Death Watch II: Caspases and Apoptosis

Caspase Related Reagents Courtesy of Bingren Hu, Queen's Medical Center, Hawaii. Provided by Cell Signaling TechnologyConfocal micrograph of double immunostaining for cleaved caspase-3 (green) and propidium iodide (red) in newborn rat brain tissue. This section shows control and transient cerebral ischemia. Editor's Note: This is the second article in our two-part series on cell death. The first part: J. Cortese, "Death watch I: Cytotoxicity detection," The Scientist, 15[5]:26, March 5, 2001. Cells die. This basic, two-word statement is fraught with unanswered questions that are loaded with complexity: What events and signals trigger cell death? What is the mechanism by which cell death occurs? How is death regulated, and why does that regulation sometimes go haywire? These are important questions, and thousands of researchers are actively working to tackle them. Cells can die either through necrosis or apoptosis. Necrosis is unplanned cell death that is usually triggered by toxic stress or profound cellular damage. It can affect surrounding cells, and it is usually accompanied by an inflammatory response.1 Apoptosis, on the other hand, is a normal, planned, and regulated event that leads to morphological changes hours after the cell biochemically commits itself to this pathway.2 During apoptosis, the nucleus condenses and breaks down (karyorrhexis), phosphatidylserine (PS) is exposed to the extracellular media, targeting cells for removal, and the cell shrinks and fragments into membrane-bound apoptotic bodies that are rapidly taken up by macrophages.1-3 Some cells detach from surrounding tissue to help their removal.2 Several techniques used to measure cytotoxicity, necrosis, and apoptosis have recently been discussed in The Scientist.1 However, this field has been expanding so rapidly--MEDLINE counted 6,700 articles including the word "apoptosis" in the year 2000 alone--that the topic is worth another look. This article focuses on the central phenomenon in apoptosis--specific proteolysis by the caspase family, which accounts for one-third of the publications in this field. Angels of Death Caspases (cysteine-dependent, aspartate-specific proteases) are endoproteases that are integral to the disassembly of the cell.4 These enzymes exist in the cell as inactive precursors that are activated by proteolytic cleavage and subsequent heterodimerization. Once activated, they trans-activate other caspases by proteolytic cleavage.3,5 This creates a proteolytic cascade, similar to that of blood clotting factors, that spreads outwardly and destroys key proteins, leading to apoptosis.6 Caspases may generally be classified into two groups: cytokine-processing enzymes and apoptosis-related enzymes. The first caspase discovered, the Interleukin-1b; Converting Enzyme (ICE or caspase-1), is in the former group; caspases-4, -5, and -11-14 are also involved in cytokine-processing.4,6-8 The apoptosis-related group includes caspases-2, -3, and -6-10. Apoptotic "initiators" (caspases-2 and -8-10) can initiate the apoptotic cascade. In contrast, apoptotic "effectors" (caspases-3, -6, and -7) carry out proteolytic events leading directly to cellular breakdown. Caspase proteins have three discrete domains--an N-terminal prodomain, a large subunit (which contains the active-site cysteine residue), and a small subunit. Initiators have long N-terminal prodomains that can couple them to extracellular receptors via adaptor molecules, allowing them to respond to extracellular stimuli.8 Generally speaking, that response involves effector activation. Although there are general similarities between family members, and some caspase family members (e.g., caspase-14) relate structurally to both the apoptotic and cytokine-processing subgroups, the functional dichotomy between the two classes of caspases is strong.9 According to Donald W. Nicholson, senior director of the Merck Frosst Center for Therapeutic Research in Montreal, "there is no compelling evidence for implicating cytokine-processing caspases in apoptosis." Caspase activation is required for development of the complete apoptotic phenotype.2 But there is some disagreement as to whether caspase activity is really the best marker for apoptosis. For example, it might be preferable to examine morphological markers such as nuclear condensation, or even biochemical indexes such as PS exposure and DNA fragmentation. Mohanish Deshmukh, an assistant professor of Neurosciences at the University of North Carolina, has been probing the mechanistic aspects of the role of caspases in neuronal cell death. He considers caspase activation "an apoptotic marker as good as any other one" and explains that "activated caspases are predominantly responsible for inducing apoptotic