In Vitro Physical and Functional Interaction Assays to Examine the Binding of Progranulin Derivative Atsttrin to TNFR2 and Its Anti-TNFα Activity

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2020 Nov 13

PMID 33185871

Citations 5

Authors

Wenyu Fu

Aubryanna Hettinghouse

Chuan-Ju Liu

Affiliations

Soon will be listed here.

Abstract

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.

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References

Jules J, Feng X . In vitro investigation of the roles of the proinflammatory cytokines tumor necrosis factor-α and interleukin-1 in murine osteoclastogenesis. Methods Mol Biol. 2014; 1155:109-23. DOI: 10.1007/978-1-4939-0669-7_10. View

Tian Q, Zhao Y, Mundra J, Gonzalez-Gugel E, Jian J, Uddin S . Three TNFR-binding domains of PGRN act independently in inhibition of TNF-alpha binding and activity. Front Biosci (Landmark Ed). 2014; 19(7):1176-85. PMC: 4410860. DOI: 10.2741/4274. View

Faustman D . TNF, TNF inducers, and TNFR2 agonists: A new path to type 1 diabetes treatment. Diabetes Metab Res Rev. 2017; 34(1). DOI: 10.1002/dmrr.2941. View

Chen G, Goeddel D . TNF-R1 signaling: a beautiful pathway. Science. 2002; 296(5573):1634-5. DOI: 10.1126/science.1071924. View

Vezina A, Vaillancourt-Jean E, Albarao S, Annabi B . Mesenchymal stromal cell ciliogenesis is abrogated in response to tumor necrosis factor-α and requires NF-κB signaling. Cancer Lett. 2013; 345(1):100-5. DOI: 10.1016/j.canlet.2013.11.021. View

Thurner L, Stoger E, Fadle N, Klemm P, Regitz E, Kemele M . Proinflammatory progranulin antibodies in inflammatory bowel diseases. Dig Dis Sci. 2014; 59(8):1733-42. DOI: 10.1007/s10620-014-3089-3. View

Ding H, Wei J, Zhao Y, Liu Y, Liu L, Cheng L . Progranulin derived engineered protein Atsttrin suppresses TNF-α-mediated inflammation in intervertebral disc degenerative disease. Oncotarget. 2018; 8(65):109692-109702. PMC: 5752552. DOI: 10.18632/oncotarget.22766. View

Noguchi T, Ebina K, Hirao M, Kawase R, Ohama T, Yamashita S . Progranulin plays crucial roles in preserving bone mass by inhibiting TNF-α-induced osteoclastogenesis and promoting osteoblastic differentiation in mice. Biochem Biophys Res Commun. 2015; 465(3):638-43. DOI: 10.1016/j.bbrc.2015.08.077. View

Guo F, Lai Y, Tian Q, Lin E, Kong L, Liu C . Granulin-epithelin precursor binds directly to ADAMTS-7 and ADAMTS-12 and inhibits their degradation of cartilage oligomeric matrix protein. Arthritis Rheum. 2010; 62(7):2023-36. PMC: 2902708. DOI: 10.1002/art.27491. View

10.

Sedger L, McDermott M . TNF and TNF-receptors: From mediators of cell death and inflammation to therapeutic giants - past, present and future. Cytokine Growth Factor Rev. 2014; 25(4):453-72. DOI: 10.1016/j.cytogfr.2014.07.016. View

11.

Tang W, Lu Y, Tian Q, Zhang Y, Guo F, Liu G . The growth factor progranulin binds to TNF receptors and is therapeutic against inflammatory arthritis in mice. Science. 2011; 332(6028):478-84. PMC: 3104397. DOI: 10.1126/science.1199214. View

12.

Wei J, Fu W, Ding Y, Hettinghouse A, Lendhey M, Schwarzkopf R . Progranulin derivative Atsttrin protects against early osteoarthritis in mouse and rat models. Arthritis Res Ther. 2017; 19(1):280. PMC: 5735869. DOI: 10.1186/s13075-017-1485-8. View

13.

Thurner L, Zaks M, Preuss K, Fadle N, Regitz E, Ong M . Progranulin antibodies entertain a proinflammatory environment in a subgroup of patients with psoriatic arthritis. Arthritis Res Ther. 2013; 15(6):R211. PMC: 3978758. DOI: 10.1186/ar4406. View

14.

Zhou B, Li H, Liu J, Xu L, Guo Q, Sun H . Progranulin induces adipose insulin resistance and autophagic imbalance via TNFR1 in mice. J Mol Endocrinol. 2015; 55(3):231-43. DOI: 10.1530/JME-15-0075. View

15.

Kruse J, Williams R, Seng J . Considering a Relational Model for Depression in Women with Postpartum Depression. Int J Childbirth. 2015; 4(3):151-168. PMC: 4334160. DOI: 10.1891/2156-5287.4.3.151. View

16.

Jian J, Li G, Hettinghouse A, Liu C . Progranulin: A key player in autoimmune diseases. Cytokine. 2016; 101:48-55. PMC: 5303690. DOI: 10.1016/j.cyto.2016.08.007. View

17.

Liu J, Li H, Zhou B, Xu L, Kang X, Yang W . PGRN induces impaired insulin sensitivity and defective autophagy in hepatic insulin resistance. Mol Endocrinol. 2015; 29(4):528-41. PMC: 5414747. DOI: 10.1210/me.2014-1266. View

18.

Bateman A, Belcourt D, Bennett H, Lazure C, Solomon S . Granulins, a novel class of peptide from leukocytes. Biochem Biophys Res Commun. 1990; 173(3):1161-8. DOI: 10.1016/s0006-291x(05)80908-8. View

19.

Liu C . Progranulin: a promising therapeutic target for rheumatoid arthritis. FEBS Lett. 2011; 585(23):3675-80. PMC: 3162080. DOI: 10.1016/j.febslet.2011.04.065. View

20.

Wang Q, Xia Q, Wu Y, Zhang X, Wen F, Chen X . 3D-Printed Atsttrin-Incorporated Alginate/Hydroxyapatite Scaffold Promotes Bone Defect Regeneration with TNF/TNFR Signaling Involvement. Adv Healthc Mater. 2015; 4(11):1701-8. DOI: 10.1002/adhm.201500211. View