Search

The article presents information on various research papers which will be published in the forthcoming issue of the "European Journal of Biochemistry". Some of the papers discusses are "Enzymatic Synthesis of Neolactotetraosylceramide by the N-Acctyllactosamine Synthase of Human Serum". "Rate of Transplantation and Kinetics of Processing of Newly Synthesized Molecules of Two Major Outer-Membrane Proteins, the OmpA and OmpF Proteins of Escherichia Coli," by the researchers I. Crowlesmith and K. Gamon.

Lists articles about biochemistry. "Impact of altered protein structures on the intracellular traffic of a mutated vasopressin precursor from Brattleboro rats," by H. Schmale et al; "Direct electron transfer of redox proteins at the bare classy carbon electrode," by W.R. Hagen; "Molecular cloning of preproacrosin and analysis of its expression pattern in spermatogenesis," by I.M. Adham; "Reduced temperature does not prevent transport of lysosomal integral membrane proteins from endoplasmic reticulum and through the Golgi system to lysosomes," by P.G. Morales; Others.

Drug transporters are membrane proteins that provide protection for organisms against natural toxic products and fungicides. In plant pathogens, drug transporters function in baseline sensitivity to fungicides, multidrug resistance (MDR) and virulence on host plants. This paper describes drug transporters of the filamentous fungi Aspergillus nidulans (Eidam) Winter, Botrytis cinerea Pers and Mycosphaerella graminicola (Fückel) Schroter that function in fungicide sensitivity and resistance. The fungi possess ATP-binding cassette (ABC) drug transporters that mediate MDR to fungicides in laboratory mutants. Similar mutants are not pronounced in field resistance to most classes of fungicide but may play a role in resistance to azoles. MDR may also explain historical cases of resistance to aromatic hydrocarbon fungicides and dodine. In clinical situations, MDR development in Candida albicans (Robin) Berkhout mediated by ABC transporters in patients suffering from candidiasis is common after prolonged treatment with azoles. Factors that can explain this striking difference between agricultural and clinical situations are discussed. Attention is also paid to the risk of MDR development in plant pathogens in the future. Finally, the paper describes the impact of fungal drug transporters on drug discovery. [ABSTRACT FROM AUTHOR]

Background and Purpose: Cystic fibrosis (CF) is a debilitating disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which codes for a Cl-/HCO3 - channel. F508del, the most common CF-associated mutation, causes both gating and biogenesis defects in the CFTR protein. This paper describes the optimization of two fluorescence assays, capable of measuring CFTR function and cellular localization, and their use in a pilot drug screen.Experimental Approach: HEK293 cells expressing YFP-F508del-CFTR, in which halide sensitive YFP is tagged to the N-terminal of CFTR, were used to screen a small library of compounds based on the VX-770 scaffold. Cells expressing F508del-CFTR-pHTomato, in which a pH sensor is tagged to the fourth extracellular loop of CFTR, were used to measure CFTR plasma membrane exposure following chronic treatment with the novel potentiators.Key Results: Active compounds with efficacy ~50% of VX-770, micromolar potency, and structurally distinct from VX-770 were identified in the screen. The F508del-CFTR-pHTomato assay suggests that the hit compound MS131A, unlike VX-770, does not decrease membrane exposure of F508del-CFTR.Conclusions and Implications: Most known potentiators have a negative influence on F508del-CFTR biogenesis/stability, which means membrane exposure needs to be monitored early during the development of drugs targeting CFTR. The combined use of the two fluorescence assays described here provides a useful tool for the identification of improved potentiators and correctors. The assays could also prove useful for basic scientific investigations on F508del-CFTR, and other CF-causing mutations. [ABSTRACT FROM AUTHOR]

To date, various affinity‐based protein labeling probes have been developed and applied in biological research to modify endogenous proteins in cell lysates and on the cell surface. However, the reactive groups on the labeling probes are also the cause of probe instability and nonselective labeling in a more complex environment, e. g. intracellular and in vivo. Here, we show that labeling probes composed of a sterically stabilized difluorophenyl pivalate can achieve efficient and selective labeling of endogenous proteins on the cell surface, inside living cells and in vivo. As compared with the existing protein labeling probes, probes with the difluorophenyl pivalate exhibit several advantages, including long‐term stability in stock solutions, resistance to enzymatic hydrolysis and can be customized easily with diverse fluorophores and protein ligands. With this probe design, endogenous hypoxia biomarker in living cells and nude mice were successfully labeled and validated by in vivo, ex vivo, and immunohistochemistry imaging. [ABSTRACT FROM AUTHOR]

Dynamic properties of proteins have crucial roles in understanding protein function and molecular mechanism within cells. In this paper, we combined total internal reflection fluorescence microscopy with oblique illumination fluorescence microscopy to observe directly the movement and localization of membrane-anchored green fluorescence proteins in living cells. Total internal reflect illumination allowed the observation of proteins in the cell membrane of living cells since the penetrate depth could be adjusted to about 80 nm, and oblique illumination allowed the observation of proteins both in the cytoplasm and apical membrane, which made this combination a promising tool to investigate the dynamics of proteins through the whole cell. Not only individual protein molecule tracks have been analyzed quantitatively but also cumulative probability distribution function analysis of ensemble trajectories has been done to reveal the mobility of proteins. Finally, single particle tracking has acted as a compensation for single molecule tracking. All the results exhibited green fluorescence protein dynamics within cytoplasm, on the membrane and from cytoplasm to plasma membrane. [ABSTRACT FROM AUTHOR]

Crystallization of proteins from a purified protein solution remains a bottleneck in the structure determination pipeline. In this paper the crystallization problem is addressed using a microfluidic device capable of determining detailed protein precipitation diagrams using less than 10 µL of protein sample. Based on the experimentally determined protein phase behavior, a crystallization screen can be designed to accommodate the physical chemistry of the particular protein target. Such a tailor-made crystallization screen has a high probability of yielding crystallization hits. The approach is applied to two different proteins: the calcium pump (SERCA), an eukaryotic integral membrane protein, and UMP kinase, a prokaryotic soluble kinase. Protein phase behavior is mapped for both proteins and tailor-made crystallization screens are designed for the two proteins resulting in about 50% crystallization probability per experiment. This illustrates the power of using microfluidic devices for detailed characterization of protein phase behavior prior to crystallization trials. [ABSTRACT FROM AUTHOR]

1. The abundance of tyrosine sulfate in secretory proteins and in various classes of cellular proteins has been quantified and compared to protein-bound carbohydrate sulfate. 2. HepG2 cells and fibroblasts, two cell types showing only the constitutive pathway of secretion, and PC12 cells, which show both the constitutive and the regulated pathway of secretion, were subjected to pulse-chase and/ or long-term labelling with [35S]sulfate and [³H]tyrosine, followed by analysis of proteins in the cells and medium. Under both conditions of labelling, 65-92% of the protein-bound tyrosine sulfate and 44-84% of the protein bound carbohydrate sulfate were found to be secretory. In HepG2 cells, the frequency of sulfation of tyrosine residues, which can be determined independently from protein abundance and the rate of protein synthesis, was 8-22 times higher in proteins secreted into the medium than in cellular proteins. 3. All cell lines studied contained significant amounts, not only of carbohydrate sulfate, but also of tyrosine sulfate in specific cellular proteins. As shown for fibroblasts, these tyrosine-sulfated proteins were retained within the cells for at least 100 min of chase following a pulse with [35S]sulfate and were almost completely recovered in a light membrane fraction after subcellular fractionation. 4. Lysosomes were found to contain small, but significant, amounts of protein-bound tyrosine sulfate in addition to protein-bound carbohydrate sulfate. Protein-bound tyrosine sulfate in lysosomes reached a peak at 20 min of chase and rapidly disappeared thereafter, whereas protein-bound carbohydrate sulfate accumulated after 20 min of chase. Examination of the known sequences of eleven lysosomal enzymes revealed the presence of potential tyrosine sulfation sites in five of them. 5. Our results show that secretory proteins are the most abundant, but not exclusive, in vivo substrates for tyrosine sulfation and suggest the presence of soluble tyrosine-sulfated proteins in lysosomes and other, as yet unidentified, organelles of the secretory pathway. In the following paper in this journal we describe the abundance of tyrosine sulfate in integral membrane proteins. [ABSTRACT FROM AUTHOR]

