Electrostatic recognition in substrate binding to serine proteases

Serine proteases of the Chymotrypsin family are structurally very similar but have very different substrate preferences. This study investigates a set of 9 different proteases of this family comprising proteases that prefer substrates containing positively charged amino acids, negatively charged amino acids, and uncharged amino acids with varying degree of specificity. Here, we show that differences in electrostatic substrate preferences can be predicted reliably by electrostatic molecular interaction fields employing customized GRID probes. Thus, we are able to directly link protease structures to their electrostatic substrate preferences. Additionally, we present a new metric that measures similarities in substrate preferences focusing only on electrostatics. It efficiently compares these electrostatic substrate preferences between different proteases. This new metric can be interpreted as the electrostatic part of our previously developed substrate similarity metric. Consequently, we suggest, that substrate recognition in terms of electrostatics and shape complementarity are rather orthogonal aspects of substrate recognition. This is in line with a 2‐step mechanism of protein‐protein recognition suggested in the literature.     

Decomposing multiple dimensions of stability in global change experiments

Ecological stability is the central framework to understand an ecosystem's ability to absorb or recover from environmental change. Recent modelling and conceptual work suggests that stability is a multidimensional construct comprising different response aspects. Using two freshwater mesocosm experiments as case studies, we show how the response to single perturbations can be decomposed in different stability aspects (resistance, resilience, recovery, temporal stability) for both ecosystem functions and community composition. We find that extended community recovery is tightly connected to a nearly complete recovery of the function (biomass production), whereas systems with incomplete recovery of the species composition ranged widely in their biomass compared to controls. Moreover, recovery was most complete when either resistance or resilience was high, the latter associated with low temporal stability around the recovery trend. In summary, no single aspect of stability was sufficient to reflect the overall stability of the system.     

Warming and oligotrophication cause shifts in freshwater phytoplankton communities

While there is a lot of data on interactive effects of eutrophication and warming, to date, we lack data to generate reliable predictions concerning possible effects of nutrient decrease and temperature increase on community composition and functional responses. In recent years, a wide‐ranging trend of nutrient decrease (re‐oligotrophication) was reported for freshwater systems. Small lakes and ponds, in particular, show rapid responses to anthropogenic pressures and became model systems to investigate single as well as synergistic effects of warming and fertilization in situ and in experiments. Therefore, we set up an experiment to investigate the single as well as the interactive effects of nutrient reduction and gradual temperature increase on a natural freshwater phytoplankton community, using an experimental indoor mesocosm setup. Biomass production initially increased with warming but decreased with nutrient depletion. If nutrient supply was constant, biomass increased further, especially under warming conditions. Under low nutrient supply, we found a sharp transition from initially positive effects of warming to negative effects when resources became scarce. Warming reduced phytoplankton richness and evenness, whereas nutrient reduction at ambient temperature had positive effects on diversity. Our results indicate that temperature effects on freshwater systems will be altered by nutrient availability. These interactive effects of energy increase and resource decrease have major impacts on biodiversity and ecosystem function and thus need to be considered in environmental management plans.     

Two Novel Human Members of an Emerging Mammalian Gene Family Related to Mono-ADP-Ribosylating Bacterial Toxins

Mono-ADP-ribosylation is one of the posttranslational protein modifications regulating cellular metabolism, e.g., nitrogen fixation, in prokaryotes. Several bacterial toxins mono-ADP-ribosylate and inactivate specific proteins in their animal hosts. Recently, two mammalian GPI-anchored cell surface enzymes with similar activities were cloned (designated ART1 and ART2). We have now identified six related expressed sequence tags (ESTs) in the public database and cloned the two novel human genes from which these are derived (designatedART3andART4). The deduced amino acid sequences of the predicted gene products show 28% sequence identity to one another and 32–41% identity vs the muscle and T cell enzymes. They contain signal peptide sequences characteristic of GPI anchorage. Southern Zoo blot analyses suggest the presence of related genes in other mammalian species. By PCR screening of somatic cell hybrids and byin situhybridization, we have mapped the two genes to human chromosomes 4p14–p15.1 and 12q13.2–q13.3. Northern blot analyses show that these genes are specifically expressed in testis and spleen, respectively. Comparison of genomic and cDNA sequences reveals a conserved exon/intron structure, with an unusually large exon encoding the predicted mature membrane proteins. Secondary structure prediction analyses indicate conserved motifs and amino acid residues consistent with a common ancestry of this emerging mammalian enzyme family and bacterial mono(ADP-ribosyl)transferases. It is possible that the four human gene family members identified so far represent the “tip of an iceberg,” i.e., a larger family of enzymes that influences the function of target proteins via mono-ADP-ribosylation.     

Methicillin-resistant Staphylococcus aureus Bacterial Nitric-oxide Synthase Affects Antibiotic Sensitivity and Skin Abscess Development

Staphylococcus aureus infections present an enormous global health concern complicated by an alarming increase in antibiotic resistance. S. aureus is among the few bacterial species that express nitric-oxide synthase (bNOS) and thus can catalyze NO production from l-arginine. Here we generate an isogenic bNOS-deficient mutant in the epidemic community-acquired methicillin-resistant S. aureus (MRSA) USA300 clone to study its contribution to virulence and antibiotic susceptibility. Loss of bNOS increased MRSA susceptibility to reactive oxygen species and host cathelicidin antimicrobial peptides, which correlated with increased MRSA killing by human neutrophils and within neutrophil extracellular traps. bNOS also promoted resistance to the pharmaceutical antibiotics that act on the cell envelope such as vancomycin and daptomycin. Surprisingly, bNOS-deficient strains gained resistance to aminoglycosides, suggesting that the role of bNOS in antibiotic susceptibility is more complex than previously observed in Bacillus species. Finally, the MRSA bNOS mutant showed reduced virulence with decreased survival and smaller abscess generation in a mouse subcutaneous infection model. Together, these data indicate that bNOS contributes to MRSA innate immune and antibiotic resistance phenotypes. Future development of specific bNOS inhibitors could be an attractive option to simultaneously reduce MRSA pathology and enhance its susceptibility to commonly used antibiotics.