Nachweis Estrogen-induzierter Genexpression in Fischen und Hepatycytenprimärkulturen als Marker für endokrin wirksame Substanzen in der Umwelt

Vitellogenin und Estrogenrezeptoren stehen in der Leber von Fischen unter estrogener Kontrolle und werden in männlichen Individuen nur gering exprimiert. Durch exogene Applikation Estrogen-wirksamer Stoffe wird jedoch auch bei Männchen eine Expression induziert, so dass beide Gene als Marker für estrogene Wirkungen dienen können. Unter dieser Voraussetzung wurde in der vorliegenden Dissertation ein Nachweissystem für die Expression beider Gene in Hepatocytenkulturen aus der Regenbogenforelle entwickelt. In einem ersten Bewertungsansatz wurde das estrogene Potential ausgewählter Xenoestrogene erfasst. Ebenso konnte in einer Stichprobe bei 6 Abwässern aus dem Schweizer Mittelland die Anwendbarkeit der Methode auf komplexe Lösungen nachgewiesen werden. Für eine grundlegende Beurteilung des estrogenen Potentials einer Substanz sind jedoch In vivo-Untersuchungen unerlässlich, da nur die Belastung eines intakten Organismus Aufschluss über Bioakkumulation, metabolische Umsetzung und Exkretion gibt. Zur Etablierung geeigneter Nachweissysteme zur Expression Estrogen-regulierter Gene in den beiden Modellfischarten Medaka und Zebrabärbling wurde zunächst Sequenzinformation über die cDNAs von Vitellogeninen beider Fischarten sowie Estrogenrezeptor des Zebrabärblings ermittelt. Mit Hilfe dieser Daten wurden auf semiquantitativer RT-PCR basierende Nachweissysteme für diese Genprodukte sowie für Estrogenreptor und Choriogenin H des Medakas bzw. ZP2 des Zebrabärblings entwickelt. Anschließend erfolgten vergleichende Studien zur zeitlichen und dosisabhängigen Expression dieser Gene nach Exposition verschiedener Xenoestrogene. Bei der Auswertung der Ergebnisse waren distinkte Unterschiede in der Empfindlichkeit beider Fischarten für die einzelnen Modellsubstanzen festzustellen. Für die Bewertung der Emission endokrin wirksamer Substanzen in das Freiland ergibt sich hieraus die Konsequenz, dass signifikante Artunterschiede in der estrogenen Sensibilität zu berücksichtigen sind

Peroxisomal ACBD4 interacts with VAPB and promotes ER-peroxisome associations

Cooperation between cellular organelles such as mitochondria, peroxisomes and the ER is essential for a variety of important and diverse metabolic processes. Effective communication and metabolite exchange requires physical linkages between the organelles, predominantly in the form of organelle contact sites. At such contact sites organelle membranes are brought into close proximity by the action of molecular tethers, which often consist of specific protein pairs anchored in the membrane of the opposing organelles. Currently numerous tethering components have been identified which link the ER with multiple other organelles but knowledge of the factors linking the ER with peroxisomes is limited. Peroxisome-ER interplay is important because it is required for the biosynthesis of unsaturated fatty acids, ether-phospholipids and sterols with defects in these functions leading to severe diseases. Here, we characterize acyl-CoA binding domain protein 4 (ACBD4) as a tail-anchored peroxisomal membrane protein which interacts with the ER protein, vesicle-associated membrane protein-associated protein–B (VAPB) to promote peroxisome-ER associations

Dual role of USP 30 in controlling basal pexophagy and mitophagy

USP 30 is an integral protein of the outer mitochondrial membrane that counteracts PINK 1 and Parkin‐dependent mitophagy following acute mitochondrial depolarisation. Here, we use two distinct mitophagy reporter systems to reveal tonic suppression by USP 30, of a PINK 1‐dependent component of basal mitophagy in cells lacking detectable Parkin. We propose that USP 30 acts upstream of PINK 1 through modulation of PINK 1‐substrate availability and thereby determines the potential for mitophagy initiation. We further show that a fraction of endogenous USP 30 is independently targeted to peroxisomes where it regulates basal pexophagy in a PINK 1‐ and Parkin‐independent manner. Thus, we reveal a critical role of USP 30 in the clearance of the two major sources of ROS in mammalian cells and in the regulation of both a PINK 1‐dependent and a PINK 1‐independent selective autophagy pathway

Mice with a deficiency in Peroxisomal Membrane Protein 4 (PXMP4) display mild changes in hepatic lipid metabolism

Peroxisomes play an important role in the metabolism of a variety of biomolecules, including lipids and bile acids. Peroxisomal Membrane Protein 4 (PXMP4) is a ubiquitously expressed peroxisomal membrane protein that is transcriptionally regulated by peroxisome proliferator-activated receptor α (PPARα), but its function is still unknown. To investigate the physiological function of PXMP4, we generated a Pxmp4 knockout (Pxmp4(−/−)) mouse model using CRISPR/Cas9-mediated gene editing. Peroxisome function was studied under standard chow-fed conditions and after stimulation of peroxisomal activity using the PPARα ligand fenofibrate or by using phytol, a metabolite of chlorophyll that undergoes peroxisomal oxidation. Pxmp4(−/−) mice were viable, fertile, and displayed no changes in peroxisome numbers or morphology under standard conditions. Also, no differences were observed in the plasma levels of products from major peroxisomal pathways, including very long-chain fatty acids (VLCFAs), bile acids (BAs), and BA intermediates di- and trihydroxycholestanoic acid. Although elevated levels of the phytol metabolites phytanic and pristanic acid in Pxmp4(−/−) mice pointed towards an impairment in peroxisomal α-oxidation capacity, treatment of Pxmp4(−/−) mice with a phytol-enriched diet did not further increase phytanic/pristanic acid levels. Finally, lipidomic analysis revealed that loss of Pxmp4 decreased hepatic levels of the alkyldiacylglycerol class of neutral ether lipids, particularly those containing polyunsaturated fatty acids. Together, our data show that while PXMP4 is not critical for overall peroxisome function under the conditions tested, it may have a role in the metabolism of (ether)lipids

