Loads Bias Genetic and Signaling Switches in Synthetic and Natural Systems

PubMed Central

Medford, June; Prasad, Ashok

2014-01-01

Biological protein interactions networks such as signal transduction or gene transcription networks are often treated as modular, allowing motifs to be analyzed in isolation from the rest of the network. Modularity is also a key assumption in synthetic biology, where it is similarly expected that when network motifs are combined together, they do not lose their essential characteristics. However, the interactions that a network module has with downstream elements change the dynamical equations describing the upstream module and thus may change the dynamic and static properties of the upstream circuit even without explicit feedback. In this work we analyze the behavior of a ubiquitous motif in gene transcription and signal transduction circuits: the switch. We show that adding an additional downstream component to the simple genetic toggle switch changes its dynamical properties by changing the underlying potential energy landscape, and skewing it in favor of the unloaded side, and in some situations adding loads to the genetic switch can also abrogate bistable behavior. We find that an additional positive feedback motif found in naturally occurring toggle switches could tune the potential energy landscape in a desirable manner. We also analyze autocatalytic signal transduction switches and show that a ubiquitous positive feedback switch can lose its switch-like properties when connected to a downstream load. Our analysis underscores the necessity of incorporating the effects of downstream components when understanding the physics of biochemical network motifs, and raises the question as to how these effects are managed in real biological systems. This analysis is particularly important when scaling synthetic networks to more complex organisms. PMID:24676102

Human HDAC7 Harbors a Class IIa Histone Deacetylase-specific Zinc Binding Motif and Cryptic Deacetylase Activity*S⃞

PubMed Central

Schuetz, Anja; Min, Jinrong; Allali-Hassani, Abdellah; Schapira, Matthieu; Shuen, Michael; Loppnau, Peter; Mazitschek, Ralph; Kwiatkowski, Nick P.; Lewis, Timothy A.; Maglathin, Rebecca L.; McLean, Thomas H.; Bochkarev, Alexey; Plotnikov, Alexander N.; Vedadi, Masoud; Arrowsmith, Cheryl H.

2008-01-01

Histone deacetylases (HDACs) are protein deacetylases that play a role in repression of gene transcription and are emerging targets in cancer therapy. Here, we characterize the structure and enzymatic activity of the catalytic domain of human HDAC7 (cdHDAC7). Although HDAC7 normally exists as part of a multiprotein complex, we show that cdHDAC7 has a low level of deacetylase activity which can be inhibited by known HDAC inhibitors. The crystal structures of human cdHDAC7 and its complexes with two hydroxamate inhibitors are the first structures of the catalytic domain of class IIa HDACs and demonstrate significant differences with previously reported class I and class IIb-like HDAC structures. We show that cdHDAC7 has an additional class IIa HDAC-specific zinc binding motif adjacent to the active site which is likely to participate in substrate recognition and protein-protein interaction and may provide a site for modulation of activity. Furthermore, a different active site topology results in modified catalytic properties and in an enlarged active site pocket. Our studies provide mechanistic insights into class IIa HDACs and facilitate the design of specific modulators. PMID:18285338

Human HDAC7 harbors a class IIa histone deacetylase-specific zinc binding motif and cryptic deacetylase activity.

PubMed

Schuetz, Anja; Min, Jinrong; Allali-Hassani, Abdellah; Schapira, Matthieu; Shuen, Michael; Loppnau, Peter; Mazitschek, Ralph; Kwiatkowski, Nick P; Lewis, Timothy A; Maglathin, Rebecca L; McLean, Thomas H; Bochkarev, Alexey; Plotnikov, Alexander N; Vedadi, Masoud; Arrowsmith, Cheryl H

2008-04-25

Histone deacetylases (HDACs) are protein deacetylases that play a role in repression of gene transcription and are emerging targets in cancer therapy. Here, we characterize the structure and enzymatic activity of the catalytic domain of human HDAC7 (cdHDAC7). Although HDAC7 normally exists as part of a multiprotein complex, we show that cdHDAC7 has a low level of deacetylase activity which can be inhibited by known HDAC inhibitors. The crystal structures of human cdHDAC7 and its complexes with two hydroxamate inhibitors are the first structures of the catalytic domain of class IIa HDACs and demonstrate significant differences with previously reported class I and class IIb-like HDAC structures. We show that cdHDAC7 has an additional class IIa HDAC-specific zinc binding motif adjacent to the active site which is likely to participate in substrate recognition and protein-protein interaction and may provide a site for modulation of activity. Furthermore, a different active site topology results in modified catalytic properties and in an enlarged active site pocket. Our studies provide mechanistic insights into class IIa HDACs and facilitate the design of specific modulators.

p63 threonine phosphorylation signals the interaction with the WW domain of the E3 ligase Itch

PubMed Central

Melino, Sonia; Bellomaria, Alessia; Nepravishta, Ridvan; Paci, Maurizio; Melino, Gerry

2014-01-01

Both in epithelial development as well as in epithelial cancers, the p53 family member p63 plays a crucial role acting as a master transcriptional regulator. P63 steady state protein levels are regulated by the E3 ubiquitin ligase Itch, via a physical interaction between the PPxY consensus sequence (PY motif) of p63 and one of the 4 WW domains of Itch; this substrate recognition process leads to protein-ubiquitylation and p63 proteasomal degradation. The interaction of the WW domains, a highly compact protein-protein binding module, with the short proline-rich sequences is therefore a crucial regulatory event that may offer innovative potential therapeutic opportunity. Previous molecular studies on the Itch-p63 recognition have been performed in vitro using the Itch-WW2 domain and the peptide interacting fragment of p63 (pep63), which includes the PY motif. Itch-WW2-pep63 interaction is also stabilized in vitro by the conformational constriction of the S-S cyclization in the p63 peptide. The PY motif of p63, as also for other proteins, is characterized by the nearby presence of a (T/S)P motif, which is a potential recognition site of the WW domain of the IV group present in the prolyl-isomerase Pin1. In this study, we demonstrate, by in silico and spectroscopical studies using both the linear pep63 and its cyclic form, that the threonine phosphorylation of the (T/S)PPPxY motif may represent a crucial regulatory event of the Itch-mediated p63 ubiquitylation, increasing the Itch-WW domains-p63 recognition event and stabilizing in vivo the Itch-WW-p63 complex. Moreover, our studies confirm that the subsequently trans/cis proline isomerization of (T/S)P motif by the Pin1 prolyl-isomerase, could modulate the E3-ligase interaction, and that the (T/S)pPtransPPxY motif represent the best conformer for the ItchWW-(T/S)PPPxY motif recognition. PMID:25485500

p63 threonine phosphorylation signals the interaction with the WW domain of the E3 ligase Itch.

PubMed

Melino, Sonia; Bellomaria, Alessia; Nepravishta, Ridvan; Paci, Maurizio; Melino, Gerry

2014-01-01

Both in epithelial development as well as in epithelial cancers, the p53 family member p63 plays a crucial role acting as a master transcriptional regulator. P63 steady state protein levels are regulated by the E3 ubiquitin ligase Itch, via a physical interaction between the PPxY consensus sequence (PY motif) of p63 and one of the 4 WW domains of Itch; this substrate recognition process leads to protein-ubiquitylation and p63 proteasomal degradation. The interaction of the WW domains, a highly compact protein-protein binding module, with the short proline-rich sequences is therefore a crucial regulatory event that may offer innovative potential therapeutic opportunity. Previous molecular studies on the Itch-p63 recognition have been performed in vitro using the Itch-WW2 domain and the peptide interacting fragment of p63 (pep63), which includes the PY motif. Itch-WW2-pep63 interaction is also stabilized in vitro by the conformational constriction of the S-S cyclization in the p63 peptide. The PY motif of p63, as also for other proteins, is characterized by the nearby presence of a (T/S)P motif, which is a potential recognition site of the WW domain of the IV group present in the prolyl-isomerase Pin1. In this study, we demonstrate, by in silico and spectroscopical studies using both the linear pep63 and its cyclic form, that the threonine phosphorylation of the (T/S)PPPxY motif may represent a crucial regulatory event of the Itch-mediated p63 ubiquitylation, increasing the Itch-WW domains-p63 recognition event and stabilizing in vivo the Itch-WW-p63 complex. Moreover, our studies confirm that the subsequently trans/cis proline isomerization of (T/S)P motif by the Pin1 prolyl-isomerase, could modulate the E3-ligase interaction, and that the (T/S)pPtransPPxY motif represent the best conformer for the ItchWW-(T/S)PPPxY motif recognition.

The MiiA motif is a common marker present in polytopic surface proteins of oral and urinary tract invasive bacteria.

PubMed

Martín-Galiano, Antonio J

2017-04-01

Many surface virulence factors of bacterial pathogens show mosaicism and confounding phylogenetic origin. The Streptococcus gordonii platelet-binding GspB protein, the Streptococcus sanguinis SrpA adhesin and the Streptococcus pneumoniae DiiA protein, share an imperfect 27-residue motif. Given the disparate domain architectures of these proteins and its association to invasive disease, this motif was named MiiA from Multiarchitecture invasion-involved motif A. MiiA is predicted to adopt a beta-sheet folding, probably related to the Ig-like fold, with a symmetrical positioning of two conserved aspartic residues. A specific hidden Markov model profiling MiiA was built, which specifically detected the motif in proteins from 58 species, mainly in cell-wall proteins from Gram-positive bacteria. These proteins contained one to ten MiiA motifs, which were embedded within larger repeat units of 70-82 residues. MiiA motifs combined to other domains and elements such as coiled-coils and low-complexity regions. The species carrying MiiA-proteins included commensals from the urogenital tract and the oral cavity, which can cause opportunistic endocarditis and sepsis. Intra-protein MiiA repeats showed a complex mixture of orthologal, paralogal and inter-species relationships, suggestive of a multistep origin. Presence of these repeats in proteins involved in oligosaccharide recognition and lifestyle of species suggest a putative function for MiiA repeats in sugars binding, probably those present in receptors of epithelial and blood cells. MiiA modules appear to have been transferred horizontally between species co-habiting in the same niche to create their own MiiA-containing determinants. The present work provides a global study and a catalog of potential MiiA virulence factors that should be analyzed experimentally. Copyright © 2017 Elsevier B.V. All rights reserved.

HRD Motif as the Central Hub of the Signaling Network for Activation Loop Autophosphorylation in Abl Kinase.

PubMed

La Sala, Giuseppina; Riccardi, Laura; Gaspari, Roberto; Cavalli, Andrea; Hantschel, Oliver; De Vivo, Marco

2016-11-08

A number of structural factors modulate the activity of Abelson (Abl) tyrosine kinase, whose deregulation is often related to oncogenic processes. First, only the open conformation of the Abl kinase domain's activation loop (A-loop) favors ATP binding to the catalytic cleft. In this regard, the trans-autophosphorylation of the Y412 residue, which is located along the A-loop, favors the stability of the open conformation, in turn enhancing Abl activity. Another key factor for full Abl activity is the formation of active conformations of the catalytic DFG motif in the Abl kinase domain. Furthermore, binding of the SH2 domain to the N-lobe of the Abl kinase was recently demonstrated to have a long-range allosteric effect on the stabilization of the A-loop open state. Intriguingly, these distinct structural factors imply a complex signal transmission network for controlling the A-loop's flexibility and conformational preference for optimal Abl function. However, the exact dynamical features of this signal transmission network structure remain unclear. Here, we report on microsecond-long molecular dynamics coupled with enhanced sampling simulations of multiple Abl model systems, in the presence or absence of the SH2 domain and with the DFG motif flipped in two ways (in or out conformation). Through comparative analysis, our simulations augment the interpretation of the existing Abl experimental data, revealing a dynamical network of interactions that interconnect SH2 domain binding with A-loop plasticity and Y412 autophosphorylation in Abl. This signaling network engages the DFG motif and, importantly, other conserved structural elements of the kinase domain, namely, the EPK-ELK H-bond network and the HRD motif. Our results show that the signal propagation for modulating the A-loop spatial localization is highly dependent on the HRD motif conformation, which thus acts as the central hub of this (allosteric) signaling network controlling Abl activation and function.

A cis-regulatory module activating transcription in the suspensor contains five cis-regulatory elements

DOE PAGES

Henry, Kelli F.; Kawashima, Tomokazu; Goldberg, Robert B.

2015-03-22

Little is known about the molecular mechanisms by which the embryo proper and suspensor of plant embryos activate specific gene sets shortly after fertilization. We analyzed the upstream region of the Scarlet Runner Bean ( Phaseolus coccineus) G564 gene in order to understand how genes are activated specifically in the suspensor during early embryo development. Previously, we showed that a 54-bp fragment of the G564 upstream region is sufficient for suspensor transcription and contains at least three required cis-regulatory sequences, including the 10-bp motif (5'-GAAAAGCGAA-3'), the 10 bp-like motif (5'-GAAAAACGAA-3'), and Region 2 motif (partial sequence 5'-TTGGT-3'). Here, we usemore » site-directed mutagenesis experiments in transgenic tobacco globularstage embryos to identify two additional cis-regulatory elements within the 54-bp cis-regulatory module that are required for G564 suspensor transcription: the Fifth motif (5'-GAGTTA-3') and a third 10-bp-related sequence (5'-GAAAACCACA-3'). Further deletion of the 54-bp fragment revealed that a 47-bp fragment containing the five motifs (the 10-bp, 10-bp-like, 10-bp-related, Region 2 and Fifth motifs) is sufficient for suspensor transcription, and represents a cis-regulatory module. A consensus sequence for each type of motif was determined by comparing motif sequences shown to activate suspensor transcription. Phylogenetic analyses suggest that the regulation of G564 is evolutionarily conserved. Lastly, a homologous cis-regulatory module was found upstream of the G564 ortholog in the Common Bean (Phaseolus vulgaris), indicating that the regulation of G564 is evolutionarily conserved in closely related bean species.« less

A cis-regulatory module activating transcription in the suspensor contains five cis-regulatory elements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Henry, Kelli F.; Kawashima, Tomokazu; Goldberg, Robert B.

Little is known about the molecular mechanisms by which the embryo proper and suspensor of plant embryos activate specific gene sets shortly after fertilization. We analyzed the upstream region of the Scarlet Runner Bean ( Phaseolus coccineus) G564 gene in order to understand how genes are activated specifically in the suspensor during early embryo development. Previously, we showed that a 54-bp fragment of the G564 upstream region is sufficient for suspensor transcription and contains at least three required cis-regulatory sequences, including the 10-bp motif (5'-GAAAAGCGAA-3'), the 10 bp-like motif (5'-GAAAAACGAA-3'), and Region 2 motif (partial sequence 5'-TTGGT-3'). Here, we usemore » site-directed mutagenesis experiments in transgenic tobacco globularstage embryos to identify two additional cis-regulatory elements within the 54-bp cis-regulatory module that are required for G564 suspensor transcription: the Fifth motif (5'-GAGTTA-3') and a third 10-bp-related sequence (5'-GAAAACCACA-3'). Further deletion of the 54-bp fragment revealed that a 47-bp fragment containing the five motifs (the 10-bp, 10-bp-like, 10-bp-related, Region 2 and Fifth motifs) is sufficient for suspensor transcription, and represents a cis-regulatory module. A consensus sequence for each type of motif was determined by comparing motif sequences shown to activate suspensor transcription. Phylogenetic analyses suggest that the regulation of G564 is evolutionarily conserved. Lastly, a homologous cis-regulatory module was found upstream of the G564 ortholog in the Common Bean (Phaseolus vulgaris), indicating that the regulation of G564 is evolutionarily conserved in closely related bean species.« less

A cis-regulatory module activating transcription in the suspensor contains five cis-regulatory elements.

PubMed

Henry, Kelli F; Kawashima, Tomokazu; Goldberg, Robert B

2015-06-01

Little is known about the molecular mechanisms by which the embryo proper and suspensor of plant embryos activate specific gene sets shortly after fertilization. We analyzed the upstream region of the Scarlet Runner Bean (Phaseolus coccineus) G564 gene in order to understand how genes are activated specifically in the suspensor during early embryo development. Previously, we showed that a 54-bp fragment of the G564 upstream region is sufficient for suspensor transcription and contains at least three required cis-regulatory sequences, including the 10-bp motif (5'-GAAAAGCGAA-3'), the 10 bp-like motif (5'-GAAAAACGAA-3'), and Region 2 motif (partial sequence 5'-TTGGT-3'). Here, we use site-directed mutagenesis experiments in transgenic tobacco globular-stage embryos to identify two additional cis-regulatory elements within the 54-bp cis-regulatory module that are required for G564 suspensor transcription: the Fifth motif (5'-GAGTTA-3') and a third 10-bp-related sequence (5'-GAAAACCACA-3'). Further deletion of the 54-bp fragment revealed that a 47-bp fragment containing the five motifs (the 10-bp, 10-bp-like, 10-bp-related, Region 2 and Fifth motifs) is sufficient for suspensor transcription, and represents a cis-regulatory module. A consensus sequence for each type of motif was determined by comparing motif sequences shown to activate suspensor transcription. Phylogenetic analyses suggest that the regulation of G564 is evolutionarily conserved. A homologous cis-regulatory module was found upstream of the G564 ortholog in the Common Bean (Phaseolus vulgaris), indicating that the regulation of G564 is evolutionarily conserved in closely related bean species.

The arrestin-1 finger loop interacts with two distinct conformations of active rhodopsin.

PubMed

Elgeti, Matthias; Kazmin, Roman; Rose, Alexander S; Szczepek, Michal; Hildebrand, Peter W; Bartl, Franz J; Scheerer, Patrick; Hofmann, Klaus Peter

2018-03-23

Signaling of the prototypical G protein-coupled receptor (GPCR) rhodopsin through its cognate G protein transducin (G t ) is quenched when arrestin binds to the activated receptor. Although the overall architecture of the rhodopsin/arrestin complex is known, many questions regarding its specificity remain unresolved. Here, using FTIR difference spectroscopy and a dual pH/peptide titration assay, we show that rhodopsin maintains certain flexibility upon binding the "finger loop" of visual arrestin (prepared as synthetic peptide ArrFL-1). We found that two distinct complexes can be stabilized depending on the protonation state of E3.49 in the conserved (D)ERY motif. Both complexes exhibit different interaction modes and affinities of ArrFL-1 binding. The plasticity of the receptor within the rhodopsin/ArrFL-1 complex stands in contrast to the complex with the C terminus of the G t α-subunit (GαCT), which stabilizes only one specific substate out of the conformational ensemble. However, G t α-subunit binding and both ArrFL-1-binding modes involve a direct interaction to conserved R3.50, as determined by site-directed mutagenesis. Our findings highlight the importance of receptor conformational flexibility and cytoplasmic proton uptake for modulation of rhodopsin signaling and thereby extend the picture provided by crystal structures of the rhodopsin/arrestin and rhodopsin/ArrFL-1 complexes. Furthermore, the two binding modes of ArrFL-1 identified here involve motifs of conserved amino acids, which indicates that our results may have elucidated a common modulation mechanism of class A GPCR-G protein/-arrestin signaling. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Mechanistic insights into avian reovirus p17-modulated suppression of cell-cycle CDK/cyclin complexes and enhancement of p53 and cyclin H interaction.

PubMed

Chiu, Hung-Chuan; Huang, Wei-Ru; Liao, Tsai-Ling; Chi, Pei-I; Nielsen, Brent L; Liu, Jyung-Hurng; Liu, Hung-Jen

2018-06-15

The avian reovirus (ARV) p17 protein is a nucleocytoplasmic shuttling protein. Although we have demonstrated that p17 causes cell growth retardation via activation of p53, the precise mechanisms remains unclear. This is the first report that ARV p17 possesses broad inhibitory effects on cell-cycle CDKs, cyclins, CDK/cyclin complexes, and CDK activating kinase (CAK) activity in various mammalian, avian, and cancer cell lines. Suppression of CDK activity by p17 occurs by direct binding to CDKs, cyclins, and CDK/cyclin complexes, transcriptional downregulation of CDKs, cytoplasmic retention of CDKs and cyclins, and inhibition of CAK activity by promoting p53/cyclin H interaction. p17 binds to CDK/cyclin except for CDK1/cyclin B1 and CDK7/cyclin H complexes. We have determined that the negatively charged 151 LAVxDxDxE/DDGADPN 165 motif in cyclin B1 interacts with a positively charged region of CDK1. p17 mimics the cyclin B1 sequence to compete for CDK1 binding. The PSTAIRE motif is not required for interaction of CDK1/cyclin B1, but is required for other CDK/cyclin complexes. p17 interacts with cyclins by its cyclin-binding motif 125 RXL 127 Sequence and mutagenic analyses of p17 indicated that a 140 WXFD 143 motif and residues D113 and K122 in p17 are critical for CDK2 and CDK6 binding, leading to their sequestration in the cytoplasm. Exogenous expression of p17 significantly enhanced virus replication while p17 mutants with low binding ability to cell-cycle CDKs have no effect on virus yield, suggesting that p17 inhibits cell growth and the cell cycle benefiting virus replication. An in vivo tumorigenesis assay also showed a significant reduction in tumor size. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Phosphorylation-Dependent Regulation of Ryanodine Receptors

PubMed Central

Marx, Steven O.; Reiken, Steven; Hisamatsu, Yuji; Gaburjakova, Marta; Gaburjakova, Jana; Yang, Yi-Ming; Rosemblit, Nora; Marks, Andrew R.

2001-01-01

Ryanodine receptors (RyRs), intracellular calcium release channels required for cardiac and skeletal muscle contraction, are macromolecular complexes that include kinases and phosphatases. Phosphorylation/dephosphorylation plays a key role in regulating the function of many ion channels, including RyRs. However, the mechanism by which kinases and phosphatases are targeted to ion channels is not well understood. We have identified a novel mechanism involved in the formation of ion channel macromolecular complexes: kinase and phosphatase targeting proteins binding to ion channels via leucine/isoleucine zipper (LZ) motifs. Activation of kinases and phosphatases bound to RyR2 via LZs regulates phosphorylation of the channel, and disruption of kinase binding via LZ motifs prevents phosphorylation of RyR2. Elucidation of this new role for LZs in ion channel macromolecular complexes now permits: (a) rapid mapping of kinase and phosphatase targeting protein binding sites on ion channels; (b) predicting which kinases and phosphatases are likely to regulate a given ion channel; (c) rapid identification of novel kinase and phosphatase targeting proteins; and (d) tools for dissecting the role of kinases and phosphatases as modulators of ion channel function. PMID:11352932

Structural analysis of the recognition of the negative regulator NmrA and DNA by the zinc finger from the GATA-type transcription factor AreA.

PubMed

Kotaka, Masayo; Johnson, Christopher; Lamb, Heather K; Hawkins, Alastair R; Ren, Jingshan; Stammers, David K

2008-08-29

Amongst the most common protein motifs in eukaryotes are zinc fingers (ZFs), which, although largely known as DNA binding modules, also can have additional important regulatory roles in forming protein:protein interactions. AreA is a transcriptional activator central to nitrogen metabolism in Aspergillus nidulans. AreA contains a GATA-type ZF that has a competing dual recognition function, binding either DNA or the negative regulator NmrA. We report the crystal structures of three AreA ZF-NmrA complexes including two with bound NAD(+) or NADP(+). The molecular recognition of AreA ZF-NmrA involves binding of the ZF to NmrA via hydrophobic and hydrogen bonding interactions through helices alpha1, alpha6 and alpha11. Comparison with an earlier NMR solution structure of AreA ZF-DNA complex by overlap of the AreA ZFs shows that parts of helices alpha6 and alpha11 of NmrA are positioned close to the GATA motif of the DNA, mimicking the major groove of DNA. The extensive overlap of DNA with NmrA explains their mutually exclusive binding to the AreA ZF. The presence of bound NAD(+)/NADP(+) in the NmrA-AreaA ZF complex, however, causes minimal structural changes. Thus, any regulatory effects on AreA function mediated by the binding of oxidised nicotinamide dinucleotides to NmrA in the NmrA-AreA ZF complex appear not to be modulated via protein conformational rearrangements.

Salicylic acid suppresses jasmonic acid signaling downstream of SCFCOI1-JAZ by targeting GCC promoter motifs via transcription factor ORA59.

PubMed

Van der Does, Dieuwertje; Leon-Reyes, Antonio; Koornneef, Annemart; Van Verk, Marcel C; Rodenburg, Nicole; Pauwels, Laurens; Goossens, Alain; Körbes, Ana P; Memelink, Johan; Ritsema, Tita; Van Wees, Saskia C M; Pieterse, Corné M J

2013-02-01

Antagonism between the defense hormones salicylic acid (SA) and jasmonic acid (JA) plays a central role in the modulation of the plant immune signaling network, but the molecular mechanisms underlying this phenomenon are largely unknown. Here, we demonstrate that suppression of the JA pathway by SA functions downstream of the E3 ubiquitin-ligase Skip-Cullin-F-box complex SCF(COI1), which targets JASMONATE ZIM-domain transcriptional repressor proteins (JAZs) for proteasome-mediated degradation. In addition, neither the stability nor the JA-induced degradation of JAZs was affected by SA. In silico promoter analysis of the SA/JA crosstalk transcriptome revealed that the 1-kb promoter regions of JA-responsive genes that are suppressed by SA are significantly enriched in the JA-responsive GCC-box motifs. Using GCC:GUS lines carrying four copies of the GCC-box fused to the β-glucuronidase reporter gene, we showed that the GCC-box motif is sufficient for SA-mediated suppression of JA-responsive gene expression. Using plants overexpressing the GCC-box binding APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factors ERF1 or ORA59, we found that SA strongly reduces the accumulation of ORA59 but not that of ERF1. Collectively, these data indicate that the SA pathway inhibits JA signaling downstream of the SCF(COI1)-JAZ complex by targeting GCC-box motifs in JA-responsive promoters via a negative effect on the transcriptional activator ORA59.

Finding specific RNA motifs: Function in a zeptomole world?

PubMed Central

KNIGHT, ROB; YARUS, MICHAEL

2003-01-01

We have developed a new method for estimating the abundance of any modular (piecewise) RNA motif within a longer random region. We have used this method to estimate the size of the active motifs available to modern SELEX experiments (picomoles of unique sequences) and to a plausible RNA World (zeptomoles of unique sequences: 1 zmole = 602 sequences). Unexpectedly, activities such as specific isoleucine binding are almost certainly present in zeptomoles of molecules, and even ribozymes such as self-cleavage motifs may appear (depending on assumptions about the minimal structures). The number of specified nucleotides is not the only important determinant of a motif’s rarity: The number of modules into which it is divided, and the details of this division, are also crucial. We propose three maxims for easily isolated motifs: the Maxim of Minimization, the Maxim of Multiplicity, and the Maxim of the Median. These maxims together state that selected motifs should be small and composed of as many separate, equally sized modules as possible. For evenly divided motifs with four modules, the largest accessible activity in picomole scale (1–1000 pmole) pools of length 100 is about 34 nucleotides; while for zeptomole scale (1–1000 zmole) pools it is about 20 specific nucleotides (50% probability of occurrence). This latter figure includes some ribozymes and aptamers. Consequently, an RNA metabolism apparently could have begun with only zeptomoles of RNA molecules. PMID:12554865

Stability and structural properties of gene regulation networks with coregulation rules.

PubMed

Warrell, Jonathan; Mhlanga, Musa

2017-05-07

Coregulation of the expression of groups of genes has been extensively demonstrated empirically in bacterial and eukaryotic systems. Such coregulation can arise through the use of shared regulatory motifs, which allow the coordinated expression of modules (and module groups) of functionally related genes across the genome. Coregulation can also arise through the physical association of multi-gene complexes through chromosomal looping, which are then transcribed together. We present a general formalism for modeling coregulation rules in the framework of Random Boolean Networks (RBN), and develop specific models for transcription factor networks with modular structure (including module groups, and multi-input modules (MIM) with autoregulation) and multi-gene complexes (including hierarchical differentiation between multi-gene complex members). We develop a mean-field approach to analyse the dynamical stability of large networks incorporating coregulation, and show that autoregulated MIM and hierarchical gene-complex models can achieve greater stability than networks without coregulation whose rules have matching activation frequency. We provide further analysis of the stability of small networks of both kinds through simulations. We also characterize several general properties of the transients and attractors in the hierarchical coregulation model, and show using simulations that the steady-state distribution factorizes hierarchically as a Bayesian network in a Markov Jump Process analogue of the RBN model. Copyright © 2017. Published by Elsevier Ltd.

Identification of Human Lineage-Specific Transcriptional Coregulators Enabled by a Glossary of Binding Modules and Tunable Genomic Backgrounds.

PubMed

Mariani, Luca; Weinand, Kathryn; Vedenko, Anastasia; Barrera, Luis A; Bulyk, Martha L

2017-09-27

Transcription factors (TFs) control cellular processes by binding specific DNA motifs to modulate gene expression. Motif enrichment analysis of regulatory regions can identify direct and indirect TF binding sites. Here, we created a glossary of 108 non-redundant TF-8mer "modules" of shared specificity for 671 metazoan TFs from publicly available and new universal protein binding microarray data. Analysis of 239 ENCODE TF chromatin immunoprecipitation sequencing datasets and associated RNA sequencing profiles suggest the 8mer modules are more precise than position weight matrices in identifying indirect binding motifs and their associated tethering TFs. We also developed GENRE (genomically equivalent negative regions), a tunable tool for construction of matched genomic background sequences for analysis of regulatory regions. GENRE outperformed four state-of-the-art approaches to background sequence construction. We used our TF-8mer glossary and GENRE in the analysis of the indirect binding motifs for the co-occurrence of tethering factors, suggesting novel TF-TF interactions. We anticipate that these tools will aid in elucidating tissue-specific gene-regulatory programs. Copyright © 2017 Elsevier Inc. All rights reserved.

Computational and experimental analysis of short peptide motifs for enzyme inhibition.

PubMed

Fu, Jinglin; Larini, Luca; Cooper, Anthony J; Whittaker, John W; Ahmed, Azka; Dong, Junhao; Lee, Minyoung; Zhang, Ting

2017-01-01

The metabolism of living systems involves many enzymes that play key roles as catalysts and are essential to biological function. Searching ligands with the ability to modulate enzyme activities is central to diagnosis and therapeutics. Peptides represent a promising class of potential enzyme modulators due to the large chemical diversity, and well-established methods for library synthesis. Peptides and their derivatives are found to play critical roles in modulating enzymes and mediating cellular uptakes, which are increasingly valuable in therapeutics. We present a methodology that uses molecular dynamics (MD) and point-variant screening to identify short peptide motifs that are critical for inhibiting β-galactosidase (β-Gal). MD was used to simulate the conformations of peptides and to suggest short motifs that were most populated in simulated conformations. The function of the simulated motifs was further validated by the experimental point-variant screening as critical segments for inhibiting the enzyme. Based on the validated motifs, we eventually identified a 7-mer short peptide for inhibiting an enzyme with low μM IC50. The advantage of our methodology is the relatively simplified simulation that is informative enough to identify the critical sequence of a peptide inhibitor, with a precision comparable to truncation and alanine scanning experiments. Our combined experimental and computational approach does not rely on a detailed understanding of mechanistic and structural details. The MD simulation suggests the populated motifs that are consistent with the results of the experimental alanine and truncation scanning. This approach appears to be applicable to both natural and artificial peptides. With more discovered short motifs in the future, they could be exploited for modulating biocatalysis, and developing new medicine.

Input-Specific Gain Modulation by Local Sensory Context Shapes Cortical and Thalamic Responses to Complex Sounds.

PubMed

Williamson, Ross S; Ahrens, Misha B; Linden, Jennifer F; Sahani, Maneesh

2016-07-20

Sensory neurons are customarily characterized by one or more linearly weighted receptive fields describing sensitivity in sensory space and time. We show that in auditory cortical and thalamic neurons, the weight of each receptive field element depends on the pattern of sound falling within a local neighborhood surrounding it in time and frequency. Accounting for this change in effective receptive field with spectrotemporal context improves predictions of both cortical and thalamic responses to stationary complex sounds. Although context dependence varies among neurons and across brain areas, there are strong shared qualitative characteristics. In a spectrotemporally rich soundscape, sound elements modulate neuronal responsiveness more effectively when they coincide with sounds at other frequencies, and less effectively when they are preceded by sounds at similar frequencies. This local-context-driven lability in the representation of complex sounds-a modulation of "input-specific gain" rather than "output gain"-may be a widespread motif in sensory processing. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Plasticity of signaling and mate choice in a trilling species of the Mecopoda complex (Orthoptera: Tettigoniidae).

PubMed

Krobath, I; Römer, H; Hartbauer, M

2017-01-01

Males of a trilling species in the Mecopoda complex produce conspicuous calling songs that consist of two motifs: an amplitude-modulated motif with alternating loud and soft segments (AM-motif) and a continuous, high-intensity trill. The function of these song motifs for female attraction and competition between males was investigated. We tested the hypothesis that males modify their signaling behavior depending on the social environment (presence/absence of females or rival males) when they compete for mates. Therefore, we analyzed acoustic signaling of males in three different situations: (1) solo singing, (2) acoustic interaction with another male, and (3) singing in the presence of a female. In addition, the preference of females for these song motifs and further song parameters was studied in two-choice experiments. As expected, females showed a preference for conspicuous and loud song elements, but nevertheless, males increased the proportion of the AM-motif in the presence of a female. In acoustic interactions, males reduced bout duration significantly compared to both other situations. However, song bouts in this situation still overlapped more than expected by chance, which indicates intentionally simultaneous singing. A multivariate statistical analysis revealed that the proportion of the AM-motif and the duration of loud segments within the AM-motif allow a reliable prediction of whether males sing in isolation, compete with another male, or sing in the presence of a female. These results indicate that the AM-motif plays a dominant role especially in close-range courtship and that males are challenged in finding a balance between attracting females and saving energy during repeated acoustic interactions. Males of acoustic insects often produce conspicuous calling songs that have a dual function in male-male competition and mate attraction. High signal amplitudes and signal rates are associated with high energetic costs for signal production. We would therefore predict that males adjust their signaling behavior according to their perception of the social context. Here we studied signal production and mate choice in a katydid, where males switch between loud and soft song segments in a dynamic way. Additionally, we examined the attractiveness of different song elements in female choice tests. Our results show how males of this katydid deal with the conflict of remaining attractive for females and competing with a costly signal with rivals.

Recognition of p63 by the E3 ligase ITCH: Effect of an ectodermal dysplasia mutant.

PubMed

Bellomaria, A; Barbato, Gaetano; Melino, G; Paci, M; Melino, Sonia

2010-09-15

The E3 ubiquitin ligase Itch mediates the degradation of the p63 protein. Itch contains four WW domains which are pivotal for the substrate recognition process. Indeed, this domain is implicated in several signalling complexes crucially involved in human diseases including Muscular Dystrophy, Alzheimer's Disease and Huntington Disease. WW domains are highly compact protein-protein binding modules that interact with short proline-rich sequences. The four WW domains present in Itch belong to the Group I type, which binds polypeptides with a PY motif characterized by a PP xY consensus sequence, where x can be any residue. Accordingly, the Itch-p63 interaction results from a direct binding of Itch-WW2 domain with the PY motif of p63. Here, we report a structural analysis of the Itch-p63 interaction by fluorescence, CD and NMR spectroscopy. Indeed, we studied the in vitro interaction between Itch-WW2 domain and p63(534-551), an 18-mer peptide encompassing a fragment of the p63 protein including the PY motif. In addition, we evaluated the conformation and the interaction with Itch-WW2 of a site specific mutant of p63, I549T, that has been reported in both Hay-Wells syndrome and Rapp-Hodgkin syndrome. Based on our results, we propose an extended PP xY motif for the Itch recognition motif (P-P-P-Y-x(4)-[ST]-[ILV]), which includes these C-terminal residues to the PP xY motif.

The TIE1 Transcriptional Repressor Links TCP Transcription Factors with TOPLESS/TOPLESS-RELATED Corepressors and Modulates Leaf Development in Arabidopsis[W

PubMed Central

Tao, Qing; Guo, Dongshu; Wei, Baoye; Zhang, Fan; Pang, Changxu; Jiang, Hao; Zhang, Jinzhe; Wei, Tong; Gu, Hongya; Qu, Li-Jia; Qin, Genji

2013-01-01

Leaf size and shape are mainly determined by coordinated cell division and differentiation in lamina. The CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors are key regulators of leaf development. However, the mechanisms that control TCP activities during leaf development are largely unknown. We identified the TCP Interactor containing EAR motif protein1 (TIE1), a novel transcriptional repressor, as a major modulator of TCP activities during leaf development. Overexpression of TIE1 leads to hyponastic and serrated leaves, whereas disruption of TIE1 causes epinastic leaves. TIE1 is expressed in young leaves and encodes a transcriptional repressor containing a C-terminal EAR motif, which mediates interactions with the TOPLESS (TPL)/TOPLESS-RELATED (TPR) corepressors. In addition, TIE1 physically interacts with CIN-like TCPs. We propose that TIE1 regulates leaf size and morphology by inhibiting the activities of TCPs through recruiting the TPL/TPR corepressors to form a tertiary complex at early stages of leaf development. PMID:23444332

Motifs, modules and games in bacteria.

PubMed

Wolf, Denise M; Arkin, Adam P

2003-04-01

Global explorations of regulatory network dynamics, organization and evolution have become tractable thanks to high-throughput sequencing and molecular measurement of bacterial physiology. From these, a nascent conceptual framework is developing, that views the principles of regulation in term of motifs, modules and games. Motifs are small, repeated, and conserved biological units ranging from molecular domains to small reaction networks. They are arranged into functional modules, genetically dissectible cellular functions such as the cell cycle, or different stress responses. The dynamical functioning of modules defines the organism's strategy to survive in a game, pitting cell against cell, and cell against environment. Placing pathway structure and dynamics into an evolutionary context begins to allow discrimination between those physical and molecular features that particularize a species to its surroundings, and those that provide core physiological function. This approach promises to generate a higher level understanding of cellular design, pathway evolution and cellular bioengineering.

Motifs, modules and games in bacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wolf, Denise M.; Arkin, Adam P.

2003-04-01

Global explorations of regulatory network dynamics, organization and evolution have become tractable thanks to high-throughput sequencing and molecular measurement of bacterial physiology. From these, a nascent conceptual framework is developing, that views the principles of regulation in term of motifs, modules and games. Motifs are small, repeated, and conserved biological units ranging from molecular domains to small reaction networks. They are arranged into functional modules, genetically dissectible cellular functions such as the cell cycle, or different stress responses. The dynamical functioning of modules defines the organism's strategy to survive in a game, pitting cell against cell, and cell against environment.more » Placing pathway structure and dynamics into an evolutionary context begins to allow discrimination between those physical and molecular features that particularize a species to its surroundings, and those that provide core physiological function. This approach promises to generate a higher level understanding of cellular design, pathway evolution and cellular bioengineering.« less

Molecular Basis for Phosphorylation-dependent SUMO Recognition by the DNA Repair Protein RAP80.

PubMed

Anamika; Spyracopoulos, Leo

2016-02-26

Recognition and repair of double-stranded DNA breaks (DSB) involves the targeted recruitment of BRCA tumor suppressors to damage foci through binding of both ubiquitin (Ub) and the Ub-like modifier SUMO. RAP80 is a component of the BRCA1 A complex, and plays a key role in the recruitment process through the binding of Lys(63)-linked poly-Ub chains by tandem Ub interacting motifs (UIM). RAP80 also contains a SUMO interacting motif (SIM) just upstream of the tandem UIMs that has been shown to specifically bind the SUMO-2 isoform. The RAP80 tandem UIMs and SIM function collectively for optimal recruitment of BRCA1 to DSBs, although the molecular basis of this process is not well understood. Using NMR spectroscopy, we demonstrate that the RAP80 SIM binds SUMO-2, and that both specificity and affinity are enhanced through phosphorylation of the canonical CK2 site within the SIM. The affinity increase results from an enhancement of electrostatic interactions between the phosphoserines of RAP80 and the SIM recognition module within SUMO-2. The NMR structure of the SUMO-2·phospho-RAP80 complex reveals that the molecular basis for SUMO-2 specificity is due to isoform-specific sequence differences in electrostatic SIM recognition modules. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Selective ligand activity at Nur/retinoid X receptor complexes revealed by dimer-specific bioluminescence resonance energy transfer-based sensors

PubMed Central

Giner, Xavier C; Cotnoir-White, David; Mader, Sylvie; Lévesque, Daniel

2017-01-01

Retinoid X receptors (RXR) play a role as master regulators due to their capacity to form heterodimers with other nuclear receptors. Accordingly, retinoid signaling is involved in multiple biological processes, including development, cell differentiation, metabolism and cell death. However, the role and functions of RXR in different heterodimer complexes remain unsolved, mainly because most RXR drugs (called rexinoids) are not selective to specific heterodimer complexes. This also strongly limits the use of rexinoids for specific therapeutic approaches. In order to better characterize rexinoids at specific nuclear receptor complexes, we have developed and optimized luciferase protein complementation-based Bioluminescence Resonance Energy Transfer (BRET) assays, which can directly measure recruitment of a co-activator motif fused to yellow fluorescent protein (YFP) by specific nuclear receptor dimers. To validate the assays, we compared rexinoid modulation of co-activator recruitment by RXR homodimer, and heterodimers Nur77/RXR and Nurr1/RXR. Results reveal that some rexinoids display selective co-activator recruitment activities with homo- or hetero-dimer complexes. In particular, SR11237 (BMS649) has increased potency for recruitment of co-activator motif and transcriptional activity with the Nur77/RXR heterodimer compared to other complexes. This technology should prove useful to identify new compounds with specificity for individual dimeric species formed by nuclear receptors. PMID:26148973

Salicylic Acid Suppresses Jasmonic Acid Signaling Downstream of SCFCOI1-JAZ by Targeting GCC Promoter Motifs via Transcription Factor ORA59[C][W][OA

PubMed Central

Van der Does, Dieuwertje; Leon-Reyes, Antonio; Koornneef, Annemart; Van Verk, Marcel C.; Rodenburg, Nicole; Pauwels, Laurens; Goossens, Alain; Körbes, Ana P.; Memelink, Johan; Ritsema, Tita; Van Wees, Saskia C.M.; Pieterse, Corné M.J.

2013-01-01

Antagonism between the defense hormones salicylic acid (SA) and jasmonic acid (JA) plays a central role in the modulation of the plant immune signaling network, but the molecular mechanisms underlying this phenomenon are largely unknown. Here, we demonstrate that suppression of the JA pathway by SA functions downstream of the E3 ubiquitin-ligase Skip-Cullin-F-box complex SCFCOI1, which targets JASMONATE ZIM-domain transcriptional repressor proteins (JAZs) for proteasome-mediated degradation. In addition, neither the stability nor the JA-induced degradation of JAZs was affected by SA. In silico promoter analysis of the SA/JA crosstalk transcriptome revealed that the 1-kb promoter regions of JA-responsive genes that are suppressed by SA are significantly enriched in the JA-responsive GCC-box motifs. Using GCC:GUS lines carrying four copies of the GCC-box fused to the β-glucuronidase reporter gene, we showed that the GCC-box motif is sufficient for SA-mediated suppression of JA-responsive gene expression. Using plants overexpressing the GCC-box binding APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factors ERF1 or ORA59, we found that SA strongly reduces the accumulation of ORA59 but not that of ERF1. Collectively, these data indicate that the SA pathway inhibits JA signaling downstream of the SCFCOI1-JAZ complex by targeting GCC-box motifs in JA-responsive promoters via a negative effect on the transcriptional activator ORA59. PMID:23435661

NF-Y Binding Site Architecture Defines a C-Fos Targeted Promoter Class

PubMed Central

Haubrock, Martin; Hartmann, Fabian; Wingender, Edgar

2016-01-01

ChIP-seq experiments detect the chromatin occupancy of known transcription factors in a genome-wide fashion. The comparisons of several species-specific ChIP-seq libraries done for different transcription factors have revealed a complex combinatorial and context-specific co-localization behavior for the identified binding regions. In this study we have investigated human derived ChIP-seq data to identify common cis-regulatory principles for the human transcription factor c-Fos. We found that in four different cell lines, c-Fos targeted proximal and distal genomic intervals show prevalences for either AP-1 motifs or CCAAT boxes as known binding motifs for the transcription factor NF-Y, and thereby act in a mutually exclusive manner. For proximal regions of co-localized c-Fos and NF-YB binding, we gathered evidence that a characteristic configuration of repeating CCAAT motifs may be responsible for attracting c-Fos, probably provided by a nearby AP-1 bound enhancer. Our results suggest a novel regulatory function of NF-Y in gene-proximal regions. Specific CCAAT dimer repeats bound by the transcription factor NF-Y define this novel cis-regulatory module. Based on this behavior we propose a new enhancer promoter interaction model based on AP-1 motif defined enhancers which interact with CCAAT-box characterized promoter regions. PMID:27517874

Molecular population dynamics of DNA structures in a bcl-2 promoter sequence is regulated by small molecules and the transcription factor hnRNP LL

PubMed Central

Cui, Yunxi; Koirala, Deepak; Kang, HyunJin; Dhakal, Soma; Yangyuoru, Philip; Hurley, Laurence H.; Mao, Hanbin

2014-01-01

Minute difference in free energy change of unfolding among structures in an oligonucleotide sequence can lead to a complex population equilibrium, which is rather challenging for ensemble techniques to decipher. Herein, we introduce a new method, molecular population dynamics (MPD), to describe the intricate equilibrium among non-B deoxyribonucleic acid (DNA) structures. Using mechanical unfolding in laser tweezers, we identified six DNA species in a cytosine (C)-rich bcl-2 promoter sequence. Population patterns of these species with and without a small molecule (IMC-76 or IMC-48) or the transcription factor hnRNP LL are compared to reveal the MPD of different species. With a pattern recognition algorithm, we found that IMC-48 and hnRNP LL share 80% similarity in stabilizing i-motifs with 60 s incubation. In contrast, IMC-76 demonstrates an opposite behavior, preferring flexible DNA hairpins. With 120–180 s incubation, IMC-48 and hnRNP LL destabilize i-motifs, which has been previously proposed to activate bcl-2 transcriptions. These results provide strong support, from the population equilibrium perspective, that small molecules and hnRNP LL can modulate bcl-2 transcription through interaction with i-motifs. The excellent agreement with biochemical results firmly validates the MPD analyses, which, we expect, can be widely applicable to investigate complex equilibrium of biomacromolecules. PMID:24609386

Evolution of Drosophila ribosomal protein gene core promoters.

PubMed

Ma, Xiaotu; Zhang, Kangyu; Li, Xiaoman

2009-03-01

The coordinated expression of ribosomal protein genes (RPGs) has been well documented in many species. Previous analyses of RPG promoters focus only on Fungi and mammals. Recognizing this gap and using a comparative genomics approach, we utilize a motif-finding algorithm that incorporates cross-species conservation to identify several significant motifs in Drosophila RPG promoters. As a result, significant differences of the enriched motifs in RPG promoter are found among Drosophila, Fungi, and mammals, demonstrating the evolutionary dynamics of the ribosomal gene regulatory network. We also report a motif present in similar numbers of RPGs among Drosophila species which does not appear to be conserved at the individual RPG gene level. A module-wise stabilizing selection theory is proposed to explain this observation. Overall, our results provide significant insight into the fast-evolving nature of transcriptional regulation in the RPG module.

Evolution of Drosophila ribosomal protein gene core promoters

PubMed Central

Ma, Xiaotu; Zhang, Kangyu; Li, Xiaoman

2011-01-01

The coordinated expression of ribosomal protein genes (RPGs) has been well documented in many species. Previous analyses of RPG promoters focus only on Fungi and mammals. Recognizing this gap and using a comparative genomics approach, we utilize a motif-finding algorithm that incorporates cross-species conservation to identify several significant motifs in Drosophila RPG promoters. As a result, significant differences of the enriched motifs in RPG promoter are found among Drosophila, Fungi, and mammals, demonstrating the evolutionary dynamics of the ribosomal gene regulatory network. We also report a motif present in similar numbers of RPGs among Drosophila species which does not appear to be conserved at the individual RPG gene level. A module-wise stabilizing selection theory is proposed to explain this observation. Overall, our results provide significant insight into the fast-evolving nature of transcriptional regulation in the RPG module. PMID:19059316

A Novel Protein Interaction between Nucleotide Binding Domain of Hsp70 and p53 Motif

PubMed Central

Elengoe, Asita; Naser, Mohammed Abu; Hamdan, Salehhuddin

2015-01-01

Currently, protein interaction of Homo sapiens nucleotide binding domain (NBD) of heat shock 70 kDa protein (PDB: 1HJO) with p53 motif remains to be elucidated. The NBD-p53 motif complex enhances the p53 stabilization, thereby increasing the tumor suppression activity in cancer treatment. Therefore, we identified the interaction between NBD and p53 using STRING version 9.1 program. Then, we modeled the three-dimensional structure of p53 motif through homology modeling and determined the binding affinity and stability of NBD-p53 motif complex structure via molecular docking and dynamics (MD) simulation. Human DNA binding domain of p53 motif (SCMGGMNR) retrieved from UniProt (UniProtKB: P04637) was docked with the NBD protein, using the Autodock version 4.2 program. The binding energy and intermolecular energy for the NBD-p53 motif complex were −0.44 Kcal/mol and −9.90 Kcal/mol, respectively. Moreover, RMSD, RMSF, hydrogen bonds, salt bridge, and secondary structure analyses revealed that the NBD protein had a strong bond with p53 motif and the protein-ligand complex was stable. Thus, the current data would be highly encouraging for designing Hsp70 structure based drug in cancer therapy. PMID:26098630

A Novel Protein Interaction between Nucleotide Binding Domain of Hsp70 and p53 Motif.

PubMed

Elengoe, Asita; Naser, Mohammed Abu; Hamdan, Salehhuddin

2015-01-01

Currently, protein interaction of Homo sapiens nucleotide binding domain (NBD) of heat shock 70 kDa protein (PDB: 1HJO) with p53 motif remains to be elucidated. The NBD-p53 motif complex enhances the p53 stabilization, thereby increasing the tumor suppression activity in cancer treatment. Therefore, we identified the interaction between NBD and p53 using STRING version 9.1 program. Then, we modeled the three-dimensional structure of p53 motif through homology modeling and determined the binding affinity and stability of NBD-p53 motif complex structure via molecular docking and dynamics (MD) simulation. Human DNA binding domain of p53 motif (SCMGGMNR) retrieved from UniProt (UniProtKB: P04637) was docked with the NBD protein, using the Autodock version 4.2 program. The binding energy and intermolecular energy for the NBD-p53 motif complex were -0.44 Kcal/mol and -9.90 Kcal/mol, respectively. Moreover, RMSD, RMSF, hydrogen bonds, salt bridge, and secondary structure analyses revealed that the NBD protein had a strong bond with p53 motif and the protein-ligand complex was stable. Thus, the current data would be highly encouraging for designing Hsp70 structure based drug in cancer therapy.

Structural complexity of Dengue virus untranslated regions: cis-acting RNA motifs and pseudoknot interactions modulating functionality of the viral genome

PubMed Central

Sztuba-Solinska, Joanna; Teramoto, Tadahisa; Rausch, Jason W.; Shapiro, Bruce A.; Padmanabhan, Radhakrishnan; Le Grice, Stuart F. J.

2013-01-01

The Dengue virus (DENV) genome contains multiple cis-acting elements required for translation and replication. Previous studies indicated that a 719-nt subgenomic minigenome (DENV-MINI) is an efficient template for translation and (−) strand RNA synthesis in vitro. We performed a detailed structural analysis of DENV-MINI RNA, combining chemical acylation techniques, Pb2+ ion-induced hydrolysis and site-directed mutagenesis. Our results highlight protein-independent 5′–3′ terminal interactions involving hybridization between recognized cis-acting motifs. Probing analyses identified tandem dumbbell structures (DBs) within the 3′ terminus spaced by single-stranded regions, internal loops and hairpins with embedded GNRA-like motifs. Analysis of conserved motifs and top loops (TLs) of these dumbbells, and their proposed interactions with downstream pseudoknot (PK) regions, predicted an H-type pseudoknot involving TL1 of the 5′ DB and the complementary region, PK2. As disrupting the TL1/PK2 interaction, via ‘flipping’ mutations of PK2, previously attenuated DENV replication, this pseudoknot may participate in regulation of RNA synthesis. Computer modeling implied that this motif might function as autonomous structural/regulatory element. In addition, our studies targeting elements of the 3′ DB and its complementary region PK1 indicated that communication between 5′–3′ terminal regions strongly depends on structure and sequence composition of the 5′ cyclization region. PMID:23531545

Combinations of various CpG motifs cloned into plasmid backbone modulate and enhance protective immunity of viral replicon DNA anthrax vaccines.

PubMed

Yu, Yun-Zhou; Ma, Yao; Xu, Wen-Hui; Wang, Shuang; Sun, Zhi-Wei

2015-08-01

DNA vaccines are generally weak stimulators of the immune system. Fortunately, their efficacy can be improved using a viral replicon vector or by the addition of immunostimulatory CpG motifs, although the design of these engineered DNA vectors requires optimization. Our results clearly suggest that multiple copies of three types of CpG motifs or combinations of various types of CpG motifs cloned into a viral replicon vector backbone with strong immunostimulatory activities on human PBMC are efficient adjuvants for these DNA vaccines to modulate and enhance protective immunity against anthrax, although modifications with these different CpG forms in vivo elicited inconsistent immune response profiles. Modification with more copies of CpG motifs elicited more potent adjuvant effects leading to the generation of enhanced immunity, which indicated a CpG motif dose-dependent enhancement of antigen-specific immune responses. Notably, the enhanced and/or synchronous adjuvant effects were observed in modification with combinations of two different types of CpG motifs, which provides not only a contribution to the knowledge base on the adjuvant activities of CpG motifs combinations but also implications for the rational design of optimal DNA vaccines with combinations of CpG motifs as "built-in" adjuvants. We describe an efficient strategy to design and optimize DNA vaccines by the addition of combined immunostimulatory CpG motifs in a viral replicon DNA plasmid to produce strong immune responses, which indicates that the CpG-modified viral replicon DNA plasmid may be desirable for use as vector of DNA vaccines.

PhyloGibbs-MP: Module Prediction and Discriminative Motif-Finding by Gibbs Sampling

PubMed Central

Siddharthan, Rahul

2008-01-01

PhyloGibbs, our recent Gibbs-sampling motif-finder, takes phylogeny into account in detecting binding sites for transcription factors in DNA and assigns posterior probabilities to its predictions obtained by sampling the entire configuration space. Here, in an extension called PhyloGibbs-MP, we widen the scope of the program, addressing two major problems in computational regulatory genomics. First, PhyloGibbs-MP can localise predictions to small, undetermined regions of a large input sequence, thus effectively predicting cis-regulatory modules (CRMs) ab initio while simultaneously predicting binding sites in those modules—tasks that are usually done by two separate programs. PhyloGibbs-MP's performance at such ab initio CRM prediction is comparable with or superior to dedicated module-prediction software that use prior knowledge of previously characterised transcription factors. Second, PhyloGibbs-MP can predict motifs that differentiate between two (or more) different groups of regulatory regions, that is, motifs that occur preferentially in one group over the others. While other “discriminative motif-finders” have been published in the literature, PhyloGibbs-MP's implementation has some unique features and flexibility. Benchmarks on synthetic and actual genomic data show that this algorithm is successful at enhancing predictions of differentiating sites and suppressing predictions of common sites and compares with or outperforms other discriminative motif-finders on actual genomic data. Additional enhancements include significant performance and speed improvements, the ability to use “informative priors” on known transcription factors, and the ability to output annotations in a format that can be visualised with the Generic Genome Browser. In stand-alone motif-finding, PhyloGibbs-MP remains competitive, outperforming PhyloGibbs-1.0 and other programs on benchmark data. PMID:18769735

COPS: Detecting Co-Occurrence and Spatial Arrangement of Transcription Factor Binding Motifs in Genome-Wide Datasets

PubMed Central

Lohmann, Ingrid

2012-01-01

In multi-cellular organisms, spatiotemporal activity of cis-regulatory DNA elements depends on their occupancy by different transcription factors (TFs). In recent years, genome-wide ChIP-on-Chip, ChIP-Seq and DamID assays have been extensively used to unravel the combinatorial interaction of TFs with cis-regulatory modules (CRMs) in the genome. Even though genome-wide binding profiles are increasingly becoming available for different TFs, single TF binding profiles are in most cases not sufficient for dissecting complex regulatory networks. Thus, potent computational tools detecting statistically significant and biologically relevant TF-motif co-occurrences in genome-wide datasets are essential for analyzing context-dependent transcriptional regulation. We have developed COPS (Co-Occurrence Pattern Search), a new bioinformatics tool based on a combination of association rules and Markov chain models, which detects co-occurring TF binding sites (BSs) on genomic regions of interest. COPS scans DNA sequences for frequent motif patterns using a Frequent-Pattern tree based data mining approach, which allows efficient performance of the software with respect to both data structure and implementation speed, in particular when mining large datasets. Since transcriptional gene regulation very often relies on the formation of regulatory protein complexes mediated by closely adjoining TF binding sites on CRMs, COPS additionally detects preferred short distance between co-occurring TF motifs. The performance of our software with respect to biological significance was evaluated using three published datasets containing genomic regions that are independently bound by several TFs involved in a defined biological process. In sum, COPS is a fast, efficient and user-friendly tool mining statistically and biologically significant TFBS co-occurrences and therefore allows the identification of TFs that combinatorially regulate gene expression. PMID:23272209

Trend Motif: A Graph Mining Approach for Analysis of Dynamic Complex Networks

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jin, R; McCallen, S; Almaas, E

2007-05-28

Complex networks have been used successfully in scientific disciplines ranging from sociology to microbiology to describe systems of interacting units. Until recently, studies of complex networks have mainly focused on their network topology. However, in many real world applications, the edges and vertices have associated attributes that are frequently represented as vertex or edge weights. Furthermore, these weights are often not static, instead changing with time and forming a time series. Hence, to fully understand the dynamics of the complex network, we have to consider both network topology and related time series data. In this work, we propose a motifmore » mining approach to identify trend motifs for such purposes. Simply stated, a trend motif describes a recurring subgraph where each of its vertices or edges displays similar dynamics over a userdefined period. Given this, each trend motif occurrence can help reveal significant events in a complex system; frequent trend motifs may aid in uncovering dynamic rules of change for the system, and the distribution of trend motifs may characterize the global dynamics of the system. Here, we have developed efficient mining algorithms to extract trend motifs. Our experimental validation using three disparate empirical datasets, ranging from the stock market, world trade, to a protein interaction network, has demonstrated the efficiency and effectiveness of our approach.« less

2,6-Diiminopiperidin-1-ol: an overlooked motif relevant to uranyl and transition metal binding on poly(amidoxime) adsorbents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kennedy, Zachary C.; Cardenas, Allan Jay P.; Corbey, Jordan F.

