Recombinant Human Caspase-8 Protein

Caspase-8 (Cysteine-aspartic acid protease 8/Casp8a; also named MCH5, FLICA and MACH alpha1) is a 28 kDa member of the peptidase C14A family of enzymes (1, 2, 3). It is widely expressed and is considered an initiating caspase for the apoptotic cascade (4). Caspase-8 acts on a wide variety of substrates, including procaspases‑3, 4, 6, 7, 9 and 10, c‑FLIP_L and procaspase-8 itself (1, 5  6). Human procaspase‑8a is a 54‑56 kDa, 479 amino acid (aa) protein (4, 7, 8, 9). It contains two N‑terminal death domains (aa 1‑177), followed by a catalytic site that utilizes His317Gly318 plus Cys360. Normally, it is an inactive, cytosolic monomer (1, 10, 11). But following death‑domain (DD) containing receptor oligomerization, Caspase‑8 is recruited to the death-inducing signaling complex (DISC) that forms around the death domains of the oligomerized receptor (12). FADD/CAP-1 is recruited first, followed by procaspase‑8/CAP‑4 and, possibly, c‑FLIP_L and procaspase‑10 (12). The recruitment, or concentration, of procaspase-8 induces homodimerization. This act alone is sufficient for activation. However, the activity level is modest at best, and appears to be directed towards either itself, or c‑FLIP_L, which is known to form a functional heterodimer with procaspase‑8 (5, 11). When directed towards itself, autocleavage occurs first between Asp374Ser375, generating a 43 kDa (p43) N‑terminal (aa 1‑374) and an 11 kDa C‑terminal (aa 375‑479) fragment. The C‑terminus is further cleaved between Asp384Leu385 to generate a mature p10 subunit (aa 385‑479). The p43 subunit is next cleaved twice, once between Asp216Ser217, and again between Asp210Ser211 to generate a 26 kDa DD‑containing prodomain (aa 1‑210) with an additional 18 kDa mature p18 subunit (aa 217‑374) (12). p18 and p10 noncovalently associate to form a 28 kDa heterodimer, which subsequently associates with another p18:p10 heterodimer to form an active, mature caspase‑8 molecule. This leaves the DISC to act on downstream apoptotic procaspases. In the event procaspase‑8 comes to the DISC complexed with c-FLIP_L, c-FLIP_L will be cleaved by procaspase‑8, generating a p43 fragment that is analogous to the Caspase‑8 p43 subunit. This fragment, however, appears not to be an intermediate in a proteolytic cascade. Rather, it serves as a functional subunit, interacting with TRAF2 and activating NF kappaB. This may account for many of the nonapoptotic activities associated with Caspase‑8 (5, 6, 13). Mature human and mouse Caspase‑8a heterodimers are 73% aa identical (14).