Endothelial ROBO4 suppresses PTGS2/COX-2 expression and inflammatory diseases

Tanaka M., Shirakura K., Takayama Y., et al. Endothelial ROBO4 suppresses PTGS2/COX-2 expression and inflammatory diseases. Communications Biology 7, 599 (2024); https://doi.org/10.1038/s42003-024-06317-z .Accumulating evidence suggests that endothelial cells can be useful therapeutic targets. One of the potential targets is an endothelial cell-specific protein, Roundabout4 (ROBO4). ROBO4 has been shown to ameliorate multiple diseases in mice, including infectious diseases and sepsis. However, its mechanisms are not fully understood. In this study, using RNA-seq analysis, we found that ROBO4 downregulates prostaglandin-endoperoxide synthase 2 (PTGS2), which encodes cyclooxygenase-2. Mechanistic analysis reveals that ROBO4 interacts with IQ motif-containing GTPase-activating protein 1 (IQGAP1) and TNF receptor-associated factor 7 (TRAF7), a ubiquitin E3 ligase. In this complex, ROBO4 enhances IQGAP1 ubiquitination through TRAF7, inhibits prolonged RAC1 activation, and decreases PTGS2 expression in inflammatory endothelial cells. In addition, Robo4-deficiency in mice exacerbates PTGS2-associated inflammatory diseases, including arthritis, edema, and pain. Thus, we reveal the molecular mechanism by which ROBO4 suppresses the inflammatory response and vascular hyperpermeability, highlighting its potential as a promising therapeutic target for inflammatory diseases

Dynamic movement of the Golgi unit and its glycosylation enzyme zones

Harada A., Kunii M., Kurokawa K., et al. Dynamic movement of the Golgi unit and its glycosylation enzyme zones. Nature Communications 15, 4514 (2024); https://doi.org/10.1038/S41467-024-48901-1.Knowledge on the distribution and dynamics of glycosylation enzymes in the Golgi is essential for better understanding this modification. Here, using a combination of CRISPR/Cas9 knockin technology and super-resolution microscopy, we show that the Golgi complex is assembled by a number of small ‘Golgi units’ that have 1-3 μm in diameter. Each Golgi unit contains small domains of glycosylation enzymes which we call ‘zones’. The zones of N- and O-glycosylation enzymes are colocalised. However, they are less colocalised with the zones of a glycosaminoglycan synthesizing enzyme. Golgi units change shapes dynamically and the zones of glycosylation enzymes rapidly move near the rim of the unit. Photobleaching analysis indicates that a glycosaminoglycan synthesizing enzyme moves between units. Depletion of giantin dissociates units and prevents the movement of glycosaminoglycan synthesizing enzymes, which leads to insufficient glycosaminoglycan synthesis. Thus, we show the structure-function relationship of the Golgi and its implications in human pathogenesis

Insight into the Regulation of Glycan Synthesis in Drosophila Chaoptin Based on Mass Spectrometry

BACKGROUND: A variety of N-glycans attached to protein are known to involve in many important biological functions. Endoplasmic reticulum (ER) and Golgi localized enzymes are responsible to this template-independent glycan synthesis resulting glycoforms at each asparagine residues. The regulation mechanism such glycan synthesis remains largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: In order to investigate the relationship between glycan structure and protein conformation, we analyzed a glycoprotein of Drosophila melanogaster, chaoptin (Chp), which is localized in photoreceptor cells and is bound to the cell membrane via a glycosylphosphatidylinositol anchor. Detailed analysis based on mass spectrometry revealed the presence of 13 N-glycosylation sites and the composition of the glycoform at each site. The synthetic pathway of glycans was speculated from the observed glycan structures and the composition at each N-glycosylation site, where the presence of novel routes were suggested. The distribution of glycoforms on a Chp polypeptide suggested that various processing enzymes act on the exterior of Chp in the Golgi apparatus, although virtually no enzyme can gain access to the interior of the horseshoe-shaped scaffold, hence explaining the presence of longer glycans within the interior. Furthermore, analysis of Chp from a mutant (RNAi against dolichyl-phosphate alpha-d-mannosyltransferase), which affects N-glycan synthesis in the ER, revealed that truncated glycan structures were processed. As a result, the distribution of glycoforms was affected for the high-mannose-type glycans only, whereas other types of glycans remained similar to those observed in the control and wild-type. CONCLUSIONS/SIGNIFICANCE: These results indicate that glycan processing depends largely on the backbone structure of the parent polypeptide. The information we obtained can be applied to other members of the LRR family of proteins