2016-01-01

Glutardiamidoxime, a structural motif on sorbents used in uranium extraction from seawater, was discovered to cyclize in situ at room temperature to 2,6-diimino-piperidin-1-ol in the presence of uranyl nitrate. The new diimino motif was also generated when exposed to competing transition metals Cu(II) and Ni(II). Multinuclear μ-O bridged U(VI), Cu(II), and Ni(II) complexes featuring bound diimino ligands were isolated. A Cu(II) complex with the historically relevant cyclic imide dioxime motif is also reported for structural comparison to the reported diimino complexes.

Neural complexity: A graph theoretic interpretation

NASA Astrophysics Data System (ADS)

Barnett, L.; Buckley, C. L.; Bullock, S.

2011-04-01

One of the central challenges facing modern neuroscience is to explain the ability of the nervous system to coherently integrate information across distinct functional modules in the absence of a central executive. To this end, Tononi [Proc. Natl. Acad. Sci. USA.PNASA60027-842410.1073/pnas.91.11.5033 91, 5033 (1994)] proposed a measure of neural complexity that purports to capture this property based on mutual information between complementary subsets of a system. Neural complexity, so defined, is one of a family of information theoretic metrics developed to measure the balance between the segregation and integration of a system’s dynamics. One key question arising for such measures involves understanding how they are influenced by network topology. Sporns [Cereb. Cortex53OPAV1047-321110.1093/cercor/10.2.127 10, 127 (2000)] employed numerical models in order to determine the dependence of neural complexity on the topological features of a network. However, a complete picture has yet to be established. While De Lucia [Phys. Rev. EPLEEE81539-375510.1103/PhysRevE.71.016114 71, 016114 (2005)] made the first attempts at an analytical account of this relationship, their work utilized a formulation of neural complexity that, we argue, did not reflect the intuitions of the original work. In this paper we start by describing weighted connection matrices formed by applying a random continuous weight distribution to binary adjacency matrices. This allows us to derive an approximation for neural complexity in terms of the moments of the weight distribution and elementary graph motifs. In particular, we explicitly establish a dependency of neural complexity on cyclic graph motifs.

Complexation of imidazopyridine-based cations with a 24-crown-8 ether host: [2]pseudorotaxane and partially threaded structures.

PubMed

Moreno-Olivares, Surisadai I; Cervantes, Ruy; Tiburcio, Jorge

2013-11-01

A new series of linear molecules derived from 1,2-bis(imidazopyridin-2-yl)ethane can fully or partially penetrate the cavity of the dibenzo-24-crown-8 macrocycle to produce a new family of host-guest complexes. Protonation or alkylation of the nitrogen atoms on the pyridine rings led to an increase in the guest total positive charge up to 4+ and simultaneously generated two new recognition sites (pyridinium motifs) that are in competition with the 1,2-bis(benzimidazole)ethane motif for the crown ether. The relative position of the pyridine ring and the chemical nature of the N-substituent determined the preferred motif and the host-guest complex geometry: (i) for linear guests with relatively bulky groups (i.e., a benzyl substituent), the 1,2-bis(benzimidazole)ethane motif is favored, leading to a fully threaded complex with a [2]pseudorotaxane geometry; (ii) for small substituents, such as -H and -CH3 groups, regardless of the guest shape, the pyridinium motifs are preferred, leading to external partially threaded complexes in a 2:1 host to guest stoichiometry.

Molecular population dynamics of DNA structures in a bcl-2 promoter sequence is regulated by small molecules and the transcription factor hnRNP LL.

PubMed

Cui, Yunxi; Koirala, Deepak; Kang, HyunJin; Dhakal, Soma; Yangyuoru, Philip; Hurley, Laurence H; Mao, Hanbin

2014-05-01

Minute difference in free energy change of unfolding among structures in an oligonucleotide sequence can lead to a complex population equilibrium, which is rather challenging for ensemble techniques to decipher. Herein, we introduce a new method, molecular population dynamics (MPD), to describe the intricate equilibrium among non-B deoxyribonucleic acid (DNA) structures. Using mechanical unfolding in laser tweezers, we identified six DNA species in a cytosine (C)-rich bcl-2 promoter sequence. Population patterns of these species with and without a small molecule (IMC-76 or IMC-48) or the transcription factor hnRNP LL are compared to reveal the MPD of different species. With a pattern recognition algorithm, we found that IMC-48 and hnRNP LL share 80% similarity in stabilizing i-motifs with 60 s incubation. In contrast, IMC-76 demonstrates an opposite behavior, preferring flexible DNA hairpins. With 120-180 s incubation, IMC-48 and hnRNP LL destabilize i-motifs, which has been previously proposed to activate bcl-2 transcriptions. These results provide strong support, from the population equilibrium perspective, that small molecules and hnRNP LL can modulate bcl-2 transcription through interaction with i-motifs. The excellent agreement with biochemical results firmly validates the MPD analyses, which, we expect, can be widely applicable to investigate complex equilibrium of biomacromolecules. © 2014 The Author(s). Published by Oxford University Press [on behalf of Nucleic Acids Research].

20-Hydroxyecdysone (20E) Primary Response Gene E93 Modulates 20E Signaling to Promote Bombyx Larval-Pupal Metamorphosis*

PubMed Central

Liu, Xi; Dai, Fangyin; Guo, Enen; Li, Kang; Ma, Li; Tian, Ling; Cao, Yang; Zhang, Guozheng; Palli, Subba R.; Li, Sheng

2015-01-01

As revealed in a previous microarray study to identify genes regulated by 20-hydroxyecdysone (20E) and juvenile hormone (JH) in the silkworm, Bombyx mori, E93 expression in the fat body was markedly low prior to the wandering stage but abundant during larval-pupal metamorphosis. Induced by 20E and suppressed by JH, E93 expression follows this developmental profile in multiple silkworm alleles. The reduction of E93 expression by RNAi disrupted 20E signaling and the 20E-induced autophagy, caspase activity, and cell dissociation in the fat body. Reducing E93 expression also decreased the expression of the 20E-induced pupal-specific cuticle protein genes and prevented growth and differentiation of the wing discs. Importantly, the two HTH domains in E93 are critical for inducing the expression of a subset of 20E response genes, including EcR, USP, E74, Br-C, and Atg1. By contrast, the LLQHLL and PLDLSAK motifs in E93 inhibit its transcriptional activity. E93 binds to the EcR-USP complex via a physical association with USP through its LLQHLL motif; and this association is enhanced by 20E-induced EcR-USP interaction, which attenuates the transcriptional activity of E93. E93 acts through the two HTH domains to bind to GAGA-containing motifs present in the Atg1 promoter region for inducing gene expression. In conclusion, E93 transcriptionally modulates 20E signaling to promote Bombyx larval-pupal metamorphosis. PMID:26378227

20-Hydroxyecdysone (20E) Primary Response Gene E93 Modulates 20E Signaling to Promote Bombyx Larval-Pupal Metamorphosis.

PubMed

Liu, Xi; Dai, Fangyin; Guo, Enen; Li, Kang; Ma, Li; Tian, Ling; Cao, Yang; Zhang, Guozheng; Palli, Subba R; Li, Sheng

2015-11-06

As revealed in a previous microarray study to identify genes regulated by 20-hydroxyecdysone (20E) and juvenile hormone (JH) in the silkworm, Bombyx mori, E93 expression in the fat body was markedly low prior to the wandering stage but abundant during larval-pupal metamorphosis. Induced by 20E and suppressed by JH, E93 expression follows this developmental profile in multiple silkworm alleles. The reduction of E93 expression by RNAi disrupted 20E signaling and the 20E-induced autophagy, caspase activity, and cell dissociation in the fat body. Reducing E93 expression also decreased the expression of the 20E-induced pupal-specific cuticle protein genes and prevented growth and differentiation of the wing discs. Importantly, the two HTH domains in E93 are critical for inducing the expression of a subset of 20E response genes, including EcR, USP, E74, Br-C, and Atg1. By contrast, the LLQHLL and PLDLSAK motifs in E93 inhibit its transcriptional activity. E93 binds to the EcR-USP complex via a physical association with USP through its LLQHLL motif; and this association is enhanced by 20E-induced EcR-USP interaction, which attenuates the transcriptional activity of E93. E93 acts through the two HTH domains to bind to GAGA-containing motifs present in the Atg1 promoter region for inducing gene expression. In conclusion, E93 transcriptionally modulates 20E signaling to promote Bombyx larval-pupal metamorphosis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Modeling and Docking Studies on Novel Mutants (K71L and T204V) of the ATPase Domain of Human Heat Shock 70 kDa Protein 1

PubMed Central

Elengoe, Asita; Naser, Mohammed Abu; Hamdan, Salehhuddin

2014-01-01

The purpose of exploring protein interactions between human adenovirus and heat shock protein 70 is to exploit a potentially synergistic interaction to enhance anti-tumoral efficacy and decrease toxicity in cancer treatment. However, the protein interaction of Hsp70 with E1A32 kDa of human adenovirus serotype 5 remains to be elucidated. In this study, two residues of ATPase domain of human heat shock 70 kDa protein 1 (PDB: 1 HJO) were mutated. 3D mutant models (K71L and T204V) using PyMol software were then constructed. The structures were evaluated by PROCHECK, ProQ, ERRAT, Verify 3D and ProSA modules. All evidence suggests that all protein models are acceptable and of good quality. The E1A32 kDa motif was retrieved from UniProt (P03255), as well as subjected to docking interaction with NBD, K71L and T204V, using the Autodock 4.2 program. The best lowest binding energy value of −9.09 kcal/mol was selected for novel T204V. Moreover, the protein-ligand complex structures were validated by RMSD, RMSF, hydrogen bonds and salt bridge analysis. This revealed that the T204V-E1A32 kDa motif complex was the most stable among all three complex structures. This study provides information about the interaction between Hsp70 and the E1A32 kDa motif, which emphasizes future perspectives to design rational drugs and vaccines in cancer therapy. PMID:24758925

Distinct modes of recruitment of the CCR4-NOT complex by Drosophila and vertebrate Nanos.

PubMed

Raisch, Tobias; Bhandari, Dipankar; Sabath, Kevin; Helms, Sigrun; Valkov, Eugene; Weichenrieder, Oliver; Izaurralde, Elisa

2016-05-02

Nanos proteins repress the expression of target mRNAs by recruiting effector complexes through non-conserved N-terminal regions. In vertebrates, Nanos proteins interact with the NOT1 subunit of the CCR4-NOT effector complex through a NOT1 interacting motif (NIM), which is absent in Nanos orthologs from several invertebrate species. Therefore, it has remained unclear whether the Nanos repressive mechanism is conserved and whether it also involves direct interactions with the CCR4-NOT deadenylase complex in invertebrates. Here, we identify an effector domain (NED) that is necessary for the Drosophila melanogaster (Dm) Nanos to repress mRNA targets. The NED recruits the CCR4-NOT complex through multiple and redundant binding sites, including a central region that interacts with the NOT module, which comprises the C-terminal domains of NOT1-3. The crystal structure of the NED central region bound to the NOT module reveals an unanticipated bipartite binding interface that contacts NOT1 and NOT3 and is distinct from the NIM of vertebrate Nanos. Thus, despite the absence of sequence conservation, the N-terminal regions of Nanos proteins recruit CCR4-NOT to assemble analogous repressive complexes. © 2016 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

Function, dynamics and evolution of network motif modules in integrated gene regulatory networks of worm and plant.

PubMed

Defoort, Jonas; Van de Peer, Yves; Vermeirssen, Vanessa

2018-06-05

Gene regulatory networks (GRNs) consist of different molecular interactions that closely work together to establish proper gene expression in time and space. Especially in higher eukaryotes, many questions remain on how these interactions collectively coordinate gene regulation. We study high quality GRNs consisting of undirected protein-protein, genetic and homologous interactions, and directed protein-DNA, regulatory and miRNA-mRNA interactions in the worm Caenorhabditis elegans and the plant Arabidopsis thaliana. Our data-integration framework integrates interactions in composite network motifs, clusters these in biologically relevant, higher-order topological network motif modules, overlays these with gene expression profiles and discovers novel connections between modules and regulators. Similar modules exist in the integrated GRNs of worm and plant. We show how experimental or computational methodologies underlying a certain data type impact network topology. Through phylogenetic decomposition, we found that proteins of worm and plant tend to functionally interact with proteins of a similar age, while at the regulatory level TFs favor same age, but also older target genes. Despite some influence of the duplication mode difference, we also observe at the motif and module level for both species a preference for age homogeneity for undirected and age heterogeneity for directed interactions. This leads to a model where novel genes are added together to the GRNs in a specific biological functional context, regulated by one or more TFs that also target older genes in the GRNs. Overall, we detected topological, functional and evolutionary properties of GRNs that are potentially universal in all species.

Helix–hairpin–helix motifs confer salt resistance and processivity on chimeric DNA polymerases

PubMed Central

Pavlov, Andrey R.; Belova, Galina I.; Kozyavkin, Sergei A.; Slesarev, Alexei I.

2002-01-01

Helix–hairpin–helix (HhH) is a widespread motif involved in sequence-nonspecific DNA binding. The majority of HhH motifs function as DNA-binding modules with typical occurrence of one HhH motif or one or two (HhH)2 domains in proteins. We recently identified 24 HhH motifs in DNA topoisomerase V (Topo V). Although these motifs are dispensable for the topoisomerase activity of Topo V, their removal narrows the salt concentration range for topoisomerase activity tenfold. Here, we demonstrate the utility of Topo V's HhH motifs for modulating DNA-binding properties of the Stoffel fragment of TaqDNA polymerase and Pfu DNA polymerase. Different HhH cassettes fused with either NH2 terminus or COOH terminus of DNA polymerases broaden the salt concentration range of the polymerase activity significantly (up to 0.5 M NaCl or 1.8 M potassium glutamate). We found that anions play a major role in the inhibition of DNA polymerase activity. The resistance of initial extension rates and the processivity of chimeric polymerases to salts depend on the structure of added HhH motifs. Regardless of the type of the construct, the thermal stability of chimeric Taq polymerases increases under the optimal ionic conditions, as compared with that of TaqDNA polymerase or its Stoffel fragment. Our approach to raise the salt tolerance, processivity, and thermostability of Taq and Pfu DNA polymerases may be applied to all pol1- and polB-type polymerases, as well as to other DNA processing enzymes. PMID:12368475

Fast social-like learning of complex behaviors based on motor motifs.

PubMed

Calvo Tapia, Carlos; Tyukin, Ivan Y; Makarov, Valeri A

2018-05-01

Social learning is widely observed in many species. Less experienced agents copy successful behaviors exhibited by more experienced individuals. Nevertheless, the dynamical mechanisms behind this process remain largely unknown. Here we assume that a complex behavior can be decomposed into a sequence of n motor motifs. Then a neural network capable of activating motor motifs in a given sequence can drive an agent. To account for (n-1)! possible sequences of motifs in a neural network, we employ the winnerless competition approach. We then consider a teacher-learner situation: one agent exhibits a complex movement, while another one aims at mimicking the teacher's behavior. Despite the huge variety of possible motif sequences we show that the learner, equipped with the provided learning model, can rewire "on the fly" its synaptic couplings in no more than (n-1) learning cycles and converge exponentially to the durations of the teacher's motifs. We validate the learning model on mobile robots. Experimental results show that the learner is indeed capable of copying the teacher's behavior composed of six motor motifs in a few learning cycles. The reported mechanism of learning is general and can be used for replicating different functions, including, for example, sound patterns or speech.

Fast social-like learning of complex behaviors based on motor motifs

NASA Astrophysics Data System (ADS)

Calvo Tapia, Carlos; Tyukin, Ivan Y.; Makarov, Valeri A.

2018-05-01

Social learning is widely observed in many species. Less experienced agents copy successful behaviors exhibited by more experienced individuals. Nevertheless, the dynamical mechanisms behind this process remain largely unknown. Here we assume that a complex behavior can be decomposed into a sequence of n motor motifs. Then a neural network capable of activating motor motifs in a given sequence can drive an agent. To account for (n -1 )! possible sequences of motifs in a neural network, we employ the winnerless competition approach. We then consider a teacher-learner situation: one agent exhibits a complex movement, while another one aims at mimicking the teacher's behavior. Despite the huge variety of possible motif sequences we show that the learner, equipped with the provided learning model, can rewire "on the fly" its synaptic couplings in no more than (n -1 ) learning cycles and converge exponentially to the durations of the teacher's motifs. We validate the learning model on mobile robots. Experimental results show that the learner is indeed capable of copying the teacher's behavior composed of six motor motifs in a few learning cycles. The reported mechanism of learning is general and can be used for replicating different functions, including, for example, sound patterns or speech.

μ2-Dependent endocytosis of N-cadherin is regulated by β-catenin to facilitate neurite outgrowth.

PubMed

Chen, Yi-Ting; Tai, Chin-Yin

2017-05-01

Circuit formation in the brain requires neurite outgrowth throughout development to establish synaptic contacts with target cells. Active endocytosis of several adhesion molecules facilitates the dynamic exchange of these molecules at the surface and promotes neurite outgrowth in developing neurons. The endocytosis of N-cadherin, a calcium-dependent adhesion molecule, has been implicated in the regulation of neurite outgrowth, but the mechanism remains unclear. Here, we identified that a fraction of N-cadherin internalizes through clathrin-mediated endocytosis (CME). Two tyrosine-based motifs in the cytoplasmic domain of N-cadherin recognized by the μ2 subunit of the AP-2 adaptor complex are responsible for CME of N-cadherin. Moreover, β-catenin, a core component of the N-cadherin adhesion complex, inhibits N-cadherin endocytosis by masking the 2 tyrosine-based motifs. Removal of β-catenin facilitates μ2 binding to N-cadherin, thereby increasing clathrin-mediated N-cadherin endocytosis and neurite outgrowth without affecting the steady-state level of surface N-cadherin. These results identify and characterize the mechanism controlling N-cadherin endocytosis through β-catenin-regulated μ2 binding to modulate neurite outgrowth. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A role for the PDZ-binding domain of the coxsackie B virus and adenovirus receptor (CAR) in cell adhesion and growth.

PubMed

Excoffon, Katherine J D Ashbourne; Hruska-Hageman, Alesia; Klotz, Michael; Traver, Geri L; Zabner, Joseph

2004-09-01

The coxsackie and adenovirus receptor (CAR) plays a role in viral infection, maintenance of the junction adhesion complex in polarized epithelia, and modulation of cellular growth properties. As a viral receptor, the C-terminus appears to play no role indicating that the major function of CAR is to tether the virus to the cell. By contrast, the C-terminus is known to play a role in cellular localization and probably has a significant function in CAR-mediated adhesion and cell growth properties. We hypothesized that the CAR PDZ (PSD-95/Disc-large/ZO-1) binding motif interacts with PDZ-domain-containing proteins to modulate the cellular phenotype. CAR was modified by deleting the last four amino acids (CARDeltaGSIV) and evaluated for cell-cell adhesion in polarized primary human airway epithelia and growth characteristics in stably transfected L-cells. Although ablation of the CAR PDZ-binding motif did not affect adenoviral infection, it did have a significant effect both on cell-cell adhesion and on cell growth. Expression of CARDeltaGSIV failed to increase the transepithelial resistance in polarized epithelia to the same degree as wild-type CAR and failed to act as a growth modulator in L-cells. Furthermore, we provide evidence for three new CAR interacting partners, including MAGI-1b, PICK1 and PSD-95. CAR appears to interact with several distinct PDZ-domain-containing proteins and may exert its biological function through these interactions.

Co-regulation analysis of co-expressed modules under cold and pathogen stress conditions in tomato.

PubMed

Abedini, Davar; Rashidi Monfared, Sajad

2018-06-01

A primary mechanism for controlling the development of multicellular organisms is transcriptional regulation, which carried out by transcription factors (TFs) that recognize and bind to their binding sites on promoter region. The distance from translation start site, order, orientation, and spacing between cis elements are key factors in the concentration of active nuclear TFs and transcriptional regulation of target genes. In this study, overrepresented motifs in cold and pathogenesis responsive genes were scanned via Gibbs sampling method, this method is based on detection of overrepresented motifs by means of a stochastic optimization strategy that searches for all possible sets of short DNA segments. Then, identified motifs were checked by TRANSFAC, PLACE and Soft Berry databases in order to identify putative TFs which, interact to the motifs. Several cis/trans regulatory elements were found using these databases. Moreover, cross-talk between cold and pathogenesis responsive genes were confirmed. Statistical analysis was used to determine distribution of identified motifs on promoter region. In addition, co-regulation analysis results, illustrated genes in pathogenesis responsive module are divided into two main groups. Also, promoter region was crunched to six subareas in order to draw the pattern of distribution of motifs in promoter subareas. The result showed the majority of motifs are concentrated on 700 nucleotides upstream of the translational start site (ATG). In contrast, this result isn't true in another group. In other words, there was no difference between total and compartmentalized regions in cold responsive genes.

Interactions of the Human LIP5 Regulatory Protein with Endosomal Sorting Complexes Required for Transport*♦

PubMed Central

Skalicky, Jack J.; Arii, Jun; Wenzel, Dawn M.; Stubblefield, William-May B.; Katsuyama, Angela; Uter, Nathan T.; Bajorek, Monika; Myszka, David G.; Sundquist, Wesley I.

2012-01-01

The endosomal sorting complex required for transport (ESCRT) pathway remodels membranes during multivesicular body biogenesis, the abscission stage of cytokinesis, and enveloped virus budding. The ESCRT-III and VPS4 ATPase complexes catalyze the membrane fission events associated with these processes, and the LIP5 protein helps regulate their interactions by binding directly to a subset of ESCRT-III proteins and to VPS4. We have investigated the biochemical and structural basis for different LIP5-ligand interactions and show that the first microtubule-interacting and trafficking (MIT) module of the tandem LIP5 MIT domain binds CHMP1B (and other ESCRT-III proteins) through canonical type 1 MIT-interacting motif (MIM1) interactions. In contrast, the second LIP5 MIT module binds with unusually high affinity to a novel MIM element within the ESCRT-III protein CHMP5. A solution structure of the relevant LIP5-CHMP5 complex reveals that CHMP5 helices 5 and 6 and adjacent linkers form an amphipathic “leucine collar” that wraps almost completely around the second LIP5 MIT module but makes only limited contacts with the first MIT module. LIP5 binds MIM1-containing ESCRT-III proteins and CHMP5 and VPS4 ligands independently in vitro, but these interactions are coupled within cells because formation of stable VPS4 complexes with both LIP5 and CHMP5 requires LIP5 to bind both a MIM1-containing ESCRT-III protein and CHMP5. Our studies thus reveal how the tandem MIT domain of LIP5 binds different types of ESCRT-III proteins, promoting assembly of active VPS4 enzymes on the polymeric ESCRT-III substrate. PMID:23105106

Ubiquitin recognition by FAAP20 expands the complex interface beyond the canonical UBZ domain

PubMed Central

Wojtaszek, Jessica L.; Wang, Su; Kim, Hyungjin; Wu, Qinglin; D'Andrea, Alan D.; Zhou, Pei

2014-01-01

FAAP20 is an integral component of the Fanconi anemia core complex that mediates the repair of DNA interstrand crosslinks. The ubiquitin-binding capacity of the FAAP20 UBZ is required for recruitment of the Fanconi anemia complex to interstrand DNA crosslink sites and for interaction with the translesion synthesis machinery. Although the UBZ–ubiquitin interaction is thought to be exclusively encapsulated within the ββα module of UBZ, we show that the FAAP20–ubiquitin interaction extends beyond such a canonical zinc-finger motif. Instead, ubiquitin binding by FAAP20 is accompanied by transforming a disordered tail C-terminal to the UBZ of FAAP20 into a rigid, extended β-loop that latches onto the complex interface of the FAAP20 UBZ and ubiquitin, with the invariant C-terminal tryptophan emanating toward I44Ub for enhanced binding specificity and affinity. Substitution of the C-terminal tryptophan with alanine in FAAP20 not only abolishes FAAP20–ubiquitin binding in vitro, but also causes profound cellular hypersensitivity to DNA interstrand crosslink lesions in vivo, highlighting the indispensable role of the C-terminal tail of FAAP20, beyond the compact zinc finger module, toward ubiquitin recognition and Fanconi anemia complex-mediated DNA interstrand crosslink repair. PMID:25414354

Functional cis-regulatory modules encoded by mouse-specific endogenous retrovirus

PubMed Central

Sundaram, Vasavi; Choudhary, Mayank N. K.; Pehrsson, Erica; Xing, Xiaoyun; Fiore, Christopher; Pandey, Manishi; Maricque, Brett; Udawatta, Methma; Ngo, Duc; Chen, Yujie; Paguntalan, Asia; Ray, Tammy; Hughes, Ava; Cohen, Barak A.; Wang, Ting

2017-01-01

Cis-regulatory modules contain multiple transcription factor (TF)-binding sites and integrate the effects of each TF to control gene expression in specific cellular contexts. Transposable elements (TEs) are uniquely equipped to deposit their regulatory sequences across a genome, which could also contain cis-regulatory modules that coordinate the control of multiple genes with the same regulatory logic. We provide the first evidence of mouse-specific TEs that encode a module of TF-binding sites in mouse embryonic stem cells (ESCs). The majority (77%) of the individual TEs tested exhibited enhancer activity in mouse ESCs. By mutating individual TF-binding sites within the TE, we identified a module of TF-binding motifs that cooperatively enhanced gene expression. Interestingly, we also observed the same motif module in the in silico constructed ancestral TE that also acted cooperatively to enhance gene expression. Our results suggest that ancestral TE insertions might have brought in cis-regulatory modules into the mouse genome. PMID:28348391

Inferring Mechanisms of Compensation from E-MAP and SGA Data Using Local Search Algorithms for Max Cut

NASA Astrophysics Data System (ADS)

Leiserson, Mark D. M.; Tatar, Diana; Cowen, Lenore J.; Hescott, Benjamin J.

A new method based on a mathematically natural local search framework for max cut is developed to uncover functionally coherent module and BPM motifs in high-throughput genetic interaction data. Unlike previous methods which also consider physical protein-protein interaction data, our method utilizes genetic interaction data only; this becomes increasingly important as high-throughput genetic interaction data is becoming available in settings where less is known about physical interaction data. We compare modules and BPMs obtained to previous methods and across different datasets. Despite needing no physical interaction information, the BPMs produced by our method are competitive with previous methods. Biological findings include a suggested global role for the prefoldin complex and a SWR subcomplex in pathway buffering in the budding yeast interactome.

Inferring mechanisms of compensation from E-MAP and SGA data using local search algorithms for max cut.

PubMed

Leiserson, Mark D M; Tatar, Diana; Cowen, Lenore J; Hescott, Benjamin J

2011-11-01

A new method based on a mathematically natural local search framework for max cut is developed to uncover functionally coherent module and BPM motifs in high-throughput genetic interaction data. Unlike previous methods, which also consider physical protein-protein interaction data, our method utilizes genetic interaction data only; this becomes increasingly important as high-throughput genetic interaction data is becoming available in settings where less is known about physical interaction data. We compare modules and BPMs obtained to previous methods and across different datasets. Despite needing no physical interaction information, the BPMs produced by our method are competitive with previous methods. Biological findings include a suggested global role for the prefoldin complex and a SWR subcomplex in pathway buffering in the budding yeast interactome.

Phosphorylation of the Usher syndrome 1G protein SANS controls Magi2-mediated endocytosis.

PubMed

Bauß, Katharina; Knapp, Barbara; Jores, Pia; Roepman, Ronald; Kremer, Hannie; Wijk, Erwin V; Märker, Tina; Wolfrum, Uwe

2014-08-01

The human Usher syndrome (USH) is a complex ciliopathy with at least 12 chromosomal loci assigned to three clinical subtypes, USH1-3. The heterogeneous USH proteins are organized into protein networks. Here, we identified Magi2 (membrane-associated guanylate kinase inverted-2) as a new component of the USH protein interactome, binding to the multifunctional scaffold protein SANS (USH1G). We showed that the SANS-Magi2 complex assembly is regulated by the phosphorylation of an internal PDZ-binding motif in the sterile alpha motif domain of SANS by the protein kinase CK2. We affirmed Magi2's role in receptor-mediated, clathrin-dependent endocytosis and showed that phosphorylated SANS tightly regulates Magi2-mediated endocytosis. Specific depletions by RNAi revealed that SANS and Magi2-mediated endocytosis regulates aspects of ciliogenesis. Furthermore, we demonstrated the localization of the SANS-Magi2 complex in the periciliary membrane complex facing the ciliary pocket of retinal photoreceptor cells in situ. Our data suggest that endocytotic processes may not only contribute to photoreceptor cell homeostasis but also counterbalance the periciliary membrane delivery accompanying the exocytosis processes for the cargo vesicle delivery. In USH1G patients, mutations in SANS eliminate Magi2 binding and thereby deregulate endocytosis, lead to defective ciliary transport modules and ultimately disrupt photoreceptor cell function inducing retinal degeneration. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

New partner proteins containing novel internal recognition motif for human Glutaminase Interacting Protein (hGIP)

PubMed Central

Zencir, Sevil; Banerjee, Monimoy; Dobson, Melanie J.; Ayaydin, Ferhan; Fodor, Elfrieda Ayaydin; Topcu, Zeki; Mohanty, Smita

2013-01-01

Regulation of gene expression in cells is mediated by protein-protein, DNA-protein and receptor-ligand interactions. PDZ (PSD-95/Discs-large/ZO-1) domains are protein–protein interaction modules. PDZ-containing proteins function in the organization of multi-protein complexes controlling spatial and temporal fidelity of intracellular signaling pathways. In general, PDZ proteins possess multiple domains facilitating distinct interactions. The human Glutaminase Interacting Protein (hGIP) is an unusual PDZ protein comprising entirely of a single PDZ domain and plays pivotal roles in many cellular processes through its interaction with the C-terminus of partner proteins. Here, we report the identification by yeast two-hybrid screening of two new hGIP-interacting partners, DTX1 and STAU1. Both proteins lack the typical C-terminal PDZ recognition motif but contain a novel internal hGIP recognition motif recently identified in a phage display library screen. Fluorescence resonance energy transfer and confocal microscopy analysis confirmed the in vivo association of hGIP with DTX1 and STAU1 in mammalian cells validating the previous discovery of S/T-X-V/L-D as a consensus internal motif for hGIP recognition. Similar to hGIP, DTX1 and STAU1 have been implicated in neuronal function. Identification of these new interacting partners furthers our understanding of GIP-regulated signaling cascades and these interactions may represent potential new drug targets in humans. PMID:23395680

RNA motif search with data-driven element ordering.

PubMed

Rampášek, Ladislav; Jimenez, Randi M; Lupták, Andrej; Vinař, Tomáš; Brejová, Broňa

2016-05-18

In this paper, we study the problem of RNA motif search in long genomic sequences. This approach uses a combination of sequence and structure constraints to uncover new distant homologs of known functional RNAs. The problem is NP-hard and is traditionally solved by backtracking algorithms. We have designed a new algorithm for RNA motif search and implemented a new motif search tool RNArobo. The tool enhances the RNAbob descriptor language, allowing insertions in helices, which enables better characterization of ribozymes and aptamers. A typical RNA motif consists of multiple elements and the running time of the algorithm is highly dependent on their ordering. By approaching the element ordering problem in a principled way, we demonstrate more than 100-fold speedup of the search for complex motifs compared to previously published tools. We have developed a new method for RNA motif search that allows for a significant speedup of the search of complex motifs that include pseudoknots. Such speed improvements are crucial at a time when the rate of DNA sequencing outpaces growth in computing. RNArobo is available at http://compbio.fmph.uniba.sk/rnarobo .

Thioredoxin reductase regulates AP-1 activity as well as thioredoxin nuclear localization via active cysteines in response to ionizing radiation.

PubMed

Karimpour, Shervin; Lou, Junyang; Lin, Lilie L; Rene, Luis M; Lagunas, Lucio; Ma, Xinrong; Karra, Sreenivasu; Bradbury, C Matthew; Markovina, Stephanie; Goswami, Prabhat C; Spitz, Douglas R; Hirota, Kiichi; Kalvakolanu, Dhananjaya V; Yodoi, Junji; Gius, David

2002-09-12

A recently identified class of signaling factors uses critical cysteine motif(s) that act as redox-sensitive 'sulfhydryl switches' to reversibly modulate specific signal transduction cascades regulating downstream proteins with similar redox-sensitive sites. For example, signaling factors such as redox factor-1 (Ref-1) and transcription factors such as the AP-1 complex both contain redox-sensitive cysteine motifs that regulate activity in response to oxidative stress. The mammalian thioredoxin reductase-1 (TR) is an oxidoreductase selenocysteine-containing flavoprotein that also appears to regulate multiple downstream intracellular redox-sensitive proteins. Since ionizing radiation (IR) induces oxidative stress as well as increases AP-1 DNA-binding activity via the activation of Ref-1, the potential roles of TR and thioredoxin (TRX) in the regulation of AP-1 activity in response to IR were investigated. Permanently transfected cell lines that overexpress wild type TR demonstrated constitutive increases in AP-1 DNA-binding activity as well as AP-1-dependent reporter gene expression, relative to vector control cells. In contrast, permanently transfected cell lines expressing a TR gene with the active site cysteine motif deleted were unable to induce AP-1 activity or reporter gene expression in response to IR. Transient genetic overexpression of either the TR wild type or dominant-negative genes demonstrated similar results using a transient assay system. One mechanism through which TR regulates AP-1 activity appears to involve TRX sub-cellular localization, with no change in the total TRX content of the cell. These results identify a novel function of the TR enzyme as a signaling factor in the regulation of AP-1 activity via a cysteine motif located in the protein.

Mismatch and G-Stack Modulated Probe Signals on SNP Microarrays

PubMed Central

Binder, Hans; Fasold, Mario; Glomb, Torsten

2009-01-01

Background Single nucleotide polymorphism (SNP) arrays are important tools widely used for genotyping and copy number estimation. This technology utilizes the specific affinity of fragmented DNA for binding to surface-attached oligonucleotide DNA probes. We analyze the variability of the probe signals of Affymetrix GeneChip SNP arrays as a function of the probe sequence to identify relevant sequence motifs which potentially cause systematic biases of genotyping and copy number estimates. Methodology/Principal Findings The probe design of GeneChip SNP arrays enables us to disentangle different sources of intensity modulations such as the number of mismatches per duplex, matched and mismatched base pairings including nearest and next-nearest neighbors and their position along the probe sequence. The effect of probe sequence was estimated in terms of triple-motifs with central matches and mismatches which include all 256 combinations of possible base pairings. The probe/target interactions on the chip can be decomposed into nearest neighbor contributions which correlate well with free energy terms of DNA/DNA-interactions in solution. The effect of mismatches is about twice as large as that of canonical pairings. Runs of guanines (G) and the particular type of mismatched pairings formed in cross-allelic probe/target duplexes constitute sources of systematic biases of the probe signals with consequences for genotyping and copy number estimates. The poly-G effect seems to be related to the crowded arrangement of probes which facilitates complex formation of neighboring probes with at minimum three adjacent G's in their sequence. Conclusions The applied method of “triple-averaging” represents a model-free approach to estimate the mean intensity contributions of different sequence motifs which can be applied in calibration algorithms to correct signal values for sequence effects. Rules for appropriate sequence corrections are suggested. PMID:19924253

Coevolved Mutations Reveal Distinct Architectures for Two Core Proteins in the Bacterial Flagellar Motor

PubMed Central

Pandini, Alessandro; Kleinjung, Jens; Rasool, Shafqat; Khan, Shahid

2015-01-01

Switching of bacterial flagellar rotation is caused by large domain movements of the FliG protein triggered by binding of the signal protein CheY to FliM. FliG and FliM form adjacent multi-subunit arrays within the basal body C-ring. The movements alter the interaction of the FliG C-terminal (FliGC) “torque” helix with the stator complexes. Atomic models based on the Salmonella entrovar C-ring electron microscopy reconstruction have implications for switching, but lack consensus on the relative locations of the FliG armadillo (ARM) domains (amino-terminal (FliGN), middle (FliGM) and FliGC) as well as changes during chemotaxis. The generality of the Salmonella model is challenged by the variation in motor morphology and response between species. We studied coevolved residue mutations to determine the unifying elements of switch architecture. Residue interactions, measured by their coevolution, were formalized as a network, guided by structural data. Our measurements reveal a common design with dedicated switch and motor modules. The FliM middle domain (FliMM) has extensive connectivity most simply explained by conserved intra and inter-subunit contacts. In contrast, FliG has patchy, complex architecture. Conserved structural motifs form interacting nodes in the coevolution network that wire FliMM to the FliGC C-terminal, four-helix motor module (C3-6). FliG C3-6 coevolution is organized around the torque helix, differently from other ARM domains. The nodes form separated, surface-proximal patches that are targeted by deleterious mutations as in other allosteric systems. The dominant node is formed by the EHPQ motif at the FliMMFliGM contact interface and adjacent helix residues at a central location within FliGM. The node interacts with nodes in the N-terminal FliGc α-helix triad (ARM-C) and FliGN. ARM-C, separated from C3-6 by the MFVF motif, has poor intra-network connectivity consistent with its variable orientation revealed by structural data. ARM-C could be the convertor element that provides mechanistic and species diversity. PMID:26561852

The Crc and Hfq proteins of Pseudomonas putida cooperate in catabolite repression and formation of ribonucleic acid complexes with specific target motifs.

PubMed

Moreno, Renata; Hernández-Arranz, Sofía; La Rosa, Ruggero; Yuste, Luis; Madhushani, Anjana; Shingler, Victoria; Rojo, Fernando

2015-01-01

The Crc protein is a global regulator that has a key role in catabolite repression and optimization of metabolism in Pseudomonads. Crc inhibits gene expression post-transcriptionally, preventing translation of mRNAs bearing an AAnAAnAA motif [the catabolite activity (CA) motif] close to the translation start site. Although Crc was initially believed to bind RNA by itself, this idea was recently challenged by results suggesting that a protein co-purifying with Crc, presumably the Hfq protein, could account for the detected RNA-binding activity. Hfq is an abundant protein that has a central role in post-transcriptional gene regulation. Herein, we show that the Pseudomonas putida Hfq protein can recognize the CA motifs of RNAs through its distal face and that Crc facilitates formation of a more stable complex at these targets. Crc was unable to bind RNA in the absence of Hfq. However, pull-down assays showed that Crc and Hfq can form a co-complex with RNA containing a CA motif in vitro. Inactivation of the hfq or the crc gene impaired catabolite repression to a similar extent. We propose that Crc and Hfq cooperate in catabolite repression, probably through forming a stable co-complex with RNAs containing CA motifs to result in inhibition of translation initiation. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

KIRMES: kernel-based identification of regulatory modules in euchromatic sequences.

PubMed

Schultheiss, Sebastian J; Busch, Wolfgang; Lohmann, Jan U; Kohlbacher, Oliver; Rätsch, Gunnar

2009-08-15

Understanding transcriptional regulation is one of the main challenges in computational biology. An important problem is the identification of transcription factor (TF) binding sites in promoter regions of potential TF target genes. It is typically approached by position weight matrix-based motif identification algorithms using Gibbs sampling, or heuristics to extend seed oligos. Such algorithms succeed in identifying single, relatively well-conserved binding sites, but tend to fail when it comes to the identification of combinations of several degenerate binding sites, as those often found in cis-regulatory modules. We propose a new algorithm that combines the benefits of existing motif finding with the ones of support vector machines (SVMs) to find degenerate motifs in order to improve the modeling of regulatory modules. In experiments on microarray data from Arabidopsis thaliana, we were able to show that the newly developed strategy significantly improves the recognition of TF targets. The python source code (open source-licensed under GPL), the data for the experiments and a Galaxy-based web service are available at http://www.fml.mpg.de/raetsch/suppl/kirmes/.

INCENP Centromere and Spindle Targeting: Identification of Essential Conserved Motifs and Involvement of Heterochromatin Protein HP1

PubMed Central

Ainsztein, Alexandra M.; Kandels-Lewis, Stefanie E.; Mackay, Alastair M.; Earnshaw, William C.

1998-01-01

The inner centromere protein (INCENP) has a modular organization, with domains required for chromosomal and cytoskeletal functions concentrated near the amino and carboxyl termini, respectively. In this study we have identified an autonomous centromere- and midbody-targeting module in the amino-terminal 68 amino acids of INCENP. Within this module, we have identified two evolutionarily conserved amino acid sequence motifs: a 13–amino acid motif that is required for targeting to centromeres and transfer to the spindle, and an 11–amino acid motif that is required for transfer to the spindle by molecules that have targeted previously to the centromere. To begin to understand the mechanisms of INCENP function in mitosis, we have performed a yeast two-hybrid screen for interacting proteins. These and subsequent in vitro binding experiments identify a physical interaction between INCENP and heterochromatin protein HP1Hsα. Surprisingly, this interaction does not appear to be involved in targeting INCENP to the centromeric heterochromatin, but may instead have a role in its transfer from the chromosomes to the anaphase spindle. PMID:9864353

The Reconstruction of Condition-Specific Transcriptional Modules Provides New Insights in the Evolution of Yeast AP-1 Proteins

PubMed Central

Goudot, Christel; Etchebest, Catherine

2011-01-01

AP-1 proteins are transcription factors (TFs) that belong to the basic leucine zipper family, one of the largest families of TFs in eukaryotic cells. Despite high homology between their DNA binding domains, these proteins are able to recognize diverse DNA motifs. In yeasts, these motifs are referred as YRE (Yap Response Element) and are either seven (YRE-Overlap) or eight (YRE-Adjacent) base pair long. It has been proposed that the AP-1 DNA binding motif preference relies on a single change in the amino acid sequence of the yeast AP-1 TFs (an arginine in the YRE-O binding factors being replaced by a lysine in the YRE-A binding Yaps). We developed a computational approach to infer condition-specific transcriptional modules associated to the orthologous AP-1 protein Yap1p, Cgap1p and Cap1p, in three yeast species: the model yeast Saccharomyces cerevisiae and two pathogenic species Candida glabrata and Candida albicans. Exploitation of these modules in terms of predictions of the protein/DNA regulatory interactions changed our vision of AP-1 protein evolution. Cis-regulatory motif analyses revealed the presence of a conserved adenine in 5′ position of the canonical YRE sites. While Yap1p, Cgap1p and Cap1p shared a remarkably low number of target genes, an impressive conservation was observed in the YRE sequences identified by Yap1p and Cap1p. In Candida glabrata, we found that Cgap1p, unlike Yap1p and Cap1p, recognizes YRE-O and YRE-A motifs. These findings were supported by structural data available for the transcription factor Pap1p (Schizosaccharomyces pombe). Thus, whereas arginine and lysine substitutions in Cgap1p and Yap1p proteins were reported as responsible for a specific YRE-O or YRE-A preference, our analyses rather suggest that the ancestral yeast AP-1 protein could recognize both YRE-O and YRE-A motifs and that the arginine/lysine exchange is not the only determinant of the specialization of modern Yaps for one motif or another. PMID:21695268

Inferring Mechanisms of Compensation from E-MAP and SGA Data Using Local Search Algorithms for Max Cut

PubMed Central

Leiserson, Mark D.M.; Tatar, Diana; Cowen, Lenore J.

2011-01-01

Abstract A new method based on a mathematically natural local search framework for max cut is developed to uncover functionally coherent module and BPM motifs in high-throughput genetic interaction data. Unlike previous methods, which also consider physical protein-protein interaction data, our method utilizes genetic interaction data only; this becomes increasingly important as high-throughput genetic interaction data is becoming available in settings where less is known about physical interaction data. We compare modules and BPMs obtained to previous methods and across different datasets. Despite needing no physical interaction information, the BPMs produced by our method are competitive with previous methods. Biological findings include a suggested global role for the prefoldin complex and a SWR subcomplex in pathway buffering in the budding yeast interactome. PMID:21882903

Recurring sequence-structure motifs in (βα)8-barrel proteins and experimental optimization of a chimeric protein designed based on such motifs.

PubMed

Wang, Jichao; Zhang, Tongchuan; Liu, Ruicun; Song, Meilin; Wang, Juncheng; Hong, Jiong; Chen, Quan; Liu, Haiyan

2017-02-01

An interesting way of generating novel artificial proteins is to combine sequence motifs from natural proteins, mimicking the evolutionary path suggested by natural proteins comprising recurring motifs. We analyzed the βα and αβ modules of TIM barrel proteins by structure alignment-based sequence clustering. A number of preferred motifs were identified. A chimeric TIM was designed by using recurring elements as mutually compatible interfaces. The foldability of the designed TIM protein was then significantly improved by six rounds of directed evolution. The melting temperature has been improved by more than 20°C. A variety of characteristics suggested that the resulting protein is well-folded. Our analysis provided a library of peptide motifs that is potentially useful for different protein engineering studies. The protein engineering strategy of using recurring motifs as interfaces to connect partial natural proteins may be applied to other protein folds. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular Investigations of the Structure and Function of the Protein Phosphatase 1:Spinophilin:Inhibitor-2 Heterotrimeric Complex

PubMed Central

Dancheck, Barbara; Ragusa, Michael J.; Allaire, Marc; Nairn, Angus C.; Page, Rebecca; Peti, Wolfgang

2011-01-01

Regulation of the major ser/thr phosphatase Protein Phosphatase 1 (PP1) is controlled by a diverse array of targeting and inhibitor proteins. Though many PP1 regulatory proteins share at least one PP1 binding motif, usually the RVxF motif, it was recently discovered that certain pairs of targeting and inhibitor proteins bind PP1 simultaneously to form PP1 heterotrimeric complexes. To date, structural information for these heterotrimeric complexes, and, in turn, how they direct PP1 activity is entirely lacking. Using a combination of NMR spectroscopy, biochemistry and small angle X-ray scattering (SAXS), we show that major structural rearrangements in both spinophilin (targeting) and Inhibitor-2 (I-2, inhibitor) are essential for the formation of the heterotrimeric PP1:spinophilin:I-2 (PSI) complex. The RVxF motif of I-2 is released from PP1 during the formation of PSI, making the less prevalent SILK motif of I-2 essential for complex stability. The release of the I-2 RVxF motif allows for enhanced flexibility of both I-2 and spinophilin in the heterotrimeric complex. In addition, we used inductively coupled plasma atomic emission spectroscopy to show that PP1 contains two metals in both heterodimeric complexes (PP1:spinophilin and PP1:I2) and PSI, demonstrating that PSI retains the biochemical characteristics of the PP1:I2 holoenzyme. Finally, we combined the NMR and biochemical data with SAXS and molecular dynamics simulations to generate a structural model of the full heterotrimeric PSI complex. Collectively, these data reveal the molecular events that enable PP1 heterotrimeric complexes to exploit both the targeting and inhibitory features of the PP1-regulatory proteins to form multi-functional PP1 holoenzymes. PMID:21218781

Unique structural modulation of a non-native substrate by cochaperone DnaJ.

PubMed

Tiwari, Satyam; Kumar, Vignesh; Jayaraj, Gopal Gunanathan; Maiti, Souvik; Mapa, Koyeli

2013-02-12

The role of bacterial DnaJ protein as a cochaperone of DnaK is strongly appreciated. Although DnaJ unaccompanied by DnaK can bind unfolded as well as native substrate proteins, its role as an individual chaperone remains elusive. In this study, we demonstrate that DnaJ binds a model non-native substrate with a low nanomolar dissociation constant and, more importantly, modulates the structure of its non-native state. The structural modulation achieved by DnaJ is different compared to that achieved by the DnaK-DnaJ complex. The nature of structural modulation exerted by DnaJ is suggestive of a unique unfolding activity on the non-native substrate by the chaperone. Furthermore, we demonstrate that the zinc binding motif along with the C-terminal substrate binding domain of DnaJ is necessary and sufficient for binding and the subsequent binding-induced structural alterations of the non-native substrate. We hypothesize that this hitherto unknown structural alteration of non-native states by DnaJ might be important for its chaperoning activity by removing kinetic traps of the folding intermediates.

Conservation of RNA chaperone activity of the human La-related proteins 4, 6 and 7.

PubMed

Hussain, Rawaa H; Zawawi, Mariam; Bayfield, Mark A

2013-10-01

The La module is a conserved tandem arrangement of a La motif and RNA recognition motif whose function has been best characterized in genuine La proteins. The best-characterized substrates of La proteins are pre-tRNAs, and previous work using tRNA mediated suppression in Schizosaccharomyces pombe has demonstrated that yeast and human La enhance the maturation of these using two distinguishable activities: UUU-3'OH-dependent trailer binding/protection and a UUU-3'OH independent activity related to RNA chaperone function. The La module has also been identified in several conserved families of La-related proteins (LARPs) that engage other RNAs, but their mode of RNA binding and function(s) are not well understood. We demonstrate that the La modules of the human LARPs 4, 6 and 7 are also active in tRNA-mediated suppression, even in the absence of stable UUU-3'OH trailer protection. Rather, the capacity of these to enhance pre-tRNA maturation is associated with RNA chaperone function, which we demonstrate to be a conserved activity for each hLARP in vitro. Our work reveals insight into the mechanisms by which La module containing proteins discriminate RNA targets and demonstrates that RNA chaperone activity is a conserved function across representative members of the La motif-containing superfamily.

Analysis of remote synchronization in complex networks

NASA Astrophysics Data System (ADS)

Gambuzza, Lucia Valentina; Cardillo, Alessio; Fiasconaro, Alessandro; Fortuna, Luigi; Gómez-Gardeñes, Jesus; Frasca, Mattia

2013-12-01

A novel regime of synchronization, called remote synchronization, where the peripheral nodes form a phase synchronized cluster not including the hub, was recently observed in star motifs [Bergner et al., Phys. Rev. E 85, 026208 (2012)]. We show the existence of a more general dynamical state of remote synchronization in arbitrary networks of coupled oscillators. This state is characterized by the synchronization of pairs of nodes that are not directly connected via a physical link or any sequence of synchronized nodes. This phenomenon is almost negligible in networks of phase oscillators as its underlying mechanism is the modulation of the amplitude of those intermediary nodes between the remotely synchronized units. Our findings thus show the ubiquity and robustness of these states and bridge the gap from their recent observation in simple toy graphs to complex networks.

Papillomavirus E6 oncoproteins

PubMed Central

Vande Pol, Scott B.; Klingelhutz, Aloysius J.

2013-01-01

Papillomaviruses induce benign and malignant epithelial tumors, and the viral E6 oncoprotein is essential for full transformation. E6 contributes to transformation by associating with cellular proteins, docking on specific acidic LXXLL peptide motifs found on the associated cellular proteins. This review examines insights from recent studies of human and animal E6 proteins that determine the three-dimensional structure of E6 when bound to acidic LXXLL peptides. The structure of E6 is related to recent advances in the purification and identification of E6 associated protein complexes. These E6 protein-complexes, together with other proteins that bind to E6, alter a broad array of biological outcomes including modulation of cell survival, cellular transcription, host cell differentiation, growth factor dependence, DNA damage responses, and cell cycle progression. PMID:23711382

Chromatin Protein HP1α Interacts with the Mitotic Regulator Borealin Protein and Specifies the Centromere Localization of the Chromosomal Passenger Complex*

PubMed Central

Liu, Xing; Song, Zhenwei; Huo, Yuda; Zhang, Jiahai; Zhu, Tongge; Wang, Jianyu; Zhao, Xuannv; Aikhionbare, Felix; Zhang, Jiancun; Duan, Hequan; Wu, Jihui; Dou, Zhen; Shi, Yunyu; Yao, Xuebiao

2014-01-01

Accurate mitosis requires the chromosomal passenger protein complex (CPC) containing Aurora B kinase, borealin, INCENP, and survivin, which orchestrates chromosome dynamics. However, the chromatin factors that specify the CPC to the centromere remain elusive. Here we show that borealin interacts directly with heterochromatin protein 1α (HP1α) and that this interaction is mediated by an evolutionarily conserved PXVXL motif in the C-terminal borealin with the chromo shadow domain of HP1α. This borealin-HP1α interaction recruits the CPC to the centromere and governs an activation of Aurora B kinase judged by phosphorylation of Ser-7 in CENP-A, a substrate of Aurora B. Consistently, modulation of the motif PXVXL leads to defects in CPC centromere targeting and aberrant Aurora B activity. On the other hand, the localization of the CPC in the midzone is independent of the borealin-HP1α interaction, demonstrating the spatial requirement of HP1α in CPC localization to the centromere. These findings reveal a previously unrecognized but direct link between HP1α and CPC localization in the centromere and illustrate the critical role of borealin-HP1α interaction in orchestrating an accurate cell division. PMID:24917673

Substantial conformational change mediated by charge-triad residues of the death effector domain in protein-protein interactions.

PubMed

Twomey, Edward C; Cordasco, Dana F; Kozuch, Stephen D; Wei, Yufeng

2013-01-01

Protein conformational changes are commonly associated with the formation of protein complexes. The non-catalytic death effector domains (DEDs) mediate protein-protein interactions in a variety of cellular processes, including apoptosis, proliferation and migration, and glucose metabolism. Here, using NMR residual dipolar coupling (RDC) data, we report a conformational change in the DED of the phosphoprotein enriched in astrocytes, 15 kDa (PEA-15) protein in the complex with a mitogen-activated protein (MAP) kinase, extracellular regulated kinase 2 (ERK2), which is essential in regulating ERK2 cellular distribution and function in cell proliferation and migration. The most significant conformational change in PEA-15 happens at helices α2, α3, and α4, which also possess the highest flexibility among the six-helix bundle of the DED. This crucial conformational change is modulated by the D/E-RxDL charge-triad motif, one of the prominent structural features of DEDs, together with a number of other electrostatic and hydrogen bonding interactions on the protein surface. Charge-triad motif promotes the optimal orientation of key residues and expands the binding interface to accommodate protein-protein interactions. However, the charge-triad residues are not directly involved in the binding interface between PEA-15 and ERK2.

Binding of serum response factor to cystic fibrosis transmembrane conductance regulator CArG-like elements, as a new potential CFTR transcriptional regulation pathway

PubMed Central

René, Céline; Taulan, Magali; Iral, Florence; Doudement, Julien; L'Honoré, Aurore; Gerbon, Catherine; Demaille, Jacques; Claustres, Mireille; Romey, Marie-Catherine

2005-01-01

CFTR expression is tightly controlled by a complex network of ubiquitous and tissue-specific cis-elements and trans-factors. To better understand mechanisms that regulate transcription of CFTR, we examined transcription factors that specifically bind a CFTR CArG-like motif we have previously shown to modulate CFTR expression. Gel mobility shift assays and chromatin immunoprecipitation analyses demonstrated the CFTR CArG-like motif binds serum response factor both in vitro and in vivo. Transient co-transfections with various SRF expression vector, including dominant-negative forms and small interfering RNA, demonstrated that SRF significantly increases CFTR transcriptional activity in bronchial epithelial cells. Mutagenesis studies suggested that in addition to SRF other co-factors, such as Yin Yang 1 (YY1) previously shown to bind the CFTR promoter, are potentially involved in the CFTR regulation. Here, we show that functional interplay between SRF and YY1 might provide interesting perspectives to further characterize the underlying molecular mechanism of the basal CFTR transcriptional activity. Furthermore, the identification of multiple CArG binding sites in highly conserved CFTR untranslated regions, which form specific SRF complexes, provides direct evidence for a considerable role of SRF in the CFTR transcriptional regulation into specialized epithelial lung cells. PMID:16170155

RHIM-based protein:protein interactions in anti-microbial defence against programmed cell death by necroptosis.

PubMed

Baker, Max O D G; Shanmugam, Nirukshan; Pham, Chi L L; Strange, Merryn; Steain, Megan; Sunde, Margaret

2018-05-05

The Receptor-interacting protein kinase Homotypic Interaction Motif (RHIM) is an amino acid sequence that mediates multiple protein:protein interactions in the mammalian programmed cell death pathway known as necroptosis. At least one key RHIM-based complex has been shown to have a functional amyloid fibril structure, which provides a stable hetero-oligomeric platform for downstream signaling. RHIMs and related motifs are present in immunity-related proteins across nature, from viruses to fungi to metazoans. Necroptosis is a hallmark feature of cellular clearance of infection. For this reason, numerous pathogens, including viruses and bacteria, have developed varied methods to modulate necroptosis, focusing on inhibiting RHIM:RHIM interactions, and thus their downstream cell death effects. This review will discuss current understanding of RHIM:RHIM interactions in normal cellular activation of necroptosis, from a structural and cell biology perspective. It will compare the mechanisms by which pathogens subvert these interactions in order to maintain their replicative and infective cycles and consider the similarities between RHIMs and other functional amyloid-forming proteins associated with cell death and innate immunity. It will discuss the implications of the heteromeric nature and structure of RHIM-based amyloid complexes in the context of other functional amyloids. Copyright © 2018. Published by Elsevier Ltd.

Control of Recombination Directionality by the Listeria Phage A118 Protein Gp44 and the Coiled-Coil Motif of Its Serine Integrase.

PubMed

Mandali, Sridhar; Gupta, Kushol; Dawson, Anthony R; Van Duyne, Gregory D; Johnson, Reid C

2017-06-01

The serine integrase of phage A118 catalyzes integrative recombination between attP on the phage and a specific attB locus on the chromosome of Listeria monocytogenes , but it is unable to promote excisive recombination between the hybrid attL and attR sites found on the integrated prophage without assistance by a recombination directionality factor (RDF). We have identified and characterized the phage-encoded RDF Gp44, which activates the A118 integrase for excision and inhibits integration. Gp44 binds to the C-terminal DNA binding domain of integrase, and we have localized the primary binding site to be within the mobile coiled-coil (CC) motif but distinct from the distal tip of the CC that is required for recombination. This interaction is sufficient to inhibit integration, but a second interaction involving the N-terminal end of Gp44 is also required to activate excision. We provide evidence that these two contacts modulate the trajectory of the CC motifs as they extend out from the integrase core in a manner dependent upon the identities of the four att sites. Our results support a model whereby Gp44 shapes the Int-bound complexes to control which att sites can synapse and recombine. IMPORTANCE Serine integrases mediate directional recombination between bacteriophage and bacterial chromosomes. These highly regulated site-specific recombination reactions are integral to the life cycle of temperate phage and, in the case of Listeria monocytogenes lysogenized by A118 family phage, are an essential virulence determinant. Serine integrases are also utilized as tools for genetic engineering and synthetic biology because of their exquisite unidirectional control of the DNA exchange reaction. Here, we identify and characterize the recombination directionality factor (RDF) that activates excision and inhibits integration reactions by the phage A118 integrase. We provide evidence that the A118 RDF binds to and modulates the trajectory of the long coiled-coil motif that extends from the large carboxyl-terminal DNA binding domain and is postulated to control the early steps of recombination site synapsis. Copyright © 2017 American Society for Microbiology.

Functional interaction of proliferating cell nuclear antigen with MSH2-MSH6 and MSH2-MSH3 complexes.

