共查询到8条相似文献,搜索用时 15 毫秒
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Expression and characterization of the first kunitz domain of human tissue factor pathway inhibitor-2 总被引:15,自引:0,他引:15
Kong D Ma D Bai H Guo H Cai X Mo W Tang Q Song H 《Biochemical and biophysical research communications》2004,324(4):1179-1185
Human tissue factor pathway inhibitor-2 (hTFPI-2) has three kunitz domains whose structure and function are unclear. We expressed the first kunitz domain of hTFPI-2 (hTFPI-2/KD1) as functional form using Pichia pastoris and investigated its characterization. In the experiment, hTFPI-2/KD1 can inhibit the plasmin and trypsin activity and the Ki of hTFPI-2/KD1 towards plasmin (30nM) and trypsin (50nM) was determined as 10 and 7nM by chromogenic assay, respectively. hTFPI-2/KD1 can also inhibit MMP-2 and MMP-9 in zymography assay. Furthermore, the inhibition of hTFPI-2/KD1 to the Matrigel invasion by HT-1080 is also described. This study provides a method to produce hTFPI-2/KD1 efficiently and some insights into the structure and function of hTFPI-2/KD1. 相似文献
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Su YC Miller TN Navaneetham D Schoonmaker RT Sinha D Walsh PN 《The Journal of biological chemistry》2011,286(36):31904-31914
To select residues in coagulation factor XIa (FXIa) potentially important for substrate and inhibitor interactions, we examined the crystal structure of the complex between the catalytic domain of FXIa and the Kunitz protease inhibitor (KPI) domain of a physiologically relevant FXIa inhibitor, protease nexin 2 (PN2). Six FXIa catalytic domain residues (Glu(98), Tyr(143), Ile(151), Arg(3704), Lys(192), and Tyr(5901)) were subjected to mutational analysis to investigate the molecular interactions between FXIa and the small synthetic substrate (S-2366), the macromolecular substrate (factor IX (FIX)) and inhibitor PN2KPI. Analysis of all six Ala mutants demonstrated normal K(m) values for S-2366 hydrolysis, indicating normal substrate binding compared with plasma FXIa; however, all except E98A and K192A had impaired values of k(cat) for S-2366 hydrolysis. All six Ala mutants displayed deficient k(cat) values for FIX hydrolysis, and all were inhibited by PN2KPI with normal values of K(i) except for K192A, and Y5901A, which displayed increased values of K(i). The integrity of the S1 binding site residue, Asp(189), utilizing p-aminobenzamidine, was intact for all FXIa mutants. Thus, whereas all six residues are essential for catalysis of the macromolecular substrate (FIX), only four (Tyr(143), Ile(151), Arg(3704), and Tyr(5901)) are important for S-2366 hydrolysis; Glu(98) and Lys(192) are essential for FIX but not S-2366 hydrolysis; and Lys(192) and Tyr(5901) are required for both inhibitor and macromolecular substrate interactions. 相似文献
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Lina Zhu ;Jiping Wang ;Jingui Mu ;Huijun Wang ;Chenqi Zhang ;Jue Wang ;Xingang Liu ;Xiaomin Yan ;Linsen Dai ;Duan Ma 《Acta biochimica et biophysica Sinica》2009,(11):948-954
Human tissue factor pathway inhibitor-2 (hTFPI-2) is a serine protease inhibitor and its inhibitory activity is enhanced by heparin. The Kunitz domain 3 and C- terminal of hTFPI-2 (hTFPI-2/KD3C), which has the activity toward heparin calcium, have been successfully expressed in Pichia pastoris and purified by SP- Sepharose and heparin-Sepharose chromatography. The Fourier transformed infrared spectroscopy (FTIR), Raman spectroscopy, and circular dichroism (CD) experiment results implied that hTFPI-2/KD3C contained small contents of α-helix and β-strand, but large amounts of random coil and two kinds of disulfide bonds, gauche-gauche-gauche (ggg) and trans-gauchetrans (tgt). The interaction of hTFPI-2/KD3C with heparin calcium was investigated by CD. It was found that heparin calcium induced β-strands in hTFPI-2/ KD3C to different extents depending on the ratio of hTFPI-2/KD3C and heparin calcium. 相似文献
