Identification Of Peptide Inhibitors Targeting The Interaction Between Ari13b And Smo

Background:The Hedgehog（Hh）signaling pathway was first discovered in 1980 by Nüsslein and Wieschaus in the study of genetic mutations that affect the development of drosophila somatic nodes.Recent studies have found that the Hh signaling pathway not only plays an important role in the process of development,its abnormal activation is involved in the occurrence and development of many kinds of malignant tumors.Abnormal activation of Hh signal pathway has been found in malignant tumors such as lung cancer,and some Hh signal pathway inhibitors have been used in the clinical treatment of some cancers.Smoothened is a crucial signal transduction element in Hh signaling pathway,mediates signal transduction in primary cilium and activates the expression of a series of downstream target genes.Hh pathway plays a‘switch’role.Inhibitors targeting Smo have been put into clinical use and have been widely used in the clinical treatment of many kinds of cancer.It is found that the mutation of Smo often leads to the resistance of tumor to Smo inhibitors,which greatly reduces its efficacy.Therefore,it is of great significance to find new targets to inhibit the activity of Hh signaling pathway.Arl13b,a member of the Arl（Arf related subfamily of the small GTP enzyme）Ras superfamily,is located on the primary cilia and is the most important ciliated protein.It is involved in the formation of cilia and the maintenance of ciliated function,such as vesicle transport and cell differentiation.Cell movement and cytoskeleton formation suggest that Arl13b may be closely related to the Sonic Hh signaling pathway,which is highly dependent on primary ciliates.The interaction between Arl13b and Smo can be used as a new target to inhibit the Hh signaling pathway.Targeting the interaction between Arl13b and Smo can be used in the treatment of cancer,therefore the question of Smo mutation resistence can be solved.Purpose:1.To verify the direct interaction between Arl13b and Smo2.To find out the smallest fragment of Arl13b interacting with Smo,then design and synthesize polypeptides3.To verify the effect of Arl13b small molecular peptides on Hh pathway.4.To study the synergistic effect of Arl13b peptide and Smo inhibitor Methods:1.The pMAL^TM protein fusion purification system was used to induce and purify the MBP-Smo 550-787aa,and the MBP-Pulldownwith His-Arl13bΔ19 protein was used to verify the direct interaction between the two proteins.On the one hand,it was transient transfected with Arl13b;On the other hand,the stable expression of Arl13b was accompanied by Hh inhibitor.The activity of Hh pathway and the localization of Smo on cell membrane and primary cilium were detected by Arl13b.2.In function,the effects of knockout Arl13b on cell proliferation,invasion and tumor formation were detected.3.I-TASSER website was used to predict the secondary structure of Arl13b.According to its secondary structure,the fragment plasmid was constructed according to its secondary structure,and the minimum segment of interaction between Arl13b and Smo was determined by MBP-Pulldown.4.Arl13b fragment plasmid was transfected into 293T and treated with circular target.The effect of Arl13b fragment plasmid on Hh pathway activity was detected,and the significance of this synergistic effect on the proliferation of gastric cancer cells was further examined.Results:1.Arl13b can promote the aggregation of Smo on the surface of cell membrane and primary cilium through direct interaction with Smo,thus activating Hh pathway,while down-regulation of Arl13b can inhibit cell proliferation and invade tumor formation.2.The minimal segment of interaction between Arl13b and Smo was successfully predicted and constructed,and the Arl13b segment not only inhibited the activity of Hh pathway,but also synergistically inhibited the activity of Hh pathway with Smo inhibitor.It can provide a new target for the treatment of tumor activated by Shh and solve the problem of drug resistance of Smo inhibitor alone.