Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 Deficiency Reduces Insulin Sensitivity in High-Fat Diet-Fed Mice

Adipose tissue inflammation is considered an important contributor to insulin resistance. Mitogen-activated protein kinase-activated protein kinase 2 (MK2) is a major downstream target of p38 MAPK and enhances inflammatory processes. In line with the role of MK2 as contributor to inflammation, MK2^−/− mice are protected against inflammation in different disease models. Therefore, MK2 is considered an attractive therapeutic target for the treatment of chronic inflammatory diseases. This study tested the impact of MK2-deficiency on high-fat diet (HFD)-induced adipose tissue inflammation and insulin resistance. After feeding MK2^−/− and WT control mice a HFD (60% energy from fat) for 24 weeks, body weight was not different between groups. Also, liver weight and the amount of abdominal fat remained unchanged. However, in MK2^−/− mice plasma cholesterol levels were significantly increased. Surprisingly, macrophage infiltration in adipose tissue was not altered. However, adipose tissue macrophages were more skewed to the inflammatory M1 phenotype in MK2^−/− mice. This differerence in macrophage polarization did however not translate in significantly altered expression levels of Mcp-1, Tnfα and Il6. Glucose and insulin tolerance tests demonstrated that MK2^−/− mice had a significantly reduced glucose tolerance and increased insulin resistance. Noteworthy, the expression of the insulin-responsive glucose transporter type 4 (GLUT4) in adipose tissue of MK2^−/− mice was reduced by 55% (p<0.05) and 33% (p<0.05) on the mRNA and protein level, respectively, compared to WT mice. In conclusion, HFD-fed MK2^−/− display decreased glucose tolerance and increased insulin resistance compared to WT controls. Decreased adipose tissue expression of GLUT4 might contribute to this phenotype. The data obtained in this study indicate that clinical use of MK2 inhibitors has to be evaluated with caution, taking potential metabolic adverse effects into account.     

Vacuole biogenesis and protein transport to the plant vacuole: A comparison with the yeast vacuole and the mammalian lysosome

Summary The vacuole is often termed the lytic compartment of the plant cell. The yeast cell also possesses a vacuole containing acid hydrolases. In animal cells these enzymes are localized in the lysosome. Recent research suggests that there is good reason to regard these organelles as homologous in terms of protein transport. Although sorting motifs for the recognition of vacuolar proteins within the endomembrane system differ between the three organelles, there is an underlying similarity in targeting determinants in the cytoplasmic tails of Golgi-based receptors. In all three cases these determinants appear to interact with adaptins of clathrin-coated vesicles which ferry their cargo first of all to an endosomal compartment. The situation in sorting and targeting of plant vacuolar proteins is complicated by the fact that storage and lytic vacuoles may exist together in the same cell. The origin of these two types of vacuole is also a matter of some uncertanity.Abbrevations AP assembly protein - ALP alkaline phosphatase - ARF adenosine diphosphate ribosylation factor - BiP immunoglobulin binding protein - CCV clathrin coated vesicle - CPY carboxypeptidase-Y - DPAP dipeptidyl aminopeptidase - ER endoplasmic reticulum - GApp Golgi apparatus - LAMPs lysosomal associated membrane protein(s) - LAP lysosomal acid phosphatase - LIMPs lysosomal integral membrane protein(s) - MPRs mannosyl 6-phosphate receptors - MVB multivesicular bodies - NSF N-ethylmaleimide sensitive fusion (protein) - PAT phosphinotricine acetyltransferase - PB protein body - PHA phytohemagglutinin - PM plasma membrane - PSV protein storage vacuole - SNAPs soluble NSF attachment protein(s) - SNAREs SNAP receptor(s) - TGN trans Golgi network - TIP tonoplast integral protein - VPS vacuolar protein sorting - ZIO zinc iodide/osmium     

Construction and Rescue of a Molecular Clone of Deformed Wing Virus (DWV)

European honey bees are highly important in crop pollination, increasing the value of global agricultural production by billions of dollars. Current knowledge about virulence and pathogenicity of Deformed wing virus (DWV), a major factor in honey bee colony mortality, is limited. With this study, we close the gap between field research and laboratory investigations by establishing a complete in vitro model for DWV pathogenesis. Infectious DWV was rescued from a molecular clone of a DWV-A genome that induces DWV symptoms such as crippled wings and discoloration. The expression of DWV proteins, production of infectious virus progeny, and DWV host cell tropism could be confirmed using newly generated anti-DWV monoclonal antibodies. The recombinant RNA fulfills Koch’s postulates circumventing the need of virus isolation and propagation of pure virus cultures. In conclusion, we describe the development and application of a reverse genetics system for the study of DWV pathogenesis.     

