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Journal: Biochemistry


Beta-arrestins regulate G protein-coupled receptor signaling as competitive inhibitors and protein adaptors. Low molecular weight biased ligands that bind receptors and discriminate between the G protein dependent arm and beta-arrestin, clathrin-associated arm of receptor signaling are considered therapeutically valuable as a result of this distinctive pharmacological behavior. Other than receptor agonists, compounds that activate beta-arrestins are not available. We show that within minutes of exposure to the cationic triphenylmethane dyes malachite green and brilliant green, tissue culture cells recruit beta-arrestins to clathrin scaffolds in a receptor-activation independent manner. In the presence of these compounds G protein signaling is inhibited, ERK and GSK3β signaling are preserved, and the recruitment of the beta2-adaptin, AP2 adaptor complex to clathrin as well as transferrin internalization are reduced. Moreover, malachite green binds beta-arrestin2-GFP coated immunotrap beads relative to GFP only coated beads. Triphenylmethane dyes are FDA approved for topical use on newborns as components of triple-dye preparations, and are not approved but used effectively as aqueous antibiotics in fish husbandry. As possible carcinogens their chronic ingestion in food preparations, particularly through farmed fish, is discouraged in the US and Europe. Our results indicate triphenylmethane dyes as a result of novel pharmacology may have additional roles as beta-arrestin/clathrin pathway signaling modulators in both pharmacology research and clinical therapy.

Concepts: Pharmacology, Signal transduction, Cell membrane, Receptor antagonist, Membrane biology, Dye, G protein, Triarylmethane dyes


Mimetics of conformational protein epitopes have broad applications, but have been difficult to identify using conventional peptide phage display. The tenth type III domain of human fibronectin (FNfn10) has two extended, randomizable surface-exposed loops and might be more amenable to the identification of such mimetics. We therefore selected a library of FNfn10 clones, randomized in both loops (15 residues in all), for binding to monoclonal antibodies (MAb) that recognize the HIV-1 envelope glycoprotein. Anti-idiotypic monobodies (αIMs) mimicking both “linear” epitopes (2F5 and 4E10 MAbs) and conformational epitopes (b12 and VRC01 MAbs) were generated. αIMs selected against 2F5 and 4E10 frequently displayed sequence homology to the corresponding linear native epitopes. In the case of b12 and VRC01, we expected that the two constrained loop domains of FNfn10 would both contribute to complex conformational interactions with target antibodies. However, mutagenesis studies revealed differences from this simple model An αIM selected against b12 was found to bind its cognate antibody via only a few residues within the BC loop of FNfn10, with minimal contribution from the FG loop. Unexpectedly, this was sufficient to generate a protein that engaged its cognate antibody in a manner very similar to HIV-1 Env, and with a strong K(D) (43 nM). In contrast, an αIM selected against VRC01 engaged its cognate antibody in a manner that was dependent on both BC and FG loop sequences. Overall, these data suggest that the FNfn10 scaffold can be used to identify complex structures that mimic conformational protein epitopes. ααα

Concepts: Immune system, Antibody, Monoclonal antibodies, Immunohistochemistry, Cambridge Antibody Technology


Most non-coding RNAs function properly only when folded into complex 3D structures, but the experimental determination of these structures remains challenging. Understanding of primary miRNA maturation is currently limited by a lack of solved structures for non-processed forms of the RNA. SHAPE chemistry efficiently determines RNA secondary structural information with single-nucleotide resolution, providing constraints suitable for input into the MC-Pipeline software for refinement of 3D structure models. Here we combine these approaches to analyze three structurally diverse primary miRNAs, revealing deviations from canonical dsRNA structure in the stem adjacent to the Drosha cut site for all three. The necessity of these deformable sites for efficient processing is demonstrated through Drosha processing assays. The structure models generated herein support the hypothesis that deformable sequences spaced roughly once per turn of A-form helix, created by non-canonical structure elements, combine with the necessary single-stranded RNA:double-stranded RNA junction to define the correct Drosha cleavage site.

