Analysis of the effect of potato carboxypeptidase inhibitor pro-sequence on the folding of the mature protein.

Protein folding can be modulated in vivo by many factors. While chaperones act as folding catalysts and show broad substrate specificity, some pro-peptides specifically facilitate the folding of the mature protein to which they are bound. Potato carboxypeptidase inhibitor (PCI), a 39-residue protein carboxypeptidase inhibitor, is synthesized in vivo as a precursor protein that includes a 27-residue N-terminal and a seven-residue C-terminal pro-regions. In this work the disulfide-coupled folding of mature PCI in vitro has been compared with that of the same protein extended with either the N-terminal pro-sequence (ProNtPCI) or both N- and C-terminal pro-sequences (ProPCI), and also with the N-terminal pro-sequence in trans (ProNt + PCI). No significant differences can be observed in the folding kinetics or efficiencies of all these molecules. In addition, in vivo folding studies in Escherichia coli have been performed using wild-type PCI and three PCI mutant forms with and without the N-terminal pro-sequence, the mutations had been previously reported to affect folding of the PCI mature form. The extent to which the 'native-like' form was secreted to the media by each construction was not affected by the presence of the N-terminal pro-sequence. These results indicate that PCI does not depend on the N-terminal pro-sequence for its folding in both, in vitro and in vivo in E. coli. However, structural analysis by spectroscopy, hydrogen exchange and limited proteolysis by mass spectrometry, indicate the capability of such N-terminal pro-sequence to fold within the precursor form.     

Isolation and re-association of the subunits from the pro-(carboxypeptidase A)–pro-(proteinase E) binary complex from pig pancreas

The component subunits of the pro-(carboxypeptidase A)–pro-(proteinase E) binary complex from pig pancreas were separated with a high recovery (80–95%) of their original potential activity. The isolated subunits and the reconstituted complex have properties similar to those of the corresponding natural species. The tryptic activation course of the pro-(carboxypeptidase A) subunit is substantially modified when bound to pro-(proteinase E), whereas the activation of pro-(proteinase E) is not dependent on this association.     

Architecture and function of metallopeptidase catalytic domains

The cleavage of peptide bonds by metallopeptidases (MPs) is essential for life. These ubiquitous enzymes participate in all major physiological processes, and so their deregulation leads to diseases ranging from cancer and metastasis, inflammation, and microbial infection to neurological insults and cardiovascular disorders. MPs cleave their substrates without a covalent intermediate in a single‐step reaction involving a solvent molecule, a general base/acid, and a mono‐ or dinuclear catalytic metal site. Most monometallic MPs comprise a short metal‐binding motif (HEXXH), which includes two metal‐binding histidines and a general base/acid glutamate, and they are grouped into the zincin tribe of MPs. The latter divides mainly into the gluzincin and metzincin clans. Metzincins consist of globular ～130–270‐residue catalytic domains, which are usually preceded by N‐terminal pro‐segments, typically required for folding and latency maintenance. The catalytic domains are often followed by C‐terminal domains for substrate recognition and other protein–protein interactions, anchoring to membranes, oligomerization, and compartmentalization. Metzincin catalytic domains consist of a structurally conserved N‐terminal subdomain spanning a five‐stranded β‐sheet, a backing helix, and an active‐site helix. The latter contains most of the metal‐binding motif, which is here characteristically extended to HEXXHXXGXX(H,D). Downstream C‐terminal subdomains are generally shorter, differ more among metzincins, and mainly share a conserved loop—the Met‐turn—and a C‐terminal helix. The accumulated structural data from more than 300 deposited structures of the 12 currently characterized metzincin families reviewed here provide detailed knowledge of the molecular features of their catalytic domains, help in our understanding of their working mechanisms, and form the basis for the design of novel drugs.     

The poor health of deep-water species in the context of fishing activity and a warming climate: will populations of Molva species rebuild or collapse?

Many deep-water fish populations, being K-selected species, have little resilience to overexploitation and may be at serious risk of depletion as a consequence. Sea warming represents an additional threat. In this study, the condition, or health, of several populations of common ling (Molva molva), blue ling (Molva dypterygia) and Mediterranean or Spanish ling (Molva macrophthalma) inhabiting different areas in the North Atlantic and the Mediterranean was evaluated, to shed light on the challenges these deep-water species are facing in the context of fishing activity and a warming climate. The data on the condition of Molva populations which are analysed here have been complemented with data on abundance and, for the southernmost species (Mediterranean ling), with two other health indicators (parasitism and hepato-somatic index). Despite some exceptions (e.g., common ling in Icelandic waters), this study shows that the condition of many populations of Molva species in the northeastern Atlantic and the Mediterranean Sea has worsened, a trend which, in recent decades, has usually been found to be accompanied by a decline in their abundance. In addition, the poor health status of most populations of common ling, blue ling and Mediterranean ling considered in this analysis points to a lower sustainability of these populations in the future. Overall, the health status and abundance of Molva populations in the northeastern Atlantic and the Mediterranean suggest that only some populations located in the North Atlantic may be able to rebuild, whereas the populations in southern North Atlantic and the Mediterranean, which are probably most at risk from sea warming, are facing serious difficulties in doing so. In the context of fisheries and global warming, this study's results strongly indicate that management bodies need to consider the health status of many of the populations of Molva species, particularly in southern European waters, before implementing their decisions.     

