ABSTRACT
The RNA-binding protein TRIM71/LIN-41 is a phylogenetically conserved developmental regulator that functions in mammalian stem cell reprogramming, brain development, and cancer. TRIM71 recognizes target mRNAs through hairpin motifs and silences them through molecular mechanisms that await identification. Here, we uncover that TRIM71 represses its targets through RNA-supported interaction with TNRC6/GW182, a core component of the miRNA-induced silencing complex (miRISC). We demonstrate that AGO2, TRIM71, and UPF1 each recruit TNRC6 to specific sets of transcripts to silence them. As cellular TNRC6 levels are limiting, competition occurs among the silencing pathways, such that the loss of AGO proteins or of AGO binding to TNRC6 enhances the activities of the other pathways. We conclude that a miRNA-like silencing activity is shared among different mRNA silencing pathways and that the use of TNRC6 as a central hub provides a means to integrate their activities.
Subject(s)
Argonaute Proteins , MicroRNAs , Animals , Argonaute Proteins/genetics , Argonaute Proteins/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Protein Binding , Stem Cells/metabolism , Mammals/metabolismABSTRACT
The Sec61 complex forms a protein-conducting channel in the endoplasmic reticulum membrane that is required for secretion of soluble proteins and production of many membrane proteins. Several natural and synthetic small molecules specifically inhibit Sec61, generating cellular effects that are useful for therapeutic purposes, but their inhibitory mechanisms remain unclear. Here we present near-atomic-resolution structures of human Sec61 inhibited by a comprehensive panel of structurally distinct small molecules-cotransin, decatransin, apratoxin, ipomoeassin, mycolactone, cyclotriazadisulfonamide and eeyarestatin. All inhibitors bind to a common lipid-exposed pocket formed by the partially open lateral gate and plug domain of Sec61. Mutations conferring resistance to the inhibitors are clustered at this binding pocket. The structures indicate that Sec61 inhibitors stabilize the plug domain in a closed state, thereby preventing the protein-translocation pore from opening. Our study provides the atomic details of Sec61-inhibitor interactions and the structural framework for further pharmacological studies and drug design.
Subject(s)
Endoplasmic Reticulum , Membrane Proteins , Humans , Endoplasmic Reticulum/metabolism , Membrane Proteins/metabolism , Protein Transport/physiology , SEC Translocation Channels/antagonists & inhibitors , SEC Translocation Channels/chemistry , SEC Translocation Channels/metabolismABSTRACT
The ability to directly uncover the contributions of genes to a given phenotype is fundamental for biology research. However, ostensibly homogeneous cell populations exhibit large clonal variance that can confound analyses and undermine reproducibility. Here we used genome-saturated mutagenesis to create a biobank of over 100,000 individual haploid mouse embryonic stem (mES) cell lines targeting 16,970 genes with genetically barcoded, conditional and reversible mutations. This Haplobank is, to our knowledge, the largest resource of hemi/homozygous mutant mES cells to date and is available to all researchers. Reversible mutagenesis overcomes clonal variance by permitting functional annotation of the genome directly in sister cells. We use the Haplobank in reverse genetic screens to investigate the temporal resolution of essential genes in mES cells, and to identify novel genes that control sprouting angiogenesis and lineage specification of blood vessels. Furthermore, a genome-wide forward screen with Haplobank identified PLA2G16 as a host factor that is required for cytotoxicity by rhinoviruses, which cause the common cold. Therefore, clones from the Haplobank combined with the use of reversible technologies enable high-throughput, reproducible, functional annotation of the genome.
