Profiling the Protein Targets of Unmodified Bio‐Active Molecules with Drug Affinity Responsive Target Stability and Liquid Chromatography/Tandem Mass Spectrometry

Identifying the target proteins of bioactive small molecules is a key step in understanding mode‐of‐action of the drug and addressing the underlying mechanisms responsible for a particular phenotype. Proteomics has been successfully used to elucidate the target protein profiles of unmodified and ligand‐modified bioactive small molecules. In the latter approach, compounds can be modified via click chemistry and combined with activity‐based protein profiling. Target proteins are then enriched by performing a pull‐down with the modified ligand. Methods that utilize unmodified bioactive small molecules include the cellular thermal shift assay, thermal proteome profiling, stability of proteins from rates of oxidation, and the drug affinity responsive target stability (DARTS) determination (or read‐out). This review highlights recent proteomic approaches utilizing data‐dependent analysis and data‐independent analysis to identify target proteins by DARTS. When combined with liquid chromatography/tandem mass spectrometry, DARTS enables the identification of proteins that bind to drug molecules that leads to a conformational change in the target protein(s). In addition, an effective strategy is proposed for selecting the target protein(s) from within the pool of analyzed candidates. With additional complementary methods, the biologically relevant target proteins that bind to the small bio‐active molecules can be further validated.     

Insights into PG‐binding,conformational change,and dimerization of the OmpA C‐terminal domains from Salmonella enterica serovar Typhimurium and Borrelia burgdorferi

Salmonella enterica serovar Typhimurium can induce both humoral and cell‐mediated responses when establishing itself in the host. These responses are primarily stimulated against the lipopolysaccharide and major outer membrane (OM) proteins. OmpA is one of these major OM proteins. It comprises a N‐terminal eight‐stranded β‐barrel transmembrane domain and a C‐terminal domain (OmpA^CTD). The OmpA^CTD and its homologs are believed to bind to peptidoglycan (PG) within the periplasm, maintaining bacterial osmotic homeostasis and modulating the permeability and integrity of the OM. Here we present the first crystal structures of the OmpA^CTD from two pathogens: S. typhimurium (STOmpA^CTD) in open and closed forms and causative agent of Lyme Disease Borrelia burgdorferi (BbOmpA^CTD), in closed form. In the open form of STOmpA^CTD, an aspartate residue from a long β2‐α3 loop points into the binding pocket, suggesting that an anion group such as a carboxylate group from PG is favored at the binding site. In the closed form of STOmpA^CTD and in the structure of BbOmpA^CTD, a sulfate group from the crystallization buffer is tightly bound at the binding site. The differences between the closed and open forms of STOmpA^CTD, suggest a large conformational change that includes an extension of α3 helix by ordering a part of β2‐α3 loop. We propose that the sulfate anion observed in these structures mimics the carboxylate group of PG when bound to STOmpA^CTD suggesting PG‐anchoring mechanism. In addition, the binding of PG or a ligand mimic may enhance dimerization of STOmpA^CTD, or possibly that of full length STOmpA.     

Activation of ras signaling pathway by 8-oxoguanine DNA glycosylase bound to its excision product, 8-oxoguanine

8-Oxo-7,8-dihydroguanine (8-oxoG), arguably the most abundant base lesion induced in mammalian genomes by reactive oxygen species, is repaired via the base excision repair pathway that is initiated with the excision of 8-oxoG by OGG1. Here we show that OGG1 binds the 8-oxoG base with high affinity and that the complex then interacts with canonical Ras family GTPases to catalyze replacement of GDP with GTP, thus serving as a guanine nuclear exchange factor. OGG1-mediated activation of Ras leads to phosphorylation of the mitogen-activated kinases MEK1,2/ERK1,2 and increasing downstream gene expression. These studies document for the first time that in addition to its role in repairing oxidized purines, OGG1 has an independent guanine nuclear exchange factor activity when bound to 8-oxoG.     

Human apolipoprotein E genotypes differentially modify house dust mite-induced airway disease in mice

Apolipoprotein E (apoE) is an endogenous negative regulator of airway hyperreactivity (AHR) and mucous cell metaplasia in experimental models of house dust mite (HDM)-induced airway disease. The gene encoding human apoE is polymorphic, with three common alleles (ε2, ε3, and ε4) reflecting single amino acid substitutions at amino acids 112 and 158. The objective of this study was to assess whether the human apoE alleles modify airway responses to repeated nasal HDM challenges. Mice expressing the human apoE ε2 (huApoE2), ε3 (huApoE3), or ε4 (huApoE4) alleles received nasal HDM challenges, and airway responses were compared with mice expressing the endogenous murine apoE gene (muApoE). huApoE3 mice displayed significant reductions in AHR, mucous cell metaplasia, and airway inflammation compared with muApoE mice. The attenuated severity of airway inflammation in huApoE3 mice was associated with reductions in lung mRNA levels of Th2 and Th17 cytokines, as well as chemokines (CCL7, CCL11, CCL24). huApoE4 mice had an intermediate phenotype, with attenuated AHR and IgE production, compared with muApoE mice, whereas airway inflammation and mucous cell metaplasia were not reduced. In contrast, HDM-induced airway responses were not modified in mice expressing the huApoE2 allele. We conclude that the polymorphic huApoE alleles differentially modulate HDM-induced airway disease, which can be stratified, in rank order of increasing disease severity, ε3 < ε4 < ε2. These results raise the possibility that the polymorphic apoE alleles may modify disease severity in human asthma.     

