Adhesion Molecules-28

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Development-223

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Gene Expression-178

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Chapter category: Apoptosis

Other Methods of Caspase Activity Monitoring

Chapter authors:
Hubert Hug, Christof Burek and Marek Los

Caspases (Cysteine-Aspart-ases) are important effector molecules involved in apoptosis, though some of them can also participate in other physiological processes such as activation of proinflammatory cytokines and/or possibly regulation of cell activation and proliferation.^1,2 For detailed information regarding the protease family please refer to the first chapter of this book. Since the discovery of the prototype death protease Ced-3, in Caenorhabditis elegans³ more than thirteen mammalian and invertebrate caspases have been described.⁴ They can be divided into different sub-classes, based on structural similarities of either prodomain or their catalytic sub-units. Some of these structural similarities correlate with substrate specificity as indicated below (see Table1). All mammalian caspases described so far are specific for aspartic acid in the P₁ position of their substrates. Caspases exist as latent zymogens that contain an N-terminal precursor domain followed by the region that forms the two subunits of the catalytic domain after proteolytic processing. The core of the catalytic center of these enzymes is formed by the conserved amino acid sequence: QACXG (for caspase-8, -10 X = Q; for caspase-9 X = G, for most of other known caspases X = R). The cysteine at the core of this peptide directly participates in catalysis and defines these proteases as cysteine proteases. The pro-forms of caspases are activated by proteolytic cleavage at specific aspartic residues (Fig.1). Usually, an initial cleavage event occurs which separates the carboxy-terminal "short" subunit of the protease from the rest of the molecule, allowing assembly of an active protease that autocatalytically cleaves off its N-terminal prodomain to generate the mature active enzyme. The proteolytic mechanism of caspase activation allows detailed detection of activation of single caspases by Western blotting. It is important to note that some pro-caspases can be cleaved by other proteases without the formation of an active subunit. Alternative cleavage products created this way remain inactive and they may mislead inexperienced investigators. Once activated, many caspases can propagate proteolytic activation of other family members by processing their pro-forms through cleavage at specified aspartic acid residues. The kinetics and mass balances of caspase activation and inhibition have been modeled and the model could be useful in the developing of new strategies to detect caspase activity.⁵ A family of cellular inhibitors of caspases (IAPs) has been identified⁶⁸ (see Chapter 5 for more details). In addition, some viruses are known to produce caspase inhibitory proteins such as CrmA of poxviruses and p35 of the insect baculoviruses (more information in chapter 6). Several synthetic peptidyl inhibitors of caspases have been designed by taking advantage of the known specificity of caspases for certain substrates (see Table 1). Usually, in such an inhibitor the P₁ aspartate is modified by chloro-, fluoro-methyl-keton (cmk-, fmk-), or an aldehyde group. The aldehyde-based compounds are reversible inhibitors, whereas the cmk and fmkbased reagents form covalent interactions with the active-site-cysteine of caspases and thus are irreversible inhibitors. Tetra-peptides are good inhibitors in vitro, but they usually poorly penetrate through cellular membranes. Therefore, they are more useful for caspase blockage in cellular extracts. For inhibition of caspases in intact cells tripeptide compounds, especially benzyoxyl-carbony-Val-Ala-Asp-CH₂F (zVAD-fmk), were proven to be effective and useful as a control or reference substance for caspase research. The cellular and/or viral caspase inhibitors mentioned above often strongly influence the detected activation and/or activity of caspases. In this chapter we describe several methods to detect caspases as well as caspase activity in cells and cell extracts.

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