1. The abundance of tyrosine sulfate in membrane proteins was quantified in four different cell lines and compared to that in soluble cellular and secreted proteins. 2. Upon metabolic labelling of HepG2, Ltk-, AtT20 and PC12 cells with [35S]sulfate or [³H]tyrosine, a fraction enriched in integral membrane proteins was found to contain small, but significant, amounts of protein-bound tyrosine sulfate (up to 2.5% of the total cellular plus secreted protein-bound tyrosine sulfate). On the other hand, the frequency of sulfation of tyrosine residues of membrane proteins was within the same order of magnitude as that of secreted proteins, indicating that the low abundance of tyrosine sulfate in membrane proteins was largely a reflection of the low abundance of these proteins themselves. Consistent with this conclusion were the results of an analysis showing that 14 out of 32 selected membrane-spanning proteins contain potential tyrosine sulfation sites. 3. In HepG2 cells, three tyrosine-sulfated integral membrane glycoproteins of molecular mass 100, 125 and 150 kDa were identified. Characterization of the 150-kDa tyrosine-sulfated membrane protein revealed that it was protected from proteolysis in intact cells, suggesting a localization in an intracellular organelle. 4. Together with the results reported in the preceding paper in this journal, our data suggest that tyrosine sulfation occurs in various classes of trans-Golgi-derived proteins, soluble as well as membrane, and extracellularly exposed as well as intracellularly retained, proteins. This suggests that tyrosine sulfation may have a variety of physiological functions, depending on the individual tyrosine-sulfated protein or protein class. [ABSTRACT FROM AUTHOR]

Brain spectrin, or fodrin, a major protein of the subaxolemmal cytoskeleton, associates specifically in in vitro assays with the 70-kDa neurofilament subunit (NF-L) and with glial filaments from pig spinal cord. As an initial approach to the identification of the fodrin-binding proteins, a crude preparation of neurofilaments was resolved by electrophoresis on SDS/polyacrylamide gels and then transferred to nitrocellulose paper, which was 'blotted' with 125I-fodrin. A significant binding of fodrin was observed on polypeptides of 70 kDa, 52 kDa and 20 kDa. These polypeptides were further purified and identified respectively as the NF-L subunit of neurofilaments, the glial fibrillary acidic protein (GFP) and the myelin basic protein. The binding of fodrin to NF-L was reversible and concentration-dependent. The ability of the pure NF-L and GFP to form filaments was used to quantify their association with fodrin. a) The binding of fodrin to reassembled NF-L was saturable with a stoichiometry of 1 mol fodrin bound/ 50 ± 10 mol NF-L and an apparent dissociation constant Kd = 4.3 × 10-7 M. b) The binding involved the N-terminal domain of the polypeptide chain derived from the [2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine] cleavage of NF-L. c) Binding occurred optimally at physiological pH (6.8-7.2) and salt concentrations (50 mM). d) Interestingly, calmodulin, a Ca2+-binding protein, which has been shown to bind to fodrin, was found to reinforce the binding of fodrin to the NF-L, at Ca2+ physiological concentrations. The binding of fodrin to pure neurofilaments was not affected by the presence of the 200-kDa (NF-H) and the 160-kDa (NF-M) subunits. The apparent dissociation constant for the binding of fodrin to NF-L in the pure NF was 1.0 × 10-6 M with I mol fodrin bound/80 ± 10 mol NF-L. Moreover, the binding of fodrin to GFP, demonstrated in blot assays, was confirmed by cosedimentation experiments. The apparent dissociation constant Kd for the fodrin binding was 2.8 × 10-7 M and the maximum binding was I mol fodrin/55 ± 10 mol GFP. [ABSTRACT FROM AUTHOR]

Secretion of haemolysin (HlyA) is secA independent, but depends upon two accessory membrane proteins, HlyB and HlyD. encoded by the hly determinant. A fourth (cytoplasmic) protein, HlyC, is required to activate HlyA post-translationally, but has no role in export. Deletion studies have previously shown that the HlyA molecule contains a targeting signal close to the C-terminus which specifically directs its secretion to the medium. This targeting signal has been variously located within the terminal 27, 53, 60 or 113 amino acids. In this paper, we have sought to confirm the presence of a C-terminal targeting signal and to analyse the specificity of the Hly transport system through fusion of C-terminal fragments of HlyA to heterologous polypeptides. A C-terminal fragment (23 kDa) of HlyA. when fused at the C-terminus, efficiently promoted the secretion of the eukaryotic protein prochymosin (PCM) to the medium via HlyB and HlyD. This result is in contrast to previous findings that prochymosin, preceded by the alkaline phosphatase signal sequence, cannot be translocated across the Escherichia coli inner membrane. The HlyA targeting domain was also used to secrete to the medium varying portions of chloramphenicol acetyltransferase (CAT) and 98 per cent of the β-galactosidase (LacZ) molecule (both E. coli cytoplasmic proteins). In the case of the PCM and CAT fusions the efficiency of secretion was reduced as the proportion of the PCM and CAT molecule increased. This result is consistent with inhibition of secretion through the irreversible folding of the larger passenger protein fragments, or the occlusion of the HlyA targeting signal by upstream sequences. Analysis of the nature of the C-terminal domain promoting secretion of prochymosin, demonstrated that shortening the signal domain from 218 to 113 amino acids significantly reduced the efficiency of secretion. This result may also reflect the importance of maintaining an independent... [ABSTRACT FROM AUTHOR]

Until now, no completely effective parasite‐specific drugs or vaccines have been approved for the treatment of cryptosporidiosis. Through the separation and identification of the sporozoite membrane protein of Cryptosporidium parvum (C. parvum), 20 related proteins were obtained. Among them, a calmodulin‐like protein (CML) has a similar functional domain‐exchange factor hand (EF‐hand) motif as calmodulin proteins (CaMs), so it may play a similarly important role in the invasion process. A 663 bp full gene encoding the C. parvum calmodulin‐like protein (CpCML) was inserted in pET28a vector and expressed in Escherichia coli. An immunofluorescence assay showed that CpCML was mainly located on the surface of the sporozoites. Three‐week‐old female BALB/c mice were used for modelling the immunoreactions and immunoprotection of recombinant CpCML (rCpCML) against artificial Cryptosporidium tyzzeri infections. The results indicated a significantly increased in anti‐CpCML antibody response, which was induced by the immunized recombinant protein. Compared to rP23 (recombinant P23), GST6P‐1 (expressed by pGEX‐6P‐1 transfected E. coli), GST4T‐1 (expressed by pGEX‐4T‐1 transfected E. coli), glutathione (GSH), adjuvant and blank control groups, rCpCML‐immunized mice produced specific spleen cell proliferation in addition to different production levels of IL‐2, IFN‐γ, TNF‐α, IL‐4 and IL‐5. Additionally, immunization with rCpCML led to 34.08% reduction of oocyst shedding in C. tyzzeri infected mice faeces which was similar to rP23. These results suggest that CpCML may be developed as a potential vaccine candidate antigen against cryptosporidiosis. [ABSTRACT FROM AUTHOR]

Summary: To acquire phosphorus, cyanobacteria use the typical bacterial ABC‐type phosphate transporter, which is composed of a periplasmic high‐affinity phosphate‐binding protein PstS and a channel formed by two transmembrane proteins PstC and PstA. A putative pstS gene was identified in the genomes of cyanophages that infect the unicellular marine cyanobacteria Prochlorococcus and Synechococcus. However, it has not been determined whether the cyanophage PstS protein is functional during infection to enhance the phosphate uptake rate of host cells. Here we showed that the cyanophage P‐SSM2 PstS protein was abundant in the infected Prochlorococcus NATL2A cells and the host phosphate uptake rate was enhanced after infection. This is consistent with our biochemical and structural analyses showing that the phage PstS protein is indeed a high‐affinity phosphate‐binding protein. We further modelled the complex structure of phage PstS with host PstCA and revealed three putative interfaces that may facilitate the formation of a chimeric ABC transporter. Our results provide insights into the molecular mechanism by which cyanophages enhance the phosphate uptake rate of cyanobacteria. Phosphate acquisition by infected bacteria can increase the phosphorus contents of released cellular debris and virus particles, which together constitute a significant proportion of the marine dissolved organic phosphorus pool. [ABSTRACT FROM AUTHOR]

