Project description:piRNAs are 26-30nt germ-line specific small non-coding RNAs that have evolutionarily conserved function in mobile genetic element silencing and maintenance of genome integrity. It has been shown that Drosophila Hsp70/90 Organizing Protein Homolog (Hop) – a co-chaperone interacts with piRNA binding protein Piwi and mediates silencing of phenotypic variations. However, it is not known if Hop has a direct role in piRNA biogenesis and transposon silencing. Here, we show that knock down of Hop in the germ-line nurse cells (GLKD) of Drosophila ovaries leads to activation of transposable elements. Females without germ-line Hop can lay eggs but the eggs do not hatch into larvae. GLKD of Hop leads to accumulation of γ-H2Av foci in the germline indicating increased DNA damage in the ovary. We also show that Hop is required for efficient piRNA biogenesis. Based on these results, we conclude that Hop is a critical component of piRNA pathway and it maintains genome integrity by silencing transposable elements.
Project description:The hop plant, Humulus lupulus L., contains an exceptionally high content of secondary metabolites, the hop iso-α-acids, which possess a range of beneficial properties including antiseptic action. Studies performed on the mode of action of hop iso-α-acids have hitherto been restricted to lactic acid bacteria. The present study investigates molecular mechanisms of hop iso-α-acid resistance in the model eukaryote Saccharomyces cerevisiae. Growth inhibition occurred at concentrations of hop iso-α-acids that were an order of magnitude higher than those found with hop-tolerant prokaryotes. Chemostat-based transcriptome analysis and phenotype screening of the S. cerevisiae haploid gene deletion collection were used as complementary methods to screen for genes involved in hop iso-α-acids detoxification and tolerance. Further analysis of deletion mutants confirmed that yeast tolerance to hop iso-α-acids involves two major processes: active export of iso-α-acids across the plasma membrane and active proton pumping into the vacuole by the V-ATPase to enable vacuolar sequestration of iso-α-acids. Furthermore, iso-α-acids were shown to affect cellular metal homeostasis by acting as strong zinc and iron chelator.
Project description:Folding of stringent clients requires transfer from Hsp70 to Hsp90. The co-chaperone Hop physically connects the chaperone machineries. Here we define its role from the remodeling of Hsp70/40-client complexes to the mechanism of client transfer and the conformational switching from stalled to active client-processing states of Hsp90. We show that Hsp70 together with Hsp40 completely unfolds a stringent client, the glucocorticoid receptor ligand binding domain (GR-LBD) in large assemblies. Hop remodels these for efficient transfer onto Hsp90. As p23 enters, Hsp70 leaves the complex via switching between binding sites in Hop. To proceed to client folding, current concepts assume that Hop dissociates and the co-chaperone p23 stabilizes the Hsp90 closed state. In contrast, we show that p23 directly interacts with Hop, relieves the stalling Hsp90-Hop interaction and closes Hsp90. This reaction allows folding of the client and is thus the key regulatory step for the progression of the chaperone cycle. To study the interaction between the different proteins, especially p23 and the DP2 domain of Hop, we performed crosslinking-MS.
Project description:Nucleic acids in wastewater provide a rich source of data for detection and surveillance of microbes. We have longitudinally collected 116 RNA samples from a wastewater treatment plant in Berlin/Germany, from March 2021 to July 2022, and 24 DNA samples from May to July 2022. We tracked human astroviruses, enteroviruses, noroviruses and adenoviruses over time to the level of strains or even individual nucleotide variations, showing how detailed human pathogens can be observed using wastewater. For respiratory pathogens, a broad enrichment panel enabled us to detect waves of RSV, influenza, or common cold coronaviruses in high agreement with clinical data. By applying a profile Hidden Markov Model-based search for novel viruses, we identified more than 100 thousand novel transcript assemblies likely not belonging to known virus species, thus substantially expanding our knowledge of virus diversity. Phylogenetic analysis is shown for bunyaviruses and parvoviruses. Finally, we identify Hundreds of novel protein sequences for CRISPR-associated proteins such as Transposase B, a class of small RNA-guided DNA editing enzymes. Taken together, we present a longitudinal and deep investigation into wastewater-derived genomic sequencing data that underlines the value of sewage surveillance for public health, planetary virome research, and biotechnological potential.
Project description:The hop plant, Humulus lupulus L., contains an exceptionally high content of secondary metabolites, the hop iso-α-acids, which possess a range of beneficial properties including antiseptic action. Studies performed on the mode of action of hop iso-α-acids have hitherto been restricted to lactic acid bacteria. The present study investigates molecular mechanisms of hop iso-α-acid resistance in the model eukaryote Saccharomyces cerevisiae. Growth inhibition occurred at concentrations of hop iso-α-acids that were an order of magnitude higher than those found with hop-tolerant prokaryotes. Chemostat-based transcriptome analysis and phenotype screening of the S. cerevisiae haploid gene deletion collection were used as complementary methods to screen for genes involved in hop iso-α-acids detoxification and tolerance. Further analysis of deletion mutants confirmed that yeast tolerance to hop iso-α-acids involves two major processes: active export of iso-α-acids across the plasma membrane and active proton pumping into the vacuole by the V-ATPase to enable vacuolar sequestration of iso-α-acids. Furthermore, iso-α-acids were shown to affect cellular metal homeostasis by acting as strong zinc and iron chelator. Experiment Overall Design: Two complementary genome-wide approaches were employed to investigate cellular responses of S. cerevisiae to hop extracts enriched in iso-α-acids. Microarray transcriptome analysis was performed on chemostat cultures of an S. cerevisiae reference strain grown in the presence and absence of iso-α-acids. In addition, screening of the nearly complete set of yeast open reading frame (ORF) haploid knock-outs generated by the Saccharomyces Genome Deletion Project (SGDP) (Open Biosystems) identified the mutants with increased hop sensitivity. Subsequently, involvement of selected genes and cellular processes in hop acid sensitivity and tolerance was analyzed by construction and detailed analysis of selected mutant strains.
Project description:Ulcerative colitis is a chronic inflammatory disorder for which a definitive cure is still missing. This is characterized by an overwhelming inflammatory milieu in the colonic tract where a composite set of immune and non-immune cells orchestrate its pathogenesis. Over the last years, a growing body of evidence has been pinpointing gut virome dysbiosis as underlying its progression. Nonetheless, its role during the early phases of chronic inflammation is far from being fully defined. Here we show the gut virome-associated Hepatitis B virus protein X, most likely acquired after an event of zoonotic spillover, to be associated with the early stages of ulcerative colitis and to induce colonic inflammation in mice. It acts as a transcriptional regulator in epithelial cells, provoking barrier leakage and altering mucosal immunity at the level of both innate and adaptive immunity. This study paves the way to the comprehension of the aetiopathogenesis of intestinal inflammation and encourages further investigations of the virome as a trigger also in other scenarios. Moreover, it provides a brand-new standpoint that looks at the virome as a target for tailored treatments, blocking the early phases of chronic inflammation and possibly leading to better disease management.