A heme-responsive regulator controls synthesis of staphyloferrin B in Staphylococcus aureus [Gene Regulation]

November 3rd, 2015 by

Staphylococcus aureus possesses a multitude of mechanisms by which it can obtain iron during growth under iron starvation conditions. It expresses an effective heme acquisition system (the iron-regulated surface determinant system), it produces two carboxylate-type siderophores staphyloferrin A (SA) and staphyloferrin B (SB), and it expresses transporters for many other siderophores that it itself does not synthesize. The Ferric Uptake Regulator, or Fur, protein regulates expression of genes encoding all of these systems. Mechanisms of fine-tuning expression of iron-regulated genes, beyond simple iron-regulation via Fur, have not been uncovered in this organism. Here, we identify the ninth gene of the sbn operon, sbnI, as encoding a ParB/Spo0J-like protein that is required for expression of genes in the sbn operon from sbnD onwards. Expression of sbnD-I is drastically decreased in an sbnI mutant, and the mutant does not synthesize detectable SB during early phases of growth. Thus, SB-mediated iron acquisition is impaired in an sbnI mutant strain. We show that the protein forms dimers and tetramers in solution and binds to DNA within the sbnC coding region. Moreover, we show that SbnI binds heme, and that heme-bound SbnI does not bind DNA. Last, we show that providing exogenous heme to S. aureus growing in an iron-free medium results in delayed synthesis of SB. This is the first study in S. aureus that identifies a DNA-binding regulatory protein that senses heme to control gene expression for siderophore synthesis.

HIV-1-Tat Inhibits SC35-Mediated Tau Exon 10 Inclusion through Upregulation of DYRK1A Kinase [Signal Transduction]

November 3rd, 2015 by

The HIV-1 transactivator protein Tat is implicated in the neuronal damage that contributes to neurocognitive impairment affecting people living with HIV/AIDS. Aberrant splicing of Tau exon 10 results in tauopathies characterized by alterations in the proportion of Tau isoforms containing three (3R) or four (4R) microtubule-binding repeats. The splicing factor SC35/SRSF2 binds to nuclear RNA and facilitates the incorporation of exon 10 in the Tau molecule. Here, we utilized clinical samples, an animal model, and neuronal cell cultures and found that Tat promotes Tau 3R upregulation though increased levels of phosphorylated SC35, which is retained in nuclear speckles. This mechanism involved Tat-mediated increased expression of DYRK1A and was prevented by DYRK1A silencing. In addition, we found that Tat associates with Tau RNA, further demonstrating that Tat interferes with host RNA metabolism in the absence of viral infection. Altogether, our data unravel a novel mechanism of Tat-mediated neuronal toxicity through dysregulation of the SC35-dependent alternative splicing of Tau exon 10. Furthermore, the increased immunostaining of DYRK1A in HIV+ brains without pathology points at dysregulation of DYRK1A as an early event in the neuronal complications of HIV infection.

Exosome adherence and internalization by hepatic stellate cells triggers sphingosine 1-phosphate dependent migration [Cell Biology]

November 3rd, 2015 by

Exosomes are cell-derived extracellular vesicles thought to promote intercellular communication by delivering specific content to target cells. The aim of this study was to determine whether endothelial cell (EC) derived exosomes could regulate the phenotype of hepatic stellate cells (HSC). Initial microarray studies showed fibroblast growth factor-2 induced a 2.4-fold increase in mRNA levels of sphingosine kinase 1 (SK1). Exosomes derived from an SK1 overexpressing EC line increased HSC migration by 3.2-fold. Migration was not conferred by the dominant negative SK1 exosome. Incubation of HSC with exosomes was also associated with an 8.3-fold increased phosphorylation of AKT and 2.5-fold increased migration. Exosomes were found to express the matrix protein and integrin ligand fibronectin (FN) by Western blot and transmission electron microscopy. Blockade of FN-integrin interaction with a CD29 neutralizing antibody or the RGD peptide attenuated exosome-induced HSC AKT phosphorylation and migration. Inhibitions of endocytosis with transfection of dynamin siRNA, dominant negative dynamin GTPase construct Dyn2K44A, or by the pharmacological inhibitor Dynasore, significantly attenuated exosome-induced AKT phosphorylation. SK1 levels were increased in serum exosomes derived from mice with experimental liver fibrosis and SK1 mRNA levels were upregulated in human liver cirrhosis patient samples by 2.5-fold. Finally, S1PR2 inhibition protected mice from CCl4-induced liver fibrosis. Thus, EC-derived SK1-containing exosomes regulate HSC signaling and migration through FN-integrin dependent exosome adherence and dynamin dependent exosome internalization. These findings advance our understanding of EC/HSC crosstalk and identify exosomes as a potential target to attenuate pathobiology signals.

