Bush K T, Goldberg A L, Nigam S K
Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 1997 Apr 4;272(14):9086-92. doi: 10.1074/jbc.272.14.9086.
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, by 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 degrees C). Thus, these agents may have applications in protecting against cell injury.
错误折叠的蛋白质在胞质溶胶中积累会导致热休克蛋白的表达增加，而此类蛋白质在内质网（ER）中的积累会刺激许多内质网驻留蛋白的表达，其中大多数作为分子伴侣发挥作用。最近，已鉴定出蛋白酶体抑制剂，其可阻断异常胞质和内质网相关蛋白的快速降解。因此，我们测试了这些试剂是否通过导致异常蛋白的积累，可能刺激胞质热休克蛋白和/或内质网分子伴侣的表达，从而诱导热耐受性。将Madin-Darby犬肾细胞暴露于各种蛋白酶体抑制剂，包括肽醛（MG132、MG115、N-乙酰-亮氨酰-亮氨酰-正亮氨酸）和乳胞素，抑制了短命蛋白的降解，并显著增加了编码胞质热休克蛋白（Hsp70、多聚泛素）和内质网伴侣蛋白（BiP、Grp94、ERp72）的mRNA水平，Northern印迹分析表明了这一点。然而，半胱氨酸蛋白酶抑制剂（E64）、丝氨酸蛋白酶抑制剂（亮抑酶肽）或金属蛋白酶抑制剂（1,10-菲咯啉）对这些mRNA水平没有影响。肽醛抑制剂诱导这些mRNA的相对效力与其对蛋白酶体的效力相关。此外，MG132醛基的还原降低了其对蛋白水解的抑制作用，并在很大程度上阻止了这些mRNA的诱导。尽管用蛋白酶体抑制剂处理导致mRNA水平迅速升高（早在用MG132处理后2小时），但即使在处理18小时后，这些抑制剂也未检测到对总蛋白合成、总蛋白分泌内质网形态或内质网腔蛋白保留的影响。总之，这些发现表明蛋白酶体功能的抑制由于足够量的异常蛋白的积累和/或关键调节因子（如热休克因子）降解的抑制而诱导热休克蛋白和内质网伴侣蛋白。由于热休克蛋白的表达可以保护细胞免受热损伤，我们测试了蛋白酶体抑制剂是否也能赋予热耐受性。用MG132处理细胞仅2小时，就显著提高了经受高温（高达46摄氏度）的细胞的存活率。因此，这些试剂可能在预防细胞损伤方面有应用价值。
