Allantopyrone A interferes with multiple components of the TNF receptor 1 complex and blocks RIP1 modifications in the TNF-α-induced signaling pathway

Abstract

Allantopyrone A is a fungal metabolite that uniquely possesses two α,β-unsaturated carbonyl moieties. We recently reported that allantopyrone A inhibited the nuclear factor-κB (NF-κB) signaling pathway induced by tumor necrosis factor (TNF)-α in human lung carcinoma A549 cells. In the present study, the mechanism by which allantopyrone A inhibits the TNF-α-induced signaling pathway was investigated in more detail. Allantopyrone A blocked extensive modifications to receptor-interacting protein 1 (RIP1) in the TNF receptor 1 (TNF-R1) complex. Allantopyrone A augmented the high-MW bands of TNF-R1, TNF receptor-associated factor 2, RIP1, the NF-κB subunit RelA and inhibitor of NF-κB kinase β in A549 cells, suggesting that it binds to and promotes the crosslinking of these proteins. The extracellular cysteine-rich domains of TNF-R1 were crosslinked by allantopyrone A more preferentially than its intracellular portion. The present results demonstrate that allantopyrone A interferes with multiple components of the TNF-R1 complex and blocks RIP1 modifications in the TNF-α-induced NF-κB signaling pathway.

Cucurbitacin B Down-Regulates TNF Receptor 1 Expression and Inhibits the TNF-α-Dependent Nuclear Factor κB Signaling Pathway in Human Lung Adenocarcinoma A549 Cells

“…We previously reported that allantopyrone A, possessing two α,β-unsaturated carbonyl moieties, binds and crosslinks multiple proteins, including TNF-R1, which blocks the TNF-α-induced NF-κB pathway [47]. Allantopyrone A directly bound to TNF-R1 because it reduced the reactivity of the anti-TNF-R1 antibody to an epitope containing a cysteine residue in the extracellular CRD [47]. Western blotting using different antibodies recognizing TNF-R1 excluded the possibility that cucurbitacin B bound to TNF-R1 in a similar manner to allantopyrone A.…”

“…A pCR3 expression vector encoding C-terminal FLAG-tagged human TNF-R1 was previously described [47]. A pGL4.22[luc2CP/Puro] vector containing the ICAM-1 promoter (−1604 to +40) was previously described [55].…”

Isopanduratin A Inhibits Tumor Necrosis Factor (TNF)-α-Induced Nuclear Factor κB Signaling Pathway by Promoting Extracellular Signal-Regulated Kinase-Dependent Ectodomain Shedding of TNF Receptor 1 in Human Lung Adenocarcinoma A549 Cells

“…The pCR3 expression vector encoding C-terminal FLAG-tagged human full-length TNF-R1 was previously described [42]. A549 cells were transfected with the pCR3 expression vector encoding TNF-R1-FLAG by the lipofection method using HilyMax transfection reagent (Dojindo, Kumamoto, Japan).…”

“…Allantopyrone A is a natural metabolite produced by the endophytic fungus Allantophomopsis lycopodiana KS-97 and contains two α,β-unsaturated carbonyl moieties [44], which are reactive to cysteinyl thiol groups. We previously showed that allantopyrone A, which directly bound to TNF-R1, possibly via cysteine residues, reduced the TNF-R1 reactivity of H-5, which appeared to decrease TNF-R1 bands on Western blots [42]. To exclude this possibility, we transfected A549 cells with an expression vector encoding C-terminal FLAG-tagged TNF-R1.…”

“…inyl thiol groups. We previously showed that allantopyrone A, which directly bound to TNF-R1, possibly via cysteine residues, reduced the TNF-R1 reactivity of H-5, which appeared to decrease TNF-R1 bands on Western blots [42]. To exclude this possibility, we transfected A549 cells with an expression vector encoding C-terminal FLAG-tagged TNF-R1.…”