Catalytic Promiscuity of O-GlcNAc Transferase Enables Unexpected Metabolic Engineering of Cytoplasmic Proteins with 2-Azido-2-deoxy-glucose

O-GlcNAc transferase (OGT) catalyzes the installation of N-acetylglucosamine (GlcNAc) O-linked to nucleocytoplasmic proteins (O-GlcNAc) within multicellular eukaryotes. OGT shows surprising tolerance for structural changes in the sugar component of its nucleotide sugar donor substrate, uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Here, we find that OGT uses UDP-glucose to install O-linked glucose (O-Glc) onto proteins only 25-fold less efficiently than O-GlcNAc. Spurred by this observation, we show that OGT transfers 2-azido-2-deoxy-d-glucose (GlcAz) in vitro from UDP-GlcAz to proteins. Further, feeding cells with per-O-acetyl GlcAz (AcGlcAz), in combination with inhibition or inducible knockout of OGT, shows OGT-dependent modification of nuclear and cytoplasmic proteins with O-GlcAz as detected using microscopy, immunoblot, and proteomics. We find that O-GlcAz is reversible within cells, and an unidentified cellular enzyme exists to cleave O-Glc that can also process O-GlcAz. We anticipate that AcGlcAz will prove to be a useful tool to study the O-GlcNAc modification. We also speculate that, given the high concentration of UDP-Glc within certain mammalian tissues, O-Glc may exist within mammals and serve as a physiologically relevant modification

Fault Activity Control on Upper Jurassic-Early Cretaceous Wedges in the Snorre Fault Block and its Surrounding Area

Master's thesis in Petroleum Geosciences EngineeringIn this study, the question” how fault activity controlled the infill and geometry of the Upper Jurassic-Lower Cretaceous wedges in the Snorre Fault Block and surrounding area” is addressed. To achieve the goal, three-dimensional seismic data, core description and well log data are used. The study improves the understanding of the tectonostratigraphy evolution of the wedges in the Snorre Area. The result of the study shows that two wedges are present in the hanging wall of main faults in the study area. The main wedge is located in a large area along the hanging wall of Inner Snorre Fault. Well correlation shows that the Upper Jurassic-Lower Cretaceous stratigraphy of the wedges includes the Heather Formation, the Draupne Formation and the Cromer Knoll Group. Seismic facies analysis, time-thickness and time-structural maps reveals that development of the initially segmented major faults of the study area greatly influenced the internal character, the thickness and the geometry of the wedges along their strike and in perpendicular direction to the strike. In addition, fault activity led to rotation and tilting of hanging wall and footwall areas. The study shows that the Upper Jurassic-Lower Cretaceous succession of the wedges have possible potential for the hydrocarbon exploration purposes

Mapping the enzyme specificities of intestinal maltase-glucoamylase and sucrase-isomaltase

In humans, maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) are the small intestinal glucosidases responsible for catalyzing the last glucose-releasing step in starch digestion. MGAM and SI are each composed of duplicated catalytic domains, N-terminal membrane domains (ntMGAM and ntSI) and C-terminal luminal domains (ctMGAM and ctSI). They display complementary substrate specificities for the mixture of short, linear and branched oligosaccharide substrates that typically make up terminal starch-digestion products. As they are involved in the breakdown of dietary starch and sugars into glucose, regulating their activities with α-glucosidase inhibitors is an attractive approach to control blood glucose levels for the prevention and treatment of type-2 diabetes.This thesis work deals with mapping (determination of selectivity and specificity) of MGAM and SI with synthetic inhibitors. The syntheses and enzymatic evaluation of sulfonium-ion glucosidase inhibitors, with potent inhibitory activities against intestinal glucosidases are the main topics of this thesis. First, an alternative route for the synthesis of kotalanol, a naturally-occurring sulfonium-ion glucosidase inhibitor isolated from Salacia reticulata, and its 6\u27-epimer are described, and the inhibitory activities of these compounds against ntMGAM are reported. Second, the total syntheses of de-O-sulfonated ponkoranol, another naturally-occurring sulfonium-ion glucosidase inhibitor isolated from the same species, its 5\u27-epimer, and their selenium analogues are described. The synthetic route is also extended to obtain 3\u27-O-methylponkoranol. The inhibitory activities of these latter compounds against the four human intestinal glucosidase enzymes, ntMGAM, ctMGAM, ntSI, and ctSI are examined. Finally, from the structural studies of ntMGAM, it was postulated that ctMGAM might have an extended binding site compared to ntMGAM, which favours binding of longer inhibitors such as acarbose (an antidiabetic drug that is currently in use for the treatment of type-2 diabetes). Based on this difference, the syntheses of candidate inhibitors containing maltose extensions at 3\u27- and 5\u27- of de-O-sulfonated ponkoranol are described. The inhibition of maltose hydrolysis suggests that selective inhibition of one enzyme unite over the others is possible despite relatively small structural changes in the inhibitor. This panel of inhibitors can now be used to turn off certain enzymes while probing the action of others with respect to starch digestion

