Small molecules that inhibit the late stage of Munc13-4-dependent secretory granule exocytosis in mast cells.

Ca2+-dependent secretory granule fusion with the plasma membrane is the final step for the exocytic release of inflammatory mediators, neuropeptides, and peptide hormones. Secretory cells use a similar protein machinery at late steps in the regulated secretory pathway, employing protein isoforms from the Rab, Sec1/Munc18, Munc13/CAPS, SNARE, and synaptotagmin protein families. However, no small-molecule inhibitors of secretory granule exocytosis that target these proteins are currently available but could have clinical utility. Here we utilized a high-throughput screen of a 25,000-compound library that identified 129 small-molecule inhibitors of Ca2+-triggered secretory granule exocytosis in RBL-2H3 mast cells. These inhibitors broadly fell into six different chemical classes, and follow-up permeable cell and liposome fusion assays identified the target for one class of these inhibitors. A family of 2-aminobenzothiazoles (termed benzothiazole exocytosis inhibitors or bexins) was found to inhibit mast cell secretory granule fusion by acting on a Ca2+-dependent, C2 domain-containing priming factor, Munc13-4. Our findings further indicated that bexins interfere with Munc13-4-membrane interactions and thereby inhibit Munc13-4-dependent membrane fusion. We conclude that bexins represent a class of specific secretory pathway inhibitors with potential as therapeutic agents.

Bead-linked transposomes enable a normalization-free workflow for NGS library preparation.

BACKGROUND: Transposome-based technologies have enabled the streamlined production of sequencer-ready DNA libraries; however, current methods are highly sensitive to the amount and quality of input nucleic acid. RESULTS: We describe a new library preparation technology (Nextera DNA Flex) that utilizes a known concentration of transposomes conjugated directly to beads to bind a fixed amount of DNA, and enables direct input of blood and saliva using an integrated extraction protocol. We further report results from libraries generated outside the standard parameters of the workflow, highlighting novel applications for Nextera DNA Flex, including human genome builds and variant calling from below 1 ng DNA input, customization of insert size, and preparation of libraries from short fragments and severely degraded FFPE samples. Using this bead-linked library preparation method, library yield saturation was observed at an input amount of 100 ng. Preparation of libraries from a range of species with varying GC levels demonstrated uniform coverage of small genomes. For large and complex genomes, coverage across the genome, including difficult regions, was improved compared with other library preparation methods. Libraries were successfully generated from amplicons of varying sizes (from 50 bp to 11 kb), however, a decrease in efficiency was observed for amplicons smaller than 250 bp. This library preparation method was also compatible with poor-quality DNA samples, with sequenceable libraries prepared from formalin-fixed paraffin-embedded samples with varying levels of degradation. CONCLUSIONS: In contrast to solution-based library preparation, this bead-based technology produces a normalized, sequencing-ready library for a wide range of DNA input types and amounts, largely obviating the need for DNA quantitation. The robustness of this bead-based library preparation kit and flexibility of input DNA facilitates application across a wide range of fields.

The SEQC2 epigenomics quality control (EpiQC) study.

BACKGROUND: Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmental processes, maintain cellular lineage specification, and can define or stratify types of cancer and other diseases. However, the wide variety of approaches available to interrogate these modifications has created a need for harmonized materials, methods, and rigorous benchmarking to improve genome-wide methylome sequencing applications in clinical and basic research. Here, we present a multi-platform assessment and cross-validated resource for epigenetics research from the FDA's Epigenomics Quality Control Group. RESULTS: Each sample is processed in multiple replicates by three whole-genome bisulfite sequencing (WGBS) protocols (TruSeq DNA methylation, Accel-NGS MethylSeq, and SPLAT), oxidative bisulfite sequencing (TrueMethyl), enzymatic deamination method (EMSeq), targeted methylation sequencing (Illumina Methyl Capture EPIC), single-molecule long-read nanopore sequencing from Oxford Nanopore Technologies, and 850k Illumina methylation arrays. After rigorous quality assessment and comparison to Illumina EPIC methylation microarrays and testing on a range of algorithms (Bismark, BitmapperBS, bwa-meth, and BitMapperBS), we find overall high concordance between assays, but also differences in efficiency of read mapping, CpG capture, coverage, and platform performance, and variable performance across 26 microarray normalization algorithms. CONCLUSIONS: The data provided herein can guide the use of these DNA reference materials in epigenomics research, as well as provide best practices for experimental design in future studies. By leveraging seven human cell lines that are designated as publicly available reference materials, these data can be used as a baseline to advance epigenomics research.

