anisomycin and benzyloxycarbonylleucyl-leucyl-leucine-aldehyde

Mutations in PINK1 (PTEN-induced putative kinase 1) are associated with autosomal recessive early-onset Parkinson's disease. Full-length PINK1 (PINK1-l) has been extensively studied in mitophagy; however, the functions of the short form of PINK1 (PINK1-s) remain poorly understood. Here, we report that PINK1-s is recruited to ribosome fractions after short-term inhibition of proteasomes. The expression of PINK1-s greatly inhibits protein synthesis even without proteasomal stress. Mechanistically, PINK1-s phosphorylates the translation elongation factor eEF1A1 during proteasome inhibition. The expression of the phosphorylation mimic mutation eEF1A1S396E rescues protein synthesis defects and cell viability caused by PINK1 knockout. These findings implicate an important role for PINK1-s in protecting cells against proteasome stress through inhibiting protein synthesis.

Topics: Amino Acid Sequence; Anisomycin; Cell Line; Cell Survival; Cysteine Proteinase Inhibitors; Epithelial Cells; HEK293 Cells; Humans; Leupeptins; Peptide Elongation Factor 1; Phosphorylation; Proteasome Endopeptidase Complex; Protein Biosynthesis; Protein Kinases; Protein Processing, Post-Translational; Protein Synthesis Inhibitors; Proteolysis; Ribosomes; RNA, Small Interfering

We investigated the in vivo role of protein degradation during intermediate (ITM) and long-term memory (LTM) in Aplysia using an operant learning paradigm. The proteasome inhibitor MG-132 inhibited the induction and molecular consolidation of LTM with no effect on ITM. Remarkably, maintenance of steady-state protein levels through inhibition of protein synthesis using either anisomycin or rapamycin in conjunction with proteasome inhibition permitted the formation of robust 24 h LTM. Our studies suggest a primary role for proteasomal activity in facilitation of gene transcription for LTM and raise the possibility that synaptic mechanisms are sufficient to sustain 24 h memory.

Topics: Analysis of Variance; Animals; Anisomycin; Aplysia; Conditioning, Operant; Cysteine Proteinase Inhibitors; Leupeptins; Memory, Long-Term; Proteasome Endopeptidase Complex; Time Factors

Expression of synaptic plasticity involves the translation of mRNA into protein and, probably, active protein degradation via the proteasome pathway. Here, we report on the rapid activation of synthesis and degradation of a probe protein with the induction of long-term potentiation (LTP) in the hippocampal Schaffer collateral CA1 pathway. The proteasome inhibitor MG132 significantly reduced the field EPSP slope potentiation and LTP maintenance without acutely affecting basal synaptic transmission. To visualize protein dynamics, CA1 pyramidal cells of hippocampal slices were transfected with Semliki Forest virus particles expressing a recombinant RNA. This RNA contained the coding sequence for a degradable green fluorescence protein with a nuclear localization signal (NLS-d1EGFP) followed by a 3'- untranslated region dendritic targeting sequence. NLS-d1EGFP fluorescence remained stable in the low-frequency test stimulation but increased with LTP induction in the cell body and in most dendritic compartments of CA1 neurons. Applying anisomycin, a protein synthesis inhibitor, caused NLS-d1EGFP levels to decline; a proteasome inhibitor MG132 reversed this effect. In the presence of anisomycin, LTP induction accelerated the degradation of NLS-d1EGFP. When both inhibitors were present, NLS-d1EGFP levels remained unaffected by LTP induction. Moreover, LTP-induced acceleration of NLS-d1EGFP synthesis was blocked by rapamycin, which is consistent with the involvement of dendritic mammalian target of rapamycin in LTP-triggered translational activity. Our results clearly demonstrate that LTP induction not only leads to a rapid increase in the rate of protein synthesis but also accelerates protein degradation via the proteasome system.

