Effective Use of Empirical Data for Virtual Screening against APJR GPCR Receptor.

Alzheimer's disease is a neurodegenerative disorder incompatible with normal daily activity, affecting one in nine people. One of its potential targets is the apelin receptor (APJR), a G-protein coupled receptor, which presents considerably high expression levels in the central nervous system. In silico studies of APJR drug-like molecule binding are in small numbers while high throughput screenings (HTS) are already sufficiently many to devise efficient drug design strategies. This presents itself as an opportunity to optimize different steps in future large scale virtual screening endeavours. Here, we ran a first stage docking simulation against a library of 95 known binders and 3829 generated decoys in an effort to improve the rescoring stage. We then analyzed receptor binding site structure and ligands binding poses to describe their interactions. As a result, we devised a simple and straightforward virtual screening Stage II filtering score based on search space extension followed by a geometric estimation of the ligand-binding site fitness. Having this score, we used an ensemble of receptors generated by Hamiltonian Monte Carlo simulation and reported the results. The improvements shown herein prove that our ensemble docking protocol is suited for APJR and can be easily extrapolated to other GPCRs.

Characterization and expression analysis of Galnts in developing Strongylocentrotus purpuratus embryos.

Mucin-type O-glycosylation is a ubiquitous posttranslational modification in which N-Acetylgalactosamine (GalNAc) is added to the hydroxyl group of select serine or threonine residues of a protein by the family of UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferases (GalNAc-Ts; EC 2.4.1.41). Previous studies demonstrate that O-glycosylation plays essential roles in protein function, cell-cell interactions, cell polarity and differentiation in developing mouse and Drosophila embryos. Although this type of protein modification is highly conserved among higher eukaryotes, little is known about this family of enzymes in echinoderms, basal deuterostome relatives of the chordates. To investigate the potential role of GalNAc-Ts in echinoderms, we have begun the characterization of this enzyme family in the purple sea urchin, S. purpuratus. We have fully or partially cloned a total of 13 genes (SpGalnts) encoding putative sea urchin SpGalNAc-Ts, and have confirmed enzymatic activity of five recombinant proteins. Amino acid alignments revealed high sequence similarity among sea urchin and mammalian glycosyltransferases, suggesting the presence of putative orthologues. Structural models underscored these similarities and helped reconcile some of the substrate preferences observed. Temporal and spatial expression of SpGalnt transcripts, was studied by whole-mount in situ hybridization. We found that many of these genes are transcribed early in developing embryos, often with restricted expression to the endomesodermal region. Multicolor fluorescent in situ hybridization (FISH) demonstrated that transcripts encoding SpGalnt7-2 co-localized with both Endo16 (a gene expressed in the endoderm), and Gcm (a gene expressed in secondary mesenchyme cells) at the early blastula stage, 20 hours post fertilization (hpf). At late blastula stage (28 hpf), SpGalnt7-2 message co-expresses with Gcm, suggesting that it may play a role in secondary mesenchyme development. We also discovered that morpholino-mediated knockdown of SpGalnt13 transcripts, results in a deficiency of embryonic skeleton and neurons, suggesting that mucin-type O-glycans play essential roles during embryonic development in S. purpuratus.

Cross-talk between Dopachrome Tautomerase and Caveolin-1 Is Melanoma Cell Phenotype-specific and Potentially Involved in Tumor Progression.

l-Dopachrome tautomerase (l-DCT), also called tyrosinase-related protein-2 (TRP-2), is a melanoma antigen overexpressed in most chemo-/radiotherapeutic stress-resistant tumor clones, and caveolin-1 (CAV1) is a main regulator of numerous signaling processes. A structural and functional relationship between DCT and CAV1 is first presented here in two human amelanotic melanoma cell lines, derived from vertical growth phase (MelJuSo) and metastatic (SKMel28) melanomas. DCT co-localizes at the plasma membrane with CAV1 and Cavin-1, another molecular marker for caveolae in both cell phenotypes. Our novel structural model proposed for the DCT-CAV1 complex, in addition to co-immunoprecipitation and mass spectrometry data, indicates a possible direct interaction between DCT and CAV1. The CAV1 control on DCT gene expression, DCT post-translational processing, and subcellular distribution is cell phenotype-dependent. DCT is a modulator of CAV1 stability and supramolecular assembly in both cell phenotypes. During autocrine stimulation, the expressions of DCT and CAV1 are oppositely regulated; DCT increases while CAV1 decreases. Sub-confluent MelJuSo clones DCT(high)/CAV1(low) are proliferating and acquire fibroblast-like morphology, forming massive, confluent clusters as demonstrated by immunofluorescent staining and TissueFAXS quantitative image cytometry analysis. CAV1 down-regulation directly contributes to the expansion of MelJuSo DCT(high) subtype. CAV1 involved in the perpetuation of cell phenotype-overexpressing anti-stress DCT molecule supports the concept that CAV1 functions as a tumor suppressor in early stages of melanoma. DCT is a regulator of the CAV1-associated structures and is possibly a new molecular player in CAV1-mediated processes in melanoma.