Subject(s)
Biological Specimen Banks , Genomics/methods , Haploidy , Mouse Embryonic Stem Cells/metabolism , Mutation , Animals , Blood Vessels/cytology , Cell Lineage/genetics , Common Cold/genetics , Common Cold/virology , Genes, Essential/genetics , Genetic Testing , HEK293 Cells , Homozygote , Humans , Mice , Mouse Embryonic Stem Cells/cytology , Neovascularization, Physiologic/genetics , Phospholipases A2, Calcium-Independent/genetics , Phospholipases A2, Calcium-Independent/metabolism , Rhinovirus/pathogenicityABSTRACT
The identification of activating mutations in NOTCH1 in 50% of T cell acute lymphoblastic leukemia has generated interest in elucidating how these mutations contribute to oncogenic transformation and in targeting the pathway. A phenotypic screen identified compounds that interfere with trafficking of Notch and induce apoptosis via an endoplasmic reticulum (ER) stress mechanism. Target identification approaches revealed a role for SLC39A7 (ZIP7), a zinc transport family member, in governing Notch trafficking and signaling. Generation and sequencing of a compound-resistant cell line identified a V430E mutation in ZIP7 that confers transferable resistance to the compound NVS-ZP7-4. NVS-ZP7-4 altered zinc in the ER, and an analog of the compound photoaffinity labeled ZIP7 in cells, suggesting a direct interaction between the compound and ZIP7. NVS-ZP7-4 is the first reported chemical tool to probe the impact of modulating ER zinc levels and investigate ZIP7 as a novel druggable node in the Notch pathway.
Subject(s)
Cation Transport Proteins/genetics , Endoplasmic Reticulum Stress/physiology , Receptor, Notch1/genetics , Animals , Apoptosis , Carrier Proteins/metabolism , Cation Transport Proteins/metabolism , Cation Transport Proteins/physiology , Cell Line , Cell Transformation, Neoplastic , Endoplasmic Reticulum/physiology , Humans , Mutation , Protein Transport , Receptor, Notch1/physiology , Signal Transduction , Zinc/metabolismABSTRACT
A chemicogenetic screen was performed in budding yeast mutants that have a weakened replication stress response. This identified an inhibitor of target of rapamycin (TOR) complexes 1 and 2 that selectively enhances the sensitivity of sgs1Δ cells to hydroxyurea and camptothecin. More importantly, the inhibitor has strong synthetic lethality in combination with either the break-inducing antibiotic Zeocin or ionizing radiation, independent of the strain background. Lethality correlates with a rapid fragmentation of chromosomes that occurs only when TORC2, but not TORC1, is repressed. Genetic inhibition of Tor2 kinase, or its downstream effector kinases Ypk1/Ypk2, conferred similar synergistic effects in the presence of Zeocin. Given that Ypk1/Ypk2 controls the actin cytoskeleton, we tested the effects of actin modulators latrunculin A and jasplakinolide. These phenocopy TORC2 inhibition on Zeocin, although modulation of calcineurin-sensitive transcription does not. These results implicate TORC2-mediated actin filament regulation in the survival of low levels of DNA damage.
Subject(s)
Genomic Instability , Multiprotein Complexes/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , TOR Serine-Threonine Kinases/genetics , Transcription Factors/genetics , Actins/antagonists & inhibitors , Actins/metabolism , Bleomycin/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Chromosomes/drug effects , Chromosomes/genetics , Chromosomes/radiation effects , DNA Damage/genetics , DNA Replication/drug effects , DNA Replication/radiation effects , Genomic Instability/drug effects , Genomic Instability/radiation effects , Glycogen Synthase Kinase 3/metabolism , Mechanistic Target of Rapamycin Complex 2 , Multiprotein Complexes/antagonists & inhibitors , Multiprotein Complexes/metabolism , Radiation, Ionizing , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Saccharomyces cerevisiae Proteins/metabolism , Signal Transduction/drug effects , Signal Transduction/radiation effects , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/metabolism , Thiazolidines/pharmacology , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolismABSTRACT
Natural Products (NPs) are molecular' special equipment ' that impart survival benefits on their producers in nature. Due to their evolved functions to modulate biology these privileged metabolites are substantially represented in the drug market and are continuing to contribute to the discovery of innovative medicines such as the recently approved semi-synthetic derivative of the bacterial alkaloid staurosporin in oncology indications. The innovation of low molecular weight compounds in modern drug discovery is built on rapid progress in chemical, molecular biological, pharmacological and data sciences, which together provide a rich understanding of disease-driving molecular interactions and how to modulate them. NPs investigated in these pharmaceutical research areas create new perspectives on their chemical and biological features and thereby new chances to advance medical research. New methods in analytical chemistry linked with searchable NP-databases solved the issue of reisolation and enabled targeted and efficient access to novel molecules from nature. Cheminformatics delivers high resolution descriptions of NPs and explores the substructures that systematically map NP-chemical space by sp³-enriched fragments. Whole genome sequencing has revealed the existence of collocated gene clusters that form larger functional entities together with proximate resistance factors thus avoiding self-inhibition of the encoded metabolites. The analysis of bacterial and fungal genes provides tantalizing glimpses of new compound-target pairs of therapeutic value. Furthermore, a dedicated investigation of structurally unique, selectively active NPs in chemical biology demonstrates their extraordinary power as shuttles between new biological target spaces of pharmaceutical relevance.
