The DenA/DEN1 Interacting Phosphatase DipA Controls Septa Positioning and Phosphorylation-Dependent Stability of Cytoplasmatic DenA/DEN1 during Fungal Development

DenA/DEN1 and the COP9 signalosome (CSN) represent two deneddylases which remove the ubiquitin-like Nedd8 from modified target proteins and are required for distinct fungal developmental programmes. The cellular DenA/DEN1 population is divided into a nuclear and a cytoplasmatic subpopulation which is especially enriched at septa. DenA/DEN1 stability control mechanisms are different for the two cellular subpopulations and depend on different physical interacting proteins and the C-terminal DenA/DEN1 phosphorylation pattern. Nuclear DenA/DEN1 is destabilized during fungal development by five of the eight CSN subunits which target nuclear DenA/DEN1 for degradation. DenA/DEN1 becomes stabilized as a phosphoprotein at S243/S245 during vegetative growth, which is necessary to support further asexual development. After the initial phase of development, the newly identified cytoplasmatic DenA/DEN1 interacting phosphatase DipA and an additional developmental specific C-terminal phosphorylation site at serine S253 destabilize DenA/DEN1. Outside of the nucleus, DipA is co-transported with DenA/DEN1 in the cytoplasm between septa and nuclei. Deletion of dipA resulted in increased DenA/DEN1 stability in a strain which is unresponsive to illumination. The mutant strain is dysregulated in cytokinesis and impaired in asexual development. Our results suggest a dual phosphorylation-dependent DenA/DEN1 stability control with stabilizing and destabilizing modifications and physical interaction partner proteins which function as control points in the nucleus and the cytoplasm.     

siRNA Screen Identifies Trafficking Host Factors that Modulate Alphavirus Infection

Little is known about the repertoire of cellular factors involved in the replication of pathogenic alphaviruses. To uncover molecular regulators of alphavirus infection, and to identify candidate drug targets, we performed a high-content imaging-based siRNA screen. We revealed an actin-remodeling pathway involving Rac1, PIP5K1- α, and Arp3, as essential for infection by pathogenic alphaviruses. Infection causes cellular actin rearrangements into large bundles of actin filaments termed actin foci. Actin foci are generated late in infection concomitantly with alphavirus envelope (E2) expression and are dependent on the activities of Rac1 and Arp3. E2 associates with actin in alphavirus-infected cells and co-localizes with Rac1–PIP5K1-α along actin filaments in the context of actin foci. Finally, Rac1, Arp3, and actin polymerization inhibitors interfere with E2 trafficking from the trans-Golgi network to the cell surface, suggesting a plausible model in which transport of E2 to the cell surface is mediated via Rac1- and Arp3-dependent actin remodeling.     

How to monitor environmental pressures of a circular economy: An assessment of indicators

Understanding how a circular economy (CE) can reduce environmental pressures from economic activities is crucial for policy and practice. Science provides a range of indicators to monitor and assess CE activities. However, common CE activities, such as recycling and eco‐design, are contested in terms of their contribution to environmental sustainability. This article assesses whether and to what extent current approaches to assess CE activities sufficiently capture environmental pressures to monitor progress toward environmental sustainability. Based on a material flow perspective, we show that most indicators do not capture environmental pressures related to the CE activities they address. Many focus on a single CE activity or process, which does not necessarily contribute to increased environmental sustainability overall. Based on these results, we suggest complementing CE management indicators with indicators capturing basic environmental pressures related to the respective CE activity. Given the conceptual linkage between CE activities, resource extraction, and waste flows, we suggest that a resource‐based footprint approach accounting for major environmental inputs and outputs is necessary—while not sufficient—to assess the environmental sustainability of CE activities. As footprint approaches can be used across scales, they could aid the challenging process of developing indicators for monitoring progress toward an environmentally sustainable CE at the European, national, and company levels.     

