The Drosophila AWP1 ortholog Doctor No regulates JAK/STAT signaling for left-right asymmetry in the gut by promoting receptor endocytosis.

Many organs of Drosophila show stereotypical left-right (LR) asymmetry; however, the underlying mechanisms remain elusive. Here, we have identified an evolutionarily conserved ubiquitin-binding protein, AWP1/Doctor No (Drn), as a factor required for LR asymmetry in the embryonic anterior gut. We found that drn is essential in the circular visceral muscle cells of the midgut for JAK/STAT signaling, which contributes to the first known cue for anterior gut lateralization via LR asymmetric nuclear rearrangement. Embryos homozygous for drn and lacking its maternal contribution showed phenotypes similar to those with depleted JAK/STAT signaling, suggesting that Drn is a general component of JAK/STAT signaling. Absence of Drn resulted in specific accumulation of Domeless (Dome), the receptor for ligands in the JAK/STAT signaling pathway, in intracellular compartments, including ubiquitylated cargos. Dome colocalized with Drn in wild-type Drosophila. These results suggest that Drn is required for the endocytic trafficking of Dome, which is a crucial step for activation of JAK/STAT signaling and the subsequent degradation of Dome. The roles of AWP1/Drn in activating JAK/STAT signaling and in LR asymmetric development may be conserved in various organisms.

Left-right Myosin-Is, Myosin1C, and Myosin1D exhibit distinct single molecule behaviors on the plasma membrane of Drosophila macrophages.

Left-right (LR) asymmetry is crucial for animal development, particularly in Drosophila where LR-asymmetric morphogenesis of organs hinges on cellular-level chirality, termed cell chirality. In this species, two class I myosins, Myosin1D (Myo1D), and Myosin1C (Myo1C), respectively determine dextral (wild type) and sinistral (mirror image) cell chirality. Previous studies demonstrated Myo1D's ability to propel F-actin in leftward circles during in vitro gliding assays, suggesting its mechanochemical role in defining dextral chirality. Conversely, Myo1C propels F-actin without exhibiting LR-directional preference in this assay, suggesting at other properties governing sinistral chirality. Given the interaction of Myo1D and Myo1C with the membrane, we hypothesized that differences in their membrane behaviors might be critical in dictating their dextral or sinistral activities. In this study, employing single-molecule imaging analyses, we investigated the dynamic behaviors of Myo1D and Myo1C on the plasma membrane. Our findings revealed that Myo1C exhibits a significantly greater proportion of slow-diffusing population compared to Myo1D. Importantly, this characteristic was contingent upon both head and tail domains of Myo1C. The distinct diffusion patterns of Myo1D and Myo1C did not exert mutual influence on each other. This divergence in membrane diffusion between Myo1D and Myo1C may be crucial for dictating cell and organ chirality.

Collective nuclear behavior shapes bilateral nuclear symmetry for subsequent left-right asymmetric morphogenesis in Drosophila.

Proper organ development often requires nuclei to move to a specific position within the cell. To determine how nuclear positioning affects left-right (LR) development in the Drosophila anterior midgut (AMG), we developed a surface-modeling method to measure and describe nuclear behavior at stages 13-14, captured in three-dimensional time-lapse movies. We describe the distinctive positioning and a novel collective nuclear behavior by which nuclei align LR symmetrically along the anterior-posterior axis in the visceral muscles that overlie the midgut and are responsible for the LR-asymmetric development of this organ. Wnt4 signaling is crucial for the collective behavior and proper positioning of the nuclei, as are myosin II and the LINC complex, without which the nuclei fail to align LR symmetrically. The LR-symmetric positioning of the nuclei is important for the subsequent LR-asymmetric development of the AMG. We propose that the bilaterally symmetrical positioning of these nuclei may be mechanically coupled with subsequent LR-asymmetric morphogenesis.

Suppression of Mast Cell Activation by GPR35: GPR35 Is a Primary Target of Disodium Cromoglycate.

