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1.  Dissecting direct and indirect genetic effects on chronic obstructive pulmonary disease (COPD) susceptibility 
Human genetics  2013;132(4):431-441.
Cigarette smoking is the major environmental risk factor for chronic obstructive pulmonary disease (COPD). Genome-wide association studies have provided compelling associations for three loci with COPD. In this study, we aimed to estimate direct, i.e., independent from smoking, and indirect effects of those loci on COPD development using mediation analysis. We included a total of 3,424 COPD cases and 1,872 unaffected controls with data on two smoking-related phenotypes: lifetime average smoking intensity and cumulative exposure to tobacco smoke (pack years). Our analysis revealed that effects of two linked variants (rs1051730 and rs8034191) in the AGPHD1/CHRNA3 cluster on COPD development are significantly, yet not entirely, mediated by the smoking-related phenotypes. Approximately 30 % of the total effect of variants in the AGPHD1/CHRNA3 cluster on COPD development was mediated by pack years. Simultaneous analysis of modestly (r2 = 0.21) linked markers in CHRNA3 and IREB2 revealed that an even larger (~42 %) proportion of the total effect of the CHRNA3 locus on COPD was mediated by pack years after adjustment for an IREB2 single nucleotide polymorphism. This study confirms the existence of direct effects of the AGPHD1/CHRNA3, IREB2, FAM13A and HHIP loci on COPD development. While the association of the AGPHD1/CHRNA3 locus with COPD is significantly mediated by smoking-related phenotypes, IREB2 appears to affect COPD independently of smoking.
doi:10.1007/s00439-012-1262-3
PMCID: PMC3600068  PMID: 23299987
2.  Acetylation of the Response Regulator RcsB Controls Transcription from a Small RNA Promoter 
Journal of Bacteriology  2013;195(18):4174-4186.
Nε-lysine acetylation was recently discovered on many bacterial proteins that function in diverse cellular processes. Thus, many questions remain unanswered. For example, what mechanisms regulate lysine acetylation? Does acetylation affect physiology? To help answer these questions, we studied the Escherichia coli response regulator and transcription factor RcsB, which is reported to be acetylated in vitro. To characterize RcsB acetylation, we monitored transcription from the rprA promoter, which requires RcsB. The conventional view is that RcsB is activated by phosphorylation through either the Rcs phosphorelay or acetyl phosphate. We affirmed that rprA transcription requires phosphorylated RcsB and showed that acetyl-phosphate (AcP) is a phosphoryl group donor to RcsB. However, a mutant that accumulates AcP (ackA) exhibited a reduction in rprA transcription instead of the predicted increase. rprA transcription also diminished in the cobB mutant, which lacks the only known E. coli protein deacetylase. This suggests the existence of an inhibitory mechanism that involves lysine acetylation, a supposition supported by the observation that RcsB isolated from the ackA or cobB mutant was hyperacetylated. Finally, we used a genetic approach to identify an AckA- and CobB-sensitive lysine (Lys-154) that controls RcsB activity. We propose that acetylation inhibits RcsB activity and that some of this inhibition acts through the acetylation of Lys-154.
doi:10.1128/JB.00383-13
PMCID: PMC3754749  PMID: 23852870
3.  Structural characterization of a hypothetical protein: a potential agent involved in trimethylamine metabolism in Catenulispora acidiphila 
Catenulispora acidiphila is a newly identified lineage of actinomycetes that produces antimicrobial activities and represents a promising source of novel antibiotics and secondary metabolites. Among the discovered protein coding genes, 68 % were assigned a putative function, while the remaining 32 % are genes encoding “hypothetical” proteins. Caci_0382 is one of the “hypothetical” proteins that has very few homologs. Sequence analysis shows that the protein belongs to the NTF2-like protein family. The structure of Caci_0382 demonstrates that it shares the same fold and has a similar active site as limonene-1,2-epoxide hydrolase, which suggests that it may have a related function. Using a fluorescence thermal shift assay, we identified stabilizing compounds that suggest potential natural ligands of Caci_0382. Using this information, we determined the crystal structure in complex with trimethylamine to provide a better understanding of the function of this uncharacterized protein.
Electronic supplementary material
The online version of this article (doi:10.1007/s10969-014-9176-z) contains supplementary material, which is available to authorized users.
doi:10.1007/s10969-014-9176-z
PMCID: PMC3955178  PMID: 24562475
Trimethylamine; NTF2-like superfamily; Caci_0382; X-ray crystal structure; Structural genomics
4.  Discovery of Selective Inhibitors of the Clostridium difficile Dehydroquinate Dehydratase 
PLoS ONE  2014;9(2):e89356.