QSAR Approaches Applied to Antidepressants Induced Neurogenesis--in vivo and in silico Applications.

Brain presents very complex advanced protective mechanisms. However, these mechanisms occasionally fail due to risk factors represented by genetic, environmental or social stress and consequently, severe psychiatric disorders such as depression, schizophrenia or psychotic depression are induced. Under such circumstances, latest strategies in experimental and in silico neuroscience consider essential to identify new applications of already clinically-approved drugs for the treatment of psychiatric disorders but also as promoters of neurogenesis and neurites outgrowth. Results of recent studies suggested that antidepressants are able to induce neurogenesis and neurites outgrowth by their agonistic effects on 5-hydroxytryptamine receptor (5-HT), especially 5-HT 1A, and sigma1 receptor (σ1R), but many molecular aspects of these processes are still unclear. Here we present structural aspects of molecular complexes (5-HT 1A and σ1R and their ligands) revealed by experimental and in silico studies. Here we present the chemical structures-biological activity relationship (SAR) of these molecules revealed by recent experimental and in silico studies, offering a new perspective on the antidepressants mechanism as neurogenesis and neurites outgrowth promoters.

Decapping Scavenger (DcpS) enzyme: advances in its structure, activity and roles in the cap-dependent mRNA metabolism.

Decapping Scavenger (DcpS) enzyme rids eukaryotic cells of short mRNA fragments containing the 5' mRNA cap structure, which appear in the 3'→5' mRNA decay pathway, following deadenylation and exosome-mediated turnover. The unique structural properties of the cap, which consists of 7-methylguanosine attached to the first transcribed nucleoside by a triphosphate chain (m(7)GpppN), guarantee its resistance to non-specific exonucleases. DcpS enzymes are dimers belonging to the Histidine Triad (HIT) superfamily of pyrophosphatases. The specific hydrolysis of m(7)GpppN by DcpS yields m(7)GMP and NDP. By precluding inhibition of other cap-binding proteins by short m(7)GpppN-containing mRNA fragments, DcpS plays an important role in the cap-dependent mRNA metabolism. Over the past decade, lots of new structural, biochemical and biophysical data on DcpS has accumulated. We attempt to integrate these results, referring to DcpS enzymes from different species. Such a synergistic characteristic of the DcpS structure and activity might be useful for better understanding of the DcpS catalytic mechanism, its regulatory role in gene expression, as well as for designing DcpS inhibitors of potential therapeutic application, e.g. in spinal muscular atrophy.

Value of dopachrome tautomerase detection in the assessment of melanocytic tumors.

Dopachrome tautomerase (DCT) and tyrosinase (Tyr) are melanogenic enzymes and structurally related melanosomal proteins. The present study investigates DCT expression comparatively with Tyr, the most tested melanoma biomarker, aiming to evaluate DCT potential in the assessment of melanocytic tumors and gain insights into the molecular and pathological characterization of DCT-phenotype in tumor progression. DCT and Tyr are simultaneously analyzed in melanoma cell lines by semiquantitative RT-PCR, western blot, and N-glycan analysis, and in cell populations of melanocytic tumors by immunohistofluorescence using a novel anti-hDCT antibody against an extended sequence within DCT luminal domain. DCT, unlike Tyr, is fully processed along the secretory pathway in both pigmented and amelanotic melanoma cells. In 53 nevi and 116 primary malignant melanomas, 81% and 52%, respectively, are DCT+/Tyr+, showing that DCT is a stable antigen, retained by most tumors and partially expressed in Tyr-negative cell populations. The DCT/Tyr disjunction is a process correlated with melanocyte neoplastic transformation and malignant progression. A tumor architecture--DCT-phenotype-containing DCT+/Tyr- cell populations selected into the innermost dermis from double-positive cells is detected in 35% of DCT+/Tyr+ specimens. The DCT-phenotype is associated with enhanced neurotization in benign nevi and with ulceration in thin malignant melanomas. The intradermal DCT+/Tyr- clones in superficial melanomas acquire the expression and specific subcellular distribution of unfavorable prognostic markers. DCT assessment shows specific antigen patterns with potential significance in the outcome of melanocytic lesions, connecting DCT, a mediator of a melanoma stress-resistant pathway, and an antiapoptotic molecule to DCT- phenotypes that are possibly more stable and stress resistant.

