Overexpression of proteasome β5 subunit increases the amount of assembled proteasome and confers ameliorated response to oxidative stress and higher survival rates
Overexpression of proteasome β5 subunit increases the amount of assembled proteasome and confers ameliorated response to oxidative stress and higher survival rates
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DOI：
10.1074/jbc.m413007200
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发表时间：
2005-03-25
影响因子：
4.8
通讯作者：
Gonos, ES
中科院分区：
文献类型：
--
作者：
Chondrogianni, N;Tzavelas, C;Gonos, ES
蛋白酶体是主要的细胞蛋白水解机制，负责正常和受损蛋白质的降解。蛋白酶体在维持细胞稳态中起着重要作用。蛋白酶体功能的改变在包括衰老在内的各种生物现象中都有记录。我们最近发现，衰老的人成纤维细胞中蛋白酶体活性的下降与β型亚单位的下调有关。在这项研究中，我们通过开发和进一步表征许多不同的过表达β(5)亚基的人类细胞系，遵循了我们的初步观察。在WI38/T和HL60细胞中，β(5)亚基的稳定过表达导致其他β型亚基水平升高，三种蛋白酶体活性水平均升高。免疫沉淀实验表明，稳定克隆中组装的蛋白酶体水平增加。凝胶过滤分析显示，记录的较高水平的蛋白酶体组装与“自由”（未整合）β型亚基的有效整合直接相关，这些亚基被鉴定为在载体转染的细胞中积累。为了支持这一观点，我们还发现β(5)转染细胞中蛋白酶体成熟蛋白水平较低，从而揭示了与载体转染细胞相比，这些细胞中蛋白酶体组装的速度/水平有所增加。功能研究表明，β(5)-过表达的细胞系在接受各种氧化剂处理后可提高存活率。此外，我们证明这种增加的存活率是由于氧化应激后更高的降解率。最后，由于氧化被认为是导致衰老和衰老的主要因素，我们在原代IMR90人成纤维细胞中过度表达β(5)亚基，并观察到衰老延迟4-5倍。总之，这些数据证明了蛋白酶体基因上调后的表型效应，并为更好地理解蛋白酶体调控提供了见解。
The proteasome is the major cellular proteolytic machinery responsible for the degradation of both normal and damaged proteins. Proteasomes play a fundamental role in retaining cellular homeostasis. Alterations of proteasome function have been recorded in various biological phenomena including aging. We have recently shown that the decrease in proteasome activity in senescent human fibroblasts relates to the down-regulation of beta-type subunits. In this study we have followed our preliminary observation by developing and further characterizing a number of different human cell lines overexpressing the beta(5) subunit. Stable overexpression of the beta(5) subunit in WI38/T and HL60 cells resulted in elevated levels of other beta-type subunits and increased levels of all three proteasome activities. Immunoprecipitation experiments have shown increased levels of assembled proteasomes in stable clones. Analysis by gel filtration has revealed that the recorded higher level of proteasome assembly is directly linked to the efficient integration of "free" (not integrated) beta-type subunits identified to accumulate in vector-transfected cells. In support we have also found low proteasome maturation protein levels in beta(5) transfectants, thus revealing an increased rate/level of proteasome assembly in these cells as opposed to vector-transfected cells. Functional studies have shown that beta(5)-overexpressing cell lines confer enhanced survival following treatment with various oxidants. Moreover, we demonstrate that this increased rate of survival is due to higher degradation rates following oxidative stress. Finally, because oxidation is considered to be a major factor that contributes to aging and senescence, we have overexpressed the beta(5) subunit in primary IMR90 human fibroblasts and observed a delay of senescence by 4-5 population doublings. In summary, these data demonstrate the phenotypic effects following genetic up-regulation of the proteasome and provide insights toward a better understanding of proteasome regulation.