Several natural phytochemicals from Chinese traditional fermented food-pickled Raphanus sativus L.: Purification and characterization.

In this study, we aimed to isolate and identify the bioactive compounds from 5-year pickled radish. The pickled radish was extracted with methanol or ethyl acetate. Sephadex LH-20, normal phase and reverse phase silica gel column chromatography were used for separation and purification, combined with thin layer chromatography (TLC), high performance liquid chromatography (HPLC), electrospray mass spectrometry (ESI-MS), nuclear magnetic resonance spectroscopy (NMR) technology for structural identification. The results showed that 6 compounds were separated and purified from methanol and ethyl acetate extracts of 5-year-old pickled radish. The structures were identified as 5-hydroxymethylfurfural, ß-sitosterol, ß-sitosterol-3-O-glucose glycosides, α-linolenic acid, 1-monopalmitin and chaenomic acid A. Using molecular docking, it was determined that ß-sitosterol and its derivative ß-sitosterol-3-O-glucose glycosides have high affinity for five antioxidant enzymes, and there were multiple hydrogen bonds between them. These results indicated that pickled radishes might be used as an important source of natural chemical substances.

Other Types of Glycosylation.

O-Linked glycosylation such as O-fucose, O-glucose, and O-N-acetylglucosamine are considered to be unusual. As suggested by the high levels of evolutional conservation, these O-glycans are fundamentally important for life. In the last two decades, our understanding of the importance of these glycans has greatly advanced. In particular, identification of the glycosyltransferases responsible for the biosynthesis of these glycans has accelerated basic research on the functional significance and molecular mechanisms by which these O-glycans regulate protein functions as well as clinical research on human diseases due to changes in these types of O-glycosylation. Notably, Notch receptor signaling is modified with and regulated by these types of O-glycans. Here, we summarize the current view of the structures and the significance of these O-glycans mainly in the context of Notch signaling regulation and human diseases.

POGLUT2 and POGLUT3 O-glucosylate multiple EGF repeats in fibrillin-1, -2, and LTBP1 and promote secretion of fibrillin-1.

Fibrillin-1 (FBN1) is the major component of extracellular matrix microfibrils, which are required for proper development of elastic tissues, including the heart and lungs. Through protein-protein interactions with latent transforming growth factor (TGF) ß-binding protein 1 (LTBP1), microfibrils regulate TGF-ß signaling. Mutations within the 47 epidermal growth factor-like (EGF) repeats of FBN1 cause autosomal dominant disorders including Marfan Syndrome, which is characterized by disrupted TGF-ß signaling. We recently identified two novel protein O-glucosyltransferases, Protein O-glucosyltransferase 2 (POGLUT2) and 3 (POGLUT3), that modify a small fraction of EGF repeats on Notch. Here, using mass spectral analysis, we show that POGLUT2 and POGLUT3 also modify over half of the EGF repeats on FBN1, fibrillin-2 (FBN2), and LTBP1. While most sites are modified by both enzymes, some sites show a preference for either POGLUT2 or POGLUT3. POGLUT2 and POGLUT3 are homologs of POGLUT1, which stabilizes Notch proteins by addition of O-glucose to Notch EGF repeats. Like POGLUT1, POGLUT2 and 3 can discern a folded versus unfolded EGF repeat, suggesting POGLUT2 and 3 are involved in a protein folding pathway. In vitro secretion assays using the N-terminal portion of recombinant FBN1 revealed reduced FBN1 secretion in POGLUT2 knockout, POGLUT3 knockout, and POGLUT2 and 3 double-knockout HEK293T cells compared with wild type. These results illustrate that POGLUT2 and 3 function together to O-glucosylate protein substrates and that these modifications play a role in the secretion of substrate proteins. It will be interesting to see how disease variants in these proteins affect their O-glucosylation.

Flavonoid compounds of buah merah (Pandanus conoideus Lamk) as a potent SARS-CoV-2 main protease inhibitor: in silico approach.

