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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 20003.,china 2 graduate school of the chinese academy of sciences,shanghai 20003.,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 20003.,china 5 Laboratory of Proteomics,institute of biochemistry and cell biology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 20003.,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

Expression of Monomeric Insulin Precursor in Pichia pastoris and Its Conversion into Monomeric B27 Lys Destripeptide Insulin by Tryptic Hydrolysis

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Acta Biochimica et Biophysica Sinica 2005年第4期37卷 234-240页

作者： Jin-Guo DING~(1,3) Jian FEI~2 Da-Fu CUI~1 You-Shang ZHANG~1 ~1institute of biochemistry and cell biology,and ~2Model Organism research center,shanghai institutes for Biologicol sciences, chinese academy of sciences,shanghai 20003.,china ~3graduate school of the chinese academy of sciences,shanghai 20003.,china institute of biochemistry and cell biology Graduate School of the Chinese Academy of Sciences Shanghai 200031 China

Monomeric B27 Lys destripeptide insulin (B27 Lys DTrI) was designed and produced from itsprecursor expressed in Pichia pastoris through tryptic hydrolysis instead of the less efficient *** monomeric B27 Lys DTrI precursor (MIP) was purified from a cultured medium *** by a combination of hydrophobic,size-exclusion,and ion-exchange *** purifiedMIP was converted,by tryptic hydrolysis,to B27 Lys DTrI,which was then purified by ion-exchangechromatography to homogeneity as assessed by native gel electrophoresis,HPLC,amino acid composition,and electrospray mass-spectrometric analysis.B27 Lys DTrI exhibited superior monomeric properties insize-exclusion *** yield of MIP was 200 mg per liter of culture,and the overall yield ofpurified B27 Lys DTrI from the crude MIP was 70%.The in vivo biological activity of B27 Lys DTrI asdetermined by the mouse convulsion assay was 21 U/mg,identical to that obtained by semisynthesis.

关键词： monomeric B27 Lys destripeptide insulin tryptic hydrolysis Pichia pastoris

Characterization of Function of Three Domains in Dishevelled-1: DEP Domain is Responsible for Membrane Translocation of Dishevelled-1

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cell research 2004年第4期14卷 324-330页

作者： Wei Jun PAN, Shu Zhao PANG, Tao HUANG, Hui Yun GUO, Dianqing WU, Lin LI Laboratory of Molecular cell biology, institute of biochemistry and cell biology, shanghai institutes for biological sciences, chinese academy of sciences, graduate school of the chinese academy of sciences, 320 Yueyang Rd, shanghai 20003. College of Life Science, Jilin University, Changchun 130061, china. Department of Genetics and Developmental biology, University of Connecticut Health center, Farmington, Connecticut 06030, USA. 1Laboratory of Molecular cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Graduate School of the Chinese Academy of Sciences 320 Yueyang Rd Shanghai 200031. 2College of Life Science Jilin University Changchun 130061 China. 1Laboratory of Molecular cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Graduate School of the Chinese Academy of Sciences 320 Yueyang Rd Shanghai 200031. 2College of Life Science Jilin University Changchun 130061 China. 3Department of Genetics and Developmental biology University of Connecticut Health Center Farmington Connecticut 06030 USA. 1Laboratory of Molecular cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Graduate School of the Chinese Academy of Sciences 320 Yueyang Rd Shanghai 200031.

Wnt signaling plays an important role in embryogenesis and tumorgenesis. Although the mechanism about how Wnts transduce their signaling from receptor frizzled (Fz) to cytosol has not been understood, dishevelled (Dvl) protein was considered as the intersection of Wnt signal traffic. In this study, we characterized the function of three domains (DIX, PDZ and DEP) of Dvl-1 in canonical Wnt signal transduction and Dvl-1 membrane translocation. It was found both DIX and DEP domain were sufficient to block Wnt-3a-induced LEF-1 transcriptional activity and free cytosol β-catenin accumulation; whereas PDZ domain and a functional mutant form of DEP domain (DEP-KM) had no effect on canonical Wnt signaling. In addition, when cotransfected with Fz-7, DEP domain, but not DIX, PDZ or DEP-KM, translocated and co-localized with Fz-7 to the plasma membrane, which was similar to Dvl-1. Furthermore, it was DEP domain that could block Fz-7-induced membrane translocation of Dvl-1 via a possible competitive mechanism. Thes

