Detection Of PARP Activity And Screening Of Inhibitors

Objective:Stroke is the second most common cause of death after coronary heart disease,but there are few effective treatments for stroke.Recently,TIPARP has been found to be involved in the occurrence of ischemic stroke,especially in the activation of astrocytes and autophagy,which can be used as a potential therapeutic target for stroke.The development of small molecule inhibitors against TIPARP is of great significance.TIPARP is a mono-ADP-ribosyl transfer PARP.Poly-ADP-ribose polymerase(PARP)family,also known as ADP-ribosyl transferase diphtheria toxin-like(ARTD)family,consisting of 17 members.PARP1 is a founding member and the most distinctive member of the PARP family.PARP1 catalyzes the transfer of PAR to receptor proteins and plays an important role in cell proliferation,protein degradation,transcription,RNA biology,and cellular stress responses(such as DNA damage).PARP inhibitors represent a promising cancer treatment strategy,and several FDA approved drugs have been marketed,including Olaparib,Rucaparib,and Niraparib.Most family members except PARP1 have MARylation activity.TIPARP,as a mono-ADP-ribosyltransferase,has not yet a reliable and low-cost enzyme activity detection method for high-throughput screening of its small molecule inhibitors.This requires to develop and improve the detection methods for quantifying the activity of various mono-ADP-ribosyltransferases,including TIPARP,providing a simple,fast,and low-cost method for high-throughput screening of drugs.Method 1:Here we propose two optimized methods for detecting TIPARP activity based on the remaining substrate of the enzyme reaction system or Macrodomain2 of PARP14.The first method based on the conversion of the remaining substrate NAD~+of the enzymatic reaction system into fluorescent compounds that can be used to detect mono-ART activity,screen and identify inhibitors.This method has previously been applied to ARTD1 and ARTD5,sirtuins and bacterial toxins,ARTD7(PARP15),and ARTD10(PARP10).Here,we optimized it to detect TIPARP enzyme activity.First,we constructed PARP1 and TIPARP prokaryotic expression vectors,expressed and purified them,and used qualitative and quantitative analysis with SDS-PAGE and western blot for subsequent testing.Because the protein is originally expressed by human,TIPARP in E.coli is insoluble,we therefore added MBP,GST or SUMO tag proteins to the N-terminus of TIPARP for expression plasmid design.After Ni-NTA affinity chromatography purification,protein concentration and purity were analyzed by SDS-PAGE and Coomassie blue staining.Next,we tested the catalytic activity of PARP1CD and MBP-TIPARP for different reaction time and different enzyme concentrations by NAD~+chemical quantification method.First,a standard curve was generated using different concentrations of NAD~+.After testing the effects of different enzyme concentrations and different incubation times,we chose the appropriate enzyme concentration and incubation time for inhibitor screening.Method 2:Macrodomain2 and Macrodomain3 of PARP14(ARTD8)are described as domains that only bind to MARylated protein,and importantly,they do not exhibit hydrolase activity.We fused the green fluorescent protein(Neon Green)to the C-terminus of PARP14 Macrodomain2.This fusion protein can tightly bind to free or linked ADP-ribose in vitro or in vivo.Next,in order to establish a method for detecting TIPARP activity based on Macrodomain2 of PARP14,we constructed a fusion plasmid of Macrodomain2 and Neon Green(M2-Neon Green),and expressed and purified the fusion protein in E.coli.TIPARP was transiently transfected in 293T cells and stained with PAR/MAR antibody or M2-Neon Green fusion protein.Next,we tried to observe the MAR event in living cells.We synthesized the eukaryotic expression plasmids of TIPARP and M2-Neon Green,and then co-transfected both into 293T cells.After 48h,they were directly observed under the microscope without staining.Result 1:The purity of PARP1CD can reach more than 90%,and the yield is about15mg/L,which is enough for enzyme activity testing and high-throughput screening of inhibitors.After induced expression and purification of various versions of TIPARP,the yield of MBP-TIAPRP is relatively high,about 0.4 mg/L.We performed western blot detection of PARP1CD and MBP-TIPARP,and found that both enzymes were self-modified.Unlike PARP1CD,there was no significant change in molecular weight of MBP-TIPARP after the reaction,indicating that these protein molecules did not form polymers,reflecting its mono-ADP-ribosyltransferase activity.The fluorescence measured at 430nm showed a good linear relationship in the range of 0～10μM.After the enzyme was added to the reaction system,the conversion rate of NAD~+showed a time and dose dependent increase.These data indicate that the enzyme activity of MBP-TIPARP can be quantified by the chemical quantification method of NAD~+.After testing the effects of different enzyme concentrations and different incubation times,the enzyme concentration used for inhibitor screening was selected to be 100n M,and the reaction time was 2 hours.The inhibition rates of several different inhibitors we tested on PARP1CD and MBP-TIPARP are quite different,which indicates that although PARP1CD and MBP-TIPARP belong to the PARP family,the interaction between the inhibitor and the enzyme catalytic domain may be different.This indicates that it is possible to develop specific inhibitors targeting TIPARP in the future.Result 2:The results showed that both the PAR/MAR antibody and the M2-Neon Green fusion protein exhibited specific signals in the 293T cell line overexpressing TIPARP.Under the microscope,M2-Neon Green was dispersed in cells co-transfected with the control vector plasmid,and condensed spots of M2-Neon Green signal were observed in the cells co-transfected with TIPARP.These results indicate that the overexpression of TIPARP induces the formation of green fluorescent protein(M2-Neon Green)spots in cells,which makes M2-Neon Green a possible sensor for detecting TIPARP catalytic activity in vivo.Conclusion:In order to develop a mono-ART activity detection method,we took TIPARP as the research object,established a NAD~+chemical quantitative analysis method and an M2-Neon Green probe designed based on human PARP 14macrodomain2 for detecting TIPARP activity in vitro and in vivo.The M2-Neon Green probe not only provides a simple,fast and low-cost method for high-throughput screening of drugs at the cellular level,but also provides a convenient and effective tool for detecting MAR signals in vivo in the future.