A novel approach for the characterisation of proteoglycans and biosynthetic enzymes in a snail model

Proteoglycans encompass a heterogeneous group of glycoconjugates where proteins are substituted with linear, highly negatively charged glycosaminoglycan chains. Sulphated glycosaminoglycans are ubiquitous to the animal kingdom of the Eukarya domain. Information on the distribution and characterisation of proteoglycans in invertebrate tissues is limited and restricted to a few species. By the use of multidimensional protein identification technology and immunohistochemistry, this study shows for the first time the presence and tissue localisation of different proteoglycans, such as perlecan, aggrecan, and heparan sulphate proteoglycan, amongst others, in organs of the gastropoda Achatina fulica. Through a proteomic analysis of Golgi proteins and immunohistochemistry of tissue sections, we detected the machinery involved in glycosaminoglycan biosynthesis, related to polymer formation (polymerases), as well as secondary modifications (sulphation and uronic acid epimerization). Therefore, this work not only identifies both the proteoglycan core proteins and glycosaminoglycan biosynthetic enzymes in invertebrates but also provides a novel method for the study of glycosaminoglycan and proteoglycan evolution. (C) 2011 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)NIHUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilUniv Texas El Paso, Dept Biol Sci, Border Biomed Res Ctr, El Paso, TX 79912 USAUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilNIH: 2G12RR008124-16A1NIH: 2G12RR008124-16A1S1Web of Scienc

Multistep Regulation of TXNIP Expression under Oxidative Stress Conditions: A role for microRNAs

Universidade Federal de São Paulo, São Paulo, BrazilKyoto Univ, Kyoto 6068501, JapanUniversidade Federal de São Paulo, São Paulo, BrazilWeb of Scienc

Nitrosative/Oxidative Stress Conditions Regulate Thioredoxin-Interacting Protein (TXNIP) Expression and Thioredoxin-1 (TRX-1) Nuclear Localization

<div><p>Thioredoxin (TRX-1) is a multifunctional protein that controls the redox status of other proteins. TRX-1 can be found in the extracellular milieu, cytoplasm and nucleus, and it has distinct functions in each environment. Previously, we studied the intracellular localization of TRX-1 and its relationship with the activation of the p21Ras - ERK1/2 MAP Kinases signaling pathway. In situations where this pathway was activated by stress conditions evoked by a nitrosothiol, S-nitroso-N-acetylpenicillamine (SNAP), TRX-1 accumulated in the nuclear compartment due to nitrosylation of p21Ras and activation of downstream ERK1/2 MAP kinases. Presently, we demonstrate that ERK1/2 MAP Kinases activation and spatial distribution within cells trigger TRX-1 nuclear translocation through down-regulation of the physiological inhibitor of TRX-1, Thioredoxin Interacting Protein (TXNIP). Once activated by the oxidants, SNAP and H₂O₂, the ERK1/2 MAP kinases migrate to the nucleus. This is correlated with down-regulation of TXNIP. In the presence of the MEK inhibitors (PD98059 or UO126), or in cells transfected with the Protein Enriched in Astrocytes (PEA-15), a cytoplasmic anchor of ERK1/2 MAP kinases, TRX-1 nuclear migration and TXNIP down-regulation are no longer observed in cells exposed to oxidants. On the other hand, over-expression of TXNIP abolishes nuclear migration of TRX-1 under nitrosative/oxidative stress conditions, whereas gene silencing of TXNIP facilitates nuclear migration even in the absence of stress conditions. Studies based on the TXNIP promoter support this regulation. In conclusion, changes in TRX-1 compartmentalization under nitrosative/oxidative stress conditions are dependent on the expression levels of TXNIP, which are regulated by cellular compartmentalization and activation of the ERK1/2 MAP kinases. </p> </div

Schematic representation of TRX-1 and the ERK1/2 MAP kinases nuclear translocation stimulated by nitrosative/oxidative stress conditions.

<p>(A) The Ras-Raf-MEK-ERK 1/2 signaling pathway is activated in HeLa wild-type cells after exposure to 0.5 mM SNAP or 0.5 mM H₂O₂ (1). Under these conditions expression of TXNIP is down-regulated at mRNA and protein levels (2) and TRX-1 and the ERK 1/2 MAP kinases, independently of each other, migrate to the nuclear compartment (3). (B) Nuclear migration of TRX-1 and the ERK 1/2 MAP kinases is prevented in three situations: HeLa cells pre-incubated with the MEK inhibitor PD98059 (4), HeLa cells over-expressing the cytoplasmic anchor of ERK 1/2 MAP kinases – PEA-15 (5), HeLa cells over-expressing the physiological inhibitor of TRX-1, TXNIP (6).</p

Determination of sub-cellular compartmentalization of TRX-1 in HeLa cells transiently over-expressing PEA-15; TXNIP; TRX-1 and exposed to 0.5 mM H₂O₂.

