| During metamorphosis of insects there is major rescuplturing of various organs and tissues by programmed cell death (PCD). In Manduca sexta (tobacco hornworm), involution of the labial gland follows an autophagic pathway towards PCD (also referred to as Type II PCD). We confirm, by specific labeling with monodansyl cadaverine, that vacuoles seen in EM images are indeed autophagic. The integrity of the cytoskeleton is proteolytically compromised by proteases other than caspases coincident with the accumulation of autophagic vacuoles. There is no obvious mitochondrial involvement, as evidenced by lack of mitochondrial membrane permeability and cytochrome c release. We were unable to detect activation of caspases throughout most of the destruction of the gland. We assayed for cathepsins B & L, proteasomal protease, and caspases -1, -2, -3, and -6 using fluorometric substrates and aldehyde inhibitors. Degradation of FR-AMC and RR-AMC, preferential substrates for cathepsin B & L and cathepsin B respectively, were the highest activities measured, increasing both in amount and in concentration as the gland involuted. However, cleavage of YVAD-AMC (preferential substrate for caspase-1) and DEVD-AMC (substrate for caspases-3 & -7) was barely detectable and did not change with metamorphosis. On a per-unit-protein basis, digestion of these caspase substrates was less than 0.02% of that seen in induced apoptosis in vertebrate embryos. Cleavage of VDVAD-AFC (substrate for caspase -2) and VEID-AFC (substrate for caspase -6) was also assessed but no specific activity was detected. Exogenously added caspase substrate, polyadenosyl ribose phosphorylase (PARP), is cleaved in labial gland extracts, but only at an acidic pH of 5.5-6.0, and into fragments different from those generated by caspases (confirmed by N-terminal sequencing). The cysteine protease inhibitor leupeptin inhibited PARP cleavage, but the caspase inhibitors YVAD-CMK and DEVD-CHO did not. However, potential caspase fragments of PARP are seen following the addition of exogenous cytochrome c and dATP. Thus although apoptotic machinery is conserved and functional, this form of cell death occurs independently of caspases. We postulate that lysosomal proteases play the major proteolytic role similar to the caspase cascade seen in apoptosis. |
