benzyloxycarbonylleucyl-leucyl-leucine-aldehyde and calpain-inhibitor-2

Gunn rat is a hyperbilirubinemic rat strain that is inherently deficient in the activity of UDP-glucuronosyltransferase form 1A1 (UGT1A1). A premature termination codon is predicted to produce truncated UGT1 proteins that lack the COOH-terminal 116 amino acids in Gunn rat. Pulse-chase experiments using primary cell cultures showed that the truncated UGT1A1 protein in Gunn rat hepatocytes was synthesized similarly to wild-type UGT1A1 protein in normal Wistar rat hepatocytes. However, the truncated UGT1A1 protein was degraded rapidly with a half-life of about 50 min, whereas the wild-type UGT1A1 protein had a much longer half-life of about 10 h. The rapid degradation of truncated UGT1A1 protein was inhibited partially but not completely by treating Gunn rat hepatocytes with proteasome inhibitors such as carbobenzoxy-Leu-Leu-leucinal and lactacystin. By contrast, neither the lysosomal cysteine protease inhibitor nor the calpain inhibitor slowed the degradation. Our findings show that the absence of UGT1 protein from Gunn rat hepatocytes is due to rapid degradation of the truncated UGT1 protein by the proteasome and elucidate the molecular basis underlying the deficiency in bilirubin glucuronidation.

Topics: Acetylcysteine; Animals; Cells, Cultured; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Glucuronosyltransferase; Glycoproteins; Half-Life; Hepatocytes; Leupeptins; Male; Monosaccharide Transport Proteins; Multienzyme Complexes; Mutation; Oligopeptides; Proteasome Endopeptidase Complex; Rats; Rats, Gunn; Rats, Wistar; RNA, Messenger; Subcellular Fractions

Recently we found a clearly reduced basal level of wt p53 protein in PARP-deficient cells. Interestingly, PARP deficiency affected only regularly spliced (RS) wt p53. No significant difference of the p53 transcription rate was observed between wt and PARP-lacking cells. To clarify whether the reduction of RS p53 protein is due to a lower translation rate or rather to its instability in the absence of functional PARP, we investigated the effect of the inhibition of proteasome activity and nuclear export on the p53 level. The p53 half-life was approximately eight-fold decreased in PARP-lacking cells. Surprisingly, treatment with three proteasome inhibitors increased RS p53 in normal but not in PARP-deficient cells. However, the inhibition of nuclear export resulted in a considerable accumulation of RS p53 in the latter. Therefore, we decided to increase concentrations of the inhibitors. Their higher concentrations strongly affected viability of normal, but not of PARP-deficient cells, about 70% of MEFs died. Interestingly, higher concentrations of proteasome inhibitors resulted in the appearance of RS p53 in PARP-lacking fibroblasts. Reconstitution of PARP-deficient cells with PARP restored the normal susceptibility to proteasome inhibitors thereby unequivocally demonstrating that the enhanced cytotoxicity of proteasome inhibitors and their action on p53 level depends on the presence of functional PARP.

Topics: Acetylcysteine; Animals; Cell Line; Cell Nucleus; Cell Survival; Cells, Cultured; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Fatty Acids, Unsaturated; Fibroblasts; Fluorescent Dyes; Humans; Immunoblotting; Indoles; Leupeptins; Mice; Mice, Knockout; Microscopy, Fluorescence; Multienzyme Complexes; Oligopeptides; Phenotype; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proteasome Endopeptidase Complex; Proteins; Time Factors; Tumor Suppressor Protein p53

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), the key regulatory enzyme in the mevalonate (MVA) pathway, is rapidly degraded in mammalian cells supplemented with sterols or MVA. This accelerated turnover was blocked by N-acetyl-leucyl-leucyl-norleucinal (ALLN), MG-132, and lactacystin, and to a lesser extent by N-acetyl-leucyl-leucyl-methional (ALLM), indicating the involvement of the 26 S proteasome. Proteasome inhibition led to enhanced accumulation of high molecular weight polyubiquitin conjugates of HMGR and of HMGal, a chimera between the membrane domain of HMGR and beta-galactosidase. Importantly, increased amounts of polyubiquitinated HMGR and HMGal were observed upon treating cells with sterols or MVA. Cycloheximide inhibited the sterol-stimulated degradation of HMGR concomitantly with a marked reduction in polyubiquitination of the enzyme. Inhibition of squalene synthase with zaragozic acid blocked the MVA- but not sterol-stimulated ubiquitination and degradation of HMGR. Thus, similar to yeast, the ubiquitin-proteasome pathway is involved in the metabolically regulated turnover of mammalian HMGR. Yet, the data indicate divergence between yeast and mammals and suggest distinct roles for sterol and nonsterol metabolic signals in the regulated ubiquitination and degradation of mammalian HMGR.

