Multiple functions of aromatic-carbohydrate interactions in a processive cellulase examined with molecular simulation

Proteins employ aromatic residues for carbohydrate binding in a wide range of biological functions. Glycoside hydrolases, which are ubiquitous in nature, typically exhibit tunnels, clefts, or pockets lined with aromatic residues for processing carbohydrates. Mutation of these aromatic residues often results in significant activity differences on insoluble and soluble substrates. However, the thermodynamic basis and molecular level role of these aromatic residues remain unknown. Here, we calculate the relative ligand binding free energy by mutating tryptophans in the Trichoderma reesei family 6 cellulase (Cel6A) to alanine. Removal of aromatic residues near the catalytic site has little impact on the ligand binding free energy, suggesting that aromatic residues immediately upstream of the active site are not directly involved in binding, but play a role in the glucopyranose ring distortion necessary for catalysis. Removal of aromatic residues at the entrance and exit of the Cel6A tunnel, however, dramatically impacts the binding affinity, suggesting that these residues play a role in chain acquisition and product stabilization, respectively. The roles suggested from differences in binding affinity are confirmed by molecular dynamics and normal mode analysis. Surprisingly, our results illustrate that aromatic-carbohydrate interactions vary dramatically depending on the position in the enzyme tunnel. As aromatic-carbohydrate interactions are present in all carbohydrate-active enzymes, these results have implications for understanding protein structure-function relationships in carbohydrate metabolism and recognition, carbon turnover in nature, and protein engineering strategies for biomass utilization. Generally, these results suggest that nature employs aromatic-carbohydrate interactions with a wide range of binding affinities for diverse functions.     

A Digital Framework to Build,Visualize and Analyze a Gene Expression Atlas with Cellular Resolution in Zebrafish Early Embryogenesis

A gene expression atlas is an essential resource to quantify and understand the multiscale processes of embryogenesis in time and space. The automated reconstruction of a prototypic 4D atlas for vertebrate early embryos, using multicolor fluorescence in situ hybridization with nuclear counterstain, requires dedicated computational strategies. To this goal, we designed an original methodological framework implemented in a software tool called Match-IT. With only minimal human supervision, our system is able to gather gene expression patterns observed in different analyzed embryos with phenotypic variability and map them onto a series of common 3D templates over time, creating a 4D atlas. This framework was used to construct an atlas composed of 6 gene expression templates from a cohort of zebrafish early embryos spanning 6 developmental stages from 4 to 6.3 hpf (hours post fertilization). They included 53 specimens, 181,415 detected cell nuclei and the segmentation of 98 gene expression patterns observed in 3D for 9 different genes. In addition, an interactive visualization software, Atlas-IT, was developed to inspect, supervise and analyze the atlas. Match-IT and Atlas-IT, including user manuals, representative datasets and video tutorials, are publicly and freely available online. We also propose computational methods and tools for the quantitative assessment of the gene expression templates at the cellular scale, with the identification, visualization and analysis of coexpression patterns, synexpression groups and their dynamics through developmental stages.     

Tumor-associated macrophages in breast cancer: distinct subsets, distinct functions

Macrophages display remarkable plasticity, allowing these cells to adapt to changing microenvironments and perform functions as diverse as tissue development and homeostasis, inflammation, pathogen clearance and wound healing. Macrophage activation can be triggered by Th1 cytokines and pathogen-associated or endogenous danger signals, leading to the formation of classically activated or M1 macrophages. On the other hand, anti-inflammatory mediators, including IL-4, IL-10, TGF-β and M-CSF, induce diverse anti-inflammatory types of macrophages, known under the generic term M2. In human breast carcinomas, tumor-associated macrophage (TAM) density correlates with poor prognosis. In mouse models of breast cancer, eliminating macrophages from the tumor site, either via genetic or therapeutic means, results in retarded tumor progression. Over the years, multiple signals from the mammary tumor microenvironment have been reported to influence the TAM phenotype and TAM have been propagated as anti-inflammatory M2-like cells. Recent developments point to the existence of at least two distinct TAM subpopulations in mammary tumors, based on a differential expression of markers such as CD206 or MHC II and different in vivo behaviour: perivascular, migratory TAM which are less M2-like, and sessile TAM found at tumor-stroma borders and/or hypoxic regions that resemble more M2-like or "trophic" macrophages. Hence, a further refinement of the molecular and functional heterogeneity of TAM is an avenue for further research, with a potential impact on the usefulness of these cells as therapeutic targets.     

