Bidirectional, iterative approach to the structural delineation of the functional "Chemoprint" in GPR40 for agonist recognition

GPR40, free fatty acid receptor 1 (FFAR1), is a member of the GPCR superfamily and a possible target for the treatment of type 2 diabetes. In this work, we conducted a bidirectional iterative investigation, including computational modeling and site-directed mutagenesis, aimed at delineating amino acid residues forming the functional "chemoprint" of GPR40 for agonist recognition. The computational and experimental studies revolved around the recognition of the potent synthetic agonist GW9508. Our experimentally supported model suggested that H137(4.56), R183(5.39), N244(6.55), and R258(7.35) are directly involved in interactions with the ligand. We have proposed a polarized NH-pi interaction between H137(4.56) and GW9508 as one of the contributing forces leading to the high potency of GW9508. The modeling approach presented in this work provides a general strategy for the exploration of receptor-ligand interactions in G-protein coupled receptors beginning prior to acquisition of experimental data.

CD4+ T Cells Targeting Dominant and Cryptic Epitopes from Bacillus anthracis Lethal Factor

Anthrax is an endemic infection in many countries, particularly in the developing world. The causative agent, Bacillus anthracis, mediates disease through the secretion of binary exotoxins. Until recently, research into adaptive immunity targeting this bacterial pathogen has largely focused on the humoral response to these toxins. There is, however, growing recognition that cellular immune responses involving IFNγ producing CD4+ T cells also contribute significantly to a protective memory response. An established concept in adaptive immunity to infection is that during infection of host cells, new microbial epitopes may be revealed, leading to immune recognition of so called 'cryptic' or 'subdominant' epitopes. We analyzed the response to both cryptic and immunodominant T cell epitopes derived from the toxin component lethal factor and presented by a range of HLA-DR alleles. Using IFNγ-ELISpot assays we characterized epitopes that elicited a response following immunization with synthetic peptide and the whole protein and tested their capacities to bind purified HLA-DR molecules in vitro. We found that DR1 transgenics demonstrated T cell responses to a greater number of domain III cryptic epitopes than other HLA-DR transgenics, and that this pattern was repeated with the immunodominant epitopes, as a greater proportion of these epitopes induced a T cell response when presented within the context of the whole protein. Immunodominant epitopes LF457-476 and LF467-487 were found to induce a T cell response to the peptide, as well as to the whole native LF protein in DR1 and DR15, but not in DR4 transgenics. The analysis of Domain I revealed the presence of several unique cryptic epitopes all of which showed a strong to moderate relative binding affinity to HLA-DR4 molecules. However, none of the cryptic epitopes from either domain III or I displayed notably high binding affinities across all HLA-DR alleles assayed. These responses were influenced by the specific HLA alleles presenting the peptide, and imply that construction of future epitope string vaccines which are immunogenic across a wide range of HLA alleles could benefit from a combination of both cryptic and immunodominant anthrax epitopes.

Molecularly imprinted polymer based on MWCNTs-QDs as fluorescent biomimetic sensor for specific recognition of target protein

A novel molecularly imprinted optosensing material based on multi-walled carbon nanotube-quantum dots (MWCNT-QDs) has been designed and synthesized for its high selectivity, sensitivity and specificity in the recognition of a target protein bovine serum albumin (BSA). Molecularly imprinted polymer coated MWCNT-QDs using BSA as the template (BMIP-coated MWCNT-QDs) exhibits a fast mass-transfer speed with a response time of 25 min. It is found that the BSA as a target protein can significantly quench the luminescence of BMIP-coated MWCNT-QDs in a concentration-dependent manner that is best described by a Stem-Volmer equation. The K-SV for BSA is much higher than bovine hemoglobin and lysozyme, implying a highly selective recognition of the BMIP-coated MWCNT-QDs to BSA. Under optimal conditions, the relative fluorescence intensity of BMIP-coated MWCNT-QDs decreases linearly with the increasing target protein BSA in the concentration range of 5.0 x 10(-7)-35.0 x 10(-7) M with a detection limit of 80 nM.

