Motivated proteins: a web application for studying small three-dimensional protein motifs

<b>BACKGROUND:</b> Small loop-shaped motifs are common constituents of the three-dimensional structure of proteins. Typically they comprise between three and seven amino acid residues, and are defined by a combination of dihedral angles and hydrogen bonding partners. The most abundant of these are alphabeta-motifs, asx-motifs, asx-turns, beta-bulges, beta-bulge loops, beta-turns, nests, niches, Schellmann loops, ST-motifs, ST-staples and ST-turns.We have constructed a database of such motifs from a range of high-quality protein structures and built a web application as a visual interface to this. <b>DESCRIPTION:</b> The web application, Motivated Proteins, provides access to these 12 motifs (with 48 sub-categories) in a database of over 400 representative proteins. Queries can be made for specific categories or sub-categories of motif, motifs in the vicinity of ligands, motifs which include part of an enzyme active site, overlapping motifs, or motifs which include a particular amino acid sequence. Individual proteins can be specified, or, where appropriate, motifs for all proteins listed. The results of queries are presented in textual form as an (X)HTML table, and may be saved as parsable plain text or XML. Motifs can be viewed and manipulated either individually or in the context of the protein in the Jmol applet structural viewer. Cartoons of the motifs imposed on a linear representation of protein secondary structure are also provided. Summary information for the motifs is available, as are histograms of amino acid distribution, and graphs of dihedral angles at individual positions in the motifs. <b>CONCLUSION:</b> Motivated Proteins is a publicly and freely accessible web application that enables protein scientists to study small three-dimensional motifs without requiring knowledge of either Structured Query Language or the underlying database schem

MSH3 polymorphisms and protein levels affect CAG repeat instability in huntington's disease mice

Expansions of trinucleotide CAG/CTG repeats in somatic tissues are thought to contribute to ongoing disease progression through an affected individual's life with Huntington's disease or myotonic dystrophy. Broad ranges of repeat instability arise between individuals with expanded repeats, suggesting the existence of modifiers of repeat instability. Mice with expanded CAG/CTG repeats show variable levels of instability depending upon mouse strain. However, to date the genetic modifiers underlying these differences have not been identified. We show that in liver and striatum the R6/1 Huntington's disease (HD) (CAG)~100 transgene, when present in a congenic C57BL/6J (B6) background, incurred expansion-biased repeat mutations, whereas the repeat was stable in a congenic BALB/cByJ (CBy) background. Reciprocal congenic mice revealed the Msh3 gene as the determinant for the differences in repeat instability. Expansion bias was observed in congenic mice homozygous for the B6 Msh3 gene on a CBy background, while the CAG tract was stabilized in congenics homozygous for the CBy Msh3 gene on a B6 background. The CAG stabilization was as dramatic as genetic deficiency of Msh2. The B6 and CBy Msh3 genes had identical promoters but differed in coding regions and showed strikingly different protein levels. B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability. The DHFR protein, which is divergently transcribed from a promoter shared by the Msh3 gene, did not show varied levels between mouse strains. Thus, naturally occurring MSH3 protein polymorphisms are modifiers of CAG repeat instability, likely through variable MSH3 protein stability. Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with polymorphisms of DNA repair genes may have prognostic implications for various repeat-associated diseases

Requirement for the N-Terminal Coiled-Coil Domain for Expression and Function, but not Subunit Interaction of, the ADPR-Activated TRPM2 Channel

Transient receptor potential melastatin 2 (TRPM2) proteins form multiple-subunit complexes, most likely homotetramers, which operate as Ca2+-permeable, nonselective cation channels activated by intracellular ADP-ribose (ADPR) and oxidative stress. Each TRPM2 channel subunit is predicted to contain two coiled-coil (CC) domains, one in the N-terminus and the other in the C-terminus. Our recent study has shown that the C-terminal CC domain plays an important, but not exclusive, role in the TRPM2 channel assembly. This study aimed to examine the potential role of the N-terminal CC domain. Domain deletion dramatically reduced protein expression and abolished ADPR-evoked currents but did not alter the subunit interaction. Deletion of both CC domains strongly attenuated the subunit interaction, confirming that the C-terminal CC domain is critical in the subunit interaction. Glutamine substitutions into individual hydrophobic residues at positions a and d in the heptad repeats to disrupt the CC formation had no effect on protein expression, subunit interaction, or ADPR-evoked currents. Mutation of Ile658 to glutamine, which did not perturb the CC formation, decreased ADPR-evoked currents without affecting protein expression, subunit interaction, or membrane trafficking. These results collectively suggest the requirement for the N-terminal CC domain for protein expression and function, but not subunit interaction, of the TRPM2 channel

