BackgroundMyocardial infarction (MI) is a major cause of heart failure. The carboxypeptidase cathepsin A is a novel target in the treatment of cardiac failure. We aim to show that recently developed inhibitors of the protease cathepsin A attenuate post-MI heart failure.MethodsMice were subjected to permanent left anterior descending artery (LAD) ligation or sham operation. 24 h post–surgery, LAD-ligated animals were treated with daily doses of the cathepsin A inhibitor SAR1 or placebo. After 4 weeks, the three groups (sham, MI-placebo, MI-SAR1) were evaluated.ResultsCompared to sham-operated animals, placebo-treated mice showed significantly impaired cardiac function and increased plasma BNP levels. Cathepsin A inhibition prevented the increase of plasma BNP levels and displayed a trend towards improved cardiac functionality. Proteomic profiling was performed for the three groups (sham, MI-placebo, MI-SAR1). More than 100 proteins were significantly altered in placebo-treated LAD ligation compared to the sham operation, including known markers of cardiac failure as well as extracellular/matricellular proteins. This ensemble constitutes a proteome fingerprint of myocardial infarction induced by LAD ligation in mice. Cathepsin A inhibitor treatment normalized the marked increase of the muscle stress marker CA3 as well as of Igγ 2b and fatty acid synthase. For numerous further proteins, cathepsin A inhibition partially dampened the LAD ligation-induced proteome alterations.ConclusionsOur proteomic and functional data suggest that cathepsin A inhibition has cardioprotective properties and support a beneficial effect of cathepsin A inhibition in the treatment of heart failure after myocardial infarction.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0907-8) contains supplementary material, which is available to authorized users.

The human fungal pathogen Candida albicans is constantly exposed to environmental challenges impacting the cell wall. Signaling pathways coordinate stress adaptation and are essential for commensalism and virulence. The transcription factors Sko1, Cas5, and Rlm1 control the response to cell wall stress caused by the antifungal drug caspofungin. Here, we expand the Sko1 and Rlm1 transcriptional circuit and demonstrate that Rlm1 activates Sko1 cell wall stress signaling. Caspofungin-induced transcription of SKO1 and several Sko1dependent cell wall integrity genes are attenuated in an rlm1Δ/Δ mutant strain when compared to the treated wild-type strain but not in a cas5Δ/Δ mutant strain. Genome-wide chromatin immunoprecipitation (ChIP-seq) results revealed numerous Sko1 and Rlm1 directly bound target genes in the presence of caspofungin that were undetected in previous gene expression studies. Notable targets include genes involved in cell wall integrity, osmolarity, and cellular aggregation, as well as several uncharacterized genes. Interestingly, we found that Rlm1 does not bind to the upstream intergenic region of SKO1 in the presence of caspofungin, indicating that Rlm1 indirectly controls caspofungin-induced SKO1 transcription. In addition, we discovered that caspofungin-induced SKO1 transcription occurs through selfactivation. Based on our ChIP-seq data, we also discovered an Rlm1 consensus motif unique to C. albicans. For Sko1, we found a consensus motif similar to the known Sko1 motif for Saccharomyces cerevisiae. Growth assays showed that SKO1 overexpression suppressed caspofungin hypersensitivity in an rlm1Δ/Δ mutant strain. In addition, overexpression of the glycerol phosphatase, RHR2, suppressed caspofungin hypersensitivity specifically in a sko1Δ/Δ mutant strain. Our findings link the Sko1 and Rlm1 signaling pathways, identify new biological roles for Sko1 and Rlm1, and highlight the complex dynamics underlying cell wall signaling.

Nicotinic acetylcholine receptors (nAChRs) are neuromuscular proteins responsible for muscle contraction upon binding with chemical stimulant acetylcholine (ACh). The α-neurotoxins of snake mimic the structure of ACh and attacks nAChRs, which block the flow of ACh and leads to numbness and paralysis. The toxin-binding site of alpha subunit in the nAChRs is highly conserved throughout chordate lineages with few exceptions in resistance organisms. In this study, we have analyzed the sequence and structures of toxin-binding/resistant nAChRs and their interaction stability with toxins through molecular docking and molecular dynamics simulation (MDS). We have reported the potential glycosylation residues within the toxin-binding cleft adding sugar moieties through N-linked glycosylation in resistant organisms. Residue variations at key positions alter the secondary structure of binding cleft, which might interfere with toxin binding and it could be one of the possible explanations for the resistance to snake venoms. Analysis of nAChR-α-neurotoxin complexes has confirmed the key interacting residues. In addition, drastic variation in the binding stability of Mongoose nAChR-α-Bungarotoxin (α-BTX) and human nAChR-α-BTX complexes were found at specific phase of MDS. Our findings suggest that specific mutations in the binding site of toxin are potentially preventing the formation of stable complex of receptor-toxin, which might lead to mechanism of resistance. This in silico study on the binding cleft of nAChR and the findings of interacting residues will assist in designing potential inhibitors as therapeutic targets.

