Discovery of small molecules that activate RNA-methylation through cooperative binding to the METTL3/14/WTAP complex active site

Chemical modifications of RNA provide an additional, epitranscriptomic, level of control over cellular functions. N-6-methylated adenosines (m6As) are found in several types of RNA, and their amounts are regulated by methyltransferases and demethylases. One of the most important enzymes catalyzing generation of m6A on mRNA is the trimer N-6-methyltransferase METTL3-14-WTAP complex. Its activity has been linked to such critical biological processes as cell differentiation, proliferation, and death. We used in silico-based discovery to identify small-molecule ligands that bind to METTL3-14-WTAP and determined experimentally their binding affinity and kinetics, as well as their effect on enzymatic function. We show that these ligands serve as activators of the METTL3-14-WTAP complex

Metabolomic Profiling of Extracellular Vesicles and Alternative Normalization Methods Reveal Enriched Metabolites and Strategies to Study Prostate Cancer-Related Changes

Body fluids are a rich source of extracellular vesicles (EVs), which carry cargo derived from the secreting cells. So far, biomarkers for pathological conditions have been mainly searched from their protein, (mi) RNA, DNA and lipid cargo. Here, we explored the small molecule metabolites from urinary and platelet EVs relative to their matched source samples. As a proof-of-concept study of intra-EV metabolites, we compared alternative normalization methods to profile urinary EVs from prostate cancer patients before and after prostatectomy and from healthy controls. Methods: We employed targeted ultra-performance liquid chromatography-tandem mass spectrometry to profile over 100 metabolites in the isolated EVs, original urine samples and platelets. We determined the enrichment of the metabolites in the EVs and analyzed their subcellular origin, pathways and relevant enzymes or transporters through data base searches. EV-and urine-derived factors and ratios between metabolites were tested for normalization of the metabolomics data. Results: Approximately 1 x 10(10) EVs were sufficient for detection of metabolite profiles from EVs. The profiles of the urinary and platelet EVs overlapped with each other and with those of the source materials, but they also contained unique metabolites. The EVs enriched a selection of cytosolic metabolites including members from the nucleotide and spermidine pathways, which linked to a number of EV-resident enzymes or transporters. Analysis of the urinary EVs from the patients indicated that the levels of glucuronate, D-ribose 5-phosphate and isobutyryl-L-carnitine were 2-26-fold lower in all pre-prostatectomy samples compared to the healthy control and post-prostatectomy samples (p <0.05). These changes were only detected from EVs by normalization to EV-derived factors or with metabolite ratios, and not from the original urine samples. Conclusions: Our results suggest that metabolite analysis of EVs from different samples is feasible using a high-throughput platform and relatively small amount of sample material. With the knowledge about the specific enrichment of metabolites and normalization methods, EV metabolomics could be used to gain novel biomarker data not revealed by the analysis of the original EV source materials.Peer reviewe

Improved Characterization of EV Preparations Based on Protein to Lipid Ratio and Lipid Properties.

In recent years the study of extracellular vesicles has gathered much scientific and clinical interest. As the field is expanding, it is becoming clear that better methods for characterization and quantification of extracellular vesicles as well as better standards to compare studies are warranted. The goal of the present work was to find improved parameters to characterize extracellular vesicle preparations. Here we introduce a simple 96 well plate-based total lipid assay for determination of lipid content and protein to lipid ratios of extracellular vesicle preparations from various myeloid and lymphoid cell lines as well as blood plasma. These preparations included apoptotic bodies, microvesicles/microparticles, and exosomes isolated by size-based fractionation. We also investigated lipid bilayer order of extracellular vesicle subpopulations using Di-4-ANEPPDHQ lipid probe, and lipid composition using affinity reagents to clustered cholesterol (monoclonal anti-cholesterol antibody) and ganglioside GM1 (cholera toxin subunit B). We have consistently found different protein to lipid ratios characteristic for the investigated extracellular vesicle subpopulations which were substantially altered in the case of vesicular damage or protein contamination. Spectral ratiometric imaging and flow cytometric analysis also revealed marked differences between the various vesicle populations in their lipid order and their clustered membrane cholesterol and GM1 content. Our study introduces for the first time a simple and readily available lipid assay to complement the widely used protein assays in order to better characterize extracellular vesicle preparations. Besides differentiating extracellular vesicle subpopulations, the novel parameters introduced in this work (protein to lipid ratio, lipid bilayer order, and lipid composition), may prove useful for quality control of extracellular vesicle related basic and clinical studies