Plasmodium falciparum merozoite surface protein 2: epitope mapping and biological activity analysis of specific antibodies

Background: Plasmodium falciparum(P. falciparum) merozoite surfaceprotein 2 (MSP-2) is one of bloodstage proteins that are associated withprotection from malaria. MSP-2 consistsof a highly polymorphic centralrepeat region flanked by a dimorphicregion that defines the two allelicfamilies, 3D7 and FC27; N- and Cterminalregions are conserved domains.Long synthetic peptides (LSP)representing the two allelic familiesof MSP-2 and constant regions arerecognized by sera from donors livingin endemic areas; and specific antibodies(Abs) are associated with protectionand active in antibody dependentcellular inhibition (ADCI) in vitro.However, the fine specificity ofAb response to the two allelic familiesof MSP-2 is unknown. Methods: Peptidesrepresenting dimorphic regionof 3D7 and FC27 families and theirC-terminal (common fragment to thetwo families) termed 3D7-D (88 aa),FC27-D (48 aa) and C (40 aa) respectivelywere synthesized. Overlapping20 mer peptides covering dimorphicand constant regions of two familieswere also synthesized for epitopemapping. Human sera were obtainedfrom donors living in malaria endemicareas. SpecificDand CregionsAbs were purified from single or poolhuman sera. Sera from mice were obtainedafter immunization with thetwo families LSP mixture in three differentadjuvants: alhydrogel (Alum),Glucopyranosyl Lipid Adjuvant-Stableoil-in-water Emulsion (GLA-SE)and Virosome. For ADCI, P. falciparum(strain 3D7) parasite wasmaintained in culture at 0.5% parasitemiaand 4% hematocrit in air tightbox at love oxygen (2%) and 37 ºC.Results: We identified several epitopesfrom the dimorphic and constantregions of both families of MSP-2, inmice and humans (adults and children).In human, most recognizedepitopes were the same in differentendemic regions for each domain ofthe two families of MSP-2. In mice,the differential recognition of epitopewas depending on the strain of mouseand interestingly on the adjuvantused. GLA-SE and alum as adjuvantswere more often associated with therecognition of multiple epitopes thanvirosomes. Epitope-specific Abs recognizednative merozoites of P.falciparum and were active in ADCIto block development of parasite.Conclusion: The delineation of a limitednumber of epitopes could be exploitedto develop MSP-2 vaccinesactive on both allelic families ofMSP-2.

Dictyostelium discoideum as an expression host for the circumsporozoite protein of Plasmodium falciparum.

We have used the cellular slime mold, Dictyostelium discoideum (Dd), to express the Plasmodium falciparum circumsporozoite protein (CS), a potential component of a subunit vaccine against malaria. This was accomplished via an expression vector based on the discoidin I-encoding gene promoter, in which we linked a sequence coding for a Dd leader peptide to the almost complete CS coding region (pEDII-CS). CS production at both the mRNA and protein levels is induced by starving cells in a simple phosphate buffer. Variation in pH or cell density does not seem to influence CS synthesis. CS-producing cells can be grown either on their normal substrate, bacteria, or on a semi-synthetic media, without affecting CS accumulation level. The CS produced in Dd seems similar to the natural parasite protein as judged by its size and epitope recognition by a panel of monoclonal antibodies. We constructed a second expression vector in which the CS is under the control of a Dd ras promoter. CS accumulation can then be induced by external addition of cAMP. Such a tightly regulated promoter may allow expression of proteins potentially toxic to the cell. Thus, Dd could be a useful eukaryotic system to produce recombinant proteins, in particular from human or animal parasites like P. falciparum.