Mast cell stabilizers, including disodium cromoglycate (DSCG), were found to have potential as the agonists of an orphan G protein-coupled receptor, GPR35, although it remains to be determined whether GPR35 is expressed in mast cells and involved in suppression of mast cell degranulation. Our purpose in this study is to verify the expression of GPR35 in mast cells and to clarify how GPR35 modulates the degranulation. We explored the roles of GPR35 using an expression system, a mast cell line constitutively expressing rat GPR35, peritoneal mast cells, and bone marrow-derived cultured mast cells. Immediate allergic responses were assessed using the IgE-mediated passive cutaneous anaphylaxis (PCA) model. Various known GPR35 agonists, including DSCG and newly designed compounds, suppressed IgE-mediated degranulation. GPR35 was expressed in mature mast cells but not in immature bone marrow-derived cultured mast cells and the rat mast cell line. Degranulation induced by antigens was significantly downmodulated in the mast cell line stably expressing GPR35. A GPR35 agonist, zaprinast, induced a transient activation of RhoA and a transient decrease in the amount of filamentous actin. GPR35 agonists suppressed the PCA responses in the wild-type mice but not in the GPR35-/- mice. These findings suggest that GPR35 should prevent mast cells from undergoing degranulation induced by IgE-mediated antigen stimulation and be the primary target of mast cell stabilizers. SIGNIFICANCE STATEMENT: The agonists of an orphan G protein-coupled receptor, GPR35, including disodium cromoglycate, were found to suppress degranulation of rat and mouse mature mast cells, and their antiallergic effects were abrogated in the GPR35-/- mice, indicating that the primary target of mast cell stabilizers should be GPR35.

Inhibition of Lipopolysaccharide-Induced Inflammatory Signaling by Soft Coral-Derived Prostaglandin A2 in RAW264.7 Cells.

Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria and causes inflammatory diseases. We searched MeOH extracts of collected marine organisms for inhibitors of LPS-induced nitric oxide (NO) production in RAW264.7 cells and identified prostaglandin A2 (PGA2) as an active compound from the MeOH extract of the soft coral Lobophytum sp. PGA2 inhibited the production of NO and reduced the expression of inducible NO synthase (iNOS) in LPS-stimulated RAW264.7 cells. Although short preincubation with PGA2 did not inhibit LPS-induced degradation and resynthesis of IκBα, the suppressive effect of PGA2 was observed only after a prolonged incubation period prior to LPS treatment. In addition, PGA2-inhibited NO production was negated by the addition of the EP4 antagonist L161982. Thus, PGA2 was identified as an inhibitor of LPS-induced inflammatory signaling in RAW264.7 cells.

Antiapoptotic activity of methyl (3R,4E,6Z,15E)-3-hydroxyoctadecatrienoate in human cervical carcinoma HeLa cells.

Inhibitors of thapsigargin-induced cell death in human cervical carcinoma HeLa cells were screened among the metabolites of marine organisms. The MeOH extract of the cyanobacterium Rivularia sp. was found to exhibit inhibitory activity. Column chromatography purification was used to isolate methyl (3R,4E,6Z,15E)-3-hydroxyoctadecatrienoate (MHO) as the active compound. MHO was determined to inhibit apoptotic stimuli-induced cell death in HeLa cells.

E and ID proteins regulate cell chirality and left-right asymmetric development in Drosophila.

How left-right (LR) asymmetric forms in the animal body is a fundamental problem in Developmental Biology. Although the mechanisms for LR asymmetry are well studied in some species, they are still poorly understood in invertebrates. We previously showed that the intrinsic LR asymmetry of cells (designated as cell chirality) drives LR asymmetric development in the Drosophila embryonic hindgut, although the machinery of the cell chirality formation remains elusive. Here, we found that the Drosophila homologue of the Id gene, extra macrochaetae (emc), is required for the normal LR asymmetric morphogenesis of this organ. Id proteins, including Emc, are known to interact with and inhibit E-box-binding proteins (E proteins), such as Drosophila Daughterless (Da). We found that the suppression of da by wild-type emc was essential for cell chirality formation and for normal LR asymmetric development of the embryonic hindgut. Myosin ID (MyoID), which encodes the Drosophila Myosin ID protein, is known to regulate cell chirality. We further showed that Emc-Da regulates cell chirality formation, in which Emc functions upstream of or parallel to MyoID. Abnormal Id-E protein regulation is involved in various human diseases. Our results suggest that defects in cell shape may contribute to the pathogenesis of such diseases.

