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Effects of ionic strength on the catalysis and stability of prolyl oligopeptidase.

ABSTRACT: Prolyl oligopeptidase is the prototype of a new serine protease family, unrelated to trypsin and subtilisin. In contrast with these proteases, prolyl oligopeptidase is remarkably sensitive to ionic strength, being more active in the presence of high concentrations of salt. The enzyme has two catalytic forms, which interconvert with changing pH. To reveal the structural bases of these phenomena, the effects of 0.5 M NaCl on the stability of the enzyme were investigated by studying its denaturation as a function of pH, temperature, and urea concentration. The three independent methods have unequivocally demonstrated that denaturation of the enzyme is promoted in the presence of NaCl. Furthermore, destabilization of the low-pH form by urea is more significant than that of the high-pH form. Examination of the fluorescence emission spectra of various denatured forms indicates that the enzyme is not fully unfolded in 8 M urea, nor at acidic pH. The tryptophan residues in the acid-denatured state are mainly buried. The results are interpreted in terms of the decay of the protective water shell at the higher ionic strength. The higher enthalpy and entropy of activation for heat denaturation provide further evidence that a more ordered water structure stabilizes the protein in the absence of salt. The biphasic kinetics obtained with denaturation by heat and urea suggest that the enzyme has two domains of different stabilities.

SUBMITTER: Polgar L

PROVIDER: S-EPMC1136254 | biostudies-other | 1995 Nov

REPOSITORIES: biostudies-other

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Project description:BackgroundProlyl oligopeptidase (POP) is a serine endopeptidase that is widely distributed in vivo, particularly in the liver. Significant changes in functional mitochondrial proteins involved with mitochondrial oxidoreductases/transporters and nucleic acid binding proteins were observed after POP inhibition in the liver, which suggested a role of POP in regulating liver energy metabolism. Steatosis in nonalcoholic fatty liver disease (NAFLD) is associated with disturbances in lipid and energy metabolism in hepatocytes. Here, we aimed to study the effect of POP on hepatocyte steatosis.MethodsThe human liver cell line L02 was used to investigate the biological effects of POP. An in vitro cell model of steatosis was successfully induced with oleic acid and palmitic acid. L02 cells were also subjected to S17092 (a POP inhibitor) at different concentrations for 24 or 48 h. Ac-SDKP levels and POP activity were measured to assess the rate of inhibition of POP by S17092. The POP gene and protein expression levels were detected using real-time PCR and Western blots, respectively. Oil red O staining was performed and the triglyceride levels in the L02 cells were also measured. Cell proliferation and apoptosis were detected using CCK-8 and flow cytometry, respectively. The expression of genes involved in lipid metabolism was detected using real-time PCR. The effects of POP inhibition on LC3B II were detected by Western blot.ResultsCompared with the control, the POP mRNA levels increased by approximately 30%, and the POP protein levels increased by almost 60% in the steatotic L02 cells. After S17092 (0.026~130 ?M) incubation for 24 or 48 h, cell proliferation was significantly decreased in the free fatty acid (FFA)-treated cells at 26-130 ?M; however, S17092 did not affect the proliferation of L02 cells after 24 h of incubation with S17092 at 0.026-65 ?M without FFA treatment. S17092 treatment (13 and 26 ?M) also elicited no significant effect on apoptosis in normal L02 cells, but FFA treatment increased cell apoptosis, which was attenuated by S17092 incubation. S17092 treatment inhibited intracellular POP activity and decreased the AcSDKP level at the concentration of 0.026-26 ?M. After treatment with FFA for 24 h, oil red O staining revealed significant lipid accumulation in the cells in the model group compared with the controls; however, lipid accumulation was suppressed after the administration of S17092 (13 and 26 ?M). Accordingly, the triglyceride levels in the FFA-treated cells were approximately 5-fold greater than those of the controls and were decreased by approximately 25% and 45% after the administration of S17092 at 13 and 26 ?M, respectively. The mRNA levels of FASN, PPAR-?, and SREBP-1c were higher in the FFA-treated cells than in the normal controls, and all of these levels were significantly inhibited in the presence of S17092 at both 13 and 26 ?M. S17092 treatment did not affect LC3B II in the FFA-treated cells compared with FFA treatment alone.ConclusionThe expression of POP increases with hepatocyte steatosis, and POP inhibitors can significantly reduce intracellular lipid accumulation, which might be related to the inhibition of genes involved in lipid synthesis.

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Effects of ionic strength on the catalysis and stability of prolyl oligopeptidase.

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