Regulation of Monocarboxylic Acid Transporter 1 Trafficking by the Canonical Wnt/{beta}-catenin Pathway in Rat Brain Endothelial Cells, Requiring a Crosstalk with Notch Signaling [Signal Transduction]

February 12th, 2016 by Liu, Z., Sneve, M., Haroldson, T. A., Smith, J. P., Drewes, L. R.

The transport of monocarboxylate fuels, such as lactate, pyruvate and ketone bodies, across brain endothelial cells is mediated by monocarboxylic acid transporter 1 (MCT1). Although the canonical Wnt/β-catenin pathway is required for rodent blood-brain barrier (BBB) development and for the expression of associated nutrient transporters, the role of this pathway in regulation of brain endothelial MCT1 is unknown. Here, we report expression of nine members of the frizzled receptor family by the RBE4 rat brain endothelial cell line. Furthermore, activation of the canonical Wnt/β-catenin pathway in RBE4 cells via nuclear β-catenin signaling with lithium chloride (LiCl) does not alter brain endothelial Mct1 mRNA, but increases the amount of MCT1 transporter protein. Plasma membrane biotinylation studies and confocal microscopic examination of mCherry-tagged MCT1 indicate that increased transporter results from reduced MCT1 trafficking from the plasma membrane via the endosomal/lysosomal pathway and is facilitated by decreased MCT1 ubiquitination following LiCl treatment. Inhibition of the Notch pathway by the γ-secretase inhibitor, DAPT, negated the upregulation of MCT1 by LiCl, thus demonstrating a crosstalk between the canonical Wnt/β-catenin and Notch pathways. Our results are important because they show for the first time the regulation of MCT1 in cerebrovascular endothelial cells by the multi-functional canonical Wnt/β-catenin and Notch signaling pathways.
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Role of Intrinsic Protein Disorder in the Function and Interactions of the Transcriptional Coactivators CREB-Binding Protein (CBP) and p300 [Signal Transduction]

February 5th, 2016 by Dyson, H. J., Wright, P. E.

The transcriptional coactivators CBP and p300 undergo a particularly rich set of interactions with disordered and partly-ordered partners, as a part of their ubiquitous role in facilitating transcription of genes. CBP and p300 contain a number of small structured domains that provide scaffolds for the interaction of disordered transactivation domains from a wide variety of partners including p53, HIF-1&alpha, NF-&kappaB and STAT proteins, and are the targets for the interactions of disordered viral proteins that compete with cellular factors to disrupt signaling and subvert the cell cycle. The functional diversity of the CBP/p300 interactome provides an excellent example of the power of intrinsic disorder to facilitate the complexity of living systems.
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Thumb Site 2 Inhibitors of Hepatitis C Viral RNA-Dependent RNA Polymerase Allosterically Block the Transition from Initiation to Elongation [RNA]

February 5th, 2016 by Li, J., Johnson, K. A.

Replication of the Hepatitis C viral genome is catalyzed by the NS5B RNA-dependent RNA polymerase catalyzes, which is a major target of antiviral drugs currently in the clinic. Prior studies established that initiation of RNA replication could be facilitated by starting with a dinucleotide (pGG). Here we establish conditions for efficient initiation from GTP to form the dinucleotide and subsequent intermediates leading to highly processive elongation, and we examine the effects of four classes of nonnucleoside inhibitors on each step of the reaction. We show that palm site inhibitors block initiation starting from GTP but not when starting from pGG. In addition we show that nonnucleoside inhibitors binding to thumb site-2 (NNI2) lead to the accumulation of abortive intermediates 3-5 nucleotides in length. Our kinetic analysis shows that NNI2 do not significantly block initiation or elongation of RNA synthesis; rather they block the transition from initiation to elongation, which is thought to proceed with significant structural rearrangement of the enzyme-RNA complex including displacement of the β-loop from the active site. Direct measurement in single turnover kinetic studies show that pyrophosphate release is faster than the chemistry step, which appears to be rate-limiting during processive synthesis. These results reveal important new details to define the steps involved in initiation and elongation during viral RNA replication, establish the allosteric mechanisms by which NNI2 inhibitors act, and point the way to the design of more effective allosteric inhibitors that exploit this new information.
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Design principles involving protein disorder facilitate specific substrate selection and degradation by the ubiquitin-proteasome system [Protein Synthesis and Degradation]

February 5th, 2016 by Guharoy, M., Bhowmick, P., Tompa, P.

