Human pro-Tumor Necrosis Factor Is a Homotrimer

Abstract

The structure of human transmembrane pro-TNF-alpha was studied both in intact cell systems and in an in vitro translation system. In intact cell systems (LPS-induced THP-1 and TNF cDNA-transfected COS-7), a trimer of pro-TNF was detected after chemical cross-linking based on its molecular weight in Western blotting analysis. The trimer was shown to be a TNF-specific protein and could be partially cleaved to 26-kDa pro-TNF monomers by cleaving the cross-linkers. The trimeric structure was assembled intracellularly, because it could be detected in both the in vitro microsomal translation system and in THP-1 cells coincident with the appearance of pro-TNF in the cell lysate, prior to secretion of mature TNF. To further analyze the relationship between the trimeric structure and the biological activity of pro-TNF, we characterized several noncleavable pro-TNF deletion mutants. We observed a correlation between expression of TNF cytotoxicity in a juxtacrine fashion and detection of trimer. Thus, human pro-TNF-alpha, like the secreted mature TNF-alpha, has trimeric structure which is assembled intracellularly before transport to the cell surface and is apparently required for mediating its biologic activity.

Tumor necrosis factor signaling

“…TRAF2 has therefore a dual role in TNF-induced ASK1 activation, firstly as an inducer of Trx-ASK1 dissociation by ROS induction and secondly, as an activating oligomerization scaffold for ASK1. In agreement with the existence of parallel TRAF2-dependent, JNK-activating pathways (TRAF2-GCKs-MEKK1 versus TRAF2-ROS-ASK1), it has been found that Trx-(SH) 2 completely blocks TRAF2-ASK1 interaction, but has only a partial inhibitory effect on TRAF2-mediated JNK activation. 159 TNF not only robustly activates the JNK-inducing MAP kinase cascade, but also the p38-MAPK signalling cascade.…”

“…1,2 From this membrane-integrated form the soluble homotrimeric cytokine (sTNF) is released via proteolytic cleavage by the metalloprotease TNF alpha converting enzyme (TACE). 3 The soluble 51 kDa trimeric sTNF tends to dissociate at concentrations below the nanomolar range, thereby losing its bioactivity.…”

Production of Recombinant Porcine Tumor Necrosis Factor Alpha in a Baculovirus Expression System.

“…There are two forms of hTNF-α, a 26 kDa pro-TNF-α (the precursor of mature TNF-α) and a 17 kDa mature TNF-α [8,16,18]. Pro-TNF-α is a transmembrane protein with its N-terminus locating inside the cytoplasm and C-terminus outside of the cell.…”

Desenvolvimento de anti-hTNFα terapêutico.

“…Após clivagem por metaloproteases extracelulares entre os aminoácidos alanina76 e valina77 (BLACK et al, 1997) a forma madura solúvel de 157 aa (~17 kDa) é secretada, sendo monomérica e biologicamente inativa (TANG; HUNG; KLOSTERGAARD, 1996). A forma ativa compreende um homotrímero composto pelos monômeros de 17 kDa, embora haja envolvimento do TNFα transmembrânico na resposta inflamatória, igualmente existente como homotrímero formado por monômeros não clivados de 26 kDa (PEREZ et al, 1990;TANG;HUNG;KLOSTERGAARD, 1996).…”

“…A forma ativa compreende um homotrímero composto pelos monômeros de 17 kDa, embora haja envolvimento do TNFα transmembrânico na resposta inflamatória, igualmente existente como homotrímero formado por monômeros não clivados de 26 kDa (PEREZ et al, 1990;TANG;HUNG;KLOSTERGAARD, 1996). Descrita por Carswell e colaboradores, 1975, em experimentos de regressão e "necrose" de sarcomas, a presença do TNFα foi demonstrada em soro murino após infecção experimental com bacillus Calmette-Guérin (BCG) sensibilizado com endotoxina (CARSWELL et al, 1975).…”