A vibrant and healthy gut flora is essential for preventing the proliferation of Clostridium difficile, a pathogenic bacterium that causes severe gastrointestinal symptoms. In fact, most C. difficile infections (CDIs) occur after broad-spectrum antibiotic treatment, which, by eradicating the commensal gut bacteria, allows its spores to proliferate. Hence, a C. difficile specific antibiotic that spares the gut flora would be highly beneficial in treating CDI. Towards this goal, we set out to discover small molecule inhibitors of the C. difficile enzyme dehydroquinate dehydratase (DHQD). DHQD is the 3rd of seven enzymes that compose the shikimate pathway, a metabolic pathway absent in humans, and is present in bacteria as two phylogenetically and mechanistically distinct types. Using a high-throughput screen we identified three compounds that inhibited the type I C. difficile DHQD but not the type II DHQD from Bacteroides thetaiotaomicron, a highly represented commensal gut bacterial species. Kinetic analysis revealed that the compounds inhibit the C. difficile enzyme with Ki values ranging from 10 to 20 µM. Unexpectedly, kinetic and biophysical studies demonstrate that inhibitors also exhibit selectivity between type I DHQDs, inhibiting the C. difficile but not the highly homologous Salmonella enterica DHQD. Therefore, the three identified compounds seem to be promising lead compounds for the development of C. difficile specific antibiotics.
doi:10.1371/journal.pone.0089356
PMCID: PMC3931744  PMID: 24586713
5.  Comparison of serum, EDTA plasma and P100 plasma for luminex-based biomarker multiplex assays in patients with chronic obstructive pulmonary disease in the SPIROMICS study 
Background
As a part of the longitudinal Chronic Obstructive Pulmonary Disease (COPD) study, Subpopulations and Intermediate Outcome Measures in COPD study (SPIROMICS), blood samples are being collected from 3200 subjects with the goal of identifying blood biomarkers for sub-phenotyping patients and predicting disease progression. To determine the most reliable sample type for measuring specific blood analytes in the cohort, a pilot study was performed from a subset of 24 subjects comparing serum, Ethylenediaminetetraacetic acid (EDTA) plasma, and EDTA plasma with proteinase inhibitors (P100™).
Methods
105 analytes, chosen for potential relevance to COPD, arranged in 12 multiplex and one simplex platform (Myriad-RBM) were evaluated in duplicate from the three sample types from 24 subjects. The reliability coefficient and the coefficient of variation (CV) were calculated. The performance of each analyte and mean analyte levels were evaluated across sample types.
Results
20% of analytes were not consistently detectable in any sample type. Higher reliability and/or smaller CV were determined for 12 analytes in EDTA plasma compared to serum, and for 11 analytes in serum compared to EDTA plasma. While reliability measures were similar for EDTA plasma and P100 plasma for a majority of analytes, CV was modestly increased in P100 plasma for eight analytes. Each analyte within a multiplex produced independent measurement characteristics, complicating selection of sample type for individual multiplexes.
Conclusions
There were notable detectability and measurability differences between serum and plasma. Multiplexing may not be ideal if large reliability differences exist across analytes measured within the multiplex, especially if values differ based on sample type. For some analytes, the large CV should be considered during experimental design, and the use of duplicate and/or triplicate samples may be necessary. These results should prove useful for studies evaluating selection of samples for evaluation of potential blood biomarkers.
doi:10.1186/1479-5876-12-9
PMCID: PMC3928911  PMID: 24397870
Chronic obstructive pulmonary disease; COPD; SPIROMICS; Biomarkers; Blood analytes; Multiplex assays; P100 plasma; Serum; EDTA plasma; Pilot study
6.  Crystal structure of a type II dehydroquinate dehydratase-like protein from Bifidobacterium longum 
Journal of structural and functional genomics  2013;14(1):10.1007/s10969-013-9149-7.
Dehydroquinate dehydratase (DHQD) catalyzes the third step in the biosynthetic shikimate pathway. Here we identify a Bifidobacterium longum protein with high sequence homology to type II DHQDs but no detectable DHQD activity under standard assay conditions. A crystal structure reveals that the B. longum protein adopts a DHQD-like tertiary structure but a distinct quaternary state. Apparently forming a dimer, the B. longum protein lacks the active site aspartic acid contributed from a neighboring protomer in the type II DHQD dodecamer. Relating to the absence of protein–protein interactions established in the type II DHQD dodecameric assembly, substantial conformational changes distinguish the would-be active site of the B. longum protein. As B. longum possess no other genes with homology to known DHQDs, these findings imply a unique DHQD activity within B. longum.
doi:10.1007/s10969-013-9149-7
PMCID: PMC3878977  PMID: 23539270
Post-translational activation; Quaternary structure; Shikimate pathway; X-ray crystal structure; Structural genomics
7.  Correction: A Fluorescence-Based Thermal Shift Assay Identifies Inhibitors of Mitogen Activated Protein Kinase Kinase 4 
PLoS ONE  2013;8(12):10.1371/annotation/5edeb1de-b76c-4fd9-a3f6-0f1cf45e3905.
doi:10.1371/annotation/5edeb1de-b76c-4fd9-a3f6-0f1cf45e3905
PMCID: PMC3875687
8.  Systemic Steroid Exposure Is Associated with Differential Methylation in Chronic Obstructive Pulmonary Disease 
Rationale: Systemic glucocorticoids are used therapeutically to treat a variety of medical conditions. Epigenetic processes such as DNA methylation may reflect exposure to glucocorticoids and may be involved in mediating the responses and side effects associated with these medications.