death with all its hallmarks." Although caspase-independent and caspase-resistant cell death may occur in some experimental systems, it is becoming clear that in most experimental settings, caspase activation is the point at which full-blown apoptosis becomes irreversible.10 Caspases are therefore the "trigger" of the suicidal gun; once they are fully active, not much, if anything, can be done to save the cell. Licensed to Kill There are two main pathways leading to caspase activation. The "extrinsic" pathway involves transmembrane, extracellular receptor-dependent activation of caspase-8 (and possibly -10). In the "intrinsic" pathway, release of cytochrome c and interaction with Apaf-1 leads to dATP-dependent activation of caspase-9, and subsequent cascading caspase activation.2,4 Courtesy of Biosource InternationalImmunohistochemical detection of cleaved caspase-9 in staurosporine-treated HeLa cells. The extrinsic pathway is mediated by cell surface death receptors (containing cytoplasmic "Death Domains", DDs) from the tumor necrosis factor (TNF)/neuronal growth factor (NGF) family.6,9 This family of receptors includes CD95 (Fas, Apo1), TNFR1, and DR3 (Apo3, Wsl1). After ligand binding these trimeric receptors recruit DD-binding adaptor proteins such as FADD (Fas receptor-associated DD), in turn recruiting and activating caspase-8. Caspase-8 then activates the apoptotic effectors such as caspases-3 and -7, initiating apoptosis.9 The intrinsic pathway occurs in mitochondria, which can receive similar or different death signals than Fas/TNF receptors.11 An initial event is the release of cytochrome c from mitochondria, probably mediated by Bax (a channel-forming, mitochondrial outer membrane protein) and inhibited by members of the Bcl-2 protein family.12,13 A mega-channel comprised of Bax and other putative channel proteins forms across the mitochondrial membranes, leading to a permeability transition, release of cytochrome c into the cytosol, and impairment of mitochondrial function.11,14 Cytosolic cytochrome c interacts with Apaf-1 protein, dATP, and multiple molecules of pro-caspase-9 to generate an active complex--the "apoptosome"--that activates caspase-9.5,11,14 As in the extrinsic pathway, activation of caspase-9 leads to a caspase cascade that induces apoptosis. These pathways can--in a given cell type--compete with each other, be activated sequentially or independently, and even "cross-talk" in subtle ways. For instance, active caspase-8 can cleave Bid, a Bcl-2-related protein whose proteolytic product sends a signal to mitochondria to release even more cytochrome c.6,9 The positive feedback between cytochrome c release and caspase-9 activation amplifies apoptotic induction. Close to 100 caspase-sensitive substrates are broken down during apoptosis.2,3 Examples include PARP (a poly(ADP-ribose) polymerase), which is a useful indicator of apoptosis, and lamins, structural proteins from the nuclear envelope.7 Cell morphology is affected by cleavage of cytoskeletal proteins such as gelsolin, fodrin, and actin, leading to plasma membrane blebbing.2,7 Another key substrate is the Inhibitor of Caspase-activated DNase (ICAD), whose proteolysis promotes the endonuclease activity of CAD, and leads to apoptotic DNA fragmentation.2,3,8 These DNA fragments can be detected as a ladder pattern in DNA electrophoresis or in situ in the TUNEL assay (TdT-mediated dUTP Nick-End Labeling assay).2 Killing the Killers Caspases recognize a tetrapeptide sequence (e.g., DEVD (caspases-3, -7, and -10) and WEHD (caspases-1, -4, and -5)) within their targets.3,15 Researchers have used these defined substrate motifs as a starting point for the development of caspase inhibitors.15 Aldehyde derivatives of inhibitory tetrapeptides have a slowly reversible effect on caspase activity, whereas chloro-, fluoro-, and acetoxy-methylketone derivatives (FMKs) are irreversible inhibitors. The tripeptide VAD, usually modified as a fluoromethylketone, is active against most caspases and is being tested as a possible therapeutic agent in neurodegenerative diseases characterized by uncontrolled apoptosis.15 Unfortunately, VAD-FMK is unstable and can react nonspecifically with other proteins. Courtesy of Biosource InternationalSchematic representation of effector caspase activation pathways. According to the Merck Frosst Center's Don Nicholson, these peptide inhibitors are "promiscuous, but not usually labeled as such." He cautions that "there is no real pan-caspase inhibitor; what is currently available are poly-caspase inhibitors." Therefore, care should be taken to compare the effects of several inhibitors (with different specificity) in biological assay systems, and, if possible, to cross-check these results with alternative means for detecting caspase