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Rational redesign of inhibitors of furin/kexin processing proteases by electrostatic mutations

Acta Pharmacologica Sinica 2004年第12期25卷 146-152页

作者： Xiao-hui CAI, Qing ZHANG, Da-fu DING2 Key laboratory of proteomics, Institute of biochemistry and cell biology, shanghai institutes for biological sciences, chinese academy of sciences, graduate school of the chinese academy of sciences, shanghai 2.003., china Key laboratory of proteomics Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Graduate School of the Chinese Academy of Sciences Shanghai 200031 China

AIM: To model the three-dimensional structure and investigatethe interaction mechanism ofthe proprotein convertase furin/kexin and their inhibitors (eglin c mutants). METHODS: The three-dimensional complex structures of furin/ kexin with its inhibitors, eglin c mutants, were generated by modeller program using the newly published X-ray crystallographical structures of mouse furin and yeast kexin as templates. The electrostatic interaction energy of each complex was calculated and the results were compared with the experimentally determined inhibition con- stants to find the correlation between them. RESULTS: High quality models of furin/kexin-eglin c mutants were obtained and used for calculation of the electrostatic interaction energies between the proteases and their inhibitors. The calculated electrostaticenergies of interaction showeda linear correlation to the experimental inhibition constants. CONCLUSION: The modeled structures give good explanations of the specificity of eglin c mutants to furin/ kexin. The electrostatic interactions play important roles in inhibitory activity of eglin c mutants to furin/kexin. The results presented here provided quantitative structural and functional information concerning the role of the charge- charge interactions in the binding of furin/kexin and their inhibitors.

关键词： furin kexin eglin c drug design electrostatic mutation

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Detection of Tumor Marker CA125 in Ovarian Carcinoma Using Quantum Dots

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Acta Biochimica et Biophysica Sinica 2004年第10期36卷 681-686页

作者： Hui-Zhi WANG, Hai-Yan WANG, Ru-Qiang LIANG, and Kang-Cheng RUAN* Key laboratory of proteomics, Institute of biochemistry and cell biology, shanghai institutes for biological sciences,chinese academy of sciences, graduate school of the chinese academy of sciences, shanghai 2.003., china Key laboratory of proteomics Institute of Biochemistry and Cell BiologyShanghai Institutes for Biological SciencesChinese Academy of SciencesGraduate School of the Chinese Academy of SciencesShanghai 200031China

Semiconductor quantum dots (QDs) offer several advantages over organic dyes in fluores- cence-imaging applications, such as higher quantum yield, exceptional photostability, and a narrow, tunable, and symmetric emission spectrum. To explore whether QDs could specifically and effectively label tumor markers and be used in immunohistochemistry as a novel type of fluorescent probe, we used quantum dots with maximum emission wavelength 605 nm (QD605) to detect the ovarian carcinoma marker CA125 in specimens of different types (fixed cells, tissue sections, and xenograft piece). Additionally, we compared the photostability of QD signals with that of a conventional organic dye, FITC. All labeling signals of QDs were found to be more specific and brighter than those of FITC. Moreover, the QDs exhibited exceptional photostability during continuous illumination for 1 h by a high-intensity laser (Ar laser power 100 mW) at 488 nm, while the FITC signals faded very quickly and became undetectable after 24 min of illumination. These results indicate that QD-based probes can offer substantial advantages over existing fluorophores in many applications, and can be used effectively in immunohistochemistry as a novel class of fluorescent probes.

关键词： quantum dots detection of tumor marker CA125 ovarian carcinoma

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Positive Charges on Lysine Residues of the Extrinsic 18 kDa Protein Are Important to Its Electrostatic Interaction with Spinach Photosystem Ⅱ Membranes

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Acta Biochimica et Biophysica Sinica 2005年第11期37卷 737-742页

作者： Jin-Peng GAO Zhen-Hua YONG Feng ZHANG Kang-Cheng RUAN Chun-He XU Gen-Yun CHEN Institute of Plant Physiology & Ecology.shanghai institutes for biological sciences,chinese academy of sciences,shanghai 2.003.,china laboratory of proteomics,Institute of biochemistry and cell biology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 2.003.,china College of sciences,Zhejiang University.Hangzhou 310029,china graduate school of the chinese academy of sciences,shanghai 2.003.,china Institute of Plant Physiology & Ecology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200032 China graduate school of the chinese academy of sciencesshanghai 2.003. China College of sciences Zhejiang UniversityHangzhou 310029 China laboratory of proteomics Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200031 China

