alpha5 subunit in Trypanosoma brucei proteasome can self-assemble to form a cylinder of four stacked heptamer rings.
alpha5 subunit in Trypanosoma brucei proteasome can self-assemble to form a cylinder of four stacked heptamer rings.
复制标题
布氏锥虫蛋白酶体中的α5亚基可以自组装形成由四个堆叠的七聚体环组成的圆柱体。
DOI：
10.1042/0264-6021:3440349
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Wang,CC
中科院分区：
文献类型：
--
作者：
Yao,Y;Toth,CR;Huang,L;Wong,ML;Dias,P;Burlingame,AL;Coffino,P;Wang,CC
蛋白酶体在催化原核生物和真核生物的蛋白质降解中起着中心作用。20s蛋白酶体构成了它们的催化核心。为了研究布鲁氏锥虫20s蛋白酶体的结构，我们用二维凝胶电泳分离了一个27 kDa的亚基蛋白，估计pI为4.7，并对其进行了质谱分析。该蛋白的色氨酸序列与大鼠α5亚基相同。使用抗t血清。bruce20s蛋白酶体筛选aT。b. rhodesienseλ表达cDNA文库，获得一个全长蛋白cDNA克隆，编码246个氨基酸残基，计算分子量为27174 Da， pI为4.71。与大鼠和酵母蛋白酶体α5亚基分别具有50.0%和46.3%的序列一致性，且与2D凝胶纯化蛋白的质谱分析获得的肽段序列完全匹配。因此该蛋白被命名为α5亚基ofT。bruce20s蛋白酶体（TbPSA5）。在质粒转化大肠杆菌中表达的重组蛋白以27 kDa的单体形式和聚合形式存在，估计分子质量在190至800 kDa之间。在电子显微镜下，最高度聚合的形式呈现四层七聚体环的圆柱体外观，估计外径为14.5 nm，长度为18 nm，可被抗- T免疫沉淀。bruce20s蛋白酶体抗血清。鉴于已有文献记载的古细菌蛋白酶体α亚基自组装成双七聚体环，以及红红红球菌20 S蛋白酶体的两个α亚基自发组装。布鲁氏α亚基可能反映了原核生物和原始真核生物（如锥虫）所共有的蛋白酶体生物发生的共同特征，但在酵母和哺乳动物等高级真核生物中却明显缺失。
The proteasomes have a central role in catalysing protein degradation among both prokaryotes and eukaryotes. The 20 S proteasome constitutes their catalytic core. In studying the structure ofTrypanosoma brucei20 S proteasomes, we isolated by two-dimensional (2D) gel electrophoresis a 27 kDa subunit protein with an estimated pI of 4.7 and subjected it to mass spectrometric analysis. A tryptic peptide sequence from the protein was found identical with that of the rat α5 subunit. With the use of antiserum againstT. brucei20 S proteasomes to screen aT. b. rhodesienseλ expression cDNA library, we obtained a cDNA clone encoding a full-length protein of 246 amino acid residues with a calculated molecular mass of 27174 Da and a pI of 4.71. It bears 50.0% and 46.3% sequence identity with rat and yeast proteasome subunit α5 respectively, and matches all the peptide sequences derived from MS of the 2D gel-purified protein. The protein is thus designated the α5 subunit ofT. brucei20 S proteasome (TbPSA5). The recombinant protein, expressed in plasmid-transformedEscherichia coli, was found in a 27 kDa monomer form as well as polymerized forms with estimated molecular masses ranging from 190 to 800 kDa. Under the electron microscope, the most highly polymerized forms bear the appearance of cylinders of four-stacked heptamer rings with an estimated outer diameter of 14.5 nm and a length of 18 nm, which were immunoprecipitable by anti-(T. brucei20 S proteasome) antiserum. In view of the documented self-assembly of the archaeon proteasome α subunit into double heptamer rings and the spontaneous assembly of the two α subunits from the 20 S proteasome ofRhodococcus erythropolis, the self-assembly of theT. bruceiα subunit might reflect a common feature of proteasome biogenesis shared by prokaryotes and primitive eukaryotes such as the trypanosomes but apparently lost among the higher forms of eukaryote such as the yeast and the mammals.