More effective DPP4 inhibitors as antidiabetics based on sitagliptin applied QSAR and clinical methods.

Xanthine-based molecules such as serine protease dipeptidyl peptidase 4 (DPP4) inhibitors are compounds often used in improving glycemic control in type 2 diabetic patients and also used for their effects as mild stimulants and as bronchodilators, notably in treating asthma symptoms. Here, we aim to better understand the molecular features affecting activity of xanthine-based DPP4 inhibitors such as sitagliptin and related compounds and use these features to de novo predict improved sitagliptin derivatives. To this end, we performed a clinical study to examine the efficacy and safety of once-daily 100 mg oral sitagliptin as monotherapy in Romanian patients with type 2 diabetes. This study indicates that sitagliptin effectively decreases the glycemic level and provides very good glycemic equilibrium. To predict putative new drugs with identical pharmacological effects at lower dosages, we generate QSAR models based on compound series containing 35 DPP4 inhibitors. We establish that the physicochemical parameters critical for DPP4 inhibitory activity are: hydrophobicity described by the logarithm of the octanol/water partition coefficient, counts of rotatable bonds, hydrogen bond donor and acceptor atoms, and topological polar surface area. The predictive power of our QSAR models is indicated by significant values of statistical coefficients: cross-validated correlation q2 (0.77), fitted correlation coefficient r2 (0.85) and standard error of prediction (0.34). Based on the established QSAR equations, we propose and analyse 19 new sitagliptin derivatives with possibly improved pharmacological effect as DPP4 inhibitors.

Analysis of decapping scavenger cap complex using modified cap analogs reveals molecular determinants for efficient cap binding.

Decapping scavenger (DcpS) assists in precluding inhibition of cap-binding proteins by hydrolyzing cap species remaining after mRNA 3'→5' degradation. Its significance was reported in splicing, translation initiation and microRNA turnover. Here we examine the structure and binding mode of DcpS from Caenorhabditis elegans (CeDcpS) using a large collection of chemically modified methylenebis(phosphonate), imidodiphosphate and phosphorothioate cap analogs. We determine that CeDcpS is a homodimer and propose high accuracy structural models of apo- and m(7) GpppG-bound forms. The analysis of CeDcpS regioselectivity uncovers that the only site of hydrolysis is located between the ß and γ phosphates. Structure-affinity relationship studies of cap analogs for CeDcpS reveal molecular determinants for efficient cap binding: a strong dependence on the type of substituents in the phosphate chain, and reduced binding affinity for either methylated hydroxyl groups of m(7) Guo or an extended triphosphate chain. Docking analysis of cap analogs in the CeDcpS active site explains how both phosphate chain mobility and the orientation in the cap-binding pocket depend on the number of phosphate groups, the substituent type and the presence of the second nucleoside. Finally, the comparison of CeDcpS with its well known human homolog provides general insights into DcpS-cap interactions.

Characterization of the anti-HBV activity of HLP1-23, a human lactoferrin-derived peptide.

Lactoferrin (Lf) was shown to exhibit its antiviral activity at an early phase of viral infection and a mechanism whereby the protein interacts with host cell surface molecules has been suggested. In this study, human Lf (HLf) and seven HLf-derived synthetic peptides (HLP) corresponding to the N-terminal domain of the native protein (1-47 amino acids sequence) were assayed for their capacity to prevent hepatitis B virus (HBV) infection and replication using the HepaRG and HepG2.2.2.15 cell lines. Of the series tested, four peptides showed 40-75% inhibition of HBV infection in HepaRG cells, HLP1-23 , containing the GRRRR cationic cluster, being the most potent. Interestingly, this cluster is one of the two glycosaminoglycan binding sites of the native HLf involved in its antiviral activity; however, the mechanism of the HLP1-23 action was different from that of the full-length protein, the peptide inhibiting HBV infection when pre-incubated with the virus, while no effect was observed on the target cells. It is suggested that the cationic cluster is sufficient for the peptide to interact stably with negatively charged residues on the virion envelope, while the absence of the second glycosaminoglycan binding site prevents its efficient attachment to the cells. In conclusion, this peptide may constitute a non-toxic approach for potential clinical applications in inhibiting HBV entry by neutralizing the viral particles.

Symmetry-restrained molecular dynamics simulations improve homology models of potassium channels.

