Functional characterization of monomeric GTPase Rab1 in the secretory pathway of Leishmania [Microbiology]

October 23rd, 2015 by Bahl, S., Parashar, S., Malhotra, H., Raje, M., Mukhopadhyay, A.

Leishmania secretes large number of their effectors to the extracellular milieu. However, regulation of secretory pathway in Leishmania is not well characterized. Here, we report the cloning, expression and characterization of Rab1 homologue from Leishmania. We have found that Ld-Rab1 localizes in Golgi in Leishmania. To understand the role of Ld-Rab1 in the secretory pathway of Leishmania, we have generated transgenic parasites overexpressing Ld-GFP-Rab1:WT, Ld-GFP-Rab1:Q67L, a GTPase deficient dominant-positive mutant of Rab1 and Ld-GFP-Rab1:S22N, a GDP locked dominant-negative mutant of Rab1. Surprisingly, our results have shown that overexpression of Ld-GFP-Rab1:Q67L or Ld-GFP-Rab1:S22N does not disrupt the trafficking and localization of HbR in Leishmania. To determine whether Rab1 dependent secretory pathway is conserved in parasites, we have analyzed the role of Ld-Rab1 in the secretion of secretory acid phosphatase (SAP) and Ld-gp63 in Leishmania. Our results have shown that overexpression of Ld-GFP-Rab1:Q67L or Ld-GFP-Rab1:S22N significantly inhibits the secretion of SAP by Leishmania. We have also found that overexpression of Ld-GFP-Rab1:Q67L or Ld-GFP-Rab1:S22N retains Ld-RFP-gp63 in Golgi and blocks the secretion of Ld-gp63 whereas the trafficking of Ld-RFP-gp63 in Ld-GFP-Rab1:WT expressed cells is unaltered in comparison to control cells. Taken together, our results have shown that Rab1 regulated secretory pathway is well conserved and HbR trafficking follows Rab1 independent secretory pathway in Leishmania.

Fasting and systemic insulin signaling regulate phosphorylation of brain proteins that modulate cell morphology and link to neurological disorders [Neurobiology]

October 23rd, 2015 by

Diabetes is strongly associated with cognitive decline, but the molecular reasons are unknown. We found that fasting and peripheral insulin promote phosphorylation and dephosphorylation, respectively, of specific residues on brain proteins that included cytoskeletal regulators such as slit-robo GTPase-activating protein 3 (srGAP3) and microtubule affinity-regulating protein kinases (MARKs), whose deficiency or dysregulation are linked to neurological disorders. Fasting activates protein kinase A (PKA) but not PKB/Akt signaling in the brain, and PKA can phosphorylate the purified srGAP3. The phosphorylation of srGAP3 and MARKs were increased when PKA signaling was activated in primary neurons. Knockdown of PKA decreased phosphorylation of srGAP3. Furthermore, WAVE1, an A-kinase anchoring protein (AKAP), can form a complex with srGAP3 and PKA in the brain of fasted mice to facilitate the phosphorylation of srGAP3 by PKA. Although brain cells have insulin receptors, our findings are inconsistent with the down-regulation of phosphorylation of target proteins being mediated by insulin signaling within the brain. Rather, our findings infer that systemic insulin through a yet unknown mechanism inhibits PKA or protein kinase(s) with similar specificity and/or activates an unknown phosphatase in the brain. Ser858 of srGAP3 was identified as a key regulatory residue, whose phosphorylation by PKA enhanced the GAP activity of srGAP3 towards its substrate Rac1 in cells, thereby inhibiting the action of this GTPase in cytoskeletal regulation. Our findings reveal novel mechanisms linking peripheral insulin sensitivity with cytoskeletal remodelling in neurons, which may help to explain the association of diabetes with neurological disorders such as Alzheimer's disease (AD).
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Coupling Between Nutrient Availability and Thyroid Hormone Activation [Signal Transduction]

