Membrane-associated DegP in Bordetella chaperones a repeat-rich secretory protein

The chaperone/protease DegP belongs to the HtrA superfamily and is involved in protein quality control in the periplasm of Gram-negative bacteria. In Escherichia coli, typical substrates are unfolded or misfolded globular proteins that trigger the rearrangement of inactive DegP hexamers into substrate-sequestering 12- or 24-mers 'cages' for refolding or degradation. In Bordetella pertussis, DegP(Bp) facilitates, in addition, the secretion of FHA, a long β-helical adhesin that passes through the periplasm in an extended conformation. We show that DegP(Bp) exists as soluble trimers and as a membrane-associated form. Different substrates interact differently with the distinct forms of DegP(Bp), and membrane-associated DegP(Bp) has high affinity for non-native FHA. Unlike more globular substrates, FHA does not efficiently mediate rearrangement of trimers into proteolytically active, short-lived dodecamers. In contrast to these dodecamers, membrane-associated DegP(Bp) is not committed to substrate degradation, although it is proteolytically competent. In B. pertussis, membrane-associated DegP(Bp) thus represents a specific functional form serving as a holding chaperone for client proteins including FHA. If FHA secretion is impaired, membrane-associated DegP(Bp) participates in its degradation. This form of DegP(Bp) is appropriate to handle substrates unsuitable to be sequestered in cages or non-folded, secretory proteins that must not be degraded.     

Gender differences in UV-induced inflammation and immunosuppression in mice reveal male unresponsiveness to UVA radiation

Immunosuppression attributed mainly to the UVB (290-320 nm) waveband is a prerequisite for skin cancer development in mice and humans. The contribution of UVA (320-400 nm) is controversial, but in mice UVA irradiation has been found to antagonise immunosuppression by UVB. In other studies of photoimmune regulation, protection mediated via oestrogen receptor-β signalling was identified as a normal endogenous defence in mice, and was shown to depend on UVA irradiation. A gender bias in photoimmune responsiveness was thus suggested, and is tested in this study by comparing the UV-induced inflammatory and immune responses in male and female hairless mice. We report that male mice, which show greater skin thickness than females, developed a less intense but slower resolving sunburn inflammatory oedema, correlated with reduced epidermal expression of pro-inflammatory IL-6 than females following solar simulated UV (SSUV, 290-400 nm) exposure. On the other hand, the contact hypersensitivity reaction (CHS) was more severely suppressed by SSUV in males, correlated with increased epidermal expression of immunosuppressive IL-10. Exposure to the UVB waveband alone, or to cis-urocanic acid, suppressed CHS equally in males and females. However, whereas UVA irradiation induced immunoprotection against either UVB or cis-urocanic acid in females, this protection was significantly reduced or abrogated in males. The results indicate that males are compromised by a relative unresponsiveness to the photoimmune protective effects of UVA, alone or as a component of SSUV. This could explain the known gender bias in skin cancer development in both mice and humans.     

Monthly to interannual variability of microbial eukaryote assemblages at four depths in the eastern North Pacific

The monthly, seasonal and interannual variability of microbial eukaryote assemblages were examined at 5 m, the deep chlorophyll maximum, 150 m and 500 m at the San Pedro Ocean Time-series station (eastern North Pacific). The depths spanned transitions in temperature, light, nutrients and oxygen, and included a persistently hypoxic environment at 500 m. Terminal restriction fragment length polymorphism was used for the analysis of 237 samples that were collected between September 2000 and December 2010. Spatiotemporal variability patterns of microeukaryote assemblages indicated the presence of distinct shallow and deep communities at the SPOT station, presumably reflecting taxa that were specifically adapted for the conditions in those environments. Community similarity values between assemblages collected 1 month apart at each depth ranged between ∼20% and ∼84% (averages were ∼50–59%). The assemblage at 5 m was temporally more dynamic than deeper assemblages and also displayed substantial interannual variability during the first ∼3 years of the study. Evidence of seasonality was detected for the microbial eukaryote assemblage at 5 m between January 2008 and December 2010 and at 150 m between September 2000 and December 2003. Seasonality was not detected for assemblages at the deep chlorophyll a maximum, which varied in depth seasonally, or at 500 m. Microbial eukaryote assemblages exhibited cyclical patterns in at least 1 year at each depth, implying an annual resetting of communities. Substantial interannual variability was detected for assemblages at all depths and represented the largest source of temporal variability in this temperate coastal ecosystem.     