BackgroundIn quantitative proteomics, peptide mapping is a valuable approach to combine positional quantitative information with topographical and domain information of proteins. Quantitative proteomic analysis of cell surface shedding is an exemplary application area of this approach.ResultsWe developed ImproViser ( http://www.improviser.uni-freiburg.de) for fully automated peptide mapping of quantitative proteomics data in the protXML data. The tool generates sortable and graphically annotated output, which can be easily shared with further users. As an exemplary application, we show its usage in the proteomic analysis of regulated intramembrane proteolysis.ConclusionImproViser is the first tool to enable automated peptide mapping of the widely-used protXML format.

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Mycobacterium tuberculosis , the primary causative agent of tuberculosis, kills more humans than any other infectious bacterium. Yet 40% of its genome is functionally uncharacterized, leaving much about the genetic basis of its resistance to antibiotics, capacity to withstand host immunity, and basic metabolism yet undiscovered.

Each decade, billions are invested in Tuberculosis (TB) research to further characterize M. tuberculosis pathogenesis. Despite this investment, nearly half of the 4,031 M. tuberculosis protein-coding genes lack descriptive annotation in community databases, due largely to incomplete reconciliation with the literature and a lack of structure-based methods for functional inference. We coin the term "hypotheticome" as the set of genes in an organism without known function. For M. tuberculosis' hypotheticome, we compiled the set of genes lacking functional assignment in the most frequently used Mycobacteria annotation database through systematic, exhaustive manual literature curation and 3Dprotein structure-based inference, and reconciled these annotations with frequented functional databases, creating a comprehensive M. tuberculosis functional knowledge-base. In doing so, we also introduce standard usage of qualifying adjectives based on quantitative measures of certainty with the hope that this approach is adopted in choosing qualifiers for future functional assignments.Through these methods we functionally annotated 41.3% of the M. tuberculosis hypotheticome, and provide insight into its pathogenesis, antibiotic-resistance, and virulence. Processes implicated in the unique lifestyle of M. tuberculosis of long-term persistence and obligate pathogenesis in genotoxic host microenvironmentslipid metabolism, polyketide biosynthesis, and membrane transport and effluxwere overrepresented in our annotation. Our structural similarity approach unturned proteins that appear critical in host-interaction through apparent host mimicry, particularly involving the phagosome and vesicle-mediated transport, as well as putative structural analogs for highly mutable protein classes, including dozens of PE/PPE family proteins which are major players at the host-pathogen interface, and sixteen potential efflux pumps which are integral to M. tuberculosis drug tolerance. Hypotheses drawn from these proteins' function may help characterize the onset of latency and identify therapeutic targets. A unified annotation is essential for clear communication about M. tuberculosis. These improvements provide the most comprehensive M. tuberculosis genome annotation to date, and the approach presented can be applied to systematically annotate the genome of other organisms. We provide our novel annotations in General Feature Format with Enzyme Commission and Gene Ontology terms for integration into existing annotation frameworks.

De novo assembly has become commonplace for microbial organisms, increasing the demand for reliable genome annotation. Ab initio annotation is not an ideal approach for closely related strains due to suboptimal matching of the short or hypervariable genomic features that reference-based annotation transfer can overcome through identification of conserved synteny. At the same time, reference-based annotation methods leave gaps in the annotation where structural variations introduce unique sequence. We present Hybran, a hybrid reference-based and ab initio prokaryotic genome annotation pipeline that transfers features from a curated reference annotation and supplements unannotated regions with ab initio predictions. It builds on existing tools to create initial annotations using both approaches, then compares and resolves them to produce the hybrid annotation. With this pipeline, full advantage is taken of the community's experimental efforts on reference strains to propagate as many known features as possible without sacrificing best-effort ab initio predictions for the remaining unannotated loci. Genome annotation performed in this way can facilitate comparative genomics and the investigation of evolutionary dynamics in microbial populations. Hybran is freely available at https://lpcdrp.gitlab.io/hybran