The executioners sing a new song: killer caspases activate microglia

Activation of microglia and inflammation-mediated neurotoxicity are suggested to have key roles in the pathogenesis of several neurodegenerative disorders. We recently published an article in Nature revealing an unexpected role for executioner caspases in the microglia activation process. We showed that caspases 8 and 3/7, commonly known to have executioner roles for apoptosis, can promote microglia activation in the absence of death. We found these caspases to be activated in microglia of PD and AD subjects. Inhibition of this signaling pathway hindered microglia activation and importantly reduced neurotoxicity in cell and animal models of disease. Here we review evidence suggesting that microglia can have a key role in the pathology of neurodegenerative disorders. We discuss possible underlying mechanisms regulating their activation and neurotoxic effect. We focus on the provocative hypothesis that caspase inhibition can be neuroprotective by targeting the microglia rather than the neurons themselves.     

TNFalpha-induced IEC-6 cell apoptosis requires activation of ICE caspases whereas complete inhibition of the caspase cascade leads to necrotic cell death.

Tumor necrosis factor (TNF)alpha is considered to play a key pathogenetic role in inflammatory bowel diseases. In this study we analyzed the mechanisms by which TNFalpha induces intestinal epithelial cell apoptosis. TNFalpha alone, and more potently in combination with IFNgamma, induced a high degree of IEC-6 cell apoptosis. This effect was more than 100-fold stronger if both of the TNF-R were stimulated, compared to stimulation of the p55-TNF-R alone, indicating an important apoptosis enhancing effect of the p75-TNF-R. TNFalpha-induced apoptosis required activation of ICE caspases and was completely abolished by its inhibitor, zVAD-fmk. Specific inhibition of caspase-3 with zDEVD-fmk did not alter the effect of TNFalpha. Western blot analyses confirmed that caspase-3 was not activated in response to TNFalpha. In the presence of complete inhibition of the caspase cascade with zVAD-fmk (>/=50 microM), TNFalpha induced cell necrosis rather than apoptosis. Our data reveal that TNFalpha can trigger enterocyte cell death via apoptosis or necrosis, depending upon the activation or blockade of specific 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。     

Caspase activation, inhibition, and reactivation: a mechanistic view

Caspases, a unique family of cysteine proteases, execute programmed cell death (apoptosis). Caspases exist as inactive zymogens in cells and undergo a cascade of catalytic activation at the onset of apoptosis. The activated caspases are subject to inhibition by the inhibitor-of-apoptosis (IAP) family of proteins. This inhibition can be effectively removed by diverse proteins that share an IAP-binding tetrapeptide motif. Recent structural and biochemical studies have revealed the underlying molecular mechanisms for these processes in mammals and in Drosophila. This paper reviews these latest advances.     

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.     

Proteasome inhibition activates the mitochondrial pathway of apoptosis in human CD4+ T cells

We have previously shown that inhibition of the proteolytic activity of the proteasome induces apoptosis and suppresses essential functions of activated human CD4⁺ T cells, and we report now the detailed mechanisms of apoptosis following proteasome inhibition in these cells. Here we show that proteasome inhibition by bortezomib activates the mitochondrial pathway of apoptosis in activated CD4⁺ T cells by disrupting the equilibrium of pro‐apoptotic and anti‐apoptotic proteins at the outer mitochondrial membrane (OMM) and by inducing the generation of reactive oxygen species (ROS). Proteasome inhibition leads to accumulation of pro‐apoptotic proteins PUMA, Noxa, Bim and p53 at the OMM. This event provokes mitochondrial translocation of activated Bax and Bak homodimers, which induce loss of mitochondrial membrane potential (ΔΨm). Breakdown of ΔΨm is followed by rapid release of pro‐apoptotic Smac/DIABLO and HtrA2 from mitochondria, whereas release of cytochrome c and AIF is delayed. Cytoplasmic Smac/DIABLO and HtrA2 antagonize IAP‐mediated inhibition of partially activated caspases, leading to premature activation of caspase‐3 followed by activation of caspase‐9. Our data show that proteasome inhibition triggers the mitochondrial pathway of apoptosis by activating mutually independent apoptotic pathways. These results provide novel insights into the mechanisms of apoptosis induced by proteasome inhibition in activated T cells and underscore the future use of proteasome inhibitors for immunosuppression. J. Cell. Biochem. 108: 935–946, 2009. © 2009 Wiley‐Liss, Inc.     

