Apoptosis-associated caspase activation assays☆
Introduction
Caspases are a family of intracellular cysteine proteases. Caspase 1, the founding member of the family, was originally identified based on its ability to cleave pro-interleukin-1β to the mature cytokine. Subsequent studies identified caspases 2–14 (numbered in order of cloning) and demonstrated roles for caspases 2, 3, 6, 7, 8, 9, and 10 during apoptosis [1]. Biochemical and crystallographic analyses have demonstrated that members of the caspase family share several features, including a classical histidine/cysteine catalytic dyad and a strong preference for cleavage at the C-terminal side of aspartate [1], [2] or, in some cases, glutamate [3], [4] residues.
Each caspase gene encodes a precursor that contains a prodomain, a large subunit and a small subunit. Based on their functions, apoptotic caspases are now classified as initiator caspases, which transduce various signals into proteolytic activity, and effector caspases, which cleave most of the more than 400 substrates that are degraded in cells undergoing apoptosis [1], [5], [6]. These two classes of caspases differ in not only their functions, but also their quaternary structure, requirements for activation, and abundance.
In their zymogen forms, initiator caspases are present as monomers in the cytoplasm [7]. There are two classical pathways for activating these proteases. In the extrinsic pathway, ligation causes cell surface receptors such as CD95/Fas to bind and oligomerize the cytoplasmic adaptor molecule FADD [8]. The subsequent binding of the prodomains of procaspases 8 and/or 10 to FADD leads to presumed oligomerization of these procaspases [9], causing a conformational change that results in acquisition of enzymatic activity [7], [10]. In the intrinsic pathway, release of cytochrome c from mitochondria results in dATP- or ATP-dependent oligomerization of the cytoplasmic scaffolding molecule Apaf-1 (apoptotic protease activating factor-1), which in turn binds and presumably oligomerizes the procaspase 9 zymogen [9], [11], leading to a conformational change at its active site that results in acquisition of enzymatic activity [12].
Procaspases 8, 9 and 10 in turn appear to participate in activation of downstream effector caspases, which exist as inactive zymogen dimers within the cytoplasm [7]. Effector caspases are activated by proteolytic cleavages at specific aspartate residues [1], [13]. These cleavages separate the large and small subunits from each other [13], while simultaneously causing a conformational change that displaces a peptide loop that occludes the active site of the inactive effector caspase zymogens [14]. One current model suggests that caspases 8, 9, and 10 are able to proteolytically activate procaspases 3 and 7, which are in turn responsible for proteolytically activating procaspase 6 [1]. Alternative activation schemes, however, have also been proposed. For example, caspase 1 reportedly activates caspase 6 to initiate apoptosis in neurons [15].
Other caspases appear to participate in apoptosis following specific stimuli. Procaspase 2 has recently been reported to be the initiator caspase when cells respond to heat shock [16]. Once activated, caspase 2 cleaves and activates the proapoptotic Bcl-2 member Bid, thereby triggering the intrinsic pathway, with release of cytochrome c, activation of caspase 9, and subsequent activation of effector caspases [17]. At present, the biochemical basis for the heat shock-induced activation of the caspase 2 zymogen remains to be more fully defined.
Because various initiator caspases are activated by different signals, identifying the caspases that are activated and their order of activation after treatment of cells with a particular stimulus can provide insight into the lethal signaling that is induced by that stimulus. Toward this end, the caspase activation process can be followed using assays for cleavage of suitable fluorogenic or chromogenic substrates, immunoblotting with monospecific antibodies, immunochemistry with conformation-sensitive antibodies, or affinity labeling with reactive substrate analogs. In the sections that follow, we outline these various approaches, describe recent improvements in these methods, and review the strengths as well as potential limitations of some of the assays.
Section snippets
Overview
Conceptually, the most straightforward method for determining whether caspases have been activated in apoptotic cells is to assay for their ability to cleave model substrates. This approach became feasible after the identification of sequences that are cleaved in various caspase substrates during apoptosis [reviewed in 1] and the development of synthetic substrates containing the appropriate peptides coupled to fluorogenic or chromogenic leaving groups [13], [18]. Because caspases are
Overview
Because activated caspases cleave intracellular polypeptides, it is possible to follow caspase activation by assessing the integrity of these substrates. Thus, immunoblotting remains an important approach for determining whether caspases have been activated. The availability of sera that specifically recognize caspase cleavage products (so-called “anti-neoepitope antibodies”) has enhanced the usefulness of this approach. Because the zymogen forms of effector caspases are cleaved during their
Overview
Because caspases undergo conformational changes upon activation [14], it should in principle be possible to generate immunological reagents that recognize only the active conformations of various caspases. Consistent with this view, antibodies that recognize the active conformation of caspase 3 have been described [46]. Because SDS–PAGE destroys polypeptide secondary and tertiary structure, these reagents are not useful for immunoblotting but can be utilized for detecting active caspases by
Overview
Another approach for the detection of active caspases involves the covalent modification of the caspase active site with a substrate-like molecule that contains both an inhibitory group and a reporter moiety. Covalent binding of such an affinity label facilitates subsequent detection of the activated caspase by immunoblotting, fluorescence microscopy or flow cytometry depending upon the reporter group. A number of variations on the basic molecular structure of the affinity label have been
Summary
In summary, a variety of techniques to assess caspase activation are now available. Because each of these techniques has limitations or can lead to equivocal results, we currently recommend that at least two complementary approaches be applied to determine whether caspases have been activated.
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Supported in part by a Grant from the NIH (R01 CA69008). S.-H.L. is a recipient of a studentship from the Mayo Foundation. W.C.E. is a Principal Fellow of the Wellcome Trust.