Sequence Determinants for Regulated Degradation of Yeast 3-Hydroxy-3-Methylglutaryl-CoA Reductase, an Integral Endoplasmic Reticulum Membrane Protein

The degradation rate of 3-hydroxy-3-methylglutaryl CoA reductase (HMG-R), a key enzyme of the mevalonate pathway, is regulated through a feedback mechanism by the mevalonate pathway. To discover the intrinsic determinants involved in the regulated degradation of the yeast HMG-R isozyme Hmg2p, we replaced small regions of the Hmg2p transmembrane domain with the corresponding regions from the other, stable yeast HMG-R isozyme Hmg1p. When the first 26 amino acids of Hmg2p were replaced with the same region from Hmg1p, Hmg2p was stabilized. The stability of this mutant was not due to mislocalization, but rather to an inability to be recognized for degradation. When amino acid residues 27–54 of Hmg2p were replaced with those from Hmg1p, the mutant was still degraded, but its degradation rate was poorly regulated. The degradation of this mutant was still dependent on the first 26 amino acid residues and on the function of the HRD genes. These mutants showed altered ubiquitination levels that were well correlated with their degradative phenotypes. Neither determinant was sufficient to impart regulated degradation to Hmg1p. These studies provide evidence that there are sequence determinants in Hmg2p necessary for degradation and optimal regulation, and that independent processes may be involved in Hmg2p degradation and its regulation.     

Adrenergic modulation of Escherichia coli O157:H7 adherence to the colonic mucosa

Enteric neurotransmitters can modulate the biodefensive functions of the intestinal mucosa, but their role in mucosal interactions with enteropathogens is not well defined. Here we tested the hypothesis that norepinephrine (NE) modulates interactions between enterohemorrhagic Escherichia coli O157:H7 (EHEC) and the colonic epithelium. Mucosal sheets from porcine distal colon were mounted in Ussing chambers. Drugs and an inoculum of either Shiga toxin-negative or -positive EHEC were added to the contraluminal and luminal bathing medium, respectively. After 90 min, adherent bacteria were quantified by an adherence assay and by immunohistochemical methods; short-circuit current (I(sc)) was measured continuously to assess changes in active ion transport. NE-treated tissues exhibited concentration-dependent increases in I(sc) and EHEC adherence. NE did not alter adherence of a rodent-adapted, noninfectious E. coli strain or two porcine-adapted non-O157 E. coli strains. The actions of NE on EHEC adherence but not I(sc) were prevented by the alpha-adrenergic antagonist yohimbine and the PKA activator Sp-8-bromoadenosine-3',5'-cyclic monophosphorothioate. Like NE, the PKA inhibitor Rp-8-bromoadenosine-3',5'-cyclic monophosphorothioate or indirectly acting sympathomimetic agents increased EHEC adherence. Nerve fibers immunoreactive for the NE-synthesizing enzymes tyrosine hydroxylase and dopamine beta-hydroxylase appeared to innervate the colonic epithelium. EHEC-like immunoreactivity on the colonic surface had the appearance of bacterial microcolonies and increased after NE treatment by a phentolamine-sensitive mechanism. Through interactions with alpha(2)-adrenergic receptors, NE appears to increase EHEC adherence to the colonic mucosa. Changes in sympathetic neural outflow may alter intestinal susceptibility to infection.     

Peptide-based biomaterials for protease-enhanced drug delivery

Controlled delivery of drugs in response to environments has the potential of targeting therapies and personalized treatments. Here, we described self-assembled peptide sequences that release therapeutic payloads upon specific interaction with disease-associated proteases. The core peptide sequence consists of a protease cleavable region flanked by two self-assembly motifs. In aqueous solution, the peptides self-assemble as a gel scaffold. With treatment of the model preparations with the appropriate protease, the matrix can be degraded in a controlled fashion, where the degradation rate is fine-tuned by varying the peptide compositions. Protease-mediated drug release was demonstrated by enzymatic treatment of a model therapeutic peptide incorporated into the optimized matrix. Our results suggest that this type of material may have far-reaching applications for functionally targeted drug delivery.     

Seasonal and diel patterns in Pacific white-sided dolphin (Lagenorhynchus obliquidens) pulsed calls near Barkley Canyon

Evaluation of temporal patterns in offshore marine mammal activity through visual data collection can be impaired by light and weather conditions and the cost of ship time. The conditions-independence and cost-efficacy of acoustic data collection make it popular for researching the temporal patterns of wild dolphins. However, Pacific white-sided dolphins (PWSDs) north of Southern California remain under-researched. This study evaluates diel and seasonal patterns in PWSD communication signals detected in 480 days of near-continuous passive acoustic data collected over 20 months from the Barkley Canyon node of Ocean Networks Canada's NEPTUNE observatory, offshore Vancouver Island, British Columbia, Canada. In this study, a random forest classifier is used to identify PWSD pulsed calls and false positives are manually removed. The resulting acoustic presence-absence data set is evaluated for seasonal and diel patterns using generalized additive mixed models, considering masking effects from ambient noise. PWSDs are found to be vocally active during all diel periods year-round, with high summer and low spring activity. Seasonally dependent diel patterns are identified, with PWSD pulsed calls exhibiting no diel pattern in spring, a slight increase in vocal activity during dusk and night in fall and winter, and a notable increase during night and dawn in summer.     