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Schmidt AE Chand HS Cascio D Kisiel W Bajaj SP 《The Journal of biological chemistry》2005,280(30):27832-27838
Kunitz domain 1 (KD1) of tissue factor pathway inhibitor-2 inhibits trypsin, plasmin, and factor VIIa (FVIIa)/tissue factor with Ki values of 13, 3, and 1640 nM, respectively. To investigate the molecular specificity of KD1, crystals of the complex of KD1 with bovine beta-trypsin were obtained that diffracted to 1.8 A. The P1 residue Arg-15 (bovine pancreatic trypsin inhibitor numbering) in KD1 interacts with Asp-189 (chymotrypsin numbering) and with the carbonyl oxygens of Gly-219 and Ogamma of Ser-190. Leu-17, Leu-18, Leu-19, and Leu-34 in KD1 make van der Waals contacts with Tyr-39, Phe-41, and Tyr-151 in trypsin, forming a hydrophobic interface. Molecular modeling indicates that this complementary hydrophobic patch is composed of Phe-37, Met-39, and Phe-41 in plasmin, whereas in FVIIa/tissue factor, it is essentially absent. Arg-20, Tyr-46, and Glu-39 in KD1 interact with trypsin through ordered water molecules. In contrast, insertions in the 60-loop in plasmin and FVIIa allow Arg-20 of KD1 to directly interact with Glu-60 in plasmin and Asp-60 in FVIIa. Moreover, Tyr-46 in KD1 electrostatically interacts with Lys-60A and Arg-60D in plasmin and Lys-60A in FVIIa. Glu-39 in KD1 interacts directly with Arg-175 of the basic patch in plasmin, whereas in FVIIa, such interactions are not possible. Thus, the specificity of KD1 for plasmin is attributable to hydrophobic and direct electrostatic interactions. For trypsin, hydrophobic interactions are intact, and electrostatic interactions are weak, whereas for FVIIa, hydrophobic interactions are missing, and electrostatic interactions are partially intact. These findings provide insight into the protease selectivity of KD1. 相似文献
5.
The conformation of Kunitz domain 1 of human tissue factor pathway inhibitor-2 (hTFPI-2/KD1) has been studied by Fourier transform infrared spectroscopy, circular dichroism, and Raman spectroscopy. It was found that hTFPI-2/KD1 contained approximately 17% s-helices, 24% β-strands, 46% random coils, 13% β-turns, and two kinds of disulfide bonds (ggg and tgt) at 25℃. The detailed conformational changes of the heated protein observed by Fourier transform infrared spectroscopy, circular dichroism and Raman spectroscopy revealed that hTFPI-2/KD1 was thermally stable. However, KD1 could form an intermediate form at high temperature, then return to its normal conformation when the temperature was lowered. Activity assays also showed that hTFPI-2/KD1 was able to keep its inhibitory activity on plasmin after being heated to 80℃ for 5min. 相似文献
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Danoy P Sonoda E Lathrop M Takeda S Matsuda F 《Biochemical and biophysical research communications》2007,352(3):763-768
Homologous recombination, a major double strand break repair pathway, plays critical roles in maintaining genome stability. Genetic polymorphisms in HR genes have been implicated in cancer risk. We report a novel assay system for evaluating polymorphisms in human homologous recombination genes using a panel of chicken DT40 repair mutants. We established mutant cell lines complemented with either wild-type or variant cDNAs of three human genes, RAD51, XRCC2, and XRCC3, and assessed their sensitivity to cisplatin and mitomycin C. DT40 mutants complemented with RAD51 coding and 5'UTR variants, and with a XRCC3 coding variant showed equivalent sensitivity as those with wild-type cDNAs. Interestingly, Xrcc2(-/-) DT40 cells complemented with variant XRCC2 (R188H) were more tolerant to cisplatin than those with wild-type XRCC2. Considering that the XRCC2 (R188H) allele reduces risk to epithelial ovarian cancer, the increased XRCC2 activity with the R188H polymorphism may have clinical benefit in preventing cancer risk. 相似文献