Identification of RNA-binding proteins in macrophages by interactome capture

病原体成分，如细菌脂多糖(LPS)，激活Toll样受体4(TLR4)，通过不同的下游途径诱导丝裂原激活的激酶(MAPKs)和NFkappaB刺激炎症细胞因子的表达。虽然这些介质对对抗和协调细胞感染反应是必不可少的，但它们的过度表达会导致全身炎症和败血症，这是除心血管疾病和癌症之外，全球第三大死亡原因。重要的是，转录后调控TLR4下游信号分子的表达有助于严密调控巨噬细胞炎性细胞因子的合成。新的证据强调了RNA结合蛋白(RBPs)在先天免疫反应的转录后控制中的作用。通过对脂多糖诱导和未处理的小鼠RAW 264.7巨噬细胞的RBP与RNA的相互作用组捕获、细胞裂解、多腺化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，并表征了其对内毒素刺激的反应。我们的数据揭示了巨噬细胞RNA相互作用组的402个蛋白质，包括91个以前未知的限制性商业惯例。与已发表的核糖核酸相互作用的比较发现，到目前为止有32个限制性商业惯例是RAW 264.7巨噬细胞所特有的。在这些蛋白质中，有19种与生化活动有关，与RNA没有直接关系。其中，具有胞浆前列腺素E2合成酶3(PTGES3)活性的HSP90辅助伴侣P23和造血细胞特异性LYN底物1(HCLS1或HS1)被确认为新的巨噬细胞RBPs。我们启动了紫外线交联和免疫沉淀(CLIP)，并结合mRNAs的深度测序，与新鉴定的巨噬细胞限制性商业惯例共同纯化。在此基础上，我们将生成mRNA-蛋白质相互作用图来识别编码TLR4下游信号分子或它们的调节器的特定mRNAs，并将分析识别出的靶mRNAs在体外和体内是如何被限制性商业惯例转录后调控的。在SPP 1935中，该项目将有助于扩大哺乳动物的RBP谱系，并将识别巨噬细胞mRNPs，这些mRNPs是调节和执行内毒素诱导的TLR4信号通路和先天性免疫反应的主要候选者。有关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作用的理解，并将提供关于它们作为治疗靶点的潜在知识，以预防全身炎症和脓毒症。

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.