Subject(s)
Biological Products , Databases, Factual , Drug Discovery , Drug IndustryABSTRACT
Pooled CRISPR screens are a powerful tool for assessments of gene function. However, conventional analysis is based exclusively on the relative abundance of integrated single guide RNAs (sgRNAs) between populations, which does not discern distinct phenotypes and editing outcomes generated by identical sgRNAs. Here we present CRISPR-UMI, a single-cell lineage-tracing methodology for pooled screening to account for cell heterogeneity. We generated complex sgRNA libraries with unique molecular identifiers (UMIs) that allowed for screening of clonally expanded, individually tagged cells. A proof-of-principle CRISPR-UMI negative-selection screen provided increased sensitivity and robustness compared with conventional analysis by accounting for underlying cellular and editing-outcome heterogeneity and detection of outlier clones. Furthermore, a CRISPR-UMI positive-selection screen uncovered new roadblocks in reprogramming mouse embryonic fibroblasts as pluripotent stem cells, distinguishing reprogramming frequency and speed (i.e., effect size and probability). CRISPR-UMI boosts the predictive power, sensitivity, and information content of pooled CRISPR screens.
Subject(s)
CRISPR-Cas Systems/genetics , Cell Lineage/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , Gene Editing , RNA, Guide, Kinetoplastida , Single-Cell Analysis/methods , Animals , Cells, Cultured , Fibroblasts/cytology , Gene Knockout Techniques , Genetic Vectors , Mice , Pluripotent Stem Cells/cytology , Retroviridae/genetics , Signal-To-Noise RatioABSTRACT
Kendomycin is a small-molecule natural product that has gained significant attention due to reported cytotoxicity against pathogenic bacteria and fungi as well as a number of cancer cell lines. Despite significant biomedical interest and attempts to reveal its mechanism of action, the cellular target of kendomycin remains disputed. Herein it is shown that kendomycin induces cellular responses indicative of cation stress comparable to the effects of established iron chelators. Furthermore, addition of excess iron and copper attenuated kendomycin cytotoxicity in bacteria, yeast, and mammalian cells. Finally, NMR analysis demonstrated a direct interaction with cations, corroborating a close link between the observed kendomycin polypharmacology across different species and modulation of iron and/or copper levels.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antibiotics, Antineoplastic/pharmacology , Antifungal Agents/pharmacology , Bacteria/drug effects , Chelating Agents/pharmacology , Fungi/drug effects , Rifabutin/analogs & derivatives , Cations , Cell Line , Copper/metabolism , Iron/metabolism , Leupeptins/pharmacology , Microbial Sensitivity Tests , Mutagenesis , Rifabutin/pharmacology , Yeasts/drug effectsABSTRACT
Cladosporin, a natural product known for decades, has recently been discovered to display potent and selective antiplasmodial activity by inhibition of lysyl-tRNA synthetase. It was subjected to a panel of oxidative biotransformations with one fungal and two actinomycetes strains, as well as a triple mutant bacterial CYP102A1, yielding eight, mostly hydroxylated, derivatives. These new compounds covered a wide chemical space and contained two pairs of epimers in the tetrahydropyran ring. Although less potent than the parent compound, all analogues showed activity in a cell-based synthetase assay, thus demonstrating uptake and on-target activity in living cells with varying degrees of selectivity for the enzyme lysyl-tRNA synthetase from Plasmodium falciparum and highlighting sites suitable for synthesis of future cladosporin analogues. Compounds with adjacent hydroxy functions showed different MS/MS fragmentation that can be explained in terms of an, in some cases, regioselective loss of water followed by a retro-Diels-Alder reaction.
