of Peaks 1–3. (F–I) Mass spectra of Peak 1, Peak 2 and Peak 3. Under Peak 3, two main species were identified as the fragments Ac-EYM(ox)FM(ox)QKYPHTHLVQ (species c) and Ac-EYM(ox)FM(ox)QKYPHTHL (species d). The species identified are reported on the mass spectra.
Figure 4
Figure 4
Scheme of main cleavage site of APEH on GDF11[48-64]ox.
See this image and copyright information in PMC

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