Taxonomical considerations and molecular phylogeny of the closely related genera Bitylenchus, Sauertylenchus and Tylenchorhynchus (Nematoda: Telotylenchinae), with one new and four known species from Iran

During several nematological surveys in cultivated and natural habitats in Khuzestan and Zanjan provinces of Iran, a new species, Bitylenchus parvulus n. sp., two new records for Iran – namely, Tylenchorhynchus agri and Tylenchorhynchus graciliformis – and a population of Bitylenchus parvus and one of Sauertylenchus maximus were recovered and characterized based upon morphological and molecular approaches. The new species is characterized by lip region with five to seven annuli, stylet 17.7 (17.0–18.5) μm long, sub-cylindrical tail narrowing abruptly near terminus giving a bluntly digitate shape to the tail tip, cuticle near anterior part of vulva wrinkled and post-rectal sac occupies whole of tail cavity. The phylogenetic analyses were carried out using molecular data from D2–D3 expansion segments of large ribosomal subunit (28S rRNA) for all studied species and the partial small ribosomal subunit (18S rRNA) for the new species. The representatives of Bitylenchus and Sauertylenchus formed distinct clades from Tylenchorhynchus members, supporting the hypothesis in which Bitylenchus and Sauertylenchus could be considered as valid genera, but rejecting the ‘large-genus’ concept for Tylenchorhynchus. Also, Sauertylenchus ibericus was proposed as a junior synonym of S. maximus based on the results from morphological and phylogenetic analysis. Furthermore, an identification key for all known species included in the three genera Bitylenchus, Tylenchorhynchus and Sauertylenchus is presented herein. The number of transverse annuli on the lip region and presence/absence of post-rectal sac were considered as the main diagnostic characters for classifying the species into seven groups, and other morphological and morphometric characters were subsequently used for distinguishing species in each group.J.E.P.R. acknowledges the Spanish Ministry of Economy and Competitiveness for the ‘Ramon y Cajal’ Fellowship RYC-2017-22228

Direct One-Step Fluorescent Labeling of O-GlcNAc-Modified Proteins in Live Cells Using Metabolic Intermediates

The modification of proteins with O-linked N-acetylglucosamine (O-GlcNAc) by the enzyme O-GlcNAc transferase (OGT) has emerged as an important regulator of cellular physiology. Metabolic labeling strategies to monitor O-GlcNAcylation in cells has proven of great value for uncovering the molecular roles of O-GlcNAc. These strategies rely on two-step labeling procedures, which limits the scope of experiments that can be performed. Here we report on the creation of fluorescent uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) analogues in which the N-acyl group of glucosamine is modified with a suitable linker and fluorophore. Using human OGT we show these donor sugar substrates permit direct monitoring of OGT activity on protein substrates in vitro. We show that feeding cells with a corresponding fluorescent metabolic precursor for the last step of the hexosamine biosynthetic pathway (HBP) leads to its metabolic assimilation and labeling of O-GlcNAcylated proteins within live cells. This one-step metabolic feeding strategy permits labeling of O-GlcNAcylated proteins with a fluorescent glucosamine-nitrobenzoxadiazole (GlcN-NBD) conjugate that accumulates in a time and dose dependent manner. Since no genetic engineering of cells is required, we anticipate this strategy should be generally amenable to studying the roles of O-GlcNAc in cellular phys-iology as well as to gain an improved understanding of the regulation of OGT within cells. The further expansion of this one-step in-cell labeling strategy should enable performing a range of experiments including two-colour pulse chase ex-periments and monitoring OGT activity on specific protein substrate in live cells

The CNS in inbred transgenic models of 4-repeat Tauopathy develops consistent tau seeding capacity yet focal and diverse patterns of protein deposition

