Regulated cell death: signaling and mechanisms.

Cell turnover is a fundamental feature in metazoans. Cells can die passively, as a consequence of severe damage to their structural integrity, or actively, owing to a more confined biological disruption such as DNA damage. Passive cell death is uncontrolled and often harmful to the organism. In contrast, active cell death is tightly regulated and serves to support the organism's life. Apoptosis-the primary form of regulated cell death-is relatively well defined. Necroptosis-an alternative, distinct kind of regulated cell death discovered more recently-is less well understood. Apoptosis and necroptosis can be triggered either from within the cell or by extracellular stimuli. Certain signaling components, including several death ligands and receptors, can regulate both processes. Whereas apoptosis is triggered and executed via intracellular proteases called caspases, necroptosis is suppressed by caspase activity. Here we highlight current understanding of the key signaling mechanisms that control regulated cell death.

Dennd1a, a susceptibility gene for polycystic ovary syndrome, is essential for mouse embryogenesis.

BACKGROUND: The DENND1A has been identified as a guanine nucleotide exchange factor for small GTPase Rab35, which functions in endocytic trafficking to mediate the recycling of selective cargos. Genetic alterations within the DENND1A gene have been implicated in human disease such as polycystic ovary syndrome (PCOS). However, the role of DENND1A in developmental and reproductive processes is largely unknown. RESULTS: Using Dennd1a gene knockout mice, we uncovered that homogeneous Dennd1a-/- mutants died around embryonic day (E) 14.5. The brain of Dennd1a-/- embryos exhibited defects, partially attributed to the dysregulation of cell division and survival in the telencephalon. The transcription of Fgf8 mRNA was ectopically elevated in the dorsal midline of telencephalon, concomitant with a decrease of active ß-catenin and Axin2 in the brain of Dennd1a-/- embryos. During liver morphogenesis, the ablation of Dennd1a impaired hepatic cell proliferation, the differentiation of hepatocyte, and hepatic hematopoiesis. In addition, loss of Dennd1a also affected the development of primordial germ cells. CONCLUSIONS: We demonstrate that Dennd1a, a susceptibility gene for PCOS, is essential for embryogenesis, probably through the mediation of endocytic recycling of selective cargos that are involved in cell signaling crucial for the development of multiple embryonic organ systems.

The resurrection of the PIDDosome - emerging roles in the DNA-damage response and centrosome surveillance.

The PIDDosome is often used as the alias for a multi-protein complex that includes the p53-induced death domain protein 1 (PIDD1), the bipartite linker protein CRADD (also known as RAIDD) and the pro-form of an endopeptidase belonging to the caspase family, i.e. caspase-2. Yet, PIDD1 variants can also interact with a number of other proteins that include RIPK1 (also known as RIP1) and IKBKG (also known as NEMO), PCNA and RFC5, as well as nucleolar components such as NPM1 or NCL. This promiscuity in protein binding is facilitated mainly by autoprocessing of the full-length protein into various fragments that contain different structural domains. As a result, multiple responses can be mediated by protein complexes that contain a PIDD1 domain. This suggests that PIDD1 acts as an integrator for multiple types of stress that need instant attention. Examples are various types of DNA lesion but also the presence of extra centrosomes that can foster aneuploidy and, ultimately, promote DNA damage. Here, we review the role of PIDD1 in response to DNA damage and also highlight novel functions of PIDD1, such as in centrosome surveillance and scheduled polyploidisation as part of a cellular differentiation program during organogenesis.

The role of DENND1A and CYP19A1 gene variants in individual susceptibility to obesity in Turkish population-a preliminary study.

