The patterned structure of galactoglucomannan suggests it may bind to cellulose in seed mucilage

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOThe interaction between mannan polysaccharides and cellulose microfibrils contributes to cell wall properties in some vascular plants, but the molecular arrangement of mannan in the cell wall and the nature of the molecular bonding between mannan and cell178310111026FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO2013/08293-7The authors would like to acknowledge Prof. George Lomonossoff (John Innes Centre, UK), who developed the pEAQ-HyperTrans expression system used in this study. Plant Bioscience Limited supplied the pEAQ-HT vector that was used in this wor

Tomographic Evidence for Continuous Turnover of Golgi Cisternae in Pichia pastoris

The budding yeast Pichia pastoris contains ordered Golgi stacks next to discrete transitional endoplasmic reticulum (tER) sites, making this organism ideal for structure–function studies of the secretory pathway. Here, we have used P. pastoris to test various models for Golgi trafficking. The experimental approach was to analyze P. pastoris tER-Golgi units by using cryofixed and freeze-substituted cells for electron microscope tomography, immunoelectron microscopy, and serial thin section analysis of entire cells. We find that tER sites and the adjacent Golgi stacks are enclosed in a ribosome-excluding “matrix.” Each stack contains three to four cisternae, which can be classified as cis, medial, trans, or trans-Golgi network (TGN). No membrane continuities between compartments were detected. This work provides three major new insights. First, two types of transport vesicles accumulate at the tER-Golgi interface. Morphological analysis indicates that the center of the tER-Golgi interface contains COPII vesicles, whereas the periphery contains COPI vesicles. Second, fenestrae are absent from cis cisternae, but are present in medial through TGN cisternae. The number and distribution of the fenestrae suggest that they form at the edges of the medial cisternae and then migrate inward. Third, intact TGN cisternae apparently peel off from the Golgi stacks and persist for some time in the cytosol, and these “free-floating” TGN cisternae produce clathrin-coated vesicles. These observations are most readily explained by assuming that Golgi cisternae form at the cis face of the stack, progressively mature, and ultimately dissociate from the trans face of the stack

3D electron tomographic and biochemical analysis of ER, Golgi and trans Golgi network membrane systems in stimulated Venus flytrap (Dionaea muscipula) glandular cells

Background: The insect-trapping leaves of Dionaea muscipula provide a model for studying the secretory pathway of an inducible plant secretory system. The leaf glands were induced with bovine serum albumin to secrete proteases that were characterized via zymogram activity gels over a 6-day period. The accompanying morphological changes of the endoplasmic reticulum (ER) and Golgi were analyzed using 3D electron tomography of glands preserved by highpressure freezing/freeze substitution methods. Results: Secretion of multiple cysteine and aspartic proteases occurred biphasically. The majority of the Golgi was organized in clusters consisting of 3–6 stacks surrounded by a cage-like system of ER cisternae. In these clusters, all Golgi stacks were oriented with their cis-most C1 cisterna facing an ER export site. The C1 Golgi cisternae varied in size and shape consistent with the hypothesis that they form de novo. Following induction, the number of ER-bound polysomes doubled, but no increase in COPII vesicles was observed. Golgi changes included a reduction in the number of cisternae per stack and a doubling of cisternal volume without increased surface area. Polysaccharide molecules that form the sticky slime cause swelling of the trans and trans Golgi network (TGN) cisternae. Peeling of the trans-most cisternae gives rise to free TGN cisternae. One day after gland stimulation, the free TGNs were frequently associated with loose groups of oriented actin-like flaments which were not seen in any other samples. Conclusions: These fndings suggest that the secretory apparatus of resting gland cells is “overbuilt” to enable the cells to rapidly up-regulate lytic enzyme production and secretion in response to prey trapping.Instituto de Fisiología Vegeta

3D electron tomographic and biochemical analysis of ER, Golgi and trans Golgi network membrane systems in stimulated Venus flytrap (Dionaea muscipula) glandular cells

Abstract Background The insect-trapping leaves of Dionaea muscipula provide a model for studying the secretory pathway of an inducible plant secretory system. The leaf glands were induced with bovine serum albumin to secrete proteases that were characterized via zymogram activity gels over a 6-day period. The accompanying morphological changes of the endoplasmic reticulum (ER) and Golgi were analyzed using 3D electron tomography of glands preserved by high-pressure freezing/freeze substitution methods. Results Secretion of multiple cysteine and aspartic proteases occurred biphasically. The majority of the Golgi was organized in clusters consisting of 3–6 stacks surrounded by a cage-like system of ER cisternae. In these clusters, all Golgi stacks were oriented with their cis-most C1 cisterna facing an ER export site. The C1 Golgi cisternae varied in size and shape consistent with the hypothesis that they form de novo. Following induction, the number of ER-bound polysomes doubled, but no increase in COPII vesicles was observed. Golgi changes included a reduction in the number of cisternae per stack and a doubling of cisternal volume without increased surface area. Polysaccharide molecules that form the sticky slime cause swelling of the trans and trans Golgi network (TGN) cisternae. Peeling of the trans-most cisternae gives rise to free TGN cisternae. One day after gland stimulation, the free TGNs were frequently associated with loose groups of oriented actin-like filaments which were not seen in any other samples. Conclusions These findings suggest that the secretory apparatus of resting gland cells is “overbuilt” to enable the cells to rapidly up-regulate lytic enzyme production and secretion in response to prey trapping

