Interactive Pathway: Death of a Cell - Apoptosis


Activation and Inhibition of Apoptosis
Douglas K. Miller, Ph.D.
Department of Immunology and Rheumatology
Merck Research Laboratories
Rahway, NJ 07065

Apoptosis Overview:
Apoptotic cell death has been characterized by a progressive series of morphological and biochemical changes ranging from the appearance on cell surfaces of phosphatidylserine, to proteolytic cleavage of numerous intracellular proteins, to nuclear condensation and fragmentation and the cleavage of DNA into nucleosomal fragments.1 Principally two mechanisms have been identified in mammalian cells for the induction of apoptosis: agents that lead to the perturbation of mitochondria, resulting in the leakage of cytochrome C and the activation of apoptosis; or agents that directly activate a family of death receptors leading to the activation of a parallel apoptotic cascade. Proapoptotic agents such as the activation of p53, growth factor withdrawal, irradiation, activated oxygen, and cytotoxic drugs induce their effects through the mitochondrial pathway.2 The mammalian proteins involved in this pathway have to a large extent been identified as homologs of the C. elegans genes originally identified as necessary for apoptotic control.3 Three proteins acted at the core of this pathway: Ced-3, Ced-4, and Ced-9. While Ced-3 was shown to be essential for the execution of apoptosis, the cytoplasmic protein Ced-4 was identified as an adapter protein that activated Ced-3.4 The mitochondrial protein Ced-9 in turn controlled the activation of Ced-4.5 Ced-3 produced the cellular cleavages necessary to induce apoptosis, and this protein was later shown to be a caspase.6

Role of Caspases:
Caspases are a family of cysteinyl endoproteases that cleave after Asp residues. In contrast to the case with C. elegans, there are fourteen or more caspases currently known in mammals, and they are divided into 2 broad phylogenetic categories based upon their sequence alignment: those that are related to Ced-3 and those that are related to caspase-1 (Interleukin-1 converting enzyme). Evidence clearly links those members in the Ced-3 subclass to the initiation and propagation of apoptosis (including such members as caspases-2, -3, 6, -7, 8, -9, and -10), whereas members within the caspase-1 subfamily (such as caspases-1, -4, and -5) are probably involved in proteolytic activation, such as those previously observed for IL-1b or interleukin-18.7,8

Caspase Structure:
Caspases are found in relatively large amounts as inactive precursors within the cytoplasm. Following a proapoptotic stimulus, they are sequentially processed into a precursor, noncatalytic domain and two catalytic subunits: a large subunit ca 17-20 kDa containing the active site Cys, and a smaller 10 kDa subunit.9,10 Active caspases are composed of a dimer of these two subunits. This catalytic tetramer contains two active sites, each of which is characterized by a deep pocket binding the Asp (P1) found at the cleavage site together with a broader, shallower irregular pocket that binds the preceding three amino acids (P2-P4), a pocket unique for each caspase member recognized.11,12 Proapoptotic caspases can be generally divided into subclasses with either long prodomains or short prodomains present on the nonactivated precursors. The long prodomains in the first class contain protein motifs, such as CARD (caspase recruitment domains, found in caspase-9 and -2) or DED (death effector domains, found in caspase-8 and -10). These protein domains are ball-shaped structures distinguished by discreet surface charged patches that interact with adaptor proteins containing similarly structured, but complementary charged, protein motifs.13 The specific interaction of caspase and activator protein promotes the formation of a multimeric complex that is necessary to bring two caspase precursors together to activate each other and produce the active tetramer.14,15

In mammalian cells, it is caspase-9 that functionally corresponds to Ced-3 during its activation. The activation of caspase-9 is controlled by the cytoplasmic adaptor protein Apaf-1 (apoptosis activating factor 1), the mammalian protein corresponding to Ced-4. This interaction is mediated by the CARD domains of Caspase-9 and Apaf-1, just as occurs with Ced-3 and Ced-4.16 To enable the caspase activation, Apaf-1 must first have cytochrome C bound to it and undergo ATP dependent oligomerization prior to caspase-9 binding.17 The source of the necessary cytochrome C is leakage from apoptotically damaged mitochondria. Control of that cytochrome C release is mediated by the mitochondrial protein Bcl-2, which is highly homologous to Ced-9.18-20 The central role of this Apaf-1/caspase- 9/cytochrome C complex in promoting apoptosis via this pathway has resulted in it being termed the "Apoptosome".21

