Pair Of Motifs Research Articles

Calcium-Induced Conformational Dynamics of the C-Domain of Tetrahymena Cytoskeletal Protein Tcb2

Tcb2 is a protein found in the cytoskeleton of the protozoan Tetrahymena thermophila. The calcium binding protein has been hypothesized to play roles involved with ciliary movement, pronuclear exchange, as well as cytoskeletal contractions triggered by Calcium binding. Solution NMR studies showed that although the C-domain of Tcb2 shares a common structure with other calcium binding proteins (two EF-hand paired motifs), the conformational change as a result of Calcium binding differs from Calmodulin.

A Novel Insecticidal Spider Peptide that Affects the Mammalian Voltage-Gated Ion Channel hKv1.5.

Spider venoms include various peptide toxins that modify the ion currents, mainly of excitable insect cells. Consequently, scientific research on spider venoms has revealed a broad range of peptide toxins with different pharmacological properties, even for mammal species. In this work, thirty animal venoms were screened against hKv1.5, a potential target for atrial fibrillation therapy. The whole venom of the spider Oculicosa supermirabilis, which is also insecticidal to house crickets, caused voltage-gated potassium ion channel modulation in hKv1.5. Therefore, a peptide from the spider O. supermirabilis venom, named Osu1, was identified through HPLC reverse-phase fractionation. Osu1 displayed similar biological properties as the whole venom; so, the primary sequence of Osu1 was elucidated by both of N-terminal degradation and endoproteolytic cleavage. Based on its primary structure, a gene that codifies for Osu1 was constructed de novo from protein to DNA by reverse translation. A recombinant Osu1 was expressed using a pQE30 vector inside the E. coli SHuffle expression system. recombinant Osu1 had voltage-gated potassium ion channel modulation of human hKv1.5, and it was also as insecticidal as the native toxin. Due to its novel primary structure, and hypothesized disulfide pairing motif, Osu1 may represent a new family of spider toxins.

Computational modeling suggests binding-induced expansion of Epsin disordered regions upon association with AP2.

Intrinsically disordered regions (IDRs) are prevalent in the eukaryotic proteome. Common functional roles of IDRs include forming flexible linkers or undergoing allosteric folding-upon-binding. Recent studies have suggested an additional functional role for IDRs: generating steric pressure on the plasma membrane during endocytosis, via molecular crowding. However, in order to accomplish useful functions, such crowding needs to be regulated in space (e.g., endocytic hotspots) and time (e.g., during vesicle formation). In this work, we explore binding-induced regulation of IDR steric volume. We simulate the IDRs of two proteins from Clathrin-mediated endocytosis (CME) to see if their conformational spaces are regulated via binding-induced expansion. Using Monte-Carlo computational modeling of excluded volumes, we generate large conformational ensembles (3 million) for the IDRs of Epsin and Eps15 and dock the conformers to the alpha subunit of Adaptor Protein 2 (AP2α), their CME binding partner. Our results show that as more molecules of AP2α are bound, the Epsin-derived ensemble shows a significant increase in global dimensions, measured as the radius of Gyration (RG) and the end-to-end distance (EED). Unlike Epsin, Eps15-derived conformers that permit AP2α binding at one motif were found to be more likely to accommodate binding of AP2α at other motifs, suggesting a tendency toward co-accessibility of binding motifs. Co-accessibility was not observed for any pair of binding motifs in Epsin. Thus, we speculate that the disordered regions of Epsin and Eps15 perform different roles during CME, with accessibility in Eps15 allowing it to act as a recruiter of AP2α molecules, while binding-induced expansion of the Epsin disordered region could impose steric pressure and remodel the plasma membrane during vesicle formation.

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Broń jako wyznacznik prestiżu, rekwizyt rytuału oraz świadectwo kontaktów w Barbaricum w młodszym okresie przedrzymskim i w okresie wpływów rzymskich

DeepYY1: a deep learning approach to identify YY1-mediated chromatin loops.