activation (see Sidebar). The pharmaceutical industry is currently very interested in obtaining truly specific caspase inhibitors, because of their potential use in the treatment of neurodegenerative diseases.16 Other macromolecular caspase inhibitors also have been identified, including the baculovirus p35 protein and the serpin-like cowpox protein CrmA (cytokine response modifier A).7,8 Both inhibit TNF- and CD95-induced apoptosis; CrmA is more restricted, inhibiting mostly caspases-1 and -8, whereas p35 inhibits apoptosis triggered by many signal transduction pathways. A family of Inhibitors of Apoptosis (IAPs) that associates with cytoplasmic domains of TNF receptors and caspases has also been identified. These inhibitors are selective for caspases-2, -3, and -7, and may bind to procaspase-9.6,15 Other protein inhibitors are pseudocaspases such as c-FLIP, a FADD-like ICE-inhibiting protein, which competes with procaspase-8 for FADD binding.9 The study of cell death is not merely academic: apoptosis--and caspases--have been implicated in disease. Insufficient cell death may lead to some cancers and pathological lymphocytosis.10 Inappropriate, excessive apoptosis has been implicated in spinal cord-dependent muscular atrophy and ALS, and it may underlie many neurodegenerative diseases.10 For example, caspases cleave the amyloid-b precursor protein and huntingtin, which are abnormally proteolyzed in Alzheimer's and Huntington's diseases, respectively.3,7 Perhaps progress in selective control of apoptosis with novel caspase activators and inhibitors could lead to new "apoptotic therapies" in a few years.10,15 Jorge Cortese (jorge_cortese@mindspring.com) is a freelance writer in Durham, N.C. References 1. J. Cortese, "Death watch I: Cytotoxicity detection," The Scientist, 15[5]:26, March 5, 2001. 2. A. Saraste, K. Pulkki, "Morphologic and biochemical hallmarks of apoptosis," Cardiovascular Research, 45[3]:528-37, Feb., 2000. 3. B.B. Wolf, "Suicidal tendencies: Apoptotic cell death by caspase family proteinases," Journal of Biological Chemistry, 274[29]:20049-52, 1999. 4. H.R. Stennicke, G.S. Salvesen, "Caspases--controlling intracellular signals by protease zymogen activation," Biochimica et Biophysica Acta, 1477[1-2]:299-306, March 7, 2000. 5. H.R. Stennicke, G.S. Salvesen, "Catalytic properties of the caspases," Cell Death and Differentiation, 6[11]:1054-9, 1999. 6. I. Budihardjo et al., "Biochemical pathways of caspase activation during apoptosis," Annual Review of Cell and Developmental Biology, 15:269-90, 1999. 7. G.M. Cohen, "Caspases: The executioners of apoptosis," Biochemical Journal, 326[Pt. 1]:1-16, 1997. 8. N.A. Thornberry, Y. Lazebnik, "Caspases: Enemies within," Science, 281[5381]:1312-6, 1998. 9. E.A. Slee et al., "Serial killers: Ordering caspase activation events in apoptosis," Cell Death and Differentiation, 6[11]:1067-74, 1999. 10.N.A. Thornberry, "Caspases: A decade of death research," Cell Death and Differentiation, 6[11]:1023-7, 1999. 11.D. Green, G. Kroemer, "The central executioners of apoptosis: Caspases or mitochondria?," Trends in Cell Biology, 8[7]:267-71, 1998. 12.M. Saito et al., "BAX-dependent transport of cytochrome c reconstituted in pure liposomes," Nature Cell Biology, 2[8]:553-5, Aug., 2000. 13.A. Kelekar, C.B. Thompson, "Bcl-2-family proteins: The role of the BH3 domain in apoptosis," Trends in Cell Biology, 8[8]:324-30, 1998. 14.A.P Halestrap et al., "Mitochondria and cell death," Biochemical Society Transactions, 28[2]:170-7, Feb., 2000. 15.D.W. Nicholson, "Caspase structure, proteolytic substrates, and function during apoptotic cell death," Cell Death and Differentiation, 6[11]:1028-42, 1999. 16.D. W. Nicholson, "From bench to clinic with apoptosis-based therapeutic agents," Nature, 407[6805]:810-6, Oct. 12, 2000. Killer Tools A number of companies have developed tools for apoptosis/caspase research. Some of these are profiled here. For a more comprehensive listing, visit our Web site. DNA Degradation: One of the defining characteristics of apoptotic cells is the degradation of genomic DNA. The Death Check™ I Assay System from Promega Corp. of Madison, Wis., combines the TUNEL assay with a colorimetric caspase assay to detect two different apoptotic events. The APO-BRDU™ and APO-DIRECT™ Kits from BD Biosciences-Pharmingen of San Diego are both two-color TUNEL assays. The former kit incorporates 5-bromo-2'-deoxyuridine into DNA breakpoints, which are then detected with a labeled antibody. The APO-DIRECT kit detects DNA breaks directly using FITC-labeled nucleotides. Caspase Substrates: Tetrapeptide substrates for most caspases, conveniently linked to fluorogenic or chromogenic probes, are now commercially available. They can be used to detect caspase activities in customized assays, and