Ubiquitination of inducible nitric oxide synthase is required for its degradation
Ubiquitination of inducible nitric oxide synthase is required for its degradation
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DOI：
10.1073/pnas.192345199
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发表时间：
2002-09-17
影响因子：
11.1
通讯作者：
Eissa, NT
中科院分区：
文献类型：
--
作者：
Kolodziejski, PJ;Musial, A;Eissa, NT
诱导型一氧化氮合酶（iNOS）负责响应炎症介质从L-精氨酸合成一氧化氮（NO）。我们以前已经表明，NOS是通过26 S蛋白酶体降解。靶向蛋白质进行蛋白酶体降解可能需要或可能不需要它们与多泛素链的共价连接（泛素化）。此外，蛋白质的泛素化可以起到信号蛋白水解以外的功能。在这种情况下，它是不知道是否NOS是受泛素化或是否泛素化是必需的降解。在这项研究中，我们表明，诱导型一氧化氮合酶，表达在HEK 293细胞或诱导的原代支气管上皮细胞，A549细胞，或小鼠巨噬细胞，是受泛素化。为了研究iNOS泛素化是否是其降解所必需的，HEK 293 T细胞与含有人iNOS和显性负性泛素突变体K48 R的cDNA的质粒共转染。K48 R泛素化的破坏导致iNOS降解的抑制。ts 20是一种突变细胞系，其含有在高温下失活的热不稳定的泛素活化酶（Ell），从而防止泛素化。在非允许温度（40 ℃）下孵育稳定表达人iNOS的ts 20细胞导致NOS降解的抑制和iNOS的显著积累。这些研究表明，诱导型一氧化氮合酶是受泛素化和泛素化是必需的，其降解。
Inducible nitric oxide synthase (iNOS) is responsible for nitric oxide (NO) synthesis from L-arginine in response to inflammatory mediators. We have previously shown that NOS is degraded through the 26S proteasome. Targeting of proteins for proteasomal degradation may or may not require their covalent linkage to multiubiquitin chains (ubiquitination). In addition, ubiquitination of a protein can serve functions other than signaling proteolysis. In this context, it is not known whether NOS is subject to ubiquitination or whether ubiquitination is required for its degradation. In this study, we show that iNOS, expressed in HEK293 cells or induced in primary bronchial epithelial cells, A549 cells, or murine macrophages, is subject to ubiquitination. To investigate whether iNOS ubiquitination is required for its degradation, HEK293T cells were cotransfected with plasmids containing cDNAs of human iNOS and of the dominant negative ubiquitin mutant K48R. Disruption of ubiquitination by K48R ubiquitin resulted in inhibition of iNOS degradation. ts20 is a mutant cell line that contains a thermolabile ubiquitin-activating enzyme (Ell) that is inactivated at elevated temperature, preventing ubiquitination. Incubation of ts20 cells, stably expressing human iNOS, at the nonpermissive temperature (40degreesC) resulted in inhibition of NOS degradation and marked accumulation of iNOS. These studies indicate that iNOS is subject to ubiquitination and that ubiquitination is required for its degradation.