Concepts: DNA, Gene, RNA, Structure, MicroRNA, Hierarchy, System, Non-coding RNA


Class II fructose 1,6-bisphosphate aldolases (FBA; E.C. comprise one of two families of aldolases. Instead of forming a Schiff-base intermediate using an ε-amino group of a lysine side chain, class II FBAs utilize Zn(II) to stabilize a proposed hydroxyenolate intermediate (HEI) in the reversible cleavage of fructose 1,6-bisphosphate forming glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). As class II FBAs has been shown to be essential in pathogenic bacteria, focus has been placed on these enzymes as potential antibacterial targets. Although structural studies on class II FBAs from Mycobacterium tuberculosis (MtFBA), other bacteria and protozoa have been reported, the structure of the active site loop responsible for catalyzing the protonation/deprotonation steps of the reaction for class II FBAs has not yet been observed. We therefore utilized the potent class II FBA inhibitor phosphoglycolohydroxamate (PGH) as a mimic of the HEI/DHAP bound form of the enzyme and determined the X-ray structure of MtFBA-PGH complex to 1.58 Å. Remarkably, we are able to observe well-defined electron density for the previously elusive active site loop of MtFBA trapped in a catalytically competent orientation. Utilization of this structural information plus site-directed mutagenesis and kinetic studies conducted on a series of residues within the active-site loop revealed that E169 facilitates a water mediated deprotonation/protonation step of the MtFBA reaction mechanism. Also, secondary isotope effects on MtFBA and catalytically relevant mutants were used to probe the effect of loop flexibility on catalytic efficiency. Additionally, we also reveal the structure of MtFBA in its holoenzyme form.

Concepts: Bacteria, Enzyme, Tuberculosis, Pathogen, Mycobacterium, Mycobacterium tuberculosis, Glyceraldehyde 3-phosphate, Dihydroxyacetone phosphate


Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a relatively new imaging modality that allows mapping of a wide range of biomolecules within a thin tissue section. The technology uses a laser beam to directly desorb and ionize molecules from discrete locations on the tissue that are subsequently recorded in a mass spectrometer. IMS is distinguished by the capability to directly measure molecules in situ ranging from small metabolites to proteins, reporting hundreds to thousands of expression patterns from a single imaging experiment. This article reviews recent advances in IMS technology, applications, and experimental strategies that allow it to significantly aid in the discovery and understanding of molecular processes in biological and clinical samples.

Concepts: Molecular biology, Mass spectrometry, Chemistry, Proteomics, Ion source, Matrix-assisted laser desorption/ionization, MALDI imaging, Mass spectrometry imaging


A series of substrate analogues has been used to determine which chemical moieties of the substrate, phosphoenolpyruvate (PEP) contribute to the allosteric inhibition of rabbit muscle pyruvate kinase (M1-PYK) by phenylalanine. Replacing the carboxyl group of the substrate with a methyl alcohol, or removing the phosphate altogether, greatly reduces substrate affinity. However, removal of the carboxyl group is the only modification tested that removes the ability to allosterically reduce Phe binding. From this, it can be concluded that the carboxyl group of PEP is responsible for energetic coupling with Phe binding in the allosteric sites.

Concepts: Enzyme kinetics, Allosteric regulation, Alcohol, Adenosine triphosphate, Enzyme, Functional group, Ligand, Carboxylic acid


Biological membranes are exposed to a number of chemical and physical stresses that may alter the structure of the lipid bilayer in such a way that the permeability barrier to hydrophilic molecules and ions is degraded. These stresses include amphiphilic molecules involved in metabolism and signaling, highly charged polyamines, membrane permeating peptides, and mechanical and osmotic stresses. As annexins are known to bind to lipid headgroups in the presence of calcium and increase the order of the bilayer lipids, this study addressed whether this activity of annexins provides a potential benefit to the membrane of protecting the bilayer against disruptions of this nature, or can promote restoration of the permeability barrier after damage by such agents. The release of carboxyfluorescein from large unilamellar vesicles composed of lipids characteristically present in the inner leaflet of cell membranes (PS/PE/PC/cholesterol) was used to measure membrane permeability. It was determined that in the presence of calcium, annexin A5 reduced baseline leakage from the vesicles and reduced or reversed damage due to arachidonic acid, lysophosphatidic acid, lysophosphatidylcholine, diacylglycerol, monoacylglycerol, spermidine, amyloid beta, amylin, and osmotic shock. Annexin A6 was also able to provide membrane protection in many, but not all of these cases. In a cell, it is likely annexins would move to sites of breakdown of the permeability barrier because of the calcium-dependent promotion of the binding of annexins to membranes at sites of calcium entry. Because of the fundamental importance to life of maintaining the permeability barrier of the cell membrane, it is proposed here that this property of annexins may represent a critical, primordial activity that explains their great evolutionary conservation and abundant expression in most cells.