SUMMARY: The phytohormone abscisic acid (ABA) regulates ion channel activity and stomatal movement in response to drought stress. Cellular ABA levels change depending on cellular and environmental conditions via modulation of its biosynthesis, catabolism and transport. Although factors involved in ABA biosynthesis and degradation have been studied extensively, how ABA transporters are modulated to fine‐tune ABA levels, especially under drought stress, remains elusive. Here, we show that Arabidopsis thaliana SORTING NEXIN 2 (SNX2) proteins play a critical role in endosomal trafficking of the ABA exporter ATP BINDING CASETTE G25 (ABCG25) via direct interaction at endosomes, leading to its degradation in the vacuole. In agreement, snx2a and snx2b mutant plants showed enhanced recycling of GFP‐ABCG25 from early endosomes to the plasma membrane and higher accumulation of GFP‐ABCG25. Phenotypically, snx2a and snx2b plants were highly sensitive to exogenous ABA and displayed enhanced ABA‐mediated inhibition of inward K+ currents and ABA‐mediated activation of slow anion currents in guard cells, resulting in an increased tolerance to drought stress. Based on these results, we propose that SNX2 proteins play a crucial role in stomatal movement and tolerance to drought stress by modulating the endosomal trafficking of ABCG25 and thus cellular ABA levels. Significance Statement: Increasing evidence supports a role for sorting nexins (SNXs) in intracellular trafficking of membrane proteins; however, the role of SNX2 proteins in endomembrane protein trafficking remains elusive, and it is not clear whether and how they might function in response to abiotic stress. Here, we show that Arabidopsis SNX2a and SNX2b play a crucial role in stomatal movement and tolerance to drought stress via direct interaction with the abscisic acid exporter ABCG25 and modulation of its endosomal trafficking. [ABSTRACT FROM AUTHOR]

MCCs are linear invaginations of the yeast plasma membrane that form stable membrane microdomains. Although over 20 proteins are localized in the MCCs, it is not well understood how these proteins coordinately maintain normal MCC function. Pil1 is a core eisosome protein and is responsible for MCC‐invaginated structures. In addition, six‐tetraspan membrane proteins (6‐Tsp) are localized in the MCCs and classified into two families, the Sur7 family and Nce102 family. To understand the coordinated function of these MCC proteins, single and multiple deletion mutants of Pil1 and 6‐Tsp were generated and their MCC structure and growth under various stresses were investigated. Genetic interaction analysis revealed that the Sur7 family and Nce102 function in stress tolerance and normal eisosome assembly, respectively, by cooperating with Pil1. To further understand the role of MCCs/eisosomes in stress tolerance, we screened for suppressor mutants using the SDS‐sensitive phenotype of pil1Δ 6‐tspΔ cells. This revealed that SDS sensitivity is caused by hyperactivation of Tor kinase complex 2 (TORC2)‐Ypk1 signaling. Interestingly, inhibition of sphingolipid metabolism, a well‐known downstream pathway of TORC2‐Ypk1 signaling, did not rescue the SDS‐sensitivity of pil1Δ 6‐tspΔ cells. These results suggest that Pil1 and 6‐Tsp cooperatively regulate TORC2 signaling during the stress response. [ABSTRACT FROM AUTHOR]

Protein fractionation and purification remain challenging in membrane separation. Herein, a pH‐responsive membrane was developed by grafting the brush‐like poly(dimethylaminoethyl methacrylate) (PDMAEMA) via ATRP onto the ethylene vinyl alcohol copolymer (EVAL) membrane for protein fractionation. The PDMAEMA brushes are distributed in all pore channels of the membrane. Based on the electrostatic and sieving properties of the as‐prepared pH‐responsive membrane, three milk proteins are successfully separated from their mixture. The α‐Lactalbumin (α‐LA) preferentially permeates the membrane from the mixture at pH 3.2, and the bovine serum albumin (BSA) and lactoferrin (LF) can be separated at pH 6.4 based on the selective adsorption. This work proposes a promising candidate for protein separation by a pH‐responsive membrane. [ABSTRACT FROM AUTHOR]

Background and Purpose: beta-Arrestins are critical scaffold proteins that shape spatiotemporal signalling from seven transmembrane domain receptors (7TMRs). Here, we study the association between neuropeptide Y (NPY) receptors and beta-arrestin2, using bimolecular fluorescence complementation (BiFC) to directly report underlying protein-protein interactions.Experimental Approach: Y1 receptors were tagged with a C-terminal fragment, Yc, of yellow fluorescent protein (YFP), and beta-arrestin2 fused with the complementary N-terminal fragment, Yn. After Y receptor-beta-arrestin association, YFP fragment refolding to regenerate fluorescence (BiFC) was examined by confocal microscopy in transfected HEK293 cells. Y receptor/beta-arrestin2 BiFC responses were also quantified by automated imaging and granularity analysis.Key Results: NPY stimulation promoted association between Y1-Yc and beta-arrestin2-Yn, and the specific development of BiFC in intracellular compartments, eliminated when using non-interacting receptor and arrestin mutants. Responses developed irreversibly and were slower than for downstream Y1 receptor-YFP internalization, a consequence of delayed maturation and stability of complemented YFP. However, beta-arrestin2 BiFC measurements delivered appropriate ligand pharmacology for both Y1 and Y2 receptors, and demonstrated higher affinity of Y1 compared to Y2 receptors for beta-arrestin2. Receptor mutagenesis combined with beta-arrestin2 BiFC revealed that alternative arrangements of Ser/Thr residues in the Y1 receptor C tail could support beta-arrestin2 association, and that Y2 receptor-beta-arrestin2 interaction was enhanced by the intracellular loop mutation H155P.Conclusions and Implications: The BiFC approach quantifies Y receptor ligand pharmacology focused on the beta-arrestin2 pathway, and provides insight into mechanisms of beta-arrestin2 recruitment by activated and phosphorylated 7TMRs, at the level of protein-protein interaction. [ABSTRACT FROM AUTHOR]

Background and Purpose: We have investigated the effect of deletions of a postsynaptic density, disc large and zo-1 protein (PDZ) motif at the end of the COOH-terminus of the rat A(2B) adenosine receptor on intracellular trafficking following long-term exposure to the agonist 5'-(N-ethylcarboxamido)-adenosine.Experimental Approach: The trafficking of the wild type A(2B) adenosine receptor and deletion mutants expressed in Chinese hamster ovary cells was studied using an enzyme-linked immunosorbent assay in combination with immunofluorescence microscopy.Key Results: The wild type A(2B) adenosine receptor and deletion mutants were all extensively internalized following prolonged treatment with NECA. The intracellular compartment through which the Gln(325)-stop receptor mutant, which lacks the Type II PDZ motif found in the wild type receptor initially trafficked was not the same as the wild type receptor. Expression of dominant negative mutants of arrestin-2, dynamin or Eps-15 inhibited internalization of wild type and Leu(330)-stop receptors, whereas only dominant negative mutant dynamin inhibited agonist-induced internalization of Gln(325)-stop, Ser(326)-stop and Phe(328)-stop receptors. Following internalization, the wild type A(2B) adenosine receptor recycled rapidly to the cell surface, whereas the Gln(325)-stop receptor did not recycle.Conclusions and Implications: Deletion of the COOH-terminus of the A(2B) adenosine receptor beyond Leu(330) switches internalization from an arrestin- and clathrin-dependent pathway to one that is dynamin dependent but arrestin and clathrin independent. The presence of a Type II PDZ motif appears to be essential for arrestin- and clathrin-dependent internalization, as well as recycling of the A(2B) adenosine receptor following prolonged agonist addition. [ABSTRACT FROM AUTHOR]