Synthesis of novel purine nucleosides towards a selective inhibition of human butyrylcholinesterase

The search for new and potent cholinesterase inhibitors is an ongoing quest mobilizing many organic chemistry groups around the world as these molecules have been shown to treat the late symptoms of Alzheimer’s disease as well as to act as neuroprotecting agents. In this work, we disclose the synthesis of novel 2-acetamidopurine nucleosides and, for the first time, regioselective N⁷-glycosylation with 2-acetamido-6-chloropurine, promoted by trimethylsilyl triflate, was accomplished by tuning the reaction conditions (acetonitrile as solvent, 65 °C, 5 h) starting from 1-acetoxy bicyclic glycosyl donors, or by direct coupling of a methyl glucopyranoside with the nucleobase to obtain only N⁷ nucleosides in reasonable yield (55–60%). The nucleosides as well as their sugar precursors were screened for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition. While none of the compounds tested inhibited AChE, remarkably, some of the N⁷ nucleosides and sugar bicyclic derivatives showed potent inhibition towards BChE. Nanomolar inhibition was obtained for one compound competing well with rivastigmine, a drug currently in use for the treatment of Alzheimer’s disease. Experimental results showed that the presence of benzyl groups on the carbohydrate scaffold and the N⁷-linked purine nucleobase were necessary for strong BChE inactivation. A preliminary evaluation of the acute cytotoxicity of the elongated bicyclic sugar precursors and nucleosides was performed indicating low values, in the same order of magnitude as those of rivastigmine.     

H. pylori selectively blocks EGFR endocytosis via the non-receptor kinase c-Abl and CagA

Helicobacter pylori infection is a primary cause of peptic ulcers and is associated with gastric carcinogenesis. The H. pylori -induced pathophysiology may be linked to the deregulation of EGFR signalling. Elevated mucosal levels of EGF and the EGFR have been found in antral gastric biopsies of H. pylori -infected patients. A critical mechanism for regulating EGFR signalling is ligand-induced endocytosis. The internalized receptor recycles back to the plasma membrane for continued signalling or is targeted for degradation terminating receptor signalling. Here, we show that H. pylori blocks EGFR endocytosis and receptor degradation upon prolonged infection of gastric epithelial cells. Moreover, this inhibition occurs via a CagA-dependent, but CagA phosphorylation-independent activation of the non-receptor kinase c-Abl, which in turn phosphorylates the EGFR target site pY1173. This suggests a novel CagA-induced host cell response that is independent of CagA tyrosine phosphorylation. Our data indicate an intriguing strategy of H. pylori in host cell manipulations by altering selective receptor populations via a CagA-dependent endocytic mechanism. Furthermore, we identified a new role for c-Abl in phosphorylation of the EGFR target site pY1173 during H. pylori infection.     

Reduced Expression of CD45 Protein-tyrosine Phosphatase Provides Protection against Anthrax Pathogenesis