Role of Phosphorylation of the Oxysterol Binding Protein (OSBP) in (PI(4)P) and Sterol-Containing Membrane Recognition and Binding

Studies have demonstrated that the oxysterol binding protein (OSBP) acts as a phosphatidylinositol phosphate (PIP)-sterol exchanger at membrane contact sites (MCS) of the endoplasmic reticulum (ER) and Golgi. OSBP is known to pick up phosphatidylinositol-4-phosphate (PI(4)P) from the ER, transfer it to the trans-Golgi in exchange for a cholesterol molecule that is then transferred from the trans-Golgi to the ER. Upon further examination of this pathway by Ridgway et al. (1), it appeared that phosphorylation of OSBP played a role in the localization of OSBP. The dephosphorylation state of OSBP was linked to Golgi localization and the depletion of cholesterol at the ER. To mimic the phosphorylated state of OSBP, the mutant OSBP-S5E was designed by Ridgway et al. (1). The lipid and sterol recognition by wt-OSBP and its phosphomimic mutant OSBP-S5E were investigated using immobilized lipid bilayers and dual polarization interferometry (DPI). DPI is a technique in which the protein binding affinity to immobilized lipid bilayers is measured and the binding behavior is examined through real time. Lipid bilayers containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and varying concentrations of PI(4)Ps or sterols (cholesterol or 25-hydroxycholesterol) were immobilized on a silicon nitride chip. It was determined that wt-OSBP binds differently to PI(4)P-containing bilayers compared to OSBP-S5E. The binding behavior suggested that wt-OSBP extracts PI(4)P and the change in the binding behavior, in the case of OSBP-S5E, suggested that the phosphorylation of OSBP may prevent the recognition and/or extraction of PI(4)P. In the presence of sterols, the overall binding behavior of OSBP, regardless of phosphorylation state, was fairly similar. The maximum specific bound mass of OSBP to sterols did not differ as the concentration of sterols increased. However, comparing the maximum specific bound mass of OSBP to cholesterol with oxysterol (25-hydroxycholesterol), OSBP displayed nearly a 2-fold increase in bound mass. With the absence of the wt-OSBP-PI(4)P binding behavior, it can be speculated that the sterols were not extracted. In addition, the binding behavior of OSBP was further tested using a fluorescence based binding assay. Using 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (22-NBD cholesterol), wt-OSBP a one site binding dissociation constant Kd, of 15 ± 1.4 nM was determined. OSBP-S5E did not bind to 22-NBD cholesterol and Kd value was not obtained.

Intrinsic disorder prediction from the analysis of multiple protein fold recognition models

Motivation: Intrinsic protein disorder is functionally implicated in numerous biological roles and is, therefore, ubiquitous in proteins from all three kingdoms of life. Determining the disordered regions in proteins presents a challenge for experimental methods and so recently there has been much focus on the development of improved predictive methods. In this article, a novel technique for disorder prediction, called DISOclust, is described, which is based on the analysis of multiple protein fold recognition models. The DISOclust method is rigorously benchmarked against the top.ve methods from the CASP7 experiment. In addition, the optimal consensus of the tested methods is determined and the added value from each method is quantified. Results: The DISOclust method is shown to add the most value to a simple consensus of methods, even in the absence of target sequence homology to known structures. A simple consensus of methods that includes DISOclust can significantly outperform all of the previous individual methods tested.

