The Role Of IER3IP1 On Secretion,Folding And Degradation Of (Pre) Proinsulin

Object: The infants with multiple irrelevant pedigrees in the clinic were found a disease called MEDS syndrome has developed because of an autosomal homozygous mutation in the IER3IP1(immediate early response 3 interacting protein 1)gene.The manifestations include neonatal diabetes,along with primary microcephaly with simplified gyral pattern,associated with severe infantile epileptic encephalopathy.At present,the research on the function of this protein is mainly summarized as two points: 1.When the endoplasmic reticulum cannot maintain steady state,it will cause endoplasmic reticulum stress,and endoplasmic reticulum stress activates unfolded protein reaction(UPR),trying to restore stability.The lack of IER3IP1 inhibits the UPR response and leads to the uncontrollable endoplasmic reticulum stress.2.IER3IP1 is homologous to the yos1 p protein in yeast and yos1 p cooperates with cop II(the coat protein)and participates in the transport of secretory proteins from the endoplasmic reticulum to Golgi,so inferred IER3IP1 may also have the same function as yos1 p.This experiment mainly started from IER3IP1 causing neonatal diabetes,focusing on its effects on(pre)proinsulin biosynthesis and endoplasmic reticulum homeostasis.First,we determined that IERIP1 is highly conserved in human,rat and mouse species.Secondly,studied the effect of IER3IP1 on the biosynthesis of insulin precursors.Thirdly,because IER3IP1 is located in the endoplasmic reticulum,it can be speculated that IER3IP1 may act on the ER quality control system.Therefore,this experiment predetected its effect on the endoplasmic reticulum-associated degradation(ERAD)and detected the endoplasmic reticulum.Based on the above hypothesis,this study designs experimental protocols.Materials and Methods:(1)First of all,in order to prove the IER3IP1 genes in various species of highly conservative,the experiment designed the primers according mice and rat IER3IP1 c DNA on PUNMED website,then extracted the ins-1(rat islet cells)and MIN6 cells(mice islet cell)m RNA,reverse transcription for c DNA,with two kinds of primers using PCR amplification c DNA,run the DNA gel,cut the ban,recycling,extracting DNA and sequencing.(2)The IER3IP1 gene in HEK293 T cells was knocked out by using crispr-cas9 technology,and the protein level of IER3IP1 was detected by western blotting,which verified the successful knockout.(3)The wild type HEK293 T cells and IER3IP1 cells were transfected with the mutant signal peptide R6C(the sixth position of the proinsulin signal peptide was mutated from arginine to cysteine,which affected the efficiency of the preproinsulin translocated into ER),observed the ratio of the preproinsulin and proinsulin.(4)Wild type HEK293 T cells in 12-well plates and IER3IP1-/-HEK293 T cells were transferred into wild-type insulin plasmids,respectively.1)proteins in cells and supernatant were collected,and the proportion of proinsulin in cells and supernatant in each group was compared.2)collected cell proteins and divided into two equal portions,one of which was added to 100 n M DTT,measured misfolded proteins in each group.Similarly,wild type preproparathyroid plasmid tagged with Myc was transfected to observe whether IER3IP1 affected the secretion of other secretory proteins.(5)Wild-type HEK293 T cells were respectively transferred into wild-type insulin plasmids and Akita plasmids.Immunoprecipitation technology was used to conduct immunoprecipitation with home-made proinsulin antibodies.The immunoprecipitated proteins were tested by western blotting to verify whether IER3IP1 directly binds to proinsulin and facilitates its transport.(6)Wild type and IER3IP1-/-HEK 293 T cell were transfected Akita plasmid(the proinsulin plasmid that A chain seventh cysteine mutated into tyrosine),constructed a cell model of endoplasmic reticulum stress caused by misfolding proinsulin artificially,and examined endoplasmic reticulum stress related proteins in these two kinds of cell lines.(7)Wild type and IER3IP-/-HEK293 T cells were transfected Akita and R6 C respectively,after 24 h,divided into two wells in each group,one of the well was treated with cyclohexan(CHX)for 5 hours in Akita group,30 min in R6 C group.observe the Akita proinsulin and R6 C preproinsulin degradation.Results: 1.Through the sequencing of rat and mouse IER3IP1 c DNA and the comparison with human IER3IP1 gene,it was found that this gene was highly conserved in species.2.The IER3IP1 gene in HEK293 T cells was knocked out by crispr-cas9 technology,and the protein level of IER3IP1 in the cells was significantly reduced by western blotting 3.IER3IP1 deletion does not affect the translocation of preproinsulin to endoplasmic reticulum.4.By comparing the protein amounts of wild-type proinsulin and wild-type proparathyroid hormone in HEK293 T cells,IER3IP1-/-HEK293 T cells and supernatant,it was found that IER3IP1 is not only involved in the secretion of insulin,but also in the secretion of other secreted proteins.5.It was found that IER3IP1 neither binds to wild-type proinsulin nor directly binds to mutated AKITA by means of immunoprecipitation.6.In the IER3IP1-/-HEK923 T,the degradation of misfolded Akita proinsulin was reduced,but there was no significant effect on the degradation of R6 C preproinsulin.Conclusion: 1.This experiment verified the high conservation of IER3IP1 among various species.2.The HEK293 T cell line with IER3IP1 protein knockout was successfully established.3.Because of deficiency of IER3IP1,proinsulin secretion decreased and misfolding proinsulin accumulated,and secretion of other secretory proteins proparathyroid protein also decreased.4.IER3IP1 neither binds to wild-type proinsulin nor to misfolded proinsulin.5.The knockout of IER3IP1 increased endoplasmic reticulum stress,but the activation of endoplasmic reticulum stress decreased when misfolded proteins appeared in cells.6.IER3IP1 plays a role in the endoplasmic reticulum associated degradation pathway.Lack of IER3IP1 can slow down the degradation of proteins and accumulate misfolded proteins in the endoplasmic reticulum.However,it had no significant effect on the degradation of proteins that did not enter the endoplasmic reticulum.This indicated that IER3IP1 was only related to the endoplasmic reticulum associated degradation.