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1.
Molecules ; 26(16)2021 Aug 12.
Article in English | MEDLINE | ID: mdl-34443478

ABSTRACT

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.


Subject(s)
Apelin Receptors/chemistry , High-Throughput Screening Assays/methods , Molecular Docking Simulation/methods , Receptors, G-Protein-Coupled/metabolism , Apelin/analogs & derivatives , Apelin/chemistry , Binding Sites , Biomimetics , Drug Design , Humans , Ligands , Peptides/chemistry , Protein Binding
2.
PLoS One ; 12(4): e0176479, 2017.
Article in English | MEDLINE | ID: mdl-28448610

ABSTRACT

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.


Subject(s)
Embryo, Nonmammalian/metabolism , Gene Expression Profiling , Strongylocentrotus purpuratus/embryology , Strongylocentrotus purpuratus/genetics , Acetylgalactosamine/metabolism , Amino Acid Sequence , Animals , Gene Knockdown Techniques , Models, Molecular , Mucins/metabolism , Muscle, Skeletal/embryology , Muscle, Skeletal/metabolism , Neurons/metabolism , Protein Conformation , RNA, Messenger/genetics , RNA, Messenger/metabolism , Strongylocentrotus purpuratus/cytology , Strongylocentrotus purpuratus/metabolism
3.
J Biol Chem ; 291(24): 12481-12500, 2016 Jun 10.
Article in English | MEDLINE | ID: mdl-27053106

ABSTRACT

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.


Subject(s)
Caveolin 1/genetics , Cell Proliferation/genetics , Gene Expression Regulation, Neoplastic , Intramolecular Oxidoreductases/genetics , Blotting, Western , Caveolae/metabolism , Caveolin 1/metabolism , Cell Line, Tumor , Humans , Intramolecular Oxidoreductases/metabolism , Melanoma/genetics , Melanoma/metabolism , Melanoma/pathology , Microscopy, Confocal , Microscopy, Fluorescence , Phenotype , Protein Binding , Protein Processing, Post-Translational , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction
4.
Mini Rev Med Chem ; 16(3): 230-40, 2015.
Article in English | MEDLINE | ID: mdl-26349492

ABSTRACT

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.


Subject(s)
Antidepressive Agents/adverse effects , Models, Biological , Neurogenesis , Quantitative Structure-Activity Relationship , Antidepressive Agents/pharmacology , Humans , Neurogenesis/drug effects , Protein Binding/drug effects , Receptor, Serotonin, 5-HT1A/metabolism , Receptors, sigma/metabolism , Sigma-1 Receptor
5.
Int J Mol Sci ; 15(11): 21381-400, 2014 Nov 18.
Article in English | MEDLINE | ID: mdl-25411801

ABSTRACT

Overexpression of mitotic arrest-deficient proteins Mad1 and Mad2, two components of spindle assembly checkpoint, is a risk factor for chromosomal instability (CIN) and a trigger of many genetic disorders. Mad2 transition from inactive open (O-Mad2) to active closed (C-Mad2) conformations or Mad2 binding to specific partners (cell-division cycle protein 20 (Cdc20) or Mad1) were targets of previous pharmacogenomics studies. Here, Mad2 binding to Cdc20 and the interconversion rate from open to closed Mad2 were predicted and the molecular features with a critical contribution to these processes were determined by extending the quantitative structure-activity relationship (QSAR) method to large-size proteins such as Mad2. QSAR models were built based on available published data on 23 Mad2 mutants inducing CIN-related functional changes. The most relevant descriptors identified for predicting Mad2 native and mutants action mechanism and their involvement in genetic disorders are the steric (van der Waals area and solvent accessible area and their subdivided) and energetic van der Waals energy descriptors. The reliability of our QSAR models is indicated by significant values of statistical coefficients: Cross-validated correlation q2 (0.53-0.65) and fitted correlation r2 (0.82-0.90). Moreover, based on established QSAR equations, we rationally design and analyze nine de novo Mad2 mutants as possible promoters of CIN.


Subject(s)
Genetic Diseases, Inborn/genetics , Mad2 Proteins/genetics , Mutation/genetics , Carcinogenesis/genetics , Cdc20 Proteins/genetics , Gene Expression Regulation/genetics , Humans , M Phase Cell Cycle Checkpoints/genetics , Protein Structure, Tertiary/genetics , Quantitative Structure-Activity Relationship
6.
Biochim Biophys Acta ; 1839(6): 452-62, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24742626

ABSTRACT

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.


