Many physiologic processes are controlled through the activation of G protein-coupled receptors (GPCRs) by regulatory peptides, making peptide GPCRs particularly useful targets for major human ...diseases such as diabetes and cancer. Peptide GPCRs are also being evaluated as next-generation targets for the development of novel antiparasite agents and insecticides in veterinary medicine and agriculture. Resolution of crystal structures for several peptide GPCRs has advanced our understanding of peptide-receptor interactions and fueled interest in correlating peptide heterogeneity with receptor-binding properties. In this review, the knowledge of recently crystalized peptide-GPCR complexes, previously accumulated peptide structure-activity relationship studies, receptor mutagenesis, and sequence alignment are integrated to better understand peptide binding to the transmembrane cavity of class A GPCRs. Using SAR data, we show that peptide class A GPCRs can be divided into groups with distinct hydrophilic residues. These characteristic residues help explain the preference of a receptor to bind the C-terminal free carboxyl group, the C-terminal amidated group, or the N-terminal ammonium group of peptides.
Level of reactive species in blood is an important pathogenic factor in diabetes mellitus leading to dysfunctions of vascular endothelial and smooth muscle cells and coagulation system abnormality. A ...massive release of reactive species (respiratory burst), catalyzed by NADPH oxidase in blood phagocytes, is not well understood in diabetes. The work aimed to study kinetics of response to microbial particles in blood to specify changes in regulatory mechanisms of generation of reactive species in patients with type 2 diabetes. Production of reactive species in blood and isolated granulocytes was measured by luminol-dependent chemiluminescence. Respiratory burst was initiated by serum opsonized zymosan in blood samples and phorbol ester in cell samples. Kinetic parameters were calculated from experimental kinetic curves of chemiluminescence intensity. ROC curve analysis and mathematical modeling were used to reveal the most significant predictors and clarify specific mechanisms of NADPH oxidase activation. It was shown that kinetic parameters of response to opsonized zymosan (lag-time, response rate, amplitude, production of reactive species) were higher in blood of patients than controls. Amplitude and response rate were the most statistically significant predictors for distinguishing patients and controls at high glucose. It indicated NADPH oxidase activation was the target of hyperglycemia. Mathematical modeling showed hyperglycemia increased stability of NADPH oxidase complex, decreased synchronization of its assembling and elevated neutrophil capacity to phagocytosis in patients. Weak or no dependence of response kinetics on ionomycin concentration was shown in patients indicating changed Ca2+-dependent mechanism of NADPH oxidase activation. Hyperglycemia in type 2 diabetes causes disturbances in mechanisms of NADPH oxidase activation associated with both phagocytosis and the state of intracellular signaling systems, including Ca2+-dependent. We suggest that NADPH oxidase in blood granulocytes can be a promising target for clinical intervention improving management of diabetic complications associated with inflammation.
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•Generation of reactive species was more intensive in blood of patients vs controls.•Amplitude and response rate of respiratory burst were significant predictors.•Modeling showed changes in stability and assembly of NADPH oxidase in diabetes.•Granulocyte capacity to phagocytosis enhanced in patients in model.•Ca2+-signaling in generation of reactive species by granulocytes impaired in T2DM.
The excessive amount of reactive species under chronic inflammation, which are accompanied by an increase body temperature, lead to diabetic complications. Phagocyte NADPH oxidase is the key enzyme ...in these processes. The role of high temperature in its regulation in diabetes is not clear. The aim was to investigate the effect of high temperature on NADPH-oxidase-dependent generation of reactive species in diabetic patients. Chemiluminescent method was applied to assess respiratory burst kinetics initiated by opsonized zymosan in blood or phorbol ester in isolated granulocytes. Analyzing ROC curves, the main predictors and changes in stages of activation of NADPH oxidase were determined. Phosphoisoforms of p47phox and p67phox were quantified by immunoblotting. Response to opsonized zymosan was lower in all subjects at 40 °C vs 37 °C, its kinetic parameters (except Tmax) were higher in blood of patients vs controls. Response rate was the main significant predictor to distinguish groups of subjects at 40 °C indicating NADPH oxidase upregulation in diabetes. Ca2+-dependent generation of reactive species by cells increased in both groups at 40 °C vs 37 °C, kinetic parameters were higher in patients. Initial phospho-p47phox level was higher in patient cells vs ones in controls. It was increased by ionomycin, phorbol ester, or 40 °C in control cells and unchanged in patient ones. Phospho-p67phox level was unchangeable in intact cells of healthy donors and patients at both temperatures. Excessive amounts of reactive species in patient cells were the consequence of granulocyte priming due to p47phox phosphorylation. Thus, high temperature decreased phagocytosis- and enhanced Ca2+-dependent generation of reactive species making the differences between controls and patients less pronounced. The effect of temperature on the generation of reactive species in blood granulocytes is associated with activity of NADPH oxidase that can be a prospective therapeutic target for pathologies accompanied by inflammation including type 2 diabetes.
