Mining of caspase-7 substrates using a degradomic approach

Caspases play critical roles in the execution of apoptosis. Caspase-3 and caspase-7 are closely related in sequence as well as in substrate specificity. The two caspases have overlapping substrate specificities with special preference for the DEVD motif. However, they are targeted to different subcellular locations during apoptosis, implying the existence of substrates specific for one or other caspase. To identify new caspase-7 substrates, we digested cell lysates obtained from the caspase-3-deficient MCF-7 cell line with purified recombinant caspase-7, and analyzed spots that disappeared or decreased by 2-DE (we refer to this as the caspase-7 degradome). Several proteins with various cellular functions underwent caspase-7- dependent proteolysis. The substrates of capase-7 identified by the degradomic approach were rather different from those of caspase-3 (Proteomics, 4, 3429-3435, 2004). Among the candidate substrates, we confirmed that Valosin-containing protein (VCP) was cleaved by both capspase-7 and caspase-3 in vitro and during apoptosis. Cleavage occurred at both DELD(307) and DELD(580). The degradomic study yielded several candidate caspase-7 substrates and their further analysis should provide valuables clues to the functions of caspase-7 during apoptosis.     

Caspases in cell survival, proliferation and differentiation

Caspases, a family of evolutionarily, conserved cysteinyl proteases, mediate both apoptosis and inflammation through aspartate-specific cleavage of a wide number of cellular substrates. Most substrates of apoptotic caspases have been conotated with cellular dismantling, while inflammatory caspases mediate the proteolytic activation of inflammatory cytokines. Through detailed functional analysis of conditional caspase-deficient mice or derived cells, caspase biology has been extended to cellular responses such as cell differentiation, proliferation and NF-kappaB activation. Here, we discuss recent data indicating that non-apoptotic functions of caspases involve proteolysis exerted by their catalytic domains as well as non-proteolytic functions exerted by their prodomains. Homotypic oligomerization motifs in the latter mediate the recruitment of adaptors and effectors that modulate NF-kappaB activation. The non-apoptotic functions of caspases suggest that they may become activated independently of--or without--inducing an apoptotic cascade. Moreover, the existence of non-catalytic caspase-like molecules such as human caspase-12, c-FLIP and CARD-only proteins further supports the non-proteolytic functions of caspases in the regulation of cell survival, proliferation, differentiation and inflammation.     

Identification of the novel substrates for caspase-6 in apoptosis using proteomic approaches

Apoptosis, programmed cell death, is a process involved in the development and maintenance of cell homeostasis in multicellular organisms. It is typically accompanied by the activation of a class of cysteine proteases called caspases. Apoptotic caspases are classified into the initiator caspases and the executioner caspases, according to the stage of their action in apoptotic processes. Although caspase-3, a typical executioner caspase, has been studied for its mechanism and substrates, little is known of caspase-6, one of the executioner caspases. To understand the biological functions of caspase-6, we performed proteomics analyses, to seek for novel caspase-6 substrates, using recombinant caspase-6 and HepG2 extract. Consequently, 34 different candidate proteins were identified, through 2-dimensional electrophoresis/MALDI-TOF analyses. Of these identified proteins, 8 proteins were validated with in vitro and in vivo cleavage assay. Herein, we report that HAUSP, Kinesin5B, GEP100, SDCCAG3 and PARD3 are novel substrates for caspase-6 during apoptosis. [BMB Reports 2013; 46(12): 588-593]     

Mechanisms of Gasdermin Family Members in Inflammasome Signaling and Cell Death

The Gasdermin (GSDM) family consists of Gasdermin A (GSDMA), Gasdermin B (GSDMB), Gasdermin C (GSDMC), Gasdermin D (GSDMD), Gasdermin E (GSDME) and Pejvakin (PJVK). GSDMD is activated by inflammasome-associated inflammatory caspases. Cleavage of GSDMD by human or mouse caspase-1, human caspase-4, human caspase-5, and mouse caspase-11 liberates the N-terminal effector domain from the C-terminal inhibitory domain. The N-terminal domain oligomerizes in the cell membrane and forms a pore of 10–16?nm in diameter, through which substrates of a smaller diameter, such as interleukin-1β and interleukin-18, are secreted. The increasing abundance of membrane pores ultimately leads to membrane rupture and pyroptosis, releasing the entire cellular content. Other than GSDMD, the N-terminal domain of all GSDMs, with the exception of PJVK, have the ability to form pores. There is evidence to suggest that GSDMB and GSDME are cleaved by apoptotic caspases. Here, we review the mechanistic functions of GSDM proteins with respect to their expression and signaling profile in the cell, with more focused discussions on inflammasome activation and cell death.     

Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways

Caspases orchestrate the controlled demise of a cell after an apoptotic signal through specific protease activity and cleavage of many substrates altering protein function and ensuring apoptosis proceeds efficiently. Comparing a variety of substrates of each apoptotic caspase (2, 3, 6, 7, 8, 9 and 10) showed that the cleavage sites had a general motif, sometimes specific for one caspase, but other times specific for several caspases. Using commercially available short peptide-based substrates and inhibitors the promiscuity for different cleavage motifs was indicated, with caspase-3 able to cleave most substrates more efficiently than those caspases to which the substrates are reportedly specific. In a cell-free system, immunodepletion of caspases before or after cytochrome c-dependent activation of the apoptosome indicated that the majority of activity on synthetic substrates was dependent on caspase-3, with minor roles played by caspases-6 and -7. Putative inhibitors of individual caspases were able to abolish all cytochrome c-induced caspase activity in a cell-free system and inhibit apoptosis in whole cells through the extrinsic and intrinsic pathways, raising issues regarding the use of such inhibitors to define relevant caspases and pathways. Finally, caspase activity in cells lacking caspase-9 displayed substrate cleavage activity of a putative caspase-9-specific substrate underlining the lack of selectivity of peptide-based substrates and inhibitors of caspases.     

Yeast two-hybrid screening using constitutive-active caspase-7 as bait in the identification of PA28gamma as an effector caspase substrate

Caspase-3 and -7 represent executioner/effector caspases that directly cause apoptotic morphological changes by cleaving various death substrates. The substrates for caspases generally interact with active caspases, but not with inactive zymogens of caspase or procaspases. Here, to isolate proteins that interact with caspase-7, we established a yeast two-hybrid screening system using reversed-caspase-7, a constitutive active mutant of caspase-7 as a bait plasmid. Screening of an adult brain cDNA library led to isolation of proteasome activator 28 subunit, PA28gamma. In vitro translates of PA28gamma were cleaved by both recombinant caspase-3 and -7. Mutagenesis of potential cleavage site DGLD80 to EGLE80 completely abolished caspase-mediated cleavage. Moreover, endogenous PA28gamma was cleaved during not only Fas-induced apoptosis of HeLa cells, but also cisplatin-induced cell death of MCF7 cells, which are devoid of caspase-3. These findings indicate that PA28gamma is an endogenous substrate for caspase-3 and -7 and that yeast two-hybrid screening using reversed-caspase is a novel and useful approach to clone substrates for effector caspases.     

Non-apoptotic Functions of Caspase-3 in Nervous Tissue

Some enzymes that have been recognized as "apoptotic" so far may be involved in important cellular processes not necessarily related to cell death in nervous tissue. The activity of caspase-3, an "apoptotic" enzyme, can be measured in normally functioning neurons. The results reported by several groups point to the possibility that caspases may be involved in nervous tissue function as top enzymes in the regulatory proteolytic cascade. A concept on a new mechanism of synaptic plasticity modulation involving caspase-3 has been formulated postulating a specific role of caspase-3 in normal brain functioning. The idea of synaptic plasticity modulation by caspase-3 is in line with data reported recently. For example, caspase-3 is possibly involved in the long-term potentiation (LTP) phenomenon since proteins that are key players of molecular mechanisms of LTP induction and maintenance are caspase-3 substrates. Experimental results on blocking LTP by a caspase-3 inhibitor confirm this concept.     

Anamorsin,a Novel Caspase-3 Substrate in Neurodegeneration

Activated caspases play a central role in the execution of apoptosis by cleaving endogenous substrates. Here, we developed a high throughput screening method to identify novel substrates for caspase-3 in a neuronal cell line. Critical steps in our strategy consist of two-dimensional electrophoresis-based protein separation and in vitro caspase-3 incubation of immobilized proteins to sort out direct substrates. Among 46 putative substrates identified in MN9D neuronal cells, we further evaluated whether caspase-3-mediated cleavage of anamorsin, a recently recognized cell death-defying factor in hematopoiesis, is a general feature of apoptosis. In vitro and cell-based cleavage assays indicated that anamorsin was specifically cleaved by caspase-3 but not by other caspases, generating 25- and 10-kDa fragments. Thus, in apoptosis of neuronal and non-neuronal cells induced by various stimuli including staurosporine, etoposide, or 6-hydroxydopamine, the cleavage of anamorsin was found to be blocked in the presence of caspase inhibitor. Among four tetrapeptide consensus DXXD motifs existing in anamorsin, we mapped a specific cleavage site for caspase-3 at DSVD²⁰⁹↓L. Intriguingly, the 25-kDa cleaved fragment of anamorsin was also detected in post-mortem brains of Alzheimer and Parkinson disease patients. Although the RNA interference-mediated knockdown of anamorsin rendered neuronal cells more vulnerable to staurosporine treatment, reintroduction of full-length anamorsin into an anamorsin knock-out stromal cell line made cells resistant to staurosporine-induced caspase activation, indicating the antiapoptotic function of anamorsin. Taken together, our approach seems to be effective to identify novel substrates for caspases and has the potential to provide meaningful insights into newly identified substrates involved in neurodegenerative processes.     