Organellar Proteomics Reveals Golgi Arginine Dimethylation

The Golgi complex functions to posttranslationally modify newly synthesized proteins and lipids and to sort them to their sites of function. In this study, a stacked Golgi fraction was isolated by classical cell fractionation, and the protein complement (the Golgi proteome) was characterized using multidimensional protein identification technology. Many of the proteins identified are known residents of the Golgi, and 64% of these are predicted transmembrane proteins. Proteins localized to other organelles also were identified, strengthening reports of functional interfacing between the Golgi and the endoplasmic reticulum and cytoskeleton. Importantly, 41 proteins of unknown function were identified. Two were selected for further analysis, and Golgi localization was confirmed. One of these, a putative methyltransferase, was shown to be arginine dimethylated, and upon further proteomic analysis, arginine dimethylation was identified on 18 total proteins in the Golgi proteome. This survey illustrates the utility of proteomics in the discovery of novel organellar functions and resulted in 1) a protein profile of an enriched Golgi fraction; 2) identification of 41 previously uncharacterized proteins, two with confirmed Golgi localization; 3) the identification of arginine dimethylated residues in Golgi proteins; and 4) a confirmation of methyltransferase activity within the Golgi fraction

The Patterned Structure of Galactoglucomannan Suggests It May Bind to Cellulose in Seed Mucilage.

The interaction between mannan polysaccharides and cellulose microfibrils contributes to cell wall properties in some vascular plants, but the molecular arrangement of mannan in the cell wall and the nature of the molecular bonding between mannan and cellulose remain unknown. Previous studies have shown that mannan is important in maintaining Arabidopsis (Arabidopsis thaliana) seed mucilage architecture, and that Cellulose Synthase-Like A2 (CSLA2) synthesizes a glucomannan backbone, which Mannan α-Galactosyl Transferase1 (MAGT1/GlycosylTransferase-Like6/Mucilage Related10) might decorate with single α-Gal branches. Here, we investigated the ratio and sequence of Man and Glc and the arrangement of Gal residues in Arabidopsis mucilage mannan using enzyme sequential digestion, carbohydrate gel electrophoresis, and mass spectrometry. We found that seed mucilage galactoglucomannan has a backbone consisting of the repeating disaccharide [4)-β-Glc-(1,4)-β-Man-(1,], and most of the Man residues in the backbone are substituted by single α-1,6-Gal. CSLA2 is responsible for the synthesis of this patterned glucomannan backbone and MAGT1 catalyses the addition of α-Gal. In vitro activity assays revealed that MAGT1 transferred α-Gal from UDP-Gal only to Man residues within the CSLA2 patterned glucomannan backbone acceptor. These results indicate that CSLAs and galactosyltransferases are able to make precisely defined galactoglucomannan structures. Molecular dynamics simulations suggested this patterned galactoglucomannan is able to bind stably to some hydrophilic faces and to hydrophobic faces of cellulose microfibrils. A specialization of the biosynthetic machinery to make galactoglucomannan with a patterned structure may therefore regulate the mode of binding of this hemicellulose to cellulose fibrils

The role of IKKβ in Venezuelan equine encephalitis virus infection.

Venezuelan equine encephalitis virus (VEEV) belongs to the genus Alphavirus, family Togaviridae. VEEV infection is characterized by extensive inflammation and studies from other laboratories implicated an involvement of the NF-κB cascade in the in vivo pathology. Initial studies indicated that at early time points of VEEV infection, the NF-κB complex was activated in cells infected with the TC-83 strain of VEEV. One upstream kinase that contributes to the phosphorylation of p65 is the IKKβ component of the IKK complex. Our previous studies with Rift valley fever virus, which exhibited early activation of the NF-κB cascade in infected cells, had indicated that the IKKβ component underwent macromolecular reorganization to form a novel low molecular weight form unique to infected cells. This prompted us to investigate if the IKK complex undergoes a comparable macromolecular reorganization in VEEV infection. Size-fractionated VEEV infected cell extracts indicated a macromolecular reorganization of IKKβ in VEEV infected cells that resulted in formation of lower molecular weight complexes. Well-documented inhibitors of IKKβ function, BAY-11-7082, BAY-11-7085 and IKK2 compound IV, were employed to determine whether IKKβ function was required for the production of infectious progeny virus. A decrease in infectious viral particles and viral RNA copies was observed with inhibitor treatment in the attenuated and virulent strains of VEEV infection. In order to further validate the requirement of IKKβ for VEEV replication, we over-expressed IKKβ in cells and observed an increase in viral titers. In contrast, studies carried out using IKKβ(-/-) cells demonstrated a decrease in VEEV replication. In vivo studies demonstrated that inhibitor treatment of TC-83 infected mice increased their survival. Finally, proteomics studies have revealed that IKKβ may interact with the viral protein nsP3. In conclusion, our studies have revealed that the host IKKβ protein may be critically involved in VEEV replication

