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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{3. 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 3.0029,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{3. 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,Lys13. （or Lys13.） 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 {3.,and ~2Model Organism {3. center,shanghai institutes for Biologicol sciences, chinese academy of sciences,shanghai 20003.,{13. ~3.14} school of the chinese academy of sciences,shanghai 20003.,{13. institute of biochemistry and cell {3. 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,{3., {3. academy of sciences, graduate school of the chinese academy of sciences, 3.0 Yueyang Rd, shanghai 20003. College of Life Science, Jilin University, Changchun 13.061, china. Department of Genetics and Developmental biology, University of Connecticut Health center, Farmington, Connecticut 0603., 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. 3.epartment 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-3.-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

Dynamic Proteomics Based on Multiplex Strategies

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Dynamic Proteomics Based on Multiplex Strategies

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

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,{3. academy of sciences, 3.0 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 {3., integrated

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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,{3., {3. academy of sciences, shanghai 20003., china. ~2Department of Urology, Tongji Hospital, Tongji University, 3.9 Xincun Road, shanghai 200065, china. ~3.ew Drug R & D center, North china Pharmaceutical Group Corporation, 3.8 Heping East Road, Shijia Zhuang 050015, Hebei, china. ~4school of life sciences, Fudan University, 220 Handan Road, shanghai 20043., 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.

LARGE-SCALE QUANTITATIVE PROTEOMIC research THE INTERDEPENDENCE BETWEEN cell CYCLE AND cell DIFFERENTIATION USING PRE-ADIPOCYTE, 3.3.L1 cell MODEL

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LARGE-SCALE QUANTITATIVE PROTEOMIC RESEARCH THE INTERDEPENDE...

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

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,{3. academy of sciences, shanghai, 20003., china

Pre-adipocyte 3.3.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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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...

Receptor tyrosine kinases positively regulate BACE activity and Amyloid-β production through enhancing BACE internalization

中国蛋白质组学第三届学术大会

作者： Cheng-Jian Tu Jie Dai Su-Jun Li Quan-Hu Sheng Wen-Jun Deng Qi-Chang Xia Rong Zeng research center for proteome analysis, Key Lab of Proteomics, institute of biochemistry and cell biology, shanghai institutes for biological sciences,{3. academy of sciences, shanghai, 200013., china

Immobilized pH gradients isoelectric focusing (IPG-IEF) is the first dimension typically used in two-dimensional gel electrophoresis (2-DE). It can also be used on its own in conjunction with tandem mass spectrometry (MS/MS) for the analysis of proteins. Here, we described a strategy combining isoelectric focusing in immobilized pH gradient strips, and mass spectrometry to create a new high-throughput and sensitive detection method. Protein mixture is separated by in-gel IEF, then the entire strip is cut into a set of gel

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cell research 2007年第5期17卷 389-401页

作者： Lin Zou1,2 Zhu Wang1 Li Shen1,3.Guo Bin Bao1 Tian Wang1 Jiu Hong Kang1 Gang Pei1 1 Laboratory of Molecular cell biology, institute of biochemistry and cell biology,{3., {3. academy of sciences, shanghai 20003., china 2 Clinical Molecular Medical center, Children's Hospital, Chongqing University of Medical Sicences, Chongqing 400011, china 3.11}, shanghai 20003., china LaboratoryofMolecularcellbiology InstituteofBiochemistryandCellBiologyShanghaiInstitutesforBiologicalSciencesChineseAcademyofSciencesShanghai200031China ClinicalMolecularMedicalcenter Children'sHospitalChongqing

Amyloid-β (AP) peptide, the primary constituent of senile plaques in Alzheimer's disease (AD), is generated byβ-secretase- and γ-secretase-mediated sequential proteolysis of the amyloid precursor protein (APP). The aspartic pro-tease, β -site APP cleavage enzyme (BACE), has been identified as the main P-secretase in brain but the regulation of itsactivity is largely unclear. Here, we demonstrate that both BACE activity and subsequent Aβ production are enhancedafter stimulation of receptor tyrosine kinases (RTKs), such as the receptors for epidermal growth factor (EGF) and nervegrowth factor (NGF), in cultured cells as well as in mouse hippocampus. Furthermore, stimulation of RTKs also inducesBACE internalization into endosomes and Golgi apparatus. This enhancement of BACE activity and Aβ production uponRTK activation could be specifically inhibited by Src family kinase inhibitors and by depletion of endogenous c-Src withRNAi, and could be mimicked by over-expressed c-Src. Moreover, blockage of BACE internalization by a dominantnegative form of Rab5 also abolished the enhancement of BACE activity and Aβ production, indicating the requirementof BACE internalization for the enhanced activity. Taken together, our study presents evidence that BACE activity andAβ production are under the regulation of RTKs and this is achieved via RTK-stimulated BACE internalization, andsuggests that an aberration of such regulation might contribute to pathogenic Aβ production.

关键词： β-site APP cleavage enzyme RTK Amyloid-β Src