Objectives: To test the hypothesis that differences in DNA methylation are associated with current systemic steroid use.
Methods: We obtained DNA methylation data at 27,578 CpG sites in 14,475 genes throughout the genome in two large, independent cohorts: the International COPD Genetics Network (ndiscovery = 1,085) and the Boston Early Onset COPD study (nreplication = 369). Sites were tested for association with current systemic steroid use using generalized linear mixed models.
Measurements and Main Results: A total of 511 sites demonstrated significant differential methylation by systemic corticosteroid use in all three of our primary models. Pyrosequencing validation confirmed robust differential methylation at CpG sites annotated to genes such as SLC22A18, LRP3, HIPK3, SCNN1A, FXYD1, IRF7, AZU1, SIT1, GPR97, ABHD16B, and RABGEF1. Functional annotation clustering demonstrated significant enrichment in intrinsic membrane components, hemostasis and coagulation, cellular ion homeostasis, leukocyte and lymphocyte activation and chemotaxis, protein transport, and responses to nutrients.
Conclusions: Our analyses suggest that systemic steroid use is associated with site-specific differential methylation throughout the genome. Differentially methylated CpG sites were found in biologically plausible and previously unsuspected pathways; these genes and pathways may be relevant in the development of novel targeted therapies.
doi:10.1164/rccm.201207-1280OC
PMCID: PMC3622442  PMID: 23065012
DNA methylation; glucocorticoids; chronic obstructive pulmonary disease
9.  A Fluorescence-Based Thermal Shift Assay Identifies Inhibitors of Mitogen Activated Protein Kinase Kinase 4 
PLoS ONE  2013;8(12):e81504.
Prostate cancer (PCa) is the second highest cause of cancer death in United States males. If the metastatic movement of PCa cells could be inhibited, then mortality from PCa could be greatly reduced. Mitogen-activated protein kinase kinase 4 (MAP2K4) has previously been shown to activate pro-invasion signaling pathways in human PCa. Recognizing that MAP2K4 represents a novel and validated therapeutic target, we sought to develop and characterize an efficient process for the identification of small molecules that target MAP2K4. Using a fluorescence-based thermal shift assay (FTS) assay, we first evaluated an 80 compound library of known kinase inhibitors, thereby identifying 8 hits that thermally stabilized MAP2K4 in a concentration dependent manner. We then developed an in vitro MAP2K4 kinase assay employing the biologically relevant downstream substrates, JNK1 and p38 MAPK, to evaluate kinase inhibitory function. In this manner, we validated the performance of our initial FTS screen. We next applied this approach to a 2000 compound chemically diverse library, identified 7 hits, and confirmed them in the in vitro kinase assay. Finally, by coupling our structure-activity relationship data to MAP2K4's crystal structure, we constructed a model for ligand binding. It predicts binding of our identified inhibitory compounds to the ATP binding pocket. Herein we report the creation of a robust inhibitor-screening platform with the ability to inform the discovery and design of new and potent MAP2K4 inhibitors.
doi:10.1371/journal.pone.0081504
PMCID: PMC3855329  PMID: 24339940
10.  The crystal structures of the α-subunit of the α2β2 tetrameric Glycyl-tRNA synthetase 
Aminoacyl-tRNA synthetases (AARSs) are ligases (EC.6.1.1.-) that catalyze the acylation of amino acids to their cognate tRNAs in the process of translating genetic information from mRNA to protein. Their amino acid and tRNA specificity are crucial for correctly translating the genetic code. Glycine is the smallest amino acid and the glycyl-tRNA synthetase (GlyRS) belongs to Class II AARSs. The enzyme is unusual because it can assume different quaternary structures. In eukaryotes, archaebac-teria and some bacteria, it forms an α2 homodimer. In some bacteria, GlyRS is an α2β2 heterotetramer and shows a distant similarity to α2 GlyRSs. The human pathogen eubacterium Campylobacter jejuni GlyRS (CjGlyRS) is an α2β2 heterotetramer and is similar to Escherichia coli GlyRS; both are members of Class IIc AARSs. The two-step aminoacylation reaction of tetrameric GlyRSs requires the involvement of both α- and β-subunits. At present, the structure of the GlyRS α2β2 class and the details of the enzymatic mechanism of this enzyme remain unknown. Here we report the crystal structures of the catalytic α-subunit of CjGlyRS and its complexes with ATP, and ATP and glycine. These structures provide detailed information on substrate binding and show evidence for a proposed mechanism for amino acid activation and the formation of the glycyl-adenylate intermediate for Class II AARSs.
doi:10.1007/s10969-012-9142-6
PMCID: PMC3691008  PMID: 23054484
Gly-tRNA synthetase; Catalytic subunit; ATP binding; Glycine binding
11.  Bacillus anthracis Inosine 5′-Monophosphate Dehydrogenase in Action: The First Bacterial Series of Structures of Phosphate Ion-, Substrate-, and Product-Bound Complexes 
Biochemistry  2012;51(31):10.1021/bi300511w.
