Effect Of INS-gene Mutations On Proinsulin Folding And Secretion

Object: Monogenic diabetes is a special type of diabetes caused by a single genetic mutation.Taking insulin gene mutation as an example,it has been found that more than 50 insulin gene mutations can cause early-onset diabetes.The Department of Endocrinology and Metabolism of General Hospital of Tianjin Medical University,conducted a monogenic diabetes screening.In order to explore the pathogenic mechanism of insulin gene mutations,this experiment studied its folding and secretory functions.Secondly,previous studies have shown that misfolded proinsulin caused by mutation may have an abnormal effect on the wild-type proinsulin,affecting its folding processing and secretion,thereby further aggravating the lack of insulin.This phenomenon is called dominant negative effect of mutant proinsulin on wild-type proinsulin.The three new mutations may also have a dominant negative effect.Materials and Methods: First,the wild-type proinsulin/mutant proinsulin overexpression plasmid with Myc tag was made and transfected into 293 T cells separately.After transfection for 48 hours,the protein in the cells and medium was collected,and count the proinsulin content and calculate its secretion rate,at the same time,the protein in the cell is divided into two parts,half of which is untreated,and the other half is added with a reducing reagent to open the disulfide bond,and then compare the misfolding of different proinsulin.Secondly,the Myc-tagged proinsulin was co-transfected into 293 T cells with untagged wild-type proinsulin.After transfection for 48 hours,the protein in the cells was collected with mild cell lysis buffer,and 10% of the total lysis was used as a control.90% of the protein is immunoprecipitated.All of the protein was added with reducing reagent to open the disulfide bond.The interaction between Myc-tagged proinsulin and untagged wild-type proinsulin was investigated by calculating the ratio of untagged wild-type proinsulin in 10% total lysis to that in immunoprecipitation.Then,the Myc-tagged proinsulin was transfected into 293 T cells together with the untagged wild-type proinsulin.After transfection for 48 hours,the proteins in the cells and the medium were collected to calculate the secretion rate of the untagged wild-type proinsulin.At the same time,the protein in the cell is divided into two parts,half of which is untreated,and the other half is added with a reducing reagent to open the disulfide bond,and the misfolding condition is observed.Finally,the Myc-tagged wild-type proinsulin or mutant proinsulin were separately transfected into ins1 cells,and after 24 hours of transfection,cell slides were performed,and immunofluorescence was performed after 48 hours of transfection.Myc+TGN38 or Myc+INS were co-stained to observe the localization of wild-type proinsulin or mutant proinsulin in cells and its effect on the synthesis of endogenous insulin in ins1 cell.Results: 1.Different clinical manifestations of insulin mutations vary in severity 2.Insulin gene mutations can lead to increased misfolding of mutant proinsulin and decreased secretion capacity.3.Mutant proinsulin interacts with wild-type proinsulin.4.Mutant proinsulin can affect the folding and secretion of co-expressed wild-type proinsulin.5.Mutant proinsulin is mainly localized in the endoplasmic reticulum and can affect the synthesis of endogenous insulin of ins1 cell.Conclusion: The newly discovered mutations(C43Y,R46 X and R55C)can interact with wild-type proinsulin.The C43 Y mutant proinsulin and R55 C mutant proinsulin can form a mismatched disulfide bond with wild-type proinsulin and affect folding and processing of wild-type proinsulin.Thereby mutant proinsulin can reduce the synthesis of insulin and lead to the occurrence and development of diabetes.