Maternal almondex, a neurogenic gene, is required for proper subcellular Notch distribution in early Drosophila embryogenesis.

Notch signaling plays crucial roles in the control of cell fate and physiology through local cell-cell interactions. The core processes of Notch signal transduction are well established, but the mechanisms that fine-tune the pathway in various developmental and post-developmental contexts are less clear. Drosophila almondex, which encodes an evolutionarily conserved double-pass transmembrane protein, was identified in the 1970s as a maternal-effect gene that regulates Notch signaling in certain contexts, but its mechanistic function remains obscure. In this study, we examined the role of almondex in Notch signaling during early Drosophila embryogenesis. We found that in addition to being required for lateral inhibition in the neuroectoderm, almondex is also partially required for Notch signaling-dependent single-minded expression in the mesectoderm. Furthermore, we found that almondex is required for proper subcellular Notch receptor distribution in the neuroectoderm, specifically during mid-stage 5 development. The absence of maternal almondex during this critical window of time caused Notch to accumulate abnormally in cells in a mesh-like pattern. This phenotype did not include any obvious change in subcellular Delta ligand distribution, suggesting that it does not result from a general vesicular-trafficking defect. Considering that dynamic Notch trafficking regulates signal output to fit the specific context, we speculate that almondex may facilitate Notch activation by regulating intracellular Notch receptor distribution during early embryogenesis.

Inhibitory effects of biseokeaniamide A against lipopolysaccharide-induced signal transduction.

Lipopolysaccharides (LPS) are associated with various inflammatory diseases; therefore, the inhibition of LPS-induced nitric oxide (NO) production may have extensive therapeutic applications. We searched for inhibitors of NO production in the LPS-stimulated murine macrophage-like cell line RAW264.7 from MeOH extracts of marine organisms. The MeOH extract of the marine cyanobacterium Okeania sp., collected in Okinawa, Japan, showed inhibitory activity. Biseokeaniamide A was isolated from the MeOH extract by chromatographic separation. Biseokeaniamide A inhibited NO production without cytotoxicity. It reduced inducible nitric oxide synthase levels and suppressed the expression of IL-1ß in LPS-stimulated RAW264.7 cells. Biseokeaniamide A did not inhibit IκBα degradation but inhibited IκBα expression. Thus, biseokeaniamide A, a naturally occurring lipopeptide, was identified as a selective inhibitor of LPS signal transduction.

Selective Inhibition of Spindle Microtubules by a Tubulin-Binding Quinazoline Derivative.

In the research field of tubulin-binding agents for the development of anticancer agents, hidden targets are emerging as a problem in understanding the exact mechanisms of actions. The quinazoline derivative 1-(4-methoxyphenyl)-1-(quinazolin-4-yl)ethan-1-ol (PVHD121) has anti-cell proliferative activity and inhibits tubulin polymerization by binding to the colchicine site of tubulin. However, the molecular mechanism of action of PVHD121 in cells remains unclear. Here, we demonstrate that PVHD121 delays mitotic entry and efficiently causes mitotic arrest with spindle checkpoint activation, leading to subsequent cell death. The dominant phenotype induced by PVHD121 was aberrant spindles with robust microtubules and unseparated centrosomes. The microtubules were radially distributed, and their ends appeared to adhere to kinetochores, and not to centrosomes. Extensive inhibition by high concentrations of PVHD121 eliminated all microtubules from cells. PVHD277 [1-(4-methoxyphenyl)-1-(2-morpholinoquinazolin-4-yl)ethan-1-ol], a PVHD121 derivative with fluorescence, tended to localize close to the centrosomes when cells prepared to enter mitosis. Our results show that PVHD121 is an antimitotic agent that selectively disturbs microtubule formation at centrosomes during mitosis. This antimitotic activity can be attributed to the targeting of centrosome maturation in addition to the interference with microtubule dynamics. Due to its unique bioactivity, PVHD121 is a potential tool for studying the molecular biology of mitosis and a potential lead compound for the development of anticancer agents. SIGNIFICANCE STATEMENT: Many tubulin-binding agents have been developed as potential anticancer agents. The aim of this study was to understand the subcellular molecular actions of a quinazoline derivative tubulin-binding agent, 1-(4-methoxyphenyl)-1-(quinazolin-4-yl)ethan-1-ol (PVHD121). As expected from its binding activity to tubulin, PVHD121 caused aberrant spindles and inhibited mitotic progression. However, in addition to tubulin, PVHD121 also targeted an unexpected biomolecule involved in centrosome maturation. Due to targeting the biomolecule just before entering mitosis, PVHD121 preferentially inhibited centrosome-derived microtubules rather than chromosome-derived microtubules during spindle formation. This study not only revealed the molecular action of PVHD121 in cells but also emphasized the importance of considering possible tubulin-independent effects of tubulin-binding agents via hidden targeted biomolecules for future use.

