Localization of neurotransmitters and calcium binding proteins to neurons of salamander and mudpuppy retinas

We wished to identify the different types of retinal neurons on the basis of their content of neuroactive substances in both larval tiger salamander and mudpuppy retinas, favored species for electrophysiological investigation. Sections and wholemounts of retinas were labeled by immunocytochemical methods to demonstrate three calcium binding protein species and the common neurotransmitters, glycine, GABA and acetylcholine. Double immunostained sections and single labeled wholemount retinas were examined by confocal microscopy. Immunostaining patterns appeared to be the same in salamander and mudpuppy. Double and single cones, horizontal cells, some amacrine cells and ganglion cells were strongly calbindin-immunoreactive (IR). Calbindin-IR horizontal cells colocalized GABA. Many bipolar cells, horizontal cells, some amacrine cells and ganglion cells were strongly calretinin-IR. One type of horizontal cell and an infrequently occurring amacrine cell were parvalbumin-IR. Acetylcholine as visualized by ChAT-immunoreactivity was seen in a mirror-symmetric pair of amacrine cells that colocalized GABA and glycine. Glycine and GABA colocalized with calretinin, calbindin and occasionally with parvalbumin in amacrine cells. (C) 2001 Elsevier Science Ltd. All rights reserved.

Binding and functional studies with the growth hormone receptor antagonist, B2036-PEG (pegvisomant), reveal effects of pegylation and evidence that it binds to a receptor dimers

GH actions are dependent on receptor dimerization. The GH receptor antagonist, B2036-PEG, has been developed for treating acromegaly. B2036 has mutations in site 1 to enhance receptor binding and in site 2 to block receptor dimerization. Pegylation (B2036-PEG) increases half-life and lowers immunogenicity, but high concentrations are required to control insulin-like growth factor-I levels. We examined antagonist structure and function and the impact of pegylation on biological efficacy. Unpegylated B2036 had a 4.5-fold greater affinity for GH binding protein (GHBP) than GH but similar affinity for membrane receptor. Pegylation substantially reduced membrane binding affinity and receptor antagonism, as assessed by a transcription assay, by 39- and 20-fold, respectively. GHBP reduced antagonist activity of unpegylated B2036 but did not effect antagonism by B2036-PEG. B2036 down-regulated receptors, and membrane binding sites doubled in the presence of dimerization-blocking antibodies, suggesting that B2036 binds to a receptor dimer. It is concluded that the high concentration requirement of B2036-PEG for clinical efficacy relates to pegylation, which decreases binding to membrane receptor but has the advantages of reduced clearance, immunogenicity, and interactions with GHBP. Our studies suggest that B2036 binds to a receptor dimer and induces internalization but not signaling.

UV-induced hyperphosphorylation of replication protein a depends on DNA replication and expression of ATM protein

Exposure to DNA-damaging agents triggers signal transduction pathways that are thought to play a role in maintenance of genomic stability. A key protein in the cellular processes of nucleotide excision repair, DNA recombination, and DNA double-strand break repair is the single-stranded DNA binding protein, RPA. We showed previously that the p34 subunit of RPA becomes hyperphosphorylated as a delayed response (4-8 h) to UV radiation (10-30 J/m(2)). Here we show that UV-induced RPA-p34 hyperphosphorylation depends on expression of ATM, the product of the gene mutated in the human genetic disorder ataxia telangiectasia (A-T). UV-induced RPA-p34 hyperphosphorylation was not observed in A-T cells, but this response was restored by ATM expression. Furthermore, purified ATM kinase phosphorylates the p34 subunit of RPA complex in vitro at many of the same sites that are phosphorylated in vivo after UV radiation. Induction of this DNA damage response was also dependent on DNA replication; inhibition of DNA replication by aphidicolin prevented induction of RPA-p34 hyperphosphorylation by UV radiation. We postulate that this pathway is triggered by the accumulation of aberrant DNA replication intermediates, resulting from DNA replication fork blockage by UV photoproducts. Further, we suggest that RPA-p34 is hyperphosphorylated as a participant in the recombinational postreplication repair of these replication products. Successful resolution of these replication intermediates reduces the accumulation of chromosomal aberrations that would otherwise occur as a consequence of UV radiation.