To determine the contribution of charged amino acids to binding with the photosystem Ⅱcomplex(PSⅡ),the amino or carboxyl groups of the extrinsic 18 kDa protein were modified with N-succinimidyl propionate(NSP)or glycine methyl ester(GME)in the presence of a water-soluble carbodiimide,*** on isoelectric point shift,4-10 and 10-14 amino groups were modified in the presence of2 and 4 mM NSP,***,3-4 carboxyl groups were modified by reaction with *** of negatively charged carboxyl groups with GME did not alter the binding activity of theextrinsic 18 kDa ***,the NSP-modified 18 kDa protein,in which the positively charged aminogroups had been modified to uncharged methyl esters,failed to bind with the PSⅡ membrane in the presenceof the extrinsic 23 kDa *** defect can not be attributed to structural or conformational alterationsimposed by chemical modification,as the fluorescence and circular dichroism spectra among native,GME-and NSP-modified extrinsic 18 kDa proteins were ***,we have concluded that the positive chargesof lysyl residues in the extrinsic 18 kDa protein are important for its interaction with PSⅡ membranes in thepresence of the extrinsic 23 kDa ***,it was found that the negative charges of carboxylgroups of this protein did not participate in binding with the extrinsic 23 kDa protein associated with PSⅡmembranes.

关键词： extrinsic 18 kDa protein circular dichroism electrostatic interaction chemical modification

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Identification and Characterization of GH Receptor and Serum GH-binding Protein in chinese Sturgeon (Acipenser sinensis)

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Acta Biochimica et Biophysica Sinica 2004年第12期36卷 811-816页

作者： Zhi-Yong LIAO1,2 and Shang-Quan ZHU2* 1College of Life sciences, Jiangxi Normal University, Nanchang 330027, china 2.ey laboratory of proteomics biochemistry and cell biology, shanghai institutes for biological sciences, chinese academy of sciences, graduate school of the chinese academy of sciences, shanghai 2.003., china College of Life sciences Jiangxi Normal UniversityNanchang 330027 China Key laboratory of proteomics biochemistry and cell biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Graduate School of the Chinese Academy of SciencesShanghai 200031 China

chinese sturgeon, a kind of cartilage ganoid, has a history of over one billion years and it is called the living fossil of aquatic biology since it keeps some evolutionary trace. Here, we characterized the growth hormone receptor (GHR) and serum growth hormone binding protein (GHBP) of chinese sturgeon. It was shown that GHR was expressed in various tissues, mainly in hepatic, kidney and intestine tissues. GHR on the hepatic membrane has high and specific affinity for bream GH (brGH) and Scatchard analysis of the binding data showed a single class of high affinity binding site with an association constant Ka of 3.1×109 M–1. A specific band around 94 kD was detected by SDS-PAGE in cross-linking studies of membrane receptors. After incubation of chinese sturgeon serum with 125I-brGH, a 125I-brGH-GHBP complex was identified by Sephadex G-75, indicating that in the serum exists GHBP specially binding to brGH.

关键词： chinese sturgeon growth hormone receptor growth hormone binding protein ligand binding

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Six Specific Lysihe Residues are Crucial in Maintaining the Structure and Function of Soluble Manganese Stabilizing Protein

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Acta Biochimica et Biophysica Sinica 2006年第9期 611-619页

作者： Jin-Peng GAO~(1,2#) Feng ZHANG~(1,3#) Li ZHANG~4 Yin-Long GUO~4 Kang-Cheng RUAN~5 De-An JIANG~3* Chun-He XU~1 1 Institute of Plant Physiology and Ecology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 2.003.,china 2 graduate school of the chinese academy of sciences,shanghai 2.003.,china 3 College of Life sciences,Zhejiang University,Hangzhou 310029,china 4 shanghai Mass Spectrometry Center,shanghai Institute of Organic Chemistry,chinese academy of sciences,shanghai 2.003.,china 5 laboratory of proteomics,Institute of biochemistry and cell biology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 2.003.,china

