Affinity labelling provides an alternative approach for demonstrating that caspases have been activated. This approach is based on the observation that the catalytic cysteines in active caspases can be covalently modified by reaction with a variety of agents (reviewed in ref. 39). Reactions with aldehydes and nitriles are reversible, whereas reactions with chloromethyl, fluoro-methyl, or acyloxymethyl ketones are irreversible. Coupling of these reactive groups to suitable peptides provides potential enzyme inhibitors as well as affinity labelling reagents.
Two reagents currently in widespread use, 7V-(acetyltyrosinylvalinyl-V£-biotinyllysyl) aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone [Figure 3\ abbreviated to YVK(bio)D-aomk] and V-(iY™-benzyloxycarbonylglutamyl-VE-biotinyllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone [abbreviated to Z-EK(bio)D-aomk], will be discussed to illustrate essential features of the affinity labelling reagents. These molecules contain peptide sequences that direct them to caspases, acyloxymethyl ketone moieties to permit covalent coupling to the caspase active site, and biotin groups to allow detection of polypeptides that have been derivatized. Each of these features plays an important role in determining the properties of these reagents.
The presence of the polypeptide moiety enhances the affinity of each reagent for certain proteases. YVK(bio)D-aomk contains a YVKD polypeptide that preferentially binds to caspase-1. Z-EK(bio)D-aomk contains the E-X-D motif found in many polypeptides at sites cleaved by caspases-3, -6, and -7. At least four amino acids (or amino acid-like moieties) to the amino terminal side of the reactive group are required to achieve relatively high affinity to caspases (34, 38). In both YVK(bio)D-aomk and Z-EK(bio)D-aomk, the Pt amino acid is an aspartate, a group that is absolutely required for binding to caspases (1). Because the P2 and P3 side chains point away from the active site of the enzyme, the bulky biotin moiety is tolerated in the P2 position (1, 40). The group in the P4 position, on the other hand, is tightly bound to the enzyme. The corresponding subsite on caspase-3 is positively charged and prefers acidic amino acids, whereas the subsite on caspase-1 accommodates bulkier groups like tyrosine. As described below, however, this selectivity is relative rather than absolute.
The acyloxymethylketone group is employed for covalent modification of the caspases because of its somewhat lower reactivity (and presumed greater selectivity) than chloromethyl or fluoromethyl ketones (39, 40). When working with purified caspases, where selectivity is less of an issue, it might be
10: Methods for detecting proteolysis during apoptosis in intact cells
possible to use affinity labelling reagents with the more reactive groups. In whole-cell lysates, however, chloromethyl ketones exhibit relatively promiscuous reactivity with cysteine proteases (41) in addition to their well-established, but more selective, reactivity with serine proteases. Likewise, fluoromethyl ketones are used to derivatize a variety of sulfhydryl proteases. Because of the widespread reactivity of these types of molecules with polypeptides containing activated cysteine residues, the possibility must be borne in mind that peptide-eoupled acyloxymethyl ketones can potentially react with other enzymes, not just caspases.
The presence of biotin in the affinity labelling reagent permits the detection of polypeptides that have been covalently modified (e.g. by blotting with streptavidin). Unfortunately, endogenous biotinylated polypeptides will also be labelled by streptavidin (18), limiting the usefulness of this approach in immunohistochemical studies. Reagents with different detection groups remain to be synthesized.
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