Regulation of TNF biosynthesis by MK2/3: Role of MK2/3-dependent expression and modification of TTP and its interplay with further ARE-binding proteins and co-factors

MK2/3 对 TNF 生物合成的调节：MK2/3 依赖性表达和 TTP 修饰的作用及其与其他 ARE 结合蛋白和辅因子的相互作用

• 批准号: 227410360
• 负责人: Professor Dr. Matthias Gaestel
• 金额: --
• 依托单位: Institut für Zellbiochemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2014-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/227410360?language=en
• 关键词: Regulation; TNF; biosynthesis; MK2; Role

TNF is a master cytokine of inflammatory signaling of macrophages. Its biosynthesis is tightly controlled to allow rapid secretion but also to avoid delay or leakiness in its down-regulation, which could result in exaggerated or persistent inflammation. The levels of regulation comprise transcription, processing, nuclear export and stability of the TNF mRNA, translation of pro-TNF and shedding of TNF at the cell membrane. The p38 MAPK/MK2/3 pathway regulates TNF-biosynthesis mainly at the translational level depending on the AU-rich element (ARE) in the 3’-UTR of TNF mRNA, which binds to p38- and MK2/3-substrates such as hnRNP A0, tristetraprolin (TTP), and KSRP. So far the molecular mechanisms regulating ARE-dependent translation of pro-TNF via phosphorylation are not understood. The ARE-binding MK2/3-substrate TTP is mainly held responsible for regulation of the stability of specific mRNAs. Its role in translational regulation and a possible switch in its function between regulation of stability and translation of mRNA have remained uncharacterized to date.We have generated macrophage cell lines representing the MK2/3-deficient and MK2-rescued genotype and have obtained evidence that TTP is involved in MK2-dependent translational regulation of TNF mRNA and also of TTP’s own mRNA. We have successfully demonstrated transcriptional regulation of the TTP gene by SRF and identified the SRF-cofactor MRTF-A (MAL, MKL1) as a substrate of MK2. Furthermore, we have characterized components of the proteasome as substrates of MK2 and interaction partners of TTP. Based on this preliminary work, the scientific objective of the project proposed is the elucidation of the molecular mechanisms of p38/MK2/3-dependent translational regulation of TNF biosynthesis with specific regard to the role of TTP and other ARE-binding proteins and regulation of their expression and activity. In addition, the role of TTP in regulated mRNA decay will be further analyzed.The identification and characterization of components influencing MK2- and TTP-dependent translation in macrophages will comprise sub-cellular fractionation and polysome profiling in combination with the analysis of the distribution of relevant mRNA-ARE-binding proteins and the effects of their knockdown on the translation of TNF- as well as TTP-mRNA. The effects of these proteins on cap-dependent translation and on ARE-binding of TTP will be analyzed by reporter and in vitro systems. The role of the specific phosphorylations of TTP by MK2 will be characterized by rescue of TTP-deficient macrophages with phosphorylation-site mutants of TTP. In addition, further biochemical and genetic analysis of the regulation of TTP expression, modification and degradation will be performed.Fulfilling the aims of this project will contribute to the understanding of major principles of post-transcriptional regulation of gene expression in inflammation.

TNF是巨噬细胞炎症信号传导的主要细胞因子。它的生物合成受到严格控制，以允许快速分泌，但也避免其下调的延迟或泄漏，这可能导致过度或持续的炎症。调节水平包括TNF mRNA的转录、加工、核输出和稳定性，pro-TNF的翻译和TNF在细胞膜上的脱落。p38 MAPK/MK2/3途径主要在翻译水平上调节TNF生物合成，这取决于TNF mRNA的3 '-UTR中的富含AU的元件（ARE），其结合p38-和MK2/3-底物，如hnRNP A0、tristetraprolin（TTP）和KSRP。到目前为止，通过磷酸化调节ARE依赖性pro-TNF翻译的分子机制尚不清楚。ARE结合MK 2/3底物TTP主要负责调节特定mRNA的稳定性。它在翻译调节和可能的开关之间的稳定性和mRNA的翻译调节其功能的作用仍然uncharacterized到data.We产生了巨噬细胞系代表MK2/3缺陷和MK2拯救的基因型，并已获得的证据表明，TTP参与MK2依赖的TNF mRNA的翻译调节，也TTP的自己的mRNA。我们已经成功地证明了TTP基因的转录调控SRF和确定的SRF-辅因子MRTF-A（MAL，MKL 1）作为MK2的底物。此外，我们的特点是组成部分的蛋白酶体作为底物的MK2和TTP的相互作用的合作伙伴。基于这一初步工作，该项目的科学目标是阐明TNF生物合成的p38/MK 2/3依赖性翻译调节的分子机制，特别是TTP和其他ARE结合蛋白的作用及其表达和活性的调节。此外，TTP在调节mRNA衰变中的作用将进一步分析。影响巨噬细胞中MK 2和TTP依赖性翻译的组分的鉴定和表征将包括亚细胞分级分离和多核糖体分析，以及相关mRNA-ARE结合蛋白的分布分析和其敲低对TNF-α和TTP-mRNA翻译的影响。这些蛋白质对TTP的帽依赖性翻译和ARE结合的影响将通过报告基因和体外系统进行分析。MK2对TTP的特异性磷酸化作用的特征在于用TTP的磷酸化位点突变体拯救TTP缺陷的巨噬细胞。此外，还将对TTP的表达、修饰和降解的调控进行进一步的生物化学和遗传学分析，实现本项目的目的将有助于理解炎症中基因表达的转录后调控的主要原理。