Pleiotropic Functions of Tumor Suppressor WWOX in Normal and Cancer Cells [Molecular Bases of Disease]

October 23rd, 2015 by

WW domain-containing oxidoreductase (WWOX), originally marked as a likely tumor suppressor gene, has over the years become recognized for its role in a much wider range of cellular activities. Phenotypic effects displayed in animal studies, along with resolution of WWOX's architecture, fold, and binding partners, point to the protein's multifaceted biological functions. Results from a series of complementary experiments seem to indicate WWOX's involvement in metabolic regulation. More recently, clinical studies involving cases of severe encephalopathy suggest WWOX also plays a part in controlling CNS development, further expanding our understanding of the breadth and complexity of WWOX behavior. Here we present a short overview of the various approaches taken to study this dynamic gene, emphasizing the most recent findings regarding WWOX's metabolic- and CNS-associated functions and their underlying molecular basis.

Probing the Mec1/ATR Checkpoint Activation Mechanism with Small Peptides [Signal Transduction]

October 23rd, 2015 by

Yeast Mec1, the ortholog of human ATR, is the apical protein kinase that initiates the cell cycle checkpoint in response to DNA damage and replication stress. The basal activity of Mec1 kinase is activated by cell cycle phase-specific activators. Three distinct activators stimulate Mec1 kinase using an intrinsically disordered domain of the protein. These are the Ddc1 subunit of the 9-1-1 checkpoint clamp (ortholog of human and S. pombe Rad9), the replication initiator Dpb11 (ortholog of human TopBP1 and S. pombe Cut5), and the multifunctional nuclease/helicase Dna2. Here, we use small peptides to determine the requirements for Mec1 activation. For Ddc1, we identify two essential aromatic amino acids in a hydrophobic environment that when fused together are proficient activators. Using this increased insight, we have been able to identify homologous motifs in S. pombe Rad9 that can activate Mec1. Furthermore, we show that a 9-amino acid Dna2-based peptide is sufficient for Mec1 activation. Studies with mutant activators suggest that binding of an activator to Mec1 is a two-step process, the first step involving the obligatory binding of essential aromatic amino acids to Mec1, followed by an enhancement in binding energy through interactions with neighboring sequences.

The Stable Interaction Between Signal-Peptidase LepB of Escherichia coli and Nuclease Bacteriocins Promotes Toxin Entry into the Cytoplasm [Microbiology]

October 23rd, 2015 by Mora, L., Moncoq, K., England, P., Oberto, J., de Zamaroczy, M.

LepB is a key membrane component of the cellular secretion machinery, which releases secreted proteins into the periplasm by cleaving the inner membrane-bound leader. We showed that LepB is also an essential component of the machinery hijacked by the tRNase colicin D for its import. Here we demonstrate that this non-catalytic activity of LepB is to promote the association of the central domain of colicin D with the inner membrane, prior to the FtsH-dependent proteolytic processing and translocation of the toxic tRNase domain into the cytoplasm. The novel structural role of LepB results in a stable interaction with colicin D, with a stoichiometry of 1:1 and a nanomolar Kd determined in vitro. LepB provides a chaperone-like function for the penetration of several nuclease-type bacteriocins into target cells. The colicin-LepB interaction is shown to require only a short peptide sequence within the central domain of these bacteriocins and to involve residues present in the short C-terminal Box E of LepB. Genomic screening identified the conserved LepB binding motif in colicin-like ORFs from thirteen additional bacterial species. These findings establish a new paradigm for the functional adaptability of an essential inner-membrane enzyme.
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The PTK7 and ROR2 receptors interact in the vertebrate WNT/PCP pathway [Cell Biology]

October 23rd, 2015 by

The non-canonical WNT/Planar Cell Polarity (WNT/PCP) pathway plays important roles in morphogenetic processes in vertebrates. Among WNT/PCP components, Protein Tyrosine Kinase 7 (PTK7) is a tyrosine kinase receptor lacking catalytic activity with poorly defined functions. Here we show that PTK7 associates with receptor tyrosine kinase-like orphan receptor 2 (ROR2) to form a heterodimeric complex in mammalian cells. We demonstrate that PTK7 and ROR2 physically and functionally interact with the non-canonical WNT5A ligand leading to JNK activation and cell movements. In the Xenopus embryo, Ptk7 functionally interacts with Ror2 to regulate protocadherin papc expression and morphogenesis. Furthermore, we show that Ptk7 is required for papc activation induced by Wnt5a. Interestingly, we find that Wnt5a stimulates the release of the tagged Ptk7 intracellular domain, which can translocate into the nucleus and activate papc expression. This study reveals novel molecular mechanisms of action of PTK7 in non-canonical WNT/PCP signalling that may promote cell and tissue movements.

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.