October 23rd, 2015 by

The activity of the thyroid gland is stimulated by food availability via leptin-induced TRH/TSH expression. Here we show that food availability also stimulates thyroid hormone activation by accelerating conversion of T4-to-T3 via type 2 deiodinase (D2) in mouse skeletal muscle and in a cell model transitioning from 0.1 to 10% FBS. The underlying mechanism is transcriptional de-repression of DIO2 through the mTORC2 pathway as defined in rictor knock down cells. In cells kept in 0.1% FBS there is DIO2 inhibition via FOXO1 binding to DIO2 promoter. Repression of DIO2 by FOXO1 was confirmed using its specific inhibitor AS1842856 or adenoviral infection of constitutively active FOXO1. ChIP studies indicate that 4h after 10% FBS-containing media FOXO1 binding markedly decreases and DIO2 promoter is activated. Studies in the insulin-receptor FOXO1 KO mouse indicate that insulin is a key signaling molecule in this process. We conclude that FOXO1 represses DIO2 during fasting and de-repression occurs via nutritional activation of the PI3K-mTORC2-Akt pathway.

ER stress induces SIRT1 expression via the PI3K-Akt-GSK3{beta} signaling pathway and promotes hepatocellular inȷury [Cell Biology]

October 23rd, 2015 by

Sirtuin 1 (SIRT1), a NAD(+)-dependent histone deacetylase, plays crucial roles in various biological processes including longevity, stress response, and cell survival. Endoplasmic reticulum (ER) stress is caused by dysfunction of ER homeostasis, and exacerbates various diseases including diabetes, fatty liver, and chronic obstructive pulmonary disease. While several reports have shown that SIRT1 negatively regulates ER stress and ER stress-induced responses in vitro and in vivo, the effect of ER stress on SIRT1 is less explored. In this study, we showed that ER stress induced SIRT1 expression in vitro and in vivo. We further determined the molecular mechanisms of how ER stress induces SIRT1 expression. Surprisingly, the conventional ER stress-activated transcription factors XBP1, ATF4 and ATF6 seem to be dispensable for SIRT1 induction. Based on inhibitor screening experiments with SIRT1 promoter, we found that the PI3K-Akt-GSK3β signaling pathway is required for SIRT1 induction by ER stress. Moreover, we showed that pharmacological inhibition of SIRT1 by EX527 inhibited the ER stress-induced cellular death in vitro and severe hepatocellular injury in vivo, indicating a detrimental role of SIRT1 in ER stress-induced damage responses. Collectively, these data suggests that SIRT1 expression is up-regulated by ER stress and contributes to ER stress-induced cellular damage.

D-Amino Acid Probes for Penicillin Binding Protein-based Bacterial Surface Labeling [Enzymology]

October 23rd, 2015 by Fura, J. M., Kearns, D., Pires, M. M.

Peptidoglycan is an essential and highly conserved mesh structure that surrounds bacterial cells. It plays a critical role in retaining a defined cell shape and, in the case of pathogenic Gram-positive bacteria, it lies at the interface between bacterial cells and the host organisms. Intriguingly, bacteria can metabolically incorporate unnatural D-amino acids into the peptidoglycan stem peptide directly from the surrounding media, a process mediated by penicillin binding proteins (PBPs). Metabolic peptidoglycan remodeling via unnatural D-amino acids has provided unique insight into peptidoglycan biosynthesis of live bacteria and has also served as the basis of a synthetic immunology strategy with potential therapeutic implications. A striking feature of this process is the vast promiscuity displayed by PBPs in tolerating entirely unnatural sidechains. Yet, the chemical space and physical features of this sidechain promiscuity has not been systematically determined. In this report, we designed and synthesized a library of variants displaying diverse sidechains to comprehensively establish the tolerability of unnatural D-amino acids by PBPs in both Gram-positive and Gram-negative organisms. In addition, nine Bacillus subtilis PBP-null mutants were evaluated with the goal of identifying a potential primary PBP responsible for unnatural D-amino acid incorporation and gaining insight into temporal control of PBP activity. Together, we have empirically established the scope of physical parameters that govern metabolic incorporation of unnatural D-amino acids into bacterial peptidoglycan.