Subject(s)
Antimalarials/metabolism , Drug Discovery , Enzyme Inhibitors/metabolism , Isocoumarins/metabolism , Lysine-tRNA Ligase/antagonists & inhibitors , Malaria, Falciparum/drug therapy , Bacteria/metabolism , Biotransformation , Fungi/metabolism , Plasmodium falciparum/enzymology , Structure-Activity RelationshipABSTRACT
Selective and specific inhibitors of Plasmodium falciparum lysyl-tRNA synthetase represent promising therapeutic antimalarial avenues. Cladosporin was identified as a potent P.â falciparum lysyl-tRNA synthetase inhibitor, with an activity against parasite lysyl-tRNA synthetase >100-fold more potent than that of the activity registered against the human enzyme. Despite its compelling activity, cladosporin exhibits poor oral bioavailability; a critical requirement for antimalarial drugs. Thus, the quest to develop metabolically stable cladosporin-derived analogues, while retaining similar selectivity and potency to that of the natural compound, has begun. Chemogenomic profiling of a designed library allowed an entirely innovative structure-activity relationship study to be initiated; this shed light on structural evidence of a privileged scaffold with a unique activity against tRNA synthetases.
Subject(s)
Antimalarials/chemical synthesis , Drug Discovery , Enzyme Inhibitors/chemical synthesis , Isocoumarins/chemical synthesis , Lysine-tRNA Ligase/antagonists & inhibitors , Malaria, Falciparum/drug therapy , Humans , Plasmodium falciparum/enzymology , Structure-Activity RelationshipABSTRACT
Tim17 and Tim23 are the main subunits of the TIM23 complex, one of the two major essential mitochondrial inner-membrane protein translocon machineries (TIMs). No chemical probes that specifically inhibit TIM23-dependent protein import were known to exist. Here we show that the natural product stendomycin, produced by Streptomyces hygroscopicus, is a potent and specific inhibitor of the TIM23 complex in yeast and mammalian cells. Furthermore, stendomycin-mediated blockage of the TIM23 complex does not alter normal processing of the major regulatory mitophagy kinase PINK1, but TIM23 is required to stabilize PINK1 on the outside of mitochondria to initiate mitophagy upon membrane depolarization.
Subject(s)
Mitochondrial Proteins/metabolism , Peptides/pharmacology , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Antimicrobial Cationic Peptides , Dose-Response Relationship, Drug , HeLa Cells , Humans , Membrane Transport Proteins/metabolism , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondrial Precursor Protein Import Complex Proteins , Molecular Structure , Peptides/chemistry , Protein Transport/drug effects , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
Invasive infections by fungal pathogens cause more deaths than malaria worldwide. We found the ergoline compound NGx04 in an antifungal screen, with selectivity over mammalian cells. High-resolution chemogenomics identified the lipid transfer protein Sec14p as the target of NGx04 and compound-resistant mutations in Sec14p define compound-target interactions in the substrate binding pocket of the protein. Beyond its essential lipid transfer function in a variety of pathogenic fungi, Sec14p is also involved in secretion of virulence determinants essential for the pathogenicity of fungi such as Cryptococcus neoformans, making Sec14p an attractive antifungal target. Consistent with this dual function, we demonstrate that NGx04 inhibits the growth of two clinical isolates of C. neoformans and that NGx04-related compounds have equal and even higher potency against C. neoformans. Furthermore NGx04 analogues showed fungicidal activity against a fluconazole resistant C. neoformans strain. In summary, we present genetic evidence that NGx04 inhibits fungal Sec14p and initial data supporting NGx04 as a novel antifungal starting point.
