ER Stress-Induced eIF2-alpha Phosphorylation Underlies Sensitivity of Striatal Neurons to Pathogenic Huntingtin

A hallmark of Huntington's disease is the pronounced sensitivity of striatal neurons to polyglutamine-expanded huntingtin expression. Here we show that cultured striatal cells and murine brain striatum have remarkably low levels of phosphorylation of translation initiation factor eIF2 alpha, a stress-induced process that interferes with general protein synthesis and also induces differential translation of pro-apoptotic factors. EIF2 alpha phosphorylation was elevated in a striatal cell line stably expressing pathogenic huntingtin, as well as in brain sections of Huntington's disease model mice. Pathogenic huntingtin caused endoplasmic reticulum (ER) stress and increased eIF2 alpha phosphorylation by increasing the activity of PKR-like ER-localized eIF2 alpha kinase (PERK). Importantly, striatal neurons exhibited special sensitivity to ER stress-inducing agents, which was potentiated by pathogenic huntingtin. We could strongly reduce huntingtin toxicity by inhibiting PERK. Therefore, alteration of protein homeostasis and eIF2 alpha phosphorylation status by pathogenic huntingtin appears to be an important cause of striatal cell death. A dephosphorylated state of eIF2 alpha has been linked to cognition, which suggests that the effect of pathogenic huntingtin might also be a source of the early cognitive impairment seen in patients

Trans-sialylation: a strategy used to incorporate sialic acid into oligosaccharides

Sialic acid, as a component of cell surface glycoconjugates, plays a crucial role in recognition events. Efficient synthetic methods are necessary for the supply of sialosides in enough quantities for biochemical and immunological studies. Enzymatic glycosylations obviate the steps of protection and deprotection of the constituent monosaccharides required in a chemical synthesis. Sialyl transferases with CMP-Neu5Ac as an activated donor were used for the construction of α2-3 or α2-6 linkages to terminal galactose or N-acetylgalactosamine units. trans-Sialidases may transfer sialic acid from a sialyl glycoside to a suitable acceptor and specifically construct a Siaα2-3Galp linkage. The trans-sialidase of Trypanosoma cruzi (TcTS), which fulfills an important role in the pathogenicity of the parasite, is the most studied one. The recombinant enzyme was used for the sialylation of β-galactosyl oligosaccharides. One of the main advantages of trans-sialylation is that it circumvents the use of the high energy nucleotide. Easily available glycoproteins with a high content of sialic acid such as fetuin and bovine κ-casein-derived glycomacropeptide (GMP) have been used as donor substrates. Here we review the trans-sialidase from various microorganisms and describe their application for the synthesis of sialooligosaccharides.Fil: De Lederkremer, Rosa M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Giorgi, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Agusti, Rosalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentin

Biosíntesis de glicoproteínas en Mucor Rouxii : Metilación in vivo de residuos de manosa en oligosacáridos con unión N-glicosídica a asparagina

Fil:Lederkremer, Gerardo Zelmar. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Protein Misfolding and ER Stress in Huntington's Disease

Increasing evidence in recent years indicates that protein misfolding and aggregation, leading to ER stress, are central factors of pathogenicity in neurodegenerative diseases. This is particularly true in Huntington's disease (HD), where in contrast with other disorders, the cause is monogenic. Mutant huntingtin interferes with many cellular processes, but the fact that modulation of ER stress and of the unfolded response pathways reduces the toxicity, places these mechanisms at the core and gives hope for potential therapeutic approaches. There is currently no effective treatment for HD and it has a fatal outcome a few years after the start of symptoms of cognitive and motor impairment. Here we will discuss recent findings that shed light on the mechanisms of protein misfolding and aggregation that give origin to ER stress in neurodegenerative diseases, focusing on Huntington's disease, on the cellular response and on how to use this knowledge for possible therapeutic strategies

Synthesis of the hexasaccharide from Trypanosoma cruzi mucins with the Galp(1 → 2)Galf unit constructed with a superarmed thiogalactopyranosyl donor

Hexasaccharide β-D-Galp-(1→ 2)-[β-D-Galp-(1 → 3)]-β-D-Galp-(1 → 6)-[β-D-Galp-(1 → 2)-β-D-Galf-(1 → 4)]-D-GlcNAc (1) was found O-linked in mucins of Trypanosoma cruzi epimastigotes and metacyclic trypomatigotes. Studies on the biological pathways and functionalities of the mucin oligosaccharides are prompted in order to understand the interactions of these molecules with the insect host. Trisaccharide constituent β-D-Galp-(1 → 2)-β-D-Galf-(1 → 4)-D-GlcNAc was constructed from the reducing to the non-reducing end. We discuss the difficulties to introduce a Galp unit at the O-2 position of a partially protected galactofuranosyl unit which were overcome using an anchimerically superarmed donor. By this route and employing a [3 + 3] nitrilium convergent approach hexasaccharide 1 was synthesized in moderate yield.Fil: Kashiwagi, Gustavo Adolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; ArgentinaFil: Cori Calizaya, Carmen Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; ArgentinaFil: de Lederkremer, Rosa M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; ArgentinaFil: Gallo, Carola. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentin

