Species-Specific Inhibition of Necroptosis by HCMV UL36

Viral infection activates cellular antiviral defenses including programmed cell death (PCD). Many viruses, particularly those of the Herpesviridae family, encode cell death inhibitors that antagonize different forms of PCD. While some viral inhibitors are broadly active in cells of different species, others have species-specific functions, probably reflecting the co-evolution of the herpesviruses with their respective hosts. Human cytomegalovirus (HCMV) protein UL36 is a dual cell death pathway inhibitor. It blocks death receptor-dependent apoptosis by inhibiting caspase-8 activation, and necroptosis by binding to the mixed lineage kinase domain-like (MLKL) protein and inducing its degradation. While UL36 has been shown to inhibit apoptosis in human and murine cells, the specificity of its necroptosis-inhibiting function has not been investigated. Here we show that UL36 interacts with both human and murine MLKL, but has a higher affinity for human MLKL. When expressed by a recombinant mouse cytomegalovirus (MCMV), UL36 caused a modest reduction of murine MLKL levels but did not inhibit necroptosis in murine cells. These data suggest that UL36 inhibits necroptosis, but not apoptosis, in a species-specific manner, similar to ICP6 of herpes simplex virus type 1 and MC159 of molluscum contagiosum virus. Species-specific necroptosis inhibition might contribute to the narrow host range of these viruses.     

RIP and FADD: two "death domain"-containing proteins can induce apoptosis by convergent, but dissociable, pathways.

With use of the yeast two-hybrid system, the proteins RIP and FADD/MORT1 have been shown to interact with the "death domain" of the Fas receptor. Both of these proteins induce apoptosis in mammalian cells. Using receptor fusion constructs, we provide evidence that the self-association of the death domain of RIP by itself is sufficient to elicit apoptosis. However, both the death domain and the adjacent alpha-helical region of RIP are required for the optimal cell killing induced by the overexpression of this gene. By contrast, FADD's ability to induce cell death does not depend on crosslinking. Furthermore, RIP and FADD appear to activate different apoptotic pathways since RIP is able to induce cell death in a cell line that is resistant to the apoptotic effects of Fas, tumor necrosis factor, and FADD. Consistent with this, a dominant negative mutant of FADD, lacking its N-terminal domain, blocks apoptosis induced by RIP but not by FADD. Since both pathways are blocked by CrmA, the interleukin 1 beta converting enzyme family protease inhibitor, these results suggest that FADD and RIP can act along separable pathways that nonetheless converge on a member of the interleukin 1 beta converting enzyme family of cysteine proteases.     

MRIT, a novel death-effector domain-containing protein, interacts with caspases and BclXL and initiates cell death

Activation of the cascade of proteolytic caspases has been identified as the final common pathway of apoptosis in diverse biological systems. We have isolated a gene, termed MRIT, that possesses overall sequence homology to FLICE (MACH), a large prodomain caspase that links the aggregated complex of the death domain receptors of the tumor necrosis factor receptor family to downstream caspases. However, unlike FLICE, the C-terminal domain of MRIT lacks the caspase catalytic consensus sequence QAC(R/Q)G. Nonetheless MRIT activates caspase-dependent death. Using yeast two-hybrid assays, we demonstrate that MRIT associates with caspases possessing large and small prodomains (FLICE, and CPP32/YAMA), as well as with the adaptor molecule FADD. In addition, MRIT simultaneously and independently interacts with BclX_L and FLICE in mammalian cells. Thus, MRIT is a mammalian protein that interacts simultaneously with both caspases and a Bcl-2 family member.     

Autophagy in the myocardium: Dying for survival?

