PREDICT modeling and in-silico screening for G-protein coupled receptors

G-protein coupled receptors (GPCRs) are a major group of drug targets for which only one x-ray structure is known (the nondrugable rhodopsin), limiting the application of structure-based drug discovery to GPCRs. In this paper we present the details of PREDICT, a new algorithmic approach for modeling the 3D structure of GPCRs without relying on homology to rhodopsin. PREDICT, which focuses on the transmembrane domain of GPCRs, starts from the primary sequence of the receptor, simultaneously optimizing multiple 'decoy' conformations of the protein in order to find its most stable structure, culminating in a virtual receptor-ligand complex. In this paper we present a comprehensive analysis of three PREDICT models for the dopamine D2, neurokinin NK1, and neuropeptide Y Y1 receptors. A shorter discussion of the CCR3 receptor model is also included. All models were found to be in good agreement with a large body of experimental data. The quality of the PREDICT models, at least for drug discovery purposes, was evaluated by their successful utilization in in-silico screening. Virtual screening using all three PREDICT models yielded enrichment factors 9-fold to 44-fold better than random screening. Namely, the PREDICT models can be used to identify active small-molecule ligands embedded in large compound libraries with an efficiency comparable to that obtained using crystal structures for non-GPCR targets.     

Prediction and classification of human G-protein coupled receptors based on support vector machines

A computational system for the prediction and classification of human G-protein coupled receptors （GPCRs） has been developed based on the support vector machine （SVM） method and protein sequence information. The feature vectors used to develop the SVM prediction models consist of statistically significant features selected from single amino acid, dipeptide, and tripeptide compositions of protein sequences. Furthermore, the length distribution difference between GPCRs and non-GPCRs has also been exploited to improve the prediction performance. The testing results with annotated human protein sequences demonstrate that this system can get good performance for both prediction and classification of human GPCRs.     

Influences of illumination intensity on the nearest neighbour distance in coho salmon Oncorhynchus kisutch

Effects of illumination intensity on the nearest neighbour distance (NND) of coho salmon in an experimental aquarium were studied under illumination intensities ranging from 0 to 4000 Ix. The NNDs in sighted (intact) fish were divided into three groups according to the illumination intensities; the largest [1.15 times fork length (f.l.)] at 0 1x, the medium (0.78 to 0.84 f.l.) at 0.01 to 0.4 1x, and the least (0.60 to 0.66 f.l.) at 4–4000 1x. The fact that NNDs under 4 1x or higher illumination conditions were significantly smaller than those under 0.01 to 0.4 1x conditions suggests a change from photopic to scotopic vision. Blinded fish exhibited constant NNDs irrespective of illumination intensity and the value was similar to that of sighted fish under the 0 1x condition. The NNDs of blinded fish and sighted fish exposed to 0 1x were larger than the simulated NND calculated by hypothesizing that fish distribute randomly in the experimental aquarium. These results indicate that NND in a coho school is maintained primarily by vision, however, the NND is not determined by vision alone but by integrated senses.     

Location and nature of the residues important for ligand recognition in G-protein coupled receptors

The overall structure of the biogenic amine subclass of the G-protein-coupled receptors, and of their ligand binding sites, is discussed with the aim of highlighting the major structural features of these receptors that are responsible for ligand recognition. A comparison is made between biogenic amine receptors, peptide receptors of the rhodopsin class, and the secretin receptors which all have peptide ligands. The question of where the peptide ligands bind, whether at extracellular sites or within the transmembrane helix bundle, is discussed. The suitability of the rhodopsin crystal structure as a template for construction of homology models is discussed and it is concluded that there are many reasons why a caution should be issued against using it uncritically.     