关键词： Wnt frizzled-7 dishevelled DIX PDZ DEP membrane translocation signal transduction

Impaired reproduction in transgenic mice overexpressing γ-aminobutyric acid transporter Ⅰ (GAT1)

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cell research 2004年第1期14卷 54-59页

作者： Jia Hua HU~(1,*), Jin Fu ZHANG~(2,*), Ying Hua MA~1, Jie JIANG~1, Na YANG~1, Xin Bo LI~3, Zhi Guang YU CHI~4, Jian FEI~(1,5,**), Li He GUO~(1,**) ~1Laboratory of Molecular cell biology, institute of biochemistry and cell biology, shanghai institutes for biological sciences, chinese academy of sciences, shanghai 20003., china. ~2Department of Urology, Tongji Hospital, Tongji University, 389 Xincun Road, shanghai 200065, china. ~3New Drug R & D center, North china Pharmaceutical Group Corporation, 388 Heping East Road, Shijia Zhuang 050015, Hebei, china. ~4school of life sciences, Fudan University, 220 Handan Road, shanghai 200433, china. ~5shanghai research center for Biomodel Organism, shanghai 201203, china. Laboratory of Molecular cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China.

It is well documented that γ-aminobutyric acid (GABA) system existed in reproductive organs. Recent researches showed that GABA_A and GABA_B receptors were present in testis and sperm, and might mediate the acrosome reaction induced by GABA and progesterone. GABA transporter Ⅰ (GAT1) also existed in testis and sperm, but its physiological function was unknown. In the present study, we used GAT1 overexpressing mice to explore GAT1 function in male reproductive system. We found that the expression level of GAT1 continuously increased in wild-type mouse testis from 1 month to 2 months after birth. GAT1 overexpression in mouse affected testis development, which embodied reduced testis mass and slowed spermatogenesis in transgenic mice. Moreover, transgenic mice showed increase of the percentage of broken sperm. The further study revealed that the reproductive capacity was impaired in GAT1 overexpressing mice. In addition, testosterone level was significantly low in transgenic mice compared with that in wi

关键词： GABA GAT1 testes sperm reproduction transgenic.

Dynamic Proteomics Based on Multiplex Strategies

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Dynamic Proteomics Based on Multiplex Strategies

LARGE-SCALE QUANTITATIVE PROTEOMIC research THE INTERDEPENDENCE BETWEEN cell CYCLE AND cell DIFFERENTIATION USING PRE-ADIPOCYTE, 3T3-L1 cell MODEL

中国蛋白质组学第三届学术大会

作者： Rong Zeng research center for proteome analysis, Key Lab of Proteomics, institute of biochemistry and cell biology, shanghai institutes for biological sciences, chinese academy of sciences, 320 YueYang Road, shanghai, 20003., china

The proteomes in a cell and tissue display a very dynamic mode which involve in the dynamic expression, modification, location and interaction. The different features of proteome can also interact with each other and finally determine the cellular activities and functions. Therefore, the multiplex strategies were necessary discover the dynamics of proteome. In qualitative analysis, integrated

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LARGE-SCALE QUANTITATIVE PROTEOMIC RESEARCH THE INTERDEPENDE...

