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Arrestin proteins bind to active, phosphorylated G-protein-coupled receptors (GPCRs), thereby preventing G-protein coupling, triggering receptor internalization and affecting various downstream signalling pathways.sup.1,2. Although there is a wealth of structural information detailing the interactions between GPCRs and G proteins, less is known about how arrestins engage GPCRs. Here we report a cryo-electron microscopy structure of full-length human neurotensin receptor 1 (NTSR1) in complex with truncated human [beta]-arrestin 1 ([beta]arr1([DELTA]CT)). We find that phosphorylation of NTSR1 is critical for the formation of a stable complex with [beta]arr1([DELTA]CT), and identify phosphorylated sites in both the third intracellular loop and the C terminus that may promote this interaction. In addition, we observe a phosphatidylinositol-4,5-bisphosphate molecule forming a bridge between the membrane side of NTSR1 transmembrane segments 1 and 4 and the C-lobe of arrestin. Compared with a structure of a rhodopsin-arrestin-1 complex, in our structure arrestin is rotated by approximately 85° relative to the receptor. These findings highlight both conserved aspects and plasticity among arrestin-receptor interactions. A cryo-electron microscopy structure of the neurotensin receptor 1 in complex with [beta]-arrestin 1 is reported., Author(s): Weijiao Huang [sup.1] , Matthieu Masureel [sup.1] , Qianhui Qu [sup.1] [sup.2] , John Janetzko [sup.1] , Asuka Inoue [sup.3] , Hideaki E. Kato [sup.1] [sup.6] , Michael J. [...]

After activation by an agonist, G-protein-coupled receptors (GPCRs) recruit [beta]-arrestin, which desensitizes heterotrimeric G-protein signalling and promotes receptor endocytosis.sup.1. Additionally, [beta]-arrestin directly regulates many cell signalling pathways that can induce cellular responses distinct from that of G proteins.sup.2. In contrast to G proteins, for which there are many high-resolution structures in complex with GPCRs, the molecular mechanisms underlying the interaction of [beta]-arrestin with GPCRs are much less understood. Here we present a cryo-electron microscopy structure of [beta]-arrestin 1 ([beta]arr1) in complex with M2 muscarinic receptor (M2R) reconstituted in lipid nanodiscs. The M2R-[beta]arr1 complex displays a multimodal network of flexible interactions, including binding of the N domain of [beta]arr1 to phosphorylated receptor residues and insertion of the finger loop of [beta]arr1 into the M2R seven-transmembrane bundle, which adopts a conformation similar to that in the M2R-heterotrimeric G.sub.o protein complex.sup.3. Moreover, the cryo-electron microscopy map reveals that the C-edge of [beta]arr1 engages the lipid bilayer. Through atomistic simulations and biophysical, biochemical and cellular assays, we show that the C-edge is critical for stable complex formation, [beta]arr1 recruitment, receptor internalization, and desensitization of G-protein activation. Taken together, these data suggest that the cooperative interactions of [beta]-arrestin with both the receptor and the phospholipid bilayer contribute to its functional versatility. A cryo-electron microscopy structure of [beta]-arrestin 1 in complex with the M2 muscarinic receptor reconstituted in lipid nanodiscs is reported., Author(s): Dean P. Staus [sup.1] [sup.2] , Hongli Hu [sup.3] [sup.8] , Michael J. Robertson [sup.3] [sup.4] , Alissa L. W. Kleinhenz [sup.1] [sup.2] [sup.9] , Laura M. Wingler [sup.1] [...]

