巨噬细胞是检测和清除病原体并通过激活表面受体启动炎症反应的重要免疫前哨,包括Toll样受体(TLRs)。活化的TLR采用复杂的细胞运输和信号传导途径来启动炎性细胞因子程序的转录。我们之前已经表明Rab8a被多个TLRs激活,并通过募集效应子PI3Kγ调节下游Akt/mTOR信号传导,但目前尚不清楚TLR途径中Rab8a激活所需的鸟嘌呤核苷酸交换因子(GEF)。使用GST亲和下拉法和质谱分析,我们确定了一个Rab8特定的GEF,Grab,作为LPS激活的巨噬细胞中的Rab8a结合伴侣。免疫共沉淀和荧光显微镜显示GRAB和结构相似的GEF,Rabin8经历LPS诱导的与Rab8a的结合,并定位在细胞表面皱褶和大黄体上,它们与Rab8a介导的信号传导位点重合。使用CRISPR-Cas9介导的GRAB敲除(KO)细胞系进行Rab核苷酸激活测定,Rabin8和双KOs显示两种GEF都有助于TLR4诱导的Rab8aGTP负荷,但不是膜招募。此外,用双KO测量信号谱和活细胞成像显示,在TLR4驱动的炎症过程中,任一GEF单独地足以介导巨粒PI3Kγ依赖性Akt/mTOR信号传导,表明这些蛋白质之间存在冗余关系。因此,GRAB和Rabin8都被揭示为TLR信号传导和炎症程序的Rab8a核苷酸交换的关键正调节因子。这些GEF可用作操纵炎症的潜在靶标。缩写:TLR:Toll样受体;OCRL:Lowe蛋白的眼脑肾综合征;PI3Kγ:磷酸肌醇-3-激酶γ;LPS:脂多糖;GEF:鸟嘌呤核苷酸交换因子;GST:谷胱甘肽S-转移酶;BMMPH:骨髓来源的巨噬细胞;pleckstrin同源性;GAP:GTP酶激活蛋白1家族:低GDP结合蛋白1。
Macrophages are important immune sentinels that detect and clear pathogens and initiate inflammatory responses through the activation of surface receptors, including Toll-like receptors (TLRs). Activated TLRs employ complex cellular trafficking and signalling pathways to initiate transcription for inflammatory cytokine programs. We have previously shown that Rab8a is activated by multiple TLRs and regulates downstream Akt/mTOR signalling by recruiting the effector PI3Kγ, but the guanine nucleotide exchange factors (GEF) canonically required for Rab8a activation in TLR pathways is not known. Using GST affinity pull-downs and mass spectrometry analysis, we identified a Rab8 specific GEF, GRAB, as a Rab8a binding partner in LPS-activated macrophages. Co-immunoprecipitation and fluorescence microscopy showed that both GRAB and a structurally similar GEF, Rabin8, undergo LPS-inducible binding to Rab8a and are localised on cell surface ruffles and macropinosomes where they coincide with sites of Rab8a mediated signalling. Rab nucleotide activation assays with CRISPR-Cas9 mediated knock-out (KO) cell lines of GRAB, Rabin8 and double KOs showed that both
GEFs contribute to TLR4 induced Rab8a GTP loading, but not membrane recruitment. In addition, measurement of signalling profiles and live cell imaging with the double KOs revealed that either GEF is individually sufficient to mediate PI3Kγ-dependent Akt/mTOR signalling at macropinosomes during TLR4-driven inflammation, suggesting a redundant relationship between these proteins. Thus, both GRAB and Rabin8 are revealed as key positive regulators of Rab8a nucleotide exchange for TLR signalling and inflammatory programs. These
GEFs may be useful as potential targets for manipulating inflammation. Abbreviations: TLR: Toll-like Receptor; OCRL: oculocerebrorenal syndrome of Lowe protein; PI3Kγ: phosphoinositol-3-kinase gamma; LPS: lipopolysaccharide; GEF: guanine nucleotide exchange factor; GST: glutathione S-transferases; BMMs: bone marrow derived macrophages; PH: pleckstrin homology; GAP: GTPase activating protein; ABCA1: ATP binding cassette subfamily A member 1; GDI: GDP dissociation inhibitor; LRP1: low density lipoprotein receptor-related protein 1.