Inosine 5′-monophosphate dehydrogenase (IMPDH) catalyzes the first unique step of the GMP branch of the purine nucleotide biosynthetic pathway. This enzyme is found in organisms of all three kingdoms. IMPDH inhibitors have broad clinical applications in cancer treatment, as antiviral drugs and as immunosuppressants, and have also displayed antibiotic activity. We have determined three crystal structures of Bacillus anthracis IMPDH, in a phosphate ion-bound (termed “apo”) form and in complex with its substrate, inosine 5′-monophosphate (IMP), and product, xanthosine 5′-monophosphate (XMP). This is the first example of a bacterial IMPDH in more than one state from the same organism. Furthermore, for the first time for a prokaryotic enzyme, the entire active site flap, containing the conserved Arg-Tyr dyad, is clearly visible in the structure of the apoenzyme. Kinetic parameters for the enzymatic reaction were also determined, and the inhibitory effect of XMP and mycophenolic acid (MPA) has been studied. In addition, the inhibitory potential of two known Cryptosporidium parvum IMPDH inhibitors was examined for the B. anthracis enzyme and compared with those of three bacterial IMPDHs from Campylobacter jejuni, Clostridium perfringens, and Vibrio cholerae. The structures contribute to the characterization of the active site and design of inhibitors that specifically target B. anthracis and other microbial IMPDH enzymes.
doi:10.1021/bi300511w
PMCID: PMC3836674  PMID: 22788966
12.  LigSearch: a knowledge-based web server to identify likely ligands for a protein target 
LigSearch is a web server for identifying ligands likely to bind to a given protein. It can be accessed at http://www.ebi.ac.uk/thornton-srv/databases/LigSearch.
Identifying which ligands might bind to a protein before crystallization trials could provide a significant saving in time and resources. LigSearch, a web server aimed at predicting ligands that might bind to and stabilize a given protein, has been developed. Using a protein sequence and/or structure, the system searches against a variety of databases, combining available knowledge, and provides a clustered and ranked output of possible ligands. LigSearch can be accessed at http://www.ebi.ac.uk/thornton-srv/databases/LigSearch.
doi:10.1107/S0907444913022294
PMCID: PMC3852652  PMID: 24311580
LigSearch; ligand prediction
13.  Nitric oxide synthase polymorphisms, gene expression and lung function in chronic obstructive pulmonary disease 
Background
Due to the pleiotropic effects of nitric oxide (NO) within the lungs, it is likely that NO is a significant factor in the pathogenesis of chronic obstructive pulmonary disease (COPD). The aim of this study was to test for association between single nucleotide polymorphisms (SNPs) in three NO synthase (NOS) genes and lung function, as well as to examine gene expression and protein levels in relation to the genetic variation.
Methods
One SNP in each NOS gene (neuronal NOS (NOS1), inducible NOS (NOS2), and endothelial NOS (NOS3)) was genotyped in the Lung Health Study (LHS) and correlated with lung function. One SNP (rs1800779) was also analyzed for association with COPD and lung function in four COPD case–control populations. Lung tissue expression of NOS3 mRNA and protein was tested in individuals of known genotype for rs1800779. Immunohistochemistry of lung tissue was used to localize NOS3 expression.
Results
For the NOS3 rs1800779 SNP, the baseline forced expiratory volume in one second in the LHS was significantly higher in the combined AG + GG genotypic groups compared with the AA genotypic group. Gene expression and protein levels in lung tissue were significantly lower in subjects with the AG + GG genotypes than in AA subjects. NOS3 protein was expressed in the airway epithelium and subjects with the AA genotype demonstrated higher NOS3 expression compared with AG and GG individuals. However, we were not able to replicate the associations with COPD or lung function in the other COPD study groups.
Conclusions
Variants in the NOS genes were not associated with lung function or COPD status. However, the G allele of rs1800779 resulted in a decrease of NOS3 gene expression and protein levels and this has implications for the numerous disease states that have been associated with this polymorphism.
doi:10.1186/1471-2466-13-64
PMCID: PMC3827989  PMID: 24192154
Chronic obstructive pulmonary disease; Nitric oxide synthase; Polymorphism; Gene expression
14.  Characterization of Fibrinogen Binding by Glycoproteins Srr1 and Srr2 of Streptococcus agalactiae* 
The Journal of Biological Chemistry  2013;288(50):35982-35996.
Background: The serine-rich repeat glycoproteins Srr1 and Srr2 are surface adhesins of Streptococcus agalactiae important for pathogenicity.
Results: Both Srrs bind tandem repeats of the fibrinogen Aα chain, but Srr2 has greater affinity explained by structure-function analysis of the Srrs.
Conclusion: A dock, lock, and latch mechanism describes the Srr-fibrinogen interaction.
Significance: The higher affinity of Srr2 may contribute to the hypervirulence of Srr2-expressing strains.