The RNA World Hypothesis posits that the first self-replicating molecules were RNAs. RNA self-replicases are, in general, assumed to have employed nucleotide 5'-polyphosphates (or their analogues) as substrates for RNA polymerization. The mechanism by which these substrates might be synthesized with sufficient abundance to supply a growing and evolving population of RNAs is problematic for evolutionary hypotheses because non-enzymatic synthesis and assembly of nucleotide 5'-triphosphates (or other analogously activated phosphodiester species) is inherently difficult. However, nucleotide 2',3'-cyclic phosphates are also phosphodiesters, and are the natural and abundant products of RNA degradation. These have previously been dismissed as viable substrates for prebiotic RNA synthesis. We propose that the arguments for their dismissal are based on a flawed assumption, and that nucleotide 2',3'-cyclic phosphates in fact possess several significant, advantageous properties that indeed make them particularly viable substrates for prebiotic RNA synthesis. An RNA World hypothesis based upon the polymerization of nucleotide 2',3'-cyclic phosphates possesses additional explanatory power in that it accounts for the observed ribozyme "fossil record", suggests a viable mechanism for substrate transport across lipid vesicle boundaries of primordial proto-cells, circumvents the problems of substrate scarcity and implausible synthetic pathways, provides for a primitive but effective RNA replicase editing mechanism, and definitively explains why RNA, rather than DNA, must have been the original catalyst. Finally, our analysis compels us to propose that a fundamental and universal property that drives the evolution of living systems, as well as pre-biotic replicating molecules (be they composed of RNA or protein), is that they exploit chemical reactions that already possess competing kinetically-preferred and thermodynamically-preferred pathways in a manner that optimizes the balance between the two types of pathways.

Immunostimulatory cytokines can enhance anti-tumor immunity and are part of the therapeutic armamentarium for cancer treatment. We have previously reported that post-transplant lymphoma patients have an acquired deficiency of signal transducer and activator of transcription 4, which results in defective IFNγ production during clinical immunotherapy. With the goal of further improving cytokine-based immunotherapy, we examined the effects of a soybean peptide called lunasin that synergistically works with cytokines on natural killer (NK) cells. Peripheral blood mononuclear cells of healthy donors and post-transplant lymphoma patients were stimulated with or without lunasin in the presence of IL-12 or IL-2. NK activation was evaluated, and its tumoricidal activity was assessed using in vitro and in vivo tumor models. Chromatin immunoprecipitation assay was performed to evaluate the histone modification of gene loci that are regulated by lunasin and cytokine. Adding lunasin to IL-12- or IL-2-stimulated NK cells demonstrated synergistic effects in the induction of IFNG and GZMB involved in cytotoxicity. The combination of lunasin and cytokines (IL-12 plus IL-2) was capable of restoring IFNγ production by NK cells from post-transplant lymphoma patients. In addition, NK cells stimulated with lunasin plus cytokines displayed higher tumoricidal activity than those stimulated with cytokines alone using in vitro and in vivo tumor models. The underlying mechanism responsible for the effects of lunasin on NK cells is likely due to epigenetic modulation on target gene loci. Lunasin represents a different class of immune modulating agent that may augment the therapeutic responses mediated by cytokine-based immunotherapy.

The bark of Pinus radiata offers an underutilized source of high-value renewable chemicals such as extractable polyphenols and lipophilic compounds (waxes and suberin). Here, the depolymerization and extraction of suberin from P. radiata bark and its repolymerization to form novel polyesters are reported. Three different strategies were evaluated for repolymerization of the suberin monomers, with starting materials and products characterized using chemical and thermal analysis techniques. The inclusion of comonomer (1,12-dodecanediol) to provide stoichiometric balance improved the conversion, product yield, solubility and increased molecular weight. Enzymatic polymerization conditions gave the highest yield, while the highest molecular weight was achieved using titanium butoxide, demonstrating that polymerization conditions could be varied to target desired product properties. Products were hydrophobic, as shown by contact angles, ϴ ≥ 90° after 30 s. This work highlights opportunities for utilizing suberin to add value to a P. radiata bark biorefinery concept. Potential future applications include its use as a starting material for novel bio-based polymers that can serve as water-repellent surfaces and coatings, replacing established products derived from fossil resources. [ABSTRACT FROM AUTHOR]