PubMed

Clark, A B; Valle, F; Drotschmann, K; Gary, R K; Kunkel, T A

2000-11-24

Eukaryotic DNA mismatch repair requires the concerted action of several proteins, including proliferating cell nuclear antigen (PCNA) and heterodimers of MSH2 complexed with either MSH3 or MSH6. Here we report that MSH3 and MSH6, but not MSH2, contain N-terminal sequence motifs characteristic of proteins that bind to PCNA. MSH3 and MSH6 peptides containing these motifs bound PCNA, as did the intact Msh2-Msh6 complex. This binding was strongly reduced when alanine was substituted for conserved residues in the motif. Yeast strains containing alanine substitutions in the PCNA binding motif of Msh6 or Msh3 had elevated mutation rates, indicating that these interactions are important for genome stability. When human MSH3 or MSH6 peptides containing the PCNA binding motif were added to a human cell extract, mismatch repair activity was inhibited at a step preceding DNA resynthesis. Thus, MSH3 and MSH6 interactions with PCNA may facilitate early steps in DNA mismatch repair and may also be important for other roles of these eukaryotic MutS homologs.

Space-related pharma-motifs for fast search of protein binding motifs and polypharmacological targets

PubMed Central

2012-01-01

Background To discover a compound inhibiting multiple proteins (i.e. polypharmacological targets) is a new paradigm for the complex diseases (e.g. cancers and diabetes). In general, the polypharmacological proteins often share similar local binding environments and motifs. As the exponential growth of the number of protein structures, to find the similar structural binding motifs (pharma-motifs) is an emergency task for drug discovery (e.g. side effects and new uses for old drugs) and protein functions. Results We have developed a Space-Related Pharmamotifs (called SRPmotif) method to recognize the binding motifs by searching against protein structure database. SRPmotif is able to recognize conserved binding environments containing spatially discontinuous pharma-motifs which are often short conserved peptides with specific physico-chemical properties for protein functions. Among 356 pharma-motifs, 56.5% interacting residues are highly conserved. Experimental results indicate that 81.1% and 92.7% polypharmacological targets of each protein-ligand complex are annotated with same biological process (BP) and molecular function (MF) terms, respectively, based on Gene Ontology (GO). Our experimental results show that the identified pharma-motifs often consist of key residues in functional (active) sites and play the key roles for protein functions. The SRPmotif is available at http://gemdock.life.nctu.edu.tw/SRP/. Conclusions SRPmotif is able to identify similar pharma-interfaces and pharma-motifs sharing similar binding environments for polypharmacological targets by rapidly searching against the protein structure database. Pharma-motifs describe the conservations of binding environments for drug discovery and protein functions. Additionally, these pharma-motifs provide the clues for discovering new sequence-based motifs to predict protein functions from protein sequence databases. We believe that SRPmotif is useful for elucidating protein functions and drug discovery. PMID:23281852

Space-related pharma-motifs for fast search of protein binding motifs and polypharmacological targets.

PubMed

Chiu, Yi-Yuan; Lin, Chun-Yu; Lin, Chih-Ta; Hsu, Kai-Cheng; Chang, Li-Zen; Yang, Jinn-Moon

2012-01-01

To discover a compound inhibiting multiple proteins (i.e. polypharmacological targets) is a new paradigm for the complex diseases (e.g. cancers and diabetes). In general, the polypharmacological proteins often share similar local binding environments and motifs. As the exponential growth of the number of protein structures, to find the similar structural binding motifs (pharma-motifs) is an emergency task for drug discovery (e.g. side effects and new uses for old drugs) and protein functions. We have developed a Space-Related Pharmamotifs (called SRPmotif) method to recognize the binding motifs by searching against protein structure database. SRPmotif is able to recognize conserved binding environments containing spatially discontinuous pharma-motifs which are often short conserved peptides with specific physico-chemical properties for protein functions. Among 356 pharma-motifs, 56.5% interacting residues are highly conserved. Experimental results indicate that 81.1% and 92.7% polypharmacological targets of each protein-ligand complex are annotated with same biological process (BP) and molecular function (MF) terms, respectively, based on Gene Ontology (GO). Our experimental results show that the identified pharma-motifs often consist of key residues in functional (active) sites and play the key roles for protein functions. The SRPmotif is available at http://gemdock.life.nctu.edu.tw/SRP/. SRPmotif is able to identify similar pharma-interfaces and pharma-motifs sharing similar binding environments for polypharmacological targets by rapidly searching against the protein structure database. Pharma-motifs describe the conservations of binding environments for drug discovery and protein functions. Additionally, these pharma-motifs provide the clues for discovering new sequence-based motifs to predict protein functions from protein sequence databases. We believe that SRPmotif is useful for elucidating protein functions and drug discovery.

Integration of G protein α (Gα) signaling by the regulator of G protein signaling 14 (RGS14).

PubMed

Brown, Nicole E; Goswami, Devrishi; Branch, Mary Rose; Ramineni, Suneela; Ortlund, Eric A; Griffin, Patrick R; Hepler, John R

2015-04-03

RGS14 contains distinct binding sites for both active (GTP-bound) and inactive (GDP-bound) forms of Gα subunits. The N-terminal regulator of G protein signaling (RGS) domain binds active Gαi/o-GTP, whereas the C-terminal G protein regulatory (GPR) motif binds inactive Gαi1/3-GDP. The molecular basis for how RGS14 binds different activation states of Gα proteins to integrate G protein signaling is unknown. Here we explored the intramolecular communication between the GPR motif and the RGS domain upon G protein binding and examined whether RGS14 can functionally interact with two distinct forms of Gα subunits simultaneously. Using complementary cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive Gαi1-GDP at the plasma membrane and that free cytosolic RGS14 is recruited to the plasma membrane by activated Gαo-AlF4(-). Bioluminescence resonance energy transfer studies showed that RGS14 adopts different conformations in live cells when bound to Gα in different activation states. Hydrogen/deuterium exchange mass spectrometry revealed that RGS14 is a very dynamic protein that undergoes allosteric conformational changes when inactive Gαi1-GDP binds the GPR motif. Pure RGS14 forms a ternary complex with Gαo-AlF4(-) and an AlF4(-)-insensitive mutant (G42R) of Gαi1-GDP, as observed by size exclusion chromatography and differential hydrogen/deuterium exchange. Finally, a preformed RGS14·Gαi1-GDP complex exhibits full capacity to stimulate the GTPase activity of Gαo-GTP, demonstrating that RGS14 can functionally engage two distinct forms of Gα subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals in host CA2 hippocampal neurons to modulate synaptic plasticity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

TFIID TAF6-TAF9 Complex Formation Involves the HEAT Repeat-containing C-terminal Domain of TAF6 and Is Modulated by TAF5 Protein*

PubMed Central

Scheer, Elisabeth; Delbac, Frédéric; Tora, Laszlo; Moras, Dino; Romier, Christophe

2012-01-01

The general transcription factor TFIID recognizes specifically the core promoter of genes transcribed by eukaryotic RNA polymerase II, nucleating the assembly of the preinitiation complex at the transcription start site. However, the understanding in molecular terms of TFIID assembly and function remains poorly understood. Histone fold motifs have been shown to be extremely important for the heterodimerization of many TFIID subunits. However, these subunits display several evolutionary conserved noncanonical features when compared with histones, including additional regions whose role is unknown. Here we show that the conserved additional C-terminal region of TFIID subunit TAF6 can be divided into two domains: a small middle domain (TAF6M) and a large C-terminal domain (TAF6C). Our crystal structure of the TAF6C domain from Antonospora locustae at 1.9 Å resolution reveals the presence of five conserved HEAT repeats. Based on these data, we designed several mutants that were introduced into full-length human TAF6. Surprisingly, the mutants affect the interaction between TAF6 and TAF9, suggesting that the formation of the complex between these two TFIID subunits do not only depend on their histone fold motifs. In addition, the same mutants affect even more strongly the interaction between TAF6 and TAF9 in the context of a TAF5-TAF6-TAF9 complex. Expression of these mutants in HeLa cells reveals that most of them are unstable, suggesting their poor incorporation within endogenous TFIID. Taken together, our results suggest that the conserved additional domains in histone fold-containing subunits of TFIID and of co-activator SAGA are important for the assembly of these complexes. PMID:22696218

A novel protein-protein interaction in the RES (REtention and Splicing) complex.

PubMed

Tripsianes, Konstantinos; Friberg, Anders; Barrandon, Charlotte; Brooks, Mark; van Tilbeurgh, Herman; Seraphin, Bertrand; Sattler, Michael

2014-10-10

The retention and splicing (RES) complex is a conserved spliceosome-associated module that was shown to enhance splicing of a subset of transcripts and promote the nuclear retention of unspliced pre-mRNAs in yeast. The heterotrimeric RES complex is organized around the Snu17p protein that binds to both the Bud13p and Pml1p subunits. Snu17p exhibits an RRM domain that resembles a U2AF homology motif (UHM) and Bud13p harbors a Trp residue reminiscent of an UHM-ligand motif (ULM). It has therefore been proposed that the interaction between Snu17p and Bud13p resembles canonical UHM-ULM complexes. Here, we have used biochemical and NMR structural analysis to characterize the structure of the yeast Snu17p-Bud13p complex. Unlike known UHMs that sequester the Trp residue of the ULM ligand in a hydrophobic pocket, Snu17p and Bud13p utilize a large interaction surface formed around the two helices of the Snu17p domain. In total 18 residues of the Bud13p ligand wrap around the Snu17p helical surface in an U-turn-like arrangement. The invariant Trp(232) in Bud13p is located in the center of the turn, and contacts surface residues of Snu17p. The structural data are supported by mutational analysis and indicate that Snu17p provides an extended binding surface with Bud13p that is notably distinct from canonical UHM-ULM interactions. Our data highlight structural diversity in RRM-protein interactions, analogous to the one seen for nucleic acid interactions. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Crystallographic and Computational Studies of a Class II MHC Complex with a Nonconforming Peptide: HLA-DRA/DRB3*0101

NASA Astrophysics Data System (ADS)

Parry, Christian S.; Gorski, Jack; Stern, Lawrence J.

2003-03-01

The stable binding of processed foreign peptide to a class II major histocompatibility (MHC) molecule and subsequent presentation to a T cell receptor is a central event in immune recognition and regulation. Polymorphic residues on the floor of the peptide binding site form pockets that anchor peptide side chains. These and other residues in the helical wall of the groove determine the specificity of each allele and define a motif. Allele specific motifs allow the prediction of epitopes from the sequence of pathogens. There are, however, known epitopes that do not satisfy these motifs: anchor motifs are not adequate for predicting epitopes as there are apparently major and minor motifs. We present crystallographic studies into the nature of the interactions that govern the binding of these so called nonconforming peptides. We would like to understand the role of the P10 pocket and find out whether the peptides that do not obey the consensus anchor motif bind in the canonical conformation observed in in prior structures of class II MHC-peptide complexes. HLA-DRB3*0101 complexed with peptide crystallized in unit cell 92.10 x 92.10 x 248.30 (90, 90, 90), P41212, and the diffraction data is reliable to 2.2ÅWe are complementing our studies with dynamical long time simulations to answer these questions, particularly the interplay of the anchor motifs in peptide binding, the range of protein and ligand conformations, and water hydration structures.

Single site mutations in the hetero-oligomeric Mrp antiporter from alkaliphilic Bacillus pseudofirmus OF4 that affect Na+/H+ antiport activity, sodium exclusion, individual Mrp protein levels, or Mrp complex formation.

PubMed

Morino, Masato; Natsui, Shinsuke; Ono, Tomohiro; Swartz, Talia H; Krulwich, Terry A; Ito, Masahiro

2010-10-01

Mrp systems are widely distributed and structurally complex cation/proton antiporters. Antiport activity requires hetero-oligomeric complexes of all six or seven hydrophobic Mrp proteins (MrpA-MrpG). Here, a panel of site-directed mutants in conserved or proposed motif residues was made in the Mrp Na(+)(Li(+))/H(+) antiporter from an alkaliphilic Bacillus. The mutant operons were expressed in antiporter-deficient Escherichia coli KNabc and assessed for antiport properties, support of sodium resistance, membrane levels of each Mrp protein, and presence of monomeric and dimeric Mrp complexes. Antiport did not depend on a VFF motif or a conserved tyrosine pair, but a role for a conserved histidine in a potential quinone binding site of MrpA was supported. The importance of several acidic residues for antiport was confirmed, and the importance of additional residues was demonstrated (e.g. three lysine residues conserved across MrpA, MrpD, and membrane-bound respiratory Complex I subunits (NuoL/M/N)). The results extended indications that MrpE is required for normal membrane levels of other Mrp proteins and for complex formation. Moreover, mutations in several other Mrp proteins lead to greatly reduced membrane levels of MrpE. Thus, changes in either of the two Mrp modules, MrpA-MrpD and MrpE-MrpG, influence the other. Two mutants, MrpB-P37G and MrpC-Q70A, showed a normal phenotype but lacked the MrpA-MrpG monomeric complex while retaining the dimeric hetero-oligomeric complex. Finally, MrpG-P81A and MrpG-P81G mutants exhibited no antiport activity but supported sodium resistance and a low [Na(+)](in). Such mutants could be used to screen hypothesized but uncharacterized sodium efflux functions of Mrp apart from Na(+) (Li(+))/H(+) antiport.

Genomic Context Analysis of de Novo STXBP1 Mutations Identifies Evidence of Splice Site DNA-Motif Associated Hotspots.

PubMed

Uddin, Mohammed; Woodbury-Smith, Marc; Chan, Ada J S; Albanna, Ammar; Minassian, Berge; Boelman, Cyrus; Scherer, Stephen W

2018-03-28

Mutations within STXBP1 have been associated with a range of neurodevelopmental disorders implicating the pleotropic impact of this gene. Although the frequency of de novo mutations within STXBP1 for selective cohorts with early onset epileptic encephalopathy is more than 1%, there is no evidence for a hotspot within the gene. In this study, we analyzed the genomic context of de novo STXBP1 mutations to examine whether certain motifs indicated a greater risk of mutation. Through a comprehensive context analysis of 136 de novo /rare mutation (SNV/Indels) sites in this gene, strikingly 26.92% of all SNV mutations occurred within 5bp upstream or downstream of a 'GTA' motif ( P < 0.0005). This implies a genomic context modulated mutagenesis. Moreover, 51.85% (14 out of 27) of the 'GTA' mutations are splicing compared to 14.70% (20 out of 136) of all reported mutations within STXBP1 We also noted that 11 of these 14 'GTA' associated mutations are de novo in origin. Our analysis provides strong evidence of DNA motif modulated mutagenesis for STXBP1 de novo splicing mutations. Copyright © 2018 Uddin et al.

Minireview: Role of Intracellular Scaffolding Proteins in the Regulation of Endocrine G Protein-Coupled Receptor Signaling

PubMed Central

Walther, Cornelia

2015-01-01

The majority of hormones stimulates and mediates their signal transduction via G protein-coupled receptors (GPCRs). The signal is transmitted into the cell due to the association of the GPCRs with heterotrimeric G proteins, which in turn activates an extensive array of signaling pathways to regulate cell physiology. However, GPCRs also function as scaffolds for the recruitment of a variety of cytoplasmic protein-interacting proteins that bind to both the intracellular face and protein interaction motifs encoded by GPCRs. The structural scaffolding of these proteins allows GPCRs to recruit large functional complexes that serve to modulate both G protein-dependent and -independent cellular signaling pathways and modulate GPCR intracellular trafficking. This review focuses on GPCR interacting PSD95-disc large-zona occludens domain containing scaffolds in the regulation of endocrine receptor signaling as well as their potential role as therapeutic targets for the treatment of endocrinopathies. PMID:25942107

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Yong; Choi, Mihwa; Cavey, Greg

The orphan nuclear receptor steroidogenic factor 1 (SF-1) regulates the differentiation and function of endocrine glands. Although SF-1 is constitutively active in cell-based assays, it is not known whether this transcriptional activity is modulated by ligands. Here, we describe the 1.5 {angstrom} crystal structure of the SF-1 ligand binding domain in complex with an LXXLL motif from a coregulator protein. The structure reveals the presence of a phospholipid ligand in a surprisingly large pocket ({approx}1600 {angstrom}{sup 3}), with the receptor adopting the canonical active conformation. The bound phospholipid is readily exchanged and modulates SF-1 interactions with coactivators. Mutations designed tomore » reduce the size of the SF-1 pocket or to disrupt hydrogen bonds with the phospholipid abolish SF-1/coactivator interactions and significantly reduce SF-1 transcriptional activity. These findings provide evidence that SF-1 is regulated by endogenous ligands and suggest an unexpected relationship between phospholipids and endocrine development and function.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fukumoto, Yasunori, E-mail: fukumoto@faculty.chiba-u.jp; Ikeuchi, Masayoshi; Nakayama, Yuji

ATR-dependent DNA damage checkpoint is the major DNA damage checkpoint against UV irradiation and DNA replication stress. The Rad17–RFC and Rad9–Rad1–Hus1 (9–1–1) complexes interact with each other to contribute to ATR signaling, however, the precise regulatory mechanism of the interaction has not been established. Here, we identified a conserved sequence motif, KYxxL, in the AAA+ domain of Rad17 protein, and demonstrated that this motif is essential for the interaction with the 9–1–1 complex. We also show that UV-induced Rad17 phosphorylation is increased in the Rad17 KYxxL mutants. These data indicate that the interaction with the 9–1–1 complex is not required formore » Rad17 protein to be an efficient substrate for the UV-induced phosphorylation. Our data also raise the possibility that the 9–1–1 complex plays a negative regulatory role in the Rad17 phosphorylation. We also show that the nucleotide-binding activity of Rad17 is required for its nuclear localization. - Highlights: • We have identified a conserved KYxxL motif in Rad17 protein. • The KYxxL motif is crucial for the interaction with the 9–1–1 complex. • The KYxxL motif is dispensable or inhibitory for UV-induced Rad17 phosphorylation. • Nucleotide binding of Rad17 is required for its nuclear localization.« less

Functional Analysis of Light-harvesting-like Protein 3 (LIL3) and Its Light-harvesting Chlorophyll-binding Motif in Arabidopsis*

PubMed Central

Takahashi, Kaori; Takabayashi, Atsushi; Tanaka, Ayumi; Tanaka, Ryouichi

2014-01-01

The light-harvesting complex (LHC) constitutes the major light-harvesting antenna of photosynthetic eukaryotes. LHC contains a characteristic sequence motif, termed LHC motif, consisting of 25–30 mostly hydrophobic amino acids. This motif is shared by a number of transmembrane proteins from oxygenic photoautotrophs that are termed light-harvesting-like (LIL) proteins. To gain insights into the functions of LIL proteins and their LHC motifs, we functionally characterized a plant LIL protein, LIL3. This protein has been shown previously to stabilize geranylgeranyl reductase (GGR), a key enzyme in phytol biosynthesis. It is hypothesized that LIL3 functions to anchor GGR to membranes. First, we conjugated the transmembrane domain of LIL3 or that of ascorbate peroxidase to GGR and expressed these chimeric proteins in an Arabidopsis mutant lacking LIL3 protein. As a result, the transgenic plants restored phytol-synthesizing activity. These results indicate that GGR is active as long as it is anchored to membranes, even in the absence of LIL3. Subsequently, we addressed the question why the LHC motif is conserved in the LIL3 sequences. We modified the transmembrane domain of LIL3, which contains the LHC motif, by substituting its conserved amino acids (Glu-171, Asn-174, and Asp-189) with alanine. As a result, the Arabidopsis transgenic plants partly recovered the phytol-biosynthesizing activity. However, in these transgenic plants, the LIL3-GGR complexes were partially dissociated. Collectively, these results indicate that the LHC motif of LIL3 is involved in the complex formation of LIL3 and GGR, which might contribute to the GGR reaction. PMID:24275650

Conservation of RNA chaperone activity of the human La-related proteins 4, 6 and 7

PubMed Central

Hussain, Rawaa H.; Zawawi, Mariam; Bayfield, Mark A.

2013-01-01

The La module is a conserved tandem arrangement of a La motif and RNA recognition motif whose function has been best characterized in genuine La proteins. The best-characterized substrates of La proteins are pre-tRNAs, and previous work using tRNA mediated suppression in Schizosaccharomyces pombe has demonstrated that yeast and human La enhance the maturation of these using two distinguishable activities: UUU-3′OH-dependent trailer binding/protection and a UUU-3′OH independent activity related to RNA chaperone function. The La module has also been identified in several conserved families of La-related proteins (LARPs) that engage other RNAs, but their mode of RNA binding and function(s) are not well understood. We demonstrate that the La modules of the human LARPs 4, 6 and 7 are also active in tRNA-mediated suppression, even in the absence of stable UUU-3′OH trailer protection. Rather, the capacity of these to enhance pre-tRNA maturation is associated with RNA chaperone function, which we demonstrate to be a conserved activity for each hLARP in vitro. Our work reveals insight into the mechanisms by which La module containing proteins discriminate RNA targets and demonstrates that RNA chaperone activity is a conserved function across representative members of the La motif-containing superfamily. PMID:23887937

A Logical OR Redundancy within the Asx-Pro-Asx-Gly Type 1 {Beta}-Turn Motif

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jihun; Dubey, Vikash Kumar; Longo, Lian M.

2008-04-19

Turn secondary structure is essential to the formation of globular protein architecture. Turn structures are, however, much more complex than either {alpha}-helix or {beta}-sheet, and the thermodynamics and folding kinetics are poorly understood. Type I {beta}-turns are the most common type of reverse turn, and they exhibit a statistical consensus sequence of Asx-Pro-Asx-Gly (where Asx is Asp or Asn). A comprehensive series of individual and combined Asx mutations has been constructed within three separate type I 3:5 G1 bulge {beta}-turns in human fibroblast growth factor-1, and their effects on structure, stability, and folding have been determined. The results show amore » fundamental logical OR relationship between the Asx residues in the motif, involving H-bond interactions with main-chain amides within the turn. These interactions can be modulated by additional interactions with residues adjacent to the turn at positions i + 4 and i + 6. The results show that the Asx residues in the turn motif make a substantial contribution to the overall stability of the protein, and the Asx logical OR relationship defines a redundant system that can compensate for deleterious point mutations. The results also show that the stability of the turn is unlikely to be the prime determinant of formation of turn structure in the folding transition state.« less

Evolutionary dynamics of a conserved sequence motif in the ribosomal genes of the ciliate Paramecium.

PubMed

Catania, Francesco; Lynch, Michael

2010-05-04

In protozoa, the identification of preserved motifs by comparative genomics is often impeded by difficulties to generate reliable alignments for non-coding sequences. Moreover, the evolutionary dynamics of regulatory elements in 3' untranslated regions (both in protozoa and metazoa) remains a virtually unexplored issue. By screening Paramecium tetraurelia's 3' untranslated regions for 8-mers that were previously found to be preserved in mammalian 3' UTRs, we detect and characterize a motif that is distinctly conserved in the ribosomal genes of this ciliate. The motif appears to be conserved across Paramecium aurelia species but is absent from the ribosomal genes of four additional non-Paramecium species surveyed, including another ciliate, Tetrahymena thermophila. Motif-free ribosomal genes retain fewer paralogs in the genome and appear to be lost more rapidly relative to motif-containing genes. Features associated with the discovered preserved motif are consistent with this 8-mer playing a role in post-transcriptional regulation. Our observations 1) shed light on the evolution of a putative regulatory motif across large phylogenetic distances; 2) are expected to facilitate the understanding of the modulation of ribosomal genes expression in Paramecium; and 3) reveal a largely unexplored--and presumably not restricted to Paramecium--association between the presence/absence of a DNA motif and the evolutionary fate of its host genes.

Synthetic Oligodeoxynucleotides (ODN) Containing Suppressive TTAGGG Motifs Inhibit AIM2 Inflammasome Activation

PubMed Central

Kaminski, John J.; Schattgen, Stefan A.; Tzeng, Te-Chen; Bode, Christian; Klinman, Dennis M.; Fitzgerald, Katherine A.

2013-01-01

Synthetic oligodeoxynucleotides comprised of the immunosuppressive motif TTAGGG block TLR9 signaling, prevent STAT1 and STAT4 phosphorylation and attenuate a variety of inflammatory responses in vivo. Here, we demonstrate that such suppressive oligodeoxynucleotides (sup ODN) abrogate activation of cytosolic nucleic acid sensing pathways. Pretreatment of dendritic cells and macrophages with the suppressive ODN-A151 abrogated type I IFN, TNFα and ISG induction in response to cytosolic dsDNA. In addition, A151 abrogated caspase-1-dependent IL-1β and IL-18 maturation in dendritic cells stimulated with dsDNA and murine cytomegalovirus (MCMV). Inhibition was dependent on A151’s phosphorothioate backbone while substitution of the guanosine residues for adenosine negatively affected potency. A151 mediates these effects by binding to AIM2 in a manner that is competitive with immune-stimulatory DNA and as a consequence prevents AIM2 inflammasome complex formation. Collectively, these findings reveal a new route by which suppressive ODNs modulate the immune system and unveil novel applications for suppressive ODNs in the treatment of infectious and autoimmune diseases. PMID:23986531

Control of mitotic chromosome condensation by the fission yeast transcription factor Zas1.

PubMed

Schiklenk, Christoph; Petrova, Boryana; Kschonsak, Marc; Hassler, Markus; Klein, Carlo; Gibson, Toby J; Haering, Christian H

2018-05-07

Although the formation of rod-shaped chromosomes is vital for the correct segregation of eukaryotic genomes during cell divisions, the molecular mechanisms that control the chromosome condensation process have remained largely unknown. Here, we identify the C 2 H 2 zinc-finger transcription factor Zas1 as a key regulator of mitotic condensation dynamics in a quantitative live-cell microscopy screen of the fission yeast Schizosaccharomyces pombe By binding to specific DNA target sequences in their promoter regions, Zas1 controls expression of the Cnd1 subunit of the condensin protein complex and several other target genes, whose combined misregulation in zas1 mutants results in defects in chromosome condensation and segregation. Genetic and biochemical analysis reveals an evolutionarily conserved transactivation domain motif in Zas1 that is pivotal to its function in gene regulation. Our results suggest that this motif, together with the Zas1 C-terminal helical domain to which it binds, creates a cis/trans switch module for transcriptional regulation of genes that control chromosome condensation. © 2018 Schiklenk et al.

Identification, characterization, and expression analysis of calmodulin and calmodulin-like genes in grapevine (Vitis vinifera) reveal likely roles in stress responses.

PubMed

Vandelle, Elodie; Vannozzi, Alessandro; Wong, Darren; Danzi, Davide; Digby, Anne-Marie; Dal Santo, Silvia; Astegno, Alessandra

2018-06-04

Calcium (Ca 2+ ) is an ubiquitous key second messenger in plants, where it modulates many developmental and adaptive processes in response to various stimuli. Several proteins containing Ca 2+ binding domain have been identified in plants, including calmodulin (CaM) and calmodulin-like (CML) proteins, which play critical roles in translating Ca 2+ signals into proper cellular responses. In this work, a genome-wide analysis conducted in Vitis vinifera identified three CaM- and 62 CML-encoding genes. We assigned gene family nomenclature, analyzed gene structure, chromosomal location and gene duplication, as well as protein motif organization. The phylogenetic clustering revealed a total of eight subgroups, including one unique clade of VviCaMs distinct from VviCMLs. VviCaMs were found to contain four EF-hand motifs whereas VviCML proteins have one to five. Most of grapevine CML genes were intronless, while VviCaMs were intron rich. All the genes were well spread among the 19 grapevine chromosomes and displayed a high level of duplication. The expression profiling of VviCaM/VviCML genes revealed a broad expression pattern across all grape organs and tissues at various developmental stages, and a significant modulation in biotic stress-related responses. Our results highlight the complexity of CaM/CML protein family also in grapevine, supporting the versatile role of its different members in modulating cellular responses to various stimuli, in particular to biotic stresses. This work lays the foundation for further functional and structural studies on specific grapevine CaMs/CMLs in order to better understand the role of Ca 2+ -binding proteins in grapevine and to explore their potential for further biotechnological applications. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

A Bioinformatics Approach for Detecting Repetitive Nested Motifs using Pattern Matching.

PubMed

Romero, José R; Carballido, Jessica A; Garbus, Ingrid; Echenique, Viviana C; Ponzoni, Ignacio

2016-01-01

The identification of nested motifs in genomic sequences is a complex computational problem. The detection of these patterns is important to allow the discovery of transposable element (TE) insertions, incomplete reverse transcripts, deletions, and/or mutations. In this study, a de novo strategy for detecting patterns that represent nested motifs was designed based on exhaustive searches for pairs of motifs and combinatorial pattern analysis. These patterns can be grouped into three categories, motifs within other motifs, motifs flanked by other motifs, and motifs of large size. The methodology used in this study, applied to genomic sequences from the plant species Aegilops tauschii and Oryza sativa , revealed that it is possible to identify putative nested TEs by detecting these three types of patterns. The results were validated through BLAST alignments, which revealed the efficacy and usefulness of the new method, which is called Mamushka.

FPGA implementation of motifs-based neuronal network and synchronization analysis

NASA Astrophysics Data System (ADS)

Deng, Bin; Zhu, Zechen; Yang, Shuangming; Wei, Xile; Wang, Jiang; Yu, Haitao

2016-06-01

Motifs in complex networks play a crucial role in determining the brain functions. In this paper, 13 kinds of motifs are implemented with Field Programmable Gate Array (FPGA) to investigate the relationships between the networks properties and motifs properties. We use discretization method and pipelined architecture to construct various motifs with Hindmarsh-Rose (HR) neuron as the node model. We also build a small-world network based on these motifs and conduct the synchronization analysis of motifs as well as the constructed network. We find that the synchronization properties of motif determine that of motif-based small-world network, which demonstrates effectiveness of our proposed hardware simulation platform. By imitation of some vital nuclei in the brain to generate normal discharges, our proposed FPGA-based artificial neuronal networks have the potential to replace the injured nuclei to complete the brain function in the treatment of Parkinson's disease and epilepsy.

Reduction potentials of heterometallic manganese–oxido cubane complexes modulated by redox-inactive metals

PubMed Central

Tsui, Emily Y.; Agapie, Theodor

2013-01-01

Understanding the effect of redox-inactive metals on the properties of biological and heterogeneous water oxidation catalysts is important both fundamentally and for improvement of future catalyst designs. In this work, heterometallic manganese–oxido cubane clusters [MMn3O4] (M = Sr2+, Zn2+, Sc3+, Y3+) structurally relevant to the oxygen-evolving complex (OEC) of photosystem II were prepared and characterized. The reduction potentials of these clusters and other related mixed metal manganese–tetraoxido complexes are correlated with the Lewis acidity of the apical redox-inactive metal in a manner similar to a related series of heterometallic manganese–dioxido clusters. The redox potentials of the [SrMn3O4] and [CaMn3O4] clusters are close, which is consistent with the observation that the OEC is functional only with one of these two metals. Considering our previous studies of [MMn3O2] moieties, the present results with more structurally accurate models of the OEC ([MMn3O4]) suggest a general relationship between the reduction potentials of heterometallic oxido clusters and the Lewis acidities of incorporated cations that applies to diverse structural motifs. These findings support proposals that one function of calcium in the OEC is to modulate the reduction potential of the cluster to allow electron transfer. PMID:23744039

High-throughput analysis of peptide binding modules

PubMed Central

Liu, Bernard A.; Engelmann, Brett; Nash, Piers D.

2014-01-01

Modular protein interaction domains that recognize linear peptide motifs are found in hundreds of proteins within the human genome. Some protein interaction domains such as SH2, 14-3-3, Chromo and Bromo domains serve to recognize post-translational modification of amino acids (such as phosphorylation, acetylation, methylation etc.) and translate these into discrete cellular responses. Other modules such as SH3 and PDZ domains recognize linear peptide epitopes and serve to organize protein complexes based on localization and regions of elevated concentration. In both cases, the ability to nucleate specific signaling complexes is in large part dependent on the selectivity of a given protein module for its cognate peptide ligand. High throughput analysis of peptide-binding domains by peptide or protein arrays, phage display, mass spectrometry or other HTP techniques provides new insight into the potential protein-protein interactions prescribed by individual or even whole families of modules. Systems level analyses have also promoted a deeper understanding of the underlying principles that govern selective protein-protein interactions and how selectivity evolves. Lastly, there is a growing appreciation for the limitations and potential pitfalls of high-throughput analysis of protein-peptide interactomes. This review will examine some of the common approaches utilized for large-scale studies of protein interaction domains and suggest a set of standards for the analysis and validation of datasets from large-scale studies of peptide-binding modules. We will also highlight how data from large-scale studies of modular interaction domain families can provide insight into systems level properties such as the linguistics of selective interactions. PMID:22610655

Carbohydrate Recognition by an Architecturally Complex α-N-Acetylglucosaminidase from Clostridium perfringens

PubMed Central

Ficko-Blean, Elizabeth; Stuart, Christopher P.; Suits, Michael D.; Cid, Melissa; Tessier, Matthew; Woods, Robert J.; Boraston, Alisdair B.

2012-01-01

CpGH89 is a large multimodular enzyme produced by the human and animal pathogen Clostridium perfringens. The catalytic activity of this exo-α-d-N-acetylglucosaminidase is directed towards a rare carbohydrate motif, N-acetyl-β-d-glucosamine-α-1,4-d-galactose, which is displayed on the class III mucins deep within the gastric mucosa. In addition to the family 89 glycoside hydrolase catalytic module this enzyme has six modules that share sequence similarity to the family 32 carbohydrate-binding modules (CBM32s), suggesting the enzyme has considerable capacity to adhere to carbohydrates. Here we suggest that two of the modules, CBM32-1 and CBM32-6, are not functional as carbohydrate-binding modules (CBMs) and demonstrate that three of the CBMs, CBM32-3, CBM32-4, and CBM32-5, are indeed capable of binding carbohydrates. CBM32-3 and CBM32-4 have a novel binding specificity for N-acetyl-β-d-glucosamine-α-1,4-d-galactose, which thus complements the specificity of the catalytic module. The X-ray crystal structure of CBM32-4 in complex with this disaccharide reveals a mode of recognition that is based primarily on accommodation of the unique bent shape of this sugar. In contrast, as revealed by a series of X-ray crystal structures and quantitative binding studies, CBM32-5 displays the structural and functional features of galactose binding that is commonly associated with CBM family 32. The functional CBM32s that CpGH89 contains suggest the possibility for multivalent binding events and the partitioning of this enzyme to highly specific regions within the gastrointestinal tract. PMID:22479408

Mutations in the Putative Zinc-Binding Motif of UL52 Demonstrate a Complex Interdependence between the UL5 and UL52 Subunits of the Human Herpes Simplex Virus Type 1 Helicase/Primase Complex

PubMed Central

Chen, Yan; Carrington-Lawrence, Stacy D.; Bai, Ping; Weller, Sandra K.

2005-01-01

Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase-primase (UL5/8/52) complex. UL5 contains seven motifs found in helicase superfamily 1, and UL52 contains conserved motifs found in primases. The contributions of each subunit to the biochemical activities of the complex, however, remain unclear. We have previously demonstrated that a mutation in the putative zinc finger at UL52 C terminus abrogates not only primase but also ATPase, helicase, and DNA-binding activities of a UL5/UL52 subcomplex, indicating a complex interdependence between the two subunits. To test this hypothesis and to further investigate the role of the zinc finger in the enzymatic activities of the helicase-primase, a series of mutations were constructed in this motif. They differed in their ability to complement a UL52 null virus: totally defective, partial complementation, and potentiating. In this study, four of these mutants were studied biochemically after expression and purification from insect cells infected with recombinant baculoviruses. All mutants show greatly reduced primase activity. Complementation-defective mutants exhibited severe defects in ATPase, helicase, and DNA-binding activities. Partially complementing mutants displayed intermediate levels of these activities, except that one showed a wild-type level of helicase activity. These data suggest that the UL52 zinc finger motif plays an important role in the activities of the helicase-primase complex. The observation that mutations in UL52 affected helicase, ATPase, and DNA-binding activities indicates that UL52 binding to DNA via the zinc finger may be necessary for loading UL5. Alternatively, UL5 and UL52 may share a DNA-binding interface. PMID:15994803

Mutations in the putative zinc-binding motif of UL52 demonstrate a complex interdependence between the UL5 and UL52 subunits of the human herpes simplex virus type 1 helicase/primase complex.

PubMed

Chen, Yan; Carrington-Lawrence, Stacy D; Bai, Ping; Weller, Sandra K

2005-07-01

Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase-primase (UL5/8/52) complex. UL5 contains seven motifs found in helicase superfamily 1, and UL52 contains conserved motifs found in primases. The contributions of each subunit to the biochemical activities of the complex, however, remain unclear. We have previously demonstrated that a mutation in the putative zinc finger at UL52 C terminus abrogates not only primase but also ATPase, helicase, and DNA-binding activities of a UL5/UL52 subcomplex, indicating a complex interdependence between the two subunits. To test this hypothesis and to further investigate the role of the zinc finger in the enzymatic activities of the helicase-primase, a series of mutations were constructed in this motif. They differed in their ability to complement a UL52 null virus: totally defective, partial complementation, and potentiating. In this study, four of these mutants were studied biochemically after expression and purification from insect cells infected with recombinant baculoviruses. All mutants show greatly reduced primase activity. Complementation-defective mutants exhibited severe defects in ATPase, helicase, and DNA-binding activities. Partially complementing mutants displayed intermediate levels of these activities, except that one showed a wild-type level of helicase activity. These data suggest that the UL52 zinc finger motif plays an important role in the activities of the helicase-primase complex. The observation that mutations in UL52 affected helicase, ATPase, and DNA-binding activities indicates that UL52 binding to DNA via the zinc finger may be necessary for loading UL5. Alternatively, UL5 and UL52 may share a DNA-binding interface.

Structure of Rot, a global regulator of virulence genes in Staphylococcus aureus.

PubMed

Zhu, Yuwei; Fan, Xiaojiao; Zhang, Xu; Jiang, Xuguang; Niu, Liwen; Teng, Maikun; Li, Xu

2014-09-01

Staphylococcus aureus is a highly versatile pathogen that can infect human tissue by producing a large arsenal of virulence factors that are tightly regulated by a complex regulatory network. Rot, which shares sequence similarity with SarA homologues, is a global regulator that regulates numerous virulence genes. However, the recognition model of Rot for the promoter region of target genes and the putative regulation mechanism remain elusive. In this study, the 1.77 Å resolution X-ray crystal structure of Rot is reported. The structure reveals that two Rot molecules form a compact homodimer, each of which contains a typical helix-turn-helix module and a β-hairpin motif connected by a flexible loop. Fluorescence polarization results indicate that Rot preferentially recognizes AT-rich dsDNA with ~30-base-pair nucleotides and that the conserved positively charged residues on the winged-helix motif are vital for binding to the AT-rich dsDNA. It is proposed that the DNA-recognition model of Rot may be similar to that of SarA, SarR and SarS, in which the helix-turn-helix motifs of each monomer interact with the major grooves of target dsDNA and the winged motifs contact the minor grooves. Interestingly, the structure shows that Rot adopts a novel dimerization model that differs from that of other SarA homologues. As expected, perturbation of the dimer interface abolishes the dsDNA-binding ability of Rot, suggesting that Rot functions as a dimer. In addition, the results have been further confirmed in vivo by measuring the transcriptional regulation of α-toxin, a major virulence factor produced by most S. aureus strains.

Conserved Non-Coding Regulatory Signatures in Arabidopsis Co-Expressed Gene Modules

PubMed Central

Spangler, Jacob B.; Ficklin, Stephen P.; Luo, Feng; Freeling, Michael; Feltus, F. Alex

2012-01-01

Complex traits and other polygenic processes require coordinated gene expression. Co-expression networks model mRNA co-expression: the product of gene regulatory networks. To identify regulatory mechanisms underlying coordinated gene expression in a tissue-enriched context, ten Arabidopsis thaliana co-expression networks were constructed after manually sorting 4,566 RNA profiling datasets into aerial, flower, leaf, root, rosette, seedling, seed, shoot, whole plant, and global (all samples combined) groups. Collectively, the ten networks contained 30% of the measurable genes of Arabidopsis and were circumscribed into 5,491 modules. Modules were scrutinized for cis regulatory mechanisms putatively encoded in conserved non-coding sequences (CNSs) previously identified as remnants of a whole genome duplication event. We determined the non-random association of 1,361 unique CNSs to 1,904 co-expression network gene modules. Furthermore, the CNS elements were placed in the context of known gene regulatory networks (GRNs) by connecting 250 CNS motifs with known GRN cis elements. Our results provide support for a regulatory role of some CNS elements and suggest the functional consequences of CNS activation of co-expression in specific gene sets dispersed throughout the genome. PMID:23024789

Conserved non-coding regulatory signatures in Arabidopsis co-expressed gene modules.

PubMed

Spangler, Jacob B; Ficklin, Stephen P; Luo, Feng; Freeling, Michael; Feltus, F Alex

2012-01-01

Complex traits and other polygenic processes require coordinated gene expression. Co-expression networks model mRNA co-expression: the product of gene regulatory networks. To identify regulatory mechanisms underlying coordinated gene expression in a tissue-enriched context, ten Arabidopsis thaliana co-expression networks were constructed after manually sorting 4,566 RNA profiling datasets into aerial, flower, leaf, root, rosette, seedling, seed, shoot, whole plant, and global (all samples combined) groups. Collectively, the ten networks contained 30% of the measurable genes of Arabidopsis and were circumscribed into 5,491 modules. Modules were scrutinized for cis regulatory mechanisms putatively encoded in conserved non-coding sequences (CNSs) previously identified as remnants of a whole genome duplication event. We determined the non-random association of 1,361 unique CNSs to 1,904 co-expression network gene modules. Furthermore, the CNS elements were placed in the context of known gene regulatory networks (GRNs) by connecting 250 CNS motifs with known GRN cis elements. Our results provide support for a regulatory role of some CNS elements and suggest the functional consequences of CNS activation of co-expression in specific gene sets dispersed throughout the genome.

Beyond Atg8 binding: The role of AIM/LIR motifs in autophagy.

PubMed

Fracchiolla, Dorotea; Sawa-Makarska, Justyna; Martens, Sascha

2017-05-04

Selective macroautophagy/autophagy mediates the selective delivery of cytoplasmic cargo material via autophagosomes into the lytic compartment for degradation. This selectivity is mediated by cargo receptor molecules that link the cargo to the phagophore (the precursor of the autophagosome) membrane via their simultaneous interaction with the cargo and Atg8 proteins on the membrane. Atg8 proteins are attached to membrane in a conjugation reaction and the cargo receptors bind them via short peptide motifs called Atg8-interacting motifs/LC3-interacting regions (AIMs/LIRs). We have recently shown for the yeast Atg19 cargo receptor that the AIM/LIR motifs also serve to recruit the Atg12-Atg5-Atg16 complex, which stimulates Atg8 conjugation, to the cargo. We could further show in a reconstituted system that the recruitment of the Atg12-Atg5-Atg16 complex is sufficient for cargo-directed Atg8 conjugation. Our results suggest that AIM/LIR motifs could have more general roles in autophagy.

ATRIAL NATRIURETIC FACTOR RECEPTOR GUANYLATE CYCLASE SIGNALING: NEW ATP- REGULATED TRANSDUCTION MOTIF

PubMed Central

Duda, Teresa; Bharill, Shashank; Wojtas, Ireneusz; Yadav, Prem; Gryczynski, Ignacy; Gryczynski, Zygmunt; Sharma, Rameshwar K.

2010-01-01

ANF-RGC$ membrane guanylate cyclase is the receptor for the hypotensive peptide hormones, atrial natriuretic factor (ANF) and type B natriuretic peptide (BNP). It is a single transmembrane spanning protein. Binding the hormone to the extracellular domain activates its intracellular catalytic domain. This results in accelerated production of cyclic GMP, a second messenger in controlling blood pressure, cardiac vasculature and fluid secretion. ATP is the obligatory transducer of the ANF signal. It works through its ATP regulated module, ARM, which is juxtaposed to the C-terminal side of the transmembrane domain. Upon interaction, ATP induces a cascade of temporal and spatial changes in the ARM, which, finally, result in activation of the catalytic module. Although the exact nature and the details of these changes are not known, some of these have been stereographed in the simulated three-dimensional model of the ARM and validated biochemically. Through comprehensive techniques ofsteady-state, time-resolved tryptophan fluorescence and Forster Resonance Energy Transfer (FRET), site-directed and deletion-mutagenesis, and reconstitution, the present study validates and explains themechanism of the model-based predicted transduction role of the ARM’s structural motif, 669WTAPELL675. This motif is critical in the ATP-dependent ANF signaling. Molecular modeling shows that ATP binding exposes the 669WTAPELL675 motif, the exposure, in turn, facilitates its interaction and activation of the catalytic module. These principles of the model have been experimentally validated. This knowledge brings us a step closer to our understanding of the mechanism by which the ATP-dependent spatial changes within the ARM cause ANF signaling of ANF-RGC. PMID:19137266

Structural Basis for Carbohydrate Recognition and Anti-inflammatory Modulation by Gastrointestinal Nematode Parasite Toxascaris leonina Galectin*

PubMed Central

Hwang, Eun Young; Jeong, Mi Suk; Park, Sang Kyun; Ha, Sung Chul; Yu, Hak Sun; Jang, Se Bok

2016-01-01

Toxascaris leonina galectin (Tl-gal) is a galectin-9 homologue protein isolated from an adult worm of the canine gastrointestinal nematode parasite, and Tl-gal-vaccinated challenge can inhibit inflammation in inflammatory bowel disease-induced mice. We determined the first X-ray structures of full-length Tl-gal complexes with carbohydrates (lactose, N-acetyllactosamine, lacto-N-tetraose, sialyllactose, and glucose). Bonds were formed on concave surfaces of both carbohydrate recognition domains (CRDs) in Tl-gal. All binding sites were found in the HXXXR and WGXEER motifs. Charged Arg61/Arg196 and Glu80/Glu215 on the conserved motif of Tl-gal N-terminal CRD and C-terminal CRD are critical amino acids for recognizing carbohydrate binding, and the residues can affect protein folding and structure. The polar amino acids His, Asn, and Trp are also important residues for the interaction with carbohydrates through hydrogen bonding. Hemagglutination activities of Tl-gal were inhibited by interactions with carbohydrates and mutations. We found that the mutation of Tl-gal (E80A/E215A) at the carbohydrate binding region induced protein aggregation and could be caused in many diseases. The short linker region between the N-terminal and C-terminal CRDs of Tl-gal was very stable against proteolysis and maintained its biological activity. This structural information is expected to elucidate the carbohydrate recognition mechanism of Tl-gal and improve our understanding of anti-inflammatory mediators and modulators of immune response. PMID:27742836

Motif mismatches in microsatellites: insights from genome-wide investigation among 20 insect species.

PubMed

Behura, Susanta K; Severson, David W

2015-02-01

We present a detailed genome-wide comparative study of motif mismatches of microsatellites among 20 insect species representing five taxonomic orders. The results show that varying proportions (∼15-46%) of microsatellites identified in these species are imperfect in motif structure, and that they also vary in chromosomal distribution within genomes. It was observed that the genomic abundance of imperfect repeats is significantly associated with the length and number of motif mismatches of microsatellites. Furthermore, microsatellites with a higher number of mismatches tend to have lower abundance in the genome, suggesting that sequence heterogeneity of repeat motifs is a key determinant of genomic abundance of microsatellites. This relationship seems to be a general feature of microsatellites even in unrelated species such as yeast, roundworm, mouse and human. We provide a mechanistic explanation of the evolutionary link between motif heterogeneity and genomic abundance of microsatellites by examining the patterns of motif mismatches and allele sequences of single-nucleotide polymorphisms identified within microsatellite loci. Using Drosophila Reference Genetic Panel data, we further show that pattern of allelic variation modulates motif heterogeneity of microsatellites, and provide estimates of allele age of specific imperfect microsatellites found within protein-coding genes. © The Author 2014. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Integrative analyses shed new light on human ribosomal protein gene regulation

PubMed Central

Li, Xin; Zheng, Yiyu; Hu, Haiyan; Li, Xiaoman

2016-01-01

Ribosomal protein genes (RPGs) are important house-keeping genes that are well-known for their coordinated expression. Previous studies on RPGs are largely limited to their promoter regions. Recent high-throughput studies provide an unprecedented opportunity to study how human RPGs are transcriptionally modulated and how such transcriptional regulation may contribute to the coordinate gene expression in various tissues and cell types. By analyzing the DNase I hypersensitive sites under 349 experimental conditions, we predicted 217 RPG regulatory regions in the human genome. More than 86.6% of these computationally predicted regulatory regions were partially corroborated by independent experimental measurements. Motif analyses on these predicted regulatory regions identified 31 DNA motifs, including 57.1% of experimentally validated motifs in literature that regulate RPGs. Interestingly, we observed that the majority of the predicted motifs were shared by the predicted distal and proximal regulatory regions of the same RPGs, a likely general mechanism for enhancer-promoter interactions. We also found that RPGs may be differently regulated in different cells, indicating that condition-specific RPG regulatory regions still need to be discovered and investigated. Our study advances the understanding of how RPGs are coordinately modulated, which sheds light to the general principles of gene transcriptional regulation in mammals. PMID:27346035

Integrative analyses shed new light on human ribosomal protein gene regulation.

PubMed

Li, Xin; Zheng, Yiyu; Hu, Haiyan; Li, Xiaoman

2016-06-27

Ribosomal protein genes (RPGs) are important house-keeping genes that are well-known for their coordinated expression. Previous studies on RPGs are largely limited to their promoter regions. Recent high-throughput studies provide an unprecedented opportunity to study how human RPGs are transcriptionally modulated and how such transcriptional regulation may contribute to the coordinate gene expression in various tissues and cell types. By analyzing the DNase I hypersensitive sites under 349 experimental conditions, we predicted 217 RPG regulatory regions in the human genome. More than 86.6% of these computationally predicted regulatory regions were partially corroborated by independent experimental measurements. Motif analyses on these predicted regulatory regions identified 31 DNA motifs, including 57.1% of experimentally validated motifs in literature that regulate RPGs. Interestingly, we observed that the majority of the predicted motifs were shared by the predicted distal and proximal regulatory regions of the same RPGs, a likely general mechanism for enhancer-promoter interactions. We also found that RPGs may be differently regulated in different cells, indicating that condition-specific RPG regulatory regions still need to be discovered and investigated. Our study advances the understanding of how RPGs are coordinately modulated, which sheds light to the general principles of gene transcriptional regulation in mammals.

Evolutionary dynamics of a conserved sequence motif in the ribosomal genes of the ciliate Paramecium

PubMed Central

2010-01-01

Background In protozoa, the identification of preserved motifs by comparative genomics is often impeded by difficulties to generate reliable alignments for non-coding sequences. Moreover, the evolutionary dynamics of regulatory elements in 3' untranslated regions (both in protozoa and metazoa) remains a virtually unexplored issue. Results By screening Paramecium tetraurelia's 3' untranslated regions for 8-mers that were previously found to be preserved in mammalian 3' UTRs, we detect and characterize a motif that is distinctly conserved in the ribosomal genes of this ciliate. The motif appears to be conserved across Paramecium aurelia species but is absent from the ribosomal genes of four additional non-Paramecium species surveyed, including another ciliate, Tetrahymena thermophila. Motif-free ribosomal genes retain fewer paralogs in the genome and appear to be lost more rapidly relative to motif-containing genes. Features associated with the discovered preserved motif are consistent with this 8-mer playing a role in post-transcriptional regulation. Conclusions Our observations 1) shed light on the evolution of a putative regulatory motif across large phylogenetic distances; 2) are expected to facilitate the understanding of the modulation of ribosomal genes expression in Paramecium; and 3) reveal a largely unexplored--and presumably not restricted to Paramecium--association between the presence/absence of a DNA motif and the evolutionary fate of its host genes. PMID:20441586

Noncoding RNA danger motifs bridge innate and adaptive immunity and are potent adjuvants for vaccination

PubMed Central

Wang, Lilin; Smith, Dan; Bot, Simona; Dellamary, Luis; Bloom, Amy; Bot, Adrian

2002-01-01

The adaptive immune response is triggered by recognition of T and B cell epitopes and is influenced by “danger” motifs that act via innate immune receptors. This study shows that motifs associated with noncoding RNA are essential features in the immune response reminiscent of viral infection, mediating rapid induction of proinflammatory chemokine expression, recruitment and activation of antigen-presenting cells, modulation of regulatory cytokines, subsequent differentiation of Th1 cells, isotype switching, and stimulation of cross-priming. The heterogeneity of RNA-associated motifs results in differential binding to cellular receptors, and specifically impacts the immune profile. Naturally occurring double-stranded RNA (dsRNA) triggered activation of dendritic cells and enhancement of specific immunity, similar to selected synthetic dsRNA motifs. Based on the ability of specific RNA motifs to block tolerance induction and effectively organize the immune defense during viral infection, we conclude that such RNA species are potent danger motifs. We also demonstrate the feasibility of using selected RNA motifs as adjuvants in the context of novel aerosol carriers for optimizing the immune response to subunit vaccines. In conclusion, RNA-associated motifs produced during viral infection bridge the early response with the late adaptive phase, regulating the activation and differentiation of antigen-specific B and T cells, in addition to a short-term impact on innate immunity. PMID:12393853

Organization of feed-forward loop motifs reveals architectural principles in natural and engineered networks.

PubMed

Gorochowski, Thomas E; Grierson, Claire S; di Bernardo, Mario

2018-03-01

Network motifs are significantly overrepresented subgraphs that have been proposed as building blocks for natural and engineered networks. Detailed functional analysis has been performed for many types of motif in isolation, but less is known about how motifs work together to perform complex tasks. To address this issue, we measure the aggregation of network motifs via methods that extract precisely how these structures are connected. Applying this approach to a broad spectrum of networked systems and focusing on the widespread feed-forward loop motif, we uncover striking differences in motif organization. The types of connection are often highly constrained, differ between domains, and clearly capture architectural principles. We show how this information can be used to effectively predict functionally important nodes in the metabolic network of Escherichia coli . Our findings have implications for understanding how networked systems are constructed from motif parts and elucidate constraints that guide their evolution.

Organization of feed-forward loop motifs reveals architectural principles in natural and engineered networks

PubMed Central

Grierson, Claire S.

2018-01-01

Network motifs are significantly overrepresented subgraphs that have been proposed as building blocks for natural and engineered networks. Detailed functional analysis has been performed for many types of motif in isolation, but less is known about how motifs work together to perform complex tasks. To address this issue, we measure the aggregation of network motifs via methods that extract precisely how these structures are connected. Applying this approach to a broad spectrum of networked systems and focusing on the widespread feed-forward loop motif, we uncover striking differences in motif organization. The types of connection are often highly constrained, differ between domains, and clearly capture architectural principles. We show how this information can be used to effectively predict functionally important nodes in the metabolic network of Escherichia coli. Our findings have implications for understanding how networked systems are constructed from motif parts and elucidate constraints that guide their evolution. PMID:29670941

pKa Modulation of the Acid/Base Catalyst within GH32 and GH68: A Role in Substrate/Inhibitor Specificity?

PubMed Central

Yuan, Shuguang; Le Roy, Katrien; Venken, Tom; Lammens, Willem; Van den Ende, Wim; De Maeyer, Marc

2012-01-01

Glycoside hydrolases of families 32 (GH32) and 68 (GH68) belong to clan GH-J, containing hydrolytic enzymes (sucrose/fructans as donor substrates) and fructosyltransferases (sucrose/fructans as donor and acceptor substrates). In GH32 members, some of the sugar substrates can also function as inhibitors, this regulatory aspect further adding to the complexity in enzyme functionalities within this family. Although 3D structural information becomes increasingly available within this clan and huge progress has been made on structure-function relationships, it is not clear why some sugars bind as inhibitors without being catalyzed. Conserved aspartate and glutamate residues are well known to act as nucleophile and acid/bases within this clan. Based on the available 3D structures of enzymes and enzyme-ligand complexes as well as docking simulations, we calculated the pKa of the acid-base before and after substrate binding. The obtained results strongly suggest that most GH-J members show an acid-base catalyst that is not sufficiently protonated before ligand entrance, while the acid-base can be fully protonated when a substrate, but not an inhibitor, enters the catalytic pocket. This provides a new mechanistic insight aiming at understanding the complex substrate and inhibitor specificities observed within the GH-J clan. Moreover, besides the effect of substrate entrance on its own, we strongly suggest that a highly conserved arginine residue (in the RDP motif) rather than the previously proposed Tyr motif (not conserved) provides the proton to increase the pKa of the acid-base catalyst. PMID:22662155

Structural Analysis of the Complex between Penta-EF-Hand ALG-2 Protein and Sec31A Peptide Reveals a Novel Target Recognition Mechanism of ALG-2

PubMed Central

Takahashi, Takeshi; Kojima, Kyosuke; Zhang, Wei; Sasaki, Kanae; Ito, Masaru; Suzuki, Hironori; Kawasaki, Masato; Wakatsuki, Soichi; Takahara, Terunao; Shibata, Hideki; Maki, Masatoshi

2015-01-01

ALG-2, a 22-kDa penta-EF-hand protein, is involved in cell death, signal transduction, membrane trafficking, etc., by interacting with various proteins in mammalian cells in a Ca2+-dependent manner. Most known ALG-2-interacting proteins contain proline-rich regions in which either PPYPXnYP (type 1 motif) or PXPGF (type 2 motif) is commonly found. Previous X-ray crystal structural analysis of the complex between ALG-2 and an ALIX peptide revealed that the peptide binds to the two hydrophobic pockets. In the present study, we resolved the crystal structure of the complex between ALG-2 and a peptide of Sec31A (outer shell component of coat complex II, COPII; containing the type 2 motif) and found that the peptide binds to the third hydrophobic pocket (Pocket 3). While amino acid substitution of Phe85, a Pocket 3 residue, with Ala abrogated the interaction with Sec31A, it did not affect the interaction with ALIX. On the other hand, amino acid substitution of Tyr180, a Pocket 1 residue, with Ala caused loss of binding to ALIX, but maintained binding to Sec31A. We conclude that ALG-2 recognizes two types of motifs at different hydrophobic surfaces. Furthermore, based on the results of serial mutational analysis of the ALG-2-binding sites in Sec31A, the type 2 motif was newly defined. PMID:25667979

Single-molecule DNA unzipping reveals asymmetric modulation of a transcription factor by its binding site sequence and context

PubMed Central

Rudnizky, Sergei; Khamis, Hadeel; Malik, Omri; Squires, Allison H; Meller, Amit; Melamed, Philippa

2018-01-01

Abstract Most functional transcription factor (TF) binding sites deviate from their ‘consensus’ recognition motif, although their sites and flanking sequences are often conserved across species. Here, we used single-molecule DNA unzipping with optical tweezers to study how Egr-1, a TF harboring three zinc fingers (ZF1, ZF2 and ZF3), is modulated by the sequence and context of its functional sites in the Lhb gene promoter. We find that both the core 9 bp bound to Egr-1 in each of the sites, and the base pairs flanking them, modulate the affinity and structure of the protein–DNA complex. The effect of the flanking sequences is asymmetric, with a stronger effect for the sequence flanking ZF3. Characterization of the dissociation time of Egr-1 revealed that a local, mechanical perturbation of the interactions of ZF3 destabilizes the complex more effectively than a perturbation of the ZF1 interactions. Our results reveal a novel role for ZF3 in the interaction of Egr-1 with other proteins and the DNA, providing insight on the regulation of Lhb and other genes by Egr-1. Moreover, our findings reveal the potential of small changes in DNA sequence to alter transcriptional regulation, and may shed light on the organization of regulatory elements at promoters. PMID:29253225

Computational Analyses of Synergism in Small Molecular Network Motifs

PubMed Central

Zhang, Yili; Smolen, Paul; Baxter, Douglas A.; Byrne, John H.

