Novel regulation of the synthesis of AMPA receptor subunit GluA1 by carnitine palmitoyltransferase 1C (CPT1C) in the hippocampus [Neurobiology]

September 3rd, 2015 by

The regulation of AMPA-type receptor (AMPAR) abundance in the postsynaptic membrane is an important mechanism involved in learning and memory formation. Recent data suggest that one of the constituents of the AMPAR complex is carnitine palmitoyltransferase 1C (CPT1C), a brain-specific isoform located in the endoplasmic reticulum of neurons. Previous results had demonstrated that CPT1C deficiency disrupted spine maturation in hippocampal neurons and impaired spatial learning, but the role of CPT1C in AMPAR physiology had remained mostly unknown. In the present study we show that CPT1C binds GluA1 and GluA2 and that the three proteins have the same expression profile during neuronal maturation. Moreover, in hippocampal neurons of CPT1C knockout (KO) mice, AMPAR-mediated miniature excitatory postsynaptic currents and synaptic levels of AMPAR subunits GluA1 and GluA2 are significantly reduced. We show that AMPAR expression is dependent on CPT1C levels because total protein levels of GluA1 and GluA2 are decreased in CPT1C KO neurons and are increased in CPT1C overexpressing neurons while other synaptic proteins remain unaltered. Notably, mRNA levels of AMPARs remained unchanged in those cultures, indicating that CPT1C is post-transcriptionally involved. We demonstrate that CPT1C is directly involved in the de novo synthesis of GluA1 and not in protein degradation. Moreover, in CPT1C KO cultured neurons, GluA1 synthesis after chemical long-term depression was clearly diminished, and BDNF treatment was unable to phosphorylate mTOR and stimulate GluA1 protein synthesis. These data newly identify CPT1C as a regulator of AMPAR translation efficiency and therefore also synaptic function in the hippocampus.
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A Role for Widely Interspaced Zinc Finger (WIZ) in Retention of the G9a Methyltransferase on Chromatin [Genomics and Proteomics]

September 3rd, 2015 by

G9a and GLP lysine methyltransferases form a heterodimeric complex that is responsible for the majority of histone H3 lysine 9 mono- and di-methylation (H3K9me1/me2). Widely Interspaced Zinc finger (WIZ) associates with the G9a/GLP protein complex, but its role in mediating lysine methylation is poorly defined. Here, we show that WIZ regulates global H3K9me2 levels by facilitating the interaction of G9a with chromatin. Disrupting the association of G9a/GLP with chromatin by depleting WIZ results in altered gene expression and protein-protein interactions that are distinguishable from that of small molecule-based inhibition of G9a/GLP in support of discrete functions of the G9a/GLP/WIZ/chromatin complex in addition to H3K9me2 methylation.

Altered Lipid Synthesis by Lack of Yeast Pah1 Phosphatidate Phosphatase Reduces Chronological Life Span [Lipids]

September 3rd, 2015 by

In Saccharomyces cerevisiae, Pah1 phosphatidate phosphatase, which catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol, plays a crucial role in the synthesis of the storage lipid triacylglycerol. This evolutionarily conserved enzyme also plays a negative regulatory role in controlling de novo membrane phospholipid synthesis through its consumption of phosphatidate. We found that the pah1Δ mutant was defective in the utilization of non-fermentable carbon sources, but not in oxidative phosphorylation; the mutant did not exhibit major changes in oxygen consumption rate, mitochondrial membrane potential, F1FO-ATP synthase activity, or gross mitochondrial morphology. The pah1Δ mutant contained an almost normal complement of major mitochondrial phospholipids with some alterations in molecular species. Although oxidative phosphorylation was not compromised in the pah1Δ mutant, the cellular levels of ATP in quiescent cells were reduced by two-fold, inversely correlating with a four-fold increase in membrane phospholipids. In addition, the quiescent pah1Δ mutant cells had three-fold higher levels of mitochondrial superoxide and cellular lipid hydroperoxides, had reduced activities of superoxide dismutase 2 and catalase, and were hypersensitive to hydrogen peroxide. Consequently, the pah1Δ mutant had a shortened chronological life span. In addition, the loss of Tsa1 thioredoxin peroxidase caused a synthetic growth defect with the pah1Δ mutation. The shortened chronological life span of the pah1Δ mutant along with its growth defect on non-fermentable carbon sources and hypersensitivity to hydrogen peroxide was suppressed by the loss of Dgk1 diacylglycerol kinase, indicating that the underpinning of pah1Δ mutant defects was the excess synthesis of membrane phospholipids.

YscU/FlhB of Yersinia pseudotuberculosis harbors a C-terminal T3S signal [Microbiology]

September 3rd, 2015 by Login, F. H., Wolf-Watz, H.

All type III secretion system (T3SS) harbors a member of the YscU/FlhB family of proteins that is characterized by an auto-proteolytic process that occurs at a conserved cytoplasmic NPTH motif. We have previously demonstrated that YscUCC, the C-terminal peptide generated by auto-proteolysis of Yersinia pseudotuberculosis YscU, is secreted by the T3SS when bacteria are grown in Ca2+-depleted medium at 37°C. Here, we investigated the secretion of this early T3S-substrate and showed that YscUCC encompasses a specific C-terminal T3S signal within the 15 last residues (U15). U15 promoted C-terminal secretion of reporter proteins like GST and YopE lacking its native secretion signal. Similarly to the "classical" N-terminal secretion signal, U15 interacted with the ATPase YscN. Although U15 is critical for YscUCC secretion, deletion of the C-terminal secretion signal of YscUCC did neither affect Yop secretion nor Yop translocation. However, these deletions resulted in increased secretion of YscF, the needle subunit. Thus, these results suggest that YscU via its C-terminal secretion signal is involved in regulation of the YscF secretion.

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.