The serine-rich repeat glycoproteins of Gram-positive bacteria comprise a large family of cell wall proteins. Streptococcus agalactiae (group B streptococcus, GBS) expresses either Srr1 or Srr2 on its surface, depending on the strain. Srr1 has recently been shown to bind fibrinogen, and this interaction contributes to the pathogenesis of GBS meningitis. Although strains expressing Srr2 appear to be hypervirulent, no ligand for this adhesin has been described. We now demonstrate that Srr2 also binds human fibrinogen and that this interaction promotes GBS attachment to endothelial cells. Recombinant Srr1 and Srr2 bound fibrinogen in vitro, with affinities of KD = 2.1 × 10−5 and 3.7 × 10−6 m, respectively, as measured by surface plasmon resonance spectroscopy. The binding site for Srr1 and Srr2 was localized to tandem repeats 6–8 of the fibrinogen Aα chain. The structures of both the Srr1 and Srr2 binding regions were determined and, in combination with mutagenesis studies, suggest that both Srr1 and Srr2 interact with a segment of these repeats via a “dock, lock, and latch” mechanism. Moreover, properties of the latch region may account for the increased affinity between Srr2 and fibrinogen. Together, these studies identify how greater affinity of Srr2 for fibrinogen may contribute to the increased virulence associated with Srr2-expressing strains.
doi:10.1074/jbc.M113.513358
PMCID: PMC3861647  PMID: 24165132
Bacterial Adhesion; Bacterial Pathogenesis; Fibrinogen; Protein Crystallization; Streptococcus; Streptococcus agalactiae; Prot
15.  Structural and functional characterization of microcin C resistance peptidase MccF from Bacillus anthracis 
Journal of molecular biology  2012;420(0):366-383.
Microcin C (McC) is heptapeptide-adenylate antibiotic produced by Escherichia coli strains carrying the mccABCDEF gene cluster encoding enzymes, in addition to the heptapeptide structural gene mccA, necessary for McC biosynthesis and self-immunity of the producing cell. The heptapeptide facilitates McC transport into susceptible cells, where it is processed releasing a non-hydrolyzable aminoacyl adenylate that inhibits an essential aminoacyl-tRNA synthetase. The self-immunity gene mccF encodes a specialized serine-peptidase that cleaves an amide bond connecting the peptidyl or aminoacyl moieties of, respectively, intact and processed McC with the nucleotidyl moiety. Most mccF orthologs from organisms other than E. coli are not linked to the McC biosynthesis gene cluster. Here, we show that a protein product of one such gene, MccF from Bacillus anthracis (BaMccF), is able to cleave intact and processed McC and we present a series of structures of this protein. Structural analysis of apo-BaMccF and its AMP-complex reveal specific features of MccF-like peptidases that allow them to interact with substrates containing nucleotidyl moieties. Sequence analyses and phylogenetic reconstructions suggest that several distinct subfamilies form the MccF clade of the large S66 family of bacterial serine peptidases. We show that various representatives of the MccF clade can specifically detoxify non-hydrolyzable aminoacyl adenylates differing in their aminoacyl moieties. We hypothesize that bacterial mccF genes serve as a source of bacterial antibiotic resistance.
doi:10.1016/j.jmb.2012.04.011
PMCID: PMC3690760  PMID: 22516613
MccF; serine peptidase; nucleophilic elbow; catalytic triad (Ser-His-Glu); substrate binding loop
16.  Cigarette smoking behaviors and time since quitting are associated with differential DNA methylation across the human genome 
Human Molecular Genetics  2012;21(13):3073-3082.
The impact of cigarette smoking can persist for extended periods following smoking cessation and may involve epigenetic reprogramming. Changes in DNA methylation associated with smoking may help to identify molecular pathways that contribute to the latency between exposure and disease onset. Cross-sectional cohort data from subjects in the International COPD Genetics Network (n = 1085) and the Boston Early-Onset COPD study (n = 369) were analyzed as the discovery and replication cohorts, respectively. Genome-wide methylation data on 27 578 CpG sites in 14 475 genes were obtained on DNA from peripheral blood leukocytes using the Illumina HumanMethylation27K Beadchip in both cohorts. We identified 15 sites significantly associated with current smoking, 2 sites associated with cumulative smoke exposure, and, within the subset of former smokers, 3 sites associated with time since quitting cigarettes. Two loci, factor II receptor-like 3 (F2RL3) and G-protein-coupled receptor 15 (GPR15), were significantly associated in all three analyses and were validated by pyrosequencing. These findings (i) identify a novel locus (GPR15) associated with cigarette smoking and (ii) suggest the existence of dynamic, site-specific methylation changes in response to smoking which may contribute to the extended risks associated with cigarette smoking that persist after cessation.
doi:10.1093/hmg/dds135
PMCID: PMC3373248  PMID: 22492999
17.  Development of Novel In Vivo Chemical Probes to Address CNS Protein Kinase Involvement in Synaptic Dysfunction 
PLoS ONE  2013;8(6):e66226.
