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]     

Regulation of cell proliferation and survival: Convergence of protein kinases and caspases

Intricate networks of protein kinases are intimately involved in the regulation of cellular events related to cell proliferation and survival. In addition to protein kinases, cells also contain networks of proteases including aspartic-acid directed caspases organized in cascades that play a major role in the regulation of cell survival through their involvement in the initiation and execution phases of apoptosis. Perturbations in regulatory protein kinase and caspase networks induce alterations in cell survival and frequently accompany transformation and tumorigenesis. Furthermore, recent studies have documented that caspases or their substrates are subject to phosphorylation in cells illustrating a potential convergence of protein kinase and caspase signaling pathways. Interestingly, a number of caspase substrates are protected from cleavage when they are phosphorylated at sites that are adjacent to caspase cleavage sites. While it is theoretically possible that many distinct protein kinases could protect proteins from caspase-mediated cleavage, protein kinase CK2 is of particular interest because acidic amino acids, including aspartic acid residues that are recognized by caspases, are its dominant specificity determinants.     

Interactome disassembly during apoptosis occurs independent of caspase cleavage

Protein–protein interaction networks (interactomes) define the functionality of all biological systems. In apoptosis, proteolysis by caspases is thought to initiate disassembly of protein complexes and cell death. Here we used a quantitative proteomics approach, protein correlation profiling (PCP), to explore changes in cytoplasmic and mitochondrial interactomes in response to apoptosis initiation as a function of caspase activity. We measured the response to initiation of Fas‐mediated apoptosis in 17,991 interactions among 2,779 proteins, comprising the largest dynamic interactome to date. The majority of interactions were unaffected early in apoptosis, but multiple complexes containing known caspase targets were disassembled. Nonetheless, proteome‐wide analysis of proteolytic processing by terminal amine isotopic labeling of substrates (TAILS) revealed little correlation between proteolytic and interactome changes. Our findings show that, in apoptosis, significant interactome alterations occur before and independently of caspase activity. Thus, apoptosis initiation includes a tight program of interactome rearrangement, leading to disassembly of relatively few, select complexes. These early interactome alterations occur independently of cleavage of these protein by caspases.     

Uncovering early markers of cardiac disease by proteomics: avoiding (heart) failure!

Histone post-translational modifications (PTMs) comprise one of the most intricate nuclear signaling networks that govern gene expression in a long-term and dynamic fashion. These PTMs are considered to be ‘epigenetic’ or heritable from one cell generation to the next and help establish genomic expression patterns. While much of the analyses of histones have historically been performed using site-specific antibodies, these methods are replete with technical obstacles (i.e., cross-reactivity and epitope occlusion). Mass spectrometry-based proteomics has begun to play a significant role in the interrogation of histone PTMs, revealing many new aspects of these modifications that cannot be easily determined with standard biological approaches. Here, we review the accomplishments of mass spectrometry in the histone field, and outline the future roadblocks that must be overcome for mass spectrometry-based proteomics to become the method of choice for chromatin biologists.     

Breaking the histone code with quantitative mass spectrometry

Histone post-translational modifications (PTMs) comprise one of the most intricate nuclear signaling networks that govern gene expression in a long-term and dynamic fashion. These PTMs are considered to be 'epigenetic' or heritable from one cell generation to the next and help establish genomic expression patterns. While much of the analyses of histones have historically been performed using site-specific antibodies, these methods are replete with technical obstacles (i.e., cross-reactivity and epitope occlusion). Mass spectrometry-based proteomics has begun to play a significant role in the interrogation of histone PTMs, revealing many new aspects of these modifications that cannot be easily determined with standard biological approaches. Here, we review the accomplishments of mass spectrometry in the histone field, and outline the future roadblocks that must be overcome for mass spectrometry-based proteomics to become the method of choice for chromatin biologists.     

Molecular ordering of the caspase activation cascade initiated by the cytotoxic T lymphocyte/natural killer (CTL/NK) protease granzyme B

Granzyme B is a major cytotoxic T lymphocyte/natural killer (CTL/NK) granule protease that can activate members of the caspase family of cysteine proteases through processing of caspase zymogens. However, the molecular order and relative importance of caspase activation events that occur in target cells during granzyme B-initiated apoptosis has not been established. Here, we have examined the hierarchy of granzyme B-initiated caspase activation events using a cell-free system where all caspases are present at physiological levels. We show that granzyme B initiates a two-tiered caspase activation cascade involving seven caspases, where caspase-3 is required for the second tier of caspase activation events. Using a two-dimensional gel-based proteomics approach we have also examined the scale of granzyme B-initiated alterations to the proteome in the presence or absence of effector caspase-3 or -7. These studies indicate that granzyme B targets a highly restricted range of substrates and orchestrates cellular demolition largely through activation of caspase-3.     