Benchmarking consensus model quality assessment for protein fold recognition

Background: Selecting the highest quality 3D model of a protein structure from a number of alternatives remains an important challenge in the field of structural bioinformatics. Many Model Quality Assessment Programs (MQAPs) have been developed which adopt various strategies in order to tackle this problem, ranging from the so called "true" MQAPs capable of producing a single energy score based on a single model, to methods which rely on structural comparisons of multiple models or additional information from meta-servers. However, it is clear that no current method can separate the highest accuracy models from the lowest consistently. In this paper, a number of the top performing MQAP methods are benchmarked in the context of the potential value that they add to protein fold recognition. Two novel methods are also described: ModSSEA, which based on the alignment of predicted secondary structure elements and ModFOLD which combines several true MQAP methods using an artificial neural network. Results: The ModSSEA method is found to be an effective model quality assessment program for ranking multiple models from many servers, however further accuracy can be gained by using the consensus approach of ModFOLD. The ModFOLD method is shown to significantly outperform the true MQAPs tested and is competitive with methods which make use of clustering or additional information from multiple servers. Several of the true MQAPs are also shown to add value to most individual fold recognition servers by improving model selection, when applied as a post filter in order to re-rank models. Conclusion: MQAPs should be benchmarked appropriately for the practical context in which they are intended to be used. Clustering based methods are the top performing MQAPs where many models are available from many servers; however, they often do not add value to individual fold recognition servers when limited models are available. Conversely, the true MQAP methods tested can often be used as effective post filters for re-ranking few models from individual fold recognition servers and further improvements can be achieved using a consensus of these methods.

Recognition of sequence-information in synthetic copolymer chains by a conformationally-constrained tweezer molecule

A novel type of tweezer molecule containing electron-rich 2-pyrenyloxy arms has been designed to exploit intramolecular hydrogen bonding in stabilising a preferred conformation for supramolecular complexation to complementary sequences in aromatic copolyimides. This tweezer-conformation is demonstrated by single-crystal X-ray analyses of the tweezer molecule itself and of its complex with an aromatic diimide model-compound. In terms of its ability to bind selectively to polyimide chains, the new tweezer molecule shows very high sensitivity to sequence effects. Thus, even low concentrations of tweezer relative to diimide units (<2.5 mol%) are sufficient to produce dramatic, sequence-related splittings of the pyromellitimide proton NMR resonances. These induced resonance-shifts arise from ring-current shielding of pyromellitimide protons by the pyrenyloxy arms of the tweezer-molecule, and the magnitude of such shielding is a function of the tweezer-binding constant for any particular monomer sequence. Recognition of both short-range and long-range sequences is observed, the latter arising from cumulative ring-current shielding of diimide protons by tweezer molecules binding at multiple adjacent sites on the copolymer chain.

Inhibition of synaptic transmission and G protein modulation by synthetic CaV2.2 Ca2+ channel peptides

Abstract: Modulation of presynaptic voltage-dependent Ca+ channels is a major means of controlling neurotransmitter release. The CaV 2.2 Ca2+ channel subunit contains several inhibitory interaction sites for Gβγ subunits, including the amino terminal (NT) and I–II loop. The NT and I–II loop have also been proposed to undergo a G protein-gated inhibitory interaction, whilst the NT itself has also been proposed to suppress CaV 2 channel activity. Here, we investigate the effects of an amino terminal (CaV 2.2[45–55]) ‘NT peptide’ and a I–II loop alpha interaction domain (CaV 2.2[377–393]) ‘AID peptide’ on synaptic transmission, Ca2+ channel activity and G protein modulation in superior cervical ganglion neurones (SCGNs). Presynaptic injection of NT or AID peptide into SCGN synapses inhibited synaptic transmission and also attenuated noradrenaline-induced G protein modulation. In isolated SCGNs, NT and AID peptides reduced whole-cell Ca2+ current amplitude, modified voltage dependence of Ca2+ channel activation and attenuated noradrenaline-induced G protein modulation. Co-application of NT and AID peptide negated inhibitory actions. Together, these data favour direct peptide interaction with presynaptic Ca2+ channels, with effects on current amplitude and gating representing likely mechanisms responsible for inhibition of synaptic transmission. Mutations to residues reported as determinants of Ca2+ channel function within the NT peptide negated inhibitory effects on synaptic transmission, Ca2+ current amplitude and gating and G protein modulation. A mutation within the proposed QXXER motif for G protein modulation did not abolish inhibitory effects of the AID peptide. This study suggests that the CaV 2.2 amino terminal and I–II loop contribute molecular determinants for Ca2+ channel function; the data favour a direct interaction of peptides with Ca2+ channels to inhibit synaptic transmission and attenuate G protein modulation. Non-technical summary: Nerve cells (neurones) in the body communicate with each other by releasing chemicals (neurotransmitters) which act on proteins called receptors. An important group of receptors (called G protein coupled receptors, GPCRs) regulate the release of neurotransmitters by an action on the ion channels that let calcium into the cell. Here, we show for the first time that small peptides based on specific regions of calcium ion channels involved in GPCR signalling can themselves inhibit nerve cell communication. We show that these peptides act directly on calcium channels to make them more difficult to open and thus reduce calcium influx into native neurones. These peptides also reduce GPCR-mediated signalling. This work is important in increasing our knowledge about modulation of the calcium ion channel protein; such knowledge may help in the development of drugs to prevent signalling in pathways such as those involved in pain perception.