Hydrogen peroxide-mediated necrosis induction in HUVECs is associated with an atypical pattern of caspase-3 cleavage

Oxidative stress, continuously exerted during chronic inflammation, has been implicated as a major causative agent of cellular dysfunction and cell death. In the present study, we investigated the impact of oxidative stress on the mode of cell death in HUVECs using H2O2 as a model reagent. We found that the predominant form of cell death was necrosis. Necrosis induction was accompanied by a distinct mode of caspase-3 cleavage, yielding a 29-kDa fragment. While inhibition of caspases could not prevent the generation of the 29-kDa fragment, general protease inhibitors, such as leupeptin and LLNL, proved to be effective in inhibiting the distinct processing pattern of caspase-3. These results suggest that caspases can act as substrates for non-caspase proteases in cells primed for necrosis induction. Thus, the pattern of caspase-3 cleavage might reflect the proteolytic system engaged in the cell death machinery in HUVECs.     

Cleavage of the Bloom's syndrome gene product during apoptosis by caspase-3 results in an impaired interaction with topoisomerase IIIalpha

In higher eukaryotes, the integration of signals triggered in response to certain types of stress can result in programmed cell death. Central to these events is the sequential activation of a cascade of proteinases known as caspases. The final activated effector caspases of this cascade digest a number of cellular proteins, in some cases increasing their enzymatic activity, in others destroying their function. Of the proteins shown to be targets for caspase-mediated proteolysis, a surprisingly large proportion are proteins involved in the signalling or repair of DNA damage. Here we investigate whether BLM, the product of the gene mutated in Bloom’s syndrome, a human autosomal disease characterised by cancer predisposition and sunlight sensitivity, is cleaved during apoptosis. BLM interacts with topoisomerase IIIα and has been proposed to play an important role in maintaining genomic integrity through its roles in DNA repair and replication. We show that BLM is cleaved during apoptosis by caspase-3 and reveal that the main cleavage site is located at the junction between the N-terminal and central helicase domains of BLM. Proteolytic cleavage by caspase-3 produces a 120 kDa fragment, which contains the intact helicase domain and three smaller fragments, the relative amounts of which depend on time of incubation with caspase-3. The 120 kDa fragment retains the helicase activity of the intact BLM protein. However, its interaction with topoisomerase IIIα is severely impaired. Since the BLM–topoisomerase interaction is believed to be necessary for many of the replication and recombination functions of BLM, we suggest that caspase-3 cleavage of BLM could alter the localisation and/or function of BLM and that these changes may be important in the process of apoptosis.     

Direct cleavage by the calcium-activated protease calpain can lead to inactivation of caspases

Caspases, a unique family of cysteine proteases involved in cytokine activation and in the execution of apoptosis can be sub-grouped according to the length of their prodomain. Long prodomain caspases such as caspase-8 and caspase-9 are believed to act mainly as upstream caspases to cleave downstream short prodomain caspases such as caspases-3 and -7. We report here the identification of caspases as direct substrates of calcium-activated proteases, calpains. Calpains cleave caspase-7 at sites distinct from those of the upstream caspases, generating proteolytically inactive fragments. Caspase-8 and caspase-9 can also be directly cleaved by calpains. Two calpain cleavage sites in caspase-9 have been identified by N-terminal sequencing of the cleaved products. Cleavage of caspase-9 by calpain generates truncated caspase-9 that is unable to activate caspase-3 in cell lysates. Furthermore, direct cleavage of caspase-9 by calpain blocks dATP and cytochrome-c induced caspase-3 activation. Therefore our results suggest that calpains may act as negative regulators of caspase processing and apoptosis by effectively inactivating upstream caspases.     

Proteases for cell suicide: functions and regulation of caspases.

