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991.
Tomohiro Kimura Krishna Vukoti Diane L. Lynch Dow P. Hurst Alan Grossfield Michael C. Pitman Patricia H. Reggio Alexei A. Yeliseev Klaus Gawrisch 《Proteins》2014,82(3):452-465
The global fold of human cannabinoid type 2 (CB2) receptor in the agonist‐bound active state in lipid bilayers was investigated by solid‐state 13C‐ and 15N magic‐angle spinning (MAS) NMR, in combination with chemical‐shift prediction from a structural model of the receptor obtained by microsecond‐long molecular dynamics (MD) simulations. Uniformly 13C‐ and 15N‐labeled CB2 receptor was expressed in milligram quantities by bacterial fermentation, purified, and functionally reconstituted into liposomes. 13C MAS NMR spectra were recorded without sensitivity enhancement for direct comparison of Cα, Cβ, and C?O bands of superimposed resonances with predictions from protein structures generated by MD. The experimental NMR spectra matched the calculated spectra reasonably well indicating agreement of the global fold of the protein between experiment and simulations. In particular, the 13C chemical shift distribution of Cα resonances was shown to be very sensitive to both the primary amino acid sequence and the secondary structure of CB2. Thus the shape of the Cα band can be used as an indicator of CB2 global fold. The prediction from MD simulations indicated that upon receptor activation a rather limited number of amino acid residues, mainly located in the extracellular Loop 2 and the second half of intracellular Loop 3, change their chemical shifts significantly (≥1.5 ppm for carbons and ≥5.0 ppm for nitrogens). Simulated two‐dimensional 13Cα(i)? 13C?O(i) and 13C?O(i)? 15NH(i + 1) dipolar‐interaction correlation spectra provide guidance for selective amino acid labeling and signal assignment schemes to study the molecular mechanism of activation of CB2 by solid‐state MAS NMR. Proteins 2014; 82:452–465. © 2013 Wiley Periodicals, Inc. 相似文献
992.
High‐throughput sequencing technologies are now allowing us to study patterns of community assembly for diverse microbial assemblages across environmental gradients and during succession. Here we discuss potential explanations for similarities and differences in bacterial and fungal community assembly patterns along a soil chronosequence in the foreland of a receding glacier. Although the data are not entirely conclusive, they do indicate that successional trajectories for bacteria and fungi may be quite different. Recent empirical and theoretical studies indicate that smaller microbes (like most bacteria) are less likely to be dispersal limited than are larger microbes – which could result in a more deterministic community assembly pattern for bacteria during primary succession. Many bacteria are also better adapted (than are fungi) to life in barren, early‐successional sediments in that some can fix nitrogen and carbon from the atmosphere – traits not possessed by any fungi. Other differences between bacteria and fungi are discussed, but it is apparent from this and other recent studies of microbial succession that we are a long way from understanding the mechanistic underpinnings of microbial community assembly during ecosystem succession. We especially need a better understanding of global and regional patterns of microbial dispersal and what environmental factors control the development of microbial communities in complex natural systems. 相似文献
993.
