In moths, pheromone-binding proteins (PBPs) are responsible for the transport of the hydrophobic pheromones to the membrane-bound receptors across the aqueous sensillar lymph. We report here that recombinant Antheraea polyphemus PBP1 (ApolPBP1) picks up hydrophobic molecule(s) endogenous to the Escherichia coli expression host that keeps the protein in the "open" (bound) conformation at high pH but switches to the "closed" (free) conformation at low pH. This finding has bearing on the solution structures of undelipidated lepidopteran moth PBPs determined thus far. Picking up a hydrophobic molecule from the host expression system could be a common feature for lipid-binding proteins. Thus, delipidation is critical for bacterially expressed lipid-binding proteins. We have shown for the first time that the delipidated ApolPBP1 exists primarily in the closed form at all pH levels. Thus, current views on the pH- Chemoreception in insects is mediated via sensing of a variety of small, volatile organic compounds. Chemoreception plays a critical role not only in the regulation of the most fundamental chemosensory behaviors in insects but also for intraspecies communication. Pheromone is a chemical signal that triggers a natural behavioral response in another member within the same species. In lepidopteran insects, sex pheromones produced by females are detected by males of the same species with extreme sensitivity and selectivity. These hydrophobic compounds are transported to the membrane-bound receptors (ion channels) (1, 2) across the aqueous sensillar lymph by the pheromone-binding proteins (PBPs).2 PBPs are small, acidic proteins, highly soluble in water, with a molecular mass of 14 -16 kDa. The first PBP to be identified, cloned, and expressed in the bacterial system was that from the giant silk moth Antheraea polyphemus (3, 4). Since then, PBPs have been isolated from at least eight moth species that share about 50% sequence identity, with six conserved cysteine residues forming three disulfide bridges that are important for the formation of the hydrophobic binding pocket (5).Moth PBPs are known to undergo a dramatic conformational switch with a change in pH, which has been proposed to be necessary for the release of ligand at lower pH near the membrane-bound receptors (ion channels) (6 -11). The current view on insect PBPs is that the unliganded protein exists in two conformations as follows: form A (PBP A ), the acidic or "ligand releasing" conformation that exists at low pH, and form B (PBP B ), the basic or "ligand binding" conformation that exists at high pH (7). The low and high pH conformations have been determined by solution NMR for PBPs from two representative moths, Bombyx mori (BmorPBP) and Antheraea polyphemus (ApolPBP1) (8 -11), which are believed to represent the "free" or "unliganded" form of the two proteins. High pH conformations of both "ligand-bound" and free forms of BmorPBP have also been determined by x-ray crystallography (12, 13).The conformation of BmorPBP B in solution at pH 6.5 (9) cons...
Pheromone-binding proteins (PBPs) in lepidopteran moths selectively transport the hydrophobic pheromone molecules across the sensillar lymph to trigger the neuronal response. Moth PBPs are known to bind ligand at physiological pH and release it at acidic pH while undergoing a conformational change. Two molecular switches are considered to play a role in this mechanism: (i) Protonation of His70 and His95 situated at one end of binding pocket, and (ii) Switch of the unstructured C-terminus at the other end of the binding pocket to a helix that enters the pocket. We have reported previously the role of the histidine-driven switch in ligand release for Antheraea polyphemus PBP1 (ApolPBP1). Here we show that the C-terminus plays a role in ligand release and binding mechanism of ApolPBP1. The C-terminus truncated mutants of ApolPBP1 (ApolPBP1ΔP129-V142 and ApolPBP1H70A/H95AΔP129-V142) exist only in the bound conformation at all pH levels, and they fail to undergo pH- or ligand- dependent conformational switch. Although these proteins could bind ligands even at acidic pH unlike the wild-type ApolPBP1, they had ~4 fold reduced affinity towards the ligand at both acidic and physiological pH than that of ApolPBP1wt and ApolPBP1H70A/H95A. Thus, apart from helping in the ligand-release at acidic pH, the C-terminus in ApolPBP1 also plays an important role in ligand binding and/or locking the ligand in the binding pocket. Our results are in stark contrast to those reported for BmorPBP and AtraPBP, where C-terminus truncated proteins had similar or increased pheromone-binding affinity at any pH.