Abstract Background MAPT mutations cause neurodegenerative diseases such as frontotemporal dementia but, strikingly, patients with the same mutation may have different clinical phenotypes. Methods Given heterogeneities observed in a transgenic (Tg) mouse line expressing low levels of human (2 N, 4R) P301L Tau, we backcrossed founder stocks of mice to C57BL/6Tac, 129/SvEvTac and FVB/NJ inbred backgrounds to discern the role of genetic versus environmental effects on disease-related phenotypes. Results Three inbred derivatives of a TgTauP301L founder line had similar quality and steady-state quantity of Tau production, accumulation of abnormally phosphorylated 64–68 kDa Tau species from 90 days of age onwards and neuronal loss in aged Tg mice. Variegation was not seen in the pattern of transgene expression and seeding properties in a fluorescence-based cellular assay indicated a single “strain” of misfolded Tau. However, in other regards, the aged Tg mice were heterogeneous; there was incomplete penetrance for Tau deposition despite maintained transgene expression in aged animals and, for animals with Tau deposits, distinctions were noted even within each subline. Three classes of rostral deposition in the cortex, hippocampus and striatum accounted for 75% of pathology-positive mice yet the mean ages of mice scored as class I, II or III were not significantly different and, hence, did not fit with a predictable progression from one class to another defined by chronological age. Two other patterns of Tau deposition designated as classes IV and V, occurred in caudal structures. Other pathology-positive Tg mice of similar age not falling within classes I-V presented with focal accumulations in additional caudal neuroanatomical areas including the locus coeruleus. Electron microscopy revealed that brains of Classes I, II and IV animals all exhibit straight filaments, but with coiled filaments and occasional twisted filaments apparent in Class I. Most strikingly, Class I, II and IV animals presented with distinct western blot signatures after trypsin digestion of sarkosyl-insoluble Tau. Conclusions Qualitative variations in the neuroanatomy of Tau deposition in genetically constrained slow models of primary Tauopathy establish that non-synchronous, focal events contribute to the pathogenic process. Phenotypic diversity in these models suggests a potential parallel to the phenotypic variation seen in P301L patients

Evaluation of BIRC6 Expression in Oral Squamous Cell Carcinoma, Epithelial Dysplasia, Lichen Planus with and without Dysplasia, and Hyperkeratosis

Background: BIRC6, regarded as the pivotal member of the inhibitor of the apoptosis (IAP) family, has been linked to the development of different types of cancer in humans. The objective of this study was to examine the expression of BIRC6 in various oral conditions, including OLP with dysplasia (OLPD), hyperkeratosis (HK), OLP, epithelial dysplasia (ED), and oral squamous cell carcinoma (OSCC), to investigate its potential involvement in the development of OSCC and the pathogenesis and malignant transformation of OLP, which is known as a precancerous condition. Methods: In this retrospective cross-sectional study, 99 cases, consisting of 19 cases of OSCC, 21 cases of ED, 23 cases of OLP, 20 cases of OLPD, and 16 cases of HK as the control group, were investigated regarding BIRC6 expression by immunohistochemical staining. After that, the immunohistochemical expression of BIRC6 in the epithelial compartment was analyzed. Statistical analysis was performed to investigate the relationship between the expression of BIRC6 and clinicopathological variables. The statistical analysis of the data involved the use of one-way ANOVA, post hoc Tukey, Kruskal–Wallis, Chi-square, Spearman’s correlation, and Mann–Whitney tests. The significance level was set at p p = 0.00). The average total staining score was remarkably greater in OSCC and dysplastic lesions compared with HK (p = 0.00, p = 0.00). Conclusions: While the current study suggested that BIRC6 may play a role in the tumorigenesis of OSCC, its role in the malignant transformation of OLP has yet to be definitively established

Additional file 9: Figure S20. of The CNS in inbred transgenic models of 4-repeat Tauopathy develops consistent tau seeding capacity yet focal and diverse patterns of protein deposition

Undigested P3 fraction assessed with CP13 and PHF1 antibodies. A schematic of antibody epitopes is presented. Blot represents P3 fraction from 3 animals of classes I, II and IV. Class I mice at ages 587, 662, and 646 days left to right, class II animals at ages 735, 592, and 658 days left to right, and class IV mice at ages 530, 466, and 639 days left to right. For both blots, 5 μg of total protein was loaded on the gel. Antibody: CP13 (1/500) and PHF1 (1/500). (TIFF 199 kb

Additional file 3: Figures S2-S5. of The CNS in inbred transgenic models of 4-repeat Tauopathy develops consistent tau seeding capacity yet focal and diverse patterns of protein deposition

Class I mice. These figures represent the counterparts of Fig. 4. stained with MC1, CP27, RZ3 and PHF1 antibodies, respectively. (ZIP 2909 kb