Victor Kim Adam Davey Alejandro P Comellas Meilan K Han George Washko Carlos H Martinez David Lynch Jin Hwa Lee Edwin K Silverman James D Crapo Barry J Make Gerard J Criner 《Respiratory research》2014,15(1):52
Background
Chronic bronchitis (CB) has been related to poor outcomes in Chronic Obstructive Pulmonary Disease (COPD). From a clinical standpoint, we have shown that subjects with CB in a group with moderate to severe airflow obstruction were younger, more likely to be current smokers, male, Caucasian, had worse health related quality of life, more dyspnea, and increased exacerbation history compared to those without CB. We sought to further refine our clinical characterization of chronic bronchitics in a larger cohort and analyze the CT correlates of CB in COPD subjects. We hypothesized that COPD patients with CB would have thicker airways and a greater history of smoking, acute bronchitis, allergic rhinitis, and occupational exposures compared to those without CB.Methods
We divided 2703 GOLD 1–4 subjects in the Genetic Epidemiology of COPD (COPDGene®) Study into two groups based on symptoms: chronic bronchitis (CB+, n = 663, 24.5%) and no chronic bronchitis (CB-, n = 2040, 75.5%). Subjects underwent extensive clinical characterization, and quantitative CT analysis to calculate mean wall area percent (WA%) of 6 segmental airways was performed using VIDA PW2 (http://www.vidadiagnostics.com). Square roots of the wall areas of bronchi with internal perimeters 10 mm and 15 mm (Pi10 and Pi15, respectively), % emphysema, %gas trapping, were calculated using 3D Slicer (http://www.slicer.org).Results
There were no differences in % emphysema (11.4 ± 12.0 vs. 12.0 ± 12.6%, p = 0.347) or % gas trapping (35.3 ± 21.2 vs. 36.3 ± 20.6%, p = 0.272) between groups. Mean segmental WA% (63.0 ± 3.2 vs. 62.0 ± 3.1%, p < 0.0001), Pi10 (3.72 ± 0.15 vs. 3.69 ± 0.14 mm, p < 0.0001), and Pi15 (5.24 ± 0.22 vs. 5.17 ± 0.20, p < 0.0001) were greater in the CB + group. Greater percentages of gastroesophageal reflux, allergic rhinitis, histories of asthma and acute bronchitis, exposures to dusts and occupational exposures, and current smokers were seen in the CB + group. In multivariate binomial logistic regression, male gender, Caucasian race, a lower FEV1%, allergic rhinitis, history of acute bronchitis, current smoking, and increased airway wall thickness increased odds for having CB.Conclusions
Histories of asthma, allergic rhinitis, acute bronchitis, current smoking, a lower FEV1%, Caucasian race, male gender, and increased airway wall thickness are associated with CB. These data provide clinical and radiologic correlations to the clinical phenotype of CB. 相似文献994.
Emily S Wan Peter J Castaldi Michael H Cho John E Hokanson Elizabeth A Regan Barry J Make Terri H Beaty MeiLan K Han Jeffrey L Curtis Douglas Curran-Everett David A Lynch Dawn L DeMeo James D Crapo Edwin K Silverman The COPDGene Investigators 《Respiratory research》2014,15(1)
Background
Preserved Ratio Impaired Spirometry (PRISm), defined as a reduced FEV1 in the setting of a preserved FEV1/FVC ratio, is highly prevalent and is associated with increased respiratory symptoms, systemic inflammation, and mortality. Studies investigating quantitative chest tomographic features, genetic associations, and subtypes in PRISm subjects have not been reported.Methods
Data from current and former smokers enrolled in COPDGene (n = 10,192), an observational, cross-sectional study which recruited subjects aged 45–80 with ≥10 pack years of smoking, were analyzed. To identify epidemiological and radiographic predictors of PRISm, we performed univariate and multivariate analyses comparing PRISm subjects both to control subjects with normal spirometry and to subjects with COPD. To investigate common genetic predictors of PRISm, we performed a genome-wide association study (GWAS). To explore potential subgroups within PRISm, we performed unsupervised k-means clustering.Results
The prevalence of PRISm in COPDGene is 12.3%. Increased dyspnea, reduced 6-minute walk distance, increased percent emphysema and decreased total lung capacity, as well as increased segmental bronchial wall area percentage were significant predictors (p-value <0.05) of PRISm status when compared to control subjects in multivariate models. Although no common genetic variants were identified on GWAS testing, a significant association with Klinefelter’s syndrome (47XXY) was observed (p-value < 0.001). Subgroups identified through k-means clustering include a putative “COPD-subtype”, “Restrictive-subtype”, and a highly symptomatic “Metabolic-subtype”.Conclusions
PRISm subjects are clinically and genetically heterogeneous. Future investigations into the pathophysiological mechanisms behind and potential treatment options for subgroups within PRISm are warranted.Trial registration
Clinicaltrials.gov Identifier: NCT000608764.Electronic supplementary material
The online version of this article (doi:10.1186/s12931-014-0089-y) contains supplementary material, which is available to authorized users. 相似文献995.
996.
997.