Itaconate is produced from the mitochondrial TCA cycle enzyme aconitase decarboxylase (encoded by immune responsive gene1; Irg1) that exerts immunomodulatory function in myeloid cells. However, the role of the Irg1/itaconate pathway in dendritic cells (DC)-mediated airway inflammation and adaptive immunity to inhaled allergens, which are the primary antigen-presenting cells in allergic asthma, remains largely unknown. House dust mite (HDM)-challenged Irg1−/− mice displayed increases in eosinophilic airway inflammation, mucous cell metaplasia, and Th2 cytokine production with a mechanism involving impaired mite antigen presentations by DC. Adoptive transfer of HDM-pulsed DC from Irg1-deficient mice into naïve WT mice induced a similar phenotype of elevated type 2 airway inflammation and allergic sensitization. Untargeted metabolite analysis of HDM-pulsed DC revealed itaconate as one of the most abundant polar metabolites that potentially suppress mitochondrial oxidative damage. Furthermore, the immunomodulatory effect of itaconate was translated in vivo, where intranasal administration of 4-octyl itaconate 4-OI following antigen priming attenuated the manifestations of HDM-induced airway disease and Th2 immune response. Taken together, these data demonstrated for the first time a direct regulatory role of the Irg1/itaconate pathway in DC for the development of type 2 airway inflammation and suggest a possible therapeutic target in modulating allergic asthma.
Lepidopteran male moths have an extraordinarily sensitive olfactory system that is capable of detecting and responding to minute amounts of female-secreted pheromones over great distances. Pheromone-binding proteins (PBPs) in male antennae ferry the hydrophobic ligand across the aqueous lymph to the olfactory receptor neuron triggering the response. PBPs bind ligands at physiological pH of the lymph and release them at acidic pH near the receptor while undergoing a conformational change. In Anthereae polyphemus PBP1, ligand binding to the hydrophobic pocket and its release is regulated by two biological gates: His70 and His95 at one end of the pocket and C-terminus tail at the other end. Interestingly, in Asian corn borer Ostrinia furnacalis PBP2 (OfurPBP2), critical residues for ligand binding and release are substituted in both biological gates. The impact of these substitutions on the ligand binding and release mechanism in OfurPBP2 is not known. We report here overexpression of soluble OfurPBP2 and structural characterization at high and low pH by circular dichroism (CD) and NMR. Ligand binding and ab initio model development were carried out with fluorescence and small-angle X-ray scattering (SAXS) respectively. OfurPBP2 in solution at pH 6.5 is homogeneous, well-folded and has a compact globular shape.
A large number of cellular processes are mediated by protein-protein interactions, often specified by particular protein binding modules. PDZ domains are an important class of protein-protein interaction modules that typically bind to the C-terminus of target proteins. These domains act as a scaffold where signaling molecules are linked to a multiprotein complex. Human Glutaminase Interacting Protein (GIP), also known as Tax Interacting Protein, is unique among PDZ domain containing proteins since it is composed almost exclusively of a single PDZ domain rather than one of many domains as part of a larger protein. GIP plays pivotal roles in cellular signaling, protein scaffolding and cancer pathways via its interaction with the C-terminus of a growing list of partner proteins. We have identified novel internal motifs that are recognized by GIP through combinatorial phage library screening. Leu and Asp residues in the consensus sequence were identified to be critical for binding to GIP through site-directed mutagenesis studies. Structure-based models of GIP bound to two different surrogate peptides determined from NMR constraints revealed that the binding pocket is flexible enough to accommodate either the smaller carboxylate (COO−) group of a C-terminal recognition motif or the bulkier aspartate side chain (CH2 COO−) of an internal motif. The non-canonical ILGF loop in GIP moves in for the C-terminal motif but moves out for the internal recognition motifs, allowing binding to different partner proteins. One of the peptides co-localizes with GIP within human glioma cells indicating that GIP might be a potential target for anti-cancer therapeutics.
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