The modulation of cellular processes by small molecule inhibitors, gene inactivation, or targeted knockdown strategies combined with phenotypic screens are powerful approaches to delineate complex cellular pathways and to identify key players involved in disease pathogenesis. Using chemical genetic screening, we tested a library of known phosphatase inhibitors and identified several compounds that protected Bacillus anthracis infected macrophages from cell death. The most potent compound was assayed against a panel of sixteen different phosphatases of which CD45 was found to be most sensitive to inhibition. Testing of a known CD45 inhibitor and antisense phosphorodiamidate morpholino oligomers targeting CD45 also protected B. anthracis-infected macrophages from cell death. However, reduced CD45 expression did not protect anthrax lethal toxin (LT) treated macrophages, suggesting that the pathogen and independently added LT may signal through distinct pathways. Subsequent, in vivo studies with both gene-targeted knockdown of CD45 and genetically engineered mice expressing reduced levels of CD45 resulted in protection of mice after infection with the virulent Ames B. anthracis. Intermediate levels of CD45 expression were critical for the protection, as mice expressing normal levels of CD45 or disrupted CD45 phosphatase activity or no CD45 all succumbed to this pathogen. Mechanism-based studies suggest that the protection provided by reduced CD45 levels results from regulated immune cell homeostasis that may diminish the impact of apoptosis during the infection. To date, this is the first report demonstrating that reduced levels of host phosphatase CD45 modulate anthrax pathogenesis.Interactions between microbes and immune cells play a critical role in microbial pathogenesis. Many pathogenic organisms exploit the host immune machinery and subsequently modulate cell function, signaling, migration, and cytoskeleton rearrangement. Hence, identifying host cellular components with which microbes interact will allow for a more comprehensive understanding of microbial pathogenesis, define common strategies used by multiple pathogens, and elucidate unique tactics evolved by individual species to help establish infections or evade host innate responses. Another interesting aspect of infection is that diverse pathogens seem to target common cellular pathways (1, 2). Thus, identifying host targets exploited by multiple pathogens will be useful in the development of broad-spectrum host-oriented therapeutics and vaccines.Protein kinases and phosphatases regulate a range of cellular responses to external and internal stimuli, including cell proliferation, metabolism, and apoptosis. Aberrant kinase and/or phosphatase activities underlie many different types of pathological conditions from cancer to infectious diseases. Protein kinases have been extensively investigated as targets for drug discovery. In addition, phosphatases are now being recognized as important regulators of many biological processes. In particular, there is an increasing interest in protein-tyrosine phosphatases (PTPs)³ as drug targets (3–8) because immune cells express a remarkably high proportion of the 107 PTP genes in the human genome (9) and also due to the growing number of human diseases discovered to be associated with PTP abnormalities (9–11). The involvement of cellular and bacterial PTPs during intracellular microbial pathogenesis has been a topic of significant interest (2, 12, 13). The bacterial PTP YopH, secreted by Yersinia pestis, interferes with the host adhesion-regulated signaling pathway via dephosphorylation of selective tyrosine-phosphorylated proteins (14). Activation of host PTPs after infection with bacteria or their virulence factors has been demonstrated for a diverse group of microorganisms such as Mycobacterium tuberculosis and Leishmania donovani (13). Specific mechanistic models of how PTPs contribute to the development of infection and disease progression by highly lethal organisms still remain unclear.Bacillus anthracis, a Gram-positive spore-forming bacterium, is the etiologic agent of anthrax. The lethal toxin (LT) produced by B. anthracis can cleave host cell mitogen-activated protein kinase kinases (MAPKK), thereby affecting the immune response and the host ability to fight the infection (15, 16). Macrophages are the primary targets of anthrax LT. However, macrophages from only certain strains of mice are susceptible to LT-mediated cell death (17, 18). To date, there is no known direct relation between MAPKK cleavage and LT-induced macrophage cell death, as LT-resistant macrophages exhibit MAPKK cleavage (19–21). This suggests that another cellular target(s) may play a role in anthrax pathogenesis.Previously, using a chemical genetic approach, we identified a class of Cdc25 inhibitors that protected macrophages from cell death induced by anthrax LT (22). Although Cdc25 was not the cellular target, induction of anti-apoptotic responses by the compounds via either the MAPK-dependent or -independent pathways was responsible for the protective phenotype.In the present study we investigated if the previously identified phosphatase inhibitors (22) and their analogs produced any phenotypic changes in the B. anthracis infection model. Two compounds that previously protected LT-treated macrophages (22) also protect B. anthracis-infected macrophages. Subsequent in vitro phosphatase profiling studies identified CD45, a previously unknown target of one of the small molecules, as the most sensitive enzyme to the inhibitor. We then investigated the effect of CD45 reduction in anthrax pathogenesis both in cells and in vivo by using antisense phosphorodiamidate morpholino oligomers and mice engineered to express reduced levels of CD45.