Kodierungsvielfalt der Phospholipid-Hydroperoxid Glutathion Peroxidase Untersuchungen zur Expressionsregulation des Enzyms in tierischen Geweben

Selenhaltige Glutathion-Peroxidasen bilden eine Familie antioxidativer Enzyme, die in der Lage sind organische und anorganische Hydroperoxide in die entsprechenden Hydroxide zu reduzieren. Unter den beschriebenen Glutathion-Peroxidasen nimmt die Phospholipid-Hydroperoxid Glutathion-Peroxidase (phGPx) eine besondere Rolle ein, da sie als einziger Vertreter in der Lage ist, neben freien Hydroperoxiden auch komplexe Esterlipid-Hydroperoxide zu reduzieren. Durch die Nutzung alternativer Startkodons im Exon 1 kann die phGPx als mitochondriale oder zytosolische Form exprimiert werden. Ein alternatives Exon 1 im phGPx-Gen definiert eine weitere Glutathion-Peroxidase (snGPx), die zuerst in den Kernen der späten Spermatiden identifiziert wurde. Bei einer detaillierten Untersuchung der Gewebeverteilung der snGPx im Rahmen dieser Arbeit konnte diese Isoform außer im Hoden auch in der Niere nachgewiesen werden. Hier wurde sie im Zytosol interstitieller Nierenzellen gefunden und belegt erstmals eine somatische Expression der snGPx. Während die phGPx in vielen Geweben exprimiert wird, wurde die snGPx nur im Hoden, in der Niere und in humanen embryonalen Nierenzellen detektiert. Um mögliche Erklärungen für diese gewebespezifische Expressionsregulation zu finden, wurden im Rahmen dieser Arbeit die potentiellen Promotorregionen für die phGPx und snGPx untersucht. Für die Bildung der phGPx-Isoformen wurde die Hauptpromotoraktivität im Exon 1a nachgewiesen. Dagegen zeigt der potentielle Promotor (Intron 1a) für die snGPx keine eigenständige Aktivität. Es konnte jedoch ein deutlich hemmender Einfluss auf die Aktivität des phGPx-Promotors nachgewiesen werden. Diese Ergebnisse weisen auf die Existenz eines gemeinsamen ph/snGPx-Promotors hin, der durch negativ regulatorische Sequenzen im Intron 1a beeinflusst werden kann. DNase-Footprint Analyse führte zur Identifizierung von DNA/Protein-Kontaktpunkten im Intron 1a und im alternativen Exon 1 der snGPx-Isoform. Es wurde eine Bindung der trans-regulatorischen Proteine SP1, EGR1, WT1, SREBP1 und USF1 sowie von Proteinen der CREB- und GATA-Familie nachgewiesen. Des weiteren konnte die in vivo-Bindung von EGR1 und SREBP1 durch Chromatin Immunopräzipitation belegt werden. Neben dem murinen ph/snGPx-Gen wurden zwei prozessierte phGPx-Pseudogene identifiziert. Für das Pseudogen 1, welches im Vergleich zur phGPx-cDNA nur zwei stumme Punktmutationen enthält, konnte eine gewebespezifische Expression nachgewiesen werden. Dieses Ergebnis erweitert die ohnehin schon große Transkriptionsvielfalt des ph/snGPx-Gens

Systemic deficiency of mouse arachidonate 15‐lipoxygenase induces defective erythropoiesis and transgenic expression of the human enzyme rescues this phenotype

Arachidonic acid 15-lipoxygenases (ALOX15) are lipid peroxidizing enzymes, which has previously been implicated in the maturational breakdown of intracellular organelles and plasma membrane remodeling during reticulocyte-erythrocyte transition. Conventional Alox15−/− mice are viable, develop normally but do not exhibit a major defective erythropoietic phenotype. To characterize the putative in vivo relevance of Alox15 for red blood cell development, we explored the impact of systemic inactivation of the Alox15 gene on mouse erythropoiesis. We found that Alox15−/− mice exhibited reduced erythrocyte counts, elevated reticulocyte counts and red cell hyperchromia. The structure of the plasma membrane of Alox15−/− erythrocytes is altered and a significant share of the red cells was present as echinocytes and/or acanthocytes. An increased share of the Alox15−/− erythrocytes cells were annexin V positive, which indicates a loss of plasma membrane asymmetry. Erythrocytes of Alox15−/− mice were more susceptible to osmotic hemolysis and exhibited a reduced ex vivo life span. When we transgenically expressed human ALOX15 in Alox15−/− mice under the control of the aP2 promoter the defective erythropoietic system was rescued and the impaired osmotic resistance was normalized. Together these data suggest the involvement Alox15 in the maturational remodeling of the plasma membrane during red cell development

