calpain and carbobenzoxy-leucyl-leucyl-norvalinal

We have studied specific effects of proteasome inhibition on cell cycle progression. To this end, the protease inhibitors MG115, calpain inhibitor I, and calpain inhibitor II, which display differential inhibitory effects on proteasomes, were used. Cell kinetic studies using bromodeoxyuridine pulse labeling revealed a complete block of G1/S and metaphase transitions and a delayed progression through S phase in cell cultures treated with 54 microM of MG115. Calpain inhibitor I in similar concentrations displayed a fivefold lower effect on cell cycle kinetics. Calpain inhibitor II and MG2M, which is a structural analogue of MG115, had no effect on the cell cycle. The inhibitory effect of MG115 treatment was reversible, because the cell cycle was immediately resumed when the MG115-containing culture medium was replaced by fresh culture medium. Because ubiquitinated proteins accumulated after MG115 treatment, it was confirmed that ubiquitin-dependent protein degradation, and thus proteasomal activity were blocked. By comparison of biochemical and in vitro proteasome inhibition experiments, it was hypothesized that chymotrypsin-like activity of proteasomes may play an important role in cell cycle kinetics.

Topics: Animals; Bromodeoxyuridine; Calpain; Cell Cycle; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Glycoproteins; Humans; Kinetics; Leupeptins; Microscopy, Phase-Contrast; Multienzyme Complexes; Oligopeptides; Protease Inhibitors; Proteasome Endopeptidase Complex; Tumor Cells, Cultured

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

Multiple cell adhesion proteins are up-regulated in vascular endothelial cells in response to TNF alpha and other inflammatory cytokines. This increase in cell adhesion gene expression is thought to require the transcription factor NF-kappa B. Here, we show that peptide aldehyde inhibitors of the proteasome, a multicatalytic protease recently shown to be required for the activation of NF-kappa B, block TNF alpha induction of the leukocyte adhesion molecules E-selectin, VCAM-1, and ICAM-1. Striking functional consequences of this inhibition were observed in analyses of leukocyte-endothelial interactions under defined flow conditions. Lymphocyte attachment to TNF alpha-treated endothelial monolayers was totally blocked, while neutrophil attachment was partially reduced but transmigration was essentially prevented.

Topics: Base Sequence; Calpain; Cell Adhesion; Cell Adhesion Molecules; Cells, Cultured; Cysteine Endopeptidases; E-Selectin; Endothelium, Vascular; Gene Expression; Humans; In Vitro Techniques; Intercellular Adhesion Molecule-1; Interleukin-8; Leukocytes; Leupeptins; Molecular Sequence Data; Multienzyme Complexes; NF-kappa B; Oligodeoxyribonucleotides; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins; RNA, Messenger; Time Factors; Transcription Factor RelB; Transcription Factors; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1