sq-23377 and lactacystin

Targeting of the cyclin-dependent kinase inhibitor p27(Kip1) for proteolysis has been thought to be mediated by Skp2, the F-box protein component of an SCF ubiquitin ligase complex. Degradation of p27(Kip1) at the G(0)-G(1) transition of the cell cycle has now been shown to proceed normally in Skp2(-/-) lymphocytes, whereas p27(Kip1) proteolysis during S-G(2) phases is impaired in these Skp2-deficient cells. Degradation of p27(Kip1) at the G(0)-G(1) transition was blocked by lactacystin, a specific proteasome inhibitor, suggesting that it is mediated by the ubiquitin-proteasome pathway. The first cell cycle of stimulated Skp2(-/-) lymphocytes appeared normal, but the second cycle was markedly inhibited, presumably as a result of p27(Kip1) accumulation during S-G(2) phases of the first cell cycle. Polyubiquitination of p27(Kip1) in the nucleus is dependent on Skp2 and phosphorylation of p27(Kip1) on threonine 187. However, polyubiquitination activity was also detected in the cytoplasm of Skp2(-/-) cells, even with a threonine 187 --> alanine mutant of p27(Kip1) as substrate. These results suggest that a polyubiquitination activity in the cytoplasm contributes to the early phase of p27(Kip1) degradation in a Skp2-independent manner, thereby promoting cell cycle progression from G(0) to G(1).

Topics: Acetylcysteine; Animals; Cell Cycle Proteins; Cell Fractionation; Cells, Cultured; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Flow Cytometry; G1 Phase; G2 Phase; Ionomycin; Ionophores; Lymphocytes; Mice; Mice, Knockout; Models, Biological; Multienzyme Complexes; Proteasome Endopeptidase Complex; Recombinant Fusion Proteins; S Phase; S-Phase Kinase-Associated Proteins; Tumor Suppressor Proteins; Ubiquitin

To develop a system for continuous evaluation of proteolytic activity in human lens epithelium and to characterize factors of importance for the regulation of proteolytic activity in lens epithelial cells.. Human lens epithelial cells were obtained during cataract surgery. Capsule epithelium specimens consisted of the central parts of the anterior capsule and the underlying lens epithelium. The sample, with the cell-permeable substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin, was placed in a chamber, which was placed in a thermostat-controlled aluminum block. Fluorescence changes were continuously measured by the fiber optics of the luminometer, which was placed 5 mm above the buffer surface.. After administration of substrate to the medium overlying the cells, the substrate was degraded at a relatively slow rate. Approximately 10 picomoles of amino-4-methylcoumarin were formed per minute. A significant increase of proteolytic activity could be observed after application of 1 microM ionomycin or 2 microM thapsigargin. No leakage of lactate dehydrogenase from the cells was observed during these procedures. Basal proteolytic activity was totally inhibited by the proteasome inhibitor lactacystin. Lactacystin also attenuated the response to ionomycin and thapsigargin.. Human lens epithelium responds to increased Ca levels from external or internal stores with an increased proteolytic activity that may be mediated by calpain, by the proteasome, or by both. This calcium-dependent change in proteolytic activity may be of importance in the development of cataract.

Topics: Acetylcysteine; Calcium; Calpain; Cell Membrane Permeability; Cell Survival; Coumarins; Cysteine Endopeptidases; Enzyme Inhibitors; Epithelium; Humans; Ionomycin; L-Lactate Dehydrogenase; Lens, Crystalline; Multienzyme Complexes; Oligopeptides; Proteasome Endopeptidase Complex; Substrate Specificity; Thapsigargin

We characterized kinetic and biochemical changes during glucocorticoid (GC)-induced apoptosis of immature CD8+CD4+ double-positive (DP) thymocytes. A GC analog dexamethasone (Dex) induced rapid apoptotic commitment and a transient up-regulation of the NF-kappaB/RelA-p50-binding activity in DP cells. This required an early activation of proteasome, as judged by the ability of a specific proteasomal inhibitor, lactacystine, to delay apoptosis and to suppress Dex-dependent NF-kappaB activation. Dex-induced apoptotic commitment was preceded by the rapid (3 h) cleavage of both a typical caspase substrate, poly(ADP-ribose) polymerase (PARP), and of nuclear transcription factors AP-1, NF-kappaB p50-p50 and NUR-77. By contrast, phorbol myristate acetate (PMA) and/or ionomycin-induced apoptosis had much slower kinetics, were preceded by an early increase of NF-kappaB/RelA-p50, AP-1 and NUR-77 activities, and were insensitive to proteasome inhibition. Both the transgenic Bcl-2 and zVAD-fmk, an inhibitor of caspases, affected all features of Dex-induced apoptosis in a similar fashion, by inhibiting cell death and PARP cleavage, and by stabilizing AP-1, NF-kappaB p50-p50 and NUR-77 levels. Furthermore, Bcl-2 prevented Dex-induced RelA-p50 activation. However, a higher gene dosage of the transgenic Bcl-2 was required for protection against Dex, compared to the PMA and/or ionomycin-induced apoptosis. These findings highlight the unique mechanistic features of GC-induced apoptosis.

Topics: Acetylcysteine; Animals; Apoptosis; Caspase Inhibitors; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dexamethasone; Female; Ionomycin; Ionophores; Mice; Mice, Inbred C57BL; Multienzyme Complexes; NF-kappa B; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-bcl-2; Tetradecanoylphorbol Acetate; Transcription Factor RelA; Transcription Factors; Transgenes; Up-Regulation