Diverse Family Of Proteins Research Articles

PocketZebra: a web-server for automated selection and classification of subfamily-specific binding sites by bioinformatic analysis of diverse protein families.

The new web-server pocketZebra implements the power of bioinformatics and geometry-based structural approaches to identify and rank subfamily-specific binding sites in proteins by functional significance, and select particular positions in the structure that determine selective accommodation of ligands. A new scoring function has been developed to annotate binding sites by the presence of the subfamily-specific positions in diverse protein families. pocketZebra web-server has multiple input modes to meet the needs of users with different experience in bioinformatics. The server provides on-site visualization of the results as well as off-line version of the output in annotated text format and as PyMol sessions ready for structural analysis. pocketZebra can be used to study structure–function relationship and regulation in large protein superfamilies, classify functionally important binding sites and annotate proteins with unknown function. The server can be used to engineer ligand-binding sites and allosteric regulation of enzymes, or implemented in a drug discovery process to search for potential molecular targets and novel selective inhibitors/effectors. The server, documentation and examples are freely available at http://biokinet.belozersky.msu.ru/pocketzebra and there are no login requirements.

Experimental validation of FINDSITE(comb) virtual ligand screening results for eight proteins yields novel nanomolar and micromolar binders.

BackgroundIdentification of ligand-protein binding interactions is a critical step in drug discovery. Experimental screening of large chemical libraries, in spite of their specific role and importance in drug discovery, suffer from the disadvantages of being random, time-consuming and expensive. To accelerate the process, traditional structure- or ligand-based VLS approaches are combined with experimental high-throughput screening, HTS. Often a single protein or, at most, a protein family is considered. Large scale VLS benchmarking across diverse protein families is rarely done, and the reported success rate is very low. Here, we demonstrate the experimental HTS validation of a novel VLS approach, FINDSITEcomb, across a diverse set of medically-relevant proteins.ResultsFor eight different proteins belonging to different fold-classes and from diverse organisms, the top 1% of FINDSITEcomb’s VLS predictions were tested, and depending on the protein target, 4%-47% of the predicted ligands were shown to bind with μM or better affinities. In total, 47 small molecule binders were identified. Low nanomolar (nM) binders for dihydrofolate reductase and protein tyrosine phosphatases (PTPs) and micromolar binders for the other proteins were identified. Six novel molecules had cytotoxic activity (<10 μg/ml) against the HCT-116 colon carcinoma cell line and one novel molecule had potent antibacterial activity.ConclusionsWe show that FINDSITEcomb is a promising new VLS approach that can assist drug discovery.

Novel fluorescent protein from Hydnophora rigida possesses green emission

Fluorescent proteins are a family of proteins capable of producing fluorescence at various specific wavelengths of ultra violet light. We have previously reported the identification and characterization of a novel cyan fluorescent protein (HriCFP) from a reef coral species, Hydnophora rigida. In search of new members of the diverse family of fluorescent proteins, here we report a new green fluorescent protein (HriGFP) from H. rigida. HriGFP was identified, cloned, expressed in Escherichia coli and purified to homogeneity by metal affinity and size exclusion chromatography. The dynamic light scattering and gel filtration experiments suggested the presence of monomers in solution. The peptide mass fingerprint on the purified protein established the identity of HriGFP. HriGFP had excitation peak at 507nm and emission peak at 527nm. HriGFP was similar to HriCFP except the last 16 amino acid sequence at the C-terminal; however, they have shown least similarity with other known fluorescent proteins. Moreover the computational model suggests that HriGFP is a globular protein which consists of 6 α-helices and 3 β-sheets. Taken together our results suggested that HriGFP is a novel naturally occurring fluorescent protein that exists as a monomer in solution.

Myosin chaperones

The folding and assembly of myosin motor proteins is essential for most movement processes at the cellular, but also at the organism level. Importantly, myosins, which represent a very diverse family of proteins, require the activity of general and specialized folding factors to develop their full motor function. The activities of the myosin-specific UCS (UNC-45/Cro1/She4) chaperones range from assisting acto-myosin dependent transport processes to scaffolding multi-subunit chaperone complexes, which are required to assemble myofilaments. Recent structure-function studies revealed the structural organization of TPR (tetratricopeptide repeat)-containing and TPR-less UCS chaperones. The observed structural differences seem to reflect the specialized and remarkably versatile working mechanisms of myosin-directed chaperones, as will be discussed in this review.

