Abstract
Alix/AIP1 regulates cell death in a way involving interactions with the calcium-binding protein ALG-2 and with proteins of ESCRT (endosomal sorting complex required for transport). Using mass spectrometry we identified caspase-8 among proteins co-immunoprecipitating with Alix in dying neurons. We next demonstrated that Alix and ALG-2 interact with pro-caspase-8 and that Alix forms a complex with the TNFalpha receptor-1 (TNF-R1), depending on its capacity to bind ESCRT proteins. Thus, Alix and ALG-2 may allow the recruitment of pro-caspase-8 onto endosomes containing TNF-R1, a step thought to be necessary for activation of the apical caspase. In line with this, expression of Alix deleted of its ALG-2-binding site (AlixDeltaALG-2) significantly reduced TNF-R1-induced cell death, without affecting endocytosis of the receptor. In a more physiological setting, we found that programmed cell death of motoneurons, which can be inhibited by AlixDeltaALG-2, is regulated by TNF-R1. Taken together, these results highlight Alix and ALG-2 as new actors of the TNF-R1 pathway.
Highlights
In some cases such as for neurotrophin-bound Trk receptors, activated receptors continue signaling inside endosomes [2]
Dele- act with pro-caspase-8, was capable of co-immunoprecipitating tion of the prodomain of pro-caspase-8 containing the two with TNF␣ receptor-1 (TNF-R1) (Fig. 4C). This underscores the fact that binding death effector domains (DEDs) abolished the capacity of the of Alix to a TNF-R1 complex is independent of ALG-2 and of caspase to interact with Alix (Fig. 2B)
We previously showed that overexpressed Alix is sufficient to activate caspases and apoptosis and that this activity requires binding to ALG-2
Summary
Reagents and Antibodies—Human recombinant TNF␣FLAG was from Alexis Biochemicals (Covalab). Magnetic Isolation of Endosomes Containing TNF-R1 Bound to TNF␣—HEK 293 cells transfected with TNF-R1 and AlixMyc were incubated in a total volume of 1 ml of cold D-PBS (0.9 mM CaCl2, 0.493 mM MgCl2, 2.67 mM KCl, 1.47 mM KH2PO4, 138 mM NaCl, 8 mM Na2HPO4) containing 3% bovine serum albumin, 100 ng/ml TNF␣-FLAG, and 10 g/ml anti-FLAG monoclonal M1 for 1 h at 4 °C. They were washed twice in cold D-PBS and incubated for 1 h at 4 °C in 1 ml of cold D-PBS containing 50 l of protein G microbeads (MACS Protein G Microbeads, MACS Molecular, Miltenyi Biotec). Solubilization of endosomes was performed using a solution of 8% sucrose, 3 mM imidazole, pH 7.4, containing 0.5% Triton X-100
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