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sequential activations form the foundation for apoptotic cascades.
The catalytic activity of an effector caspase is enhanced by several
orders of magnitude by intrachain cleavage mediated by a specifi c
initiator caspase. Hence, separation of denatured protein lysates
isolated from apoptotic cells by sodium dodecyl sulfate polyacryl-
amide gel electrophoresis (SDS-PAGE) and taking advantage of
specifi c antibodies in western blotting is one broadly used approach
to discriminate processed caspases from their respective zymogens
(Fig. 1 ). However, the activation process of initiator caspases has
additional layers of complexity. A characteristic feature within this
group of proteases is the presence of homotypic CARD (caspase-
recruitment domain) or DED (death effector domain) sequence
motifs in their N-terminal pro-domains, modules that are important
for formation of relevant activity platform complexes in the cell [ 2 ].pro LS SS -53/55 kDpro LS SS -48 kDpro LS SS-41/43 kD-17 kD-36 kD-17 kDpro LS SSGADPH-19 kD-17 kD-35 kD
-30 kDcaspase-8caspase-2caspase-3caspase-712Fig. 1 Typical appearances of pro- and processed caspases in western blotting and a schematic illustration of
their amino acid tertiary structure, connecting immunoblot signals from cleaved fragments to their corre-
sponding subdomains. Western blotting was performed as described in the main text using protein lysates
from control HCT116 cells ( lane 1 ) and HCT116 cells incubated in the presence of 5-FU (5-fl uorouracil, 768 μM)
for 24 h ( lane 2 ). Antibodies for immunodetection of caspase-8, caspase-2, cleaved caspase-3 and caspase-7
were used. Glyceraldehyde 3-phosphate dehydrogenase (G3PDH) was used as a control for equal loading of
samples. The asterisk indicates a nonspecifi c band detected with the anti-cleaved caspase-3 pAb. Abbreviations:
pro , N-terminal pro-domain, LS large subunit (p17), SS small subunit (p12)
Magnus Olsson and Boris Zhivotovskyhttp://www.ebook3000.com