一氧化氮诱导凋亡过程中的蛋白酶激活：聚（ADP - 核糖）聚合酶与U1 - 70kDa裂解的比较

Protease activation during nitric oxide-induced apoptosis: comparison between poly(ADP-ribose) polymerase and U1-70kDa cleavage.

作者信息

Messmer U K, Reimer D M, Brüne B

机构信息

University of Erlangen-Nürnberg, Faculty of Medicine, Department of Medicine IV, Erlangen, Germany.

出版信息

Eur J Pharmacol. 1998 May 22;349(2-3):333-43. doi: 10.1016/s0014-2999(98)00189-7.

DOI:10.1016/s0014-2999(98)00189-7

PMID:9671115

Abstract

Nitric oxide (NO) promotes apoptotic cell death in the mouse macrophage cell line RAW 264.7 and in the human promyelocytic leukaemia cell line U937, which exemplifies p53-dependent and p53-independent executive death pathways. Here, we followed the cleavage of two caspase substrates during NO-intoxication, assaying poly(ADP-ribose) polymerase and U1-70kDa small ribonucleoprotein (U1-70kDa) degradation. By using pharmacological inhibitors, we found that Z-aspartyl-2,6-dichlorobenzoyloxymethylketone (Z-Asp-CH2-DCB; 100 microM), a caspase-like protease inhibitor, completely blocked S-nitrosoglutathione (GSNO)-induced apoptosis in both RAW 264.7 and U937 cells (IC50 = 50 microM for RAW 264.7 macrophages vs. IC50 = 33 microM for U937 cells). Notably, a characterized caspase-3 (Ac-DEVD-CHO) inhibitor left NO-induced DNA fragmentation and the appearance of an apoptotic morphology unaltered, although completely blocking caspase-3 activity. However, Z-Asp-CH2-DCB suppressed protease-mediated U1-70kDa cleavage and DNA fragmentation in parallel. In contrast, poly(ADP-ribose) polymerase cleavage in U937 cells was only delayed by Z-Asp-CH2-DCB, while poly(ADP-ribose) polymerase digestion in RAW 264.7 macrophages proceeded unaltered. We further compared U1-70kDa and poly(ADP-ribose) polymerase cleavage in stably Bcl-2 transfected RAW 264.7 macrophages. Rbcl2-2, a Bcl-2 overexpressing clone, suppressed DNA fragmentation and U1-70kDa digestion in response to GSNO, although allowing delayed but complete poly(ADP-ribose) polymerase degradation. Conclusively, poly(ADP-ribose) polymerase cleavage not causatively coincided with the appearance of other apoptotic parameters. Our results suggest that NO-induced apoptosis demands a Z-Asp-CH2-DCB inhibitable caspase activity, most likely distinct from caspase-3 and caspase-1. NO-mediated executive apoptotic signaling results in U1-70kDa and poly(ADP-ribose) polymerase cleavage. Whereas U1-70kDa digestion closely correlates to the occurrence of apoptotic parameters such as DNA fragmentation or an apoptotic morphology, poly(ADP-ribose) polymerase-breakdown does not.

摘要

一氧化氮（NO）可促进小鼠巨噬细胞系RAW 264.7和人早幼粒细胞白血病细胞系U937的凋亡性细胞死亡，这两种细胞系分别代表了p53依赖性和p53非依赖性的程序性死亡途径。在此，我们追踪了NO中毒过程中两种半胱天冬酶底物的裂解情况，检测了聚（ADP-核糖）聚合酶和U1-70kDa小核糖核蛋白（U1-70kDa）的降解。通过使用药理学抑制剂，我们发现Z-天冬氨酰-2,6-二氯苄氧基甲基酮（Z-Asp-CH2-DCB；100 microM），一种类半胱天冬酶蛋白酶抑制剂，能完全阻断RAW 264.7和U937细胞中S-亚硝基谷胱甘肽（GSNO）诱导的凋亡（RAW 264.7巨噬细胞的IC50 = 50 microM，而U937细胞的IC50 = 33 microM）。值得注意的是，一种已鉴定的半胱天冬酶-3（Ac-DEVD-CHO）抑制剂虽然完全阻断了半胱天冬酶-3的活性，但并未改变NO诱导的DNA片段化及凋亡形态的出现。然而，Z-Asp-CH2-DCB能同时抑制蛋白酶介导的U1-70kDa裂解和DNA片段化。相反，Z-Asp-CH2-DCB仅延迟了U937细胞中聚（ADP-核糖）聚合酶的裂解，而RAW 264.7巨噬细胞中聚（ADP-核糖）聚合酶的降解过程未受影响。我们进一步比较了稳定转染Bcl-2的RAW 264.7巨噬细胞中U1-70kDa和聚（ADP-核糖）聚合酶的裂解情况。Rbcl2-2，一个过表达Bcl-2的克隆，能抑制GSNO诱导的DNA片段化和U1-70kDa降解，尽管允许聚（ADP-核糖）聚合酶的降解延迟但完全发生。总之，聚（ADP-核糖）聚合酶的裂解与其他凋亡参数的出现并无因果关联。我们的结果表明，NO诱导的凋亡需要一种可被Z-Asp-CH2-DCB抑制的半胱天冬酶活性，很可能不同于半胱天冬酶-3和半胱天冬酶-1。NO介导的程序性凋亡信号传导导致U1-70kDa和聚（ADP-核糖）聚合酶的裂解。虽然U1-70kDa的降解与DNA片段化或凋亡形态等凋亡参数的出现密切相关，但聚（ADP-核糖）聚合酶的降解并非如此。