反距离加权插值法在污染场地评价中的应用

确定污染场地的土壤修复范围在实际操作中存在很大的难度.本文以北方某废弃农药厂为例,结合专家判定采样法与网格采样法,采用地统计学方法中的反距离加权空间插值法,分两种情形将大于土壤环境基准值的区域划定为治理范围.结果表明,由于国家土壤环境二级标准主要适用于农业生产的土地,依据反距离加权空间插值后,以此标准确定的治理范围较大,而基于健康风险水平阈值所确定的修复范围和修复成本较小,较合理且经济可行.采用风险评价的反距离加权插值法确定污染场地的污染范围,为今后的场地评价和土壤修复提供了思路.     

The effect of heterotachy in multigene analysis using the neighbor joining method

Sequence alignments of multiple genes are routinely used to infer phylogenetic relationships among species. The analysis of their concatenation is more likely to give correct results under an assumption of homotachy (i.e., the evolutionary rates within lineages in each of the concatenated genes are constant during evolution). Here, we examine how the violation of homotachy (i.e., presence of within-site rate variation, called heterotachy) distorts species phylogenies. A theoretical examination has been conducted using a four taxon case and the neighbor joining (NJ) method, concluding that NJ recovers the incorrect tree when concatenated genes exhibit heterotachy. The application of average and weighted-average distance approaches, where gene boundaries are kept intact, overcomes the detrimental effect of heterotachy in multigene analysis using the NJ method.     

A method for estimating the nearest neighbor base-pair content of RNAs using CD and absorption spectroscopy.

CD and absorption spectra are sensitive to the secondary structure of RNAs. By fitting the spectra contained in our basis set to the CD and absorption spectra of an RNA of known sequence, we could determine the fractions of base pairs, the fractions of each of the nearest neighbor base pairs, and the fractions of the single-stranded nucleotides in that RNA. The basis set included 58 CD and 58 absorption spectra. The fitting was done with a guided selection routine. The estimated error was about 0.05 for predicting the fractions of the nearest neighbor base pairs, 0.06 for predicting the fractions of A.U, G.C, and G.U base pairs, and 0.04 for predicting the fractions of the single-stranded nucleotides.     

Severe limitations of the FEve metric of functional evenness and some alternative metrics

The metric of functional evenness FEve is an example of how approaches to conceptualizing and measuring functional variability may go astray. This index has several critical conceptual and practical drawbacks:

1. Different values of the FEve index for the same community can be obtained if the species have unequal species abundances; this result is highly likely if most of the traits are categorical.
2. Very minor differences in even one pairwise distance can result in very different values of FEve.
3. FEve uses only a fraction of the information contained in the matrix of species distances. Counterintuitively, this can cause very similar FEve scores for communities with substantially different patterns of species dispersal in trait space.
4. FEve is a valid metric only if all species have exactly the same abundances. However, the meaning of FEve in such an instance is unclear as the purpose of the metric is to measure the variability of abundances in trait space.

We recommend not using the FEve metric in studies of functional variability. Given the wide usage of FEve index over the last decade, the validity of the conclusions based on those estimates is in question. Instead, we suggest three alternative metrics that combine variability in species distances in trait space with abundance in various ways. More broadly, we recommend that researchers think about which community properties (e.g., trait distances of a focus species to the nearest neighbor or all other species, variability of pairwise interactions between species) they want to measure and pick from among the appropriate metrics.     

Classification of G-protein coupled receptors by alignment-independent extraction of principal chemical properties of primary amino acid sequences

We have developed an alignment-independent method for classification of G-protein coupled receptors (GPCRs) according to the principal chemical properties of their amino acid sequences. The method relies on a multivariate approach where the primary amino acid sequences are translated into vectors based on the principal physicochemical properties of the amino acids and transformation of the data into a uniform matrix by applying a modified autocross-covariance transform. The application of principal component analysis to a data set of 929 class A GPCRs showed a clear separation of the major classes of GPCRs. The application of partial least squares projection to latent structures created a highly valid model (cross-validated correlation coefficient, Q(2) = 0.895) that gave unambiguous classification of the GPCRs in the training set according to their ligand binding class. The model was further validated by external prediction of 535 novel GPCRs not included in the training set. Of the latter, only 14 sequences, confined in rapidly expanding GPCR classes, were mispredicted. Moreover, 90 orphan GPCRs out of 165 were tentatively identified to GPCR ligand binding class. The alignment-independent method could be used to assess the importance of the principal chemical properties of every single amino acid in the protein sequences for their contributions in explaining GPCR family membership. It was then revealed that all amino acids in the unaligned sequences contributed to the classifications, albeit to varying extent; the most important amino acids being those that could also be determined to be conserved by using traditional alignment-based methods.     