Evaluation of recombinant caspase specificity by competitive substrates

The specificity of 10 recombinant caspases was investigated using a set of competitive substrates. The caspase activity was determined by high-performance liquid chromatography using highly fluorescent peptides containing 2-aminoacridone (AMAC) as reporting group. The sequences of the used substrates were designed according to literature data for being specific for 10 of the caspases. The described approach allows the concentration changes of several substrates to be monitored simultaneously in a single sample. Because the substrates are in competitive conditions, the preferences of particular caspases to given peptide sequences are most clearly demonstrated. In the studied competitive assay conditions, all tested caspases except caspase 2 exhibit activity toward more than one substrate. None of the used peptide sequences was found to be highly specific for a defined caspase. The results obtained indicate that there is well-expressed group specificity among the caspases.     

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.     

Targeted disruption of caspase genes in mice: what they tell us about the functions of individual caspases in apoptosis

Cysteine proteases of the caspase family are crucial mediators of apoptosis. All mammalian cells contain a large number of caspases. Although many caspases are activated in a cell committed to apoptosis, recent data from caspase gene knockout mice suggest that individual caspases may be involved in the cell and stimulus-specific pathways of cell death. The gene disruption studies also establish the functional hierarchy between two structurally distinct classes of caspases. The present review discusses these recent findings and elaborates on how these mutant mouse models have helped the understanding of the mechanisms that govern programmed cell death in the immune and other systems.     

Site-specific analysis of bacterial phosphoproteomes

Protein phosphorylation on serine, threonine and tyrosine is established as an important regulatory modification in bacteria. A growing number of studies employing mass spectrometry-based proteomics report large protein phosphorylation datasets, providing precise evidence for in-vivo phosphorylation that is especially suitable for functional follow-up. Here, we provide an overview of the strategies currently used in bacterial phosphoproteomics, with an emphasis on gel-free proteomics and approaches that enable global detection of phosphorylation sites in bacterial proteins. The proteomics technology has matured sufficiently to permit routine characterization of phosphoproteomes and phosphopeptides with high sensitivity; we argue that the next challenge in the field will be the large-scale detection of protein kinase and phosphatase substrates and their integration into regulatory networks of the bacterial cell.     

Constitutive death of platelets leading to scavenger receptor-mediated phagocytosis. A caspase-independent cell clearance program

Apoptosis is a physiological program for the deletion of cells in which caspases govern events leading to safe clearance by phagocytes. However, a growing weight of evidence now suggests that not all forms of programmed cell death are caspase-dependent. We now report a complete and constitutive but caspase-independent program for the specific phagocytic clearance of intact effete platelets, anucleated blood cells of critical importance in health and disease. Platelets aged in vitro not only exhibited increased expression of proapoptotic Bak and Bax but also evidenced constitutive diminution of function such as decreased aggregation to ADP, which was accelerated by culture in the absence of plasma. This abrogation of cell function in plasma-deprived platelets was associated with morphological and biochemical features similar to those of granulocyte apoptosis, that is, cytoplasmic condensation, plasma membrane changes including exposure of phosphatidylserine and the granule protein P-selectin, and recognition by phagocyte scavenger receptors. However, and in contrast with constitutive death of other inflammatory blood cells by apoptosis, these events were not affected by caspase inhibitors, nor was there evidence of caspase-3 activation either by hydrolysis of analog peptide substrates or Western blot analysis, serving to emphasize that neither programmed cell death nor clearance by phagocytes need involve caspases.     

Establishing a blueprint for CED-3-dependent killing through identification of multiple substrates for this protease

Genetic studies have established that the cysteine protease CED-3 plays a central role in coordinating programmed cell death in Caenorhabditis elegans. However, it remains unclear how CED-3 activation results in cell death because few substrates for this protease have been described. We have used a global proteomics approach to seek substrates for CED-3 and have identified 22 worm proteins that undergo CED-3-dependent proteolysis. Proteins that were found to be substrates for CED-3 included the cytoskeleton proteins actin, myosin light chain, and tubulin, as well as proteins involved in ATP synthesis, cellular metabolism, and chaperone function. We estimate that approximately 3% of the C. elegans proteome is susceptible to CED-3-dependent proteolysis. Notably, the endoplasmic reticulum chaperone calreticulin, which has been implicated in the recognition of apoptotic cells by phagocytes, was cleaved by CED-3 and was also cleaved by human caspases during apoptosis. Inhibitors of caspase activity blocked the appearance of calreticulin on the surface of apoptotic cells, suggesting a mechanism for the surface display of calreticulin during apoptosis. Further analysis of these substrates is likely to yield important insights into the mechanism of killing by CED-3 and its human caspase counterparts.     