Subject(s)
Carrier Proteins/chemistry , Cryptococcosis/drug therapy , Cryptococcus neoformans/drug effects , Ergolines/pharmacology , Lipid Metabolism/drug effects , Antifungal Agents/pharmacology , Carrier Proteins/genetics , Cryptococcosis/microbiology , Cryptococcus neoformans/genetics , Cryptococcus neoformans/pathogenicity , Ergolines/chemistry , Humans , Microbial Sensitivity Tests , Protein Conformation , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/geneticsABSTRACT
Limited detoxification capacity often directs aggregation-prone, potentially hazardous, misfolded proteins to be deposited in designated cytosolic compartments known as 'aggresomes'. The roles of aggresomes as cellular quality control centers, and the cellular origin of the deposits contained within these structures, remain to be characterized. Here, we utilized the observation that the prion protein (PrP, also known as PRNP) accumulates in aggresomes following the inhibition of folding chaperones, members of the cyclophilin family, to address these questions. We found that misfolded PrP molecules must pass through the endoplasmic reticulum (ER) in order to be deposited in aggresomes, that the Golgi plays no role in this process and that cytosolic PrP species are not deposited in pre-existing aggresomes. Prior to their deposition in the aggresome, PrP molecules lose the ER localization signal and have to acquire a GPI anchor. Our discoveries indicate that PrP aggresomes are cytosolic overflow deposition centers for the ER quality control mechanisms and highlight the importance of these structures for the maintenance of protein homeostasis within the ER.
Subject(s)
Cytosol/metabolism , Endoplasmic Reticulum/metabolism , Prion Proteins/metabolism , Protein Aggregates , Animals , CHO Cells , Cricetinae , Cricetulus , Cyclosporine/pharmacology , Cytosol/drug effects , Endoplasmic Reticulum/drug effects , Glycosylation , Glycosylphosphatidylinositols/metabolism , Golgi Apparatus/drug effects , Golgi Apparatus/metabolism , Mice, Inbred C57BL , Mice, Knockout , Models, Biological , Protein Aggregates/drug effects , Protein Folding/drug effectsABSTRACT
A new cyclic decadepsipeptide was isolated from Chaetosphaeria tulasneorum with potent bioactivity on mammalian and yeast cells. Chemogenomic profiling in S. cerevisiae indicated that the Sec61 translocon complex, the machinery for protein translocation and membrane insertion at the endoplasmic reticulum, is the target. The profiles were similar to those of cyclic heptadepsipeptides of a distinct chemotype (including HUN-7293 and cotransin) that had previously been shown to inhibit cotranslational translocation at the mammalian Sec61 translocon. Unbiased, genome-wide mutagenesis followed by full-genome sequencing in both fungal and mammalian cells identified dominant mutations in Sec61p (yeast) or Sec61α1 (mammals) that conferred resistance. Most, but not all, of these mutations affected inhibition by both chemotypes, despite an absence of structural similarity. Biochemical analysis confirmed inhibition of protein translocation into the endoplasmic reticulum of both co- and post-translationally translocated substrates by both chemotypes, demonstrating a mechanism independent of a translating ribosome. Most interestingly, both chemotypes were found to also inhibit SecYEG, the bacterial Sec61 translocon homolog. We suggest 'decatransin' as the name for this new decadepsipeptide translocation inhibitor.
Subject(s)
Biological Products/pharmacology , Endoplasmic Reticulum/drug effects , Membrane Proteins/metabolism , Protein Transport/drug effects , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Animals , Ascomycota/metabolism , COS Cells , Cells, Cultured , Chlorocebus aethiops , HCT116 Cells , Humans , Membrane Proteins/antagonists & inhibitors , Peptides, Cyclic/pharmacology , Polymorphism, Single Nucleotide/genetics , SEC Translocation Channels , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & developmentABSTRACT
Unbiased phenotypic screens enable identification of small molecules that inhibit pathogen growth by unanticipated mechanisms. These small molecules can be used as starting points for drug discovery programs that target such mechanisms. A major challenge of the approach is the identification of the cellular targets. Here we report GNF7686, a small molecule inhibitor of Trypanosoma cruzi, the causative agent of Chagas disease, and identification of cytochrome b as its target. Following discovery of GNF7686 in a parasite growth inhibition high throughput screen, we were able to evolve a GNF7686-resistant culture of T. cruzi epimastigotes. Clones from this culture bore a mutation coding for a substitution of leucine by phenylalanine at amino acid position 197 in cytochrome b. Cytochrome b is a component of complex III (cytochrome bc1) in the mitochondrial electron transport chain and catalyzes the transfer of electrons from ubiquinol to cytochrome c by a mechanism that utilizes two distinct catalytic sites, QN and QP. The L197F mutation is located in the QN site and confers resistance to GNF7686 in both parasite cell growth and biochemical cytochrome b assays. Additionally, the mutant cytochrome b confers resistance to antimycin A, another QN site inhibitor, but not to strobilurin or myxothiazol, which target the QP site. GNF7686 represents a promising starting point for Chagas disease drug discovery as it potently inhibits growth of intracellular T. cruzi amastigotes with a half maximal effective concentration (EC50) of 0.15 µM, and is highly specific for T. cruzi cytochrome b. No effect on the mammalian respiratory chain or mammalian cell proliferation was observed with up to 25 µM of GNF7686. Our approach, which combines T. cruzi chemical genetics with biochemical target validation, can be broadly applied to the discovery of additional novel drug targets and drug leads for Chagas disease.