Utilization of Dioxygen by Carotenoid Cleavage Oxygenases [Protein Structure and Folding]

October 23rd, 2015 by

Carotenoid cleavage oxygenases (CCOs) are non-heme, Fe(II)-dependent enzymes that participate in biologically important metabolic pathways involving carotenoids, apocarotenoids including retinoids, stilbenes and related compounds. CCOs typically catalyze the cleavage of non-aromatic double bonds by dioxygen (O2) to form aldehyde or ketone products. Expressed only in vertebrates, the RPE65 sub-group of CCOs catalyzes a non-canonical reaction consisting of concerted ester cleavage and trans-cis isomerization of all-trans-retinyl esters. It remains unclear whether the former group of CCOs function as mono- or di-oxygenases. Additionally, a potential role for O2 in catalysis by the RPE65 group of CCOs has not been evaluated to date. Here, we investigated the pattern of oxygen incorporation into apocarotenoid products of Synechocystis apocarotenoid oxygenase (ACO). Reactions performed in the presence of 18O-labeled water and 18O2 revealed an unambiguous dioxygenase pattern of O2 incorporation into the reaction products. Substitution of Ala for Thr at position 136 of ACO, a site predicted to govern the mono- vs. dioxygenase tendency of CCOs, greatly reduced enzymatic activity without altering the dioxygenase labeling pattern. Reevaluation of the oxygen-labeling pattern of the resveratrol-cleaving CCO, NOV2, previously reported to be a monooxygenase, using a purified enzyme sample revealed that it too is a dioxygenase. We also demonstrated that bovine RPE65 is not dependent on O2 for its cleavage/isomerase activity. In conjunction with prior research, the results of this study resolve key issues regarding the utilization of O2 by CCOs and indicate that dioxygenase activity is a feature common amongst double bond-cleaving CCOs.

The Blockade of NF-{kappa}B Activation by a Specific Inhibitory Peptide has a Strong Neuroprotective Role in a Sprague-Dawley Rat Kernicterus Model [Neurobiology]

October 23rd, 2015 by Li, M., Song, S., Li, S., Feng, J., Hua, Z.

Kernicterus, the permanent nerve damage occurs as a result of bilirubin precipitation, still occurs worldwide and may lead to death or permanent neurological impairments. However, the underlying mechanisms remain unclear and effective therapeutic strategies are lacking. The present study aims to investigate the activation of nuclear factor kappa B (NF-κB) and to identify the effect of NF-κB inhibition on the newborn rat Kernicterus model. The NF-κB essential modifier-binding domain peptide (NBD), coupled with the HIV trans-activator of transcription peptide (TAT) was used to inhibit NF-κB. NF-κB was significantly activated in the cerebrum at 1 and 3 h (P < 0.05) after the model was established, as measured by the electrophoretic mobility shift assay (EMSA). NF-κB activation was inhibited by intraperitoneal administration of TAT-NBD. The general conditions of the TAT-NBD-treated rats were improved, meanwhile, these rats performed much better on the neurological evaluation, the rotarod test and the Morris water maze test (P<0.05) than the vehicle-treated rats at 28 days. Furthermore, the morphology of the nerve cells was better preserved in the TAT-NBD group, and displayed less neurodegeneration and astrocytosis. Simultaneously, apoptosis in the brain was attenuated, and the levels of the TNF-α and IL-1β proteins were decreased (P < 0.01). These results suggested that NF-κB was activated and inhibition of NF-κB activation by TAT-NBD not only attenuates the acute neurotoxicity, apoptosis and inflammation, but also improved the long-term neurobehavioral impairments in the Kernicterus model rats in vivo. Thus, inhibiting NF-κB activation might be a potential therapeutic approach for Kernicterus.
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The Protein Interaction of RNA Helicase B (RhlB) and Polynucleotide Phosphorylase (PNPase) Contributes to the Homeostatic Control of Cysteine in Escherichia coli [Microbiology]

October 22nd, 2015 by Tseng, Y.-T., Chiou, N.-T., Gogiraju, R., Lin-Chao, S.