Topics: 2-Amino-5-phosphonovalerate; Animals; Animals, Newborn; Anisomycin; CHO Cells; Cloning, Molecular; Cricetinae; Cricetulus; Cysteine Proteinase Inhibitors; Diagnostic Imaging; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Green Fluorescent Proteins; Hippocampus; In Vitro Techniques; Leupeptins; Long-Term Potentiation; Male; Membrane Glycoproteins; Microtubule-Associated Proteins; Neurons; Nuclear Localization Signals; Patch-Clamp Techniques; Perforant Pathway; Protein Biosynthesis; Protein Synthesis Inhibitors; Proteins; Rats; Rats, Wistar; Sirolimus; Synapses; Time Factors; Transfection; Viral Envelope Proteins

Genetic deletion of fragile X mental retardation protein (FMRP) has been shown to enhance mGluR-dependent long-term depression (LTD). Herein, we demonstrate that mGluR-LTD induces a transient, translation-dependent increase in FMRP that is rapidly degraded by the ubiquitin-proteasome pathway. Moreover, proteasome inhibitors abolished mGluR-LTD, and LTD was absent in mice that overexpress human FMRP. Neither translation nor proteasome inhibitors blocked the augmentation of mGluR-LTD in FMRP-deficient mice. In addition, mGluR-LTD is associated with rapid increases in the protein levels of FMRP target mRNAs in wild-type mice. Interestingly, the basal levels of these proteins were elevated and their synthesis was improperly regulated during mGluR-LTD in FMRP-deficient mice. Our findings indicate that hippocampal mGluR-LTD requires the rapid synthesis and degradation of FMRP and that mGluR-LTD triggers the synthesis of FMRP binding mRNAs. These findings indicate that the translation, ubiquitination, and proteolysis of FMRP functions as a dynamic regulatory system for controlling synaptic plasticity.

Topics: Animals; Animals, Newborn; Anisomycin; Benzoates; Blotting, Western; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Drug Interactions; Excitatory Amino Acid Antagonists; Fluorescent Antibody Technique; Fragile X Mental Retardation Protein; Glycine; In Vitro Techniques; Leupeptins; Long-Term Synaptic Depression; Male; Methoxyhydroxyphenylglycol; Mice; Mice, Knockout; Microtubule-Associated Proteins; Models, Biological; Proteasome Endopeptidase Complex; Protein Biosynthesis; Protein Synthesis Inhibitors; Pyridines; Receptors, Metabotropic Glutamate; RNA, Messenger; Signal Transduction

Exposure of the human breast epithelial cell line MCF10A to > or = 1 microg/ml cycloheximide (CHX)-induced accumulations of CYP1A1 mRNA 6-fold greater than that achieved with only 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Cotreatment with CHX and TCDD caused superinduction of CYP1A1 with accumulations of CYP1A1 mRNA 30-fold greater than that achieved with only TCDD. Similar results were obtained with the protein translation inhibitors anisomycin (ANS) and puromycin (PUR). Intra- and interinhibitor comparisons of dose/concentration response curves demonstrated the absence of a quantitative relationship between [3H]leucine incorporation and CYP1A1 induction/superinduction. The inducing/superinducing activities of CHX were suppressed by coincubation with the aryl hydrocarbon receptor (AhR) antagonists alpha-naphthoflavone and 3'-methoxy-4'-nitroflavone (PD168641). Electrophoretic mobility shift assays demonstrated that nuclear extracts from CHX-treated and CHX + TCDD cotreated cultures formed approximately 58 and approximately 340% of the AhR/DNA complexes obtained with TCDD-treated cultures, respectively. In contrast, rat liver extracts did not form AhR/DNA complexes after in vitro transformation with CHX. AhR turnover in TCDD-treated hepatoma 1c1c7 cultures was suppressed by cotreatment with CHX. In contrast, CHX or ANS treatment of MCF10A cultures induced AhR loss and enhanced AhR loss in cultures cotreated with TCDD. Cotreatment with N-benzoyloxycarbonyl-(Z)-Leu-Leu-leucinal (MG132) but not leptomycin B suppressed AhR loss. Hence, in MCF10A cells, CHX is not an AhR agonist but can superinduce CYP1A1 via an AhR-dependent mechanism; CYP1A1 superinduction by translation inhibitors is neither quantitatively related to effects on protein synthesis nor due to a generalized prevention of AhR proteolysis, and proteasome-mediated degradation of the activated AhR can occur in the nucleus.