Autophagy is an intracellular phenomenon in which a cell digests its own constituents. Autophagy is well conserved in nature from lower eukaryotes to mammals and has been attributed to disparate physiological events - including cell death, the mechanism of which is different from apoptosis. However, unlike in apoptosis, in which a family of cysteine proteases (caspases) and a number of other regulatory proteins have been identified and characterized, the mechanism of autophagic cell death remains unclear. In addition, the general mechanisms by which autophagy is initiated and modulated are just emerging, and several lines of evidence indicate that activated class I phosphatidylinositol 3-kinase and mammalian target of rapamycin (mTOR) inhibit autophagy, while class III phosphatidylinositol 3-kinase acts as a facilitator. Autophagy has been attributed to a number of cardiac disorders, such as ischemic cardiomyopathy, cardiac hypertrophy, hemochromatosis and myocardial aging. Induction of ventricular hypertrophy is associated with decreased autophagy, whereas it is enhanced during the regression of hypertrophy. Induction of acute cardiotoxicity by the anticancer drug anthracycline is also associated with massive cardiomyocyte loss due to autophagy (and apoptosis). Myocyte loss due to autophagy has also been reported during progression from compensated hypertrophy to heart failure in a pressure-overloaded model. Although the depth and dimension of the regulatory network that modulates autophagy in mammalian cells has yet to emerge, existing evidence suggests that it is an integral part of maintaining cellular metabolism, organelle homeostasis and redox equilibrium. Thus, it is a likely possibility that autophagy plays a crucial role in maintaining healthy myocytes in the myocardium.     

Selective activation of JNK1 is necessary for the anti-apoptotic activity of hILP

The balance between the inductive signals and endogenous anti-apoptotic mechanisms determines whether or not programmed cell death occurs. The widely expressed inhibitor of apoptosis gene family includes three closely related mammalian proteins: c-IAP1, c-IAP2, and hILP. The anti-apoptotic properties of these proteins have been linked to caspase inhibition. Here we show that one member of this group, hILP, inhibits interleukin-1β-converting enzyme-induced apoptosis via a mechanism dependent on the selective activation of c-Jun N-terminal kinase 1. These data demonstrate that apoptosis can be inhibited by an endogenous cellular protein by a mechanism that requires the activation of a single member of the mitogen-activating protein kinase family.     

Apoptosis in the intestinal epithelium: its relevance in normal and pathophysiological conditions

Apoptosis is now recognized as an important process responsible for maintenance of the cellular balance between proliferation and death. Apoptosis is distinct from necrosis in that it is a programmed form of cell death and occurs without any accompanying inflammation. This form of cell death can be induced by a wide range of cellular signals, which leads to activation of cell death machinery within the cell and is characterized by distinct morphological changes. Apoptosis is especially relevant in the gastrointestinal tract, as the mammalian intestinal mucosa undergoes a process of continual cell turnover that is essential for maintenance of normal function. Cell proliferation is confined to the crypts, while differentiation occurs during a rapid, orderly migration up to the villus. The differentiated enterocytes, which make up the majority of the cells, then undergo a process of programmed cell death (apoptosis). Although apoptosis is essential for the maintenance of normal gut epithelial function, dysregulated apoptosis is seen in a number of pathological conditions in the gastrointestinal tract. The cellular mechanisms regulating this tightly regimented process have not been clearly defined and this topic represents an area of active investigation as delineation of this process will lead to a better understanding of normal gut mucosal growth.     

Apoptosis: programmed cell death at a molecular level

OBJECTIVES: To characterize cell surface receptors, their ligands, and their proteins in the 2 major pathways of apoptosis; the components that promote/suppress these interactions; the noninflammatory removal of apoptotic bodies by dendritic cells; and methods of assay in studies of cell death. To describe: how deregulation of apoptosis may contribute to autoimmunity, cancer, and neurodegenerative disorders and strategies some viruses have evolved that interfere with the host's apoptotic pathways. METHODS: The authors reviewed and compiled literature on the extrinsic (tumor necrosis factor [TNF] receptor superfamily and ligands) and intrinsic (mitochondria-associated) apoptotic pathways, the pro- and antiapoptotic proteins of the B-cell follicular lymphoma (Bcl)-2 family, the nuclear factor (NF)-kappaB family of proteins, commonly used laboratory methods to distinguish apoptosis from necrosis, the recognition and removal by phagocytosis of apoptotic cells by dendritic cells, and viral strategies to avoid a host's apoptotic response. RESULTS: The 2 major pathways of apoptosis are (1). FasL and other TNF superfamily ligands induce trimerization of cell-surface death receptors and (2). perturbated mitochondria release cytochrome c, the flavoprotein apoptosis-inducing factor, and second mitochondria-derived activator of caspases/DIABLO (a protein that directly neutralizes inhibitors of apoptotic proteins and activates proteases). Catalytically inactive cysteine proteases, called caspases, and other proteases are activated, ultimately leading to cell death with characteristic cellular chromatin condensation and DNA cleavage to fragments of approximately 180 bp. The inhibitory/promoting action of Bcl-2 family members is involved in the release of cytochrome c, an essential factor for the mitochondrial-associated pathway. A balance between inhibition/promotion determines a cell's fate. The NF-kappaB family in the cytoplasm of cells activates various genes carrying the NF-kappaB response element, such as members of the inhibitor of apoptotic proteins family. A few of the more common methods to detect apoptotic cell death are described, which use immunochemical, morphologic and flow cytometric methods, and genetic markers. Exposed phosphatidylserine at the outer leaflet of the plasma membrane of the apoptotic cell serves as a possible receptor for phagocytosis by immature dendritic cells. These cells phagocytize both apoptotic and necrotic cells, but only the latter induce maturation to become fully functional antigen-presenting cells. Viral inhibitors of apoptosis allow increased virus replication in cells, possibly resulting in their oncogenicity. CONCLUSIONS: Balanced apoptosis is crucial in development and homeostasis, and all multicellular organisms have a physiologically programmed continuum of pathways to apoptotic cell death. Further studies of the control at the molecular level of key components and promoters/suppressors of apoptosis may provide better approaches to treatment of autoimmune diseases, malignancies, and neurodegenerative disorders. Many important questions remain regarding the advantages of modifying apoptotic programs in clinical situations.     

