Effects Of Cell Environment On The Signaling Pathways Of Cytokines Sharing ?C:the Study Of The Interaction Between ?C And Lipid Of Plasma Membrane And That Between ?C And PRDX2

The ?C family of cytokines consists of IL-2,IL-4,IL-7,IL-9,IL-15,and IL-21,members of I-type cytokines with four ?-helical bundle structures.Their common feature is that all of which signal through specific receptor complexes sharing the common ?receptor chain.As a result of this shared receptor usage,?C cytokines share some similar biological functions in regulating the immune response and the homeostasis of the lymphatic system.The common gamma chain acts as a non-redundant receptor subunit in the signal transduction of ?C family cytokines thought it can not bind to cytokines alone.What's more,?C interact with cytokines mainly through non-polar interactions so their combination is weak.Studies have shown that cell membranes can influence receptor-mediated signaling processes by altering receptor aggregation,altering the conformation of receptors,and inhibiting or initiating phosphorylation of the intracellular domain of receptors.In addition,a certerin protein usually perform their own functions by forming complexes with other proteins,that is,the functional phenotype of a certain protein is achieved through the interaction with other proteins.Therefore,in order to have a deeper understanding of the role of ?C in the process of ?C-mediated signal transduction,it is necessary to investigate the interaction between the intracellular domain of ?C and cell membrane as well as identify the proteins that can interact with yc under physiological conditions.Based on the above discussion,the main contents of this paper are as follows:1.We successfully constructed the 6His-SUMO-ICD vector using molecular cloning techniques and meanwhile we heterologously express the protein in the E.coli expression system.The experimental results show that the SUMO fusion tag can increases the solubility of target protein in a certain extent,but the effect is not satisfactory.Since the fusion tag has a certerin influence on the function and structure of the target protein,it is necessary to use SUMO protease to remove the 6His-SUMO tag in subsequent experiments.Taken together,the relevant cDNAs of human ?C intracellular domain were cloned into the pET-28a vector downstream of 6His tag coding sequences via 5'NdeI and 3'XhoI.Similary,we used the E.coli prokaryotic expression system to induce the expression of 6His-ICD.The inclusion bodies we obtained exist in an insoluble form which has no biological activity.Then we performed dilution renaturation and dialysis refolding.Finally,we conducted further purification using FPLC to obtain high purity and correctly folded protein samples for subsequent experiments.2.We use phospholipids to simulate cell membranes at physiological pH and demonstrated that the intracellular domain of ?C can specifically binds to acidic phospholipids by liquid NMR,circular dichroism,and intrinsic fluorescence of proteins.Furthermore,we found that the above specific binding process is reversible.And we successfully constructed four mutants such as W310A,R285A+R289A,K294A,and K315A by site-directed mutagenesis in vitro and obtained high purity and biological activity of mutant protein.Further studies indicate that W310 in the cytoplasmic domain of ?C mainly contribute to it intrinsic fluorescence.Besides R285+R289 and K294 have no significant effect on the interaction between ?C-ICD and acidic lipids,while K315 has a great influence on which.Namely,when ?C-ICD binds to acidic phospholipids,other forces such as hydrophobic interactions may exsit except for electrostatic interactions.The kinetic simulation data of the previous research group showed that the residues between E284 and G316 in the periplasmic membrane region of ?C have stong and stable interactions with the POPG bilayer during the simulation.Obviously,the kinetic simulation results are consistent with the experimental results.3.We combined co-immunoprecipitation with the fixation of protein complexes in a cell by formaldehyde cross-linking to preliminary explore the interaction between ?C and PRDX2 under physiological conditions.Western blotting confirmed that both ?C and PRDX2 were expressed in co-transfected HEK293T cells,then the co-eluted immunoprecipitated complexes can be denatured by boiled and separated by SDS-PAGE and identified afterwards one by one by western blot.Since both ?C and PRDX2 were detected in the co-immunoprecipitation complex,we preliminarily thought that they could interact with each other in the cells.Subsequent our groups will further experiment to eliminate the possibility of false positive,while using bimolecular fluorescence complementation(BiFC)to further verify.