Protein Signaling Domains Laboratory, Department of Biological Sciences, Virginia Tech, Blacksburg, VA 24061, USA.
Biochem J. 2011 May 1;435(3):597-608. doi: 10.1042/BJ20102160.
TLRs (Toll-like receptors) provide a mechanism for host defence immune responses. Activated TLRs lead to the recruitment of adaptor proteins to their cytosolic tails, which in turn promote the activation of IRAKs (interleukin-1 receptor-associated kinases). IRAKs act upon their transcription factor targets to influence the expression of genes involved in the immune response. Tollip (Toll-interacting protein) modulates IRAK function in the TLR signalling pathway. Tollip is multimodular, with a conserved C2 domain of unknown function. We found that the Tollip C2 domain preferentially interacts with phosphoinositides, most notably with PtdIns3P (phosphatidylinositol 3-phosphate) and PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate), in a Ca2+-independent manner. However, NMR analysis demonstrates that the Tollip C2 domain binds Ca2+, which may be required to target the membrane interface. NMR and lipid-protein overlay analyses suggest that PtdIns3P and PtdIns(4,5)P2 share interacting residues in the protein. Kinetic studies reveal that the C2 domain reversibly binds PtdIns3P and PtdIns(4,5)P2, with affinity values in the low micromolar range. Mutational analysis identifies key PtdIns3P- and PtdIns(4,5)P2-binding conserved basic residues in the protein. Our findings suggest that basic residues of the C2 domain mediate membrane targeting of Tollip by interaction with phosphoinositides, which contribute to the observed partition of the protein in different subcellular compartments.
TLRs（Toll-like receptors）为宿主防御免疫反应提供了一种机制。激活的 TLR 导致衔接蛋白募集到它们的胞质尾部，这反过来又促进了 IRAKs（白细胞介素 1 受体相关激酶）的激活。IRAKs 作用于其转录因子靶标，影响参与免疫反应的基因的表达。Tollip（Toll-interacting protein）调节 TLR 信号通路中的 IRAK 功能。Tollip 是多结构域的，具有一个保守的未知功能的 C2 结构域。我们发现，Tollip C2 结构域优先与磷酸肌醇相互作用，特别是与 PtdIns3P（磷脂酰肌醇 3-磷酸）和 PtdIns（4,5）P2（磷脂酰肌醇 4,5-二磷酸），以 Ca2+-非依赖性方式。然而，NMR 分析表明，Tollip C2 结构域结合 Ca2+，这可能是靶向膜界面所必需的。NMR 和脂质-蛋白覆盖分析表明，PtdIns3P 和 PtdIns（4,5）P2 在蛋白质中共享相互作用残基。动力学研究表明，C2 结构域可逆地结合 PtdIns3P 和 PtdIns（4,5）P2，亲和力值在低微摩尔范围内。突变分析确定了蛋白质中与 PtdIns3P 和 PtdIns（4,5）P2 结合的关键保守碱性残基。我们的发现表明，C2 结构域的碱性残基通过与磷酸肌醇的相互作用介导 Tollip 的膜靶向，这有助于观察到该蛋白在不同亚细胞隔室中的分布。