Background and Purpose: In previous studies investigating cross-talk of signalling between prostaglandin (PG)E(2) receptor (EP) and the TPalpha and TPbeta isoforms of the human thromboxane (TX)A(2) receptor (TP), 17-phenyl trinor PGE(2)-induced desensitization of TP receptor signalling through activation of the AH6809 and SC19220-sensitive EP(1) subtype of the EP receptor family, in a cell-specific manner. Here, we sought to further investigate that cross-talk in human erythroleukaemic (HEL) 92.1.7 cells.Experimental Approach: Specificity of 17-phenyl trinor PGE(2) signalling and its possible cross-talk with signalling by TPalpha/TPbeta receptors endogenously expressed in HEL cells was examined through assessment of agonist-induced inositol 1,4,5-trisphosphate (IP)(3) generation and intracellular calcium ([Ca(2+)](i)) mobilization.Key Results: While 17-Phenyl trinor PGE(2) led to activation of phospholipase (PL)Cbeta to yield increases in IP(3) generation and [Ca(2+)](i), it did not desensitize but rather augmented that signalling in response to subsequent stimulation with the TXA(2) mimetic U46619. Furthermore, the augmentation was reciprocal. Signalling by 17-phenyl trinor PGE(2) was found to occur through AH6809- and SC19920-insensitive, Pertussis toxin-sensitive, G(i)/G(betagamma)-dependent activation of PLCbeta. Further pharmacological investigation using selective EP receptor subtype agonists and antagonists confirmed that 17-phenyl trinor PGE(2)-mediated signalling and reciprocal cross-talk with the TP receptors occurred through the EP(3), rather than the EP(1), EP(2) or EP(4) receptor subtype in HEL cells.Conclusions and Implications: The EP(1) and EP(3) subtypes of the EP receptor family mediated intermolecular cross-talk to differentially regulate TP receptor-mediated signalling whereby activation of EP(1) receptors impaired or desensitized, while that of EP(3) receptors augmented signalling through TPalpha/TPbeta receptors, in a cell type-specific manner. [ABSTRACT FROM AUTHOR]

Atomic-resolution structures from small proteins have recently been deter- mined from high-quality powder diffraction patterns using a combination of stereochemical restraints and Rietveld refinement [Von Dreele (2007), J. Appl. Cryst. 40, 133-143; Margiolaki et al. (2007), J. Am. Chem. Soc. 129, 11865- 11871]. While powder diffraction data have been obtained from batch samples of small crystal-suspensions, which are exposed to X-rays for long periods of time and undergo significant radiation damage, the proof-of-concept that protein powder diffraction data from nanocrystals of a membrane protein can be obtained using a continuous microjet is shown. This flow-focusing aerojet has been developed to deliver a solution of hydrated protein nanocrystals to an X-ray beam for diffraction analysis. This method requires neither the crushing of larger polycrystalline samples nor any techniques to avoid radiation damage such as cryocooling. Apparatus to record protein powder diffraction in this manner has been commissioned, and in this paper the first powder diffraction patterns from a membrane protein, photosystem I, with crystallite sizes of less than 500 nm are presented. These preliminary patterns show the lowest-order reflections, which agree quantitatively with theoretical calculations of the powder profile. The results also serve to test our aerojet injector system, with future application to femtosecond diffraction in free-electron X-ray laser schemes, and for serial crystallography using a single-file beam of aligned hydrated molecules. [ABSTRACT FROM AUTHOR]

A high-throughput robotic system has been developed for crystallizing membrane proteins using lipidic mesophases. It incorporates commercially available components and is relatively inexpensive. The crystallization robot uses standard automated liquid-handlers and a specially built device for accurately and reproducibly delivering nanolitre volumes of highly viscous protein/lipid mesophases. Under standard conditions, the robot uses just 20 nl protein solution, 30 nl lipid and 1 ml precipitant solution. 96 wells can be set up using the robot in 13 min. Trials are performed in specially designed 96-well glass plates. The slim (<2 mm high) plates have exquisite optical properties and are well suited for the detection of microcrystals and for birefringence-free imaging between crossed polarizers. Quantitative evaluation of the crystallization progress is performed using an automated imaging system. The optics, in combination with the slim crystallization plates, enables in-focus imaging of the entire well volume in a single shot such that a 96-well plate can be imaged in just 4.5 min. The performance characteristics of the robotic system and the versatility of the crystallization robot in performing vapor-diffusion, microbatch and bicelle crystallizations of membrane and soluble proteins are described. [ABSTRACT FROM AUTHOR]

The structure of the conjugative coupling protein TrwBΔN70 from Escherichia coli plasmid R388 was solved using two crystal forms. This large multimeric membrane protein of 437 residues per monomer is involved in cell-to-cell single-strand DNA transfer. Diffraction data to 2.4 Å were available from trigonal crystals obtained from ammonium sulfate and to 2.5 Å from monoclinic crystals grown from tartrate. A single tantalum bromide (Ta6Br122+) derivative of the trigonal form, which presented a protein hexamer with C6 local symmetry in the asymmetric unit, was used in a three-wavelength MAD experiment to achieve 4.5 Å resolution for initial phases. Sixfold averaging and phase extension increased the effective phasing resolution and eventually produced a straight-forwardly traceable electron-density map. The monoclinic structure was solved by molecular replacement, i.e. a hexamer of the trigonal form was used as a search model. Two such hexamers are present in the asymmetric unit. [ABSTRACT FROM AUTHOR]

Background: Glomerular endothelial cells (GECs) are directly exposed to circulating viral particles in the glomerulus. Although viral infections may trigger the development of acute kidney injury or the worsening of pre‐existing chronic kidney disease, the specific molecular mechanisms underlying antiviral reactions via the activation of endothelial Toll‐like receptor 3 signaling in the kidney remain to be determined. Interferon (IFN)‐induced transmembrane protein 1 (IFITM1), a member of interferon‐stimulated gene protein family, is involved in the prevention of viral entry into cerebral vascular endothelial cells, respiratory epithelial cells, and endometrium. However, as far as we are aware, the implication of IFITM1 associated with viral infections in GECs has not been investigated to date. Methods: Cultured, normal human GECs were treated with polyinosinic‐polycytidylic acid (poly IC), a synthesized viral double‐stranded RNA, then the expression of IFITM1 was examined by quantitative real‐time reverse transcription‐polymerase chain reaction and western blotting. To further elucidate the poly IC‐induced signaling pathway, the cells were applied to RNA interference against IFN‐β, nuclear factor‐κB p65, and IFN regulatory factor 3. We also conducted an immunofluorescence study to examine endothelial IFITM1 expression in biopsy specimens from patients with chronic kidney disease. Results: We found that the activation of Toll‐like receptor 3 induced endothelial expression of IFITM1, and that this involved IFN regulatory factor 3 and IFN‐β, but not nuclear factor‐κB. Intense endothelial IFITM1 immunoreactivity was observed in biopsy specimens from patients with lupus nephritis. Conclusions: Antiviral reaction‐related endothelial expression of IFITM1 may be involved, at least in part, in the development of particularly in lupus nephritis. Further detailed studies of the implication of interferon stimulated genes, including IFITM1 in GECs are needed. [ABSTRACT FROM AUTHOR]

Early signalling events in response to elicitation include reversible protein phosphorylation and re‐localization of plasma membrane (PM) proteins. Oligogalacturonides (OGs) are a class of damage‐associated molecular patterns (DAMPs) that act as endogenous signals to activate the plant immune response. Previous data on early phosphoproteome changes in Arabidopsis thaliana upon OG perception uncovered the immune‐related phospho‐regulation of several membrane proteins, among which PCaP1, a PM‐anchored protein with actin filament–severing activity, was chosen for its potential involvement in OG‐ and flagellin‐triggered responses. Here, we demonstrate that PCaP1 is required for late, but not early, responses induced by OGs and flagellin. Moreover, pcap1 mutants, unlike the wild type, are impaired in the recovery of full responsiveness to a second treatment with OGs performed 24 h after the first one. Localization studies on PCaP1 upon OG treatment in plants expressing a functional PCaP1‐GFP fusion under the control of PCaP1 promoter revealed fluorescence on the PM, organized in densely packed punctate structures, previously reported as microdomains. Fluorescence was found to be associated also with endocytic vesicles, the number of which rapidly increased after OG treatment, suggesting both an endocytic turnover of PCaP1 for maintaining its homeostasis at the PM and an OG‐induced endocytosis. The A. thaliana plasma membrane (PM)‐anchored protein, PCaP1, is involved in elicitor‐induced protection against the necrotrophic fungus B. cinerea. (GFP)‐PCaP1 organization into PM microdomains and its oligogalacturonide‐induced endocytosis point to a role of PCaP1 and its turn‐over in the sensing/transduction response to elicitors. [ABSTRACT FROM AUTHOR]

