Zehn Jahre PRAC – der Ausschuss für Risikobewertung im Bereich der Pharmakovigilanz

Im Juli 2022 jährte sich zum zehnten Mal das Inkrafttreten der sogenannten neuen Pharmakovigilanzgesetzgebung, die im Dezember 2010 vom Europäischen Parlament und Rat der Europäischen Union verabschiedet wurde. Nach außen sichtbarstes Zeichen war die Einrichtung des Ausschusses für Risikobewertung im Bereich der Pharmakovigilanz (Pharmacovigilance Risk Assessment Committee, PRAC) als neues wissenschaftliches Gremium der Europäischen Arzneimittelagentur EMA. Zielsetzung war es, die für alle Fragestellungen in der Pharmakovigilanz erforderliche Expertise und die notwendigen Ressourcen auf europäischer Ebene zur Verfügung zu stellen. Dies ist rückblickend sicherlich gelungen. Aus Anlass des zurückliegenden Jubiläums möchte der folgende Artikel über den PRAC und insbesondere seine aktuellen Aufgaben informieren und dabei exemplarisch das Thema Fluorchinolone beleuchten

Crystallization and preliminary characterization of a novel haem-binding protein of Streptomyces reticuli

The haem-binding protein HbpS from Streptomyces reticuli was crystallized and diffraction data were collected to a maximal resolution of 2.25 Å

Iron Binding at Specific Sites within the Octameric HbpS Protects Streptomycetes from Iron-Mediated Oxidative Stress

<p>The soil bacterium Streptomyces reticuli secretes the octameric protein HbpS that acts as a sensory component of the redox-signalling pathway HbpS-SenS-SenR. This system modulates a genetic response on iron-and haem-mediated oxidative stress. Moreover, HbpS alone provides this bacterium with a defence mechanism to the presence of high concentrations of iron ions and haem. While the protection against haem has been related to its haem-binding and haem-degrading activity, the interaction with iron has not been studied in detail. In this work, we biochemically analyzed the iron-binding activity of a set of generated HbpS mutant proteins and present evidence showing the involvement of one internal and two exposed D/EXXE motifs in binding of high quantities of ferrous iron, with the internal E78XXE81 displaying the tightest binding. We additionally show that HbpS is able to oxidize ferrous to ferric iron ions. Based on the crystal structure of both the wild-type and the mutant HbpS-D78XXD81, we conclude that the local arrangement of the side chains from the glutamates in E78XXE81 within the octameric assembly is a pre-requisite for interaction with iron. The data obtained led us to propose that the exposed and the internal motif build a highly specific route that is involved in the transport of high quantities of iron ions into the core of the HbpS octamer. Furthermore, physiological studies using Streptomyces transformants secreting either wild-type or HbpS mutant proteins and different redox-cycling compounds led us to conclude that the iron-sequestering activity of HbpS protects these soil bacteria from the hazardous side effects of peroxide-and iron-based oxidative stress.</p>

The three-component signalling system HbpS-SenS-SenR as an example of a redox sensing pathway in bacteria

The two-component system SenS-SenR and the extracellular HbpS protein of the cellulose degrader Streptomyces reticuli have been shown to act in concert as a novel system which detects redox stress. In vivo and in vitro experiments have led to the hypothesis that HbpS binds and degrades heme, communicating the extracellular presence of heme and oxidative stress to the membrane-embedded sensor histidine kinase SenS via a bound iron. The response regulator SenR would then up-regulate downstream signalling cascades, leading to the appropriate gene expression levels for bacterial survival in an oxidative environment. Sequence analysis has shown that homologs of HbpS and SenS-SenR exist in a number of ecologically and medically relevant bacterial species, suggesting the existence of a previously undescribed bacterial oxidative stress-response pathway common to both Gram-negative and Gram-positive bacteria. The presented report reviews the current knowledge of the function of this novel protein family consisting of an accessory protein and its cognate two-component system, which could be more properly described as a three-component system

Transcriptional Regulation of furA and katG upon Oxidative Stress in Mycobacterium smegmatis

The DNA region upstream of katG in Mycobacterium smegmatis was cloned and sequenced. The furA gene, highly homologous to Mycobacterium tuberculosis furA, mapped in this region. The furA-katG organization appears to be conserved among several mycobacteria. The transcription pattern of furA and katG in M. smegmatis upon oxidative stress was analyzed by Northern blotting and primer extension. Although transcription of both furA and katG was induced upon oxidative stress, transcripts covering both genes could not be identified either by Northern blotting or by reverse transcriptase PCR. Specific transcripts and 5′ ends were identified for furA and katG, respectively. By cloning M. smegmatis and M. tuberculosis DNA regions upstream of a reporter gene, we demonstrated the presence of two promoters, pfurA, located immediately upstream of the furA gene, and pkatG, located within the terminal part of the furA coding sequence. Transcription from pfurA was induced upon oxidative stress. A 23-bp sequence overlapping the pfurA −35 region is highly conserved among mycobacteria and streptomycetes and might be involved in controlling pfurA activity. Transcription from a cloned pkatG, lacking the upstream pfurA region, was not induced upon oxidative stress, suggesting a cis-acting regulatory role of this region

Applications of Structural Biology and Bioinformatics in the Investigation of Oxidative Stress-Related Processes

Reactive oxygen species (ROS)-mediated dysfunction of certain biological processes is implicated in different diseases in humans, including cardiovascular, cancer, or neurodegenerative disorders. Not only human cells and tissues are affected by ROS but also all other biological systems, including plants and microorganisms. Primary targets of ROS are proteins, lipids, and nucleic acids. Modifications of these macromolecules result mostly in the start of signalling cascades between proteins, proteins and DNA, DNA and RNA, proteins and RNA, proteins and lipids within single cell compartments, entire cells, or tissues. In this chapter, basics of tools of structural biology (i.e., X-ray crystallography, NMR, and EPR spectroscopy) as well as bioinformatics are presented. These tools are explained as well as how they can be applied in the analysis of ROS-mediated modifications within macromolecules and systems, and perspectives are discussed