CAPS and Munc13 utilize distinct PIP2-linked mechanisms to promote vesicle exocytosis.

Phosphoinositides provide compartment-specific signals for membrane trafficking. Plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) is required for Ca(2+)-triggered vesicle exocytosis, but whether vesicles fuse into PIP2-rich membrane domains in live cells and whether PIP2 is metabolized during Ca(2+)-triggered fusion were unknown. Ca(2+)-dependent activator protein in secretion 1 (CAPS-1; CADPS/UNC31) and ubMunc13-2 (UNC13B) are PIP2-binding proteins required for Ca(2+)-triggered vesicle exocytosis in neuroendocrine PC12 cells. These proteins are likely effectors for PIP2, but their localization during exocytosis had not been determined. Using total internal reflection fluorescence microscopy in live cells, we identify PIP2-rich membrane domains at sites of vesicle fusion. CAPS is found to reside on vesicles but depends on plasma membrane PIP2 for its activity. Munc13 is cytoplasmic, but Ca(2+)-dependent translocation to PIP2-rich plasma membrane domains is required for its activity. The results reveal that vesicle fusion into PIP2-rich membrane domains is facilitated by sequential PIP2-dependent activation of CAPS and PIP2-dependent recruitment of Munc13. PIP2 hydrolysis only occurs under strong Ca(2+) influx conditions sufficient to activate phospholipase Cη2 (PLCη2). Such conditions reduce CAPS activity and enhance Munc13 activity, establishing PLCη2 as a Ca(2+)-dependent modulator of exocytosis. These studies provide a direct view of the spatial distribution of PIP2 linked to vesicle exocytosis via regulation of lipid-dependent protein effectors CAPS and Munc13.

Frizzled receptors signal through G proteins.

Frizzled receptors have long been thought to couple to G proteins but biochemical evidence supporting such an interaction has been lacking. Here we expressed mammalian Wnt-Frizzled fusion proteins in Saccharomyces cerevisiae and tested the receptors' ability to activate the yeast mitogen-activated protein kinase (MAPK) pathway via heterotrimeric G proteins. Our results show that Frizzled receptors can interact with Gαi, Gαq, and Gαs proteins, thus confirming that Frizzled functions as a G protein coupled receptor (GPCR). However, the activity level of Frizzled-mediated G protein signaling was much lower than that of a typical GPCR and, surprisingly, was highest when coupled to Gαs. The Frizzled/Gαs interaction was further established in vivo as Drosophila expressing a loss-of-function Gαs allele rescued the photoreceptor differentiation phenotype of Frizzled mutant flies. Together, these data point to an important role for Frizzled as a nontraditional GPCR that preferentially couples to Gαs heterotrimeric G proteins.

Munc13-4 reconstitutes calcium-dependent SNARE-mediated membrane fusion.