G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies,in the total absence of detergent

G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A_2A receptor (A_2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A_2AR–SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (∼5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A_2AR controls. The A_2AR–SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR–SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([³H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms.     

Arginine (CGC) codon targeting in the human prostacyclin receptor gene (PTGIR) and G-protein coupled receptors (GPCR)

The human prostacyclin receptor (hIP) has recently been recognized as an important seven transmembrane G-protein coupled receptor that plays critical roles in atheroprevention and cardioprotection. To date, four non-synonymous genetic variants have been identified, two of which occur at the same Arg amino acid position (R212H, R212C). This observation instigated further genetic screening for prostacyclin receptor variants on 1455 human genomic samples. A total of 31 distinct genetic variants were detected, with 6 (19%) involving Arg residues. Distinct differences in location and frequencies of genetic variants were noted between Caucasian, Asian, Hispanic and African Americans, with the most changes noted in the Asian cohort. From the sequencing results, three Arg-targeted changes at the same 212 position within the third cytoplasmic loop of the human prostacyclin (hIP) receptor were detected: 1) R212C (CGC-->TGC), 2) R212H (CGC-->CAC), and 3) R212R (CGC-->CGT). Three additional Arg codon variants (all exhibiting the same CGC to TGC change) were also detected, R77C, R215C, and R279C. Analysis (GPCR and SNP databases) of 200 other GPCRs, with recorded non-synonymous mutations, confirmed a high frequency of Arg-targeted missense mutations, particularly within the important cytoplasmic domain. Preferential nucleotide changes (at Arg codons), were observed involving cytosine (C) to thymine (T) (pyrimidine to pyrimidine), as well as guanine (G) to adenine (A) (purine to purine) (p<0.001, Pearson's goodness-of-fit test). Such targeting of Arg residues, leading to significant changes in coding amino acid size and/or charge, may have potentially-important structural and evolutionary implications on the hIP and GPCRs in general. In the case of the human prostacyclin receptor, such alterations may reduce the cardio-, vasculo-, and cytoprotective effects of prostacyclin.     

Role of phospholipase D2 in the agonist-induced and constitutive endocytosis of G-protein coupled receptors

We have recently shown that the mu-opioid receptor [MOR1, also termed mu-opioid peptide (MOP) receptor] is associated with the phospholipase D2 (PLD2), a phospholipid-specific phosphodiesterase located in the plasma membrane. We further demonstrated that, in human embryonic kidney (HEK) 293 cells co-expressing MOR1 and PLD2, treatment with (D-Ala2, Me Phe4, Glyol5)enkephalin (DAMGO) led to an increase in PLD2 activity and an induction of receptor endocytosis, whereas morphine, which does not induce opioid receptor endocytosis, failed to activate PLD2. In contrast, a C-terminal splice variant of the mu-opioid receptor (MOR1D, also termed MOP(1D)) exhibited robust endocytosis in response to both DAMGO and morphine treatment. We report here that MOR1D also mediates an agonist-independent (constitutive) PLD2-activation facilitating agonist-induced and constitutive receptor endocytosis. Inhibition of PLD2 activity by over-expression of a dominant negative PLD2 (nPLD2) blocked the constitutive PLD2 activation and impaired the endocytosis of MOR1D receptors. Moreover, we provide evidence that the endocytotic trafficking of the delta-opioid receptor [DOR, also termed delta-opioid peptide (DOP) receptor] and cannabinoid receptor isoform 1 (CB1) is also mediated by a PLD2-dependent pathway. These data indicate the generally important role for PLD2 in the regulation of agonist-dependent and agonist-independent G protein-coupled receptor (GPCR) endocytosis.     

