Cholesterol and lipid microdomains stabilize the postsynapse at the neuromuscular junction

Stabilization and maturation of synapses are important for development and function of the nervous system. Previous studies have implicated cholesterol-rich lipid microdomains in synapse stabilization, but the underlying mechanisms remain unclear. We found that cholesterol stabilizes clusters of synaptic acetylcholine receptors (AChRs) in denervated muscle in vivo and in nerve-muscle explants. In paralyzed muscles, cholesterol triggered maturation of nerve sprout-induced AChR clusters into pretzel shape. Cholesterol treatment also rescued a specific defect in AChR cluster stability in cultured src(-/-);fyn(-/-) myotubes. Postsynaptic proteins including AChRs, rapsyn, MuSK and Src-family kinases were strongly enriched in lipid microdomains prepared from wild-type myotubes. Microdomain disruption by cholesterol-sequestering methyl-beta-cyclodextrin disassembled AChR clusters and decreased AChR-rapsyn interaction and AChR phosphorylation. Amounts of microdomains and enrichment of postsynaptic proteins into microdomains were decreased in src(-/-);fyn(-/-) myotubes but rescued by cholesterol treatment. These data provide evidence that cholesterol-rich lipid microdomains and SFKs act in a dual mechanism in stabilizing the postsynapse: SFKs enhance microdomain-association of postsynaptic components, whereas microdomains provide the environment for SFKs to maintain interactions and phosphorylation of these components.     

Co-delivery of antigen and IL-12 by Venezuelan equine encephalitis virus replicon particles enhances antigen-specific immune responses and antitumor effects

We recently demonstrated that Venezuelan equine encephalitis virus-based replicon particle (VRPs) encoding tumor antigens could break tolerance in the immunomodulatory environment of advanced cancer. We hypothesized that local injection of VRP-expressing interleukin-12 (IL-12) at the site of injections of VRP-based cancer vaccines would enhance the tumor-antigen-specific T cell and antibody responses and antitumor efficacy. Mice were immunized with VRP encoding the human tumor-associated antigen, carcinoembryonic antigen (CEA) (VRP-CEA(6D)), and VRP-IL-12 was also administered at the same site or at a distant location. CEA-specific T cell and antibody responses were measured. To determine antitumor activity, mice were implanted with MC38-CEA-2 cells and immunized with VRP-CEA with and without VRP-IL-12, and tumor growth and mouse survival were measured. VRP-IL-12 greatly enhanced CEA-specific T cell and antibody responses when combined with VRP-CEA(6D) vaccination. VRP-IL-12 was superior to IL-12 protein at enhancing immune responses. Vaccination with VRP-CEA(6D) plus VRP-IL-12 was superior to VRP-CEA(6D) or VRP-IL-12 alone in inducing antitumor activity and prolonging survival in tumor-bearing mice. Importantly, local injection of VRP-IL-12 at the VRP-CEA(6D) injection site provided more potent activation of CEA-specific immune responses than that of VRP-IL-12 injected at a distant site from the VRP-CEA injections. Together, this study shows that VRP-IL-12 enhances vaccination with VRP-CEA(6D) and was more effective at activating CEA-specific T cell responses when locally expressed at the vaccine site. Clinical trials evaluating the adjuvant effect of VRP-IL-12 at enhancing the immunogenicity of cancer vaccines are warranted.     

Oxidative Damage in Muscular Dystrophy Correlates with the Severity of the Pathology: Role of Glutathione Metabolism

Muscular dystrophies (MDs) such as Duchenne muscular dystrophy (DMD), sarcoglycanopathy (Sgpy) and dysferlinopathy (Dysfy) are recessive genetic neuromuscular diseases that display muscle degeneration. Although these MDs have comparable endpoints of muscle pathology, the onset, severity and the course of these diseases are diverse. Different mechanisms downstream of genetic mutations might underlie the disparity in these pathologies. We surmised that oxidative damage and altered antioxidant function might contribute to these differences. The oxidant and antioxidant markers in the muscle biopsies from patients with DMD (n = 15), Sgpy (n = 15) and Dysfy (n = 15) were compared to controls (n = 10). Protein oxidation and lipid peroxidation was evident in all MDs and correlated with the severity of pathology, with DMD, the most severe dystrophic condition showing maximum damage, followed by Sgpy and Dysfy. Oxidative damage in DMD and Sgpy was attributed to the depletion of glutathione (GSH) and lowered antioxidant activities while loss of GSH peroxidase and GSH-S-transferase activities was observed in Dysfy. Lower GSH level in DMD was due to lowered activity of gamma-glutamyl cysteine ligase, the rate limiting enzyme in GSH synthesis. Similar analysis in cardiotoxin (CTX) mouse model of MD showed that the dystrophic muscle pathology correlated with GSH depletion and lipid peroxidation. Depletion of GSH prior to CTX exposure in C2C12 myoblasts exacerbated oxidative damage and myotoxicity. We deduce that the pro and anti-oxidant mechanisms could be correlated to the severity of MD and might influence the dystrophic pathology to a different extent in various MDs. On a therapeutic note, this could help in evolving novel therapies that offer myoprotection in MD.     