<p>HeLa wild-type cells were seeded in complete medium and 24 h later were transiently transfected with the following plasmids pcDNA3_PEA-15, pcDNA3_TXNIP, and pEGFP-C1_TRX-1. After 16 h, transiently transfected cultures were divided to perform either western blot analysis, to confirm over expression of designated inserts, or to perform immunofluorescence-based analysis to determine TRX-1 sub cellular localization after exposure to 0.5 mM H₂O₂. (<b>A</b>) HeLa cells transfected with pcDNA3_PEA-15. Left panel: western blot analysis of PEA-15 expression. Right panel: Immunofluorescence image of TRX-1 cellular location. (<b>B</b>) HeLa cells transfected with pcDNA3_TXNIP. Left panel: western blot analysis of TXNIP expression. Right panel: Immunofluorescence image of TRX-1 cellular location (<b>C</b>) HeLa cells transfected with pEGFP-C1_TRX-. Left panel: western blot analysis of TRX-1 expression. Right panel: Immunofluorescence image of TRX-1 cellular location. Red arrows indicate TRX-1 nuclear accumulation.</p

SNAP and H₂O₂ -modulated TXNIP mRNA and protein levels in wild-type Hela cells and in HeLa cells expressing PEA-15.

<p>(<b>A</b>) Relative levels of TXNIP mRNA determined by quantitative real-time PCR in HeLa cells wild-type, in HeLa cells expressing PEA-15, and in HeLa cells wild-type pre-treated with 50μM of PD98059 for 30 min as indicated (see Materials and Methods for details). All cell lines were treated with 0.5mM SNAP or 0.5mM H₂O₂ for 2 h at 37°C. Values are reported in the bar graphs and expressed as means ± S.D. (n = 3, *<i>p</i> < 0.05; **<i>p</i> < 0.01). (<b>B</b>) wild-type HeLa cells and HeLa cells expressing PEA-15 were incubated with SNAP (0.5 mM) or H₂O₂ (0.5 mM) for 2 h at 37°C. Cells were lysed and expression levels of TXNIP were examined by western blotting. Western blot results are representative of four independent experiments. Histogram represents the ratio between the densitometric values of the protein bands corresponding to TXNIP and of β-actin (Lower panel). </p

Confocal microscopy based analysis to determine a time-dependent sub-cellular localization of TRX-1 in HeLa cells.

<p>(<b>A</b>) Wild-type HeLa cells, HeLa cells expressing PEA-15, and HeLa cells expressing TXNIP were plated two days prior to experiment. Transient transfections with pEGFP-C1_TRX-1 were performed on day one. On day two, cells were assayed for TRX-1 sub-cellular localization. Cells were kept in a heated stage chamber (37°C) of the confocal microscope, and a first scanning was recorded to serve as control. After that, media was replaced and H₂O₂ was added to a final concentration of 0.5 mM. Subsequent scanning were performed at every 15 min up to 2 h. Bar = 20µm. (<b>B</b>) Cells were transfected with pEGFP-C1-TRX-1, treated with SNAP (0.5 mM) for 2 h at 37°C and submitted to indirect immunofluorescence for GFP, to show the localization of TRX-1 in HeLa cells. Red arrows indicate TRX-1 nuclear accumulation.</p

TXNIP promoter construction and regulation of its expression by oxidative and nitrosative stress.

<p>(<b>A</b>) Scheme representing the molecular cloning of TXNIP’s promoter region replacing the pEGFP-C1 CMV’s promoter region. (<b>B</b>) HeLa wild-type cells were transiently transfected with the TXNIP promoter construction and relative levels of GFP mRNA were determined by real-time PCR. (<b>C</b>) HeLa cells permanently transfected with the different TXNIP promoter constructs were stimulated with H₂O₂ (0.5 mM) or SNAP (0.5 mM). Treatments stimulated strong suppression of GFP expression only in cells transfected with the –2056 base pairs TXNIP promoter construct. Values are reported in the bar graphs and expressed as means ± S.D. (n = 3, *<i>p</i><0.05).</p