Project description:Using our eletrophile delivery platform in zebrafish embryos (termed "Z-REX"), we launched a Z-REX-coupled RNA-seq screen to identify genes whose expression is significantly changed following Keap1-engagement with selective native lipid-derived electrophiles housing covalently-reactive motifs. The electrophiles tested were 4-hydroxynonenal (HNE) and 4-hydroxydodecenal (HDE).
Project description:KEAP1, a cytoplasmic repressor of the oxidative stress responsive transcription factor NRF2, senses the presence of electrophilic agents by modification of its sensor cysteine residues. In addition to xenobiotics, several reactive metabolites have been shown to covalently modify key cysteines on KEAP1, although the full repertoire of these molecules and their respective modifications remains undefined. Here, we report the discovery of sAKZ692, a small molecule identified by high throughput screening that stimulates NRF2 transcriptional activity in cells by inhibiting the glycolytic enzyme pyruvate kinase. sAKZ692 treatment promotes the buildup of glyceraldehyde 3-phosphate, a metabolite which leads to S-lactate modification of cysteine sensor residues of KEAP1, resulting in NRF2 dependent transcription. This work identifies a novel posttranslational modification of cysteine derived from a reactive central carbon metabolite and helps further define the complex relationship between metabolism and the oxidative stress sensing machinery of the cell.
Project description:Zebrafish (Danio rerio) model system have used widespread vertebrate investigations for genetic and cell biological analyses, and is suitable for small molecular screens such as chemical, toxicity and drug in order to use for human diseases and drug discovery . Recently, These powerful zebrafish model increasingly apply to human metabolic disease such as obesity and diabetes and toxicology. Despite a lot of advantages, proteomics research at zebrafish has received little interest in comparison with genetic and biological research using histology and in situ hybridization. Protein lysine acetylation is one of the most known post-translational modifications with dynamic and reversibly controlled by lysine acetyltransferase such as histone acetyltransferases and lysine deacetylase such as histone deacetylases and sirtuins family.Also, during the past year, global lysine acetylome studies using MS-based proteomics approach was in diverse species such as human, mouse, E. coli, Yeast and plants. Based on global acetylome data, our understanding of the roles of lysine acetylation in various cellular processes has increased. . The aim of this study was to identify Lysine acetylation in zebrafish embryos and determine the homology from Human at modified site level. Here we showed the global lysine acetylation study in Zebrafish embryos using MS-based zebrafish embryos.
Project description:Thallium (Tl) is a trace metal element used in the electronics, semiconductor and electro-optical industries. With the development of high-tech industries, thallium severely pollutes the aquatic environment. The purpose of this study was to evaluate the cardiotoxicity and developmental toxicity of Tl by using vertebrate model zebrafish embryos. RNA-seq was performed on wild type zebrafish embryos exposed to 0, 200, and 800 ppb Tl from 6 to 48 hpf. The transcriptomic profile revealed molecular understanding regarding the cardiovascular and developmental toxicity of Tl, providing valuable information for risk assessment of the emerging contaminant thallium.
Project description:Glycolysis is a master regulator of cellular energy, synthesis and redox regulation, however the mechanisms that underlie the responses to and regulation of changing glucose metabolism are incompletely understood. The NRF2-antioxidant response element (ARE) pathway serves as a central regulator of cellular stress by sensing and eliminating 2 chemically reactive species in the cell. A phenotypic high-throughput chemical screen for novel activators of NRF2 signaling identified an inhibitor of the glycolytic enzyme PGK1. Metabolomic and proteomic profiling experiments revealed that inhibition of PGK1 results in the accumulation of central glycolytic intermediates, as well as the reactive dicarbonyl metabolite methylglyoxal (MGx). MGx selectively modifies reactive residues on KEAP1, resulting in the formation of a covalent KEAP1 homodimer, accumulation of NRF2 and activation of the NRF2 transcriptional program. Proteomic and NMR experiments verified that KEAP1 homodimerization is mediated by a novel post-translational modification, termed MICA, formed by the reaction of methylglyoxal with proximal cysteine and arginine residues. The identified PGK1 inhibitor series was found to be efficacious in a NRF2-dependent mouse model of UV-damage, demonstrating that activation of KEAP1- NRF2 signaling through this mechanism is physiologically relevant. Together, these results demonstrate the existence of direct inter-pathway communication between glycolysis and the KEAP1-NRF2 transcriptional program, which is mediated by a reactive metaboliteinduced post-translational modification (rmPTM). In addition, they provide new insight into metabolic regulation of cell stress response, and a potential therapeutic axis for controlling the cytoprotective antioxidant response in numerous human diseases.
Project description:We set up a zebrafish model to study EVs in vivo by light microscopy, using CD63-pHluorin as a marker. To verify if exosomes from zebrafish have a comparable composition as human exosomes, and to validate the use of CD63 as a marker, we first analysed the compositon of EVs isolated from in vitro cultured fibroblasts (AB.9 ATCC), and could detect the presence of conventional exosome markers, including (zebrafish) CD63. In live embryos, we could track a population of exosomes from their source (the yolk syncytial layer (YSL)) to their final destination by live microscopy. To assess the composition of these exosomes, we dissociated YSL:CD63-pHluorin expressing 3dpf embryos and isolated exosomes from the supernatant and enriched for EVs from the YSL. This was compared to a background of total EVs isolated the control fish (non-expressing).
Project description:The zebrafish (Danio rerio) is a popular animal model in studies of vertebrate development and organogenesis. Recent research has shown a similarity of approximately 70% between the human and zebrafish genomes and of 84% in human disease-causing genes, specifically. Zebrafish embryos have a number of desirable features, including transparency, a large size, and rapid embryogenesis. Protein phosphorylation is a well-known post-translational modification (PTM), which performs various biological functions. Recent mass spectrometry (MS) developments have enabled the study of global phosphorylation patterns by using MS-based proteomics coupled with TiO2 phosphopeptide enrichment. In the present study, we identified 3,500 non-redundant phosphorylation sites on 2,166 phosphoproteins and 1,564 quantified phosphoproteins in zebrafish embryos.