The protein Yin Yang 1 (YY1) could form dimers that facilitate the interaction between active enhancers and promoter-proximal elements. YY1-mediated enhancer-promoter interaction is the general feature of mammalian gene control. Recently, some computational methods have been developed to characterize the interactions between DNA elements by elucidating important features of chromatin folding; however, no computational methods have been developed for identifying the YY1-mediated chromatin loops. In this study, we developed a deep learning algorithm named DeepYY1 based on word2vec to determine whether a pair of YY1 motifs would form a loop. The proposed models showed a high prediction performance (AUCs$\ge$0.93) on both training datasets and testing datasets in different cell types, demonstrating that DeepYY1 has an excellent performance in the identification of the YY1-mediated chromatin loops. Our study also suggested that sequences play an important role in the formation of YY1-mediated chromatin loops. Furthermore, we briefly discussed the distribution of the replication origin site in the loops. Finally, a user-friendly web server was established, and it can be freely accessed at http://lin-group.cn/server/DeepYY1.

Deep Learning of Sequence Patterns for CCCTC-Binding Factor-Mediated Chromatin Loop Formation

The three-dimensional (3D) organization of the human genome is of crucial importance for gene regulation, and the CCCTC-binding factor (CTCF) plays an important role in chromatin interactions. However, it is still unclear what sequence patterns in addition to CTCF motif pairs determine chromatin loop formation. To discover the underlying sequence patterns, we have developed a deep learning model, called DeepCTCFLoop, to predict whether a chromatin loop can be formed between a pair of convergent or tandem CTCF motifs using only the DNA sequences of the motifs and their flanking regions. Our results suggest that DeepCTCFLoop can accurately distinguish the CTCF motif pairs forming chromatin loops from the ones not forming loops. It significantly outperforms CTCF-MP, a machine learning model based on word2vec and boosted trees, when using DNA sequences only. Furthermore, we show that DNA motifs binding to several transcription factors, including ZNF384, ZNF263, ASCL1, SP1, and ZEB1, may constitute the complex sequence patterns for CTCF-mediated chromatin loop formation. DeepCTCFLoop has also been applied to disease-associated sequence variants to identify candidates that may disrupt chromatin loop formation. Therefore, our results provide useful information for understanding the mechanism of 3D genome organization and may also help annotate and prioritize the noncoding sequence variants associated with human diseases.

Asymmetric Conservation within Pairs of Co-Occurred Motifs Mediates Weak Direct Binding of Transcription Factors in ChIP-Seq Data.

(1) Background: Transcription factors (TFs) are main regulators of eukaryotic gene expression. The cooperative binding to genomic DNA of at least two TFs is the widespread mechanism of transcription regulation. Cooperating TFs can be revealed through the analysis of co-occurrence of their motifs. (2) Methods: We applied the motifs co-occurrence tool (MCOT) that predicted pairs of spaced or overlapped motifs (composite elements, CEs) for a single ChIP-seq dataset. We improved MCOT capability for the prediction of asymmetric CEs with one of the participating motifs possessing higher conservation than another does. (3) Results: Analysis of 119 ChIP-seq datasets for 45 human TFs revealed that almost for all families of TFs the co-occurrence with an overlap between motifs of target TFs and more conserved partner motifs was significantly higher than that for less conserved partner motifs. The asymmetry toward partner TFs was the most clear for partner motifs of TFs from the ETS (E26 Transformation Specific) family. (4) Conclusion: Co-occurrence with an overlap of less conserved motif of a target TF and more conserved motifs of partner TFs explained a substantial portion of ChIP-seq data lacking conserved motifs of target TFs. Among other TF families, conservative motifs of TFs from ETS family were the most prone to mediate interaction of target TFs with its weak motifs in ChIP-seq.

Regulation of Cell Type-Specific Immunity Networks in Arabidopsis Roots.

While root diseases are among the most devastating stresses in global crop production, our understanding of root immunity is still limited relative to our knowledge of immune responses in leaves. Considering that root performance is based on the concerted functions of its different cell types, we undertook a cell type-specific transcriptome analysis to identify gene networks activated in epidermis, cortex, and pericycle cells of Arabidopsis (Arabidopsis thaliana) roots challenged with two immunity elicitors, the bacterial flagellin-derived flg22 and the endogenous Pep1 peptide. Our analyses revealed distinct immunity gene networks in each cell type. To further substantiate our understanding of regulatory patterns underlying these cell type-specific immunity networks, we developed a tool to analyze paired transcription factor binding motifs in the promoters of cell type-specific genes. Our study points toward a connection between cell identity and cell type-specific immunity networks that might guide cell types in launching immune response according to the functional capabilities of each cell type.

