Tebbi Ali, Guittet Olivier, Tuphile Karine, Cabrié Aimeric, Lepoivre Michel
From the Université Paris Sud, Institute of Molecular and Cellular Biochemistry and Biophysics, UMR 8619, 91405 Orsay, France, CNRS, 91405 Orsay, France, and Department of Virology, Institut Pasteur, Pathogenesis of Hepatitis B Virus, 75015 Paris, France.
From the Université Paris Sud, Institute of Molecular and Cellular Biochemistry and Biophysics, UMR 8619, 91405 Orsay, France, CNRS, 91405 Orsay, France, and.
J Biol Chem. 2015 May 29;290(22):14077-90. doi: 10.1074/jbc.M115.649640. Epub 2015 Apr 15.
Ribonucleotide reductase (RnR) is a key enzyme synthesizing deoxyribonucleotides for DNA replication and repair. In mammals, the R1 catalytic subunit forms an active complex with either one of the two small subunits R2 and p53R2. Expression of R2 is S phase-specific and required for DNA replication. The p53R2 protein is expressed throughout the cell cycle and in quiescent cells where it provides dNTPs for mitochondrial DNA synthesis. Participation of R2 and p53R2 in DNA repair has also been suggested. In this study, we investigated the fate of the RnR subunits during apoptosis. The p53R2 protein was cleaved in a caspase-dependent manner in K-562 cells treated with inhibitors of the Bcr-Abl oncogenic kinase and in HeLa 229 cells incubated with TNF-α and cycloheximide. The cleavage site was mapped between Asp(342) and Asn(343). Caspase attack released a C-terminal p53R2 peptide of nine residues containing the conserved heptapeptide essential for R1 binding. As a consequence, the cleaved p53R2 protein was inactive. In vitro, purified caspase-3 and -8 could release the C-terminal tail of p53R2. Knocking down these caspases, but not caspase-2, -7, and -10, also inhibited p53R2 cleavage in cells committed to die via the extrinsic death receptor pathway. The R2 subunit was subjected to caspase- and proteasome-dependent proteolysis, which was prevented by siRNA targeting caspase-8. Knocking down caspase-3 was ineffective. Protein R1 was not subjected to degradation. Adding deoxyribonucleosides to restore dNTP pools transiently protected cells from apoptosis. These data identify RnR activity as a prosurvival function inactivated by proteolysis during apoptosis.
核糖核苷酸还原酶（RnR）是一种为DNA复制和修复合成脱氧核糖核苷酸的关键酶。在哺乳动物中，R1催化亚基与两个小亚基R2和p53R2中的任何一个形成活性复合物。R2的表达具有S期特异性，是DNA复制所必需的。p53R2蛋白在整个细胞周期以及静止细胞中均有表达，在这些细胞中它为线粒体DNA合成提供脱氧核苷酸三磷酸（dNTP）。也有人提出R2和p53R2参与DNA修复。在本研究中，我们调查了凋亡过程中RnR亚基的命运。在用Bcr-Abl致癌激酶抑制剂处理的K-562细胞以及用肿瘤坏死因子-α（TNF-α）和环己酰亚胺孵育的HeLa 229细胞中，p53R2蛋白以半胱天冬酶依赖性方式被切割。切割位点位于天冬氨酸（Asp）342和天冬酰胺（Asn）343之间。半胱天冬酶攻击释放出一个九肽的C末端p53R2肽段，该肽段包含与R1结合所必需的保守七肽。因此，被切割的p53R2蛋白无活性。在体外，纯化的半胱天冬酶-3和-8可以释放p53R2的C末端尾巴。敲低这些半胱天冬酶，但不包括半胱天冬酶-2、-7和-10，也能抑制通过外源性死亡受体途径走向死亡的细胞中的p53R2切割。R2亚基经历了半胱天冬酶和蛋白酶体依赖性蛋白水解，而靶向半胱天冬酶-8的小干扰RNA（siRNA）可阻止这种水解。敲低半胱天冬酶-3无效。R1蛋白未发生降解。添加脱氧核苷以恢复dNTP库可短暂保护细胞免于凋亡。这些数据表明RnR活性是一种在凋亡过程中因蛋白水解而失活的促生存功能。