PNPase, one of the major enzymes with 3'-to-5' single-stranded RNA (ssRNA) degradation and processing activities, can interact with the RNA helicase RhlB independently of RNA degradosome formation in E. coli. Here, we report that loss of interaction between RhlB and PNPase impacts cysteine homeostasis in E. coli. By random mutagenesis, we identified a mutant RhlBP238L that loses 75% of its ability to interact with PNPase, but retains normal interaction with RNase E and RNA, in addition to exhibiting normal helicase activity. Applying microarray analyses to an E. coli strain with impaired RNA degradosome formation, we investigated the biological consequences of a weakened interaction between RhlB and PNPase. We found significant increases in 11 out of 14 genes involved in cysteine biosynthesis. Subsequent Northern blot analyses showed that the upregulated transcripts were the result of stabilization of the cysB transcript encoding a transcriptional activator for the cys operons. Furthermore, Northern blots of PNPase or RhlB mutants showed that RhlB-PNPase plays both a catalytic and structural role in regulating cysB degradation. Cells expressing the RhlBP238L mutant exhibited an increase in intracellular cysteine and an enhanced anti-oxidative response. Collectively, this study suggests a mechanism by which bacteria use the PNPase-RhlB exosome-like complex to combat oxidative stress by modulating cysB mRNA degradation.
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Enhancing autophagy with drugs or lung-directed gene therapy reverses the pathological effects of respiratory epithelial cell proteinopathy [Protein Synthesis and Degradation]

October 22nd, 2015 by

Recent studies have shown that autophagy mitigates the pathological effects of proteinopathies in the liver, heart and skeletal muscle but this has not been investigated for proteinopathies that affect the lung. This may be due at least in part to the lack of an animal model robust enough for spontaneous pathological effects from proteinopathies even though several rare proteinopathies, surfactant protein A and C deficiencies, cause severe pulmonary fibrosis. In this report we show that the PiZ mouse, transgenic for the common misfolded variant α1-antitrypsin Z (ATZ), is a model of respiratory epithelial cell proteinopathy with spontaneous pulmonary fibrosis. Intracellular accumulation of misfolded ATZ in respiratory epithelial cells of the PiZ model resulted in activation of autophagy, leukocyte infiltration and spontaneous pulmonary fibrosis severe enough to elicit functional restrictive deficits. Treatment with autophagy enhancer drugs or lung-directed gene transfer of TFEB, a master transcriptional activator of the autophagolysosomal system, reversed these proteotoxic consequences. We conclude that this mouse is an excellent model of respiratory epithelial proteinopathy with spontaneous pulmonary fibrosis, that autophagy is an important endogenous proteostasis mechanism and an attractive target for therapy.
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Store-Operated Ca2+ Entry Mediated by Orai1 and TRPC1 participates to Insulin Secretion in Rat Beta-cells [Cell Biology]

October 22nd, 2015 by

Store-Operated Ca2+ Channels (SOCs) are voltage-independent Ca2+ channels activated upon depletion of the endoplasmic reticulum (ER) Ca2+ stores. Early studies suggest the contribution of such channels to Ca2+ homeostasis in insulin-secreting pancreatic β-cells. However, their composition and contribution to glucose-stimulated insulin secretion (GSIS) remains unclear. In this study, ER Ca2+ depletion triggered by acetylcholine (ACh) or thapsigargin (Tg) stimulated the formation of a ternary complex composed of Orai1, TRPC1 and STIM1, the key proteins involved in the formation of SOCs. Ca2+ imaging further revealed that Orai1 and TRPC1 are required to form functional SOCs and that these channels are activated by STIM1 in response to Tg or ACh. Pharmacological SOCs inhibition or dominant-negative blockade of Orai1 or TRPC1 using the specific pore mutants Orai1-E106D or TRPC1-F562A impaired GSIS in rat β-cells and fully blocked the potentiating effect of ACh on secretion. In contrast, pharmacological or dominant-negative blockade of TRPC3 had no effect on extracellular Ca2+ entry and GSIS. Finally, we observed that prolonged exposure to supraphysiological glucose concentration impaired SOCs function without altering the expression levels of STIM1, Orai1 and TRPC1. We conclude that Orai1 and TRPC1, which form SOCs regulated by STIM1, play a key role in the effect of ACh on GSIS, a process which may be impaired in type 2 diabetes.