Cytoplasmic and periplasmic proteomic signatures of exponentially growing cells of the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC125

The psychrophilic model bacterium Pseudoalteromonas haloplanktis is characterized by remarkably fast growth rates under low-temperature conditions in a range from 5°C to 20°C. In this study the proteome of cellular compartments, the cytoplasm and periplasm, of P. haloplanktis strain TAC125 was analyzed under exponential growth conditions at a permissive temperature of 16°C. By means of two-dimensional protein gel electrophoresis and mass spectrometry, a first inventory of the most abundant cytoplasmic and periplasmic proteins expressed in a peptone-supplemented minimal medium was established. By this approach major enzymes of the amino acid catabolism of this marine bacterium could be functionally deduced. The cytoplasmic proteome showed a predominance of amino acid degradation pathways and tricarboxylic acid (TCA) cycle enzymes but also the protein synthesis machinery. Furthermore, high levels of cold acclimation and oxidative stress proteins could be detected at this moderate growth temperature. The periplasmic proteome was characterized by a significant abundance of transporters, especially of highly expressed putative TonB-dependent receptors. This high capacity for protein synthesis, efficient amino acid utilization, and substrate transport may contribute to the fast growth rates of the copiotrophic bacterium P. haloplanktis in its natural environments.     

Importance of predation by white shrimp Litopenaeus setiferus on estuarine subtidal macrobenthos

Macrobenthic invertebrates are an important food source for higher trophic levels in freshwater and marine habitats, yet the importance of predation impacting regular seasonal changes in macrobenthos abundance remains unclear. Benthic invertebrates and transient marine species in temperate estuaries display inverse patterns of seasonal abundance, suggesting a link between predation and summer macrobenthos abundance minima. We conducted manipulative caging experiments to test the importance of predation by white shrimp (Litopenaeus setiferus) in regulating estuarine subtidal macrobenthos densities. We predicted greater declines in macrobenthos densities with increased shrimp densities due to predation rather than disturbance and macrobenthos emigration. Using these field and laboratory data, we estimate whether white shrimp predation can significantly contribute to the macrobenthos seasonal abundance minima observed in long-term monitoring data.White shrimp predation was measured in the field using 7-d predator enclosure/exclusion experiments. Within the uppermost 0-2 cm of sediment, total macrobenthos densities decreased within shrimp enclosure cages using 12 or 36 shrimp m^− 2. Laboratory experiments distinguished between the effects of shrimp predation versus shrimp disturbance and macrobenthos emigration. Shrimp predation significantly reduced macrobenthos densities, while effects of shrimp disturbance and macrobenthos emigration were not significant in these experiments. Despite the impacts from other ambient predators and other abiotic factors, white shrimp were clearly capable of driving subtidal macrobenthos from their annual maximum density in winter/spring to their summertime minimum density.     

A trafficking pathway for anthocyanins overlaps with the endoplasmic reticulum-to-vacuole protein-sorting route in Arabidopsis and contributes to the formation of vacuolar inclusions

Plants produce a very large number of specialized compounds that must be transported from their site of synthesis to the sites of storage or disposal. Anthocyanin accumulation has provided a powerful system to elucidate the molecular and cellular mechanisms associated with the intracellular trafficking of phytochemicals. Benefiting from the unique fluorescent properties of anthocyanins, we show here that in Arabidopsis (Arabidopsis thaliana), one route for anthocyanin transport to the vacuole involves vesicle-like structures shared with components of the secretory pathway. By colocalizing the red fluorescence of the anthocyanins with green fluorescent protein markers of the endomembrane system in Arabidopsis seedlings, we show that anthocyanins are also sequestered to the endoplasmic reticulum and to endoplasmic reticulum-derived vesicle-like structures targeted directly to the protein storage vacuole in a Golgi-independent manner. Moreover, our results indicate that vacuolar accumulation of anthocyanins does not depend solely on glutathione S-transferase activity or ATP-dependent transport mechanisms. Indeed, we observed a dramatic increase of anthocyanin-filled subvacuolar structures, without a significant effect on total anthocyanin levels, when we inhibited glutathione S-transferase activity, or the ATP-dependent transporters with vanadate, a general ATPase inhibitor. Taken together, these results provide evidence for an alternative novel mechanism of vesicular transport and vacuolar sequestration of anthocyanins in Arabidopsis.     