Subject(s)
Endoribonucleases/chemistry , Endoribonucleases/metabolism , RNA Caps/chemistry , RNA, Messenger/metabolism , Endoribonucleases/genetics , Humans , RNA Cap-Binding Proteins/metabolism , RNA Caps/metabolism
7.
Melanoma Res ; 24(3): 219-36, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24709887

ABSTRACT

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.


Subject(s)
Biomarkers, Tumor/metabolism , Intramolecular Oxidoreductases/metabolism , Melanocytes/enzymology , Melanoma/enzymology , Nevus, Pigmented/enzymology , Skin Neoplasms/enzymology , Biomarkers, Tumor/genetics , Fluorescent Antibody Technique , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , HEK293 Cells , HeLa Cells , Humans , Intramolecular Oxidoreductases/genetics , Melanocytes/pathology , Melanoma/genetics , Melanoma/pathology , Monophenol Monooxygenase/metabolism , Nevus, Pigmented/genetics , Nevus, Pigmented/pathology , Phenotype , Predictive Value of Tests , Prognosis , RNA Interference , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Transfection
8.
Curr Comput Aided Drug Des ; 10(3): 237-49, 2014.
Article in English | MEDLINE | ID: mdl-25756669

ABSTRACT

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.


Subject(s)
Diabetes Mellitus, Type 2/drug therapy , Dipeptidyl-Peptidase IV Inhibitors/pharmacology , Hypoglycemic Agents/pharmacology , Pyrazines/pharmacology , Triazoles/pharmacology , Blood Glucose/drug effects , Dipeptidyl-Peptidase IV Inhibitors/adverse effects , Dipeptidyl-Peptidase IV Inhibitors/chemistry , Drug Design , Female , Humans , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Hypoglycemic Agents/adverse effects , Hypoglycemic Agents/chemistry , Male , Middle Aged , Models, Molecular , Pyrazines/adverse effects , Pyrazines/chemistry , Quantitative Structure-Activity Relationship , Romania , Sitagliptin Phosphate , Treatment Outcome , Triazoles/adverse effects , Triazoles/chemistry
9.
FEBS J ; 280(24): 6508-27, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24119043

ABSTRACT

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.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , Gene Expression Regulation , Pyrophosphatases/metabolism , RNA Cap Analogs/metabolism , RNA Caps/metabolism , RNA, Messenger/metabolism , Amino Acid Sequence , Animals , Binding Sites , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans Proteins/genetics , Catalytic Domain , Chromatography, Gel , Chromatography, High Pressure Liquid , Humans , Hydrolysis , Models, Molecular , Molecular Sequence Data , Pyrophosphatases/chemistry , Pyrophosphatases/genetics , RNA Cap Analogs/chemistry , RNA Cap Analogs/genetics , RNA Caps/chemistry , RNA Caps/genetics , RNA, Messenger/genetics
10.
J Med Virol ; 85(5): 780-8, 2013 May.
Article in English | MEDLINE | ID: mdl-23508903

ABSTRACT

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.


Subject(s)
Antiviral Agents/pharmacology , Hepatitis B virus/drug effects , Lactoferrin/pharmacology , Peptide Fragments/pharmacology , Cell Line , Hepatitis B virus/physiology , Hepatocytes/virology , Humans , Virus Internalization/drug effects , Virus Replication/drug effects
11.
J Enzyme Inhib Med Chem ; 28(2): 350-9, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23116520

ABSTRACT

Dithiocarbamates (DTC) are promising compounds with potential applications in antitumoral and glaucoma therapy. Our aim is to understand molecular features affecting DTC interaction with carbonic anhydrases (CAs), zinc-containing enzymes maintaining acid-base balance in blood and other tissues. To this end, we generate QSAR models based on a compound series containing 25 DTC, inhibitors of four human (h) CAs isoforms: hCA I, II, IX and XII. We establish that critical physicochemical parameters for DTC inhibitory activity are: hydrophobic, electronic, steric, topological and shape. The predictive power of our QSAR models is indicated by significant values of statistical coefficients: cross-validated correlation q(2) (0.55-0.73), fitted correlation r(2) (0.75-0.84) and standard error of prediction (0.47-0.23). Based on the established QSAR equations, we analyse 22 new DTC derivatives and identify DTC dicarboxilic acids derivatives and their esters as potentially improved inhibitors of CA I, II, IX and XII.