Analysis of Spatial Distribution of Vesicles in Presynaptic Terminals of the Hippocampus in vivo and in vitro

A comparative morphometric analysis of electron photomicrographs of the presynaptic terminals in cultured in vitro for 7 days CA1 hippocampal slices from 7-day-old rats and in the hippocampi of 14-day-old rats was conducted. As compared with the terminals of intact neurons, the terminals of cultured cells were larger and contained a greater amount of synaptic vesicles, which, however, to a lesser extent were united in clusters. Distributions of the vesicle profiles in cultured slices were characterized by a greater distance to the nearest neighbor. Obviously, such structural features should be taken into account when interpreting data obtained in electrophysiological studies on hippocampal slice cultures.     

Calmodulin interacts with the cytoplasmic tails of the parathyroid hormone 1 receptor and a sub-set of class b G-protein coupled receptors

Parathyroid hormone (PTH) binds to its receptor (PTH 1 receptor, PTH1R) and activates multiple pathways. The PTH1R, a class b GPCR, contains consensus calmodulin-binding motifs. The PTH1R cytoplasmic tail interacts with calmodulin in a calcium-dependent manner via the basic 1-5-8-14 motif. Calcium-dependent calmodulin interactions with the cytoplasmic tails of receptors for PTH 2, vasoactive intestinal peptide, pituitary adenylate cyclase activating peptide, corticotropin releasing hormone, calcitonin, and the glucagon-like peptides 1 and 2 are demonstrated. The cytoplasmic tails of the secretin receptor and the growth hormone releasing hormone receptor either interact poorly or not at all with calmodulin, respectively. Fluphenazine, a calmodulin antagonist, enhances PTH-mediated accumulation of total inositol phosphates, suggesting that calmodulin regulates signaling via phospholipase C.     

Activation of ERK, JNK, Akt, and G-protein coupled signaling by hybrid angiotensin II AT1/bradykinin B2 receptors expressed in HEK-293 cells

Bradykinin (BK) and angiotensin II (AngII) often have opposite roles in cardiovascular diseases. Our aim here was to construct hybrid receptors which bind AngII but signal as BK. Various sequences of the intracellular face of the AngII type I receptor, AT1R, were replaced with corresponding sequences from the bradykinin B2 receptor (BKB2R). The hybrids demonstrated a number of signaling characteristics of the BKB2R. For example, the hybrids demonstrated BK as opposed to AngII like phosphorylation of Akt and JNK. The hybrids containing the BKB2R intracellular loop 2 (IC2) displayed minimal G-protein, Galphai/Galphaq, linked signaling. Computer based molecular models suggested that Ser-Met-Gly from the IC2 of the BKB2R is detrimental for the Galphai/Galphaq coupled functions of this hybrid. The return of Lys-Ser-Arg of the AT1R to this hybrid led to almost full recovery of Galphai and Galphaq activation. The design and production of AT1/BKB2 hybrid receptors is a potential approach in the treatment of hypertension related diseases where the presence of AngII, its AT1 receptor and the consequent signal transduction has proven detrimental.     

"In vivo" intraneuronal trafficking of G protein coupled receptors in the striatum: regulation by dopaminergic and cholinergic environment

