Laboratory of Biological Cancer Therapy, Xuzhou Medical College, Xuzhou, Jiangsu 221002, P.R. China.
J Interferon Cytokine Res. 2012 Dec;32(12):575-82. doi: 10.1089/jir.2012.0055. Epub 2012 Oct 18.
Melanoma differentiation-associated gene-7/interleukin-24 (MDA-7/IL-24) uniquely takes on multiple anticancer functions, such as direct tumor cell cytotoxicity, immune stimulation, and antiangiogenic activities. MDA-7/IL-24 protein levels depend on proteasome degradation. Western blotting and coimmunoprecipitation analyses verified that the MDA-7/IL-24 protein was ubiquitinated and degraded by the 26S proteasome in Hela cells, which was confirmed by protein accumulation treated with proteasome inhibitor MG132. MDA-7/IL-24 contains 10 lysine sites: K63, K69, K78, K119, K123, K136, K179, K189, K203, and K206. Site-directed mutagenesis in these sites reveals that lysine 123 is the major internal lysine involvement of MDA-7/IL-24 ubiquitination. Our results further demonstrated that the mutation of lysine 123 to arginine led to the inhibition of ubiquitin-mediated degradation and the recovery of MDA-7/IL-24 protein level. At the same time, the apoptosis-inducing activity of K123R mutant was obviously stronger than wild-type MDA-7/IL-24 detected by 4'-6-diamidino-2-phenylindole (DAPI) assay and annexin V analysis. In addition, K123R mutant tilted the balance between antiapoptotic protein Bcl-2 and proapoptotic protein Bax, which subsequently induced the cleavage of caspase signaling cascade and initiated cell apoptosis. Together, lysine 123 is mainly implicated in the ubiquitination and degradation of MDA-7/IL-24. Inhibition of degradation and ubiquitination of MDA-7/IL-24 through mutation of lysine 123 result in enhanced stability of MDA-7/IL-24 and exhibits persistent tumor suppression activity compared with the wild type.
黑色素瘤分化相关基因 7/白细胞介素 24（MDA-7/IL-24）具有多种抗癌功能，如直接杀伤肿瘤细胞、免疫刺激和抗血管生成作用。MDA-7/IL-24 蛋白水平依赖于蛋白酶体降解。Western blot 和 coimmunoprecipitation 分析证实 MDA-7/IL-24 蛋白在 Hela 细胞中被 26S 蛋白酶体泛素化和降解，这一过程在使用蛋白酶体抑制剂 MG132 处理后可观察到蛋白积累得到证实。MDA-7/IL-24 含有 10 个赖氨酸位点：K63、K69、K78、K119、K123、K136、K179、K189、K203 和 K206。对这些位点进行定点突变表明赖氨酸 123 是 MDA-7/IL-24 泛素化的主要内部赖氨酸参与位点。我们的结果进一步表明，赖氨酸 123 突变为精氨酸导致泛素介导的降解抑制和 MDA-7/IL-24 蛋白水平的恢复。同时，通过 4'-6-二脒基-2-苯基吲哚（DAPI）检测和膜联蛋白 V 分析，K123R 突变体诱导的细胞凋亡活性明显强于野生型 MDA-7/IL-24。此外，K123R 突变体使抗凋亡蛋白 Bcl-2 和促凋亡蛋白 Bax 之间的平衡倾斜，随后诱导 caspase 信号级联的切割，引发细胞凋亡。总之，赖氨酸 123 主要参与 MDA-7/IL-24 的泛素化和降解。通过突变赖氨酸 123 抑制 MDA-7/IL-24 的降解和泛素化导致 MDA-7/IL-24 的稳定性增强，并表现出比野生型更强的持续肿瘤抑制活性。
