Dirk De Valck

The zinc finger protein A20 interacts with a novel anti-apoptotic protein which is cleaved by specific caspases

A20 is a Cys2/Cys2 zinc finger protein which is induced by a variety of inflammatory stimuli and which has been characterized as an inhibitor of cell death by a yet unknown mechanism. In order to clarify its molecular mechanism of action, we used the yeast two-hybrid system to screen for proteins that interact with A20. A cDNA fragment was isolated which encoded a portion of a novel protein (TXBP151), which was recently found to be a human T-cell leukemia virus type-I (HTLV-I) Tax-binding protein. The full-length 2386 bp TXBP151 mRNA encodes a protein of 86 kDa. Like A20, overexpression of TXBP151 could inhibit apoptosis induced by tumour necrosis factor (TNF) in NIH3T3 cells. Moreover, transfection of antisense TXBP151 partially abolished the anti-apoptotic effect of A20. Furthermore, apoptosis induced by TNF or CD95 (Fas/APO-1) was associated with proteolysis of TXBP151. This degradation could be inhibited by the broad-spectrum caspase inhibitor zVAD-fmk or by expression of the cowpox virus-derived inhibitor CrmA, suggesting that TXBP151 is a novel substrate for caspase family members. TXBP151 was indeed found to be specifically cleaved in vitro by members of the caspase-3-like subfamily, viz. caspase-3, caspase-6 and caspase-7. Thus TXBP151 appears to be a novel A20-binding protein which might mediate the anti-apoptotic activity of A20, and which can be processed by specific caspases.

A20, an inhibitor of cell death, self-associates by its zinc finger domain.

A20 is a primary response gene which is induced after monocyte adherence or cytokine stimulation of a variety of cells. The A20 protein belongs to a novel class of Cys2/Cys2 zinc finger proteins, and has been characterized as an inhibitor of both apoptotic and necrotic cell death. In order to clarify its molecular mechanism of action, we used the yeast‐based‐two‐hybrid system to screen for A20‐associated proteins. Here we report that A20 is able to self‐associate, and demonstrate that the latter interaction is mediated by its zinc finger domain.

Differential activation of phospholipases during necrosis or apoptosis: A comparative study using tumor necrosis factor and anti‐Fas antibodies

Phospholipases generate important secondary messengers in several cellular processes, including cell death. Tumor necrosis factor (TNF) can induce two distinct modes of cell death, viz. necrosis and apoptosis. Here we demonstrate that phospholipase D (PLD) and cytosolic phospholipase A2 (cPLA2) are differentially activated during TNF‐induced necrosis or apoptosis. Moreover, a comparative study using TNF and anti‐Fas antibodies as cell death stimuli showed that PLD and cPLA2 are specifically activated by TNF. These results indicate that both the mode of cell death and the type of death stimulus determine the potential role of phospholipases as generators of secondary messengers. J. Cell. Biochem. 71:392–399, 1998.

FUNCTIONAL CHARACTERIZATION OF THE PRODOMAIN OF INTERLEUKIN-1BETA -CONVERTING ENZYME

Interleukin-1β-converting enzyme (ICE) has been identified as the main protease responsible for maturation of the prodomain of interleukin-1β. Recently, it was shown to belong to a larger gene family, members of which play an important role in programmed cell death. A common feature of the ICE family proteases is the presence of a prodomain that has been hypothesized to keep the enzyme in an inactive form. Expression analysis in yeast revealed autocatalytic degradation of p45ICE, but not of p30ICE lacking a prodomain. We further demonstrate that p45ICE, in which the critical cysteine has been mutated, is still able to dimerize in vivo. Dimerization requires the prodomain and occurs prior to autoprocessing. These results provide evidence for a regulatory role of the prodomain of ICE.

Tumor Necrosis Factor Cytotoxicity is Associated with Activation of Cellular Phospholipases

Tumor Necrosis Factor (TNF), a cytokine primarily produced by activated macrophages, exerts a broad range of activities on different cell types, mostly related to inflammation and immunomodulation (reviewed in Camussi et al., 1991; Fiers, 1991; Aggarwal and Vilcek, 1992). Among these, the most interesting feature of TNF is its selective toxicity for many tumor cells, leaving normal cells unaffected. Remarkably, the selective killing of transformed cells is often much more pronounced in combination with synergizing agents [e.g. interferon-γ (Williamson et al., 1983), LiC1 (Beyaert et al., 1989)], making this molecule a good candidate for future cancer therapy development.