Serine-threonine protein kinases are critical to CNS function, yet there is a dearth of highly selective, CNS-active kinase inhibitors for in vivo investigations. Further, prevailing assumptions raise concerns about whether single kinase inhibitors can show in vivo efficacy for CNS pathologies, and debates over viable approaches to the development of safe and efficacious kinase inhibitors are unsettled. It is critical, therefore, that these scientific challenges be addressed in order to test hypotheses about protein kinases in neuropathology progression and the potential for in vivo modulation of their catalytic activity. Identification of molecular targets whose in vivo modulation can attenuate synaptic dysfunction would provide a foundation for future disease-modifying therapeutic development as well as insight into cellular mechanisms. Clinical and preclinical studies suggest a critical link between synaptic dysfunction in neurodegenerative disorders and the activation of p38αMAPK mediated signaling cascades. Activation in both neurons and glia also offers the unusual potential to generate enhanced responses through targeting a single kinase in two distinct cell types involved in pathology progression. However, target validation has been limited by lack of highly selective inhibitors amenable to in vivo use in the CNS. Therefore, we employed high-resolution co-crystallography and pharmacoinformatics to design and develop a novel synthetic, active site targeted, CNS-active, p38αMAPK inhibitor (MW108). Selectivity was demonstrated by large-scale kinome screens, functional GPCR agonist and antagonist analyses of off-target potential, and evaluation of cellular target engagement. In vitro and in vivo assays demonstrated that MW108 ameliorates beta-amyloid induced synaptic and cognitive dysfunction. A serendipitous discovery during co-crystallographic analyses revised prevailing models about active site targeting of inhibitors, providing insights that will facilitate future kinase inhibitor design. Overall, our studies deliver highly selective in vivo probes appropriate for CNS investigations and demonstrate that modulation of p38αMAPK activity can attenuate synaptic dysfunction.
doi:10.1371/journal.pone.0066226
PMCID: PMC3694096  PMID: 23840427
18.  Variable DNA Methylation Is Associated with Chronic Obstructive Pulmonary Disease and Lung Function 
Rationale: Chronic obstructive pulmonary disease (COPD) is associated with local (lung) and systemic (blood) inflammation and manifestations. DNA methylation is an important regulator of gene transcription, and global and specific gene methylation marks may vary with cigarette smoke exposure.
Objectives: To perform a comprehensive assessment of methylation marks in DNA from subjects well phenotyped for nonneoplastic lung disease.
Methods: We conducted array-based methylation screens, using a test-replication approach, in two family-based cohorts (n = 1,085 and 369 subjects).
Measurements and Main Results: We observed 349 CpG sites significantly associated with the presence and severity of COPD in both cohorts. Seventy percent of the associated CpG sites were outside of CpG islands, with the majority of CpG sites relatively hypomethylated. Gene ontology analysis based on these 349 CpGs (330 genes) suggested the involvement of a number of genes responsible for immune and inflammatory system pathways, responses to stress and external stimuli, as well as wound healing and coagulation cascades. Interestingly, our observations include significant, replicable associations between SERPINA1 hypomethylation and COPD and lower average lung function phenotypes (combined P values: COPD, 1.5 × 10−23; FEV1/FVC, 1.5 × 10−35; FEV1, 2.2 × 10−40).
Conclusions: Genetic and epigenetic pathways may both contribute to COPD. Many of the top associations between COPD and DNA methylation occur in biologically plausible pathways. This large-scale analysis suggests that DNA methylation may be a biomarker of COPD and may highlight new pathways of COPD pathogenesis.
doi:10.1164/rccm.201108-1382OC
PMCID: PMC3297093  PMID: 22161163
chronic obstructive pulmonary disease; epigenetics; DNA methylation; smoking
19.  A genome-wide association study of COPD identifies a susceptibility locus on chromosome 19q13 
Human Molecular Genetics  2011;21(4):947-957.
The genetic risk factors for chronic obstructive pulmonary disease (COPD) are still largely unknown. To date, genome-wide association studies (GWASs) of limited size have identified several novel risk loci for COPD at CHRNA3/CHRNA5/IREB2, HHIP and FAM13A; additional loci may be identified through larger studies. We performed a GWAS using a total of 3499 cases and 1922 control subjects from four cohorts: the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE); the Normative Aging Study (NAS) and National Emphysema Treatment Trial (NETT); Bergen, Norway (GenKOLS); and the COPDGene study. Genotyping was performed on Illumina platforms with additional markers imputed using 1000 Genomes data; results were summarized using fixed-effect meta-analysis. We identified a new genome-wide significant locus on chromosome 19q13 (rs7937, OR = 0.74, P = 2.9 × 10−9). Genotyping this single nucleotide polymorphism (SNP) and another nearby SNP in linkage disequilibrium (rs2604894) in 2859 subjects from the family-based International COPD Genetics Network study (ICGN) demonstrated supportive evidence for association for COPD (P = 0.28 and 0.11 for rs7937 and rs2604894), pre-bronchodilator FEV1 (P = 0.08 and 0.04) and severe (GOLD 3&4) COPD (P = 0.09 and 0.017). This region includes RAB4B, EGLN2, MIA and CYP2A6, and has previously been identified in association with cigarette smoking behavior.