Takahiko Sasaki Kelsey L. Lynch Caitlin V. Mueller Steven Friedman Michael Freitag Zachary A. Lewis 《Eukaryotic cell》2014,13(8):990-1000
In response to genotoxic stress, ATR and ATM kinases phosphorylate H2A in fungi and H2AX in animals on a C-terminal serine. The resulting modified histone, called γH2A, recruits chromatin-binding proteins that stabilize stalled replication forks or promote DNA double-strand-break repair. To identify genomic loci that might be prone to replication fork stalling or DNA breakage in Neurospora crassa, we performed chromatin immunoprecipitation (ChIP) of γH2A followed by next-generation sequencing (ChIP-seq). γH2A-containing nucleosomes are enriched in Neurospora heterochromatin domains. These domains are comprised of A·T-rich repetitive DNA sequences associated with histone H3 methylated at lysine-9 (H3K9me), the H3K9me-binding protein heterochromatin protein 1 (HP1), and DNA cytosine methylation. H3K9 methylation, catalyzed by DIM-5, is required for normal γH2A localization. In contrast, γH2A is not required for H3K9 methylation or DNA methylation. Normal γH2A localization also depends on HP1 and a histone deacetylase, HDA-1, but is independent of the DNA methyltransferase DIM-2. γH2A is globally induced in dim-5 mutants under normal growth conditions, suggesting that the DNA damage response is activated in these mutants in the absence of exogenous DNA damage. Together, these data suggest that heterochromatin formation is essential for normal DNA replication or repair. 相似文献
998.
Amphotericin B is a medically important antifungal antibiotic that is produced by Streptomyces nodosus. Genetic manipulation of this organism has led to production of the first amphotericin analogues by engineered biosynthesis. Here, these studies were extended by sequencing the chromosomal regions flanking the amphotericin polyketide synthase genes, and by refining the phage KC515 transduction method for disruption and replacement of S. nodosus genes. A hybrid vector was constructed from KC515 DNA and the Escherichia coli plasmid pACYC177. This vector replicated as a plasmid in E. coli and the purified DNA yielded phage plaques on Streptomyces lividans after polyethylene glycol (PEG)-mediated transfection of protoplasts. The left flank of the amphotericin gene cluster was found to include amphRI, RII, RIII and RIV genes that are similar to regulatory genes in other polyene biosynthetic gene clusters. One of these regulatory genes, amphRI, was found to have a homologue, amphRVI, located in the right flank at a distance of 127 kbp along the chromosome. However, disruption of amphRVI using the hybrid vector had no effect on the yield of amphotericin obtained from cultures grown on production medium. The hybrid vector was also used for precise deletion of the DNA coding for two modules of the AmphC polyketide synthase protein. Analysis by UV spectrophotometry revealed that the deletion mutant produced a novel pentaene, with reduced antifungal activity but apparently greater water-solubility than amphotericin B. This shows the potential for use of the new vector in engineering of this and other biosynthetic pathways in Streptomyces. 相似文献
999.
The efficacy of agonists at Cys-loop ion channel receptors is determined by the rate they isomerize receptors to a pre-open flip state. Once the flip state is reached, the shut-open reaction is similar for low and high efficacy agonists. The present study sought to identify a conformational change associated with the closed-flip transition in the α1-glycine receptor. We employed voltage-clamp fluorometry to compare ligand-binding domain conformational changes induced by the following agonists, listed from highest to lowest affinity and efficacy: glycine > β-alanine > taurine. Voltage-clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. Agonist affinity and efficacy correlated inversely with maximum fluorescence magnitudes at labeled residues in ligand-binding domain loops D and E, suggesting that large conformational changes in this region preclude efficacious gating. However, agonist affinity and efficacy correlated directly with maximum fluorescence magnitudes from a label attached to A52C in loop 2, near the transmembrane domain interface. Because glycine experiences the largest affinity increase between closed and flip states, we propose that the magnitude of this fluorescence signal is directly proportional to the agonist affinity increase. In contrast, labeled residues in loops C, F, and the pre-M1 domain yielded agonist-independent fluorescence responses. Our results support the conclusion that a closed-flip conformation change, with a magnitude proportional to the agonist affinity increase from closed to flip states, occurs in the microenvironment of Ala-52.Glycine receptors (GlyRs)3 are pentameric chloride-selective ion channels that mediate fast inhibitory neurotransmission (1). They are members of the Cys-loop receptor family that includes the prototypical nicotinic acetylcholine receptor (nAChR), the γ-aminobutyric acid type-A receptors (GABAARs), and serotonin type-3 receptors (5-HT3Rs). Recent structural studies have provided a wealth of information on the structure and function of this receptor family (2–6). In Cys-loop receptors, the ligand-binding domain (LBD) preceding the four transmembrane helices consists of two twisted β-sheets. The inner (vestibule facing) β-sheet comprises seven β-strands, while the outer β-sheet is formed by three β-strands (3). The ligand binding site is located at the interface of adjacent subunits and is lined by six domains: three loops from the principal and the complementary sides, termed A-C and D-F, respectively (3).GlyRs are activated by endogenous amino acid agonists in the following order of efficacy: glycine > β-alanine > taurine (7, 8). As these amino acids share considerable structural similarity (Fig. 1A), they are likely to compete for the same binding site (9–11). A recent ground-breaking study on an intermediate pre-open state, the so-called “flip” state (12), has provided new insights into the mechanism of partial agonism in Cys-loop receptors (13). This study suggested that agonist efficacy depends on the ability of the agonist to convert the inert agonist-bound receptor to the pre-open flip state. Once the flip state is reached, the shut-open reaction is similar for high and low efficacy agonists. To date there is, however, very little information concerning the structural basis for the lower efficacies of partial agonists. To address this, the present study employed the voltage-clamp fluorometry (VCF) technique (14) to compare the conformational changes induced by glycine, β-alanine, and taurine at various positions in the GlyR LBD.Open in a separate windowFIGURE 1.A, structures of glycine, β-alanine, and taurine. B, model of the LBD, based on carbomylcholine-bound AChBP (PDB code 1uv6). The inner β-sheet is displayed in red, the outer β-sheet in blue. Connecting loops are shown in gray. Colored balls represent approximate locations of selected residues labeled in regions flanking the outer β-sheet (black, G181C in loop F; N203C in loop C; Q219C in the pre-M1 domain) and in the inner β-sheet (yellow, L127C in loop E; Q67C in loop D; A52C in loop 2).VCF involves tethering of an environmentally sensitive fluorophore to a cysteine engineered into a domain of interest. If ligand-binding and/or channel opening leads to a changed dielectric environment surrounding the fluorophore, a change in quantum yield or emission spectrum can be detected. VCF was first employed on voltage-gated potassium channels (15) and has since provided a wealth of information on Cys-loop receptor structure and function (16–23). Here we employ VCF to identify an agonist-specific conformational change that may control or reflect the rate at which the GlyR isomerizes to the flip state. 相似文献
1000.
Yong Chen Chongguang Chen Evangelia Kotsikorou Diane L. Lynch Patricia H. Reggio Lee-Yuan Liu-Chen 《The Journal of biological chemistry》2009,284(3):1673-1685
We demonstrated previously that the protein GEC1 (glandular epithelial cell
1) bound to the human κ opioid receptor (hKOPR) and promoted cell
surface expression of the receptor by facilitating its trafficking along the
secretory pathway. Here we showed that three hKOPR residues
(Phe345, Pro346, and Met350) and seven GEC1
residues (Tyr49, Val51, Leu55,
Thr56, Val57, Phe60, and Ile64)
are indispensable for the interaction. Modeling studies revealed that the
interaction was mediated via direct contacts between the kinked hydrophobic
fragment in hKOPR C-tail and the curved hydrophobic surface in GEC1 around the
S2 β-strand. Intramolecular Leu44-Tyr109
interaction in GEC1 was important, likely by maintaining its structural
integrity. Microtubule binding mediated by the GEC1 N-terminal domain was
essential for the GEC1 effect. Expression of GEC1 also increased cell surface
levels of the GluR1 subunit and the prostaglandin EP3.f receptor, which have
FPXXM and FPXM sequences, respectively. With its widespread
distribution in the nervous system and its predominantly hydrophobic
interactions, GEC1 may have chaperone-like effects for many cell surface
proteins along the biosynthesis pathway.κ opioid receptor
(KOPR)2 is one of the
three major types of opioid receptors mediating effects of opioid drugs and
endogenous opioid peptides. Stimulation of KOPR generates many effects in
vivo, for example antinociception (especially for visceral chemical pain,
antipruritis, and water diuresis
(1). The KOPR agonist
nalfurafine (TRK-820) is used clinically in Sweden for the treatment of uremic
pruritus in kidney dialysis patients
(2). Because KOPR agonists
produce profound sedative effects, it has been proposed that KOPR agonists may
be useful in treating mania, antagonists as anti-depressants, and partial
agonists for the management of mania depression
(3). KOPR antagonists may also
be useful for curbing cocaine craving and as anti-anxiety drugs
(4,
5).KOPR, a member of the rhodopsin subfamily of the seven-transmembrane
receptor superfamily, is coupled preferentially to pertussis toxin-sensitive G
proteins, namely Gi/o proteins
(6). KOPR has been found to
interact with several non-G protein-binding partners, such as
Na+,H+-exchanger regulatory
factor-1/ezrin-radixin-moesin-binding phosphoprotein-50 and the δ opioid
receptor. These interactions have influence on signal transduction and
trafficking of the receptor
(7–9).
