I know most of caspases have their own substrate, especially recognize a subset of peptide sequence. I found some references for caspase-1, -3, -4, -6, -7 and -8.
Is there anyone who could provide information about caspase-9 and so on?
You need to be very careful about using these substrates, especially in intact cells or cell lysates, because they can be cleaved by many caspases, not just the caspases they are claimed to be specific for. It really depends on the relative concentration of each caspase in the mixture. The dangers of using such substrates in cell lysates where several caspases are present is demonstrated in the following papers
McStay GP, Salvesen GS, Green DR. (2008) Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways. Cell Death Differ. 2008 Feb;15(2):322-31.
and Walsh JG et al., (2011) J. Biol. Chem. Sep 16;286(37):32513-24.
@ Seamus. What is your opinion on the Puri, A.W., et al., Nat Chem Biol Paper last July? What are the specificities of such caspase-tagging compounds and do you think they will make it to market? Not that I want to run gels the rest of my life, but it seems to be a new? approach to follow caspase activation?
With overlapping specificities for substrates for multiple proteinases, there are ways to overcome this. For metalloproteinases, we used MEMRAS, where multiple reagents such as different substrates with different specificities, and inhibitors are used simultsneously. Each reaxtion generates an equation with unknowns ( in qthis case, enzyme concentrations for the proteinases of interest). The equations are solved simultaneously to give active enzyme concentrations.
The best part is that now, at MIT, they have developed a program called proteolytically matrix analysis, that can determine active enzyme concentrations for any system even when proteolytically impurities are present. So maybe you can speak to Doug Lauffenburger at MIT. If the substrate is selective enough, then this approach is not needed.
My theory for making selective substrates for caspases is to use the amino acids surrounding the cleavage site in its own prodomain and to make it into a FRET substrate. It should be as long as possible, ie at least 12 amino acids.
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