When manganese stabilizing protein （MSP） was treated with 0.5mM N-succinimidyl propionate（NSP）,the rebinding ability and oxygen-releasing capabilities of the modified MSP were not altered,in spiteof changes of MSP surface Lys ***,far-ultraviolet circular dichroism and intrinsicfluorescence spectra analysis revealed that 0.5 mM NSP-modified MSP retained most of its native secondaryand tertiary *** of the sites of NSP modification by Staphylococcus V8protease digestion ofthe modified protein,as well as analysis by matrix-assisted laser desorption ionization-time of flight massspectrometry,indicated that seven Lys residues were *** results suggested that these residues arenot absolutely essential to the structure and function of ***,when the NSP concentration wasincreased to 4mM,the modified MSP was unable to bind photosystem Ⅱ and completely lost its *** far-ultraviolet circular dichroism and intrinsic fluorescence spectra analysis revealed a clearconformational change in MSP after 4 mM NSP treatment,suggesting that some Lys residues are involved inmaintaining the structure and function of *** by matrix-assisted laser desorption ionization-timeof flight mass spectrometry indicated that another six Lys residues,namely Lys20,Lys101,Lys196,Lys207,Lys130 （or Lys137） and Lys66 （or Lys76）,were modified by 4 mM ***,these six Lys residuesare crucial in maintaining the structure and function of soluble MSP.

关键词： chemical modification fluorescence spectrum circular dichroism spectrum manganese stabilizing protein Lys residue

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Searching for Potential Drug Targets in Two-component and Phosphorelay Signal-transduction Systems using Three-dimensional Cluster Analysis

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Acta Biochimica et Biophysica Sinica 2005年第5期37卷 293-302页

作者： Xiao-Hui CAI Qing ZHANG Shuo-Yong SHI Da-Fu DING Key laboratory of Proteonmics,Institute of biochemistry and cell biology,Stanghai institutes for biological sciences, chinese academy of sciences,graduate school of the chinese academy of sciences, shanghai 2.003.,china Key laboratory of proteomics Institute of Biochemistry and Cell BiologyShanghai Institutes for Biological SciencesChinese Academy of SciencesGraduate School of the Chinese Academy of SciencesShanghai 200031China

Two-component and phosphorelay signal transduction systems are central components in thevirulence and antimicrobial resistance responses of a number of bacterial and fungal pathogens;in somecases,these systems are essential for bacterial growth and ***,we analyze in detail the conservedsurface residue clusters in the phosphotransferase domain of histidine kinases and the regulatory domain ofresponse regulators by using complex structure-based three-dimensional cluster *** also investigatethe protein-protein interactions that these residue clusters participate *** Spo0B-Spo0F complex structurewas used as the reference structure,and the multiple aligned sequences of phosphotransferases and responseregulators were paired *** results show that a contiguous conserved residue cluster isformed around the active site,which crosses the interface of histidine kinases and response *** residue clusters of phosphotransferase and the regulatory domains are directly involved in thefunctional implementation of two-component signal transduction systems and are good targets for thedevelopment of novel antimicrobial agents.

关键词： two-component system phosphorelay histidine kinase response regulator three-dimensional cluster analysis

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pH-Dependent Stability of EGX, A Multi-functional cellulase from Mollusca, Ampullaria crossean

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Acta Biochimica et Biophysica Sinica 2004年第9期36卷 603-608页

作者： Wen-Ying LI2, Ji WANG1, Yan-Hong LI1, Ming DING1, Gen-Jun XU1,3, Lan-Ying LIU2, and Fu-Kun ZHAO1,3* 1Key laboratory of proteomics, Institute of biochemistry and cell biology, shanghai institutes for biological sciences, the chinese academy of sciences,graduate school of the chinese academy of sciences, shanghai 2.003., china 2College of Life Science, Jilin University, Changchun 130023, china College of Life Science, Zhejiang University of Science and Technology, Hangzhou 310018, china Key laboratory ofproteomics Institute of Biochemistry and Cell BiologyShanghai Institutes for Biological Sciencesthe Chinese Academy of SciencesGraduate School of the Chinese Academy of SciencesShanghai 200031China