Comprehensive genome-wide analysis of WDR superfamily of Plasmodium falciparum, cloning and production of PfWDR protein involved in chromatin remodelling

Background: WD40 repeats (WDRs) proteins comprise one of the largest and functionally diverse protein families in eukaryotes. WD40 gene family is of special interest because of its diverse functionality and mutidomain context; however this family remains uncharacterized in P. falciparum the most virulent species of human malaria. The present study gives a comprehensive in silico analysis of PfWDR gene family and cloning and production of a PfWD40 gene involved in chromatin assembly. Methods & Materials: PfWDR genes were extracted from PlasmoDB v 9.0 by gene text search, BLASTp and HMM search. Domain architecture of extracted genes was drawn by SMART and Pfam. Subcellular localization prediction was carried by Euk-mPLoc 2.0 server, PATS, Mitoprot and predict NLS. Expression of all PfWDR genes was analysed using MeV software as per transcriptome and proteome data available at PlasmoDB. Human homologs were assigned by BLASTp and literature search. Evolutionary relationships were drawn using phylip by NJ method. Further, one of WDR genes i.e. the chromatin assembly factor 1 (PfCAF-1) was amplified, cloned in pETM 11 vector and expressed in E. Coli B834 cellls. Expressed protein was purified by Ni-NTA chromatography. Results: In our present analysis, we identified 80WDR genes in P. falciparum and classified them in two families and twenty one subfamilies based on domain composition. While based on their functions,we categorized PfWDRproteins in eleven classes. By subcellular localization prediction, PfWDR proteins were found to be targeted to various cellular compartments verifying their diversified functions. Approximately fifty PfWD40 genes were predicted to have human homologs. Importantly, we were able to annotate some of PfWD40 genes based on their orthologs in other species. TheORF (1341bp) of the PfCAF-1was amplified and cloned in pETM 11.When transformed E. coli B834 cells were induced at 16 ◦Cwith 0.25mM IPTG, 50% of expressed protein was found to be in soluble form. The recombinant protein was purified and identity was confirmed by western blotting. Conclusion: This study provides a genomic glimpse of PfWDR gene family. The purified recombinant PfCAF-1 can be further exploited for the functional and structural characterization.

Highly reinforced structure of a C-terminal dimerization domain in von Willebrand factor

AbstractThe C-terminal cystine knot (CK) (CTCK) domain in von Willebrand factor (VWF) mediates dimerization of proVWF in the endoplasmic reticulum and is essential for long multimers required for hemostatic function. The CTCK dimer crystal structure reveals highly elongated monomers with 2 β-ribbons and 4 intra-chain disulfides, including 3 in the CK. Dimerization buries an extensive interface of 1500 Å2 corresponding to 32% of the surface of each monomer and forms a super β-sheet and 3 inter-chain disulfides. The shape, dimensions, and N-terminal connections of the crystal structure agree perfectly with previous electron microscopic images of VWF dimeric bouquets with the CTCK dimer forming a down-curved base. The dimer interface is suited to resist hydrodynamic force and disulfide reduction. CKs in each monomer flank the 3 inter-chain disulfides, and their presence in β-structures with dense backbone hydrogen bonds creates a rigid, highly crosslinked interface. The structure reveals the basis for von Willebrand disease phenotypes and the fold and disulfide linkages for CTCK domains in diverse protein families involved in barrier function, eye and inner ear development, insect coagulation and innate immunity, axon guidance, and signaling in extracellular matrices.

