Combination of correctors rescue {Delta}F508-CFTR by reducing its association with Hsp 40 and 27 [Cell Biology]

September 2nd, 2015 by

Correcting the processing of ΔF508 -CFTR, the most common mutation in cystic fibrosis, is the major goal in the development of new therapies for this disease. Here, we determined whether ΔF508 could be rescued by a combination of small-molecule correctors, and identified the mechanism by which correctors rescue the trafficking mutant of CFTR. We transfected Cos-7 cells with ΔF508 , created HEK-293 stably expressing ΔF508 and utilized CFBE41o- cell lines stably transduced with ΔF508. As shown previously, ΔF508 expressed less protein, was unstable at physiological temperature, and rapidly degraded. When the cells were treated with the combination C18+C4 the mature C-band was expressed at the cell surface. After treatment with C18+C4, we saw a lower rate of protein disappearance after translation was stopped with cycloheximide. To understand how this rescue occurs, we evaluated the change in the binding of proteins involved in endoplasmic reticulum associated degradation (ERAD), such as Hsp 27 (HspB1) and 40(DnaJ). We saw a dramatic reduction in binding to the heat shock proteins Hsp27, and 40 following combined corrector therapy. siRNA experiments confirmed that a reduction in Hsp27 or Hsp40 rescued CFTR in the ΔF508 mutant, but the rescue was not additive or synergistic with C4+18 treatment, indicating these correctors shared a common pathway for rescue involving a network of ERAD proteins.

Competing Lipid-Protein and Protein-Protein Interactions Determine Clustering and Gating Patterns in KcsA [Molecular Biophysics]

September 2nd, 2015 by

There is increasing evidence to support the notion that membrane proteins, instead of being isolated components floating in a fluid lipid environment, can be assembled into supramolecular complexes that take part in a variety of cooperative cellular functions. The interplay between lipid-protein and protein-protein interactions is expected to be a determinant factor in the assembly and dynamics of such membrane complexes. Here we report on a role of anionic phospholipids on determining the extent of clustering of KcsA, a model potassium channel. Assembly/disassembly of channel clusters occurs, at least partly, as a consequence of competing lipid-protein and protein-protein interactions at non-annular lipid binding sites on the channel surface and brings about profound changes in the gating properties of the channel. Our results suggest that these latter effects of anionic lipids are mediated via the W67-E71-D80 inactivation triad within the channel structure and its bearing on the selectivity filter.

Crystal structure and mutational analysis of isomaltodextranase, a member of glycoside hydrolase family 27 [Protein Structure and Folding]

September 1st, 2015 by

Arthrobacter globiformis T6 isomaltodextranse (AgIMD) is an enzyme that liberates isomaltose from the non-reducing end of a polymer of glucose, dextran. AgIMD is classified as a member of glycoside hydrolase family (GH) 27, which comprises mainly α-galactosidases and α-N-acetylgalactosaminidases, whereas AgIMD does not show α-galactosidase or α-N-acetylgalactosaminidase activities. Here we determined the crystal structure of AgIMD. AgIMD consists of three domains: A, C, and D. Domains A and C are identified as a (β/α)8-barrel catalytic domain and an antiparallel β-structure, respectively, both of which are commonly found in GH27 enzymes. However, domain A of AgIMD has subdomain B, loop-1, and loop-2, all of which are not found in GH27 human α-galactosidase. AgIMD in a complex with trisaccharide panose shows that Asp207, a residue in loop-1, is involved in subsite +1. Kinetic parameters of the wild-type and mutant enzymes for a small synthetic saccharide, p-nitrophenyl α-isomaltoside, and the polysaccharide, dextran, were compared, showing that Asp207 is important for the catalysis of dextran. Domain D is classified as carbohydrate-binding module (CBM) 35, and an isomaltose molecule is seen in this domain in the AgIMD-isomaltose complex. Domain D is highly homologous to CBM35 domains found in GH31 and GH66 enzymes. The results here indicate that some features found in GH13, 31, and 66 enzymes, such as subdomain B, residues at subsite +1, and the CBM35 domain, are also observed in the GH27 enzyme, AgIMD, and thus provide insights into the evolutionary relationships among GH13, 27, 31, 36, and 66 enzymes.

