Copper-dependent co-internalization of the prion protein and glypican-1

Heparan sulfate chains have been found to be associated with amyloid deposits in a number of diseases including transmissible spongiform encephalopathies. Diverse lines of evidence have linked proteoglycans and their glycosaminoglycan chains, and especially heparan sulfate, to the metabolism of the prion protein isoforms. Glypicans are a family of glycosylphosphatidylinositol-anchored, heparan sulfate-containing, cell-associated proteoglycans. Cysteines in glypican-1 can become nitrosylated by endogenously produced nitric oxide. When glypican-1 is exposed to a reducing agent, such as ascorbate, nitric oxide is released and autocatalyses deaminative cleavage of heparan sulfate chains. These processes take place while glypican-1 recycles via a non-classical, caveolin-associated pathway. We have previously demonstrated that prion protein provides the Cu2+ ions required to nitrosylate thiol groups in the core protein of glypican-1. By using confocal immunofluorescence microscopy and immunomagnetic techniques, we now show that copper induces co-internalization of prion protein and glypican-1 from the cell surface to perinuclear compartments. We find that prion protein is controlling both the internalization of glypican-1 and its nitric oxide-dependent autoprocessing. Silencing glypican-1 expression has no effect on copper-stimulated prion protein endocytosis, but in cells expressing a prion protein construct lacking the copper binding domain internalization of glypican-1 is much reduced and autoprocessing is abrogated. We also demonstrate that heparan sulfate chains of glypican-1 are poorly degraded in prion null fibroblasts. The addition of either Cu2+ ions, nitric oxide donors, ascorbate or ectopic expression of prion protein restores heparan sulfate degradation. These results indicate that the interaction between glypican-1 and Cu2+-loaded prion protein is required both for co-internalization and glypican-1 self-pruning.     

Mouse ApoM displays an unprecedented seven-stranded lipocalin fold: folding decoy or alternative native fold?

Mouse apolipoprotein M (m-apoM) displays a 79% sequence identity to human apolipoprotein M (h-apoM). Both proteins are apolipoproteins associated with high-density lipoproteins, with similar anticipated biological functions. The structure of h-apoM has recently been determined by X-ray crystallography, which revealed that h-apoM displays, as expected, a lipocalin-like fold characterized by an eight-stranded β?barrel that encloses an internal fatty-acid-binding site. Surprisingly, this is not true for m-apoM. After refolding from inclusion bodies, the crystal structure of m-apoM (reported here at 2.5 Å resolution) displays a novel yet unprecedented seven-stranded β-barrel structure. The fold difference is not caused by a mere deletion of a single β-strand; instead, β-strands E and F are removed and replaced by a single β-strand A′ formed from residues from the N-terminus. Molecular dynamics simulations suggest that m-apoM is able to adopt both a seven-stranded barrel structure and an eight-stranded barrel structure in solution, and that both folds are comparably stable. Thermal unfolding simulations identify the position where β-strand exchange occurs as the weak point of the β-barrel. We wonder whether the switch in topology could have a biological function and could facilitate ligand release, since it goes hand in hand with a narrowing of the barrel diameter. Possibly also, the observed conformation represents an on-pathway or off-pathway folding intermediate of apoM. The difference in fold topology is quite remarkable, and the fold promiscuity observed for m-apoM might possibly provide a glimpse at potential cross-points during the evolution of β-barrels.     

Establishment of heparan sulphate deficient primary endothelial cells from EXT-1<Superscript>flox/flox</Superscript> mouse lungs and sprouting aortas

Angiogenesis is a hallmark of expanding tissue e.g. during embryogenesis and wound healing in physiology as well as in diseases such as cancer and atherosclerosis. Key steps of the angiogenic process involve growth factor-mediated stimulation of endothelial cell sprouting and tube formation. Heparan sulphate proteoglycans (HSPGs) have been implicated as important co-receptors of several pro-angiogenic proteins. The importance of HSPGs in physiology was underscored by the finding that knockout of the gene encoding HS polymerase, EXT-1, resulted in early embryonic lethality. Here, we describe the establishment of HS-deficient endothelial cells from sprouting aortas as well as from the lungs of EXT-1^flox/flox mice. Recombination of the loxP-flanked EXT-1 locus by Cre-expressing adenovirus was demonstrated at the mRNA level. Moreover, depletion of HS polysaccharides was verified by flow cytometry and fluorescence microscopy methodology using phage display-derived anti-HS antibodies. In summary, we provide a genetic model to unravel the functional role of HSPGs specifically in primary endothelial cells during early steps of angiogenesis. Our studies are applicable to most loxP-based transgenic mouse strains, and may thus be of general importance in the angiogenesis field.     