Identification of Genes Required for Neural-Specific Glycosylation Using Functional Genomics

Glycosylation plays crucial regulatory roles in various biological processes such as development, immunity, and neural functions. For example, α1,3-fucosylation, the addition of a fucose moiety abundant in Drosophila neural cells, is essential for neural development, function, and behavior. However, it remains largely unknown how neural-specific α1,3-fucosylation is regulated. In the present study, we searched for genes involved in the glycosylation of a neural-specific protein using a Drosophila RNAi library. We obtained 109 genes affecting glycosylation that clustered into nine functional groups. Among them, members of the RNA regulation group were enriched by a secondary screen that identified genes specifically regulating α1,3-fucosylation. Further analyses revealed that an RNA–binding protein, second mitotic wave missing (Swm), upregulates expression of the neural-specific glycosyltransferase FucTA and facilitates its mRNA export from the nucleus. This first large-scale genetic screen for glycosylation-related genes has revealed novel regulation of fucTA mRNA in neural cells

In Vivo RNAi-Based Screens: Studies in Model Organisms

RNA interference (RNAi) is a technique widely used for gene silencing in organisms and cultured cells, and depends on sequence homology between double-stranded RNA (dsRNA) and target mRNA molecules. Numerous cell-based genome-wide screens have successfully identified novel genes involved in various biological processes, including signal transduction, cell viability/death, and cell morphology. However, cell-based screens cannot address cellular processes such as development, behavior, and immunity. Drosophila and Caenorhabditis elegans are two model organisms whose whole bodies and individual body parts have been subjected to RNAi-based genome-wide screening. Moreover, Drosophila RNAi allows the manipulation of gene function in a spatiotemporal manner when it is implemented using the Gal4/UAS system. Using this inducible RNAi technique, various large-scale screens have been performed in Drosophila, demonstrating that the method is straightforward and valuable. However, accumulated results reveal that the results of RNAi-based screens have relatively high levels of error, such as false positives and negatives. Here, we review in vivo RNAi screens in Drosophila and the methods that could be used to remove ambiguity from screening results

Localization of inositol 1,4,5-trisphosphate receptors in the rat kidney

Localization of inositol 1,4,5-trisphosphate receptors in the rat kidney. Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) serve as intracellular calcium release channels involved in signal transduction of various hormones in the kidney. Molecular cloning studies have shown that there are three types of IP3R, designated type 1, type 2, and type 3. To characterize their localizations in the rat kidney, we employed immunohistochemical studies using type-specific monoclonal antibodies that were raised against the 15 C-terminal amino acids of each type of IP3R. Type 1 was detected in glomerular mesangial cells and vascular smooth muscle cells. Type 2 was expressed exclusively in intercalated cells of collecting ducts from the cortex to the inner medulla. Type 3 was expressed in vascular smooth muscle cells, glomerular mesangial cells, and some cells of cortical collecting ducts, probably principal cells. As to the subcellular distribution, type 1 and type 2 showed a homogenous distribution in the cytoplasm, whereas type 3 was present mainly in the basolateral portion of the cytoplasm. These results indicate that IP3R isoforms were expressed in a cell-specific manner. The heterogeneous subcellular localizations among the IP3R types suggests compartmentalization of distinct IP3-sensitive Ca2+ pools

Balanced ubiquitylation and deubiquitylation of Frizzled regulate cellular responsiveness to Wg/Wnt

Wingless (Wg)/Wnt has been proposed to exert various functions as a morphogen depending on the levels of its signalling. Therefore, not just the concentration of Wg/Wnt, but also the responsiveness of Wg/Wnt-target cells to the ligand, must have a crucial function in controlling cellular outputs. Here, we show that a balance of ubiquitylation and deubiquitylation of the Wg/Wnt receptor Frizzled determines the cellular responsiveness to Wg/Wnt both in mammalian cells and in Drosophila, and that the cell surface level of Frizzled is regulated by deubiquitylating enzyme UBPY/ubiquitin-specific protease 8 (USP8). Although ubiquitylated Frizzled underwent lysosomal trafficking and degradation, UBPY/USP8-dependent deubiquitylation led to recycling of Frizzled to the plasma membrane, thereby elevating its surface level. Importantly, a gain and loss of UBPY/USP8 function led to up- and down-regulation, respectively, of canonical Wg/Wnt signalling. These results unveil a novel mechanism that regulates the cellular responsiveness to Wg/Wnt by controlling the cell surface level of Frizzled