Topics: Animals; beta-Galactosidase; Bridged Bicyclo Compounds, Heterocyclic; Cell Line; Cholesterol; Cricetinae; Cycloheximide; Cysteine Proteinase Inhibitors; Farnesyl-Diphosphate Farnesyltransferase; Humans; Hydroxycholesterols; Hydroxymethylglutaryl CoA Reductases; Leupeptins; Lovastatin; Oligopeptides; Peptide Hydrolases; Precipitin Tests; Proteasome Endopeptidase Complex; Recombinant Fusion Proteins; Tricarboxylic Acids; Ubiquitins

Proteasomes interact with a variety of macromolecular ligands that modulate their ability to degrade peptide and protein substrates. The effector PA28 increases the peptidase activities of proteasomes whereas HSP90 and alpha-crystallin inhibit a peptide-hydrolyzing activity. Four monoclonal antibodies were used as probes to detect conformational changes of proteasome subunits. Conformational changes in alpha- or beta-subunits were found upon binding PA28, HSP90, alpha-crystallin, and the substrate casein but not with the peptide substrate analogs calpain inhibitor 1 (Ac-Leu-Leu-norleucinal), calpain inhibitor 2 (Ac-Leu-Leu-methioninal), or MG 132 (N-Cbz-Leu-Leu-leucinal).

Topics: Animals; Antibodies, Monoclonal; Antibody Affinity; Binding Sites, Antibody; Caseins; Crystallins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme-Linked Immunosorbent Assay; Glycoproteins; HSP90 Heat-Shock Proteins; Humans; Leupeptins; Ligands; Multienzyme Complexes; Muscle Proteins; Oligopeptides; Proteasome Endopeptidase Complex; Protein Conformation; Proteins

The transcription factors p53 and E2F-1 play important roles in the control of cell cycle progression. In transient transfection experiments, expression of E2F-1, other E2F family members, or p53 squelched transcription from cotransfected plasmids in a dose-dependent manner. Although the proteasome inhibitors MG-132 and lactacystin markedly increased the level of expression of E2F-1 and p53, these inhibitors completely alleviated squelching by both proteins. Several observations indicate MG-132 alleviates squelching by influencing the conformation of newly synthesized p53 and E2F-1:MG-132 increased the fraction of wild type p53 bound by a monoclonal antibody which preferentially recognizes mutant conformers of p53, increased binding of hsp70 to p53 and inhibited nuclear accumulation of both p53 and E2F-1, but not the pocket protein p107. The protease inhibitors ALLN and ALLM did not influence expression of E2F-1 or p53, nor did they alleviate squelching by either transcription factor. Because MG-132 and lactacycstin are highly specific inhibitors of the proteasome protease, our results suggest that the proteasome influences post-translational processes involved in proper folding and cytoplasmic clearing of E2F-1 and p53.

Topics: Acetylcysteine; Animals; beta-Galactosidase; Blotting, Northern; Carrier Proteins; Cell Cycle Proteins; CHO Cells; Cricetinae; Cycloheximide; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; Leupeptins; Oligopeptides; Protein Synthesis Inhibitors; Retinoblastoma-Binding Protein 1; RNA, Messenger; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transfection; Tumor Suppressor Protein p53; Ubiquitins

The molecular components of the quality control system that rapidly degrades abnormal membrane and secretory proteins have not been identified. The cystic fibrosis transmembrane conductance regulator (CFTR) is an integral membrane protein to which this quality control is stringently applied; approximately 75% of the wild-type precursor and 100% of the delta F508 CFTR variant found in most CF patients are rapidly degraded before exiting from the ER. We now show that this ER degradation is sensitive to inhibitors of the cytosolic proteasome, including lactacystin and certain peptide aldehydes. One of the latter compounds, MG-132, also completely blocks the ATP-dependent conversion of the wild-type precursor to the native folded form that enables escape from degradation. Hence, CFTR and presumably other intrinsic membrane proteins are substrates for proteasomal degradation during their maturation within the ER.

Topics: Acetylcysteine; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; CHO Cells; Cricetinae; Cysteine Endopeptidases; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Endopeptidases; Endoplasmic Reticulum; Humans; Leupeptins; Multienzyme Complexes; Oligopeptides; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Recombinant Fusion Proteins; Ubiquitins