A poly(thymine)-melamine duplex for the assembly of DNA nanomaterials.

The diversity of DNA duplex structures is limited by a binary pair of hydrogen-bonded motifs. Here we show that poly(thymine) self-associates into antiparallel, right-handed duplexes in the presence of melamine, a small molecule that presents a triplicate set of the hydrogen-bonding face of adenine. X-ray crystallography shows that in the complex two poly(thymine) strands wrap around a helical column of melamine, which hydrogen bonds to thymine residues on two of its three faces. The mechanical strength of the thymine-melamine-thymine triplet surpasses that of adenine-thymine base pairs, which enables a sensitive detection of melamine at 3 pM. The poly(thymine)-melamine duplex is orthogonal to native DNA base pairing and can undergo strand displacement without the need for overhangs. Its incorporation into two-dimensional grids and hybrid DNA-small-molecule polymers highlights the poly(thymine)-melamine duplex as an additional tool for DNA nanotechnology.

Protein interactions studies of the small TbTims chaperons in the mitochondrion of Trypanosoma brucei

Trypanosoma brucei causes a neglected tropical disease known as trypanosomiasis that causes death in population of Sub‐Sahara Africa. T. brucei possesses a single mitochondrion and like other eukaryotes it imports most of its proteins into the mitochondria, however mitochondrial protein import machinery is divergent in this parasite, which makes it a useful target to control this disease. Nucleus‐encoded proteins are imported into T. brucei mitochondrion via two major heteromeric protein complexes in the outer and inner membrane, ATOM and TbTIM, respectively. Besides the membrane integral protein components of these complexes, TbTIM is also peripherally associated with 6 small TbTims (Tim9, 10, 8/13, 11, 12, and 13). At least three of these small TbTims (TbTim8/13, 11, & 12) are unique for T. brucei. Knock‐down of any of these 6 small TbTims destabilized the TbTIM17 complex therefore these proteins are essential in T. brucei. In fungi and human, the small Tims are soluble proteins that form hetero‐hexameric complexes. Each small Tims possesses two pairs of CX3C motifs that are required for proper folding of the monomers by formation of the two intramolecular disulfide bonds. T. brucei small Tims also possess these conserved motifs, except for TbTim12, which has only one pair. However, it is not clear how small TbTims interact with each other and what type of complex structure they form. Thus, our goal for these studies is to find the direct interaction pattern for the small TbTims. To complete this goal, we use yeast two hybrid (Y2H) analysis. Results showed that although all small TbTims can interact with one another, some of these interactions are stronger than others. Overall, we found that TbTim8/13 interacts strongly with TbTim9, TbTim10, and TbTim8/13 while TbTim13 interacts strongly with TbTim13. Based on these results we proposed 3 possible model for small TbTim heterohexameric complexes; 1) random order, and 2) defined order, and 3) modular with different sub‐complexes. We are now testing these models by structure‐modeling and site‐directed mutagenesis studies.Support or Funding InformationThis work is supported by the following NIH grants: 5RO1AI125666, 2SC1GM081146, and R25 GM05994

Christian motifs and images in A. G. Rumyantsev lyrical cycle “At The Black Doorstep”

This article is dedicated to examination of Christian imagery in lyrical cycle “At The Black Doorstep” by the Buryat poet A.G. Rumyantsev. The subject of this research is semantic correlation of the key images and motifs of the cycle with the Christian values. The object of this research is literary comprehension of Christian symbolism in a lyrical text. The central theme in the cycle – tragedy of a father who lost his son – is reflected in the motif pairs “darkness – light”, “curse – forgiveness”, as well as theme of apocalypses and reference to the images of natural world. Methodology is based on the axiological principle, according to which the defining in artistic consciousness of the poet is his personal values and priorities. For determination of Cristian code of the images and motifs, the author applied structural-semiotic method. The scientific novelty consists in carrying out a motif and imagery analysis of A. G. Rumyantsev lyrical cycle “At The Black Doorstep” from the perspective of Christian symbolism. A conclusion is formulated that Christian imagery encompasses a recurring for the entire cycle theme of soul revival of a lyrical character, a path he walked from personal apocalypses and inner darkness to light through acceptance and forgiveness.

