Recombinant Deg/HtrA proteases from Synechocystis sp. PCC 6803 differ in substrate specificity, biochemical characteristics and mechanism

Cyanobacteria require efficient protein-quality-control mechanisms to survive under dynamic, often stressful, environmental conditions. It was reported that three serine proteases, HtrA (high temperature requirement A), HhoA (HtrA homologue A) and HhoB (HtrA homologue B), are important for survival of Synechocystis sp. PCC 6803 under high light and temperature stresses and might have redundant physiological functions. In the present paper, we show that all three proteases can degrade unfolded model substrates, but differ with respect to cleavage sites, temperature and pH optima. For recombinant HhoA, and to a lesser extent for HtrA, we observed an interesting shift in the pH optimum from slightly acidic to alkaline in the presence of Mg2+ and Ca2+ ions. All three proteases formed different homo-oligomeric complexes with and without substrate, implying mechanistic differences in comparison with each other and with the well-studied Escherichia coli orthologues DegP (degradation of periplasmic proteins P) and DegS. Deletion of the PDZ domain decreased, but did not abolish, the proteolytic activity of all three proteases, and prevented substrate-induced formation of complexes higher than trimers by HtrA and HhoA. In summary, biochemical characterization of HtrA, HhoA and HhoB lays the foundation for a better understanding of their overlapping, but not completely redundant, stress-resistance functions in Synechocystis sp. PCC 6803

RNA-Seq and metabolic flux analysis of Tetraselmis sp. M8 during nitrogen starvation reveals a two-stage lipid accumulation mechanism

To map out key lipid-related pathways that lead to rapid triacylglyceride accumulation in oleaginous microalgae, RNA-Seq was performed with Tetraselmis sp. M8 at 24 h after exhaustion of exogenous nitrogen to reveal molecular changes during early stationary phase. Further gene expression profiling by quantitative real-time PCR at 16–72 h revealed a distinct shift in expression of the fatty acid/triacylglyceride biosynthesis and β-oxidation pathways, when cells transitioned from log-phase into early-stationary and stationary phase. Metabolic reconstruction modeling combined with real-time PCR and RNA-Seq gene expression data indicates that the increased lipid accumulation is a result of a decrease in lipid catabolism during the early-stationary phase combined with increased metabolic fluxes in lipid biosynthesis during the stationary phase. During these two stages, Tetraselmis shifts from reduced lipid consumption to active lipid production. This process appears to be independent from DGAT expression, a key gene for lipid accumulation in microalgae

OSIRISv1.2: A named entity recognition system for sequence variants of genes in biomedical literature

<p>Abstract</p> <p>Background</p> <p>Single Nucleotide Polymorphisms, among other type of sequence variants, constitute key elements in genetic epidemiology and pharmacogenomics. While sequence data about genetic variation is found at databases such as dbSNP, clues about the functional and phenotypic consequences of the variations are generally found in biomedical literature. The identification of the relevant documents and the extraction of the information from them are hampered by the large size of literature databases and the lack of widely accepted standard notation for biomedical entities. Thus, automatic systems for the identification of citations of allelic variants of genes in biomedical texts are required.</p> <p>Results</p> <p>Our group has previously reported the development of OSIRIS, a system aimed at the retrieval of literature about allelic variants of genes <url>http://ibi.imim.es/osirisform.html</url>. Here we describe the development of a new version of OSIRIS (OSIRISv1.2, <url>http://ibi.imim.es/OSIRISv1.2.html</url>) which incorporates a new entity recognition module and is built on top of a local mirror of the MEDLINE collection and HgenetInfoDB: a database that collects data on human gene sequence variations. The new entity recognition module is based on a pattern-based search algorithm for the identification of variation terms in the texts and their mapping to dbSNP identifiers. The performance of OSIRISv1.2 was evaluated on a manually annotated corpus, resulting in 99% precision, 82% recall, and an F-score of 0.89. As an example, the application of the system for collecting literature citations for the allelic variants of genes related to the diseases intracranial aneurysm and breast cancer is presented.</p> <p>Conclusion</p> <p>OSIRISv1.2 can be used to link literature references to dbSNP database entries with high accuracy, and therefore is suitable for collecting current knowledge on gene sequence variations and supporting the functional annotation of variation databases. The application of OSIRISv1.2 in combination with controlled vocabularies like MeSH provides a way to identify associations of biomedical interest, such as those that relate SNPs with diseases.</p

Development of a cost-effective ovine antibody-based therapy against SARS-CoV-2 infection and contribution of antibodies specific to the spike subunit proteins.