Mitochondria,the killer organelles and their weapons

Apoptosis is a cell-autonomous mode of death that is activated to eradicate superfluous, damaged, mutated, or aged cells. In addition to their role as the cell's powerhouse, mitochondria play a central role in the control of apoptosis. Thus, numerous pro-apoptotic molecules act on mitochondria and provoke the permeabilization of mitochondrial membranes. Soluble proteins contained in the mitochondrial intermembrane space are released through the outer membrane and participate in the organized destruction of the cell. Several among these lethal proteins can activate caspases, a class of cysteine proteases specifically activated in apoptosis, whereas others act in a caspase-independent fashion, by acting as nucleases (e.g., endonuclease G), nuclease activators (e.g., apoptosis-inducing factor), or serine proteases (e.g., Omi/HtrA2). In addition, mitochondria can generate reactive oxygen species, following uncoupling and/or inhibition of the respiratory chain. The diversity of mitochondrial factors participating in apoptosis emphasizes the central role of these organelles in apoptosis control and unravels novel mechanisms of cell death execution.     

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.     

The kinder side of killer proteases: Caspase activation contributes to neuroprotection and CNS remodeling

Caspases are a family of cysteine proteases that are expressed as inactive zymogens and undergo proteolytic maturation in a sequential manner in which initiator caspases cleave and activate the effector caspases 3, 6 and 7. Effector caspases cleave structural proteins, signaling molecules, DNA repair enzymes and proteins which inhibit apoptosis. Activation of effector, or executioner, caspases has historically been viewed as a terminal event in the process of programmed cell death. Emerging evidence now suggests a broader role for activated caspases in cellular maturation, differentiation and other non-lethal events. The importance of activated caspases in normal cell development and signaling has recently been extended to the CNS where these proteases have been shown to contribute to axon guidance, synaptic plasticity and neuroprotection. This review will focus on the adaptive roles activated caspases in maintaining viability, the mechanisms by which caspases are held in check so as not produce apoptotic cell death and the ramifications of these observations in the treatment of neurological disorders.     

The inflammatory caspases: key players in the host response to pathogenic invasion and sepsis

Caspases are cysteinyl-aspartate-specific proteinases known for their role in apoptosis (cell death or apoptotic caspases) and proinflammatory cytokine maturation (inflammatory caspases). The inflammatory caspases were among the first to be discovered, but only recently have the mechanisms leading to their activation and inhibition begun to be elucidated. In this review, we examine the biochemistry, substrates, and function of this unique family of inflammatory proteases, highlight the most recent findings regarding their regulatory mechanisms, and discuss what remains to be understood about their roles in health and disease.     

Activation of Bak in ultrasound-induced, JNK- and p38-independent apoptosis and its inhibition by Bcl-2

The molecular mechanisms underlying ultrasound-induced apoptosis remain poorly understood. We have demonstrated that in Jurkat cells, the over-expression of the anti-apoptotic protein Bcl-2 inhibited ultrasound-induced apoptosis, but not necrosis. Inhibition of caspase activity also protected the cells from apoptosis, but not from necrosis, showing the involvement of different mechanisms in ultrasound-induced apoptosis and necrosis. Bak, a pro-apoptotic member of the Bcl-2 family proteins, was activated by ultrasound and its activation was completely inhibited by Bcl-2 over-expression, but not by caspase inhibition. Antioxidant N-acetyl cysteine did not protect the cells from ultrasound-induced apoptosis or necrosis, nor did the inhibition of either c-Jun N-terminal kinase or p38, key factors in the radical oxygen species (ROS)-mediated cell stress response, suggesting that ROS do not play a crucial role in ultrasound-induced apoptosis. Our results confirm that ultrasound induces apoptosis via a pathway that involves Bak, Bcl-2, and caspases, but not ROS.     

Caspase- and serine protease-dependent apoptosis by the death domain of FADD in normal epithelial cells

The adapter protein FADD consists of two protein interaction domains: a death domain and a death effector domain. The death domain binds to activated death receptors such as Fas, whereas the death effector domain binds to procaspase 8. An FADD mutant, which consists of only the death domain (FADD-DD), inhibits death receptor-induced apoptosis. FADD-DD can also activate a mechanistically distinct, cell type-specific apoptotic pathway that kills normal but not cancerous prostate epithelial cells. Here, we show that this apoptosis occurs through activation of caspases 9, 3, 6, and 7 and a serine protease. Simultaneous inhibition of caspases and serine proteases prevents FADD-DD-induced death. Inhibition of either pathway alone does not prevent cell death but does affect the morphology of the dying cells. Normal prostate epithelial cells require both the caspase and serine protease inhibitors to efficiently prevent apoptosis in response to TRAIL. In contrast, the serine protease inhibitor does not affect TRAIL-induced death in prostate tumor cells suggesting that the FADD-DD-dependent pathway can be activated by TRAIL. This apoptosis pathway is activated in a cell type-specific manner that is defective in cancer cells, suggesting that this pathway may be targeted during cancer development.     

