Mechanisms of Enhancer-mediated Hormonal Control of Vitamin D Receptor Gene Expression in Target Cells [Gene Regulation]

October 25th, 2015 by Lee, S. M., Meyer, M. B., Benkusky, N. A., O'Brien, C. A., Pike, J. W.

The biological actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are mediated by the vitamin D receptor (VDR) whose expression in bone cells is regulated positively by 1,25(OH)2D3, retinoic acid and parathyroid hormone through both intergenic and intronic enhancers. In this report, we used ChIP-seq analysis to confirm the presence of these Vdr gene enhancers in mesenchymal-derived bone cells and then describe the epigenetic histone landscape that span the Vdr locus. Using bacterial artificial chromosome-minigene stable cell lines, CRISPR/Cas9 enhancer-deleted daughter cell lines, transient transfection/mutagenesis analyses and transgenic mice, we confirm the functionality of these bone cell enhancers in vivo as well as in vitro. We also identified VDR binding sites across the Vdr gene locus in kidney and intestine using ChIP-seq analysis, revealing that that only one of the bone cell-type enhancers bound VDR in kidney tissue and none were occupied by the VDR in the intestine, consistent with weak or absent regulation by the 1,25(OH)2D3 hormone in these tissues, respectively. However, a number of additional sites of VDR binding unique to either kidney or intestine were present further upstream of the Vdr gene, suggesting the potential for alternative regulatory loci. Importantly, virtually all of these regions retained histone signatures consistent with those of enhancers and exhibited unique DNase I hypersensitivity profiles that reflected the potential for chromatin access. These studies define mechanisms associated with hormonal regulation of the Vdr and hint at the differential nature of VDR binding activity at the Vdr gene in different primary target tissues in vivo.

ROS mediates p300-dependent STAT1 interaction with PPAR{gamma} in CD36 expression and foam cell formation [Lipids]

October 25th, 2015 by Kotla, S., Rao, G. N.

Previously we have demonstrated that 15(S)-HETE induces CD36 expression involving STAT1. Many studies have shown that PPARγ mediates CD36 expression. Therefore, we asked the question whether these transcriptional factors interact with each other in the regulation of CD36 expression by 15(S)-HETE. Here we show that STAT1 interacts with PPARγ in the induction of CD36 expression and foam cell formation by 15(S)-HETE. In addition, using molecular biological approaches such as EMSA, supershift EMSA, ChIP, re-ChIP and promoter-reporter gene assays, we demonstrate that the STAT1 and PPARγ complex binds to STAT binding site at -107 nt in the CD36 promoter and enhances its activity. Furthermore, the interaction of STAT1 with PPARγ depends on STAT1 acetylation, which is mediated by p300. In addition, our findings show that ROS-dependent Syk and Pyk2 stimulation is required for p300 tyrosine phosphorylation and activation. Together, these results demonstrate that an interaction between p300, STAT1 and PPARγ is required for 15(S)-HETE-induced CD36 expression, oxLDL uptake and foam cell formation, critical events underlying the pathogenesis of atherosclerosis.

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.

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.

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.