Modulation of cell death by Bcl-xL through caspase interaction

The caspases are cysteine proteases that have been implicated in the execution of programmed cell death in organisms ranging from nematodes to humans. Many members of the Bcl-2 family, including Bcl-x_L, are potent inhibitors of programmed cell death and inhibit activation of caspases in cells. Here, we report a direct interaction between caspases and Bcl-x_L. The loop domain of Bcl-x_L is cleaved by caspases in vitro and in cells induced to undergo apoptotic death after Sindbis virus infection or interleukin 3 withdrawal. Mutation of the caspase cleavage site in Bcl-x_L in conjunction with a mutation in the BH1 homology domain impairs the death-inhibitory activity of Bcl-x_L, suggesting that interaction of Bcl-x_L with caspases may be an important mechanism of inhibiting cell death. However, once Bcl-x_L is cleaved, the C-terminal fragment of Bcl-x_L potently induces apoptosis. Taken together, these findings indicate that the recognition/cleavage site of Bcl-x_L may facilitate protection against cell death by acting at the level of caspase activation and that cleavage of Bcl-x_L during the execution phase of cell death converts Bcl-x_L from a protective to a lethal protein.     

BAR: An apoptosis regulator at the intersection of caspases and Bcl-2 family proteins

Two major pathways for induction of apoptosis have been identified-intrinsic and extrinsic. The extrinsic pathway is represented by tumor necrosis factor family receptors, which utilize protein interaction modules known as death domains and death effector domains (DEDs) to assemble receptor signaling complexes that recruit and activate certain caspase-family cell death proteases, namely procaspases-8 and -10. The intrinsic pathway for apoptosis involves the participation of mitochondria, which release caspase-activating proteins. Bcl-2 family proteins govern this mitochondria-dependent apoptosis pathway, with proteins such as Bax functioning as inducers and proteins such as Bcl-2 and Bcl-X(L) serving as suppressors of cell death. An apoptosis regulator, BAR, was identified by using a yeast-based screen for inhibitors of Bax-induced cell death. The BAR protein contains a SAM domain, which is required for its interactions with Bcl-2 and Bcl-X(L) and for suppression of Bax-induced cell death in both mammalian cells and yeast. In addition, BAR contains a DED-like domain responsible for its interaction with DED-containing procaspases and suppression of Fas-induced apoptosis. Furthermore, BAR can bridge procaspase-8 and Bcl-2 into a protein complex. The BAR protein is anchored in intracellular membranes where Bcl-2 resides. BAR therefore may represent a scaffold protein capable of bridging two major apoptosis pathways.     