Time-resolved studies of IsdG identify molecular signposts along the non-canonical heme oxygenase pathway [Microbiology]

November 3rd, 2015 by

IsdGs are heme monooxygenases that break open the tetrapyrrole, releasing the iron and thereby allowing bacteria expressing this protein to use heme as a nutritional iron source. Little is currently known about the mechanism by which IsdGs degrade heme, though the products differ from those generated by canonical heme oxygenases. A synthesis of time resolved techniques including in proteo mass spectrometry and conventional and stopped flow UV/vis was used in conjunction with analytical methods to define the reaction steps mediated by IsdG from Staphylococcus aureus and their time scales. An apparent meso -(hydr)oxyheme (forming with k = 0.6 min-1, pH 7.4, 10 mM ascorbate, 10 μM IsdG-heme, 22 °C) was identified as a likely common intermediate with the canonical heme oxygenases (HOs). Unlike HOs, this intermediate does not form with added H2O2, nor does it convert to verdoheme and CO. Rather, the next observable intermediates (k = 0.16 min-1) were a set of formyl-oxo-bilin isomers, similar to the mycobilin products of the IsdG homolog from Mycobacterium tuberculosis (MhuD). These converted in separate fast and slow phases to β-/δ-staphylobilin isomers and formaldehyde (CH2O). Controlled release of this unusual C1 product may support IsdG′s dual role as both an oxygenase and a sensor of heme availability in S. aureus.

Immunometabolism; cellular metabolism turns immune regulator [Metabolism]

November 3rd, 2015 by Loftus, R. M., Finlay, D. K.

Immune cells are highly dynamic in terms of their growth, proliferation and effector functions as they respond to immunological challenges. Different immune cells can adopt distinct metabolic configurations that allow the cell to balance its requirements for energy, molecular biosynthesis and longevity. However, in addition to facilitating immune cell responses, it is now becoming clear that cellular metabolism has direct roles in regulating immune cell function. This review article describes the distinct metabolic signatures of key immune cells, explains how these metabolic setups facilitate immune function and discusses the emerging evidence that intracellular metabolism has an integral role in controlling immune responses.

SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMPK-mTOR Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells [Metabolism]

November 3rd, 2015 by

Mitochondrial oxidative-phosphorylation produces most of the energy in aerobic cells by coupling respiration to the production of ATP. Mitochondrial uncouplers, which reduce the proton-gradient across the mitochondrial inner membrane, create a futile cycle of nutrient oxidation without generating ATP. Regulation of mitochondrial dysfunction and associated cellular bioenergetics has been recently identified as promising targets for anticancer therapy. Here, we show that SR4 is a novel mitochondrial uncoupler that causes dose-dependent increase in mitochondrial respiration and dissipation of mitochondrial-membrane-potential (MMP) in HepG2 hepatocarcinoma cells. These effects were reversed by the recoupling agent 6-ketocholestanol, but not cyclosporin A, and were non-existent in mitochondrial-DNA depleted HepG2 (po) cells. In isolated mouse liver mitochondria, SR4 similarly increased oxygen consumption independent of adenine nucleotide translocase and uncoupling proteins, decreases MMP, and promotes swelling of valinomycin-treated mitochondria in potassium acetate media. Mitochondrial uncoupling in HepG2 cells by SR4 results in the reduction of cellular ATP production, increased ROS production, activation of the energy-sensing enzyme AMPK, and inhibition of acetyl-CoA carboxylase and mammalian target of rapamycin (mTOR) signaling pathways, leading to cell cycle arrest and apoptosis. Global analysis of SR4-associated differential gene expression confirms these observations, including significant induction of apoptotic genes and down regulation of cell cycle, mitochondrial and oxidative-phosphorylation pathway transcripts at 24 h post treatment. Collectively, our studies demonstrate that SR4's previously reported indirect activation of AMPK and in-vitro anticancer properties, as well as its beneficial effects in both animal xenograft and obese-mice models could be a direct consequence of its mitochondrial uncoupling activity.
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Hepatocyte Nuclear Factor 4{alpha} Controls Iron Metabolism and Regulates Transferrin Receptor 2 in Mouse Liver [Metabolism]

November 2nd, 2015 by

Iron is an essential element in biological systems, but excess iron promotes the formation of reactive oxygen species, resulting in cellular toxicity. Several iron-related genes are highly expressed in the liver, a tissue in which hepatocyte nuclear factor 4α (HNF4α) plays a critical role in controlling gene expression. Therefore, the role of hepatic HNF4α in iron homeostasis was examined using liver-specific HNF4α-null mice (Hnf4aΔH mice). Hnf4aΔH mice exhibit hypoferremia and a significant change in hepatic gene expression. Notably, the expression of transferrin receptor 2 (Tfr2) mRNA was markedly decreased in Hnf4aΔH mice. Promoter analysis of the Tfr2 gene showed that the basal promoter was located at a GC-rich region upstream of the transcription start site, a region that can be transactivated in an HNF4α-independent manner. HNF4α-dependent expression of Tfr2 was mediated by a proximal promoter containing two HNF4α binding sites located between the transcription start site and the translation start site. Both the GC-rich region of the basal promoter and the HNF4α binding sites were required for maximal transactivation. Moreover, siRNA knockdown of HNF4α suppressed Tfr2 expression in human HCC cells. These results suggest that Tfr2 is a novel target gene for HNF4α and hepatic HNF4α plays a critical role in iron homeostasis.