Identification of IRF8, TMEM39A, and IKZF3-ZPBP2 as susceptibility loci for systemic lupus erythematosus in a large-scale multiracial replication study

Systemic lupus erythematosus (SLE) is a chronic heterogeneous autoimmune disorder characterized by the loss of tolerance to self-antigens and dysregulated interferon responses. The etiology of SLE is complex, involving both heritable and environmental factors. Candidate-gene studies and genome-wide association (GWA) scans have been successful in identifying new loci that contribute to disease susceptibility; however, much of the heritable risk has yet to be identified. In this study, we sought to replicate 1,580 variants showing suggestive association with SLE in a previously published GWA scan of European Americans; we tested a multiethnic population consisting of 7,998 SLE cases and 7,492 controls of European, African American, Asian, Hispanic, Gullah, and Amerindian ancestry to find association with the disease. Several genes relevant to immunological pathways showed association with SLE. Three loci exceeded the genome-wide significance threshold: interferon regulatory factor 8 (IRF8; rs11644034; p_meta-Euro = 2.08 × 10⁻¹⁰), transmembrane protein 39A (TMEM39A; rs1132200; p_meta-all = 8.62 × 10⁻⁹), and 17q21 (rs1453560; p_meta-all = 3.48 × 10⁻¹⁰) between IKAROS family of zinc finger 3 (AIOLOS; IKZF3) and zona pellucida binding protein 2 (ZPBP2). Fine mapping, resequencing, imputation, and haplotype analysis of IRF8 indicated that three independent effects tagged by rs8046526, rs450443, and rs4843869, respectively, were required for risk in individuals of European ancestry. Eleven additional replicated effects (5 × 10⁻⁸ < p_meta-Euro < 9.99 × 10⁻⁵) were observed with CFHR1, CADM2, LOC730109/IL12A, LPP, LOC63920, SLU7, ADAMTSL1, C10orf64, OR8D4, FAM19A2, and STXBP6. The results of this study increase the number of confirmed SLE risk loci and identify others warranting further investigation.     

ECTOCELLULAR GLUCOSIDASE AND PEPTIDASE ACTIVITY OF THE MIXOTROPHIC DINOFLAGELLATE PROROCENTRUM MINIMUM (DINOPHYCEAE)1

The activities of the enzymes α‐ and β‐glucosidase, and leucine aminopeptidase were measured in cultures of the dinoflagellate Prorocentrum minimum (Pavill.) J. Schiller and in field samples collected during dinoflagellate blooms occurring in tributaries of the Chesapeake Bay, Maryland, USA. Activities were measured using fluorogenic artificial substrates and partitioned among the >5 μm size fraction, small microbes fraction (0.1–5 μm), and dissolved phase (<0.1 μm). P. minimum and most other photosynthetic dinoflagellates are >5 μm in size and thus can be separated from the small microbes fraction, which contains most bacteria. Little to no glucosidase activity was detected associated with the >5 μm size fraction in cultures or in field samples, with most of the activity (67% to 93% in cultures, 54% to 100% in field samples) in the small microbes size fraction for both α and β glucosidase. In contrast, 67% to 90% of the total leucine aminopeptidase (LAP) activity in cultures was measured in the >5 μm fraction. Within a culture, LAP activity in the size fraction containing P. minimum decreased in response to ammonium and urea additions, but not in response to nitrate. In field samples, LAP activity was positively correlated with dinoflagellate abundance and chl a, and negatively correlated with ammonium concentration. During blooms, up to 34% of LAP activity was associated with the >5 μm fraction, indicating that when abundant, dinoflagellates may make a substantial contribution to ectocellular LAP activity in the water column.     

Acoustic characteristics, early experience, and endocrine status interact to modulate female zebra finches' behavioral responses to songs

Female songbirds use male songs as an important criterion for mate selection. Properties of male songs are thought to indicate the male's quality as a potential mate. Song preferences in female zebra finches are known to be influenced by two factors--early auditory experience and the acoustic characteristics of males' songs. Studies often investigate song preferences by priming females with estrogen. However, estrogenic influences on song preferences have not been studied. We investigated the relative influence of early auditory experience, acoustic features of songs, and estrogen availability on song responsiveness in female zebra finches. Juvenile female zebra finches were tutored for 10 days with 40 songs per day with one of three acoustically different song types--simple songs, long-bout songs or complex songs. A fourth group of females was untutored. Aside from this brief song exposure, females were raised and maintained without exposure to male songs. During adulthood, females' behavioral responses to the three song types were tested under three hormone conditions--untreated, estradiol-treated and 1,4,6-androstatriene-3,17-dione (ATD)-treated (to lower endogenous estrogen). Based on the results of our study, four conclusions can be drawn. First, song responsiveness in female zebra finches is strongly affected by minimal early acoustic experience. Second, inexperienced female zebra finches are inherently biased to respond more to complex songs over other song types Third, although female zebra finches are inherently biased to respond more to complex songs, early acoustic experience may either reinforce or weaken this inherent responsiveness to complex songs. Fourth, estrogen selectively accentuates song responsiveness in acoustically-experienced female zebra finches.     

