Interactions between Glutathione S-Transferase P1, Tumor Necrosis Factor, and Traffic-Related Air Pollution for Development of Childhood Allergic Disease

BACKGROUND: Air pollutants may induce airway inflammation and sensitization due to generation of reactive oxygen species. The genetic background to these mechanisms could be important effect modifiers. OBJECTIVE: Our goal was to assess interactions between exposure to air pollution and single nucleotide polymorphisms (SNPs) in the beta2-adrenergic receptor (ADRB2), glutathione S-transferase P1 (GSTP1), and tumor necrosis factor (TNF) genes for development of childhood allergic disease. METHODS: In a birth cohort originally of 4,089 children, we assessed air pollution from local traffic using nitrogen oxides (traffic NO(x)) as an indicator based on emission databases and dispersion modeling and estimated individual exposure through geocoding of home addresses. We measured peak expiratory flow rates and specific IgE for inhalant and food allergens at 4 years of age, and selected children with asthma symptoms up to 4 years of age (n = 542) and controls (n = 542) for genotyping. RESULTS: Interaction effects on allergic sensitization were indicated between several GSTP1 SNPs and traffic NO(x) exposure during the first year of life (p(nominal) < 0.001-0.06). Children with Ile105Val/Val105Val genotypes were at increased risk of sensitization to any allergen when exposed to elevated levels of traffic NO(x) (for a difference between the 5th and 95th percentile of exposure: odds ratio = 2.4; 95% confidence interval, 1.0-5.3). In children with TNF-308 GA/AA genotypes, the GSTP1-NO(x) interaction effect was even more pronounced. We observed no conclusive interaction effects for ADRB2. CONCLUSION: The effect of air pollution from traffic on childhood allergy appears to be modified by GSTP1 and TNF variants, supporting a role of genes controlling the antioxidative system and inflammatory response in allergy

Differential Scanning Fluorometry Signatures as Indicators of Enzyme Inhibitor Mode of Action: Case Study of Glutathione S-Transferase

Differential scanning fluorometry (DSF), also referred to as fluorescence thermal shift, is emerging as a convenient method to evaluate the stabilizing effect of small molecules on proteins of interest. However, its use in the mechanism of action studies has received far less attention. Herein, the ability of DSF to report on inhibitor mode of action was evaluated using glutathione S-transferase (GST) as a model enzyme that utilizes two distinct substrates and is known to be subject to a range of inhibition modes. Detailed investigation of the propensity of small molecule inhibitors to protect GST from thermal denaturation revealed that compounds with different inhibition modes displayed distinct thermal shift signatures when tested in the presence or absence of the enzyme's native co-substrate glutathione (GSH). Glutathione-competitive inhibitors produced dose-dependent thermal shift trendlines that converged at high compound concentrations. Inhibitors acting via the formation of glutathione conjugates induced a very pronounced stabilizing effect toward the protein only when GSH was present. Lastly, compounds known to act as noncompetitive inhibitors exhibited parallel concentration-dependent trends. Similar effects were observed with human GST isozymes A1-1 and M1-1. The results illustrate the potential of DSF as a tool to differentiate diverse classes of inhibitors based on simple analysis of co-substrate dependency of protein stabilization

THE X-RAY CRYSTAL-STRUCTURE OF THE CATALYTIC DOMAIN OF HUMAN NEUTROPHIL COLLAGENASE INHIBITED BY A SUBSTRATE-ANALOG REVEALS THE ESSENTIALS FOR CATALYSIS AND SPECIFICITY

BODE W, REINEMER P, HUBER R, KLEINE T, SCHNIERER S, Tschesche H. THE X-RAY CRYSTAL-STRUCTURE OF THE CATALYTIC DOMAIN OF HUMAN NEUTROPHIL COLLAGENASE INHIBITED BY A SUBSTRATE-ANALOG REVEALS THE ESSENTIALS FOR CATALYSIS AND SPECIFICITY. EMBO JOURNAL. 1994;13(6):1263-1269.Matrix metalloproteinases are a family of zinc endopeptidases involved in tissue remodelling. They have been implicated in various disease processes including tumour invasion and joint destruction. These enzymes consist of several domains, which are responsible for latency, catalysis and substrate recognition. Human neutrophil collagenase (PMNL-CL, MMP-8) represents one of the two 'interstitial' collagenases that cleave triple helical collagens types I, II and III. Its 163 residue catalytic domain (Met80 to Gly242) has been expressed in Escherichia coli and crystallized as a non-covalent complex with the inhibitor Pro-Leu-Gly-hydroxylamine. The 2.0 angstrom crystal structure reveals a spherical molecule with a shallow active-site cleft separating a smaller C-terminal subdomain from a bigger N-terminal domain, composed of a rive-stranded beta-sheet, two alpha-helices, and bridging loops. The inhibitor mimics the unprimed (P1-P3) residues of a substrate; primed (P1'-P3') peptide substrate residues should bind in an extended conformation, with the bulky P1' side-chain fitting into the deep hydrophobic S1' subsite. Modelling experiments with collagen show that the scissile strand of triple-helical collagen must be freed to fit the subsites. The catalytic zinc ion is situated at the bottom of the active-site cleft and is penta-coordinated by three histidines and by both hydroxamic acid oxygens of the inhibitor. In addition to the catalytic zinc, the catalytic domain harbours a second, non-exchangeable zinc ion and two calcium ions, which are packed against the top of the beta-sheet and presumably function to stabilize the catalytic domain. The polypeptide folding and in particular the zinc environment of the collagenase catalytic domain bear a close resemblance to the astacins and the snake venom metalloproteinases

Unexpected active-site flexibility in the structure of human neutrophil elastase in complex with a new dihydropyrimidone inhibitor

Human neutrophil elastase (HNE), a trypsin-type serine protease, is of pivotal importance in the onset and progression of chronic obstructive pulmonary disease (COPD). COPD encompasses a group of slowly progressive respiratory disorders and is a major medical problem and the fifth leading cause of death worldwide. HNE is a major target for the development of compounds that inhibit the progression of long-term lung function decline in COPD patients. Here, we present the three-dimensional structure of a potent dihydropyrimidone inhibitor (DHPI) non-covalently bound to HNE at a resolution of 2.0 Å. The inhibitor binds to the active site in a unique orientation addressing S1 and S2 subsites of the protease. To facilitate further analysis of this binding mode, we determined the structure of the uncomplexed enzyme at a resolution of 1.86 Å. Detailed comparisons of the HNE:DHPI complex with the uncomplexed HNE structure and published structures of other elastase:inhibitor complexes revealed that binding of DHPI leads to large conformational changes in residues located in the S2 subsite. The rearrangement of residues Asp95-Leu99B creates a deep, well-defined cavity, which is filled by the P2 moiety of the inhibitor molecule to almost perfect shape complementarity. The shape of the S2 subsite in complex with DHPI clearly differs from all other observed HNE structures. The observed structural flexibility of the S2 subsite is a key feature for the understanding of the binding mode of DHPIs in general and the development of new HNE selective inhibitors