Antibodies against SARS-CoV-2 are important to generate protective immunity, with convalescent plasma one of the first therapies approved. An alternative source of polyclonal antibodies suitable for upscaling would be more amendable to regulatory approval and widespread use. In this study, sheep were immunised with SARS-CoV-2 whole spike protein or one of the subunit proteins: S1 and S2. Once substantial antibody titres were generated, plasma was collected and samples pooled for each antigen. Non-specific antibodies were removed via affinity-purification to yield candidate products for testing in a hamster model of SARS-CoV-2 infection. Affinity-purified polyclonal antibodies to whole spike, S1 and S2 proteins were evaluated for in vitro for neutralising activity against SARS-CoV-2 Wuhan-like virus (Australia/VIC01/2020) and a recent variant of concern, B.1.1.529 BA.1 (Omicron), antibody-binding, complement fixation and phagocytosis assays were also performed. All antibody preparations demonstrated an effect against SARS-CoV-2 disease in the hamster model of challenge, with those raised against the S2 subunit providing the most promise. A rapid, cost-effective therapy for COVID-19 was developed which provides a source of highly active immunoglobulin specific to SARS-CoV-2 with multi-functional activity

Deg-Proteasen in Arabidopsis thaliana

Proteasen, auch Peptidasen genannt, sind Enzyme zur Hydrolyse von Peptidbindungen. Sie sind an vielen zellulären Prozessen beteiligt, u.a. an der Qualitätskontrolle von Proteinen, an der Aufnahme von Nährstoffen, an der posttranslationalen Modifizierung von Proteinvorstufen, und sind außerdem verantwortlich für viele zelluläre Signalübertragungsvorgänge. Deg-Proteasen (auch HtrA-Proteasen genannt) bilden eine Familie ATP-unabhängiger Serinendopeptidasen und kommen in fast jedem Organismus vor. Zusätzlich zu ihrer Proteasendomäne besitzen die meisten Deg-Proteasen ein oder mehrere PDZ-Domänen zur Protein-Protein-Interaktion. Das vollständig sequenzierte Genom des pflanzlichen Modellorganismus Arabidopsis thaliana enthält 16 Gene für Deg-Proteasen, über deren Rolle im Organismus jedoch kaum etwas bekannt ist. Die Grundlage der hier vorliegenden Arbeit sind meine Untersuchungen an vier Deg-Proteasen aus dieser Pflanze.DEG15 ist, wie wir zeigen konnten, eine peroxisomale Protease. Sie ist verantwortlich für das Prozessieren von Proteinen, welche ein PTS2 (Peroxisomal Targeting Signal 2) Signal Peptid enthalten, ein Vorgang, der nur in höheren Eukaryoten vorkommt. Durch unsere Analyse von Arabidopsis-deg15-Knock-Out-Mutanten konnten wir zeigen, daß diese Pflanzen einen Defekt in der Beta-Oxidation von Hormonvorstufen besitzen. Dies ist ein erster Hinweis auf die Wichtigkeit dieses Prozesses für den Organismus. Das entsprechende DEG15 ähnliche Protein aus Säugetieren wurde als eine neue Art von Cysteinprotease klassifiziert, doch wir konnten durch Mutagenesestudien beweisen, daß DEG15 tatsächlich eine Serinprotease ist.DEG7 gehört zu einer Gruppe von Deg-Proteasen, welche nur in Pflanzen und Pilzen vorkommen. Wir erkannten, daß dieses Protein aufgrund seiner ungewöhnlichen Domänenstruktur einen neuartigen Oligomerisierungs-Mechanismus besitzt. Außerdem konnten wir zeigen, daß DEG7 aus Arabidopsis, anders als das entsprechende Protein aus der Bäckerhefe Saccharomyces cerevisiae, nicht an der Durchführung des Programmierten Zelltods beteiligt ist. Zusätzlich identifizierten wir aus einer Arabidopsis-cDNA-Bibliothek mehrere Gene, welche für potentielle DEG7 Interaktionspartner kodieren.In dieser Arbeit konnten wir außerdem DEG9 als die erste Protease im Nukleolus identifizieren, welche keinen Teil des Ubiquitin-Proteasom-Systems darstellt. Normalerweise liegt sie in unseren in-vitro-Versuchen als Hexamer vor, doch ist ihr Oligomerisierungsgrad abhängig von dem Vorhandensein ihrer PDZ-Domäne. Arabidopsis Pflanzen, welche kein DEG9-Protein besitzen, zeigten keine phänotypischen Veränderungen gegenüber Wildtyp-Pflanzen. Gleiches gilt für Pflanzen mit einem erhöhten Level and DEG9.Frühere Arbeiten in unserem Labor haben DEG2 als ein chloroplastidäres Enzym identifizert, welches unter Lichtstress für den Abbau des D1-Proteins aus dem Photosystem II verantwortlich ist. Da diese Arbeiten auf in-vitro-Daten beruhten, haben wir untersucht, in welchem Maße der D1-Abbau in Arabidopsis-Linien ohne DEG2 beeinträchtigt ist. Da diese deg2-Knock-Out-Pflanzen im Vergleich zu Wildtyp-Pflanzen keinen Unterschied zeigten, entwickelten wir ein erweitertes Modell des D1-Abbaus und der daran beteiligten Proteasen