Combined inhibitor free-energy landscape and structural analysis reports on the mannosidase conformational coordinate

Mannosidases catalyze the hydrolysis of a diverse range of polysaccharides and glycoconjugates, and the various sequence-based mannosidase families have evolved ingenious strategies to overcome the stereoelectronic challenges of mannoside chemistry. Using a combination of computational chemistry, inhibitor design and synthesis, and X-ray crystallography of inhibitor/enzyme complexes, it is demonstrated that mannoimidazole-type inhibitors are energetically poised to report faithfully on mannosidase transition-state conformation, and provide direct evidence for the conformational itinerary used by diverse mannosidases, including β-mannanases from families GH26 and GH113. Isofagomine-type inhibitors are poor mimics of transition-state conformation, owing to the high energy barriers that must be crossed to attain mechanistically relevant conformations, however, these sugar-shaped heterocycles allow the acquisition of ternary complexes that span the active site, thus providing valuable insight into active-site residues involved in substrate recognition

A Secreted Form of the Asialoglycoprotein Receptor, sH2a, as a Novel Potential Noninvasive Marker for Liver Fibrosis

Background and Aim: The human asialoglycoprotein receptor is a membrane heterooligomer expressed exclusively in hepatocytes. A soluble secreted form, sH2a, arises, not by shedding at the cell surface, but by intracellular cleavage of its membrane-bound precursor, which is encoded by an alternatively spliced form of the receptor H2 subunit. Here we determined and report that sH2a, present at constant levels in serum from healthy individuals is altered upon liver fibrosis, reflecting the status of hepatocyte function. Methods: We measured sH2a levels in serum using a monoclonal antibody and an ELISA assay that we developed, comparing with routine liver function markers. We compared blindly pretreatment serum samples from a cohort of 44 hepatitis C patients, which had METAVIR-scored biopsies, with 28 healthy individuals. Results: sH2a levels varied minimally for the healthy individuals (150621 ng/ml), whereas the levels deviated from this normal range increasingly in correlation with fibrosis stage. A simple algorithm combining sH2a levels with those of alanine aminotransferase allowed prediction of fibrosis stage, with a very high area under the ROC curve of 0.86

Sphingolipids of the mycopathogen Sporothrix schenckii: identification of a glycosylinositol phosphorylceramide with novel core GlcNH(2)alpha 1 -> 2Ins motif

Acidic glycosphingolipid components were extracted from the yeast form of the dimorphic mycopathogen Sporothrix schenckii. Two minor and the major fraction from the yeast form (Ss-Y1, -Y2, and -Y6. respectively) have been isolated. By a combination of 1- and 2-D H-1-nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and gas chromatography/mass spectrometry (GC/MS). Ss-Y6 was determined to be triglycosylinositol phosphorylceramide with a novel glycan structure, Man alpha1 --> 3Man alpha1 --> 6GlcNH(2)alpha1 --> 2Ins1-P-1Cer (where Ins = myo-inositol, P = phosphodiester), While the GlcNH(2)alpha1 --> 6Ins1-P-motif is found widely distributed in eukaryotic GPI anchors, the linkage GlcNH(2)alpha1 --> 2Insl-P- has not been previously observed in any glycolipid, Ss-Y1 and Ss-Y2 were both found to have the known glycan structure Man alpha1 --> 3Man alpha1 --> 2Ins1-P-1Cer, Together with the results of a prior study [Toledo et al, (2001) Biochem, Biophys. Res. Commun, 280, 19-24] which showed that the mycelium form expresses GIPCs with the structures Man alpha1 --> 6Ins1-P-1Cer and Man alpha1 --> 3Man alpha1 --> 6Ins1-P-1Cer, these results demonstrate that S, schenckii can synthesize glycosylinositol phosphorylceramides with at least three different core Linkages, (C) 2001 Federation of European Biochemical Societies, Published by Elsevier Science B.V. All rights reserved.Univ Georgia, Dept Biochem & Mol Biol, Athens, GA 30602 USAUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biochem, BR-04023900 São Paulo, BrazilUniv Georgia, Complex Carbohydrate Res Ctr, Athens, GA 30602 USAUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biochem, BR-04023900 São Paulo, BrazilWeb of Scienc