Does autophagy contribute to cell death?

Autophagy (specifically macroautophagy) is an evolutionarily conserved catabolic process where the cytoplasmic contents of a cell are sequestered within double membrane vacuoles, called autophagosomes, and subsequently delivered to the lysosome for degradation. Autophagy can function as a survival mechanism in starving cells. At the same time, extensive autophagy is commonly observed in dying cells, leading to its classification as an alternative form of programmed cell death. The functional contribution of autophagy to cell death has been a subject of great controversy. However, several recent loss-of-function studies of autophagy (atg) genes have begun to address the roles of autophagy in both cell death and survival. Here, we review the emerging evidence in favor of and against autophagic cell death, discuss the possible roles that autophagic degradation might play in dying cells, and identify salient issues for future investigation.     

A proteome approach defines protective functions of tobacco leaf trichomes

The leaf surface of most terrestrial plants is covered with plant hairs called trichomes. These epidermal appendages are thought to contribute to many aspects of plant defense against biotic and abiotic stresses in a variety of species. Trichome development has been intensively studied in Arabidopsis, and the phytochemical composition of trichomes was analyzed in a number of plant species. However, comparatively little is known of the proteins expressed. We therefore initiated a proteome approach to better define the cellular mechanisms operating in plant trichomes using two-dimensional gel electrophoresis to separate proteins of whole leaves and isolated trichomes. Tobacco was chosen due to the presence of glandular trichomes involved in the secretion of defense compounds. Comparative image analysis of the protein patterns indicated a number of spots, which were highly enriched in trichomes relative to leaves. These spots were excised for identification by mass spectrometry. The results showed that among the proteins specifically enriched in trichomes, the components of stress defense responses were strongly represented. The high expression of stress-related proteins was verified by Western blotting. Superoxide dismutase isoforms were additionally analyzed by activity staining. Our results demonstrate feasibility of the proteome approach to elucidate the cell biology of plant trichomes.     

His374 of wheat endoxylanase inhibitor TAXI-I stabilizes complex formation with glycoside hydrolase family 11 endoxylanases

Wheat endoxylanase inhibitor TAXI-I inhibits microbial glycoside hydrolase family 11 endoxylanases. Crystallographic data of an Aspergillus niger endoxylanase-TAXI-I complex showed His374 of TAXI-I to be a key residue in endoxylanase inhibition. Its role in enzyme-inhibitor interaction was further investigated by site-directed mutagenesis of His374 into alanine, glutamine or lysine. Binding kinetics and affinities of the molecular interactions between A. niger, Bacillus subtilis, Trichoderma longibrachiatumendoxylanases and wild-type TAXI-I and TAXI-I His374 mutants were determined by surface plasmon resonance analysis. Enzyme-inhibitor binding was in accordance with a simple 1 : 1 binding model. Association and dissociation rate constants of wild-type TAXI-I towards the endoxylanases were in the range between 1.96 and 36.1 x 10(4)m(-1) x s(-1) and 0.72-3.60 x 10(-4) x s(-1), respectively, resulting in equilibrium dissociation constants in the low nanomolar range. Mutation of TAXI-I His374 to a variable degree reduced the inhibition capacity of the inhibitor mainly due to higher complex dissociation rate constants (three- to 80-fold increase). The association rate constants were affected to a smaller extent (up to eightfold decrease). Substitution of TAXI-I His374 therefore strongly affects the affinity of the inhibitor for the enzymes. In addition, the results show that His374 plays a critical role in the stabilization of the endoxylanase-TAXI-I complex rather than in the docking of inhibitor onto enzyme.     

An AIF orthologue regulates apoptosis in yeast

Apoptosis-inducing factor (AIF), a key regulator of cell death, is essential for normal mammalian development and participates in pathological apoptosis. The proapoptotic nature of AIF and its mode of action are controversial. Here, we show that the yeast AIF homologue Ynr074cp controls yeast apoptosis. Similar to mammalian AIF, Ynr074cp is located in mitochondria and translocates to the nucleus of yeast cells in response to apoptotic stimuli. Purified Ynr074cp degrades yeast nuclei and plasmid DNA. YNR074C disruption rescues yeast cells from oxygen stress and delays age-induced apoptosis. Conversely, overexpression of Ynr074cp strongly stimulates apoptotic cell death induced by hydrogen peroxide and this effect is attenuated by disruption of cyclophilin A or the yeast caspase YCA1. We conclude that Ynr074cp is a cell death effector in yeast and rename it AIF-1 (Aif1p, gene AIF1).     