Open-Access-Publikationen mit OS-APS medienneutral und mit automatisiertem Corporate Design erstellen: Anforderungserhebung, Schlussfolgerungen für den Publikationsworkflow und Stand der Umsetzung

Die Universitätsbibliothek Erlangen-Nürnberg, die Universitäts- und Landesbibliothek Sachsen-Anhalt und das Technologieunternehmen SciFlow in Berlin entwickeln gemeinsam die „Open Source Academic Publishing Suite (OS-APS)“, die es Verlagen ermöglichen soll, E-Books einfach und automatisiert ihren Formatvorgaben entsprechend zu erstellen. OS-APS soll einen XML-basierten Single-Source-Publishing-Workflow ermöglichen. Die Software wird Import-, Editier- und Exportfunktionen integrieren, sodass die in Verlagen üblichen Manuskriptformate (z.B. DOCX oder ODT) mit wenigen Mausklicks importiert, analysiert und in das gewünschte Format (z.B. EPUB, PDF, HTML oder XML) im Corporate Design des Verlags umgewandelt werden können. Darüber hinaus werden die Publikationsplattformen Open Journal Systems (OJS) und Open Monograph Press (OMP) sowie die Repositoriumssoftware DSpace an OS-APS angebunden. Um sich im Vorfeld ein Bild von den Anforderungen der Verlage an die Software zu machen, wurde zu Beginn des Projekts eine Anforderungsanalyse in Form einer Online-Umfrage durchgeführt. Dabei wurden Fragen zu Manuskripteingang und -bearbeitung, Publikationsformaten und Zukunftspotenzialen im Verlagswesen gestellt. Die Ergebnisse dieser Umfrage sind in die Entwicklung mit eingeflossen. Im Rahmen des vorliegenden Papers erfolgt zunächst eine kurze Einführung in die Grundlagen des Publizierens mit XML, bevor das BMBF-geförderte Projekt OS-APS inklusive des angedachten Workflows und der einzelnen Softwarekomponenten vorgestellt wird. Im Anschluss daran werden die Ergebnisse der Anforderungsanalyse sowie die daraus entstandenen Schlussfolgerungen für den Workflow ausgeführt. Abschließend wird auf die Einbeziehung der Community und die nächsten anstehenden Entwicklungsschritte eingegangen.The University Library of Erlangen-Nuremberg, the University and State Library of Saxony-Anhalt and the technology company SciFlow in Berlin are jointly developing the “Open Source Academic Publishing Suite (OS-APS)”, which is supposed to enable publishers to create e-books easily and automatically according to their style guidelines. OS-APS will enable an XML-based single-source publishing workflow. The software will integrate import, editing and export functions so that the manuscript formats that commonly occur in publishing houses (e.g., DOCX or ODT) can be imported, analysed and converted into the desired format (e.g., EPUB, PDF, HTML or XML) in the publisher’s corporate design with just a few mouse clicks. In addition, OS-APS will connect to the publication platforms Open Journal Systems (OJS) and Open Monograph Press (OMP) as well as the repository software DSpace. In order to get an idea of the publishers’ requirements for the software in advance, a requirements analysis in the form of an online survey was conducted at the beginning of the project. Questions were asked about manuscript submission and processing, publication formats and future potential in publishing. The results of this survey have been incorporated into the development. This paper begins with a brief introduction to the basics of publishing with XML before presenting the BMBF-funded project OS-APS, including the envisaged workflow and the individual software components. This is followed by the results of the requirements elicitation and the resulting conclusions for the workflow. Finally, the involvement of the community and the next steps regarding the development are discusse

Molecular biology of glutathione peroxidase 4: from genomic structure to developmental expression and neural function

