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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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

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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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.

Structural basis of Ribosomal S6 Kinase 1 (RSK1) inhibition by S100B Protein: modulation of the Extracellular Signal-regulated Kinase (ERK) signaling cascade in a calcium-dependent way [Protein Structure and Folding]

November 2nd, 2015 by

Mitogen-activated protein kinases (MAPK) promote MAPK activated protein kinase (MAPKAPK) activation. In the MAPK pathway responsible to cell growth, ERK2 initiates the first phosphorylation event on RSK1, which is inhibited by calcium-binding S100 proteins in malignant melanomas. Here we present a detailed in vitro biochemical and structural characterization of the S100B-RSK1 interaction. The calcium-dependent binding of S100B to the calcium/calmodulin dependent protein kinase (CaMK)-type domain of RSK1 is reminiscent to the better known binding of calmodulin to CaMKII. Although S100B-RSK1 and the calmodulin-CAMKII system are clearly distinct functionally, they demonstrate how unrelated intracellular Ca2+ binding proteins could influence the activity of CaMK domain containing protein kinases. Our crystallographic, small angle X-ray scattering (SAXS) and NMR analysis revealed that S100B forms a ″fuzzy″ complex with RSK1 peptide ligands. Based on fast-kinetics experiments we conclude that the binding involves both conformation selection and induced fit steps. Knowledge of the structural basis of this interaction could facilitate therapeutic targeting of melanomas.
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Integrin {alpha}V{beta}5-mediated removal of apoptotic cell debris by the eye lens and its inhibition by UV-light exposure [Cell Biology]

November 2nd, 2015 by Chauss, D., Brennan, L. A., Bakina, O., Kantorow, M.

Accumulation of apoptotic material is toxic and associated with cataract and other disease states. Identification of mechanisms that prevent accumulation of apoptotic debris is important towards establishing the etiology of these diseases. The ocular lens is routinely assaulted by UV-light that causes lens cell apoptosis and is associated with cataract formation. To date, no molecular mechanism for removal of toxic apoptotic debris has been identified in the lens. Vesicular debris within lens cells exposed to UV-light has been observed raising speculation that lens cells themselves could act as phagocytes to remove toxic apoptotic debris. However, phagocytosis has not been confirmed as a function of the intact eye lens and no mechanism for lens phagocytosis has been established. Here, we demonstrate that the eye lens is capable of phagocytizing extracellular lens cell debris. Using high-throughput RNA sequencing and bioinformatics analysis we establish that lens epithelial cells express members of the integrin αVβ5-mediated phagocytosis pathway and that internalized cell debris co-localizes with αVβ5 and with RAB7 and RILP that are required for phagosome maturation and fusion with lysosomes. We demonstrate that the αVβ5 receptor is required for lens epithelial cell phagocytosis and that UV-light treatment of lens epithelial cells results in damage to the αVβ5 receptor with concomitant loss of phagocytosis. These data suggest that loss of αVβ5-mediated phagocytosis by the eye lens could result in accumulation of toxic cell debris that could contribute to UV light-induced cataract formation.

Regulation of Focal Adhesion Dynamics and Cell Motility by EB2 and Hax1 Complex [Signal Transduction]

November 2nd, 2015 by Liu, H., Yue, J., Huang, H., Gou, X., Chen, S.-Y., Zhao, Y., Wu, X.

Cell migration is a fundamental cellular process, requiring integrated activities of cytoskeleton, membrane, and cell/ECM adhesions. Many cytoskeletal activities rely on microtubule filaments. It has been speculated that microtubules can serve as tracks to deliver proteins essential for focal adhesion turnover. Three microtubule end-binding proteins (EB1, EB2 and EB3) in mammalian cells can track the plus ends of growing microtubules. EB1 and EB3 together can regulate microtubule dynamics by promoting microtubule growth and suppressing catastrophe; while in contrast, EB2 does not play a direct role in microtubule dynamic instability, and little is known about EB2 cellular function. By quantitative proteomics, we identified mammalian HAX1 (HCLS1 associated protein X-1), which associates with EB2 specifically. Knockdown of HAX1 and EB2 in skin epidermal cells stabilizes focal adhesions and impairs epidermal migration in vitro and in vivo. Our results further demonstrate that cell motility and focal adhesion turnover requires interaction between Hax1 and EB2. Together, our findings provide new insights for this critical cellular process, suggesting that EB2 association with Hax1 plays a significant role in focal adhesion turnover and epidermal migration.