Drosophila flies high over the Asia-Pacific: Report on the Fourth Asia-Pacific Drosophila Research Conference.

The Fourth Asia-Pacific Drosophila Research Conference (APDRC4) was held at the convention center of Osaka University, Osaka, Japan, on May 8-11, 2017. Derived from the Japanese Drosophila Research Conference, the APDRC visited its home for the first time since its launch in 2011 with APDRC1 in Taipei, followed by APDRC2 in Seoul and APDRC3 in Beijing. There were 344 participants from 18 countries, more than half of whom were from abroad (Data S1). Two keynote speakers, Drs. Henry Sun and Daisuke Yamamoto, who have had rich science careers, gave overviews of their research. In addition, 14 invited speakers who are highly regarded in their fields introduced their new findings. Thirty-four oral presenters, many of them young investigators and students, were selected from the general participants to report their exciting results. During the conference, many stimulating questions and discussions were shared. Furthermore, 176 posters were presented, which also inspired enthusiastic discussions. In addition to the scientific presentations, a mixer and banquet enabled further intercommunion among the researchers (Figure b, e). During the conference, it was decided that the next Asia-Pacific Drosophila Research Conference (APDRC5) would be in Pune, India, in 2020. Thus, APDRC4 successfully achieved its mission to facilitate Drosophila research in the Asia-Pacific region.

Cyclic analogue of S-benzylisothiourea that suppresses kynurenine production without inhibiting indoleamine 2,3-dioxygenase activity.

Kynurenine is biosynthesised from tryptophan catalysed by indoleamine 2,3-dioxygenase (IDO). The abrogation of kynurenine production is considered a promising therapeutic target for immunological cancer treatment. In the course of our IDO inhibitor programme, formal cyclisation of the isothiourea moiety of the IDO inhibitor 1 afforded the 5-Cl-benzimidazole derivative 2b-6, which inhibited both recombinant human IDO (rhIDO) activity and cellular kynurenine production. Further derivatisation of 2b-6 provided the potent inhibitor of cellular kynurenine production 2i (IC50 = 0.34 µM), which unexpectedly exerted little effect on the enzymatic activity of rhIDO. Elucidation of the mechanism of action revealed that compound 2i suppresses IDO expression at the protein level by inhibiting STAT1 expression in IFN-γ-treated A431 cells. The kynurenine-production inhibitor 2i is expected to be a promising starting point for a novel approach to immunological cancer treatment.

Structural Development of Cell-Penetrating Peptides Containing Cationic Proline Derivatives.

We designed and synthesized a series of cell-penetrating peptides containing cationic proline derivatives (ProGu) that exhibited responsive changes in their secondary structures to the cellular environment. Effects of the peptide length and steric arrangement of the side chain in cationic proline derivatives [Pro4SGu and Pro4RGu] on their secondary structures and cell membrane permeability were investigated. Moreover, peptides 3 and 8 exhibited efficient intracellular delivery of plasmid DNA.

Crystal Structure of LSD1 in Complex with 4-[5-(Piperidin-4-ylmethoxy)-2-(p-tolyl)pyridin-3-yl]benzonitrile.