Turbidity Hampers Mate Choice in a Pipefish

European coastal waters have in recent years become more turbid as algal growth has increased, probably due to eutrophication, global warming and changes in fish communities. Turbidity reduces visibility, and such changes may in turn affect animal behaviour as well as evolutionary processes that are dependent on visual stimuli. In this study we experimentally manipulated water visibility and olfactory cues to investigate mate choice using the sex role‐reversed broad‐nosed pipefish Syngnathus typhle as our study organism. We show that males spent significantly longer time assessing females when they had access to full visual cues, compared to when visibility was reduced. Presence or absence of olfactory cues from females did not affect mate choice, suggesting that the possible use of smell could not make up for a reduction in visibility. This implies that mate choice is environmentally dependent and that an increased turbidity may affect processes of sexual selection through an impaired possibility for visually based mate choice.     

Apolipoprotein M associates to lipoproteins through its retained signal peptide

Apolipoprotein M (apoM) is predominantly associated with HDL. In this study, it was investigated whether apoM's uncleaved signal peptide is necessary for the protein's ability to associate with lipoproteins. ApoM with a cleavable signal peptide, Q22A, was expressed, together with wild-type apoM, in HEK293 cells. On size-exclusion chromatography, the elution profile of wild-type apoM was similar to that of human HDL-associated plasma apoM. In contrast, the size of the Q22A mutant corresponded to free, unassociated apoM. This strongly indicates that the signal peptide is indeed necessary for apoM's ability to associate with lipid.     

The low density lipoprotein receptor prevents secretion of dense apoB100-containing lipoproteins from the liver

The assembly and secretion of very low density lipoproteins (VLDL) require microsomal triglyceride transfer protein (MTP). Recent evidence also suggests a role for the low density lipoprotein (LDL) receptor in this process. However, the relative importance of MTP in the two steps of VLDL assembly and the specific role of the LDL receptor still remain unclear. To further investigate the role of MTP and the LDL receptor in VLDL assembly, we bred mice harboring "floxed" Mttp alleles (Mttpflox/flox) and a Cre transgene on a low-density lipoprotein receptor-deficient background to generate mice with double deficiency in the liver (Ldlr-/- MttpDelta/Delta). In contrast to the plasma of Ldlr+/+ MttpDelta/Delta mice, the plasma of Ldlr-/- MttpDelta/Delta mice contained apoB100. Accordingly, Ldlr-/- MttpDelta/Delta but not Ldlr+/+ MttpDelta/Delta hepatocytes secreted apoB100-containing lipoprotein particles. The secreted lipoproteins were of LDL and HDL sizes but no VLDL-sized lipoproteins could be detected. These findings indicate that hepatic LDL receptors function as "gatekeepers" targeting dense apoB100-containing lipoproteins for degradation. In addition, these results suggest that very low levels of MTP are insufficient to mediate the second step but sufficient for the first step of VLDL assembly.     

Use of RNA interference and complementation to study the function of the Drosophila and human 26S proteasome subunit S13

