Engineering the substrate specificity of ADP-ribosyltransferases for identifying direct protein targets.
Engineering the substrate specificity of ADP-ribosyltransferases for identifying direct protein targets.
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DOI：
10.1021/ja412897a
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发表时间：
2014-04-09
影响因子：
15
通讯作者：
Cohen MS
中科院分区：
文献类型：
--
作者：
Carter-O'Connell I;Jin H;Morgan RK;David LL;Cohen MS
二磷酸腺苷核糖基转移酶(ARTDS；在人类中为ARTD1-17)在正常生理和疾病中都是细胞功能的关键调节因子。这些酶将ADP-核糖部分从其底物烟酰胺腺嘌呤二核苷酸(NAD+)转移到目标蛋白质的氨基酸上。人类17个ARTD之间的功能冗余和重叠的靶点特异性使得在细胞环境中识别单个ARTD家族成员的直接靶点是一个巨大的挑战。在这里，我们描述了用于识别单个ARTD的直接蛋白质靶点的NAD+模拟工程ARTD对的合理设计。在对C-5位含取代基的烟酰胺类似物进行的初步抑制剂研究的指导下，我们合成了一个正交的NAD+变异体，发现它可作为几种工程ARTD(ARTD 1、−2和ARTD 6)的底物，而不是它们的野生型对应物。比较核提取液中ARTD1(PARP1)和ARTD2(PARP2)的靶向图谱，突出了半互补但不同的蛋白质靶向。利用亲和纯化和串联质谱法，我们鉴定了42个直接ARTD1靶点和301个直接ARTD2靶点。这为识别单个ARTD家族成员的直接蛋白质靶标提供了一种强大的新技术，这将有助于研究从ARTD激活到给定细胞反应的途径。
Adenosine diphosphate ribosyltransferases (ARTDs; ARTD1–17 in humans) are emerging as critical regulators of cell function in both normal physiology and disease. These enzymes transfer the ADP-ribose moiety from its substrate, nicotinamide adenine dinucleotide (NAD+), to amino acids of target proteins. The functional redundancy and overlapping target specificities among the 17 ARTDs in humans make the identification of direct targets of individual ARTD family members in a cellular context a formidable challenge. Here we describe the rational design of orthogonal NAD+ analogue-engineered ARTD pairs for the identification of direct protein targets of individual ARTDs. Guided by initial inhibitor studies with nicotinamide analogues containing substituents at the C-5 position, we synthesized an orthogonal NAD+ variant and found that it is used as a substrate for several engineered ARTDs (ARTD1, −2, and −6) but not their wild-type counterparts. Comparing the target profiles of ARTD1 (PARP1) and ARTD2 (PARP2) in nuclear extracts highlighted the semi-complementary, yet distinct, protein targeting. Using affinity purification followed by tandem mass spectrometry, we identified 42 direct ARTD1 targets and 301 direct ARTD2 targets. This represents a powerful new technique for identifying direct protein targets of individual ARTD family members, which will facilitate studies delineating the pathway from ARTD activation to a given cellular response.
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影响因子：
14.9
作者：
Gagné JP;Pic E;Isabelle M;Krietsch J;Ethier C;Paquet E;Kelly I;Boutin M;Moon KM;Foster LJ;Poirier GG
通讯作者：
Poirier GG
影响因子：
16
作者：
Krishnakumar R;Kraus WL
通讯作者：
Kraus WL
影响因子：
48
作者：
Zhang, Yajie;Wang, Jianqi;Yu, Yonghao
通讯作者：
Yu, Yonghao
影响因子：
2.9
作者：
Karlberg, Tobias;Hammarstrom, Martin;Schuler, Herwig
通讯作者：
Schuler, Herwig
影响因子：
4
作者：
Lindgren, Anders E. G.;Karlberg, Tobias;Schueler, Herwig
通讯作者：
Schueler, Herwig