title: Studies of binding of tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) to fibroblast growth factor inducible 14 (Fn14).
abstract: To perform highly sensitive cellular binding studies with TNF-like weak inducer of apoptosis (TWEAK), we developed a bioluminescent variant of soluble TWEAK (GpL-FLAG-TNC-TWEAK) by fusing it genetically to the C terminus of the luciferase of Gaussia princeps (GpL). Equilibrium binding studies on human (HT1080 and HT29) and murine (Renca and B16) cell lines at 37 °C revealed high affinities of human TWEAK from 53 to 112 pm. The dissociation rate constant of the TWEAK-Fn14 interaction was between 0.48×10(-3) s(-1) (HT29) and 0.58×10(-3) s(-1) (HT1080) for the human molecules, and the association rate constant obtained was 3.3×10(6) m(-1) s(-1) for both cell lines. It has been shown previously that oligomerization of soluble TWEAK trimers results in enhanced Fn14-mediated activation of the classical NFκB pathway. Binding studies with GpL-FLAG-TNC-TWEAK trimers oligomerized by help of a FLAG tag-specific antibody gave no evidence for a major increase in Fn14 occupancy by oligomerized ligand despite strongly enhanced induction of the NFκB target IL8. Thus, aggregated complexes of soluble TWEAK and Fn14 have a higher intrinsic activity to stimulate the classical NFκB pathway and qualitatively differ from isolated trimeric TWEAK-Fn14 complexes. Furthermore, determination of IL8 induction as a function of occupied activated receptors revealed that the intrinsic capability of TNFR1 to stimulate the classical NFκB pathway and IL8 production was ∼100-fold higher than Fn14. Thus, although ∼25 activated TNFR1 trimers were sufficient to trigger half-maximal IL8 production, more than 2500 cell-bound oligomerized TWEAK trimers were required to elicit a similar response.
authors.affiliation: Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Würzburg, Röntgenring 11, 97070 Würzburg, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Fick
authors.affiliation: Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Würzburg, Röntgenring 11, 97070 Würzburg, Germany.
authors.forename: Isabell
authors.initials: I
authors.lastname: Lang
authors.affiliation: Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Würzburg, Röntgenring 11, 97070 Würzburg, Germany.
authors.forename: Viktoria
authors.initials: V
authors.lastname: Schäfer
authors.affiliation: Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Würzburg, Röntgenring 11, 97070 Würzburg, Germany.
authors.forename: Axel
authors.initials: A
authors.lastname: Seher
authors.affiliation: Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Würzburg, Röntgenring 11, 97070 Würzburg, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Trebing
authors.affiliation: Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Würzburg, Röntgenring 11, 97070 Würzburg, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Weisenberger
authors.affiliation: Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Würzburg, Röntgenring 11, 97070 Würzburg, Germany. Electronic address: harald.wajant@mail.uni-wuerzburg.de.
authors.forename: Harald
authors.initials: H
authors.lastname: Wajant
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D015214:Antigens, Differentiation, Myelomonocytic / Q000378:metabolism; D002460:Cell Line; D000074049:Cytokine TWEAK; D005786:Gene Expression Regulation; D006801:Humans; D007249:Inflammation / Q000378:metabolism / Q000473:pathology; D008156:Luciferases / Q000235:genetics; D051379:Mice; D011485:Protein Binding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011956:Receptors, Cell Surface / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism*; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism; D015398:Signal Transduction; D012995:Solubility; D000074050:TWEAK Receptor; D048069:Tumor Necrosis Factors / Q000737:chemistry / Q000378:metabolism*; D000098902:CD163 Antigen
chemical_list: D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D000098902:CD163 Antigen; D000074049:Cytokine TWEAK; D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; C000613297:TNFRSF12A protein, human; C109743:TNFSF12 protein, human; D000074050:TWEAK Receptor; C000613299:Tnfrsf12a protein, mouse; D048069:Tumor Necrosis Factors; D008156:Luciferases
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