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

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，以使其快速分泌，但也避免其下调的延迟或泄漏，从而可能导致夸大或持续的炎症。调控水平包括TNF mRNA的转录、加工、核输出和稳定性、TNF前的翻译和TNF在细胞膜上的脱落。p38 MAPK/MK2/3通路主要在翻译水平上通过TNF mRNA 3 ' - utr中的富au元素（ARE）调控TNF生物合成，该元素与p38-和MK2/3底物结合，如hnRNP A0、tristetrprolin （TTP）和KSRP。到目前为止，通过磷酸化调节肿瘤坏死因子前的are依赖性翻译的分子机制尚不清楚。结合的mk2 /3底物TTP主要负责调节特定mrna的稳定性。它在翻译调节中的作用以及它在调节mRNA的稳定性和翻译之间可能的功能转换至今仍未被表征。我们已经生成了代表mk2 /3缺陷和mk2拯救基因型的巨噬细胞系，并获得了TTP参与mk2依赖的TNF mRNA和TTP自身mRNA的翻译调节的证据。我们已经成功地证明了SRF对TTP基因的转录调控，并鉴定出SRF辅助因子MRTF-A （MAL, MKL1）是MK2的底物。此外，我们已经鉴定了蛋白酶体的组分作为MK2的底物和TTP的相互作用伙伴。在此基础上，本项目提出的科学目标是阐明p38/ mk2 /3依赖翻译调控TNF生物合成的分子机制，特别是TTP和其他are结合蛋白的作用及其表达和活性的调控。此外，我们将进一步分析TTP在调控mRNA衰变中的作用。巨噬细胞中影响MK2-和ttp依赖性翻译的成分的鉴定和表征将包括亚细胞分离和多聚体分析，并结合分析相关mrna结合蛋白的分布及其敲除对TNF-和TTP-mRNA翻译的影响。这些蛋白对帽依赖性翻译和TTP的ARE-binding的影响将通过报告细胞和体外系统进行分析。MK2对TTP特异性磷酸化的作用将以TTP磷酸化位点突变体拯救TTP缺陷巨噬细胞为特征。此外，还将对TTP表达、修饰和降解的调控进行进一步的生化和遗传分析。完成该项目的目标将有助于理解炎症中基因表达转录后调控的主要原理。

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.