Isolation and characterization of platelet-derived extracellular vesicles

Background: Platelet-derived extracellular vesicles (EVs) participate, for example, in haemostasis, immunity and development. Most studies of platelet EVs have targeted microparticles, whereas exosomes and EV characterization under various conditions have been less analyzed. Studies have been hampered by the difficulty in obtaining EVs free from contaminating cells and platelet remnants. Therefore, we optimized an EV isolation protocol and compared the quantity and protein content of EVs induced by different agonists. Methods: Platelets isolated with iodixanol gradient were activated by thrombin and collagen, lipopolysaccharide (LPS) or Ca2+ ionophore. Microparticles and exosomes were isolated by differential centrifugations. EVs were quantitated by nanoparticle tracking analysis (NTA) and total protein. Size distributions were determined by NTA and electron microscopy. Proteomics was used to characterize the differentially induced EVs. Results: The main EV populations were 100–250 nm and over 90% were <500 nm irrespective of the activation. However, activation pathways differentially regulated the quantity and the quality of EVs, which also formed constitutively. Thrombogenic activation was the most potent physiological EV-generator. LPS was a weak inducer of EVs, which had a selective protein content from the thrombogenic EVs. Ca2+ ionophore generated a large population of protein-poor and unselectively packed EVs. By proteomic analysis, EVs were highly heterogeneous after the different activations and between the vesicle subpopulations. Conclusions: Although platelets constitutively release EVs, vesiculation can be increased, and the activation pathway determines the number and the cargo of the formed EVs. These activation-dependent variations render the use of protein content in sample normalization invalid. Since most platelet EVs are 100–250 nm, only a fraction has been analyzed by previously used methods, for example, flow cytometry. As the EV subpopulations could not be distinguished and large vesicle populations may be lost by differential centrifugation, novel methods are required for the isolation and the differentiation of all EVs

Ability of Platelet-Derived Extracellular Vesicles to Promote Neutrophil-Endothelial Cell Interactions.

We tested the ability of platelet-derived extracellular vesicles (PEV) to promote adhesion of flowing neutrophils to endothelial cells (EC). PEV were collected from platelets stimulated with collagen-related peptide, and differential centrifugation was used to collect larger vesicles enriched for platelet membrane microvesicles (PMV) or smaller vesicles enriched for platelet exosomes (Pexo). Vesicle binding and resultant activation of neutrophils and EC were assessed by flow cytometry. Flow-based adhesion assays assessed binding of neutrophils directly to deposited vesicles or to EC, after neutrophils or EC had been treated with vesicles. PEV bound efficiently to neutrophils or EC, with resultant upregulation of activation markers. Binding was Ca-dependent and dominantly mediated by CD62P for neutrophils or by integrins for EC. Deposited PEV supported mainly transient attachments of flowing neutrophils through CD62P and some stable adhesion through CXC-chemokines. Neutrophil adhesion to EC was promoted when either cell was pre-treated with PEV, although the effect was less prominent when EC were pre-activated with tumor necrosis factor-α. The pro-adhesive effects on neutrophils could largely be attributed to the larger PMV rather than Pexo. Thus, surface-bound PEV can capture flowing neutrophils, while PEV also activate neutrophils and EC to promote interactions. PEV may potentiate inflammatory responses after tissue injury