Conformational modulation of sequence recognition in synthetic macromolecules

The different triplet sequences in high molecular weight aromatic copolyimides comprising pyromellitimide units ("I") flanked by either ether-ketone ("K") or ether-sulfone residues ("S") show different binding strengths for pyrene-based tweezer-molecules. Such molecules bind primarily to the diimide unit through complementary π-π-stacking and hydrogen bonding. However, as shown by the magnitudes of 1H NMR complexation shifts and tweezer-polymer binding constants, the triplet "SIS" binds tweezer-molecules more strongly than "KIS" which in turn bind such molecules more strongly than "KIK". Computational models for tweezer-polymer binding, together with single-crystal X-ray analyses of tweezer-complexes with macrocyclic ether-imides, reveal that the variations in binding strength between the different triplet sequences arise from the different conformational preferences of aromatic rings at diarylketone and diarylsulfone linkages. These preferences determine whether or not chain-folding and secondary π−π-stacking occurs between the arms of the tweezermolecule and the 4,4'-biphenylene units which flank the central diimide residue.

What are the baselines for protein fold recognition?

What constitutes a baseline level of success for protein fold recognition methods? As fold recognition benchmarks are often presented without any thought to the results that might be expected from a purely random set of predictions, an analysis of fold recognition baselines is long overdue. Given varying amounts of basic information about a protein—ranging from the length of the sequence to a knowledge of its secondary structure—to what extent can the fold be determined by intelligent guesswork? Can simple methods that make use of secondary structure information assign folds more accurately than purely random methods and could these methods be used to construct viable hierarchical classifications?

Activation of G-protein-coupled receptor 30 is sufficient to enhance spatial recognition memory in ovariectomized rats

In ovariectomized rats, administration of estradiol, or selective estrogen receptor agonists that activate either the alpha or beta isoforms, have been shown to enhance spatial cognition on a variety of learning and memory tasks, including those that capitalize on the preference of rats to seek out novelty. Although the effects of the putative estrogen G-protein-coupled receptor 30 (GPR30) on hippocampus-based tasks have been reported using food-motivated tasks, the effects of activation of GPR30 receptors on tasks that depend on the preference of rats to seek out spatial novelty remain to be determined. Therefore, the aim of the current study was to determine if short-term treatment of ovariectomized rats with G-1, an agonist for GPR30, would mimic the effects on spatial recognition memory observed following short-term estradiol treatment. In Experiment 1, ovariectomized rats treated with a low dose (1mug) of estradiol 48h and 24h prior to the information trial of a Y-maze task exhibited a preference for the arm associated with the novel environment on the retention trial conducted 48h later. In Experiment 2, treatment of ovariectomized rats with G-1 (25mug) 48h and 24h prior to the information trial of a Y-maze task resulted in a greater preference for the arm associated with the novel environment on the retention trial. Collectively, the results indicated that short-term treatment of ovariectomized rats with a GPR30 agonist was sufficient to enhance spatial recognition memory, an effect that also occurred following short-term treatment with a low dose of estradiol.