Human TNFRSF1A Knockout Cell Line-HEK293

Tumor necrosis factor (TNF) receptor 1 (TNFR1, also known as tumor necrosis factor receptor superfamily member 1A (TNFRSF1A) ) plays a key role in mediating TNF-induced downstream signaling and regulating inflammatory responses. Recent studies have shown that TNFR1 activation involves conformational rearrangements of preligand-assembled receptor dimers and that targeting receptor conformational dynamics is a viable strategy to modulate TNFR1 signaling. Here, researchers used a combination of biophysical, biochemical, and cellular assays and molecular dynamics simulations to show that an anti-inflammatory peptide (FKCRRWQWRMKK), referred to as FKC, allosterically inhibits TNFR1 activation by altering the conformational state of receptor dimers without blocking receptor-ligand interactions or disrupting receptor dimerization. The researchers also demonstrated the efficacy of FKC by showing that the peptide inhibited TNFR1 signaling in HEK293 cells and reduced inflammation in mice injected with intraperitoneal TNF. Mechanistically, FKC binds to the TNFR1 cysteine-rich domain (CRD2/3) and perturbs the conformational dynamics required for receptor activation. Importantly, FKC increased the open frequency of CRD2/3 and CRD4 in receptor dimers and induced conformational opening of the receptor cytoplasmic region. Altogether, these data provide evidence for the feasibility of targeting conformationally active regions of TNFR1 and open new avenues for receptor-specific inhibition of TNFR1 signaling.

To investigate whether FKC could functionally inhibit TNFR1 signaling, the researchers examined its effects on pathways downstream of the receptor, including IκBα degradation and NF-κB activation (Figure 1A). In HEK293 cells, TNF induced not only IκBα phosphorylation (Figure 1B) but also IκBα degradation (Figure 1C). Correspondingly, TNF increased p65 phosphorylation, which was blocked by FKC (Figure 1D), demonstrating the inhibitory effect of the peptide on TNFR1 downstream signaling. As an important control, the expression levels of p65 (Figure 1E) and TNFR1 (Figure 1F) were found to be unaffected by FKC. In HEK293 cells with endogenous wild-type TNFR1 (TNFR1 WT), TNF stimulated NF-κB activation to five-fold above basal levels (Figure 1G). FKC inhibited NF-κB activation in TNFR1 WT HEK293 cells in a dose-dependent manner (Figure 1H), consistent with results observed in TNFR1 FRET analysis. To examine whether the inhibitory effect of FKC requires TNFR1, the researchers tested the effect of FKC in TNFR1 knockout (TNFR1 KO) HEK293 cells. TNFR1 KO cells have basal NF-κB activity of approximately 20% of the relative luciferase level, similar to unstimulated TNFR1 WT cells. First, TNF did not stimulate NF-κB activation in TNFR1 KO cells (Figure 1G), confirming the lack of TNFR1 signaling. Importantly, FKC did not attenuate basal NF-κB activity in TNFR1 KO cells, indicating its specificity in inhibiting TNFR1 signaling (Figure 1I).

Figure 1. FKC inhibits TNFR1 signaling in a receptor-specific manner. (Zeng J, et al., 2024)