2014-01-01

Cellular functions and responses to stimuli are controlled by complex regulatory networks that comprise a large diversity of molecular components and their interactions. However, achieving an intuitive understanding of the dynamical properties and responses to stimuli of these networks is hampered by their large scale and complexity. To address this issue, analyses of regulatory networks often focus on reduced models that depict distinct, reoccurring connectivity patterns referred to as motifs. Previous modeling studies have begun to characterize the dynamics of small motifs, and to describe ways in which variations in parameters affect their responses to stimuli. The present study investigates how variations in pairs of parameters affect responses in a series of ten common network motifs, identifying concurrent variations that act synergistically (or antagonistically) to alter the responses of the motifs to stimuli. Synergism (or antagonism) was quantified using degrees of nonlinear blending and additive synergism. Simulations identified concurrent variations that maximized synergism, and examined the ways in which it was affected by stimulus protocols and the architecture of a motif. Only a subset of architectures exhibited synergism following paired changes in parameters. The approach was then applied to a model describing interlocked feedback loops governing the synthesis of the CREB1 and CREB2 transcription factors. The effects of motifs on synergism for this biologically realistic model were consistent with those for the abstract models of single motifs. These results have implications for the rational design of combination drug therapies with the potential for synergistic interactions. PMID:24651495

A generic motif discovery algorithm for sequential data.

PubMed

Jensen, Kyle L; Styczynski, Mark P; Rigoutsos, Isidore; Stephanopoulos, Gregory N

2006-01-01

Motif discovery in sequential data is a problem of great interest and with many applications. However, previous methods have been unable to combine exhaustive search with complex motif representations and are each typically only applicable to a certain class of problems. Here we present a generic motif discovery algorithm (Gemoda) for sequential data. Gemoda can be applied to any dataset with a sequential character, including both categorical and real-valued data. As we show, Gemoda deterministically discovers motifs that are maximal in composition and length. As well, the algorithm allows any choice of similarity metric for finding motifs. Finally, Gemoda's output motifs are representation-agnostic: they can be represented using regular expressions, position weight matrices or any number of other models for any type of sequential data. We demonstrate a number of applications of the algorithm, including the discovery of motifs in amino acids sequences, a new solution to the (l,d)-motif problem in DNA sequences and the discovery of conserved protein substructures. Gemoda is freely available at http://web.mit.edu/bamel/gemoda

Identification and preliminary characterization of a protein motif related to the zinc finger.

PubMed Central

Lovering, R; Hanson, I M; Borden, K L; Martin, S; O'Reilly, N J; Evan, G I; Rahman, D; Pappin, D J; Trowsdale, J; Freemont, P S

1993-01-01

We have identified a protein motif, related to the zinc finger, which defines a newly discovered family of proteins. The motif was found in the sequence of the human RING1 gene, which is proximal to the major histocompatibility complex region on chromosome six. We propose naming this motif the "RING finger" and it is found in 27 proteins, all of which have putative DNA binding functions. We have synthesized a peptide corresponding to the RING1 motif and examined a number of properties, including metal and DNA binding. We provide evidence to support the suggestion that the RING finger motif is the DNA binding domain of this newly defined family of proteins. Images Fig. 1 Fig. 4 PMID:7681583

Improved prediction of MHC class I and class II epitopes using a novel Gibbs sampling approach.

PubMed

Nielsen, Morten; Lundegaard, Claus; Worning, Peder; Hvid, Christina Sylvester; Lamberth, Kasper; Buus, Søren; Brunak, Søren; Lund, Ole

2004-06-12

Prediction of which peptides will bind a specific major histocompatibility complex (MHC) constitutes an important step in identifying potential T-cell epitopes suitable as vaccine candidates. MHC class II binding peptides have a broad length distribution complicating such predictions. Thus, identifying the correct alignment is a crucial part of identifying the core of an MHC class II binding motif. In this context, we wish to describe a novel Gibbs motif sampler method ideally suited for recognizing such weak sequence motifs. The method is based on the Gibbs sampling method, and it incorporates novel features optimized for the task of recognizing the binding motif of MHC classes I and II. The method locates the binding motif in a set of sequences and characterizes the motif in terms of a weight-matrix. Subsequently, the weight-matrix can be applied to identifying effectively potential MHC binding peptides and to guiding the process of rational vaccine design. We apply the motif sampler method to the complex problem of MHC class II binding. The input to the method is amino acid peptide sequences extracted from the public databases of SYFPEITHI and MHCPEP and known to bind to the MHC class II complex HLA-DR4(B1*0401). Prior identification of information-rich (anchor) positions in the binding motif is shown to improve the predictive performance of the Gibbs sampler. Similarly, a consensus solution obtained from an ensemble average over suboptimal solutions is shown to outperform the use of a single optimal solution. In a large-scale benchmark calculation, the performance is quantified using relative operating characteristics curve (ROC) plots and we make a detailed comparison of the performance with that of both the TEPITOPE method and a weight-matrix derived using the conventional alignment algorithm of ClustalW. The calculation demonstrates that the predictive performance of the Gibbs sampler is higher than that of ClustalW and in most cases also higher than that of the TEPITOPE method.

Rarely at rest

PubMed Central

Hardwick, Steven W.; Luisi, Ben F.

2013-01-01

RNA helicases are compact, machine-like proteins that can harness the energy of nucleoside triphosphate binding and hydrolysis to dynamically remodel RNA structures and protein-RNA complexes. Through such activities, helicases participate in virtually every process associated with the expression of genetic information. Often found as components of multi-enzyme assemblies, RNA helicases facilitate the processivity of RNA degradation, the remodeling of protein interactions during maturation of structured RNA precursors, and fidelity checks of RNA quality. In turn, the assemblies modulate and guide the activities of the helicases. We describe the roles of RNA helicases with a conserved “DExD/H box” sequence motif in representative examples of such machineries from bacteria, archaea and eukaryotes. The recurrent occurrence of such helicases in complex assemblies throughout the course of evolution suggests a common requirement for their activities to meet cellular demands for the dynamic control of RNA metabolism. PMID:23064154

Cis-acting elements in its 3′ UTR mediate post-transcriptional regulation of KRAS

PubMed Central

Kim, Minlee; Kogan, Nicole; Slack, Frank J.

2016-01-01

Multiple RNA-binding proteins and non-coding RNAs, such as microRNAs (miRNAs), are involved in post-transcriptional gene regulation through recognition motifs in the 3′ untranslated region (UTR) of their target genes. The KRAS gene encodes a key signaling protein, and its messenger RNA (mRNA) contains an exceptionally long 3′ UTR; this suggests that it may be subject to a highly complex set of regulatory processes. However, 3′ UTR-dependent regulation of KRAS expression has not been explored in detail. Using extensive deletion and mutational analyses combined with luciferase reporter assays, we have identified inhibitory and stabilizing cis-acting regions within the KRAS 3′ UTR that may interact with miRNAs and RNA-binding proteins, such as HuR. Particularly, we have identified an AU-rich 49-nt fragment in the KRAS 3′ UTR that is required for KRAS 3′ UTR reporter repression. This element contains a miR-185 complementary element, and we show that overexpression of miR-185 represses endogenous KRAS mRNA and protein in vitro. In addition, we have identified another 49-nt fragment that is required to promote KRAS 3′ UTR reporter expression. These findings indicate that multiple cis-regulatory motifs in the 3′ UTR of KRAS finely modulate its expression, and sequence alterations within a binding motif may disrupt the precise functions of trans-regulatory factors, potentially leading to aberrant KRAS expression. PMID:26930719

AP2 adaptor complex mediates bile salt export pump internalization and modulates its hepatocanalicular expression and transport function.

PubMed

Hayashi, Hisamitsu; Inamura, Kaori; Aida, Kensuke; Naoi, Sotaro; Horikawa, Reiko; Nagasaka, Hironori; Takatani, Tomozumi; Fukushima, Tamio; Hattori, Asami; Yabuki, Takashi; Horii, Ikuo; Sugiyama, Yuichi

2012-06-01

The bile salt export pump (BSEP) mediates the biliary excretion of bile salts and its dysfunction induces intrahepatic cholestasis. Reduced canalicular expression of BSEP resulting from the promotion of its internalization is one of the causes of this disease state. However, the molecular mechanism underlying BSEP internalization from the canalicular membrane (CM) remains unknown. We have shown previously that 4-phenylbutyrate (4PBA), a drug used for ornithine transcarbamylase deficiency (OTCD), inhibited internalization and subsequent degradation of cell-surface-resident BSEP. The current study found that 4PBA treatment decreased significantly the expression of α- and μ2-adaptin, both of which are subunits of the AP2 adaptor complex (AP2) that mediates clathrin-dependent endocytosis, in liver specimens from rats and patients with OTCD, and that BSEP has potential AP2 recognition motifs in its cytosolic region. Based on this, the role of AP2 in BSEP internalization was explored further. In vitro analysis with 3×FLAG-human BSEP-expressing HeLa cells and human sandwich-culture hepatocytes indicates that the impairment of AP2 function by RNA interference targeting of α-adaptin inhibits BSEP internalization from the plasma membrane and increases its cell-surface expression and transport function. Studies using immunostaining, coimmunoprecipitation, glutathione S-transferase pulldown assay, and time-lapse imaging show that AP2 interacts with BSEP at the CM through a tyrosine motif at the carboxyl terminus of BSEP and mediates BSEP internalization from the CM of hepatocytes. AP2 mediates the internalization and subsequent degradation of CM-resident BSEP through direct interaction with BSEP and thereby modulates the canalicular expression and transport function of BSEP. This information should be useful for understanding the pathogenesis of severe liver diseases associated with intrahepatic cholestasis. Copyright © 2012 American Association for the Study of Liver Diseases.

HIV-1 Vpu Antagonizes CD317/Tetherin by Adaptor Protein-1-Mediated Exclusion from Virus Assembly Sites

PubMed Central

Pujol, François M.; Laketa, Vibor; Schmidt, Florian; Mukenhirn, Markus; Müller, Barbara; Boulant, Steeve; Grimm, Dirk; Keppler, Oliver T.

2016-01-01

ABSTRACT The host cell restriction factor CD317/tetherin traps virions at the surface of producer cells to prevent their release. The HIV-1 accessory protein Vpu antagonizes this restriction. Vpu reduces the cell surface density of the restriction factor and targets it for degradation; however, these activities are dispensable for enhancing particle release. Instead, Vpu has been suggested to antagonize CD317/tetherin by preventing recycling of internalized CD317/tetherin to the cell surface, blocking anterograde transport of newly synthesized CD317/tetherin, and/or displacing the restriction factor from virus assembly sites at the plasma membrane. At the molecular level, antagonism relies on the physical interaction of Vpu with CD317/tetherin. Recent findings suggested that phosphorylation of a diserine motif enables Vpu to bind to adaptor protein 1 (AP-1) trafficking complexes via two independent interaction motifs and to couple CD317/tetherin to the endocytic machinery. Here, we used a panel of Vpu proteins with specific mutations in individual interaction motifs to define which interactions are required for antagonism of CD317/tetherin. Impairing recycling or anterograde transport of CD317/tetherin to the plasma membrane was insufficient for antagonism. In contrast, excluding CD317/tetherin from HIV-1 assembly sites depended on Vpu motifs for interaction with AP-1 and CD317/tetherin and correlated with antagonism of the particle release restriction. Consistently, interference with AP-1 function or its expression blocked these Vpu activities. Our results define displacement from HIV-1 assembly sites as active principle of CD317/tetherin antagonism by Vpu and support a role of tripartite complexes between Vpu, AP-1, and CD317/tetherin in this process. IMPORTANCE CD317/tetherin poses an intrinsic barrier to human immunodeficiency virus type 1 (HIV-1) replication in human cells by trapping virus particles at the surface of producer cells and thereby preventing their release. The viral protein Vpu antagonizes this restriction, and molecular interactions with the restriction factor and adaptor protein complex 1 (AP-1) were suggested to mediate this activity. Vpu modulates intracellular trafficking of CD317/tetherin and excludes the restriction factor from HIV-1 assembly sites at the plasma membrane, but the relative contribution of these effects to antagonism remain elusive. Using a panel of Vpu mutants, as well as interference with AP-1 function and expression, we show here that Vpu antagonizes CD317/tetherin by blocking its recruitment to viral assembly sites in an AP-1-dependent manner. These results refine our understanding of the molecular mechanisms of CD317/tetherin antagonism and suggest complexes of Vpu with the restriction factor and AP-1 as targets for potential therapeutic intervention. PMID:27170757

Grafting of functional motifs onto protein scaffolds identified by PDB screening--an efficient route to design optimizable protein binders.

PubMed

Tlatli, Rym; Nozach, Hervé; Collet, Guillaume; Beau, Fabrice; Vera, Laura; Stura, Enrico; Dive, Vincent; Cuniasse, Philippe

2013-01-01

Artificial miniproteins that are able to target catalytic sites of matrix metalloproteinases (MMPs) were designed using a functional motif-grafting approach. The motif corresponded to the four N-terminal residues of TIMP-2, a broad-spectrum protein inhibitor of MMPs. Scaffolds that are able to reproduce the functional topology of this motif were obtained by exhaustive screening of the Protein Data Bank (PDB) using STAMPS software (search for three-dimensional atom motifs in protein structures). Ten artificial protein binders were produced. The designed proteins bind catalytic sites of MMPs with affinities ranging from 450 nm to 450 μm prior to optimization. The crystal structure of one artificial binder in complex with the catalytic domain of MMP-12 showed that the inter-molecular interactions established by the functional motif in the artificial binder corresponded to those found in the MMP-14-TIMP-2 complex, albeit with some differences in geometry. Molecular dynamics simulations of the ten binders in complex with MMP-14 suggested that these scaffolds may allow partial reproduction of native inter-molecular interactions, but differences in geometry and stability may contribute to the lower affinity of the artificial protein binders compared to the natural protein binder. Nevertheless, these results show that the in silico design method used provides sets of protein binders that target a specific binding site with a good rate of success. This approach may constitute the first step of an efficient hybrid computational/experimental approach to protein binder design. © 2012 The Authors Journal compilation © 2012 FEBS.

Structural insight into the interaction of proteins containing NPF, DPF, and GPF motifs with the C-terminal EH-domain of EHD1

PubMed Central

Kieken, Fabien; Jović, Marko; Tonelli, Marco; Naslavsky, Naava; Caplan, Steve; Sorgen, Paul L

2009-01-01

Eps15 homology (EH)-domain containing proteins are regulators of endocytic membrane trafficking. EH-domain binding to proteins containing the tripeptide NPF has been well characterized, but recent studies have shown that EH-domains are also able to interact with ligands containing DPF or GPF motifs. We demonstrate that the three motifs interact in a similar way with the EH-domain of EHD1, with the NPF motif having the highest affinity due to the presence of an intermolecular hydrogen bond. The weaker affinity for the DPF and GPF motifs suggests that if complex formation occurs in vivo, they may require high ligand concentrations, the presence of successive motifs and/or specific flanking residues. PMID:19798736

Structural basis for catalytic activation by the human ZNF451 SUMO E3 ligase

DOE PAGES

Cappadocia, Laurent; Pichler, Andrea; Lima, Christopher D.

2015-11-02

E3 protein ligases enhance transfer of ubiquitin-like (Ubl) proteins from E2 conjugating enzymes to substrates by stabilizing the thioester-charged E2~Ubl in a closed configuration optimally aligned for nucleophilic attack. In this paper, we report biochemical and structural data that define the N-terminal domain of the Homo sapiens ZNF451 as the catalytic module for SUMO E3 ligase activity. The ZNF451 catalytic module contains tandem SUMO-interaction motifs (SIMs) bridged by a Pro-Leu-Arg-Pro (PLRP) motif. The first SIM and PLRP motif engage thioester-charged E2~SUMO while the next SIM binds a second molecule of SUMO bound to the back side of E2. We showmore » that ZNF451 is SUMO2 specific and that SUMO modification of ZNF451 may contribute to activity by providing a second molecule of SUMO that interacts with E2. Finally, our results are consistent with ZNF451 functioning as a bona fide SUMO E3 ligase.« less

Structural basis for catalytic activation by the human ZNF451 SUMO E3 ligase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cappadocia, Laurent; Pichler, Andrea; Lima, Christopher D.

E3 protein ligases enhance transfer of ubiquitin-like (Ubl) proteins from E2 conjugating enzymes to substrates by stabilizing the thioester-charged E2~Ubl in a closed configuration optimally aligned for nucleophilic attack. In this paper, we report biochemical and structural data that define the N-terminal domain of the Homo sapiens ZNF451 as the catalytic module for SUMO E3 ligase activity. The ZNF451 catalytic module contains tandem SUMO-interaction motifs (SIMs) bridged by a Pro-Leu-Arg-Pro (PLRP) motif. The first SIM and PLRP motif engage thioester-charged E2~SUMO while the next SIM binds a second molecule of SUMO bound to the back side of E2. We showmore » that ZNF451 is SUMO2 specific and that SUMO modification of ZNF451 may contribute to activity by providing a second molecule of SUMO that interacts with E2. Finally, our results are consistent with ZNF451 functioning as a bona fide SUMO E3 ligase.« less

Identifying DNA-binding proteins using structural motifs and the electrostatic potential

PubMed Central

Shanahan, Hugh P.; Garcia, Mario A.; Jones, Susan; Thornton, Janet M.

2004-01-01

Robust methods to detect DNA-binding proteins from structures of unknown function are important for structural biology. This paper describes a method for identifying such proteins that (i) have a solvent accessible structural motif necessary for DNA-binding and (ii) a positive electrostatic potential in the region of the binding region. We focus on three structural motifs: helix–turn-helix (HTH), helix–hairpin–helix (HhH) and helix–loop–helix (HLH). We find that the combination of these variables detect 78% of proteins with an HTH motif, which is a substantial improvement over previous work based purely on structural templates and is comparable to more complex methods of identifying DNA-binding proteins. Similar true positive fractions are achieved for the HhH and HLH motifs. We see evidence of wide evolutionary diversity for DNA-binding proteins with an HTH motif, and much smaller diversity for those with an HhH or HLH motif. PMID:15356290

Some properties of three αB-crystallin mutants carrying point substitutions in the C-terminal domain and associated with congenital diseases.

PubMed

Gerasimovich, Evgeniia S; Strelkov, Sergei V; Gusev, Nikolai B

2017-11-01

Physico-chemical properties of G154S, R157H and A171T mutants of αB-crystallin (HspB5) associated with congenital human diseases including certain myopathies and cataract were investigated. Oligomers formed by G154S and A171T mutants have the size and apparent molecular weight indistinguishable from those of the wild-type HspB5, whereas the size of oligomers formed by R157H mutant is slightly smaller. All mutants are less thermostable and start to aggregate at a lower temperature than the wild-type protein. All mutants effectively interact with a triple phosphomimicking mutant of HspB1 and form large heterooligomeric complexes of similar composition. All mutants interact with HspB6 forming heterooligomeric complexes with size and composition dependent on the molar ratio of two proteins. The wild-type HspB5 and its G154S and A171T mutants form only high molecular weight (300-450 kDa) heterooligomeric complexes with HspB6, whereas the R157H mutant forms both high and low (∼120 kDa) molecular weight complexes. The wild-type HspB5 and its G154S and A171T mutants form two types of heterooligomers with HspB4, whereas R157H mutant effectively forms only one type of heterooligomers with HspB4. G154S and A171T mutants have lower chaperone-like activity than the wild-type protein when subfragment S1 of myosin or β L -crystallin are used as a model substrates. With these substrates, the R157H mutant shows equal or higher chaperone activity than the wild-type HspB5. We hypothesize that the mutations in the C-terminal region modulate the binding of the IP(I/V) motif to the core α-crystallin domain. The R157H mutation is located in the immediate proximity of this motif. Such modulation could cause altered interaction of HspB5 with partners and substrates and eventually lead to pathological processes. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Discovering Hematopoietic Mechanisms Through Genome-Wide Analysis of GATA Factor Chromatin Occupancy

PubMed Central

Fujiwara, Tohru; O'Geen, Henriette; Keles, Sunduz; Blahnik, Kimberly; Linnemann, Amelia K.; Kang, Yoon-A; Choi, Kyunghee; Farnham, Peggy J.; Bresnick, Emery H.

2009-01-01

SUMMARY GATA factors interact with simple DNA motifs (WGATAR) to regulate critical processes, including hematopoiesis, but very few WGATAR motifs are occupied in genomes. Given the rudimentary knowledge of mechanisms underlying this restriction, and how GATA factors establish genetic networks, we used ChIP-seq to define GATA-1 and GATA-2 occupancy genome-wide in erythroid cells. Coupled with genetic complementation analysis and transcriptional profiling, these studies revealed a rich collection of targets containing a characteristic binding motif of greater complexity than WGATAR. GATA factors occupied loci encoding multiple components of the Scl/TAL1 complex, a master regulator of hematopoiesis and leukemogenic target. Mechanistic analyses provided evidence for cross-regulatory and autoregulatory interactions among components of this complex, including GATA-2 induction of the hematopoietic corepressor ETO-2 and an ETO-2 negative autoregulatory loop. These results establish fundamental principles underlying GATA factor mechanisms in chromatin and illustrate a complex network of considerable importance for the control of hematopoiesis. PMID:19941826

A Non-Invasive NMR Method Based on Histidine Imidazoles to Analyze the pH-Modulation of Protein-Nucleic Acid Interfaces.

PubMed

Cruz-Gallardo, Isabel; Del Conte, Rebecca; Velázquez-Campoy, Adrián; García-Mauriño, Sofía M; Díaz-Moreno, Irene

2015-05-11

A useful (2) J(N-H) coupling-based NMR spectroscopic approach is proposed to unveil, at the molecular level, the contribution of the imidazole groups of histidines from RNA/DNA-binding proteins on the modulation of binding to nucleic acids by pH. Such protonation/deprotonation events have been monitored on the single His96 located at the second RNA/DNA recognition motif (RRM2) of T-cell intracellular antigen-1 (TIA-1) protein. The pKa values of the His96 ionizable groups were substantially higher in the complexes with short U-rich RNA and T-rich DNA oligonucleotides than those of the isolated TIA-1 RRM2. Herein, the methodology applied to determine changes in pKa of histidine side chains upon DNA/RNA binding, gives valuable information to understand the pH effect on multidomain DNA/RNA-binding proteins that shuttle among different cellular compartments. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bioinformatic Analysis Reveals Conservation of Intrinsic Disorder in the Linker Sequences of Prokaryotic Dual-family Immunophilin Chaperones.

PubMed

Barik, Sailen

2018-01-01

The two classical immunophilin families, found essentially in all living cells, are: cyclophilin (CYN) and FK506-binding protein (FKBP). We previously reported a novel class of immunophilins that are natural chimera of these two, which we named dual-family immunophilin (DFI). The DFIs were found in either of two conformations: CYN-linker-FKBP (CFBP) or FKBP-3TPR-CYN (FCBP). While the 3TPR domain can serve as a flexible linker between the FKBP and CYN modules in the FCBP-type DFI, the linker sequences in the CFBP-type DFIs are relatively short, diverse in sequence, and contain no discernible motif or signature. Here, I present several lines of computational evidence that, regardless of their primary structure, these CFBP linkers are intrinsically disordered. This report provides the first molecular foundation for the model that the CFBP linker acts as an unstructured, flexible loop, allowing the two flanking chaperone modules function independently while linked in cis , likely to assist in the folding of multisubunit client complexes.

CPI motif interaction is necessary for capping protein function in cells

PubMed Central

Edwards, Marc; McConnell, Patrick; Schafer, Dorothy A.; Cooper, John A.

2015-01-01

Capping protein (CP) has critical roles in actin assembly in vivo and in vitro. CP binds with high affinity to the barbed end of actin filaments, blocking the addition and loss of actin subunits. Heretofore, models for actin assembly in cells generally assumed that CP is constitutively active, diffusing freely to find and cap barbed ends. However, CP can be regulated by binding of the ‘capping protein interaction' (CPI) motif, found in a diverse and otherwise unrelated set of proteins that decreases, but does not abolish, the actin-capping activity of CP and promotes uncapping in biochemical experiments. Here, we report that CP localization and the ability of CP to function in cells requires interaction with a CPI-motif-containing protein. Our discovery shows that cells target and/or modulate the capping activity of CP via CPI motif interactions in order for CP to localize and function in cells. PMID:26412145

Structure-Function Relationship of the Bik1-Bim1 Complex.

PubMed

Stangier, Marcel M; Kumar, Anil; Chen, Xiuzhen; Farcas, Ana-Maria; Barral, Yves; Steinmetz, Michel O

2018-04-03

In budding yeast, the microtubule plus-end tracking proteins Bik1 (CLIP-170) and Bim1 (EB1) form a complex that interacts with partners involved in spindle positioning, including Stu2 and Kar9. Here, we show that the CAP-Gly and coiled-coil domains of Bik1 interact with the C-terminal ETF peptide of Bim1 and the C-terminal tail region of Stu2, respectively. The crystal structures of the CAP-Gly domain of Bik1 (Bik1CG) alone and in complex with an ETF peptide revealed unique, functionally relevant CAP-Gly elements, establishing Bik1CG as a specific C-terminal phenylalanine recognition domain. Unlike the mammalian CLIP-170-EB1 complex, Bik1-Bim1 forms ternary complexes with the EB1-binding motifs SxIP and LxxPTPh, which are present in diverse proteins, including Kar9. Perturbation of the Bik1-Bim1 interaction in vivo affected Bik1 localization and astral microtubule length. Our results provide insight into the role of the Bik1-Bim1 interaction for cell division, and demonstrate that the CLIP-170-EB1 module is evolutionarily flexible. Copyright © 2018 Elsevier Ltd. All rights reserved.

MotifNet: a web-server for network motif analysis.

PubMed

Smoly, Ilan Y; Lerman, Eugene; Ziv-Ukelson, Michal; Yeger-Lotem, Esti

2017-06-15

Network motifs are small topological patterns that recur in a network significantly more often than expected by chance. Their identification emerged as a powerful approach for uncovering the design principles underlying complex networks. However, available tools for network motif analysis typically require download and execution of computationally intensive software on a local computer. We present MotifNet, the first open-access web-server for network motif analysis. MotifNet allows researchers to analyze integrated networks, where nodes and edges may be labeled, and to search for motifs of up to eight nodes. The output motifs are presented graphically and the user can interactively filter them by their significance, number of instances, node and edge labels, and node identities, and view their instances. MotifNet also allows the user to distinguish between motifs that are centered on specific nodes and motifs that recur in distinct parts of the network. MotifNet is freely available at http://netbio.bgu.ac.il/motifnet . The website was implemented using ReactJs and supports all major browsers. The server interface was implemented in Python with data stored on a MySQL database. estiyl@bgu.ac.il or michaluz@cs.bgu.ac.il. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

IndeCut evaluates performance of network motif discovery algorithms.

PubMed

Ansariola, Mitra; Megraw, Molly; Koslicki, David

2018-05-01

Genomic networks represent a complex map of molecular interactions which are descriptive of the biological processes occurring in living cells. Identifying the small over-represented circuitry patterns in these networks helps generate hypotheses about the functional basis of such complex processes. Network motif discovery is a systematic way of achieving this goal. However, a reliable network motif discovery outcome requires generating random background networks which are the result of a uniform and independent graph sampling method. To date, there has been no method to numerically evaluate whether any network motif discovery algorithm performs as intended on realistically sized datasets-thus it was not possible to assess the validity of resulting network motifs. In this work, we present IndeCut, the first method to date that characterizes network motif finding algorithm performance in terms of uniform sampling on realistically sized networks. We demonstrate that it is critical to use IndeCut prior to running any network motif finder for two reasons. First, IndeCut indicates the number of samples needed for a tool to produce an outcome that is both reproducible and accurate. Second, IndeCut allows users to choose the tool that generates samples in the most independent fashion for their network of interest among many available options. The open source software package is available at https://github.com/megrawlab/IndeCut. megrawm@science.oregonstate.edu or david.koslicki@math.oregonstate.edu. Supplementary data are available at Bioinformatics online.

Detection of core-periphery structure in networks based on 3-tuple motifs

NASA Astrophysics Data System (ADS)

Ma, Chuang; Xiang, Bing-Bing; Chen, Han-Shuang; Small, Michael; Zhang, Hai-Feng

2018-05-01

Detecting mesoscale structure, such as community structure, is of vital importance for analyzing complex networks. Recently, a new mesoscale structure, core-periphery (CP) structure, has been identified in many real-world systems. In this paper, we propose an effective algorithm for detecting CP structure based on a 3-tuple motif. In this algorithm, we first define a 3-tuple motif in terms of the patterns of edges as well as the property of nodes, and then a motif adjacency matrix is constructed based on the 3-tuple motif. Finally, the problem is converted to find a cluster that minimizes the smallest motif conductance. Our algorithm works well in different CP structures: including single or multiple CP structure, and local or global CP structures. Results on the synthetic and the empirical networks validate the high performance of our method.

Structural Basis for Carbohydrate Recognition and Anti-inflammatory Modulation by Gastrointestinal Nematode Parasite Toxascaris leonina Galectin.

PubMed

Hwang, Eun Young; Jeong, Mi Suk; Park, Sang Kyun; Ha, Sung Chul; Yu, Hak Sun; Jang, Se Bok

2016-12-02

Toxascaris leonina galectin (Tl-gal) is a galectin-9 homologue protein isolated from an adult worm of the canine gastrointestinal nematode parasite, and Tl-gal-vaccinated challenge can inhibit inflammation in inflammatory bowel disease-induced mice. We determined the first X-ray structures of full-length Tl-gal complexes with carbohydrates (lactose, N-acetyllactosamine, lacto-N-tetraose, sialyllactose, and glucose). Bonds were formed on concave surfaces of both carbohydrate recognition domains (CRDs) in Tl-gal. All binding sites were found in the HXXXR and WGXEER motifs. Charged Arg 61 /Arg 196 and Glu 80 /Glu 215 on the conserved motif of Tl-gal N-terminal CRD and C-terminal CRD are critical amino acids for recognizing carbohydrate binding, and the residues can affect protein folding and structure. The polar amino acids His, Asn, and Trp are also important residues for the interaction with carbohydrates through hydrogen bonding. Hemagglutination activities of Tl-gal were inhibited by interactions with carbohydrates and mutations. We found that the mutation of Tl-gal (E80A/E215A) at the carbohydrate binding region induced protein aggregation and could be caused in many diseases. The short linker region between the N-terminal and C-terminal CRDs of Tl-gal was very stable against proteolysis and maintained its biological activity. This structural information is expected to elucidate the carbohydrate recognition mechanism of Tl-gal and improve our understanding of anti-inflammatory mediators and modulators of immune response. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of a novel mitotic phosphorylation motif associated with protein localization to the mitotic apparatus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Feng; Camp, David G.; Gritsenko, Marina A.

2007-11-16

The chromosomal passenger complex (CPC) is a critical regulator of chromosome, cytoskeleton and membrane dynamics during mitosis. Here, we identified phosphopeptides and phosphoprotein complexes recognized by a phosphorylation specific antibody that labels the CPC using liquid chromatography coupled to mass spectrometry. A mitotic phosphorylation motif (PX{G/T/S}{L/M}[pS]P or WGL[pS]P) was identified in 11 proteins including Fzr/Cdh1 and RIC-8, two proteins with potential links to the CPC. Phosphoprotein complexes contained known CPC components INCENP, Aurora-B and TD-60, as well as SMAD2, 14-3-3 proteins, PP2A, and Cdk1, a likely kinase for this motif. Protein sequence analysis identified phosphorylation motifs in additional proteins includingmore » SMAD2, Plk3 and INCENP. Mitotic SMAD2 and Plk3 phosphorylation was confirmed using phosphorylation specific antibodies, and in the case of Plk3, phosphorylation correlates with its localization to the mitotic apparatus. A mutagenesis approach was used to show INCENP phosphorylation is required for midbody localization. These results provide evidence for a shared phosphorylation event that regulates localization of critical proteins during mitosis.« less

Hydrophobic Motif Phosphorylation Coordinates Activity and Polar Localization of the Neurospora crassa Nuclear Dbf2-Related Kinase COT1

PubMed Central

Maerz, Sabine; Dettmann, Anne

2012-01-01

Nuclear Dbf2p-related (NDR) kinases and associated proteins are recognized as a conserved network that regulates eukaryotic cell polarity. NDR kinases require association with MOB adaptor proteins and phosphorylation of two conserved residues in the activation segment and hydrophobic motif for activity and function. We demonstrate that the Neurospora crassa NDR kinase COT1 forms inactive dimers via a conserved N-terminal extension, which is also required for the interaction of the kinase with MOB2 to generate heterocomplexes with basal activity. Basal kinase activity also requires autophosphorylation of the COT1-MOB2 complex in the activation segment, while hydrophobic motif phosphorylation of COT1 by the germinal center kinase POD6 fully activates COT1 through induction of a conformational change. Hydrophobic motif phosphorylation is also required for plasma membrane association of the COT1-MOB2 complex. MOB2 further restricts the membrane-associated kinase complex to the hyphal apex to promote polar cell growth. These data support an integrated mechanism of NDR kinase regulation in vivo, in which kinase activation and cellular localization of COT1 are coordinated by dual phosphorylation and interaction with MOB2. PMID:22451488

Detecting Statistically Significant Communities of Triangle Motifs in Undirected Networks

DTIC Science & Technology

2016-04-26

REPORT TYPE Final 3. DATES COVERED (From - To) 15 Oct 2014 to 14 Jan 2015 4. TITLE AND SUBTITLE Detecting statistically significant clusters of...extend the work of Perry et al. [6] by developing a statistical framework that supports the detection of triangle motif-based clusters in complex...priori, the need for triangle motif-based clustering . 2. Developed an algorithm for clustering undirected networks, where the triangle con guration was

Molecular Interaction Between Smurfl WW2 Domain and PPXY Motifs of Smadl, Smad5, and Smad6-Modeling and Analysis.

PubMed

Sangadala, Sreedhara; Rao Metpally, Raghu Prasad; B Reddy, Boojala Vijay

2007-08-01

Abstract The ubiquitin-proteasome proteolytic pathway is essential for various important biological processes including cell cycle progression, gene transcription, and signal transduction. One of the important regulatory mechanisms by which the bone-inducing activity of the bone morphogenetic protein (BMP) signaling is modulated involves ubiquitin-mediated proteasomal degradation. The BMP induced receptor signal is transmitted intracellularly by phosphorylation of Smad proteins by the activated receptor I. The phosphorylated Smads 1, 5, and 8 (R-Smads) oligomerize with the co-Smad (Smad4). The complex, thus, formed translocates to the nucleus and interacts with other cofactors to regulate the expression of downstream target genes. R-Smads contain PPXY motif in the linker region that interacts with Smad ubiquitin regulatory factor 1 (Smurf1), an E3 ubiquitin ligase that catalyzes ubiquitination of target proteins for proteasomal degradation. Smurf1 contains a HECT domain, a C2 domain, and 2 WW domains (WW1, WW2). The PPXY motif in target proteins and its interaction with Smurf1 may form the basis for regulation of steady-state levels of Smads in controlling BMP-responsiveness of cells. Here, we present a homology-based model of the Smurf1 WW2 domain and the target octa-peptides containing PPXY motif of Smurf1- interacting Smads. We carried out docking of Smurf1 WW2 domain with the PPXY motifs of Smadl, Smad5, and Smad6 and identified the key amino acid residues involved in interaction. Furthermore, we present experimental evidence that WW2 domain of Smurf1 does indeed interact with the Smad proteins and that the deletion of WW2 domain of Smurf1 results in loss of its binding to Smads using the purified recombinant proteins. Finally, we also present data confirming that the deletion of WW2 domain in Smurf1 abolishes its ubiquitination activity on Smad1 in an in vitro ubiquitination assay. It shows that the interaction between the WW domain and Smad PPXY motif is a key step in Smurf1-mediated ubiquitination of its natural targets such as Smad1, Smad5, and Smad6. This work facilitates further strategies to unravel the biological function of such interactions and help in designing effective mimetic compounds that either mimic or disrupt the specific interaction.

Molecular interaction between Smurf1 WW2 domain and PPXY motifs of Smad1, Smad5, and Smad6--modeling and analysis.

PubMed

Sangadala, Sreedhara; Metpally, Raghu Prasad Rao; Reddy, Boojala Vijay B

2007-08-01

The ubiquitin-proteasome proteolytic pathway is essential for various important biological processes including cell cycle progression, gene transcription, and signal transduction. One of the important regulatory mechanisms by which the bone-inducing activity of the bone morphogenetic protein (BMP) signaling is modulated involves ubiquitin-mediated proteasomal degradation. The BMP induced receptor signal is transmitted intracellularly by phosphorylation of Smad proteins by the activated receptor I. The phosphorylated Smads 1, 5, and 8 (R-Smads) oligomerize with the co-Smad (Smad4). The complex, thus, formed translocates to the nucleus and interacts with other cofactors to regulate the expression of downstream target genes. R-Smads contain PPXY motif in the linker region that interacts with Smad ubiquitin regulatory factor 1 (Smurf1), an E3 ubiquitin ligase that catalyzes ubiquitination of target proteins for proteasomal degradation. Smurf1 contains a HECT domain, a C2 domain, and 2 WW domains (WW1, WW2). The PPXY motif in target proteins and its interaction with Smurf1 may form the basis for regulation of steady-state levels of Smads in controlling BMP-responsiveness of cells. Here, we present a homology-based model of the Smurf1 WW2 domain and the target octa-peptides containing PPXY motif of Smurf1-interacting Smads. We carried out docking of Smurf1 WW2 domain with the PPXY motifs of Smad1, Smad5, and Smad6 and identified the key amino acid residues involved in interaction. Furthermore, we present experimental evidence that WW2 domain of Smurf1 does indeed interact with the Smad proteins and that the deletion of WW2 domain of Smurf1 results in loss of its binding to Smads using the purified recombinant proteins. Finally, we also present data confirming that the deletion of WW2 domain in Smurf1 abolishes its ubiquitination activity on Smad1 in an in vitro ubiquitination assay. It shows that the interaction between the WW domain and Smad PPXY motif is a key step in Smurf1-mediated ubiquitination of its natural targets such as Smad1, Smad5, and Smad6. This work facilitates further strategies to unravel the biological function of such interactions and help in designing effective mimetic compounds that either mimic or disrupt the specific interaction.

Novel functions of CCM1 delimit the relationship of PTB/PH domains.

PubMed

Zhang, Jun; Dubey, Pallavi; Padarti, Akhil; Zhang, Aileen; Patel, Rinkal; Patel, Vipulkumar; Cistola, David; Badr, Ahmed

2017-10-01

Three NPXY motifs and one FERM domain in CCM1 makes it a versatile scaffold protein for tethering the signaling components together within the CCM signaling complex (CSC). The cellular role of CCM1 protein remains inadequately expounded. Both phosphotyrosine binding (PTB) and pleckstrin homology (PH) domains were recognized as structurally related but functionally distinct domains. By utilizing molecular cloning, protein binding assays and RT-qPCR to identify novel cellular partners of CCM1 and its cellular expression patterns; by screening candidate PTB/PH proteins and subsequently structurally simulation in combining with current X-ray crystallography and NMR data to defined the essential structure of PTB/PH domain for NPXY-binding and the relationship among PTB, PH and FERM domain(s). We identified a group of 28 novel cellular partners of CCM1, all of which contain either PTB or PH domain(s), and developed a novel classification system for these PTB/PH proteins based on their relationship with different NPXY motifs of CCM1. Our results demonstrated that CCM1 has a wide spectrum of binding to different PTB/PH proteins and perpetuates their specificity to interact with certain PTB/PH domains through selective combination of three NPXY motifs. We also demonstrated that CCM1 can be assembled into oligomers through intermolecular interaction between its F3 lobe in FERM domain and one of the three NPXY motifs. Despite being embedded in FERM domain as F3 lobe, F3 module acts as a fully functional PH domain to interact with NPXY motif. The most salient feature of the study was that both PTB and PH domains are structurally and functionally comparable, suggesting that PTB domain is likely evolved from PH domain with polymorphic structural additions at its N-terminus. A new β1A-strand of the PTB domain was discovered and new minimum structural requirement of PTB/PH domain for NPXY motif-binding was determined. Based on our data, a novel theory of structure, function and relationship of PTB, PH and FERM domains has been proposed, which extends the importance of the NPXY-PTB/PH interaction on the CSC signaling and/or other cell receptors with great potential pointing to new therapeutic strategies. The study provides new insight into the structural characteristics of PTB/PH domains, essential structural elements of PTB/PH domain required for NPXY motif-binding, and function and relationship among PTB, PH and FERM domains. Copyright © 2017 Elsevier B.V. All rights reserved.

DNA sequence determinants controlling affinity, stability and shape of DNA complexes bound by the nucleoid protein Fis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hancock, Stephen P.; Stella, Stefano; Cascio, Duilio

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequencesmore » in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. Lastly, the affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.« less

DNA sequence determinants controlling affinity, stability and shape of DNA complexes bound by the nucleoid protein Fis

DOE PAGES

Hancock, Stephen P.; Stella, Stefano; Cascio, Duilio; ...

2016-03-09

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequencesmore » in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. Lastly, the affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.« less

Polycomb-like proteins link the PRC2 complex to CpG islands

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Haojie; Liefke, Robert; Jiang, Junyi

The Polycomb repressive complex 2 (PRC2) mainly mediates transcriptional repression1,2 and has essential roles in various biological processes including the maintenance of cell identity and proper differentiation. Polycomb-like (PCL) proteins, such as PHF1, MTF2 and PHF19, are PRC2-associated factors that form sub-complexes with PRC2 core components3, and have been proposed to modulate the enzymatic activity of PRC2 or the recruitment of PRC2 to specific genomic loci4,5,6,7,8,9,10,11,12,13. Mammalian PRC2-binding sites are enriched in CG content, which correlates with CpG islands that display a low level of DNA methylation14. However, the mechanism of PRC2 recruitment to CpG islands is not fully understood.more » Here we solve the crystal structures of the N-terminal domains of PHF1 and MTF2 with bound CpG-containing DNAs in the presence of H3K36me3-containing histone peptides. We show that the extended homologous regions of both proteins fold into a winged-helix structure, which specifically binds to the unmethylated CpG motif but in a completely different manner from the canonical winged-helix DNA recognition motif. We also show that the PCL extended homologous domains are required for efficient recruitment of PRC2 to CpG island-containing promoters in mouse embryonic stem cells. Our research provides the first, to our knowledge, direct evidence to demonstrate that PCL proteins are crucial for PRC2 recruitment to CpG islands, and further clarifies the roles of these proteins in transcriptional regulation in vivo.« less

Complex Interplay among DNA Modification, Noncoding RNA Expression and Protein-Coding RNA Expression in Salvia miltiorrhiza Chloroplast Genome

PubMed Central

Chen, Haimei; Zhang, Jianhui; Yuan, George; Liu, Chang

2014-01-01

Salvia miltiorrhiza is one of the most widely used medicinal plants. As a first step to develop a chloroplast-based genetic engineering method for the over-production of active components from S. miltiorrhiza, we have analyzed the genome, transcriptome, and base modifications of the S. miltiorrhiza chloroplast. Total genomic DNA and RNA were extracted from fresh leaves and then subjected to strand-specific RNA-Seq and Single-Molecule Real-Time (SMRT) sequencing analyses. Mapping the RNA-Seq reads to the genome assembly allowed us to determine the relative expression levels of 80 protein-coding genes. In addition, we identified 19 polycistronic transcription units and 136 putative antisense and intergenic noncoding RNA (ncRNA) genes. Comparison of the abundance of protein-coding transcripts (cRNA) with and without overlapping antisense ncRNAs (asRNA) suggest that the presence of asRNA is associated with increased cRNA abundance (p<0.05). Using the SMRT Portal software (v1.3.2), 2687 potential DNA modification sites and two potential DNA modification motifs were predicted. The two motifs include a TATA box–like motif (CPGDMM1, “TATANNNATNA”), and an unknown motif (CPGDMM2 “WNYANTGAW”). Specifically, 35 of the 97 CPGDMM1 motifs (36.1%) and 91 of the 369 CPGDMM2 motifs (24.7%) were found to be significantly modified (p<0.01). Analysis of genes downstream of the CPGDMM1 motif revealed the significantly increased abundance of ncRNA genes that are less than 400 bp away from the significantly modified CPGDMM1motif (p<0.01). Taking together, the present study revealed a complex interplay among DNA modifications, ncRNA and cRNA expression in chloroplast genome. PMID:24914614

Complex interplay among DNA modification, noncoding RNA expression and protein-coding RNA expression in Salvia miltiorrhiza chloroplast genome.

PubMed

Chen, Haimei; Zhang, Jianhui; Yuan, George; Liu, Chang

2014-01-01

Salvia miltiorrhiza is one of the most widely used medicinal plants. As a first step to develop a chloroplast-based genetic engineering method for the over-production of active components from S. miltiorrhiza, we have analyzed the genome, transcriptome, and base modifications of the S. miltiorrhiza chloroplast. Total genomic DNA and RNA were extracted from fresh leaves and then subjected to strand-specific RNA-Seq and Single-Molecule Real-Time (SMRT) sequencing analyses. Mapping the RNA-Seq reads to the genome assembly allowed us to determine the relative expression levels of 80 protein-coding genes. In addition, we identified 19 polycistronic transcription units and 136 putative antisense and intergenic noncoding RNA (ncRNA) genes. Comparison of the abundance of protein-coding transcripts (cRNA) with and without overlapping antisense ncRNAs (asRNA) suggest that the presence of asRNA is associated with increased cRNA abundance (p<0.05). Using the SMRT Portal software (v1.3.2), 2687 potential DNA modification sites and two potential DNA modification motifs were predicted. The two motifs include a TATA box-like motif (CPGDMM1, "TATANNNATNA"), and an unknown motif (CPGDMM2 "WNYANTGAW"). Specifically, 35 of the 97 CPGDMM1 motifs (36.1%) and 91 of the 369 CPGDMM2 motifs (24.7%) were found to be significantly modified (p<0.01). Analysis of genes downstream of the CPGDMM1 motif revealed the significantly increased abundance of ncRNA genes that are less than 400 bp away from the significantly modified CPGDMM1motif (p<0.01). Taking together, the present study revealed a complex interplay among DNA modifications, ncRNA and cRNA expression in chloroplast genome.

Leucine zipper motif in RRS1 is crucial for the regulation of Arabidopsis dual resistance protein complex RPS4/RRS1

PubMed Central

Narusaka, Mari; Toyoda, Kazuhiro; Shiraishi, Tomonori; Iuchi, Satoshi; Takano, Yoshitaka; Shirasu, Ken; Narusaka, Yoshihiro

2016-01-01

Arabidopsis thaliana leucine-rich repeat-containing (NLR) proteins RPS4 and RRS1, known as dual resistance proteins, confer resistance to multiple pathogen isolates, such as the bacterial pathogens Pseudomonas syringae and Ralstonia solanacearum and the fungal pathogen Colletotrichum higginsianum. RPS4 is a typical Toll/interleukin 1 Receptor (TIR)-type NLR, whereas RRS1 is an atypical TIR-NLR that contains a leucine zipper (LZ) motif and a C-terminal WRKY domain. RPS4 and RRS1 are localised near each other in a head-to-head orientation. In this study, direct mutagenesis of the C-terminal LZ motif in RRS1 caused an autoimmune response and stunting in the mutant. Co-immunoprecipitation analysis indicated that full-length RPS4 and RRS1 are physically associated with one another. Furthermore, virus-induced gene silencing experiments showed that hypersensitive-like cell death triggered by RPS4/LZ motif-mutated RRS1 depends on EDS1. In conclusion, we suggest that the RRS1-LZ motif is crucial for the regulation of the RPS4/RRS1 complex. PMID:26750751

Common fold in helix–hairpin–helix proteins

PubMed Central

Shao, Xuguang; Grishin, Nick V.

2000-01-01

Helix–hairpin–helix (HhH) is a widespread motif involved in non-sequence-specific DNA binding. The majority of HhH motifs function as DNA-binding modules, however, some of them are used to mediate protein–protein interactions or have acquired enzymatic activity by incorporating catalytic residues (DNA glycosylases). From sequence and structural analysis of HhH-containing proteins we conclude that most HhH motifs are integrated as a part of a five-helical domain, termed (HhH)2 domain here. It typically consists of two consecutive HhH motifs that are linked by a connector helix and displays pseudo-2-fold symmetry. (HhH)2 domains show clear structural integrity and a conserved hydrophobic core composed of seven residues, one residue from each α-helix and each hairpin, and deserves recognition as a distinct protein fold. In addition to known HhH in the structures of RuvA, RadA, MutY and DNA-polymerases, we have detected new HhH motifs in sterile alpha motif and barrier-to-autointegration factor domains, the α-subunit of Escherichia coli RNA-polymerase, DNA-helicase PcrA and DNA glyco­s­y­lases. Statistically significant sequence similarity of HhH motifs and pronounced structural conservation argue for homology between (HhH)2 domains in different protein families. Our analysis helps to clarify how non-symmetric protein motifs bind to the double helix of DNA through the formation of a pseudo-2-fold symmetric (HhH)2 functional unit. PMID:10908318

Scalable Performance Environments for Parallel Systems

NASA Technical Reports Server (NTRS)

Reed, Daniel A.; Olson, Robert D.; Aydt, Ruth A.; Madhyastha, Tara M.; Birkett, Thomas; Jensen, David W.; Nazief, Bobby A. A.; Totty, Brian K.

1991-01-01

As parallel systems expand in size and complexity, the absence of performance tools for these parallel systems exacerbates the already difficult problems of application program and system software performance tuning. Moreover, given the pace of technological change, we can no longer afford to develop ad hoc, one-of-a-kind performance instrumentation software; we need scalable, portable performance analysis tools. We describe an environment prototype based on the lessons learned from two previous generations of performance data analysis software. Our environment prototype contains a set of performance data transformation modules that can be interconnected in user-specified ways. It is the responsibility of the environment infrastructure to hide details of module interconnection and data sharing. The environment is written in C++ with the graphical displays based on X windows and the Motif toolkit. It allows users to interconnect and configure modules graphically to form an acyclic, directed data analysis graph. Performance trace data are represented in a self-documenting stream format that includes internal definitions of data types, sizes, and names. The environment prototype supports the use of head-mounted displays and sonic data presentation in addition to the traditional use of visual techniques.

Multiple TPR motifs characterize the Fanconi anemia FANCG protein.

PubMed

Blom, Eric; van de Vrugt, Henri J; de Vries, Yne; de Winter, Johan P; Arwert, Fré; Joenje, Hans

2004-01-05

The genome protection pathway that is defective in patients with Fanconi anemia (FA) is controlled by at least eight genes, including BRCA2. A key step in the pathway involves the monoubiquitylation of FANCD2, which critically depends on a multi-subunit nuclear 'core complex' of at least six FANC proteins (FANCA, -C, -E, -F, -G, and -L). Except for FANCL, which has WD40 repeats and a RING finger domain, no significant domain structure has so far been recognized in any of the core complex proteins. By using a homology search strategy comparing the human FANCG protein sequence with its ortholog sequences in Oryzias latipes (Japanese rice fish) and Danio rerio (zebrafish) we identified at least seven tetratricopeptide repeat motifs (TPRs) covering a major part of this protein. TPRs are degenerate 34-amino acid repeat motifs which function as scaffolds mediating protein-protein interactions, often found in multiprotein complexes. In four out of five TPR motifs tested (TPR1, -2, -5, and -6), targeted missense mutagenesis disrupting the motifs at the critical position 8 of each TPR caused complete or partial loss of FANCG function. Loss of function was evident from failure of the mutant proteins to complement the cellular FA phenotype in FA-G lymphoblasts, which was correlated with loss of binding to FANCA. Although the TPR4 mutant fully complemented the cells, it showed a reduced interaction with FANCA, suggesting that this TPR may also be of functional importance. The recognition of FANCG as a typical TPR protein predicts this protein to play a key role in the assembly and/or stabilization of the nuclear FA protein core complex.

Deciphering the combinatorial architecture of a Drosophila homeotic gene enhancer

PubMed Central

Drewell, Robert A.; Nevarez, Michael J.; Kurata, Jessica S.; Winkler, Lauren N.; Li, Lily; Dresch, Jacqueline M.

2013-01-01

Summary In Drosophila, the 330 kb bithorax complex regulates cellular differentiation along the anterio-posterior axis during development in the thorax and abdomen and is comprised of three homeotic genes: Ultrabithorax, abdominal-A, and Abdominal-B. The expression of each of these genes is in turn controlled through interactions between transcription factors and a number of cis-regulatory modules in the neighboring intergenic regions. In this study, we examine how the sequence architecture of transcription factor binding sites mediates the functional activity of one of these cis-regulatory modules. Using computational, mathematical modeling and experimental molecular genetic approaches we investigate the IAB7b enhancer, which regulates Abdominal-B expression specifically in the presumptive seventh and ninth abdominal segments of the early embryo. A cross-species comparison of the IAB7b enhancer reveals an evolutionarily conserved signature motif containing two FUSHI-TARAZU activator transcription factor binding sites. We find that the transcriptional repressors KNIRPS, KRUPPEL and GIANT are able to restrict reporter gene expression to the posterior abdominal segments, using different molecular mechanisms including short-range repression and competitive binding. Additionally, we show the functional importance of the spacing between the two FUSHI-TARAZU binding sites and discuss the potential importance of cooperativity for transcriptional activation. Our results demonstrate that the transcriptional output of the IAB7b cis-regulatory module relies on a complex set of combinatorial inputs mediated by specific transcription factor binding and that the sequence architecture at this enhancer is critical to maintain robust regulatory function. PMID:24514265

The crystal structure of the Sox4 HMG domain-DNA complex suggests a mechanism for positional interdependence in DNA recognition.

PubMed

Jauch, Ralf; Ng, Calista K L; Narasimhan, Kamesh; Kolatkar, Prasanna R

2012-04-01

It has recently been proposed that the sequence preferences of DNA-binding TFs (transcription factors) can be well described by models that include the positional interdependence of the nucleotides of the target sites. Such binding models allow for multiple motifs to be invoked, such as principal and secondary motifs differing at two or more nucleotide positions. However, the structural mechanisms underlying the accommodation of such variant motifs by TFs remain elusive. In the present study we examine the crystal structure of the HMG (high-mobility group) domain of Sox4 [Sry (sex-determining region on the Y chromosome)-related HMG box 4] bound to DNA. By comparing this structure with previously solved structures of Sox17 and Sox2, we observed subtle conformational differences at the DNA-binding interface. Furthermore, using quantitative electrophoretic mobility-shift assays we validated the positional interdependence of two nucleotides and the presence of a secondary Sox motif in the affinity landscape of Sox4. These results suggest that a concerted rearrangement of two interface amino acids enables Sox4 to accommodate primary and secondary motifs. The structural adaptations lead to altered dinucleotide preferences that mutually reinforce each other. These analyses underline the complexity of the DNA recognition by TFs and provide an experimental validation for the conceptual framework of positional interdependence and secondary binding motifs.

ELM: the status of the 2010 eukaryotic linear motif resource

PubMed Central

Gould, Cathryn M.; Diella, Francesca; Via, Allegra; Puntervoll, Pål; Gemünd, Christine; Chabanis-Davidson, Sophie; Michael, Sushama; Sayadi, Ahmed; Bryne, Jan Christian; Chica, Claudia; Seiler, Markus; Davey, Norman E.; Haslam, Niall; Weatheritt, Robert J.; Budd, Aidan; Hughes, Tim; Paś, Jakub; Rychlewski, Leszek; Travé, Gilles; Aasland, Rein; Helmer-Citterich, Manuela; Linding, Rune; Gibson, Toby J.

2010-01-01

Linear motifs are short segments of multidomain proteins that provide regulatory functions independently of protein tertiary structure. Much of intracellular signalling passes through protein modifications at linear motifs. Many thousands of linear motif instances, most notably phosphorylation sites, have now been reported. Although clearly very abundant, linear motifs are difficult to predict de novo in protein sequences due to the difficulty of obtaining robust statistical assessments. The ELM resource at http://elm.eu.org/ provides an expanding knowledge base, currently covering 146 known motifs, with annotation that includes >1300 experimentally reported instances. ELM is also an exploratory tool for suggesting new candidates of known linear motifs in proteins of interest. Information about protein domains, protein structure and native disorder, cellular and taxonomic contexts is used to reduce or deprecate false positive matches. Results are graphically displayed in a ‘Bar Code’ format, which also displays known instances from homologous proteins through a novel ‘Instance Mapper’ protocol based on PHI-BLAST. ELM server output provides links to the ELM annotation as well as to a number of remote resources. Using the links, researchers can explore the motifs, proteins, complex structures and associated literature to evaluate whether candidate motifs might be worth experimental investigation. PMID:19920119

World Color Survey color naming reveals universal motifs and their within-language diversity

PubMed Central

Lindsey, Delwin T.; Brown, Angela M.

2009-01-01

We analyzed the color terms in the World Color Survey (WCS) (www.icsi.berkeley.edu/wcs/), a large color-naming database obtained from informants of mostly unwritten languages spoken in preindustrialized cultures that have had limited contact with modern, industrialized society. The color naming idiolects of 2,367 WCS informants fall into three to six “motifs,” where each motif is a different color-naming system based on a subset of a universal glossary of 11 color terms. These motifs are universal in that they occur worldwide, with some individual variation, in completely unrelated languages. Strikingly, these few motifs are distributed across the WCS informants in such a way that multiple motifs occur in most languages. Thus, the culture a speaker comes from does not completely determine how he or she will use color terms. An analysis of the modern patterns of motif usage in the WCS languages, based on the assumption that they reflect historical patterns of color term evolution, suggests that color lexicons have changed over time in a complex but orderly way. The worldwide distribution of the motifs and the cooccurrence of multiple motifs within languages suggest that universal processes control the naming of colors. PMID:19901327

TFII-I regulates target genes in the PI-3K and TGF-β signaling pathways through a novel DNA binding motif.

PubMed

Segura-Puimedon, Maria; Borralleras, Cristina; Pérez-Jurado, Luis A; Campuzano, Victoria

2013-09-25

General transcription factor (TFII-I) is a multi-functional protein involved in the transcriptional regulation of critical developmental genes, encoded by the GTF2I gene located on chromosome 7q11.23. Haploinsufficiency at GTF2I has been shown to play a major role in the neurodevelopmental features of Williams-Beuren syndrome (WBS). Identification of genes regulated by TFII-I is thus critical to detect molecular determinants of WBS as well as to identify potential new targets for specific pharmacological interventions, which are currently absent. We performed a microarray screening for transcriptional targets of TFII-I in cortex and embryonic cells from Gtf2i mutant and wild-type mice. Candidate genes with altered expression were verified using real-time PCR. A novel motif shared by deregulated genes was found and chromatin immunoprecipitation assays in embryonic fibroblasts were used to document in vitro TFII-I binding to this motif in the promoter regions of deregulated genes. Interestingly, the PI3K and TGFβ signaling pathways were over-represented among TFII-I-modulated genes. In this study we have found a highly conserved DNA element, common to a set of genes regulated by TFII-I, and identified and validated novel in vivo neuronal targets of this protein affecting the PI3K and TGFβ signaling pathways. Overall, our data further contribute to unravel the complexity and variability of the different genetic programs orchestrated by TFII-I. © 2013 Elsevier B.V. All rights reserved.