The S13 subunit (also called Pad1, Rpn11, and MPR1) is a component of the 19S complex, a regulatory complex essential for the ubiquitin-dependent proteolytic activity of the 26S proteasome. To address the functional role of S13, we combined double-stranded RNA interference (RNAi) against the Drosophila proteasome subunit DmS13 with expression of wild-type and mutant forms of the homologous human gene, HS13. These studies show that DmS13 is essential for 26S function. Loss of the S13 subunit in metazoan cells leads to increased levels of ubiquitin conjugates, cell cycle defects, DNA overreplication, and apoptosis. In vivo assays using short-lived proteasome substrates confirmed that the 26S ubiquitin-dependent degradation pathway is compromised in S13-depleted cells. In complementation experiments using Drosophila cell lines expressing HS13, wild-type HS13 was found to fully rescue the knockdown phenotype after DmS13 RNAi treatment, while an HS13 containing mutations (H113A-H115A) in the proposed isopeptidase active site was unable to rescue. A mutation within the conserved MPN/JAMM domain (C120A) abolished the ability of HS13 to rescue the Drosophila cells from apoptosis or DNA overreplication. However, the C120A mutant was found to partially restore normal levels of ubiquitin conjugates. The S13 subunit may possess multiple functions, including a deubiquitinylating activity and distinct activities essential for cell cycle progression that require the conserved C120 residue.     

Frequency Dependence of Host Plant Choice within and Between Patches: A Large Cage Experiment

Oviposition preference is considered to be one of the most important factors behind patterns of host use among herbivorous insects. However, preference is defined as host plant choice under equal host abundance and availability, and it is likely that frequency-dependent effects will alter the actual pattern of host use beyond what preference trials reveals. The effects of such alterations are poorly known but could be important for the understanding of specialization and host shifts. We investigated how changes in frequency of a preferred and a less preferred host affected movement patterns and egg deposition within and among patches in a polyphagous butterfly, Polygonia c-album. Two experiments were carried out in large (8 × 30 m) outdoor cages, artificially divided into distinct patches with different frequencies of the two hosts: one that allowed for limited movement between patches and one that did not. There was a clear effect of frequency on patch selection; females spent more time in and laid more eggs in patches with a high frequency of the preferred host, which will potentially have a large effect on host use by modifying encounter rates in favor of the preferred host. However, there was no significant frequency-dependent plant choice within patches in any experiment. Instead, results indicate that females are distributing their eggs among plants species according to specific likelihoods of oviposition, independent of encounter rates, which is compatible with a strategy of risk-spreading.Co-ordinating editor: N. Yamamura     

Feeding, prey selection and prey encounter mechanisms in the heterotrophic dinoflagellate Noctiluca scintillans

The heterotrophic dinoflagellate Noctiluca scintillans has anegligible swimming ability and feeds predominantly on immobileprey. How, then, does it encounter prey? Noctiluca scintillansis positively buoyant and, therefore, we hypothesized that itintercepts prey particles during ascent and/or that microscaleshear brings it into contact with prey. Noctiluca scintillanshas a specific carbon content 1–2 orders of magnitudeless than that typical for protists and, thus, an inflated volume.It also has a density slightly less than that of the ambientwater and therefore ascends at high velocities (-1 m h^–1).In stagnant water, clearance rates of latex spheres (5–80µm) increased approximately with prey particle size squared.This scaling is consistent with N.scintillans being an interceptionfeeder. However, absolute clearance rates were substantiallylower than those predicted by modeling N.scintillans both asa spherical and as a cylindrical collector. The latter modelassumes that prey particles are collected on the string of mucusthat may form at the tip of the tentacle. Feeding, growth andprey selection experiments all demonstrated that diatoms arecleared at substantially higher rates than latex beads and otherphytoplankters, particularly dinoflagellates. We propose thatdiatoms stick more efficiently than latex beads to the mucusof N.scintillans and that dinoflagellates reduce fatal contactbehaviorally. We conclude that N.scintillans is an interceptionfeeder and that the high ascent velocity accounts for encounterswith prey. However, the flow field around the cell-mucus complexis too complicated to be described accurately by simple geometricmodels. Fluid shear (0.7–1.8 s^–1 had a negativeimpact on feeding rates, which were much less than predictedby models. Noctiluca scintillans can survive starvation forlong periods (>3 weeks), it can grow at low concentrationsof prey (-15 µg C l^–1), but growth saturates onlyat very high prey concentrations of 500–1000 µgC l^–1 or more. We demonstrate how the functional biologyof N.scintillans is consistent with its spatial and seasonaldistribution, which is characterized by persistence in the plankton,blooms in association with high concentrations of diatoms, andsurface accumulation during quiescent periods or exponentialdecline in abundance with depth during periods of turbulentmixing.