Caspases are a large family of evolutionarily conserved proteases found from Caenorhabditis elegans to humans. Although the first caspase was identified as a processing enzyme for interleukin-1beta, genetic and biochemical data have converged to reveal that many caspases are key mediators of apoptosis, the intrinsic cell suicide program essential for development and tissue homeostasis. Each caspase is a cysteine aspartase; it employs a nucleophilic cysteine in its active site to cleave aspartic acid peptide bonds within proteins. Caspases are synthesized as inactive precursors termed procaspases; proteolytic processing of procaspase generates the tetrameric active caspase enzyme, composed of two repeating heterotypic subunits. Based on kinetic data, substrate specificity, and procaspase structure, caspases have been conceptually divided into initiators and effectors. Initiator caspases activate effector caspases in response to specific cell death signals, and effector caspases cleave various cellular proteins to trigger apoptosis. Adapter protein-mediated oligomerization of procaspases is now recognized as a universal mechanism of initiator caspase activation and underlies the control of both cell surface death receptor and mitochondrial cytochrome c-Apaf-1 apoptosis pathways. Caspase substrates have bene identified that induce each of the classic features of apoptosis, including membrane blebbing, cell body shrinkage, and DNA fragmentation. Mice deficient for caspase genes have highlighted tissue- and signal-specific pathways for apoptosis and demonstrated an independent function for caspase-1 and -11 in cytokine processing. Dysregulation of caspases features prominently in many human diseases, including cancer, autoimmunity, and neurodegenerative disorders, and increasing evidence shows that altering caspase activity can confer therapeutic benefits.     

Early caspase-3 activation independent of apoptosis is required for cellular function

A number of pro-apoptotic stimuli induce the activation of caspase-9, an initiator protease that activates executioner caspases, such as caspase-3, leading to the development of programmed cell death. Here we demonstrate that cell (platelets and pancreatic acinar cells) stimulation with agonists induces a bimodal activation of caspase-3. The early caspase-3 activation occurs within 1 min of stimulation and is independent on caspase-9 or mitochondrial cytochrome c release suggesting that is a non-apoptotic event. The ability of agonists to induce early activation of caspase-3 is similar to that observed for other physiological processes. Activation of caspase-3 by physiological concentrations of cellular agonists, including thrombin or CCK-8, is independent of rises in cytosolic calcium concentration but requires PKC activation, and is necessary for agonist-induced activation of the tyrosine kinases Btk and pp60src and for several cellular functions, including store-operated calcium entry, platelet aggregation, or pancreatic secretion. Thus, early activation of caspase-3 seems to be a non-apoptotic event required for cellular function.     

Plasticity of S2-S4 specificity pockets of executioner caspase-7 revealed by structural and kinetic analysis

Many protein substrates of caspases are cleaved at noncanonical sites in comparison to the recognition motifs reported for the three caspase subgroups. To provide insight into the specificity and aid in the design of drugs to control cell death, crystal structures of caspase-7 were determined in complexes with six peptide analogs (Ac-DMQD-Cho, Ac-DQMD-Cho, Ac-DNLD-Cho, Ac-IEPD-Cho, Ac-ESMD-Cho, Ac-WEHD-Cho) that span the major recognition motifs of the three subgroups. The crystal structures show that the S2 pocket of caspase-7 can accommodate diverse residues. Glu is not required at the P3 position because Ac-DMQD-Cho, Ac-DQMD-Cho and Ac-DNLD-Cho with varied P3 residues are almost as potent as the canonical Ac-DEVD-Cho. P4 Asp was present in the better inhibitors of caspase-7. However, the S4 pocket of executioner caspase-7 has alternate regions for binding of small branched aliphatic or polar residues similar to those of initiator caspase-8. The observed plasticity of the caspase subsites agrees very well with the reported cleavage of many proteins at noncanonical sites. The results imply that factors other than the P4-P1 sequence, such as exosites, contribute to the in vivo substrate specificity of caspases. The novel peptide binding site identified on the molecular surface of the current structures is suggested to be an exosite of caspase-7. These results should be considered in the design of selective small molecule inhibitors of this pharmacologically important protease.     