Post-translational Down-regulation of Melanoma antigen-A11 (MAGE-A11) by Human p14-ARF Tumor Suppressor [Molecular Bases of Disease]

September 1st, 2015 by Minges, J. T., Grossman, G., Zhang, P., Kafri, T., Wilson, E. M.

X-linked primate-specific melanoma antigen-A11 (MAGE-A11) is a human androgen receptor (AR) coactivator and proto-oncogene expressed at low levels in normal human reproductive tract tissues and at higher levels in castration-resistant prostate cancer where it is required for androgen-dependent cell growth. In this report we show that MAGE-A11 is targeted for degradation by human p14-ARF, a tumor suppressor expressed from an alternative reading frame of the p16 cyclin-dependent kinase inhibitor INK4a/ARF gene. MAGE-A11 degradation by the proteasome was mediated by an interaction with p14-ARF, and was independent of lysine ubiquitination. A dose-dependent inverse relationship between MAGE-A11 and p14-ARF correlated with p14-ARF inhibition of the MAGE-A11-induced increase in androgen-dependent AR transcriptional activity and constitutive activity of a splice variant-like AR. Reciprocal stabilization between MAGE-A11 and AR did not protect against degradation promoted by p14-ARF. p14-ARF prevented MAGE-A11 interaction with the E2F1 oncoprotein, and inhibited the MAGE-A11-induced increase in E2F1 transcriptional activity. Post-translational down-regulation of MAGE-A11 promoted by p14-ARF was independent of HDM2, the human homologue of mouse double minute 2, an E3 ubiquitin ligase inhibited by p14-ARF. However, MAGE-A11 had a stabilizing effect on HDM2 in the absence or presence of p14-ARF, and cooperated with HDM2 to increase E2F1 transcriptional activity in the absence of p14-ARF. We conclude that degradation of MAGE-A11 promoted by the human p14-ARF tumor suppressor contributes to low levels of MAGE-A11 in nontransformed cells, and that higher levels of MAGE-A11 associated with low p14-ARF increases AR and E2F1 transcriptional activity and promotes the development of castration-resistant prostate cancer.

A Novel Role of Proline Oxidase in HIV-1 Envelope Glycoprotein Induced Neuronal Autophagy [Neurobiology]

September 1st, 2015 by Pandhare, J., Dash, S., Jones, B., Villalta, F., Dash, C.

Proline oxidase (POX) catalytically converts proline to pyrroline-5-carboxylate (P5C). This catabolic conversion generates reactive oxygen species (ROS) that triggers cellular signaling cascades including autophagy and apoptosis. This study for the first time demonstrates a role of POX in HIV-1 envelope glycoprotein (gp120) induced neuronal autophagy. HIV-1 gp120 is a neurotoxic factor and involved in HIV-1 associated neurological disorders (HAND). However, the mechanism of gp120-mediated neurotoxicity remains unclear. Using SH-SY5Y neuroblastoma cells as a model; this study demonstrates that gp120 treatment induced POX expression and catalytic activity. Concurrently, gp120 also increased intracellular ROS levels. However, increased ROS had a minimal effect on neuronal apoptosis. Further investigation indicated that the immediate cellular response to increased ROS paralleled with induction of autophagy markers, beclin 1 and LC3-II. These data lead to the hypothesis that neuronal autophagy is activated as a cellular protective response to the toxic effects of gp120. A direct and functional role of POX in gp120 mediated neuronal autophagy was examined by inhibition and over-expression studies. Inhibition of POX activity by a competitive inhibitor-dehydroproline decreased ROS levels concomitant with reduced neuronal autophagy. Conversely, overexpression of POX in neuronal cells increased ROS levels and activated ROS-dependent autophagy. Mechanistic studies suggest that gp120 induces POX by targeting p53. Luciferase reporter assays confirm that p53 drives POX transcription. Furthermore, data demonstrate that gp120 induces p53 via binding to the CXCR4 co-receptor. Collectively, these results demonstrate a novel role of POX as a stress response metabolic regulator in HIV-1 gp120 associated neuronal autophagy.