Dimer formation through domain swapping in the crystal structure of the Grb2-SH2-Ac-pYVNV complex.

PubMed

Schiering, N; Casale, E; Caccia, P; Giordano, P; Battistini, C

2000-11-07

Src homology 2 (SH2) domains are key modules in intracellular signal transduction. They link activated cell surface receptors to downstream targets by binding to phosphotyrosine-containing sequence motifs. The crystal structure of a Grb2-SH2 domain-phosphopeptide complex was determined at 2.4 A resolution. The asymmetric unit contains four polypeptide chains. There is an unexpected domain swap so that individual chains do not adopt a closed SH2 fold. Instead, reorganization of the EF loop leads to an open, nonglobular fold, which associates with an equivalent partner to generate an intertwined dimer. As in previously reported crystal structures of canonical Grb2-SH2 domain-peptide complexes, each of the four hybrid SH2 domains in the two domain-swapped dimers binds the phosphopeptide in a type I beta-turn conformation. This report is the first to describe domain swapping for an SH2 domain. While in vivo evidence of dimerization of Grb2 exists, our SH2 dimer is metastable and a physiological role of this new form of dimer formation remains to be demonstrated.

Regulatory elements of Caenorhabditis elegans ribosomal protein genes

PubMed Central

2012-01-01

Background Ribosomal protein genes (RPGs) are essential, tightly regulated, and highly expressed during embryonic development and cell growth. Even though their protein sequences are strongly conserved, their mechanism of regulation is not conserved across yeast, Drosophila, and vertebrates. A recent investigation of genomic sequences conserved across both nematode species and associated with different gene groups indicated the existence of several elements in the upstream regions of C. elegans RPGs, providing a new insight regarding the regulation of these genes in C. elegans. Results In this study, we performed an in-depth examination of C. elegans RPG regulation and found nine highly conserved motifs in the upstream regions of C. elegans RPGs using the motif discovery algorithm DME. Four motifs were partially similar to transcription factor binding sites from C. elegans, Drosophila, yeast, and human. One pair of these motifs was found to co-occur in the upstream regions of 250 transcripts including 22 RPGs. The distance between the two motifs displayed a complex frequency pattern that was related to their relative orientation. We tested the impact of three of these motifs on the expression of rpl-2 using a series of reporter gene constructs and showed that all three motifs are necessary to maintain the high natural expression level of this gene. One of the motifs was similar to the binding site of an orthologue of POP-1, and we showed that RNAi knockdown of pop-1 impacts the expression of rpl-2. We further determined the transcription start site of rpl-2 by 5’ RACE and found that the motifs lie 40–90 bases upstream of the start site. We also found evidence that a noncoding RNA, contained within the outron of rpl-2, is co-transcribed with rpl-2 and cleaved during trans-splicing. Conclusions Our results indicate that C. elegans RPGs are regulated by a complex novel series of regulatory elements that is evolutionarily distinct from those of all other species examined up until now. PMID:22928635

Analysis of the Isolated SecA DEAD Motor Suggests a Mechanism for Chemical-Mechanical Coupling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nithianantham, Stanley; Shilton, Brian H

The preprotein cross-linking domain and C-terminal domains of Escherichia coli SecA were removed to create a minimal DEAD motor, SecA-DM. SecA-DM hydrolyzes ATP and has the same affinity for ADP as full-length SecA. The crystal structure of SecA-DM in complex with ADP was solved and shows the DEAD motor in a closed conformation. Comparison with the structure of the E. coli DEAD motor in an open conformation (Protein Data Bank ID 2FSI) indicates main-chain conformational changes in two critical sequences corresponding to Motif III and Motif V of the DEAD helicase family. The structures that the Motif III and Motifmore » V sequences adopt in the DEAD motor open conformation are incompatible with the closed conformation. Therefore, when the DEAD motor makes the transition from open to closed, Motif III and Motif V are forced to change their conformations, which likely functions to regulate passage through the transition state for ATP hydrolysis. The transition state for ATP hydrolysis for the SecA DEAD motor was modeled based on the conformation of the Vasa helicase in complex with adenylyl imidodiphosphate and RNA (Protein Data Bank ID 2DB3). A mechanism for chemical-mechanical coupling emerges, where passage through the transition state for ATP hydrolysis is hindered by the conformational changes required in Motif III and Motif V, and may be promoted by binding interactions with the preprotein substrate and/or other translocase domains and subunits.« less

Analysis of the Isolated SecA DEAD Motor Suggests a Mechanism for Chemical-Mechanical Coupling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nithianantham, Stanley; Shilton, Brian H

2011-09-28

The preprotein cross-linking domain and C-terminal domains of Escherichia coli SecA were removed to create a minimal DEAD motor, SecA-DM. SecA-DM hydrolyzes ATP and has the same affinity for ADP as full-length SecA. The crystal structure of SecA-DM in complex with ADP was solved and shows the DEAD motor in a closed conformation. Comparison with the structure of the E. coli DEAD motor in an open conformation (Protein Data Bank ID 2FSI) indicates main-chain conformational changes in two critical sequences corresponding to Motif III and Motif V of the DEAD helicase family. The structures that the Motif III and Motifmore » V sequences adopt in the DEAD motor open conformation are incompatible with the closed conformation. Therefore, when the DEAD motor makes the transition from open to closed, Motif III and Motif V are forced to change their conformations, which likely functions to regulate passage through the transition state for ATP hydrolysis. The transition state for ATP hydrolysis for the SecA DEAD motor was modeled based on the conformation of the Vasa helicase in complex with adenylyl imidodiphosphate and RNA (Protein Data Bank ID 2DB3). A mechanism for chemical-mechanical coupling emerges, where passage through the transition state for ATP hydrolysis is hindered by the conformational changes required in Motif III and Motif V, and may be promoted by binding interactions with the preprotein substrate and/or other translocase domains and subunits.« less

RNF11 is a multifunctional modulator of growth factor receptor signalling and transcriptional regulation.

PubMed

Azmi, Peter; Seth, Arun

2005-11-01

Our laboratory has found that the 154aa RING finger protein 11 (RNF11), has modular domains and motifs including a RING-H2 finger domain, a PY motif, an ubiquitin interacting motif (UIM), a 14-3-3 binding sequence and an AKT phosphorylation site. RNF11 represents a unique protein with no other known immediate family members yet described. Comparative genetic analysis has shown that RNF11 is highly conserved throughout evolution. This may indicate a conserved and non-redundant role for the RNF11 protein. Molecular binding assays using RNF11 have shown that RNF11 has important roles in growth factor signalling, ubiquitination and transcriptional regulation. RNF11 has been shown to interact with HECT-type E3 ubiquitin ligases Nedd4, AIP4, Smurf1 and Smurf2, as well as with Cullin1, the core protein in the multi-subunit SCF E3 ubiquitin ligase complex. Work done in our laboratory has shown that RNF11 is capable of antagonizing Smurf2-mediated inhibition of TGFbeta signalling. Furthermore, RNF11 is capable of degrading AMSH, a positive regulator of both TGFbeta and EGFR signalling pathways. Recently, we have found that RNF11 can directly enhance TGFbeta signalling through a direct association with Smad4, the common signal transducer and transcription factor in the TGFbeta, BMP, and Activin pathways. Through its association with Smad4 and other transcription factors, RNF11 may have a role in direct transcriptional regulation. Our laboratory and others have found nearly 80 protein interactions for RNF11, placing RNF11 at the cross-roads of cell signalling and transcriptional regulation. RNF11 is highly expressed in breast tumours. Deregulation of RNF11 function may prove to be harmful to patient therapeutic outcomes. RNF11 may therefore provide a novel target for cancer therapeutics. The purpose of this review is to discuss the role of RNF11 in cell signalling and transcription factor modulation with special attention given to the ubiquitin-proteasomal pathway, TGFbeta pathway and EGFR pathway.

Silencing of OsGRX17 in rice improves drought stress tolerance by modulating ROS accumulation and stomatal closure

USDA-ARS?s Scientific Manuscript database

Glutaredoxins (GRXs) have emerged as key mediators in plant responses to environmental stimuli by modulating redox-dependent signaling pathways. Here, we report that RNAi-mediated suppression of the rice gene OsGRXS17, encoding a monothiol GRX with a CGFS-type active site motif, elevates H2O2 produc...

Regulatory elements involved in tax-mediated transactivation of the HTLV-I LTR.

PubMed

Seeler, J S; Muchardt, C; Podar, M; Gaynor, R B

1993-10-01

HTLV-I is the etiologic agent of adult T-cell leukemia. In this study, we investigated the regulatory elements and cellular transcription factors which function in modulating HTLV-I gene expression in response to the viral transactivator protein, tax. Transfection experiments into Jurkat cells of a variety of site-directed mutants in the HTLV-1 LTR indicated that each of the three motifs A, B, and C within the 21-bp repeats, the binding sites for the Ets family of proteins, and the TATA box all influenced the degree of tax-mediated activation. Tax is also able to activate gene expression of other viral and cellular promoters. Tax activation of the IL-2 receptor and the HIV-1 LTR is mediated through NF-kappa B motifs. Interestingly, sequences in the 21-bp repeat B and C motifs contain significant homology with NF-kappa B regulatory elements. We demonstrated that an NF-kappa B binding protein, PRDII-BF1, but not the rel protein, bound to the B and C motifs in the 21-bp repeat. PRDII-BF1 was also able to stimulate activation of HTLV-I gene expression by tax. The role of the Ets proteins on modulating tax activation was also studied. Ets 1 but not Ets 2 was capable of increasing the degree of tax activation of the HTLV-I LTR. These results suggest that tax activates gene expression by either direct or indirect interaction with several cellular transcription factors that bind to the HTLV-I LTR.

Structural and biochemical analysis of Bcl-2 interaction with the hepatitis B virus protein HBx.

PubMed

Jiang, Tianyu; Liu, Minhao; Wu, Jianping; Shi, Yigong

2016-02-23

HBx is a hepatitis B virus protein that is required for viral infectivity and replication. Anti-apoptotic Bcl-2 family members are thought to be among the important host targets of HBx. However, the structure and function of HBx are poorly understood and the molecular mechanism of HBx-induced carcinogenesis remains unknown. In this study, we report biochemical and structural characterization of HBx. The recombinant HBx protein contains metal ions, in particular iron and zinc. A BH3-like motif in HBx (residues 110-135) binds Bcl-2 with a dissociation constant of ∼193 μM, which is drastically lower than that for a canonical BH3 motif from Bim or Bad. Structural analysis reveals that, similar to other BH3 motifs, the BH3-like motif of HBx adopts an amphipathic α-helix and binds the conserved BH3-binding groove on Bcl-2. Unlike the helical Bim or Bad BH3 motif, the C-terminal portion of the bound HBx BH3-like motif has an extended conformation and makes considerably fewer interactions with Bcl-2. These observations suggest that HBx may modulate Bcl-2 function in a way that is different from that of the classical BH3-only proteins.

Self-assembled 1D infinite inorganic [2]catenane and 2D sheet framework with calix[8]phenylazoimidazole and [4+4]metallomacrocyclic motifs based on silver and ditopic bis(imidazolyl)methane ligands

NASA Astrophysics Data System (ADS)

Jin, Tianqi; Zhou, Junqiang; Pan, Yangyang; Huang, Yu; Jin, Chuanming

2018-05-01

Three novel supramolecular complexes, [Ag4(2-mBIM)4](ClO4)4(H2O) (1), [Ag2(2-mBIM)2](PF6)2 (2) and [Ag2(PA-BIM)2](ClO4)2(CH2Cl2) (3) (2-mBIM = bis(2-methyl- imidazol-1-yl)methane; PA-BIM = 1,1-bis[(2-phenylazo)imidazol-1-yl]methane), have been prepared and structurally characterized. The reported complexes bear [4+4]metallomacrocyclic motifs comprising four silver atoms and four ditopic bis(imidazolyl)methane ligands. Complex 1 exhibits a rare 1D infinite inorganic [2]catenane structure, which was self-assembled by the interlocking action of [4+4]metallomacrocyclic units. Complex 2 is a 2D layered supramolecular motif containing [4+4]macrometallacycle units with π-π interaction between imidazole rings. Complex 3 has a 2D sheet supramolecular framework through Ag-Ag interactions in [4+4]macrometallacyclic calix [8]phenylazoimidazole with a nanocavity. The results suggest that the bisimidazolium ligands and anions play crucial roles in the formation of the different host structures. The thermal stability and photoluminescence spectra of the synthesized complexes have also been discussed.

Redundant CArG Box Cis-motif Activity Mediates SHATTERPROOF2 Transcriptional Regulation during Arabidopsis thaliana Gynoecium Development

PubMed Central

Sehra, Bhupinder; Franks, Robert G.

2017-01-01

In the Arabidopsis thaliana seed pod, pod shatter and seed dispersal properties are in part determined by the development of a longitudinally orientated dehiscence zone (DZ) that derives from cells of the gynoecial valve margin (VM). Transcriptional regulation of the MADS protein encoding transcription factors genes SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2) are critical for proper VM identity specification and later on for DZ development. Current models of SHP1 and SHP2 regulation indicate that the transcription factors FRUITFULL (FUL) and REPLUMLESS (RPL) repress these SHP genes in the developing valve and replum domains, respectively. Thus the expression of the SHP genes is restricted to the VM. FUL encodes a MADS-box containing transcription factor that is predicted to act through CArG-box containing cis-regulatory motifs. Here we delimit functional modules within the SHP2 cis-regulatory region and examine the functional importance of CArG box motifs within these regulatory regions. We have characterized a 2.2kb region upstream of the SHP2 translation start site that drives early and late medial domain expression in the gynoecium, as well as expression within the VM and DZ. We identified two separable, independent cis-regulatory modules, a 1kb promoter region and a 700bp enhancer region, that are capable of giving VM and DZ expression. Our results argue for multiple independent cis-regulatory modules that support SHP2 expression during VM development and may contribute to the robustness of SHP2 expression in this tissue. Additionally, three closely positioned CArG box motifs located in the SHP2 upstream regulatory region were mutated in the context of the 2.2kb reporter construct. Mutating simultaneously all three CArG boxes caused a moderate de-repression of the SHP2 reporter that was detected within the valve domain, suggesting that these CArG boxes are involved in SHP2 repression in the valve. PMID:29085379

Subtle Changes in Motif Positioning Cause Tissue-Specific Effects on Robustness of an Enhancer's Activity

PubMed Central

Erceg, Jelena; Saunders, Timothy E.; Girardot, Charles; Devos, Damien P.; Hufnagel, Lars; Furlong, Eileen E. M.

2014-01-01

Deciphering the specific contribution of individual motifs within cis-regulatory modules (CRMs) is crucial to understanding how gene expression is regulated and how this process is affected by sequence variation. But despite vast improvements in the ability to identify where transcription factors (TFs) bind throughout the genome, we are limited in our ability to relate information on motif occupancy to function from sequence alone. Here, we engineered 63 synthetic CRMs to systematically assess the relationship between variation in the content and spacing of motifs within CRMs to CRM activity during development using Drosophila transgenic embryos. In over half the cases, very simple elements containing only one or two types of TF binding motifs were capable of driving specific spatio-temporal patterns during development. Different motif organizations provide different degrees of robustness to enhancer activity, ranging from binary on-off responses to more subtle effects including embryo-to-embryo and within-embryo variation. By quantifying the effects of subtle changes in motif organization, we were able to model biophysical rules that explain CRM behavior and may contribute to the spatial positioning of CRM activity in vivo. For the same enhancer, the effects of small differences in motif positions varied in developmentally related tissues, suggesting that gene expression may be more susceptible to sequence variation in one tissue compared to another. This result has important implications for human eQTL studies in which many associated mutations are found in cis-regulatory regions, though the mechanism for how they affect tissue-specific gene expression is often not understood. PMID:24391522

Process-based network decomposition reveals backbone motif structure

PubMed Central

Wang, Guanyu; Du, Chenghang; Chen, Hao; Simha, Rahul; Rong, Yongwu; Xiao, Yi; Zeng, Chen

2010-01-01

A central challenge in systems biology today is to understand the network of interactions among biomolecules and, especially, the organizing principles underlying such networks. Recent analysis of known networks has identified small motifs that occur ubiquitously, suggesting that larger networks might be constructed in the manner of electronic circuits by assembling groups of these smaller modules. Using a unique process-based approach to analyzing such networks, we show for two cell-cycle networks that each of these networks contains a giant backbone motif spanning all the network nodes that provides the main functional response. The backbone is in fact the smallest network capable of providing the desired functionality. Furthermore, the remaining edges in the network form smaller motifs whose role is to confer stability properties rather than provide function. The process-based approach used in the above analysis has additional benefits: It is scalable, analytic (resulting in a single analyzable expression that describes the behavior), and computationally efficient (all possible minimal networks for a biological process can be identified and enumerated). PMID:20498084

Matrix metalloproteinases and epidermal wound repair.

PubMed

Martins, Vera L; Caley, Matthew; O'Toole, Edel A

2013-02-01

Epidermal wound healing is a complex and highly coordinated process where several different cell types and molecules, such as growth factors and extracellular matrix (ECM) components, play an important role. Among the many proteins that are essential for the restoration of tissue integrity is the metalloproteinase (MMP) family. MMPs can act on ECM and non-ECM components affecting degradation and modulation of the ECM, growth-factor activation and cell-cell and cell-matrix signalling. MMPs are secreted by different cell types such as keratinocytes, fibroblasts and inflammatory cells at different stages and locations during wound healing, thereby regulating this process in a very coordinated and controlled way. In this article, we review the role of MMPs and their inhibitors (TIMPs), as well as the disintegrin and metalloproteinase with the thrombospondin motifs (ADAMs) family, in epithelial wound repair.

Multiple interactions drive adaptor-mediated recruitment of the ubiquitin ligase rsp5 to membrane proteins in vivo and in vitro.

PubMed

Sullivan, James A; Lewis, Michael J; Nikko, Elina; Pelham, Hugh R B

2007-07-01

Recognition of membrane proteins by the Nedd4/Rsp5 ubiquitin ligase family is a critical step in their targeting to the multivesicular body pathway. Some substrates contain "PY" motifs (PPxY), which bind to WW domains in the ligase. Others lack PY motifs and instead rely on adaptors that recruit the ligase to them. To investigate the mechanism of adaptor-mediated ubiquitination, we have characterized the interactions between the adaptor Bsd2, the ubiquitin ligase Rsp5, and the membrane proteins Cps1, Tre1, and Smf1 from Saccharomyces cerevisiae. We have reconstituted adaptor-mediated modification of Cps1 and Tre1 in vitro, and we show that two PY motifs in Bsd2 and two WW domains (WW2 and WW3) in Rsp5 are crucial for this. The binding of a weak noncanonical DMAPSY motif in Bsd2 to WW3 is an absolute requirement for Bsd2 adaptor function. We show that sorting of the manganese transporter Smf1, which requires both Bsd2 and Tre1, depends upon two PY motifs in Bsd2 and one motif in Tre1 but only two WW domains in Rsp5. We suggest that sequential assembly of first a Bsd2/Rsp5 complex, then a Tre1/Bsd2/Rsp5 complex followed by a rearrangement of PY-WW interactions is required for the ubiquitination of Smf1.

Multiple Interactions Drive Adaptor-Mediated Recruitment of the Ubiquitin Ligase Rsp5 to Membrane Proteins In Vivo and In Vitro

PubMed Central

Sullivan, James A.; Lewis, Michael J.; Nikko, Elina

2007-01-01

Recognition of membrane proteins by the Nedd4/Rsp5 ubiquitin ligase family is a critical step in their targeting to the multivesicular body pathway. Some substrates contain “PY” motifs (PPxY), which bind to WW domains in the ligase. Others lack PY motifs and instead rely on adaptors that recruit the ligase to them. To investigate the mechanism of adaptor-mediated ubiquitination, we have characterized the interactions between the adaptor Bsd2, the ubiquitin ligase Rsp5, and the membrane proteins Cps1, Tre1, and Smf1 from Saccharomyces cerevisiae. We have reconstituted adaptor-mediated modification of Cps1 and Tre1 in vitro, and we show that two PY motifs in Bsd2 and two WW domains (WW2 and WW3) in Rsp5 are crucial for this. The binding of a weak noncanonical DMAPSY motif in Bsd2 to WW3 is an absolute requirement for Bsd2 adaptor function. We show that sorting of the manganese transporter Smf1, which requires both Bsd2 and Tre1, depends upon two PY motifs in Bsd2 and one motif in Tre1 but only two WW domains in Rsp5. We suggest that sequential assembly of first a Bsd2/Rsp5 complex, then a Tre1/Bsd2/Rsp5 complex followed by a rearrangement of PY–WW interactions is required for the ubiquitination of Smf1. PMID:17429078

The Methionine-aromatic Motif Plays a Unique Role in Stabilizing Protein Structure*

PubMed Central

Valley, Christopher C.; Cembran, Alessandro; Perlmutter, Jason D.; Lewis, Andrew K.; Labello, Nicholas P.; Gao, Jiali; Sachs, Jonathan N.

2012-01-01

Of the 20 amino acids, the precise function of methionine (Met) remains among the least well understood. To establish a determining characteristic of methionine that fundamentally differentiates it from purely hydrophobic residues, we have used in vitro cellular experiments, molecular simulations, quantum calculations, and a bioinformatics screen of the Protein Data Bank. We show that approximately one-third of all known protein structures contain an energetically stabilizing Met-aromatic motif and, remarkably, that greater than 10,000 structures contain this motif more than 10 times. Critically, we show that as compared with a purely hydrophobic interaction, the Met-aromatic motif yields an additional stabilization of 1–1.5 kcal/mol. To highlight its importance and to dissect the energetic underpinnings of this motif, we have studied two clinically relevant TNF ligand-receptor complexes, namely TRAIL-DR5 and LTα-TNFR1. In both cases, we show that the motif is necessary for high affinity ligand binding as well as function. Additionally, we highlight previously overlooked instances of the motif in several disease-related Met mutations. Our results strongly suggest that the Met-aromatic motif should be exploited in the rational design of therapeutics targeting a range of proteins. PMID:22859300

An experimental test of a fundamental food web motif.

PubMed

Rip, Jason M K; McCann, Kevin S; Lynn, Denis H; Fawcett, Sonia

2010-06-07

Large-scale changes to the world's ecosystem are resulting in the deterioration of biostructure-the complex web of species interactions that make up ecological communities. A difficult, yet crucial task is to identify food web structures, or food web motifs, that are the building blocks of this baroque network of interactions. Once identified, these food web motifs can then be examined through experiments and theory to provide mechanistic explanations for how structure governs ecosystem stability. Here, we synthesize recent ecological research to show that generalist consumers coupling resources with different interaction strengths, is one such motif. This motif amazingly occurs across an enormous range of spatial scales, and so acts to distribute coupled weak and strong interactions throughout food webs. We then perform an experiment that illustrates the importance of this motif to ecological stability. We find that weak interactions coupled to strong interactions by generalist consumers dampen strong interaction strengths and increase community stability. This study takes a critical step by isolating a common food web motif and through clear, experimental manipulation, identifies the fundamental stabilizing consequences of this structure for ecological communities.

Motif formation and industry specific topologies in the Japanese business firm network

NASA Astrophysics Data System (ADS)

Maluck, Julian; Donner, Reik V.; Takayasu, Hideki; Takayasu, Misako

2017-05-01

Motifs and roles are basic quantities for the characterization of interactions among 3-node subsets in complex networks. In this work, we investigate how the distribution of 3-node motifs can be influenced by modifying the rules of an evolving network model while keeping the statistics of simpler network characteristics, such as the link density and the degree distribution, invariant. We exemplify this problem for the special case of the Japanese Business Firm Network, where a well-studied and relatively simple yet realistic evolving network model is available, and compare the resulting motif distribution in the real-world and simulated networks. To better approximate the motif distribution of the real-world network in the model, we introduce both subgraph dependent and global additional rules. We find that a specific rule that allows only for the merging process between nodes with similar link directionality patterns reduces the observed excess of densely connected motifs with bidirectional links. Our study improves the mechanistic understanding of motif formation in evolving network models to better describe the characteristic features of real-world networks with a scale-free topology.

Motif discovery with data mining in 3D protein structure databases: discovery, validation and prediction of the U-shape zinc binding ("Huf-Zinc") motif.

PubMed

Maurer-Stroh, Sebastian; Gao, He; Han, Hao; Baeten, Lies; Schymkowitz, Joost; Rousseau, Frederic; Zhang, Louxin; Eisenhaber, Frank

2013-02-01

Data mining in protein databases, derivatives from more fundamental protein 3D structure and sequence databases, has considerable unearthed potential for the discovery of sequence motif--structural motif--function relationships as the finding of the U-shape (Huf-Zinc) motif, originally a small student's project, exemplifies. The metal ion zinc is critically involved in universal biological processes, ranging from protein-DNA complexes and transcription regulation to enzymatic catalysis and metabolic pathways. Proteins have evolved a series of motifs to specifically recognize and bind zinc ions. Many of these, so called zinc fingers, are structurally independent globular domains with discontinuous binding motifs made up of residues mostly far apart in sequence. Through a systematic approach starting from the BRIX structure fragment database, we discovered that there exists another predictable subset of zinc-binding motifs that not only have a conserved continuous sequence pattern but also share a characteristic local conformation, despite being included in totally different overall folds. While this does not allow general prediction of all Zn binding motifs, a HMM-based web server, Huf-Zinc, is available for prediction of these novel, as well as conventional, zinc finger motifs in protein sequences. The Huf-Zinc webserver can be freely accessed through this URL (http://mendel.bii.a-star.edu.sg/METHODS/hufzinc/).

Genome-wide colonization of gene regulatory elements by G4 DNA motifs

PubMed Central

Du, Zhuo; Zhao, Yiqiang; Li, Ning

2009-01-01

G-quadruplex (or G4 DNA), a stable four-stranded structure found in guanine-rich regions, is implicated in the transcriptional regulation of genes involved in growth and development. Previous studies on the role of G4 DNA in gene regulation mostly focused on genomic regions proximal to transcription start sites (TSSs). To gain a more comprehensive understanding of the regulatory role of G4 DNA, we examined the landscape of potential G4 DNA (PG4Ms) motifs in the human genome and found that G4 motifs, not restricted to those found in the TSS-proximal regions, are bias toward gene-associated regions. Significantly, analyses of G4 motifs in seven types of well-known gene regulatory elements revealed a constitutive enrichment pattern and the clusters of G4 motifs tend to be colocalized with regulatory elements. Considering our analysis from a genome evolutionary perspective, we found evidence that the occurrence and accumulation of certain progenitors and canonical G4 DNA motifs within regulatory regions were progressively favored by natural selection. Our results suggest that G4 DNA motifs are ‘colonized’ in regulatory regions, supporting a likely genome-wide role of G4 DNA in gene regulation. We hypothesize that G4 DNA is a regulatory apparatus situated in regulatory elements, acting as a molecular switch that can modulate the role of the host functional regions, by transition in DNA structure. PMID:19759215

Stem/Progenitor Cell Proteoglycans Decorated with 7-D-4, 4-C-3 and 3-B-3(-) Chondroitin Sulphate Motifs Are Morphogenetic Markers Of Tissue Development.

PubMed

Hayes, Anthony J; Smith, Susan M; Caterson, Bruce; Melrose, James

2018-06-11

This study reviewed the occurrence of chondroitin sulphate (CS) motifs 4-C-3, 7-D-4 and 3-B-3(-) which are expressed by progenitor cells in tissues undergoing morphogenesis. These motifs have a transient early expression pattern during tissue development and also appear in mature tissues during pathological remodeling and attempted repair processes by activated adult stem cells. The CS motifs are information and recognition modules, which may regulate cellular behavior and delineate stem cell niches in developmental tissues. One of the difficulties in determining the precise role of stem cells in tissue development and repair processes is their short engraftment period and the lack of specific markers, which differentiate the activated stem cell lineages from the resident cells. The CS sulphation motifs 7-D-4, 4-C-3 and 3-B-3 (-) decorate cell surface proteoglycans on activated stem/progenitor cells and appear to identify these cells in transitional areas of tissue development and in tissue repair and may be applicable to determining a more precise role for stem cells in tissue morphogenesis. This article is protected by copyright. All rights reserved. © 2018 AlphaMed Press.

Regulation of polycystin-1 ciliary trafficking by motifs at its C-terminus and polycystin-2 but not by cleavage at the GPS site

PubMed Central

Su, Xuefeng; Wu, Maoqing; Yao, Gang; El-Jouni, Wassim; Luo, Chong; Tabari, Azadeh; Zhou, Jing

2015-01-01

ABSTRACT Failure to localize membrane proteins to the primary cilium causes a group of diseases collectively named ciliopathies. Polycystin-1 (PC1, also known as PKD1) is a large ciliary membrane protein defective in autosomal dominant polycystic kidney disease (ADPKD). Here, we developed a large set of PC1 expression constructs and identified multiple sequences, including a coiled-coil motif in the C-terminal tail of PC1, regulating full-length PC1 trafficking to the primary cilium. Ciliary trafficking of wild-type and mutant PC1 depends on the dose of polycystin-2 (PC2, also known as PKD2), and the formation of a PC1–PC2 complex. Modulation of the ciliary trafficking module mediated by the VxP ciliary-targeting sequence and Arf4 and Asap1 does not affect the ciliary localization of full-length PC1. PC1 also promotes PC2 ciliary trafficking. PC2 mutations truncating its C-terminal tail but not those changing the VxP sequence to AxA or impairing the pore of the channel, leading to a dead channel, affect PC1 ciliary trafficking. Cleavage at the GPCR proteolytic site (GPS) of PC1 is not required for PC1 trafficking to cilia. We propose a mutually dependent model for the ciliary trafficking of PC1 and PC2, and that PC1 ciliary trafficking is regulated by multiple cis-acting elements. As all pathogenic PC1 mutations tested here are defective in ciliary trafficking, ciliary trafficking might serve as a functional read-out for ADPKD. PMID:26430213

Regulation of polycystin-1 ciliary trafficking by motifs at its C-terminus and polycystin-2 but not by cleavage at the GPS site.

PubMed

Su, Xuefeng; Wu, Maoqing; Yao, Gang; El-Jouni, Wassim; Luo, Chong; Tabari, Azadeh; Zhou, Jing

2015-11-15

Failure to localize membrane proteins to the primary cilium causes a group of diseases collectively named ciliopathies. Polycystin-1 (PC1, also known as PKD1) is a large ciliary membrane protein defective in autosomal dominant polycystic kidney disease (ADPKD). Here, we developed a large set of PC1 expression constructs and identified multiple sequences, including a coiled-coil motif in the C-terminal tail of PC1, regulating full-length PC1 trafficking to the primary cilium. Ciliary trafficking of wild-type and mutant PC1 depends on the dose of polycystin-2 (PC2, also known as PKD2), and the formation of a PC1-PC2 complex. Modulation of the ciliary trafficking module mediated by the VxP ciliary-targeting sequence and Arf4 and Asap1 does not affect the ciliary localization of full-length PC1. PC1 also promotes PC2 ciliary trafficking. PC2 mutations truncating its C-terminal tail but not those changing the VxP sequence to AxA or impairing the pore of the channel, leading to a dead channel, affect PC1 ciliary trafficking. Cleavage at the GPCR proteolytic site (GPS) of PC1 is not required for PC1 trafficking to cilia. We propose a mutually dependent model for the ciliary trafficking of PC1 and PC2, and that PC1 ciliary trafficking is regulated by multiple cis-acting elements. As all pathogenic PC1 mutations tested here are defective in ciliary trafficking, ciliary trafficking might serve as a functional read-out for ADPKD. © 2015. Published by The Company of Biologists Ltd.

MAPK14/p38α-dependent modulation of glucose metabolism affects ROS levels and autophagy during starvation.

PubMed

Desideri, Enrico; Vegliante, Rolando; Cardaci, Simone; Nepravishta, Ridvan; Paci, Maurizio; Ciriolo, Maria Rosa

2014-09-01

Increased glycolytic flux is a common feature of many cancer cells, which have adapted their metabolism to maximize glucose incorporation and catabolism to generate ATP and substrates for biosynthetic reactions. Indeed, glycolysis allows a rapid production of ATP and provides metabolic intermediates required for cancer cells growth. Moreover, it makes cancer cells less sensitive to fluctuations of oxygen tension, a condition usually occurring in a newly established tumor environment. Here, we provide evidence for a dual role of MAPK14 in driving a rearrangement of glucose metabolism that contributes to limiting reactive oxygen species (ROS) production and autophagy activation in condition of nutrient deprivation. We demonstrate that MAPK14 is phosphoactivated during nutrient deprivation and affects glucose metabolism at 2 different levels: on the one hand, it increases SLC2A3 mRNA and protein levels, resulting in a higher incorporation of glucose within the cell. This event involves the MAPK14-mediated enhancement of HIF1A protein stability. On the other hand, MAPK14 mediates a metabolic shift from glycolysis to the pentose phosphate pathway (PPP) through the modulation of PFKFB3 (6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase 3) degradation by the proteasome. This event requires the presence of 2 distinct degradation sequences, KEN box and DSG motif Ser273, which are recognized by 2 different E3 ligase complexes. The mutation of either motif increases PFKFB3 resistance to starvation-induced degradation. The MAPK14-driven metabolic reprogramming sustains the production of NADPH, an important cofactor for many reduction reactions and for the maintenance of the proper intracellular redox environment, resulting in reduced levels of ROS. The final effect is a reduced activation of autophagy and an increased resistance to nutrient deprivation.

A systematic analysis of a mi-RNA inter-pathway regulatory motif

PubMed Central

2013-01-01

Background The continuing discovery of new types and functions of small non-coding RNAs is suggesting the presence of regulatory mechanisms far more complex than the ones currently used to study and design Gene Regulatory Networks. Just focusing on the roles of micro RNAs (miRNAs), they have been found to be part of several intra-pathway regulatory motifs. However, inter-pathway regulatory mechanisms have been often neglected and require further investigation. Results In this paper we present the result of a systems biology study aimed at analyzing a high-level inter-pathway regulatory motif called Pathway Protection Loop, not previously described, in which miRNAs seem to play a crucial role in the successful behavior and activation of a pathway. Through the automatic analysis of a large set of public available databases, we found statistical evidence that this inter-pathway regulatory motif is very common in several classes of KEGG Homo Sapiens pathways and concurs in creating a complex regulatory network involving several pathways connected by this specific motif. The role of this motif seems also confirmed by a deeper review of other research activities on selected representative pathways. Conclusions Although previous studies suggested transcriptional regulation mechanism at the pathway level such as the Pathway Protection Loop, a high-level analysis like the one proposed in this paper is still missing. The understanding of higher-level regulatory motifs could, as instance, lead to new approaches in the identification of therapeutic targets because it could unveil new and “indirect” paths to activate or silence a target pathway. However, a lot of work still needs to be done to better uncover this high-level inter-pathway regulation including enlarging the analysis to other small non-coding RNA molecules. PMID:24152805

Form and function in gene regulatory networks: the structure of network motifs determines fundamental properties of their dynamical state space.

PubMed

Ahnert, S E; Fink, T M A

2016-07-01

Network motifs have been studied extensively over the past decade, and certain motifs, such as the feed-forward loop, play an important role in regulatory networks. Recent studies have used Boolean network motifs to explore the link between form and function in gene regulatory networks and have found that the structure of a motif does not strongly determine its function, if this is defined in terms of the gene expression patterns the motif can produce. Here, we offer a different, higher-level definition of the 'function' of a motif, in terms of two fundamental properties of its dynamical state space as a Boolean network. One is the basin entropy, which is a complexity measure of the dynamics of Boolean networks. The other is the diversity of cyclic attractor lengths that a given motif can produce. Using these two measures, we examine all 104 topologically distinct three-node motifs and show that the structural properties of a motif, such as the presence of feedback loops and feed-forward loops, predict fundamental characteristics of its dynamical state space, which in turn determine aspects of its functional versatility. We also show that these higher-level properties have a direct bearing on real regulatory networks, as both basin entropy and cycle length diversity show a close correspondence with the prevalence, in neural and genetic regulatory networks, of the 13 connected motifs without self-interactions that have been studied extensively in the literature. © 2016 The Authors.

Phosphatidylinositol-4-kinase type II alpha contains an AP-3-sorting motif and a kinase domain that are both required for endosome traffic.

PubMed

Craige, Branch; Salazar, Gloria; Faundez, Victor

2008-04-01

The adaptor complex 3 (AP-3) targets membrane proteins from endosomes to lysosomes, lysosome-related organelles and synaptic vesicles. Phosphatidylinositol-4-kinase type II alpha (PI4KIIalpha) is one of several proteins possessing catalytic domains that regulate AP-3-dependent sorting. Here we present evidence that PI4KIIalpha uniquely behaves both as a membrane protein cargo as well as an enzymatic regulator of adaptor function. In fact, AP-3 and PI4KIIalpha form a complex that requires a dileucine-sorting motif present in PI4KIIalpha. Mutagenesis of either the PI4KIIalpha-sorting motif or its kinase-active site indicates that both are necessary to interact with AP-3 and properly localize PI4KIIalpha to LAMP-1-positive endosomes. Similarly, both the kinase activity and the sorting signal present in PI4KIIalpha are necessary to rescue endosomal PI4KIIalpha siRNA-induced mutant phenotypes. We propose a mechanism whereby adaptors use canonical sorting motifs to selectively recruit a regulatory enzymatic activity to restricted membrane domains.

Binding Modes of Teixobactin to Lipid II: Molecular Dynamics Study.

PubMed

Liu, Yang; Liu, Yaxin; Chan-Park, Mary B; Mu, Yuguang

2017-12-08

Teixobactin (TXB) is a newly discovered antibiotic targeting the bacterial cell wall precursor Lipid II (L II ). In the present work, four binding modes of TXB on L II were identified by a contact-map based clustering method. The highly flexible binary complex ensemble was generated by parallel tempering metadynamics simulation in a well-tempered ensemble (PTMetaD-WTE). In agreement with experimental findings, the pyrophosphate group and the attached first sugar subunit of L II are found to be the minimal motif for stable TXB binding. Three of the four binding modes involve the ring structure of TXB and have relatively higher binding affinities, indicating the importance of the ring motif of TXB in L II recognition. TXB-L II complexes with a ratio of 2:1 are also predicted with configurations such that the ring motif of two TXB molecules bound to the pyrophosphate-MurNAc moiety and the glutamic acid residue of one L II , respectively. Our findings disclose that the ring motif of TXB is critical to L II binding and novel antibiotics can be designed based on its mimetics.

Analysis of an artificial zinc finger epigenetic modulator: widespread binding but limited regulation

PubMed Central

Grimmer, Matthew R.; Stolzenburg, Sabine; Ford, Ethan; Lister, Ryan; Blancafort, Pilar; Farnham, Peggy J.

2014-01-01

Artificial transcription factors (ATFs) and genomic nucleases based on a DNA binding platform consisting of multiple zinc finger domains are currently being developed for clinical applications. However, no genome-wide investigations into their binding specificity have been performed. We have created six-finger ATFs to target two different 18 nt regions of the human SOX2 promoter; each ATF is constructed such that it contains or lacks a super KRAB domain (SKD) that interacts with a complex containing repressive histone methyltransferases. ChIP-seq analysis of the effector-free ATFs in MCF7 breast cancer cells identified thousands of binding sites, mostly in promoter regions; the addition of an SKD domain increased the number of binding sites ∼5-fold, with a majority of the new sites located outside of promoters. De novo motif analyses suggest that the lack of binding specificity is due to subsets of the finger domains being used for genomic interactions. Although the ATFs display widespread binding, few genes showed expression differences; genes repressed by the ATF-SKD have stronger binding sites and are more enriched for a 12 nt motif. Interestingly, epigenetic analyses indicate that the transcriptional repression caused by the ATF-SKD is not due to changes in active histone modifications. PMID:25122745

Molecular basis for the wide range of affinity found in Csr/Rsm protein-RNA recognition.

PubMed

Duss, Olivier; Michel, Erich; Diarra dit Konté, Nana; Schubert, Mario; Allain, Frédéric H-T

2014-04-01

The carbon storage regulator/regulator of secondary metabolism (Csr/Rsm) type of small non-coding RNAs (sRNAs) is widespread throughout bacteria and acts by sequestering the global translation repressor protein CsrA/RsmE from the ribosome binding site of a subset of mRNAs. Although we have previously described the molecular basis of a high affinity RNA target bound to RsmE, it remains unknown how other lower affinity targets are recognized by the same protein. Here, we have determined the nuclear magnetic resonance solution structures of five separate GGA binding motifs of the sRNA RsmZ of Pseudomonas fluorescens in complex with RsmE. The structures explain how the variation of sequence and structural context of the GGA binding motifs modulate the binding affinity for RsmE by five orders of magnitude (∼10 nM to ∼3 mM, Kd). Furthermore, we see that conformational adaptation of protein side-chains and RNA enable recognition of different RNA sequences by the same protein contributing to binding affinity without conferring specificity. Overall, our findings illustrate how the variability in the Csr/Rsm protein-RNA recognition allows a fine-tuning of the competition between mRNAs and sRNAs for the CsrA/RsmE protein.

Bio-mimicking of Proline-Rich Motif Applied to Carbon Nanotube Reveals Unexpected Subtleties Underlying Nanoparticle Functionalization

NASA Astrophysics Data System (ADS)

Zhang, Yuanzhao; Jimenez-Cruz, Camilo A.; Wang, Jian; Zhou, Bo; Yang, Zaixing; Zhou, Ruhong

2014-11-01

Here, we report computational studies of the SH3 protein domain interacting with various single-walled carbon nanotubes (SWCNT) either bare or functionalized by mimicking the proline-rich motif (PRM) ligand (PPPVPPRR) and compare it to the SH3-PRM complex binding. With prolines or a single arginine attached, the SWCNT gained slightly on specificity when compared with the bare control, whereas with multi-arginine systems the specificity dropped dramatically to our surprise. Although the electrostatic interaction provided by arginines is crucial in the recognition between PRM and SH3 domain, our results suggest that attaching multiple arginines to the SWCNT has a detrimental effect on the binding affinity. Detailed analysis of the MD trajectories found two main factors that modulate the specificity of the binding: the existence of competing acidic patches at the surface of SH3 that leads to ``trapping and clamping'' by the arginines, and the rigidity of the SWCNT introducing entropic penalties in the proper binding. Further investigation revealed that the same ``clamping'' phenomenon exits in the PRM-SH3 system, which has not been reported in previous literature. The competing effects between nanoparticle and its functionalization components revealed by our model system should be of value to current and future nanomedicine designs.

Bio-mimicking of Proline-Rich Motif Applied to Carbon Nanotube Reveals Unexpected Subtleties Underlying Nanoparticle Functionalization

PubMed Central

Zhang, Yuanzhao; Jimenez-Cruz, Camilo A.; Wang, Jian; Zhou, Bo; Yang, Zaixing; Zhou, Ruhong

2014-01-01

Here, we report computational studies of the SH3 protein domain interacting with various single-walled carbon nanotubes (SWCNT) either bare or functionalized by mimicking the proline-rich motif (PRM) ligand (PPPVPPRR) and compare it to the SH3-PRM complex binding. With prolines or a single arginine attached, the SWCNT gained slightly on specificity when compared with the bare control, whereas with multi-arginine systems the specificity dropped dramatically to our surprise. Although the electrostatic interaction provided by arginines is crucial in the recognition between PRM and SH3 domain, our results suggest that attaching multiple arginines to the SWCNT has a detrimental effect on the binding affinity. Detailed analysis of the MD trajectories found two main factors that modulate the specificity of the binding: the existence of competing acidic patches at the surface of SH3 that leads to “trapping and clamping” by the arginines, and the rigidity of the SWCNT introducing entropic penalties in the proper binding. Further investigation revealed that the same “clamping” phenomenon exits in the PRM-SH3 system, which has not been reported in previous literature. The competing effects between nanoparticle and its functionalization components revealed by our model system should be of value to current and future nanomedicine designs. PMID:25427563

Novel structural features drive DNA binding properties of Cmr, a CRP family protein in TB complex mycobacteria.

PubMed

Ranganathan, Sridevi; Cheung, Jonah; Cassidy, Michael; Ginter, Christopher; Pata, Janice D; McDonough, Kathleen A

2018-01-09

Mycobacterium tuberculosis (Mtb) encodes two CRP/FNR family transcription factors (TF) that contribute to virulence, Cmr (Rv1675c) and CRPMt (Rv3676). Prior studies identified distinct chromosomal binding profiles for each TF despite their recognizing overlapping DNA motifs. The present study shows that Cmr binding specificity is determined by discriminator nucleotides at motif positions 4 and 13. X-ray crystallography and targeted mutational analyses identified an arginine-rich loop that expands Cmr's DNA interactions beyond the classical helix-turn-helix contacts common to all CRP/FNR family members and facilitates binding to imperfect DNA sequences. Cmr binding to DNA results in a pronounced asymmetric bending of the DNA and its high level of cooperativity is consistent with DNA-facilitated dimerization. A unique N-terminal extension inserts between the DNA binding and dimerization domains, partially occluding the site where the canonical cAMP binding pocket is found. However, an unstructured region of this N-terminus may help modulate Cmr activity in response to cellular signals. Cmr's multiple levels of DNA interaction likely enhance its ability to integrate diverse gene regulatory signals, while its novel structural features establish Cmr as an atypical CRP/FNR family member. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

The Crystal Structure of a Cardiovirus RNA-Dependent RNA Polymerase Reveals an Unusual Conformation of the Polymerase Active Site

PubMed Central

Vives-Adrian, Laia; Lujan, Celia; Oliva, Baldo; van der Linden, Lonneke; Selisko, Barbara; Coutard, Bruno; Canard, Bruno; van Kuppeveld, Frank J. M.

2014-01-01

ABSTRACT Encephalomyocarditis virus (EMCV) is a member of the Cardiovirus genus within the large Picornaviridae family, which includes a number of important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for viral genome replication. In this study, we report the X-ray structures of two different crystal forms of the EMCV RdRp determined at 2.8- and 2.15-Å resolution. The in vitro elongation and VPg uridylylation activities of the purified enzyme have also been demonstrated. Although the overall structure of EMCV 3Dpol is shown to be similar to that of the known RdRps of other members of the Picornaviridae family, structural comparisons show a large reorganization of the active-site cavity in one of the crystal forms. The rearrangement affects mainly motif A, where the conserved residue Asp240, involved in ribonucleoside triphosphate (rNTP) selection, and its neighbor residue, Phe239, move about 10 Å from their expected positions within the ribose binding pocket toward the entrance of the rNTP tunnel. This altered conformation of motif A is stabilized by a cation-π interaction established between the aromatic ring of Phe239 and the side chain of Lys56 within the finger domain. Other contacts, involving Phe239 and different residues of motif F, are also observed. The movement of motif A is connected with important conformational changes in the finger region flanked by residues 54 to 63, harboring Lys56, and in the polymerase N terminus. The structures determined in this work provide essential information for studies on the cardiovirus RNA replication process and may have important implications for the development of new antivirals targeting the altered conformation of motif A. IMPORTANCE The Picornaviridae family is one of the largest virus families known, including many important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for picornavirus genome replication and a validated target for the development of antiviral therapies. Solving the X-ray structure of the first cardiovirus RdRp, EMCV 3Dpol, we captured an altered conformation of a conserved motif in the polymerase active site (motif A) containing the aspartic acid residue involved in rNTP selection and binding. This altered conformation of motif A, which interferes with the correct positioning of the rNTP substrate in the active site, is stabilized by a number of residues strictly conserved among picornaviruses. The rearrangements observed suggest that this motif A segment is a dynamic element that can be modulated by external effectors, either activating or inhibiting enzyme activity, and this type of modulation appears to be general to all picornaviruses. PMID:24600002

The crystal structure of a cardiovirus RNA-dependent RNA polymerase reveals an unusual conformation of the polymerase active site.

PubMed

Vives-Adrian, Laia; Lujan, Celia; Oliva, Baldo; van der Linden, Lonneke; Selisko, Barbara; Coutard, Bruno; Canard, Bruno; van Kuppeveld, Frank J M; Ferrer-Orta, Cristina; Verdaguer, Núria

2014-05-01

Encephalomyocarditis virus (EMCV) is a member of the Cardiovirus genus within the large Picornaviridae family, which includes a number of important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for viral genome replication. In this study, we report the X-ray structures of two different crystal forms of the EMCV RdRp determined at 2.8- and 2.15-Å resolution. The in vitro elongation and VPg uridylylation activities of the purified enzyme have also been demonstrated. Although the overall structure of EMCV 3Dpol is shown to be similar to that of the known RdRps of other members of the Picornaviridae family, structural comparisons show a large reorganization of the active-site cavity in one of the crystal forms. The rearrangement affects mainly motif A, where the conserved residue Asp240, involved in ribonucleoside triphosphate (rNTP) selection, and its neighbor residue, Phe239, move about 10 Å from their expected positions within the ribose binding pocket toward the entrance of the rNTP tunnel. This altered conformation of motif A is stabilized by a cation-π interaction established between the aromatic ring of Phe239 and the side chain of Lys56 within the finger domain. Other contacts, involving Phe239 and different residues of motif F, are also observed. The movement of motif A is connected with important conformational changes in the finger region flanked by residues 54 to 63, harboring Lys56, and in the polymerase N terminus. The structures determined in this work provide essential information for studies on the cardiovirus RNA replication process and may have important implications for the development of new antivirals targeting the altered conformation of motif A. The Picornaviridae family is one of the largest virus families known, including many important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for picornavirus genome replication and a validated target for the development of antiviral therapies. Solving the X-ray structure of the first cardiovirus RdRp, EMCV 3Dpol, we captured an altered conformation of a conserved motif in the polymerase active site (motif A) containing the aspartic acid residue involved in rNTP selection and binding. This altered conformation of motif A, which interferes with the correct positioning of the rNTP substrate in the active site, is stabilized by a number of residues strictly conserved among picornaviruses. The rearrangements observed suggest that this motif A segment is a dynamic element that can be modulated by external effectors, either activating or inhibiting enzyme activity, and this type of modulation appears to be general to all picornaviruses.

Discovering Sequence Motifs with Arbitrary Insertions and Deletions

PubMed Central

Frith, Martin C.; Saunders, Neil F. W.; Kobe, Bostjan; Bailey, Timothy L.

2008-01-01

Biology is encoded in molecular sequences: deciphering this encoding remains a grand scientific challenge. Functional regions of DNA, RNA, and protein sequences often exhibit characteristic but subtle motifs; thus, computational discovery of motifs in sequences is a fundamental and much-studied problem. However, most current algorithms do not allow for insertions or deletions (indels) within motifs, and the few that do have other limitations. We present a method, GLAM2 (Gapped Local Alignment of Motifs), for discovering motifs allowing indels in a fully general manner, and a companion method GLAM2SCAN for searching sequence databases using such motifs. glam2 is a generalization of the gapless Gibbs sampling algorithm. It re-discovers variable-width protein motifs from the PROSITE database significantly more accurately than the alternative methods PRATT and SAM-T2K. Furthermore, it usefully refines protein motifs from the ELM database: in some cases, the refined motifs make orders of magnitude fewer overpredictions than the original ELM regular expressions. GLAM2 performs respectably on the BAliBASE multiple alignment benchmark, and may be superior to leading multiple alignment methods for “motif-like” alignments with N- and C-terminal extensions. Finally, we demonstrate the use of GLAM2 to discover protein kinase substrate motifs and a gapped DNA motif for the LIM-only transcriptional regulatory complex: using GLAM2SCAN, we identify promising targets for the latter. GLAM2 is especially promising for short protein motifs, and it should improve our ability to identify the protein cleavage sites, interaction sites, post-translational modification attachment sites, etc., that underlie much of biology. It may be equally useful for arbitrarily gapped motifs in DNA and RNA, although fewer examples of such motifs are known at present. GLAM2 is public domain software, available for download at http://bioinformatics.org.au/glam2. PMID:18437229

Phosphorylation of PPP(S/T)P motif of the free LRP6 intracellular domain is not required to activate the Wnt/beta-catenin pathway and attenuate GSK3beta activity.

PubMed

Beagle, Brandon; Mi, Kaihong; Johnson, Gail V W

2009-11-01

The canonical Wnt/beta-catenin signaling pathway plays a critical role in numerous physiological and pathological processes. LRP6 is an essential co-receptor for Wnt/beta-catenin signaling; as transduction of the Wnt signal is strongly dependent upon GSK3beta-mediated phosphorylation of multiple PPP(S/T)P motifs within the membrane-anchored LRP6 intracellular domain. Previously, we showed that the free LRP6 intracellular domain (LRP6-ICD) can activate the Wnt/beta-catenin pathway in a beta-catenin and TCF/LEF-1 dependent manner, as well as interact with and attenuate GSK3beta activity. However, it is unknown if the ability of LRP6-ICD to attenuate GSK3beta activity and modulate activation of the Wnt/beta-catenin pathway requires phosphorylation of the LRP6-ICD PPP(S/T)P motifs, in a manner similar to the membrane-anchored LRP6 intracellular domain. Here we provide evidence that the LRP6-ICD does not have to be phosphorylated at its PPP(S/T)P motif by GSK3beta to stabilize endogenous cytosolic beta-catenin resulting in activation of TCF/LEF-1 and the Wnt/beta-catenin pathway. LRP6-ICD and a mutant in which all 5 PPP(S/T)P motifs were changed to PPP(A)P motifs equivalently interacted with and attenuated GSK3beta activity in vitro, and both constructs inhibited the in situ GSK3beta-mediated phosphorylation of beta-catenin and tau to the same extent. These data indicate that the LRP6-ICD attenuates GSK3beta activity similar to other GSK3beta binding proteins, and is not a result of it being a GSK3beta substrate. Our findings suggest the functional and regulatory mechanisms governing the free LRP6-ICD may be distinct from membrane-anchored LRP6, and that release of the LRP6-ICD may provide a complimentary signaling cascade capable of modulating Wnt-dependent gene expression. (c) 2009 Wiley-Liss, Inc.

3D extracellular matrix interactions modulate tumour cell growth, invasion and angiogenesis in engineered tumour microenvironments.

PubMed

Taubenberger, Anna V; Bray, Laura J; Haller, Barbara; Shaposhnykov, Artem; Binner, Marcus; Freudenberg, Uwe; Guck, Jochen; Werner, Carsten

2016-05-01

Interactions between tumour cells and extracellular matrix proteins of the tumour microenvironment play crucial roles in cancer progression. So far, however, there are only a few experimental platforms available that allow us to study these interactions systematically in a mechanically defined three-dimensional (3D) context. Here, we have studied the effect of integrin binding motifs found within common extracellular matrix (ECM) proteins on 3D breast (MCF-7) and prostate (PC-3, LNCaP) cancer cell cultures, and co-cultures with endothelial and mesenchymal stromal cells. For this purpose, matrix metalloproteinase-degradable biohybrid poly(ethylene) glycol-heparin hydrogels were decorated with the peptide motifs RGD, GFOGER (collagen I), or IKVAV (laminin-111). Over 14days, cancer spheroids of 100-200μm formed. While the morphology of poorly invasive MCF-7 and LNCaP cells was not modulated by any of the peptide motifs, the aggressive PC-3 cells exhibited an invasive morphology when cultured in hydrogels comprising IKVAV and GFOGER motifs compared to RGD motifs or nonfunctionalised controls. PC-3 (but not MCF-7 and LNCaP) cell growth and endothelial cell infiltration were also significantly enhanced in IKVAV and GFOGER presenting gels. Taken together, we have established a 3D culture model that allows for dissecting the effect of biochemical cues on processes relevant to early cancer progression. These findings provide a basis for more mechanistic studies that may further advance our understanding of how ECM modulates cancer cell invasion and how to ultimately interfere with this process. Threedimensional in vitro cancer models have generated great interest over the past decade. However, most models are not suitable to systematically study the effects of environmental cues on cancer development and progression. To overcome this limitation, we have developed an innovative hydrogel platform to study the interactions between breast and prostate cancer cells and extracellular matrix ligands relevant to the tumour microenvironment. Our results show that hydrogels with laminin- and collagen-derived adhesive peptides induce a malignant phenotype in a cell-line specific manner. Thus, we have identified a method to control the incorporation of biochemical cues within a three dimensional culture model and anticipate that it will help us in better understanding the effects of the tumour microenvironment on cancer progression. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Unexpected Heterodivalent Recruitment of NOS1AP to nNOS Reveals Multiple Sites for Pharmacological Intervention in Neuronal Disease Models.

PubMed

Li, Li-Li; Melero-Fernandez de Mera, Raquel M; Chen, Jia; Ba, Wei; Kasri, Nael Nadif; Zhang, Mingjie; Courtney, Michael J

2015-05-13

The protein NOS1AP/CAPON mediates signaling from a protein complex of NMDA receptor, PSD95 and nNOS. The only stroke trial for neuroprotectants that showed benefit to patients targeted this ternary complex. NOS1AP/nNOS interaction regulates small GTPases, iron transport, p38MAPK-linked excitotoxicity, and anxiety. Moreover, the nos1ap gene is linked to disorders from schizophrenia, post-traumatic stress disorder, and autism to cardiovascular disorders and breast cancer. Understanding protein interactions required for NOS1AP function, therefore, has broad implications for numerous diseases. Here we show that the interaction of NOS1AP with nNOS differs radically from the classical PDZ docking assumed to be responsible. The NOS1AP PDZ motif does not bind nNOS as measured by multiple methods. In contrast, full-length NOS1AP forms an unusually stable interaction with nNOS. We mapped the discrepancy between full-length and C-terminal PDZ motif to a novel internal region we call the ExF motif. The C-terminal PDZ motif, although neither sufficient nor necessary for binding, nevertheless promotes the stability of the complex. It therefore potentially affects signal transduction and suggests that functional interaction of nNOS with NOS1AP might be targetable at two distinct sites. We demonstrate that excitotoxic pathways can be regulated, in cortical neuron and organotypic hippocampal slice cultures from rat, either by the previously described PDZ ligand TAT-GESV or by the ExF motif-bearing region of NOS1AP, even when lacking the critical PDZ residues as long as the ExF motif is intact and not mutated. This previously unrecognized heterodivalent interaction of nNOS with NOS1AP may therefore provide distinct opportunities for pharmacological intervention in NOS1AP-dependent signaling and excitotoxicity. Copyright © 2015 the authors 0270-6474/15/357349-16$15.00/0.

Identification and characterization of a calmodulin binding domain in the plasma membrane Ca2+-ATPase from Trypanosoma equiperdum.

