Studies of Binding of Tumor Necrosis Factor (TNF)-like Weak Inducer of Apoptosis (TWEAK) to Fibroblast Growth Factor Inducible 14 (Fn14)

Abstract

Background: TWEAK and its receptor Fn14 are targets in oncology and autoimmunity. Results: Ligand oligomerization has no major effect on Fn14-TWEAK interaction but strongly enhances TWEAK-induced IL8 production. Conclusion: Avidity is irrelevant for TWEAK trimer binding to Fn14 but required for robust IL8 induction. Significance: Enhanced activity of oligomerized TWEAK trimers is not related to an avidity-related increase in Fn14 occupancy.

Fibroblast Growth Factor Inducible (Fn14)-specific Antibodies Concomitantly Display Signaling Pathway-specific Agonistic and Antagonistic Activity

“…pCMV-SPORT6 expression vectors encoding Fc␥ receptors were purchased from Source Bioscience (Nottingham, UK). Production and purification of Flag-TWEAK, GpL-Flag-TNC-TWEAK, and Fc-Flag-TWEAK have already been described previously (7,43). NIK-, IB␣-, and p-IB␣-specific antibodies were ordered from Cell Signaling (Frankfurt, Germany); PE-conjugated antiFc␥R1a (anti-CD64), anti-Fc␥R3a (anti-CD16), and unconjugated anti-Fc␥R2a (anti-CD32A) and anti-Fc␥R2b (anti-CD32B) were from Santa Cruz Biotechnology; and anti-tubulin was from Dunn Labortechnik (Asbach, Germany).…”

TWEAK Inhibits TRAF2-Mediated CD40 Signaling by Destabilization of CD40 Signaling Complexes

“…To confirm this by an independent method and also to gain insight into the stability of the CD40L-CD40 interaction, we also performed ligand binding studies. We recently demonstrated for TWEAK and the related ligands TNF and CD95L that these molecules can be labeled by genetic engineering using GpL as a reporter domain (36,37). We therefore fused in a similar approach the GpL domain to CD40L and used the resulting GpL-Flag-CD40L fusion protein for cellular binding studies.…”

Binding Studies of TNF Receptor Superfamily (TNFRSF) Receptors on Intact Cells

“…Furthermore, soluble CD40L, which benefits only moderately from ligand oligomerization, has a relatively poor affinity of 7.1 nM, whereas EDA-A1, which interacts with EDAR with an affinity of 0.05 nM, still gains activity upon oligomerization (33). Indeed, we recently addressed the relevance of ligand oligomerization for affinity by help of GpL fusion proteins for soluble TWEAK, which poorly stimulate Fn14-mediated induction of the classical NFB target IL8, and for soluble CD95L, which fails to trigger robust apoptosis induction (9,10). In these two cases, we noticed no major effect of ligand oligomerization on receptor occupancy and apparent affinity.…”

“…Cloning, Production, and Purification of Recombinant Proteins-Cloning of GpL-FLAG-TNC-TNF, GpL-FLAG-TNC-TWEAK, and GpL-fLAG-TNC-CD95L has been described elsewhere (9,10). The expression plasmids encoding the GpL-FLAG-TNC variants of the other TNF ligands used in this study have been obtained by exchange of the TNF-encoding fragment of the pCR3-based GpL-FLAG-TNC-TNF expression plasmid with PCR amplicons encoding THD-encompassing soluble versions of the other TNFSF ligand types ( Table 2) by help of flanking EcoRI (5Ј end) and XbaI (3Ј end) restriction sites.…”

“…We recently took advantage of this fact and generated GpL fusion proteins of the TNFSF ligands CD95L, TNF, and TWEAK for cellular binding studies (9,10). These GpL-TNFSF ligand fusion proteins were proved to be highly traceable and functionally not distinguishable from their conventional counterparts.…”