Clickable NAD analogues for labeling substrate proteins of poly(ADP-ribose) polymerases.
Clickable NAD analogues for labeling substrate proteins of poly(ADP-ribose) polymerases.
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DOI：
10.1021/ja101588r
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发表时间：
2010-07-14
影响因子：
15
通讯作者：
Lin H
中科院分区：
文献类型：
--
作者：
Jiang H;Kim JH;Frizzell KM;Kraus WL;Lin H
多聚腺苷二磷酸核糖聚合酶（Poly（ADP-ribose）polymerases，PARP）催化多个腺苷二磷酸核糖（Adenine diphosphate ribose，ADP-ribose）单元从烟酰胺腺嘌呤二核苷酸（nicotinamide adenine dinucleotide，NAD）转移到底物蛋白。人类有17种PARP。已知几种PARP，如PARP-1和端锚聚合酶-1，在DNA修复、转录、有丝分裂和端粒长度维持中起重要作用。为了在分子水平上更好地理解PARP的功能，有必要知道PARP修饰的底物蛋白。在这里，我们报告了可点击的NAD类似物，可用于标记PARP底物蛋白。可点击的NAD类似物具有末端炔基，其允许荧光或亲和标签与底物蛋白缀合。使用该方法，PARP-1和端锚聚合酶-1底物蛋白通过荧光标签标记并在SDS-PAGE凝胶上可视化。使用生物素亲和标签，我们能够分离和鉴定总共79种蛋白质，这些蛋白质被鉴定为潜在的PARP-1底物。这些包括已知的PARP-1底物蛋白，包括组蛋白和异质核核糖核蛋白。在最近的蛋白质组学研究中，约40%的蛋白质也被鉴定为潜在的PARP-1底物。在鉴定的潜在底物中，我们进一步证明了微管蛋白和三种线粒体蛋白TRAP 1（TNF受体相关蛋白1）、柠檬酸合酶和GDH（谷氨酸脱氢酶1）是PARP-1的体外底物。这些结果表明，点击NAD类似物是有用的标记，胶内检测，分离和鉴定PARP的底物蛋白，并将有助于了解PARP的生物学功能。
Poly(ADP-ribose) polymerases (PARPs) catalyze the transfer of multiple adenine diphosphate ribose (ADP-ribose) units from nicotinamide adenine dinucleotide (NAD) to substrate proteins. There are seventeen PARPs in humans. Several PARPs, such as PARP-1 and Tankyrase-1, are known to play important roles in DNA repair, transcription, mitosis, and telomere length maintenance. To better understand the functions of PARPs at a molecular level, it is necessary to know what substrate proteins PARPs modify. Here we report clickable NAD analogs that can be used to label PARP substrate proteins. The clickable NAD analogs have a terminal alkyne group which allows the conjugation of fluorescent or affinity tags to the substrate proteins. Using this method, PARP-1 and tankyrase-1 substrate proteins were labeled by a fluorescent tag and visualized on SDS-PAGE gel. Using a biotin affinity tag, we were able to isolate and identify a total of 79 proteins were identified as potential PARP-1 substrates. These include known PARP-1 substrate proteins, including histones and heterogeneous nuclear ribonucleoproteins. About 40% of the proteins were also identified in recent proteomic studies as potential PARP-1 substrates. Among the identified potential substrates, we further demonstrated that tubulin and three mitochondrial proteins, TRAP1 (TNF receptor-associated protein 1), citrate synthase, and GDH (glutamate dehydrogenase 1), are substrates of PARP-1 in vitro. These results demonstrate that the clickable NAD analog is useful for labeling, in-gel detection, isolation, and identification of the substrate proteins of PARPs and will help to understand the biological functions of PARPs.
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影响因子：
2.9
作者：
KRUPITZA, G;CERUTTI, P
通讯作者：
CERUTTI, P
影响因子：
15
作者：
Jiang H;Congleton J;Liu Q;Merchant P;Malavasi F;Lee HC;Hao Q;Yen A;Lin H
通讯作者：
Lin H
影响因子：
14.9
作者：
Gagne, Jean-Philippe;Isabelle, Maxim;Lo, Ken Sin;Bourassa, Sylvie;Hendzel, Michael J.;Dawson, Valina L.;Dawson, Ted M.;Poirier, Guy G.
通讯作者：
Poirier, Guy G.
影响因子：
7.5
作者：
Kraus, W. Lee
通讯作者：
Kraus, W. Lee
影响因子：
56.9
作者：
Dynek, JN;Smith, S
通讯作者：
Smith, S