Inhibition of lipid synthesis through activation of AMP kinase: an additional mechanism for the hypolipidemic effects of berberine

The alkaloid drug berberine (BBR) was recently described to decrease plasma cholesterol and triglycerides (TGs) in hypercholesterolemic patients by increasing expression of the hepatic low density lipoprotein receptor (LDLR). Using HepG2 human hepatoma cells, we found that BBR inhibits cholesterol and TG synthesis in a similar manner to the AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide 1-beta-ribofuranoside (AICAR). Significant increases in AMPK phosphorylation and AMPK activity were observed when the cells were incubated with BBR. Activation of AMPK was also demonstrated by measuring the phosphorylation of acetyl-CoA carboxylase, a substrate of AMPK, correlated with a subsequent increase in fatty acid oxidation. All of these effects were abolished by the mitogen-activated protein kinase kinase inhibitor PD98059. Treatment of hyperlipidemic hamsters with BBR decreased plasma LDL cholesterol and strongly reduced fat storage in the liver. These findings indicate that BBR, in addition to upregulating the LDLR, inhibits lipid synthesis in human hepatocytes through the activation of AMPK. These effects could account for the strong reduction of plasma TGs observed with this drug in clinical trials.     

Identification of ILK as a new partner of the ADAM12 disintegrin and metalloprotease in cell adhesion and survival

Based on its shedding and binding activities, the disintegrin and metalloprotease 12 (ADAM12) has been implicated in cell signaling. Here we investigate the intracellular protein interaction network of the transmembrane ADAM12L variant using an integrative approach. We identify the integrin-linked kinase (ILK) as a new partner for ADAM12L cellular functions. We demonstrate that ADAM12L coimmunoprecipitates with ILK in cells and that its cytoplasmic tail is required for this interaction. In human cultured hepatic stellate cells (HSCs), which express high levels of endogenous ADAM12L and ILK, the two proteins are redistributed to focal adhesions upon stimulation of a β1 integrin-dependent pathway. We show that down-regulation of ADAM12L in HSCs leads to cytoskeletal disorganization and loss of adhesion. Conversely, up-regulation of ADAM12L induces the Akt Ser-473 phosphorylation-dependent survival pathway via stimulation of β1 integrins and activation of phosphoinositide 3-kinase (PI3K). Depletion of ILK inhibits this effect, which is independent of ADAM12L proteolytic activity and involves its cytoplasmic domain. We further demonstrate that overexpression of ADAM12L promotes kinase activity from ILK immunoprecipitates. Our data suggest a new role for ADAM12L in mediating the functional association of ILK with β1 integrin to regulate cell adhesion/survival through a PI3K/Akt signaling pathway.     

Algal Remodeling in a Ubiquitous Planktonic Photosymbiosis

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Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases

We describe the use of zinc-finger nucleases (ZFNs) for somatic and germline disruption of genes in zebrafish (Danio rerio), in which targeted mutagenesis was previously intractable. ZFNs induce a targeted double-strand break in the genome that is repaired to generate small insertions and deletions. We designed ZFNs targeting the zebrafish golden and no tail/Brachyury (ntl) genes and developed a budding yeast-based assay to identify the most active ZFNs for use in vivo. Injection of ZFN-encoding mRNA into one-cell embryos yielded a high percentage of animals carrying distinct mutations at the ZFN-specified position and exhibiting expected loss-of-function phenotypes. Over half the ZFN mRNA-injected founder animals transmitted disrupted ntl alleles at frequencies averaging 20%. The frequency and precision of gene-disruption events observed suggest that this approach should be applicable to any loci in zebrafish or in other organisms that allow mRNA delivery into the fertilized egg.     

Biofilm-Forming Abilities of Shiga Toxin-Producing Escherichia coli Isolates Associated with Human Infections

Forming biofilms may be a survival strategy of Shiga toxin-producing Escherichia coli to enable it to persist in the environment and the food industry. Here, we evaluate and characterize the biofilm-forming ability of 39 isolates of Shiga toxin-producing Escherichia coli isolates recovered from human infection and belonging to seropathotypes A, B, or C. The presence and/or production of biofilm factors such as curli, cellulose, autotransporter, and fimbriae were investigated. The polymeric matrix of these biofilms was analyzed by confocal microscopy and by enzymatic digestion. Cell viability and matrix integrity were examined after sanitizer treatments. Isolates of the seropathotype A (O157:H7 and O157:NM), which have the highest relative incidence of human infection, had a greater ability to form biofilms than isolates of seropathotype B or C. Seropathotype A isolates were unique in their ability to produce cellulose and poly-N-acetylglucosamine. The integrity of the biofilms was dependent on proteins. Two autotransporter genes, ehaB and espP, and two fimbrial genes, z1538 and lpf2, were identified as potential genetic determinants for biofilm formation. Interestingly, the ability of several isolates from seropathotype A to form biofilms was associated with their ability to agglutinate yeast in a mannose-independent manner. We consider this an unidentified biofilm-associated factor produced by those isolates. Treatment with sanitizers reduced the viability of Shiga toxin-producing Escherichia coli but did not completely remove the biofilm matrix. Overall, our data indicate that biofilm formation could contribute to the persistence of Shiga toxin-producing Escherichia coli and specifically seropathotype A isolates in the environment.