Three-dimensional modelling of honeybee venom allergenic proteases: relation to allergenicity

Api SI and Api SII are serine proteases of the honeybee venom containing allergenic determinants. Each protease consists of two structural modules: an N-terminal CUB (Api SI) or a clip domain (Api SII) and a C-terminal serine protease-like (SPL) domain. Both domains are connected with a linker peptide. The knowledge about the structure and function of Api SI and Api SII is limited mainly to their amino acid sequences. We constructed 3-D models of the two proteases using their amino acid sequences and crystallographic coordinates of related proteins. The models of the SPL domains were built using the structure of the prophenoloxidase-activating factor (PPAF)-II as a template. For modelling of the Api SI CUB domain the coordinates of porcine spermadhesin PSP-I were used. The models revealed the catalytic and substrate-binding sites and the negatively charged residue responsible for the trypsin-like activity. IgE-binding and antigenic sites in the two allergens were predicted using the models and programs based on the structure of known epitopes. Api SI and Api SII show structural and functional similarity to the members of the PPAF-II family. Most probably, they are part of the defence system of Apis mellifera.     

Ubiquitination of the peroxisomal targeting signal type 1 receptor, Pex5p, suggests the presence of a quality control mechanism during peroxisomal matrix protein import

PEX genes encode proteins (peroxins) that are required for the biogenesis of peroxisomes. One of these peroxins, Pex5p, is the receptor for matrix proteins with a type 1 peroxisomal targeting signal (PTS1), which shuttles newly synthesized proteins from the cytosol into the peroxisome matrix. We observed that in various Saccharomyces cerevisiae pex mutants disturbed in the early stages of PTS1 import, the steady-state levels of Pex5p are enhanced relative to wild type controls. Furthermore, we identified ubiquitinated forms of Pex5p in deletion mutants of those PEX genes that have been implicated in recycling of Pex5p from the peroxisomal membrane into the cytosol. Pex5p ubiquitination required the presence of the ubiquitin-conjugating enzyme Ubc4p and the peroxins that are required during early stages of PTS1 protein import. Finally, we provide evidence that the proteasome is involved in the turnover of Pex5p in wild type yeast cells, a process that requires Ubc4p and occurs at the peroxisomal membrane. Our data suggest that during receptor recycling a portion of Pex5p becomes ubiquitinated and degraded by the proteasome. We propose that this process represents a conserved quality control mechanism in peroxisome biogenesis.     

Solution structure of the Mesorhizobium loti K1 channel cyclic nucleotide‐binding domain in complex with cAMP

Cyclic nucleotide‐sensitive ion channels, known as HCN and CNG channels, are crucial in neuronal excitability and signal transduction of sensory cells. HCN and CNG channels are activated by binding of cyclic nucleotides to their intracellular cyclic nucleotide‐binding domain (CNBD). However, the mechanism by which the binding of cyclic nucleotides opens these channels is not well understood. Here, we report the solution structure of the isolated CNBD of a cyclic nucleotide‐sensitive K⁺ channel from Mesorhizobium loti. The protein consists of a wide anti‐parallel β‐roll topped by a helical bundle comprising five α‐helices and a short 3₁₀‐helix. In contrast to the dimeric arrangement (‘dimer‐of‐dimers’) in the crystal structure, the solution structure clearly shows a monomeric fold. The monomeric structure of the CNBD supports the hypothesis that the CNBDs transmit the binding signal to the channel pore independently of each other.     

Evaluation of promoter sequences for the secretory production of a Clostridium thermocellum cellulase in Paenibacillus polymyxa

Due to their high secretion capacity, Gram-positive bacteria from the genus Bacillus are important expression hosts for the high-yield production of enzymes in industrial biotechnology; however, to date, strains from only few Bacillus species are used for enzyme production at industrial scale. Herein, we introduce Paenibacillus polymyxa DSM 292, a member of a different genus, as a novel host for secretory protein production. The model gene cel8A from Clostridium thermocellum was chosen as an easily detectable reporter gene with industrial relevance to demonstrate heterologous expression and secretion in P. polymyxa. The yield of the secreted cellulase Cel8A protein was increased by optimizing the expression medium and testing several promoter sequences in the expression plasmid pBACOV. Quantitative mass spectrometry was used to analyze the secretome in order to identify promising new promoter sequences from the P. polymyxa genome itself. The most abundantly secreted host proteins were identified, and the promoters regulating the expression of their corresponding genes were selected. Eleven promoter sequences were cloned and tested, including well-characterized promoters from Bacillus subtilis and Bacillus megaterium. The best result was achieved with the promoter for the hypothetical protein PPOLYM_03468 from P. polymyxa. In combination with the optimized expression medium, this promoter enabled the production of 5475 U/l of Cel8A, which represents a 6.2-fold increase compared to the reference promoter P_aprE. The set of promoters described in this work covers a broad range of promoter strengths useful for heterologous expression in the new host P. polymyxa.

