Mbonye Uri R, Wada Masayuki, Rieke Caroline J, Tang Hui-Yuan, Dewitt David L, Smith William L
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, USA.
J Biol Chem. 2006 Nov 24;281(47):35770-8. doi: 10.1074/jbc.M608281200. Epub 2006 Sep 25.
Cyclooxygenase (COX) isoforms catalyze the committed step in prostaglandin biosynthesis. The primary structures of COX-1 and COX-2 are very similar except that COX-2 has a 19-amino acid (19-AA) segment of unknown function located just inside its C terminus. Here we provide evidence that the major role of the 19-AA cassette is to mediate entry of COX-2 into the ER-associated degradation system that transports ER proteins to the cytoplasm. COX-1 is constitutively expressed in many cells, whereas COX-2 is usually expressed inducibly and transiently. In murine NIH/3T3 fibroblasts, we find that COX-2 protein is degraded with a half-life (t(1/2)) of about 2 h, whereas COX-1 is reasonably stable (t(1/2) > 12 h); COX-2 degradation is retarded by 26 S proteasome inhibitors. Similarly, COX-1 expressed heterologously in HEK293 cells is quite stable (t(1/2) > 24 h), whereas COX-2 expressed heterologously is degraded with a t(1/2) of approximately 5 h, and its degradation is slowed by proteasome inhibitors. A deletion mutant of COX-2 was prepared lacking 18 residues of the 19-AA cassette. This mutant retains native COX-2 activity but, unlike native COX-2, is stable in HEK293 cells. Conversely, inserting the COX-2 19-AA cassette near the C terminus of COX-1 yields a mutant ins594-612 COX-1 that is unstable (t(1/2) approximately 3 h). Mutation of Asn-594, an N-glycosylation site at the beginning of the 19-AA cassette, stabilizes both COX-2 and ins594-612 COX-1; nonetheless, COX mutants that are glycosylated at Asn-594 but lack the remainder of the 19-amino acid cassette (i.e. del597-612 COX-2 and ins594-596 COX-1) are stable. Thus, although glycosylation of Asn-594 is necessary for COX-2 degradation, at least part of the remainder of the 19-AA insert is also required. Finally, kifunensine, a mannosidase inhibitor that can block entry of ER proteins into the ER-associated degradation system, retards COX-2 degradation.
环氧化酶（COX）同工型催化前列腺素生物合成的关键步骤。COX-1和COX-2的一级结构非常相似，只是COX-2在其C末端内侧有一段19个氨基酸（19-AA）的功能未知片段。在此我们提供证据表明，19-AA片段的主要作用是介导COX-2进入将内质网（ER）蛋白转运至细胞质的内质网相关降解系统。COX-1在许多细胞中组成性表达，而COX-2通常是诱导性和短暂性表达。在小鼠NIH/3T3成纤维细胞中，我们发现COX-2蛋白的半衰期（t(1/2)）约为2小时，而COX-1相当稳定（t(1/2) > 12小时）；COX-2的降解受到26S蛋白酶体抑制剂的抑制。同样，在HEK293细胞中异源表达的COX-1相当稳定（t(1/2) > 24小时），而异源表达的COX-2的半衰期约为5小时，其降解也因蛋白酶体抑制剂而减缓。制备了一个缺失19-AA片段中18个残基的COX-2缺失突变体。该突变体保留了天然COX-2的活性，但与天然COX-2不同的是，它在HEK293细胞中是稳定的。相反，在COX-1的C末端附近插入COX-2的19-AA片段会产生一个不稳定的突变体ins594-612 COX-1（t(1/2)约为3小时）。19-AA片段起始处的N-糖基化位点Asn-594发生突变后，COX-2和ins594-612 COX-1都变得稳定；然而，在Asn-594处发生糖基化但缺少19个氨基酸片段其余部分的COX突变体（即del597-612 COX-2和ins594-596 COX-1）是稳定的。因此，虽然Asn-594的糖基化对于COX-2的降解是必需的，但19-AA插入片段的其余部分至少有一部分也是必需的。最后，甘露糖苷酶抑制剂 kifunensine可阻止内质网蛋白进入内质网相关降解系统，它能抑制COX-2的降解。
