Transduction of the TAT-FLIP Fusion Protein Results in Transient Resistance to Fas-induced Apoptosis in Vivo

Abstract

Although tightly regulated programmed cell death (apoptosis) possesses great importance for tissue homeostasis, several pathologic processes are associated with organ failure due to adversely activated cell apoptosis. Transient increase in apoptosis has been shown to cause organ damage during fulminant hepatitis B, autoimmune diseases, ischemia-reperfusion injury, sepsis, or allograft rejection. A defined and temporary inhibition of cell apoptosis may therefore be of high clinical relevance. Activation of death receptors results in caspase-8 recruitment to the death-inducing signaling complex, which initiates the apoptotic process through cleavage of caspase-8 and downstream substrates. This initial step may be inhibited by the caspase-8 inhibitor FLIP (FLICE inhibitory protein). To specifically inhibit the initiation of death receptor-mediated apoptosis we constructed a fusion protein containing FLIP fused N-terminally to the human immunodeficiency virus TAT domain. This TAT domain allows the fusion protein to cross the cell membrane and thus makes the Because of severe organ damage due to misregulated apoptosis in various diseases, intense studies have focused on apoptosis signaling pathways triggered by death receptors and their ligands including the Fas ligand, the tumor necrosis factor, and the tumor necrosis factor-related apoptosisinducing ligand (1-3). Fas (APO-1/CD95) surface expression does not necessarily render cells susceptible to Fas ligandinduced apoptosis because of counteracting physiological cellular inhibitors. One of these regulators of death receptormediated apoptosis was termed FLIP 1 (for FLICE inhibitory protein), which is predominantly expressed in lymphoid tissues (4, 5). Cross-linking of Fas-sensitive cells by the Fas ligand or an agonistic antibody induces apoptosis through procaspase-8 recruitment to the Fas-mediated death-inducing signaling complex (DISC), where procaspase-8 is cleaved to initiate apoptosis through a systematic cleavage of downstream substrates. The recruitment of the caspase-8 inhibitor cFLIP into the DISC prevents the cleavage of procaspase-8, resulting in concomitantly reduced apoptosis (6).Multiple splice variants of cFLIP have been reported, but to date only a long and a short form, designated cFLIP L and cFLIP S , respectively, could be detected on a protein level. It has been shown that, in the presence of cFLIP S , procaspase-8 is recruited into the DISC but remains unprocessed (7). Thus FLIP S appears to be a good candidate to block apoptosis in death receptor-mediated caspase-8 dependent pathways.In previous investigations it has been demonstrated that protein transduction is a powerful tool for introducing fulllength proteins into cells without the help of viral or chemical transporters (8,9). The principle of protein transduction originates from the biology of various viruses. In vivo analysis of the transduction properties of the HIV TAT domain demonstrated that almost all cells within the body, even those protected by the blood-brain barrier, were targ...

Functional Characterization of a Chimeric Soluble Fas Ligand Polymer with In Vivo Anti-Tumor Activity

“…Using experiments in mouse, we detected no liver damage after intravenous injection. Although these findings seem in contradiction with data showing that Fas engagement in mice induce an acute liver injury, it is noteworthy that these reports used in fact the anti-Fas JO2 agonistic antibody and not FasL [3], [29], [30], [31], [32]. The liver destruction observed following injection of anti-Fas antibodies may simply be the consequence of an antibody-dependent cell-mediated cytotoxicity reaction [33], as the production of inflammatory cytokines by Fc receptor–bearing Kupffer cells has been observed [34].…”

Effective Blockage of Both the Extrinsic and Intrinsic Pathways of Apoptosis in Mice by TAT-crmA

“…Maximal intracellular presence was detected within 10 min of incubation at 37°C. After 30 min the concentration of TAT-crmA decreased slowly but remained detectable for Ͼ3 h. These data are consistent with the kinetics of other previously published TAT proteins (17). for 5 min completely blocked cleavage of procaspase-8 for at least 5 h and resulted in significantly reduced activation after 7 h. We further analyzed the activity of caspase-3 (Fig.…”

The taming of the cell penetrating domain of the HIV Tat: Myths and realities

“…Subsequently, several other studies have also reported effective biodistribution of therapeutically important proteins in vivo by using PTD [52,[69][70][71][72][73][74][75]. In a previous study, however, no PTD mediated transduction in brain was observed, [46] possibly because beta-gal was chemically conjugated to PTD instead of recombinant fusion protein.…”