Detrital traits affect substitutability of a range‐expanding foundation species across latitude

Climate‐driven range shifts of foundation species could alter ecosystem processes and community composition by providing different resources than resident foundation species. Along the US Atlantic coast, the northward expanding foundation species, black mangrove Avicennia germinans, is replacing the dominant salt marsh foundation species, marsh cordgrass Spartina alterniflora. These species have distinct detrital attributes that ostensibly provide different resources to epifauna. We experimentally examined how detritus of these species affects decomposition and community composition in different habitat contexts at regional and local scales. First, we manipulated detritus identity (Avicennia, Spartina) at 13 sites across a 5° latitudinal gradient spanning mangrove, mixed marsh‐mangrove and salt marsh habitats. Across latitude, we found that Avicennia detritus decomposed 2–4 times faster than Spartina detritus, suggesting that detrital turnover will increase with mangrove expansion. Epifaunal abundance and richness increased 2–7 times from south to north (mangrove to salt marsh) and were equivalent between Avicennia and Spartina detritus except for crabs, a dominant taxonomic group that preferred Spartina detritus. Second, to examine the whether changing habitat context affected regional patterns, we manipulated detritus identity and surrounding habitat type (mangrove, salt marsh) at a single mixed site, also including inert mimics to separate structural and nutritional roles of detritus. Epifaunal richness was similar between the two detrital types, but crabs were 2–7 times more abundant in Spartina detritus due to its structural attributes. Surrounding habitat type did not influence decomposition rate or community patterns, which suggests that latitudinal influences, not surrounding habitat, drove the regional community patterns in the first experiment. Overall, mangrove expansion could alter epifaunal communities due to the lower structural value and faster turnover of mangrove detritus. As species shift with changing climate, understanding foundation species substitutability is critical to predict community change, but we must account for concomitant environmental changes that also modify communities.     

Synthesis and in vitro platelet aggregation and TP receptor binding studies on bicyclic 5,8-ethanooctahydroisoquinolines and 5,8-ethanotetrahydroisoquinolines

Eighteen novel bicyclic 1-substituted benzyl octahydro- and tetrahydroisoquinolines were synthesized and evaluated for human thromboxane A(2)/prostaglandin H(2) (TP) receptor affinity and antagonism of TP receptor-mediated platelet aggregation. In both cases, potency depended more on the presence of methoxy groups on the 1-benzyl moiety than on nitrogen substitution or extent of oxidation of the isoquinoline ring system. The most potent of the bicyclic compounds retained the 5,8-ethanooctahydroisoquinoline ring structure of the parent molecule (1) and required the 3,4,5-trimethoxybenzyl substitution pattern found in the well-characterized tetrahydroisoquinoline antiplatelet agent trimetoquinol. Differences in nitrogen substituent SAR were noted between the mono-methoxylated compounds and the 3,4,5-trimethoxybenzyl derivatives.     

Is there a cold shock response in the Antarctic psychrophile Pseudoalteromonas haloplanktis?

The growth behavior and the proteomic response after a cold shock were investigated in the psychrophilic Antarctic bacterium Pseudoalteromonas haloplanktis. Remarkably, no cold-induced proteins were observed in the proteome, whereas some key proteins were repressed. This suggests noticeable differences in the cold shock response between a true psychrophile and mesophiles.     

Order and disorder in large multi-site docking proteins of the Gab family--implications for signalling complex formation and inhibitor design strategies

Large multi-site docking (LMD) proteins of the Gab, IRS, FRS, DOK and Cas families consist of one or two folded N-terminal domains, followed by a predominantly disordered C-terminal extension. Their primary function is to provide a docking platform for signalling molecules (including PI3K, PLC, Grb2, Crk, RasGAP, SHP2) in intracellular signal transmission from activated cell-surface receptors, to which they become coupled. A detailed analysis of the structural nature and intrinsic disorder propensity of LMD proteins, with Gab proteins as specific examples, is presented. By primary sequence analysis and literature review the varying levels of disorder and hidden order are predicted, revealing properties and a physical architecture that help to explain their biological function and characteristics, common for network hub proteins. The virulence factor, CagA, from Helicobacter pylori is able to mimic Gab function once injected by this human pathogen into stomach epithelial cells. Its predicted differential structure is compared to Gab1 with respect to its functional mimicry. Lastly, we discuss how LMD proteins, in particular Gab1 and Gab2, and their protein partners, such as SH2 and SH3 domain-containing adaptors like Grb2, might qualify for future anti-cancer strategies in developing protein-protein interaction (PPI) inhibitors towards binary interactors consisting of an intrinsically disordered epitope and a structured domain surface.     

