Identification And Functional Study Of The Interaction Between Angiogenin And PLSCR1

Angiogenin(ANG) potentiates angiogenesis and tumor cell proliferation.However, the underlied molecular mechanisms remain elusive.Since the protein-protein interaction plays essential role in the biological events,exploring the ANG-interacted proteins could be an effective pathway to elucidate those ANG-mediated processes.Yeast two-hybrid screen is one of the widely used methods to search for unknown interacting proteins.Taking ANG as the bait,the original fellowers had successfully screened a human heart cDNA library in the yeast two-hybrid system,and indentified several ANG-interacted proteins.In order to find out more ANG-interacted molecules, here we continue to carry out the yeast two-hybrid screen with a human liver cDNA library.8 potential ANG-interacted proteins were identified,4 of which had also been identified from the human heart cDNA library screen.These newly identified ANG-interacted proteins could help us to further understand the molecular mechanisms of ANG.Phospholipid scramblase 1(PLSCR1) is one of the novel identified ANG-interacted proteins.It is an endofacial membrane protein and named after its phospholipid scramblase activity.More recently,PLSCR1 was found to translocate into the nuclei under certain conditions,though its nuclear function remains unclear.The interactions identified from yeast two-hybrid screen may be false positive. Therefore,the interaction between PLSCR1 and ANG need to be confirmed by other methods.The results from GST pull-down and in vivo co-immunoprecipitaion assays revealed that these two proteins did interact with each other both in vitro and in vivo. Thereafter,the endocellular co-localization and interaction of PLSCR1 and ANG was further explored.When ectopically expressed in HeLa cells,the flurescent protein-tagged PLSCR1(CFP-tagged) and ANG(YFP-tagged) were found to co-localize and physically interact with each other in the nuclei.The nuclear interaction was also observed from the HA-tagged PLSCR1 and native ANG by using indirect immunoflurence detection and nuclear coimmunoprecipitaiton analysis.These results revealed that PLSCR1 and ANG interact in the nuclei of HeLa cells.The nuclear localized ANG could promote the rRNA transcription,a process thought to be essential for ANG to exert its angiogenic and tumorigenic activites. However,the mechanism and regulation of ANG-enhancing rRNA transcription remain elusive.As a physical nuclear interacting partner of ANG,PLSCR1 was then explored for its influence on ANG-mediated rRNA transcription.We found that the PLSCR1 protein level was positively correlated with the rRNA transcription activity in HeLa cells;and moreover,PLSCR1 enhanced the rRNA transcription level only when ANG was available.The current data thus demonstrated that PLSCR1 could positively regulate the ANG-enhancing rRNA transcription at least in HeLa cells.Therefore,innovative conclusions could be drawn base on the data:1.Granulin(GRN),Fibronectinl(FN1),ERAD-associated E3 ubiquitin-protein ligase HRD1(HRD1),vascular endothelial statin(VE-statin),latent transforming growth factor beta binding protein 3(LTBP3),PLSCR1,Sprouty1(Spry1) and chordin(CHRD) are 8 potential ANG-interacted proteins indentified from the yeast two-hybrid screen with a human liver cDNA library as the prey.2.PLSCR1 could directly interact with ANG in HeLa cells.3.The interaction between PLSCR1 and ANG could take place in the nuclei of HeLa cells.4.PLSCR1 could upregulate the ANG-enhancing rRNA transcription in HeLa cells.