Subject(s)
Antifungal Agents/pharmacology , Chagas Disease/drug therapy , Chagas Disease/microbiology , Cytochromes b/metabolism , Trypanosoma cruzi/drug effects , Animals , Antimycin A/metabolism , Chagas Disease/genetics , Cytochromes b/genetics , Electron Transport/drug effects , Electron Transport/immunology , Genomics , Mice , Mitochondria/drug effects , Mitochondria/metabolism , Mutation , Oxygen Consumption/drug effects , Trypanosoma cruzi/isolation & purification , Trypanosoma cruzi/metabolismABSTRACT
High-throughput screening (HTS) is an integral part of early drug discovery. Herein, we focused on those small molecules in a screening collection that have never shown biological activity despite having been exhaustively tested in HTS assays. These compounds are referred to as 'dark chemical matter' (DCM). We quantified DCM, validated it in quality control experiments, described its physicochemical properties and mapped it into chemical space. Through analysis of prospective reporter-gene assay, gene expression and yeast chemogenomics experiments, we evaluated the potential of DCM to show biological activity in future screens. We demonstrated that, despite the apparent lack of activity, occasionally these compounds can result in potent hits with unique activity and clean safety profiles, which makes them valuable starting points for lead optimization efforts. Among the identified DCM hits was a new antifungal chemotype with strong activity against the pathogen Cryptococcus neoformans but little activity at targets relevant to human safety.
Subject(s)
Antifungal Agents/pharmacology , Cryptococcus neoformans/drug effects , Drug Discovery , High-Throughput Screening Assays , Antifungal Agents/chemistry , Microbial Sensitivity Tests , Molecular Structure , Structure-Activity RelationshipABSTRACT
Malaria continues to be one of the most devastating human diseases despite many efforts to limit its spread by prevention of infection or by pharmaceutical treatment of patients. We have conducted a screen for antiplasmodial compounds by using a natural product library. Here we report on cyclomarinâ A as a potent growth inhibitor of Plasmodium falciparum and the identification of its molecular target, diadenosine triphosphate hydrolase (PfAp3Aase), by chemical proteomics. Using a biochemical assay, we could show that cyclomarinâ A is a specific inhibitor of the plasmodial enzyme but not of the closest human homologue hFHIT. Co-crystallisation experiments demonstrate a unique binding mode of the inhibitor. One molecule of cyclomarinâ A binds a dimeric PfAp3Aase and prevents the formation of the enzymeâ substrate complex. These results validate PfAp3Aase as a new drug target for the treatment of malaria. We have previously elucidated the structurally unrelated regulatory subunit ClpC1 of the ClpP protease as the molecular target of cyclomarinâ A in Mycobacterium tuberculosis. Thus, cyclomarinâ A is a rare example of a natural product with two distinct and specific modes of action.