Genetic variation and covariation in floral allocation of two species of Schiedea with contrasting levels of sexual dimorphism

The evolution of sexual dimorphism depends in part on the additive genetic variance-covariance matrices within females, within males, and across the sexes. We investigated quantitative genetics of floral biomass allocation in females and hermaphrodites of gynodioecious Schiedea adamantis (Caryophyllaceae). The G-matrices within females (G(f)), within hermaphrodites (G(m)), and between sexes (B) were compared to those for the closely related S. salicaria, which exhibits a lower frequency of females and less-pronounced sexual dimorphism. Additive genetic variation was detected in all measured traits in S. adamantis, with narrow-sense heritability from 0.34-1.0. Female allocation and floral size traits covaried more tightly than did those traits with allocation to stamens. Between-sex genetic correlations were all <1, indicating sex-specific expression of genes. Common principal-components analysis detected differences between G(f) and G(m) , suggesting potential for further independent evolution of the sexes. The two species of Schiedea differed in G(m) and especially so in G(f) , with S. adamantis showing greater genetic variation in capsule mass and tighter genetic covariation between female allocation traits and flower size in females. Despite greater sexual dimorphism in S. adamantis, genetic correlations between the two sexes (standardized elements of B) were similar to correlations between sexes in S. salicaria.     

Effect of Phosphorylation on EGFR Dimer Stability Probed by Single-Molecule Dynamics and FRET/FLIM

Deregulation of epidermal growth factor receptor (EGFR) signaling has been correlated with the development of a variety of human carcinomas. EGF-induced receptor dimerization and consequent trans- auto-phosphorylation are among the earliest events in signal transduction. Binding of EGF is thought to induce a conformational change that consequently unfolds an ectodomain loop required for dimerization indirectly. It may also induce important allosteric changes in the cytoplasmic domain. Despite extensive knowledge on the physiological activation of EGFR, the effect of targeted therapies on receptor conformation is not known and this particular aspect of receptor function, which can potentially be influenced by drug treatment, may in part explain the heterogeneous clinical response among cancer patients. Here, we used Förster resonance energy transfer/fluorescence lifetime imaging microscopy (FRET/FLIM) combined with two-color single-molecule tracking to study the effect of ATP-competitive small molecule tyrosine kinase inhibitors (TKIs) and phosphatase-based manipulation of EGFR phosphorylation on live cells. The distribution of dimer on-times was fitted to a monoexponential to extract dimer off-rates (k_off). Our data show that pretreatment with gefitinib (active conformation binder) stabilizes the EGFR ligand-bound homodimer. Overexpression of EGFR-specific DEP-1 phosphatase was also found to have a stabilizing effect on the homodimer. No significant difference in the k_off of the dimer could be detected when an anti-EGFR antibody (425 Snap single-chain variable fragment) that allows for dimerization of ligand-bound receptors, but not phosphorylation, was used. These results suggest that both the conformation of the extracellular domain and phosphorylation status of the receptor are involved in modulating the stability of the dimer. The relative fractions of these two EGFR subpopulations (interacting versus free) were obtained by a fractional-intensity analysis of ensemble FRET/FLIM images. Our combined imaging approach showed that both the fraction and affinity (surrogate of conformation at a single-molecule level) increased after gefitinib pretreatment or DEP-1 phosphatase overexpression. Using an EGFR mutation (I706Q, V948R) that perturbs the ability of EGFR to dimerize intracellularly, we showed that a modest drug-induced increase in the fraction/stability of the EGFR homodimer may have a significant biological impact on the tumor cell’s proliferation potential.     

Adaptation mechanism of the aspartate receptor: electrostatics of the adaptation subdomain play a key role in modulating kinase activity

The aspartate receptor of the Escherichia coli and Salmonella typhimurium chemotaxis pathway generates a transmembrane signal that regulates the activity of the cytoplasmic kinase CheA. Previous studies have identified a region of the cytoplasmic domain that is critical to receptor adaptation and kinase regulation. This region, termed the adaptation subdomain, contains a high density of acidic residues, including specific glutamate residues that serve as receptor adaptation sites. However, the mechanism of signal propagation through this region remains poorly understood. This study uses site-directed mutagenesis to neutralize each acidic residue within the subdomain to probe the hypothesis that electrostatics in this region play a significant role in the mechanism of kinase activation and modulation. Each point mutant was tested for its ability to regulate chemotaxis in vivo and kinase activity in vitro. Four point mutants (D273N, E281Q, D288N, and E477Q) were found to superactivate the kinase relative to the wild-type receptor, and all four of these kinase-activating substitutions are located along the same intersubunit interface as the adaptation sites. These activating substitutions retained the wild-type ability of the attractant-occupied receptor to inhibit kinase activity. When combined in a quadruple mutant (D273N/E281Q/D288N/E477Q), the four charge-neutralizing substitutions locked the receptor in a kinase-superactivating state that could not be fully inactivated by the attractant. Similar lock-on character was observed for a charge reversal substitution, D273R. Together, these results implicate the electrostatic interactions at the intersubunit interface as a major player in signal transduction and kinase regulation. The negative charge in this region destabilizes the local structure in a way that enhances conformational dynamics, as detected by disulfide trapping, and this effect is reversed by charge neutralization of the adaptation sites. Finally, two substitutions (E308Q and E463Q) preserved normal kinase activation in vitro but blocked cellular chemotaxis in vivo, suggesting that these sites lie within the docking site of an adaptation enzyme, CheR or CheB. Overall, this study highlights the importance of electrostatics in signal transduction and regulation of kinase activity by the cytoplasmic domain of the aspartate receptor.