Subject(s)
Carbonic Anhydrase Inhibitors/pharmacology , Carbonic Anhydrases/metabolism , Computer Simulation , Quantitative Structure-Activity Relationship , Thiocarbamates/pharmacology , Algorithms , Carbonic Anhydrase Inhibitors/chemical synthesis , Carbonic Anhydrase Inhibitors/chemistry , Carbonic Anhydrases/isolation & purification , Dose-Response Relationship, Drug , Humans , Models, Molecular , Molecular Structure , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/isolation & purification , Protein Isoforms/metabolism , Thiocarbamates/chemical synthesis , Thiocarbamates/chemistry
12.
Mol Biosyst ; 8(5): 1418-25, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22373544

ABSTRACT

Antidepressants and antipsychotics are psychiatric agents used for the treatment of various types of psychiatric diseases. Although currently among the most commonly prescribed drugs, their effectiveness and adverse effects are the topic of many studies and controversial claims. Here we generate QSAR models based on compounds series including 20 drugs recommended for two critical psychiatric diseases: depression and schizophrenia and we use these QSAR models to predict the biological activity of these 20 antidepressants and antipsychotics. We establish the membrane ions' contributions (sodium, potassium, calcium and iron) mediated by water to the antagonism of these drugs at the 5-HT1A receptor. The reliability of our QSAR models in predicting compounds activity is indicated by significant values for cross-validated correlation q² (0.60-0.76) and fitted correlation r² (0.96-0.98) coefficients. Our results indicate that potassium, calcium and iron play a key role for the antagonistic activity of drugs at the 5-HT1A receptor. Moreover, based on the established QSAR equations, we analysed 24 new escitalopram derivatives as possibly improved antidepressants targeting the 5-HT1A receptor. We identified that the presence of methyl groups and hydrogen atoms improves antidepressant activity while the simultaneous presence of ethyl, propyl or halogens decreased drastically antidepressant activity at the 5-HT1A site.


Subject(s)
Antipsychotic Agents/pharmacology , Cell Membrane/metabolism , Ions/pharmacology , Quantitative Structure-Activity Relationship , Receptor, Serotonin, 5-HT1A/metabolism , Antidepressive Agents/chemistry , Antidepressive Agents/pharmacology , Catalytic Domain , Citalopram/chemistry , Citalopram/pharmacology , Models, Molecular , Serotonin 5-HT1 Receptor Antagonists/chemistry , Serotonin 5-HT1 Receptor Antagonists/pharmacology , Water
13.
Mol Biosyst ; 8(2): 587-94, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22086548

ABSTRACT

Antimicrobial peptides are drugs used against a wide range of pathogens which present a great advantage: in contrast with antibiotics they do not develop resistance. The wide spectrum of antimicrobial peptides advertises them in the research and pharmaceutical industry as attractive starting points for obtaining new, more effective analogs. Here we predict the antimicrobial activity against Bacillus subtilis (expressed as minimal inhibitory concentration values) for 33 mastoparan analogs and their new derivatives by a non-aligned 3D-QSAR (quantitative structure-activity relationship) method. We establish the contribution to antimicrobial activity of molecular descriptors (hydrophobicity, hydrogen bond donor and steric), correlated with contributions from the membrane environment (sodium, potassium, chloride). Our best QSAR models show significant cross-validated correlation q(2) (0.55-0.75), fitted correlation r(2) (greater than 0.90) coefficients and standard error of prediction SDEP (less than 0.250). Moreover, based on our most accurate 3D-QSAR models, we propose nine new mastoparan analogs, obtained by computational mutagenesis, some of them predicted to have significantly improved antimicrobial activity compared to the parent compound.


Subject(s)
Bacillus subtilis/drug effects , Peptides/pharmacology , Quantitative Structure-Activity Relationship , Wasp Venoms/pharmacology , Anti-Infective Agents/pharmacology , Chlorides/chemistry , Computer Simulation , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Intercellular Signaling Peptides and Proteins , Microbial Sensitivity Tests , Models, Molecular , Potassium/chemistry , Sodium/chemistry
14.
Channels (Austin) ; 5(1): 23-33, 2011.
Article in English | MEDLINE | ID: mdl-21084863

ABSTRACT

Mechanosensitive TREK channels belong to the family of K2P channels, a family of widely distributed, well modulated channels that uniquely have two similar or identical subunits, each with two TM1-P-TM2 motifs. Our goal is to build viable structural models of TREK channels, as representatives of K2P channels family. The structures available to be used as templates belong to the 2TM channels superfamily. These have low sequence similarity and different structural features: four symmetrically arranged subunits, each having one TM1-P-TM2 motif. Our model building strategy used two subunits of the template (KcsA) to build one subunit of the target (TREK-1). Our models of the Closed channel were adjusted to differ substantially from those of the template, e.g., TM2 of the 2nd repeat is near the axis of the pore whereas TM2 of the 1st repeat is far from the axis. Segments linking the two repeats and immediately following the last TM segment were modeled ab initio as α-helices based on helical periodicities of hydrophobic and hydrophilic residues, highly conserved and poorly conserved residues, and statistically related positions from multiple sequence alignments. The models were further refined by two-fold symmetry-constrained MD simulations using a protocol we developed previously. We also built models of the Open state and suggest a possible tension-activated gating mechanism characterized by helical motion with two-fold symmetry. Our models are consistent with deletion/truncation mutagenesis and thermodynamic analysis of gating described in the accompanying paper.