Combination of RGD Compound and Low-Dose Paclitaxel Induces Apoptosis in Human Glioblastoma Cells

) peptide, to human glioblastoma U87MG cells with combination of low dose Paclitaxel (PTX) pre-treatment to augment therapeutic activity for RGD peptide-induced apoptosis. peptide induced U87MG programmed cell death. The increased expression of PTX-induced integrin-αvβ3 was correlated with the enhanced apoptosis in U87MG cells.This study provides a novel concept of targeting integrin-αvβ3 with RGD peptides in combination with low-dose PTX pre-treatment to improve efficiency in human glioblastoma treatment

NetTurnP – Neural Network Prediction of Beta-turns by Use of Evolutionary Information and Predicted Protein Sequence Features

UNLABELLED: β-turns are the most common type of non-repetitive structures, and constitute on average 25% of the amino acids in proteins. The formation of β-turns plays an important role in protein folding, protein stability and molecular recognition processes. In this work we present the neural network method NetTurnP, for prediction of two-class β-turns and prediction of the individual β-turn types, by use of evolutionary information and predicted protein sequence features. It has been evaluated against a commonly used dataset BT426, and achieves a Matthews correlation coefficient of 0.50, which is the highest reported performance on a two-class prediction of β-turn and not-β-turn. Furthermore NetTurnP shows improved performance on some of the specific β-turn types. In the present work, neural network methods have been trained to predict β-turn or not and individual β-turn types from the primary amino acid sequence. The individual β-turn types I, I', II, II', VIII, VIa1, VIa2, VIba and IV have been predicted based on classifications by PROMOTIF, and the two-class prediction of β-turn or not is a superset comprised of all β-turn types. The performance is evaluated using a golden set of non-homologous sequences known as BT426. Our two-class prediction method achieves a performance of: MCC=0.50, Qtotal=82.1%, sensitivity=75.6%, PPV=68.8% and AUC=0.864. We have compared our performance to eleven other prediction methods that obtain Matthews correlation coefficients in the range of 0.17-0.47. For the type specific β-turn predictions, only type I and II can be predicted with reasonable Matthews correlation coefficients, where we obtain performance values of 0.36 and 0.31, respectively. CONCLUSION: The NetTurnP method has been implemented as a webserver, which is freely available at http://www.cbs.dtu.dk/services/NetTurnP/. NetTurnP is the only available webserver that allows submission of multiple sequences

Optimized Hydrophobic Interactions and Hydrogen Bonding at the Target-Ligand Interface Leads the Pathways of Drug-Designing

Weak intermolecular interactions such as hydrogen bonding and hydrophobic interactions are key players in stabilizing energetically-favored ligands, in an open conformational environment of protein structures. However, it is still poorly understood how the binding parameters associated with these interactions facilitate a drug-lead to recognize a specific target and improve drugs efficacy. To understand this, comprehensive analysis of hydrophobic interactions, hydrogen bonding and binding affinity have been analyzed at the interface of c-Src and c-Abl kinases and 4-amino substituted 1H-pyrazolo [3, 4-d] pyrimidine compounds.In-silico docking studies were performed, using Discovery Studio software modules LigandFit, CDOCKER and ZDOCK, to investigate the role of ligand binding affinity at the hydrophobic pocket of c-Src and c-Abl kinase. Hydrophobic and hydrogen bonding interactions of docked molecules were compared using LigPlot program. Furthermore, 3D-QSAR and MFA calculations were scrutinized to quantify the role of weak interactions in binding affinity and drug efficacy.The in-silico method has enabled us to reveal that a multi-targeted small molecule binds with low affinity to its respective targets. But its binding affinity can be altered by integrating the conformationally favored functional groups at the active site of the ligand-target interface. Docking studies of 4-amino-substituted molecules at the bioactive cascade of the c-Src and c-Abl have concluded that 3D structural folding at the protein-ligand groove is also a hallmark for molecular recognition of multi-targeted compounds and for predicting their biological activity. The results presented here demonstrate that hydrogen bonding and optimized hydrophobic interactions both stabilize the ligands at the target site, and help alter binding affinity and drug efficacy