Single nucleotide polymorphisms (SNPs), the most common genetic variations in human genome, can manage the predisposition of certain complex diseases or situations such as obesity. Genetic polymorphisms also play an important role as they can impact a population's susceptibility to being overweight or obese and developing related chronic complications such as hypertension, coronary heart disease, diabetes and cancer. The present study comprised of 193 unrelated healthy volunteers (120 females and 73 males) with Turkish origin. Only female adolescents (n = 110) were divided into 2 categories according to their BMI values as overweight (BMI ≥ 25) and normal (18.5 < BMI < 25) according to WHO classification. Genomic DNA was isolated from venous blood samples and genotyping of DENND1A rs10818854 and CYP19A1 rs2414096 variants was performed on Roche Light Cycler 2.0 Real-Time PCR platform. Serum hormone levels were analyzed by Electrochemiluminescent Immunoassay (ECLIA; Roche diagnostics). The genotype distributions were consistent with the Hardy-Weinberg equilibrium for both SNPs in the studied population (p > 0.05). The genotype distribution of DENND1A rs10818854 was determined for the first time in Turkish population and the variant allele frequency was found as 0.095. According to reduced sex hormone-binding globulin levels and increased free androgen index in the present study, obesity was linked with hyperandrogenism in female subjects. Both polymorphisms were investigated as potential genetic susceptibility markers for obesity and neither DENND1A nor CYP19A1 showed any associations.

Reconstitution of the death-inducing signaling complex reveals a substrate switch that determines CD95-mediated death or survival.

The death-inducing signaling complex (DISC) is critical for initiation of death-receptor-mediated apoptosis; however, paradoxically, CD95 also signals for cell survival. Here, we reconstitute a functional DISC using only purified CD95, FADD, and procaspase-8 and unveil a two-step activation mechanism involving both dimerization and proteolytic cleavage of procaspase-8 that is obligatory for death-receptor-induced apoptosis. Initially, dimerization yields active procaspase-8 with a very restricted substrate repertoire, limited to itself or c-FLIP. Proteolytic cleavage is then required to fully activate caspase-8, thereby permitting DISC-mediated cleavage of the critical exogenous apoptotic substrates, caspase-3 and Bid. This switch in catalytic activity and substrate range is a key determinant of DISC signaling, as cellular expression of noncleavable procaspase-8 mutants, which undergo DISC-mediated oligomerization, but not cleavage, fails to initiate CD95-induced apoptosis. Thus, using the reconstituted DISC, we have delineated a crucial two-step activation mechanism whereby activated death receptor complexes can trigger death or survival.

Low expression of PIDD is associated with cell proliferation and apoptosis in hepatocellular carcinoma.

p53-induced death domain protein (PIDD) facilitates p53-dependent apoptosis through the interaction with components of the death receptor signaling pathways. However, the role of PIDD in hepatocellular carcinoma (HCC) development remains unknown. In this study, we investigated the expression pattern of PIDD in clinical HCC samples and adjacent non-cancerous tissues using immunohistochemistrical and Western blot analyses. The results showed that PIDD was lowly expressed in HCC tissues and HCC cell lines, compared with the adjacent non-tumorous tissues and LO2 normal hepatocytes. In addition, clinicopathological analysis showed that the expression of PIDD was closely related with multiple clinicopathological variables, such as American Joint Committee on Cancer (AJCC) stage, AFP, and poor prognosis of HCC. Univariate and multivariate survival analyses demonstrated that PIDD could serve as an independent prognostic factor to predict the survival of HCC patients. We used serum starvation-refeeding experiment to explore the involvement of PIDD in HCC cell cycle regulation. We found that PIDD was accumulated in growth-arrested HCC cells and was progressively decreased when cells entered into S phase. Moreover, flow cytometry and cell counting kit-8 (CCK-8) assays indicated that depleting the expression of PIDD could facilitate cell cycle progression and accelerate cell proliferation in HepG2 cells, while overexpression of PIDD could result in cell cycle arrest at G1 phase and hinder the cell proliferation in Hep3B cells. Finally, flow cytometry revealed that overexpression of PIDD slightly increased the apoptosis of HCC cells. Taken together, we concluded that PIDD may be a valuable prognostic marker and promising therapeutic target of HCC.

Death effecter domain for the assembly of death-inducing signaling complex.