RhoGEFs in Cell Motility: Novel Links Between Rgnef and Focal Adhesion Kinase

Rho guanine exchange factors (GEFs) are a large, diverse family of proteins defined by their ability to catalyze the exchange of GDP for GTP on small GTPase proteins such as Rho family members. GEFs act as integrators from varied intra- and extracellular sources to promote spatiotemporal activity of Rho GTPases that control signaling pathways regulating cell proliferation and movement. Here we review recent studies elucidating roles of RhoGEF proteins in cell motility. Emphasis is placed on Dbl-family GEFs and connections to development, integrin signaling to Rho GTPases regulating cell adhesion and movement, and how these signals may enhance tumor progression. Moreover, RhoGEFs have additional domains that confer distinctive functions or specificity. We will focus on a unique interaction between Rgnef (also termed Arhgef28 or p190RhoGEF) and focal adhesion kinase (FAK), a non-receptor tyrosine kinase that controls migration properties of normal and tumor cells. This Rgnef-FAK interaction activates canonical GEF-dependent RhoA GTPase activity to govern contractility and also functions as a scaffold in a GEF-independent manner to enhance FAK activation. Recent studies have also brought to light the importance of specific regions within the Rgnef pleckstrin homology (PH) domain for targeting the membrane. As revealed by ongoing Rgnef-FAK investigations, exploring GEF roles in cancer will yield fundamental new information on the molecular mechanisms promoting tumor spread and metastasis.

Oxygen Sensitivity, Potassium Channels, and Regulation of Placental Vascular Tone

The human fetoplacental vasculature is a low-resistance circulation with deoxygenated arterial relative to venous blood. The placenta lacks neuronal innervation suggesting that local physical (e.g., oxygenation; flow rate), paracrine (e.g., endothelial cell nitric oxide), and circulating (e.g., angiotensin II) factors will contribute to blood flow regulation in small fetoplacental vessels. Oxygenation (specifically hypoxia) has received particular attention. At the macro-level, hypoxic challenge increases vascular resistance, but the data's physiological relevance remains questionable. K(+) channels are a diverse family of proteins known to play important roles in the normal physiological functions of endothelial and smooth muscle cells of a variety of vascular beds. K(+) channels are categorized by their predicted transmembrane structure or gating properties. A small number of perfused placental cotyledon and isolated blood vessels studies have assessed K(+) channel activity. Specific activator/inhibitor application suggests functional voltage-gated channels, whereas toxin inhibitor studies have documented KCa channel activity. Pharmacological KATP channel activation significantly dilates preconstricted placental arteries and veins. There is a paucity of cell subtype-specific expression studies of placental K(+) channels. This review focuses on the roles of K(+) channels and oxygenation in controlling reactivity of small fetoplacental blood vessels.

The Activation Dynamics of Class C Gpcrs Revealed by Single Molecule FRET

G-protein-coupled receptors (GPCRs) are the largest and most diverse family of membrane signaling proteins. Binding of an extracellular signal to a GPCR activates G protein, which in turn modulates the activity of different downstream cellular effectors. Recent biochemical, pharmacological and biophysical studies suggest that GPCRs do not simply act as on-off switches but instead exist in multiple conformational and functional states where they may signal to distinct G proteins and other partners. However, despite recent advances, many aspects of signal transduction through GPCRs is still not well understood. Here we report a novel functional assay based on single molecule FRET to probe real-time conformational dynamics of isolated receptors. Using our assay, we studied metabotropic glutamate receptors (mGluRs), dimeric members of class C GPCR family, which are involved in modulation of synaptic transmission and excitability of nerve cells. Experiments on mGluR2 and mGluR3 homodimers reveal multiple stable conformational states. Strikingly, in spite of more than 70% sequence identity between mGluR2 and mGluR3, we observe substantial differences in their inherent dynamics and basal activity in absence of ligand. Our approach should make it possible to directly quantify how drugs stabilize conformational states of receptors and provide new insights into the molecular mechanisms of GPCR signaling.

