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首页> 美国卫生研究院文献>Journal of Bacteriology >Biochemical and Physical Properties of the Methanococcus jannaschii 20S Proteasome and PAN a Homolog of the ATPase (Rpt) Subunits of the Eucaryal 26S Proteasome
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Biochemical and Physical Properties of the Methanococcus jannaschii 20S Proteasome and PAN a Homolog of the ATPase (Rpt) Subunits of the Eucaryal 26S Proteasome

机译：詹氏甲烷球菌20S蛋白酶体和PAN（真核26S蛋白酶体ATPase（Rpt）亚基的同源物）的生化和物理特性

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The 20S proteasome is a self-compartmentalized protease which degrades unfolded polypeptides and has been purified from eucaryotes, gram-positive actinomycetes, and archaea. Energy-dependent complexes, such as the 19S cap of the eucaryal 26S proteasome, are assumed to be responsible for the recognition and/or unfolding of substrate proteins which are then translocated into the central chamber of the 20S proteasome and hydrolyzed to polypeptide products of 3 to 30 residues. All archaeal genomes which have been sequenced are predicted to encode proteins with up to ∼50% identity to the six ATPase subunits of the 19S cap. In this study, one of these archaeal homologs which has been named PAN for proteasome-activating nucleotidase was characterized from the hyperthermophile Methanococcus jannaschii. In addition, the M. jannaschii 20S proteasome was purified as a 700-kDa complex by in vitro assembly of the α and β subunits and has an unusually high rate of peptide and unfolded-polypeptide hydrolysis at 100°C. The 550-kDa PAN complex was required for CTP- or ATP-dependent degradation of β-casein by archaeal 20S proteasomes. A 500-kDa complex of PAN(Δ1–73), which has a deletion of residues 1 to 73 of the deduced protein and disrupts the predicted N-terminal coiled-coil, also facilitated this energy-dependent proteolysis. However, this deletion increased the types of nucleotides hydrolyzed to include not only ATP and CTP but also ITP, GTP, TTP, and UTP. The temperature optimum for nucleotide (ATP) hydrolysis was reduced from 80°C for the full-length protein to 65°C for PAN(Δ1–73). Both PAN protein complexes were stable in the absence of ATP and were inhibited by N-ethylmaleimide and p-chloromercuriphenyl-sulfonic acid. Kinetic analysis reveals that the PAN protein has a relatively high Vmax for ATP and CTP hydrolysis of 3.5 and 5.8 μmol of Pi per min per mg of protein as well as a relatively low affinity for CTP and ATP with Km values of 307 and 497 μM compared to other proteins of the AAA family. Based on electron micrographs, PAN and PAN(Δ1–73) apparently associate with the ends of the 20S proteasome cylinder. These results suggest that the M. jannaschii as well as related archaeal 20S proteasomes require a nucleotidase complex such as PAN to mediate the energy-dependent hydrolysis of folded-substrate proteins and that the N-terminal 73 amino acid residues of PAN are not absolutely required for this reaction.
机译：20S蛋白酶体是一种自我分隔的蛋白酶，可降解未折叠的多肽，并已从真核生物，革兰氏阳性放线菌和古细菌中纯化。能量依赖的复合物，如真核26S蛋白酶体的19S帽，被认为负责底物蛋白的识别和/或展开，然后将其转移到20S蛋白酶体的中心腔中并水解为3的多肽产物至30个残基。预测所有已测序的古细菌基因组将编码与19S帽的六个ATPase亚基具有约50％的同一性的蛋白质。在这项研究中，这些古细菌的同系物之一被称为PAN，用于蛋白酶体激活核苷酸酶，其特征在于超嗜热型甲烷球菌。另外，通过体外组装α和β亚基，将詹氏甲烷球菌20S蛋白酶体纯化为700-kDa复合物，并且在100℃下具有异常高的肽水解率和未折叠的多肽水解率。 550 kDa PAN复合物是古细菌20S蛋白酶体依赖CTP或ATP依赖性降解β-酪蛋白的必需条件。一个500 kDa的PAN（Δ1-73）复合物，具有推定蛋白质的1至73位残基的缺失，并破坏了预测的N末端卷曲螺旋，也促进了这种依赖能量的蛋白水解。但是，这种缺失增加了水解的核苷酸类型，不仅包括ATP和CTP，还包括ITP，GTP，TTP和UTP。核苷酸（ATP）水解的最佳温度从全长蛋白的80°C降低到PAN（Δ1-73）的65°C。两种PAN蛋白复合物在没有ATP的情况下都是稳定的，并被N-乙基马来酰亚胺和对氯巯基苯基磺酸抑制。动力学分析表明，PAN蛋白对于ATP和CTP水解的相对最高Vmax为每毫克蛋白每分钟3.5和5.8μmolPi，并且对CTP和ATP的亲和力相对较低，Km值分别为307和497μM AAA家族的其他蛋白质。根据电子显微照片，PAN和PAN（Δ1-73）显然与20S蛋白酶体圆柱体的末端相关。这些结果表明，詹氏甲烷球菌和相关的古细菌20S蛋白酶体需要核苷酸酶复合物（例如PAN）来介导折叠底物蛋白质的能量依赖性水解，并且PAN的N末端73个氨基酸残基不是绝对必需的对于这个反应。

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