Death-inducing signaling complex (DISC) is a platform for the activation of initiator caspase in extrinsic apoptosis. Assembly of DISC is accomplished by two different types of homotypic interaction: one is between death domains (DDs) of a death receptor and FADD, and the other is between death effecter domains (DEDs) of FADD, procaspase-8/-10 and cFLIP. Recent biochemical investigations on the stoichiometry of DISC have revealed that single-DED-containing FADD exists in DISC in a substantially lower abundance than the sum of tandem-DEDs-containing components that are procaspase-8 and cFLIP. In addition, the homology models of the tandem DEDs in procaspase-8 and cFLIP show that two different interaction faces, H1-H4 face and H2-H5 face, are exposed for possible inter-molecular DED-DED interactions. These recent findings led to a proposal of the DED chain model for the interactions between FADD, procaspase-8 and cFLIP in DISC. This emerging view provides new insights on the topology of DED-DED network in DISC and furthermore on how procaspase-8 and cFLIP cluster for dimerization and proteolytic activation.

Activation and assembly of the inflammasomes through conserved protein domain families.

Inflammasomes are oligomeric protein complexes assembled through interactions among the death domain superfamily members, in particular the CARD and PYD domains. Recent progress has shed lights on how the ASC PYD can polymerize to form filaments using multiple domain:domain interfaces, and how the caspase4 CARD can recognize LPS to activate the non-classical inflammasome pathway. Comprehensive understanding of the molecular mechanisms of inflammasome activation and assembly require more extensive structural and biophysical dissection of the inflammasome components and complexes, in particular additional CARD or PYD filaments. Because of the variations in death domain structures and complexes observed so far, future work will undoubtedly shed lights on the mechanisms of inflammasome assembly as well as more surprises on the versatile structure and function of the death domain superfamily.

Tandem DEDs and CARDs suggest novel mechanisms of signaling complex assembly.

Apoptosis is an important process to maintain cellular homeostasis. Deregulated apoptosis has linked to a number of diseases, such as inflammatory diseases, neurodegenerative disorder, and cancers. A major signaling complex in the death receptor signaling pathway leading to apoptosis is death-induced signaling complex (DISC), which is regulated mainly by death effector domain (DED)-containing proteins. There are seven DED-containing proteins in human, including FADD, c-FLIP, caspase-8, caspase-10, DEDD, DEDD2, and PEA-15. The main players in DISC formation employ tandem DEDs for regulating signaling complex formation. The regulatory mechanism of signaling complex formation is important and yet remains unclear. Interestingly, three caspase recruitment domain (CARD)-containing members, which belong to the same DD superfamily as DED-containing proteins, also contains similar tandem CARDs. Recent structural studies have shown that tandem CARDs are essential for the formation of a helical signaling complex. This review summarizes recent structural studies on DED-containing proteins and especially discusses the studies on tandem DEDs and tandem CARDs, which suggest new mechanisms of signaling complex assembly.

Exercise-induced leukocyte apoptosis.

Physical exercise is well known to affect leukocyte numbers and function. While regular exercise training has been shown to enhance specific immune functions, acute bouts of intensive exercise often lead to a pro-inflammatory response accompanied by a transient lymphocytopenia and neutrophilia. It can be assumed, that lymphocytopenia can be attributed at least partially to an enhanced lymphocyte apoptosis. In contrast, regulation of neutrophil apoptosis after exercise remains controversial since studies demonstrated both an up-regulation as well as a down-regulation of cell death. However, these discrepancies may be due to differences in exercise protocols, subjects' fitness levels, and to different methodological approaches. Two major signalling pathways of exercise induced apoptosis have been identified. First the external receptor mediated pathway using death receptors, and second the internal, oxidative-mediated pathway which encompasses the mitochondria. Potential apoptosis modulating mediators are reactive oxygen species (ROS), glucocorticoids and cytokines which are part of the systemic inflammatory response evoked after acute intensive exercise. Finally, the physiological impact and clinical relevance of leukocyte apoptosis will be discussed. On the one hand, exercise-induced apoptosis might be a mechanism to remove activated and potentially autoreactive immune cells. On the other hand, apoptosis might be a regulatory mechanism which is necessary for tissue reorganization and adaptational training processes.

