MG132

Abstract: In eukaryotic cells, the ubiquitin–proteasome pathway is the major mechanism for the targeted degradation of proteins with short half-lives. The covalent attachment of ubiquitin to lysine residues of targeted proteins is a signal for the recognition and rapid degradation by the proteasome, a large multi-subunit protease. In this report, we demonstrate that the human estrogen receptor (ER) protein is rapidly degraded in mammalian cells in an estradiol-dependent manner. The treatment of mammalian cells with the proteasome inhibitor MG132 inhibits activity of the proteasome and blocks ER degradation, suggesting that ER protein is turned over through the ubiquitin–proteasome pathway. In addition, we show that in vitro ER degradation depends on ubiquitin-activating E1 enzyme (UBA) and ubiquitin-conjugating E2 enzymes (UBCs), and the proteasome inhibitors MG132 and lactacystin block ER protein degradation in vitro. Furthermore, the UBA/UBCs and proteasome inhibitors promote the accumulation of higher molecular weight forms of ER. The UBA and UBCs, which promote ER degradation in vitro, have no significant effect on human progesterone receptor and human thyroid hormone receptor β proteins.

Abstract: Brassinosteroids (BRs) are a class of steroid hormones essential for normal growth and development in plants. BR signaling involves the cell-surface receptor BRI1, the glycogen synthase kinase-3-like kinase BIN2 as a negative regulator, and nuclear proteins BZR1 and BZR2/BES1 as positive regulators. The interactions among these components remain unclear. Here we report that BRs induce dephosphorylation and accumulation of BZR1 protein. Experiments using a proteasome inhibitor, MG132, suggest that phosphorylation of BZR1 increases its degradation by the proteasome machinery. BIN2 directly interacts with BZR1 in yeast two-hybrid assays, phosphorylates BZR1 in vitro, and negatively regulates BZR1 protein accumulation in vivo. These results strongly suggest that BIN2 phosphorylates BZR1 and targets it for degradation and that BR signaling causes BZR1 dephosphorylation and accumulation by inhibiting BIN2 activity.

Abstract: The accumulation of misfolded proteins in the cytosol leads to increased expression of heat-shock proteins, while accumulation of such proteins in the endoplasmic reticulum (ER) stimulates the expression of many ER resident proteins, most of which function as molecular chaperones. Recently, inhibitors of the proteasome have been identified that can block the rapid degradation of abnormal cytosolic and ER-associated proteins. We therefore tested whether these agents, y causing the accumulation of abnormal proteins, might stimulate the expression of cytosolic heat-shock proteins and/or ER molecular chaperones and thereby induce thermotolerance. Exposure of Madin-Darby canine kidney cells to various proteasome inhibitors, including the peptide aldehydes (MG132, MG115, N-acetyl-leucyl-leucyl-norleucinal) and lactacystin, inhibited the degradation of short-lived proteins and increased markedly the levels of mRNAs encoding cytosolic heat-shock proteins (Hsp70, polyubiquitin) and ER chaperones (BiP, Grp94, ERp72), as shown by Northern blot analysis. However, inhibitors of cysteine proteases (E64), serine proteases (leupeptin), or metalloproteases (1,10-phenanthroline) had no effect on the levels of these mRNAs. The relative efficacies of the peptide aldehyde inhibitors in inducing these mRNAs correlated with their potencies against the proteasome. Furthermore, reduction of the aldehyde group of MG132 decreased its inhibitory effect on proteolysis and largely prevented the induction of these mRNAs. Although treatment with the proteasome inhibitors caused rapid increases in mRNA levels (as early as 2 h after treatment with MG132), the inhibitors did not detectably affect total protein synthesis, total protein secretion, ER morphology, or the retention of ER-lumenal proteins, even after 18 h of treatment. Together, the findings suggest that inhibition of proteasome function induces heat-shock proteins and ER chaperones due to the accumulation of sufficient amounts of abnormal proteins and/or the inhibition of degradation of a key regulatory factor (e.g. heat-shock factor). Since expression of heat-shock proteins can protect cells from thermal injury, we tested whether the proteasome inhibitors might also confer thermotolerance. Treatment of cells with MG132 for as little as 2 h, markedly increased the survival of cells subjected to high temperatures (up to 46°C). Thus, these agents may have applications in protecting against cell injury.

Abstract: Skeletal muscle atrophy and weakness are thought to be stimulated by tumor necrosis factor alpha (TNF-alpha) in a variety of chronic diseases. However, little is known about the direct effects of TNF-alpha on differentiated skeletal muscle cells or the signaling mechanisms involved. We have tested the effects of TNF-alpha on the mouse-derived C2C12 muscle cell line and on primary cultures from rat skeletal muscle. TNF-alpha treatment of differentiated myotubes stimulated time- and concentration-dependent reductions in total protein content and loss of adult myosin heavy chain (MHCf) content; these changes were evident at low TNF-alpha concentrations (1-3 ng/ml) that did not alter muscle DNA content and were not associated with a decrease in MHCf synthesis. TNF-alpha activated binding of nuclear factor kappaB (NF-kappaB) to its targeted DNA sequence and stimulated degradation of I-kappaBalpha, an NF-kappaB inhibitory protein. TNF-alpha stimulated total ubiquitin conjugation whereas a 26S proteasome inhibitor (MG132 10-40 microM) blocked TNF-alpha activation of NF-kappaB. Catalase 1 kU/ml inhibited NF-kappaB activation by TNF-alpha; exogenous hydrogen peroxide 200 microM activated NF-kappaB and stimulated I-kappaBalpha degradation. These data demonstrate that TNF-alpha directly induces skeletal muscle protein loss, that NF-kappaB is rapidly activated by TNF-alpha in differentiated skeletal muscle cells, and that TNF-alpha/NF-kappaB signaling in skeletal muscle is regulated by endogenous reactive oxygen species.

Abstract: Doxorubicin is among the most effective and widely used anticancer drugs in the clinic. However, cardiotoxicity is one of the life-threatening side effects of doxorubicin-based therapy. Dexrazoxane (Zinecard, also known as ICRF-187) has been used in the clinic as a cardioprotectant against doxorubicin cardiotoxicity. The molecular basis for doxorubicin cardiotoxicity and the cardioprotective effect of dexrazoxane, however, is not fully understood. In the present study, we showed that dexrazoxane specifically abolished the DNA damage signal gamma-H2AX induced by doxorubicin, but not camptothecin or hydrogen peroxide, in H9C2 cardiomyocytes. Doxorubicin-induced DNA damage was also specifically abolished by the proteasome inhibitors bortezomib and MG132 and much reduced in top2beta(-/-) mouse embryonic fibroblasts (MEF) compared with TOP2beta(+/+) MEFs, suggesting the involvement of proteasome and DNA topoisomerase IIbeta (Top2beta). Furthermore, in addition to antagonizing Top2 cleavage complex formation, dexrazoxane also induced rapid degradation of Top2beta, which paralleled the reduction of doxorubicin-induced DNA damage. Together, our results suggest that dexrazoxane antagonizes doxorubicin-induced DNA damage through its interference with Top2beta, which could implicate Top2beta in doxorubicin cardiotoxicity. The specific involvement of proteasome and Top2beta in doxorubicin-induced DNA damage is consistent with a model in which proteasomal processing of doxorubicin-induced Top2beta-DNA covalent complexes exposes the Top2beta-concealed DNA double-strand breaks.