Caspases,Paracaspases, and Metacaspases Methods and Protocols

23

Here, a simple protocol is described to characterize the cleavage

of a natural substrate by a caspase in vitro. The procedure uses a

cell lysate as a source of substrate, but the same protocol is suitable

for a recombinant protein. For recombinant substrates, it is useful

to label the protein with fluorescein using an amine or a sulfhydryl

reacting reagent such as N-hydroxysuccinimide ester (NHS) or

N-ethylmaleimide (NEM)-fluorescein, respectively. This step will

enable a more sensitive measurement of cleavage rates and render

antibody use unnecessary. However, it is essential to verify that the

labeling strategy used does not affect caspase cleavage. Such an

approach has been used to study the cleavage of the Hsp90 co-

chaperone p23 by caspase-7 [ 32 ].

Lysates made from cell lines deficient in specific caspases are very

useful. For example, breast cancer carcinoma MCF-7 cells, which

are deficient in caspase-3 and caspase-10a [ 33 , 34 ], limit experi-

mental bias when studying caspase-7 and caspase-8, respectively.

Prepare large quantities of cell extracts (5–10 plates) and freeze the

lysate in small aliquots. Doing so will make it easier to compare

substrate cleavage by various caspases over several weeks or a few

months. This protocol is described for cells grown as a monolayer

in one 15 cm tissue culture dish but can easily be scaled up.

The procedure takes 1 h to be completed.

1. Rinse cells twice with 10 mL of cold PBS. Keep the tissue cul-
   ture dish on ice.


2. Detach the cells using 10 mL of cold PBS-EGTA/EDTA.
   Incubate for 5 min on ice.

3.3.1 Preparing Cytosolic
Extracts from
Mammalian Cells

IB: PARP

20 nM/30 min -

(^250150)
100
50
37
20
(^250150)
100
50
37
20
2-fold serial dilution
Casp7
Casp3
kcat,app /KM,app = 6.2 x 10^5 M-1s-1
kcat,app /KM,app = 0.8 x 10^5 M-1s-1
Fig. 5 PARP-1 cleavage by two executioner caspases. MCF-7 cell extracts were incubated for 30 min with
twofold serial dilution of the indicated recombinant caspase in executioner caspase buffer starting at 20 nM.
Apparent kcat/KM values were estimated as described in Subheading 3.3.2. Samples were analyzed by immu-
noblotting using an antibody recognizing the N-terminus of PARP. Arrows mark the point at which 50 % of
PARP-1 is cleaved. These results were originally published in the Proceedings of the National Academy of
Science of the USA [ 61 ] © National Academy of Sciences
Apoptotic Caspases Assays