Because lysine-specific demethylase 1 (LSD1) regulates the maintenance of cancer stem cell properties, small-molecule inhibitors of LSD1 are expected to be useful for the treatment of several cancers. Reversible inhibitors of LSD1 with submicromolar inhibitory potency have recently been reported, but their exact binding modes are poorly understood. In this study, we synthesized a recently reported reversible inhibitor, 4-[5-(piperidin-4-ylmethoxy)-2-(p-tolyl)pyridin-3-yl]benzonitrile, which bears a 4-piperidinylmethoxy group, a 4-methylphenyl group, and a 4-cyanophenyl group on a pyridine ring, and determined the crystal structure of LSD1 in complex with this inhibitor at 2.96 Å. We observed strong electron density for the compound, showing that its cyano group forms a hydrogen bond with Lys661, which is a critical residue in the lysine demethylation reaction located deep in the catalytic center of LSD1. The piperidine ring interacts with the side chains of Asp555 and Asn540 in two conformations, and the 4-methylphenyl group is bound in a hydrophobic pocket in the catalytic center. Our elucidation of the binding mode of this compound can be expected to facilitate the rational design of more-potent reversible LSD1 inhibitors.

Dual Roles of O-Glucose Glycans Redundant with Monosaccharide O-Fucose on Notch in Notch Trafficking.

Notch is a transmembrane receptor that mediates cell-cell interactions and controls various cell-fate specifications in metazoans. The extracellular domain of Notch contains multiple epidermal growth factor (EGF)-like repeats. At least five different glycans are found in distinct sites within these EGF-like repeats. The function of these individual glycans in Notch signaling has been investigated, primarily by disrupting their individual glycosyltransferases. However, we are just beginning to understand the potential functional interactions between these glycans. Monosaccharide O-fucose and O-glucose trisaccharide (O-glucose-xylose-xylose) are added to many of the Notch EGF-like repeats. In Drosophila, Shams adds a xylose specifically to the monosaccharide O-glucose. We found that loss of the terminal dixylose of O-glucose-linked saccharides had little effect on Notch signaling. However, our analyses of double mutants of shams and other genes required for glycan modifications revealed that both the monosaccharide O-glucose and the terminal dixylose of O-glucose-linked saccharides function redundantly with the monosaccharide O-fucose in Notch activation and trafficking. The terminal dixylose of O-glucose-linked saccharides and the monosaccharide O-glucose were required in distinct Notch trafficking processes: Notch transport from the apical plasma membrane to adherens junctions, and Notch export from the endoplasmic reticulum, respectively. Therefore, the monosaccharide O-glucose and terminal dixylose of O-glucose-linked saccharides have distinct activities in Notch trafficking, although a loss of these activities is compensated for by the presence of monosaccharide O-fucose. Given that various glycans attached to a protein motif may have redundant functions, our results suggest that these potential redundancies may lead to a serious underestimation of glycan functions.

Preorganized Cyclic α,α-Disubstituted α-Amino Acids Bearing Functionalized Side Chains That Act as Peptide-Helix Inducers.

Preorganized cyclic α,α-disubstituted α-amino acids (dAA) bearing functionalized side chains that acted as peptide-helix inducers, which could be used for solid-phase peptide synthesis, were designed and synthesized. Furthermore, a helical octapeptide with the following amino acid sequence was prepared, and its preferred conformation was analyzed based on its CD spectra: Ac-X1EYSAX2KA-NH2 (11: X1 = ApiC4N3, X2 = Ac6c). The side-chain azido functional group of peptide 11 was efficiently converted to various 1,2,3-triazole groups via Huisgen 1,3-dipolar cycloaddition reactions involving different types of alkynes. The new cyclic dAA derivatives, which combine the advantages of conformational preorganization and side-chain functional groups, should prove to be a useful tool for the further development of biologically active peptides.

O-fucose monosaccharide of Drosophila Notch has a temperature-sensitive function and cooperates with O-glucose glycan in Notch transport and Notch signaling activation.

