Synthesis of dimeric ADP-ribose and its structure with human poly(ADP-ribose) glycohydrolase.
Synthesis of dimeric ADP-ribose and its structure with human poly(ADP-ribose) glycohydrolase.
复制标题
DOI：
10.1021/ja512528p
复制
发表时间：
2015-03-18
影响因子：
15
通讯作者：
Hergenrother PJ
中科院分区：
文献类型：
--
作者：
Lambrecht MJ;Brichacek M;Barkauskaite E;Ariza A;Ahel I;Hergenrother PJ
聚（ADP-核糖基）化是一种常见的翻译后修饰，其介导多种细胞过程，包括DNA损伤修复、染色质调节、转录和凋亡。与获得均质形式的聚（ADP-核糖）（PAR）相关的困难一直是理解PAR与聚（ADP-核糖）糖水解酶（PARG）和其他结合蛋白的相互作用的障碍。在这里，我们描述了ADP-核糖二聚体的化学合成，并且我们使用该化合物获得了第一个人PARG底物-酶共晶体结构。PAR的化学合成是传统酶促合成和分馏的有吸引力的替代方案，允许获得诸如二聚ADP-核糖的产物，其已被检测到但从未从天然来源分离。此外，我们描述了炔基化二聚体的合成，并证明该化合物可用于合成PAR探针，包括生物素和荧光团标记的化合物。然后将荧光标记的ADP-核糖二聚体用于一般的基于荧光偏振的PAR-蛋白结合测定。最后，我们使用我们的合成中间体来访问各种PAR片段和评价这些化合物作为底物PARG揭示了底物识别和酶促裂解的最小功能。同质PAR寡聚体和非天然的变体产生的化学合成将允许进一步详细的结构和生化研究的相互作用的PAR与其许多蛋白质结合的合作伙伴。
Poly(ADP-ribosyl)ation is a common post-translational modification that mediates a wide variety of cellular processes including DNA damage repair, chromatin regulation, transcription, and apoptosis. The difficulty associated with accessing poly(ADP-ribose) (PAR) in a homogeneous form has been an impediment to understanding the interactions of PAR with poly(ADP-ribose) glycohydrolase (PARG) and other binding proteins. Here we describe the chemical synthesis of the ADP-ribose dimer, and we use this compound to obtain the first human PARG substrate-enzyme co-crystal structure. Chemical synthesis of PAR is an attractive alternative to traditional enzymatic synthesis and fractionation, allowing access to products such as dimeric ADP-ribose, which has been detected but never isolated from natural sources. Additionally, we describe the synthesis of an alkynylated dimer and demonstrate that this compound can be used to synthesize PAR probes including biotin and fluorophore-labeled compounds. The fluorescently labeled ADP-ribose dimer was then utilized in a general fluorescence polarization-based PAR-protein binding assay. Finally, we use intermediates of our synthesis to access various PAR fragments and evaluation of these compounds as substrates for PARG reveals the minimal features for substrate recognition and enzymatic cleavage. Homogeneous PAR oligomers and unnatural variants produced from chemical synthesis will allow for further detailed structural and biochemical studies on the interaction of PAR with its many protein binding partners.
登录
查看更多内容
影响因子：
2.7
作者：
Le Corre SS;Berchel M;Couthon-Gourvès H;Haelters JP;Jaffrès PA
通讯作者：
Jaffrès PA
影响因子：
64.8
作者：
Ahel, Ivan;Ahel, Dragana;West, Stephen C.
通讯作者：
West, Stephen C.
DOI：
10.1016/j.bbrc.2010.01.044
发表时间：
2010-02-19
影响因子：
3.1
作者：
Okita, Naoyuki;Ashizawa, Daisuke;Tanuma, Sei-ichi
通讯作者：
Tanuma, Sei-ichi
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.
DOI：
10.1073/pnas.0606526103
发表时间：
2006-11-28
影响因子：
11.1
作者：
Andrabi, Shaida A.;Kim, No Soo;Dawson, Ted M.
通讯作者：
Dawson, Ted M.