In Vitro Physical and Functional Interaction Assays to Examine the Binding of Progranulin Derivative Atsttrin to TNFR2 and Its Anti-TNFα Activity.
In Vitro Physical and Functional Interaction Assays to Examine the Binding of Progranulin Derivative Atsttrin to TNFR2 and Its Anti-TNFα Activity.
复制标题
DOI：
10.1007/978-1-0716-1130-2_8
复制
发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Liu CJ
中科院分区：
文献类型：
--
作者：
Fu W;Hettinghouse A;Liu CJ
TNFα/TNFR信号在多种炎症和自身免疫性疾病的发病机制中发挥着关键作用，抗TNFα疗法已被认为是治疗多种自身免疫性疾病的有效方法。颗粒体蛋白前体 (PGRN) 是一种多面生长因子样分子，直接与 TNFR1 和 TNFR2 结合，特别是与后者的亲和力比 TNFα 更高。 PGRN 衍生物 Atsttrin 由 PGRN 的三个 TNFR 结合域组成，在多种炎症疾病模型（包括胶原诱导的关节炎）中表现出比 PGRN 更好的治疗效果。在此，我们描述了使用 1) 基于 ELISA 的固相蛋白质-蛋白质相互作用测定法来证明 Atsttrin 与 TNFR2 的直接结合及其对 TNFα/TNFR2 相互作用的抑制的详细方法； 2) 体外破骨细胞生成的抗酒石酸酸性磷酸酶 (TRAP) 染色，以揭示 Atsttrin 的细胞抗 TNFα 活性。使用此处描述的协议，研究人员应该能够重复检测 TNFα 与 TNFR 结合的物理抑制以及 Atsttrin 和各种 TNF 抑制剂对 TNFα 活性的功能抑制。
TNFα/TNFR signaling plays a critical role in the pathogenesis of various inflammatory and autoimmune diseases, and anti-TNFα therapies have been accepted as the effective approaches for treating several autoimmune diseases. Progranulin (PGRN), a multi-faced growth-factor like molecule, directly binds to TNFR1 and TNFR2, particularly to the latter with higher affinity than TNFα. PGRN derivative Atsttrin is composed of three TNFR-binding domain of PGRN and exhibits even better therapeutic effects than PGRN in several inflammatory disease models, including collagen-induced arthritis. Herein we describe the detailed methodology of using 1) ELISA-based solid phase protein-protein interaction assay to demonstrate the direct binding of Atsttrin to TNFR2 and its inhibition of TNFα/TNFR2 interaction; and 2) tartrate-resistant acid phosphatase (TRAP) staining of in vitro osteoclastogenesis to reveal the cell-based anti-TNFα activity of Atsttrin. Using the protocol described here, the investigators should be able to reproducibly detect the physical inhibition of TNFα binding to TNFR and the functional inhibition of TNFα activity by Atsttrin and various kinds of TNF inhibitors.