拟南芥多聚ADP-核糖化修饰蛋白的富集和鉴定

多聚ADP-核糖化修饰poly(ADP-ribosyl)ation,PARylation]是一种可逆的蛋白质翻译后修饰,该修饰由多聚ADP-核糖聚合酶poly(ADP-ribose)polymerase,PARP]催化.多聚ADP-核糖化修饰蛋白主要在动物中发现,在植物中少有报道.微生物来源的蛋白质AF1521具有一个能结合多聚ADP-核糖poly(ADP-ribose),PAR]的macro结构域(macro domain),其突变蛋白AF1521-G42E失去了结合PAR的活性.我们利用AF1521蛋白对PAR的亲和作用来富集拟南芥中的PARylation蛋白质底物并进行质谱分析,同时用AF1521-G42E作为富集过程的负对照.通过这种方法,我们得到多个ADP-核糖化修饰相关蛋白,并对其中一个蛋白GRP7进行了验证.

Identification of poly(ADP-ribosyl) ated Proteins in Arabidopsis

poly(ADP-rihosyl)ation is a type of reversible posttranslational modification catalyzed by enzymes known as poly(ADP-ribose) polymerases(PARP).The protein substrates are mostly found in animals and there are rare reports in plants so far.AF1521 protein originated from microorganism possesses a macrodomain,which can recognize and interact selectively with the terminal residue of poly(ADP-ribose)(PAR),whereas the mutant protein AF1521-G42E loses the PAR-binding activity.In our study,we used AF1521 as a selective bait for enriching poly(ADP-ribosyl) ated protein substrates or proteins that interact with them in Arabidopsis.In the meantime,AF1521-G42E was used as a control of the experiment.LC-MS/MS was used to analyze the pull-down proteins.In this way,we identified a couple of possible protein substrates of poly(ADP-ribosyl) ation or proteins that related with poly(ADP-ribosyl)ated proteins,and one protein of them,GRP7,was verified by in vitro pull-down assay.