Use of fluorescently labeled caspase inhibitors as affinity labels to detect activated caspases

Activation of caspases is the key event of apoptosis and different approaches were developed to assay it. To detect their activation in situ, we applied fluorochrome labeled inhibitors of caspases (FLICA) as affinity labels of active centers of these enzymes. The FLICA ligands are fluorescein or sulforhodamine conjugated peptide-fluoromethyl ketones that covalently bind to enzymatic centers of caspases with 1:1 stoichiometry. The specificity of FLICA towards individual caspases is provided by the peptide sequence of amino acids. Exposure of live cells to FLICA results in uptake of these ligands and their binding to activated caspases; unbound FLICA is removed by cell rinse. Cells labeled with FLICA can be examined by fluorescence microscopy or subjected to quantitative analysis by cytometry. Intracellular binding sites of FLICA are consistent with known localization of caspases. Covalent binding of FLICA allowed us to identify the labeled proteins by immunoblotting: the proteins that bound individual FLICAs had molecular weight between 17 and 22 kDa, which corresponds to large subunits of the caspases. Detection of caspases activation by FLICA can be combined with other markers of apoptosis or cell cycle for multiparametric analysis. Because FLICA are caspase inhibitors they arrest the process of apoptosis preventing cell disintegration. The stathmo-apoptotic method was developed, therefore, that allows one to assay cumulative apoptotic index over long period of time and estimate the rate of cell entry into apoptosis for large cell populations. FLICA offers a rapid and convenient assay of caspases activation and can also be used to accurately estimate the incidence of apoptosis.     

Mechanisms mediating caspase activation in cell death

The initial activation of a caspase in a caspase cascade is a crucial event that determines whether a cell will ultimately undergo cell death. Although each cell contains a number of different caspases, only a small subset may be required for apoptosis in response to a specific stimulus. It now seems that each caspase cascade has two types of caspases involved, the upstream or class I caspases, and the downstream or class II caspases. Class I caspases are characterised by long amino-terminal prodomains that carry specific protein - protein interaction domains which mediate oligomerisation of caspases, often assisted by specific adaptor molecules. Oligomerisation appears to be sufficient for autocatalytic activation of class I caspases. Once the first caspase in the pathway has been activated, it processes downstream caspases initiating a cascade of amplifying events that lead to the apoptotic death of a cell. This article reviews our current understanding of mechanisms that mediate the activation of caspases.     

Detection of caspase activation in situ by fluorochrome-labeled caspase inhibitors

Apoptosis is dependent on the activation of a group of proteolytic enzymes called caspases. Caspase activation can be detected by immunoblotting using caspase-specific antibodies or by caspase activity measurement employing pro-fluorescent substrates that become fluorescent upon cleavage by the caspase. Most of these methods require the preparation of cell extracts and, therefore, are not suitable for the detection of active caspases within the living cell. Using FAM-VAD-FMK, we have developed a simple and sensitive assay for the detection of caspase activity in living cells. FAM-VAD-FMK is a carboxyfluorescein (FAM) derivative of benzyloxycarbonyl-valine-alanine-aspartic acid-fluoromethyl ketone (zVAD-FMK), which is a potent broad-spectrum inhibitor of caspases. FAM-VAD-FMK enters the cell and irreversibly binds to activated caspases. Cells containing bound FAM-VAD-FMK can be analyzed by flow cytometry, fluorescence microscopy, or a fluorescence plate reader. Using FAM-VAD-FMK, we have measured caspase activation in live non-adherent and adherent cells. We show that FAM-VAD-FMK labeled Jurkat and HeLa cells that had undergone apoptosis following treatment with camptothecin or staurosporine. Non-stimulated negative control cells were not stained. Pretreatment with the general caspase inhibitor zVAD-FMK blocked caspase-specific staining in induced Jurkat and HeLa cells. Pretreatment of staurosporine-induced Jurkat cells with FAM-VAD-FMK inhibited affinity labeling of caspase-3, -6, and -7, blocked caspase-specific cell staining, and led to the inhibition of apoptosis. In contrast, the fluorescent control inhibitor FAM-FA-FMK had no effect. Measurement of caspase activation in 96-well plates showed a 3- to 5-fold increase in FAM-fluorescence in staurosporine-treated cells compared to control cells. In summary, we show that FAM-VAD-FMK is a versatile and specific tool for detecting activated caspases in living cells.     