This contribution reports on a study using Purexa™‐MQ multimodal anion‐exchange (AEX) membranes for protein polishing at elevated solution conductivities. Dynamic binding capacities (DBC10) of bovine serum albumin (BSA), human immunoglobulins, and salmon sperm DNA (ss‐DNA) are reported for various salt types, salt concentrations, flowrates, and pH. Using 1 mg/ml BSA, DBC10 values for Purexa™‐MQ were >90 mg/ml at conductivities up to 15 mS/cm. The membranes maintained a high, salt‐tolerant BSA DBC10 of 89.8 ± 2.7 (SD) over the course of 100 bind‐elute cycles. Polishing studies with acidic and basic monoclonal antibodies at >2 kg/L loads showed that Purexa™‐MQ had higher clearance of host cell proteins and aggregate species at high conductivity (13 mS/cm) and in the presence of phosphate than other commercial AEX media. Purexa™‐MQ also had a high ss‐DNA DBC10 of 50 mg/ml at conductivities up to 15 mS/cm, markedly outperforming other commercial products. In addition to the effectiveness of Purexa™‐MQ for protein polishing at elevated solution conductivities, its unusually high binding capacity for ss‐DNA indicates potential applications for plasmid DNA purification. [ABSTRACT FROM AUTHOR]

Proteins destined to various intra‐ and extra‐cellular locations must traverse membranes most frequently in an unfolded form. When the proteins being translocated need to remain in a folded state, specialized cellular transport machinery is used. One such machine is the membrane‐bound AAA protein Bcs1 (Bcs1), which assists the iron‐sulfur protein, an essential subunit of the respiratory Complex III, across the mitochondrial inner membrane. Recent structure determinations of mouse and yeast Bcs1 in three different nucleotide states reveal its homo‐heptameric association and at least two dramatically different conformations. The apo and ADP‐bound structures are similar, both containing a large substrate‐binding cavity accessible to the mitochondrial matrix space, which contracts by concerted motion of the ATPase domains upon ATP binding, suggesting that bound substrate could then be pushed across the membrane. ATP hydrolysis drives substrate release and resets Bcs1 conformation back to the apo/ADP form. These structures shed new light on the mechanism of folded protein translocation across a membrane, provide better understanding on the assembly process of the respiratory Complex III, and correlate clinical presentations of disease‐associated mutations with their locations in the 3D structure. [ABSTRACT FROM AUTHOR]

SUMMARY: The chloroplast signal recognition particle 54 kDa (CpSRP54) protein is a member of the CpSRP pathway known to target proteins to thylakoid membranes in plants and green algae. Loss of CpSRP54 in the marine diatom Phaeodactylum tricornutum lowers the accumulation of a selection of chloroplast‐encoded subunits of photosynthetic complexes, indicating a role in the co‐translational part of the CpSRP pathway. In contrast to plants and green algae, absence of CpSRP54 does not have a negative effect on the content of light‐harvesting antenna complex proteins and pigments in P. tricornutum, indicating that the diatom CpSRP54 protein has not evolved to function in the post‐translational part of the CpSRP pathway. Cpsrp54 KO mutants display altered photophysiological responses, with a stronger induction of photoprotective mechanisms and lower growth rates compared to wild type when exposed to increased light intensities. Nonetheless, their phenotype is relatively mild, thanks to the activation of mechanisms alleviating the loss of CpSRP54, involving upregulation of chaperones. We conclude that plants, green algae, and diatoms have evolved differences in the pathways for co‐translational and post‐translational insertion of proteins into the thylakoid membranes. Significance Statement: Loss of diatom CpSRP54 results in altered photophysiological responses and lower levels of chloroplast‐encoded subunits of photosynthetic complexes, but in contrast to plants and green microalgae, absence of CpSRP54 does not have a negative impact on the level of light‐harvesting complex proteins and pigments in diatoms. By characterizing cpsrp54 KO lines in diatoms, we show that knowledge from the green lineage is not directly transferable to other groups of photosynthetic organisms. [ABSTRACT FROM AUTHOR]

The cyanogens bromide fragments of protein I, a major protein of the Escherichia coli outer cell envelope membrane, have been isolated and characterized. There appear to be two methionineserine or methionine-threonine sequences causing incomplete cleavage but complete conversion of methionine to homoserine. Largerly due to the existence of these overlapping fragments the order of 5 of the 6 fragments present could be deduced. None of the fragments exhibits any remarkable low degree of polarity, and the tryptic fingerprint of the largest fragment (comprising about 60%,of protein I) also does not show any conspicuous large fraction of lipophilic peptides. It is concluded that the domain of protein I that may be buried in the lipid phase of the outer membrane in all likelihood is not very large, and there is, in fact, no definite proof yet that protein I is a membrane protein sensu stricto. [ABSTRACT FROM AUTHOR]

Predicting protein structure from the amino acid sequence has been a challenge with theoretical and practical significance in biophysics. Despite the recent progresses elicited by improved inter‐residue contact prediction, contact‐based structure prediction has gradually reached the performance ceiling. New methods have been proposed to predict the inter‐residue distance, but unanimously by simplifying the real‐valued distance prediction into a multiclass classification problem. Here, a lightweight regression‐based distance prediction method is shown, which adopts the generative adversarial network to capture the delicate geometric relationship between residue pairs and thus could predict the continuous, real‐valued inter‐residue distance rapidly and satisfactorily. The predicted residue distance map allows quick structure modeling by the CNS suite, and the constructed models approach the same level of quality as the other state‐of‐the‐art protein structure prediction methods when tested on CASP13 targets. Moreover, this method can be used directly for the structure prediction of membrane proteins without transfer learning. [ABSTRACT FROM AUTHOR]

Background and Purpose: Osteoclasts are unique cells to absorb bone. Targeting osteoclast differentiation is a therapeutic strategy for osteolytic diseases. Natural marine products have already become important sources of new drugs. The naturally occurring nitrobenzoyl sesquiterpenoids first identified from marine fungi in 1998 are bioactive compounds with a special structure, but their pharmacological functions are largely unknown. Here, we investigated six marine fungus-derived nitrobenzoyl sesquiterpenoids on osteoclastogenesis and elucidated the mechanisms.Experimental Approach: Compounds were first tested by RANKL-induced NF-κB luciferase activity and osteoclastic TRAP assay, followed by molecular docking to characterize the structure-activity relationship. The effects and mechanisms of the most potent nitrobenzoyl sesquiterpenoid on RANKL-induced osteoclastogenesis and bone resorption were further evaluated in vitro. Micro-CT and histology analysis were used to assess the prevention of bone destruction by nitrobenzoyl sesquiterpenoids in vivo.Key Results: Nitrobenzoyl sesquiterpenoid 4, with a nitrobenzoyl moiety at C-14 and a hydroxyl group at C-9, was the most active compound on NF-κB activity and osteoclastogenesis. Consequently, nitrobenzoyl sesquiterpenoid 4 exhibited suppression of RANKL-induced osteoclastogenesis and bone resorption from 0.5 μM. It blocked RANKL-induced IκBa phosphorylation, NF-κB p65 and RelB nuclear translocation, NFATc1 activation, reduced DC-STAMP but not c-Fos expression during osteoclastogenesis in vitro. Nitrobenzoyl sesquiterpenoid 4 also ameliorated LPS-induced osteolysis in vivo.Conclusion and Implications: These results highlighted nitrobenzoyl sesquiterpenoid 4 as a novel inhibitor of osteoclast differentiation. This marine-derived sesquiterpenoid is a promising lead compound for the treatment of osteolytic diseases. [ABSTRACT FROM AUTHOR]