     

Amylase activity is associated with AMY2B copy numbers in dog: implications for dog domestication,diet and diabetes

High amylase activity in dogs is associated with a drastic increase in copy numbers of the gene coding for pancreatic amylase, AMY2B, that likely allowed dogs to thrive on a relatively starch‐rich diet during early dog domestication. Although most dogs thus probably digest starch more efficiently than do wolves, AMY2B copy numbers vary widely within the dog population, and it is not clear how this variation affects the individual ability to handle starch nor how it affects dog health. In humans, copy numbers of the gene coding for salivary amylase, AMY1, correlate with both salivary amylase levels and enzyme activity, and high amylase activity is related to improved glycemic homeostasis and lower frequencies of metabolic syndrome. Here, we investigate the relationship between AMY2B copy numbers and serum amylase activity in dogs and show that amylase activity correlates with AMY2B copy numbers. We then describe how AMY2B copy numbers vary in individuals from 20 dog breeds and find strong breed‐dependent patterns, indicating that the ability to digest starch varies both at the breed and individual level. Finally, to test whether AMY2B copy number is strongly associated with the risk of developing diabetes mellitus, we compare copy numbers in cases and controls as well as in breeds with varying diabetes susceptibility. Although we see no such association here, future studies using larger cohorts are needed before excluding a possible link between AMY2B and diabetes mellitus.     

Movement of outbreak populations of mountain pine beetle: influences of spatiotemporal patterns and climate

Insect outbreaks exert landscape-level influences, yet quantifying the relative contributions of various exogenous and endogenous factors that contribute to their pattern and spread remains elusive. We examine an outbreak of mountain pine beetle covering an 800 thousand ha area on the Chilcotin Plateau of British Columbia, Canada, during the 1970s and early 1980s. We present a model that incorporates the spatial and temporal arrangements of outbreaking insect populations, as well as various climatic factors that influence insect development. Onsets of eruptions of mountain pine beetle demonstrated landscape-level synchrony. On average, the presence of outbreaking populations was highly correlated with outbreaking populations within the nearest 18  km the same year and local populations within 6 km in the previous two years. After incorporating these spatial and temporal dependencies, we found that increasing temperatures contributed to explaining outbreak probabilities during this 15  yr outbreak. During collapse years, landscape-level synchrony declined while local synchrony values remained high, suggesting that in some areas host depletion was contributing to population decline. Model forecasts of outbreak propensity one year in advance at a 12 by 12  km scale provided 80% accuracy over the landscape, and never underestimated the occurrence of locally outbreaking populations. This model provides a flexible approach for linking temperature and insect population dynamics to spatial spread, and complements existing decision support tools for resource managers.     

Calmodulin-dependent activation and inactivation of anoctamin calcium-gated chloride channels

Calcium-dependent chloride channels serve critical functions in diverse biological systems. Driven by cellular calcium signals, the channels codetermine excitatory processes and promote solute transport. The anoctamin (ANO) family of membrane proteins encodes three calcium-activated chloride channels, named ANO 1 (also TMEM16A), ANO 2 (also TMEM16B), and ANO 6 (also TMEM16F). Here we examined how ANO 1 and ANO 2 interact with Ca²⁺/calmodulin using nonstationary current analysis during channel activation. We identified a putative calmodulin-binding domain in the N-terminal region of the channel proteins that is involved in channel activation. Binding studies with peptides indicated that this domain, a regulatory calmodulin-binding motif (RCBM), provides two distinct modes of interaction with Ca²⁺/calmodulin, one at submicromolar Ca²⁺ concentrations and one in the micromolar Ca²⁺ range. Functional, structural, and pharmacological data support the concept that calmodulin serves as a calcium sensor that is stably associated with the RCBM domain and regulates the activation of ANO 1 and ANO 2 channels. Moreover, the predominant splice variant of ANO 2 in the brain exhibits Ca²⁺/calmodulin-dependent inactivation, a loss of channel activity within 30 s. This property may curtail ANO 2 activity during persistent Ca²⁺ signals in neurons. Mutagenesis data indicated that the RCBM domain is also involved in ANO 2 inactivation, and that inactivation is suppressed in the retinal ANO 2 splice variant. These results advance the understanding of Ca²⁺ regulation in anoctamin Cl⁻ channels and its significance for the physiological function that anoctamin channels subserve in neurons and other cell types.