Unexpected Attraction of Polarotactic Water-Leaving Insects to Matt Black Car Surfaces: Mattness of Paintwork Cannot Eliminate the Polarized Light Pollution of Black Cars

The horizontally polarizing surface parts of shiny black cars (the reflection-polarization characteristics of which are similar to those of water surfaces) attract water-leaving polarotactic insects. Thus, shiny black cars are typical sources of polarized light pollution endangering water-leaving insects. A new fashion fad is to make car-bodies matt black or grey. Since rough (matt) surfaces depolarize the reflected light, one of the ways of reducing polarized light pollution is to make matt the concerned surface. Consequently, matt black/grey cars may not induce polarized light pollution, which would be an advantageous feature for environmental protection. To test this idea, we performed field experiments with horizontal shiny and matt black car-body surfaces laid on the ground. Using imaging polarimetry, in multiple-choice field experiments we investigated the attractiveness of these test surfaces to various water-leaving polarotactic insects and obtained the following results: (i) The attractiveness of black car-bodies to polarotactic insects depends in complex manner on the surface roughness (shiny, matt) and species (mayflies, dolichopodids, tabanids). (ii) Non-expectedly, the matt dark grey car finish is much more attractive to mayflies (being endangered and protected in many countries) than matt black finish. (iii) The polarized light pollution of shiny black cars usually cannot be reduced with the use of matt painting. On the basis of these, our two novel findings are that (a) matt car-paints are highly polarization reflecting, and (b) these matt paints are not suitable to repel polarotactic insects. Hence, the recent technology used to make matt the car-bodies cannot eliminate or even can enhance the attractiveness of black/grey cars to water-leaving insects. Thus, changing shiny black car painting to matt one is a disadvantageous fashion fad concerning the reduction of polarized light pollution of black vehicles.     

Females with PDHA1 gene mutations: a diagnostic challenge

Biochemical analysis was performed in muscle tissue and in fibroblasts of four unrelated females consecutively diagnosed with a 'de novo' point mutation in the PDHA1 gene. Pyruvate dehydrogenase E1 subunit deficiency was confirmed in the muscle sample of all patients, however, in three out of four cases the activity of the pyruvate dehydrogenase complex in fibroblasts showed a normal activity. A skewed inactivation was confirmed of the maternal X chromosome in fibroblasts in all children. Due to the possibility of a skewed X inactivation pattern enzyme measurements in fibroblasts are not always reliable for the diagnosis of a PDHc defect in females. Based on the overlapping features of PDHc deficiency with those of the disorders of the oxidative phosphorylation we suggest performing a fresh muscle biopsy for detailed biochemical analysis in females with a suspected pyruvate dehydrogenase deficiency, followed by molecular genetic analysis of the PDHA1 gene.     

Coronary vasoconstrictor effect of ghrelin is not mediated by growth hormone secretagogue receptor 1a type in dogs

Ghrelin (GHR) is a recently discovered endocrine regulatory peptide of gastrointestinal origin with multiple functions including cardiovascular effects. However, contradictory data are available on the vascular actions of GHR in different organs and species. The aim of this study was to characterize the direct effect of the peptide on the canine coronary bed and to evaluate the role of the growth hormone secretagogue receptor (GHS-R) in the effect of GHR on coronary arterioles. The presence of GHS-R1a and 1b subtypes in canine coronary arterioles was investigated using Western blotting and immunohistochemistry. Responses of coronary arterioles with spontaneous and elevated vascular tone (the latter evoked by the thromboxane mimetic agent U46619, 10⁻⁷-10⁻⁶ mol/l) to GHR (10⁻⁹-3 × 10⁻⁷ nmol/l) were recorded by video-microscopy as changes of vessel diameter. Positive immunostaining for both GHS-R subtypes was found in the wall of intramural arterioles. The microarteriographic study results showed that GHR alone could not elicit any significant effect on vessel diameter of arterioles with spontaneous tone. However, when vascular smooth muscle was preconstricted by the thromboxane mimetic agent U46619, administration of GHR induced further constriction (+31 ± 9% increase in contraction p < 0.01). This was not abolished by the specific blockade of GHS-R1a by d-Lys³-GHRP-6 (5 × 10⁻⁶ mol/l). The results suggest that GHR induces tone-dependent constriction of canine coronary arterioles which is mediated by a receptor other than GHS-R1a.