Study Of The Interaction Between Nucleolar Protein Def And Cysteine Protease CAPN3, And Identification Of The Def-CAPN3 Target Proteins In The Nucleolus

Nucleolus is responsible for the ribosome biogenesis,and involved in biochemical processes including cell cycle regulation,DNA damage repair and stress response.As a dense non-membrane organelle inside the nucleus,nucleolus is enriched with over 4,500 different proteins.However,the function and regulation of the majority of these nucleolar proteins are not well studied.Nucleolar protein Def(Digestive-organ expansion factor)mediates the degradation of p53 through cysteine protease calpain3(CAPN3),but the degradation mechanism is still not clear.And we also don't know whether the Def-CAPN3 pathway targets other proteins in the nucleolus in addition to p53.In this study,we first discover that CAPN3 degrades p53A138V,p53M237I,p53R248W and p53R273P,but not p53R175H.p53R175H mutation ranks first among the p53 substitution mutations in cancer samples,suggesting the importance of CAPN3 during tumorigenesis.By co-immunoprecipitation,we prove Def directly interacts with human CAPN3 to form a protein complex in the nucleolus.This interaction is conserved in zebrafish.Zebrafish Def and Capn3b(homologous protein to human CAPN3)also form a protein complex.Further investigation shows that Def recruits CAPN3 to enter the nucleolus.This process is regulated by phosphorylation modification of Def.Furthermore,the control of cell cycle progression and zebrafish early organogenesis by Def-CAPN3 is also partially dependent on p53.We generated two capn3b mutant alleles and found that p53 is highly enriched in the hepatocyte nucleoli of capn3b knock-out fish.Although homozygous capn3b knock-out mutants are viable and fertile,they display shorter length and lateral curvature phenotype in adulthood,which is similar to the limb-girdle muscular dystrophy type 2A(LGMD2A)patients caused by capn3 mutation.In order to identify other Def-CAPN3 target proteins,we compare the differentially expressed hepatocyte nucleolar proteins between capn3b knock-out fish and wild-type fish by proteomics,and identify 119 nuclear proteins upregulated(>1.45 folds)in the mutant fish.Among them 33 are nucleolar proteins.We select 8 candidates for in vivo and in vitro protein degradation experiments,proving that 7(LmnA,Nkap,Ddx18,Cdk9,Hmgb1,Tra2a,Bbof1)of them are the substrates of the Def-CAPN3 pathway.In addition,our proteomics analysis shows that Capn3b is involved in the regulation of innate immune response,wich explains why some LGMD2A patients are diagnosed with inflammation during initial disease stage.In summary,our findings demonstrate that Def-CAPN3 pathway regulates cell cycle and organogenesis by degrading target proteins in the nucleolus.To be the first zebrafish nucleolar poteomics analysis,our work helps to improve the study of zebrafish nucleolar protein function.We also identify 7 new Def-CAPN3 substrates which lays the ground for future study of the Def-CAPN3 function.Besides,the capn3b knock-out fish we generated is an excellent LGMD2A disease model,which can be used for drug screening in the future.