AK2 activates a novel apoptotic pathway through formation of a complex with FADD and caspase-10.

Mitochondrial proteins function as essential regulators in apoptosis. Here, we show that mitochondrial adenylate kinase 2 (AK2) mediates mitochondrial apoptosis through the formation of an AK2-FADD-caspase-10 (AFAC10) complex. Downregulation of AK2 attenuates etoposide- or staurosporine-induced apoptosis in human cells, but not that induced by tumour-necrosis-factor-related apoptosis-inducing ligand (TRAIL) or Fas ligand (FasL). During intrinsic apoptosis, AK2 translocates to the cytoplasm, whereas this event is diminished in Apaf-1 knockdown cells and prevented by Bcl-2 or Bcl-X(L). Addition of purified AK2 protein to cell extracts first induces activation of caspase-10 via FADD and subsequently caspase-3 activation, but does not affect caspase-8. AFAC10 complexes are detected in cells undergoing intrinsic cell death and AK2 promotes the association of caspase-10 with FADD. In contrast, AFAC10 complexes are not detected in several etoposide-resistant human tumour cell lines. Taken together, these results suggest that, acting in concert with FADD and caspase-10, AK2 mediates a novel intrinsic apoptotic pathway that may be involved in tumorigenesis.

Capsaicin induces apoptosis by generating reactive oxygen species and disrupting mitochondrial transmembrane potential in human colon cancer cell lines.

Although genetic factors are a well-known cause of colorectal cancer, environmental factors contribute more to its development. Despite advances in the fields of surgery, radiotherapy and chemotherapy, the cure rates for colon cancer have not substantially improved over the past few decades. Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), the principal pungent ingredient of hot chili pepper, has exhibited an anti-tumor effect in many cell types. However, the mechanisms responsible for the anti-tumor effect of capsaicin are not yet completely understood. In this study, we investigated whether capsaicin induces apoptosis in colon cancer cell lines. Capsaicin decreased cell viability in a dose-dependent manner in Colo320DM and LoVo cells. In addition, capsaicin produced cell morphology changes and DNA fragmentation, decreased the DNA contents, and induced phosphatidylserine translocation, which is a hallmark of apoptotic cell death. We showed that capsaicin-induced apoptosis is associated with an increase in ROS generation and a disruption of the mitochondrial transmenbrane potential. A possible mechanism of capsaicin-induced apoptosis is the activation of caspase 3, a major apoptosis-executing enzyme. Treatment with capsaicin induced a dramatic increase in caspase 3 activity, as assessed by the cleavage of Ac-DEVD-AMC, a fluorogenic substrate. In conclusion, our results clearly showed that capsaicin induced apoptosis in colon cancer cells. Although the actual mechanisms of capsaicin-induced apoptosis remain uncertain, it may be a beneficial agent for colon cancer treatment and chemoprevention.

Role of FLASH in caspase-8-mediated activation of NF-kappaB: dominant-negative function of FLASH mutant in NF-kappaB signaling pathway.

Caspase-8 is the most receptor-proximal, upstream caspase in the caspase cascade and plays a key role in cell death triggered by various death receptors. Here, we addressed the role of endogenous caspase-8 in tumor necrosis factor (TNF)-alpha-induced activation of NF-kappaB. Direct targeting of caspase-8 with siRNA and antisense (AS) approaches abolished TNF-alpha-induced activation of NF-kappaB in NIH3T3, HeLa, and HEK293 cells as determined with luciferase reporter gene and cell fractionation assays. Reconstitution of caspase-8-deficient C33A cells with processing-defective (P/D) mutant of caspase-8 sensitized the cells to TNF-alpha for NF-kappaB activation. In contrast to wild-type caspase-8, death effector domain mutant replacing Asp73 with Ala (caspase-8 (D73A)) failed to activate NF-kappaB and to bind FLICE-associated huge protein (FLASH) in vitro and in vivo. Instead, caspase-8 (D73A) mutant bound to caspase-8 and blocked NF-kappaB activation triggered by TNF-alpha and caspase-8. In addition, expression of an NF-kappaB-activating domain-deletion mutant of FLASH or transfection of FLASH AS oligonucleotides abolished TNF-alpha and caspase-8, but not phorbol 12-myristate 13-acetate, -induced activation of NF-kappaB. Further, immunoprecipitation assays showed that caspase-8 formed triple complex with TRAF2 and FLASH. Taken together, these results suggest that endogenous caspase-8 mediates TNF-alpha-induced activation of NF-kappaB via FLASH.

Inhibition of Bcl10-mediated activation of NF-kappa B by BinCARD, a Bcl10-interacting CARD protein.

