Regulation des Gehaltes der osmoprotektiven Substanz Glucosylglycerol im Cyanobakterium Synechocystis sp. PCC 6803

Das Cyanobakterium Synechocystis sp. PCC 6803 kann sowohl in Süßwasser- als auch in Seewasserhabitaten wachsen. Dabei ist unter hyperosmotischen Bedingungen die Akkumulation von Glucosylglycerol (GG) als osmoprotektive Substanz essentiell. Das Heterosid kann durch Aufnahme oder de novo-Synthese in hohen Konzentrationen im Cytoplasma akkumuliert werden, wobei es die Zelle vor einem Wasserverlust schützt, ohne Stoffwechselfunktionen negativ zu beeinflussen. Die Menge des akkumulierten GG entspricht in akklimatisierten Zellen der externen osmotischen Belastung und wird durch die Aktivität des Schlüsselenzyms der GG-Synthese (GgpS) reguliert. Als Regulator des GgpS-Proteingehaltes konnte durch Anwendung einer Transposomutagenese eine FtsH-Protease identifiziert werden, die für den Abbau von inaktivem GgpS-Protein zuständig ist. Dadurch konnte die Verbindung zwischen Protein-Qualitätskontrolle und Osmoregulation in Cyanobakterien erstmals aufgezeigt und auf der molekularen Ebene analysiert werden. Darüber hinaus wurde in der vorliegenden Arbeit ein neuer Mechanismus charakterisiert, der eine stabile, salzabhängige GG-Syntheserate sicherstellt. Dabei ist die biochemische Modulation der Aktivität des GgpS-Enzyms von entscheidender Bedeutung. Mittels in vitro-Enzymtests konnte gezeigt werden, dass die Aktivität des gereinigten GgpS-Proteins durch hohe Konzentrationen von GG, wie sie auch in der salzakklimatisierten Zelle vorliegen, und anderer osmoprotektiver Substanzen gehemmt werden konnte. Dies ließ sich auch unter in vivo-Bedingungen durch den hemmenden Effekt der Akkumulation von Trehalose durch Aufnahme bzw. de novo-Synthese nachweisen. Die durch erhöhte GG-Konzentrationen bedingte Hemmung der GgpS-Aktivität konnte durch steigende NaCl-Konzentrationen moduliert werden. Auf der Grundlage der erhaltenen Ergebnisse wurde ein Modell erstellt, in dem die GG-Syntheserate der externen Salzkonzentration angepasst und eine energetisch ungünstige Überproduktion an GG vermieden werden kann. Dabei stellt das GgpS-Protein zugleich Osmosensor und Osmoregulator dar

Mucus detachment by host metalloprotease meprin \beta requires shedding of its inactive pro-form, which is abrogated by the pathogenic protease RgpB

The host metalloprotease meprin β is required for mucin 2 (MUC2) cleavage, which drives intestinal mucus detachment and prevents bacterial overgrowth. To gain access to the cleavage site in MUC2, meprin β must be proteolytically shed from epithelial cells. Hence, regulation of meprin β shedding and activation is important for physiological and pathophysiological conditions. Here, we demonstrate that meprin β activation and shedding are mutually exclusive events. Employing ex vivo small intestinal organoid and cell culture experiments, we found that ADAM-mediated shedding is restricted to the inactive pro-form of meprin β and is completely inhibited upon its conversion to the active form at the cell surface. This strict regulation of meprin β activity can be overridden by pathogens, as demonstrated for the bacterial protease Arg-gingipain (RgpB). This secreted cysteine protease potently converts membrane-bound meprin β into its active form, impairing meprin β shedding and its function as a mucus-detaching protease

A Short Peptide Inhibitor as an Activity-Based Probe for Matriptase-2

Matriptase-2 is a type II transmembrane serine protease and a key regulator of systemic iron homeostasis. Since the activation mechanism and several features of the physiological role of matriptase-2 are not fully understood, there is strong need for analytical tools to perform tasks such as distinguishing active and inactive matriptase-2. For this purpose we present a short biotinylated peptide derivative with a chloromethyl ketone group, biotin-RQRR-CMK, as an activity-based probe for matriptase-2. Biotin-RQRR-CMK was kinetically characterized and exhibited a second-order rate constant of inactivation (kinac/Ki) of 10,800 M−1 s−1 towards the matriptase-2 activity in the supernatant of transfected human embryonic kidney (HEK) cells. Biotin-RQRR-CMK was able to label active matriptase-2, as visualized in western blot experiments. Pretreatment with aprotinin, an active-site directed inhibitor of serine proteases, protected matriptase-2 from the reaction with biotin-RQRR-CMK