Munc13-4 is a widely expressed member of the CAPS/Munc13 protein family proposed to function in priming secretory granules for exocytosis. Munc13-4 contains N- and C-terminal C2 domains (C2A and C2B) predicted to bind Ca(2+), but Ca(2+)-dependent regulation of Munc13-4 activity has not been described. The C2 domains bracket a predicted SNARE-binding domain, but whether Munc13-4 interacts with SNARE proteins is unknown. We report that Munc13-4 bound Ca(2+) and restored Ca(2+)-dependent granule exocytosis to permeable cells (platelets, mast, and neuroendocrine cells) dependent on putative Ca(2+)-binding residues in C2A and C2B. Munc13-4 exhibited Ca(2+)-stimulated SNARE interactions dependent on C2A and Ca(2+)-dependent membrane binding dependent on C2B. In an apparent coupling of membrane and SNARE binding, Munc13-4 stimulated SNARE-dependent liposome fusion dependent on putative Ca(2+)-binding residues in both C2A and C2B domains. Munc13-4 is the first priming factor shown to promote Ca(2+)-dependent SNARE complex formation and SNARE-mediated liposome fusion. These properties of Munc13-4 suggest its function as a Ca(2+) sensor at rate-limiting priming steps in granule exocytosis.

Beta2-chimaerin in cancer signaling: connecting cell adhesion and MAP kinase activation.

The chimaerins are Rac GTPase-activating proteins that bind diacylglycerol. Emerging evidence implicates beta2-chimaerin in tumor progression. Here, we discuss our recent work in Drosophila melanogaster in the context of previous studies performed in human cancer cell lines that together lend new mechanistic insight into the role of chimaerins in cancer.

Chimaerin and Rac regulate cell number, adherens junctions, and ERK MAP kinase signaling in the Drosophila eye.

The chimaerin family of Rac GTPase-activating proteins (GAPs) has been implicated in neural development and tumor progression, although the cellular mechanisms of their effects are poorly understood. To study their physiologic function, we used the Drosophila retina as a model system. Reduced expression of the fly chimaerin ortholog RhoGAP5a in the pupal eye led to an excess of interommatidial pigment cells, aberrant cell contacts, and an increase in activated ERK that localized specifically to the plasma membrane. Reducing RhoGAP5A levels suppressed the effects of disrupted EGF receptor signaling. Perturbation of Rac activity led to similar phenotypes, whereas coexpression of Rac and RhoGAP5A-dsRNAi resulted in the elimination of adherens junctions between interommatidial cells. Our results reveal a role for chimaerin in the regulation of ERK signaling and cell-cell adhesion and have implications for its participation in epithelial development and tumor progression.

C5a receptor oligomerization. II. Fluorescence resonance energy transfer studies of a human G protein-coupled receptor expressed in yeast.

Recent studies demonstrate that members of the superfamily of G protein-coupled receptors (GPCRs) form oligomers both in vitro and in vivo. The mechanisms by which GPCRs oligomerize and the roles of accessory proteins in this process are not well understood. We used disulfide-trapping experiments to show that C5a receptors, expressed in mammalian cells, reside in membranes as oligomers (Klco, J. M., Lassere, T. B., and Baranski, T. J. (2003) J. Biol. Chem. 278, 35345-35353). To begin to address how C5a receptors form oligomers, we now use fluorescence resonance energy transfer experiments on human C5a receptors expressed in the lower eukaryote Saccharomyces cerevisiae. C5a receptors tagged with variants of the green fluorescent protein display energy transfer in intact yeast, demonstrating that mammalian accessory proteins are not required for C5a receptor oligomerization. In both intact yeast cells and membrane preparations, agonist does not affect FRET efficiency, and little energy transfer is observed between the C5a receptor and a co-expressed yeast pheromone receptor (encoded by STE2), indicating that C5a receptor oligomerization is both receptor-specific and constitutive. FRET studies performed on fractionated membranes demonstrate similar levels of energy transfer between tagged C5a receptors in endoplasmic reticulum compared with plasma membrane, and urea washing of membranes has little effect on the extent of energy transfer. The oligomerization of C5a receptors expressed in yeast displays characteristics similar to those observed for other GPCRs studied in mammalian cells. This model system should prove useful for further studies to define mechanisms of oligomerization of mammalian GPCRs.