Blood pressure is regulated by an alpha1D-adrenergic receptor/dystrophin signalosome

Hypertension is a cardiovascular disease associated with increased plasma catecholamines, overactivation of the sympathetic nervous system, and increased vascular tone and total peripheral resistance. A key regulator of sympathetic nervous system function is the alpha(1D)-adrenergic receptor (AR), which belongs to the adrenergic family of G-protein-coupled receptors (GPCRs). Endogenous catecholamines norepinephrine and epinephrine activate alpha(1D)-ARs on vascular smooth muscle to stimulate vasoconstriction, which increases total peripheral resistance and mean arterial pressure. Indeed, alpha(1D)-AR KO mice display a hypotensive phenotype and are resistant to salt-induced hypertension. Unfortunately, little information exists about how this important GPCR functions because of an inability to obtain functional expression in vitro. Here, we identified the dystrophin proteins, syntrophin, dystrobrevin, and utrophin as essential GPCR-interacting proteins for alpha(1D)-ARs. We found that dystrophins complex with alpha(1D)-AR both in vitro and in vivo to ensure proper functional expression. More importantly, we demonstrate that knock-out of multiple syntrophin isoforms results in the complete loss of alpha(1D)-AR function in mouse aortic smooth muscle cells and abrogation of alpha(1D)-AR-mediated increases in blood pressure. Our findings demonstrate that syntrophin and utrophin associate with alpha(1D)-ARs to create a functional signalosome, which is essential for alpha(1D)-AR regulation of vascular tone and blood pressure.     

Variation in hydraulic conductivity of mangroves: influence of species, salinity, and nitrogen and phosphorus availability

We investigated how species identity and variation in salinity and nutrient availability influence the hydraulic conductivity of mangroves. Using a fertilization study of two species in Florida, we found that stem hydraulic conductivity expressed on a leaf area basis ( K _leaf) was significantly different among species of differing salinity tolerance, but was not significantly altered by enrichment with limiting nutrients. Reviewing data from two additional sites (Panamá and Belize), we found an overall pattern of declining leaf‐specific hydraulic conductivity ( K _leaf) with increasing salinity. Over three sites, a general pattern emerges, indicating that native stem hydraulic conductivity ( K _h) and K _leaf are less sensitive to nitrogen (N) fertilization when N limits growth, but more sensitive to phosphorus (P) fertilization when P limits growth. Processes leading to growth enhancement with N fertilization are probably associated with changes in allocation to leaf area and photosynthetic processes, whereas water uptake and transport processes could be more limiting when P limits growth. These findings suggest that whereas salinity and species identity place broad bounds on hydraulic conductivity, the effects of nutrient availability modulate hydraulic conductivity and growth in complex ways.     

Did psychrophilic enzymes really win the challenge?

Organisms living in permanently cold environments, which actually represent the greatest proportion of our planet, display at low temperatures metabolic fluxes comparable to those exhibited by mesophilic organisms at moderate temperatures. They produce cold-evolved enzymes partially able to cope with the reduction in chemical reaction rates and the increased viscosity of the medium induced by low temperatures. In most cases, the adaptation is achieved through a reduction in the activation energy, leading to a high catalytic efficiency, which possibly originates from an increased flexibility of either a selected area of or the overall protein structure. This enhanced plasticity seems in return to be responsible for the weak thermal stability of cold enzymes. These particular properties render cold enzymes particularly useful in investigating the possible relationships existing between stability, flexibility, and specific activity and make them potentially unrivaled for numerous biotechnological tasks. In most cases, however, the adaptation appears to be far from being fully achieved.