CDB15:0001513 TNF — TNFRSF1A

ABSTRACT: Membrane-bound tumour necrosis factor (TNF) has a different binding affinity to two TNF receptors (TNFR1 and TNFR2) and shows different biological activities compared with TNF-alpha. We have developed a mutein, TNF-SAM2, which has modified TNF-alpha. It has higher tumour killing activity than TNF-alpha and fewer side effects.

ABSTRACT: Tumor necrosis factor (TNF) is an important cytokine that suppresses carcinogenesis and excludes infectious pathogens to maintain homeostasis. TNF activates its two receptors [TNF receptor (TNFR) 1 and TNFR2], but the contribution of each receptor to various host defense functions and immunologic surveillance is not yet clear. Here, we used phage display techniques to generate receptor-selective TNF mutants that activate only one TNFR. These TNF mutants will be useful in the functional analysis of TNFR. Six amino acids in the receptor binding interface (near TNF residues 30, 80, and 140) were randomly mutated by polymerase chain reaction. Two phage libraries comprising over 5 million TNF mutants were constructed. By selecting the mutants without affinity for TNFR1 or TNFR2, we successfully isolated 4 TNFR2-selective candidates and 16 TNFR1-selective candidates, respectively. The TNFR1-selective candidates were highly mutated near residue 30, whereas TNFR2-selective candidates were highly mutated near residue 140, although both had conserved sequences near residues 140 and 30, respectively. This finding suggested that the phage display technique was suitable for identifying important regions for the TNF interaction with TNFR1 and TNFR2. Purified clone R1-6, a TNFR1-selective candidate, remained fully bioactive and had full affinity for TNFR1 without activating TNFR2, indicating the usefulness of the R1-6 TNF mutant in analyzing TNFR1 receptor function. To further elucidate the receptor selectivity of R1-6, we examined the structure of R1-6 by X-ray crystallography. The results suggested that R31A and R32G mutations strongly influenced electrostatic interaction with TNFR2, and that L29K mutation contributed to the binding of R1-6 to TNFR1. This phage display technique can be used to efficiently construct functional mutants for analysis of the TNF structure-function relationship, which might facilitate in silico drug design based on receptor selectivity.

ABSTRACT: Tumour necrosis factor (TNF) is a trimeric protein which signals through two membrane receptors, TNFR1 and TNFR2. Previously, we identified small molecules that inhibit human TNF by stabilising a distorted trimer and reduce the number of receptors bound to TNF from three to two. Here we present a biochemical and structural characterisation of the small molecule-stabilised TNF-TNFR1 complex, providing insights into how a distorted TNF trimer can alter signalling function. We demonstrate that the inhibitors reduce the binding affinity of TNF to the third TNFR1 molecule. In support of this, we show by X-ray crystallography that the inhibitor-bound, distorted, TNF trimer forms a complex with a dimer of TNFR1 molecules. This observation, along with data from a solution-based network assembly assay, leads us to suggest a model for TNF signalling based on TNF-TNFR1 clusters, which are disrupted by small molecule inhibitors.

ABSTRACT: The extracellular domains of the two human tumor necrosis factor (TNF) receptors critical for binding TNF-alpha were examined by deletion mapping. The ligand binding capability of full-length and truncated recombinant soluble TNF receptors (TNFRs) was assessed by ligand blot analysis and their binding affinity determined by Scatchard analysis. The results showed that deletion of the fourth cysteine-rich domain of the p55 receptor (TNFR-1) did not alter ligand binding affinity significantly. Deletion of domains 3 and 4 of TNFR-1 resulted in no ligand binding, suggesting that domain 3, but not 4, of TNFR-1 binds directly to ligand. Deletion of domain 4 of TNFR-2 resulted in drastically reduced protein yield and 3-fold reduction in ligand binding affinity, while deletion of both domains 4 and 3 yielded no protein. Thus, the domain 4 of TNFR-2, but not that of TNFR-1, appears to be involved directly in binding TNF, although it is also possible that the domain 4 of TNFR-2 is involved in the correct folding of other domains. These results suggest that the modes of interaction between TNF-alpha and its dual receptors are different, providing opportunity to modulate each receptor specifically for research and therapeutic purposes.