Compensating for Cardiac Hypertrophy: A Crucial Role for A-Kinase Anchoring Protein-Lbc

In response to mechanical load, cardiac hypertrophy (myocyte enlargement) is initially compensatory, but becomes a maladaptive process that leads to the development of heart failure. The high prevalence of pathological myocardial hypertrophy in cardiovascular disease coupled with a lack of effective treatment highlights a need for novel therapeutic strategies in this area. Recently, a diverse family of scaffold proteins termed A-Kinase Anchoring Proteins have been identified and characterized, playing multiple important roles in the heart. In a recent study using a mouse model with a partial AKAPLbc (AKAP13) gene deletion, we demonstrate a crucial in vivo role for AKAP-Lbc-protein kinase D1 (PKD1) signaling in the development of compensatory hypertrophy in response to transverse aortic constriction (TAC)-induced pressure overload and neurohumoral stimulation by AT-II/PE treatment. Overall, our results show that AKAP-Lbc-PKD1 signaling is critical for transcriptional activation during the development of compensatory hypertrophy in vivo, under conditions of pathological hypertrophy. By defining and understanding regulation, downstream signaling and function of AKAP-Lbc- PKD1 signaling under pathological conditions, studies will determine whether AKAP-Lbc-PKD1 is a possible therapeutic target for treatment of cardiac dysfunction.

Probing Heme Delivery Processes in Cytochrome c Biogenesis System I

Cytochromes c comprise a diverse and widespread family of proteins containing covalently bound heme that are central to the life of most organisms. In many bacteria and in certain mitochondria, the synthesis of cytochromes c is performed by a complex post-translational modification apparatus called System I (or cytochrome c maturation, Ccm, system). In Escherichia coli, there are eight maturation proteins, several of which are involved in heme handling, but the mechanism of heme transfer from one protein to the next is not known. Attachment of the heme to the apocytochrome occurs via a novel covalent bond to a histidine residue of the heme chaperone CcmE. The discovery of a variant maturation system (System I*) has provided a new tool for studying cytochrome c assembly because the variant CcmE functions via a cysteine residue in the place of the histidine of System I. In this work, we use site-directed mutagenesis on both maturation systems to probe the function of the individual component proteins as well as their concerted action in transferring heme to the cytochrome c substrate. The roles of CcmA, CcmC, CcmE, and CcmF in the heme delivery process are compared between Systems I and I*. We show that a previously proposed quinone-binding site on CcmF is not essential for either system. Significant differences in the heme chemistry involved in the formation of cytochromes c in the variant system add new pieces to the cytochrome c biogenesis puzzle.

The expression changes of vacuolar protein sorting 4B (VPS4B) following middle cerebral artery occlusion (MCAO) in adult rats brain hippocampus.

Vacuolar protein sorting 4 (VPS4), is a member of ATPases associated with diverse cellular activities protein family. VPS4 is composed of VPS4A and VPS4B, VPS4B plays an important role in the lysosomal degradation pathway, intracellular protein trafficking, virus budding and abscission of cytokinesis. However, information regarding its distribution and possible function in the central nervous system is limited. Therefore, we performed a middle cerebral artery occlusion (MCAO) in adult rats and detected the dynamic changes of VPS4B in hippocampus CA1 subregion. We found that the VPS4B expression was increased strongly after MCAO and reached the peak after 3 days. VPS4B mainly located in the cytoplasm of neurons, but not astrocytes and microglia. Moreover, there was a concomitant up-regulation of active caspase-3. In vitro studies indicated that the up-regulation of VPS4B may be involved in oxygen-glucose deprivation-induced PC12 cell death. And knock-down of VPS4B in cultured differentiated PC12 cells by siRNA showed that VPS4B promoted the expression of active caspase-3. Collectively, all these results and MTT assay suggested that the up-regulation of VPS4B played an important role in the pathophysiology after MCAO, and further research is needed to have a good understanding of its function and mechanism.

Genetic mapping of non-target-site resistance to a sulfonylurea herbicide (Envoke®) in Upland cotton (Gossypium hirsutum L.)

Acetolactate synthase (ALS) is responsible for a rate-limiting step in the synthesis of essential branched-chain amino acids. Resistance to ALS-inhibiting herbicides, such as trifloxysulfuron sodium (Envoke®), can be due to mutations in the target gene itself. Alternatively, plants may exhibit herbicide tolerance through reduced uptake and translocation or increased metabolism of the herbicide. The diverse family of cytochrome P450 proteins has been suggested to be a source of novel herbicide metabolism in both weed and crop plants. In this study we generated a mapping population between resistant and susceptible cotton (Gossypium hirsutum L.) cultivars. We found that both cultivars possess identical and sensitive ALS sequences; however, the segregation of resistance in the F2 progeny was consistent with a single dominant gene. Here we report the closely linked genetic markers and approximate physical location on chromosome 20 of the source of Envoke herbicide susceptibility in the cotton cultivar Paymaster HS26. There are no P450 proteins in the corresponding region of the G. raimondii Ulbr. genome, suggesting that an uncharacterized molecular mechanism is responsible for Envoke herbicide tolerance in G. hirsutum. Identification of this genetic mechanism will provide new opportunities for exploiting sulfonylurea herbicides for management of both weeds and crop plants.

