Caspase-mediated cleavage of Beclin-1 inactivates Beclin-1-induced autophagy and enhances apoptosis by promoting the release of proapoptotic factors from mitochondria

Autophagy and apoptosis are two important and interconnected stress-response mechanisms. However, the molecular interplay between these two pathways is not fully understood. To study the fate and function of autophagic proteins at the onset of apoptosis, we used a cellular model system in which autophagy precedes apoptosis. IL-3 depletion of Ba/F3 cells caused caspase (casp)-mediated cleavage of Beclin-1 and PI3KC3, two crucial components of the autophagy-inducing complex. We identified two casp cleavage sites in Beclin-1, TDVD¹³³ and DQLD¹⁴⁹, cleavage at which yields fragments lacking the autophagy-inducing capacity. Noteworthy, the C-terminal fragment, Beclin-1-C, localized predominantly at the mitochondria and sensitized the cells to apoptosis. Moreover, on isolated mitochondria, recombinant Beclin-1-C was able to induce the release of proapoptotic factors. These findings point to a mechanism by which casp-dependent generation of Beclin-1-C creates an amplifying loop enhancing apoptosis upon growth factor withdrawal.     

IP10, KC and M-CSF Are Remarkably Increased in the Brains from the Various Strains of Experimental Mice Infected with Different Scrapie Agents

Virologica Sinica - Activation of inflammatory cells and upregulations of a number of cytokines in the central nervous system (CNS) of patients with prion diseases are frequently observed. To...     

Transient infiltration of neutrophils into the thymus following whole-body X-ray irradiation in IL-10 knockout mice

IL-10 is known to suppress the inflammatory responses in a variety of experimental models. Because we previously found that whole-body X-irradiation causes massive apoptosis in the thymus and transient infiltration of neutrophils, in this study, we examined whether or not IL-10 is involved in the regulation of neutrophil infiltration upon whole-body X-ray irradiation using IL-10 knockout mice. Although IL-10 was induced in the thymus on whole-body X-ray irradiation, apoptosis of thymocytes, neutrophil infiltration, and MIP-2 and KC production in the thymus were not affected by an IL-10 deficiency. Coculturing of bone marrow-derived macrophages with late apoptotic cells caused MIP-2 production, which was also not affected by an IL-10 deficiency. These results suggest the uniqueness of the inflammatory response induced by whole-body X-ray irradiation, which does not seem to be regulated by IL-10.     

Macrophage inflammatory protein-2 (MIP-2)/CXCR2 blockade attenuates acute graft-versus-host disease while preserving graft-versus-leukemia activity

Allogenic bone marrow transplantation (BMT), an important treatment for hematological malignancies, is often complicated by graft-versus-host disease (GVHD). Suppression of GVHD is associated with the unwanted diminishment of the graft-versus-leukemia (GVL) response. The aim of this study was to maintain the benefits of GVL during GVHD suppression through isolated blockade of T-cell migration factors. To this end, we developed a murine model of B-cell leukemia, which was treated with BMT to induce GVHD. Within this model, functional blockade of MIP-2/CXCR2 was analyzed by observing proteomic, histologic and clinical variables of GVHD manifestation. Luminex assay of collected tissue identified several cytokines [granulocyte colony-stimulating factor (G-CSF), keratinocyte-derived chemokine (KC), macrophage inflammatory protein-2 (MIP-2), and interleukin-23 (IL-23)] that were upregulated during GHVD, but reduced by neutralizing the MIP-2/CXCR2 axis. In addition, donor T-cell blockade of CXCR2 combined with recipient administration of anti-MIP-2 caused a significant decrease in GVHD while preserving the GVL response. We propose that blocking the MIP-2/CXCR2 axis represents a novel strategy to separate the toxicity of GVHD from the beneficial effects of GVL after allogenic BMT.     