The ubiquitin-proteasome system (UPS) regulates diverse cellular pathways by the timely removal (or processing) of proteins. Here we review the role of structural disorder and conformational flexibility in the different aspects of degradation. First, we discuss posttranslational modifications within disordered regions that regulate E3 ligase localization, conformation and enzymatic activity, and also the role of flexible linkers in mediating ubiquitin transfer and reaction processivity. Next we review well-studied substrates and discuss that substrate elements (degrons) recognized by E3 ligases are highly disordered: short linear motifs recognized by many E3s constitute an important class of degrons and these are almost always present in disordered regions. Substrate lysines targeted for ubiquitination are also often located in neighboring regions of the E3 docking motifs and are therefore part of the disordered segment. Finally, biochemical experiments and predictions show that initiation of degradation at the 26S proteasome requires a partially unfolded region to facilitate substrate entry into the proteasomal core.
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Expanding the Range of Protein Function at the Far end of the Order-Structure Continuum [Molecular Biophysics]

February 5th, 2016 by Burger, V. M., Nolasco, D. O., Stultz, C. M.

The traditional view of the structure-function paradigm is that a protein's function is inextricably linked to a well-defined, three-dimensional structure, which is determined by the protein's primary amino acid sequence. However, it is now accepted that a number of proteins do not adopt a unique tertiary structure in solution and that some degree of disorder is required for many proteins to perform their prescribed functions. In this review we highlight how a number of protein functions are facilitated by intrinsic disorder and introduce a new protein structure taxonomy that is based on quantifiable metrics of a protein's disorder.

Nutrient-Regulated Phosphorylation of ATG13 Inhibits Starvation-Induced Autophagy [Membrane Biology]

January 22nd, 2016 by Puente, C., Hendrickson, R. C., Jiang, X.

Autophagy is a conserved catabolic process that utilizes a defined series of membrane trafficking events to generate a de novo double-membrane vesicle termed the autophagosome, which matures by fusing to the lysosome. Subsequently, the lysosome facilitates the degradation and recycling of the cytoplasmic cargo. In yeast, the upstream signals that regulate the induction of starvation-induced autophagy are clearly defined. The nutrient-sensing kinase Tor inhibits the activation of autophagy by regulating the formation of the Atg1-Atg13-Atg17 complex, through hyperphosphorylation of Atg13. However, in mammals, the ortholog complex ULK1-ATG13-FIP200 is constitutively formed. As such, the molecular mechanism by which mTOR regulates mammalian autophagy is unknown. Here we report the identification and characterization of novel nutrient-regulated phosphorylation sites on ATG13: Ser224 and Ser258. mTOR directly phosphorylates ATG13 on Ser258 while Ser224 is a putative AMPK phosphorylation site. In ATG13 knockout cells reconstituted with an unphosphorylatable mutant of ATG13, ULK1 kinase activity is more potent, and amino acid starvation induced more rapid ULK1 translocation and autophagy. Therefore, ATG13 phosphorylation plays a crucial role in autophagy regulation.

Coordinative Modulation of Chlorothricin Biosynthesis by Binding of the Glycosylated Intermediates and End Product to A Responsive Regulator ChlF1 [Gene Regulation]

January 10th, 2016 by Li, Y., Li, J., Tian, Z., Xu, Y., Zhang, J., Liu, W., Tan, H.

Chlorothricin, isolated from Streptomyces antibioticus, is a parent member of spirotetronate family of antibiotics that have long been appreciated for their remarkable biological activities. ChlF1 plays bifunctional roles in chlorothricin biosynthesis by binding to its target genes (chlJ, chlF1, chlG and chlK). The dissociation constants of ChlF1 to these genes are around 102-140 nM. A consensus sequence, 5′-GTAANNATTTAC-3′, was found in these binding sites. ChlF1 represses the transcription of chlF1, chlG and chlK but activates chlJ, which encodes a key enzyme acyl-CoA carboxyl transferase involved in the chlorothricin biosynthesis. We demonstrate that the end product chlorothricin and likewise its biosynthetic intermediates (demethylsalicycloyl chlorothricin and deschloro-chlorothricin) can act as signaling molecules to modulate the binding of ChlF1 to its target genes. Intriguingly, a correlation between the antibacterial activity and binding ability of signaling molecules to the regulator ChlF1 is clearly observed. These features of the signaling molecules are associated with the glycosylation of spirotetronate macrolide aglycone. The findings provide new insights into the TetR family regulators responding to special structure of signaling molecules, and we reveal the regulatory mini-network mediated by ChlF1 in chlorothricin biosynthesis for the first time.
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The lipid bilayer modulates the structure and function of an ATP-binding cassette exporter [Membrane Biology]

January 2nd, 2016 by Zoghbi, M. E., Cooper, R. S., Altenberg, G. A.