Paralogues of Mmp11 and Timp4 Interact during the Development of the Myotendinous Junction in the Zebrafish Embryo.

The extracellular matrix (ECM) of the myotendinous junction (MTJ) undergoes dramatic physical and biochemical remodeling during the first 48 h of development in zebrafish, transforming from a rectangular fibronectin-dominated somite boundary to a chevron-shaped laminin-dominated MTJ. Matrix metalloproteinase 11 (Mmp11, a.k.a. Stromelysin-3) is both necessary and sufficient for the removal of fibronectin at the MTJ, but whether this protease acts directly on fibronectin and how its activity is regulated remain unknown. Using immunofluorescence, we show that both paralogues of Mmp11 accumulate at the MTJ during this time period, but with Mmp11a present early and later replaced by Mmp11b. Moreover, Mmp11a also accumulates intracellularly, associated with the Z-discs of sarcomeres within skeletal muscle cells. Using the epitope-mediated MMP activation (EMMA) assay, we show that despite having a weaker paired basic amino acid motif in its propeptide than Mmp11b, Mmp11a is activated by furin, but may also be activated by other mechanisms intracellularly. One or both paralogues of tissue inhibitors of metalloproteinase-4 (Timp4) are also present at the MTJ throughout this process, and yeast two-hybrid assays reveal distinct and specific interactions between various domains of these proteins. We propose a model in which Mmp11a activity is modulated (but not inhibited) by Timp4 during early MTJ remodeling, followed by a phase in which Mmp11b activity is both inhibited and spatially constrained by Timp4 in order to maintain the structural integrity of the mature MTJ.

7C:\xa0Computational Chromosome Conformation Capture by Correlation of ChIP-seq at CTCF motifs

BackgroundKnowledge of the three-dimensional structure of the genome is necessary to understand how gene expression is regulated. Recent experimental techniques such as Hi-C or ChIA-PET measure long-range chromatin interactions genome-wide but are experimentally elaborate, have limited resolution and such data is only available for a limited number of cell types and tissues.ResultsWhile ChIP-seq was not designed to detect chromatin interactions, the formaldehyde treatment in the ChIP-seq protocol cross-links proteins with each other and with DNA. Consequently, also regions that are not directly bound by the targeted TF but interact with the binding site via chromatin looping are co-immunoprecipitated and sequenced. This produces minor ChIP-seq signals at loop anchor regions close to the directly bound site. We use the position and shape of ChIP-seq signals around CTCF motif pairs to predict whether they interact or not. We implemented this approach in a prediction method, termed Computational Chromosome Conformation Capture by Correlation of ChIP-seq at CTCF motifs (7C). We applied 7C to all CTCF motif pairs within 1 Mb in the human genome and validated predicted interactions with high-resolution Hi-C and ChIA-PET. A single ChIP-seq experiment from known architectural proteins (CTCF, Rad21, Znf143) but also from other TFs (like TRIM22 or RUNX3) predicts loops accurately. Importantly, 7C predicts loops in cell types and for TF ChIP-seq datasets not used in training.Conclusion7C predicts chromatin loops which can help to associate TF binding sites to regulated genes. Furthermore, profiling of hundreds of ChIP-seq datasets results in novel candidate factors functionally involved in chromatin looping. Our method is available as an R/Bioconductor package: http://bioconductor.org/packages/sevenC.

Studying 3D genome evolution using genomic sequence.