Mitochondrial-mediated Caspase Activation:
Bcl-2 and closely related antiapoptotic homologues such as Bcl-x exert control of mitochondrial permeability by stimulating ADP/ATP exchange, stabilizing the mitochondrial inner transmembrane potential, and preventing the opening of a permeability transition pore that enables cytochrome C release. There are similarly structured proapoptotic Bcl-2 family members such as Bax or Bak that can promote mitochondrial permeability via their direct effects on the mitochondria or indirectly through inhibition of Bcl-2.22 All of these Bcl-2 family members are present in outer mitochondrial membranes as dimers with a structure analogous to that of diptheria toxin where they directly control membrane permeability in ion channel fashion.23 In the C. elegans system the cytoplasmic protein Egl-1 has been identified as a controller of Ced-9 function.24 Likewise, in mammals similar cytoplasmic proapoptotic Bcl-2 family members such as Bad or Bik have been identified with only limited homology to Bcl-2. Following apoptotic stimulation they translocate to the mitochondria and block the protective regulatory activity of Bcl-2 by binding to the conserved Bcl-2 surface. A third type of Bcl-2 family protein is exemplified by the cytoplasmic protein BID which has limited sequence homology to Bcl-2. When BID is cleaved by activated caspase-8, it assumes a structure like Bcl-2, translocates to the mitochondria, and independently inserts and induces channel formation in the membrane.25-27

Receptor-mediated Caspase Activation:
A parallel pathway of death induction in mammals is the activation of one of at least five different cell death receptors, all members of the TNF receptor superfamily.28 The two prototypical death receptors are Fas and the type I TNF receptor. In the cytoplasmic domain of all of the death receptors there is a polypeptide domain termed the death domain (DD), which has a similar structure to that of the CARD and DED domains.29,30 When bound with TNF or homologous ligands, these death receptors bind secondary adaptor proteins such as FADD (Fas associated death domain) via interactions with their own DD.31 Since FADD also contains a DED, it can subsequently bind and activate the closely related caspase-8 and -10 via their DEDs. This receptor, adaptor protein, and caspase complex has been termed the “death inducing signaling complex” (DISC).32 A parallel activation of caspase-2 utilizes the adaptor protein RAIDD (receptor death domain). RAIDD contains both a CARD domain that binds to the corresponding CARD domain of caspase-2 as well as a DD that can bind to an as yet unidentified receptor.33,34 Hence, instead of a single round of caspase activation to generate the proapoptotic cleavages as is apparently the case in C. elegans, in mammals the pattern is to have two stages: a first stage of initiator caspases activated by variety of stimuli, and a second stage of executioner caspases activated as a final common pathway.

Caspase Function - Division of Labor:
The downstream executioner caspases, such as caspases-3, -6, and -7, have been shown to be chiefly responsible for the majority of the intracellular caspase-induced cleavages that result in cell death.8,35 With their short prodomain sequence, these downstream caspases are unable to activate themselves, and instead must be activated by other previously activated caspases. Both caspase-8 and caspase-9 have been shown to cleave all the above three caspases following their autoactivation; cleavage of caspase-3 and caspase-7 occurs first, and then caspase-3 activates caspase-6.36 - 38 All of the precursor cleavages that activate the upstream ('activator') caspases or the downstream ('effector') caspases occur at unique XXXD sites indicating that it was caspase activity itself that induced the activation. Hence, mutations of the Asp residues at the sites of cleavage as well as of the active site Cys prevent the activation of the caspases.14,39 Alternatively caspase activation of almost all of the activator and effector caspases can occur through the action of the Ser protease granzyme B. This protease, normally found in the granules of killer T cells, is largely responsible for the induction of the Asp site cleavages of proteins associated with cellular apoptosis residues.40,41 While the primary effects of granzyme B work through caspase activation, a number of caspase-sensitive targets as well as other unique proteins not normally cleaved by caspases can be cleaved directly by granzyme B.42,43

