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Degradation of oxidized or oxidatively modified proteins is an essential part of the antioxidant defenses of cells. 4-Hydroxy-2-nonenal, a major reactive aldehyde formed by lipid peroxidation, causes many types of cellular damage. It has been reported that 4-hydroxy-2-nonenal-modified proteins are degraded by the ubiquitin-proteasome pathway or, in some cases, by the lysosomal pathway. However, our previous studies using U937 cells showed that 4-hydroxy-2-nonenal-modified glyceraldehyde-3-phosphate dehydrogenase is degraded by cathepsin G. In the present study, we isolated the 4-hydroxy-2-nonenal-modified glyceraldehyde-3-phosphate dehydrogenase-degrading enzyme from rat neutrophils to an active protein fraction of 28 kDa. Using the specific antibody, the 28 kDa protein was identified as cathepsin G. Moreover, the degradation activity was inhibited by cathepsin G inhibitors. These results suggest that cathepsin G plays a crucial role in the degradation of 4-hydroxy-2-nonenal-modified glyceraldehyde-3-phosphate dehydrogenase.

Degradation of oxidized or oxidatively modified proteins is an essential part of the antioxidant defenses of cells. 4-Hydroxy-2-nonenal (HNE), a major reactive aldehyde formed by lipid peroxidation, causes many types of cellular damage. It has been reported that HNE-modified proteins are degraded by the ubiquitin-proteasome pathway or, in some cases, by the lysosomal pathway. However, our previous studies using U937 cells showed that HNE-modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is degraded by an enzyme that is sensitive to a serine protease inhibitor, diisopropyl fluorophosphate (DFP), but not a proteasome inhibitor, MG-132, and that its degradation is not catalyzed in the acidic pH range where lysosomal enzymes are active. In the present study, we purified an HNE-modified GAPDH-degrading enzyme from a U937 cell extract to a final active fraction containing two proteins of 28 kDa (P28) and 27 kDa (P27) that became labeled with [(3)H]DFP. Using peptide mass fingerprinting and a specific antibody, P28 and P27 were both identified as cathepsin G. The degradation activity was inhibited by cathepsin G inhibitors. Furthermore, a cell extract from U937 cells transfected with a cathepsin G-specific siRNA hardly degraded HNE-modified GAPDH. These results suggest that cathepsin G plays a role in the degradation of HNE-modified GAPDH.

Lipid peroxidation products such as 4-hydroxy-2-nonenal (HNE) may be responsible for various pathophysiological events under oxidative stress, since they injure cellular components such as proteins and DNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a key enzyme of glycolysis and has been reported to be a multifunctional enzyme, is one of the enzymes inhibited by HNE. Previous studies showed that GAPDH is degraded when incubated with acetylleucine chloromethyl ketone (ALCK), resulting in the liberation of a 23-kDa fragment. In this study, we examined whether GAPDH incubated with HNE or other aldehydes of lipid peroxidation products are degraded similarly to that with ALCK. The U937 cell extract was incubated with these aldehydes at 37 degrees C and analyzed by Western blotting using anti-GAPDH antibodies. Incubation with HNE or 4-hydroxy-2-hexenal (HHE) decreased GAPDH activity and GAPDH protein level, and increased the 23-kDa fragment, in time- and dose-dependent manners, but that with other aldehydes did not. Gel filtration using the Superose 6 showed that the GAPDH-degrading activity was eluted in higher molecular fractions than proteasome activity. The enzyme activity was detected at the basic range of pH and inhibited by serine protease inhibitors, diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, but not by other protease inhibitors including a proteasome inhibitor, MG-132, and a tripeptidyl peptidase II (TPP II) inhibitor, AAF-CMK. These results suggest that GAPDH modified by HNE and HHE is degraded by a giant serine protease, releasing the 23-kDa fragment, not by proteasome or TPP II.

We have previously reported that acetylleucine chloromethyl ketone (ALCK), an inhibitor of acylpeptidehydrolase, induces the inhibition and degradation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the U937 cell extract. In the present study, the process of ALCK-induced GAPDH degradation was investigated. A kinetic study revealed that GAPDH was irreversibly inhibited by ALCK. ALCK treatment induced a change in the signal intensity of GAPDH in the near-UV region of the circular dichroism (CD) spectrum, and the fluorescence intensity of GAPDH at 330 nm increased to about 10% when excited at 280 nm, suggesting that a significant conformational change of GAPDH was induced by ALCK. When the U937 cell extract was incubated with ALCK and the products were separated by SDS-polyacrylamide gel electrophoresis (PAGE), a 23-kDa fragment from GAPDH was detected by Western blotting using anti-GAPDH serum. When ALCK-treated GAPDH was incubated with protease fractions from the U937 cell extract, a 17-kDa fragment was also detected. Sequence analysis showed that the N-terminal amino acid sequence of the 23-kDa fragment was GKVKVG and that of 17-kDa fragment was RDGRGAL. Therefore, ALCK-modified GAPDH is deduced to be digested at the peptide bond Trp(195)-Arg(196). The protease activity liberating a 23-kDa fragment from ALCK-treated GAPDH was effective under the basic condition. Results suggested that ALCK binds to GAPDH to modulate the conformation of enzyme, which is susceptible to chymotrypsin-like protease activity.

We examined whether any changes were induced in cellular proteins by an inhibitor of acylpeptide hydrolase (ACPH) (EC 3.4.19.1), acetylleucine chloromethyl ketone (ALCK), which was shown in our previous report to induce apoptosis of human U937 cells. Extract prepared from U937 cells in 0.05% Triton X-100-PBS was incubated with ALCK at 37 degrees, and then analyzed using SDS-PAGE. A 36kDa protein in the cell extract was decreased markedly during the incubation period. This protein was purified and identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) by its specific enzyme activity, N-terminal amino acid sequence, and Western blotting. Incubation of purified GAPDH with ALCK resulted in a decrease of GAPDH activity, but not in a decrease in the amount of GAPDH. The ALCK-induced GAPDH decrease in the cell extract was abrogated by co-incubation with a serine protease inhibitor, diisopropyl fluorophosphate, suggesting that GAPDH was first inactivated by ALCK, and subsequently degraded by a serine protease(s). GAPDH degradation was also observed in U937 cell cultures in the presence of ALCK. The significance of GAPDH inhibition in the apoptotic process is discussed.