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.

Streptococcal 5′-nucleotidase A (S5nA), a novel group A Streptococcal virulence factor that facilitates immune evasion [Molecular Bases of Disease]

November 2nd, 2015 by

Streptococcus pyogenes is an important human pathogen that causes a wide range of diseases. Using bioinformatics analysis of the complete S. pyogenes strain SF370 genome, we have identified a novel S. pyogenes virulence factor, which we termed streptococcal 5-nucleotidase A (S5nA). A recombinant form of S5nA hydrolyzed AMP and ADP, but not ATP, to generate the immunomodulatory molecule adenosine. Michaelis-Menten kinetics revealed a Km of 9.3 nmol and a Vmax of 7550 nmol/mg/min for the substrate AMP. Furthermore, rS5nA acted synergistically with S. pyogenes nuclease A (SpnA) to generate macrophage-toxic deoxyadenosine from DNA. The enzyme showed optimal activity between pH5 and pH6.5, and between 37C and 47C. Like other 5-nucleotidases, S5nA requires divalent cations and was active in the presence of Mg2+, Ca2+ or Mn2+. However, Zn2+ inhibited the enzymatic activity. Structural modeling combined with mutational analysis revealed a highly conserved catalytic dyad, as well as conserved substrate and cation-binding sites. Recombinant S5nA significantly increased the survival of the non-pathogenic bacterium Lactococcus lactis during a human whole blood killing assay in a dose-dependent manner suggesting a role as a S. pyogenes virulence factor. In conclusion, we have identified a novel S. pyogenes enzyme with 5-nucleotidase activity and immune evasion properties.
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The C-terminal Cytosolic Region of Rim21 Senses Alterations in Plasma Membrane Lipid Composition: Insights into Sensing Mechanisms for Plasma Membrane Lipid Asymmetry [Lipids]

November 2nd, 2015 by Nishino, K., Obara, K., Kihara, A.

Yeast responds to alterations in plasma membrane lipid asymmetry and external alkalization via the sensor protein Rim21 in the Rim101 pathway. However, the sensing mechanism used by Rim21 remains unclear. Here, we found that the C-terminal cytosolic domain of Rim21 (Rim21C) fused with GFP was associated with the plasma membrane under normal conditions, but dissociated upon alterations in lipid asymmetry or external alkalization. This indicates that Rim21C contains a sensor motif. Rim21C contains multiple clusters of charged residues. Among them, three consecutive Glu residues (EEE motif) were essential for Rim21 function and dissociation of Rim21C from the plasma membrane in response to changes in lipid asymmetry. In contrast, positively charged residues adjacent to the EEE motif were required for Rim21C to associate with the membrane. We therefore propose an ″antenna hypothesis″, in which Rim21C moves to or from the plasma membrane and functions as the sensing mechanism of Rim21.
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Loss of Scribble promotes Snail translation through translocation of HuR and enhances cancer drug resistance [Signal Transduction]

November 2nd, 2015 by Zhou, Y., Chang, R., Ji, W., Wang, N., Qi, M., Xu, Y., Guo, J., Zhan, L.

Drug resistance of cancer cells to various therapeutic agents and molecular targets is a major problem facing current cancer research. The tumor suppressor gene Scribble encodes a polarity protein that is conserved between Drosophila and mammals; loss of the locus disrupts cell polarity, inhibits apoptosis, and mediates cancer process. However, the role of Scribble in drug resistance remains unknown. We show here that knockdown of Scribble enhances drug resistance by permitting accumulation of Snail, which functions as a transcription factor during the epithelial-mesenchymal transition. Then, loss of Scribble activates the mRNA binding protein HuR by facilitating translocation of HuR from the nucleus to the cytoplasm. Furthermore, we demonstrate HuR can recognize AU-rich elements (AREs) of the Snail-encoding mRNA, thereby regulating Snail translation. Moreover, Loss of Scribble induced HuR translocation mediates the accumulation of Snail via activation of the p38 MAPK pathway. Thus, this work clarifies the role of polarity protein Scribble, which is directly implicated in the regulation of developmental transcription factor Snail, and suggesting a mechanism for Scribble mediating cancer drug resistance

MicroRNA Cargo of Extracellular Vesicles from Alcohol-Exposed Monocytes Signals Naive Monocytes to Differentiate into M2 Macrophages [Signal Transduction]

November 2nd, 2015 by Saha, B., Momen-Heravi, F., Kodys, K., Szabo, G.

Membrane-coated extracellular vesicles (EVs) released by cells can serve as vehicles for delivery of biological materials and signals. Recently, we demonstrated that alcohol-treated hepatocytes crosstalk with immune cells via exosomes containing miRNAs. Here we hypothesized that alcohol-exposed monocytes can communicate with naive monocytes via EVs. We observed increased number of EVs, mostly exosomes, secreted by primary human monocytes and THP-1 monocytic cells in the presence of alcohol in a concentration- and time-dependent manner. EVs derived from alcohol-treated monocytes stimulated naive monocytes to polarize into M2-macrophages as indicated by increased surface expression of CD68 (macrophage marker), M2 markers [CD206 (mannose receptor), CD163 (scavenger receptor)], secretion of IL-10 and TGFβ, and increased phagocytic activity. miRNA profiling of the EVs derived from alcohol-treated THP-1 monocytes revealed high expression of the M2 polarizing miRNA, miR-27a. Treatment of naive monocytes with control EVs overexpressing miR-27a reproduced the effect of EVs from alcohol-treated monocytes on naive monocytes and induced M2 polarization, suggesting that the effect of alcohol EVs was mediated by miR-27a. We found that miR-27a modulated the process of phagocytosis by targeting CD206 expression on monocytes. Importantly, analysis of circulating EVs from plasma of alcoholic hepatitis patients revealed increased numbers of EVs that contained high levels of miR-27a as compared to healthy controls. Our results demonstrate first, that alcohol increases EV production in monocytes, second, alcohol-exposed monocytes communicate with naive monocytes via EVs and third, miR-27a cargo in monocyte-derived EVs can program naive monocytes to polarize into M2-macrophages.
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Chemical Modulation of Endocytic Sorting Augments Adeno-Associated Viral Transduction [Cell Biology]

November 2nd, 2015 by Berry, G. E., Asokan, A.

Intracellular trafficking of viruses can be influenced by a variety of inter-connected cellular sorting and degradation pathways involving endo-lysosomal vesicles, the ubiquitin-proteasome system, autophagy-based or ER-associated machinery. In case of recombinant adeno-associated viruses (AAV), proteasome inhibitors are known to prevent degradation of ubiquitinated AAV capsids, thereby leading to increased nuclear accumulation and transduction. However, the impact of other cellular degradation pathways on AAV trafficking is not well-understood. In the current report, we screened a panel of small molecules focused on modulating different cellular degradation pathways and identified Eeyarestatin I (EerI) as a novel reagent that enhances AAV transduction. EerI improved AAV transduction by an order of magnitude regardless of vector dose, genome architecture, cell type, or serotype. This effect was preceded by sequestration of AAV within enlarged vesicles that were dispersed throughout the cytoplasm. Specifically, EerI treatment redirected AAV particles towards large vesicles positive for late endosomal (Rab7) and lysosomal (LAMP1) markers. Notably, MG132 and EerI (proteasomal and endoplasmic reticulum-associated degradation (ERAD) inhibitors, respectively) appear to enhance AAV transduction by increasing the intracellular accumulation of viral particles in a mutually exclusive fashion. Taken together, our results expand on potential strategies to redirect recombinant AAV vectors towards more productive trafficking pathways by deregulating cellular degradation mechanisms.