PubMed

Ramírez-Iglesias, José Rubén; Pérez-Gordones, María Carolina; Del Castillo, Jesús Rafael; Mijares, Alfredo; Benaim, Gustavo; Mendoza, Marta

2018-05-09

The plasma membrane Ca 2+ -ATPase (PMCA) from trypanosomatids lacks a classical calmodulin (CaM) binding domain, although CaM stimulated activities have been detected by biochemical assays. Recently we proposed that the Trypanosoma equiperdum CaM-sensitive PMCA (TePMCA) contains a potential 1-18 CaM-binding motif at the C-terminal region of the pump. In the present study, we evaluated the potential CaM-binding motifs using CaM from Trypanosoma cruzi and either the recombinant full length TePMCA C-terminal sequence (P14) or synthetic peptides comprising different regions of the C-terminal domain. We demonstrated that P14 and a synthetic peptide corresponding to residues 1037-1062 (which contains the predicted 1-18 binding motif) competed efficiently for binding to TcCaM, exhibiting similar IC 50 s of 200 nM. A stable complex of this peptide and TcCaM was formed in the presence of Ca 2+ , as determined by native-polyacrylamide gel electrophoresis. A predicted structure obtained by molecular docking showed an interaction of the 1-18 binding motif with the Ca 2+ /CaM complex. Moreover, when the peptide was incubated with CaM and Ca 2+ , a blue shift in the tryptophan fluorescence spectrum (from 350 to 329 nm) was observed. Substitutions at W 1039 and F 1056 , strongly decreased both CaM-peptide interaction and the complex assembly. Our results demonstrated the presence of a functional 1-18 motif at the TePMCA C-terminal domain. Furthermore, on the basis of spectrofluorometric assays and the resulting structure modeled by docking we propose that the L 1042 and W 1060 residues might also participate as anchors to form a 1-4-18-22 motif. Copyright © 2018 Elsevier B.V. All rights reserved.

Modular Activating Receptors in Innate and Adaptive Immunity.

PubMed

Berry, Richard; Call, Matthew E

2017-03-14

Triggering of cell-mediated immunity is largely dependent on the recognition of foreign or abnormal molecules by a myriad of cell surface-bound receptors. Many activating immune receptors do not possess any intrinsic signaling capacity but instead form noncovalent complexes with one or more dimeric signaling modules that communicate with a common set of kinases to initiate intracellular information-transfer pathways. This modular architecture, where the ligand binding and signaling functions are detached from one another, is a common theme that is widely employed throughout the innate and adaptive arms of immune systems. The evolutionary advantages of this highly adaptable platform for molecular recognition are visible in the variety of ligand-receptor interactions that can be linked to common signaling pathways, the diversification of receptor modules in response to pathogen challenges, and the amplification of cellular responses through incorporation of multiple signaling motifs. Here we provide an overview of the major classes of modular activating immune receptors and outline the current state of knowledge regarding how these receptors assemble, recognize their ligands, and ultimately trigger intracellular signal transduction pathways that activate immune cell effector functions.

Regulatory polymorphisms modulate the expression of HLA class II molecules and promote autoimmunity

PubMed Central

Raj, Prithvi; Rai, Ekta; Song, Ran; Khan, Shaheen; Wakeland, Benjamin E; Viswanathan, Kasthuribai; Arana, Carlos; Liang, Chaoying; Zhang, Bo; Dozmorov, Igor; Carr-Johnson, Ferdicia; Mitrovic, Mitja; Wiley, Graham B; Kelly, Jennifer A; Lauwerys, Bernard R; Olsen, Nancy J; Cotsapas, Chris; Garcia, Christine K; Wise, Carol A; Harley, John B; Nath, Swapan K; James, Judith A; Jacob, Chaim O; Tsao, Betty P; Pasare, Chandrashekhar; Karp, David R; Li, Quan Zhen; Gaffney, Patrick M; Wakeland, Edward K

2016-01-01

Targeted sequencing of sixteen SLE risk loci among 1349 Caucasian cases and controls produced a comprehensive dataset of the variations causing susceptibility to systemic lupus erythematosus (SLE). Two independent disease association signals in the HLA-D region identified two regulatory regions containing 3562 polymorphisms that modified thirty-seven transcription factor binding sites. These extensive functional variations are a new and potent facet of HLA polymorphism. Variations modifying the consensus binding motifs of IRF4 and CTCF in the XL9 regulatory complex modified the transcription of HLA-DRB1, HLA-DQA1 and HLA-DQB1 in a chromosome-specific manner, resulting in a 2.5-fold increase in the surface expression of HLA-DR and DQ molecules on dendritic cells with SLE risk genotypes, which increases to over 4-fold after stimulation. Similar analyses of fifteen other SLE risk loci identified 1206 functional variants tightly linked with disease-associated SNPs and demonstrated that common disease alleles contain multiple causal variants modulating multiple immune system genes. DOI: http://dx.doi.org/10.7554/eLife.12089.001 PMID:26880555

Interconnected network motifs control podocyte morphology and kidney function.

PubMed

Azeloglu, Evren U; Hardy, Simon V; Eungdamrong, Narat John; Chen, Yibang; Jayaraman, Gomathi; Chuang, Peter Y; Fang, Wei; Xiong, Huabao; Neves, Susana R; Jain, Mohit R; Li, Hong; Ma'ayan, Avi; Gordon, Ronald E; He, John Cijiang; Iyengar, Ravi

2014-02-04

Podocytes are kidney cells with specialized morphology that is required for glomerular filtration. Diseases, such as diabetes, or drug exposure that causes disruption of the podocyte foot process morphology results in kidney pathophysiology. Proteomic analysis of glomeruli isolated from rats with puromycin-induced kidney disease and control rats indicated that protein kinase A (PKA), which is activated by adenosine 3',5'-monophosphate (cAMP), is a key regulator of podocyte morphology and function. In podocytes, cAMP signaling activates cAMP response element-binding protein (CREB) to enhance expression of the gene encoding a differentiation marker, synaptopodin, a protein that associates with actin and promotes its bundling. We constructed and experimentally verified a β-adrenergic receptor-driven network with multiple feedback and feedforward motifs that controls CREB activity. To determine how the motifs interacted to regulate gene expression, we mapped multicompartment dynamical models, including information about protein subcellular localization, onto the network topology using Petri net formalisms. These computational analyses indicated that the juxtaposition of multiple feedback and feedforward motifs enabled the prolonged CREB activation necessary for synaptopodin expression and actin bundling. Drug-induced modulation of these motifs in diseased rats led to recovery of normal morphology and physiological function in vivo. Thus, analysis of regulatory motifs using network dynamics can provide insights into pathophysiology that enable predictions for drug intervention strategies to treat kidney disease.

Interconnected Network Motifs Control Podocyte Morphology and Kidney Function

PubMed Central

Azeloglu, Evren U.; Hardy, Simon V.; Eungdamrong, Narat John; Chen, Yibang; Jayaraman, Gomathi; Chuang, Peter Y.; Fang, Wei; Xiong, Huabao; Neves, Susana R.; Jain, Mohit R.; Li, Hong; Ma’ayan, Avi; Gordon, Ronald E.; He, John Cijiang; Iyengar, Ravi

2014-01-01

Podocytes are kidney cells with specialized morphology that is required for glomerular filtration. Diseases, such as diabetes, or drug exposure that causes disruption of the podocyte foot process morphology results in kidney pathophysiology. Proteomic analysis of glomeruli isolated from rats with puromycin-induced kidney disease and control rats indicated that protein kinase A (PKA), which is activated by adenosine 3′,5′-monophosphate (cAMP), is a key regulator of podocyte morphology and function. In podocytes, cAMP signaling activates cAMP response element–binding protein (CREB) to enhance expression of the gene encoding a differentiation marker, synaptopodin, a protein that associates with actin and promotes its bundling. We constructed and experimentally verified a β-adrenergic receptor–driven network with multiple feedback and feedforward motifs that controls CREB activity. To determine how the motifs interacted to regulate gene expression, we mapped multicompartment dynamical models, including information about protein subcellular localization, onto the network topology using Petri net formalisms. These computational analyses indicated that the juxtaposition of multiple feedback and feedforward motifs enabled the prolonged CREB activation necessary for synaptopodin expression and actin bundling. Drug-induced modulation of these motifs in diseased rats led to recovery of normal morphology and physiological function in vivo. Thus, analysis of regulatory motifs using network dynamics can provide insights into pathophysiology that enable predictions for drug intervention strategies to treat kidney disease. PMID:24497609

Analysis of secondary structural elements in human microRNA hairpin precursors.

PubMed

Liu, Biao; Childs-Disney, Jessica L; Znosko, Brent M; Wang, Dan; Fallahi, Mohammad; Gallo, Steven M; Disney, Matthew D

2016-03-01

MicroRNAs (miRNAs) regulate gene expression by targeting complementary mRNAs for destruction or translational repression. Aberrant expression of miRNAs has been associated with various diseases including cancer, thus making them interesting therapeutic targets. The composite of secondary structural elements that comprise miRNAs could aid the design of small molecules that modulate their function. We analyzed the secondary structural elements, or motifs, present in all human miRNA hairpin precursors and compared them to highly expressed human RNAs with known structures and other RNAs from various organisms. Amongst human miRNAs, there are 3808 are unique motifs, many residing in processing sites. Further, we identified motifs in miRNAs that are not present in other highly expressed human RNAs, desirable targets for small molecules. MiRNA motifs were incorporated into a searchable database that is freely available. We also analyzed the most frequently occurring bulges and internal loops for each RNA class and found that the smallest loops possible prevail. However, the distribution of loops and the preferred closing base pairs were unique to each class. Collectively, we have completed a broad survey of motifs found in human miRNA precursors, highly expressed human RNAs, and RNAs from other organisms. Interestingly, unique motifs were identified in human miRNA processing sites, binding to which could inhibit miRNA maturation and hence function.

DNA motifs determining the accuracy of repeat duplication during CRISPR adaptation in Haloarcula hispanica

PubMed Central

Wang, Rui; Li, Ming; Gong, Luyao; Hu, Songnian; Xiang, Hua

2016-01-01

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) acquire new spacers to generate adaptive immunity in prokaryotes. During spacer integration, the leader-preceded repeat is always accurately duplicated, leading to speculations of a repeat-length ruler. Here in Haloarcula hispanica, we demonstrate that the accurate duplication of its 30-bp repeat requires two conserved mid-repeat motifs, AACCC and GTGGG. The AACCC motif was essential and needed to be ∼10 bp downstream from the leader-repeat junction site, where duplication consistently started. Interestingly, repeat duplication terminated sequence-independently and usually with a specific distance from the GTGGG motif, which seemingly served as an anchor site for a molecular ruler. Accordingly, altering the spacing between the two motifs led to an aberrant duplication size (29, 31, 32 or 33 bp). We propose the adaptation complex may recognize these mid-repeat elements to enable measuring the repeat DNA for spacer integration. PMID:27085805

The RCAN carboxyl end mediates calcineurin docking-dependent inhibition via a site that dictates binding to substrates and regulators

PubMed Central

Martínez-Martínez, Sara; Genescà, Lali; Rodríguez, Antonio; Raya, Alicia; Salichs, Eulàlia; Were, Felipe; López-Maderuelo, María Dolores; Redondo, Juan Miguel; de la Luna, Susana

2009-01-01

Specificity of signaling kinases and phosphatases toward their targets is usually mediated by docking interactions with substrates and regulatory proteins. Here, we characterize the motifs involved in the physical and functional interaction of the phosphatase calcineurin with a group of modulators, the RCAN protein family. Mutation of key residues within the hydrophobic docking-cleft of the calcineurin catalytic domain impairs binding to all human RCAN proteins and to the calcineurin interacting proteins Cabin1 and AKAP79. A valine-rich region within the RCAN carboxyl region is essential for binding to the docking site in calcineurin. Although a peptide containing this sequence compromises NFAT signaling in living cells, it does not inhibit calcineurin catalytic activity directly. Instead, calcineurin catalytic activity is inhibited by a motif at the extreme C-terminal region of RCAN, which acts in cis with the docking motif. Our results therefore indicate that the inhibitory action of RCAN on calcineurin-NFAT signaling results not only from the inhibition of phosphatase activity but also from competition between NFAT and RCAN for binding to the same docking site in calcineurin. Thus, competition by substrates and modulators for a common docking site appears to be an essential mechanism in the regulation of Ca2+-calcineurin signaling. PMID:19332797

The effect of orthology and coregulation on detecting regulatory motifs.

PubMed

Storms, Valerie; Claeys, Marleen; Sanchez, Aminael; De Moor, Bart; Verstuyf, Annemieke; Marchal, Kathleen

2010-02-03

Computational de novo discovery of transcription factor binding sites is still a challenging problem. The growing number of sequenced genomes allows integrating orthology evidence with coregulation information when searching for motifs. Moreover, the more advanced motif detection algorithms explicitly model the phylogenetic relatedness between the orthologous input sequences and thus should be well adapted towards using orthologous information. In this study, we evaluated the conditions under which complementing coregulation with orthologous information improves motif detection for the class of probabilistic motif detection algorithms with an explicit evolutionary model. We designed datasets (real and synthetic) covering different degrees of coregulation and orthologous information to test how well Phylogibbs and Phylogenetic sampler, as representatives of the motif detection algorithms with evolutionary model performed as compared to MEME, a more classical motif detection algorithm that treats orthologs independently. Under certain conditions detecting motifs in the combined coregulation-orthology space is indeed more efficient than using each space separately, but this is not always the case. Moreover, the difference in success rate between the advanced algorithms and MEME is still marginal. The success rate of motif detection depends on the complex interplay between the added information and the specificities of the applied algorithms. Insights in this relation provide information useful to both developers and users. All benchmark datasets are available at http://homes.esat.kuleuven.be/~kmarchal/Supplementary_Storms_Valerie_PlosONE.

The Effect of Orthology and Coregulation on Detecting Regulatory Motifs

PubMed Central

Storms, Valerie; Claeys, Marleen; Sanchez, Aminael; De Moor, Bart; Verstuyf, Annemieke; Marchal, Kathleen

2010-01-01

Background Computational de novo discovery of transcription factor binding sites is still a challenging problem. The growing number of sequenced genomes allows integrating orthology evidence with coregulation information when searching for motifs. Moreover, the more advanced motif detection algorithms explicitly model the phylogenetic relatedness between the orthologous input sequences and thus should be well adapted towards using orthologous information. In this study, we evaluated the conditions under which complementing coregulation with orthologous information improves motif detection for the class of probabilistic motif detection algorithms with an explicit evolutionary model. Methodology We designed datasets (real and synthetic) covering different degrees of coregulation and orthologous information to test how well Phylogibbs and Phylogenetic sampler, as representatives of the motif detection algorithms with evolutionary model performed as compared to MEME, a more classical motif detection algorithm that treats orthologs independently. Results and Conclusions Under certain conditions detecting motifs in the combined coregulation-orthology space is indeed more efficient than using each space separately, but this is not always the case. Moreover, the difference in success rate between the advanced algorithms and MEME is still marginal. The success rate of motif detection depends on the complex interplay between the added information and the specificities of the applied algorithms. Insights in this relation provide information useful to both developers and users. All benchmark datasets are available at http://homes.esat.kuleuven.be/~kmarchal/Supplementary_Storms_Valerie_PlosONE. PMID:20140085

Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment

PubMed Central

Barouch-Bentov, Rina; Neveu, Gregory; Xiao, Fei; Beer, Melanie; Bekerman, Elena; Schor, Stanford; Campbell, Joseph; Boonyaratanakornkit, Jim; Lindenbach, Brett; Lu, Albert; Jacob, Yves

2016-01-01

ABSTRACT Enveloped viruses commonly utilize late-domain motifs, sometimes cooperatively with ubiquitin, to hijack the endosomal sorting complex required for transport (ESCRT) machinery for budding at the plasma membrane. However, the mechanisms underlying budding of viruses lacking defined late-domain motifs and budding into intracellular compartments are poorly characterized. Here, we map a network of hepatitis C virus (HCV) protein interactions with the ESCRT machinery using a mammalian-cell-based protein interaction screen and reveal nine novel interactions. We identify HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), an ESCRT-0 complex component, as an important entry point for HCV into the ESCRT pathway and validate its interactions with the HCV nonstructural (NS) proteins NS2 and NS5A in HCV-infected cells. Infectivity assays indicate that HRS is an important factor for efficient HCV assembly. Specifically, by integrating capsid oligomerization assays, biophysical analysis of intracellular viral particles by continuous gradient centrifugations, proteolytic digestion protection, and RNase digestion protection assays, we show that HCV co-opts HRS to mediate a late assembly step, namely, envelopment. In the absence of defined late-domain motifs, K63-linked polyubiquitinated lysine residues in the HCV NS2 protein bind the HRS ubiquitin-interacting motif to facilitate assembly. Finally, ESCRT-III and VPS/VTA1 components are also recruited by HCV proteins to mediate assembly. These data uncover involvement of ESCRT proteins in intracellular budding of a virus lacking defined late-domain motifs and a novel mechanism by which HCV gains entry into the ESCRT network, with potential implications for other viruses. PMID:27803188

Classification and assessment tools for structural motif discovery algorithms.

PubMed

Badr, Ghada; Al-Turaiki, Isra; Mathkour, Hassan

2013-01-01

Motif discovery is the problem of finding recurring patterns in biological data. Patterns can be sequential, mainly when discovered in DNA sequences. They can also be structural (e.g. when discovering RNA motifs). Finding common structural patterns helps to gain a better understanding of the mechanism of action (e.g. post-transcriptional regulation). Unlike DNA motifs, which are sequentially conserved, RNA motifs exhibit conservation in structure, which may be common even if the sequences are different. Over the past few years, hundreds of algorithms have been developed to solve the sequential motif discovery problem, while less work has been done for the structural case. In this paper, we survey, classify, and compare different algorithms that solve the structural motif discovery problem, where the underlying sequences may be different. We highlight their strengths and weaknesses. We start by proposing a benchmark dataset and a measurement tool that can be used to evaluate different motif discovery approaches. Then, we proceed by proposing our experimental setup. Finally, results are obtained using the proposed benchmark to compare available tools. To the best of our knowledge, this is the first attempt to compare tools solely designed for structural motif discovery. Results show that the accuracy of discovered motifs is relatively low. The results also suggest a complementary behavior among tools where some tools perform well on simple structures, while other tools are better for complex structures. We have classified and evaluated the performance of available structural motif discovery tools. In addition, we have proposed a benchmark dataset with tools that can be used to evaluate newly developed tools.

Conserved binding of GCAC motifs by MEC-8, couch potato, and the RBPMS protein family

PubMed Central

Soufari, Heddy

2017-01-01

Precise regulation of mRNA processing, translation, localization, and stability relies on specific interactions with RNA-binding proteins whose biological function and target preference are dictated by their preferred RNA motifs. The RBPMS family of RNA-binding proteins is defined by a conserved RNA recognition motif (RRM) domain found in metazoan RBPMS/Hermes and RBPMS2, Drosophila couch potato, and MEC-8 from Caenorhabditis elegans. In order to determine the parameters of RNA sequence recognition by the RBPMS family, we have first used the N-terminal domain from MEC-8 in binding assays and have demonstrated a preference for two GCAC motifs optimally separated by >6 nucleotides (nt). We have also determined the crystal structure of the dimeric N-terminal RRM domain from MEC-8 in the unbound form, and in complex with an oligonucleotide harboring two copies of the optimal GCAC motif. The atomic details reveal the molecular network that provides specificity to all four bases in the motif, including multiple hydrogen bonds to the initial guanine. Further studies with human RBPMS, as well as Drosophila couch potato, confirm a general preference for this double GCAC motif by other members of the protein family and the presence of this motif in known targets. PMID:28003515

T:G mismatch-specific thymine-DNA glycosylase (TDG) as a coregulator of transcription interacts with SRC1 family members through a novel tyrosine repeat motif

PubMed Central

Lucey, Marie J.; Chen, Dongsheng; Lopez-Garcia, Jorge; Hart, Stephen M.; Phoenix, Fladia; Al-Jehani, Rajai; Alao, John P.; White, Roger; Kindle, Karin B.; Losson, Régine; Chambon, Pierre; Parker, Malcolm G.; Schär, Primo; Heery, David M.; Buluwela, Lakjaya; Ali, Simak

2005-01-01

Gene activation involves protein complexes with diverse enzymatic activities, some of which are involved in chromatin modification. We have shown previously that the base excision repair enzyme thymine DNA glycosylase (TDG) acts as a potent coactivator for estrogen receptor-α. To further understand how TDG acts in this context, we studied its interaction with known coactivators of nuclear receptors. We find that TDG interacts in vitro and in vivo with the p160 coactivator SRC1, with the interaction being mediated by a previously undescribed motif encoding four equally spaced tyrosine residues in TDG, each tyrosine being separated by three amino acids. This is found to interact with two motifs in SRC1 also containing tyrosine residues separated by three amino acids. Site-directed mutagenesis shows that the tyrosines encoded in these motifs are critical for the interaction. The related p160 protein TIF2 does not interact with TDG and has the altered sequence, F-X-X-X-Y, at the equivalent positions relative to SRC1. Substitution of the phenylalanines to tyrosines is sufficient to bring about interaction of TIF2 with TDG. These findings highlight a new protein–protein interaction motif based on Y-X-X-X-Y and provide new insight into the interaction of diverse proteins in coactivator complexes. PMID:16282588

Temporal motifs reveal collaboration patterns in online task-oriented networks

NASA Astrophysics Data System (ADS)

Xuan, Qi; Fang, Huiting; Fu, Chenbo; Filkov, Vladimir

2015-05-01

Real networks feature layers of interactions and complexity. In them, different types of nodes can interact with each other via a variety of events. Examples of this complexity are task-oriented social networks (TOSNs), where teams of people share tasks towards creating a quality artifact, such as academic research papers or software development in commercial or open source environments. Accomplishing those tasks involves both work, e.g., writing the papers or code, and communication, to discuss and coordinate. Taking into account the different types of activities and how they alternate over time can result in much more precise understanding of the TOSNs behaviors and outcomes. That calls for modeling techniques that can accommodate both node and link heterogeneity as well as temporal change. In this paper, we report on methodology for finding temporal motifs in TOSNs, limited to a system of two people and an artifact. We apply the methods to publicly available data of TOSNs from 31 Open Source Software projects. We find that these temporal motifs are enriched in the observed data. When applied to software development outcome, temporal motifs reveal a distinct dependency between collaboration and communication in the code writing process. Moreover, we show that models based on temporal motifs can be used to more precisely relate both individual developer centrality and team cohesion to programmer productivity than models based on aggregated TOSNs.

Temporal motifs reveal collaboration patterns in online task-oriented networks.

PubMed

Xuan, Qi; Fang, Huiting; Fu, Chenbo; Filkov, Vladimir

2015-05-01

Real networks feature layers of interactions and complexity. In them, different types of nodes can interact with each other via a variety of events. Examples of this complexity are task-oriented social networks (TOSNs), where teams of people share tasks towards creating a quality artifact, such as academic research papers or software development in commercial or open source environments. Accomplishing those tasks involves both work, e.g., writing the papers or code, and communication, to discuss and coordinate. Taking into account the different types of activities and how they alternate over time can result in much more precise understanding of the TOSNs behaviors and outcomes. That calls for modeling techniques that can accommodate both node and link heterogeneity as well as temporal change. In this paper, we report on methodology for finding temporal motifs in TOSNs, limited to a system of two people and an artifact. We apply the methods to publicly available data of TOSNs from 31 Open Source Software projects. We find that these temporal motifs are enriched in the observed data. When applied to software development outcome, temporal motifs reveal a distinct dependency between collaboration and communication in the code writing process. Moreover, we show that models based on temporal motifs can be used to more precisely relate both individual developer centrality and team cohesion to programmer productivity than models based on aggregated TOSNs.

Transient α-helices in the disordered RPEL motifs of the serum response factor coactivator MKL1

NASA Astrophysics Data System (ADS)

Mizuguchi, Mineyuki; Fuju, Takahiro; Obita, Takayuki; Ishikawa, Mitsuru; Tsuda, Masaaki; Tabuchi, Akiko

2014-06-01

The megakaryoblastic leukemia 1 (MKL1) protein functions as a transcriptional coactivator of the serum response factor. MKL1 has three RPEL motifs (RPEL1, RPEL2, and RPEL3) in its N-terminal region. MKL1 binds to monomeric G-actin through RPEL motifs, and the dissociation of MKL1 from G-actin promotes the translocation of MKL1 to the nucleus. Although structural data are available for RPEL motifs of MKL1 in complex with G-actin, the structural characteristics of RPEL motifs in the free state have been poorly defined. Here we characterized the structures of free RPEL motifs using NMR and CD spectroscopy. NMR and CD measurements showed that free RPEL motifs are largely unstructured in solution. However, NMR analysis identified transient α-helices in the regions where helices α1 and α2 are induced upon binding to G-actin. Proline mutagenesis showed that the transient α-helices are locally formed without helix-helix interactions. The helix content is higher in the order of RPEL1, RPEL2, and RPEL3. The amount of preformed structure may correlate with the binding affinity between the intrinsically disordered protein and its target molecule.

RNA Bricks—a database of RNA 3D motifs and their interactions

PubMed Central

Chojnowski, Grzegorz; Waleń, Tomasz; Bujnicki, Janusz M.

2014-01-01

The RNA Bricks database (http://iimcb.genesilico.pl/rnabricks), stores information about recurrent RNA 3D motifs and their interactions, found in experimentally determined RNA structures and in RNA–protein complexes. In contrast to other similar tools (RNA 3D Motif Atlas, RNA Frabase, Rloom) RNA motifs, i.e. ‘RNA bricks’ are presented in the molecular environment, in which they were determined, including RNA, protein, metal ions, water molecules and ligands. All nucleotide residues in RNA bricks are annotated with structural quality scores that describe real-space correlation coefficients with the electron density data (if available), backbone geometry and possible steric conflicts, which can be used to identify poorly modeled residues. The database is also equipped with an algorithm for 3D motif search and comparison. The algorithm compares spatial positions of backbone atoms of the user-provided query structure and of stored RNA motifs, without relying on sequence or secondary structure information. This enables the identification of local structural similarities among evolutionarily related and unrelated RNA molecules. Besides, the search utility enables searching ‘RNA bricks’ according to sequence similarity, and makes it possible to identify motifs with modified ribonucleotide residues at specific positions. PMID:24220091

Discovery of candidate KEN-box motifs using cell cycle keyword enrichment combined with native disorder prediction and motif conservation.

PubMed

Michael, Sushama; Travé, Gilles; Ramu, Chenna; Chica, Claudia; Gibson, Toby J

2008-02-15

KEN-box-mediated target selection is one of the mechanisms used in the proteasomal destruction of mitotic cell cycle proteins via the APC/C complex. While annotating the Eukaryotic Linear Motif resource (ELM, http://elm.eu.org/), we found that KEN motifs were significantly enriched in human protein entries with cell cycle keywords in the UniProt/Swiss-Prot database-implying that KEN-boxes might be more common than reported. Matches to short linear motifs in protein database searches are not, per se, significant. KEN-box enrichment with cell cycle Gene Ontology terms suggests that collectively these motifs are functional but does not prove that any given instance is so. Candidates were surveyed for native disorder prediction using GlobPlot and IUPred and for motif conservation in homologues. Among >25 strong new candidates, the most notable are human HIPK2, CHFR, CDC27, Dab2, Upf2, kinesin Eg5, DNA Topoisomerase 1 and yeast Cdc5 and Swi5. A similar number of weaker candidates were present. These proteins have yet to be tested for APC/C targeted destruction, providing potential new avenues of research.

T-Reg Comparator: an analysis tool for the comparison of position weight matrices

PubMed Central

Roepcke, Stefan; Grossmann, Steffen; Rahmann, Sven; Vingron, Martin

2005-01-01

T-Reg Comparator is a novel software tool designed to support research into transcriptional regulation. Sequence motifs representing transcription factor binding sites are usually encoded as position weight matrices. The user inputs a set of such weight matrices or binding site sequences and our program matches them against the T-Reg database, which is presently built on data from the Transfac [E. Wingender (2004) In Silico Biol., 4, 55–61] and Jaspar [A. Sandelin, W. Alkema, P. Engstrom, W. W. Wasserman and B. Lenhard (2004) Nucleic Acids Res., 32, D91–D94]. Our tool delivers a detailed report on similarities between user-supplied motifs and motifs in the database. Apart from simple one-to-one relationships, T-Reg Comparator is also able to detect similarities between submatrices. In addition, we provide a user interface to a program for sequence scanning with weight matrices. Typical areas of application for T-Reg Comparator are motif and regulatory module finding and annotation of regulatory genomic regions. T-Reg Comparator is available at . PMID:15980506

T-Reg Comparator: an analysis tool for the comparison of position weight matrices.

PubMed

Roepcke, Stefan; Grossmann, Steffen; Rahmann, Sven; Vingron, Martin

2005-07-01

T-Reg Comparator is a novel software tool designed to support research into transcriptional regulation. Sequence motifs representing transcription factor binding sites are usually encoded as position weight matrices. The user inputs a set of such weight matrices or binding site sequences and our program matches them against the T-Reg database, which is presently built on data from the Transfac [E. Wingender (2004) In Silico Biol., 4, 55-61] and Jaspar [A. Sandelin, W. Alkema, P. Engstrom, W. W. Wasserman and B. Lenhard (2004) Nucleic Acids Res., 32, D91-D94]. Our tool delivers a detailed report on similarities between user-supplied motifs and motifs in the database. Apart from simple one-to-one relationships, T-Reg Comparator is also able to detect similarities between submatrices. In addition, we provide a user interface to a program for sequence scanning with weight matrices. Typical areas of application for T-Reg Comparator are motif and regulatory module finding and annotation of regulatory genomic regions. T-Reg Comparator is available at http://treg.molgen.mpg.de.

Drosophila Brahma complex remodels nucleosome organizations in multiple aspects.

PubMed

Shi, Jiejun; Zheng, Meizhu; Ye, Youqiong; Li, Min; Chen, Xiaolong; Hu, Xinjie; Sun, Jin; Zhang, Xiaobai; Jiang, Cizhong

2014-09-01

ATP-dependent chromatin remodeling complexes regulate nucleosome organizations. In Drosophila, gene Brm encodes the core Brahma complex, the ATPase subunit of SWI/SNF class of chromatin remodelers. Its role in modulating the nucleosome landscape in vivo is unclear. In this study, we knocked down Brm in Drosophila third instar larvae to explore the changes in nucleosome profiles and global gene transcription. The results show that Brm knockdown leads to nucleosome occupancy changes throughout the entire genome with a bias in occupancy decrease. In contrast, the knockdown has limited impacts on nucleosome position shift. The knockdown also alters another important physical property of nucleosome positioning, fuzziness. Nucleosome position shift, gain or loss and fuzziness changes are all enriched in promoter regions. Nucleosome arrays around the 5' ends of genes are reorganized in five patterns as a result of Brm knockdown. Intriguingly, the concomitant changes in the genes adjacent to the Brahma-dependent remodeling regions have important roles in development and morphogenesis. Further analyses reveal abundance of AT-rich motifs for transcription factors in the remodeling regions. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Structural and functional aspects of winged-helix domains at the core of transcription initiation complexes.

PubMed

Teichmann, Martin; Dumay-Odelot, Hélène; Fribourg, Sébastien

2012-01-01

The winged helix (WH) domain is found in core components of transcription systems in eukaryotes and prokaryotes. It represents a sub-class of the helix-turn-helix motif. The WH domain participates in establishing protein-DNA and protein-protein-interactions. Here, we discuss possible explanations for the enrichment of this motif in transcription systems.

The heptanucleotide motif GAGACGC is a key component of a cis-acting promoter element that is critical for SnSAG1 expression in Sarcocystis neurona.

PubMed

Gaji, Rajshekhar Y; Howe, Daniel K

2009-07-01

The apicomplexan parasite Sarcocystis neurona undergoes a complex process of intracellular development, during which many genes are temporally regulated. The described study was undertaken to begin identifying the basic promoter elements that control gene expression in S. neurona. Sequence analysis of the 5'-flanking region of five S. neurona genes revealed a conserved heptanucleotide motif GAGACGC that is similar to the WGAGACG motif described upstream of multiple genes in Toxoplasma gondii. The promoter region for the major surface antigen gene SnSAG1, which contains three heptanucleotide motifs within 135 bases of the transcription start site, was dissected by functional analysis using a dual luciferase reporter assay. These analyses revealed that a minimal promoter fragment containing all three motifs was sufficient to drive reporter molecule expression, with the presence and orientation of the 5'-most heptanucleotide motif being absolutely critical for promoter function. Further studies should help to identify additional sequence elements important for promoter function and for controlling gene expression during intracellular development by this apicomplexan pathogen.

Molecular dynamics simulations of electrostatics and hydration distributions around RNA and DNA motifs

NASA Astrophysics Data System (ADS)

Marlowe, Ashley E.; Singh, Abhishek; Semichaevsky, Andrey V.; Yingling, Yaroslava G.

2009-03-01

Nucleic acid nanoparticles can self-assembly through the formation of complementary loop-loop interactions or stem-stem interactions. Presence and concentration of ions can significantly affect the self-assembly process and the stability of the nanostructure. In this presentation we use explicit molecular dynamics simulations to examine the variations in cationic distributions and hydration environment around DNA and RNA helices and loop-loop interactions. Our simulations show that the potassium and sodium ionic distributions are different around RNA and DNA motifs which could be indicative of ion mediated relative stability of loop-loop complexes. Moreover in RNA loop-loop motifs ions are consistently present and exchanged through a distinct electronegative channel. We will also show how we used the specific RNA loop-loop motif to design a RNA hexagonal nanoparticle.

Unitary circular code motifs in genomes of eukaryotes.

PubMed

El Soufi, Karim; Michel, Christian J

A set X of 20 trinucleotides was identified in genes of bacteria, eukaryotes, plasmids and viruses, which has in average the highest occurrence in reading frame compared to its two shifted frames (Michel, 2015; Arquès and Michel, 1996). This set X has an interesting mathematical property as X is a circular code (Arquès and Michel, 1996). Thus, the motifs from this circular code X, called X motifs, have the property to always retrieve, synchronize and maintain the reading frame in genes. The origin of this circular code X in genes is an open problem since its discovery in 1996. Here, we first show that the unitary circular codes (UCC), i.e. sets of one word, allow to generate unitary circular code motifs (UCC motifs), i.e. a concatenation of the same motif (simple repeats) leading to low complexity DNA. Three classes of UCC motifs are studied here: repeated dinucleotides (D + motifs), repeated trinucleotides (T + motifs) and repeated tetranucleotides (T + motifs). Thus, the D + , T + and T + motifs allow to retrieve, synchronize and maintain a frame modulo 2, modulo 3 and modulo 4, respectively, and their shifted frames (1 modulo 2; 1 and 2 modulo 3; 1, 2 and 3 modulo 4 according to the C 2 , C 3 and C 4 properties, respectively) in the DNA sequences. The statistical distribution of the D + , T + and T + motifs is analyzed in the genomes of eukaryotes. A UCC motif and its comp lementary UCC motif have the same distribution in the eukaryotic genomes. Furthermore, a UCC motif and its complementary UCC motif have increasing occurrences contrary to their number of hydrogen bonds, very significant with the T + motifs. The longest D + , T + and T + motifs in the studied eukaryotic genomes are also given. Surprisingly, a scarcity of repeated trinucleotides (T + motifs) in the large eukaryotic genomes is observed compared to the D + and T + motifs. This result has been investigated and may be explained by two outcomes. Repeated trinucleotides (T + motifs) are identified in the X motifs of low composition (cardinality less than 10) in the genomes of eukaryotes. Furthermore, identical trinucleotide pairs of the circular code X are preferentially used in the gene sequences of eukaryotes. These two results suggest that the unitary circular codes of trinucleotides may have been involved in the formation of the trinucleotide circular code X. Indeed, repeated trinucleotides in the X motifs in the genomes of eukaryotes may represent an intermediary evolution from repeated trinucleotides of cardinality 1 (T + motifs) in the genomes of eukaryotes up to the X motifs of cardinality 20 in the gene sequences of eukaryotes. Copyright © 2017 Elsevier B.V. All rights reserved.

Overexpression of TRIM25 in Lung Cancer Regulates Tumor Cell Progression.

PubMed

Qin, Ying; Cui, He; Zhang, Hua

2016-10-01

Lung cancer is one of the most common causes of cancer-related deaths worldwide. Although great efforts and progressions have been made in the study of the lung cancer in the recent decades, the mechanism of lung cancer formation remains elusive. To establish effective therapeutic methods, new targets implied in lung cancer processes have to be identified. Tripartite motif-containing 25 has been associated with ovarian and breast cancer and is thought to positively promote cell growth by targeting the cell cycle. However, whether tripartite motif-containing 25 has a function in lung cancer development remains unknown. In this study, we found that tripartite motif-containing 25 was overexpressed in human lung cancer tissues. Expression of tripartite motif-containing 25 in lung cancer cells is important for cell proliferation and migration. Knockdown of tripartite motif-containing 25 markedly reduced proliferation of lung cancer cells both in vitro and in vivo and reduced migration of lung cancer cells in vitro Meanwhile, tripartite motif-containing 25 silencing also increased the sensitivity of doxorubicin and significantly increased death and apoptosis of lung cancer cells by doxorubicin were achieved with knockdown of tripartite motif-containing 25. We also observed that tripartite motif-containing 25 formed a complex with p53 and mouse double minute 2 homolog (MDM2) in both human lung cancer tissues and in lung cancer cells and tripartite motif-containing 25 silencing increased the expression of p53. These results provide evidence that tripartite motif-containing 25 contributes to the pathogenesis of lung cancer probably by promoting proliferation and migration of lung cancer cells. Therefore, targeting tripartite motif-containing 25 may provide a potential therapeutic intervention for lung cancer. © The Author(s) 2015.

Modulation of cyclobutane thymine photodimer formation in T11-tracts in rotationally phased nucleosome core particles and DNA minicircles.

PubMed

Wang, Kesai; Taylor, John-Stephen A

2017-07-07

Cyclobutane pyrimidine dimers (CPDs) are DNA photoproducts linked to skin cancer, whose mutagenicity depends in part on their frequency of formation and deamination. Nucleosomes modulate CPD formation, favoring outside facing sites and disfavoring inward facing sites. A similar pattern of CPD formation in protein-free DNA loops suggests that DNA bending causes the modulation in nucleosomes. To systematically study the cause and effect of nucleosome structure on CPD formation and deamination, we have developed a circular permutation synthesis strategy for positioning a target sequence at different superhelix locations (SHLs) across a nucleosome in which the DNA has been rotationally phased with respect to the histone octamer by TG motifs. We have used this system to show that the nucleosome dramatically modulates CPD formation in a T11-tract that covers one full turn of the nucleosome helix at seven different SHLs, and that the position of maximum CPD formation at all locations is shifted to the 5΄-side of that found in mixed-sequence nucleosomes. We also show that an 80-mer minicircle DNA using the same TG-motifs faithfully reproduces the CPD pattern in the nucleosome, indicating that it is a good model for protein-free rotationally phased bent DNA of the same curvature as in a nucleosome, and that bending is modulating CPD formation. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Modeling of the structure and interactions of the B. anthracis antitoxin, MoxX: deletion mutant studies highlight its modular structure and repressor function

NASA Astrophysics Data System (ADS)

Chopra, Nikita; Agarwal, Shivangi; Verma, Shashikala; Bhatnagar, Sonika; Bhatnagar, Rakesh

2011-03-01

Our previous report on Bacillus anthracis toxin-antitoxin module (MoxXT) identified it to be a two component system wherein, PemK-like toxin (MoxT) functions as a ribonuclease (Agarwal S et al. JBC 285:7254-7270, 2010). The labile antitoxin (MoxX) can bind to/neutralize the action of the toxin and is also a DNA-binding protein mediating autoregulation. In this study, molecular modeling of MoxX in its biologically active dimeric form was done. It was found that it contains a conserved Ribbon-Helix-Helix (RHH) motif, consistent with its DNA-binding function. The modeled MoxX monomers dimerize to form a two-stranded antiparallel ribbon, while the C-terminal region adopts an extended conformation. Knowledge guided protein-protein docking, molecular dynamics simulation, and energy minimization was performed to obtain the structure of the MoxXT complex, which was exploited for the de novo design of a peptide capable of binding to MoxT. It was found that the designed peptide caused a decrease in MoxX binding to MoxT by 42% at a concentration of 2 μM in vitro. We also show that MoxX mediates negative transcriptional autoregulation by binding to its own upstream DNA. The interacting regions of both MoxX and DNA were identified in order to model their complex. The repressor activity of MoxX was found to be mediated by the 16 N-terminal residues that contains the ribbon of the RHH motif. Based on homology with other RHH proteins and deletion mutant studies, we propose a model of the MoxX-DNA interaction, with the antiparallel β-sheet of the MoxX dimer inserted into the major groove of its cognate DNA. The structure of the complex of MoxX with MoxT and its own upstream regulatory region will facilitate design of molecules that can disrupt these interactions, a strategy for development of novel antibacterials.

Using RNA nanoparticles with thermostable motifs and fluorogenic modules for real-time detection of RNA folding and turnover in prokaryotic and eukaryotic cells.

PubMed

Zhang, Hui; Pi, Fengmei; Shu, Dan; Vieweger, Mario; Guo, Peixuan

2015-01-01

RNA nanotechnology is an emerging field at the interface of biochemistry and nanomaterials that shows immense promise for applications in nanomedicines, therapeutics and nanotechnology. Noncoding RNAs, such as siRNA, miRNA, ribozymes, and riboswitches, play important roles in the regulation of cellular processes. They carry out highly specific functions on a compact and efficient footprint. The properties of specificity and small size make them excellent modules in the construction of multifaceted RNA nanoparticles for targeted delivery and therapy. Biological activity of RNA molecules, however, relies on their proper folding. Therefore their thermodynamic and biochemical stability in the cellular environment is critical. Consequently, it is essential to assess global fold and intracellular lifetime of multifaceted RNA nanoparticles to optimize their therapeutic effectiveness. Here, we describe a method to express and assemble stable RNA nanoparticles in cells, and to assess the folding and turnover rate of RNA nanoparticles in vitro as well as in vivo in real time using a thermostable core motif derived from pRNA of bacteriophage Phi29 DNA packaging motor and fluorogenic RNA modules.

Semaphorin4D Drives CD8+ T-Cell Lesional Trafficking in Oral Lichen Planus via CXCL9/CXCL10 Upregulations in Oral Keratinocytes.

PubMed

Ke, Yao; Dang, Erle; Shen, Shengxian; Zhang, Tongmei; Qiao, Hongjiang; Chang, Yuqian; Liu, Qing; Wang, Gang

2017-11-01

Chemokine-mediated CD8 + T-cell recruitment is an essential but not well-established event for the persistence of oral lichen planus (OLP). Semaphorin 4D (Sema4D)/CD100 is implicated in immune dysfunction, chemokine modulation, and cell migration, which are critical aspects for OLP progression, but its implication in OLP pathogenesis has not been determined. In this study, we sought to explicate the effect of Sema4D on human oral keratinocytes and its capacity to drive CD8 + T-cell lesional trafficking via chemokine modulation. We found that upregulations of sSema4D in OLP tissues and blood were positively correlated with disease severity and activity. In vitro observation revealed that Sema4D induced C-X-C motif chemokine ligand 9/C-X-C motif chemokine ligand 10 production by binding to plexin-B1 via protein kinase B-NF-κB cascade in human oral keratinocytes, which elicited OLP CD8 + T-cell migration. We also confirmed using clinical samples that elevated C-X-C motif chemokine ligand 9/C-X-C motif chemokine ligand 10 levels were positively correlated with sSema4D levels in OLP lesions and serum. Notably, we determined matrix metalloproteinase-9 as a new proteolytic enzyme for the cleavage of sSema4D from the T-cell surface, which may contribute to the high levels of sSema4D in OLP lesions and serum. Our findings conclusively revealed an amplification feedback loop involving T cells, chemokines, and Sema4D-dependent signal that promotes OLP progression. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Malfunctioning of Chaperone-Mediated Autophagy in Parkinson's Disease: Feats, Constraints, and Flaws of Modulators.

PubMed

Tripathi, Manish Kumar; Rajput, Charul; Mishra, Saumya; Rasheed, Mohd Sami Ur; Singh, Mahendra Pratap

2018-06-11

Homeostatic regulation of class II programmed cell death/autophagy for the degradation and elimination of substandard organelles and defective proteins is decisive for the survival of dopaminergic neurons. Chaperone-mediated autophagy (CMA), one of the most highly dedicated self-sacrificing events, is accountable for the partial elimination of redundant soluble cytoplasmic proteins in Parkinson's disease (PD). CMA is characterized by the selective delivery of superfluous protein containing lysine-phenylalanine-glutamate-arginine-glutamine (KFERQ)/KFERQ-like motif to the lysosome through molecular chaperones, such as heat shock cognate-70 (Hsc-70). KFERQ/KFERQ-like motif present in the poor quality cytoplasmic substrate protein and Hsc-70 complex is recognized by a janitor protein, which is referred to as the lysosome-associated membrane protein-2A (LAMP-2A). This protein is known to facilitate an entry of substrate-chaperone complex in the lumen for hydrolytic cleavage of substrate and elimination of end-products. Impaired CMA is repeatedly blamed for an accumulation of surplus soluble proteins. However, it is still an enigma if CMA is a bonus or curse for PD. Case-control studies and cellular and animal models have deciphered the contribution of impaired CMA in PD. Current article updates the role of CMA in toxicant models and recapitulates the evidences that have highlighted a link between impaired CMA and PD. Although PD is an irreversible happening and CMA is a dual edging phenomenon, it is anticipated that fine-tuning of the latter encounters the former to a certain extent. Besides, the truth, embellishment, and propaganda regarding the issue are also emphasized in the final segment of the article.

Investigation of the binding properties of a multi-modular GH45 cellulase using bioinspired model assemblies.

PubMed

Fong, Monica; Berrin, Jean-Guy; Paës, Gabriel

2016-01-01

Enzymes degrading plant biomass polymers are widely used in biotechnological applications. Their efficiency can be limited by non-specific interactions occurring with some chemical motifs. In particular, the lignin component is known to bind enzymes irreversibly. In order to determine interactions of enzymes with their substrates, experiments are usually performed on isolated simple polymers which are not representative of plant cell wall complexity. But when using natural plant substrates, the role of individual chemical and structural features affecting enzyme-binding properties is also difficult to decipher. We have designed and used lignified model assemblies of plant cell walls as templates to characterize binding properties of multi-modular cellulases. These three-dimensional assemblies are modulated in their composition using the three principal polymers found in secondary plant cell walls (cellulose, hemicellulose, and lignin). Binding properties of enzymes are obtained from the measurement of their mobility that depends on their interactions with the polymers and chemical motifs of the assemblies. The affinity of the multi-modular GH45 cellulase was characterized using a statistical analysis to determine the role played by each assembly polymer. Presence of hemicellulose had much less impact on affinity than cellulose and model lignin. Depending on the number of CBMs appended to the cellulase catalytic core, binding properties toward cellulose and lignin were highly contrasted. Model assemblies bring new insights into the molecular determinants that are responsible for interactions between enzymes and substrate without the need of complex analysis. Consequently, we believe that model bioinspired assemblies will provide relevant information for the design and optimization of enzyme cocktails in the context of biorefineries.

Conserved structural and functional aspects of the tripartite motif gene family point towards therapeutic applications in multiple diseases.

PubMed

Gushchina, Liubov V; Kwiatkowski, Thomas A; Bhattacharya, Sayak; Weisleder, Noah L

2018-05-01

The tripartite motif (TRIM) gene family is a highly conserved group of E3 ubiquitin ligase proteins that can establish substrate specificity for the ubiquitin-proteasome complex and also have proteasome-independent functions. While several family members were studied previously, it is relatively recent that over 80 genes, based on sequence homology, were grouped to establish the TRIM gene family. Functional studies of various TRIM genes linked these proteins to modulation of inflammatory responses showing that they can contribute to a wide variety of disease states including cardiovascular, neurological and musculoskeletal diseases, as well as various forms of cancer. Given the fundamental role of the ubiquitin-proteasome complex in protein turnover and the importance of this regulation in most aspects of cellular physiology, it is not surprising that TRIM proteins display a wide spectrum of functions in a variety of cellular processes. This broad range of function and the highly conserved primary amino acid sequence of family members, particularly in the canonical TRIM E3 ubiquitin ligase domain, complicates the development of therapeutics that specifically target these proteins. A more comprehensive understanding of the structure and function of TRIM proteins will help guide therapeutic development for a number of different diseases. This review summarizes the structural organization of TRIM proteins, their domain architecture, common and unique post-translational modifications within the family, and potential binding partners and targets. Further discussion is provided on efforts to target TRIM proteins as therapeutic agents and how our increasing understanding of the nature of TRIM proteins can guide discovery of other therapeutics in the future. Copyright © 2017 Elsevier Inc. All rights reserved.

At the Frontier; RXLR Effectors Crossing the Phytophthora-Host Interface.

PubMed

Bouwmeester, Klaas; Meijer, Harold J G; Govers, Francine

2011-01-01

Plants are constantly beset by pathogenic organisms. To successfully infect their hosts, plant pathogens secrete effector proteins, many of which are translocated to the inside of the host cell where they manipulate normal physiological processes and undermine host defense. The way by which effectors cross the frontier to reach the inside of the host cell varies among different classes of pathogens. For oomycete plant pathogens - like the potato late blight pathogen Phytophthora infestans - it has been shown that effector translocation to the host cell cytoplasm is dependent on conserved amino acid motifs that are present in the N-terminal part of effector proteins. One of these motifs, known as the RXLR motif, has a strong resemblance with a host translocation motif found in effectors secreted by Plasmodium species. These malaria parasites, that reside inside specialized vacuoles in red blood cells, make use of a specific protein translocation complex to export effectors from the vacuole into the red blood cell. Whether or not also oomycete RXLR effectors require a translocation complex to cross the frontier is still under investigation. For one P. infestans RXLR effector named IPI-O we have found a potential host target that could play a role in establishing the first contact between this effector and the host cell. This membrane spanning lectin receptor kinase, LecRK-I.9, interacts with IPI-O via the tripeptide RGD that overlaps with the RXLR motif. In animals, RGD is a well-known cell adhesion motif; it binds to integrins, which are membrane receptors that regulate many cellular processes and which can be hijacked by pathogens for either effector translocation or pathogen entry into host cells.

Molecular Mechanisms Controlling GLUT4 Intracellular Retention

PubMed Central

Blot, Vincent

2008-01-01

In basal adipocytes, glucose transporter 4 (GLUT4) is sequestered intracellularly by an insulin-reversible retention mechanism. Here, we analyze the roles of three GLUT4 trafficking motifs (FQQI, TELEY, and LL), providing molecular links between insulin signaling, cellular trafficking machinery, and the motifs in the specialized trafficking of GLUT4. Our results support a GLUT4 retention model that involves two linked intracellular cycles: one between endosomes and a retention compartment, and the other between endosomes and specialized GLUT4 transport vesicles. Targeting of GLUT4 to the former is dependent on the FQQI motif and its targeting to the latter is dependent on the TELEY motif. These two motifs act independently in retention, with the TELEY-dependent step being under the control of signaling downstream of the AS160 rab GTPase activating protein. Segregation of GLUT4 from endosomes, although positively correlated with the degree of basal retention, does not completely account for GLUT4 retention or insulin-responsiveness. Mutation of the LL motif slows return to basal intracellular retention after insulin withdrawal. Knockdown of clathrin adaptin protein complex-1 (AP-1) causes a delay in the return to intracellular retention after insulin withdrawal. The effects of mutating the LL motif and knockdown of AP-1 were not additive, establishing that AP-1 regulation of GLUT4 trafficking requires the LL motif. PMID:18550797

Modulation of CaV2.1 channels by neuronal calcium sensor-1 induces short-term synaptic facilitation.

PubMed

Yan, Jin; Leal, Karina; Magupalli, Venkat G; Nanou, Evanthia; Martinez, Gilbert Q; Scheuer, Todd; Catterall, William A

2014-11-01

Facilitation and inactivation of P/Q-type Ca2+ currents mediated by Ca2+/calmodulin binding to Ca(V)2.1 channels contribute to facilitation and rapid depression of synaptic transmission, respectively. Other calcium sensor proteins displace calmodulin from its binding site and differentially modulate P/Q-type Ca2 + currents, resulting in diverse patterns of short-term synaptic plasticity. Neuronal calcium sensor-1 (NCS-1, frequenin) has been shown to enhance synaptic facilitation, but the underlying mechanism is unclear. We report here that NCS-1 directly interacts with IQ-like motif and calmodulin-binding domain in the C-terminal domain of Ca(V)2.1 channel. NCS-1 reduces Ca2 +-dependent inactivation of P/Q-type Ca2+ current through interaction with the IQ-like motif and calmodulin-binding domain without affecting peak current or activation kinetics. Expression of NCS-1 in presynaptic superior cervical ganglion neurons has no effect on synaptic transmission, eliminating effects of this calcium sensor protein on endogenous N-type Ca2+ currents and the endogenous neurotransmitter release machinery. However, in superior cervical ganglion neurons expressing wild-type Ca(V)2.1 channels, co-expression of NCS-1 induces facilitation of synaptic transmission in response to paired pulses and trains of depolarizing stimuli, and this effect is lost in Ca(V)2.1 channels with mutations in the IQ-like motif and calmodulin-binding domain. These results reveal that NCS-1 directly modulates Ca(V)2.1 channels to induce short-term synaptic facilitation and further demonstrate that CaS proteins are crucial in fine-tuning short-term synaptic plasticity.

Most of the tight positional conservation of transcription factor binding sites near the transcription start site reflects their co-localization within regulatory modules.

PubMed

Acevedo-Luna, Natalia; Mariño-Ramírez, Leonardo; Halbert, Armand; Hansen, Ulla; Landsman, David; Spouge, John L

2016-11-21

Transcription factors (TFs) form complexes that bind regulatory modules (RMs) within DNA, to control specific sets of genes. Some transcription factor binding sites (TFBSs) near the transcription start site (TSS) display tight positional preferences relative to the TSS. Furthermore, near the TSS, RMs can co-localize TFBSs with each other and the TSS. The proportion of TFBS positional preferences due to TFBS co-localization within RMs is unknown, however. ChIP experiments confirm co-localization of some TFBSs genome-wide, including near the TSS, but they typically examine only a few TFs at a time, using non-physiological conditions that can vary from lab to lab. In contrast, sequence analysis can examine many TFs uniformly and methodically, broadly surveying the co-localization of TFBSs with tight positional preferences relative to the TSS. Our statistics found 43 significant sets of human motifs in the JASPAR TF Database with positional preferences relative to the TSS, with 38 preferences tight (±5 bp). Each set of motifs corresponded to a gene group of 135 to 3304 genes, with 42/43 (98%) gene groups independently validated by DAVID, a gene ontology database, with FDR < 0.05. Motifs corresponding to two TFBSs in a RM should co-occur more than by chance alone, enriching the intersection of the gene groups corresponding to the two TFs. Thus, a gene-group intersection systematically enriched beyond chance alone provides evidence that the two TFs participate in an RM. Of the 903 = 43*42/2 intersections of the 43 significant gene groups, we found 768/903 (85%) pairs of gene groups with significantly enriched intersections, with 564/768 (73%) intersections independently validated by DAVID with FDR < 0.05. A user-friendly web site at http://go.usa.gov/3kjsH permits biologists to explore the interaction network of our TFBSs to identify candidate subunit RMs. Gene duplication and convergent evolution within a genome provide obvious biological mechanisms for replicating an RM near the TSS that binds a particular TF subunit. Of all intersections of our 43 significant gene groups, 85% were significantly enriched, with 73% of the significant enrichments independently validated by gene ontology. The co-localization of TFBSs within RMs therefore likely explains much of the tight TFBS positional preferences near the TSS.

Identification and characterization of tandem repeats in exon III of dopamine receptor D4 (DRD4) genes from different mammalian species.

PubMed

Larsen, Svend Arild; Mogensen, Line; Dietz, Rune; Baagøe, Hans Jørgen; Andersen, Mogens; Werge, Thomas; Rasmussen, Henrik Berg

2005-12-01

In this study we have identified and characterized dopamine receptor D4 (DRD4) exon III tandem repeats in 33 public available nucleotide sequences from different mammalian species. We found that the tandem repeat in canids could be described in a novel and simple way, namely, as a structure composed of 15- and 12- bp modules. Tandem repeats composed of 18-bp modules were found in sequences from the horse, zebra, onager, and donkey, Asiatic bear, polar bear, common raccoon, dolphin, harbor porpoise, and domestic cat. Several of these sequences have been analyzed previously without a tandem repeat being found. In the domestic cow and gray seal we identified tandem repeats composed of 36-bp modules, each consisting of two closely related 18-bp basic units. A tandem repeat consisting of 9-bp modules was identified in sequences from mink and ferret. In the European otter we detected an 18-bp tandem repeat, while a tandem repeat consisting of 27-bp modules was identified in a sequence from European badger. Both these tandem repeats were composed of 9-bp basic units, which were closely related with the 9-bp repeat modules identified in the mink and ferret. Tandem repeats could not be identified in sequences from rodents. All tandem repeats possessed a high GC content with a strong bias for C. On phylogenetic analysis of the tandem repeats evolutionary related species were clustered into the same groups. The degree of conservation of the tandem repeats varied significantly between species. The deduced amino acid sequences of most of the tandem repeats exhibited a high propensity for disorder. This was also the case with an amino acid sequence of the human DRD4 exon III tandem repeat, which was included in the study for comparative purposes. We identified proline-containing motifs for SH3 and WW domain binding proteins, potential phosphorylation sites, PDZ domain binding motifs, and FHA domain binding motifs in the amino acid sequences of the tandem repeats. The numbers of potential functional sites varied pronouncedly between species. Our observations provide a platform for future studies of the architecture and evolution of the DRD4 exon III tandem repeat, and they suggest that differences in the structure of this tandem repeat contribute to specialization and generation of diversity in receptor function.

Identification of sequence motifs significantly associated with antisense activity.

PubMed

McQuisten, Kyle A; Peek, Andrew S

2007-06-07

Predicting the suppression activity of antisense oligonucleotide sequences is the main goal of the rational design of nucleic acids. To create an effective predictive model, it is important to know what properties of an oligonucleotide sequence associate significantly with antisense activity. Also, for the model to be efficient we must know what properties do not associate significantly and can be omitted from the model. This paper will discuss the results of a randomization procedure to find motifs that associate significantly with either high or low antisense suppression activity, analysis of their properties, as well as the results of support vector machine modelling using these significant motifs as features. We discovered 155 motifs that associate significantly with high antisense suppression activity and 202 motifs that associate significantly with low suppression activity. The motifs range in length from 2 to 5 bases, contain several motifs that have been previously discovered as associating highly with antisense activity, and have thermodynamic properties consistent with previous work associating thermodynamic properties of sequences with their antisense activity. Statistical analysis revealed no correlation between a motif's position within an antisense sequence and that sequences antisense activity. Also, many significant motifs existed as subwords of other significant motifs. Support vector regression experiments indicated that the feature set of significant motifs increased correlation compared to all possible motifs as well as several subsets of the significant motifs. The thermodynamic properties of the significantly associated motifs support existing data correlating the thermodynamic properties of the antisense oligonucleotide with antisense efficiency, reinforcing our hypothesis that antisense suppression is strongly associated with probe/target thermodynamics, as there are no enzymatic mediators to speed the process along like the RNA Induced Silencing Complex (RISC) in RNAi. The independence of motif position and antisense activity also allows us to bypass consideration of this feature in the modelling process, promoting model efficiency and reducing the chance of overfitting when predicting antisense activity. The increase in SVR correlation with significant features compared to nearest-neighbour features indicates that thermodynamics alone is likely not the only factor in determining antisense efficiency.

