Robertson J D, Datta K, Biswal S S, Kehrer J P
Division of Pharmacology, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712-1074, USA.
Biochem J. 1999 Dec 1;344 Pt 2(Pt 2):477-85.
Recent evidence supports a role for heat-shock protein 70 (hsp70) and the 26 S proteasome in regulating apoptosis, although the precise nature of their involvement is not known. In the present study, control and Bcl-x(L)-overexpressing, interleukin-3-dependent FL5.12 cell lines were treated with the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132). Basal proteasome activity appeared to be approximately 30% lower in bcl-x(L) cells compared with control cells using a substrate for the chymotrypsin-like activity. However, no difference in proteasome activity was detected using substrates for the trypsin-like or peptidylglutamyl peptide-hydrolysing activities. In addition, protein levels of the 20 S proteasome beta-subunit, as determined by Western blot analyses, were similar in control and bcl-x(L) cells, leading to the conclusion that proteasome activities were the same in these two cell lines. At 24 h after treatment with 500 nM MG132, apoptosis in bcl-x(L) cells (22%) was less than that observed in control cells (34%). Concomitantly, caspase activity in control cells, as assessed by N-acetyl-l-aspartyl-l-glutamyl-l-valyl-l-aspartyl-7-amino-4-methylcou marin (Ac-DEVD-AMC), was twice that observed in bcl-x(L) cells. By 48 h after MG132 treatment, apoptosis and caspase activity in bcl-x(L) cells were similar to those observed in control cells at 24 h. Proteasome inhibition stimulated increases in hsp70 protein levels in control and bcl-x(L) cells by 12 h, although the maximal increases found in bcl-x(L) cells were less. Blocking this induction with hsp70 antisense oligonucleotides potentiated apoptosis after treatment with MG132. Inhibiting caspase activity with a broad-spectrum caspase inhibitor, t-butoxycarbonyl-Asp(OMe)-fluoromethyl ketone, prevented MG132-induced apoptosis. The more specific caspase-3 inhibitor, Ac-DEVD-aldehyde, afforded less protection, although both inhibitors completely inhibited Ac-DEVD-AMC cleavage. These data indicate that both hsp70 and Bcl-x(L) provide some protection against proteasome inhibitor-induced apoptosis.
最近的证据支持热休克蛋白70（hsp70）和26S蛋白酶体在调节细胞凋亡中起作用，尽管它们参与的具体性质尚不清楚。在本研究中，用蛋白酶体抑制剂N-苄氧羰基（Z）-亮氨酰-亮氨酰-亮氨醛（MG132）处理对照细胞和过表达Bcl-x（L）的白细胞介素-3依赖性FL5.12细胞系。使用针对胰凝乳蛋白酶样活性的底物，与对照细胞相比，bcl-x（L）细胞中的基础蛋白酶体活性似乎低约30%。然而，使用针对胰蛋白酶样或肽基谷氨酰肽水解活性的底物未检测到蛋白酶体活性的差异。此外，通过蛋白质印迹分析测定，20S蛋白酶体β亚基的蛋白质水平在对照细胞和bcl-x（L）细胞中相似，从而得出这两种细胞系中蛋白酶体活性相同的结论。用500 nM MG132处理24小时后，bcl-x（L）细胞中的细胞凋亡率（22%）低于对照细胞（34%）。同时，通过N-乙酰-L-天冬氨酰-L-谷氨酰-L-缬氨酰-L-天冬氨酰-7-氨基-4-甲基香豆素（Ac-DEVD-AMC）评估，对照细胞中的半胱天冬酶活性是bcl-x（L）细胞中的两倍。MG132处理48小时后，bcl-x（L）细胞中的细胞凋亡和半胱天冬酶活性与24小时时对照细胞中观察到的相似。蛋白酶体抑制在12小时时刺激对照细胞和bcl-x（L）细胞中hsp70蛋白水平升高，尽管在bcl-x（L）细胞中发现的最大升高幅度较小。用hsp70反义寡核苷酸阻断这种诱导可增强MG132处理后的细胞凋亡。用广谱半胱天冬酶抑制剂叔丁氧羰基-天冬氨酸（OMe）-氟甲基酮抑制半胱天冬酶活性可预防MG132诱导的细胞凋亡。更特异的半胱天冬酶-3抑制剂Ac-DEVD-醛提供的保护较少，尽管两种抑制剂都完全抑制了Ac-DEVD-AMC的切割。这些数据表明，hsp70和Bcl-x（L）都对蛋白酶体抑制剂诱导的细胞凋亡提供了一定的保护作用。