We have studied "in vivo" neurochemically identified striatal neurons to analyze the localisation and the trafficking of dopamine and acetylcholine G protein coupled receptors (GPCR) (D1R, D2R, m2R and m4R) under the influence of neurotransmitter environment. We have identified receptors in tissue sections through immunohistochemical detection at the light and electron microscopic level. We have identified receptors in normal animals and after acute and chronic stimulations. We have quantified receptors through image analysis at the electron microscopic level in relation to various subcellular compartments. Our results demonstrate that, in normal conditions, GPCRs are mostly associated with plasma membrane of the striatal neurons, mostly at extra-synaptic sites. In certain instances (m4R; D2R), receptors have prominent localisation inside the rough endoplasmic reticulum. Our results also show that two distinct receptors for a same neurotransmitter may have distinct subcellular localisation in a same neuronal population (m2R versus m4R) and that the same neurotransmitter receptor (m4R) can have distinct localisation in distinct neuronal populations (cytoplasm versus cell surface). After acute stimulation, cell surface receptors undergo dramatic subcellular changes that involve plasma membrane depletion, internalisation in endosomes and in multivesicular bodies. Such changes are reversible after the end of the stimulation and are blocked by antagonist action. Chronic stimulation also provokes changes in subcellular localisation with specific pattern: plasma membrane depletion, and exaggerated storage of receptors in rough endoplasmic reticulum and eventually Golgi complex (D1R; m2R and m4R). Decreasing chronic receptor stimulation reverses such changes. These results demonstrate that, "in vivo", in the striatum, GPCRs undergo complex intraneuronal trafficking under the influence of neurochemical environment in conditions that dramatically modulate the number of cell surface receptors available for interaction with neurotransmitters or drugs. This confirms that "in vivo", the trafficking and the subcellular compartmentalization of GPCRs may contribute to regulate neuronal sensitivity and neuronal interactions in physiological, experimental and pathological conditions, including in therapeutic conditions.     

Effects of distance to crop rows and to conspecific neighbours on the size of Brassica napus and Veronica persica weeds

We tested the hypothesis that local competitive conditions are a determinant of the size of individual weeds in cereal crops by investigating the relationship between individual weed size and (a) distance from the crop row and (b) distance to the nearest conspecific neighbour in cereal crops. There were significant but weak effects of distance to rows of summer and winter wheat (Triticum aestivum), and distance to conspecific individuals on individuals of two weed species, Brassica napus and Veronica persica. Our results suggest that local neighbourhood competitive conditions, although detectable, have only limited effects on weed growth. Size-asymmetric competition from the crop population and plasticity in weed growth reduce the importance of a weed individual's exact location relative to crop individuals and to other weed individuals. A static, two-dimensional view of space is not sufficient to describe competitive effects because the third dimension can be the most important in competition, and because many plants can change their locations through plastic growth.Wir überprüften die Hypothese, dass lokale Konkurrenzbedingungen ein Bestimmungsfaktor für die Größe einzelner Unkräuter in Getreidefeldern sind, indem wir die Beziehung zwischen der individuellen Unkrautgröße und (a) der Distanz zu den Pflanzenreihen und (b) der Distanz zu den nächsten, artgleichen Nachbarpflanzen in Getreidefeldern untersuchten. Es gab signifikante jedoch geringfügige Effekte der Distanz zu den Reihen von Sommer- und Winterweizen (Tritium aestivum) sowie der Distanz zu den artgleichen Individuen auf die Individuen von zwei Unkrautarten, Brassica napus und Veronica persica. Unsere Ergebnisse lassen vermuten, dass Konkurrenzbedingungen in der unmittelbaren Nachbarschaft nur einen begrenzten Effekt auf das Unkrautwachstum haben, auch wenn sie wahrnehmbar sind. Größenasymmetrische Konkurrenz seitens der Getreidepopulation und die Plastizität des Pflanzenwachstums reduzieren die Bedeutung der exakten Position einer einzelnen Unkrautpflanze in Beziehung zu einzelnen Getreide- oder anderen, einzelnen Unkrautwpflanzen. Eine statische, zweidimensionale Betrachtung des Raumes reicht nicht aus, um die Konkurrenzeffekte zu beschreiben, weil die dritte Dimension die wichtigste für die Konkurrenz sein kann und weil viele Pflanzen ihre Position durch plastisches Wachstum verändern können.     