We have identified a novel CARD-containing protein from EST database. BinCARD (Bcl10-interacting protein with CARD). BinCARD was ubiquitously expressed. Co-immunoprecipitation, In vitro binding, mammalian two-hybrid, and immunostaining assays revealed that BinCARD interacted with Bcl10 through CARD. BinCARD potently suppressed NF-kappa B activation induced by Bcl10 and decreased the amounts of phosphorylated Bcl10. Mutations at the residue Leu17 or Leu65, which is highly conserved in CARD, abolished the inhibitory effects of BinCARD on both Bcl10-induced activation of NF-kappa B and phosphorylation of Bcl10. Further, expression of BinCARD inhibited Bcl10 phosphorylation induced by T cell activation signal. These results suggest that BinCARD interacts with Bcl10 to inhibit Bcl10-mediated activation of NF-kappa B and to suppress Bcl10 phosphorylation.

The Interplay between Autophagy and Aging.

Numerous studies have established a link between autophagy and aging; however, the relationship has not been clearly defined. Aging is a very complex process caused by the accumulation of various factors due to the gradual failure of cellular maintenance. Recent studies have shown that autophagy reduces the stress responses induced by starvation, reactive oxygen species, and the accumulation of intracellular proteins and organelles through cytoprotection, clearance of damaged mitochondria, and lysosomal degradation. Here, we summarize our current understanding of the relationship between autophagy and the aging process.

Overexpression of Atg5 in mice activates autophagy and extends lifespan.

Autophagy has been implicated in the ageing process, but whether autophagy activation extends lifespan in mammals is unknown. Here we show that ubiquitous overexpression of Atg5, a protein essential for autophagosome formation, extends median lifespan of mice by 17.2%. We demonstrate that moderate overexpression of Atg5 in mice enhances autophagy, and that Atg5 transgenic mice showed anti-ageing phenotypes, including leanness, increased insulin sensitivity and improved motor function. Furthermore, mouse embryonic fibroblasts cultured from Atg5 transgenic mice are more tolerant to oxidative damage and cell death induced by oxidative stress, and this tolerance was reversible by treatment with an autophagy inhibitor. Our observations suggest that the leanness and lifespan extension in Atg5 transgenic mice may be the result of increased autophagic activity.

BECN1/Beclin 1 is recruited into lipid rafts by prion to activate autophagy in response to amyloid ß 42.

Prion protein (PRNP) has been implicated in various types of neurodegenerative diseases. Although much is known about prion diseases, the function of cellular PRNP remains cryptic. Here, we show that PRNP mediates amyloid ß1­42 (Aß42)-induced autophagy activation through its interaction with BECN1. Treatment with Aß42 enhanced autophagy flux in neuronal cells. Aß42-induced autophagy activation, however, was impaired in prnp-knockout primary cortical neurons and Prnp-knockdown or prnp-knockout neuronal cells. Immunoprecipitation assays revealed that PRNP interacted with BECN1 via the BCL2-binding domain of BECN1. This interaction promoted the subcellular localization of BECN1 into lipid rafts of the plasma membrane and enhanced activity of PtdIns3K (whose catalytic subunit is termed PIK3C3, mammalian ortholog of yeast VPS34) in lipid rafts by generating PtdIns3P in response to Aß42. Further, the levels of lipid rafts that colocalized with BECN1, decreased in the brains of aged C57BL/6 mice, as did PRNP. These results suggested that PRNP interacts with BECN1 to recruit the PIK3C3 complex into lipid rafts and thus activates autophagy in response to Aß42, defining a novel role of PRNP in the regulation of autophagy.

Molecules and their functions in autophagy.

Autophagy is a self-degradation system of cellular components through an autophagosomal-lysosomal pathway. Over the last 15 yr, yeast genetic screens led to the identification of a number of genes involved in the autophagic pathway. Most of these autophagy genes are present in higher eukaryotes and regulate autophagy process for cell survival and homeostasis. Significant progress has recently been made to better understand the molecular mechanisms of the autophagy machinery. Especially, autophagy process, including the regulation of autophagy induction through mTOR and the nucleation and elongation in autophagosome formation through class III phosphatidylinositol 3-kinase complex and ubiquitin-like conjugation systems, became evident. While many unanswered questions remain to be answered, here, we summarize the recent process of autophagy with emphasis on molecules and their protein complexes along with advanced molecular mechanisms that regulate the autophagy machinery.

Compensatory activation of ERK1/2 in Atg5-deficient mouse embryo fibroblasts suppresses oxidative stress-induced cell death.