Autophagosomal membrane serves as platform for intracellular death-inducing signaling complex (iDISC)-mediated caspase-8 activation and apoptosis.

Autophagy and apoptosis are two evolutionarily conserved processes that regulate cell fate in response to cytotoxic stress. However, the functional relationship between these two processes remains far from clear. Here, we demonstrate an autophagy-dependent mechanism of caspase-8 activation and initiation of the apoptotic cascade in response to SKI-I, a pan-sphingosine kinase inhibitor, and bortezomib, a proteasome inhibitor. Autophagy is induced concomitantly with caspase-8 activation, which is responsible for initiation of the caspase cascade and the mitochondrial amplification loop that is required for full execution of apoptosis. Inhibition of autophagosome formation by depletion of Atg5 or Atg3 results in a marked suppression of caspase-8 activation and apoptosis. Although caspase-8 self-association depends on p62/SQSTM1, its self-processing requires the autophagosomal membrane. Caspase-8 forms a complex with Atg5 and colocalizes with LC3 and p62. Moreover, FADD, an adaptor protein for caspase-8 activation, associates with Atg5 on Atg16L- and LC3-positive autophagosomal membranes and loss of FADD suppresses cell death. Taken together, these results indicate that the autophagosomal membrane serves as a platform for an intracellular death-inducing signaling complex (iDISC) that recruits self-associated caspase-8 to initiate the caspase-8/-3 cascade.

Intracellular pathways of pancreatic ß-cell apoptosis in type 1 diabetes.

BACKGROUND: Apoptosis of ß cells is a feature of type 1 diabetes. It is also increasingly recognized in type 2 diabetes and islet graft rejection. METHODS: We have studied the intracellular pathways that regulate ß-cell apoptosis in type 1 and 2 diabetes. We have examined the role of Bid, a pro-apoptotic member of the Bcl-2 family, using islets from mice deficient in Bid. We also studied the Bcl-2 family molecules involved in killing by using high concentrations of reducing sugars such as glucose or ribose. RESULTS: We found that Bid-deficient islets are protected from recombinant human perforin and granzyme B, as well as from Fas-mediated killing. This makes Bid a target for protection of ß cells from multiple insults relevant to type 1 diabetes. In contrast to granzyme B and death receptor signalling, we found that islets lacking Bim or Puma were protected from glucose toxicity. CONCLUSIONS: Our data indicate that different stimuli activate different initiator molecules in the Bcl-2-regulated pathway in ß cells.

Knockdown of MADD and c-FLIP overcomes resistance to TRAIL-induced apoptosis in ovarian cancer cells.

OBJECTIVE: The clinical utility of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in the treatment of established human malignancies is limited by the development of resistance to TRAIL. We hypothesized that knockdown of map-kinase activating death domain containing protein (MADD), a TRAIL-resistance factor, may overcome TRAIL resistance in ovarian cancer cells. STUDY DESIGN: MADD expression in resected ovarian cancer specimens and cell lines was quantified with the use of polymerase chain reaction. Sensitivity of ovarian cancer cell lines to TRAIL, with or without MADD knockdown, was assessed. RESULTS: MADD is expressed at relatively higher levels in human malignant ovarian cancer tissues and cell lines, compared with normal ovarian tissues. The cell lines OVCA429 and OVCAR3 were susceptible, and cell lines CAOV-3 and SKOV-3 were resistant to TRAIL. MADD knockdown in CAOV-3 cells, but not in SKOV-3 cells, conferred TRAIL sensitivity. Knockdown of cellular Fas-associated death domain-like interleukin-1 beta-converting enzyme-inhibitory protein (c-FLIP) in SKOV-3 cells increased spontaneous and TRAIL-induced apoptosis, which was further increased on MADD knockdown. CONCLUSION: MADD/c-FLIP(L) knockdown can render TRAIL-resistant ovarian cancer cells susceptible to TRAIL.

Anoikis mediators in oral squamous cell carcinoma.