Molecular modeling of the second extracellular loop of G-protein coupled receptors and its implication on structure-based virtual screening

The current study describes the validation of high-throughput modeling procedures for the construction of the second extracellular loop (ecl2) of all nonolfactory human G Protein-coupled receptors. Our modeling flowchart is based on the alignment of essential residues determining the particular ecl2 fold observed in the bovine rhodopsin (bRho) crystal structure. For a set of GPCR targets, the dopamine D2 receptor (DRD2), adenosine A3 receptor (AA3R), and the thromboxane A2 receptor (TA2R), the implications of including ecl2 atomic coordinates is evaluated in terms of structure-based virtual screening accuracy: the suitability of the 3D models to distinguish between known antagonists and randomly chosen decoys using automated docking approaches. The virtual screening results of different models describing increasingly exhaustive receptor representations (seven helices only, seven helices and ecl2 loop, full model) have been compared. Explicit modeling of the ecl2 loop was found to be important in only one of three test cases whereas a loopless model was shown to be accurate enough in the two other receptors. An exhaustive comparison of ecl2 loops of 365 receptors to that of bRho suggests that explicit ecl2 loop modeling should be reserved to receptors where loop building can be guided by experimental restraints.     

扎龙自然保护区丹顶鹤(Grus japonensis)巢的内分布型及巢域

为了探讨丹顶鹤繁殖种群的空间分布,2002～2006年的4～5月份,在黑龙江扎龙国家级自然保护区,采用定点观察法、无样地取样法、GPS定位等研究方法和分布距离指数、最近邻体法等衡量指标对丹顶鹤(Grus japonensis)巢的内分布型及巢域进行了研究.结果表明:(1)扎龙保护区丹顶鹤巢的内分布型,I2002=2.140>2,I2003=2.048>2,I2004=2.093>2, I2006=3.263>2,均为聚集分布;(2)在假设扎龙保护区丹顶鹤巢域面积等于领域面积、巢域形状为圆形且所有个体面积大小相等的前提下,丹顶鹤的巢域为(0.510±0.019)km2,年度间有所差异,分别为2002年(0.542±0.257)km2、2003年(0.569±0.067)km2、2004年(0.557±0.054)km2、2006年(0.344±0.119)km2.分析表明,丹顶鹤对于栖息生境的整体分布和繁殖微生境质量的变化具有一定适应和应答的能力.为了更有效地验证本文的研究结果并对丹顶鹤进行保护,还有待于长期监测并进一步研究.     

A field test of the distance sampling method using Golden‐cheeked Warblers

ABSTRACT Criteria for delisting Golden‐cheeked Warblers (Dendroica chrysoparia) include protection of sufficient breeding habitat to ensure the continued existence of 1000 to 3000 singing males in each of eight recovery regions for ≥10 consecutive years. Hence, accurate abundance estimation is an integral component in the recovery of this species. I conducted a field test to determine if the distance sampling method provided unbiased abundance estimates for Golden‐cheeked Warblers and develop recommendations to improve the accuracy of estimates by minimizing the effects of violating this method's assumptions. To determine if observers could satisfy the assumptions that birds are detected at the point with certainty and at their initial locations, I compared point‐transect sampling estimates from 2‐, 3‐, 4‐, and 5‐min time intervals to actual abundance determined by intensive territory monitoring. Point‐transect abundance estimates were 15%, 29%, 43%, and 59% greater than actual abundance (N= 156) for the 2‐, 3‐, 4‐, and 5‐min intervals, respectively. Point‐transect sampling produced unbiased estimates of Golden‐cheeked Warbler abundance when counts were limited to 2 min (N= 154–207). Abundance estimates derived from point‐transect sampling were likely greater than actual abundance because observers did not satisfy the assumption that birds were detected at their initial locations due to the frequent movements and large territory sizes of male Golden‐cheeked Warblers. To minimize the effect of movement on abundance estimates, I recommend limiting counts of singing males to 2‐min per point. Counts for other species in similar habitats with similar behavior and movement patterns also should be limited to 2 min when unbiased estimates are important and conducting field tests of the point‐transect distance sampling method is not possible.