Ancient saltern metagenomics: tracking changes in microbes and their viruses from the underground to the surface

Microbial communities in hypersaline underground waters derive from ancient organisms trapped within the evaporitic salt crystals and are part of the poorly known subterranean biosphere. Here, we characterized the viral and prokaryotic assemblages present in the hypersaline springs that dissolve Triassic-Keuper evaporite rocks and feed the Añana Salt Valley (Araba/Alava, Basque Country, Spain). Four underground water samples (around 23% total salinity) with different levels of exposure to the open air were analysed by means of microscopy and metagenomics. Cells and viruses in the spring water had lower concentrations than what are normally found in hypersaline environments and seemed to be mostly inactive. Upon exposure to the open air, there was an increase in activity of both cells and viruses as well as a selection of phylotypes. The underground water was inhabited by a rich community harbouring a diverse set of genes coding for retinal binding proteins. A total of 35 viral contigs from 15 to 104 kb, representing partial or total viral genomes, were assembled and their evolutionary changes through the spring system were followed by SNP analysis and metagenomic island tracking. Overall, both the viral and the prokaryotic assemblages changed quickly upon exposure to the open air conditions.     

Affective Priming Using Facial Expressions Modulates Liking for Abstract Art

We examined the influence of affective priming on the appreciation of abstract artworks using an evaluative priming task. Facial primes (showing happiness, disgust or no emotion) were presented under brief (Stimulus Onset Asynchrony, SOA = 20ms) and extended (SOA = 300ms) conditions. Differences in aesthetic liking for abstract paintings depending on the emotion expressed in the preceding primes provided a measure of the priming effect. The results showed that, for the extended SOA, artworks were liked more when preceded by happiness primes and less when preceded by disgust primes. Facial expressions of happiness, though not of disgust, exerted similar effects in the brief SOA condition. Subjective measures and a forced-choice task revealed no evidence of prime awareness in the suboptimal condition. Our results are congruent with findings showing that the affective transfer elicited by priming biases evaluative judgments, extending previous research to the domain of aesthetic appreciation.     

Elucidating Emergence and Transmission of Multidrug-Resistant Tuberculosis in Treatment Experienced Patients by Whole Genome Sequencing

Background

Understanding the emergence and spread of multidrug-resistant tuberculosis (MDR-TB) is crucial for its control. MDR-TB in previously treated patients is generally attributed to the selection of drug resistant mutants during inadequate therapy rather than transmission of a resistant strain. Traditional genotyping methods are not sufficient to distinguish strains in populations with a high burden of tuberculosis and it has previously been difficult to assess the degree of transmission in these settings. We have used whole genome analysis to investigate M. tuberculosis strains isolated from treatment experienced patients with MDR-TB in Uganda over a period of four years.

Methods and Findings

We used high throughput genome sequencing technology to investigate small polymorphisms and large deletions in 51 Mycobacterium tuberculosis samples from 41 treatment-experienced TB patients attending a TB referral and treatment clinic in Kampala. This was a convenience sample representing 69% of MDR-TB cases identified over the four year period. Low polymorphism was observed in longitudinal samples from individual patients (2-15 SNPs). Clusters of samples with less than 50 SNPs variation were examined. Three clusters comprising a total of 8 patients were found with almost identical genetic profiles, including mutations predictive for resistance to rifampicin and isoniazid, suggesting transmission of MDR-TB. Two patients with previous drug susceptible disease were found to have acquired MDR strains, one of which shared its genotype with an isolate from another patient in the cohort.

Conclusions

Whole genome sequence analysis identified MDR-TB strains that were shared by more than one patient. The transmission of multidrug-resistant disease in this cohort of retreatment patients emphasises the importance of early detection and need for infection control. Consideration should be given to rapid testing for drug resistance in patients undergoing treatment to monitor the emergence of resistance and permit early intervention to avoid onward transmission.     

Mixtures with grass litter may hasten shrub litter decomposition after shrub encroachment into mountain grasslands

Aims

Shrub encroachment in mesic grasslands alters the identity and quality of litters entering the system. As litter from shrubs and grasses can differ in their quality, this can lead to differences in litter decomposition by the direct effect of quality, but also to litter interaction during decomposition. The objective of this study was to examine the occurrence of non-additive effects of litter mixtures on the decomposition rates of legume shrub litter (poor in P) or conifer shrub litter (poor in N) and grass litter.

Methods

In addition to single litter type litterbags for the three species, we mixed litters of each pair of possible combinations to determine the influence of each species on mass loss. Litterbags were placed in the field and collected after 1, 6, 8, 12 and 24 months. In each collection, litter of each species remaining in mixed bags was separated, dry weighed and analyzed for C, N and P.

Results

With respect to shrub litter decomposing alone, mass loss of shrub litter when mixed with grass showed a 9–10 % increase in decomposition rate for conifer and a 3 % increase for legume litter. These litter mixture effects varied with time and they were detected after a decomposition period of 1 year in legume litter and of 2 years in conifer litter.

Conclusions

Grass litter hastened conifer and legume litter decomposition in leaf litter mixtures, at least during the first stages of the process. The potential consequences of this result to alter litter accumulation patterns and thus carbon sequestration rates after shrub encroachment into grasslands will depend on whether the observed trends are maintained in the advanced decomposition stages.