The E3 ubiquitin ligase HOIL-1 induces the polyubiquitination and degradation of SOCS6 associated proteins

The suppressor of cytokine signaling (SOCS) proteins are thought to exert their function through the recruitment of interacting-proteins to the ubiquitin/proteasome degradation pathway. All SOCS proteins bind an Elongin BC E3 ubiquitin ligase complex through the common Socs-box. Here, we show that haem-oxidized IRP2 ubiquitin ligase-1 (HOIL-1), another E3 ubiquitin ligase, interacts with SOCS6. The Ubl domain of HOIL-1 and the SH2 and Socs-box domains of SOCS6 are required for the interaction. HOIL-1 expression stabilizes SOCS6 and induces the ubiquitination and degradation of proteins associated with SOCS6. These data suggest that SOCS proteins may interact with different E3 ubiquitin ligases in addition to a common Elongin BC E3 complex.     

Spectrin functions upstream of ankyrin in a spectrin cytoskeleton assembly pathway

Prevailing models place spectrin downstream of ankyrin in a pathway of assembly and function in polarized cells. We used a transgene rescue strategy in Drosophila melanogaster to test contributions of four specific functional sites in beta spectrin to its assembly and function. (1) Removal of the pleckstrin homology domain blocked polarized spectrin assembly in midgut epithelial cells and was usually lethal. (2) A point mutation in the tetramer formation site, modeled after a hereditary elliptocytosis mutation in human erythrocyte spectrin, had no detectable effect on function. (3) Replacement of repetitive segments 4-11 of beta spectrin with repeats 2-9 of alpha spectrin abolished function but did not prevent polarized assembly. (4) Removal of the putative ankyrin-binding site had an unexpectedly mild phenotype with no detectable effect on spectrin targeting to the plasma membrane. The results suggest an alternate pathway in which spectrin directs ankyrin assembly and in which some important functions of spectrin are independent of ankyrin.     

Release of extracellular membrane vesicles from microvilli of epithelial cells is enhanced by depleting membrane cholesterol

We previously reported on the occurrence of prominin-1-carrying membrane vesicles that are released into body fluids from microvilli of epithelial cells. This release has been implicated in cell differentiation. Here we have characterized these vesicles released from the differentiated Caco-2 cells. We find that in these vesicles, prominin-1 directly interacts with membrane cholesterol and is associated with a membrane microdomain. The cholesterol depletion using methyl-β-cyclodextrin resulted in a marked increase in their release, and a dramatic change in the microvillar ultrastructure from a tubular shape to a “pearling” state, with multiple membrane constrictions, suggesting a role of membrane cholesterol in vesicle release from microvilli.     

Regulation of glucose and protein metabolism in growing steers by long-chain n-3 fatty acids in muscle membrane phospholipids is dose-dependent

A previous study showed that long-chain n-3 polyunsaturated fatty acids (LCn-3PUFA; >18 carbons n-3) exert an anabolic effect on protein metabolism through the upregulation of insulin sensitivity and activation of the insulin signaling pathway. This study further delineates for the first time whether the anabolic effect of LCn-3PUFA on metabolism is dose responsive. Six steers were used to test three graded amounts of menhaden oil rich in LCn-3PUFA (0%, 2% and 4%; enteral infusions) according to a double 3 × 3 Latin square design. Treatment comparisons were made using iso-energetic substitutions of control oil for menhaden oil and using 6-week experimental periods. The LCn-3PUFA in muscle total membrane phospholipids increased from 8%, 14% to 20% as dietary menhaden oil increased. Feeding graded amounts of menhaden oil linearly decreased plasma insulin concentration (49, 35 and 25 μU/ml, P = 0.01). The insulin-stimulated amino acid disposal rates as assessed using hyperinsulinemic-euglycemic-euaminoacidemic clamps (20, 40 and 80 mU/kg per h) were linearly increased by the incremental administrations of menhaden oil from 169, 238 to 375 μmol/kg per h (P = 0.005) during the 40 mU/kg per h clamp, and from 295, 360 and 590 μmol/kg per h (P = 0.02) during the 80 mU/kg per h clamp. Glucose disposal rate responded according to a quadratic relationship with the incremental menhaden oil amounts (P < 0.05). A regression analysis showed that 47% of the amino acid disposal rates elicited during the hyperinsulinemic clamp was related to muscle membrane LCn-3PUFA content (P = 0.003). These results show for the first time that both protein and glucose metabolism respond in a dose-dependent manner to menhaden oil and to muscle membrane LCn-3PUFA.     

Genetic effects of chronic habitat fragmentation revisited: Strong genetic structure in a temperate tree, Taxus baccata (Taxaceae), with great dispersal capability

Tree species are thought to be relatively resistant to habitat fragmentation because of their longevity and their aptitude for extensive gene flow, although recent empirical studies have reported negative genetic consequences, in particular after long-term habitat fragmentation in European temperate regions. Yet the response of each species to habitat loss may differ greatly depending on their biological attributes, in particular seed dispersal ability. In this study, we used demographic and molecular data to investigate the genetic consequences of chronic habitat fragmentation in remnant populations of Taxus baccata in the Montseny Mountains, northeast Spain. The age structure of populations revealed demographic bottlenecks and recruitment events associated with exploitation and management practices. We found a strong genetic structure, both at the landscape and within-population levels. We also detected high levels of inbreeding for a strictly outcrossing species. Chronic forest fragmentation resulting from long-term exploitation in the Montseny Mountains seems the most plausible explanation for the strong genetic structure observed. Our results support the view that, contrary to some predictions, tree species are not buffered from the adverse effects of habitat fragmentation, even in the case of species with a high dispersal potential.     