HnRNP A1: The Swiss Army Knife of Gene Expression

Eukaryotic cells express a large variety of RNA binding proteins (RBPs), with diverse affinities and specificities towards target RNAs. These proteins play a crucial role in almost every aspect of RNA biogenesis, expression and function. The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a complex and diverse family of RNA binding proteins. hnRNPs display multiple functions in the processing of heterogeneous nuclear RNAs into mature messenger RNAs. hnRNP A1 is one of the most abundant and ubiquitously expressed members of this protein family. hnRNP A1 plays multiple roles in gene expression by regulating major steps in the processing of nascent RNA transcripts. The transcription, splicing, stability, export through nuclear pores and translation of cellular and viral transcripts are all mechanisms modulated by this protein. The diverse functions played by hnRNP A1 are not limited to mRNA biogenesis, but extend to the processing of microRNAs, telomere maintenance and the regulation of transcription factor activity. Genomic approaches have recently uncovered the extent of hnRNP A1 roles in the development and differentiation of living organisms. The aim of this review is to highlight recent developments in the study of this protein and to describe its functions in cellular and viral gene expression and its role in human pathologies.

Zebra: a web server for bioinformatic analysis of diverse protein families

During evolution of proteins from a common ancestor, one functional property can be preserved while others can vary leading to functional diversity. A systematic study of the corresponding adaptive mutations provides a key to one of the most challenging problems of modern structural biology – understanding the impact of amino acid substitutions on protein function. The subfamily-specific positions (SSPs) are conserved within functional subfamilies but are different between them and, therefore, seem to be responsible for functional diversity in protein superfamilies. Consequently, a corresponding method to perform the bioinformatic analysis of sequence and structural data has to be implemented in the common laboratory practice to study the structure–function relationship in proteins and develop novel protein engineering strategies. This paper describes Zebra web server – a powerful remote platform that implements a novel bioinformatic analysis algorithm to study diverse protein families. It is the first application that provides specificity determinants at different levels of functional classification, therefore addressing complex functional diversity of large superfamilies. Statistical analysis is implemented to automatically select a set of highly significant SSPs to be used as hotspots for directed evolution or rational design experiments and analyzed studying the structure–function relationship. Zebra results are provided in two ways – (1) as a single all-in-one parsable text file and (2) as PyMol sessions with structural representation of SSPs. Zebra web server is available at http://biokinet.belozersky.msu.ru/zebra.

ChemInform Abstract: Recent Developments in the Synthesis of Bioactive 2,4,6‐Trisubstituted 1,3,5‐Triazines

AbstractReview: 32 refs.

Exploring the Adenylation Domain Repertoire of Nonribosomal Peptide Synthetases Using an Ensemble of Sequence-Search Methods

The introduction of two-dimension (2D) graphs and their numerical characterization for comparative analyses of DNA/RNA and protein sequences without the need of sequence alignments is an active yet recent research topic in bioinformatics. Here, we used a 2D artificial representation (four-color maps) with a simple numerical characterization through topological indices (TIs) to aid the discovering of remote homologous of Adenylation domains (A-domains) from the Nonribosomal Peptide Synthetases (NRPS) class in the proteome of the cyanobacteria Microcystis aeruginosa. Cyanobacteria are a rich source of structurally diverse oligopeptides that are predominantly synthesized by NPRS. Several A-domains share amino acid identities lower than 20 % being a possible source of remote homologous. Therefore, A-domains cannot be easily retrieved by BLASTp searches using a single template. To cope with the sequence diversity of the A-domains we have combined homology-search methods with an alignment-free tool that uses protein four-color-maps. TI2BioP (Topological Indices to BioPolymers) version 2.0, available at http://ti2biop.sourceforge.net/ allowed the calculation of simple TIs from the protein sequences (four-color maps). Such TIs were used as input predictors for the statistical estimations required to build the alignment-free models. We concluded that the use of graphical/numerical approaches in cooperation with other sequence search methods, like multi-templates BLASTp and profile HMM, can give the most complete exploration of the repertoire of highly diverse protein families.