BiPPred: Combined sequence‐ and structure‐based prediction of peptide binding to the Hsp70 chaperone BiP

Substrate binding to Hsp70 chaperones is involved in many biological processes, and the identification of potential substrates is important for a comprehensive understanding of these events. We present a multi‐scale pipeline for an accurate, yet efficient prediction of peptides binding to the Hsp70 chaperone BiP by combining sequence‐based prediction with molecular docking and MMPBSA calculations. First, we measured the binding of 15mer peptides from known substrate proteins of BiP by peptide array (PA) experiments and performed an accuracy assessment of the PA data by fluorescence anisotropy studies. Several sequence‐based prediction models were fitted using this and other peptide binding data. A structure‐based position‐specific scoring matrix (SB‐PSSM) derived solely from structural modeling data forms the core of all models. The matrix elements are based on a combination of binding energy estimations, molecular dynamics simulations, and analysis of the BiP binding site, which led to new insights into the peptide binding specificities of the chaperone. Using this SB‐PSSM, peptide binders could be predicted with high selectivity even without training of the model on experimental data. Additional training further increased the prediction accuracies. Subsequent molecular docking (DynaDock) and MMGBSA/MMPBSA‐based binding affinity estimations for predicted binders allowed the identification of the correct binding mode of the peptides as well as the calculation of nearly quantitative binding affinities. The general concept behind the developed multi‐scale pipeline can readily be applied to other protein‐peptide complexes with linearly bound peptides, for which sufficient experimental binding data for the training of classical sequence‐based prediction models is not available. Proteins 2016; 84:1390–1407. © 2016 Wiley Periodicals, Inc.     

The role of the polo kinase Cdc5 in controlling Cdc14 localization

In budding yeast, the protein phosphatase Cdc14 controls exit from mitosis. Its activity is regulated by a competitive inhibitor Cfi1/Net1, which binds to and sequesters Cdc14 in the nucleolus. During anaphase, Cdc14 is released from its inhibitor by the action of two regulatory networks. The Cdc Fourteen Early Anaphase Release (FEAR) network initiates Cdc14 release from Cfi1/Net1 during early anaphase, and the Mitotic Exit Network (MEN) promotes Cdc14 release during late anaphase. Here, we investigate the relationship among FEAR network components and propose an order in which they function to promote Cdc14 release from the nucleolus. Furthermore, we examine the role of the protein kinase Cdc5, which is a component of both the FEAR network and the MEN, in Cdc14 release from the nucleolus. We find that overexpression of CDC5 led to Cdc14 release from the nucleolus in S phase-arrested cells, which correlated with the appearance of phosphorylated forms of Cdc14 and Cfi1/Net1. Cdc5 promotes Cdc14 phosphorylation and, by stimulating the MEN, Cfi1/Net1 phosphorylation. Furthermore, we suggest that Cdc14 release from the nucleolus only occurs when Cdc14 and Cfi1/Net1 are both phosphorylated.     

Conservation and evolutionary modifications of neuroblast expression patterns in insects

One of the major questions in evolutionary developmental neurobiology is how neuronal networks have been adapted to different morphologies and behaviour during evolution. Analyses of neurogenesis in representatives of all arthropod species have revealed evolutionary modifications of various developmental mechanisms. Among others, variations can be seen in mechanisms that are associated with changes in neural progenitor identity, which in turn determines the neuronal subtype of their progeny. Comparative analyses of the molecular processes that underlie the generation of neuronal identity might therefore uncover the steps of evolutionary changes that eventually resulted in modifications in neuronal networks. Here we address this question in the flour beetle Tribolium castaneum by analyzing and comparing the development and expression profile of neural stem cells (neuroblasts) to the published neuroblast map of the fruit fly Drosophila melanogaster. We show that substantial changes in the identity of neuroblasts have occurred during insect evolution. In almost all neuroblasts the relative positions in the ventral hemi-neuromeres are conserved; however, in over half of the neuroblasts the time of formation as well as the gene expression profile has changed. The neuroblast map presented here can be used for future comparative studies on individual neuroblast lineages in D. melanogaster and T. castaneum and additional markers and information on lineages can be added. Our data suggest that evolutionary changes in the expression profile of individual neuroblasts might have contributed to the evolution of neural diversity and subsequently to changes in neuronal networks in arthropod.     

Dubinectes infirmus, a new species of deep-water Munnopsidae (Crustacea, Isopoda, Asellota) from the Argentine Basin, South Atlantic Ocean

Dubinectes infirmussp. n., Munnopsidae, is described from the Argentine Basin, southwest Atlantic, at depths between 4586-4607 m. The new species is distinguished by a narrow rim of the pleotelson posterior margin which is not raising over its dorsal surface; article 3 of the antennula is subequal in length to article 2; distomedial lobes of male pleopod 1 are of same size as distolateral lobes; stylet of male pleopod 2 is subequal in length to protopod; uropod exopod is more than a half of endopod length. Some generic characters which are weakly pronounced in the new species or have different state are defined more precisely in the revised diagnosis of Dubinectes. The modified diagnosis of the genus, a key to the species of Dubinectes as well as the distribution of the genus are presented.