Characterization of a chemsensory protein (ASP3c) from honeybee (Apis mellifera L.) as a brood pheromone carrier

Chemosensory proteins (CSPs) are ubiquitous soluble small proteins isolated from sensory organs of a wide range of insect species, which are believed to be involved in chemical communication. We report the cloning of a honeybee CSP gene called ASP3c, as well as the structural and functional characterization of the encoded protein. The protein was heterologously secreted by the yeast Pichia pastoris using the native signal peptide. ASP3c disulfide bonds were assigned after trypsinolysis followed by chromatography and mass spectrometry combined with microsequencing. The pairing (Cys(I)-Cys(II), Cys(III)-Cys(IV)) was found to be identical to that of Schistocerca gregaria CSPs, suggesting that this pattern occurs commonly throughout the insect CSPs. CD measurements revealed that ASP3c mainly consists of alpha-helices, like other insect CSPs. Gel filtration analysis showed that ASP3c is monomeric at neutral pH. Using ASA, a fluorescent fatty acid anthroyloxy analogue as a probe, ASP3c was shown to bind specifically to large fatty acids and ester derivatives, which are brood pheromone components, in the micromolar range. It was unable to bind tested general odorants and other tested pheromones (sexual and nonsexual). This is the first report on a natural pheromonal ligand bound by a recombinant CSP with a measured affinity constant.

The development of chromatography for the characterization of protein interactions: a personal perspective

This article reviews the progress of a personal endeavour to develop chromatography as a quantitative procedure for the determination of reaction stoichiometries and equilibrium constants governing protein interactions. As well as affording insight into an aspect of chromatography with which many protein chemists are unfamiliar, it shows the way in which minor adaptations of conventional chromatographic practices have rendered the technique one of the most powerful methods available for the characterization of interactions. That pathway towards quantification is followed from the introduction of frontal gel filtration for the study of protein self-association to the characterization of ligand binding by the biosensor variant of quantitative affinity chromatography.

The Epstein-Barr virus nuclear antigen-6 protein co-localizes with EBNA-3 and survival of motor neurons protein

The Epstein-Barr virus nuclear antigen (EBNA)-6 protein is essential for Epstein-Barr virus (EBV)-induced immortalization of primary human B-lymphocytes in vitro. In this study, fusion proteins of EBNA-6 with green fluorescent protein (GFP) have been used to characterize its nuclear localization and organization within the nucleus. EBNA-6 associates with nuclear structures and in immunofluorescence demonstrate a punctate staining pattern. Herein, we show that the association of EBNA-6 with these nuclear structures was maintained throughout the cell cycle and with the use of GFP-E6 deletion mutants, that the region amino acids 733-808 of EBNA-6 contains a domain that can influence the association of EBNA-6 with these nuclear structures. Co-immunofluorescence and confocal analyses demonstrated that EBNA-6 and EBNA-3 co-localize in the nucleus of cells. Expression of EBNA-6, but not EBNA-3, caused a redistribution of nuclear survival of motor neurons protein (SMN) to the EBNA-6 containing nuclear structures resulting in co-localization of SMN with EBNA-6. (C) 2003 Elsevier Inc. All rights reserved.

The LFA-1-associated molecule PTA-1 (CD226) on T cells forms a dynamic molecular complex with protein 4.1G and human discs large

Clustering of the T cell integrin, LFA-1, at specialized regions of intercellular contact initiates integrin-mediated adhesion and downstream signaling, events that are necessary for a successful immunological response. But how clustering is achieved and sustained is not known. Here we establish that an LFA-1-associated molecule, PTA-1, is localized to membrane rafts and binds the carboxyl-terminal domain of isoforms of the actin-binding protein 4.1G. Protein 4.1 is known to associate with the membrane-associated guanylate kinase homologue, human discs large. We show that the carboxyl-terminal peptide of PTA-1 also can bind human discs large and that the presence or absence of this peptide greatly influences binding between PTA-1 and different isoforms of 4.1G. T cell stimulation with phorbol ester or PTA-1 cross-linking induces PTA-1 and 4.1G to associate tightly with the cytoskeleton, and the PTA-1 from such activated cells now can bind to the amino-terminal region of 4.1G. We propose that these dynamic associations provide the structural basis for a regulated molecular adhesive complex that serves to cluster and transport LFA-1 and associated molecules.