E2F1 transcription factor regulates O-GlcNAc transferase and O-GlcNAcase expression [Glycobiology and Extracellular Matrices]

November 2nd, 2015 by

Protein O-GlcNAcylation, which is controlled by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), has emerged as an important posttranslational modification that may factor in multiple diseases. Until recently, it was assumed that OGT/OGA protein expression was relatively constant. Several groups, including ours, have shown that OGT and/or OGA expression changes in several pathologic contexts. Yet, the cis and trans elements that regulate the expression of these enzymes remain essentially unexplored. Here, we used a reporter-based assay to analyze minimal promoters, and leveraged in silico modeling to nominate several candidate transcription factor binding sites in both Ogt and Oga. We noted multiple E2F1 binding site consensus sequences in both promoters. We performed chromatin immunoprecipitation in both human and mouse cells, and found that E2F1 bound to candidate E2F1 binding sites in both promoters. In HEK293 cells, we overexpressed E2F1, which significantly reduced OGT and OGA expression. Conversely, E2F1 deficient mouse fibroblasts had increased levels of Ogt and Oga promoters. Of the known binding partners for E2F1, we queried whether Retinoblastoma 1 (Rb1) might be involved. Rb1 deficient mouse embryonic fibroblasts showed increased levels of Ogt and Oga expression. Yet, overexpression of E2F1 in the Rb1 deficient cells did not alter Ogt and Oga expression, suggesting that Rb1 is required for E2F1-mediated suppression. In conclusion, this work identifies and validates some of the promoter elements for mouse Ogt and Oga genes. Specifically, E2F1 negatively regulates both Ogt and Oga expression in an Rb1 protein-dependent manner.

Glut4 is Sorted from a Rab10-Independent Constitutive Recycling Pathway into a Highly Insulin-Responsive Rab10-Dependent Sequestration Pathway after Adipocyte Differentiation [Metabolism]

November 2nd, 2015 by

The RabGAP AS160/TBC1D4 controls exocytosis of the insulin-sensitive glucose transporter Glut4 in adipocytes. Glut4 is internalized and recycled through a highly regulated secretory pathway in these cells. Glut4 also cycles through a slow constitutive endosomal pathway distinct from the fast transferrin (Tf) receptor recycling pathway. This slow constitutive pathway is the only Glut4 cycling pathway in undifferentiated fibroblasts. The α2-macroglobulin receptor LRP1 cycles with Glut4 and the Tf receptor, through all three exocytic pathways. To further characterize these pathways, the effects of knockdown of AS160 substrates on the trafficking kinetics of Glut4, LRP1, and the Tf receptor were measured in adipocytes and fibroblasts. Rab10 knockdown decreased cell surface Glut4 in insulin-stimulated adipocytes by 65%, but not in basal adipocytes or in fibroblasts. This decrease was due primarily to a 62% decrease in the rate constant of Glut4 exocytosis (kex), although Rab10 knockdown also caused a 1.4-fold increase in the rate constant of Glut4 endocytosis (ken). Rab10 knockdown in adipocytes also decreased cell surface LRP1 by 30% by decreasing kex 30-40%. There was no effect on LRP1 trafficking in fibroblasts, or on Tf receptor trafficking in either cell type. These data confirm that Rab10 is an AS160 substrate that limits exocytosis through the highly insulin-responsive specialized secretory pathway in adipocytes. They further show that the slow constitutive endosomal (fibroblast) recycling pathway is Rab10-independent. Thus, Rab10 is a marker for the specialized pathway in adipocytes. Interestingly, mathematical modeling shows that Glut4 traffics predominantly through the specialized, Rab10-dependent pathway both before and after insulin stimulation.
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Novel UDP-GalNAc derivative structures provide insight into the donor specificity of human blood group glycosyltransferase [Enzymology]

November 2nd, 2015 by Wagner, G. K., Pesnot, T., Palcic, M. M., Jorgensen, R.

Two closely related glycosyltranferases are responsible for the final step of the biosynthesis of ABO(H) human blood group A and B antigens. The two enzymes differ by only four amino acid residues, which determine whether the enzymes transfer GalNAc from UDP-GalNAc or Gal from UDP-Gal to the H-antigen acceptor. The enzymes belong to the class of GT-A folded enzymes, grouped as GT6 in the CAZy database, and are characterized by a single domain with a metal dependent retaining reaction mechanism. However, the exact role of the four amino acid residues in the specificity of the enzymes is still unresolved. In this study, we report the first structural information of a dual specificity cis-AB blood group glycosyltransferase in complex with a synthetic UDP-GalNAc derivative. Interestingly, the GalNAc moiety adopts an unusual yet catalytically productive conformation in the binding pocket, which is different from the "tucked under" conformation previously observed for the UDP-Gal donor. In addition, we show that this UDP-GalNAc derivative in complex with the H-antigen acceptor provokes the same unusual binding pocket closure as seen for the corresponding UDP-Gal derivative. Despite this, the two derivatives show vastly different kinetic properties. Our results provide a important structural insight into the donor substrate specificity and utilization in blood group biosynthesis, which can very likely be exploited for the development of new glycosyltransferase inhibitors and probes.