Caspase-1 promiscuity is counterbalanced by rapid inactivation of processed enzyme

Members of the caspase family of cysteine proteases coordinate the highly disparate processes of apoptosis and inflammation. However, although hundreds of substrates for the apoptosis effector caspases (caspase-3 and caspase-7) have been identified, only two confirmed substrates for the key inflammatory protease (caspase-1) are known. Whether this reflects intrinsic differences in the substrate specificity of inflammatory versus apoptotic caspases or their relative abundance in vivo is unknown. To address this issue, we have compared the specificity of caspases-1, -3, and -7 toward peptide and protein substrates. Contrary to expectation, caspase-1 displayed concentration-dependent promiscuity toward a variety of substrates, suggesting that caspase-1 specificity is maintained by restricting its abundance. Although endogenous concentrations of caspase-1 were found to be similar to caspase-3, processed caspase-1 was found to be much more labile, with a half-life of ~9 min. This contrasted sharply with the active forms of caspase-3 and caspase-7, which exhibited half-lives of 8 and 11 h, respectively. We propose that the high degree of substrate specificity displayed by caspase-1 is maintained through rapid spontaneous inactivation of this protease.     

Prediction of the tertiary structure of a caspase-9/inhibitor complex

Apoptosis, or programmed cell death, plays a central role in the development and homeostasis of an organism. The breakdown of cellular proteins in apoptosis is mediated by caspases, which comprise a highly conserved family of cysteine proteases with specificity for aspartic acid residues at the P1 positions of their substrates. Multiple lines of evidence show that caspase-9 is critical for an apoptosis pathway mediated via the mitochondria. In this study, the three-dimensional structure of the catalytic domain of caspase-9 and its interaction with the inhibitor acetyl-Asp-Val-Ala-Asp fluoromethyl ketone (Ac-DVAD-fmk) have been predicted by a segment matching modeling procedure. As expected, the predicted caspase-9 structure shows both a high similarity in the overall folding topology and remarkable differences in the surface loop regions as compared to other caspase family members such as caspase-1, -3 and -8, for which crystal structures have been determined. This kind of comparative analysis reflects the convergence-divergence duality among the caspases. Moreover, some subtle differences have been observed between caspase-9 and caspase-3 in the subsite contacts with the covalently linked inhibitor Ac-DVAD-fmk. Based on the X-ray structural analysis of caspase-8, a main chain carbonyl oxygen appears to be involved in a catalytic triad with the active site Cys and His residues. The corresponding carbonyl oxygen in caspase-9, together with other expected features of the catalytic apparatus, appears in our model. The predicted structure of caspase-9 can serve as a reference for subsite analysis relative to rational design of highly selective caspase inhibitors for therapeutic application.     

Caspases disrupt the nuclear-cytoplasmic barrier

During apoptosis, caspases, a family of proteases, disassemble a cell by cleaving a set of proteins. Caspase-3 plays a major role in the dissassembly of the nucleus by processing several nuclear substrates. The question is how caspase-3 which is usually cytoplasmic, gains access to its nuclear targets. It was suggested that caspase-3 is actively transported to the nucleus through the nuclear pores. We found that caspase-9, which is activated earlier than caspase-3, directly or indirectly inactivates nuclear transport and increases the diffusion limit of the nuclear pores. This increase allows caspase-3 and other molecules that could not pass through the nuclear pores in living cells to enter or leave the nucleus during apoptosis by diffusion. Hence, caspase-9 contributes to cell disassembly by disrupting the nuclear cytoplasmic barrier.     

The in vitro cleavage of the hAtg proteins by cell death proteases

It is becoming increasingly clear that there is crosstalk between the apoptotic and autophagic pathways, with autophagy helping to contribute to cell death by providing energy to allow the energy-requiring programmed cell death process to complete, as well as degrading cellular material in its own right. Recent evidence has suggested that Atg proteins can themselves be targets of caspases, providing potential regulation of autophagy as well as uncovering novel functions for fragments derived from Atg proteins. However, to date there has not been a detailed examination of which Atg proteins may be the targets of which death proteases. We show that the majority of human Atg (hAtg) proteins can be cleaved by calpain 1, which is activated in some apoptotic paradigms, as well as other forms of death. We also show that hAtg3 is cleaved by caspases-3, -6 and -8, hAtg6 (Beclin 1) is cleaved by caspase-3 and -6, while hAtg9, hAtg7 and the hAtg4 homologues can be cleaved by caspase-3. Cleavage of Beclin 1 was also seen in apoptosis of HeLa cells induced by staurosporine and TRAIL, along with cleavage of Atg3 and Atg4C. There were subtle effects of caspase inhibition on GFP-LC3 lipidation but more marked effects on the formation of GFP-LC3 puncta (a marker of autophagosome formation) and p62 degradation, indicating that caspase cleavage of autophagy-related proteins can affect the autophagic process. Notably we show that p62 is a target for caspase-6 and -8 cleavage.