Low-Density Lipoprotein Receptor–Related Protein-1 Is a Therapeutic Target in Acute Myocardial Infarction

Low-density lipoprotein receptor–related protein-1 (LRP1) is a ubiquitous membrane receptor functioning as a scavenger and regulatory receptor, inducing anti-inflammatory and prosurvival signals. Based on the known structure–activity of the LRP1 receptor binding site, the authors synthesized a small peptide (SP16). SP16 induced a >50% reduction in infarct size (p < 0.001) and preservation of left ventricular systolic function (p < 0.001), and treatment with an LRP1 blocking antibody eliminated the protective effects of SP16. In conclusion, LRP1 activation with SP16 given within 30 min of reperfusion during experimental acute myocardial infarction leads to a cardioprotective signal reducing infarct size and preservation of cardiac systolic function

VEEV nsP3 interaction with host IKKβ.

<p>A) IKKβ was immunoprecipitated from VEEV infected U87MGs and Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) was performed. Control immunoprecipitations were performed with an IgG antibody. Mass spectrometry analysis revealed that the viral nonstructural protein nsP3 interacted with IKKβ. B) U87MGs were transfected in a 6-well plate with 5 µg of pcDNA3.1 and VEEV_nsP3_HA for 24 and 48 hours. Cell lysates were resolved using SDS-PAGE and subsequently immunoblotted with HA and β-actin served as a loading control. C) U87MGs were transfected in duplicate with (0.2 µg) VEEV_nsP3_HA and pcDNA3.1 (control), cells were fixed after 24 hours and probed with antibodies against the endogenous IKKβ and the HA tag. Cells were subsequently incubated with appropriate secondary Alexa Fluor antibodies and the nuclei stained with DAPI. Co-localization of IKKβ with nsP3 (yellow) was observed as shown by the arrows. The co-localization was confirmed by Z-stack analysis. Panels E–H and J–M serve as examples of transfected cells in a given field of view that show co-localization of IKKβ and VEEV_nsP3_HA at 24 hours post transfection. Panels I and N are magnified images of the outlined cells in red boxes in panels H and M respectively. Co-localization was found to be approximately in 71% of cells (72 cells were counted of which 55% demonstrated expression of nsP3. Of those cells that expressed nsP3, 71% showed co-localization of both IKKβ and VEEV_nsP3_HA proteins). D) U87MGs were transfected in duplicate with (0.2 µg) VEEV_nsP3_HA and pcDNA3.1 (control); cells were treated with BAY-11-7082 (1 µM and 0.1 µM). The cells were fixed 24 hours post transfection and probed with antibodies against the endogenous IKKβ and the HA tag. Cells were subsequently incubated with appropriate secondary Alexa Fluor antibodies and the nuclei stained with DAPI. Co-localization of IKKβ with nsP3 (yellow) was observed as shown by the arrows. The co-localization was confirmed by Z-stack analysis. Panels E–H and I–L serve as examples of transfected cells in a given field of view that show co-localization of IKKβ and VEEV_nsP3_HA at 24 hours post transfection. Panel M is a magnified image of the outlined cells in red boxes in panel L. Panels N–Q and S–V are examples of transfected and treated cells in a given field of view. Panels R and W are magnified images of the outlined cells in red boxes in panels Q and V respectively. Images were taken using Nikon Eclipse TE2000-U at 60× magnification and are representative of 3 independent experiments.</p

IKKβ inhibitors decrease TC-83 replication in rat AP7 neuronal cells.

<p>A) Neurons were pre-treated with DMSO or with IKK inhibitors (1 µM) for 2 hours and 24 hours later cell viability was measured using the Cell-Titer-Glo Luminescent Cell Viability Assay. B) Neurons were pretreated with IKK inhibitors (1 µM), BAY-11-7082, BAY-11-7085 and IKK2-IV for 2 hours. Following the pretreatment, the conditioned media (media containing inhibitor) was removed and the cells infected at MOI: 1 for 1 hour. The viral inoculum was removed and the conditioned media replaced. Supernatants were collected 24 hours post-infection, and infectious viral titers were determined by plaque assay. C) Neurons were pretreated with IKK inhibitors (1 µM) for 2 hours and infected with TC-83 for 1 hour. Conditioned media was replaced after removal of the viral inoculum. Cell viability assay was performed 48 hours later using the Cell-Titer-Glo Luminescent Cell Viability Assay. The red line is representative of the base line for luminescence units, such that luminescence units above this are indicative of increased cell viability. The graphs are representative of 2 independent experiments. Error bars for the independent experiments were calculated and are represented thusly. *** p≤0.005, ** p≤0.01 and * p≤0.05.</p