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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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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Cyclable Condensation and Hierarchical Assembly of Metastable Reflectin Proteins, the Drivers of Tunable Biophotonics [Cell Biology]

December 30th, 2015 by Levenson, R., Bracken, C., Bush, N., Morse, D. E.

Reversible changes in phosphorylation of the reflectin proteins have been shown to drive the tunability of color and brightness of light reflected from specialized cells in the skin of squids and related cephalopods. We show here, using dynamic light scattering, electron microscopy, and fluorescence analyses, that reversible titration of the excess positive charges of the reflectins, comparable to that produced by phosphorylation, is sufficient to drive the reversible condensation and hierarchical assembly of these proteins. Results suggest a two-stage process in which charge neutralization first triggers condensation, resulting in the emergence of previously cryptic structures that subsequently mediate reversible, hierarchical assembly. The extent to which cyclability is seen in the in vitro formation and disassembly of complexes estimated to contain several thousand reflectin molecules suggests that intrinsic sequence- and structure-determined specificity governs the reversible condensation and assembly of the reflectins, and that these processes are thus sufficient to produce the reversible changes in refractive index, thickness and spacing of the reflectin-containing subcellular Bragg lamellae to change the brightness and color of reflected light. This molecular mechanism points to the metastability of the reflectins as the centrally important design principle governing biophotonic tunability in this system.

Inhibition of RPE65 Retinol Isomerase Activity by Inhibitors of Lipid Metabolism [Lipids]

December 30th, 2015 by Eroglu, A., Gentleman, S., Poliakov, E., Redmond, T. M.

RPE65 is the isomerase catalyzing conversion of all-trans-retinyl ester (atRE) into 11-cis-retinol in the retinal visual cycle. Crystal structures of RPE65 and site-directed mutagenesis reveal aspects of its catalytic mechanism, especially retinyl moiety isomerization, but other aspects remain to be determined. To investigate potential interactions between RPE65 and lipid metabolism enzymes, HEK293-F cells were transfected with expression vectors for visual cycle proteins and co-transfected with either fatty acyl:CoA ligases (ACSLs) 1, 3 or 6, or the SLC27A family fatty acyl-CoA synthase FATP2/SLCA27A2 to test their effect on isomerase activity. These experiments showed that RPE65 activity was reduced by co-expression of ACSLs or FATP2. Surprisingly, however, in attempting to relieve the ACSL-mediated inhibition, we discovered that triacsin C, an inhibitor of ACSLs, also potently inhibited RPE65 isomerase activity in cellulo. We found triacsin C to be a competitive inhibitor of RPE65 (IC50=500 nM). We confirmed that triacsin C competes directly with atRE by incubating membranes prepared from chicken RPE65-transfected cells with liposomes containing 0-1 μM atRE. Other inhibitors of ACSLs, had modest inhibitory effects compared to triascin C. In conclusion, we have identified an inhibitor of ACSLs as a potent inhibitor of RPE65 and which competes with the atRE substrate of RPE65 for binding. Triacsin C, with an alkenyl chain resembling, but not identical to, either acyl or retinyl chains, may compete with binding of the acyl moiety of atRE via the alkenyl moiety. Its inhibitory effect, however, may reside in its nitrosohydrazone/triazene moiety.

Recruitment of Mcm10 to Sites of Replication Initiation Requires Direct Binding to the MCM Complex [DNA and Chromosomes]

December 30th, 2015 by Douglas, M. E., Diffley, J. F. X.

Mcm10 is required for the initiation of eukaryotic DNA replication and contributes in some unknown way to the activation of the Cdc45-MCM-GINS (CMG) helicase. How Mcm10 is localised to sites of replication initiation is unclear, as current models implicate direct binding to MCM to play a role, but the details and functional importance of this interaction have not been determined. Here, we show that purified Mcm10 can bind both DNA-bound double hexamers and soluble single hexamers of MCM. The binding of Mcm10 to MCM requires the Mcm10 C-terminus. Moreover the binding site for Mcm10 on MCM includes the Mcm2 and Mcm6 subunits, and overlaps that for the loading factor Cdt1. Whether Mcm10 recruitment to replication origins depends on CMG helicase assembly has been unclear. We show that Mcm10 recruitment occurs via two modes: low affinity recruitment in the absence of CMG assembly (G1-like), and high affinity recruitment when CMG assembly takes place (S-phase-like). Mcm10 that cannot bind directly to MCM is defective in both modes of recruitment, and unable to support DNA replication. These findings indicate that Mcm10 is localised to replication initiation sites by directly binding MCM through the Mcm10 C-terminus.