Non-hematopoietic human bone marrow contains long-lasting, pluripotential mesenchymal stem cells

Mesenchymal stem cells (MSC) are considered as potential agents for reconstructive and gene-targeting therapies since they differentiate into various cell-lineages, exhibit an extended survival once injected into a host, and can easily be transfected with engineered DNA. MSC are essentially isolated from hematopoietic bone marrow (BM), a process that is rather invasive and may raise ethical concerns. In an attempt to find an alternative source, we evaluated whether non-hematopoietic (nh)BM recovered from femoral heads of patients undergoing hip arthroplasty contained MSC. Ex vivo, 99% of nhBM cells were CD45(+) leukocytes. After culture, leukocytes were replaced by a homogeneous layer of adherent CD45(-) CD14(-) CD34(-) CD11b(-) CD90(+) HLA-ABC(+) cells. Culture doubling time (mean = 4 days, range 1.6-6.7 days) was not correlated with patient age (27-81 years, n = 16). Amplified cultures supported long-term hematopoiesis, and could be differentiated in vitro into adipocytes and chondrocytes. Moreover, a small fraction of nhBM cells spontaneously expressed MyoD1 and formed myotubes, suggesting that myogenic differentiation also occurred. nhBM contained clonogenic cells whose frequency (1/13,000), doubling time (2.1 days), and maximal amplification (up to 10(6)-fold) were not age-related. All 14 clones analyzed (from five patients, ages 27-78 years) differentiated into at least one mesenchymal lineage, and 66% were bipotential (n = 8/12), or tripotential (n = 2/3). In conclusion, nhBM contains pluripotential mesenchymal progenitors which are similar to hematopoietic BM-derived MSC, and whose biological functions are not altered by aging. Furthermore, if MSC-based therapies hold their promises, nhBM may become the source of choice for responding to the increasing demand for MSC.     

Proapoptotic function of protein kinase CK2alpha" is mediated by a JNK signaling cascade

Protein kinase CK2 (formerly casein kinase II) is a tetrameric enzyme constitutively expressed in all eurakyotic tissues that plays a significant role in the regulation of cell proliferation, malignant transformation, and apoptosis. The catalytic alpha-subunit of the enzyme is known to exist in three isoforms CK2alpha, CK2alpha' and CK2alpha". CK2alpha" is highly expressed in liver compared with other tissues and is required for the normal trafficking of several hepatocellular membrane proteins. Initial studies of dengue virus infection indicated that the CK2alpha"-deficient membrane trafficking mutant cell line (Trf1) was resistant to virus-induced cell death compared with the parental human hepatoma (HuH)-7 hepatoma line. Expression of recombinant CK2alpha" in Trf1 was capable of reverting this resistant phenotype. This study was extended to TNF-alpha in addition to other stimuli of cell death in an attempt to uncover common death pathways that might be modulated by CK2alpha". Evaluation of different pathways involved in death signaling suggest that the regulation of a critical proapoptotic step in HuH-7 cells by CK2alpha" is mediated by a JNK signaling cascade.     

Phosphodiesterase type 5 inhibition improves early memory consolidation of object information

The nitric oxide (NO)-cyclic GMP (cGMP) signaling pathway is assumed to play an important role in processes underlying learning and memory. We used phosphodiesterase type 5 (PDE5) inhibitors to study the role of cGMP in object- and spatial memory. Our results and those reported in other studies indicate that elevated hippocampal cGMP levels are required to improve the memory performance of rodents in object recognition and passive avoidance learning, but not in spatial learning. The timing of treatment modulates the effects on memory and strongly supports a role for cGMP in early stages of memory formation. Alternative explanations for the improved memory performance of PDE5 inhibitors are also discussed. Immunocytochemical studies showed that in vitro slice incubations with PDE5 inhibitors increase NO-stimulated cGMP levels mainly in hippocampal varicose fibers. Reviewing the available data on the localization of the different components of the NO-cGMP signaling pathway, indicates a complex interaction between NO and cGMP, which may be independent of each other. It is discussed that further studies are needed, immunocytochemical and behavioral, to better understand the cGMP-mediated molecular mechanisms underlying memory formation.     

Mutant actins that stabilise F-actin use distinct mechanisms to activate the SRF coactivator MAL

Nuclear accumulation of the serum response factor coactivator MAL/MKL1 is controlled by its interaction with G-actin, which results in its retention in the cytoplasm in cells with low Rho activity. We previously identified actin mutants whose expression promotes MAL nuclear accumulation via an unknown mechanism. Here, we show that actin interacts directly with MAL in vitro with high affinity. We identify a further activating mutation, G15S, which stabilises F-actin, as do the activating actins S14C and V159N. The three mutants share several biochemical properties, but can be distinguished by their ability to bind cofilin, ATP and MAL. MAL interaction with actin S14C is essentially undetectable, and that with actin V159N is weakened. In contrast, actin G15S interacts more strongly with MAL than the wild-type protein. Strikingly, the nuclear accumulation of MAL induced by overexpression of actin S14C is substantially dependent on Rho activity and actin treadmilling, while that induced by actin G15S expression is not. We propose a model in which actin G15S acts directly to promote MAL nuclear entry.