Tohmé Mira, Manoury Bénédicte
INSERM, Unité 1013, Paris, France; Université Paris Descartes, Sorbonne Paris Cité, Faculté de médecine, Paris, France; INSERM, Unité 932, Institut Curie, Paris, France.
INSERM, Unité 1013, Paris, France; Université Paris Descartes, Sorbonne Paris Cité, Faculté de médecine, Paris, France.
Methods Enzymol. 2014;535:141-7. doi: 10.1016/B978-0-12-397925-4.00009-2.
Microbial pathogens are recognized through multiple, distinct receptors such as intracellular Toll-like receptors (TLRs 3, 7, 8, 9, and 13) which reside in the endosomes and lysosomes. TLRs are sensitive to chloroquine, a lysomotropic agent that neutralizes acidic compartments indicating a role for endo/lysosomal proteases for their signaling. Indeed, upon stimulation, full-length TLR7 and 9 are cleaved into a C-terminal fragment and this processing is highly dependent on a cysteine protease named asparagine endopeptidase (AEP) in dendritic cells. A recruitment and a boost in AEP activity, which was induced shortly after TLR7 and 9 stimulation, are shown to promote TLR7 and 9 cleavage and correlate with an increased acidification in endosomes and lysosomes. Moreover, mutating a putative AEP cleavage site in TLR7 or 9 strongly decreases their signaling in DCs, suggesting perhaps a direct cleavage of TLR7 and 9 by AEP. These results demonstrate that TLR7 and 9 require a proteolytic cleavage for their signaling and identified a key endocytic protease playing a critical role in this process.
微生物病原体通过多种不同的受体被识别，如存在于内体和溶酶体中的细胞内 Toll 样受体（TLR 3、7、8、9 和 13）。TLR 对氯喹敏感，氯喹是一种溶酶体亲和剂，可中和酸性区室，这表明内体/溶酶体蛋白酶在其信号传导中发挥作用。事实上，在受到刺激后，全长 TLR7 和 9 会被切割成一个 C 端片段，这种加工过程高度依赖于树突状细胞中一种名为天冬酰胺内肽酶（AEP）的半胱氨酸蛋白酶。在 TLR7 和 9 受到刺激后不久诱导的 AEP 活性的募集和增强，被证明可促进 TLR7 和 9 的切割，并与内体和溶酶体中酸化增加相关。此外，突变 TLR7 或 9 中一个假定的 AEP 切割位点会强烈降低它们在树突状细胞中的信号传导，这表明可能是 AEP 直接切割了 TLR7 和 9。这些结果表明，TLR7 和 9 的信号传导需要蛋白水解切割，并确定了一种关键的内吞蛋白酶在这一过程中发挥关键作用。