By yeast two-hybrid (Y2H) assay using the hKOPR C-tail to screen a human brain
cDNA library, we identified GEC1, also named GABAA
receptor-associated protein like 1 (GABARAPL1), to be a binding partner of
hKOPR (10).GEC1 cDNA was first cloned as an early estrogen-regulated mRNA from guinea
pig endometrial glandular epithelial cells by Pellerin et al.
(11). Subsequently, it was
cloned from other species, including human and house mouse
(12). Interestingly, the amino
acid sequences of GEC1 are completely conserved among all these species except
orangutan, in which Arg99 substitutes for His99.
Northern blot and immunoblotting analyses revealed that it has widespread
tissue distribution
(12–14).
In particular, GEC1 was found to be abundant in the central nervous system and
expressed throughout the rat brain
(14,
15). This wide tissue
distribution and the high sequence identity across species strongly suggest
that GEC1 has important biological functions in mammalian cells.Based on sequence similarity, GEC1 is classified as a member of
microtubule-associated proteins (MAPs), which also include GABAA
receptor-associated protein (GABARAP), Golgi-associated ATPase enhancer of 16
kDa (GATE16), GABARAP-like 3 (GABARAPL3), light chain 3 (LC3) of MAP 1A/1B,
and the yeast autophagy protein 8 (Atg8)
(12,
13). Among these homologues,
GEC1 share the highest identity with GABARAPL3 (93%), followed by GABARAP
(86%), GATE16 (61%), Atg8 (55%), and LC3 (∼30%).A growing body of evidence shows that this protein family is closely
related to two distinct biological functions. Studies mainly on GABARAP,
GATE16, and GEC1 indicate that they promote intracellular protein trafficking
by enhancing vesicle fusion
(10,
16–21).
In addition, they facilitate degradation of proteins and intracellular
organelles via autophagy-related pathways, which is bolstered largely by
research on Atg8 and LC3 (22,
23).We previously reported that GEC1 interacted with the hKOPR C-tail and
enhanced cell surface levels of hKOPR stably expressed in CHO cells. GEC1
expression enhances hKOPR expression through facilitating its anterograde
trafficking along the protein biosynthesis pathway without affecting
degradation of the receptor
(10). This represented the
first biological function reported for GEC1. Mansuy et al.
(24) demonstrated that GEC1
interacted with tubulin and promoted microtubule bundling in vitro,
and that green fluorescence protein-tagged GEC1 was localized in the
perinuclear vesicles with a scattered pattern. Our electron microscopic
studies in the rat brain showed that GEC1 was associated with ER, Golgi
apparatus, endosome-like vesicles, and plasma membranes and scattered in
cytoplasm in neurons (14). In
addition, N-ethylmaleimide-sensitive factor, a protein critical for
intracellular membrane-trafficking events, binds directly to GEC1
(10).In this study, we employed Y2H techniques to determine the amino acid
residues in both GEC1 and hKOPR C-tail involved in the interaction. Further
studies were then carried out in mammalian cells to examine if elimination of
the interaction affected the effect of GEC1 on hKOPR expression. In addition,
we generated a molecular model of GEC1 based on the x-ray crystal structure of
GABARAP and found that the residues involved in hKOPR binding formed
hydrophobic patches on the exterior surface of GEC1. Moreover, we found that
the cytosolic tail of AMPA receptor subunit GluR1 has the same FPXXM
motif as that found in the hKOPR C-tail to be involved in GEC1 binding and
that GEC1 expression up-regulated GluR1. 相似文献