Neuronal ClC-3 splice variants differ in subcellular localizations, but mediate identical transport functions [Molecular Biophysics]

September 4th, 2015 by Guzman, R. E., Miranda Laferte, E., Franzen, A., Fahlke, C.

ClC-3 is a member of the CLC family of anion channels and transporters, for which multiple functional properties and subcellular localizations have been reported. Since alternative splicing often results in proteins with diverse properties, we investigated to what extent alternative splicing might influence subcellular targeting and function of ClC-3. We identified three alternatively spliced ClC-3 isoforms, ClC-3a, ClC-3b and ClC-3c, in mouse brain, with ClC-3c being the predominant splice variant. Whereas ClC-3a and ClC-3b are present in late endosomes/lysosomes, ClC-3c is targeted to recycling endosomes via a novel amino-terminal isoleucine-proline (IP) motif. Surface membrane insertion of a fraction of ClC-3c transporters permitted electrophysiological characterization of this splice variant through whole-cell patch clamping on transfected mammalian cells. In contrast, neutralization of the amino-terminal dileucine-like motifs was required for functional analysis of ClC-3a and ClC-3b. Heterologous expression of ClC-3a or ClC-3b carrying mutations in amino-terminal dileucine motifs as well as WT ClC-3c in HEK293T cells resulted in outwardly rectifying Cl- currents with significant capacitive current components. We conclude that alternative splicing of Clcn3 results in proteins with different subcellular localizations, but leaves the transport function of the proteins unaffected.

Sequential Action of MalE and Maltose Allows Coupling ATP Hydrolysis to Translocation in the MalFGK2 Transporter [Enzymology]

September 3rd, 2015 by Bao, H., Dalal, K., Cytrynbaum, E., Duong, F.

ABC transporters have evolved an ATP-dependent alternating-access mechanism to transport substrates across membranes. Despite important progress especially in their structural analysis, it is still unknown how the substrate stimulates ATP hydrolysis, the hallmark of ABC transporter. In this study, we measure the ATP turnover cycle of MalFGK2 in steady and pre-steady state conditions. We show that (i) the basal ATPase activity of MalFGK2 is very low because the cleavage of ATP is rate limiting, (ii) the binding of open-state MalE to the transporter induces ATP cleavage but leaves release of Pi limiting, (iii) the additional presence of maltose stimulates release of Pi, and therefore increases the overall ATP turnover cycle. We conclude that open-state MalE stabilizes MalFGK 2 in the outward-facing conformation until maltose triggers return to the inward-facing state for substrate and Pi release. This concerted action explains why ATPase activity of MalFGK2 depends on maltose, and why MalE is essential for transport.

Airway Surface Dehydration by Growth Factor TGF-{beta} in Cystic Fibrosis is Due to Decreased Function of a Voltage-dependent Potassium Channel and Can Be Rescued by the Drug Pirfenidone [Molecular Bases of Disease]

September 3rd, 2015 by

Transforming growth factorβ1 (TGF-β1) is not only elevated in airways of cystic fibrosis (CF) patients, whose airways are characterized by abnormal ion transport and mucociliary clearance, but TGF-β1 is also associated with worse clinical outcomes. Effective mucociliary clearance (MCC) depends on adequate airway hydration, governed by ion transport. Apically expressed, large-conductance, Ca2+ and voltage-dependent K+ (BK) channels play an important role in this process. In this study, TGF-β1 decreased airway surface liquid (ASL) volume, ciliary beat frequency (CBF), and BK activity in fully differentiated cystic fibrosis bronchial epithelial (CFBE) cells by reducing mRNA expression of the BK γ subunit leucine-rich repeat-containing protein 26 (LRRC26) and its function. While LRRC26 knockdown itself reduced BK activity, LRRC26 overexpression partially reversed TGF-β1-induced BK dysfunction. TGF-β1-induced ASL volume hyperabsorption was reversed by the BK opener mallotoxin and the clinically useful TGF-β signaling inhibitor pirfenidone. The latter increased BK activity via rescue of LRRC26. Therefore, we propose that TGF-β1-induced mucociliary dysfunction in CF airways is associated with BK inactivation related to a LRRC26 decrease and is amenable to treatment with clinically useful TGF-β1 inhibitors.
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Identification and Small Molecule Inhibition of an ATF4-dependent Pathway to Age-related Skeletal Muscle Weakness and Atrophy [Metabolism]