Griseofulvin stabilizes microtubule dynamics, activates p53 and inhibits the proliferation of MCF-7 cells synergistically with vinblastine

<p>Abstract</p> <p>Background</p> <p>Griseofulvin, an antifungal drug, has recently been shown to inhibit proliferation of various types of cancer cells and to inhibit tumor growth in athymic mice. Due to its low toxicity, griseofulvin has drawn considerable attention for its potential use in cancer chemotherapy. This work aims to understand how griseofulvin suppresses microtubule dynamics in living cells and sought to elucidate the antimitotic and antiproliferative action of the drug.</p> <p>Methods</p> <p>The effects of griseofulvin on the dynamics of individual microtubules in live MCF-7 cells were measured by confocal microscopy. Immunofluorescence microscopy, western blotting and flow cytometry were used to analyze the effects of griseofulvin on spindle microtubule organization, cell cycle progression and apoptosis. Further, interactions of purified tubulin with griseofulvin were studied <it>in vitro </it>by spectrophotometry and spectrofluorimetry. Docking analysis was performed using autodock4 and LigandFit module of Discovery Studio 2.1.</p> <p>Results</p> <p>Griseofulvin strongly suppressed the dynamic instability of individual microtubules in live MCF-7 cells by reducing the rate and extent of the growing and shortening phases. At or near half-maximal proliferation inhibitory concentration, griseofulvin dampened the dynamicity of microtubules in MCF-7 cells without significantly disrupting the microtubule network. Griseofulvin-induced mitotic arrest was associated with several mitotic abnormalities like misaligned chromosomes, multipolar spindles, misegregated chromosomes resulting in cells containing fragmented nuclei. These fragmented nuclei were found to contain increased concentration of p53. Using both computational and experimental approaches, we provided evidence suggesting that griseofulvin binds to tubulin in two different sites; one site overlaps with the paclitaxel binding site while the second site is located at the αβ intra-dimer interface. In combination studies, griseofulvin and vinblastine were found to exert synergistic effects against MCF-7 cell proliferation.</p> <p>Conclusions</p> <p>The study provided evidence suggesting that griseofulvin shares its binding site in tubulin with paclitaxel and kinetically suppresses microtubule dynamics in a similar manner. The results revealed the antimitotic mechanism of action of griseofulvin and provided evidence suggesting that griseofulvin alone and/or in combination with vinblastine may have promising role in breast cancer chemotherapy.</p

Sulfhydryl Modification Induces Calcium Entry through IP3-Sensitive Store-Operated Pathway in Activation-Dependent Human Neutrophils

As the first line of host defense, neutrophils are stimulated by pro-inflammatory cytokines from resting state, facilitating the execution of immunomodulatory functions in activation state. Sulfhydryl modification has a regulatory role in a wide variety of physiological functions through mediation of signaling transductions in various cell types. Recent research suggested that two kinds of sulfhydryl modification, S-nitrosylation by exogenous nitric oxide (NO) and alkylation by N-ethylmaleimide (NEM), could induce calcium entry through a non-store-operated pathway in resting rat neutrophils and DDT1MF-2 cells, while in active human neutrophils a different process has been observed by us. In the present work, data showed that NEM induced a sharp rising of cytosolic calcium concentration ([Ca2+]c) without external calcium, followed by a second [Ca2+]c increase with readdition of external calcium in phorbol 12-myristate 13-acetate (PMA)-activated human neutrophils. Meanwhile, addition of external calcium did not cause [Ca2+]c change of Ca2+-free PMA-activated neutrophils before application of NEM. These data indicated that NEM could induce believable store-operated calcium entry (SOCE) in PMA-activated neutrophils. Besides, we found that sodium nitroprusside (SNP), a donor of exogenous NO, resulted in believable SOCE in PMA-activated human neutrophils via S-nitrosylation modification. In contrast, NEM and SNP have no effect on [Ca2+]c of resting neutrophils which were performed in suspension. Furthermore, 2-Aminoethoxydiphenyl borate, a reliable blocker of SOCE and an inhibitor of inositol 1,4,5-trisphosphate (IP3) receptor, evidently abolished SNP and NEM-induced calcium entry at 75 µM, while preventing calcium release in a concentration-dependent manner. Considered together, these results demonstrated that NEM and SNP induced calcium entry through an IP3-sensitive store-operated pathway of human neutrophils via sulfhydryl modification in a PMA-induced activation-dependent manner