The cellulase activity and stability of EGX, a multi-functional cellulase previously purified from the mollusca Ampullaria crossean, was systematically studied under different pH. The pH induced con- formation and stability change of EGX have been investigated by using the intrinsic fluorescence, ANS fluorescence and CD spectrum. It has been found that the conformation and activity of this cellulase were strongly dependent on the pH. EGX was stable for both the enzyme activity and the conformation from pH 5.6 to pH 7.4. As shown by intrinsic and ANS fluorescence, no red shift of emission maximum occurred and a negligible intensity change was observed at pH 5.6–7.4. The activity of EGX remained about 80% in pH 5.6–7.4 and obviously decreased out of side the pH range. Urea-induced changes in EGX at pH 5.4 and pH 8.0 were measured by intrinsic fluorescence and CD spectrum. At pH 5.4, a significantly red shift of emission maximum occurred when the concentration of urea was 5 M compared to the concentration was 3 M at pH 8.0. The α-helix at pH 5.4 was 40.51% in the absence of urea and 31.04% in the presence of 4 M urea. At pH 8.0 the α-helix was 7.23% in the presence of 4 M urea. The data indicated that EGX was much susceptible to urea-induced unfolding at pH 8.0 and much stable at pH 5.4. The greater pH dependent stability of EGX may allow the enzyme to adequately catalyze the hydrolysis of cellulosic materials under natural or industrial extreme conditions.

关键词： multi-functional cellulase stability Ampullaria crossean fluorescence spectroscopy pH-dependent structural change CD spectrum

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Identification and Categorization of Horizontally Transferred Genes in Prokaryotic Genomes

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Acta Biochimica et Biophysica Sinica 2005年第8期37卷 561-566页

作者： Shuo-Yong SHI~1 Xiao-Hui CAI~1 and Da-fu DING~(1,2*) Key laboratory of proteomics.Institute of biochemistry and cell biology.shanghai institutes for biological sciences,chinese academy of sciences,graduate school of the chinese academy of sciences.shanghai 2.003.,china ~2 school of Life Science & Technology.shanghai Jiaotong University.shanghai 2.003..china Keylaboratoryofproteomics InstituteofBiochemistryandCellBiologyShanghaiInstitutesforBiologicalSciencesChin

Horizontal gene transfer(HGT),a process through which genomes acquire genetic materialsfrom distantly related organisms,is believed to be one of the major forces in prokaryotic genome ***,systematic investigation is still scarce to clarify two basic issues about HGT:(1) what types ofgenes are transferred:and(2)what influence HGT events over the organization and evolution of ***-scale investigations of these two issues will advance the systematical understanding ofHGT in the context of prokaryotic genome *** investigated 82 genomes,we constructed anHGT database across broad evolutionary *** identified four function categories containing a highproportion of horizontally transferred genes:cell envelope,energy metabolism,regulatory functions,andtransport/binding *** biased function distribution indicates that HGT is not completely random;instead,it is under high selective pressure,required by function restraints in ***,wemapped the transferred genes onto the connectivity structure map of organism-specific pathways listed inKyoto Encyclopedia of Genes and Genomes(KEGG).Our results suggest that recruitment of transferredgenes into pathways is also selectively constrained because of the tuned interaction between original *** organization structures still conserve well through evolution even with the recruitment ofhorizontally transferred ***,in pathways whose organization were significantly affected byHGT events,the operon-like arrangement of transferred genes was found to be *** resultssuggest that operon plays an essential and directional role in the integration of alien genes into pathways.

关键词： genome evolution horizontal gene transfer function and pathway analysis

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Comparative proteomic analysis of proteins involved in the aggregation of mouse embryonic carcinoma P19 cells and Overexpression Wnt-1 P19 cells

Comparative proteomic analysis of proteins involved in the a...