Subject(s)
Biological Products/chemistry , Oligopeptides/chemistry , Acid Anhydride Hydrolases/antagonists & inhibitors , Acid Anhydride Hydrolases/metabolism , Antimalarials/chemistry , Antimalarials/metabolism , Antimalarials/pharmacology , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/metabolism , Binding Sites , Biological Products/metabolism , Biological Products/pharmacology , Endopeptidase Clp/antagonists & inhibitors , Endopeptidase Clp/metabolism , Humans , Inhibitory Concentration 50 , Molecular Dynamics Simulation , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/enzymology , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/metabolism , Oligopeptides/metabolism , Oligopeptides/pharmacology , Plasmodium falciparum/drug effects , Plasmodium falciparum/enzymology , Plasmodium falciparum/growth & development , Protein Binding , Protein Structure, TertiaryABSTRACT
Cultivation of myxobacteria of the Nannocystis genus led to the isolation and structure elucidation of a class of novel cyclic lactone inhibitors of elongation factorâ 1. Whole genome sequence analysis and annotation enabled identification of the putative biosynthetic cluster and synthesis process. In biological assays the compounds displayed anti-fungal and cytotoxic activity. Combined genetic and proteomic approaches identified the eukaryotic translation elongation factor 1α (EF-1α) as the primary target for this compound class. Nannocystinâ A (1) displayed differential activity across various cancer cell lines and EEF1A1 expression levels appear to be the main differentiating factor. Biochemical and genetic evidence support an overlapping binding site of 1 with the anti-cancer compound didemninâ B on EF-1α. This myxobacterial chemotype thus offers an interesting starting point for further investigations of the potential of therapeutics targeting elongation factorâ 1.
Subject(s)
Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Macrocyclic Compounds/pharmacology , Myxococcales/physiology , Neoplasms/pathology , Peptide Elongation Factor 1/antagonists & inhibitors , Antifungal Agents/chemistry , Antifungal Agents/pharmacology , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Candida albicans/drug effects , Genomics/methods , Humans , Macrocyclic Compounds/chemistry , Molecular Structure , Neoplasms/drug therapy , Peptide Elongation Factor 1/genetics , Peptide Elongation Factor 1/metabolism , Proteomics/methods , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
Ashbya gossypii grows as multinucleated and constantly elongating hyphae. Nuclei are in continuous forward and backward motion, also move during mitosis, and frequently bypass each other. Whereas these nuclear movements are well documented, comparatively little is known about the density and morphology of organelles which very likely influence these movements. To understand the three-dimensional subcellular organization of hyphae at high resolution, we performed large-scale electron tomography of the tip regions in A. gossypii. Here, we present a comprehensive space-filling model in which most membrane-limited organelles including nuclei, mitochondria, endosomes, multivesicular bodies, vacuoles, autophagosomes, peroxisomes, and vesicles are modeled. Nuclei revealed different morphologies and protrusions filled by the nucleolus. Mitochondria are very abundant and form a tubular network with a polarized spherical fraction. The organelles of the degradative pathways show a clustered organization. By analyzing vesicle-like bodies, we identified three size classes of electron-dense vesicles (â¼200, â¼150, and â¼100 nm) homogeneously distributed in the cytoplasm which most likely represent peroxisomes. Finally, coated and uncoated vesicles with approximately 40-nm diameters show a polarized distribution toward the hyphal tip with the coated vesicles preferentially localizing at the hyphal periphery.
Subject(s)
Ascomycota/ultrastructure , Hyphae/ultrastructure , Tomography, X-Ray Computed , Cell Nucleus/diagnostic imaging , Cell Nucleus/ultrastructure , Cytoplasmic Vesicles/diagnostic imaging , Cytoplasmic Vesicles/ultrastructure , Mitochondria/diagnostic imaging , Mitochondria/ultrastructure , Peroxisomes/diagnostic imaging , Peroxisomes/ultrastructureABSTRACT
Non-alcoholic fatty liver disease (NAFLD) - characterized by excess accumulation of fat in the liver - now affects one third of the world's population. As NAFLD progresses, extracellular matrix components including collagen accumulate in the liver causing tissue fibrosis, a major determinant of disease severity and mortality. To identify transcriptional regulators of fibrosis, we computationally inferred the activity of transcription factors (TFs) relevant to fibrosis by profiling the matched transcriptomes and epigenomes of 108 human liver biopsies from a deeply-characterized cohort of patients spanning the full histopathologic spectrum of NAFLD. CRISPR-based genetic knockout of the top 100 TFs identified ZNF469 as a regulator of collagen expression in primary human hepatic stellate cells (HSCs). Gain- and loss-of-function studies established that ZNF469 regulates collagen genes and genes involved in matrix homeostasis through direct binding to gene bodies and regulatory elements. By integrating multiomic large-scale profiling of human biopsies with extensive experimental validation we demonstrate that ZNF469 is a transcriptional regulator of collagen in HSCs. Overall, these data nominate ZNF469 as a previously unrecognized determinant of NAFLD-associated liver fibrosis.