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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,{2.,shanghai 2.0032.China 2.Graduate School of the {2.,shanghai 2.0032.China 3 College of Life sciences,Zhejiang University,Hangzhou 31002.,China 4 shanghai Mass Spectrometry Center,shanghai institute of Organic Chemistry,{2.,shanghai 2.0031,China 5 {12. of Proteomics,institute of biochemistry and cell biology,shanghai institutes for biological sciences,{2.,shanghai 2.0031,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 Lys2.,Lys101,Lys196,Lys2.7,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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酵母蛋白质YJL084c的磷酸化分析

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生物化学与生物物理学报 2002年第4期34卷 433-438页

作者：石晓钟敖世洲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分子生物学国家重点实验室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分子生物学国家重点实验室上海2.0031

PCL6、PCL7(PAP1)和PHO80为酵母蛋白激酶PHO85的细胞周期蛋白因子 ,同属于PHO80亚类 ,它们在蛋白质序列上有较高的同源性 ,彼此在功能上有一定程度的重叠。YLR190w为先前鉴定的PCL7 PHO85的一个底物 ,根据BLAST比较 ,YJL0 84c与YJLR190... 详细信息

PCL6、PCL7(PAP1)和PHO80为酵母蛋白激酶PHO85的细胞周期蛋白因子 ,同属于PHO80亚类 ,它们在蛋白质序列上有较高的同源性 ,彼此在功能上有一定程度的重叠。YLR190w为先前鉴定的PCL7 PHO85的一个底物 ,根据BLAST比较 ,YJL0 84c与YJLR190w在一段区域内有一定的同源性 ,并且根据已公布公共数据库 ,YJL0 84c是PCL6的一个结合蛋白。利用双杂交系统分析了YJL0 84c与这三个细胞周期蛋白因子之间的相互作用 ,表明PCL7也可以结合YJL0 84c。利用免疫共沉淀证实它们在体内的相互作用 ;通过体外GST沉降分析 ,验证它们的体外相互作用。YJL0 84c的体外翻译产物可以被PCL7 PHO85复合物磷酸化 ,进一步分别表达纯化YJL0 84c的氨基末端、中间和羧端三部分的GST融合蛋白片段 ,PCL7 PHO85复合物可以磷酸化其中间部分 ,而对N末端和C末端则未表现出磷酸化。以该中间片段蛋白质为底物 ,进一步研究高低磷条件对其磷酸化的影响。PCL7 PHO85对YJL0 84c的磷酸化受到无机磷条件的明显影响 :高磷条件下 ,磷酸化程度高 ;而低磷条件下磷酸化程度低。有意思的是 ,PCL6 PHO85免疫沉淀复合物也表现出同样的磷酸激酶特征。于是分析PHO81与这三个细胞周期因子之间相互作用。此外 ,还构建了Yjl0 84c的缺失株 ,分析了缺失的可能影响。

关键词： PCL6 PCL7 YJL084c 磷酸化酵母双杂交系统

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重组单链胰岛素在含有巯基试剂的变性剂中的解折叠

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生物化学与生物物理学报 2001年第4期33卷 431-436页

作者：郭占云乔志松冯佑民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!分子生物学国家重点实验室上海2.0031

重组单链胰岛素 (PIP)含有 3对二硫键。在含有巯基试剂的变性剂中 ,PIP产生二硫键交换从而形成一系列具有不同解折叠程度的二硫键异构体混合物。分别用高压液相色谱 (HPLC)和圆二色性 (CD)光谱分析了PIP在含有 0 .2.mol/L 2.巯基乙醇的... 详细信息

重组单链胰岛素 (PIP)含有 3对二硫键。在含有巯基试剂的变性剂中 ,PIP产生二硫键交换从而形成一系列具有不同解折叠程度的二硫键异构体混合物。分别用高压液相色谱 (HPLC)和圆二色性 (CD)光谱分析了PIP在含有 0 .2.mol/L 2.巯基乙醇的尿素和盐酸胍中的解折叠程度。PIP二硫键异构体混合物通过胰蛋白酶酶解并用质谱测定酶解片段的分子量 ,证明PIP确实产生了二硫键交换。同时还分离纯化了PIP的一种主要非天然二硫键异构体并研究了它重新折叠成天然构象的情况。观察到PIP只有一种热力学稳定的二硫键配对方式 ,PIP的非天然二硫键异构体在巯基试剂存在的条件下可以高效转化为天然二硫键配对。还将PIP解折叠和再折叠的情况与胰岛素样生长因子 I(IGF I)及胰岛素做了比较 :胰岛素和PIP只折叠成一种热力学稳定的三级结构 ,IGF I却折叠成两种热力学稳定的二硫键异构体 ;胰岛素的双链重组需缓慢进行 ,而PIP却可以快速折叠

关键词：重组单链胰岛素变性解折叠二硫键再折叠

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CaSRB9基因的克隆及其在酿酒酵母形态发生中的作用

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生物化学与生物物理学报 2002年第3期34卷 298-304页