Many proteins involved in synaptic transmission are well known, and their features, as their abundance or spatial distribution, have been analyzed in systematic studies. This has not been the case, however, for their mobility. To solve this, we analyzed the motion of 45 GFP‐tagged synaptic proteins expressed in cultured hippocampal neurons, using fluorescence recovery after photobleaching, particle tracking, and modeling. We compared synaptic vesicle proteins, endo‐ and exocytosis cofactors, cytoskeleton components, and trafficking proteins. We found that movement was influenced by the protein association with synaptic vesicles, especially for membrane proteins. Surprisingly, protein mobility also correlated significantly with parameters as the protein lifetimes, or the nucleotide composition of their mRNAs. We then analyzed protein movement thoroughly, taking into account the spatial characteristics of the system. This resulted in a first visualization of overall protein motion in the synapse, which should enable future modeling studies of synaptic physiology. Synopsis: Live imaging reveals global protein dynamics in the synaptic bouton, providing a first visualization of overall protein motion in the synapse and showing connections between various protein characteristics and mobility in vivo. Membrane proteins display lower mobility rates than soluble proteins.Proteins are less mobile in synaptic boutons than in axons.Protein mobility is strongly influenced by the protein association with synaptic vesicles.Amino acid composition, mRNA nucleotide composition, and protein lifetimes correlate with mobility parameters.Provided diffusion coefficients for 45 synaptic proteins can be used to generate models of synaptic physiology. [ABSTRACT FROM AUTHOR]

The malarial parasite Plasmodium exports its own proteins to the cell surfaces of red blood cells (RBCs) during infection. Examples of exported proteins include members of the repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR) family of proteins from Plasmodium falciparum. The presence of these parasite‐derived proteins on surfaces of infected RBCs triggers the adhesion of infected cells to uninfected cells (rosetting) and to the vascular endothelium potentially obstructing blood flow. While there is a fair amount of information on the localization of these proteins on the cell surfaces of RBCs, less is known about how they can be exported to the membrane and the topologies they can adopt during the process. The first step of export is plausibly the cotranslational insertion of proteins into the endoplasmic reticulum (ER) of the parasite, and here, we investigate the insertion of three RIFIN and two STEVOR proteins into the ER membrane. We employ a well‐established experimental system that uses N‐linked glycosylation of sites within the protein as a measure to assess the extent of membrane insertion and the topology it assumes when inserted into the ER membrane. Our results indicate that for all the proteins tested, transmembranes (TMs) 1 and 3 integrate into the membrane, so that the protein assumes an overall topology of Ncyt‐Ccyt. We also show that the segment predicted to be TM2 for each of the proteins likely does not reside in the membrane, but is translocated to the lumen. [ABSTRACT FROM AUTHOR]

In physiology, homeostasis refers to the condition where a system exhibits an optimum functional level. In contrast, any variation from this optimum is considered as a dysfunctional or pathological state. In this review, we address the proposal that a critical cholesterol level in the plasma membrane is required for the proper functioning of transmembrane proteins. Thus, membrane cholesterol depletion or enrichment produces a loss or gain of direct cholesterol-protein interaction and/or changes in the physical properties of the plasma membrane, which affect the basal or optimum activity of transmembrane proteins. Whether or not this functional switching is a generalized mechanism exhibited for all transmembrane proteins, or if it works just for an exclusive group of them, is an open question and an attractive subject to explore at a basic, pharmacological and clinical level. [ABSTRACT FROM AUTHOR]

While it is generally accepted that α‐synuclein oligomers (αSOs) play an important role in neurodegeneration in Parkinson's disease, the basis for their cytotoxicity remains unclear. We have previously shown that docosahexaenoic acid (DHA) stabilizes αSOs against dissociation without compromising their ability to colocalize with glutamatergic synapses of primary hippocampal neurons, suggesting that they bind to synaptic proteins. Here, we develop a proteomic screen for putative αSO binding partners in rat primary neurons using DHA‐stabilized human αSOs as a bait protein. The protocol involved co‐immunoprecipitation in combination with a photoactivatable heterobifunctional sulfo‐LC‐SDA crosslinker which did not compromise neuronal binding and preserved the interaction between the αSOs‐binding partners. We identify in total 29 proteins associated with DHA‐αSO of which eleven are membrane proteins, including synaptobrevin‐2B (VAMP‐2B), the sodium–potassium pump (Na+/K+ ATPase), the V‐type ATPase, the voltage‐dependent anion channel and calcium‐/calmodulin‐dependent protein kinase type II subunit gamma; only these five hits were also found in previous studies which used unmodified αSOs as bait. We also identified Rab‐3A as a target with likely disease relevance. Three out of four selected hits were subsequently validated with dot‐blot binding assays. In addition, likely binding sites on these ligands were identified by computational analysis, highlighting a diversity of possible interactions between αSOs and target proteins. These results constitute an important step in the search for disease‐modifying treatments targeting toxic αSOs. [ABSTRACT FROM AUTHOR]

Depression is a commonly occurring neuropsychiatric disease with an increasing incidence rate. Saikosaponin A (SA), a major bioactive component extracted from Radix Bupleuri, possesses anti‐malignant cell proliferation, anti‐inflammation, anti‐oxidation and liver protective effects. However, few studies have investigated SA's antidepressant effects and pharmacological mechanisms of action. Our study aimed to explore the anti‐depression effect of SA and screen the target proteins regulated by SA in a rat model of chronic unpredictable mild stress (CUMS)‐induced depression. Results showed that 8‐week CUMS combined with separation could successfully produce depressive‐like behaviours and cause a decrease of dopamine (DA) in rat hippocampus, and 4‐week administration of SA could relieve CUMS rats' depressive symptoms and up‐regulated DA content. There were 15 kinds of significant differentially expressed proteins that were detected not only between the control and CUMS groups, but also between the CUMS and SA treatment groups. Proline‐rich transmembrane protein 2 (PRRT2) was down‐regulated by CUMS while up‐regulated by SA. These findings reveal that SA may exert antidepressant effects by up‐regulating the expression level of PRRT2 and increasing DA content in hippocampus. The identification of these 15 differentially expressed proteins, including PRRT2, provides further insight into the treatment mechanism of SA for depression. [ABSTRACT FROM AUTHOR]

Background and Purpose: Although chloride channels are involved in several physiological processes and acquired diseases, the availability of compounds selectively targeting CLC proteins is limited. ClC-1 channels are responsible for sarcolemma repolarization after an action potential in skeletal muscle and have been associated with myotonia congenita and myotonic dystrophy as well as with other muscular physiopathological conditions. To date only a few ClC-1 blockers have been discovered, such as anthracene-9-carboxylic acid (9-AC) and niflumic acid (NFA), whereas no activator exists. The absence of a ClC-1 structure and the limited information regarding the binding pockets in CLC channels hamper the identification of improved modulators.Experimental Approach: Here we provide an in-depth characterization of drug binding pockets in ClC-1 through an integrated in silico and experimental approach. We first searched putative cavities in a homology model of ClC-1 built upon an eukaryotic CLC crystal structure, and then validated in silico data by measuring the blocking ability of 9-AC and NFA on mutant ClC-1 channels expressed in HEK 293 cells.Key Results: We identified four putative binding cavities in ClC-1. 9-AC appears to interact with residues K231, R421 and F484 within the channel pore. We also identified one preferential binding cavity for NFA and propose R421 and F484 as critical residues.Conclusions and Implications: This study represents the first effort to delineate the binding sites of ClC-1. This information is fundamental to discover compounds useful in the treatment of ClC-1-associated dysfunctions and might represent a starting point for specifically targeting other CLC proteins. [ABSTRACT FROM AUTHOR]

Background and Purpose: Rescue of F508del-cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the most common CF mutation, requires small molecules that overcome protein processing, stability and channel gating defects. Here, we investigate F508del-CFTR rescue by CFFT-004, a small molecule designed to independently correct protein processing and channel gating defects.Experimental Approach: Using CFTR-expressing recombinant cells and CF patient-derived bronchial epithelial cells, we studied CFTR expression by Western blotting and channel gating and stability with the patch-clamp and Ussing chamber techniques.Key Results: Chronic treatment with CFFT-004 improved modestly F508del-CFTR processing, but not its plasma membrane stability. By contrast, CFFT-004 rescued F508del-CFTR channel gating better than C18, an analogue of the clinically used CFTR corrector lumacaftor. Subsequent acute addition of CFFT-004, but not C18, potentiated F508del-CFTR channel gating. However, CFFT-004 was without effect on A561E-CFTR, a CF mutation with a comparable mechanism of CFTR dysfunction as F508del-CFTR. To investigate the mechanism of action of CFFT-004, we used F508del-CFTR revertant mutations. Potentiation by CFFT-004 was unaffected by revertant mutations, but correction was abolished by the revertant mutation G550E. These data suggest that correction, but not potentiation, by CFFT-004 might involve nucleotide-binding domain 1 of CFTR.Conclusions and Implications: CFFT-004 is a dual-acting small molecule with independent corrector and potentiator activities that partially rescues F508del-CFTR in recombinant cells and native airway epithelia. The limited efficacy and potency of CFFT-004 suggests that combinations of small molecules targeting different defects in F508del-CFTR might be a more effective therapeutic strategy than a single agent. [ABSTRACT FROM AUTHOR]

