Mouse Sir2 homolog SIRT6 is a nuclear ADP-ribosyltransferase
Mouse Sir2 homolog SIRT6 is a nuclear ADP-ribosyltransferase
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DOI：
10.1074/jbc.m413296200
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发表时间：
2005-06-03
影响因子：
4.8
通讯作者：
Guarente, L
中科院分区：
文献类型：
--
作者：
Liszt, G;Ford, E;Guarente, L
nad依赖性蛋白去乙酰化酶Sir2家族成员调节多种细胞过程，包括衰老、基因沉默和细胞分化。在这里，我们报告了遥远的哺乳动物sirr2同源物SIRT6是一个广泛表达的，主要是核蛋白。胚胎样本和多个成年组织的Northern分析显示，小鼠SIRT6 (mSIRT6) mRNA在第11天达到峰值，并在所有8个组织中持续到成年。在蛋白质水平上，mSIRT6在相同的8种组织类型中很容易检测到，其中肌肉、大脑和心脏的水平最高。利用C端和N端绿色荧光蛋白融合蛋白进行的亚细胞定位研究表明，mSIRT6-绿色荧光蛋白主要是核蛋白。使用针对两种不同mSIRT6表位的抗体的间接免疫荧光证实内源性mSIRT6也主要是核。与之前的研究结果一致，我们在mSIRT6制剂中未观察到任何NAD（+）依赖性蛋白去乙酰化酶活性。然而，纯化的重组mSIRT6确实催化了放射性标记从[P-32] NAD向mSIRT6的强烈转移。该反应需要蛋白催化核心中的两个高度保守的残基。这个反应很可能是单ADP-核糖基化，因为只有修饰后的蛋白质才能被单ADP-核糖特异性抗体识别。令人惊讶的是，我们观察到该反应的催化机制是分子内的，mSIRT6的单个分子指导它们自己的修饰。这些结果首次鉴定了系统发育分类为IV的Sir2蛋白。
Members of the Sir2 family of NAD-dependent protein deacetylases regulate diverse cellular processes including aging, gene silencing, and cellular differentiation. Here, we report that the distant mammalian Sir2 homolog SIRT6 is a broadly expressed, predominantly nuclear protein. Northern analysis of embryonic samples and multiple adult tissues revealed mouse SIRT6 (mSIRT6) mRNA peaks at day E11, persisting into adulthood in all eight tissues examined. At the protein level, mSIRT6 was readily detectable in the same eight tissue types, with the highest levels in muscle, brain, and heart. Subcellular localization studies using both C- and N- terminal green fluorescent protein fusion proteins showed mSIRT6- green fluorescent protein to be a predominantly nuclear protein. Indirect immunofluorescence using antibodies to two different mSIRT6 epitopes confirmed that endogenous mSIRT6 is also largely nuclear. Consistent with previous findings, we did not observe any NAD(+)- dependent protein deacetylase activity in preparations of mSIRT6. However, purified recombinant mSIRT6 did catalyze the robust transfer of radio-label from [P-32] NAD to mSIRT6. Two highly conserved residues within the catalytic core of the protein were required for this reaction. This reaction is most likely mono-ADP-ribosylation because only the modified form of the protein was recognized by an antibody specific to mono- ADP- ribose. Surprisingly, we observed that the catalytic mechanism of this reaction is intra-molecular, with individual molecules of mSIRT6 directing their own modification. These results provide the first characterization of a Sir2 protein from phylogenetic class IV.