Regulated expression of TATA-binding protein-related factor 3(TRF3)during early embryogenesis

中国蛋白质组学第三届学术大会

作者： Xingjun Cao, Jianhua Zhang, Quanhu Sheng, Jie Dai, Jiarui Wu, Rong Zeng research center for proteome analysis, Key Lab of Proteomics, institute of biochemistry and cell biology, shanghai institutes for biological sciences, chinese academy of sciences, shanghai, 20003., china

Pre-adipocyte 3T3-L1 cell is a kind of excellent models to study the cell differentiation. It is required about two days for the cells from the confluence to the status that enter into the GO phase and could be induced by the inducers to cell differentiation during the cell culture. In this process the mechanism how the cells retreat from the cell cycle gradually is yet unknown. Here we applied a novel quantitative proteomic strategy, SILAC

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cell research 2006年第07期16卷 8+611-621页

作者： Ye Yang~1 Jian Cao~1 Lu Huang~1 Hai Yan Fang~2 Hui Zhen Sheng~(1,2) ~1Laboratory of Stem cell biology,institute of biochemistry and cell biology,shanghai institutes.for biological sciences,chinese academy of sciences,shanghai 20003. china ~2center for Developmental biology,Xinhua Hospital,school of Medicine,shanghai Jiao Tong University,1665 Kong Jiang Road,shanghai 200092,china LaboratoryofStemcellbiology InstituteofBiochemistryandCellBiologyShanghaiInstitutesforBiologicalSciencesChineseAcademyofSciencesShanghai200031China centerforDevelopmentalbiology XinhuaHospitalSchoolofMedicine

RNA polymerase（Pol）Ⅱ transcription persists in TATA-box-binding protein（TBP）-/-mutant mouse embryos,indicat-ing TBP-independent mechanisms for Pol Ⅱ transcription in early ***-related factor3（TRF3）has beenproposed to substitute for TBP in TBP-/-mouse *** examined the expression of TRF3 in maturing oocytes andearly embryos and found that TRF3 was co-expressed with TBP in the meiotic oocytes and early embryos from the lateone-cell stage *** amounts of TBP and TRF3 changed dynamically and correlated well with *** immunoprecipitation（CHIP）assay revealed that different gene promoters in mouse embryonic stem（ES）cells recruited TRF3 and TBP *** analyses of TRF3 and TBP during cell cycle showed thatboth factors proceeded through cell cycle in a similar pace,except that TRF3 was slightly delayed than TBP in enteringthe nucleus when cells were exiting the *** from expression and biochemical analyses therefore support thehypothesis that TRF3 plays a role in early mouse *** addition,results from co-localization study suggestthat TRF3 may be also involved in Pol I transcription.

关键词： TBP TRF3 oocyte embryo ES cell NIH3T3 cell

香樟树种子中两种Ⅱ型核糖体失活蛋白凝集素活性的研究

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生物化学与生物物理进展 2004年第7期31卷 650-654页

作者：杨海燕李荣秀刘望夷中国科学院上海生命科学研究院生物化学与细胞生物学研究所中国科学院上海生命科学研究院生物工程研究中心

从香樟树成熟的种子分离到两种新的Ⅱ型核糖体失活蛋白 :新丰毒蛋白和辛纳毒蛋白 .两者A链的分子质量虽然相差近一倍 ,但B链的分子质量相同 .Ⅱ型核糖体失活蛋白的A链是RNAN 糖苷酶 ,B链是凝集素 ,A链进入细胞表现毒性在很大程度上依赖... 详细信息

从香樟树成熟的种子分离到两种新的Ⅱ型核糖体失活蛋白 :新丰毒蛋白和辛纳毒蛋白 .两者A链的分子质量虽然相差近一倍 ,但B链的分子质量相同 .Ⅱ型核糖体失活蛋白的A链是RNAN 糖苷酶 ,B链是凝集素 ,A链进入细胞表现毒性在很大程度上依赖于B链的糖结合活性和特异性 .对辛纳毒蛋白和新丰毒蛋白B链的凝集素活性进行了测定和比较 .红细胞凝集实验显示辛纳毒蛋白和新丰毒蛋白具有相同的细胞凝集活性 ,半抗原抑制实验发现它们都属于半乳糖型核糖体失活蛋白 ,荧光光谱法显示它们的糖结合常数也相同 .

关键词：新丰毒蛋白辛纳毒蛋白凝集素荧光光谱法

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High-Sensitivity analysis of Complex biological Samples by Immobilized Isoelectric Focusing Fractionation Coupled to Mass spectrometry Identification

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High-Sensitivity Analysis of Complex Biological Samples by I...