Objective: To determine the pathogenic mechanisms of autoantibodies to the cell adhesion molecule Caspr2 in acquired neuromyotonia and autoimmune encephalitis.Methods: Caspr2-positive samples were confirmed using a cell-based assay, and their IgG subtypes were determined by enzyme-linked immunosorbent assay and cell-based assay. A solid phase binding assay quantified the binding of Caspr2 to contactin-2 in the presence of Caspr2 autoantibodies. Living cultures of primary rat hippocampal neurons were incubated with Caspr2-positive or control sera, and the distribution of Caspr2-positive immunofluorescent puncta and total surface Caspr2 was quantified. HEK cells transfected to express Caspr2 were incubated with Caspr2-positive or control samples, and cell-surface biotinylation and Western blot were used to assess total, internalized, and surface levels of Caspr2.Results: We confirmed 6 samples with strong Caspr2 reactivity. IgG4 Caspr2 antibodies were present in all 6 cases. Caspr2 interacted with another cell adhesion molecule, contactin-2, with nanomolar affinity in the solid phase assay, and Caspr2 autoantibodies inhibited this interaction. Caspr2 autoantibodies did not affect the surface expression of Caspr2 in rat primary hippocampal neurons or transfected HEK cells.Interpretation: Caspr2 autoantibodies inhibit the interaction of Caspr2 with contactin-2 but do not cause internalization of Caspr2. Functional blocking of cell adhesion molecule interactions represents a potential mechanism with therapeutic implications for IgG4 autoantibodies to cell adhesion molecules in neurological diseases. Ann Neurol 2018;83:40-51. [ABSTRACT FROM AUTHOR]

Orexin-1 receptors (Ox1Rs) have been implicated in the motivation for drugs of abuse. Here, we utilized a within-session behavioral-economics threshold procedure to screen for individual differences in economic demand for the ultra-short-acting opioid remifentanil and to test whether antagonism of Ox1Rs reduces remifentanil demand. The behavioral-economics procedure revealed robust individual differences in free consumption of remifentanil (Q0 parameter; hedonic set point). Rats with low baseline Q0 (low takers) displayed high demand elasticity (α parameter; reduced responding as drug price increased indicating low motivation for drug), whereas subjects with a higher Q0 (high takers) exhibit low demand elasticity (low α) by continuing to self-administer remifentanil despite increased cost (reflecting higher motivation for drug). In a punished responding paradigm utilizing footshock, subjects that were classified as high takers at baseline withstood twice as much shock as low takers to continue self-administering remifentanil. Interestingly, Ox1R antagonism with SB-334867 reduced Q0 and increased α in low takers but not in high takers. Similarly, the Ox1R antagonist attenuated cue-induced, but not drug-induced, reinstatement of remifentanil seeking in low takers but had no significant effect on reinstatement of drug seeking in high takers. Together, these data reveal a novel role of orexins in demand for remifentanil: Ox1Rs modulate demand in low takers but not in individuals that exhibit addictive-like behaviors (high takers). Finally, the behavioral assays in this study can serve as a novel laboratory model for studying individual differences in opioid use disorders. [ABSTRACT FROM AUTHOR]

Objectives: To test the following two hypotheses: 1) different types of retainers result in distinct levels of biomarkers in gingival crevicular fluid (GCF) and 2) the retainer bonded to all mandibular anterior teeth induces more detrimental outcomes to the periodontium.Setting and Sample Population: The Department of Orthodontics at the University of Florida. The population consisted of individuals in the retention phase of orthodontic treatment.Material and Methods: This was a cross-sectional study that enrolled 36 individuals. Subjects in group 1 had retainers bonded to the mandibular canines only. Group 2 consisted of individuals having retainers bonded to all mandibular anterior teeth. Group 3 included patients using mandibular removable retainers. After clinical examination, GCF was collected from the mandibular incisor and biomarker levels were compared between the groups.Results: Plaque accumulation and gingivitis differed significantly among groups, with the highest median values in group 2 subjects. Pairwise comparison of the groups with respect to gingivitis showed significant differences between groups 1 and 2. Significant differences among groups were detected for RANKL, OPG, OPN, M-CSF, MMP-3, and MMP-9. The ratio RANKL/OPG was significantly higher in group 2 subjects, with pairwise comparisons indicating that groups 1 and 2 differed from group 3.Conclusion: An association was found between orthodontic retention groups and GCF biomarker levels, which should be further explored in longitudinal studies. The presence of retainers bonded to all anterior teeth seems to increase plaque accumulation and gingivitis. [ABSTRACT FROM AUTHOR]

A monofluoro-substituted amino acid was designed to serve as a conformationally restricted label for solid-state 19F NMR distance measurements in membrane-bound peptides. The aromatic cis and trans isomers of 1-amino-3-(4-fluorophenyl)cyclobutanecarboxylic acid were synthesized in five steps from diethyl 2-(4-fluorophenyl)propanedioate. They were incorporated into the antimicrobial peptide gramicidin S to replace a native DPhenylalanine residue. Because the Cα-tetrasubstituted amino acid cannot racemize, it showed full compatibility with solid-phase peptide synthesis protocols. According to circular dichroism analysis and molecular modeling, the 19F-labeled analogues of the known helix-inducing amino acid (1-aminocyclobutane-1-carboxylic acid) do not disrupt the peptide conformation when substituted for Phe, neither in a β-turn nor in an α-helix. [ABSTRACT FROM AUTHOR]

The article discusses a review of what the atomic force microscopy (AFM) can provide in the study of lipid-protein interactions with focus on the distribution of membrane proteins. It describes the model system exploited in AFM studies and the technical approaches where it is coupled to other biophysical techniques. It suggests that its high lateral and vertical resolution make it a preferred technique to study the interactions between lipids and proteins and progress in its coupling with other signals increases knowledge of lipid/protein interaction.

The biogenesis of melanosomes is a multistage process that requires the function of cell-type-specific and ubiquitously expressed proteins. OCA2, the product of the gene defective in oculocutaneous albinism type 2, is a melanosomal membrane protein with restricted expression pattern and a potential role in the trafficking of other proteins to melanosomes. The ubiquitous protein complexes AP-3, BLOC-1, and BLOC-2, which contain as subunits the products of genes defective in various types of Hermansky-Pudlak syndrome, have been likewise implicated in trafficking to melanosomes. We have tested for genetic interactions between mutant alleles causing deficiency in OCA2 (pink-eyed dilution unstable), AP-3 (pearl), BLOC-1 (pallid), and BLOC-2 (cocoa) in C57BL/6J mice. The pallid allele was epistatic to pink-eyed dilution, and the latter behaved as a semi-dominant phenotypic enhancer of cocoa and, to a lesser extent, of pearl. These observations suggest functional links between OCA2 and these three protein complexes involved in melanosome biogenesis. [ABSTRACT FROM AUTHOR]