Molecular analysis of the psa permease complex of Streptococcus pneumoniae

The psaBCA locus of Streptococcus pneumoniae encodes a putative ABC Mn2+-permease complex. Downstream of the operon is psaD, which may be co-transcribed and encodes a thiol peroxidase. Previously, there has been discordance concerning the phenotypic impact of mutations in the psa locus, resolution of which has been complicated by differences in mutant construction and the possibility of polar effects. Here, we constructed unmarked, in frame deletion mutants DeltapsaB, DeltapsaC, DeltapsaA, DeltapsaD, DeltapsaBC, DeltapsaBCA and DeltapsaBCAD in S. pneumoniae D39 to examine the role of each gene within the locus in Mn2+ uptake, susceptibility to oxidative stress, virulence, nasopharyngeal colonization and chain morphology. The requirement for Mn2+ for growth and transformation was also investigated for all mutants. Inductively coupled plasma mass spectrometry (ICP-MS) analysis provided the first direct evidence that PsaBCA is indeed a Mn2+ transporter. However, this study did not substantiate previous reports that the locus plays a role in choline-binding protein pro-duction or chain morphology. We also confirmed the importance of the Psa permease in systemic virulence and resistance to superoxide and hydrogen peroxide, as well as demonstrating a role in nasopharyngeal colonization for the first time. Further evi-dence is provided to support the requirement for Mn2+ supplementation for growth and transformation of DeltapsaB, DeltapsaC, DeltapsaA, DeltapsaBC, DeltapsaBCA and DeltapsaBCAD mutants. However, transformation, as well as growth, of the DeltapsaD mutant was not dependent upon Mn2+ supplementation. We also show that, apart from sensitivity to hydrogen peroxide, the DeltapsaD mutant exhibited essentially similar phenotypes to those of the wild type. Western blot analysis with a PsaD antiserum showed that deleting any of the genes upstream of psaD did not affect its expression. However, we found that deleting psaB resulted in decreased expression of PsaA relative to that in D39, whereas deleting both psaB and psaC resulted in at least wild-type levels of PsaA.

Identification and cloning of the gene encoding BmpC: an outer-membrane lipoprotein associated with Brachyspira pilosicoli membrane vesicles

The intestinal spirochaete Brachyspira pilosicoli causes colitis in a wide variety of host species. Little is known about the structure or protein constituents of the B. pilosicoli outer membrane (OM). To identify surface-exposed proteins in this species, membrane vesicles were isolated from B. pilosicoli strain 95-1000 cells by osmotic lysis in dH(2)O followed by isopycnic centrifugation in sucrose density gradients. The membrane vesicles were separated into a high-density fraction (HDMV; p = 1.18 g CM-3) and a low-density fraction (LDMV; rho=1.12 g cm(-3)). Both fractions were free of flagella and soluble protein contamination. LDMV contained predominantly OM markers (lipo-oligosaccharide and a 29 kDa B. pilosicoli OM protein) and was used as a source of antigens to produce mAbs. Five B. pilosicoli-specific mAbs reacting with proteins with molecular masses of 23, 24, 35, 61 and 79 kDa were characterized. The 23 kDa protein was only partially soluble in Triton X-114, whereas the 24 and 35 kDa proteins were enriched in the detergent phase, implying that they were integral membrane proteins or lipoproteins. All three proteins were localized to the B. pilosicoli OM by immunogold labelling using specific mAbs. The gene encoding the abundant, surface-exposed 23 kDa protein was identified by screening a B. pilosicoli 95-1000 genome library with the mAb and was expressed in Escherichia coli. Sequence analysis showed that it encoded a unique lipoprotein, designated BmpC. Recombinant BmpC partitioned predominantly in the OM fraction of E. coli strain SOLR. The mAb to BmpC was used to screen a collection of 13 genetically heterogeneous strains of B. pilosicoli isolated from five different host species. Interestingly, only strain 95-1000 was reactive with the mAb, indicating that either the surface-exposed epitope on BmpC is variable between strains or that the protein is restricted in its distribution within B. pilosicoli.

Probing the S100 protein family through genomic and functional analysis

The EF-hand superfamily of calcium binding proteins includes the S100, calcium binding protein, and troponin subfamilies. This study represents a genome, structure, and expression analysis of the S100 protein family, in mouse, human, and rat. We confirm the high level of conservation between mammalian sequences but show that four members, including S100A12, are present only in the human genome. We describe three new members of the S100 family in the three species and their locations within the S100 genomic clusters and propose a revised nomenclature and phylogenetic relationship between members of the EF-hand superfamily. Two of the three new genes were induced in bone-marrow-derived macrophages activated with bacterial lipopolysaccharide, suggesting a role in inflammation. Normal human and murine tissue distribution profiles indicate that some members of the family are expressed in a specific manner, whereas others are more ubiquitous. Structure-function analysis of the chemotactic properties of murine S100A8 and human S100A12, particularly within the active hinge domain, suggests that the human protein is the functional homolog of the murine protein. Strong similarities between the promoter regions of human S100A12 and murine S100A8 support this possibility. This study provides insights into the possible processes of evolution of the EF-hand protein superfamily. Evolution of the S100 proteins appears to have occurred in a modular fashion, also seen in other protein families such as the C2H2-type zinc-finger family. (C) 2004 Elsevier Inc. All rights reserved.