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•High temperature promoted low production of reactive species in blood.•The rate of respiratory burst was the most significant predictor.•Ca2+-dependent generation of reactive species by granulocytes was high at 40 °C.•p-p47phox was low in intact healthy donor cells and high in patient ones at 37 °C.•p-p47phox was high in healthy donor cells and unchangeable in patient ones at 40 °C.
Protein coding genes constitute only approximately 1% of the human genome but harbor 85% of the mutations with large effects on disease-related traits. Therefore, efficient strategies for selectively ...sequencing complete coding regions (i.e., "whole exome") have the potential to contribute to the understanding of rare and common human diseases. Here we report a method for whole-exome sequencing coupling Roche/NimbleGen whole exome arrays to the Illumina DNA sequencing platform. We demonstrate the ability to capture approximately 95% of the targeted coding sequences with high sensitivity and specificity for detection of homozygous and heterozygous variants. We illustrate the utility of this approach by making an unanticipated genetic diagnosis of congenital chloride diarrhea in a patient referred with a suspected diagnosis of Bartter syndrome, a renal salt-wasting disease. The molecular diagnosis was based on the finding of a homozygous missense D652N mutation at a position in SLC26A3 (the known congenital chloride diarrhea locus) that is virtually completely conserved in orthologues and paralogues from invertebrates to humans, and clinical follow-up confirmed the diagnosis. To our knowledge, whole-exome (or genome) sequencing has not previously been used to make a genetic diagnosis. Five additional patients suspected to have Bartter syndrome but who did not have mutations in known genes for this disease had homozygous deleterious mutations in SLC26A3. These results demonstrate the clinical utility of whole-exome sequencing and have implications for disease gene discovery and clinical diagnosis.
Serine protease inhibitors (serpins) regulate proteolytic events within diverse biological processes, including digestion, coagulation, inflammation and immune responses. The presence of serpins in ...Fasciola hepatica excretory-secretory products indicates that the parasite exploits these to regulate proteases encountered during its development within vertebrate hosts. Interrogation of the F. hepatica genome identified a multi-gene serpin family of seven members that has expanded by gene duplication and divergence to create an array of inhibitors with distinct specificities. We investigated the molecular properties and functions of two representatives, FhSrp1 and FhSrp2, highly expressed in the invasive newly excysted juvenile (NEJ). Consistent with marked differences in the reactive centre loop (RCL) that executes inhibitor-protease complexing, the two recombinant F. hepatica serpins displayed distinct inhibitory profiles against an array of mammalian serine proteases. In particular, rFhSrp1 efficiently inhibited kallikrein (K.sub.i = 40 nM) whilst rFhSrp2 was a highly potent inhibitor of chymotrypsin (K.sub.i = 0.07 nM). FhSrp1 and FhSrp2 are both expressed on the NEJ surface, predominantly around the oral and ventral suckers, suggesting that these inhibitors protect the parasites from the harmful proteolytic effects of host proteases, such as chymotrypsin, during invasion. Furthermore, the unusual inhibition of kallikrein suggests that rFhSrp1 modulates host responses such as inflammation and vascular permeability by interfering with the kallikrein-kinin system. A vaccine combination of rFhSrp1 and rFhSrp2 formulated in the adjuvant Montanide ISA 206VG elicited modest but non-significant protection against a challenge infection in a rat model, but did induce some protection against liver pathogenesis when compared to a control group and a group vaccinated with two well-studied vaccine candidates, F. hepatica cathepsin L2 and L3. This work highlights the importance of F. hepatica serpins to regulate host responses that enables parasite survival during infection and, coupled with the vaccine data, encourages future vaccine trials in ruminants.
Allosteric modulators are called promising candidates in G protein-coupled receptor (GPCR) drug development by displaying subtype selectivity and more specific receptor modulation. Among the ...allosteric sites known to date, cavities at the receptor–lipid interface represent an uncharacteristic binding location that raises many questions about the ligand interactions and stability, the binding site structure, and how all of these are affected by lipid molecules. In this work, we analyze interactions in the allosteric sites of the PAR2, C5aR1, and GCGR receptors in three lipid compositions using molecular dynamics simulations. In addition, we performed quantum chemical calculations involving the symmetry-adapted perturbation theory (SAPT) and the natural population analysis to quantify the strength of intermolecular interactions. We show that besides classical hydrogen bonds, weak polar interactions such as O–HC, O–Br, and long-range electrostatics with the backbone amides contribute to the stability of allosteric modulators at the receptor–lipid interface. The allosteric cavities are detectable in various membrane compositions. The availability of polar atoms for interactions in such cavities can be assessed by water molecules from simulations. Although ligand–lipid interactions are weak, lipid tails play a role in ligand binding pose stability and the size of allosteric cavities. We discuss physicochemical aspects of ligand binding at the receptor–lipid interface and suggest a compound library enriched by weak donor groups for ligand search in such sites.
