Function And Mechanism Of Isl1 And PIASy Interaction In Insulin Synthesis And Secretion

Protein-protein interactions(PPIs)are essential for all biological processes and lead to many new opinions about protein functions.It has been reported insulin gene enhancer binding protein 1(Isll)plays significant roles in regulation of insulin gene expression and secretion,but we still do not know whether there are the other proteins involved in the process together with Isll.In the study,Isll interacting proteins were identified by yeast two-hybrid system.Primary islet cells and islet ? cell line NIT cells were used to research how Isll and protein inhibitor of activated STAT 4(PIASy)mediate insulin gene expression and insulin secretion.The important results include the following points:1.We constructed a new bait in which part of Isll transcription activating domain was deleted and the autoactivation was eliminated.The new bait could be used in yeast two-hybrid system.We then built a testis cDNA library,and the titer remains 5.0×107 cfu/mL.2.Four Isll interacting proteins were screened by yeast two-hybrid system.They are protein inhibitor of activated STAT 4(PIASy),aryl-hydrocarbon receptor-interacting protein(Aip),myb-related transcription factor,partner of profilin(Mypop)and sin3-associated polypeptide 18(Sap18).3.The interaction between Isll and PIASy were then confirmed by Y2H and the coimmunoprecipitation experiment.Immunofluorescence double staining results showed Isll and PIASy colocalize in pancreas and NIT Cells.4.In cultured NIT and primary pancreatic islet cells,the insulin mRNA expression and insulin concentration were significantly enhanced in the cotransfection of Isll and PIASy overexpression vectors and significantly reduced in the cotransfection of Isl1 siRNA and PIASy siRNA.These findings demonstrate that PIASy mediates the effect of Isll on insulin gene expression and insulin secretion by proteins interaction.Both PIASy and Isll mRNA expression levels were upregulated in type 2 diabetic db/db mice with hyperinsulinemia.The results suggest that PIASy and Isll may be related with hyperinsulinism and type 2 diabetes.5.The insulin promoter activity activated by Isll and PIASy together was higher than either Isl1 or PIASy alone in dual luciferase assays.It indicates that PIASy is a transcription cofactor of Isll regulating insulin promoter activity.6.The insulin promoter activity significantly decreased for Isl11 mutant in lack of homeodomain or PIASy in lack of ring domain.The homeodomain of Isll and ring domain of PIASy are responsible for the coactivation of insulin promoter.Taken together,our research has screened four Isll interacting proteins.PIASy and Isll regulate insulin gene expression and secretion by activate insulin gene promoter in a synergetic manner.These results are helpful to find new factors involved in insulin gene expression and secretion and give new insight into pathogenesis of diabetes.