作者：周舟曹芳陈江野中国科学院上海生命科学院生物化学与细胞生物学研究所分子生物学国家重点实验室中国科学院上海生命科学院生物化学与细胞生物学研究所分子生物学国家重点实验室上海2.0031

白色念珠菌是一种重要的人体致病真菌 ,致病机制与其形态发生紧密相关。酿酒酵母Flo8因子在其形态发生中起重要作用 ,我们把白色念珠菌基因组DNA导入酿酒酵母flo8基因缺失株中 ,筛选能够互补 flo8侵入生长缺陷的基因 ,分离到了一个与酿... 详细信息

白色念珠菌是一种重要的人体致病真菌 ,致病机制与其形态发生紧密相关。酿酒酵母Flo8因子在其形态发生中起重要作用 ,我们把白色念珠菌基因组DNA导入酿酒酵母flo8基因缺失株中 ,筛选能够互补 flo8侵入生长缺陷的基因 ,分离到了一个与酿酒酵母SRB9同源的新基因 ,命名为CaSRB9。该基因全长 4998bp ,编码一种16 6 5个氨基酸的蛋白质。在双倍体酿酒酵母中CaSRB9可以部分互补MAPK途径基因缺失株以及 flo8缺失株的菌丝生长缺陷 ;在单倍体酿酒酵母中表达能够互补 flo8缺失株的侵入生长缺陷 ,但在MAPK途径基因缺失株中不能形成侵入生长

关键词：白色念珠菌 CaSRB9 FLO8 侵入生长菌丝生长

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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,{2.,{2..Graduate School of the chinese academy of sciences,chinese academy of sciences,shanghai 2.0031,China 2.College of Life Science,Zhejiang Sci-Tech University,Hangzhou 310018,China 3 Bioengineering institute,East China University of Science and Technology,shanghai 2.02.3,China 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 SciencesShanghai 200031 China Bioengineering institute East China University of Science and TechnologyShanghai 200233 China College of Life Science Zhejiang Sci-Tech UniversityHangzhou 310018 China

Two novel endo-β-1,4-glucanases,EG45 and EG2.,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 EG2. appeared as a single bandon sodium dodecylsulfate polyacrylamide gel electrophoresis with a molecular mass of 45 kDa and 2. kDa,*** optimum pH for CMC activity was 5.5 for EG45 and 4.4-4.8 for *** optimumtemperature range for EG2. 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 EG2. was acceptably stable at pH 3.0-11.0 evenwhen the incubation time was prolonged to 2. h at 30℃,whereas EG45 remained relatively stable at *** 85% of the activity of EG2. could be retained upon incubation at 60℃ for 2. ***,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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Crystal Structure of the ARL2.GTP-BART Complex Reveals a New Recognition and Binding Mode of Small GTPase with Effector

Crystal Structure of the ARL2-GTP-BART Complex Reveals a New...

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2.08年全国生物化学与分子生物学学术大会

作者： Jian-ping Ding (state key {2. of {2. biology,institute of biochemistry and cell biology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 2.0031)

ARL2.is a member of the ARF family of small GTPases but has unique biochemical and structural features and appears to have distinct function(s) different from the ARF *** is an effector of ARL2.which has been shown to...

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Epigenetic regulation in Wnt signaling

Epigenetic regulation in Wnt signaling

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第三届细胞结构与功能的信号基础研讨会

作者： Lin Li state key {2. of {2. biology,institute of biochemistry and cell biology,shanghai institutes for biological sciences, chinese academy of sciences,shanghai 2.0031,China

The canonical Wnt signaling pathway controls and regulates many developmental processes through a complex of -catenin and the HMG-box transcription factors of the LEF1/TCFs *** co-activators including histone modification enzymes are recruited intoβ-cateninLEF1/TCFs transcription complex at target gene promoters to facilitate gene *** we show that two novel components,TDG and SET8,dock at TCFs binding element(TBE) of Wnt target genes,through interacting with LEF1/TCF4,to mediate CBP-catalyzed histone H4 acetylation and establish the mono-methylation of H4K2.(H4K2.me-l) as a marker for transcriptional activation,*** detailed mechanism will be discussed then.