The Social Amoeba Dictyostelium discoideum is Highly Resistant to Polyglutamine Aggregation [Protein Structure and Folding]

September 1st, 2015 by

The expression, misfolding, and aggregation of long repetitive amino acid tracts is a major contributing factor in a number of neurodegenerative diseases, including C9ORF72 amyotrophic lateral sclerosis/Frontotemporal dementia (ALS/FTD), fragile X tremor ataxia syndrome (FXTAS), myotonic dystrophy type 1 (DM1), spinocerebellar ataxia type 8 (SCA8), and the nine polyglutamine diseases. Protein aggregation is a hallmark of each of these diseases. In model organisms, including yeast, worms, flies, mice, rats, as well as in human cells, expression of proteins with the long repetitive amino acid tracts associated with these diseases recapitulates the protein aggregation that occurs in human disease. Here we show that the model organism Dictyostelium discoideum has evolved to normally encode long polyglutamine tracts and express these proteins in a soluble form. We also show that Dictyostelium has the capacity to suppress aggregation of a polyglutamine-expanded Huntingtin construct that aggregates in other model organisms tested. Together, these data identify Dictyostelium as a novel model organism with the capacity to suppress aggregation of proteins with long polyglutamine tracts.

Myosin IIb-dependent regulation of actin dynamics is required for NMDA receptor trafficking during synaptic plasticity [Cell Biology]

September 1st, 2015 by

N-methyl-D-aspartate receptor (NMDAR) synaptic incorporation changes the number of NMDARs at synapses and is thus critical to various NMDAR-dependent brain functions. To date, the molecules involved in NMDAR trafficking and the underlying mechanisms are poorly understood. Here, we report that myosin IIb is an essential molecule in NMDAR synaptic incorporation during protein kinase C (PKC) or theta burst stimulation (TBS)-induced synaptic plasticity. Moreover, we demonstrate that myosin light chain kinase(MLCK)-dependent actin reorganization contributes to NMDAR trafficking. The findings from additional mutual occlusion experiments demonstrate that PKC and MLCK share a common signaling pathway in NMDAR-mediated synaptic regulation. Because myosin IIb is the primary substrate of MLCK and can regulate actin dynamics during synaptic plasticity, we propose that the MLCK- and myosin IIb-dependent regulation of actin dynamics is required for NMDAR trafficking during synaptic plasticity. This study provides important insights into a mechanical framework for understanding NMDAR trafficking associated with synaptic plasticity.

Disruption of Rhodopsin Dimerization with Synthetic Peptides Targeting an Interaction Interface [Membrane Biology]

September 1st, 2015 by

Though homo- and heterodimerization of G protein-coupled receptors (GPCRs) are well documented, GPCR monomers could assemble in different ways thus causing variations in the interactive interface between receptor monomers among different GPCRs. Moreover, the functional consequences of this phenomenon remain to be clarified and could be specific for different GPCRs. Synthetic peptides derived from transmembrane (TM) domains can interact with a full length GPCR, blocking dimer formation and affecting its function. Here we used peptides corresponding to TM helices of bovine rhodopsin (Rho) to investigate the Rho dimer interface and functional consequences of its disruption. Incubation of Rho with TM1, TM2, TM4 and TM5 peptides in rod outer segment (ROS) membranes shifted the resulting detergent-solubilized protein migration through a gel filtration column towards smaller molecular masses with a reduced propensity for dimer formation in a crosslinking reaction. Binding of these TM peptides to Rho was characterized by both mass spectrometry and a label-free assay, from which dissociation constants were calculated. A bioluminescence resonance energy transfer (BRET) assay revealed that the physical interaction between Rho molecules expressed in membranes of living cells was blocked by the same four TM peptides identified in our in vitro experiments. Though disruption of the Rho dimer/oligomer had no effect on the rates of G protein activation, binding of Gt to the activated receptor stabilized the dimer. However, TM peptide-induced disruption of dimer/oligomer decreased receptor stability, suggesting that Rho supramolecular organization could be essential for ROS stabilization and receptor trafficking.