ACBD5 and VAPB mediate membrane associations between peroxisomes and the ER

This is the final version of the article. Available from the publisher via the DOI in this record.Peroxisomes (POs) and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism and form tight structural associations, which were first observed in ultrastructural studies decades ago. PO–ER associations have been suggested to impact on a diverse number of physiological processes, including lipid metabolism, phospholipid exchange, metabolite transport, signaling, and PO biogenesis. Despite their fundamental importance to cell metabolism, the mechanisms by which regions of the ER become tethered to POs are unknown, in particular in mammalian cells. Here, we identify the PO membrane protein acyl-coenzyme A–binding domain protein 5 (ACBD5) as a binding partner for the resident ER protein vesicle-associated membrane protein-associated protein B (VAPB). We show that ACBD5–VAPB interaction regulates PO–ER associations. Moreover, we demonstrate that loss of PO–ER association perturbs PO membrane expansion and increases PO movement. Our findings reveal the first molecular mechanism for establishing PO–ER associations in mammalian cells and report a new function for ACBD5 in PO–ER tethering.This work was supported by grants from the Biotechnology and Biological Sciences Research Council (BB/K006231/1 and BB/ N01541X/1 to M. Schrader). J. Metz and M. Schrader are supported by a Wellcome Trust Institutional Strategic Support Award (WT097835MF and WT105618MA) and L.F. Godinho by a fellowship from Fundação para a Ciência e a Tecnologia, Portugal (SFRH/ BPD/90084/2012). M. Schrader and A.S. Azadi are supported by Marie Curie Initial Training Network action PerFuMe (316723). M. Islinger is supported by MEAMEDMA Anschubförderung, Medical Faculty Mannheim, University of Heidelberg

ACBD5 and VAPB mediate membrane associations between peroxisomes and the ER

Peroxisomes (POs) and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism and form tight structural associations, which were first observed in ultrastructural studies decades ago. PO–ER associations have been suggested to impact on a diverse number of physiological processes, including lipid metabolism, phospholipid exchange, metabolite transport, signaling, and PO biogenesis. Despite their fundamental importance to cell metabolism, the mechanisms by which regions of the ER become tethered to POs are unknown, in particular in mammalian cells. Here, we identify the PO membrane protein acyl-coenzyme A–binding domain protein 5 (ACBD5) as a binding partner for the resident ER protein vesicle-associated membrane protein-associated protein B (VAPB). We show that ACBD5–VAPB interaction regulates PO–ER associations. Moreover, we demonstrate that loss of PO–ER association perturbs PO membrane expansion and increases PO movement. Our findings reveal the first molecular mechanism for establishing PO–ER associations in mammalian cells and report a new function for ACBD5 in PO–ER tethering

Predicting the targeting of tail-anchored proteins to subcellular compartments in mammalian cells

Tail-anchored (TA) proteins contain a single transmembrane domain (TMD) at the C-terminus that anchors them to the membranes of organelles where they mediate critical cellular processes. Accordingly, mutations in genes encoding TA proteins have been identified in a number of severe inherited disorders. Despite the importance of correctly targeting a TA protein to its appropriate membrane, the mechanisms and signals involved are not fully understood. In this study, we identify additional peroxisomal TA proteins, discover more proteins that are present on multiple organelles, and reveal that a combination of TMD hydrophobicity and tail charge determines targeting to distinct organelle locations in mammals. Specifically, an increase in tail charge can override a hydrophobic TMD signal and re-direct a protein from the ER to peroxisomes or mitochondria and vice versa. We show that subtle changes in those parameters can shift TA proteins between organelles, explaining why peroxisomes and mitochondria have many of the same TA proteins. This enabled us to associate characteristic physicochemical parameters in TA proteins with particular organelle groups. Using this classification allowed successful prediction of the location of uncharacterized TA proteins for the first time

Penetration of Linezolid into Soft Tissues of Healthy Volunteers after Single and Multiple Doses

The present study tested the ability of linezolid to penetrate soft tissues in healthy volunteers. Ten healthy volunteers were subjected to linezolid drug intake at a dose of 600 mg twice a day for 3 to 5 days. The first dose was administered intravenously. All following doses were self-administered orally. The tissue penetration of linezolid was assessed by use of in vivo microdialysis. In the single-dose experiments the ratios of the area under the concentration-time curve from 0 to 8 h (AUC(0-8)) for tissue to the AUC(0-8) for free plasma were 1.4 ± 0.3 (mean ± standard deviation) and 1.3 ± 0.4 for subcutaneous adipose and muscle tissue, respectively. After multiple doses, the corresponding mean ratios were 0.9 ± 0.2 and 1.0 ± 0.5, respectively. The ratios of the AUC from 0 to 24 h (AUC(0-24)) for free linezolid in tissues to the MIC were between 50 and 100 for target pathogens with MICs between 2 and 4 mg/liter. In conclusion, the present study showed that linezolid penetrates rapidly into the interstitial space fluid of subcutaneous adipose and skeletal muscle tissues in healthy volunteers. On the basis of pharmacokinetic-pharmacodynamic calculations, we suggest that linezolid concentrations in soft tissues can be considered sufficient to inhibit the growth of many clinically relevant bacteria