doi:10.1093/hmg/ddr524
PMCID: PMC3298111  PMID: 22080838
20.  The Association of Genome-Wide Significant Spirometric Loci with Chronic Obstructive Pulmonary Disease Susceptibility 
Two recent metaanalyses of genome-wide association studies conducted by the CHARGE and SpiroMeta consortia identified novel loci yielding evidence of association at or near genome-wide significance (GWS) with FEV1 and FEV1/FVC. We hypothesized that a subset of these markers would also be associated with chronic obstructive pulmonary disease (COPD) susceptibility. Thirty-two single-nucleotide polymorphisms (SNPs) in or near 17 genes in 11 previously identified GWS spirometric genomic regions were tested for association with COPD status in four COPD case-control study samples (NETT/NAS, the Norway case-control study, ECLIPSE, and the first 1,000 subjects in COPDGene; total sample size, 3,456 cases and 1,906 controls). In addition to testing the 32 spirometric GWS SNPs, we tested a dense panel of imputed HapMap2 SNP markers from the 17 genes located near the 32 GWS SNPs and in a set of 21 well studied COPD candidate genes. Of the previously identified GWS spirometric genomic regions, three loci harbored SNPs associated with COPD susceptibility at a 5% false discovery rate: the 4q24 locus including FLJ20184/INTS12/GSTCD/NPNT, the 6p21 locus including AGER and PPT2, and the 5q33 locus including ADAM19. In conclusion, markers previously associated at or near GWS with spirometric measures were tested for association with COPD status in data from four COPD case-control studies, and three loci showed evidence of association with COPD susceptibility at a 5% false discovery rate.
doi:10.1165/rcmb.2011-0055OC
PMCID: PMC3262664  PMID: 21659657
21.  Identification of FGF7 as a novel susceptibility locus for chronic obstructive pulmonary disease 
Thorax  2011;66(12):1085-1090.
Rationale
Traditional genome-wide association studies (GWAS) of large cohort of subjects with chronic obstructive pulmonary disease (COPD) have successfully identified novel candidate genes, but several other plausible loci do not meet strict criteria for genome-wide significance after correction for multiple testing.
Objectives
We hypothesize that by applying unbiased weights derived from unique populations we can identify additional COPD susceptibility loci.
Methods
We performed a homozygosity haplotype analysis on a group of subjects with and without COPD to identify regions of conserved homozygosity (RCHH). Weights were constructed based on the frequency of these RCHH in case vs. controls, and used to adjust the P values from a large collaborative GWAS of COPD.
Results
We identified 2,318 regions of conserved homozygosity, of which 576 were significantly (P < .05) overrepresented in cases. After applying the weights constructed from these regions to a collaborative GWAS of COPD, we identified two single nucleotide polymorphisms in a novel gene (FGF7) that gained genome-wide significance by the false discovery rate method. In a follow-up analysis, both SNPs (rs12591300 and rs4480740) were significantly associated with COPD in an independent population (combined P values of 7.9E-07 and 2.8E-06 respectively). In another independent population, increased lung tissue FGF7 expression was associated with worse measures of lung function.
Conclusion
Weights constructed from a homozygosity haplotype analysis of an isolated population successfully identify novel genetic associations from a GWAS on a separate population. This method can be used to identify promising candidate genes that fail to meet strict correction for multiple testing.
doi:10.1136/thoraxjnl-2011-200017
PMCID: PMC3348619  PMID: 21921092
22.  Structure of the Type III Secretion Effector Protein ExoU in Complex with Its Chaperone SpcU 
PLoS ONE  2012;7(11):e49388.
Disease causing bacteria often manipulate host cells in a way that facilitates the infectious process. Many pathogenic gram-negative bacteria accomplish this by using type III secretion systems. In these complex secretion pathways, bacterial chaperones direct effector proteins to a needle-like secretion apparatus, which then delivers the effector protein into the host cell cytosol. The effector protein ExoU and its chaperone SpcU are components of the Pseudomonas aeruginosa type III secretion system. Secretion of ExoU has been associated with more severe infections in both humans and animal models. Here we describe the 1.92 Å X-ray structure of the ExoU–SpcU complex, a full-length type III effector in complex with its full-length cognate chaperone. Our crystallographic data allow a better understanding of the mechanism by which ExoU kills host cells and provides a foundation for future studies aimed at designing inhibitors of this potent toxin.
doi:10.1371/journal.pone.0049388
PMCID: PMC3498133  PMID: 23166655
23.  Genome-wide association study of smoking behaviors in COPD patients 
Thorax  2011;66(10):894-902.
Background
Cigarette smoking is a major risk factor for COPD and COPD severity. Previous genome-wide association studies (GWAS) have identified numerous single nucleotide polymorphisms (SNPs) associated with the number of cigarettes smoked per day (CPD) and a Dopamine Beta-Hydroxylase (DBH) locus associated with smoking cessation in multiple populations.
Objective
To identify SNPs associated with lifetime average and current CPD, age at smoking initiation, and smoking cessation in COPD subjects.
Methods
GWAS were conducted in 4 independent cohorts encompassing 3,441 ever-smoking COPD subjects (GOLD stage II or higher). Untyped SNPs were imputed using HapMap (phase II) panel. Results from all cohorts were meta-analyzed.