Topics: Anisomycin; Cycloheximide; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1A2; DNA; Enzyme Induction; Fatty Acids, Unsaturated; Humans; Leucine; Leupeptins; Polychlorinated Dibenzodioxins; Proteasome Endopeptidase Complex; Protein Biosynthesis; Puromycin; Receptors, Aryl Hydrocarbon; Tritium; Tumor Cells, Cultured

Reactive oxygen species such as hydrogen peroxide (H(2)O(2)) have taken center stage as bona fide second messengers in various signaling pathways. Here, we report the synthesis, metabolic fate, and effectiveness in modulating such pathways of a Tat-catalase conjugate. Incubation of L2 cells with Tat-catalase greatly increased cell-associated enzymatic activity, reaching close to a plateau by 30 min. The cell-associated catalase activity and antibody-detectable Tat-derivatives declined over time after changing medium, although still remaining at significantly higher levels than baseline even at 4h. While most cell-associated Tat-catalase was apparently tightly attached to the cell surface, a small fraction entered the cells as the proteasome inhibitor MG-132 slightly prevented the disappearance of the enzyme. Tat-catalase, either membrane-bound or intracellular, but not native catalase, inhibited serum-induced Elk phosphorylation and anisomycin- and/or MG-132-induced ERK phosphorylation, suggesting the involvement of H(2)O(2). Thus, Tat-catalase should be a useful tool to dissect H(2)O(2)-dependent events in signaling pathways.

Topics: Animals; Anisomycin; Catalase; Cell Line; Electrophoresis, Polyacrylamide Gel; Gene Products, tat; Hydrogen Peroxide; Kinetics; Leupeptins; Lung; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Peptides; Phosphorylation; Rats; Recombinant Fusion Proteins; Signal Transduction; Time Factors

Interactions between proteasome and cyclin-dependent kinase inhibitors have been examined in human leukemia cells in relation to induction of apoptosis. Simultaneous exposure (24 h) of U937 myelomonocytic leukemia cells to 100 nM flavopiridol and 300 nM MG-132 resulted in a marked increase in mitochondrial injury (cytochrome c, Smac/DIABLO release, loss of deltaPsi(m)), caspase activation, and synergistic induction of cell death, accompanied by a marked decrease in clonogenic potential. Similar effects were observed with other proteasome inhibitors (e.g., Bortezomib (VELCADE trade mark bortezomib or injection), lactacystin, LLnL) and cyclin-dependent kinase inhibitors (e.g., roscovitine), as well as other leukemia cell types (e.g., HL-60, Jurkat, Raji). In U937 cells, synergistic interactions between MG-132 and flavopiridol were associated with multiple perturbations in expression/activation of signaling- and survival-related proteins, including downregulation of XIAP and Mcl-1, activation of JNK and p34(cdc2), and diminished expression of p21(CIP1). The lethal effects of MG-132/flavopiridol were not reduced in leukemic cells ectopically expressing Bcl-2, but were partially attenuated in cells ectopically expressing dominant-negative caspase-8 or CrmA. Flavopiridol/proteasome inhibitor-mediated lethality was also significantly diminished by agents and siRNA blocking JNK activation. Lastly, coadministration of MG-132 with flavopiridol resulted in diminished DNA binding of NF-kappaB. Notably, pharmacologic interruption of the NF-kappaB pathway (e.g., by BAY 11-7082, PDTC, or SN-50) or molecular dysregulation of NF-kappaB (i.e., in cells ectopically expressing an IkappaBalpha super-repressor) mimicked the actions of proteasome inhibitors in promoting flavopiridol-induced mitochondrial injury, JNK activation, and apoptosis. Together, these findings indicate that proteasome inhibitors strikingly lower the apoptotic threshold of leukemic cells exposed to pharmacologic CDK inhibitors, and suggest that interruption of the NF-kappaB cytoprotective pathway and JNK activation both play key roles in this phenomenon. They also raise the possibility that combining proteasome and CDK inhibitors could represent a novel antileukemic strategy.

