Validation And Functional Study Of A New Protein Interacting With PGRN

PGRN is located on human chromosome 17q21.31.As a secreted glycoprotein molecule,the mature protein consists of 593 amino acids,including seven and one half highly conserved cysteine repeat motif.The seven and one half domains are named with paragranulin,granulin(Grn)G,F,B,A,C,D and E.Since the amino acid sequence of PGRN contained four glycosylation sites,it has an approximate molecular weight of 88 kDa.Human PGRN is widely expressed in different cells,tissues and organs,especially in those rich in many epithelial cells such as spleen,reproductive system,lung,kidney and skin.And it was also found to be highly express in certain neurons and most cancer cells.Although current research on the function of PGRN indicated that it was involved in a variety of physiological and pathological events,including nervous system development,cell proliferation,wound healing,diabetes,tumorigenesis and tumor infiltration.The mechanisms of PGRN involving in a variety of physiological and pathological activities remains unclear,however,because the biological functions of the protein in the body are carried out by its interaction with other proteins.It's very important to find the PGRN-interacting protein molecules and verify functional changes produced by the interaction between the protein molecules and PGRN are of great importance to clarify the molecular mechanism of PGRN function.In our preliminary study,some candidate molecules interacting with the PGRN have been screened out by yeast two-hybrid system and bioinformatics analysis.And one of these molecules,Dual specificity tyrosine-phosphorylation-regulated kinase 1A(DYRK1A),attract our attention.In recent years,studies have shown that DYRK1A as a kinase could regulate the expression or activity of many molecules by its phosphorylation of them,and thus it may participates in multiple signaling pathways and pathological process of disease.Coincidentally,DYRK1A function have been reported mainly in the areas of neurodegenerative diseases,The disease severity were regulated in a dose-dependent fashion by DYRK1A,studies have also reported that abnormal expression of DYRK1A affected tumor formation,malignant degree and relapse.What's more,recent article reported that DYRK1 A-haploinsufficiency induced symptoms of diabetes in mice.These results make us speculate that PGRN may regulate the role of DYRK1A in signaling pathways by affecting the interaction between DYRK1A and its substrate or influence its kinase activation,or maybe DYRK1A influences PGRN biological function by regulating its interaction with some other proteins.In this study we attempted to find the affection of PGRN and DYRK1A on cell proliferation and neuronal differentiation through investigating the interaction between them.There are two parts in this study,which is to verify the interaction of PGRN and DYRK1A,and explore the influence of this interaction on cell proliferation.Firstly,we verified the interaction between two molecules by yeast two-hybrid system,co-immunoprecipitation(Co-IP)and GST-pull down,and then validated the co-localization by using confocal laser microscopy.Then we investigated the effect of the interaction between two proteins on cell proliferation and differentiation.Finally,we established DYRK1A knockout cell lines to further study its biological function.We expected this study could provide the underlying basis for elucidating the biological functions of PGRN.The contents of the research project are as follows.(1)Validation of the interaction between PGRN and DYRK1AThe full length of DYRK1A was isolated from human cDNA library by PCR technic,and then cloned it into yeast vector.The molecular interaction was validated between PGRN and DYRK1A by yeast two-hybrid system.Firstly,the fragments with different deletion of DYRK1A were generated according to the amino acid structure,and then were used for exploring the interaction domains of the two proteins using yeast two-hybrid system.The result showed that the domain of PGRN responsible for interacting with DYRK1A was located in GmB,GrnA and GmC,and the precise region of DYRK1A necessary for interacting with PGRN resides within His-rich domain.Next,the interaction between PGRN and DYRK1A was convinced by the Co-IP in vivo and GST pull-down assay in vitro.Finally,the results of confocal laser microscopy showed that PGRN was co-localized with DYRK1A when they are co-transfected into CHO cells.Co-transfection of PGRN and DYRX1A could promote a translocation of part of DYRK1A from the nuclei into the cytoplasm,and a translocation of part of PGRN from the cytoplasm into the nuclei in CHO cells.(2)The Study on the biological function of the cells mediated by PGRN and DYRK1AFirstly,we explored the cell differentiation caused by the interaction between PGRN and DYRK1A in the SH SY5Y cells.Results showed that overexpression ofDYRK1A could promote the differentiation of SH SY5Y cells,whereas the antagonism of PGRN on the differentiation caused by DYRK1 could be observed.And the antagonism of PGRN was more obvious than the antagonism of Harmine on the inhibitory effect to differentiation of cell caused by DYRK1A.Then,we detected the effects of interaction between PGRN and DYRK1A on cell proliferation in HEK293 cells by the MTT method.The result indicated the overexpression of DYRK1A suppressed cells proliferation,but Harmine,PGRN and PGRN-BAC could impair the inhibition of cell proliferation caused by DYRK1A which result in that they could promote cell proliferation.Compared with PGRN-BAC and Harmine,the antagonism of PGRN on the cell proliferation inhibition effect caused by DYRK1A is more obvious.Next,in order to verify the molecular mechanism of effect on cell proliferation and differentiation caused by the interaction between PGRN and DYRK1A,some study was carried out from both the mRNA level and protein levels respectively.So the cell cycle related molecular P27kip1 and Cyclin D1 was chose for testing.Results showed that overexpression of PGRN did not influence the mRNA level of DYRK1A,P27kip1 and Cyclin D1 obviously,and overexpression DYRK1A also did not influence the mRNA level of P27kip1 and Cyclin D1.However,Harmine,PGRN and PGRN-BAC affected the regulation of P27kip1 and Cyclin D1 by DYRK1A,these results proved that PGRN promoted cell proliferation by influence the effect of DYRK1A to its substrate(P27kip1 and Cyclin D1),and the effect brought by full length of PGRN was stronger than it brought by Harmine and PGRN-BAC.Then,we detected the endogenous expression of DYRK1A in different cells by the Real-time PCR.The results indicated that the DYRK1A expression level was lower in tumor cell,HepG2,MCF and U87 than it in HEK293 cells,and the DYRK1A expression level was highest in SH SY5Y cells.Finally,in order to further explore the cytological function mediated by DYRK1A,the Cas9-sgRNA technology was used to establish SH SY5Y DYRK1A knock out cell line.The results showed that the sgRNA with high efficiency to DYRK1A was screened and the target sequence was cut successfully.Summary of the above research,the results obtained from the project as follows.(1)The direct interaction between PGRN and DYRKIA was validated by the co-immunoprecipitated,GST pull-down assay and confocal laser microscopy.(2)By using yeast two-hybrid system,the precise interaction region of PGRN resides within PGRN-BAC domain,and the precise domain of DYRX1A interacting with PGRN was found to be present in His-repeat tract.(3)DYRK1A promoted the differentiation in SH-SY5Y cell,well PGRN could inhibit the involved function of DYRK1A.(4)Obvious inhibitory effect of DYRK1A in HEK293 cells proliferation was verified,either PGRN or PGRN-BAC could antagonize this inhibition,PGRN was more effectively.(5)The molecular mechanism about the interaction of PGRN and DYRK1A involving cell proliferation and differentiation was investigated.It was found that PGRN promoted proliferation and inhibited differentiation of SH-SY5Y by the regulation of P27kip1 ? Cyclin D1 mediated by DYRK1A,which led to the discovery of one pathway of cell proliferation and differentiation regulated by PGRN.(6)Established DYRK1A knock-out SH-SY5Y cell line by Cas9-sgRNA technique.