Anoikis - apoptotic cell death triggered by loss of extracellular matrix (ECM) contacts - is dysregulated in many chronic debilitating and fatal diseases. Mechanisms rendering tumor cells resistant to anoikis, although not completely understood, possess significant therapeutic promise. In death receptor-mediated anoikis mechanisms, focal adhesion kinase (FAK) and receptor-interacting protein (RIP) dissociate, leading to association of RIP with Fas, formation of the death-inducing signaling complex (DISC), activation of caspase-3, and propagation of anoikis. In contrast, anoikis resistance is accomplished through constitutive activation of survival pathways that include integrin-dependent activation of FAK and extracellular-signal-regulated kinase (ERK). In addition, FAK and RIP association confers anoikis resistance by inhibiting the association of RIP with Fas and formation of the death signaling complex, which allows cells to escape anoikis. Up-regulation of CD44 also contributes to survival signals and promotes anoikis resistance. This review will focus on the roles of death receptors, prosurvival pathways, and the molecular players involved in anoikis escalation and resistance in oral squamous cell carcinoma.

E2F-Family Members Engage the PIDDosome to Limit Hepatocyte Ploidy in Liver Development and Regeneration.

E2F transcription factors control the cytokinesis machinery and thereby ploidy in hepatocytes. If or how these proteins limit proliferation of polyploid cells with extra centrosomes remains unknown. Here, we show that the PIDDosome, a signaling platform essential for caspase-2-activation, limits hepatocyte ploidy and is instructed by the E2F network to control p53 in the developing as well as regenerating liver. Casp2 and Pidd1 act as direct transcriptional targets of E2F1 and its antagonists, E2F7 and E2F8, that together co-regulate PIDDosome expression during juvenile liver growth and regeneration. Of note, whereas hepatocyte aneuploidy correlates with the basal ploidy state, the degree of aneuploidy itself is not limited by PIDDosome-dependent p53 activation. Finally, we provide evidence that the same signaling network is engaged to control ploidy in the human liver after resection. Our study defines the PIDDosome as a primary target to manipulate hepatocyte ploidy and proliferation rates in the regenerating liver.

DENND5B Regulates Intestinal Triglyceride Absorption and Body Mass.

Regulation of lipid absorption by enterocytes can influence metabolic status in humans and contribute to obesity and related complications. The intracellular steps of chylomicron biogenesis and transport from the Endoplasmic Reticulum (ER) to the Golgi complex have been described, but the mechanisms for post-Golgi transport and secretion of chylomicrons have not been identified. Using a newly generated Dennd5b-/- mouse, we demonstrate an essential role for this gene in Golgi to plasma membrane transport of chylomicron secretory vesicles. In mice, loss of Dennd5b results in resistance to western diet induced obesity, changes in plasma lipids, and reduced aortic atherosclerosis. In humans, two independent exome sequencing studies reveal that a common DENND5B variant, p.(R52K), is correlated with body mass index. These studies establish an important role for DENND5B in post-Golgi chylomicron secretion and a subsequent influence on body composition and peripheral lipoprotein metabolism.

E3 ubiquitin ligases and deubiquitinases as modulators of TRAIL-mediated extrinsic apoptotic signaling pathway.

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) initiates the extrinsic apoptotic pathway through formation of the death-inducing signaling complex (DISC), followed by activation of effector caspases. TRAIL receptors are composed of death receptors (DR4 and DR5), decoy receptors (DcR1 and DcR2), and osteoprotegerin. Among them, only DRs activate apoptotic signaling by TRAIL. Since the levels of DR expressions are higher in cancer cells than in normal cells, TRAIL selectively activates apoptotic signaling pathway in cancer cells. However, multiple mechanisms, including down-regulation of DR expression and pro-apoptotic proteins, and up-regulation of anti-apoptotic proteins, make cancer cells TRAIL-resistant. Therefore, many researchers have investigated strategies to overcome TRAIL resistance. In this review, we focus on protein regulation in relation to extrinsic apoptotic signaling pathways via ubiquitination. The ubiquitin proteasome system (UPS) is an important process in control of protein degradation and stabilization, and regulates proliferation and apoptosis in cancer cells. The level of ubiquitination of proteins is determined by the balance of E3 ubiquitin ligases and deubiquitinases (DUBs), which determine protein stability. Regulation of the UPS may be an attractive target for enhancement of TRAIL-induced apoptosis. Our review provides insight to increasing sensitivity to TRAIL-mediated apoptosis through control of post-translational protein expression. [BMB Reports 2019; 52(2): 119-126].