Subject(s)
Ion Channel Gating , Potassium Channels, Tandem Pore Domain/chemistry , Amino Acid Sequence , Computer Simulation , Databases, Protein , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Molecular Sequence Data , Potassium Channels, Tandem Pore Domain/metabolism , Protein Conformation , Protein Subunits , Structure-Activity Relationship
15.
Channels (Austin) ; 5(1): 34-42, 2011.
Article in English | MEDLINE | ID: mdl-21057213

ABSTRACT

TREK-1, a mechanosensitive K channel from the two-pore family (K(2)P), is involved in protective regulation of the resting potential in CNS neurons and other tissues. The structure of TREK-1 and the basis of its sensitivity to stretch and variety of lipid-soluble factors remain unknown. Using existing K channel structures as modeling templates, TREK-1 was envisioned as a two-fold symmetrical complex with the gate formed primarily by the centrally positioned TM2b helices of the second homologous repeat. Opening was modeled as a conical expansion of the barrel separating TM2b's accompanied by extension of TM2a helices with the cytoplasmic TM2a-TM1b connector. Seeking first experimental support to the models we have accomplished thermodynamic analysis of mouse TREK-1 gating and functional testing of several deletion mutants. The predicted increase of the channel in-plane area (ΔA) of ~5 nm(2) in models was supported by the experimental ΔA of ~4 nm(2) derived from the slope of open probability versus membrane tension in HEK-293T cells and their cytoskeleton-depleted blebs. In response to steps of suction, wild-type channel produced transient currents in cell-attached patches and mostly sustained currents upon patch excision. TREK-1 motifs not present in canonical K channels include divergent cytoplasmic N- and C-termini, and a characteristic 50-residue extracellular loop in the first homologous repeat. Deletion of the extracellular loop (Δ76-124) reduced the average current density in patches, increased spontaneous activity and generated a larger sub-population of high-conductance channels, while activation by tension augmented by arachidonic acid was fully retained. Further deletion of the C-terminal end (Δ76-124/Δ334-411) removed voltage dependency but otherwise produced no additional effect. In an attempt to generate a cysteine-free version of the channel, we mutated two remaining cysteines 159 and 219 in the transmembrane region. C219A did not compromise channel activity, whereas the C159A/S mutants were essentially inactive. Treatment with ß-mercaptoethanol suggested that none of these cysteines form functionally-important disulfides.


Subject(s)
Ion Channel Gating , Mechanotransduction, Cellular , Models, Molecular , Potassium Channels, Tandem Pore Domain/metabolism , Sequence Deletion , Thermodynamics , Animals , Cysteine , HEK293 Cells , Humans , Membrane Potentials , Mercaptoethanol/pharmacology , Mice , Mutagenesis, Site-Directed , Patch-Clamp Techniques , Potassium Channels, Tandem Pore Domain/chemistry , Potassium Channels, Tandem Pore Domain/drug effects , Potassium Channels, Tandem Pore Domain/genetics , Pressure , Protein Conformation , Protein Structure, Tertiary , Structure-Activity Relationship , Transfection
16.
Proteins ; 78(4): 932-49, 2010 Mar.
Article in English | MEDLINE | ID: mdl-19902533

ABSTRACT

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.


Subject(s)
Molecular Dynamics Simulation , Potassium Channels/chemistry , Models, Molecular , Protein Structure, Secondary , Structural Homology, Protein
17.
Mol Plant Pathol ; 8(3): 293-305, 2007 May.
Article in English | MEDLINE | ID: mdl-20507500