Abstract RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) is one the most abundant enzymes on Earth. Virtually all food webs depend on its activity to supply fixed carbon. In aerobic ...environments, RuBisCO struggles to distinguish efficiently between CO2 and O2. To compensate, organisms have evolved convergent solutions to concentrate CO2 around the active site. The genetic engineering of such inorganic carbon concentrating mechanisms (CCMs) into plants could help facilitate future global food security for humankind. In bacteria, the carboxysome represents one such CCM component, of which two independent forms exist: α and β. Cyanobacteria are important players in the planet’s carbon cycle and the vast majority of the phylum possess a β-carboxysome, including most cyanobacteria used as laboratory models. The exceptions are the exclusively marine Prochlorococcus and Synechococcus that numerically dominate open ocean systems. However, the reason why marine systems favor an α-form is currently unknown. Here, we report the genomes of 58 cyanobacteria, closely related to marine Synechococcus that were isolated from freshwater lakes across the globe. We find all these isolates possess α-carboxysomes accompanied by a form 1A RuBisCO. Moreover, we demonstrate α-cyanobacteria dominate freshwater lakes worldwide. Hence, the paradigm of a separation in carboxysome type across the salinity divide does not hold true, and instead the α-form dominates all aquatic systems. We thus question the relevance of β-cyanobacteria as models for aquatic systems at large and pose a hypothesis for the reason for the success of the α-form in nature.
Free fatty acid receptors 2 and 3 (FFA2 and FFA3) are G protein-coupled receptors for short chain free fatty acids (SCFAs). They respond to the same set of endogenous ligands but with distinct ...rank-order of potency such that acetate (C2) has been described as FFA2-selective, whereas propionate (C3) is non-selective. Although C2 was confirmed to be selective for human FFA2 over FFA3, this ligand was not selective between the mouse orthologs. Moreover, although C3 was indeed not selective between the human orthologs, it displayed clear selectivity for mouse FFA3 over mouse FFA2. This altered selectivity to C2 and C3 resulted from broad differences in SCFAs potency at the mouse orthologs. In studies to define the molecular basis for these observations, marked variation in ligand-independent constitutive activity was identified using a 35SGTPγS assay. The orthologs with higher potency for the SCFAs, human FFA2 and mouse FFA3, displayed high constitutive activity in this assay, whereas the orthologs with lower potency for the agonist ligands, mouse FFA2 and human FFA3, did not. Sequence alignments of the second extracellular loop identified single negatively charged residues in FFA2 and FFA3 not conserved between species and predicted to form ionic lock interactions with arginine residues within the FFA2 or FFA3 agonist binding pocket to regulate constitutive activity and SCFA potency. Reciprocal mutation of these residues between species orthologs resulted in the induction (or repression) of constitutive activity and in most cases also yielded corresponding changes in SCFA potency.
Background: Differences in ligand potency and selectivity are observed between human and mouse FFA2 and FFA3 orthologs.
Results: Potency differences result from differential constitutive activity between species.
Conclusion: An “ionic lock” between extracellular loop 2 and the ligand binding pocket regulates constitutive activity.
Significance: Understanding species differences in FFA2 and FFA3 function is critical to future studies with these receptors.
Premature fusion of the cranial sutures (craniosynostosis), affecting 1 in 2000 newborns, is treated surgically in infancy to prevent adverse neurologic outcomes. To identify mutations contributing ...to common non-syndromic midline (sagittal and metopic) craniosynostosis, we performed exome sequencing of 132 parent-offspring trios and 59 additional probands. Thirteen probands (7%) had damaging de novo or rare transmitted mutations in
, an inhibitor of BMP - induced osteoblast differentiation (p<10
).
mutations nonetheless showed striking incomplete penetrance (<60%). Genotypes of a common variant near
that is strongly associated with midline craniosynostosis explained nearly all the phenotypic variation in these kindreds, with highly significant evidence of genetic interaction between these loci via both association and analysis of linkage. This epistatic interaction of rare and common variants defines the most frequent cause of midline craniosynostosis and has implications for the genetic basis of other diseases.
Malaria is a life-threatening disease spread by mosquitoes. Plasmodium falciparum M1 alanyl aminopeptidase (PfM1-AAP) is a promising target for the treatment of malaria. The recently solved crystal ...structures of PfM1-AAP revealed that the buried active site can be accessed through two channel openings: a short N-terminal channel with the length of 8 Å and a long C-terminal channel with the length of 30 Å. It is unclear, however, how substrates and inhibitors migrate to the active site and a product of cleavage leaves. Here, we study the molecular mechanism of substrate and inhibitor migration to the active site and the product release using steered molecular dynamics simulations. We identified a stepwise passage of substrates and inhibitors in the C-terminal channel of PfM1-AAP, involving (I) ligand recognition at the opening of the channel, (II) ionic translation to the 'water reservoir', (III) ligand reorientation in the 'water reservoir' and (IV) passage in a suitable conformation into the active site. Endorsed by enzymatic analysis of functional recombinant PfM1-AAP and mutagenesis studies, our novel ligand-residue binding network analysis has identified the functional residues controlling ligand migration within the C-terminal channel of PfM1-AAP. Furthermore, from unbinding simulations of the Arg product we propose a charge repulsion as the driving force to expel the product out from the N-terminal channel of PfM1-AAP. Our work paves the way towards the design of a novel class of PfM1-AAP inhibitors based on preventing substrate entry to the active site.
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