BACKGROUND: COVID19 is a global pandemic that threatens all nations. As there is no effective antiviral drug for COVID19, we examined the potency of natural ingredients against the SARS-CoV-2 main protease (PDB ID 6YNQ). Buah merah is a typical fruit from Papua, Indonesia, which is known to contain high levels of carotenoids and flavonoids. The contents have been proven to be effective as antiparasitic and anti-HIV. An in silico approach to 16 metabolites of buah merah (Pandanus conoideus Lamk) was carried out using AutoDock Vina. Furthermore, the study of the dynamics of ligand-protein interactions was carried out using CABS Flex 2.0 server to determine the test ligand and receptor complexes' stability. ADMET prediction was also carried out to study the pharmacokinetic profile of potential antiviral candidates. RESULT: The docking results showed that 3 of the 16 buah merah metabolites were potent inhibitors against the SARS-CoV-2 main protease. The flavonoid compounds are quercetin 3'-glucoside, quercetin 3-O-glucose, and taxifolin 3-O-α-arabinopyranose with a binding affinity of - 9.7, - 9.3, and - 8.8, respectively, with stable ligand-protein complex. ADMET study shows that the three compounds are easily dissolved, easily absorbed orally and topically, have a high unbound fraction, low toxicity, and non-irritant. CONCLUSION: We conclude that quercetin 3'-glucoside, quercetin 3-O-glucose, and taxifolin 3-O-α-arabinopyranose can be used and improved as potential anti-SARS-CoV-2 agents in further study.

Diseases related to Notch glycosylation.

The Notch receptors are a family of transmembrane proteins that mediate direct cell-cell interactions and control numerous cell-fate specifications in humans. The extracellular domains of mammalian Notch proteins contain 29-36 tandem epidermal growth factor-like (EGF) repeats, most of which have O-linked glycan modifications: O-glucose added by POGLUT1, O-fucose added by POFUT1 and elongated by Fringe enzymes, and O-GlcNAc added by EOGT. The extracellular domain is also N-glycosylated. Mutations in the glycosyltransferases modifying Notch have been identified in several diseases, including Dowling-Degos Disease (haploinsufficiency of POFUT1 or POGLUT1), a form of limb-girdle muscular dystrophy (autosomal recessive mutations in POGLUT1), Spondylocostal Dysostosis 3 (autosomal recessive mutations in LFNG), Adams-Oliver syndrome (autosomal recessive mutations in EOGT), and some cancers (amplification, gain or loss-of-function of POFUT1, Fringe enzymes, POGLUT1, MGAT3). Here we review the characteristics of these diseases and potential molecular mechanisms.

Multiple roles for O-glycans in Notch signalling.

Notch signalling regulates a plethora of developmental processes and is also essential for the maintenance of tissue homeostasis in adults. Therefore, fine-tuning of Notch signalling strength needs to be tightly regulated. Of key importance for the regulation of Notch signalling are O-fucose, O-GlcNAc and O-glucose glycans attached to the extracellular domain of Notch receptors. The EGF repeats of the Notch receptor extracellular domain harbour consensus sites for addition of the different types of O-glycan to Ser or Thr, which takes place in the endoplasmic reticulum. Studies from Drosophila to mammals have demonstrated the multifaceted roles of O-glycosylation in regulating Notch signalling. O-glycosylation modulates different aspects of Notch signalling including recognition by Notch ligands, the strength of ligand binding, Notch receptor trafficking, stability and activation at the cell surface. Defects in O-glycosylation of Notch receptors give rise to pathologies in humans. This Review summarizes the nature of the O-glycans on Notch receptors and their differential effects on Notch signalling.

Two novel protein O-glucosyltransferases that modify sites distinct from POGLUT1 and affect Notch trafficking and signaling.

