Biochemical characterization, localization, and tissue distribution of the longer form of mouse SIRT3
Biochemical characterization, localization, and tissue distribution of the longer form of mouse SIRT3
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DOI：
10.1002/pro.50
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发表时间：
2009-03-01
期刊：
影响因子：
8
通讯作者：
Westphal, Christoph H.
中科院分区：
文献类型：
--
作者：
Jin, Lei;Galonek, Heidi;Westphal, Christoph H.
SIRT3是一种关键的线粒体蛋白脱乙酰酶，被认为在调节线粒体新陈代谢中起关键作用，但关于它的实际大小、活性所需的序列以及它的亚细胞定位一直存在很大的争议。先前克隆的小鼠SIRT3与人SIRT3的C末端有很高的序列相似性，但缺乏N端的线粒体靶向序列，在体外没有检测到脱乙酰活性。通过50个末端的快速扩增，我们克隆了小鼠SIRT3以及大鼠和兔SIRT3的全序列。重要的是，我们发现全长SIRT3蛋白仅定位于线粒体，而不是SIRT3定位于细胞核的报道。我们证明了SIRT3在体外没有去乙酰化活性，除非蛋白质被截断，这与人类的SIRT3一致。此外，我们还测定了烟酰胺和小分子SIRT3抑制剂对活性小鼠SIRT3的抑制常数和作用机制，结果表明，两种化合物的作用机制不同于多肽底物和NAD(+)。因此，对实际SIRT3序列的鉴定和鉴定应该有助于解决关于小鼠SIRT3性质的争论，并识别调控酶活性的新机制。
SIRT3 is a key mitochondrial protein deacetylase proposed to play key roles in regulating mitochondrial metabolism but there has been considerable debate about its actual size, the sequences required for activity, and its subcellular localization. A previously cloned mouse SIRT3 has high sequence similarity with the C-terminus of human SIRT3 but lacks an N-terminal mitochondrial targeting sequence and has no detectable deacetylation activity in vitro. Using 50 rapid amplification of cDNA ends, we cloned the entire sequence of mouse SIRT3, as well as rat and rabbit SIRT3. Importantly, we find that full-length SIRT3 protein localizes exclusively to the mitochondria, in contrast to reports of SIRT3 localization to the nucleus. We demonstrate that SIRT3 has no deacetylation activity in vitro unless the protein is truncated, consistent with human SIRT3. In addition, we determined the inhibition constants and mechanism of action for nicotinamide and a small molecule SIRT3 inhibitor against active mouse SIRT3 and show that the mechanisms are different for the two compounds with respect to peptide substrate and NAD(+). Thus, identification and characterization of the actual SIRT3 sequence should help resolve the debate about the nature of mouse SIRT3 and identify new mechanisms to modulate enzymatic activity.