The three dimensions (3D) genome is essential to numerous key processes such as the regulation of gene expression and the replication-timing program. In vertebrates, chromatin looping is often mediated by CTCF, and marked by CTCF motif pairs in convergent orientation. Comparative high-throughput sequencing technique (Hi-C) recently revealed that chromatin looping evolves across species. However, Hi-C experiments are complex and costly, which currently limits their use for evolutionary studies over a large number of species. Here, we propose a novel approach to study the 3D genome evolution in vertebrates using the genomic sequence only, e.g. without the need for Hi-C data. The approach is simple and relies on comparing the distances between convergent and divergent CTCF motifs by computing a ratio we named the 3D ratio or '3DR'. We show that 3DR is a powerful statistic to detect CTCF looping encoded in the human genome sequence, thus reflecting strong evolutionary constraints encoded in DNA and associated with the 3D genome. When comparing vertebrate genomes, our results reveal that 3DR which underlies CTCF looping and topologically associating domain organization evolves over time and suggest that ancestral character reconstruction can be used to infer 3DR in ancestral genomes. The R code is available at https://github.com/morphos30/PhyloCTCFLooping. Supplementary data are available at Bioinformatics online.

A single ChIP-seq dataset is sufficient for comprehensive analysis of motifs co-occurrence with MCOT package.

Recognition of composite elements consisting of two transcription factor binding sites gets behind the studies of tissue-, stage- and condition-specific transcription. Genome-wide data on transcription factor binding generated with ChIP-seq method facilitate an identification of composite elements, but the existing bioinformatics tools either require ChIP-seq datasets for both partner transcription factors, or omit composite elements with motifs overlapping. Here we present an universal Motifs Co-Occurrence Tool (MCOT) that retrieves maximum information about overrepresented composite elements from a single ChIP-seq dataset. This includes homo- and heterotypic composite elements of four mutual orientations of motifs, separated with a spacer or overlapping, even if recognition of motifs within composite element requires various stringencies. Analysis of 52 ChIP-seq datasets for 18 human transcription factors confirmed that for over 60% of analyzed datasets and transcription factors predicted co-occurrence of motifs implied experimentally proven protein-protein interaction of respecting transcription factors. Analysis of 164 ChIP-seq datasets for 57 mammalian transcription factors showed that abundance of predicted composite elements with an overlap of motifs compared to those with a spacer more than doubled; and they had 1.5-fold increase of asymmetrical pairs of motifs with one more conservative ‘leading’ motif and another one ‘guided’.

Single T Cell Sequencing Demonstrates the Functional Role of αβ TCR Pairing in Cell Lineage and Antigen Specificity.

Although structural studies of individual T cell receptors (TCRs) have revealed important roles for both the α and β chain in directing MHC and antigen recognition, repertoire-level immunogenomic analyses have historically examined the β chain alone. To determine the amount of useful information about TCR repertoire function encoded within αβ pairings, we analyzed paired TCR sequences from nearly 100,000 unique CD4+ and CD8+ T cells captured using two different high-throughput, single-cell sequencing approaches. Our results demonstrate little overlap in the healthy CD4+ and CD8+ repertoires, with shared TCR sequences possessing significantly shorter CDR3 sequences corresponding to higher generation probabilities. We further utilized tools from information theory and machine learning to show that while α and β chains are only weakly associated with lineage, αβ pairings appear to synergistically drive TCR-MHC interactions. Vαβ gene pairings were found to be the TCR feature most informative of T cell lineage, supporting the existence of germline-encoded paired αβ TCR-MHC interaction motifs. Finally, annotating our TCR pairs using a database of sequences with known antigen specificities, we demonstrate that approximately a third of the T cells possess α and β chains that each recognize different known antigens, suggesting that αβ pairing is critical for the accurate inference of repertoire functionality. Together, these findings provide biological insight into the functional implications of αβ pairing and highlight the utility of single-cell sequencing in immunogenomics.

Molecular-level origin of the carboxylate head group response to divalent metal ion complexation at the air–water interface

We exploit gas-phase cluster ion techniques to provide insight into the local interactions underlying divalent metal ion-driven changes in the spectra of carboxylic acids at the air-water interface. This information clarifies the experimental findings that the CO stretching bands of long-chain acids appear at very similar energies when the head group is deprotonated by high subphase pH or exposed to relatively high concentrations of Ca2+ metal ions. To this end, we report the evolution of the vibrational spectra of size-selected [Ca2+·RCO2-]+·(H2O) n=0to12 and RCO2-·(H2O) n=0to14 cluster ions toward the features observed at the air-water interface. Surprisingly, not only does stepwise hydration of the RCO2- anion and the [Ca2+·RCO2-]+ contact ion pair yield solvatochromic responses in opposite directions, but in both cases, the responses of the 2 (symmetric and asymmetric stretching) CO bands to hydration are opposite to each other. The result is that both CO bands evolve toward their interfacial asymptotes from opposite directions. Simulations of the [Ca2+·RCO2-]+·(H2O) n clusters indicate that the metal ion remains directly bound to the head group in a contact ion pair motif as the asymmetric CO stretch converges at the interfacial value by n = 12. This establishes that direct metal complexation or deprotonation can account for the interfacial behavior. We discuss these effects in the context of a model that invokes the water network-dependent local electric field along the C-C bond that connects the head group to the hydrocarbon tail as the key microscopic parameter that is correlated with the observed trends.