ATP-binding cassette (ABC) exporters use the energy of ATP hydrolysis to transport substrates across membranes by switching between inward- and outward-facing conformations. Essentially all structural studies of these proteins have been performed with the proteins in detergent micelles, locked in specific conformations and/or at low temperature. Here, we used luminescence resonance energy transfer (LRET) spectroscopy to study the prototypical ABC exporter MsbA reconstituted in nanodiscs,at 37°C, and while it performs ATP hydrolysis. We found major differences when comparing MsbA in these native-like conditions with double electron-electron resonance data and the crystal structure of MsbA in the open inward-facing conformation. The most striking differences include a significantly smaller separation between the nucleotide-binding domains and a larger fraction of molecules with associated nucleotide-binding domains in the nucleotide-free apo state. These studies stress the importance of studying membrane proteins in an environment that approaches physiological conditions.

Thromboxane A2 Receptor Inhibition Suppresses Multiple Myeloma Cell Proliferation by Inducing P38/JNK MAP Kinase Mediated-G2/M Progression Delay and Cell Apoptosis [Signal Transduction]

January 2nd, 2016 by Liu, Q., Tao, B., Liu, G., Chen, G., Zhu, Q., Yu, Y., , Xiong, H.

Multiple myeloma (MM) is a plasma cell malignancy without effective therapeutics. Thromboxane A2 (TxA2)/ TxA2 receptor (T prostanoid receptor, TP) modulates some carcinomas progression, however, its effects on MM cell proliferation remain unclear. In this study, we evaluated cyclooxygenase (COX) enzymes and downstream prostaglandin profiles in human myeloma cell lines RPMI-8226 and U-266, and analyzed the effects of COX-1/-2 inhibitors SC-560 and NS-398 on MM cell proliferation. Our observations implicate COX-2 is involved in modulating cell proliferation. We further incubated MM cells with prostaglandins receptors antagonists or agonists, and found only the TP antagonist, SQ29548, suppressed MM cell proliferation. TP silencing and the TP agonist, U46619, further confirmed this finding. Moreover, SQ29548 and TP silencing promoted MM cells G2/M phase delay accompanied by reducing cyclin B1/ cyclin-dependent kinase-1 (CDK1) mRNA and protein expression. Notably, cyclin B1 overexpression rescued MM cells from G2/M arrest. We also found the TP agonist activated JNK and p38 MAPK phosphorylation, and inhibitors of JNK and p38 MAPK depressed U46619-induced proliferation and cyclin B1/CDK1 protein expression. In addition, SQ29548 and TP silencing leaded to MM cells apoptotic rate increasing with improving caspase 3 activity. The knockdown of caspase 3 reversed the apoptotic rate. Taken together, our results suggest that TxA2/TP promotes MM cell proliferation by reducing cells delay at G2/M phase via elevating p38 MAPK/JNK mediated-cyclin B1/CDK1 expression, and hindering cell apoptosis. The TP inhibitor has potential as a novel agent to target kinase cascades for MM therapy.
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CXCL1/MGSA is a novel glycosaminoglycan (GAG)-binding chemokine: structural evidence for two distinct non-overlapping binding domains [Immunology]

December 31st, 2015 by Sepuru, K. M., Rajarathnam, K.

In humans, the chemokine CXCL1/MGSA (hCXCL1), plays fundamental and diverse roles in pathophysiology, from microbial killing to cancer progression, by orchestrating directed migration of immune and non-immune cells. Cellular trafficking is highly regulated and requires concentration gradients that are achieved by interactions with sulfated glycosaminoglycans (GAGs). However, very little is known regarding the structural basis underlying hCXCL1-GAG interactions. We have addressed this missing knowledge by characterizing the binding of GAG heparin oligosaccharides to hCXCL1 using nuclear magnetic resonance (NMR) spectroscopy. Binding experiments under conditions at which hCXCL1 exists as monomers and dimers indicate that the dimer is the high-affinity GAG ligand. NMR experiments and modeling studies indicate that lysine and arginine residues mediate binding, and are located in two non-overlapping domains. One domain, consisting of N-loop and C-helical residues (defined as α-domain) was also previously identified as the GAG-binding domain for the related chemokine CXCL8/IL-8. The second domain, consisting of residues from the N-terminus, 40s turn, and 3rd β-strand (defined as β-domain) is novel. Eliminating β-domain binding by mutagenesis does not perturb α-domain binding indicating two independent GAG-binding sites. It is known that N-loop and N-terminal residues mediate receptor activation, and we show that these residues are also involved in extensive GAG interactions. We also show that GAG-bound chemokine completely occludes receptor binding. We conclude that hCXCL1-GAG interactions provide stringent control over regulating chemokine levels and receptor accessibility and activation, and that chemotactic gradients mediate cellular trafficking to the target site.
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