The p40 Subunit of Interleukin (IL)-12 Promotes Stabilization and Export of the p35 Subunit IMPLICATIONS FOR IMPROVED IL-12 CYTOKINE PRODUCTION
The p40 Subunit of Interleukin (IL)-12 Promotes Stabilization and Export of the p35 Subunit IMPLICATIONS FOR IMPROVED IL-12 CYTOKINE PRODUCTION
复制标题
DOI：
10.1074/jbc.m112.436675
复制
发表时间：
2013-03-01
影响因子：
4.8
通讯作者：
Felber, Barbara K.
中科院分区：
文献类型：
--
作者：
Jalah, Rashmi;Rosati, Margherita;Felber, Barbara K.
IL-12是由p35和p40亚基组成的70-kDa异源二聚体细胞因子。为了使质粒DNA产生的细胞因子最大化，研究了在转录后和翻译后水平控制IL-12 p70生物合成的分子步骤。我们表明，RNA/密码子优化的基因序列的组合和细胞内两个亚基的相对表达水平的微调导致IL-12 p70异二聚体的产生增加。我们发现p40亚基在增强p35亚基的稳定性、细胞内运输和输出中起着关键作用。这种由p40亚基介导的翻译后调节在哺乳动物中是保守的。基于这些发现，产生了双基因表达载体，产生了两种亚基的最佳比例，导致与表达野生型序列的载体相比，人、恒河猴和鼠IL-12 p70产量增加了约1个对数。与表达野生型序列的质粒相比，这种优化的DNA质粒在小鼠体内DNA递送时也产生显著更高水平的全身生物活性IL-12。优化的鼠IL-12 DNA质粒的单次治疗性注射在小鼠B16黑色素瘤癌症模型中显示出对肿瘤发展的显著更有效的控制。因此，改进的IL-12 p70 DNA载体具有作为分子疫苗佐剂和用于癌症免疫治疗的体内应用的有希望的潜力。
IL-12 is a 70-kDa heterodimeric cytokine composed of the p35 and p40 subunits. To maximize cytokine production from plasmid DNA, molecular steps controlling IL-12p70 biosynthesis at the posttranscriptional and posttranslational levels were investigated. We show that the combination of RNA/codon-optimized gene sequences and fine-tuning of the relative expression levels of the two subunits within a cell resulted in increased production of the IL-12p70 heterodimer. We found that the p40 subunit plays a critical role in enhancing the stability, intracellular trafficking, and export of the p35 subunit. This posttranslational regulation mediated by the p40 subunit is conserved in mammals. Based on these findings, dual gene expression vectors were generated, producing an optimal ratio of the two subunits, resulting in a similar to 1 log increase in human, rhesus, and murine IL-12p70 production compared with vectors expressing the wild type sequences. Such optimized DNA plasmids also produced significantly higher levels of systemic bioactive IL-12 upon in vivo DNA delivery in mice compared with plasmids expressing the wild type sequences. A single therapeutic injection of an optimized murine IL-12 DNA plasmid showed significantly more potent control of tumor development in the B16 melanoma cancer model in mice. Therefore, the improved IL-12p70 DNA vectors have promising potential for in vivo use as molecular vaccine adjuvants and in cancer immunotherapy.