Reusable, extensible, and modifiable R scripts and Kepler workflows for comprehensive single set ChIP-seq analysis.

PubMed

Cormier, Nathan; Kolisnik, Tyler; Bieda, Mark

2016-07-05

There has been an enormous expansion of use of chromatin immunoprecipitation followed by sequencing (ChIP-seq) technologies. Analysis of large-scale ChIP-seq datasets involves a complex series of steps and production of several specialized graphical outputs. A number of systems have emphasized custom development of ChIP-seq pipelines. These systems are primarily based on custom programming of a single, complex pipeline or supply libraries of modules and do not produce the full range of outputs commonly produced for ChIP-seq datasets. It is desirable to have more comprehensive pipelines, in particular ones addressing common metadata tasks, such as pathway analysis, and pipelines producing standard complex graphical outputs. It is advantageous if these are highly modular systems, available as both turnkey pipelines and individual modules, that are easily comprehensible, modifiable and extensible to allow rapid alteration in response to new analysis developments in this growing area. Furthermore, it is advantageous if these pipelines allow data provenance tracking. We present a set of 20 ChIP-seq analysis software modules implemented in the Kepler workflow system; most (18/20) were also implemented as standalone, fully functional R scripts. The set consists of four full turnkey pipelines and 16 component modules. The turnkey pipelines in Kepler allow data provenance tracking. Implementation emphasized use of common R packages and widely-used external tools (e.g., MACS for peak finding), along with custom programming. This software presents comprehensive solutions and easily repurposed code blocks for ChIP-seq analysis and pipeline creation. Tasks include mapping raw reads, peakfinding via MACS, summary statistics, peak location statistics, summary plots centered on the transcription start site (TSS), gene ontology, pathway analysis, and de novo motif finding, among others. These pipelines range from those performing a single task to those performing full analyses of ChIP-seq data. The pipelines are supplied as both Kepler workflows, which allow data provenance tracking, and, in the majority of cases, as standalone R scripts. These pipelines are designed for ease of modification and repurposing.

RING E3 mechanism for ubiquitin ligation to a disordered substrate visualized for human anaphase-promoting complex

DOE PAGES

Brown, Nicholas G.; VanderLinden, Ryan; Watson, Edmond R.; ...

2015-03-30

For many E3 ligases, a mobile RING (Really Interesting New Gene) domain stimulates ubiquitin (Ub) transfer from a thioester-linked E2~Ub intermediate to a lysine on a remotely bound disordered substrate. One such E3 is the gigantic, multisubunit 1.2-MDa anaphase-promoting complex/cyclosome (APC), which controls cell division by ubiquitinating cell cycle regulators to drive their timely degradation. Intrinsically disordered substrates are typically recruited via their KEN-box, D-box, and/or other motifs binding to APC and a coactivator such as CDH1. On the opposite side of the APC, the dynamic catalytic core contains the cullin-like subunit APC2 and its RING partner APC11, which collaboratesmore » with the E2 UBCH10 (UBE2C) to ubiquitinate substrates. However, how dynamic RING–E2~Ub catalytic modules such as APC11–UBCH10~Ub collide with distally tethered disordered substrates remains poorly understood. In this paper, we report structural mechanisms of UBCH10 recruitment to APC CDH1 and substrate ubiquitination. Unexpectedly, in addition to binding APC11’s RING, UBCH10 is corecruited via interactions with APC2, which we visualized in a trapped complex representing an APC CDH1–UBCH10~Ub–substrate intermediate by cryo-electron microscopy, and in isolation by X-ray crystallography. To our knowledge, this is the first structural view of APC, or any cullin–RING E3, with E2 and substrate juxtaposed, and it reveals how tripartite cullin–RING–E2 interactions establish APC’s specificity for UBCH10 and harness a flexible catalytic module to drive ubiquitination of lysines within an accessible zone. Finally, we propose that multisite interactions reduce the degrees of freedom available to dynamic RING E3–E2~Ub catalytic modules, condense the search radius for target lysines, increase the chance of active-site collision with conformationally fluctuating substrates, and enable regulation.« less

Sequence, Structure, and Context Preferences of Human RNA Binding Proteins.

PubMed

Dominguez, Daniel; Freese, Peter; Alexis, Maria S; Su, Amanda; Hochman, Myles; Palden, Tsultrim; Bazile, Cassandra; Lambert, Nicole J; Van Nostrand, Eric L; Pratt, Gabriel A; Yeo, Gene W; Graveley, Brenton R; Burge, Christopher B

2018-06-07

RNA binding proteins (RBPs) orchestrate the production, processing, and function of mRNAs. Here, we present the affinity landscapes of 78 human RBPs using an unbiased assay that determines the sequence, structure, and context preferences of these proteins in vitro by deep sequencing of bound RNAs. These data enable construction of "RNA maps" of RBP activity without requiring crosslinking-based assays. We found an unexpectedly low diversity of RNA motifs, implying frequent convergence of binding specificity toward a relatively small set of RNA motifs, many with low compositional complexity. Offsetting this trend, however, we observed extensive preferences for contextual features distinct from short linear RNA motifs, including spaced "bipartite" motifs, biased flanking nucleotide composition, and bias away from or toward RNA structure. Our results emphasize the importance of contextual features in RNA recognition, which likely enable targeting of distinct subsets of transcripts by different RBPs that recognize the same linear motif. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

In Silico Molecular Modeling and Docking Studies on Novel Mutants (E229V, H225P and D230C) of the Nucleotide-Binding Domain of Homo sapiens Hsp70.

PubMed

Elengoe, Asita; Hamdan, Salehhuddin

2017-12-01

In this study, we explored the possibility of determining the synergistic interactions between nucleotide-binding domain (NBD) of Homo sapiens heat-shock 70 kDa protein (Hsp70) and E1A 32 kDa of adenovirus serotype 5 motif (PNLVP) in the efficiency of killing of tumor cells in cancer treatment. At present, the protein interaction between NBD and PNLVP motif is still unknown, but believed to enhance the rate of virus replication in tumor cells. Three mutant models (E229V, H225P and D230C) were built and simulated, and their interactions with PNLVP motif were studied. The PNLVP motif showed the binding energy and intermolecular energy values with the novel E229V mutant at -7.32 and -11.2 kcal/mol. The E229V mutant had the highest number of hydrogen bonds (7). Based on the root mean square deviation, root mean square fluctuation, hydrogen bonds, salt bridge, secondary structure, surface-accessible solvent area, potential energy and distance matrices analyses, it was proved that the E229V had the strongest and most stable interaction with the PNLVP motif among all the four protein-ligand complex structures. The knowledge of this protein-ligand complex model would help in designing Hsp70 structure-based drug for cancer therapy.

Dynamics of brain activity underlying working memory for music in a naturalistic condition.

PubMed

Burunat, Iballa; Alluri, Vinoo; Toiviainen, Petri; Numminen, Jussi; Brattico, Elvira

2014-08-01

We aimed at determining the functional neuroanatomy of working memory (WM) recognition of musical motifs that occurs while listening to music by adopting a non-standard procedure. Western tonal music provides naturally occurring repetition and variation of motifs. These serve as WM triggers, thus allowing us to study the phenomenon of motif tracking within real music. Adopting a modern tango as stimulus, a behavioural test helped to identify the stimulus motifs and build a time-course regressor of WM neural responses. This regressor was then correlated with the participants' (musicians') functional magnetic resonance imaging (fMRI) signal obtained during a continuous listening condition. In order to fine-tune the identification of WM processes in the brain, the variance accounted for by the sensory processing of a set of the stimulus' acoustic features was pruned from participants' neurovascular responses to music. Motivic repetitions activated prefrontal and motor cortical areas, basal ganglia, medial temporal lobe (MTL) structures, and cerebellum. The findings suggest that WM processing of motifs while listening to music emerges from the integration of neural activity distributed over cognitive, motor and limbic subsystems. The recruitment of the hippocampus stands as a novel finding in auditory WM. Effective connectivity and agglomerative hierarchical clustering analyses indicate that the hippocampal connectivity is modulated by motif repetitions, showing strong connections with WM-relevant areas (dorsolateral prefrontal cortex - dlPFC, supplementary motor area - SMA, and cerebellum), which supports the role of the hippocampus in the encoding of the musical motifs in WM, and may evidence long-term memory (LTM) formation, enabled by the use of a realistic listening condition. Copyright © 2014 Elsevier Ltd. All rights reserved.

SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins.

PubMed

Koch, C A; Anderson, D; Moran, M F; Ellis, C; Pawson, T

1991-05-03

Src homology (SH) regions 2 and 3 are noncatalytic domains that are conserved among a series of cytoplasmic signaling proteins regulated by receptor protein-tyrosine kinases, including phospholipase C-gamma, Ras GTPase (guanosine triphosphatase)-activating protein, and Src-like tyrosine kinases. The SH2 domains of these signaling proteins bind tyrosine phosphorylated polypeptides, implicated in normal signaling and cellular transformation. Tyrosine phosphorylation acts as a switch to induce the binding of SH2 domains, thereby mediating the formation of heteromeric protein complexes at or near the plasma membrane. The formation of these complexes is likely to control the activation of signal transduction pathways by tyrosine kinases. The SH3 domain is a distinct motif that, together with SH2, may modulate interactions with the cytoskeleton and membrane. Some signaling and transforming proteins contain SH2 and SH3 domains unattached to any known catalytic element. These noncatalytic proteins may serve as adaptors to link tyrosine kinases to specific target proteins. These observations suggest that SH2 and SH3 domains participate in the control of intracellular responses to growth factor stimulation.

Molecular basis of Kar9-Bim1 complex function during mating and spindle positioning

PubMed Central

Manatschal, Cristina; Farcas, Ana-Maria; Degen, Miriam Steiner; Bayer, Mathias; Kumar, Anil; Landgraf, Christiane; Volkmer, Rudolf; Barral, Yves; Steinmetz, Michel O.

2016-01-01

The Kar9 pathway promotes nuclear fusion during mating and spindle alignment during metaphase in budding yeast. How Kar9 supports the different outcome of these two divergent processes is an open question. Here, we show that three sites in the C-terminal disordered domain of Kar9 mediate tight Kar9 interaction with the C-terminal dimerization domain of Bim1 (EB1 orthologue). Site1 and Site2 contain SxIP motifs; however, Site3 defines a novel type of EB1-binding site. Whereas Site2 and Site3 mediate Kar9 recruitment to microtubule tips, nuclear movement, and karyogamy, only Site2 functions in spindle positioning during metaphase. Site1 in turn plays an inhibitory role during mating. Additionally, the Kar9-Bim1 complex is involved in microtubule-independent activities during mating. Together, our data reveal how multiple and partially redundant EB1-binding sites provide a microtubule-associated protein with the means to modulate its biochemical properties to promote different molecular processes during cell proliferation and differentiation. PMID:27682587

Immunomediated Pan-cancer Regulation Networks are Dominant Fingerprints After Treatment of Cell Lines with Demethylation.

PubMed

El Baroudi, Mariama; Cinti, Caterina; Capobianco, Enrico

2016-01-01

Pan-cancer studies are particularly relevant not only for addressing the complexity of the inherently observed heterogeneity but also for identifying clinically relevant features that may be common to the cancer types. Immune system regulations usually reveal synergistic modulation with other cancer mechanisms and in combination provide insights on possible advances in cancer immunotherapies. Network inference is a powerful approach to decipher pan-cancer systems dynamics. The methodology proposed in this study elucidates the impacts of epigenetic treatment on the drivers of complex pan-cancer regulation circuits involving cell lines of five cancer types. These patterns were observed from differential gene expression measurements following demethylation with 5-azacytidine. Networks were built to establish associations of phenotypes at molecular level with cancer hallmarks through both transcriptional and post-transcriptional regulation mechanisms. The most prominent feature that emerges from our integrative network maps, linking pathway landscapes to disease and drug-target associations, refers primarily to a mosaic of immune-system crosslinked influences. Therefore, characteristics initially evidenced in single cancer maps become motifs well summarized by network cores and fingerprints.

Design of a small molecule against an oncogenic noncoding RNA.

PubMed

Velagapudi, Sai Pradeep; Cameron, Michael D; Haga, Christopher L; Rosenberg, Laura H; Lafitte, Marie; Duckett, Derek R; Phinney, Donald G; Disney, Matthew D

2016-05-24

The design of precision, preclinical therapeutics from sequence is difficult, but advances in this area, particularly those focused on rational design, could quickly transform the sequence of disease-causing gene products into lead modalities. Herein, we describe the use of Inforna, a computational approach that enables the rational design of small molecules targeting RNA to quickly provide a potent modulator of oncogenic microRNA-96 (miR-96). We mined the secondary structure of primary microRNA-96 (pri-miR-96) hairpin precursor against a database of RNA motif-small molecule interactions, which identified modules that bound RNA motifs nearby and in the Drosha processing site. Precise linking of these modules together provided Targaprimir-96 (3), which selectively modulates miR-96 production in cancer cells and triggers apoptosis. Importantly, the compound is ineffective on healthy breast cells, and exogenous overexpression of pri-miR-96 reduced compound potency in breast cancer cells. Chemical Cross-Linking and Isolation by Pull-Down (Chem-CLIP), a small-molecule RNA target validation approach, shows that 3 directly engages pri-miR-96 in breast cancer cells. In vivo, 3 has a favorable pharmacokinetic profile and decreases tumor burden in a mouse model of triple-negative breast cancer. Thus, rational design can quickly produce precision, in vivo bioactive lead small molecules against hard-to-treat cancers by targeting oncogenic noncoding RNAs, advancing a disease-to-gene-to-drug paradigm.

Elucidation of Sigma Factor-Associated Networks in Pseudomonas aeruginosa Reveals a Modular Architecture with Limited and Function-Specific Crosstalk

PubMed Central

Schulz, Sebastian; Eckweiler, Denitsa; Bielecka, Agata; Nicolai, Tanja; Franke, Raimo; Dötsch, Andreas; Hornischer, Klaus; Bruchmann, Sebastian; Düvel, Juliane; Häussler, Susanne

2015-01-01

Sigma factors are essential global regulators of transcription initiation in bacteria which confer promoter recognition specificity to the RNA polymerase core enzyme. They provide effective mechanisms for simultaneously regulating expression of large numbers of genes in response to challenging conditions, and their presence has been linked to bacterial virulence and pathogenicity. In this study, we constructed nine his-tagged sigma factor expressing and/or deletion mutant strains in the opportunistic pathogen Pseudomonas aeruginosa. To uncover the direct and indirect sigma factor regulons, we performed mRNA profiling, as well as chromatin immunoprecipitation coupled to high-throughput sequencing. We furthermore elucidated the de novo binding motif of each sigma factor, and validated the RNA- and ChIP-seq results by global motif searches in the proximity of transcriptional start sites (TSS). Our integrated approach revealed a highly modular network architecture which is composed of insulated functional sigma factor modules. Analysis of the interconnectivity of the various sigma factor networks uncovered a limited, but highly function-specific, crosstalk which orchestrates complex cellular processes. Our data indicate that the modular structure of sigma factor networks enables P. aeruginosa to function adequately in its environment and at the same time is exploited to build up higher-level functions by specific interconnections that are dominated by a participation of RpoN. PMID:25780925

Engineering naturally occurring trans-acting non-coding RNAs to sense molecular signals

PubMed Central

Qi, Lei; Lucks, Julius B.; Liu, Chang C.; Mutalik, Vivek K.; Arkin, Adam P.

2012-01-01

Non-coding RNAs (ncRNAs) are versatile regulators in cellular networks. While most trans-acting ncRNAs possess well-defined mechanisms that can regulate transcription or translation, they generally lack the ability to directly sense cellular signals. In this work, we describe a set of design principles for fusing ncRNAs to RNA aptamers to engineer allosteric RNA fusion molecules that modulate the activity of ncRNAs in a ligand-inducible way in Escherichia coli. We apply these principles to ncRNA regulators that can regulate translation (IS10 ncRNA) and transcription (pT181 ncRNA), and demonstrate that our design strategy exhibits high modularity between the aptamer ligand-sensing motif and the ncRNA target-recognition motif, which allows us to reconfigure these two motifs to engineer orthogonally acting fusion molecules that respond to different ligands and regulate different targets in the same cell. Finally, we show that the same ncRNA fused with different sensing domains results in a sensory-level NOR gate that integrates multiple input signals to perform genetic logic. These ligand-sensing ncRNA regulators provide useful tools to modulate the activity of structurally related families of ncRNAs, and building upon the growing body of RNA synthetic biology, our ability to design aptamer–ncRNA fusion molecules offers new ways to engineer ligand-sensing regulatory circuits. PMID:22383579

Enrichment of Circular Code Motifs in the Genes of the Yeast Saccharomyces cerevisiae.

PubMed

Michel, Christian J; Ngoune, Viviane Nguefack; Poch, Olivier; Ripp, Raymond; Thompson, Julie D

2017-12-03

A set X of 20 trinucleotides has been found to have the highest average occurrence in the reading frame, compared to the two shifted frames, of genes of bacteria, archaea, eukaryotes, plasmids and viruses. This set X has an interesting mathematical property, since X is a maximal C3 self-complementary trinucleotide circular code. Furthermore, any motif obtained from this circular code X has the capacity to retrieve, maintain and synchronize the original (reading) frame. Since 1996, the theory of circular codes in genes has mainly been developed by analysing the properties of the 20 trinucleotides of X, using combinatorics and statistical approaches. For the first time, we test this theory by analysing the X motifs, i.e., motifs from the circular code X, in the complete genome of the yeast Saccharomyces cerevisiae . Several properties of X motifs are identified by basic statistics (at the frequency level), and evaluated by comparison to R motifs, i.e., random motifs generated from 30 different random codes R. We first show that the frequency of X motifs is significantly greater than that of R motifs in the genome of S. cerevisiae . We then verify that no significant difference is observed between the frequencies of X and R motifs in the non-coding regions of S. cerevisiae , but that the occurrence number of X motifs is significantly higher than R motifs in the genes (protein-coding regions). This property is true for all cardinalities of X motifs (from 4 to 20) and for all 16 chromosomes. We further investigate the distribution of X motifs in the three frames of S. cerevisiae genes and show that they occur more frequently in the reading frame, regardless of their cardinality or their length. Finally, the ratio of X genes, i.e., genes with at least one X motif, to non-X genes, in the set of verified genes is significantly different to that observed in the set of putative or dubious genes with no experimental evidence. These results, taken together, represent the first evidence for a significant enrichment of X motifs in the genes of an extant organism. They raise two hypotheses: the X motifs may be evolutionary relics of the primitive codes used for translation, or they may continue to play a functional role in the complex processes of genome decoding and protein synthesis.

Finding trans-regulatory genes and protein complexes modulating meiotic recombination hotspots of human, mouse and yeast.

PubMed

Wu, Min; Kwoh, Chee-Keong; Li, Xiaoli; Zheng, Jie

2014-09-11

The regulatory mechanism of recombination is one of the most fundamental problems in genomics, with wide applications in genome wide association studies (GWAS), birth-defect diseases, molecular evolution, cancer research, etc. Recombination events cluster into short genomic regions called "recombination hotspots". Recently, a zinc finger protein PRDM9 was reported to regulate recombination hotspots in human and mouse genomes. In addition, a 13-mer motif contained in the binding sites of PRDM9 is found to be enriched in human hotspots. However, this 13-mer motif only covers a fraction of hotspots, indicating that PRDM9 is not the only regulator of recombination hotspots. Therefore, the challenge of discovering other regulators of recombination hotspots becomes significant. Furthermore, recombination is a complex process. Hence, multiple proteins acting as machinery, rather than individual proteins, are more likely to carry out this process in a precise and stable manner. Therefore, the extension of the prediction of individual trans-regulators to protein complexes is also highly desired. In this paper, we introduce a pipeline to identify genes and protein complexes associated with recombination hotspots. First, we prioritize proteins associated with hotspots based on their preference of binding to hotspots and coldspots. Second, using the above identified genes as seeds, we apply the Random Walk with Restart algorithm (RWR) to propagate their influences to other proteins in protein-protein interaction (PPI) networks. Hence, many proteins without DNA-binding information will also be assigned a score to implicate their roles in recombination hotspots. Third, we construct sub-PPI networks induced by top genes ranked by RWR for various species (e.g., yeast, human and mouse) and detect protein complexes in those sub-PPI networks. The GO term analysis show that our prioritizing methods and the RWR algorithm are capable of identifying novel genes associated with recombination hotspots. The trans-regulators predicted by our pipeline are enriched with epigenetic functions (e.g., histone modifications), demonstrating the epigenetic regulatory mechanisms of recombination hotspots. The identified protein complexes also provide us with candidates to further investigate the molecular machineries for recombination hotspots. Moreover, the experimental data and results are available on our web site http://www.ntu.edu.sg/home/zhengjie/data/RecombinationHotspot/NetPipe/.

Host regulation of lysogenic decision in bacteriophage lambda: transmembrane modulation of FtsH (HflB), the cII degrading protease, by HflKC (HflA).

PubMed

Kihara, A; Akiyama, Y; Ito, K

1997-05-27

The cII gene product of bacteriophage lambda is unstable and required for the establishment of lysogenization. Its intracellular amount is important for the decision between lytic growth and lysogenization. Two genetic loci of Escherichia coli are crucial for these commitments of infecting lambda genome. One of them, hflA encodes the HflKC membrane protein complex, which has been believed to be a protease degrading the cII protein. However, both its absence and overproduction stabilized cII in vivo and the proposed serine protease-like sequence motif in HflC was dispensable for the lysogenization control. Moreover, the HflKC protein was found to reside on the periplasmic side of the plasma membrane. In contrast, the other host gene, ftsH (hflB) encoding an integral membrane ATPase/protease, is positively required for degradation of cII, since loss of its function stabilized cII and its overexpression accelerated the cII degradation. In vitro, purified FtsH catalyzed ATP-dependent proteolysis of cII and HflKC antagonized the FtsH action. These results, together with our previous finding that FtsH and HflKC form a complex, suggest that FtsH is the cII degrading protease and HflKC is a modulator of the FtsH function. We propose that this transmembrane modulation differentiates the FtsH actions to different substrate proteins such as the membrane-bound SecY protein and the cytosolic cII protein. This study necessitates a revision of the prevailing view about the host control over lambda lysogenic decision.

Extensive T-Cell Epitope Repertoire Sharing among Human Proteome, Gastrointestinal Microbiome, and Pathogenic Bacteria: Implications for the Definition of Self

PubMed Central

Bremel, Robert D.; Homan, E. Jane

2015-01-01

T-cell receptor binding to MHC-bound peptides plays a key role in discrimination between self and non-self. Only a subset, typically a pentamer, of amino acids in a MHC-bound peptide form the motif exposed to the T-cell receptor. We categorize and compare the T-cell exposed amino acid motif repertoire of the total proteomes of two groups of bacteria, comprising pathogens and gastrointestinal microbiome organisms, with the human proteome and immunoglobulins. Given the maximum 205, or 3.2 million of such motifs that bind T-cell receptors, there is considerable overlap in motif usage. We show that the human proteome, exclusive of immunoglobulins, only comprises three quarters of the possible motifs, of which 65.3% are also present in both composite bacterial proteomes. Very few motifs are unique to the human proteome. Immunoglobulin variable regions carry a broad diversity of T-cell exposed motifs (TCEMs) that provides a stratified random sample of the motifs found in pathogens, microbiome, and the human proteome. Individual bacterial genera and species vary in the content of immunoglobulin and human proteome matched motifs that they carry. Mycobacteria and Burkholderia spp carry a particularly high content of such matched motifs. Some bacteria retain a unique motif signature and motif sharing pattern with the human proteome. The implication is that distinguishing self from non-self does not depend on individual TCEMs, but on a complex and dynamic overlay of signals wherein the same TCEM may play different roles in different organisms, and the frequency with which a particular TCEM appears influences its effect. The patterns observed provide clues to bacterial immune evasion and to strategies for intervention, including vaccine design. The breadth and distinct frequency patterns of the immunoglobulin-derived peptides suggest a role of immunoglobulins in maintaining a broadly responsive T-cell repertoire. PMID:26557118

Multiple Binding Modes between HNF4[alpha] and the LXXLL Motifs of PGC-1[alpha] Lead to Full Activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rha, Geun Bae; Wu, Guangteng; Shoelson, Steven E.

2010-04-15

Hepatocyte nuclear factor 4{alpha} (HNF4{alpha}) is a novel nuclear receptor that participates in a hierarchical network of transcription factors regulating the development and physiology of such vital organs as the liver, pancreas, and kidney. Among the various transcriptional coregulators with which HNF4{alpha} interacts, peroxisome proliferation-activated receptor {gamma} (PPAR{gamma}) coactivator 1{alpha} (PGC-1{alpha}) represents a novel coactivator whose activation is unusually robust and whose binding mode appears to be distinct from that of canonical coactivators such as NCoA/SRC/p160 family members. To elucidate the potentially unique molecular mechanism of PGC-1{alpha} recruitment, we have determined the crystal structure of HNF4{alpha} in complex with amore » fragment of PGC-1{alpha} containing all three of its LXXLL motifs. Despite the presence of all three LXXLL motifs available for interactions, only one is bound at the canonical binding site, with no additional contacts observed between the two proteins. However, a close inspection of the electron density map indicates that the bound LXXLL motif is not a selected one but an averaged structure of more than one LXXLL motif. Further biochemical and functional studies show that the individual LXXLL motifs can bind but drive only minimal transactivation. Only when more than one LXXLL motif is involved can significant transcriptional activity be measured, and full activation requires all three LXXLL motifs. These findings led us to propose a model wherein each LXXLL motif has an additive effect, and the multiple binding modes by HNF4{alpha} toward the LXXLL motifs of PGC-1{alpha} could account for the apparent robust activation by providing a flexible mechanism for combinatorial recruitment of additional coactivators and mediators.« less

Evidence for the Concerted Evolution between Short Linear Protein Motifs and Their Flanking Regions

PubMed Central

Chica, Claudia; Diella, Francesca; Gibson, Toby J.

2009-01-01

Background Linear motifs are short modules of protein sequences that play a crucial role in mediating and regulating many protein–protein interactions. The function of linear motifs strongly depends on the context, e.g. functional instances mainly occur inside flexible regions that are accessible for interaction. Sometimes linear motifs appear as isolated islands of conservation in multiple sequence alignments. However, they also occur in larger blocks of sequence conservation, suggesting an active role for the neighbouring amino acids. Results The evolution of regions flanking 116 functional linear motif instances was studied. The conservation of the amino acid sequence and order/disorder tendency of those regions was related to presence/absence of the instance. For the majority of the analysed instances, the pairs of sequences conserving the linear motif were also observed to maintain a similar local structural tendency and/or to have higher local sequence conservation when compared to pairs of sequences where one is missing the linear motif. Furthermore, those instances have a higher chance to co–evolve with the neighbouring residues in comparison to the distant ones. Those findings are supported by examples where the regulation of the linear motif–mediated interaction has been shown to depend on the modifications (e.g. phosphorylation) at neighbouring positions or is thought to benefit from the binding versatility of disordered regions. Conclusion The results suggest that flanking regions are relevant for linear motif–mediated interactions, both at the structural and sequence level. More interestingly, they indicate that the prediction of linear motif instances can be enriched with contextual information by performing a sequence analysis similar to the one presented here. This can facilitate the understanding of the role of these predicted instances in determining the protein function inside the broader context of the cellular network where they arise. PMID:19584925

A novel Arg H52/Tyr H33 conservative motif in antibodies: A correlation between sequence of antibodies and antigen binding.

PubMed

Petrov, Artem; Arzhanik, Vladimir; Makarov, Gennady; Koliasnikov, Oleg

2016-08-01

Antibodies are the family of proteins, which are responsible for antigen recognition. The computational modeling of interaction between an antigen and an antibody is very important when crystallographic structure is unavailable. In this research, we have discovered the correlation between the amino acid sequence of antibody and its specific binding characteristics on the example of the novel conservative binding motif, which consists of four residues: Arg H52, Tyr H33, Thr H59, and Glu H61. These residues are specifically oriented in the binding site and interact with each other in a specific manner. The residues of the binding motif are involved in interaction strictly with negatively charged groups of antigens, and form a binding complex. Mechanism of interaction and characteristics of the complex were also discovered. The results of this research can be used to increase the accuracy of computational antibody-antigen interaction modeling and for post-modeling quality control of the modeled structures.

TALE factors use two distinct functional modes to control an essential zebrafish gene expression program.

PubMed

Ladam, Franck; Stanney, William; Donaldson, Ian J; Yildiz, Ozge; Bobola, Nicoletta; Sagerström, Charles G

2018-06-18

TALE factors are broadly expressed embryonically and known to function in complexes with transcription factors (TFs) like Hox proteins at gastrula/segmentation stages, but it is unclear if such generally expressed factors act by the same mechanism throughout embryogenesis. We identify a TALE-dependent gene regulatory network (GRN) required for anterior development and detect TALE occupancy associated with this GRN throughout embryogenesis. At blastula stages, we uncover a novel functional mode for TALE factors, where they occupy genomic DECA motifs with nearby NF-Y sites. We demonstrate that TALE and NF-Y form complexes and regulate chromatin state at genes of this GRN. At segmentation stages, GRN-associated TALE occupancy expands to include HEXA motifs near PBX:HOX sites. Hence, TALE factors control a key GRN, but utilize distinct DNA motifs and protein partners at different stages - a strategy that may also explain their oncogenic potential and may be employed by other broadly expressed TFs. © 2018, Ladam et al.

Correlated Mutation in the Evolution of Catalysis in Uracil DNA Glycosylase Superfamily

NASA Astrophysics Data System (ADS)

Xia, Bo; Liu, Yinling; Guevara, Jose; Li, Jing; Jilich, Celeste; Yang, Ye; Wang, Liangjiang; Dominy, Brian N.; Cao, Weiguo

2017-04-01

Enzymes in Uracil DNA glycosylase (UDG) superfamily are essential for the removal of uracil. Family 4 UDGa is a robust uracil DNA glycosylase that only acts on double-stranded and single-stranded uracil-containing DNA. Based on mutational, kinetic and modeling analyses, a catalytic mechanism involving leaving group stabilization by H155 in motif 2 and water coordination by N89 in motif 3 is proposed. Mutual Information analysis identifies a complexed correlated mutation network including a strong correlation in the EG doublet in motif 1 of family 4 UDGa and in the QD doublet in motif 1 of family 1 UNG. Conversion of EG doublet in family 4 Thermus thermophilus UDGa to QD doublet increases the catalytic efficiency by over one hundred-fold and seventeen-fold over the E41Q and G42D single mutation, respectively, rectifying the strong correlation in the doublet. Molecular dynamics simulations suggest that the correlated mutations in the doublet in motif 1 position the catalytic H155 in motif 2 to stabilize the leaving uracilate anion. The integrated approach has important implications in studying enzyme evolution and protein structure and function.

Promoter motifs required for c-mpl gene expression induced by thrombopoietin in CMK cells.

PubMed

Sunohara, Masataka; Sato, Iwao; Morikawa, Shigeru

2017-11-30

Thrombopoietin (TPO) and its receptor, c-Mpl, are the central regulators of megakaryocyte development and platelet production and are also crucial to regulate megakaryocytopoiesis. TPO remarkably elevated c-mpl promoter activity, while the protein kinase C (PKC) inhibitors, GF109203, H7 and Calphostin C, clearly reduced the steady level of its promoter activity.  In the present study, motifs crucial for c-mpl promoter activity induced by TPO treatment have been analyzed using a human megakaryoblastic cell line, CMK. Destruction of the -107Sp1 and the -57Sp1 sites in the c-mpl promoter enhancer region resulted in decrease of the promoter activity by 53.1% and 64.4%, respectively, and destruction of -69Ets and -28Ets elements dramatically decreased the promoter activity by 96.4% and 87.8%, respectively, while mutation of -77GATA moderately reduced the activity by 31.4%. The result was in agreement with our previous report that showed the crucial motifs in the c-mpl promoter for the promoter activity induced by PMA-treatment. This indicates that TPO-induced activation of the c-mpl promoter activity is fully modulated by transcription through a PKC-dependent pathway and the two Sp1 and two Ets motifs are crucial for the activation of the c-mpl promoter activity rather than a GATA motif in the c-mpl promoter of CMK cells.

Dipeptide frequency/bias analysis identifies conserved sites of nonrandomness shared by cysteine-rich motifs.

PubMed

Campion, S R; Ameen, A S; Lai, L; King, J M; Munzenmaier, T N

2001-08-15

This report describes the application of a simple computational tool, AAPAIR.TAB, for the systematic analysis of the cysteine-rich EGF, Sushi, and Laminin motif/sequence families at the two-amino acid level. Automated dipeptide frequency/bias analysis detects preferences in the distribution of amino acids in established protein families, by determining which "ordered dipeptides" occur most frequently in comprehensive motif-specific sequence data sets. Graphic display of the dipeptide frequency/bias data revealed family-specific preferences for certain dipeptides, but more importantly detected a shared preference for employment of the ordered dipeptides Gly-Tyr (GY) and Gly-Phe (GF) in all three protein families. The dipeptide Asn-Gly (NG) also exhibited high-frequency and bias in the EGF and Sushi motif families, whereas Asn-Thr (NT) was distinguished in the Laminin family. Evaluation of the distribution of dipeptides identified by frequency/bias analysis subsequently revealed the highly restricted localization of the G(F/Y) and N(G/T) sequence elements at two separate sites of extreme conservation in the consensus sequence of all three sequence families. The similar employment of the high-frequency/bias dipeptides in three distinct protein sequence families was further correlated with the concurrence of these shared molecular determinants at similar positions within the distinctive scaffolds of three structurally divergent, but similarly employed, motif modules.

Domain architecture of the p62 subunit from the human transcription/repair factor TFIIH deduced by limited proteolysis and mass spectrometry analysis.

PubMed

Jawhari, Anass; Boussert, Stéphanie; Lamour, Valérie; Atkinson, R Andrew; Kieffer, Bruno; Poch, Olivier; Potier, Noelle; van Dorsselaer, Alain; Moras, Dino; Poterszman, Arnaud

2004-11-16

TFIIH is a multiprotein complex that plays a central role in both transcription and DNA repair. The subunit p62 is a structural component of the TFIIH core that is known to interact with VP16, p53, Eralpha, and E2F1 in the context of activated transcription, as well as with the endonuclease XPG in DNA repair. We used limited proteolysis experiments coupled to mass spectrometry to define structural domains within the conserved N-terminal part of the molecule. The first domain identified resulted from spontaneous proteolysis and corresponds to residues 1-108. The second domain encompasses residues 186-240, and biophysical characterization by fluorescence studies and NMR analysis indicated that it is at least partially folded and thus may correspond to a structural entity. This module contains a region of high sequence conservation with an invariant FWxxPhiPhi motif (Phi representing either tyrosine or phenylalanine), which was also found in other protein families and could play a key role as a protein-protein recognition module within TFIIH. The approach used in this study is general and can be straightforwardly applied to other multidomain proteins and/or multiprotein assemblies.

Discrete and polymeric self-assembled dendrimers: Hydrogen bond-mediated assembly with high stability and high fidelity

PubMed Central

Corbin, Perry S.; Lawless, Laurence J.; Li, Zhanting; Ma, Yuguo; Witmer, Melissa J.; Zimmerman, Steven C.

2002-01-01

Hydrogen bond-mediated self-assembly is a powerful strategy for creating nanoscale structures. However, little is known about the fidelity of assembly processes that must occur when similar and potentially competing hydrogen-bonding motifs are present. Furthermore, there is a continuing need for new modules and strategies that can amplify the relatively weak strength of a hydrogen bond to give more stable assemblies. Herein we report quantitative complexation studies on a ureidodeazapterin-based module revealing an unprecedented stability for dimers of its self-complementary acceptoracceptor-donor-donor (AADD) array. Linking two such units together with a semirigid spacer that carries a first-, second-, or third-generation Fréchet-type dendron affords a ditopic structure programmed to self assemble. The specific structure that is formed depends both on the size of the dendron and the solvent, but all of the assemblies have exceptionally high stability. The largest discrete nanoscale assembly is a hexamer with a molecular mass of about 17.8 kDa. It is stabilized by 30 hydrogen bonds, including six AADD⋅DDAA contacts. The hexamer forms and is indefinitely stable in the presence of a hexamer containing six ADD⋅DAA hydrogen-bonding arrays. PMID:11917113

Benzimidazoles: an ideal privileged drug scaffold for the design of multitargeted anti-inflammatory ligands.

PubMed

Kaur, Gaganpreet; Kaur, Maninder; Silakari, Om

2014-01-01

The recent research area endeavors to discover ultimate multi-target ligands, an increasingly feasible and attractive alternative to existing mono-targeted drugs for treatment of complex, multi-factorial inflammation process which underlays plethora of debilitated health conditions. In order to improvise this option, exploration of relevant chemical core scaffold will be an utmost need. Privileged benzimidazole scaffold being historically versatile structural motif could offer a viable starting point in the search for novel multi-target ligands against multi-factorial inflammation process since, when appropriately substituted, it can selectively modulate diverse receptors, pathways and enzymes associated with the pathogenesis of inflammation. Despite this remarkable capability, the multi-target capacity of the benzimidazole scaffold remains largely unexploited. With this in focus, the present review article attempts to provide synopsis of published research to exemplify the valuable use of benzimidazole nucleus and focus on their suitability as starting scaffold to develop multi-targeted anti-inflammatory ligands.

Structural Basis for Sialoglycan Binding by the Streptococcus sanguinis SrpA Adhesin*♦

PubMed Central

Bensing, Barbara A.; Loukachevitch, Lioudmila V.; McCulloch, Kathryn M.; Yu, Hai; Vann, Kendra R.; Wawrzak, Zdzislaw; Anderson, Spencer; Chen, Xi; Sullam, Paul M.; Iverson, T. M.

2016-01-01

Streptococcus sanguinis is a leading cause of infective endocarditis, a life-threatening infection of the cardiovascular system. An important interaction in the pathogenesis of infective endocarditis is attachment of the organisms to host platelets. S. sanguinis expresses a serine-rich repeat adhesin, SrpA, similar in sequence to platelet-binding adhesins associated with increased virulence in this disease. In this study, we determined the first crystal structure of the putative binding region of SrpA (SrpABR) both unliganded and in complex with a synthetic disaccharide ligand at 1.8 and 2.0 Å resolution, respectively. We identified a conserved Thr-Arg motif that orients the sialic acid moiety and is required for binding to platelet monolayers. Furthermore, we propose that sequence insertions in closely related family members contribute to the modulation of structural and functional properties, including the quaternary structure, the tertiary structure, and the ligand-binding site. PMID:26833566

Non-essential MCM-related proteins mediate a response to DNA damage in the archaeon Methanococcus maripaludis.

PubMed

Walters, Alison D; Chong, James P J

2017-05-01

The single minichromosome maintenance (MCM) protein found in most archaea has been widely studied as a simplified model for the MCM complex that forms the catalytic core of the eukaryotic replicative helicase. Organisms of the order Methanococcales are unusual in possessing multiple MCM homologues. The Methanococcus maripaludis S2 genome encodes four MCM homologues, McmA-McmD. DNA helicase assays reveal that the unwinding activity of the three MCM-like proteins is highly variable despite sequence similarities and suggests additional motifs that influence MCM function are yet to be identified. While the gene encoding McmA could not be deleted, strains harbouring individual deletions of genes encoding each of the other MCMs display phenotypes consistent with these proteins modulating DNA damage responses. M. maripaludis S2 is the first archaeon in which MCM proteins have been shown to influence the DNA damage response.

Transcriptional modulation of some Staphylococcus aureus iron-regulated genes during growth in vitro and in a tissue cage model in vivo.

PubMed

Allard, Marianne; Moisan, Hélène; Brouillette, Eric; Gervais, Alain L; Jacques, Mario; Lacasse, Pierre; Diarra, Moussa S; Malouin, François

2006-06-01

Staphylococcus aureus can proliferate in iron-limited environments such as the mammalian host. The transcriptional profiles of 460 genes (iron-regulated, putative Fur-regulated, membrane transport, pathogenesis) obtained for S. aureus grown in iron-restricted environments in vitro and in vivo were compared in order to identify new iron-regulated genes and to evaluate their potential as possible therapeutic targets in vivo. Iron deprivation was created in vitro by 2,2-dipyridyl, and in vivo, S. aureus was grown in tissue cages implanted in mice. Bacterial RNA was obtained from each growth condition and cDNA probes were co-hybridized on DNA arrays. Thirty-six upregulated and 11 downregulated genes were commonly modulated in animals and in the low-iron medium. Real-time PCR confirmed the iron-dependent modulation of four novel genes (SACOL0161, 2170, 2369, 2431) with a Fur box motif. Some genes expressed in the dipyridyl medium were not expressed in vivo (e.g., copA, frpA, SACOL1045). Downregulated genes included an iron-storage protein gene and genes of the succinate dehydrogenase complex, reminiscent of a small RNA-dependent regulation thus far only demonstrated in Gram-negative bacteria. The expression of iron-regulated genes in distinct low-iron environments provided insight into their relative importance in vitro and in vivo and their usefulness for vaccine and drug development.

Arginine methylation promotes translation repression activity of eIF4G-binding protein, Scd6.

PubMed

Poornima, Gopalakrishna; Shah, Shanaya; Vignesh, Venkadasubramanian; Parker, Roy; Rajyaguru, Purusharth I

2016-11-02

Regulation of translation plays a critical role in determining mRNA fate. A new role was recently reported for a subset of RGG-motif proteins in repressing translation initiation by binding eIF4G1. However the signaling mechanism(s) that leads to spatial and temporal regulation of repression activity of RGG-motif proteins remains unknown. Here we report the role of arginine methylation in regulation of repression activity of Scd6, a conserved RGG-motif protein. We demonstrate that Scd6 gets arginine methylated at its RGG-motif and Hmt1 plays an important role in its methylation. We identify specific methylated arginine residues in the Scd6 RGG-motif in vivo We provide evidence that methylation augments Scd6 repression activity. Arginine methylation defective (AMD) mutant of Scd6 rescues the growth defect caused by overexpression of Scd6, a feature of translation repressors in general. Live-cell imaging of the AMD mutant revealed that it is defective in inducing formation of stress granules. Live-cell imaging and pull-down results indicate that it fails to bind eIF4G1 efficiently. Consistent with these results, a strain lacking Hmt1 is also defective in Scd6-eIF4G1 interaction. Our results establish that arginine methylation augments Scd6 repression activity by promoting eIF4G1-binding. We propose that arginine methylation of translation repressors with RGG-motif could be a general modulator of their repression activity. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Imperfect duplicate insertions type of mutations in plasmepsin V modulates binding properties of PEXEL motifs of export proteins in Indian Plasmodium vivax.

PubMed

Rawat, Manmeet; Vijay, Sonam; Gupta, Yash; Tiwari, Pramod Kumar; Sharma, Arun

2013-01-01

Plasmepsin V (PM-V) have functionally conserved orthologues across the Plasmodium genus who's binding and antigenic processing at the PEXEL motifs for export about 200-300 essential proteins is important for the virulence and viability of the causative Plasmodium species. This study was undertaken to determine P. vivax plasmepsin V Ind (PvPM-V-Ind) PEXEL motif export pathway for pathogenicity-related proteins/antigens export thereby altering plasmodium exportome during erythrocytic stages. We identify and characterize Plasmodium vivax plasmepsin-V-Ind (mutant) gene by cloning, sequence analysis, in silico bioinformatic protocols and structural modeling predictions based on docking studies on binding capacity with PEXEL motifs processing in terms of binding and accessibility of export proteins. Cloning and sequence analysis for genetic diversity demonstrates PvPM-V-Ind (mutant) gene is highly conserved among all isolates from different geographical regions of India. Imperfect duplicate insertion types of mutations (SVSE from 246-249 AA and SLSE from 266-269 AA) were identified among all Indian isolates in comparison to P.vivax Sal-1 (PvPM-V-Sal 1) isolate. In silico bioinformatics interaction studies of PEXEL peptide and active enzyme reveal that PvPM-V-Ind (mutant) is only active in endoplasmic reticulum lumen and membrane embedding is essential for activation of plasmepsin V. Structural modeling predictions based on docking studies with PEXEL motif show significant variation in substrate protein binding of these imperfect mutations with data mined PEXEL sequences. The predicted variation in the docking score and interacting amino acids of PvPM-V-Ind (mutant) proteins with PEXEL and lopinavir suggests a modulation in the activity of PvPM-V in terms of binding and accessibility at these sites. Our functional modeled validation of PvPM-V-Ind (mutant) imperfect duplicate insertions with data mined PEXEL sequences leading to altered binding and substrate accessibility of the enzyme makes it a plausible target to investigate export mechanisms for in silico virtual screening and novel pharmacophore designing.

Imperfect Duplicate Insertions Type of Mutations in Plasmepsin V Modulates Binding Properties of PEXEL Motifs of Export Proteins in Indian Plasmodium vivax

PubMed Central

Rawat, Manmeet; Vijay, Sonam; Gupta, Yash; Tiwari, Pramod Kumar; Sharma, Arun

2013-01-01

Introduction Plasmepsin V (PM-V) have functionally conserved orthologues across the Plasmodium genus who's binding and antigenic processing at the PEXEL motifs for export about 200–300 essential proteins is important for the virulence and viability of the causative Plasmodium species. This study was undertaken to determine P. vivax plasmepsin V Ind (PvPM-V-Ind) PEXEL motif export pathway for pathogenicity-related proteins/antigens export thereby altering plasmodium exportome during erythrocytic stages. Method We identify and characterize Plasmodium vivax plasmepsin-V-Ind (mutant) gene by cloning, sequence analysis, in silico bioinformatic protocols and structural modeling predictions based on docking studies on binding capacity with PEXEL motifs processing in terms of binding and accessibility of export proteins. Results Cloning and sequence analysis for genetic diversity demonstrates PvPM-V-Ind (mutant) gene is highly conserved among all isolates from different geographical regions of India. Imperfect duplicate insertion types of mutations (SVSE from 246–249 AA and SLSE from 266–269 AA) were identified among all Indian isolates in comparison to P.vivax Sal-1 (PvPM-V-Sal 1) isolate. In silico bioinformatics interaction studies of PEXEL peptide and active enzyme reveal that PvPM-V-Ind (mutant) is only active in endoplasmic reticulum lumen and membrane embedding is essential for activation of plasmepsin V. Structural modeling predictions based on docking studies with PEXEL motif show significant variation in substrate protein binding of these imperfect mutations with data mined PEXEL sequences. The predicted variation in the docking score and interacting amino acids of PvPM-V-Ind (mutant) proteins with PEXEL and lopinavir suggests a modulation in the activity of PvPM-V in terms of binding and accessibility at these sites. Conclusion/Significance Our functional modeled validation of PvPM-V-Ind (mutant) imperfect duplicate insertions with data mined PEXEL sequences leading to altered binding and substrate accessibility of the enzyme makes it a plausible target to investigate export mechanisms for in silico virtual screening and novel pharmacophore designing. PMID:23555891

A Chemokine Receptor CXCR2 Macromolecular Complex Regulates Neutrophil Functions in Inflammatory Diseases*

PubMed Central

Wu, Yanning; Wang, Shuo; Farooq, Shukkur M.; Castelvetere, Marcello P.; Hou, Yuning; Gao, Ji-Liang; Navarro, Javier V.; Oupicky, David; Sun, Fei; Li, Chunying

2012-01-01

Inflammation plays an important role in a wide range of human diseases such as ischemia-reperfusion injury, arteriosclerosis, cystic fibrosis, inflammatory bowel disease, etc. Neutrophilic accumulation in the inflamed tissues is an essential component of normal host defense against infection, but uncontrolled neutrophilic infiltration can cause progressive damage to the tissue epithelium. The CXC chemokine receptor CXCR2 and its specific ligands have been reported to play critical roles in the pathophysiology of various inflammatory diseases. However, it is unclear how CXCR2 is coupled specifically to its downstream signaling molecules and modulates cellular functions of neutrophils. Here we show that the PDZ scaffold protein NHERF1 couples CXCR2 to its downstream effector phospholipase C (PLC)-β2, forming a macromolecular complex, through a PDZ-based interaction. We assembled a macromolecular complex of CXCR2·NHERF1·PLC-β2 in vitro, and we also detected such a complex in neutrophils by co-immunoprecipitation. We further observed that the CXCR2-containing macromolecular complex is critical for the CXCR2-mediated intracellular calcium mobilization and the resultant migration and infiltration of neutrophils, as disrupting the complex with a cell permeant CXCR2-specific peptide (containing the PDZ motif) inhibited intracellular calcium mobilization, chemotaxis, and transepithelial migration of neutrophils. Taken together, our data demonstrate a critical role of the PDZ-dependent CXCR2 macromolecular signaling complex in regulating neutrophil functions and suggest that targeting the CXCR2 multiprotein complex may represent a novel therapeutic strategy for certain inflammatory diseases. PMID:22203670

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shaw, Debra J.; Morse, Robert; Todd, Adrian G.

The Ewing Sarcoma (EWS) protein is a ubiquitously expressed RNA processing factor that localises predominantly to the nucleus. However, the mechanism through which EWS enters the nucleus remains unclear, with differing reports identifying three separate import signals within the EWS protein. Here we have utilized a panel of truncated EWS proteins to clarify the reported nuclear localisation signals. We describe three C-terminal domains that are important for efficient EWS nuclear localization: (1) the third RGG-motif; (2) the last 10 amino acids (known as the PY-import motif); and (3) the zinc-finger motif. Although these three domains are involved in nuclear import,more » they are not independently capable of driving the efficient import of a GFP-moiety. However, collectively they form a complex tripartite signal that efficiently drives GFP-import into the nucleus. This study helps clarify the EWS import signal, and the identification of the involvement of both the RGG- and zinc-finger motifs has wide reaching implications.« less

Retention mechanisms for ER and Golgi membrane proteins.

PubMed

Gao, Caiji; Cai, Yi; Wang, Yejun; Kang, Byung-Ho; Aniento, Fernando; Robinson, David G; Jiang, Liwen

2014-08-01

Unless there are mechanisms to selectively retain membrane proteins in the endoplasmic reticulum (ER) or in the Golgi apparatus, they automatically proceed downstream to the plasma or vacuole membranes. Two types of coat protein complex I (COPI)-interacting motifs in the cytosolic tails of membrane proteins seem to facilitate membrane retention in the early secretory pathway of plants: a dilysine (KKXX) motif (which is typical of p24 proteins) for the ER and a KXE/D motif (which occurs in the Arabidopsis endomembrane protein EMP12) for the Golgi apparatus. The KXE/D motif is highly conserved in all eukaryotic EMPs and is additionally present in hundreds of other proteins of unknown subcellular localization and function. This novel signal may represent a new general mechanism for Golgi targeting and the retention of polytopic integral membrane proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rajan, Rakhi; Taneja, Bhupesh; Mondragón, Alfonso

Topoisomerase V is an archaeal type I topoisomerase that is unique among topoisomerases due to presence of both topoisomerase and DNA repair activities in the same protein. It is organized as an N-terminal topoisomerase domain followed by 24 tandem helix-hairpin-helix (HhH) motifs. Structural studies have shown that the active site is buried by the (HhH) motifs. Here we show that the N-terminal domain can relax DNA in the absence of any HhH motifs and that the HhH motifs are required for stable protein-DNA complex formation. Crystal structures of various topoisomerase V fragments show changes in the relative orientation of themore » domains mediated by a long bent linker helix, and these movements are essential for the DNA to enter the active site. Phosphate ions bound to the protein near the active site helped model DNA in the topoisomerase domain and show how topoisomerase V may interact with DNA.« less

Modulation of chaperone function and cochaperone interaction by novobiocin in the C-terminal domain of Hsp90: evidence that coumarin antibiotics disrupt Hsp90 dimerization.

PubMed

Allan, Rudi K; Mok, Danny; Ward, Bryan K; Ratajczak, Thomas

2006-03-17

The C-terminal domain of Hsp90 displays independent chaperone activity, mediates dimerization, and contains the MEEVD motif essential for interaction with tetratricopeptide repeat-containing immunophilin cochaperones assembled in mature steroid receptor complexes. An alpha-helical region, upstream of the MEEVD peptide, helps form the dimerization interface and includes a hydrophobic microdomain that contributes to the Hsp90 interaction with the immunophilin cochaperones and corresponds to the binding site for novobiocin, a coumarin-related Hsp90 inhibitor. Mutation of selected residues within the hydrophobic microdomain significantly impacted the chaperone function of a recombinant C-terminal Hsp90 fragment and novobiocin inhibited wild-type chaperone activity. Prior incubation of the Hsp90 fragment with novobiocin led to a direct blockade of immunophilin cochaperone binding. However, the drug had little influence on the pre-formed Hsp90-immunophilin complex, suggesting that bound cochaperones mask the novobiocin-binding site. We observed a differential effect of the drug on Hsp90-immunophilin interaction, suggesting that the immunophilins make distinct contacts within the C-terminal domain to specifically modulate Hsp90 function. Novobiocin also precluded the interaction of full-length Hsp90 with the p50(cdc37) cochaperone, which targets the N-terminal nucleotide-binding domain, and is prevalent in Hsp90 complexes with protein kinase substrates. Novobiocin therefore acts locally and allosterically to induce conformational changes within multiple regions of the Hsp90 protein. We provide evidence that coumermycin A1, a coumarin structurally related to novobiocin, interferes with dimerization of the Hsp90 C-terminal domain. Coumarin-based inhibitors then may antagonize Hsp90 function by inducing a conformation favoring separation of the C-terminal domains and release of substrate.

Tandem hnRNP A1 RNA recognition motifs act in concert to repress the splicing of survival motor neuron exon 7

PubMed Central

Beusch, Irene; Barraud, Pierre; Moursy, Ahmed; Cléry, Antoine; Allain, Frédéric Hai-Trieu

2017-01-01

HnRNP A1 regulates many alternative splicing events by the recognition of splicing silencer elements. Here, we provide the solution structures of its two RNA recognition motifs (RRMs) in complex with short RNA. In addition, we show by NMR that both RRMs of hnRNP A1 can bind simultaneously to a single bipartite motif of the human intronic splicing silencer ISS-N1, which controls survival of motor neuron exon 7 splicing. RRM2 binds to the upstream motif and RRM1 to the downstream motif. Combining the insights from the structure with in cell splicing assays we show that the architecture and organization of the two RRMs is essential to hnRNP A1 function. The disruption of the inter-RRM interaction or the loss of RNA binding capacity of either RRM impairs splicing repression by hnRNP A1. Furthermore, both binding sites within the ISS-N1 are important for splicing repression and their contributions are cumulative rather than synergistic. DOI: http://dx.doi.org/10.7554/eLife.25736.001 PMID:28650318

Programmable assembly of nanoarchitectures using genetically engineered viruses.

PubMed

Huang, Yu; Chiang, Chung-Yi; Lee, Soo Kwan; Gao, Yan; Hu, Evelyn L; De Yoreo, James; Belcher, Angela M

2005-07-01

Biological systems possess inherent molecular recognition and self-assembly capabilities and are attractive templates for constructing complex material structures with molecular precision. Here we report the assembly of various nanoachitectures including nanoparticle arrays, hetero-nanoparticle architectures, and nanowires utilizing highly engineered M13 bacteriophage as templates. The genome of M13 phage can be rationally engineered to produce viral particles with distinct substrate-specific peptides expressed on the filamentous capsid and the ends, providing a generic template for programmable assembly of complex nanostructures. Phage clones with gold-binding motifs on the capsid and streptavidin-binding motifs at one end are created and used to assemble Au and CdSe nanocrytals into ordered one-dimensional arrays and more complex geometries. Initial studies show such nanoparticle arrays can further function as templates to nucleate highly conductive nanowires that are important for addressing/interconnecting individual nanostructures.

Local complexity predicts global synchronization of hierarchically networked oscillators

NASA Astrophysics Data System (ADS)

Xu, Jin; Park, Dong-Ho; Jo, Junghyo

2017-07-01

We study the global synchronization of hierarchically-organized Stuart-Landau oscillators, where each subsystem consists of three oscillators with activity-dependent couplings. We considered all possible coupling signs between the three oscillators, and found that they can generate different numbers of phase attractors depending on the network motif. Here, the subsystems are coupled through mean activities of total oscillators. Under weak inter-subsystem couplings, we demonstrate that the synchronization between subsystems is highly correlated with the number of attractors in uncoupled subsystems. Among the network motifs, perfect anti-symmetric ones are unique to generate both single and multiple attractors depending on the activities of oscillators. The flexible local complexity can make global synchronization controllable.

Zn2+ selectively stabilizes FdU-substituted DNA through a unique major groove binding motif

PubMed Central

Ghosh, Supratim; Salsbury, Freddie R.; Horita, David A.; Gmeiner, William H.

2011-01-01

We report, based on semi-empirical calculations, that Zn2+ binds duplex DNA containing consecutive FdU–dA base pairs in the major groove with distorted trigonal bipyramidal geometry. In this previously uncharacterized binding motif, O4 and F5 on consecutive FdU are axial ligands while three water molecules complete the coordination sphere. NMR spectroscopy confirmed Zn2+ complexation occurred with maintenance of base pairing while a slight hypsochromic shift in circular dichroism (CD) spectra indicated moderate structural distortion relative to B-form DNA. Zn2+ complexation inhibited ethidium bromide (EtBr) intercalation and stabilized FdU-substituted duplex DNA (ΔTm > 15°C). Mg2+ neither inhibited EtBr complexation nor had as strong of a stabilizing effect. DNA sequences that did not contain consecutive FdU were not stabilized by Zn2+. A lipofectamine preparation of the Zn2+–DNA complex displayed enhanced cytotoxicity toward prostate cancer cells relative to the individual components prepared as lipofectamine complexes indicating the potential utility of Zn2+–DNA complexes for cancer treatment. PMID:21296761

KIR Polymorphisms Modulate Peptide-Dependent Binding to an MHC Class I Ligand with a Bw6 Motif

PubMed Central

Colantonio, Arnaud D.; Bimber, Benjamin N.; Neidermyer, William J.; Reeves, R. Keith; Alter, Galit; Altfeld, Marcus; Johnson, R. Paul; Carrington, Mary; O'Connor, David H.; Evans, David T.

