| CD44 is a kind of widely expressed transmembrane glycoproteins,which mainly mediates the interaction between extracellular matrix and intracellular,and participates in a variety of physiological and pathological activities,such as hematopoiesis,development,cellular signal transduction,inflammation,tumor,wound healing and so on.Structurally,CD44 can be divided into extracellular domain(ECD),transmembrane domain(TM)and intracellular domain(ICD).The transmembrane segment of CD44 contains two presenilin-dependent endonuclease sites,only the outer site releases soluble CD44(s CD44)under the hydrolysis of membrane-associated metalloproteinases,and the near-cytoplasmic site releases ICD under the action of ?-secretase.Because of the variable region,different CD44 subtypes have different s CD44,while ICD is relatively conservative and participates in the regulation of cellular signal transduction.Autophagy is a highly conservative metabolic process in eukaryote evolution,which plays an important role in maintaining the homeostasis of intracellular environment and ensuring intracellular material circulation and energy flow.The initiation of autophagy depends on the autophagy initiation class III phosphatidylinositol-3-kinase(Ptd Ins3K)complex.In our previous study,we found that CD44 affects the Ptd Ins3 K complex by hydrolyzing and releasing ICD,which negatively regulates autophagy of vascular endothelial cells.However,how does CD44 ICD affect the Ptd Ins3 K complex? How does it work? These questions are not yet clear.We constructed a lentiviral expression vector of CD44 ICD and found that overexpression of CD44 ICD inhibits the expression of Phosphatidylinositol-3-Kinase Catalytic Subunit Type 3(PIK3C3),Phosphoinositide-3-Kinase Regulatory Subunit 4(PIK3R4)and UV Radiation Resistance Associated(UVRAG)protein in Ptd Ins3 K complex.Using yeast two-hybrid technique,it was found that there was no direct interaction between CD44 ICD and the core components of Ptd Ins3 K complex,PIK3C3,PIK3R4,BECN1,ATG14 L and UVRAG.Further studies showed that CD44 ICD interacted directly with signal transducer and activator of transcription 3(STAT3),and the core component of STAT3 and Ptd Ins3 K complex,PIK3R4 protein,interacted directly,suggesting that CD44 ICD interacted with Ptd Ins3 K complex with STAT3 as a link.By constructing a series of truncated mutants of CD44 ICD,we found that the 18 amino acids(18ICD)between the N-terminal 18-35 of CD44 ICD is the key peptide for its inhibition of autophagy.Considering that there are two serine phosphorylation sites in 18 ICD,and phosphorylation is an important modification of protein executive function,we further designed the mutation vector for the two serine sites of 18 ICD.The results show that the first serine site of 18 ICD is necessary in the process of negative regulation of autophagy.To sum up,this study analyzed the effect of CD44 ICD on Ptd Ins3 K complex and the specific mode of action,and clarified the core sites of CD44 negative regulation of cell autophagy.The research results will provide a new theoretical basis for revealing the mechanism of autophagy.In addition,after screening,we found a new autophagy inhibitor curcumin analogue(E)-3-((E)-4-chlorobenzylidene)-5-((5-methoxy-1H-indol-3-yl)methylene)-1-methylpiperidi n-4-one(CB-2).Immunofluorescence,flow cytometry,wound healing and Transwell chamber migration experiments showed that CB-2 could induce mitochondrial damage,increase the level of intracellular reactive oxygen species and inhibit the proliferation and migration of non-small cell lung cancer.This small molecule not only provides a new tool for autophagy research,but also provides a new drug lead compound for clinical treatment of non-small cell lung cancer.Our preliminary study found that this small molecule can regulate the level of intracellular CD44,which provides a potential molecular probe for the follow-up study of the molecular mechanism of CD44 regulating autophagy. |
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