Solution structure of RING finger-like domain of retinoblastoma-binding protein-6 (RBBP6) suggests it functions as a U-box

Retinoblastoma-binding protein-6 (RBBP6) plays a facilitating role, through its RING finger-like domain, in the ubiquitination of p53 by Hdm2 that is suggestive of E4-like activity. Although the presence of eight conserved cysteine residues makes it highly probable that the RING finger-like domain coordinates two zinc ions, analysis of the primary sequence suggests an alternative classification as a member of the U-box family, the members of which do not bind zinc ions. We show here that despite binding two zinc ions, the domain adopts a homodimeric structure highly similar to those of a number of U-boxes. Zinc ions could be replaced by cadmium ions without significantly disrupting the structure or the stability of the domain, although the rate of substitution was an order of magnitude slower than any previous measurement, suggesting that the structure is particularly stable, a conclusion supported by the high thermal stability of the domain. A hallmark of U-box-containing proteins is their association with chaperones, with which they cooperate in eliminating irretrievably unfolded proteins by tagging them for degradation by the proteasome. Using a yeast two-hybrid screen, we show that RBBP6 interacts with chaperones Hsp70 and Hsp40 through its N-terminal ubiquitin-like domain. Taken together with the structural similarities to U-box-containing proteins, our data suggest that RBBP6 plays a role in chaperone-mediated ubiquitination and possibly in protein quality control.     

Lipid Droplet-Binding Protein TIP47 Regulates Hepatitis C Virus RNA Replication through Interaction with the Viral NS5A Protein

The nonstructural protein NS5A has emerged as a new drug target in antiviral therapies for Hepatitis C Virus (HCV) infection. NS5A is critically involved in viral RNA replication that takes place at newly formed membranes within the endoplasmic reticulum (membranous web) and assists viral assembly in the close vicinity of lipid droplets (LDs). To identify host proteins that interact with NS5A, we performed a yeast two-hybrid screen with the N-terminus of NS5A (amino acids 1–31), a well-studied α-helical domain important for the membrane tethering of NS5A. Our studies identified the LD-associated host protein, Tail-Interacting Protein 47 (TIP47) as a novel NS5A interaction partner. Coimmunoprecipitation experiments in Huh7 hepatoma cells confirmed the interaction of TIP47 with full-length NS5A. shRNA-mediated knockdown of TIP47 caused a more than 10-fold decrease in the propagation of full-length infectious HCV in Huh7.5 hepatoma cells. A similar reduction was observed when TIP47 was knocked down in cells harboring an autonomously replicating HCV RNA (subgenomic replicon), indicating that TIP47 is required for efficient HCV RNA replication. A single point mutation (W9A) in NS5A that disrupts the interaction with TIP47 but preserves proper subcellular localization severely decreased HCV RNA replication. In biochemical membrane flotation assays, TIP47 cofractionated with HCV NS3, NS5A, NS5B proteins, and viral RNA, and together with nonstructural viral proteins was uniquely distributed to lower-density LD-rich membrane fractions in cells actively replicating HCV RNA. Collectively, our data support a model where TIP47—via its interaction with NS5A—serves as a novel cofactor for HCV infection possibly by integrating LD membranes into the membranous web.     

Phylogenomic Analysis Reveals Deep Divergence and Recombination in an Economically Important Grapevine Virus

The evolutionary history of the exclusively grapevine (Vitis spp.) infecting, grapevine leafroll-associated virus 3 (GLRaV-3) has not been studied extensively, partly due to limited available sequence data. In this study we trace the evolutionary history of GLRaV-3, focussing on isolate GH24, a newly discovered variant. GH24 was discovered through the use of next-generation sequencing (NGS) and the whole genome sequence determined and validated with Sanger sequencing. We assembled an alignment of all 13 available whole genomes of GLRaV-3 isolates and all other publicly available GLRaV-3 sequence data. Using multiple recombination detection methods we identified a clear signal for recombination in one whole genome sequence and further evidence for recombination in two more, including GH24. We inferred phylogenetic trees and networks and estimated the ages of common ancestors of GLRaV-3 clades by means of relaxed clock models calibrated with asynchronous sampling dates. Our results generally confirm previously identified variant groups as well as two new groups (VII and VIII). Higher order groups were defined as supergroups designated A to D. Supergroup A includes variant groups I-V and supergroup B group VI and its related unclassified isolates. Supergroups C and D are less well known, including the newly identified groups VII (including isolate GH24) and VIII respectively. The inferred node ages suggest that the origins of the major groups of GLRaV-3, including isolate GH24, may have occurred prior to worldwide cultivation of grapevines, whilst the current diversity represents closely related isolates that diverged from common ancestors within the last century.     

Using root traits to understand temporal changes in biodiversity effects in grassland mixtures

Biodiversity–ecosystem functioning (BEF) studies typically show that species richness enhances community biomass, but the underlying mechanisms remain debated. Here, we combine metrics from BEF research that distinguish the contribution of dominant species (selection effects, SE) from those due to positive interactions such as resource partitioning (complementarity effects, CE) with a functional trait approach in an attempt to reveal the functional characteristics of species that drive community biomass in species mixtures. In a biodiversity experiment with 16 plant species in monocultures, 4‐species and 16‐species mixtures, we used aboveground biomass to determine the relative contributions of CE and SE to biomass production in mixtures in the second, dry year of the experiment. We also measured root traits (specific root length, root length density, root tissue density and the deep root fraction) of each species in monocultures and linked the calculated community weighted mean (CWM) trait values and trait diversity of mixtures to CE and SE. In the second year of the experiment, community biomass, CE and SE increased compared to the first year. The contribution of SE to this positive effect was greater than that of CE. The increased contribution of SE was associated with root traits: SE increased most in communities with high abundance of species with deep, thick and dense roots. In contrast, changes in CE were not related to trait diversity or CWM trait values. Together, these results suggest that increased positive effects of species richness on community biomass in a dry year were mainly driven by increased dominance of deep‐rooting species, supporting the insurance hypothesis of biodiversity. Positive CE indicates that other positive interactions did occur, but we could not find evidence that belowground resource partitioning or facilitation via root trait diversity was important for community productivity in our biodiversity experiment.