Concepts: Protein, Cell, Cell membrane, Organelle, Cell wall, Lipid, Lipid bilayer, Biological membrane


The irreversibility and autocatalytic character of amyloidogenesis and the polymorphism of amyloid fibrils underlie the phenomenon of self-propagating strains, wherein the mother seed, rather than the seeding environment, determines the properties of daughter fibrils. Here we study the formation of amyloid fibrils from bovine insulin and the recombinant Lys(B31)-Arg(B32) human insulin analog. The two polypeptides are similar enough to cross-seed but, upon spontaneous aggregation, form amyloid fibrils with distinct spectral features in the infrared amide I' band region. When bovine insulin is cross-seeded with the analog amyloid (and vice versa), the shape, absorption maximum, and even fine fingerprint features of the amide I' band are passed from the mother to daughter fibrils with a high degree of fidelity. Although the differences in primary structure between bovine insulin and the Lys(B31)-Arg(B32) analog of human insulin lie outside of the polypeptide’s critical amyloidogenic regions, they affect the secondary structure of fibrils, possibly the formation of intermolecular salt bridges, and the susceptibility to dissection and denaturation with dimethyl sulfoxide (DMSO). All these phenotypic features of mother fibrils are imprinted in daughter amyloid upon cross-seeding. Analysis of noncooperative DMSO-induced denaturation of daughter fibrils suggests that the self-propagating polymorphism underlying the emergence of new amyloid strains is encoded on the level of secondary structure. Our findings have been discussed in the context of polymorphism of fibrils, amyloid strains, and possible implications for mechanisms of amyloidogenesis.

Concepts: Protein, Protein structure, Amino acid, Insulin, Amyloid, Dimethyl sulfoxide, Eli Lilly and Company, Insulin analog


The formation of the nacre pearl in marine invertebrates represents an on-demand production of mineralization in response to an irritant or parasite threat to the mantle organ. In the Japanese pearl oyster (Pinctada fucata), this process is mediated by a 12-member protein family known as PFMG (Pinctada Fucata Mantle Gene). One of these proteins, PFGM1, has been implicated in modulating calcium carbonate crystal growth and has been reported to possess a EF-hand - like domain. In this report, we establish that the recombinant PFMG1 (rPFMG1) is an intrinsically disordered “imitator” EF-hand protein that increases the number of calcium carbonate mineral crystals that form relative to control scenarios and does not induce aragonite formation. This protein possesses a modified pseudo-EF hand sequence at the C-terminal end which exhibits low homology (30-40%) to the pseudo-EF hand mitochondrial SCaMCs buffering/solute transport proteins. This low sequence homology is the result of the inclusion of disorder-promoting amino acids and short amyloid-like aggregation-prone cross-beta strand sequences within the putative PFMG1 pseudo-EF hand sequence region. Similar to other nacre proteins, rPFMG1 oligomerizes to form amorphous, heterogeneously-sized protein oligomers and films in vitro, and this process is enhanced by Ca2+, which promotes the formation of aggregation-prone extended beta strand structure within rPFMG1. From these results, we conclude that PFMG1 forms supramolecular assemblies that play an important role in amplifying the nucleation process that is crucial for coating or neutralizing the threat to the mantle organ.

Concepts: DNA, Protein, Mineral, Calcium carbonate, Pearl oyster, Pearl, Nacre, Aragonite


Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was used to study the conformation of aggregated proteins both in vivo and in vitro. Several different protein aggregates including amyloid fibrils from several peptides and polypeptides, inclusion bodies, folding aggregates, soluble oligomers, and protein extracts from stressed cells were examined in this study. All protein aggregates demonstrate a characteristic new ß-structure with lower frequency band positions. All protein aggregates acquire this new ß-band following the aggregation process involving inter-molecular interactions. The ß-sheets, in some proteins, arise from regions of polypeptide that are helical or non-beta in the native conformation. For a given protein, all types of aggregates (e. g. inclusion bodies, folding aggregates, thermal aggregates) showed similar spectra, indicating they arose from a common partially-folded species. All the aggregates have some native-like secondary structure as well as non-periodic structure, along with the specific new ß-structure. The new ß could most likely be attributed to stronger hydrogen bonds in the intermolecular ß-sheet structure present in protein aggregates.

Concepts: Protein, Protein structure, Spectroscopy, Amino acid, Secondary structure, Tertiary structure, Peptide, Fourier transform spectroscopy