September 3rd, 2015 by

Aging reduces skeletal muscle mass and strength, but the underlying molecular mechanisms remain elusive. Here, we used mouse models to investigate molecular mechanisms of age-related skeletal muscle weakness and atrophy, as well as new potential interventions for these conditions. We identified two small molecules that significantly reduce age-related deficits in skeletal muscle strength, quality and mass: ursolic acid (a pentacyclic triterpenoid found in apples) and tomatidine (a steroidal alkaloid derived from green tomatoes). Because small molecule inhibitors can sometimes provide mechanistic insight into disease processes, we used ursolic acid and tomatidine to investigate the pathogenesis of age-related muscle weakness and atrophy. We found that ursolic acid and tomatidine generate hundreds of small positive and negative changes in mRNA levels in aged skeletal muscle, and the mRNA expression signatures of the two compounds are remarkably similar. Interestingly, a subset of the mRNAs are repressed by ursolic acid and tomatidine in aged muscle are positively regulated by the transcription factor ATF4. Based on this finding, we investigated ATF4 as a potential mediator of age-related muscle weakness and atrophy. We found that a targeted reduction in skeletal muscle ATF4 expression reduces age-related deficits in skeletal muscle strength, quality and mass, similar to ursolic acid and tomatidine. These results elucidate ATF4 as a critical mediator of age-related muscle weakness and atrophy. In addition, these results identify ursolic acid and tomatidine as potential agents and/or lead compounds for reducing ATF4 activity, weakness, and atrophy in aged skeletal muscle.

Involvement of Protein Kinase D1 in Signal Transduction from the Protein Kinase C Pathway to the Tyrosine Kinase Pathway in Response to Gonadotropin-Releasing Hormone [Cell Biology]

September 3rd, 2015 by Higa-Nakamine, S., Maeda, N., Toku, S., Yamamoto, H.

The receptor for gonadotropin-releasing hormone (GnRH) belongs to the G-protein-coupled receptors (GPCRs), and its stimulation activates extracellular signal-regulated protein kinase (ERK). We found that the transactivation of ErbB4 was involved in GnRH-induced ERK activation in immortalized GnRH neurons (GT1-7 cells). We found also that GnRH induced the cleavage of ErbB4. In the present study, we examined signal transduction for the activation of ERK and the cleavage of ErbB4 after GnRH treatment. Both ERK activation and ErbB4 cleavage were completely inhibited by YM-254890, an inhibitor of Gq/11 proteins. Down-regulation of protein kinase C (PKC) markedly decreased both ERK activation and ErbB4 cleavage. Experiments with two types of PKC inhibitors, Go 6976 and bisindolylmaleimide I, indicated that novel PKC isoforms but not conventional PKC isoforms were involved in ERK activation and ErbB4 cleavage. Our experiments indicated that the novel PKC isoforms activated protein kinase D (PKD) after GnRH treatment. Knockdown and inhibitor experiments suggested that PKD1 stimulated the phosphorylation of Pyk2 by constitutively activated Src and Fyn for ERK activation. Taken together, it is highly possible that PKD1 plays a critical role in signal transduction from the PKC pathway to the tyrosine kinase pathway. Activation of the tyrosine kinase pathway may be involved in the progression of cancer.
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Formation and repair of mismatches containing ribonucleotides and oxidized bases at repeated DNA sequences [Enzymology]

September 3rd, 2015 by Cilli, P., Minoprio, A., Bossa, C., Bignami, M., Mazzei, F.

The cellular pool of ribonucleotide triphosphates (rNTPs) is higher than that of deoxyribonucleotide triphosphates. To ensure genome stability, DNA polymerases must discriminate against rNTPs and incorporated ribonucleotides must be removed by ribonucleotide excision repair (RER). We investigated DNA polymerase beta (POL beta) capacity to incorporate ribonucleotides into trinucleotide repeated DNA sequences and the efficiency of base excision repair (BER) and RER enzymes (OGG1, MUTYH, and RNase H2) when presented with an incorrect sugar and an oxidized base. POL beta incorporated rAMP and rCMP opposite 7,8-dihydro-8-oxoguanine (8-oxodG) and extended both mispairs. In addition POL beta was able to insert and elongate an oxidized rGMP when paired with dA. We show that RNAse H2 always preserves the capacity to remove a single ribonucleotide when paired to an oxidized base or to incise an oxidized ribonucleotide in a DNA duplex. In contrast BER activity is affected by the presence of a ribonucleotide opposite an 8-oxodG. In particular MUTYH activity on 8-oxodG:rA mispairs is fully inhibited, although its binding capacity is retained. This results in the reduction of RNAse H2 incision capability of this substrate. Thus complex mispairs formed by an oxidized base and a ribonucleotide can compromise BER and RER in repeated sequences.

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.

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.

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.