The kinase activity of the Helicobacter pylori Asp-tRNA(Asn)/Glu-tRNA(Gln) amidotransferase is sensitive to distal mutations in its putative ammonia tunnel

The Helicobacter pylori (Hp) Asp-tRNA(Asn)/Glu-tRNA(Gln) amidotransferase (AdT) plays important roles in indirect aminoacylation and translational fidelity. AdT has two active sites, in two separate subunits. Kinetic studies have suggested that interdomain communication occurs between these subunits; however, this mechanism is not well understood. To explore domain-domain communication in AdT, we adapted an assay and optimized it to kinetically characterize the kinase activity of Hp AdT. This assay was applied to the analysis of a series of point mutations at conserved positions throughout the putative AdT ammonia tunnel that connects the two active sites. Several mutations that caused significant decreases in AdT's kinase activity (reduced by 55-75%) were identified. Mutations at Thr149 (37 ? distal to the GatB kinase active site) and Lys89 (located at the interface of GatA and GatB) were detrimental to AdT's kinase activity, suggesting that these mutations have disrupted interdomain communication between the two active sites. Models of wild-type AdT, a valine mutation at Thr149, and an arginine mutation at Lys89 were subjected to molecular dynamics simulations. A comparison of wild-type, T149V, and K89R AdT simulation results unmasks 59 common residues that are likely involved in connecting the two active sites.     

Aspirin Inhibits Colon Cancer Cell and Tumor Growth and Downregulates Specificity Protein (Sp) Transcription Factors

Acetylsalicylic acid (aspirin) is highly effective for treating colon cancer patients postdiagnosis; however, the mechanisms of action of aspirin in colon cancer are not well defined. Aspirin and its major metabolite sodium salicylate induced apoptosis and decreased colon cancer cell growth and the sodium salt of aspirin also inhibited tumor growth in an athymic nude mouse xenograft model. Colon cancer cell growth inhibition was accompanied by downregulation of Sp1, Sp3 and Sp4 proteins and decreased expression of Sp-regulated gene products including bcl-2, survivin, VEGF, VEGFR1, cyclin D1, c-MET and p65 (NFκB). Moreover, we also showed by RNA interference that β-catenin, an important target of aspirin in some studies, is an Sp-regulated gene. Aspirin induced nuclear caspase-dependent cleavage of Sp1, Sp3 and Sp4 proteins and this response was related to sequestration of zinc ions since addition of zinc sulfate blocked aspirin-mediated apoptosis and repression of Sp proteins. The results demonstrate an important underlying mechanism of action of aspirin as an anticancer agent and, based on the rapid metabolism of aspirin to salicylate in humans and the high salicylate/aspirin ratios in serum, it is likely that the anticancer activity of aspirin is also due to the salicylate metabolite.     

A Potent Class of GPR40 Full Agonists Engages the EnteroInsular Axis to Promote Glucose Control in Rodents

Type 2 diabetes is characterized by impaired glucose homeostasis due to defects in insulin secretion, insulin resistance and the incretin response. GPR40 (FFAR1 or FFA1) is a G-protein-coupled receptor (GPCR), primarily expressed in insulin-producing pancreatic β-cells and incretin-producing enteroendocrine cells of the small intestine. Several GPR40 agonists, including AMG 837 and TAK-875, have been disclosed, but no GPR40 synthetic agonists have been reported that engage both the insulinogenic and incretinogenic axes. In this report we provide a molecular explanation and describe the discovery of a unique and potent class of GPR40 full agonists that engages the enteroinsular axis to promote dramatic improvement in glucose control in rodents. GPR40 full agonists AM-1638 and AM-6226 stimulate GLP-1 and GIP secretion from intestinal enteroendocrine cells and increase GSIS from pancreatic islets, leading to enhanced glucose control in the high fat fed, streptozotocin treated and NONcNZO10/LtJ mouse models of type 2 diabetes. The improvement in hyperglycemia by AM-1638 was reduced in the presence of the GLP-1 receptor antagonist Ex(9–39)NH₂.     

Analysis of conformational variation in macromolecular structural models

Experimental conditions or the presence of interacting components can lead to variations in the structural models of macromolecules. However, the role of these factors in conformational selection is often omitted by in silico methods to extract dynamic information from protein structural models. Structures of small peptides, considered building blocks for larger macromolecular structural models, can substantially differ in the context of a larger protein. This limitation is more evident in the case of modeling large multi-subunit macromolecular complexes using structures of the individual protein components. Here we report an analysis of variations in structural models of proteins with high sequence similarity. These models were analyzed for sequence features of the protein, the role of scaffolding segments including interacting proteins or affinity tags and the chemical components in the experimental conditions. Conformational features in these structural models could be rationalized by conformational selection events, perhaps induced by experimental conditions. This analysis was performed on a non-redundant dataset of protein structures from different SCOP classes. The sequence-conformation correlations that we note here suggest additional features that could be incorporated by in silico methods to extract dynamic information from protein structural models.     