Determination of alpha-conotoxin binding modes on neuronal nicotinic acetylcholine receptors

alpha-Conotoxins, from cone snails, and alpha-neurotoxins, from snakes, are competitive inhibitors of nicotinic acetylcholine receptors (nAChRs) that have overlapping binding sites in the ACh binding pocket. These disulphide-rich peptides are used extensively as tools to localize and pharmacologically characterize specific nAChRs subtypes. Recently, a homology model based on the high-resolution structure of an ACh binding protein (AChBP) allowed the three-fingered alpha-neurotoxins to be docked onto the alpha7 nAChR. To investigate if alpha-conotoxins interact with the nAChR in a similar manner, we built homology models of human alpha7 and alpha3beta2 nAChRs, and performed docking simulations of alpha-conotoxins ImI, PnIB, PnIA and MII using the program GOLD. Docking revealed that alpha-conotoxins have a different mode of interaction compared with alpha-neurotoxins, with surprisingly few nAChR residues in common between their overlapping binding sites. These docking experiments show that Imi and PnIB bind to the ACh binding pocket via a small cavity located above the beta9/beta10 hairpin of the (+)alpha7 nAChR subunit. Interestingly, PnIB, PnIA and MII were found to bind in a similar location on alpha7 or alpha3beta2 receptors mostly through hydrophobic interactions, while ImI bound further from the ACh binding pocket, mostly through electrostatic interactions. These findings, which distinguish alpha-conotoxin and alpha-neurotoxin binding modes, have implications for the rational design of selective nAChR antagonists. Copyright (C) 2004 John Wiley Sons, Ltd.

AAA ATPases regulate membrane association of yeast oxysterol binding proteins and sterol metabolism

The yeast genome encodes seven oxysterol binding protein homologs, Osh1p-Osh7p, which have been implicated in regulating intracellular lipid and vesicular transport. Here, we show that both Osh6p and Osh7p interact with Vps4p, a member of the AAA ( ATPases associated with a variety of cellular activities) family. The coiled-coil domain of Osh7p was found to interact with Vps4p in a yeast two-hybrid screen and the interaction between Osh7p and Vps4p appears to be regulated by ergosterol. Deletion of VPS4 induced a dramatic increase in the membrane-associated pools of Osh6p and Osh7p and also caused a decrease in sterol esterification, which was suppressed by overexpression of OSH7. Lastly, overexpression of the coiled-coil domain of Osh7p (Osh7pCC) resulted in a multi-vesicular body sorting defect, suggesting a dominant negative role of Osh7pCC possibly through inhibiting Vps4p function. Our data suggest that a common mechanism may exist for AAA proteins to regulate the membrane association of yeast OSBP proteins and that these two protein families may function together to control subcellular lipid transport.

beta 2 subunit contribution to 4/7 alpha-conotoxin binding to the nicotinic acetylcholine receptor

The structures of acetylcholine-binding protein ( AChBP) and nicotinic acetylcholine receptor ( nAChR) homology models have been used to interpret data from mutagenesis experiments at the nAChR. However, little is known about AChBP-derived structures as predictive tools. Molecular surface analysis of nAChR models has revealed a conserved cleft as the likely binding site for the 4/7 alpha-conotoxins. Here, we used an alpha 3 beta 2 model to identify beta 2 subunit residues in this cleft and investigated their influence on the binding of alpha-conotoxins MII, PnIA, and GID to the alpha 3 beta 2 nAChR by two-electrode voltage clamp analysis. Although a beta 2-L119Q mutation strongly reduced the affinity of all three alpha-conotoxins, beta 2-F117A, beta 2-V109A, and beta 2-V109G mutations selectively enhanced the binding of MII and GID. An increased activity of alpha-conotoxins GID and MII was also observed when the beta 2-F117A mutant was combined with the alpha 4 instead of the alpha 3 subunit. Investigation of A10L-PnIA indicated that high affinity binding to beta 2-F117A, beta 2-V109A, and beta 2-V109G mutants was conferred by amino acids with a long side chain in position 10 (PnIA numbering). Docking simulations of 4/7 alpha-conotoxin binding to the alpha 3 beta 2 model supported a direct interaction between mutated nAChR residues and alpha-conotoxin residues 6, 7, and 10. Taken together, these data provide evidence that the beta subunit contributes to alpha-conotoxin binding and selectivity and demonstrate that a small cleft leading to the agonist binding site is targeted by alpha-conotoxins to block the nAChR.