Identification Of Affinity Domain Of IGFBP-7 For Insulin

Colorectal cancer(CRC)and typeâ…¡diabetes both threaten the health of human.Colorectal cancer as one of the most common gastrointestinal malignancies is the third cause of cancer-related death in western countries.In China,the incidence of colorectal carcinoma is rising from 10/10~5 to 30/10~5 in recent three decades,and its mortality ranks the 3th to 4th among all the cancer-related death,due to the changes of lifestyle and diet structure.Meanwhile,the incidence of typeâ…¡diabetes is also rising fast,from 1%in 1979 to 5%in 2002.Lots of studies confirm the association of typeâ…¡diabetes and hyperinsulinemia with the colorectal cancer,but the exact mechanism remained unclear.Therefore,it is of great significance to study the underlying mechanisms of colorectal cancer and typeâ…¡diabetes for their prevention and treatment.Insulin-like growth factor binding protein 7(IGFBP7)is screened from the cDNA subtraction library of colonic adenocarcinoma-normal mucosa by suppression subtractive hybridization(SSH) in our laboratory in 1999,which is differentially expressed in the colorectal cancer.We found that IGFBP7 might be a tumour suppressor gene in colorecatal cancer by exploring its biological effect in colorectal cancer cells,and detecting the association of its positive expression with the prognosis in colorectal cancer patients.Otherwise,we found the positive correlation between the expression of IGFBP7 and the level of fasting serum glucose.It has been verified that IGFBP7 serum concentrations were found to be increased in newly diagnosed typeâ…¡diabetic patients,which strengthens the association of IGFBP7 with insulin resistance.Compared with IGFBP 1-6,the affinity of IGFBP7 and IGFI is 5 times weaker,but 500 times stronger with insulin.Since the importance of insulin in the development of colorectal cancer and diabetes,the study of the affinity of IGFBP7 for insulin is meaningful.We used the methods of homology modeling,docking analysis, interaction energy curve analysis and high-throughput molecular field modeling to forecast the binding domain of IGFBP7 and insulin,which includes four amino regions:172-176,196-201, 217-220,235-244.Based on the results,we constructed the vectors and expressed the fragment proteinsâ… (171-244),01(1-171)and 02(244-283)to study their affinity for insulin and find the binding domain,which will lay a solid foundation for the future drug to regulate the binding. Meanwhile,we will test the mutant proteins' biological effects in colorectal cancer,and deduce the effects of binding of IGFBP7 with insulin in colorectal cancer for the next step to find an accurate target.The results show that proteinâ… and 02 both have affinity for insulin,but protein 01 doesn't have affinity for it,from which we can predict that the C-terminal of IGFBP7 protein is the binding region with insulin protein.Based on the researches of affinity of IGFBP7 to insulin,we concluded that:1.We use the methods of homology modeling,docking analysis,interaction energy curve analysis and high-throughput molecular field modeling to forecast the binding domain of IGFBP7 and insulin,which includes four regions:172-176,196-201,217-220,235-244; 2.Based on the results,we construct the vectors and express the fragment proteins: pET28a-W(32KD),pET41a-I(35KD),pET28a-01(21KD)and pET41a-02(32KD);3.The result of 4 proteins' affinity for insulin shows that proteinâ… and 02 both have affinity for insulin,but protein 01 doesn't have affinity for it,from which we can predict that the C-terminal of IGFBP7 protein is the binding region to insulin.