ABSTRACT

SUMMARY The cell wall, a strong extraprotoplasmic layer surrounding plant cells that mainly consists of a variety of polysaccharides, constitutes a major barrier for potential parasites. Plant-parasitic nematodes are well equipped to overcome this barrier as they produce and secrete cell-wall-degrading enzymes. Expression profiling of various life stages of the potato cyst nematode Globodera rostochiensis revealed a novel pectate lyase gene (Gr-pel2, 759 bp). The Gr-PEL2 protein showed highest similarity to pectate lyases from the facultative plant-parasitic nematodes Bursaphelenchus mucronatus and B. xylophilus and the soil-inhabiting saprophytic Streptomyces and Frankia species (i.e. 40-42% identity and 58-60% similarity), whereas only a remote relatedness to the previously identified Gr-PEL1 was observed (i.e. 28% identity and 43% similarity). Transient expression of Gr-pel2 in leaves of Nicotiana benthamiana resulted in severe malformations of the infiltrated tissues, not relating to maceration and soft rot symptoms. Ca(2+) is known to be essential for pectate lyase activity, and the most likely calcium-binding site was identified in the Gr-PEL2 protein by combining homology modelling of the three-dimensional structure, site-directed mutagenesis and transient expression in leaves. A highly charged cleft in Gr-PEL2, which is likely to be involved in substrate binding and which is also significantly more hydrophobic in Gr-PEL1, was shown to be essential for protein activity. Our results underline the broad spectrum of pectate lyases and cell-wall-degrading enzymes necessary for successful parasitism by cyst nematodes.

18.
J Mol Graph Model ; 25(1): 37-45, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16325439

ABSTRACT

We present here a neural networks method designed to predict biological activity based on a local representation of the ligand. The compounds of the series are represented by a vector mapping for each of four substituent properties: volume, log P, dipole moment and a simple 'steric' parameter relating to its shape. This ligand representation was tested using neural networks on a set of 42 cyclic-urea derivatives, inhibiting HIV-1 protease. The leave-one-out cross-validation using all descriptors in the input gave a correlation factor between prediction and experiment of 0.76 for the overall set and 0.88 when three outliers were left out. To rank the significance of the four descriptors, we further tested all combinations of two and three parameters for each substituent, using two disjunctive testing sets of five inhibitors. In these sets, vectors with extreme descriptor values were used either in the training or the testing set (sets A and B, respectively). The method is a very good interpolator (set A, 95+/-2% accuracy) but a less effective extrapolator (set B, 85+/-2% accuracy). Generally, the combinations including the 'steric' parameter predict better than average, while those containing the volume are less effective. The best prediction, 98.8+/-1.2%, was obtained when log P, the dipole and the steric parameter were used on set A. At the opposite end, the lowest ranked descriptor set was obtained when replacing log P with the volume, giving 92.3+/-6.7% accuracy over the set A.


Subject(s)
Combinatorial Chemistry Techniques , HIV Protease Inhibitors/chemistry , HIV Protease Inhibitors/pharmacology , Neural Networks, Computer , Quantitative Structure-Activity Relationship , Ligands , Models, Molecular , Urea/chemistry
19.
Biochem J ; 391(Pt 2): 249-59, 2005 Oct 15.
Article in English | MEDLINE | ID: mdl-15960609

ABSTRACT

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.


Subject(s)
Intramolecular Oxidoreductases/genetics , Intramolecular Oxidoreductases/metabolism , Melanins/biosynthesis , Melanocytes/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cells, Cultured , Gene Expression Regulation, Enzymologic , Melanocytes/cytology , Mice , Molecular Sequence Data , Protein Conformation , Protein Transport , Sequence Homology, Amino Acid
20.
FEBS Lett ; 579(11): 2451-7, 2005 Apr 25.
Article in English | MEDLINE | ID: mdl-15848187

ABSTRACT

Southern analysis showed that Gr-EXPB1, a functional expansin from the potato cyst nematode Globodera rostochiensis, is member of a multigene family, and EST data suggest expansins to be present in other plant parasitic nematodes as well. Homology modeling predicted that Gr-EXPB1 domain 1 (D1) has a flat beta-barrel structure with surface-exposed aromatic rings, whereas the 3D structure of Gr-EXPB1-D2 was remarkably similar to plant expansins. Gr-EXPB1 shows highest sequence similarity to two extracellular proteins from saprophytic soil-inhabiting Actinobacteria, and includes a bacterial type II carbohydrate-binding module. These results support the hypothesis that a number of pathogenicity factors of cyst nematodes is of procaryotic origin and were acquired by horizontal gene transfer.


Subject(s)
Helminth Proteins/chemistry , Helminth Proteins/metabolism , Models, Molecular , Solanum tuberosum/parasitology , Tylenchida/chemistry , Tylenchida/genetics , Amino Acid Sequence , Animals , Blotting, Southern , DNA, Complementary/genetics , Evolution, Molecular , Genome , Helminth Proteins/genetics , Molecular Sequence Data , Protein Structure, Tertiary , Sequence Alignment , Nicotiana/genetics
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