Background and Purpose: To validate a fluorescence approach for monitoring norepinephrine transporter (NET) transport rate in mature sympathetic terminals, and to determine how prejunctional muscarinic receptors affect NET rate.Experimental Approach: Confocal imaging of a fluorescent NET substrate [neurotransmitter transporter uptake assay (NTUA)] as it accumulates in the mature sympathetic nerve terminals of the mouse isolated vas deferens. Fluorescence recovery after photobleaching (FRAP), enhanced green fluorescence protein (EGFP)-transgenic mice and contraction studies were also used.Key Results: NTUA fluorescence accumulated linearly in nerve terminals, an effect that was prevented with NET inhibition with desipramine (1 microM). Such accumulation was reversed by amphetamine (10 microM), which is known to reverse the direction of transport of NET substrates. NTUA labelling was not present in cholinergic terminals (identified using EGFP fluorescence expressed in transgenic mice under a choline acetyltransferase promoter). FRAP experiments, altered nerve terminal distribution with reserpine pretreatment and co-imaging in terminals filled with a cytoplasmic marker (Alexa 594 dextran) indicated that the NTUA labelling was largely confined to vesicles within varicosities; vesicular exchange between varicosities was rare. The rate of NTUA accumulation was slower in the presence of the muscarinic agonist carbachol (10 microM) demonstrating muscarinic inhibition of NET rate.Conclusions and Implications: A straightforward protocol now exists to monitor NET transport rate at the level of the single nerve terminal varicosity, providing a useful tool to understand the physiology of NET regulation, the action of NET inhibitors on mature sympathetic terminals, dynamic vesicular tracking and to identify sympathetic terminals from mixed terminal populations in living organs. [ABSTRACT FROM AUTHOR]

Background and Purpose: Icariin may be the active ingredient in Herba Epimedii, a Chinese herb commonly used for treatment of osteoporosis. The present study aims to delineate the mechanism(s) by which icariin prevents bone loss after ovariectomy (OVX) in vivo and stimulates osteoblastic functions in vitro.Experimental Approach: Ovariectomized or sham-operated C57BL/6 mice were treated with vehicle, 17beta-oestradiol or icariin for 6 weeks. Total and trabecular bome mineral density (BMD) as well as polar stress-strain index of distal femur were measured by peripheral computed tomography. The mRNA expressions of OPG and RANKL in tibia were studied by RT-PCR. Interactions between the oestrogen receptor (ER) antagonist ICI182,780 and icariin were studied in UMR 106 cells. The functional transactivation of ERalpha and ERbeta as well as ERalpha phosphorylation by icariin were also assessed.Key Results: Icariin suppressed the loss of bone mass and strength in distal femur and increased the mRNA expression ratio of OPG/RANKL in tibia, following OVX. Icariin increased ER-dependent cell proliferation, alkaline phosphatase (ALP) activity, gene expression of OPG and the OPG/RANKL ratio in UMR 106 cells. Icariin did not activate ERE-luciferase activity in UMR 106 cells, via the ERalpha or the ERbeta-mediated pathway, but it did increase ERalpha phosphorylation at Ser118.Conclusions and Implications: Our results indicate that icariin exerts anabolic effects in bone possibly by activating ER in a ligand-independent manner. Its ability to prevent OVX-induced bone loss without inducing uterotrophic effects supports its use as an alternative regimen for management of postmenopausal osteoporosis. [ABSTRACT FROM AUTHOR]

Proteins in the YidC/Oxa1/Alb3 family have essential functions in membrane protein insertion and folding. Bacillus subtilis encodes two YidC homologs, one that is constitutively expressed (spoIIIJ/yidC1) and a second (yqjG/yidC2) that is induced in spoIIIJ mutants. Regulated induction of yidC2 allows B. subtilis to maintain capacity of the membrane protein insertion pathway. We here show that a gene located upstream of yidC2 (mifM/yqzJ) serves as a sensor of SpoIIIJ activity that regulates yidC2 translation. Decreased SpoIIIJ levels or deletion of the MifM transmembrane domain arrests mifM translation and unfolds an mRNA hairpin that otherwise blocks initiation of yidC2 translation. This regulated translational arrest and yidC2 induction require a specific interaction between the MifM C-terminus and the ribosomal polypeptide exit tunnel. MifM therefore acts as a ribosome–nascent chain complex rather than as a fully synthesized protein. B. subtilis MifM and the previously described secretion monitor SecM in Escherichia coli thereby provide examples of the parallel evolution of two regulatory nascent chains that monitor different protein export pathways by a shared molecular mechanism. [ABSTRACT FROM AUTHOR]

The CLC ‘Cl− channel’ family consists of both Cl−/H+ antiporters and Cl− channels. Although CLC channels can undergo large, conformational changes involving cooperativity between the two protein subunits, it has been hypothesized that conformational changes in the antiporters may be limited to small movements localized near the Cl− permeation pathway. However, to date few studies have directly addressed this issue, and therefore little is known about the molecular movements that underlie CLC-mediated antiport. The crystal structure of the Escherichia coli antiporter ClC-ec1 provides an invaluable molecular framework, but this static picture alone cannot depict the protein movements that must occur during ion transport. In this study we use fluorine nuclear magnetic resonance (NMR) to monitor substrate-induced conformational changes in ClC-ec1. Using mutational analysis, we show that substrate-dependent 19F spectral changes reflect functionally relevant protein movement occurring at the ClC-ec1 dimer interface. Our results show that conformational change in CLC antiporters is not restricted to the Cl− permeation pathway and show the usefulness of 19F NMR for studying conformational changes in membrane proteins of known structure. [ABSTRACT FROM AUTHOR]

Background and Purpose: In mammalian heart, connexin43 (Cx43) represents the predominant connexin in the working myocardium. As the beta-adrenoceptor is involved in many cardiac diseases, we wanted to clarify the pathway by which beta-adrenoceptor stimulation may control Cx43 expression.Experimental Approach: Cultured neonatal rat cardiomyocytes were stimulated with isoprenaline. Cx43 expression as well as activation of p38 mitogen-activated protein kinase (MAPK), p42/44 MAPK, JUN NH(2)-terminal kinase (JNK) and nuclear translocation of the transcription factors activator protein 1 (AP1) and CRE-binding protein (CREB) were investigated. Additionally, we assessed Cx43 expression and distribution in left ventricular biopsies from patients without any significant heart disease, and from patients with either congestive heart failure [dilated cardiomyopathy (DCM)] or hypertrophic cardiomyopathy (HCM).Key Results: Isoprenaline exposure caused about twofold up-regulation of Cx43 protein with a pEC(50) of 7.92 +/- 0.11, which was inhibited by propranolol, SB203580 (4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)-1H-imidazole) (p38 inhibitor), PD98059 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one) (MAPK 1 kinase inhibitor) (Alexis Biochemicals, San Diego, CA, USA) or cyclosporin A. Similar findings were obtained for Cx43 mRNA. Furthermore, Cx43 up-regulation was accompanied by phosphorylation of p38, p42/44 and JNK, and by translocation of AP1 and CREB to the nucleus. Analysis of Cx43 protein and mRNA in ventricular biopsies revealed that in patients with DCM, Cx43 content was significantly lower, and in patients with HCM, Cx43 content was significantly higher, relative to patients without any cardiomyopathy. More importantly, Cx43 distribution also changed with more Cx43 being localized at the lateral border of the cardiomyocytes.Conclusion and Implication: Beta-adrenoceptor stimulation up-regulated cardiac Cx43 expression via a protein kinase A and MAPK-regulated pathway, possibly involving AP1 and CREB. Cardiomyopathy altered Cx43 expression and distribution. [ABSTRACT FROM AUTHOR]

The mobility of membrane proteins is a critical determinant of their interaction capabilities and protein functions. The heterogeneity of cell membranes imparts different types of motion onto proteins; immobility, random Brownian motion, anomalous sub-diffusion, ‘hop’ or confined diffusion, or directed flow. Quantifying the motion of proteins therefore enables insights into the lateral organisation of cell membranes, particularly membrane microdomains with high viscosity such as lipid rafts. In this review, we examine the hypotheses and findings of three main techniques for analysing protein dynamics: fluorescence recovery after photobleaching, single particle tracking and fluorescence correlation spectroscopy. These techniques, and the physical models employed in data analysis, have become increasingly sophisticated and provide unprecedented details of the biophysical properties of protein dynamics and membrane domains in cell membranes. Yet despite these advances, there remain significant unknowns in the relationships between cholesterol-dependent lipid microdomains, protein-protein interactions, and the effect of the underlying cytoskeleton. New multi-dimensional microscopy approaches may afford greater temporal and spatial resolution resulting in more accurate quantification of protein and membrane dynamics in live cells. [ABSTRACT FROM AUTHOR]