MIF intersubunit disulfide mutant antagonist supports activation of CD74 by endogenous MIF trimer at physiologic concentrations
MIF intersubunit disulfide mutant antagonist supports activation of CD74 by endogenous MIF trimer at physiologic concentrations
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DOI：
10.1073/pnas.1221817110
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发表时间：
2013-07-02
影响因子：
11.1
通讯作者：
Lolis, Elias J.
中科院分区：
文献类型：
--
作者：
Fan, Chengpeng;Rajasekaran, Deepa;Lolis, Elias J.
巨噬细胞移动抑制因子（MIF）是一种促炎细胞因子。除了已知的受体介导的生物活性外，MIF在同源三聚体的亚基之间具有未知功能的催化位点。每个亚基为相邻的亚基贡献三条β链，以形成每个单体的核心七链β片层。已经报道了MIF单体、二聚体或三聚体，但是结合并激活MIF受体（CD 74）的活性形式仍然是一个争论的问题。半胱氨酸突变体（N110 C），通过与相邻亚基的Cys-80形成二硫键，将MIF共价锁定为三聚体，用于研究这个问题。N110 C（锁定三聚体）保留了部分催化活性和与CD 74的受体结合，但没有细胞信号传导。野生型MIF诱导的细胞信号传导、体内肺中性粒细胞积聚和肺泡通透性被5倍过量的N110 C抑制。NMR和尺寸排阻色谱与光散射表明，N110 C可以形成一个更高的顺序与一个单一的锁定三聚体的平衡低聚物。X-射线结构证实了局部构象变化，破坏了亚基界面，导致了低聚物形式的全局变化。结构还证实这些变化与部分催化和受体结合活性一致。任何潜在的单体的情况下，保留部分催化和受体结合活性，尽管在突变体中的构象（和动力学）的变化，支持内源性MIF三聚体，结合和激活CD 74在纳摩尔浓度。这一结论对治疗开发具有影响。
Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine. In addition to its known receptor-mediated biological activities, MIF possesses a catalytic site of unknown function between subunits of a homotrimer. Each subunit contributes three beta-strands to adjacent subunits to form a core seven-stranded beta-sheet for each monomer. MIF monomers, dimers, or trimers have been reported, but the active form that binds and activates the MIF receptor (CD74) is still a matter of debate. A cysteine mutant (N110C) that covalently locks MIF into a trimer by forming a disulfide with Cys-80 of an adjacent subunit is used to study this issue. Partial catalytic activity and receptor binding to CD74 are retained by N110C (locked trimer), but there is no cellular signaling. Wild-type MIF-induced cellular signaling, in vivo lung neutrophil accumulation, and alveolar permeability are inhibited with a fivefold excess of N110C. NMR and size-exclusion chromatography with light scattering reveal that N110C can form a higher-order oligomer in equilibrium with a single locked trimer. The X-ray structure confirms a local conformational change that disrupts the subunit interface and results in global changes responsible for the oligomeric form. The structure also confirms these changes are consistent for the partial catalytic and receptor binding activities. The absence of any potential monomer and the retention of partial catalytic and receptor binding activities despite changes in conformation (and dynamics) in the mutant support an endogenous MIF trimer that binds and activates CD74 at nanomolar concentrations. This conclusion has implications for therapeutic development.