Results
Several SNPs near the HLA region on chromosome 6p21 and in an intergenic region on chromosome 2q21 showed associations with age at smoking initiation, both with the lowest p=2×10−7. No SNPs were associated with lifetime average CPD, current CPD or smoking cessation with p<10−6. Nominally significant associations with candidate SNPs within alpha-nicotinic acetylcholine receptors 3/5 (CHRNA3/CHRNA5; e.g. p=0.00011 for SNP rs1051730) and Cytochrome P450 2A6 (CYP2A6; e.g. p=2.78×10−5 for a nonsynonymous SNP rs1801272) regions were observed for lifetime average CPD, however only CYP2A6 showed evidence of significant association with current CPD. A candidate SNP (rs3025343) in the DBH was significantly (p=0.015) associated with smoking cessation.
Conclusion
We identified two candidate regions associated with age at smoking initiation in COPD subjects. Associations of CHRNA3/CHRNA5 and CYP2A6 loci with CPD and DBH with smoking cessation are also likely of importance in the smoking behaviors of COPD patients.
doi:10.1136/thoraxjnl-2011-200154
PMCID: PMC3302576  PMID: 21685187
Chronic Obstructive Pulmonary Disease (COPD); Genome Wide Association study (GWAS); smoking behaviors; Single Nucleotide Polymorphism (SNP)
24.  Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria 
The structural characterization of acyl-carrier-protein synthase (AcpS) from three different pathogenic microorganisms is reported. One interesting finding of the present work is a crystal artifact related to the activity of the enzyme, which fortuitously represents an opportunity for a strategy to design a potential inhibitor of a pathogenic AcpS.
Some bacterial type II fatty-acid synthesis (FAS II) enzymes have been shown to be important candidates for drug discovery. The scientific and medical quest for new FAS II protein targets continues to stimulate research in this field. One of the possible additional candidates is the acyl-carrier-protein synthase (AcpS) enzyme. Its holo form post-translationally modifies the apo form of an acyl carrier protein (ACP), which assures the constant delivery of thioester intermediates to the discrete enzymes of FAS II. At the Center for Structural Genomics of Infectious Diseases (CSGID), AcpSs from Staphylococcus aureus (AcpSSA), Vibrio cholerae (AcpSVC) and Bacillus anthracis (AcpSBA) have been structurally characterized in their apo, holo and product-bound forms, respectively. The structure of AcpSBA is emphasized because of the two 3′,5′-adenosine diphosphate (3′,5′-ADP) product molecules that are found in each of the three coenzyme A (CoA) binding sites of the trimeric protein. One 3′,5′-ADP is bound as the 3′,5′-ADP part of CoA in the known structures of the CoA–AcpS and 3′,5′-ADP–AcpS binary complexes. The position of the second 3′,5′-ADP has never been described before. It is in close proximity to the first 3′,5′-­ADP and the ACP-binding site. The coordination of two ADPs in AcpSBA may possibly be exploited for the design of AcpS inhibitors that can block binding of both CoA and ACP.
doi:10.1107/S0907444912029101
PMCID: PMC3447402  PMID: 22993090
acyl-carrier-protein synthase; acyl carrier protein; type II fatty-acid synthesis; inhibition; 3′,5′-adenosine diphosphate; coenzyme A
25.  Genome-Wide Association Analysis of Body Mass in Chronic Obstructive Pulmonary Disease 
Cachexia, whether assessed by body mass index (BMI) or fat-free mass index (FFMI), affects a significant proportion of patients with chronic obstructive pulmonary disease (COPD), and is an independent risk factor for increased mortality, increased emphysema, and more severe airflow obstruction. The variable development of cachexia among patients with COPD suggests a role for genetic susceptibility. The objective of the present study was to determine genetic susceptibility loci involved in the development of low BMI and FFMI in subjects with COPD. A genome-wide association study (GWAS) of BMI was conducted in three independent cohorts of European descent with Global Initiative for Chronic Obstructive Lung Disease stage II or higher COPD: Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-Points (ECLIPSE; n = 1,734); Norway-Bergen cohort (n = 851); and a subset of subjects from the National Emphysema Treatment Trial (NETT; n = 365). A genome-wide association of FFMI was conducted in two of the cohorts (ECLIPSE and Norway). In the combined analyses, a significant association was found between rs8050136, located in the first intron of the fat mass and obesity–associated (FTO) gene, and BMI (P = 4.97 × 10−7) and FFMI (P = 1.19 × 10−7). We replicated the association in a fourth, independent cohort consisting of 502 subjects with COPD from COPDGene (P = 6 × 10−3). Within the largest contributing cohort of our analysis, lung function, as assessed by forced expiratory volume at 1 second, varied significantly by FTO genotype. Our analysis suggests a potential role for the FTO locus in the determination of anthropomorphic measures associated with COPD.
doi:10.1165/rcmb.2010-0294OC
PMCID: PMC3266061  PMID: 21037115
chronic obstructive pulmonary disease genetics; chronic obstructive pulmonary disease epidemiology; chronic obstructive pulmonary disease metabolism; genome-wide association study

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