The Cell Division Protein FtsZ from Streptococcus pneumoniae Exhibits a GTPase Activity Delay [Cell Biology]

September 1st, 2015 by

The cell division protein FtsZ assembles in vitro by a mechanism of cooperative association dependent on GTP, monovalent cations and Mg2+. We have analyzed the GTPase activity and assembly dynamics of Streptococcus pneumoniae FtsZ (SpnFtsZ). SpnFtsZ assembled in an apparently cooperative process, with a higher critical concentration than values reported for other FtsZ proteins. It sedimented in the presence of GTP as a high molecular mass polymer with a well-defined size and tended to form double-stranded filaments in electron microscope preparations. GTPase activity depended on K+ and Mg2+ and was inhibited by Na+. GTP hydrolysis exhibited a delay that included a lag phase followed by a GTP hydrolysis activation step, untill reaction reached the GTPase rate. The lag phase was not found in polymer assembly, suggesting a transition from an initial non-GTP-hydrolyzing polymer that switches to a GTP-hydrolyzing polymer, supporting models that explain FtsZ polymer cooperativity.

Bacteriophage-mediated glucosylation can modify lipopolysaccharide O antigens synthesized by an ABC transporter-dependent assembly mechanism [Glycobiology and Extracellular Matrices]

September 1st, 2015 by Mann, E., Ovchinnikova, O. G., King, J. D., Whitfield, C.

Lysogenic bacteriophages may encode enzymes that modify the structures of lipopolysaccharide O antigen glycans, altering the structure of the bacteriophage receptor, and resulting in serotype-conversion. This can enhance virulence and has implications for antigenic diversity and vaccine development. Side-chain glucosylation is a common modification strategy found in a number of bacterial species. To date, glucosylation has only been observed in O antigens synthesized by Wzy-dependent pathways, one of the two most prevalent O-antigen synthesis systems. Here we exploited a heterologous system to study the glucosylation-potential of a model O antigen produced in an ABC transporter-dependent system. Although O-antigen production is cryptic in E. coli K-12, due to a mutation in the synthesis genes, it possesses a prophage-glucosylation cluster, which modifies the GlcNAc residue in an α-L-Rha-(1→3)-D-GlcNAc motif found in the original O16 antigen. Raoultella terrigena ATCC 33257 produces an O antigen possessing the same disaccharide motif but its assembly uses an ABC transporter-dependent system. E. coli harboring the R. terrigena O-antigen biosynthesis genes produced an O antigen displaying reduced reactivity towards antisera raised against the native R. terrigena repeat structure, indicative of an altered chemical structure. Structural determination using NMR revealed the addition of glucose side-chains to the repeat units. O-antigen modification was dependent on a functional ABC transporter, consistent with modification in the periplasm, and was eliminated by deletion of the glucosylation genes from the E. coli chromosome, restoring native level antisera sensitivity and structure. There are therefore no intrinsic mechanistic barriers for bacteriophage-mediated O-antigen glucosylation in ABC transporter-dependent pathways.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Bacteriophage-mediated glucosylation can modify lipopolysaccharide O antigens synthesized by an ABC transporter-dependent assembly mechanism [Glycobiology and Extracellular Matrices]