O‐Methyltransferases involved in biphenyl and dibenzofuran biosynthesis

Biphenyls and dibenzofurans are the phytoalexins of the Malinae involving apple and pear. Biosynthesis of the defence compounds includes two O‐methylation reactions. cDNAs encoding the O‐methyltransferase (OMT) enzymes were isolated from rowan (Sorbus aucuparia) cell cultures after treatment with an elicitor preparation from the scab‐causing fungus, Venturia inaequalis. The preferred substrate for SaOMT1 was 3,5‐dihydroxybiphenyl, supplied by the first pathway‐specific enzyme, biphenyl synthase (BIS). 3,5‐Dihydroxybiphenyl underwent a single methylation reaction in the presence of S‐adenosyl‐l ‐methionine (SAM). The second enzyme, SaOMT2, exhibited its highest affinity for noraucuparin, however the turnover rate was greater with 5‐hydroxyferulic acid. Both substrates were only methylated at the meta‐positioned hydroxyl group. The substrate specificities of the OMTs and the regiospecificities of their reactions were rationalized by homology modeling and substrate docking. Interaction of the substrates with SAM also took place at a position other than the sulfur group. Expression of SaOMT1, SaOMT2 and SaBIS3 was transiently induced in rowan cell cultures by the addition of the fungal elicitor. While the immediate SaOMT1 products were not detectable in elicitor‐treated cell cultures, noraucuparin and noreriobofuran accumulated transiently, followed by increasing levels of the SaOMT2 products aucuparin and eriobofuran. SaOMT1, SaOMT2 and SaBIS3 were N‐ and C‐terminally fused with the super cyan fluorescent protein and a modified yellow fluorescent protein, respectively. All the fluorescent reporter fusions were localized to the cytoplasm of Nicotiana benthamiana leaf epidermis cells. A revised biosynthetic pathway of biphenyls and dibenzofurans in the Malinae is presented.     

Chemical modification-mediated optimisation of bronchodilatory activity of mepenzolate,a muscarinic receptor antagonist with anti-inflammatory activity

The treatment for patients with chronic obstructive pulmonary disease (COPD) usually involves a combination of anti-inflammatory and bronchodilatory drugs. We recently found that mepenzolate bromide (1) and its derivative, 3-(2-hydroxy-2, 2-diphenylacetoxy)-1-(3-phenoxypropyl)-1-azoniabicyclo[2.2.2]octane bromide (5), have both anti-inflammatory and bronchodilatory activities. We chemically modified 5 with a view to obtain derivatives with both anti-inflammatory and longer-lasting bronchodilatory activities. Among the synthesized compounds, (R)-(–)-12 ((R)-3-(2-hydroxy-2,2-diphenylacetoxy)-1-(3-phenylpropyl)-1-azoniabicyclo[2.2.2]octane bromide) showed the highest affinity in vitro for the human muscarinic M3 receptor (hM₃R). Compared to 1 and 5, (R)-(–)-12 exhibited longer-lasting bronchodilatory activity and equivalent anti-inflammatory effect in mice. The long-term intratracheal administration of (R)-(–)-12 suppressed porcine pancreatic elastase-induced pulmonary emphysema in mice, whereas the same procedure with a long-acting muscarinic antagonist used clinically (tiotropium bromide) did not. These results suggest that (R)-(–)-12 might be therapeutically beneficial for use with COPD patients given the improved effects seen against both inflammatory pulmonary emphysema and airflow limitation in this animal model.     

The deubiquitinase USP11 is a versatile and conserved regulator of autophagy

Autophagy is a major cellular quality control system responsible for the degradation of proteins and organelles in response to stress and damage to maintain homeostasis. Ubiquitination of autophagy-related proteins or regulatory components is important for the precise control of autophagy pathways. Here, we show that the deubiquitinase ubiquitin-specific protease 11 (USP11) restricts autophagy and that KO of USP11 in mammalian cells results in elevated autophagic flux. We also demonstrate that depletion of the USP11 homolog H34C03.2 in Caenorhabditis elegans triggers hyperactivation of autophagy and protects the animals against human amyloid-β peptide 42 aggregation-induced paralysis. USP11 coprecipitated with autophagy-specific class III phosphatidylinositol 3-kinase complex I and limited its interaction with nuclear receptor-binding factor 2, thus decreasing lipid kinase activity of class III phosphatidylinositol 3-kinase complex I and subsequent recruitment of effectors such as WD-repeat domain phosphoinositide-interacting proteins to the autophagosomal membrane. Accordingly, more WD-repeat domain phosphoinositide-interacting protein 2 puncta accumulated in USP11 KO cells. In addition, USP11 interacts with and stabilizes the serine/threonine kinase mechanistic target of rapamycin, thereby further contributing to the regulation of autophagy induction. Taken together, our data suggested that USP11 impinges on the autophagy pathway at multiple sites and that inhibiting USP11 alleviates symptoms of proteotoxicity, which is a major hallmark of neurodegenerative diseases.