leupeptins and alanyl-alanyl-phenylalanine-chloromethyl-ketone

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.

Topics: Aldehydes; Amino Acid Chloromethyl Ketones; Blotting, Western; Chromatography, Gel; Enzyme Inhibitors; Glyceraldehyde-3-Phosphate Dehydrogenases; Glycolysis; Humans; Hydrogen-Ion Concentration; Inhibitory Concentration 50; Leupeptins; Lipid Peroxidation; Oxidative Stress; Phenylmethylsulfonyl Fluoride; Proteasome Endopeptidase Complex; Sepharose; Serine Endopeptidases; Time Factors; U937 Cells

The proteasome is the primary protease used by cells for degrading proteins and generating peptide ligands for class I molecules of the major histocompatibility complex. Based on the properties of cells adapted to grow in the presence of the proteasome inhibitor 4-hydroxy-5-iodo-3-nitrophenylacetyl-Leu-Leu-leucinal-vinyl sulfone (NLVS), it was proposed that proteasomes can be replaced by alternative proteolytic systems, particularly a large proteolytic complex with a tripeptidyl peptidase II activity. Here we show that NLVS-adapted cells retain sensitivity to a number of highly specific proteasome inhibitors with regard to antigenic peptide generation, accumulation of polyubiquitinated proteins, degradation of p53, and cell viability. In addition, we show that in the same assays (with a single minor exception), NLVS-adapted cells are about as sensitive as nonselected cells to Ala-Ala-Phe-chloromethylketone, a specific inhibitor of tripeptidyl peptidase II activity. Based on these findings, we conclude that proteasomes still have essential proteolytic functions in adapted cells that are not replaced by Ala-Ala-Phe-chloromethylketone-sensitive proteases.

Topics: Amino Acid Chloromethyl Ketones; Aminopeptidases; Animals; Antigen Presentation; Antigens; Boronic Acids; Bortezomib; CD8-Positive T-Lymphocytes; Cell Survival; Cysteine Endopeptidases; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Drug Resistance; Endopeptidases; Enzyme Activation; H-2 Antigens; Leupeptins; Lymphoma, T-Cell; Mice; Multienzyme Complexes; Neoplasm Proteins; Oligopeptides; Peptide Fragments; Phenols; Protease Inhibitors; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Pyrazines; Selection, Genetic; Serine Endopeptidases; Sulfones; Thymus Neoplasms; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tyramine; Ubiquitins