to sort out multiple caspases involved in a particular system. These commercial assays are optimized for 96- or 384-well formats. Some colorimetric assays use tetrapeptide substrates covalently linked to p-nitroaniline dye, which exhibits greater absorbance at 405 nm upon release by caspase-mediated proteolysis. Similarly, fluorogenic peptides are available using coumarin dyes such as 7-amino, 4-methoxylcoumarin (AMC) and 7-amino, 4-trifluoromethylcoumarin (AFC). Overall, fluorescent measurements are more sensitive than chromogenic ones. Cell Signaling Technology Inc. (CST) of Beverly, Mass., offers a Caspase-7 Activity Assay Kit, with AFC-based detection enhanced by immunoprecipitation of this caspase. Enzyme System Products, Inc. of Livermore, Calif., has a complete set of AFC-based assays for caspases-1-10, while OncoImmunin Inc. of Gaithersburg, Md., offers cell-permeable fluorogenic substrates for caspases-1, -3/7, -6, -8, and -9, in three or more colors each. Molecular Probes Inc. of Eugene, Ore., offers the EnzCheck™ Caspase-3 Assay Kit #2, with a rhodamine-derived caspase substrate that becomes highly fluorescent after proteolysis. This kit allows for continuous monitoring of caspase activity. Targeted to flow cytometry, CaspSCREEN™ from BioVision Inc. of Palo Alto, Calif., uses (aspartyl)2-rhodamine 110 (D2R) as a caspase substrate. D2R is non-fluorescent, but fluoresces intensely after cleavage; this compound is more cell-permeable than most other fluorogenic caspase substrates. Caspase Inhibitors: A variety of tetrapeptide inhibitors, designed to exploit the substrate specificity of caspases, are now commercially available. For instance, Promega's CaspACE™ uses a fluorescein-conjugated general caspase inhibitor (FITC-VAD-FMK) as an in situ marker of caspase activity and a labeling tool for flow cytometry. Intergen® Co. of Purchase, N.Y., offers a set of kits using carboxyfluorescein (FAM)-labeled peptide inhibitors linked to FMK (CaspaTag™ Caspase Activity Kits). These kits include a general inhibitor (VAD-FMK), labeled either with FAM or sulforhodamine B (SRB), which allows for double-labeling experiments. CaspaTag, available in eight peptide sequences, can detect active caspases in live cells qualitatively by fluorescence microscopy, or quantitatively by flow cytometric or microplate-based measurements. The baculovirus protein p35 and proteins of the IAP family can also be used to inhibit caspase activity. Upstate Biotechnology of Lake Placid, N.Y., offers a CMV promoter-driven p35 expression vector. "Casputin," a fusion protein comprising inhibitory (BIR) domains from XIAP offered by BIOMOL Research Laboratories Inc. of Plymouth Meeting, Pa., is a highly specific inhibitor of caspases-3 and -7. BIOMOL also offers antisense RNA tools for caspases-3 and -7. Recombinant Caspases: Recombinant caspases are used as controls in enzymatic assays and Western blotting, or as test subjects for drug screening. Both human and nonhuman caspases-1-10 are available. Procaspases-3, -7, and -14, as well as truncated procaspase-7, are offered by BIOMOL. Anti-caspase Antibodies: Both polyclonal and monoclonal antibodies against most caspases are available from dozens of companies. These antibodies can be applied to flow cytometry, immunohistochemistry, immunoprecipitation, and Western blotting experiments. A second generation of antibodies that recognize only the cleaved forms of caspases, thus providing a test of ongoing apoptosis, is now becoming available. Examples include the anti-ACTIVE® apoptosis antibodies (caspase-3) from Promega, and those released by CST and Alexis Corp. of San Diego. ELISA-based caspase assays are produced by A. G. Scientific Inc., R&D Systems Inc. of Minneapolis, Minn., and Roche Molecular Biochemicals of Indianapolis, among others. BD Biosciences-Pharmingen offers a monoclonal antibody-based ELISA for active caspase-3. BioVision's CaspSELECT Caspase-3 and -7 Immunoassay kits use antibodies to capture the specific activity of caspase-3 and -7, individually, following induction of apoptosis. These two caspases share the same target substrate sequence, and thus their individual activities are difficult to assess using traditional fluorometric or colorimetric assays. Microarrays: Finally, a number of companies offer microarrays targeted to apoptosis (reviewed recently in The Scientist).1 Some of these arrays contain caspase genes. For instance, the GEArray™ Apoptosis-3 array from SuperArray Inc. of Bethesda, Md. (www.superarray.com), incorporates cDNA fragments of caspases-1-10 and some of their activating molecules. --Jorge Cortese 1.J. Cortese, "The array of today," The Scientist, 14[17]:25, Sept. 4, 2000.

Death Watch II: Caspases and Apoptosis

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