Molecular Mechanisms And Significances Of LRP16Binding To Poly(ADP-ribose)

Macro domain protein family is a class of highly conserved protein domain foundin diverse orgnisms. Biochemical analysis demonstrated that the majority of Macrodomain proteins are PAR binding protein. LRP16is a member of Macro domain familyand only a Macro domain was found in its C-teminal. DNA damage sensor PARP1sense the damage signal and synthesize local Poly(ADP-ribose) on itself. LRP16maybe a component of PARP1complex in DSBs and involved in the early DNA damagesignal transmission by binding to PAR. However, the key amino acid sites of LRP16that mediate LRP16recognize and bind to poly (ADP-ribose)(PAR) are not very clear.The main purpose of this study is to find out the LRP16candidate PAR-binding sitesand further explore this PAR-binding biological significance. This study is divided intothree parts.First The prediction of LRP16candidate PAR-binding sites and construction ofLRP16point mutantsObjective: To predict and find out the candidate PAR-binding sites and constructLRP16point mutants. Methods：The structure of protein LRP16was analyzed firstlyto predict the LRP16candidate PAR-binding sites and then a series of LRP16pointmutants were constructed by alanine scanning and overlap PCR method with a templatepCDNA3.1-LRP16. Results: Molecular docking and simulation showed that the keyamino acid sites of LRP16that mediate LRP16recognize and bind to poly (ADP-ribose)(PAR) are D160, I161, N174, G181, V183, D184, S268and so on and DNA seguencingshowed that the codon of the key amino mentioned above were successfully mutantedto alamine. Conclusion: Five LRP16point mutants eukaryotic expression plasmidswere constructed.Second The detection of LRP16and its point mutants PAR-binding activityObjective: To investigate LRP16and its point mutants PAR-binding activity. Methods:Five LRP16point mutants prokaryotic expression plasmids were constructed and theprotein of LRP16point mutants were expressed and purified, and their PAR bindingactivity was assayed by PAR binding assay in vitro and immunofluorescence techniquein vivo. Results: Dot blot hybridization showed that the PAR binding activity dramatically reduced when160D and161I of LRP16mutated to A respectively, butonly partial attenuation was observed when181G,183V and184D mutated to A;immunofluorescence technique showed that the co-location of LRP16I161A and PARwas reduced compared to LRP16. Conclusion: The D160and I161of LRP16are thekey sites for PAR binding.Third The functional analysis and verification of LRP16binding to PARObjective: To investigate the effects of LRP16binding to PAR on the LRP16translocation from the nuclear to cytoplasm, the formation of IKK complex and thephosphorylation level of IKKβ and further verification by small molecular compondsthat inhibit LRP16bind to PAR and PARP enzyme inhibitor. Methods: Tranfectionplasmids of pCDNA3-flag-LRP16(pCDNA3-flag), pCDNA3-flag-LRP16D160A andpCDNA3-flag-LRP16I161A in Hela cells, the translocation of LRP16from nuclear tocytoplasm was determined by immunofluorescence technique, the formation of IKKcomplex was assayed by co-immunoprecipitation and the phosphorylation level ofIKKβ was tested by western blot upon DNA damage; meanwhile, Hela cells werepretreatment with small molecular componds and PARP enzyme inhibitor and thetranslocation of LRP16from nuclear to cytoplasm and the phosphorylation level ofIKKβ was tested by western blot upon DNA damage. Results: immunofluorescencetechnique showed that ecotopic expression of pCDNA3-flag-LRP16D160A andpCDNA3-flag-LRP16I161A, the translocation of LRP16from nuclear to cytoplasmwas reduced (compared to pCDNA3-flag-LRP16); co-immunoprecipitation showed thatecotopic expression of pCDNA3-flag-LRP16D160A and pCDNA3-flag-LRP16I161A,the interactions between LRP16and PARP1, IKKβ, PKR and NEMO were attenued(compared to pCDNA3-flag-LRP16); western blot showed that ecotopic expression ofpCDNA3-flag-LRP16I161A, the phosphorylation level of IKKβ was reduced(compared to pCDNA3-flag). Furthermore, the results were further demonstrated bysmall molecular componds and PARP enzyme inhibitor. Conclusion: These resultssuggest that the D160and I161of LRP16are the key sites for PAR binding, when theywere mutanted to A respectively, their PAR binding activity was dramatically reducedand further affect the DNA damage signal nuclear-cytoplasm transmit, which lay thefoundation for elucidating the role of LRP16in the transmitting early DNA damagesignal.