Selenoproteins have been recognized as modulators of brain function and signaling. Phospholipid hydroperoxide glutathione peroxidase (GPx4/PHGPx) is a unique member of the selenium-dependent glutathione peroxidases in mammals with a pivotal role in brain development and function. GPx4 exists as a cytosolic, mitochondrial, and nuclear isoform derived from a single gene. In mice, the GPx4 gene is located on chromosome 10 in close proximity to a functional retrotransposome that is expressed under the control of captured regulatory elements. Elucidation of crystallographic data uncovered structural peculiarities of GPx4 that provide the molecular basis for its unique enzymatic properties and substrate specificity. Monomeric GPx4 is multifunctional: it acts as a reducing enzyme of peroxidized phospholipids and thiols and as a structural protein. Transcriptional regulation of the different GPx4 isoforms requires several isoform-specific cis-regulatory sequences and trans-activating factors. Cytosolic and mitochondrial GPx4 are the major isoforms exclusively expressed by neurons in the developing brain. In stark contrast, following brain trauma, GPx4 is specifically upregulated in non-neuronal cells, i.e., reactive astrocytes. Molecular approaches to genetic modification in mice have revealed an essential and isoform-specific function for GPx4 in development and disease. Here we review recent findings on GPx4 with emphasis on its molecular structure and function and consider potential mechanisms that underlie neural development and neuropathological condition

Unbalanced expression of glutathione peroxidase 4 and arachidonate 15-lipoxygenase affects acrosome reaction and In Vitro fertilization

Glutathione peroxidase 4 (Gpx4) and arachidonic acid 15 lipoxygenase (Alox15) are counterplayers in oxidative lipid metabolism and both enzymes have been implicated in spermatogenesis. However, the roles of the two proteins in acrosomal exocytosis have not been explored in detail. Here we characterized Gpx4 distribution in mouse sperm and detected the enzyme not only in the midpiece of the resting sperm but also at the anterior region of the head, where the acrosome is localized. During sperm capacitation, Gpx4 translocated to the post-acrosomal compartment. Sperm from Gpx4+/Sec46Ala mice heterozygously expressing a catalytically silent enzyme displayed an increased expression of phosphotyrosyl proteins, impaired acrosomal exocytosis after in vitro capacitation and were not suitable for in vitro fertilization. Alox15-deficient sperm showed normal acrosome reactions but when crossed into a Gpx4-deficient background spontaneous acrosomal exocytosis was observed during capacitation and these cells were even less suitable for in vitro fertilization. Taken together, our data indicate that heterozygous expression of a catalytically silent Gpx4 variant impairs acrosomal exocytosis and in vitro fertilization. Alox15 deficiency hardly impacted the acrosome reaction but when crossed into the Gpx4-deficient background spontaneous acrosomal exocytosis was induced. The detailed molecular mechanisms for the observed effects may be related to the compromised redox homeostasis

Novel Genetic Risk and Metabolic Signatures of Insulin Signaling and Androgenesis in the Anovulation of Polycystic Ovary Syndrome

