carbobenzoxy-leucyl-leucyl-norvalinal and calpastatin

Cell-permeant peptidyl aldehydes and diazomethylketones are frequently utilized as inhibitors of regulatory intracellular proteases. In the present study the specificities of several peptidyl inhibitors for purified human mu-calpain and 20 S proteasome were investigated. Acetyl-LLnL aldehyde, acetyl-LLM aldehyde, carbobenzyloxy-LLnV aldehyde (ZLLnVal), and carbobenzyloxy-LLY-diazomethyl ketone produced half-maximum inhibition of the caseinolytic activity of mu-calpain at concentrations of 1-5 x 10(-7) M. In contrast, only ZLLnVal was a reasonably potent inhibitor of the caseinolytic activity of 20 S proteasome, producing 50% inhibition at 10(-5) M. The other inhibitors were at least 10-fold less potent, producing substantial inhibition only at near saturating concentrations in the assay buffer. Further studies with ZLLnVal demonstrated that its inhibition of the proteasome was independent of casein concentration over a 25-fold range. Proteolysis of calpastatin or lysozyme by the proteasome was half-maximally inhibited by 4 and 22 microM ZLLnVal, respectively. Thus, while other studies have shown that ZLLnVal is a potent inhibitor of the hydrophobic peptidase activity of the proteasome, it appears to be a much weaker inhibitor of its proteinase activity. The ability of the cell permeant peptidyl inhibitors to inhibit growth of the yeast Saccharomyces cerevisiae was studied because this organism expresses proteasome but not calpains. Concentrations of ZLLnVal as high as 200 microM had no detectable effect on growth rates of overnight cultures. However, yeast cell lysates prepared from these cultures contained 2 microM ZLLnVal, an amount which should have been sufficient to fully inhibit hydrophobic peptidase activity of yeast proteasome. Degradation of ubiquitinylated proteins in yeast extracts by endogenous proteasome was likewise sensitive only to high concentrations of ZLLnVal. The higher sensitivity of the proteinase activity of calpains to inhibition by the cell permeant inhibitors suggests that calpain-like activities may be targets of these inhibitors in animal cells.

Topics: Acetylcysteine; Calcium-Binding Proteins; Calpain; Catalysis; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Diazomethane; Enzyme Precursors; Humans; Kinetics; Leupeptins; Multienzyme Complexes; Muramidase; Oligopeptides; Plant Proteins; Protease Inhibitors; Proteasome Endopeptidase Complex; Saccharomyces cerevisiae; Serine Proteinase Inhibitors; Ubiquitins

Nuclear factor kappaB (NF-kappaB) is a transcription factor that is critical for the inducible expression of multiple cellular and viral genes. Using the electrophoretic mobility shift assay, we demonstrated that DNA binding activity of NF-kappaB was abolished by proteolysis with mu- and m-calpains in vitro. The proteolysis of NF-kappaB by calpains and hence the abolition of its DNA binding was prevented by calpastatin, calpain inhibitor I and proteasome inhibitor. We also provided evidence that calpains degrade the C-terminal domain of NF-kappaB by Western blot using anti-NF-kappaB (p65) C-terminal antibody. These observations indicate that calpains regulate gene expression through processing of NF-kappaB.

Topics: Base Sequence; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; DNA; Glycoproteins; Humans; Leupeptins; Molecular Sequence Data; NF-kappa B; Protein Processing, Post-Translational