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Purification,Gene Cloning and Expression of an Acidic Phospholipase A2.from Agkistrodon shedaoensis Zhao

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Acta Biochimica et Biophysica Sinica 2004年第1期36卷 27-32页

作者： Qian JIN, Li-Xia YANG, Hao-Mang JIAO, Bin LU, Yu-Qun WU, and Yuan-Cong ZHOU ( key laboratory of Proteomics, {2. of {2. and Ceil biology, shanghai institutes for biological sciences, the chinese academy of sciences, shanghai 2.0031, China {2. of Snakes and Snake Venom, Dalian She Dao Hospital, Dalian 116041, China ) key laboratory of Proteomics Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences the Chinese Academy of Sciences Shanghai 200031 China institute of Snakes and Snake Venom Dalian She Dao Hospital Dalian 116041 China

A protein with the activity of phospholipase A2.named asAPLA2.was purified to homogeneity from the venom of Agkistrodon shedaoensis Zhao through DEAE-Sepharose CL-6B anion exchange column, Source S and Mono Q FPLC. Its molecular weight was estimated as 19 kD by SDS-PAGE and its pI was about 3.5 by IEF analysis. It inhibits the platelet aggregation that was induced by 1 μmol/ L ADP, and the IC50 was determined to be 6 μmol/L. Degenerate primer was designed and synthesized according to the N-terminal amino acid sequence of asAPLA2. Its full-length cDNA was cloned by RT-PCR from the total RNA extracted from the snake venom gland. According to the deduced amino acid sequence, its molecular weight and pI are determined to be 13,649 and 4.39 respectively as calculated by DNAclub and DNAstar softwares. The gene was then cloned into the expression plasmid pET-40b(+) and expressed in E. coli BL2.(DE3). Western blot analysis indicated that the expressed protein cross-reacted with the antibody against the native

关键词： Agkistrodon shedaoensis Zhao APLA2 purification cloning gene expression

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Lys2.-Linked Nonproteolytic Poly-Ubiquitination of Axin by Smurf1 Negatively Regulates Wnt/β-Catenin Signaling

Lys29-Linked Nonproteolytic Poly-Ubiquitination of Axin by S...

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2.12.国发育生物学大会

作者： Cong Fei Lin Li state key {2. of {2. biology,institute of biochemistry and cell biology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai,2.0031

The Wnt/β-catenin signaling pathway is highly conserved among eukaryotic organisms,which is essentially important for *** of the Wnt signaling pathway is linked to tumorigenesis and many other human ***,the involvement of ubiquitin-mediated regulation has been emphasized in the Wnt/β-catenin signaling *** searching for the E3 ligases for Axin protein,one of core component in the Wnt/β-catenin signaling pathway, we identified Smad ubiquitination regulatory factor 1(Smurf1) as ***,the proteolytic activity of Smurf1-mediated ubiquitination is largely concerned and well *** this work,we disclosed,for the first time,a nonproteolytic role of *** data showed that,as a novel E3 ligase of Axin,Smurfl ubiquitinates Axin via K2.-linked poly-ubiquitination,which does not affect Axin stability but Axin-LRP6 interaction and Wnt-stimulated LRP6 phosphorylation,therefore inhibits Wnt/β-catenin ***,we provided compelling evidence that the inhibitory function of Smurfl in regulating Wnt signaling is cell cycle-dependent. Conclusions:Our data demonstrated,for the first time,a nonproteolytic function of Smurfl-mediated ubiquitination. Smurfl ubiquitinates Axin via K2.-linked poly-Ub chains,which results in attenuating Axin-LRP6 interaction and thus inhibiting Wnt/β-catenin signaling,and this regulatory role is in a cell cycle-dependent manner.

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Effect of specific nucleotide G11 on cleavage activity of hairpin ribozymes in vitro

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Progress in Natural Science:Materials International 2003年第5期 27-31页

作者： SONG Yuhu, LIN Jusheng , KONG Xinjuan, LI Bin, LIU Nanzhi and JIN Youxin (1. institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China 2. state {2. {2. of molecular biology, institute of biochemistry and cell biology, shanghai institutes for biological sciences, chinese academy of sciences, shanghai 2.0031, China) institute of Liver Diseases，Tongji Hospital，Tongji Medical College，Huazhong University of Science and Technology，Wuhan 430030，China state key {2. of {2. biology，institute of biochemistry and cell biology，shanghai institutes for Biolog institute of Liver Diseases，Tongji Hospital，Tongji Medical College，Huazhong University of Science and Technology，Wuhan 430030，China state key {2. of {2. biology，institute of biochemistry and cell biology，shanghai institutes for biological sciences，chinese academy of sciences，shanghai 2.0031，China

To study the effect of specific G11 nucleotide on cleavage activity of hairpin ribozyme in vitro, the 32.-labeled pKC transcript (2.7 nt) containing hepatitis B virus core region is used as target RNA. Synthetic hairpin ribozyme genes with G11 mutationa! site are cloned into ribozyme vector p1. 5 to create pHpRz for preparation of ribozyme. Non-labeled HpRz transcripts are incubated with 32.-labeled target-RNAs under different conditions and autoradiographed after gel electrophoresis. The results show that the cleavage activity of hairpin ribozyme is closely related to the matching of the bases between hairpin ribozyme and the substrate. The G11 site is not essential to cleavage activity of hairpin ribozyme in vitro, so the selection of target sequence matching G11 site of hairpin ribozyme is not limited to this site.

关键词： hairpin ribozyme kinetic of enzyme point mutant.

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