The CASBAH: a searchable database of caspase substrates

Apoptosis is coordinated by members of the caspase family of aspartic acid-specific proteases. Other members of this protease family also play essential roles in inflammation where they participate in the maturation of pro-inflammatory cytokines. To date, almost 400 substrates for the apoptosis-associated caspases have been reported and there are likely to be hundreds more yet to be discovered. Thus, the fraction of the proteome that is degraded (the degradome) by caspases during the demolition phase of apoptosis appears to be quite substantial. Despite this, we still know surprisingly little concerning how caspases provoke some of the signature events in apoptosis, such as membrane phosphatidylserine externalization, cellular retraction, chromatin condensation and apoptotic body production. The inflammatory caspases appear to be much more specific proteases than those involved in apoptosis and only two confirmed substrates for these proteases have been described to date. Here, we have compiled a comprehensive list of caspase substrates and describe a searchable web resource (The Casbah; www.casbah.ie) which contains information pertaining to all currently known caspase substrates. We also discuss some of the unresolved issues relating to caspase-dependent events in apoptosis and inflammation.     

Regulation of NF-κB signaling by caspases and MALT1 paracaspase

Caspases are intracellular proteases that are best known for their function in apoptosis signaling. It has become evident that many caspases also function in other signaling pathways that propagate cell proliferation and inflammation, but studies on the inflammatory function of caspases have mainly been limited to caspase-1-mediated cytokine processing. Emerging evidence, however, indicates an important contribution of caspases as mediators or regulators of nuclear factor-κB (NF-κB) signaling, which plays a key role in inflammation and immunity. Much still needs to be learned about the mechanisms that govern the activation and regulation of NF-κB by caspases, and this review provides an update of this area. Whereas apoptosis signaling is dependent on the catalytic activity of caspases, they mainly act as scaffolding platforms for other signaling proteins in the case of NF-κB signaling. Caspase proteolytic activity, however, counteracts the pro-survival function of NF-κB by cleaving specific signaling molecules. A striking exception is the paracaspase mucosa-associated lymphoid tissue 1 (MALT1), whose adaptor and proteolytic activity are both needed to initiate a full blown NF-κB response in antigen-stimulated lymphocytes. Understanding the role of caspases and MALT1 in the regulation of NF-κB signaling is of high interest for therapeutic immunomodulation.     

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.     

Caspase的活化及其在细胞凋亡中的作用

Caspase是执行细胞凋亡的主要酶类,目前已鉴定的哺乳动物Caspase有14种。Caspase以酶原的形式合成,催化活性很低,必须激活以后才能发挥作用。活化的Caspase通过特异性的裂解一套底物而导致细胞凋亡。与Caspase有关的细胞凋亡通路至少有三种：线粒体／细胞色素c通路、死亡受体通路和内质网通路。Caspase总是与其抑制剂共存,以防止Caspase酶原意外激活而对正常细胞造成损伤。     

Proteolytic signaling by TNFalpha: caspase activation and IkappaB degradation

Following binding its death receptor on the plasma membrane, tumor necrosis factor (TNF) induces the receptor trimerization and recruits a number of death domain-containing molecules to form the receptor complex. The complex promotes activation of downstream caspase cascade and induces degradation of IkappaBalpha. Caspases are activated using mechanisms of oligomeration and 'self-controlled proteolysis'. According to their structures and functions, apoptosis related caspases can be divided into upstream and downstream caspases. In general, upstream caspases cleave and activate downstream caspases by proteolysis of the Asp-X site. Activated caspases then cleaved target substrates. To date, more than 70 proteins have been identified to be substrates of caspases in mammalian cells. Caspases can alter the function of their target proteins by destroying structural components of the cytoskeleton and nuclear scaffold or by removing their regulatory domains. Activation of NF-kappaB is dependent on the degradation of IkappaBalpha. IkappaB kinase (IKK) phosphorylates IkappaBalpha at the residues 32 and 36 followed by polyubiquitination at lysine 21 and 22 and subsequent degradation of the molecules by 26S proteasome. There is extensive crosstalk between the apoptotic and NF-kappaB signaling pathways that emanate from TNF-R1. On the one hand, activation of NF-kappaB can inactivate caspases; on the other hand, activated caspases can inhibit the activation of NF-kappaB. Both processes involve in proteolysis. This crosstalk may be important for maintaining the balance between the two pathways and for determining whether a cell should live or die.