Funding Information: The authors are grateful to all staff in the PCOSAct group for their effort in the collection of blood samples and clinical dataset which used in current study. Special thanks to Prof. Attila Toth from Institute of Physiological Chemistry, Dresden, Germany for the REC114 antibody. This study was supported by the National key Research and Development Program of China (2019YFC1709500); the National Collaboration Project of Critical Illness by Integrating Chinese Medicine and Western Medicine; the Project of Heilongjiang Province Innovation Team “TouYan;” the Yi-Xun Liu and Xiao-Ke Wu Academician Workstation; the Innovation Team of Reproductive Technique with Integrative Chinese Medicine and Western Medicine in Xuzhou City, China; Heilongjiang University of Chinese Medicine from the National Clinical Trial Base; Heilongjiang Provincial Clinical Research Center for Ovary Diseases; the Research Grant Council (T13-602/21-N, C5045-20EF, and 14122021); and Food and Health Bureau in Hong Kong, China (06171026). Ben Willem J. Mol is supported by a National Health and Medical Research Council (NHMRC) Investigator grant (GNT1176437). Ben Willem J. Mol reports consultancy for ObsEva and Merck and travel support from Merck. Xiaoke Wu, Yongyong Shi, and Chi Chiu Wang developed the research question and designed the study. Xiaoke Wu, Yongyong Shi, Yijuan Cao, and Chi Chiu Wang designed the analysis. Yongyong Shi and Zhiqiang Li contributed to the design of the experiment of whole-exome plus targeted SNP sequencing and the analysis, and interpreted the results. Jingshu Gao, Hui Chang, Duojia Zhang, Jing Cong, Yu Wang, Qi Wu, Xiaoxiao Han, Pui Wah Jacqueline Chung, Yiran Li, and Lin Zeng contributed to the experiment of metabolic profile and immunofluorescent staining and the analysis, and interpreted the results. Astrid Borchert and Hartmut Kuhn provided antibody support and advice. Xu Zheng and Lingxi Chen contributed to create the predictive model with deep machine learning. Jian Li, Qi Wu, Hongli Ma, Xu Zheng, and Lingxi Chen contributed to the analysis of the clinical characteristics and interpreted the results. Jian Li, Hongli Ma, Hui Chang, Jing Cong, and Chi Chiu Wang drafted the manuscript. All authors reviewed and revised the manuscript. Xiaoke Wu is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Xiaoke Wu, Chi Chiu Wang, Yijuan Cao, Jian Li, Zhiqiang Li, Hongli Ma, Jingshu Gao, Hui Chang, Duojia Zhang, Jing Cong, Yu Wang, Qi Wu, Xiaoxiao Han, Pui Wah Jacqueline Chung, Yiran Li, Xu Zheng, Lingxi Chen, Lin Zeng, Astrid Borchert, Hartmut Kuhn, Zijiang Chen, Ernest Hung Yu Ng, Elisabet Stener-Victorin, Heping Zhang, Richard S. Legro, Ben Willem J. Mol, and Yongyong Shi declare that they have no conflict of interest or financial conflicts to disclose. Funding Information: This study was supported by the National key Research and Development Program of China ( 2019YFC1709500 ); the National Collaboration Project of Critical Illness by Integrating Chinese Medicine and Western Medicine ; the Project of Heilongjiang Province Innovation Team “TouYan;” the Yi-Xun Liu and Xiao-Ke Wu Academician Workstation; the Innovation Team of Reproductive Technique with Integrative Chinese Medicine and Western Medicine in Xuzhou City , China; Heilongjiang University of Chinese Medicine from the National Clinical Trial Base ; Heilongjiang Provincial Clinical Research Center for Ovary Diseases ; the Research Grant Council ( T13-602/21-N , C5045-20EF , and 14122021 ); and Food and Health Bureau in Hong Kong, China ( 06171026 ). Publisher Copyright: © 2023Peer reviewedPublisher PD

Expression Silencing of Glutathione Peroxidase 4 in Mouse Erythroleukemia Cells Delays In Vitro Erythropoiesis

Among the eight human glutathione peroxidase isoforms, glutathione peroxidase 4 (GPX4) is the only enzyme capable of reducing complex lipid peroxides to the corresponding alcohols. In mice, corruption of the Gpx4 gene leads to embryonic lethality and more detailed expression silencing studies have implicated the enzyme in several physiological processes (e.g., embryonal cerebrogenesis, neuronal function, male fertility). Experiments with conditional knockout mice, in which expression of the Gpx4 gene was silenced in erythroid precursors, indicated a role of Gpx4 in erythropoiesis. To test this hypothesis in a cellular in vitro model we transfected mouse erythroleukemia cells with a Gpx4 siRNA construct and followed the expression kinetics of erythropoietic gene products. Our data indicate that Gpx4 is expressed at high levels in mouse erythroleukemia cells and that expression silencing of the Gpx4 gene delays in vitro erythropoiesis. However, heterozygous expression of a catalytically inactive Gpx4 mutant (Gpx4(+/Sec46Ala)) did not induce a defective erythropoietic phenotype in different in vivo and ex vivo models. These data suggest that Gpx4 plays a role in erythroid differentiation of mouse erythroleukemia cells but that heterozygous expression of a catalytically inactive Gpx4 is not sufficient to compromise in vivo and ex vivo erythropoiesis

Molecular biology of glutathione peroxidase 4: From genomic structure to developmental expression and neural function

ISSN:1431-6730ISSN:1437-431