Most crystallized homo-oligomeric ion channels are highly symmetric, which dramatically decreases conformational space and facilitates building homology models (HMs). However, in molecular dynamics (MD) simulations channels deviate from ideal symmetry and accumulate thermal defects, which complicate the refinement of HMs using MD. In this work we evaluate the ability of symmetry constrained MD simulations to improve HMs accuracy, using an approach conceptually similar to Critical Assessment of techniques for protein Structure Prediction (CASP) competition: build HMs of channels with known structure and evaluate the efficiency of proposed methods in improving HMs accuracy (measured as deviation from experimental structure). Results indicate that unrestrained MD does not improve the accuracy of HMs, instantaneous symmetrization improves accuracy but not stability of HMs during subsequent unrestrained MD, while gradually imposing symmetry constraints improves both accuracy (by 5-50%) and stability of HMs. Moreover, accuracy and stability are strongly correlated, making stability a reliable criterion in predicting the accuracy of new HMs. Proteins 2010. (c) 2009 Wiley-Liss, Inc.

Mutations in dopachrome tautomerase (Dct) affect eumelanin/pheomelanin synthesis, but do not affect intracellular trafficking of the mutant protein.

Dopachrome tautomerase (Dct) is a type I membrane protein and an important regulatory enzyme that plays a pivotal role in the biosynthesis of melanin and in the rapid metabolism of its toxic intermediates. Dct-mutant melanocytes carrying the slaty or slaty light mutations were derived from the skin of newborn congenic C57BL/6J non-agouti black mice and were used to study the effect(s) of these mutations on the intracellular trafficking of Dct and on the pigmentation of the cells. Dct activity is 3-fold lower in slaty cells compared with non-agouti black melanocytes, whereas slaty light melanocytes have a surprisingly 28-fold lower Dct activity. Homology modelling of the active site of Dct suggests that the slaty mutation [R194Q (Arg194-->Gln)] is located in the active site and may alter the ability of the enzyme to transform the substrate. Transmembrane prediction methods indicate that the slaty light mutation [G486R (Gly486-->Arg)] may result in the sliding of the transmembrane domain towards the N-terminus, thus interfering with Dct function. Chemical analysis showed that both Dct mutations increase pheomelanin and reduce eumelanin produced by melanocytes in culture. Thus the enzymatic activity of Dct may play a role in determining whether the eumelanin or pheomelanin pathway is preferred for pigment biosynthesis.

In planta secretion of a calreticulin by migratory and sedentary stages of root-knot nematode.

Esophageal secretions from endoparasitic sedentary nematodes are thought to play key roles throughout plant parasitism, in particular during the invasion of the root tissue and the initiation and maintenance of the nematode feeding site (NFS) essential for nematode development. The secretion in planta of esophageal cell-wall-degrading enzymes by migratory juveniles has been shown, suggesting a role for these enzymes in the invasion phase. Nevertheless, the secretion of an esophageal gland protein into the NFS by nematode sedentary stages has never been demonstrated. The calreticulin Mi-CRT is a protein synthesized in the esophageal glands of the root-knot nematode Meloidogyne incognita. After three-dimensional modeling of the Mi-CRT protein, a surface peptide was selected to raise specific antibodies. In planta immunolocalization showed that Mi-CRT is secreted by migratory and sedentary stage nematodes, suggesting a role for Mi-CRT throughout parasitism. During the maintenance of the NFS, the secreted Mi-CRT was localized outside the nematode at the tip of the stylet. In addition, Mi-CRT accumulation was observed along the cell wall of the giant cells that compose the feeding site, providing evidence for a nematode esophageal protein secretion into the NFS.

Statistical analysis of the protein environment of N-glycosylation sites: implications for occupancy, structure, and folding.

We recently reported statistical analysis of structural data on glycosidic linkages. Here we extend this analysis to the glycan-protein linkage, and the peptide primary, secondary, and tertiary structures around N-glycosylation sites. We surveyed 506 glycoproteins in the Protein Data Bank crystallographic database, giving 2592 glycosylation sequons (1683 occupied) and generated a database of 626 nonredundant sequons with 386 occupied. Deviations in the expected amino acid composition were seen around occupied asparagines, particularly an increased occurrence of aromatic residues before the asparagine and threonine at position +2. Glycosylation alters the asparagine side chain torsion angle distribution and reduces its flexibility. There is an elevated probability of finding glycosylation sites in which secondary structure changes. An 11-class taxonomy was developed to describe protein surface geometry around glycosylation sites. Thirty-three percent of the occupied sites are on exposed convex surfaces, 10% in deep recesses and 20% on the edge of grooves with the glycan filling the cleft. A surprisingly large number of glycosylated asparagine residues have a low accessibility. The incidence of aromatic amino acids brought into close contact with the glycan by the folding process is higher than their normal levels on the surface or in the protein core. These data have significant implications for control of sequon occupancy and evolutionary selection of glycosylation sites and are discussed in relation to mechanisms of protein fold stabilization and regional quality control of protein folding. Hydrophobic protein-glycan interactions and the low accessibility of glycosylation sites in folded proteins are common features and may be critical in mediating these functions.