Biochemical analysis of the native TRAIL death-inducing signaling complex.

The extrinsic apoptosis pathway is activated when certain members of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) are oligomerized by their cognate ligands that are members of the TNF superfamily (TNFSF). The apoptosis-inducing capacity of a member of the TNFRSF relies on the presence of a death domain (DD) in the intracellular portion of the receptor protein. Such receptors are also referred to as death receptors. Binding of a TNFSF ligand to a TNFRSF receptor that is expressed on the surface of a cell results in the formation of a receptor proximal protein complex. This protein complex is the platform for further signaling events within the cell. In case of death receptors like TNF-related apoptosis-inducing ligand receptor 1 (TRAIL-R1/DR4), TRAIL-R2 (KILLER/APO-2/DR5/TRICK), CD95 (Fas, APO-1), or TNF receptor 1 (TNF-R1), this complex is termed death-inducing signaling complex (DISC). The compositions of the various DISCs have been intensively studied in the last 12 years. For the CD95 and the TRAIL-R1/R2 DISCs, it is now clear that the adaptor protein Fas-associated DD protein (FADD) forms part of these complexes and is necessary for recruitment of the proapoptotic signaling molecules caspase-8 and caspase-10. Recruitment of these proteases allows for their activation at the DISC and subsequent induction of apoptosis. The caspase-8 homologous cellular FLICE-like inhibitory protein (cFLIP) can also be recruited to the DISC. cFLIP acts as an anti-apoptotic regulator by interfering with activation of caspases 8 and 10 at the DISC. Interestingly, treatment of TRAIL-resistant tumor cells with conventional chemotherapeutic drugs or with proteasome inhibitors renders these cells sensitive for TRAIL-induced apoptosis. By applying the methodology of the biochemical analysis of the TRAIL DISC described here, we were able to show that this sensitization is mainly due to changes in the biochemical composition of the DISC as the apoptosis-initiating protein complex of the extrinsic pathway.

Rewinding the DISC.

Fas (CD95/APO-1) belongs to the tumor necrosis factor receptor family and its signaling pathway has been extensively studied over the past 15 years. Blockade of the Fas-mediated apoptotic signal leads to abusive lymphoproliferation, auto-immunity, and an increased risk of developing lymphoma and leukemia. Fas engagement drives the formation of a complex termed DISC (death-inducing signaling complex), which contains the adaptor molecule Fas-associated protein, two members of the caspase family caspase-8 and -10, and a pseudo-caspase termed c-FLIP. According to different authors, DISC formation relies either on the redistribution of Fas into the lipid rafts or the recruitment of the actin cytoskeleton and receptor endocytosis or the production of ceramide. However, the accurate molecular ordering upstream from the formation of DISC remains very puzzling and is highly debated. Herein we review some of the factors that would potentially facilitate or limit the formation of the DISC.

Partial equilibrium approximations in apoptosis. II. The death-inducing signaling complex subsystem.

This paper is a continuation of our previous work (Huang and Yong, 2013) for simplifying the Fas signaling-induced apoptotic pathway identified by Hua et al. (2005) for human tumor T cells. The previous paper studied the downstream intracelluar-signaling subsystem, while the present one is concerned with the upstream death-inducing signaling complex (DISC) subsystem. Under the assumption that the bind of Fas-associated death domains and FLICE-inhibitory proteins to the DISC is much faster than that of the initiator procaspases, we greatly simplify the upstream subsystem from 35 reactions with 26 species to 6 reactions with 9 species by adopting the classical and recently justified partial equilibrium approximation method. Numerical simulations show that the simplified model is in an excellent agreement with the original model. Most importantly, the simplified model clearly reveals the key reactants and dominated pathways in the Fas signaling process, and thus provides new insights into the apoptosis.