Polyubiquitination of prolactin receptor stimulates its internalization, postinternalization sorting, and degradation via the lysosomal pathway

The ubiquitination of the receptor that mediates signaling induced by the polypeptide pituitary hormone prolactin (PRL) has been shown to lead to the degradation of this receptor and to the ensuing negative regulation of cellular responses to PRL. However, the mechanisms of PRL receptor (PRLr) proteolysis remain largely to be determined. Here we provide evidence that PRLr is internalized and primarily degraded via the lysosomal pathway. Ubiquitination of PRLr is essential for the rapid internalization of PRLr, which proceeds through a pathway dependent on clathrin and the assembly polypeptide 2 (AP2) adaptor complexes. Recruitment of AP2 to PRLr is stimulated by PRLr ubiquitination, which also is required for the targeting of already internalized PRLr to the lysosomal compartment. While mass spectrometry analysis revealed that both monoubiquitination and polyubiquitination (via both K48- and K63-linked chains) occur on PRLr, the results of experiments using forced expression of ubiquitin mutants indicate that PRLr polyubiquitination via K63-linked chains is important for efficient interaction of PRLr with AP2 as well as for efficient internalization, postinternalization sorting, and proteolytic turnover of PRLr. We discuss how specific ubiquitination may regulate early and late stages of endocytosis of PRLr and of related receptors to contribute to the negative regulation of the magnitude and duration of downstream signaling.     

A Study of Protein Electrochemistry on a Supported Membrane Electrode

Protein electrochemistry offers a direct method to identify and characterize biological electron transfer processes, potentially leading to commercial applications such as biosensors and diagnostic tools. However, establishing a biocompatible electrode interface that maintains the native state of the redox protein involves several challenges. In general, membrane proteins require the presence of a phospholipid bilayer to maintain their biological activity. Synthetic `biomimetic’ membranes are widely used to characterize membrane proteins, however they have seldom been applied to measurements of protein redox activity in electrochemical cells due to their inherent insulating property. In this study we demonstrate the use of the phospholipids: PC, PC/PG and PC/PG/cholesterol membrane mixtures on chemically modified (supported) gold electrode surfaces for direct protein electrochemistry. We compare the electrochemical activity of a relatively small, redox active “test protein”, cytochrome c, in the presence and absence of phospholipid on a gold electrode modified with thiol self assembled monolayers, to explore the effect of chain length and composition of the thiol on the charge coupling. Three thiols were investigated as self assembled monolayers on a gold electrode: octanethiol, mercaptopropionic and mercaptoundecanoic acid. We demonstrate here that the charge transfer efficiency of cytochrome c is better in the presence of the membrane and in addition, a superior redox response is obtained with surfaces modified with a thiol functionalised with a carboxylic acid.On leave from: Research Group on Laser Physics of the Hungarian Academy of Sciences, University of Szeged, Szeged, Hungary.Australian Peptide Conference Issue.     

Secondary metabolite as therapeutic agent from endophytic fungi Alternaria longipes strain VITN14G of mangrove plant Avicennia officinalis

Endophytic fungi, especially from mangrove plants, are rich source of secondary metabolites, which plays a major role in various pharmacological actions preferably in cancer and bacterial infections. To perceive its role in antidiabetic activity we isolated and tested the metabolites derived from a novel strain Alternaria longipes strain VITN14G obtained from mangrove plant Avicennia officinalis. The crude extract was analyzed for antidiabetic activity and subjected to column chromatography. The isolated fractions were screened in vitro for α-glucosidase and α-amylase inhibitory activities. The cytotoxicity of the isolated fractions was studied on L929 cell lines. Following which, the screened fraction 2 was allowed for structure elucidation using gas chromatography-mass spectrometry, one-dimensional, two-dimensional nuclear magnetic resonance spectroscopy, ultraviolet, and Fourier-transform infrared analysis. The binding energies of the isolated fraction 2 with glycolytic enzymes were calculated by molecular docking studies using AutoDock Vina. The isolated fraction 2 identified as 2,4,6-triphenylaniline, showed no significant difference in α-amylase inhibition rates and a significant difference of 10% in α-glucosidase inhibition rates than that of the standard drug acarbose. Further, the cytotoxicity assay of the isolated fraction 2 resulted in a cell viability of 73.96%. Supportingly, in silico studies showed 2,4,6-triphenylaniline to produce a stronger binding affinity toward the glycolytic enzyme targets. The compound 2,4,6-triphenylaniline isolated from A. longipes strain VITN14G exhibited satisfactory antidiabetic activity for type 2 diabetes in vitro, which will further be confirmed by in vivo studies. Successful outcome of the study will result in a natural substitute for existing synthetic antidiabetic drugs.