The Notch-signaling pathway is normally activated by Notch-ligand interactions. A recent structural analysis suggested that a novel O-linked hexose modification on serine 435 of the mammalian NOTCH1 core ligand-binding domain lies at the interface with its ligands. This serine occurs between conserved cysteines 3 and 4 of Epidermal Growth Factor-like (EGF) repeat 11 of NOTCH1, a site distinct from those modified by protein O-glucosyltransferase 1 (POGLUT1), suggesting that a different enzyme is responsible. Here, we identify two novel protein O-glucosyltransferases, POGLUT2 and POGLUT3 (formerly KDELC1 and KDELC2, respectively), which transfer O-glucose (O-Glc) from UDP-Glc to serine 435. Mass spectrometric analysis of NOTCH1 produced in HEK293T cells lacking POGLUT2, POGLUT3, or both genes showed that either POGLUT2 or POGLUT3 can add this novel O-Glc modification. EGF11 of NOTCH2 does not have a serine residue in the same location for this O-glucosylation, but EGF10 of NOTCH3 (homologous to EGF11 in NOTCH1 and -2) is also modified at the same position. Comparison of the sites suggests a consensus sequence for modification. In vitro assays with POGLUT2 and POGLUT3 showed that both enzymes modified only properly folded EGF repeats and displayed distinct acceptor specificities toward NOTCH1 EGF11 and NOTCH3 EGF10. Mutation of the O-Glc modification site on EGF11 (serine 435) in combination with sensitizing O-fucose mutations in EGF8 or EGF12 affected cell-surface presentation of NOTCH1 or reduced activation of NOTCH1 by Delta-like1, respectively. This study identifies a previously undescribed mechanism for fine-tuning the Notch-signaling pathway in mammals.

Regulation of Notch Function by O-Glycosylation.

The Notch receptor initiates a unique intercellular signaling pathway that is evolutionarily conserved across all metazoans and contributes to the development and maintenance of numerous tissues. Consequently, many diseases result from aberrant Notch signaling. Emerging roles for Notch in disease are being uncovered as studies reveal new information regarding various components of this signaling pathway. Notch activity is regulated at several levels, but O-linked glycosylation of Epidermal Growth Factor (EGF) repeats in the Notch extracellular domain has emerged as a major regulator that, depending on context, can increase or decrease Notch activity. Three types of O-linked glycosylation occur at consensus sequences found within the EGF repeats of Notch: O-fucosylation, O-glucosylation, and O-GlcNAcylation. Recent studies have investigated the site occupancy of these types of glycosylation and also defined specific roles for these glycans on Notch structure and function. Nevertheless, there are many functional aspects to each type of O-glycosylation that remain unclear. Here, we will discuss molecular mechanisms of how O-glycosylation regulates Notch signaling and describe disorders associated with defects in Notch O-glycosylation.

O-Glycosylation modulates the stability of epidermal growth factor-like repeats and thereby regulates Notch trafficking.

Glycosylation in the endoplasmic reticulum (ER) is closely associated with protein folding and quality control. We recently described a non-canonical ER quality control mechanism for folding of thrombospondin type 1 repeats by protein O-fucosyltransferase 2 (POFUT2). Epidermal growth factor-like (EGF) repeats are also small cysteine-rich protein motifs that can be O-glycosylated by several ER-localized enzymes, including protein O-glucosyltransferase 1 (POGLUT1) and POFUT1. Both POGLUT1 and POFUT1 modify the Notch receptor on multiple EGF repeats and are essential for full Notch function. The fact that POGLUT1 and POFUT1 can distinguish between folded and unfolded EGF repeats raised the possibility that they participate in a quality control pathway for folding of EGF repeats in proteins such as Notch. Here, we demonstrate that cell-surface expression of endogenous Notch1 in HEK293T cells is dependent on the presence of POGLUT1 and POFUT1 in an additive manner. In vitro unfolding assays reveal that addition of O-glucose or O-fucose stabilizes a single EGF repeat and that addition of both O-glucose and O-fucose enhances stability in an additive manner. Finally, we solved the crystal structure of a single EGF repeat covalently modified by a full O-glucose trisaccharide at 2.2 Å resolution. The structure reveals that the glycan fills up a surface groove of the EGF with multiple contacts with the protein, providing a chemical basis for the stabilizing effects of the glycans. Taken together, this work suggests that O-fucose and O-glucose glycans cooperatively stabilize individual EGF repeats through intramolecular interactions, thereby regulating Notch trafficking in cells.

