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Review
. 2017 Aug;24(8):1380-1389.
doi: 10.1038/cdd.2017.44. Epub 2017 May 12.

Caspases and their substrates

Olivier Julien  1 , James A Wells  1
Affiliations
Review

Caspases and their substrates

Olivier Julien et al. Cell Death Differ. 2017 Aug.

Abstract

Protease biology is intimately linked to the functional consequences of substrate cleavage events. Human caspases are a family of 12 fate-determining cysteine proteases that are best known for driving cell death, either apoptosis or pyroptosis. More recently, caspases have been shown to be involved in other cellular remodeling events as well including stem cell fate determination, spermatogenesis, and erythroid differentiation. Recent global proteomics methods enable characterization of the substrates that caspases cleave in live cells and cell extracts. The number of substrate targets identified for individual caspases can vary widely ranging from only a (few) dozen targets for caspases-4, -5, -9, and -14 to hundreds of targets for caspases-1, -2, -3, -6, -7, and -8. Proteomic studies characterizing the rates of target cleavage show that each caspase has a preferred substrate cohort that sometimes overlaps between caspases, but whose rates of cleavage vary over 500-fold within each group. Determining the functional consequences of discrete proteolytic events within the global substrate pool is a major challenge for the field. From the handful of individual targets that have been studied in detail, there are only a few so far that whose single cleavage event is capable of sparking apoptosis alone, such as cleavage of caspase-3/-7 and BIMEL, or for pyroptosis, gasdermin D. For the most part, it appears that cleavage events function cooperatively in the cell death process to generate a proteolytic synthetic lethal outcome. In contrast to apoptosis, far less is known about caspase biology in non-apoptotic cellular processes, such as cellular remodeling, including which caspases are activated, the mechanisms of their activation and deactivation, and the key substrate targets. Here we survey the progress made in global identification of caspase substrates using proteomics and the exciting new avenues these studies have opened for understanding the molecular logic of substrate cleavage in apoptotic and non-apoptotic processes.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Activation, structural features, and biological functions of caspases. Caspases are expressed as inactive cysteine proteases. (a) Caspase activation occurs upon cleavage of the pro-domain and inter-subunit linker, the latter of which is most critical. (b) The mature enzyme is formed by a dimer containing a small subunit (green), a large subunit (blue) that generates an active site on each side (inhibitor in sticks shown in orange). The figure shows a cartoon representation of caspase-7 (PDB ID 1F1J). (c) Caspase substrate identification in cells has allowed us to shed light on key biological processes mostly cell death and to a lesser extent cell remodeling and differentiation
Figure 2
Figure 2
Caspase substrates identification using proteomics approaches. Two main methods are used to identify caspase substrates: the forward and reverse techniques. The forward method involves treating a cell population with an inducer to trigger a biological process that activates endogenous caspases, such as cell death or cell differentiation. Cells are lysed, and cleaved products either captured or separated for identification using mass spectrometry. In the reverse method, one generates a cell lysate in which endogenous proteases are inhibited and adds a purified caspase of interest. After a suitable time(s), proteolytic fragments are captured, identified, and quantified using mass spectrometry
Figure 3
Figure 3
Caspases recognition motifs. (a) Background proteolysis observed in untreated cells mostly showed tryptic-like protein fragments with K or R at P1 position. (b) Apoptotic cells feature a strong enrichment for protein fragments originating from proteolysis on the C-terminal of D residues. (c) By selecting all proteolytic sites featuring a D at P1 position, a clear ‘DEVD’ consensus sequence can be observed. (d) A similar motif is obtained by selecting only proteolytic sites with an E at P1 position, suggesting caspase cleavage. Data adapted from refs ,
Figure 4
Figure 4
Rates of proteolysis of natural caspase substrates. With the advances of mass spectrometry, using targeted LC-MS/MS has become standard practice, allowing better characterization of proteolysis in complex mixtures. (a) Chromatograms of targeted transitions in SRM or PRM methods, allowing quantification of peptides captured over a time-course experiment. (b) Rates of proteolysis can be calculated for each peptides originating from proteolytic events by plotting the sum of the peak intensities (or area) as a function of time, assuming pseudo-first-order kinetics. (c) Comparison of the estimated rates of proteolysis for each cleavage sites of each caspase substrates showing up to five order of magnitude difference. Adapted from ref.
Figure 5
Figure 5
Regulation of caspase activity. Upon caspase activation, specific proteolytic events occur such as degradation of the proteasome. In non-apoptotic mechanisms, caspase activity must turn off at some point to prevent apoptosis, and inhibitor of apoptosis proteins provide a possible mechanism
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