The Interaction Between MMP26 And GRP78 In Vivo And In Vitro

Matrix metalloproteinases(MMPs)are a family of calcium ions(Ca2+)and zinc ions(Zn2+)-dependent enzymes.MMPs participate in a number of physiological processes including reproduction,development,morphogenesis,and tissue remodeling as well as several pathological conditions including arthritis,cardiovascular diseases,and cancer metastasis by their ability to degrade extracellular matrix(ECM).Human matrix metalloproteinase-26(MMP-26/endometase/matrilysin-2)is a putative biomarker for carcinomas of breast,prostate,and other cancers of epithelial origin.MMP-26 contains 261 amino acids and is expressed predominantly in the placenta and uterus.MMP-26 hydrolyzes and subsequently degrades a variety of proteins such as type IV collagen,fibronectin,fibrinogen,vitronectin,denatured collagen types I–IV,insulin-like growth factor.Expression analyses revealed that MMP-26 is detected in placenta,in endometrium and uterus,but is widely expressed in a wide range of malignant tumors in vivo and in vitro.These data together with the broad spectrum of proteolytic activity,suggest that MMP-26 may play a role in tissue-remodeling events associated to tumor progression.Immunological and proteomics approaches were employed to identify differential protein expression in MMP-26 knockdown cells.A comparison of the protein expression profiles of control and MMP-26 knockdown cells revealed nine differentially regulated proteins.Five of the proteins(heat shock protein 90,glucose-regulated protein 78(GRP78),annexin V,tropomyosin,and peroxiredoxin II)were up-regulated,while alpha-tubulin,cystatin SA-III,and beta-actin were down-regulated.Glucose-regulated protein 78(GRP78),also known as immunoglobulin heavy chain binding protein(Bip),is a member of the heat shock protein 70(HSP70)family.GRP78 is a chaperone.In the endoplasmic reticulum its biological function is to prevent newly synthesized peptide aggregation,regulate the balance of calcium ion in the endoplasmic reticulum and it isIV also involved in the transmembrane migration of the newly synthesized proteins,the mature,folding and transshipment of proteins,etc.GRP78 has been reported to be induced in different types of cancer,including breast,prostate,ovarian,renal,gastric and lung cancer,suggest that the expression of GRP78 is important to the occurrence and development of tumors.At the same time,it was found that tumor cells of overexpressing GRP78 had a certain degree of resistance to chemotherapeutic drugs,and knockout of GRP78 gene could significantly reduce the resistance of tumor cells to chemotherapeutic drugs,thus inhibiting GRP78 gene expression or inhibiting GRP78 function can as a program of cancer treatment.This suggests that we can target tumor treatment with GRP78 as the basis for the study of the interaction between MMP-26 and GRP78 in vivo and in vitro.First,we constructed a recombinant gene p GEX-4T-1-GRP78 which can expresses GRP78 protein in vitro and transformed it into express bacteria BL21(DE3)to obtain a large amount of soluble expression of GRP78 protein and purified by GST column to obtain GRP78-GST fusion Protein.The cat MMP-26 protein was expressed in the form of inclusion bodies.The natural conception and bioactivity were restored by dialysis renaturation.The activity of cat MMP-26 protein was detected by gelatin zymography and fluorescence spectrophotometry.Immunofluorescence analysis showed colocalization between the MMP-26 and GRP78.GST pull down assay analysis confirmed this interaction between MMP-26 and GRP78 in vitro.Simultaneous simulated human body temperature 37?,by immunoblotting assay we found MMP-26 can proteolytic GRP78 protein,The expression of MMP-26 protein in the cells was obtained by GFP affinity chromatography.The expression of MMP-26 and MMP-26 was prolonged by the expression of MMP-26 and sh RNA MMP-26.The expression of GRP78 protein was up-regulated and the proteolytic effect of MMP-26 on GRP78 was found.In this study,MMP-26 and GRP78 were co-located in endoplasmic reticulum.This has demonstrated the interaction of MMP-26 and GRP78 in vivo and in vivo,which will lay the foundation for exploring the relationship between MMP-26 and GRP78 and cancer.