| Kindlin protein family was a kind of vinculin discovered in earlier 50th.Till now,three sub members,kindlin-1,kindlin-2 and kindlin-3,have been identified.Kindlin family members not only share a high degree of homology,but also are highly conservative.Kindlin-1 shares 62%amino acid identity with kindlin-2.kindlin-1 shares 49%amino acid identity with kindlin-3,and kindlin-2 shares 53%amino acid identity with kindlin-3.The Kindlin proteins are composed of constant region and variation area.The costant region of kindlin family have a unique domain architecture which are composed of a centrally located FERM domain interrupted by a PH domain.The structural domain is found in a number of proteins linking the membrane to the cytoskeleton.The structural domain is composed of three subunits which are F1,F2 and F3,and the F0 domain is prior to the structure of core domain.F3ofKindlin family is most homologous to F3 of the FERM domain of talin,which both contain Phosphotyrosine-binding domain(PTB)that can be combined with β3 cytoplasmic tail region of β integrin.Although many kinds of proteins interact with cytoplasmic tail region of integrin,before the function of kindlin-2 was elucidated,only talin was testified to active the integrin,the study of mice lacking Kindlin-3 proves kindlin was the essential element.Kindlin-2 locates cell-extracellular matrix adhesion,which can interact with ILK、cell-matrix adhesion protein.Besides,Kindlin-2 locates vinculin by connecting with the cytoplasmic tail region of integrin,and links to the adhesion protein、vasodilator-stimulated phosphoprotein and filamin,being an important connection between the cell-matrix adhesion complex and the skeleton protein.Kindlin-2 play an important role in activing the integrin,signal transduction is composed by ins-outs signal and outs-ins signal.On the other hand,Kindlin-2 maybe play a key role in occurrence and development of tumor.Some papers claimed the change in expression level of kindlin-2 is related to the occurrence and development of some kinds of tumor.In many kinds of colon cancer cells,the expression level of kindlin-2 is low,even none.Moreover,the kindlin-2 is expressed normally inleiomyosarcoma,but is overexpressed in leiomyoma.Also,overexpression of kindlin-2 is related to the decrease in invasiveness of stromal tumor.But,at the same time,some studies confirmed the overexpression of kindlin-2 in malignant melanoma,and advanced the cell adhesion and migration.All those results proved kindlin-2 correlated with occurrence and development of tumor.However,the mechanism of occurrenceinvasiveness and metastasis of tumor is not known.As to the role of kindlin-2 in the occurrence and development of tumor.We started to find the protein interacting with the kindlin-2.We used the specific anti-kindlin-2 antibody to pulldown the split product of prostatic cancer pc-3 cells.The sample was analyzed by 2-D DIGE,and the result proved the protein was Prdx4 belonging to Prdx family.Prdx family is a multi-functional protein family,the most important function is resist the damage of reactive oxygen species(ROS),but the mechanism of resisting ROS is different with other antioxidant enzyme,such as superoxide dismutase,catalase,and guaiaocl peroxidase.The molecular weight of Prdx family is about 20-30KD,and Prdx family distribute almost all the living body.Until now,the family has six subunits,according to the amount of cysteine residues which involve the redox reaction,the family was divided into three kinds:typical 2-Cys-Prdx(Prdxl-4)、atypical 2-Cys-Prdx(Prdx5)and 1-Cys-Prdx(Prdx6).Furthermore,the location of them is different in tissue and cells.The function of Prdx family on resisting ROS has both sides:one side,Prdx family act as the peroxiredoxin,protecting the cells by clearing ROS,this is good to body.On the other side,when the tissue and cells were exposed to the ROS,DNA would be damaged,at the same time,the impaired mitochondria generated less ATP,making the self-restorative ability of DNA was decreased,finally resulted the mutation,but the up-expression level of Prdx made the cells free to apoptosis,then the tumor occured.More and more evidences proved the expression level of Prdx4 was connected to occurrence and development of tumor,even someone suggested regarding Prdx4 as the biomarker of prostatic cancer.Moreover,Prdx4 was overexpressed in lung cancer.In conclusion,we guess the interaction between kindling-2 and Prdx4 may play a regulating role in the occurrence and development of tumor.Through the research of the interaction between them.We would know more about the occurrence and development of tumor,and provided new ways for the therapy of tumor caused by the inflammation.To proving the prediction,we did those work:Firstly,we the plasmids pcDNA3.0-HA-Prdx4(pcDNA3.0-HA was used as the negative control)and siRNAs against human Prdx4 gene(negative control RNA was used as the negative control)were transfected into HeLa cells,respectively.The expression of Prdx4 protein was analyzed by Western blotting.The expression levels of Prdx4 protein was up-regulated in HeLa cells after transfection with pcDNA3.0-HA-Prdx4 plasmids,while it was down-regulated in HeLa cells after transfection with Prdx4 siRNAs.After then,MTT assay was performed to detect the proliferation activity of HeLa cells,The proliferation activity of HeLa cells with overexpression of Prdx4 protein was not changed as compared with those of the blank control(without transfection)and the negative control(transfected with pcDNA3.0-HA)groups(all P>0.05).But siRNA-mediated interference of Prdx4 protein expression significantly decreased the proliferation activity of HeLa cells(P<0.05).Thirdly,we used flow cytometry(FCM)assay to detect the apoptosis ratio of HeLa cells,the result displayed,under the stimulation of NaAs02,overexpression of Prdx4 protein was not changed as compared with those of the blank control(without transfection)and the negative control(transfected with pcDNA3.0-HA)groups(F＝0.418,P>0.05);but the apoptosis ratio of HeLa cells in three hPrdx4 siRNAs was increased as compared with the negative control group(transfected with the negative control RNA)(F＝8.459,P<0.05),among them,the apoptosis ratio of HeLa cells in the hPrdx4 siRNA1、2、3 and hPrdx4 siRNAl +2+3(36.85±3.02)%、(36.22± 1.73)%、(35.58±3.24)%and(46.99±1.48)%was increased(t＝2.642,P<0.05;t=2.975,P<0.05;t＝2.763,P<0.05;t＝5.930,P<0.05),as compared with the apoptosis ratio of HeLa cells in negative control group(24.75±3.45)%。At last,to testify the interaction of them,we In the m vitro binding experiment。GST-Kindllin-2 and His—Prdx4 were expressed in E coli strain BL21,then purified with GST seprose and Ni—NTA resin,respectively.When these two purified protein were accessible,the in vitro binding was performed。The results showed that GST-Kindllin-2 could be co-precipitated by His—Prdx4,which pre—bounded to the Ni.NTA resin.and the GST protein alone could not in the same condition,which suggested that Prdx4could bind to Kindllin-2 in vitro.We can have this conclusion through the experiment:1.Expressed plasmid pcDNA3.0-HA/pcDNA3.0-HA-Prdx4 or hPrdx4 siRNA was transfected into cervical cancer HeLa cells.2.Overexpression of Prdx4 has no effects on the proliferation activity and apoptosis.3.Knockdown of Prdx4 leads to the decrease of proliferation activity and the increase of apoptotic rates.4.Kindlin-2 can bind to Prdx4 in vitro. |
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