The sweet taste of death: glucose triggers apoptosis during yeast chronological aging

As time goes by, a postmitotic cell ages following a degeneration process ultimately ending in cell death. This phenomenon is evolutionary conserved and present in unicellular eukaryotes as well, making the yeast chronological aging system an appreciated model. Here, single cells die in a programmed fashion (both by apoptosis and necrosis) for the benefit of the whole population. Besides its meaning for aging and cell death research, age-induced programmed cell death represents the first experimental proof for the so-called group selection theory: Apoptotic genes became selected during evolution because of the benefits they might render to the whole cell culture and not to the individual cell. Many anti‐aging stimuli have been discovered in the yeast chronological aging system and have afterwards been confirmed in higher cells or organisms. New work from the Burhans group (this issue) now demonstrates that glucose signaling has a progeriatric effect on chronologically aged yeast cells: Glucose administration results in a diminished efficacy of cells to enter quiescence, finally causing superoxide‐mediated replication stress and apoptosis.     

A surprising sweetener from enteropathogenic Escherichia coli

Infections with enteropathogenic Escherichia coli (EPEC) are remarkably devoid of gut inflammation and necrotic damage compared to infections caused by invasive pathogens such as Salmonella and Shigella. Recently, we observed that EPEC blocks cell death using the type III secretion system (T3SS) effector NleB. NleB mediated post-translational modification of death domain containing adaptor proteins by the covalent attachment of N-acetylglucosamine (GlcNAc) to a conserved arginine in the death domain.  N-linked glycosylation of arginine has not previously been reported in mammalian cell biology and the precise biochemistry of this modification is not yet defined. Although the addition of a single GlcNAc to arginine is a seemingly slight alteration, the impact of NleB is considerable as arginine in this location is critical for death domain interactions and death receptor induced apoptosis. Hence, by blocking cell death, NleB promotes enterocyte survival and thereby prolongs EPEC attachment to the gut epithelium.     

Suppression of tumor necrosis factor-induced cell death by inhibitor of apoptosis c-IAP2 is under NF-κB control

Members of the NF-κB/Rel and inhibitor of apoptosis (IAP) protein families have been implicated in signal transduction programs that prevent cell death elicited by the cytokine tumor necrosis factor α (TNF). Although NF-κB appears to stimulate the expression of specific protective genes, neither the identities of these genes nor the precise role of IAP proteins in this anti-apoptotic process are known. We demonstrate here that NF-κB is required for TNF-mediated induction of the gene encoding human c-IAP2. When overexpressed in mammalian cells, c-IAP2 activates NF-κB and suppresses TNF cytotoxicity. Both of these c-IAP2 activities are blocked in vivo by coexpressing a dominant form of IκB that is resistant to TNF-induced degradation. In contrast to wild-type c-IAP2, a mutant lacking the C-terminal RING domain inhibits NF-κB induction by TNF and enhances TNF killing. These findings suggest that c-IAP2 is critically involved in TNF signaling and exerts positive feedback control on NF-κB via an IκB targeting mechanism. Functional coupling of NF-κB and c-IAP2 during the TNF response may provide a signal amplification loop that promotes cell survival rather than death.     

The proapoptotic function of Drosophila Hid is conserved in mammalian cells

Three genes-reaper, grim, and hid-are crucial to the regulation of programmed cell death in Drosophila melanogaster. Mutations involving all three genes virtually abolish apoptosis during development, and homozygous hid mutants die as embryos with extensive defects in apoptosis. Although Hid is central to apoptosis in Drosophila, it has no mammalian homologue identified to date. We present evidence that expression of Drosophila Hid in mammalian cells induces apoptosis. This activity is subject to regulation by inhibitors of mammalian cell death. We show that the N terminus of Hid, which is a region of homology with Reaper and Grim, is essential for Hid's function in mammalian cells. We demonstrate that Hid is localized to the mitochondria via a hydrophobic region at its C terminus and functionally interacts with BclXL. This study shows that the function of Hid as a death inducer in Drosophila is conserved in mammalian cells and argues for the existence of a mammalian homologue of this critical regulator of apoptosis.     

Elevated nitric oxide levels associated with hepatic cell apoptosis during liver injury

Hepatic injury is a major event in liver surgery such as liver transplantation and it always leads to hepatic cell apoptosis. Nitric oxide (NO) is a key signaling regulation molecule. Many researchers have shown that increased NO level can influence liver cell apoptosis by promoting or inhibiting the relative signaling pathways that are involved in the caspase family, Bax/Bcl‐2, mitochondria, oxidative stress, death receptors, and mitogen‐activated protein kinases. Elucidating the relationships between NO and hepatic cell apoptosis is necessary for ameliorating prognosis of liver surgery. This article reviews the newest research progress in the relationships between higher NO levels and hepatic cell apoptosis in liver injury.     