Blocking granule-mediated death by primary human NK cells requires both protection of mitochondria and inhibition of caspase activity

Human GraB (hGraB) preferentially induces apoptosis via Bcl-2-regulated mitochondrial damage but can also directly cleave caspases and caspase substrates in cell-free systems. How hGraB kills cells when it is delivered by cytotoxic lymphocytes (CL) and the contribution of hGraB to CL-induced death is still not clear. We show that primary human natural killer (hNK) cells, which specifically used hGraB to induce target cell death, were able to induce apoptosis of cells whose mitochondria were protected by Bcl-2. Purified hGraB also induced apoptosis of Bcl-2-overexpressing targets but only when delivered at 5- to 10-fold the concentration required to kill cells expressing endogenous Bcl-2. Caspases were critical in this process as inhibition of caspase activity permitted clonogenic survival of Bcl-2-overexpressing cells treated with hGraB or hNK cells but did not protect cells that only expressed endogenous Bcl-2. Our data therefore show that hGraB triggers caspase activation via mitochondria-dependent and mitochondria-independent mechanisms that are activated in a hierarchical manner, and that the combined effects of Bcl-2 and direct caspase inhibition can block cell death induced by hGraB and primary hNK cells.     

Caspases - controlling intracellular signals by protease zymogen activation

Animal development and homeostasis is a balance between cell proliferation and cell death. Physiologic, and sometimes pathologic, cell death - apoptosis - is driven by activation of a family of proteases known as the caspases, present in almost all nucleated animal cells. The enzymatic properties of these proteases are governed by a dominant specificity for substrates containing Asp, and by the use of a Cys side chain for catalyzing peptide bond cleavage. The primary specificity for Asp turns out to be very rare among proteases, and currently the only other known mammalian proteases with the same primary specificity is the physiological caspase activator granzyme B. Like most other proteases, the caspases are synthesized as inactive zymogens whose activation requires limited proteolysis or binding to co-factors. To transmit the apoptotic execution signal, caspase zymogens are sequentially activated through either an intrinsic or an extrinsic pathway. The activation of caspases at the apex of each pathway, the initiators, occurs by recruitment to specific adapter molecules through homophilic interaction domains, and the activated initiators directly process the executioner caspases to their catalytically active forms. In the present communication we review the different mechanisms underlying the selective activation of the caspases.     

The c-IAP-1 and c-IAP-2 proteins are direct inhibitors of specific caspases.

The inhibitor of apoptosis (IAP) family of proteins are highly conserved through evolution. However, the mechanisms by which these proteins interfere with apoptotic cell death have been enigmatic. Recently, we showed that one of the human IAP family proteins, XIAP, can bind to and potently inhibit specific cell death proteases (caspases) that function in the distal portions of the proteolytic cascades involved in apoptosis. In this study, we investigated three of the other known members of the human IAP family, c-IAP-1, c-IAP-2 and NAIP. Similarly to XIAP, in vitro binding experiments indicated that c-IAP-1 and c-IAP-2 bound specifically to the terminal effector cell death proteases, caspases-3 and -7, but not to the proximal protease caspase-8, caspases-1 or -6. In contrast, NAIP failed to bind tightly to any of these proteases. Recombinant c-IAP-1 and c-IAP-2 also inhibited the activity of caspases-3 and -7 in vitro, with estimated Kis of <=0.1 microM, whereas NAIP did not. The BIR domain-containing region of c-IAP-1 and c-IAP-2 was sufficient for inhibition of these caspases, though proteins that retained the RING domain were somewhat more potent. Utilizing a cell-free system in which caspases were activated in cytosolic extracts by addition of cytochrome c, c-IAP-1 and c-IAP-2 inhibited both the generation of caspase activities and proteolytic processing of pro-caspase-3. Similar results were obtained in intact cells when c-IAP-1 and c-IAP-2 were overexpressed by gene transfection, and apoptosis was induced by the anticancer drug, etoposide. Cleavage of c-IAP-1 or c-IAP-2 was not observed when interacting with the caspases, implying a different mechanism from the baculovirus p35 protein, the broad spectrum suicide inactivator of caspases. Taken together, these findings suggest that c-IAP-1 and c-IAP-2 function similarly to XIAP by inhibiting the distal cell death proteases, caspases-3 and -7, whereas NAIP presumably inhibits apoptosis via other targets.