The catalytic activities of the proapoptotic caspases that have been studied in detail tend to fall into two groups. The substrate specificity of the active long precursor caspases that are autocatalytic is homologous to the sequence at their activation site. For example, caspases-8, -9, or-10 prefer an aliphatic hydrophobic group such as Leu or Val; consequently, they have high activity for a peptide substrate such as IETD, and their activation can be inhibited by an IETD-aldehyde.12,44 In contrast, caspases-2, -3, and -7 prefer an Asp at the P4 residue and preferentially recognize DEVD substrates and are inhibited by DEVD aldehydes.45 The largest number of substrates cleaved during apoptosis are those that contain an Asp in the P4 position, and these are cleaved principally by caspases-3 and -7. The substrates that are cleaved targets of the executioner caspases include proteins associated with normal nuclear repair and homeostasis such as polyADP ribose polymerase (PARP); structural proteins for cytoplasmic and nuclear membranes, such as fodrin, NuMa and lamins; and a variety of signaling proteins, such as various protein kinases.35,46 A particularly important substrate is the inhibitor (ICAD/DFF45) of the nuclease responsible for the cleavage of DNA and generation of the nucleosomal fragments characteristic of apoptosis.47,48 A third caspase catalytic activity, such as that found in caspases-1, -4 or -5, recognizes a large aromatic or hydrophobic group such as Trp, and comparably structured tetrapeptides such as YVAD or WEHD are specific substrates or inhibitors with nanomolar potency.12 Because all of these tetrapeptide inhibitors are much less effective in cells than in cell-free conditions, the irreversible inhibitor benzyloxycarbonyl-V-A-D(OMe)-fluoromethylketone (Z-VAD-fmk) has been used to increase cell penetration. Lacking a P4 amino acid, Z-VAD-fmk is a pancaspase inhibitor, enabling its use as a general inhibitor for caspase associated apoptotic cleavages.49 The specificity of Z-VAD-fmk extends beyond that of caspases, however, and it can inhibit cellular cathepsins as well.50 Inhibition of total caspase activity does not, however, prevent subsequent cell death presumably because of caspase independent mitochondrial damage.51

Inhibitors of Apoptosis:
A variety of proteins have been found to inhibit the induction of apoptosis, and their presence at various stages of the apoptotic cascade indicates those sites most susceptible to inhibition. A number of viral inhibitors mimicking Bcl-2 prevent mitochondrial damage, such as the adenoviral E1B-19K protein.52 Other viral proteins prevent apoptosis by blocking stimulation of death receptors by antagonizing their activation or stimulating their turnover or inducing NF-kB stimulation (reviewed in 53). Inhibition of NF-kB activation produces a corresponding increase in apoptosis, indicating that the balance of cell viability vs cell death is maintained by the degree of NF-kB activation.54 The activation and subsequent activity of caspases are targeted by a variety of antiapoptotic proteins. The mammalian protein cFLIP (Flice (caspase-8) inhibitor protein; with homologs encoded by a variety of viruses) contains DED protein domains and an inactive caspase domain that prevents both the binding of caspase-8 to various death receptors and its activation. 55 cFLIP does not, however, prevent apoptosis induced by granzyme B or by chemotherapeutic drugs and irradiation, which are both activated by a caspase-9 dependent mechanism.56 A different class of viral proteins that inhibited caspase activation are the inhibitor of apoptosis proteins (IAPs) originally identified in baculovirus where they prevented insect cell apoptosis.57 Mammalian counterparts to the IAPs have been found associated with the activated TNF receptors.58 where they block the activation of caspase- 8, an association and activity which is upregulated by NF-kB activation.59,60 They also act downstream of mitochondrial release of cytochrome C to prevent activation of caspase-9.61,62 While the IAP proteins generally have less ability to block apoptosis by preactivated caspases, one member, XIAP, has been shown to block caspase-3 and caspase-7 activity directly via binding to one of its BIR regions.63 A third class of antiapoptotic proteins are direct inhibitors of caspase activity. These include the baculoviral inhibitor p35, a potent specific inhibitor of activated caspases that acts as a caspase pseudosubstrate.64,65 Structurally different from p35 but functionally similar are a number of pseudosubstrate inhibitors of caspases that are members of the serpin class of protease inhibitors, such as the pox viral protein CrmA or the mammalian granzyme B and caspase inhibitor PI-9.66-68

Induction of Apoptosis:
The induction of apoptosis is hence a highly regulated process that can be initiated by a variety of extracellular ligand-directed or intracellular stress-induced stimuli. Caspases act in a central role to both initiate and execute the intracellular cascade of events that result in protein and nucleic acid cleavage and ultimate cell death. The activation and the resultant activity of those caspases is modulated by a wide variety of adapter proteins and inhibitors at all stages of their involvement.

© Merck KGaA, Darmstadt, Allemagne, 2014


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