Identification of calcium-binding proteins associated with the human sperm plasma membrane

<p>Abstract</p> <p>Background</p> <p>The precise composition of the human sperm plasma membrane, the molecular interactions that define domain specific functions, and the regulation of membrane associated proteins during the capacitation process, still remain to be fully understood. Here, we investigated the repertoire of calcium-regulated proteins associated with the human sperm plasma membrane.</p> <p>Methods</p> <p>Surface specific radioiodination was combined with two-dimensional gel electrophoresis, a 45Ca-overlay assay, computer assisted image analysis and mass spectrometry to identify calcium-binding proteins exposed on the human sperm surface.</p> <p>Results</p> <p>Nine acidic 45Ca-binding sperm proteins were excised from stained preparative 2D gels and identified by mass spectrometry. Five of the calcium binding proteins; HSPA2 (HSP70-1), HSPA5 (Bip), HYOU1 (ORP150), serum amyloid P-component (SAP) and protein kinase C substrate 80K-H (80K-H) were found to be accessible to Iodo-Bead catalyzed 125I-labelling on the surface of intact human sperm. Agglutination and immunofluorescence analysis confirmed that SAP is situated on the plasma membrane of intact, motile sperm as well as permeabilized cells. Western blot analysis showed increased phosphorylation of human sperm 80K-H protein following in vitro capacitation. This is the first demonstration of the 80K-H protein in a mammalian sperm.</p> <p>Conclusion</p> <p>The presence of SAP on the surface of mature sperm implies that SAP has a physiological role in reproduction, which is thought to be in the removal of spermatozoa from the female genital tract via phagocytosis. Since 80K-H is a Ca2+-sensor recently implicated in the regulation of both inositol 1,4,5-trisphosphate receptor and transient receptor potential (TRP) cation channel activities, its detection in sperm represents the first direct signaling link between PKC and store-operated calcium channels identified in human sperm.</p

Modeling of Human Prokineticin Receptors: Interactions with Novel Small-Molecule Binders and Potential Off-Target Drugs

The Prokineticin receptor (PKR) 1 and 2 subtypes are novel members of family A GPCRs, which exhibit an unusually high degree of sequence similarity. Prokineticins (PKs), their cognate ligands, are small secreted proteins of ∼80 amino acids; however, non-peptidic low-molecular weight antagonists have also been identified. PKs and their receptors play important roles under various physiological conditions such as maintaining circadian rhythm and pain perception, as well as regulating angiogenesis and modulating immunity. Identifying binding sites for known antagonists and for additional potential binders will facilitate studying and regulating these novel receptors. Blocking PKRs may serve as a therapeutic tool for various diseases, including acute pain, inflammation and cancer.Ligand-based pharmacophore models were derived from known antagonists, and virtual screening performed on the DrugBank dataset identified potential human PKR (hPKR) ligands with novel scaffolds. Interestingly, these included several HIV protease inhibitors for which endothelial cell dysfunction is a documented side effect. Our results suggest that the side effects might be due to inhibition of the PKR signaling pathway. Docking of known binders to a 3D homology model of hPKR1 is in agreement with the well-established canonical TM-bundle binding site of family A GPCRs. Furthermore, the docking results highlight residues that may form specific contacts with the ligands. These contacts provide structural explanation for the importance of several chemical features that were obtained from the structure-activity analysis of known binders. With the exception of a single loop residue that might be perused in the future for obtaining subtype-specific regulation, the results suggest an identical TM-bundle binding site for hPKR1 and hPKR2. In addition, analysis of the intracellular regions highlights variable regions that may provide subtype specificity