Fisheries College, Jimei University, Xiamen, 361021, China.
Fisheries College, Jimei University, Xiamen, 361021, China.
Fish Shellfish Immunol. 2022 Sep;128:455-465. doi: 10.1016/j.fsi.2022.08.038. Epub 2022 Aug 18.
Toll-interacting protein (Tollip) plays an important role in the innate immune response by negative regulation of the TLR-IL-1R signaling pathway. MyD88 serves as a universal adaptor in TLR-mediated NF-κB activation. However, the regulation mechanisms of Tollip in piscine MyD88-mediated NF-κB activation is largely unknown. In the present study, the cDNA sequence of LcTollip was identified from the large yellow croaker (Larimichthys crocea). The putative LcTollip protein encoded 275 amino acid residues, containing a N-terminal TBD domain, a central C2 domain, and a C-terminal CUE domain. Quantitative PCR showed that the most predominant constitutive expression of LcTollip was detected in spleen. In addition, LcTollip transcripts enhanced significantly after LPS and poly I:C challenge (P < 0.05). Cellular localization revealed that LcTollip existed in the cytoplasm and nucleus. Furthermore, the overexpression plasmids of wild type LcTollip as well as its six domain truncated mutants of LcTollip were constructed by overlap PCR. Dual luciferase analysis showed that NF-κB activation could not be induced by overexpression of LcTollip or its domain truncated mutants alone. However, the LcMyD88-induced-NF-κB activation was significantly suppressed by overexpression with LcTollip, and the truncated mutants LcTollip-ΔTBD, LcTollip-ΔC2, LcTollip-ΔCUE and LcTollip-ΔTBDΔCUE while not by LcTollip-ΔLR and LcTollip-ΔTBDΔC2. Moreover, co-immunoprecipitation (Co-IP) assay revealed that the interaction between LcTollip and LcMyD88 was through CUE domain. More interesting, IP and immunoblotting examination of HEK293T cells co-transfected with LcMyD88, LcTollip and HA-ubiquitin showed that LcMyD88 induced a dose-dependent de-ubiquitination of LcTollip while LcTollip enhanced a dose-dependent ubiquitination of LcMyD88. However, protein degradation investigation displayed that the proteolysis and ubiquitination of LcMyD88 were not connected. Our findings suggested that the LcTollip might involve in negative regulation TLR pathway by suppressing LcMyD88-mediated immune activation and improving the ubiquitination level of LcMyD88.
Toll 相互作用蛋白 (Tollip) 通过负调控 TLR-IL-1R 信号通路，在先天免疫反应中发挥重要作用。MyD88 作为 TLR 介导的 NF-κB 激活中的通用衔接子。然而，Tollip 在鱼类 MyD88 介导的 NF-κB 激活中的调控机制在很大程度上尚不清楚。本研究从大黄鱼 (Larimichthys crocea) 中鉴定出 LcTollip 的 cDNA 序列。推定的 LcTollip 蛋白编码 275 个氨基酸残基，包含 N 端 TBD 结构域、中央 C2 结构域和 C 端 CUE 结构域。定量 PCR 显示，LcTollip 的主要组成型表达在脾脏中检测到。此外，LPS 和多聚 I:C 刺激后 LcTollip 的转录显著增强（P<0.05）。细胞定位显示 LcTollip 存在于细胞质和细胞核中。此外，通过重叠 PCR 构建了野生型 LcTollip 及其六个结构域截断突变体的过表达质粒。双荧光素酶分析表明，LcTollip 或其结构域截断突变体的过表达本身不能诱导 NF-κB 激活。然而，LcTollip 过表达显著抑制了 LcMyD88 诱导的-NF-κB 激活，而截断突变体 LcTollip-ΔTBD、LcTollip-ΔC2、LcTollip-ΔCUE 和 LcTollip-ΔTBDΔCUE 则没有，而 LcTollip-ΔLR 和 LcTollip-ΔTBDΔC2 则没有。此外，共免疫沉淀（Co-IP）试验显示，LcTollip 与 LcMyD88 之间的相互作用是通过 CUE 结构域。更有趣的是，共转染 HEK293T 细胞的 LcMyD88、LcTollip 和 HA-泛素的 IP 和免疫印迹试验表明，LcMyD88 诱导 LcTollip 的去泛素化呈剂量依赖性，而 LcTollip 增强 LcMyD88 的泛素化呈剂量依赖性。然而，蛋白降解研究显示，LcMyD88 的蛋白水解和泛素化没有关联。我们的研究结果表明，LcTollip 可能通过抑制 LcMyD88 介导的免疫激活和提高 LcMyD88 的泛素化水平来负调控 TLR 通路。
