Sirt5 deacylation activities show differential sensitivities to nicotinamide inhibition.
Sirt5 deacylation activities show differential sensitivities to nicotinamide inhibition.
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DOI：
10.1371/journal.pone.0045098
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Steegborn C
中科院分区：
文献类型：
--
作者：
Fischer F;Gertz M;Suenkel B;Lakshminarasimhan M;Schutkowski M;Steegborn C
Sirtuins是调节代谢和衰老过程的蛋白质脱酰基酶，并且七种人类同种型被认为是有吸引力的治疗靶点。Sirtuins将酰基从赖氨酸侧链转移到ADP-核糖，ADP-核糖通过烟酰胺的释放由辅底物NAD+形成，而烟酰胺又被认为是一般的Sirtuin抑制剂。然而，由于现有检测方法的缺陷，对Sirtuin调节的研究受到阻碍。在这里，我们描述了一种基于质谱的定量脱酰测定，不需要任何底物标记。使用该测定，我们表明，人Sirt 5的脱乙酰化活性具有对烟酰胺抑制的不寻常的不敏感性。相比之下，我们发现Sirt 5和Sirt 3的固有NAD+亲和力以及肽存在下的表观NAD+亲和力的值相似。结构比较和诱变鉴定了邻近Sirt 5烟酰胺结合口袋的Arg作为烟酰胺抗性的介体，并且统计序列分析沿着测试进一步的Sirtuins揭示了可能定义烟酰胺不敏感的Sirtuin脱乙酰酶家族的共进化残基网络。同样的精氨酸最近报道，使Sirt 5的一个优先脱琥珀酸酶，我们发现，这种Sirt 5的活性是高度敏感的烟酰胺抑制。Sirt 5的结构和活性数据的分析表明，精氨酸/琥珀酸相互作用是两个Sirt 5活动的差异烟酰胺敏感性的分子基础。因此，我们的研究结果表明，Sirtuin亚家族与烟酰胺不敏感的脱乙酰酶活性，并表明，决定烟酰胺敏感性的分子特征与那些占主导地位的脱酰特异性重叠，可能表明其他亚家族成员也可能更喜欢其他酰化比乙酰化。
Sirtuins are protein deacylases regulating metabolism and aging processes, and the seven human isoforms are considered attractive therapeutic targets. Sirtuins transfer acyl groups from lysine sidechains to ADP-ribose, formed from the cosubstrate NAD+ by release of nicotinamide, which in turn is assumed to be a general Sirtuin inhibitor. Studies on Sirtuin regulation have been hampered, however, by shortcomings of available assays. Here, we describe a mass spectrometry–based, quantitative deacylation assay not requiring any substrate labeling. Using this assay, we show that the deacetylation activity of human Sirt5 features an unusual insensitivity to nicotinamide inhibition. In contrast, we find similar values for Sirt5 and Sirt3 for the intrinsic NAD+ affinity as well as the apparent NAD+ affinity in presence of peptide. Structure comparison and mutagenesis identify an Arg neighboring to the Sirt5 nicotinamide binding pocket as a mediator of nicotinamide resistance, and statistical sequence analyses along with testing further Sirtuins reveal a network of coevolved residues likely defining a nicotinamide-insensitive Sirtuin deacetylase family. The same Arg was recently reported to render Sirt5 a preferential desuccinylase, and we find that this Sirt5 activity is highly sensitive to nicotinamide inhibition. Analysis of Sirt5 structures and activity data suggest that an Arg/succinate interaction is the molecular basis of the differential nicotinamide sensitivities of the two Sirt5 activities. Our results thus indicate a Sirtuin subfamily with nicotinamide-insensitive deacetylase activity and suggest that the molecular features determining nicotinamide sensitivity overlap with those dominating deacylation specificity, possibly suggesting that other subfamily members might also prefer other acylations than acetylations.
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影响因子：
56.9
作者：
Choudhary, Chunaram;Kumar, Chanchal;Mann, Matthias
通讯作者：
Mann, Matthias
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
8
作者：
Jin, Lei;Galonek, Heidi;Westphal, Christoph H.
通讯作者：
Westphal, Christoph H.
影响因子：
16
作者：
Avalos, JL;Bever, KM;Wolberger, C
通讯作者：
Wolberger, C
DOI：
10.1006/bbrc.2000.3000
发表时间：
2000-07-05
影响因子：
3.1
作者：
Frye, RA
通讯作者：
Frye, RA