Recent Developments in the Synthesis of Bioactive 2,4,6-Trisubstituted 1,3,5-Triazines

1,3,5-Triazine derivatives have widespread applications in the pharmaceutical, material, and agrochemical industries. In the biological arena, small-molecule libraries of 2,4,6-trisubstituted 1,3,5-triazines have yielded selective and potent chemical probes for diverse protein families. The structural symmetry and ease of functionalization of the 1,3,5-triazine core renders it a powerful scaffold for the rapid generation of diverse molecular libraries. Numerous synthetic routes, both solid-phase and solution-phase, have been developed to arrive at alkyl/aryl amino- and oxy-substituted triazine libraries. Here, we feature a subset of the recent synthetic advances towards generating 2,4,6-trisubstituted 1,3,5-triazines and highlight biologically active compounds that have resulted from these endeavors.

Molecular Pathways: Mitogen-Activated Protein Kinase Pathway Mutations and Drug Resistance

Receptor tyrosine kinases are a diverse family of transmembrane proteins that can activate multiple pathways upon ligation of the receptor, one of which is the series of mitogen-activated protein kinase (MAPK) signaling cascades. The MAPK pathways play critical roles in a wide variety of cancer types, from hematologic malignancies to solid tumors. Aberrations include altered expression levels and activation states of pathway components, which can sometimes be attributable to mutations in individual members. The V600E mutation of BRAF was initially described in 2002 and has been found at particularly high frequency in melanoma and certain subtypes of colorectal cancer. In the relatively short time since this discovery, a family of drugs has been developed that specifically target this mutated BRAF isoform, which, after results from phase I/II and III clinical trials, was granted U.S. Food and Drug Administration approval in August 2011. Although these drugs produce clinically meaningful increases in progression-free and overall survival, due to acquired resistance they have not improved mortality rates. New drugs targeting other members of the MAPK pathways are in clinical trials or advanced stages of development. It is hoped that combination therapies of these new drugs in conjunction with BRAF inhibitors will counteract the mechanisms of resistance and provide cures. The clinical implementation of next-generation sequencing is leading to a greater understanding of the genetic architecture of tumors, along with acquired mechanisms of drug resistance, which will guide the development of tumor-specific inhibitors and combination therapies in the future.

Cloning and construction of entry vector carrying IL-6 gene from Vietnamese chicken

Cytokines are a diverse family of proteins that play a crucial role in controlling the immune system. They determine both the type and extent of an immune response that is generated following infection with a pathogen or after vaccination. In this paper, chicken IL-6 gene (ChIL-6) was cloned and ligated in a shuttle vector, as basic to construction adenovirus vector for cytokine gene delivery in chicken for improving of their immune responses to pathogens. By PCR technique, using specific primers for chicken IL-6 gen (ChIL-6) possessing recognition sites of restriction enzymes Kpn I and Not I and Kozak sequence, the ChIL-6 was cloned into pCR2.1 vector. The gene in recombinant pCR2.1/ChIL-6 was sequenced, the obtained sequence had 726 nucleotides and was 99% similarly to the sequences in GenBank after BLASTn. The sequence has been reported to GenBank with Accession no JQ897539. For construction pShuttle/ChIL-6 vector, the cloning vector pCR2.1/ChIL-6 and pShuttle were digested by Kpn I and Not I, then the ligation reaction was performed with T4 ligase and transformed E. coli DH5α clones were selected on LB medium supplemented with ampicilin. The presence of ChIL-6 gen in pShuttle vector was verified by digestion of recombinant plasmid with Kpn I and Not I, as well as by PCR. Received results demonstrated that ChIL-6 gen was ligated into pShuttle and gave pShuttle/ChIL-6 vector.