Notch (N) is a transmembrane receptor that mediates the cell-cell interactions necessary for many cell fate decisions. N has many epidermal growth factor-like repeats that are O-fucosylated by the protein O-fucosyltransferase 1 (O-Fut1), and the O-fut1 gene is essential for N signaling. However, the role of the monosaccharide O-fucose on N is unclear, because O-Fut1 also appears to have O-fucosyltransferase activity-independent functions, including as an N-specific chaperon. Such an enzymatic activity-independent function could account for the essential role of O-fut1 in N signaling. To evaluate the role of the monosaccharide O-fucose modification in N signaling, here we generated a knock-in mutant of O-fut1 (O-fut1(R245A knock-in)), which expresses a mutant protein that lacks O-fucosyltransferase activity but maintains the N-specific chaperon activity. Using O-fut1(R245A knock-in) and other gene mutations that abolish the O-fucosylation of N, we found that the monosaccharide O-fucose modification of N has a temperature-sensitive function that is essential for N signaling. The O-fucose monosaccharide and O-glucose glycan modification, catalyzed by Rumi, function redundantly in the activation of N signaling. We also showed that the redundant function of these two modifications is responsible for the presence of N at the cell surface. Our findings elucidate how different forms of glycosylation on a protein can influence the protein's functions.

Disruption of Drosophila melanogaster lipid metabolism genes causes tissue overgrowth associated with altered developmental signaling.

Developmental patterning requires the precise interplay of numerous intercellular signaling pathways to ensure that cells are properly specified during tissue formation and organogenesis. The spatiotemporal function of many developmental pathways is strongly influenced by the biosynthesis and intracellular trafficking of signaling components. Receptors and ligands must be trafficked to the cell surface where they interact, and their subsequent endocytic internalization and endosomal trafficking is critical for both signal propagation and its down-modulation. In a forward genetic screen for mutations that alter intracellular Notch receptor trafficking in Drosophila melanogaster, we recovered mutants that disrupt genes encoding serine palmitoyltransferase and acetyl-CoA carboxylase. Both mutants cause Notch, Wingless, the Epidermal Growth Factor Receptor (EFGR), and Patched to accumulate abnormally in endosomal compartments. In mosaic animals, mutant tissues exhibit an unusual non-cell-autonomous effect whereby mutant cells are functionally rescued by secreted activities emanating from adjacent wildtype tissue. Strikingly, both mutants display prominent tissue overgrowth phenotypes that are partially attributable to altered Notch and Wnt signaling. Our analysis of the mutants demonstrates genetic links between abnormal lipid metabolism, perturbations in developmental signaling, and aberrant cell proliferation.

Deficient Notch signaling associated with neurogenic pecanex is compensated for by the unfolded protein response in Drosophila.

The Notch (N) signaling machinery is evolutionarily conserved and regulates a broad spectrum of cell-specification events, through local cell-cell communication. pecanex (pcx) encodes a multi-pass transmembrane protein of unknown function, widely found from Drosophila to humans. The zygotic and maternal loss of pcx in Drosophila causes a neurogenic phenotype (hyperplasia of the embryonic nervous system), suggesting that pcx might be involved in N signaling. Here, we established that Pcx is a component of the N-signaling pathway. Pcx was required upstream of the membrane-tethered and the nuclear forms of activated N, probably in N signal-receiving cells, suggesting that pcx is required prior to or during the activation of N. pcx overexpression revealed that Pcx resides in the endoplasmic reticulum (ER). Disruption of pcx function resulted in enlargement of the ER that was not attributable to the reduced N signaling activity. In addition, hyper-induction of the unfolded protein response (UPR) by the expression of activated Xbp1 or dominant-negative Heat shock protein cognate 3 suppressed the neurogenic phenotype and ER enlargement caused by the absence of pcx. A similar suppression of these phenotypes was induced by overexpression of O-fucosyltransferase 1, an N-specific chaperone. Taking these results together, we speculate that the reduction in N signaling in embryos lacking pcx function might be attributable to defective ER functions, which are compensated for by upregulation of the UPR and possibly by enhancement of N folding. Our results indicate that the ER plays a previously unrecognized role in N signaling and that this ER function depends on pcx activity.