Topics: Anisomycin; Antineoplastic Agents; Apoptosis; Blast Crisis; Boronic Acids; Bortezomib; Cyclin-Dependent Kinases; Cysteine Endopeptidases; Enzyme Inhibitors; Flavonoids; Gene Expression Regulation, Neoplastic; HL-60 Cells; Humans; JNK Mitogen-Activated Protein Kinases; Jurkat Cells; Leukemia, Myeloid, Acute; Leupeptins; Mitogen-Activated Protein Kinases; Multienzyme Complexes; NF-kappa B; Piperidines; Proteasome Endopeptidase Complex; Pyrazines; Tumor Cells, Cultured; U937 Cells

The role of c-Jun NH2-terminal kinase (JNK) signaling cascade in the stress-inducible phosphorylation of heat shock factor 1 (HSF1) was investigated using known agonists and antagonists of JNK. We showed that treatment of HeLa cells with MG132, a proteasome inhibitor and known INK activator, caused the transcriptional activation domain of HSF1 to be targeted and phosphorylated by JNK2 in vivo. Dose-response and time course studies of the effects of heat shock and anisomycin treatment showed a close correlation of the activation of JNK and hyperphosphorylation of HSF1. SB203580 inhibited INK at the 100 microM concentration and significantly reduced the amount of hyperphosphorylated HSF1 upon heat shock or anisomycin treatment. SB203580 and dominant-negative JNK suppress hsp70 promoter-driven reporter gene expression selectively at 45 degrees C but not at 42 degrees C heat stress, suggesting that JNK would be preferentially associated with the protective heat shock response against severe heat stress. The possibility that JNK-mediated phosphorylation of HSF1 may selectively stabilize the HSF1 protein and confers protection to cells under conditions of severe stress is discussed.

Topics: Animals; Anisomycin; Binding Sites; Cysteine Endopeptidases; DNA-Binding Proteins; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation; Heat Shock Transcription Factors; HeLa Cells; Hot Temperature; HSP70 Heat-Shock Proteins; Humans; Imidazoles; Isoenzymes; JNK Mitogen-Activated Protein Kinases; Leupeptins; Mitogen-Activated Protein Kinase 9; Mitogen-Activated Protein Kinases; Multienzyme Complexes; Phosphorylation; Promoter Regions, Genetic; Protease Inhibitors; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Protein Structure, Tertiary; Pyridines; Rabbits; Recombinant Fusion Proteins; Signal Transduction; Stress, Physiological; Transcription Factors; Transcriptional Activation; Transfection

The p53 homologue p73 efficiently activates p53-responsive genes. The well documented over-expression of p73 spliced forms in a wide variety of tumor types promoted us to elucidate the mechanisms underlying p73-mediated transcription. Using the luciferase reporter gene driven by Mdm2-minimal promoter in p53 null cells, we demonstrate that the weak transcriptional activity mediated by p73alpha was increased by the mutant form p73beta292, which by itself is transcriptionally inactive. Similarly, cooperation between p73beta and an inactive form of p73alpha increased p73beta-mediated transcriptional activities. Conversely, p73beta elicited a silencing effect on a gain of function mutant, p53(281), which by itself mediated efficient transactivation of the MDR promoter. Neither anisomycin nor actinomycin D altered p73-mediated transcriptional activities, whereas sorbitol profoundly inhibited them through a rapid proteasome-dependent degradation of p73. Our observations point to plausible scenarios in which p73, through cooperation between p73 spliced forms and suppression of gain of function mutant p53 may elicit changes in the transcription of p53 target genes that play key roles in cell growth and death.

Topics: Animals; Anisomycin; Cell Line; Dactinomycin; DNA-Binding Proteins; Fibroblasts; Gene Expression Regulation; Genes, p53; Genes, Reporter; Genes, Synthetic; Genes, Tumor Suppressor; Humans; Hypertonic Solutions; Leupeptins; Mice; Nuclear Proteins; Nucleic Acid Synthesis Inhibitors; Promoter Regions, Genetic; Protein Isoforms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Recombinant Fusion Proteins; RNA Splicing; Sorbitol; Transcriptional Activation; Tumor Protein p73; Tumor Suppressor Protein p53; Tumor Suppressor Proteins