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.

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.

Mitochondrial dysfunction has divergent, cell type-dependent effects on insulin action.

The contribution of mitochondrial dysfunction to insulin resistance is a contentious issue in metabolic research. Recent evidence implicates mitochondrial dysfunction as contributing to multiple forms of insulin resistance. However, some models of mitochondrial dysfunction fail to induce insulin resistance, suggesting greater complexity describes mitochondrial regulation of insulin action. We report that mitochondrial dysfunction is not necessary for cellular models of insulin resistance. However, impairment of mitochondrial function is sufficient for insulin resistance in a cell type-dependent manner, with impaired mitochondrial function inducing insulin resistance in adipocytes, but having no effect, or insulin sensitising effects in hepatocytes. The mechanism of mitochondrial impairment was important in determining the impact on insulin action, but was independent of mitochondrial ROS production. These data can account for opposing findings on this issue and highlight the complexity of mitochondrial regulation of cell type-specific insulin action, which is not described by current reductionist paradigms.

Avaliação do potencial antimicrobiano de Plinia glomerata (Myrtaceae) / Evaluation of the antimicrobial effects of Plinia glomerata (Myrtaceae)

A planta Plinia glomerata (Myrtaceae), popularmente conhecida como cabeludinha ou " jabuticaba-amarela" , ocorre amplamente no sul do Brasil e é cultivada como ornamental e frutífera comestível. O presente trabalho avaliou as propriedades antimicrobianas dos extratos, frações e substâncias puras isoladas da planta contra bactérias e fungos patogênicos. As concentrações inibitórias mínimas (CIM) foram determinadas através do método de diluição em ágar. As frações acetato de etila (AE) e fração aquosa (AQ) demonstraram a melhor atividade contra Staphylococcus aureus. A fração AQ também foi efetiva contra Escherichia coli. As substâncias puras, ácido 3,4,3'-trimetóxi-flavelágico-4'-O-glicosídeo e ácido 3,4,3'-trimetóxi-flavelágico foram inativas até a concentração de 500 µg/mL contra os microrganismos testados. Em relação aos resultados antifúngicos, os extratos metanólico (EM) e acetônico (EA) e a fração aquosa (AQ) mostraram boa atividade somente contra dermatófitos. Os resultados obtidos permitem concluir que a Plinia glomerata possui princípios ativos com ação antimicrobiana, sugerindo que outras substâncias da planta estão agindo contra os microrganismos indicados ou a existência de efeitos sinérgicos.

Plinia glomerata, commonly known as " yellow jaboticaba" or " cabelluda" occurs widely in south of Brazil. This work evaluated the antimicrobial properties of the extracts, fractions and isolated compounds of this plant against pathogenic microorganisms, bacteria and fungi. The minimal inhibitory concentration (MIC) was determined through the method of dilution in agar. The ethyl acetate (EtOAc) and aqueous fractions demonstrated the best activity against Staphylococcus aureus. The aqueous fraction also exhibited good activity against Escherichia coli. The pure substances 3,4,3'-trimethoxy-flavellagic-4'-O-glucose acid and 3,4,3'-trimethoxy-flavellagic acid were inactive up to 500 µg/mL. Regarding results against fungi, EA and EM extracts as well as AQ fraction showed good activity only against dermatophytes. The results suggest that P. glomerata produces active principles with antimicrobial activity, suggesting that other substances from the plant are acting against the microorganisms or the existence of synergic effects.