Osmotic stress in Synechocystis sp. PCC 6803: low tolerance towards nonionic osmotic stress results from lacking activation of glucosylglycerol accumulation

Marin K, Stirnberg M, Eisenhut M, Krämer R, Hagemann M. Osmotic stress in Synechocystis sp. PCC 6803: low tolerance towards nonionic osmotic stress results from lacking activation of glucosylglycerol accumulation. Microbiology. 2006;152(7):2023-2030.In order to compare the molecular principles of the acclimatization of bacterial cells to salt and nonionic osmotic stress, the moderately halotolerant cyanobacteriumSynechocystissp. PCC 6803 was challenged by salt (NaCl), and the osmolytes sorbitol and maltose. The physiological response towards each of the three compounds was found to be different. After salt addition, the cell volume remained unchanged, and the accumulation of the osmoprotective compound glucosylglycerol (GG) was observed after activation of the key enzyme GgpS at the biochemical and gene (ggpS) expression level. Sorbitol addition had only minor effects on the cell volume. In spite of the fact that theggpSexpression was increased, the GgpS enzyme was not activated, resulting in the absence of GG accumulation. In contrast the cells accumulated sorbitol, which served as a compatible solute and assured a certain osmotic resistance. In comparison to NaCl and sorbitol, the addition of maltose caused a strong decrease in cell volume indicating water efflux. However, no osmolyte accumulation was observed, resulting in an osmosensitive phenotype. Consequently, a successful response ofSynechocystiscells to an osmotic challenge is indicative of thede novosynthesis of GG upon salt-dependent activation of the GgpS enzyme or the uptake of external solutes

3,1-Benzothiazines, 1,4-benzodioxines, 1,4-benzoxazines as inhibitors of matriptase-2

The liver enzyme matriptase-2 is a multi-domain, transmembrane serine protease with an extracellular, C-terminal catalytic domain. Synthetic low-molecular weight inhibitors of matriptase-2 have potential as therapeutics to treat iron overload syndromes, in particular in patients with β-thalassemia. A sub-library of 64 compounds was screened for matriptase-2 inhibition and several active compounds were identified. (S)-Ethyl 2-(benzyl(3-((4-carbamidoylphenoxy)methyl)-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)amino)-2-oxoacetate ((S)-12) showed an IC$_{50}$ value of less than 10 µM. Structure-activity relationships were discussed and proposals to design new matriptase-2 inhibitors were made

3,1-Benzothiazines, 1,4-Benzodioxines and 1,4-Benzoxazines as Inhibitors of Matriptase-2: Outcome of a Focused Screening Approach

The liver enzyme matriptase-2 is a multi-domain, transmembrane serine protease with an extracellular, C-terminal catalytic domain. Synthetic low-molecular weight inhibitors of matriptase-2 have potential as therapeutics to treat iron overload syndromes, in particular in patients with β-thalassemia. A sub-library of 64 compounds was screened for matriptase-2 inhibition and several active compounds were identified. (S)-Ethyl 2-(benzyl(3-((4-carbamidoylphenoxy)methyl)-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)amino)-2-oxoacetate ((S)-12) showed an IC50 value of less than 10 µM. Structure-activity relationships were discussed and proposals to design new matriptase-2 inhibitors were made

A Novel Mechanism of Osmosensing, a Salt-dependent Protein-Nucleic Acid Interaction in the Cyanobacterium Synechocystis Species PCC 6803

The de novo synthesis of compatible solutes is an essential part of the cellular osmotic stress response. Upon an osmotic challenge, it is regulated by the immediate biochemical activation of preformed enzymes and by activation of gene expression. Whereas the transcriptional response has been investigated intensively, the mechanisms by which enzymes are activated in osmotic stress situations are still elusive. Here, we address this topic for the moderately halotolerant cyanobacterium Synechocystis sp. PCC 6803, which synthesizes glucosylglycerol as a compatible solute. The key enzyme of the glucosylglycerol pathway (GgpS) is inhibited by nucleic acids in a sequence- and length-independent manner. The protein binds DNA, RNA, and heparin via a salt-dependent electrostatic interaction with the negatively charged backbone of the polyanions. Mechanistically, DNA binding to the enzyme causes noncompetitive inhibition of GgpS activity. The interaction of the enzyme and nucleic acids under in vivo conditions is indicated by the co-purification of both after cross-linking in Synechocystis cells. We propose a novel mechanism of activity regulation by the nonspecific salt-dependent binding of an enzyme to nucleic acids