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华东六省一市生物化学与分子生物学学会2006年学术交流会

作者： Xia Gao,HongYu Tian,Cheng Wang,JianFeng lin,Li Liu, Caihong Xia,GenJun Xu,Naihe Jing FuKun Zhao (Key laboratory of proteomics, Institute of biochemistry and cell biology, shanghai institutes for biological sciences, chinese academy of sciences, shanghai,2.003., china Key laboratory of Stem cell biology and laboratory of Molecular cell biology, Institute of biochemistry and cell biology, shanghai institutes for biological sciences and graduate school of chinese academy of sciences, chinese academy of sciences, shanghai 2.003., china College of Life Science, Zhejiang Sci-Tech University,Hangzhou, 310018, china)

Mouse embryonic carcinoma P19 cells could be induced by retinoic acid (RA) to differentiate into neuroectodermal cell lineages, and this process is cell aggregation dependent. Wnt-1, the vertebrate counterpart of the Drosophila wingless gene, plays an important role in the early morphogenesis of neural tissues. Overexpression of Wnt-1 can direct embryonic carcinoma P19 cells to differentiate into neuron-like cells in the absence of retinoic acid. In this study, comparative proteomic analysis is utilized to approach the protein profiles associated with the aggregation of P19 cells and Overexpression Wnt-1 P19 cell (Wnt-l/Pl9). Image analysis of silver stained two-dimensional gels indicated that 79 protein spots had significantly differential expression patterns. 53 spots of them are related to both the P19 and the Wnt-l/Pl9 cell aggregation and 26 spots of them are uniquely related to the Wnt-1/ P19 aggregation. The differentially expressed proteins were identified by MALTI-TOF-MS/MS. Most of these proteins were identified as proteins involved in cell growth, metabolism and signal transduction, which may affect the self-renewal and pluripotency. These results provide the molecules involved in P19 cells aggregation and a novel perspective for possible mechanism underlying Wnt-1/ P19 cell neural differentiation.

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Purification and Characterization of Two Endo-β-1,4-glucanases from Mollusca,Ampullaria crossean

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Acta Biochimica et Biophysica Sinica 2005年第10期37卷 702-708页

作者： Yan-Hong LI~(1#) Rui GUO~(3#) Qiu-Yu YIN~1 Ming DING~1 Si-Liang ZHANG~3 Gen-Jun XU~(1,2) Fu-Kun ZHAO~(1,2*) 1 Key laboratory of proteomics,Institute of biochemistry and cell biology,shanghai institutes for biological sciences.graduate school of the chinese academy of sciences,chinese academy of sciences,shanghai 2.003.,china 2 College of Life Science,Zhejiang Sci-Tech University,Hangzhou 310018,china 3 Bioengineering Institute,East china University of Science and Technology,shanghai 200233,china Keylaboratoryofproteomics InstituteofBiochemistryandCellBiologyShanghaiInstitutesforBiologicalSciencesGrad

Two novel endo-β-1,4-glucanases,EG45 and EG27,were isolated from the gastric juice ofmollusca,Ampullaria crossean,by anion exchange,hydrophobic interaction,gel filtration and a secondround of anion exchange *** purified proteins EG45 and EG27 appeared as a single bandon sodium dodecylsulfate polyacrylamide gel electrophoresis with a molecular mass of 45 kDa and 27 kDa,*** optimum pH for CMC activity was 5.5 for EG45 and 4.4-4.8 for *** optimumtemperature range for EG27 was broad,between 50℃ and 60℃;for EG45 it was 50℃.The analysis on thestability of these two endo-β-1,4-glucanases showed that EG27 was acceptably stable at pH 3.0-11.0 evenwhen the incubation time was prolonged to 24 h at 30℃,whereas EG45 remained relatively stable at *** 85% of the activity of EG27 could be retained upon incubation at 60℃ for 24 ***,less than 10% residual activity of EG45 was detected at 50℃.Among different kinds of substrates,bothenzymes showed a high preference for carboxymethyl ***45,in particular,showed a carboxymethylcellulose hydrolytic activity of 146.5 IU/mg *** enzymes showed low activities to xylan (from oatspelt) and Sigmacell 101,and they were inactive to p-nitrophenyl-β-D-cellobioside,salicin and starch.

关键词： cellulase endo-β-1,4-glucanase Ampullaria crossean purification substrate specificity

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