Despite of the increasing evidence that oxidative stress may induce non-apoptotic cell death or autophagic cell death, the mechanism of this process is unclear. Here, we report a role and a down-stream molecular event of Atg5 during oxidative stress-induced cell death. Compared to wild type (WT) cells, Atg5-deficient mouse embryo fibroblasts (Atg5-/- MEFs) and Atg5 knockdown HT22 neuronal cells were more resistant to cell death induced by H2O2. On the contrary, Atg5-/- MEFs were as sensitive to tumor necrosis factor (TNF)-alpha and cycloheximide as WT cells, and were more sensitive to cell death triggered by amino acid-deprivation than WT MEFs. Treatment with H2O2 induced the recruitment of a GFP-LC3 fusion protein and conversion of LC3 I to LC3 II, correlated with the extent of autophagosome formation in WT cells, but much less in Atg5-deficient cells. Among stress kinases, ERK1/2 was markedly activated in Atg5-/- MEFs and Atg5 knockdown HT22 and SH-SY5Y neuronal cells. The inhibition of ERK1/2 by MEK1 inhibitor (PD98059) or dominant negative ERK2 enhanced the susceptibility of Atg5-/- MEFs to H2O2-induced cell death. Further, reconstitution of Atg5 sensitized Atg5-/- MEFs to H2O2 and suppressed the activation of ERK1/2. These results suggest that the inhibitory effect of Atg5 deficiency on cell death is attributable by the compensatory activation of ERK1/2 in Atg5-/- MEFs during oxidative stress-induced cell death.

Protection of cardiomyocytes from ischemic/hypoxic cell death via Drbp1 and pMe2GlyDH in cardio-specific ARC transgenic mice.

The ischemic death of cardiomyocytes is associated in heart disease and heart failure. However, the molecular mechanism underlying ischemic cell death is not well defined. To examine the function of apoptosis repressor with a caspase recruitment domain (ARC) in the ischemic/hypoxic damage of cardiomyocytes, we generated cardio-specific ARC transgenic mice using a mouse alpha-myosin heavy chain promoter. Compared with the control, the hearts of ARC transgenic mice showed a 3-fold overexpression of ARC. Langendoff preparation showed that the hearts isolated from ARC transgenic mice exhibited improved recovery of contractile performance during reperfusion. The cardiomyocytes cultured from neonatal ARC transgenic mice were significantly resistant to hypoxic cell death. Furthermore, the ARC C-terminal calcium-binding domain was as potent to protect cardiomyocytes from hypoxic cell death as ARC. Genome-wide RNA expression profiling uncovered a list of genes whose expression was changed (>2-fold) in ARC transgenic mice. Among them, expressional regulation of developmentally regulated RNA-binding protein 1 (Drbp1) or the dimethylglycine dehydrogenase precursor (pMe(2)GlyDH) affected hypoxic death of cardiomyocytes. These results suggest that ARC may protect cardiomyocytes from hypoxic cell death by regulating its downstream, Drbp1 and pMe(2)GlyDH, shedding new insights into the protection of heart from hypoxic damages.

Essential roles of Atg5 and FADD in autophagic cell death: dissection of autophagic cell death into vacuole formation and cell death.

Autophagic cell death is characterized by the accumulation of vacuoles in physiological and pathological conditions. However, its molecular event is unknown. Here, we show that Atg5, which is known to function in autophagy, contributes to autophagic cell death by interacting with Fas-associated protein with death domain (FADD). Down-regulation of Atg5 expression in HeLa cells suppresses cell death and vacuole formation induced by IFN-gamma. Inversely, ectopic expression of Atg5 using adenoviral delivery induces autophagic cell death. Deletion mapping analysis indicates that procell death activity resides in the middle and C-terminal region of Atg5. Cells harboring the accumulated vacuoles triggered by IFN-gamma or Atg5 expression become dead, and vacuole formation precedes cell death. 3-Methyladenine or expression of Atg5(K130R) mutant blocks both cell death and vacuole formation triggered by IFN-gamma, whereas benzyloxycarbonyl-VAD-fluoromethyl ketone (Z-VAD-fmk) inhibits only cell death but not vacuole formation. Atg5 interacts with FADD via death domain in vitro and in vivo, and the Atg5-mediated cell death, but not vacuole formation, is blocked in FADD-deficient cells. These results suggest that Atg5 plays a crucial role in IFN-gamma-induced autophagic cell death by interacting with FADD.

Induced inhibition of ischemic/hypoxic injury by APIP, a novel Apaf-1-interacting protein.

We describe the isolation and characterization of a new apaf-1-interacting protein (APIP) as a negative regulator of ischemic injury. APIP is highly expressed in skeletal muscle and heart and binds to the CARD of Apaf-1 in competition with caspase-9. Exogenous APIP inhibits cytochrome c-induced activation of caspase-3 and caspase-9, and suppresses cell death triggered by mitochondrial apoptotic stimuli through inhibiting the downstream activity of cytochrome c released from mitochondria. Conversely, reduction of APIP expression potentiates mitochondrial apoptosis. APIP expression is highly induced in mouse muscle affected by ischemia produced by interruption of the artery in the hindlimb and in C2C12 myotubes created by hypoxia in vitro, and the blockade of APIP up-regulation results in TUNEL-positive ischemic damage. Furthermore, forced expression of APIP suppresses ischemia/hypoxia-induced death of skeletal muscle cells. Taken together, these results suggest that APIP functions to inhibit muscle ischemic damage by binding to Apaf-1 in the Apaf-1/caspase-9 apoptosis pathway.