FragLites-Minimal, Halogenated Fragments Displaying Pharmacophore Doublets. An Efficient Approach to Druggability Assessment and Hit Generation.

Identifying ligand binding sites on proteins is a critical step in target-based drug discovery. Current approaches to this require resource-intensive screening of large libraries of lead-like or fragment molecules. Here, we describe an efficient and effective experimental approach to mapping interaction sites using a set of halogenated compounds expressing paired hydrogen-bonding motifs, termed FragLites. The FragLites identify productive drug-like interactions, which are identified sensitively and unambiguously by X-ray crystallography, exploiting the anomalous scattering of the halogen substituent. This mapping of protein interaction surfaces provides an assessment of druggability and can identify efficient start points for the de novo design of hit molecules incorporating the interacting motifs. The approach is illustrated by mapping cyclin-dependent kinase 2, which successfully identifies orthosteric and allosteric sites. The hits were rapidly elaborated to develop efficient lead-like molecules. Hence, the approach provides a new method of identifying ligand sites, assessing tractability and discovering new leads.

Kinetics of trifurcated electron flow in the decaheme bacterial proteins MtrC and MtrF

The bacterium Shewanella oneidensis has evolved a sophisticated electron transfer (ET) machinery to export electrons from the cytosol to extracellular space during extracellular respiration. At the heart of this process are decaheme proteins of the Mtr pathway, MtrC and MtrF, located at the external face of the outer bacterial membrane. Crystal structures have revealed that these proteins bind 10 c-type hemes arranged in the peculiar shape of a staggered cross that trifurcates the electron flow, presumably to reduce extracellular substrates while directing electrons to neighboring multiheme cytochromes at either side along the membrane. Especially intriguing is the design of the heme junctions trifurcating the electron flow: they are made of coplanar and T-shaped heme pair motifs with relatively large and seemingly unfavorable tunneling distances. Here, we use electronic structure calculations and molecular simulations to show that the side chains of the heme rings, in particular the cysteine linkages inserting in the space between coplanar and T-shaped heme pairs, strongly enhance electronic coupling in these two motifs. This results in an [Formula: see text]-fold speedup of ET steps at heme junctions that would otherwise be rate limiting. The predicted maximum electron flux through the solvated proteins is remarkably similar for all possible flow directions, suggesting that MtrC and MtrF shuttle electrons with similar efficiency and reversibly in directions parallel and orthogonal to the outer membrane. No major differences in the ET properties of MtrC and MtrF are found, implying that the different expression levels of the two proteins during extracellular respiration are not related to redox function.

Analysis of Structure and Dynamics in Three-Neuron Motifs.

Recurrent neural networks can produce ongoing state-to-state transitions without any driving inputs, and the dynamical properties of these transitions are determined by the neuronal connection strengths. Due to non-linearity, it is not clear how strongly the system dynamics is affected by discrete local changes in the connection structure, such as the removal, addition, or sign-switching of individual connections. Moreover, there are no suitable metrics to quantify structural and dynamical differences between two given networks with arbitrarily indexed neurons. In this work, we present such permutation-invariant metrics and apply them to motifs of three binary neurons with discrete ternary connection strengths, an important class of building blocks in biological networks. Using multidimensional scaling, we then study the similarity relations between all 3,411 topologically distinct motifs with regard to structure and dynamics, revealing a strong clustering and various symmetries. As expected, the structural and dynamical distance between pairs of motifs show a significant positive correlation. Strikingly, however, the key parameter controlling motif dynamics turns out to be the ratio of excitatory to inhibitory connections.