Identification of RNA-binding proteins in macrophages by interactome capture

病原体成分，如激活toll样受体4 （TLR4）的细菌脂多糖（LPS），通过不同的下游途径诱导丝裂原活化激酶（MAPKs）和NFkappaB，刺激炎症细胞因子的表达。尽管这些介质对对抗和协调细胞感染反应至关重要，但它们的过度表达会导致全身性炎症和败血症，这是除心血管疾病和癌症外，全球第三大死亡原因。重要的是，TLR4下游信号分子表达的转录后调控有助于严格调控巨噬细胞中炎症细胞因子的合成。新出现的证据强调了RNA结合蛋白（rbp）在先天免疫反应转录后控制中的作用。通过在LPS诱导和未处理的小鼠RAW 264.7巨噬细胞中采用rbp与RNA交联的RNA相互作用组捕获、细胞裂解、聚腺苷化RNA的寡核苷酸（dT）捕获和质谱分析，系统地鉴定了巨噬细胞rbp，并表征了它们对LPS刺激的反应。我们的数据揭示了巨噬细胞RNA相互作用组的402种蛋白质，其中包括91种以前未知的rbp。与已发表的RNA相互作用组的比较发现了迄今为止RAW 264.7巨噬细胞特有的32个rbp。其中，19种蛋白质与生物化学活动有关，与RNA没有直接关系。从这一组中，HSP90共伴侣P23被证明具有胞浆前列腺素E2合成酶3 （PTGES3）活性，以及造血细胞特异性Lyn底物1 (HCLS1或HS1)，一种造血特异性适配分子，被证实为新型巨噬细胞rbp。我们启动了紫外线交联和免疫沉淀（CLIP）结合mrna的深度测序，这些mrna与新鉴定的巨噬细胞rbp共同纯化。在此基础上，我们将生成mrna -蛋白相互作用图谱，以鉴定编码TLR4下游信号分子或其调节剂的特异性mrna，并分析鉴定出的靶mrna如何在体外和体内受到rbp的转录后调控。在SPP 1935中，该项目将有助于扩大哺乳动物RBP库，并将识别巨噬细胞mRNPs，这些mRNPs是lps诱导的TLR4信号通路和先天免疫反应的调节和执行的主要候选物。关于RBP功能的潜在分子机制的信息将促进对它们在炎症反应调节中的作用的理解，并将提供关于它们作为治疗靶点的潜力的知识，以预防全身性炎症和败血症。

Pathogen components, such as bacterial lipopolysaccharides (LPS) that activate Toll-like receptor 4 (TLR4), induce mitogen activated kinases (MAPKs) and NFkappaB through different downstream pathways to stimulate inflammatory cytokine expression. Although these mediators are essential to combat and coordinate the cellular infection response, their excessive expression causes systemic inflammation and sepsis, which are, besides cardiovascular diseases and cancer, the third leading cause of death worldwide. Importantly, post-transcriptional control of TLR4 downstream signaling molecule expression contributes to the tight regulation of inflammatory cytokine synthesis in macrophages. Emerging evidence highlights the role of RNA binding proteins (RBPs) in the post-transcriptional control of the innate immune response. By employing RNA interactome capture, which combines RBP-crosslinking to RNA in LPS-induced and untreated murine RAW 264.7 macrophages, cell lysis, oligo(dT) capture of polyadenylated RNAs and mass spectrometry analysis, macrophage RBPs were systematically identified and their response to LPS stimulation was characterized. Our data revealed 402 proteins of the macrophage RNA interactome including 91 previously unknown RBPs. A comparison with the published RNA interactomes identified 32 RBPs so far unique to RAW 264.7 macrophages. Of these, 19 proteins are linked to biochemical activities not directly related to RNA. From this group, HSP90 co-chaperone P23 that was demonstrated to exhibit cytosolic prostaglandin E2 synthase 3 (PTGES3) activity, and the hematopoietic cell-specific Lyn substrate 1 (HCLS1 or HS1), a hematopoietic specific adapter molecule, were validated as novel macrophage RBPs. We initiated UV-crosslinking and immunoprecipitation (CLIP) combined with deep sequencing of mRNAs that co-purify with novel identified macrophage RBPs. Based on that, mRNA-protein interaction maps will be generated to identify specific mRNAs encoding TLR4 downstream signaling molecules or their modulators and we will analyze how identified target mRNAs are post-transcriptionally regulated by RBPs in vitro and in vivo. Within the SPP 1935, the project will help to expand the mammalian RBP repertoire and will identify macrophage mRNPs that are prime candidates for the regulation and execution of LPS-induced TLR4 signaling pathways and the innate immune response. Information about underlying molecular mechanisms of RBP function will advance the understanding of their roles in inflammatory response modulation and will provide knowledge about their potential as therapeutic targets, to prevent systemic inflammation and sepsis.