Yersinia signals macrophages to undergo apoptosis and YopJ is necessary for this cell death

Pathogenic Yersinia spp. carry a large common plasmid that encodes a number of essential virulence determinants. Included in these factors are the Yersinia-secreted proteins called Yops. We analyzed the consequences of wild-type and mutant strains of Yersinia pseudotuberculosis interactions with the macrophage cell line RAW264.7 and murine bone marrow-derived macrophages. Wild-type Y. pseudotuberculosis kills ≈70% of infected RAW264.7 macrophages and marrow-derived macrophages after an 8-h infection. We show that the cell death mediated by Y. pseudotuberculosis is apoptosis. Mutant Y. pseudotuberculosis that do not make any Yop proteins no longer cause host cell death. Attachment to host cells via invasin or YadA is necessary for the cell death phenotype. Several Yop mutant strains that fail to express one or more Yop proteins were engineered and then characterized for their ability to cause host cell death. A mutant with a polar insertion in YpkA Ser/Thr kinase that does not express YpkA or YopJ is no longer able to cause apoptosis. In contrast, a mutant no longer making YopE or YopH (a tyrosine phosphatase) induces apoptosis in macrophages similar to wild type. When yopJ is added in trans to the ypkAyopJ mutant, the ability of this strain to signal programmed cell death in macrophages is restored. Thus, YopJ is necessary for inducing apoptosis. The ability of Y. pseudotuberculosis to promote apoptosis of macrophages in cell culture suggests that this process is important for the establishment of infection in the host and for evasion of the host immune response.     

LARD: A new lymphoid-specific death domain containing receptor regulated by alternative pre-mRNA splicing

Fas and TNF-R1 are cysteine-rich cell surface receptors related to the low-affinity nerve growth factor receptor family. Engagement of these receptors by their respective ligands, FasL and tumor necrosis factor, leads to apoptosis that is signaled through a conserved intracellular portion of the receptor termed the “death domain.” We have cloned a new member of this family, lymphocyte-associated receptor of death (LARD), which leads to spontaneous apoptosis when expressed in 293T cells. The expression of LARD is more tightly regulated than that of either Fas or TNF-R1 as it is found predominantly on lymphocytes (T and B cells) but not on macrophages or a number of transformed lymphocyte cell lines. Alternative pre-mRNA splicing generates at least 11 distinct isoforms of LARD. The full-length isoform, LARD-1, extends to include the transmembrane and death domains, whereas the other isoforms encode potentially secreted molecules. Naive B and T cells express very little LARD-1 but express combinations of the other isoforms. Upon T cell activation, a programmed change in alternative splicing occurs so that the full-length, membrane-bound LARD-1 predominates. This may have implications for the control of lymphocyte proliferation following activation.     

Mechanisms of Cancer Cell Killing by the Adenovirus E4orf4 Protein

During adenovirus (Ad) replication the Ad E4orf4 protein regulates progression from the early to the late phase of infection. However, when E4orf4 is expressed alone outside the context of the virus it induces a non-canonical mode of programmed cell death, which feeds into known cell death pathways such as apoptosis or necrosis, depending on the cell line tested. E4orf4-induced cell death has many interesting and unique features including a higher susceptibility of cancer cells to E4orf4-induced cell killing compared with normal cells, caspase-independence, a high degree of evolutionary conservation of the signaling pathways, a link to perturbations of the cell cycle, and involvement of two distinct cell death programs, in the nucleus and in the cytoplasm. Several E4orf4-interacting proteins including its major partners, protein phosphatase 2A (PP2A) and Src family kinases, contribute to induction of cell death. The various features of E4orf4-induced cell killing as well as studies to decipher the underlying mechanisms are described here. Many explanations for the cancer specificity of E4orf4-induced cell death have been proposed, but a full understanding of the reasons for the different susceptibility of cancer and normal cells to killing by E4orf4 will require a more detailed analysis of the complex E4orf4 signaling network. An improved understanding of the mechanisms involved in this unique mode of programmed cell death may aid in design of novel E4orf4-based cancer therapeutics.