Genetic Studies of Spectrin in the Larval Fat Body of Drosophila melanogaster: Evidence for a Novel Lipid Uptake Apparatus

Spectrin cytoskeleton defects produce a host of phenotypes affecting the plasma membrane, cell polarity, and secretory membrane traffic. However, many of the underlying molecular mechanisms remain unexplained by prevailing models. Here we used the larval fat body of Drosophila melanogaster as a genetic model system to further elucidate mechanisms of αβ-spectrin function. The results provide unexpected new insights into spectrin function as well as mechanisms of dietary fat uptake and storage. We show that loss of α- or β-spectrin in the fat body eliminated a population of small cortical lipid droplets and altered plasma membrane architecture, but did not affect viability of the organism. We present a novel model in which αβ-spectrin directly couples lipid uptake at the plasma membrane to lipid droplet growth in the cytoplasm. In contrast, strong overexpression of β-spectrin caused fat body atrophy and larval lethality. Overexpression of β-spectrin also perturbed transport of dietary fat from the midgut to the fat body. This hypermorphic phenotype appears to be the result of blocking secretion of the lipid carrier lipophorin from fat cells. However, this midgut phenotype was never seen with spectrin loss of function, suggesting that spectrin is not normally required for lipophorin secretion or function. The β-spectrin hypermorphic phenotype was ameliorated by co-overexpression of α-spectrin. Based on the overexpression results here, we propose that β-spectrin family members may be prone to hypermorphic effects (including effects on secretion) if their activity is not properly regulated.     

Effect of population structure corrections on the results of association mapping tests in complex maize diversity panels

Association mapping of sequence polymorphisms underlying the phenotypic variability of quantitative agronomical traits is now a widely used method in plant genetics. However, due to the common presence of a complex genetic structure within the plant diversity panels, spurious associations are expected to be highly frequent. Several methods have thus been suggested to control for panel structure. They mainly rely on ad hoc criteria for selecting the number of ancestral groups; which is often not evident for the complex panels that are commonly used in maize. It was thus necessary to evaluate the effect of the selected structure models on the association mapping results. A real maize data set (342 maize inbred lines and 12,000 SNPs) was used for this study. The panel structure was estimated using both Bayesian and dimensional reduction methods, considering an increasing number of ancestral groups. Effect on association tests depends in particular on the number of ancestral groups and on the trait analyzed. The results also show that using a high number of ancestral groups leads to an over-corrected model in which all causal loci vanish. Finally the results of all models tested were combined in a meta-analysis approach. In this way, robust associations were highlighted for each analyzed trait.     

Cell type-dependent internalization of the Escherichia coli STb enterotoxin

Previous studies have suggested that internalization of the Escherichia coli STb enterotoxin in human and rat intestinal epithelial cells is involved in STb pathogenesis, but toxin uptake in porcine jejunum epithelium, the in vivo target tissue, still remains elusive. Using flow cytometry, we studied the internalization of fluorescein isothiocyanate-labelled STb in porcine intestinal epithelial IPEC-J2 and murine fibroblast NIH-3T3 cell lines. In contrast to the selective pronase resistance of STb in NIH-3T3 cells at 37 °C, but not at 4 °C, indicative of toxin internalization, most of the toxin was pronase-sensitive at both temperatures in IPEC-J2 cells, indicating reduced uptake, but significant cell surface binding. Actin reorganization is required for STb internalization by NIH-3T3 cells, confirming STb endocytosis in these cells. The toxin receptor, sulfatide, could not explain these internalization differences because both cell lines possessed surface sulfatide and internalized antisulfatide antibodies over time at 37 °C. Inhibition of lipid rafts endocytosis, known to contain sulfatide, with methyl-β-cyclodextrin or genistein, did not influence toxin uptake by either cell line. STb internalization is therefore differentially regulated depending on the cell type, possibly by factors other than sulfatide. Although a small STb fraction could be internalized by porcine intestinal epithelial cells, our findings suggest the ability of STb to induce, from the cell surface, intracellular signalling leading to fluid secretion in porcine intestinal epithelium.     

Proteomic profile of culture filtrate from the Brazilian vaccine strain <Emphasis Type="Italic">Mycobacterium bovis</Emphasis> BCG Moreau compared to <Emphasis Type="Italic">M. bovis</Emphasis> BCG Pasteur

Background  

Bacille Calmette-Guerin (BCG) is currently the only available vaccine against tuberculosis (TB) and comprises a heterogeneous family of sub-strains with genotypic and phenotypic differences. The World Health Organization (WHO) affirms that the characterization of BCG sub-strains, both on genomic and proteomic levels, is crucial for a better comprehension of the vaccine. In addition, these studies can contribute in the development of a more efficient vaccine against TB. Here, we combine two-dimensional electrophoresis (2DE) and mass spectrometry to analyse the proteomic profile of culture filtrate proteins (CFPs) from M. bovis BCG Moreau, the Brazilian vaccine strain, comparing it to that of BCG Pasteur. CFPs are considered of great importance given their dominant immunogenicity and role in pathogenesis, being available for interaction with host cells since early infection.