The Proteomic Screening And Verification Of Core Factors LATS2 And YAP In Hippo Pathway

Background: Hippo signaling pathway coordinates cell proliferation and differentiation in the body and controls the organs size and tissue regeneration.The core kinases LATS1/2and MST1/2 inhibit the transcriptional coactivator YAP/TAZ into the nucleus after activation by upstream signal and further negatively regulate organ development and regeneration.In addition to participating in the organ size control and tissue regeneration,Hippo signaling pathway also plays an important role in biological processes such as stem cell and progenitor cell self-renewal,metabolism,immunity and tumor development.Whether LATS1/2 phosphorylates YAP is a key link in the activation and shutdown of Hippo signaling pathway,but as a tumor suppressor gene,there are few studies on the function of LATS1/2 independent of YAP.Liver is one of the largest metabolic organs in the human body,and the Hippo signaling pathway plays an important role in the hepatocellular carcinoma and regeneration of liver,but the related research on Hippo signaling pathway and metabolism is less than other regulatory areas.Therefore,we used Y2 H library which is derived from human liver tissues to screene proteins that interact with LATS2 and further study its function.As the downstream effector of Hippo pathway,YAP plays a crucial role in the function of Hippo pathway.Although relevant studies have identified direct or indirect regulatory factors(AMOT,VGLL4 and BRD4)that affect YAP activity,there are still some questions to be solved about the biological function of YAP,such as how YAP binds to epigenetic factors,and how the transcriptional activation domain of C-terminal of YAP functions.To answer these questions,we used BioID method to screen potential interaction proteins of YAP.The method is mainly based on an engineered biotin ligase BirA*,which can biotinylate adjacent proteins.Biotinylated proteins were concentrated through streptavidin beads and identified by mass spectrometry analysis Which can initially identifyYAP-adjacent proteins and potential interaction proteins.Objective: To search for proteins interacting with the core factors of the Hippo signaling pathway,LATS2 and YAP,using Y2 H and BioID respectively and further verify the function and pathology of Hippo signaling pathway.These will provide cues for studying the regulation and pathological function of Hippo signaling pathway.Methods: The DNA binding domain of Gal4 and N terminus of LATS2(N-LATS2)were constructed into a fusion plasmid,whose expression was detected by Western blot.Then,autoactivation and toxicity test were performed to confirm that the fusion plasmid could be used as a bait for Y2 H screening.The strain expressing fusion plasmid was incubated with the yeast strains library of liver tissue to screen the proteins interacting with LATS2.We acquired positive colonies from the expression of reporter genes(His3,LacZ and Leu2),then sequenced and aligned the condidator sequences in NCBI.Then the candidate genes were fused with HA tag and LATS2 were fused with Flag tag.Next,the fusion proteins were co-transfected into 293 T cells,the interaction were detected by Co-immunoprecipitation.As a transcription coactivator,YAP exists the autoactivation phenomenon in the Y2 H.Therefore,we used BioID assay to screen proteins interacting with YAP.The results were used for clustering,pathway and functional analysis.40 candidate genes were selected,and two specific shRNAs were designed for each one..The effect of candidate genes on YAP activity was detected by TBS-Dual-Luciferase Reporter Assay and the expression of downstream target genes in Hippo signaling pathway was detected by qPCR.Results: 22 proteins(PARP9,FGA,SERPINF1,PLOD1,RBP4,AZGP1,AOX1,MST122,MST127,HPX,ATP synthase 6,CP,EF1 a,WDR6,POLR1 B,HPN,NOL11,ZNF350,PSAP,FGB,NR2F6,ALDH2)interacting with LATS2 in liver tissue were screened by Y2 H.We selected PARP9,WDR6,NOL11,AZGP1,ZNF350,HPX,ALDH2 and AOX1 as candidates for Y2 H reverse comfirmation and use phosphorylation prediction tools(Http://www.phosphonet.ca/default.aspx)to predict whether they can be phosphorylated by LATS1/2.According to the results,PARP9,WDR6,NOL11,ALDH2 and AOX1 wereselected to confirm their interaction with LATS2.The results of Co-immunoprecipitation showed LATS2 interacting with ALDH2 by its N terminus.It is known that ALDH2 participates in alcohol metabolism,which suggests that LATS2 may be involved in alcohol metabolism by regulating ALDH2.Over 1000 adjacent proteins to YAP were screened by BioID assay in 293 T cells.After clustering,pathway and functional analysis,we found that the proteins associated with mechanical tension,cytoskeleton,transcriptional regulation,epigenetic may be involved in regulating YAP activity.Among these proteins,NEDD4,CBLC,HSP90AB2 P,CDK2,LATS,MST,AMOT have been reported and WDR26,STRAP,TRIM25,RANBP2,TFG,KHSRP,TES,and SEPT7 haven’t been reported.After alignment with the members of the Hippo signaling pathway included in KEGG and Biogrid,40 genes were selected as candidates for further analysis.The effect of candidates knockdown on YAP activity and downstream target genes(CTGF,CYR61,and ANKRD1)were detected by TBS-Dual-Luciferase Reporter Assay and qPCR.The results indicated that knockdown of PRDX6,TPD52,TRIM25 and TRIM33 inhibit the activity of YAP and the transcription of its target genes.Conclusion: LATS1/2 may be involved in alcohol metabolism via regulating ALDH2;Multiple YAP regulatory factors such as COR1 B,TRIM25 and TRIM33 were screened by BioID method.These results suggest there are more genes related to mechanical tension,cytoskeleton,transcriptional regulation,and epigenetics that may be involved in the regulation of YAP activity.