2011-01-01

Molecular interactions between killer immunoglobulin-like receptors (KIRs) and their MHC class I ligands play a central role in the regulation of natural killer (NK) cell responses to viral pathogens and tumors. Here we identify Mamu-A1*00201 (Mamu-A*02), a common MHC class I molecule in the rhesus macaque with a canonical Bw6 motif, as a ligand for Mamu-KIR3DL05. Mamu-A1*00201 tetramers folded with certain SIV peptides, but not others, directly stained primary NK cells and Jurkat cells expressing multiple allotypes of Mamu-KIR3DL05. Differences in binding avidity were associated with polymorphisms in the D0 and D1 domains of Mamu-KIR3DL05, whereas differences in peptide-selectivity mapped to the D1 domain. The reciprocal exchange of the third predicted MHC class I-contact loop of the D1 domain switched the specificity of two Mamu-KIR3DL05 allotypes for different Mamu-A1*00201-peptide complexes. Consistent with the function of an inhibitory KIR, incubation of lymphocytes from Mamu-KIR3DL05+ macaques with target cells expressing Mamu-A1*00201 suppressed the degranulation of tetramer-positive NK cells. These observations reveal a previously unappreciated role for D1 polymorphisms in determining the selectivity of KIRs for MHC class I-bound peptides, and identify the first functional KIR-MHC class I interaction in the rhesus macaque. The modulation of KIR-MHC class I interactions by viral peptides has important implications to pathogenesis, since it suggests that the immunodeficiency viruses, and potentially other types of viruses and tumors, may acquire changes in epitopes that increase the affinity of certain MHC class I ligands for inhibitory KIRs to prevent the activation of specific NK cell subsets. PMID:21423672

Electrochemical, spectroscopic, and photophysical properties of structurally diverse polyazine-bridged Ru(II),Pt(II) and Os(II),Ru(II),Pt(II) supramolecular motifs.

PubMed

Knoll, Jessica D; Arachchige, Shamindri M; Wang, Guangbin; Rangan, Krishnan; Miao, Ran; Higgins, Samantha L H; Okyere, Benjamin; Zhao, Meihua; Croasdale, Paul; Magruder, Katherine; Sinclair, Brian; Wall, Candace; Brewer, Karen J

2011-09-19

Five new tetrametallic supramolecules of the motif [{(TL)(2)M(dpp)}(2)Ru(BL)PtCl(2)](6+) and three new trimetallic light absorbers [{(TL)(2)M(dpp)}(2)Ru(BL)](6+) (TL = bpy = 2,2'-bipyridine or phen = 1,10-phenanthroline; M = Ru(II) or Os(II); BL = dpp = 2,3-bis(2-pyridyl)pyrazine, dpq = 2,3-bis(2-pyridyl)quinoxaline, or bpm = 2,2'-bipyrimidine) were synthesized and their redox, spectroscopic, and photophysical properties investigated. The tetrametallic complexes couple a Pt(II)-based reactive metal center to Ru and/or Os light absorbers through two different polyazine BL to provide structural diversity and interesting resultant properties. The redox potential of the M(II/III) couple is modulated by M variation, with the terminal Ru(II/III) occurring at 1.58-1.61 V and terminal Os(II/III) couples at 1.07-1.18 V versus Ag/AgCl. [{(TL)(2)M(dpp)}(2)Ru(BL)](PF(6))(6) display terminal M(dπ)-based highest occupied molecular orbitals (HOMOs) with the dpp(π*)-based lowest unoccupied molecular orbital (LUMO) energy relatively unaffected by the nature of BL. The coupling of Pt to the BL results in orbital inversion with localization of the LUMO on the remote BL in the tetrametallic complexes, providing a lowest energy charge separated (CS) state with an oxidized terminal Ru or Os and spatially separated reduced BL. The complexes [{(TL)(2)M(dpp)}(2)Ru(BL)](6+) and [{(TL)(2)M(dpp)}(2)Ru(BL)PtCl(2)](6+) efficiently absorb light throughout the UV and visible regions with intense metal-to-ligand charge transfer (MLCT) transitions in the visible at about 540 nm (M = Ru) and 560 nm (M = Os) (ε ≈ 33,000-42,000 M(-1) cm(-1)) and direct excitation to the spin-forbidden (3)MLCT excited state in the Os complexes about 720 nm. All the trimetallic and tetrametallic Ru-based supramolecular systems emit from the terminal Ru(dπ)→dpp(π*) (3)MLCT state, λ(max)(em) ≈ 750 nm. The tetrametallic systems display complex excited state dynamics with quenching of the (3)MLCT emission at room temperature to populate the lowest-lying (3)CS state population of the emissive (3)MLCT state.

Reversible Redox Activity by Ion-pH Dually Modulated Duplex Formation of i-Motif DNA with Complementary G-DNA.

PubMed

Chang, Soyoung; Kilic, Tugba; Lee, Chang Kee; Avci, Huseyin; Bae, Hojae; Oskui, Shirin Mesbah; Jung, Sung Mi; Shin, Su Ryon; Kim, Seon Jeong

2018-04-08

The unique biological features of supramolecular DNA have led to an increasing interest in biomedical applications such as biosensors. We have developed an i-motif and G-rich DNA conjugated single-walled carbon nanotube hybrid materials, which shows reversible conformational switching upon external stimuli such as pH (5 and 8) and presence of ions (Li⁺ and K⁺). We observed reversible electrochemical redox activity upon external stimuli in a quick and robust manner. Given the ease and the robustness of this method, we believe that pH- and ion-driven reversible DNA structure transformations will be utilized for future applications for developing novel biosensors.

The Calcineurin Signaling Network Evolves Via Conserved Kinase–Phosphatase Modules That Transcend Substrate Identity

PubMed Central

Bodenmiller, Bernd; Wanka, Stefanie; Landry, Christian R.; Aebersold, Ruedi; Cyert, Martha S.

2014-01-01

Summary To define the first functional network for calcineurin, the conserved Ca2+/calmodulin-regulated phosphatase, we systematically identified its substrates in S. cerevisiae using phosphoproteomics and bioinformatics, followed by co-purification and dephosphorylation assays. This study establishes new calcineurin functions and reveals mechanisms that shape calcineurin network evolution. Analyses of closely related yeasts show that many proteins were recently recruited to the network by acquiring a calcineurin-recognition motif. Calcineurin substrates in yeast and mammals are distinct due to network rewiring but surprisingly are phosphorylated by similar kinases. We postulate that co-recognition of conserved substrate features, including phosphorylation and docking motifs, preserves calcineurin-kinase opposition during evolution. One example we document is a composite docking site that confers substrate recognition by both calcineurin and MAPK. We propose that conserved kinase-phosphatase pairs define the architecture of signaling networks and allow other connections between kinases and phosphatases to develop and establish common regulatory motifs in signaling networks. PMID:24930733

Rewiring MAP kinases in Saccharomyces cerevisiae to regulate novel targets through ubiquitination.

PubMed

Groves, Benjamin; Khakhar, Arjun; Nadel, Cory M; Gardner, Richard G; Seelig, Georg

2016-08-15

Evolution has often copied and repurposed the mitogen-activated protein kinase (MAPK) signaling module. Understanding how connections form during evolution, in disease and across individuals requires knowledge of the basic tenets that govern kinase-substrate interactions. We identify criteria sufficient for establishing regulatory links between a MAPK and a non-native substrate. The yeast MAPK Fus3 and human MAPK ERK2 can be functionally redirected if only two conditions are met: the kinase and substrate contain matching interaction domains and the substrate includes a phospho-motif that can be phosphorylated by the kinase and recruit a downstream effector. We used a panel of interaction domains and phosphorylation-activated degradation motifs to demonstrate modular and scalable retargeting. We applied our approach to reshape the signaling behavior of an existing kinase pathway. Together, our results demonstrate that a MAPK can be largely defined by its interaction domains and compatible phospho-motifs and provide insight into how MAPK-substrate connections form.

Analysis of osmotin, a PR protein as metabolic modulator in plants

PubMed Central

Abdin, Malik Zainul; Kiran, Usha; Alam, Afshar

2011-01-01

Osmotin is an abundant cationic multifunctional protein discovered in cells of tobacco (Nicotiana tabacum L. var Wisconsin 38) adapted to an environment of low osmotic potential. Beside its role as osmoregulator, it provides plants protection from pathogens, hence also placed in the PRP family of proteins. The osmotin induced proline accumulation has been reported to confer tolerance against both biotic and abiotic stresses in plants including transgenic tomato and strawberry overexpressing osmotin gene. The exact mechanism of induction of proline by osmotin is however, not known till date. These observations have led us to hypothesize that osmotin could be regulating these plant responses through its involvement either as transcription factor, cell signal pathway modulator or both in plants. We have therefore, undertaken the present investigation to analyze the osmotin protein as transcription factor using bioinformatics tools. The results of available online DNA binding motif search programs revealed that osmotin does not contain DNAbinding motifs. The alignment results of osmotin protein with the protein sequence from DATF showed the homology in the range of 0-20%, suggesting that it might not contain a DNA binding motif. Further to find unique DNA-binding domain, the superimposition of osmotin 3D structure on modeled Arabidopsis transcription factors using Chimera also suggested absence of the same. However, evidence implicating osmotin in cell signaling were found during the study. With these results, we therefore, concluded that osmotin is not a transcription factor, but regulating plant responses to biotic and abiotic stresses through cell signaling. PMID:21383921

Analysis of osmotin, a PR protein as metabolic modulator in plants.

PubMed

Abdin, Malik Zainul; Kiran, Usha; Alam, Afshar

2011-01-22

Osmotin is an abundant cationic multifunctional protein discovered in cells of tobacco (Nicotiana tabacum L. var Wisconsin 38) adapted to an environment of low osmotic potential. Beside its role as osmoregulator, it provides plants protection from pathogens, hence also placed in the PRP family of proteins. The osmotin induced proline accumulation has been reported to confer tolerance against both biotic and abiotic stresses in plants including transgenic tomato and strawberry overexpressing osmotin gene. The exact mechanism of induction of proline by osmotin is however, not known till date. These observations have led us to hypothesize that osmotin could be regulating these plant responses through its involvement either as transcription factor, cell signal pathway modulator or both in plants. We have therefore, undertaken the present investigation to analyze the osmotin protein as transcription factor using bioinformatics tools. The results of available online DNA binding motif search programs revealed that osmotin does not contain DNAbinding motifs. The alignment results of osmotin protein with the protein sequence from DATF showed the homology in the range of 0-20%, suggesting that it might not contain a DNA binding motif. Further to find unique DNA-binding domain, the superimposition of osmotin 3D structure on modeled Arabidopsis transcription factors using Chimera also suggested absence of the same. However, evidence implicating osmotin in cell signaling were found during the study. With these results, we therefore, concluded that osmotin is not a transcription factor, but regulating plant responses to biotic and abiotic stresses through cell signaling.

PHEX Mimetic (SPR4-Peptide) Corrects and Improves HYP and Wild Type Mice Energy-Metabolism

PubMed Central

Zelenchuk, Lesya V.; Hedge, Anne-Marie; Rowe, Peter S. N.

2014-01-01

Context PHEX or DMP1 mutations cause hypophosphatemic-rickets and altered energy metabolism. PHEX binds to DMP1-ASARM-motif to form a complex with α5β3 integrin that suppresses FGF23 expression. ASARM-peptides increase FGF23 by disrupting the PHEX-DMP1-Integrin complex. We used a 4.2 kDa peptide (SPR4) that binds to ASARM-peptide/motif to study the DMP1-PHEX interaction and to assess SPR4 for the treatment of energy metabolism defects in HYP and potentially other bone-mineral disorders. Design Subcutaneously transplanted osmotic pumps were used to infuse SPR4-peptide or vehicle (VE) into wild-type mice (WT) and HYP-mice (PHEX mutation) for 4 weeks. Results SPR4 partially corrected HYP mice hypophosphatemia and increased serum 1.25(OH)2D3. Serum FGF23 remained high and PTH was unaffected. WT-SPR4 mice developed hypophosphatemia and hypercalcemia with increased PTH, FGF23 and 1.25(OH)2D3. SPR4 increased GAPDH HYP-bone expression 60× and corrected HYP-mice hyperglycemia and hypoinsulinemia. HYP-VE serum uric-acid (UA) levels were reduced and SPR4 infusion suppressed UA levels in WT-mice but not HYP-mice. SPR4 altered leptin, adiponectin, and sympathetic-tone and increased the fat mass/weight ratio for HYP and WT mice. Expression of perlipin-2 a gene involved in obesity was reduced in HYP-VE and WT-SPR4 mice but increased in HYP-SPR4 mice. Also, increased expression of two genes that inhibit insulin-signaling, ENPP1 and ESP, occurred with HYP-VE mice. In contrast, SPR4 reduced expression of both ENPP1 and ESP in WT mice and suppressed ENPP1 in HYP mice. Increased expression of FAM20C and sclerostin occurred with HYP-VE mice. SPR4 suppressed expression of FAM20C and sclerostin in HYP and WT mice. Conclusions ASARM peptides and motifs are physiological substrates for PHEX and modulate osteocyte PHEX-DMP1-α5β3-integrin interactions and thereby FGF23 expression. These interactions also provide a nexus that regulates bone and energy metabolism. SPR4 suppression of sclerostin and/or sequestration of ASARM-peptides improves energy metabolism and may have utility for treating familial rickets, osteoporosis, obesity and diabetes. PMID:24839967

Structural basis for the binding of tryptophan-based motifs by δ-COP

PubMed Central

Suckling, Richard J.; Poon, Pak Phi; Travis, Sophie M.; Majoul, Irina V.; Hughson, Frederick M.; Evans, Philip R.; Duden, Rainer; Owen, David J.

2015-01-01

Coatomer consists of two subcomplexes: the membrane-targeting, ADP ribosylation factor 1 (Arf1):GTP-binding βγδζ-COP F-subcomplex, which is related to the adaptor protein (AP) clathrin adaptors, and the cargo-binding αβ’ε-COP B-subcomplex. We present the structure of the C-terminal μ-homology domain of the yeast δ-COP subunit in complex with the WxW motif from its binding partner, the endoplasmic reticulum-localized Dsl1 tether. The motif binds at a site distinct from that used by the homologous AP μ subunits to bind YxxΦ cargo motifs with its two tryptophan residues sitting in compatible pockets. We also show that the Saccharomyces cerevisiae Arf GTPase-activating protein (GAP) homolog Gcs1p uses a related WxxF motif at its extreme C terminus to bind to δ-COP at the same site in the same way. Mutations designed on the basis of the structure in conjunction with isothermal titration calorimetry confirm the mode of binding and show that mammalian δ-COP binds related tryptophan-based motifs such as that from ArfGAP1 in a similar manner. We conclude that δ-COP subunits bind Wxn(1–6)[WF] motifs within unstructured regions of proteins that influence the lifecycle of COPI-coated vesicles; this conclusion is supported by the observation that, in the context of a sensitizing domain deletion in Dsl1p, mutating the tryptophan-based motif-binding site in yeast causes defects in both growth and carboxypeptidase Y trafficking/processing. PMID:26578768

Modeling gene regulatory network motifs using statecharts

PubMed Central

2012-01-01

Background Gene regulatory networks are widely used by biologists to describe the interactions among genes, proteins and other components at the intra-cellular level. Recently, a great effort has been devoted to give gene regulatory networks a formal semantics based on existing computational frameworks. For this purpose, we consider Statecharts, which are a modular, hierarchical and executable formal model widely used to represent software systems. We use Statecharts for modeling small and recurring patterns of interactions in gene regulatory networks, called motifs. Results We present an improved method for modeling gene regulatory network motifs using Statecharts and we describe the successful modeling of several motifs, including those which could not be modeled or whose models could not be distinguished using the method of a previous proposal. We model motifs in an easy and intuitive way by taking advantage of the visual features of Statecharts. Our modeling approach is able to simulate some interesting temporal properties of gene regulatory network motifs: the delay in the activation and the deactivation of the "output" gene in the coherent type-1 feedforward loop, the pulse in the incoherent type-1 feedforward loop, the bistability nature of double positive and double negative feedback loops, the oscillatory behavior of the negative feedback loop, and the "lock-in" effect of positive autoregulation. Conclusions We present a Statecharts-based approach for the modeling of gene regulatory network motifs in biological systems. The basic motifs used to build more complex networks (that is, simple regulation, reciprocal regulation, feedback loop, feedforward loop, and autoregulation) can be faithfully described and their temporal dynamics can be analyzed. PMID:22536967

An additional function of the rough endoplasmic reticulum protein complex prolyl 3-hydroxylase 1·cartilage-associated protein·cyclophilin B: the CXXXC motif reveals disulfide isomerase activity in vitro.

PubMed

Ishikawa, Yoshihiro; Bächinger, Hans Peter

2013-11-01

Collagen biosynthesis occurs in the rough endoplasmic reticulum, and many molecular chaperones and folding enzymes are involved in this process. The folding mechanism of type I procollagen has been well characterized, and protein disulfide isomerase (PDI) has been suggested as a key player in the formation of the correct disulfide bonds in the noncollagenous carboxyl-terminal and amino-terminal propeptides. Prolyl 3-hydroxylase 1 (P3H1) forms a hetero-trimeric complex with cartilage-associated protein and cyclophilin B (CypB). This complex is a multifunctional complex acting as a prolyl 3-hydroxylase, a peptidyl prolyl cis-trans isomerase, and a molecular chaperone. Two major domains are predicted from the primary sequence of P3H1: an amino-terminal domain and a carboxyl-terminal domain corresponding to the 2-oxoglutarate- and iron-dependent dioxygenase domains similar to the α-subunit of prolyl 4-hydroxylase and lysyl hydroxylases. The amino-terminal domain contains four CXXXC sequence repeats. The primary sequence of cartilage-associated protein is homologous to the amino-terminal domain of P3H1 and also contains four CXXXC sequence repeats. However, the function of the CXXXC sequence repeats is not known. Several publications have reported that short peptides containing a CXC or a CXXC sequence show oxido-reductase activity similar to PDI in vitro. We hypothesize that CXXXC motifs have oxido-reductase activity similar to the CXXC motif in PDI. We have tested the enzyme activities on model substrates in vitro using a GCRALCG peptide and the P3H1 complex. Our results suggest that this complex could function as a disulfide isomerase in the rough endoplasmic reticulum.

Crystallization and preliminary crystallographic analysis of calcium-binding protein-2 from Entamoeba histolytica and its complexes with strontium and the IQ1 motif of myosin V

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gourinath, S., E-mail: sgourinath@mail.jnu.ac.in; Padhan, Narendra; Alam, Neelima

2005-04-01

Calcium-binding protein-2 (EhCaBP2) crystals were grown using MPD as a precipitant. EhCaBP2 also crystallized in complex with strontium (replacing calcium) at similar conditions. Preliminary data for EhCaBP2 crystals in complex with an IQ motif are also reported. Calcium plays a pivotal role in the pathogenesis of amoebiasis, a major disease caused by Entamoeba histolytica. Two domains with four canonical EF-hand-containing calcium-binding proteins (CaBPs) have been identified from E. histolytica. Even though they have very high sequence similarity, these bind to different target proteins in a Ca{sup 2+}-dependent manner, leading to different functional pathways. Calcium-binding protein-2 (EhCaBP2) crystals were grown usingmore » MPD as a precipitant. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 111.74, b = 68.83, c = 113.25 Å, β = 116.7°. EhCaBP2 also crystallized in complex with strontium (replacing calcium) at similar conditions. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 69.18, b = 112.03, c = 93.42 Å, β = 92.8°. Preliminary data for EhCaBP2 crystals in complex with an IQ motif are also reported. This complex was crystallized with MPD and ethanol as precipitating agents. These crystals belong to space group P2{sub 1}, with unit-cell parameters a = 60.5, b = 69.86, c = 86.5 Å, β = 97.9°.« less

Disintegrins from Hematophagous Sources

PubMed Central

Assumpcao, Teresa C. F.; Ribeiro, José M. C.; Francischetti, Ivo M. B.

2012-01-01

Bloodsucking arthropods are a rich source of salivary molecules (sialogenins) which inhibit platelet aggregation, neutrophil function and angiogenesis. Here we review the literature on salivary disintegrins and their targets. Disintegrins were first discovered in snake venoms, and were instrumental in our understanding of integrin function and also for the development of anti-thrombotic drugs. In hematophagous animals, most disintegrins described so far have been discovered in the salivary gland of ticks and leeches. A limited number have also been found in hookworms and horseflies, and none identified in mosquitoes or sand flies. The vast majority of salivary disintegrins reported display a RGD motif and were described as platelet aggregation inhibitors, and few others as negative modulator of neutrophil or endothelial cell functions. This notably low number of reported disintegrins is certainly an underestimation of the actual complexity of this family of proteins in hematophagous secretions. Therefore an algorithm was created in order to identify the tripeptide motifs RGD, KGD, VGD, MLD, KTS, RTS, WGD, or RED (flanked by cysteines) in sialogenins deposited in GenBank database. The search included sequences from various blood-sucking animals such as ticks (e.g., Ixodes sp., Argas sp., Rhipicephalus sp., Amblyommasp.), tabanids (e.g., Tabanus sp.), bugs (e.g., Triatoma sp., Rhodnius prolixus), mosquitoes (e.g., Anopheles sp., Aedes sp., Culex sp.), sand flies (e.g., Lutzomyia sp., Phlebotomus sp.), leeches (e.g., Macrobdella sp., Placobdella sp.) and worms (e.g., Ancylostoma sp.). This approach allowed the identification of a remarkably high number of novel putative sialogenins with tripeptide motifs typical of disintegrins (>450 sequences) whose biological activity remains to be verified. This database is accessible online as a hyperlinked worksheet and displays biochemical, taxonomic, and gene ontology aspects for each putative disintegrin. It is also freely available for download (right click with the mouse) at links http://exon.niaid.nih.gov/transcriptome/RGD/RGD-Peps-WEB.xlsx (web version) and http://exon.niaid.nih.gov/transcriptome/RGD/RGD-sialogenins.zip (stand alone version). PMID:22778902

Disintegrins from hematophagous sources.

PubMed

Assumpcao, Teresa C F; Ribeiro, José M C; Francischetti, Ivo M B

2012-05-01

Bloodsucking arthropods are a rich source of salivary molecules (sialogenins) which inhibit platelet aggregation, neutrophil function and angiogenesis. Here we review the literature on salivary disintegrins and their targets. Disintegrins were first discovered in snake venoms, and were instrumental in our understanding of integrin function and also for the development of anti-thrombotic drugs. In hematophagous animals, most disintegrins described so far have been discovered in the salivary gland of ticks and leeches. A limited number have also been found in hookworms and horseflies, and none identified in mosquitoes or sand flies. The vast majority of salivary disintegrins reported display a RGD motif and were described as platelet aggregation inhibitors, and few others as negative modulator of neutrophil or endothelial cell functions. This notably low number of reported disintegrins is certainly an underestimation of the actual complexity of this family of proteins in hematophagous secretions. Therefore an algorithm was created in order to identify the tripeptide motifs RGD, KGD, VGD, MLD, KTS, RTS, WGD, or RED (flanked by cysteines) in sialogenins deposited in GenBank database. The search included sequences from various blood-sucking animals such as ticks (e.g., Ixodes sp., Argas sp., Rhipicephalus sp., Amblyommasp.), tabanids (e.g., Tabanus sp.), bugs (e.g., Triatoma sp., Rhodnius prolixus), mosquitoes (e.g., Anopheles sp., Aedes sp., Culex sp.), sand flies (e.g., Lutzomyia sp., Phlebotomus sp.), leeches (e.g., Macrobdella sp., Placobdella sp.) and worms (e.g., Ancylostoma sp.). This approach allowed the identification of a remarkably high number of novel putative sialogenins with tripeptide motifs typical of disintegrins (>450 sequences) whose biological activity remains to be verified. This database is accessible online as a hyperlinked worksheet and displays biochemical, taxonomic, and gene ontology aspects for each putative disintegrin. It is also freely available for download (right click with the mouse) at links http://exon.niaid.nih.gov/transcriptome/RGD/RGD-Peps-WEB.xlsx (web version) and http://exon.niaid.nih.gov/transcriptome/RGD/RGD-sialogenins.zip (stand alone version).

The mRNA-stabilizing factor HuR protein is targeted by β-TrCP protein for degradation in response to glycolysis inhibition.

PubMed

Chu, Po-Chen; Chuang, Hsiao-Ching; Kulp, Samuel K; Chen, Ching-Shih

2012-12-21

The mRNA-stabilizing protein HuR acts a stress response protein whose function and/or protein stability are modulated by diverse stress stimuli through posttranslational modifications. Here, we report a novel mechanism by which metabolic stress facilitates proteasomal degradation of HuR in cancer cells. In response to the glucose transporter inhibitor CG-5, HuR translocates to the cytoplasm, where it is targeted by the ubiquitin E3 ligase β-TrCP1 for degradation. The cytoplasmic localization of HuR is facilitated by PKCα-mediated phosphorylation at Ser-318 as the Ser-318 → alanine substitution abolishes the ability of the resulting HuR to bind PKCα and to undergo nuclear export. The mechanistic link between β-TrCP1 and HuR degradation was supported by the ability of ectopically expressed β-TrCP1 to mimic CG-5 to promote HuR degradation and by the protective effect of dominant negative inhibition of β-TrCP1 on HuR ubiquitination and degradation. Substrate targeting of HuR by β-TrCP1 was further verified by coimmunoprecipitation and in vitro GST pull-down assays and by the identification of a β-TrCP1 recognition site. Although HuR does not contain a DSG destruction motif, we obtained evidence that β-TrCP1 recognizes an unconventional motif, (296)EEAMAIAS(304), in the RNA recognition motif 3. Furthermore, mutational analysis indicates that IKKα-dependent phosphorylation at Ser-304 is crucial to the binding of HuR to β-TrCP1. Mechanistically, this HuR degradation pathway differs from that reported for heat shock and hypoxia, which underlies the complexity in the regulation of HuR turnover under different stress stimuli. The ability of glycolysis inhibitors to target the expression of oncogenic proteins through HuR degradation might foster novel strategies for cancer therapy.

The mRNA-stabilizing Factor HuR Protein Is Targeted by β-TrCP Protein for Degradation in Response to Glycolysis Inhibition*

PubMed Central

Chu, Po-Chen; Chuang, Hsiao-Ching; Kulp, Samuel K.; Chen, Ching-Shih

2012-01-01

The mRNA-stabilizing protein HuR acts a stress response protein whose function and/or protein stability are modulated by diverse stress stimuli through posttranslational modifications. Here, we report a novel mechanism by which metabolic stress facilitates proteasomal degradation of HuR in cancer cells. In response to the glucose transporter inhibitor CG-5, HuR translocates to the cytoplasm, where it is targeted by the ubiquitin E3 ligase β-TrCP1 for degradation. The cytoplasmic localization of HuR is facilitated by PKCα-mediated phosphorylation at Ser-318 as the Ser-318 → alanine substitution abolishes the ability of the resulting HuR to bind PKCα and to undergo nuclear export. The mechanistic link between β-TrCP1 and HuR degradation was supported by the ability of ectopically expressed β-TrCP1 to mimic CG-5 to promote HuR degradation and by the protective effect of dominant negative inhibition of β-TrCP1 on HuR ubiquitination and degradation. Substrate targeting of HuR by β-TrCP1 was further verified by coimmunoprecipitation and in vitro GST pull-down assays and by the identification of a β-TrCP1 recognition site. Although HuR does not contain a DSG destruction motif, we obtained evidence that β-TrCP1 recognizes an unconventional motif, 296EEAMAIAS304, in the RNA recognition motif 3. Furthermore, mutational analysis indicates that IKKα-dependent phosphorylation at Ser-304 is crucial to the binding of HuR to β-TrCP1. Mechanistically, this HuR degradation pathway differs from that reported for heat shock and hypoxia, which underlies the complexity in the regulation of HuR turnover under different stress stimuli. The ability of glycolysis inhibitors to target the expression of oncogenic proteins through HuR degradation might foster novel strategies for cancer therapy. PMID:23115237

Use of an Improved Matching Algorithm to Select Scaffolds for Enzyme Design Based on a Complex Active Site Model.

PubMed

Huang, Xiaoqiang; Xue, Jing; Lin, Min; Zhu, Yushan

2016-01-01

Active site preorganization helps native enzymes electrostatically stabilize the transition state better than the ground state for their primary substrates and achieve significant rate enhancement. In this report, we hypothesize that a complex active site model for active site preorganization modeling should help to create preorganized active site design and afford higher starting activities towards target reactions. Our matching algorithm ProdaMatch was improved by invoking effective pruning strategies and the native active sites for ten scaffolds in a benchmark test set were reproduced. The root-mean squared deviations between the matched transition states and those in the crystal structures were < 1.0 Å for the ten scaffolds, and the repacking calculation results showed that 91% of the hydrogen bonds within the active sites are recovered, indicating that the active sites can be preorganized based on the predicted positions of transition states. The application of the complex active site model for de novo enzyme design was evaluated by scaffold selection using a classic catalytic triad motif for the hydrolysis of p-nitrophenyl acetate. Eighty scaffolds were identified from a scaffold library with 1,491 proteins and four scaffolds were native esterase. Furthermore, enzyme design for complicated substrates was investigated for the hydrolysis of cephalexin using scaffold selection based on two different catalytic motifs. Only three scaffolds were identified from the scaffold library by virtue of the classic catalytic triad-based motif. In contrast, 40 scaffolds were identified using a more flexible, but still preorganized catalytic motif, where one scaffold corresponded to the α-amino acid ester hydrolase that catalyzes the hydrolysis and synthesis of cephalexin. Thus, the complex active site modeling approach for de novo enzyme design with the aid of the improved ProdaMatch program is a promising approach for the creation of active sites with high catalytic efficiencies towards target reactions.

Sequence-Specific Targeting of Dosage Compensation in Drosophila Favors an Active Chromatin Context

PubMed Central

Gelbart, Marnie; Tolstorukov, Michael Y.; Plachetka, Annette; Kharchenko, Peter V.; Jung, Youngsook L.; Gorchakov, Andrey A.; Larschan, Erica; Gu, Tingting; Minoda, Aki; Riddle, Nicole C.; Schwartz, Yuri B.; Elgin, Sarah C. R.; Karpen, Gary H.; Pirrotta, Vincenzo; Kuroda, Mitzi I.; Park, Peter J.

2012-01-01

The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at “entry sites” that contain a consensus sequence motif (“MSL recognition element” or MRE). However, this motif is only ∼2 fold enriched on X, and only a fraction of the motifs on X are initially targeted. Here we ask whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells (which contain MSL complex) and female Kc cells (which lack the complex), we find that the presence of active chromatin modifications, together with an elevated local GC content in the surrounding sequences, has strong predictive value for functional MSL entry sites, independent of MSL binding. We tested these sites for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. We show that ectopic MSL expression in Kc cells leads to H4K16 acetylation around these sites and a relative increase in X chromosome transcription. Collectively, our results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. Our analysis can serve as a model for identifying chromatin and local sequence features that may contribute to selection of functional protein binding sites in the genome. PMID:22570616

Use of an Improved Matching Algorithm to Select Scaffolds for Enzyme Design Based on a Complex Active Site Model

PubMed Central

Huang, Xiaoqiang; Xue, Jing; Lin, Min; Zhu, Yushan

2016-01-01

Active site preorganization helps native enzymes electrostatically stabilize the transition state better than the ground state for their primary substrates and achieve significant rate enhancement. In this report, we hypothesize that a complex active site model for active site preorganization modeling should help to create preorganized active site design and afford higher starting activities towards target reactions. Our matching algorithm ProdaMatch was improved by invoking effective pruning strategies and the native active sites for ten scaffolds in a benchmark test set were reproduced. The root-mean squared deviations between the matched transition states and those in the crystal structures were < 1.0 Å for the ten scaffolds, and the repacking calculation results showed that 91% of the hydrogen bonds within the active sites are recovered, indicating that the active sites can be preorganized based on the predicted positions of transition states. The application of the complex active site model for de novo enzyme design was evaluated by scaffold selection using a classic catalytic triad motif for the hydrolysis of p-nitrophenyl acetate. Eighty scaffolds were identified from a scaffold library with 1,491 proteins and four scaffolds were native esterase. Furthermore, enzyme design for complicated substrates was investigated for the hydrolysis of cephalexin using scaffold selection based on two different catalytic motifs. Only three scaffolds were identified from the scaffold library by virtue of the classic catalytic triad-based motif. In contrast, 40 scaffolds were identified using a more flexible, but still preorganized catalytic motif, where one scaffold corresponded to the α-amino acid ester hydrolase that catalyzes the hydrolysis and synthesis of cephalexin. Thus, the complex active site modeling approach for de novo enzyme design with the aid of the improved ProdaMatch program is a promising approach for the creation of active sites with high catalytic efficiencies towards target reactions. PMID:27243223

The RNA recognition motif domains of RBM5 are required for RNA binding and cancer cell proliferation inhibition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Lei; Zhang, Qing; Yang, Yu

Highlights: • RNA recognition motif domains of RBM5 are essential for cell proliferation inhibition. • RNA recognition motif domains of RBM5 are essential for apoptosis induction. • RNA recognition motif domains of RBM5 are essential for RNA binding. • RNA recognition motif domains of RBM5 are essential for caspase-2 alternative splicing. - Abstract: RBM5 is a known putative tumor suppressor gene that has been shown to function in cell growth inhibition by modulating apoptosis. RBM5 also plays a critical role in alternative splicing as an RNA binding protein. However, it is still unclear which domains of RBM5 are required formore » RNA binding and related functional activities. We hypothesized the two putative RNA recognition motif (RRM) domains of RBM5 spanning from amino acids 98–178 and 231–315 are essential for RBM5-mediated cell growth inhibition, apoptosis regulation, and RNA binding. To investigate this hypothesis, we evaluated the activities of the wide-type and mutant RBM5 gene transfer in low-RBM5 expressing A549 cells. We found that, unlike wild-type RBM5 (RBM5-wt), a RBM5 mutant lacking the two RRM domains (RBM5-ΔRRM), is unable to bind RNA, has compromised caspase-2 alternative splicing activity, lacks cell proliferation inhibition and apoptosis induction function in A549 cells. These data provide direct evidence that the two RRM domains of RBM5 are required for RNA binding and the RNA binding activity of RBM5 contributes to its function on apoptosis induction and cell growth inhibition.« less

Coupling of tandem Smad ubiquitination regulatory factor (Smurf) WW domains modulates target specificity.

PubMed

Chong, P Andrew; Lin, Hong; Wrana, Jeffrey L; Forman-Kay, Julie D

2010-10-26

Smad ubiquitination regulatory factor 2 (Smurf2) is an E3 ubiquitin ligase that participates in degradation of TGF-β receptors and other targets. Smurf2 WW domains recognize PPXY (PY) motifs on ubiquitin ligase target proteins or on adapters, such as Smad7, that bind to E3 target proteins. We previously demonstrated that the isolated WW3 domain of Smurf2, but not the WW2 domain, can directly bind to a Smad7 PY motif. We show here that the WW2 augments this interaction by binding to the WW3 and making auxiliary contacts with the PY motif and a novel E/D-S/T-P motif, which is N-terminal to all Smad PY motifs. The WW2 likely enhances the selectivity of Smurf2 for the Smad proteins. NMR titrations confirm that Smad1 and Smad2 are bound by Smurf2 with the same coupled WW domain arrangement used to bind Smad7. The analogous WW domains in the short isoform of Smurf1 recognize the Smad7 PY peptide using the same coupled mechanism. However, a longer Smurf1 isoform, which has an additional 26 residues in the inter-WW domain linker, is only partially able to use the coupled WW domain binding mechanism. The longer linker results in a decrease in affinity for the Smad7 peptide. Interdomain coupling of WW domains enhances selectivity and enables the tuning of interactions by isoform switching.

Coupling of tandem Smad ubiquitination regulatory factor (Smurf) WW domains modulates target specificity

PubMed Central

Chong, P. Andrew; Lin, Hong; Wrana, Jeffrey L.; Forman-Kay, Julie D.

2010-01-01

Smad ubiquitination regulatory factor 2 (Smurf2) is an E3 ubiquitin ligase that participates in degradation of TGF-β receptors and other targets. Smurf2 WW domains recognize PPXY (PY) motifs on ubiquitin ligase target proteins or on adapters, such as Smad7, that bind to E3 target proteins. We previously demonstrated that the isolated WW3 domain of Smurf2, but not the WW2 domain, can directly bind to a Smad7 PY motif. We show here that the WW2 augments this interaction by binding to the WW3 and making auxiliary contacts with the PY motif and a novel E/D-S/T-P motif, which is N-terminal to all Smad PY motifs. The WW2 likely enhances the selectivity of Smurf2 for the Smad proteins. NMR titrations confirm that Smad1 and Smad2 are bound by Smurf2 with the same coupled WW domain arrangement used to bind Smad7. The analogous WW domains in the short isoform of Smurf1 recognize the Smad7 PY peptide using the same coupled mechanism. However, a longer Smurf1 isoform, which has an additional 26 residues in the inter-WW domain linker, is only partially able to use the coupled WW domain binding mechanism. The longer linker results in a decrease in affinity for the Smad7 peptide. Interdomain coupling of WW domains enhances selectivity and enables the tuning of interactions by isoform switching. PMID:20937913

Biophysical studies and NMR structure of YAP2 WW domain - LATS1 PPxY motif complexes reveal the basis of their interaction

PubMed Central

Verma, Apoorva; Jing-Song, Fan; Finch-Edmondson, Megan L.; Velazquez-Campoy, Adrian; Balasegaran, Shanker; Sudol, Marius; Sivaraman, Jayaraman

2018-01-01

YES-associated protein (YAP) is a major effector protein of the Hippo tumor suppressor pathway, and is phosphorylated by the serine/threonine kinase LATS. Their binding is mediated by the interaction between WW domains of YAP and PPxY motifs of LATS. Their isoforms, YAP2 and LATS1 contain two WW domains and two PPxY motifs respectively. Here, we report the study of the interaction of these domains both in vitro and in human cell lines, to better understand the mechanism of their binding. We show that there is a reciprocal binding preference of YAP2-WW1 with LATS1-PPxY2, and YAP2-WW2 with LATS1-PPxY1. We solved the NMR structures of these complexes and identified several conserved residues that play a critical role in binding. We further created a YAP2 mutant by swapping the WW domains, and found that YAP2 phosphorylation at S127 by LATS1 is not affected by the spatial configuration of its WW domains. This is likely because the region between the PPxY motifs of LATS1 is unstructured, even upon binding with its partner. Based on our observations, we propose possible models for the interaction between YAP2 and LATS1. PMID:29487715

Motif structure and cooperation in real-world complex networks

NASA Astrophysics Data System (ADS)

Salehi, Mostafa; Rabiee, Hamid R.; Jalili, Mahdi

2010-12-01

Networks of dynamical nodes serve as generic models for real-world systems in many branches of science ranging from mathematics to physics, technology, sociology and biology. Collective behavior of agents interacting over complex networks is important in many applications. The cooperation between selfish individuals is one of the most interesting collective phenomena. In this paper we address the interplay between the motifs’ cooperation properties and their abundance in a number of real-world networks including yeast protein-protein interaction, human brain, protein structure, email communication, dolphins’ social interaction, Zachary karate club and Net-science coauthorship networks. First, the amount of cooperativity for all possible undirected subgraphs with three to six nodes is calculated. To this end, the evolutionary dynamics of the Prisoner’s Dilemma game is considered and the cooperativity of each subgraph is calculated as the percentage of cooperating agents at the end of the simulation time. Then, the three- to six-node motifs are extracted for each network. The significance of the abundance of a motif, represented by a Z-value, is obtained by comparing them with some properly randomized versions of the original network. We found that there is always a group of motifs showing a significant inverse correlation between their cooperativity amount and Z-value, i.e. the more the Z-value the less the amount of cooperativity. This suggests that networks composed of well-structured units do not have good cooperativity properties.

Biophysical studies and NMR structure of YAP2 WW domain - LATS1 PPxY motif complexes reveal the basis of their interaction.

PubMed

Verma, Apoorva; Jing-Song, Fan; Finch-Edmondson, Megan L; Velazquez-Campoy, Adrian; Balasegaran, Shanker; Sudol, Marius; Sivaraman, Jayaraman

2018-01-30

YES-associated protein (YAP) is a major effector protein of the Hippo tumor suppressor pathway, and is phosphorylated by the serine/threonine kinase LATS. Their binding is mediated by the interaction between WW domains of YAP and PPxY motifs of LATS. Their isoforms, YAP2 and LATS1 contain two WW domains and two PPxY motifs respectively. Here, we report the study of the interaction of these domains both in vitro and in human cell lines, to better understand the mechanism of their binding. We show that there is a reciprocal binding preference of YAP2-WW1 with LATS1-PPxY2, and YAP2-WW2 with LATS1-PPxY1. We solved the NMR structures of these complexes and identified several conserved residues that play a critical role in binding. We further created a YAP2 mutant by swapping the WW domains, and found that YAP2 phosphorylation at S127 by LATS1 is not affected by the spatial configuration of its WW domains. This is likely because the region between the PPxY motifs of LATS1 is unstructured, even upon binding with its partner. Based on our observations, we propose possible models for the interaction between YAP2 and LATS1.

New PAH gene promoter KLF1 and 3'-region C/EBPalpha motifs influence transcription in vitro.

PubMed

Klaassen, Kristel; Stankovic, Biljana; Kotur, Nikola; Djordjevic, Maja; Zukic, Branka; Nikcevic, Gordana; Ugrin, Milena; Spasovski, Vesna; Srzentic, Sanja; Pavlovic, Sonja; Stojiljkovic, Maja

2017-02-01

Phenylketonuria (PKU) is a metabolic disease caused by mutations in the phenylalanine hydroxylase (PAH) gene. Although the PAH genotype remains the main determinant of PKU phenotype severity, genotype-phenotype inconsistencies have been reported. In this study, we focused on unanalysed sequences in non-coding PAH gene regions to assess their possible influence on the PKU phenotype. We transiently transfected HepG2 cells with various chloramphenicol acetyl transferase (CAT) reporter constructs which included PAH gene non-coding regions. Selected non-coding regions were indicated by in silico prediction to contain transcription factor binding sites. Furthermore, electrophoretic mobility shift assay (EMSA) and supershift assays were performed to identify which transcriptional factors were engaged in the interaction. We found novel KLF1 motif in the PAH promoter, which decreases CAT activity by 50 % in comparison to basal transcription in vitro. The cytosine at the c.-170 promoter position creates an additional binding site for the protein complex involving KLF1 transcription factor. Moreover, we assessed for the first time the role of a multivariant variable number tandem repeat (VNTR) region located in the 3'-region of the PAH gene. We found that the VNTR3, VNTR7 and VNTR8 constructs had approximately 60 % of CAT activity. The regulation is mediated by the C/EBPalpha transcription factor, present in protein complex binding to VNTR3. Our study highlighted two novel promoter KLF1 and 3'-region C/EBPalpha motifs in the PAH gene which decrease transcription in vitro and, thus, could be considered as PAH expression modifiers. New transcription motifs in non-coding regions will contribute to better understanding of the PKU phenotype complexity and may become important for the optimisation of PKU treatment.

Protein Phosphatase 2A (PP2A)-specific Ubiquitin Ligase MID1 Is a Sequence-dependent Regulator of Translation Efficiency Controlling 3-Phosphoinositide-dependent Protein Kinase-1 (PDPK-1)*

PubMed Central

Aranda-Orgillés, Beatriz; Rutschow, Désirée; Zeller, Raphael; Karagiannidis, Antonios I.; Köhler, Andrea; Chen, Changwei; Wilson, Timothy; Krause, Sven; Roepcke, Stefan; Lilley, David; Schneider, Rainer; Schweiger, Susann

2011-01-01

We have shown previously that the ubiquitin ligase MID1, mutations of which cause the midline malformation Opitz BBB/G syndrome (OS), serves as scaffold for a microtubule-associated protein complex that regulates protein phosphatase 2A (PP2A) activity in a ubiquitin-dependent manner. Here, we show that the MID1 protein complex associates with mRNAs via a purine-rich sequence motif called MIDAS (MID1 association sequence) and thereby increases stability and translational efficiency of these mRNAs. Strikingly, inclusion of multiple copies of the MIDAS motif into mammalian mRNAs increases production of the encoded proteins up to 20-fold. Mutated MID1, as found in OS patients, loses its influence on MIDAS-containing mRNAs, suggesting that the malformations in OS patients could be caused by failures in the regulation of cytoskeleton-bound protein translation. This is supported by the observation that the majority of mRNAs that carry MIDAS motifs is involved in developmental processes and/or energy homeostasis. Further analysis of one of the proteins encoded by a MIDAS-containing mRNA, namely PDPK-1 (3-phosphoinositide dependent protein kinase-1), which is an important regulator of mammalian target of rapamycin/PP2A signaling, showed that PDPK-1 protein synthesis is significantly reduced in cells from an OS patient compared with an age-matched control and can be rescued by functional MID1. Together, our data uncover a novel messenger ribonucleoprotein complex that regulates microtubule-associated protein translation. They suggest a novel mechanism underlying OS and point at an enormous potential of the MIDAS motif to increase the efficiency of biotechnological protein production in mammalian cells. PMID:21930711

Mono- and multimeric ferrocene congeners of quinoline-based polyamines as potential antiparasitics

USDA-ARS?s Scientific Manuscript database

A series of mono- and multimeric polyamine-containing ferrocenyl complexes bearing a quinoline motif were prepared. The complexes were characterised by standard techniques. The molecular structure of the monomeric salicylaldimine derivative was elucidated using single crystal X-ray diffraction and w...

The OsSPL16-GW7 regulatory module determines grain shape and simultaneously improves rice yield and grain quality.

PubMed

Wang, Shaokui; Li, Shan; Liu, Qian; Wu, Kun; Zhang, Jianqing; Wang, Shuansuo; Wang, Yi; Chen, Xiangbin; Zhang, Yi; Gao, Caixia; Wang, Feng; Huang, Haixiang; Fu, Xiangdong

2015-08-01

The deployment of heterosis in the form of hybrid rice varieties has boosted grain yield, but grain quality improvement still remains a challenge. Here we show that a quantitative trait locus for rice grain quality, qGW7, reflects allelic variation of GW7, a gene encoding a TONNEAU1-recruiting motif protein with similarity to C-terminal motifs of the human centrosomal protein CAP350. Upregulation of GW7 expression was correlated with the production of more slender grains, as a result of increased cell division in the longitudinal direction and decreased cell division in the transverse direction. OsSPL16 (GW8), an SBP-domain transcription factor that regulates grain width, bound directly to the GW7 promoter and repressed its expression. The presence of a semidominant GW7(TFA) allele from tropical japonica rice was associated with higher grain quality without the yield penalty imposed by the Basmati gw8 allele. Manipulation of the OsSPL16-GW7 module thus represents a new strategy to simultaneously improve rice yield and grain quality.

The role of collagen charge clusters in the modulation of matrix metalloproteinase activity.

PubMed

Lauer, Janelle L; Bhowmick, Manishabrata; Tokmina-Roszyk, Dorota; Lin, Yan; Van Doren, Steven R; Fields, Gregg B

2014-01-24

Members of the matrix metalloproteinase (MMP) family selectively cleave collagens in vivo. Several substrate structural features that direct MMP collagenolysis have been identified. The present study evaluated the role of charged residue clusters in the regulation of MMP collagenolysis. A series of 10 triple-helical peptide (THP) substrates were constructed in which either Lys-Gly-Asp or Gly-Asp-Lys motifs replaced Gly-Pro-Hyp (where Hyp is 4-hydroxy-L-proline) repeats. The stabilities of THPs containing the two different motifs were analyzed, and kinetic parameters for substrate hydrolysis by six MMPs were determined. A general trend for virtually all enzymes was that, as Gly-Asp-Lys motifs were moved from the extreme N and C termini to the interior next to the cleavage site sequence, kcat/Km values increased. Additionally, all Gly-Asp-Lys THPs were as good or better substrates than the parent THP in which Gly-Asp-Lys was not present. In turn, the Lys-Gly-Asp THPs were also always better substrates than the parent THP, but the magnitude of the difference was considerably less compared with the Gly-Asp-Lys series. Of the MMPs tested, MMP-2 and MMP-9 most greatly favored the presence of charged residues with preference for the Gly-Asp-Lys series. Lys-Gly-(Asp/Glu) motifs are more commonly found near potential MMP cleavage sites than Gly-(Asp/Glu)-Lys motifs. As Lys-Gly-Asp is not as favored by MMPs as Gly-Asp-Lys, the Lys-Gly-Asp motif appears advantageous over the Gly-Asp-Lys motif by preventing unwanted MMP hydrolysis. More specifically, the lack of Gly-Asp-Lys clusters may diminish potential MMP-2 and MMP-9 collagenolytic activity. The present study indicates that MMPs have interactions spanning the P23-P23' subsites of collagenous substrates.

High-Pressure NMR and SAXS Reveals How Capping Modulates Folding Cooperativity of the pp32 Leucine-rich Repeat Protein.

PubMed

Zhang, Yi; Berghaus, Melanie; Klein, Sean; Jenkins, Kelly; Zhang, Siwen; McCallum, Scott A; Morgan, Joel E; Winter, Roland; Barrick, Doug; Royer, Catherine A

2018-04-27

Many repeat proteins contain capping motifs, which serve to shield the hydrophobic core from solvent and maintain structural integrity. While the role of capping motifs in enhancing the stability and structural integrity of repeat proteins is well documented, their contribution to folding cooperativity is not. Here we examined the role of capping motifs in defining the folding cooperativity of the leucine-rich repeat protein, pp32, by monitoring the pressure- and urea-induced unfolding of an N-terminal capping motif (N-cap) deletion mutant, pp32-∆N-cap, and a C-terminal capping motif destabilization mutant pp32-Y131F/D146L, using residue-specific NMR and small-angle X-ray scattering. Destabilization of the C-terminal capping motif resulted in higher cooperativity for the unfolding transition compared to wild-type pp32, as these mutations render the stability of the C-terminus similar to that of the rest of the protein. In contrast, deletion of the N-cap led to strong deviation from two-state unfolding. In both urea- and pressure-induced unfolding, residues in repeats 1-3 of pp32-ΔN-cap lost their native structure first, while the C-terminal half was more stable. The residue-specific free energy changes in all regions of pp32-ΔN-cap were larger in urea compared to high pressure, indicating a less cooperative destabilization by pressure. Moreover, in contrast to complete structural disruption of pp32-ΔN-cap at high urea concentration, its pressure unfolded state remained compact. The contrasting effects of the capping motifs on folding cooperativity arise from the differential local stabilities of pp32, whereas the contrasting effects of pressure and urea on the pp32-ΔN-cap variant arise from their distinct mechanisms of action. Copyright © 2018 Elsevier Ltd. All rights reserved.

Metal dependent motif transition in a self-assembled monolayer of bipyridine derivatives via coordination: An STM study.

PubMed

Wang, Yi; Yuan, Qunhui; Xu, Hongbo; Zhu, Xuefeng; Gan, Wei

2016-07-21

Low-dimensional molecular motifs with diversity developed via the on-surface chemistry are attracting growing interest for their potential in advanced nanofabrication. In this work, scanning tunneling microscopy was employed to investigate the in situ and ex situ metal coordinations between 4,4'-ditetradecyl-2,2'-bipyridine (bpy) and Zn(ii) or Cu(ii) ions at a highly oriented pyrolytic graphite (HOPG)/1-phenyloctane interface under ambient conditions. The results demonstrate that the bpy adopts a flat-lying orientation with its substituted alkyl chains in a tail-to-tail arrangement in a bpy monolayer. For the in situ coordination, the bpy/Zn(ii) and bpy/Cu(ii) complexes are aligned in edge-on fashions, wherein the bpy stands vertically on the HOPG surface and interdigitates at the alkyl chains. In the two-dimensional arrays of ex situ coordinated complexes, metal dependent motifs have been observed with Zn(ii) and Cu(ii), wherein the bipyridine moieties are parallel to the graphite surface. These results suggest that the desired on-surface coordination architectures may be achieved by the intentional selection of the metal centers.

NMR assignments of SPOC domain of the human transcriptional corepressor SHARP in complex with a C-terminal SMRT peptide.

PubMed

Mikami, Suzuka; Kanaba, Teppei; Ito, Yutaka; Mishima, Masaki

2013-10-01

The transcriptional corepressor SMRT/HDAC1-associated repressor protein (SHARP) recruits histone deacetylases. Human SHARP protein is thought to function in processes involving steroid hormone responses and the Notch signaling pathway. SHARP consists of RNA recognition motifs (RRMs) in the N-terminal region and the spen paralog and ortholog C-terminal (SPOC) domain in the C-terminal region. It is known that the SPOC domain binds the LSD motif in the C-terminal tail of corepressors silencing mediator for retinoid and thyroid receptor (SMRT)/nuclear receptor corepressor (NcoR). We are interested in delineating the mechanism by which the SPOC domain recognizes the LSD motif of the C-terminal tail of SMRT/NcoR. To this end, we are investigating the tertiary structure of the SPOC/SMRT peptide using NMR. Herein, we report on the (1)H, (13)C and (15)N resonance assignments of the SPOC domain in complex with a SMRT peptide, which contributes towards a structural understanding of the SPOC/SMRT peptide and its molecular recognition.

Structural basis of RNA folding and recognition in an AMP-RNA aptamer complex.

PubMed

Jiang, F; Kumar, R A; Jones, R A; Patel, D J

1996-07-11

The catalytic properties of RNA and its well known role in gene expression and regulation are the consequence of its unique solution structures. Identification of the structural determinants of ligand recognition by RNA molecules is of fundamental importance for understanding the biological functions of RNA, as well as for the rational design of RNA Sequences with specific catalytic activities. Towards this latter end, Szostak et al. used in vitro selection techniques to isolate RNA sequences ('aptamers') containing a high-affinity binding site for ATP, the universal currency of cellular energy, and then used this motif to engineer ribozymes with polynucleotide kinase activity. Here we present the solution structure, as determined by multidimensional NMR spectroscopy and molecular dynamics calculations, of both uniformly and specifically 13C-, 15N-labelled 40-mer RNA containing the ATP-binding motif complexed with AMP. The aptamer adopts an L-shaped structure with two nearly orthogonal stems, each capped proximally by a G x G mismatch pair, binding the AMP ligand at their junction in a GNRA-like motif.

γδ T cell receptors recognize the non-classical major histocompatibility complex (MHC) molecule T22 via conserved anchor residues in a MHC peptide-like fashion.

PubMed

Sandstrom, Andrew; Scharf, Louise; McRae, Gabrielle; Hawk, Andrew J; Meredith, Stephen C; Adams, Erin J

2012-02-17

The molecular mechanisms by which γδ T cells recognize ligand remain a mystery. The non-classical MHC molecule T22 represents the best characterized ligand for murine γδ T cells, with a motif (W … EGYEL) present in the γδ T cell receptor complementary-determining region 3δ (CDR3δ) loop mediating γδ T cell recognition of this molecule. Produced through V(D)J recombination, this loop is quite diverse, with different numbers and chemical types of amino acids between Trp and EGYEL, which have unknown functional consequences for T22 recognition. We have investigated the biophysical and structural effects of CDR3δ loop diversity, revealing a range of affinities for T22 but a common thermodynamic pattern. Mutagenesis of these CDR3δ loops defines the key anchor residues involved in T22 recognition as W … EGYEL, similar to those found for the G8 CDR3δ loop, and demonstrates that spacer residues modulate but are not required for T22 recognition. Comparison of the location of these residues in the T22 interface reveals a striking similarity to peptide anchor residues in classically presented MHC peptides, with the key Trp residue of the CDR3δ motif completing the deficient peptide-binding groove of T22. This suggests that γδ T cell recognition of T22 utilizes the conserved ligand-presenting nature of the MHC fold.

Discrete Molecular Dynamics Can Predict Helical Prestructured Motifs in Disordered Proteins

PubMed Central

Han, Kyou-Hoon; Dokholyan, Nikolay V.; Tompa, Péter; Kalmár, Lajos; Hegedűs, Tamás

2014-01-01

Intrinsically disordered proteins (IDPs) lack a stable tertiary structure, but their short binding regions termed Pre-Structured Motifs (PreSMo) can form transient secondary structure elements in solution. Although disordered proteins are crucial in many biological processes and designing strategies to modulate their function is highly important, both experimental and computational tools to describe their conformational ensembles and the initial steps of folding are sparse. Here we report that discrete molecular dynamics (DMD) simulations combined with replica exchange (RX) method efficiently samples the conformational space and detects regions populating α-helical conformational states in disordered protein regions. While the available computational methods predict secondary structural propensities in IDPs based on the observation of protein-protein interactions, our ab initio method rests on physical principles of protein folding and dynamics. We show that RX-DMD predicts α-PreSMos with high confidence confirmed by comparison to experimental NMR data. Moreover, the method also can dissect α-PreSMos in close vicinity to each other and indicate helix stability. Importantly, simulations with disordered regions forming helices in X-ray structures of complexes indicate that a preformed helix is frequently the binding element itself, while in other cases it may have a role in initiating the binding process. Our results indicate that RX-DMD provides a breakthrough in the structural and dynamical characterization of disordered proteins by generating the structural ensembles of IDPs even when experimental data are not available. PMID:24763499

The Janus Kinase (JAK) FERM and SH2 Domains: Bringing Specificity to JAK-Receptor Interactions.

PubMed

Ferrao, Ryan; Lupardus, Patrick J

2017-01-01

The Janus kinases (JAKs) are non-receptor tyrosine kinases essential for signaling in response to cytokines and interferons and thereby control many essential functions in growth, development, and immune regulation. JAKs are unique among tyrosine kinases for their constitutive yet non-covalent association with class I and II cytokine receptors, which upon cytokine binding bring together two JAKs to create an active signaling complex. JAK association with cytokine receptors is facilitated by N-terminal FERM and SH2 domains, both of which are classical mediators of peptide interactions. Together, the JAK FERM and SH2 domains mediate a bipartite interaction with two distinct receptor peptide motifs, the proline-rich "Box1" and hydrophobic "Box2," which are present in the intracellular domain of cytokine receptors. While the general sidechain chemistry of Box1 and Box2 peptides is conserved between receptors, they share very weak primary sequence homology, making it impossible to posit why certain JAKs preferentially interact with and signal through specific subsets of cytokine receptors. Here, we review the structure and function of the JAK FERM and SH2 domains in light of several recent studies that reveal their atomic structure and elucidate interaction mechanisms with both the Box1 and Box2 receptor motifs. These crystal structures demonstrate how evolution has repurposed the JAK FERM and SH2 domains into a receptor-binding module that facilitates interactions with multiple receptors possessing diverse primary sequences.

Role of sequence encoded κB DNA geometry in gene regulation by Dorsal

PubMed Central

Mrinal, Nirotpal; Tomar, Archana; Nagaraju, Javaregowda

2011-01-01

Many proteins of the Rel family can act as both transcriptional activators and repressors. However, mechanism that discerns the ‘activator/repressor’ functions of Rel-proteins such as Dorsal (Drosophila homologue of mammalian NFκB) is not understood. Using genomic, biophysical and biochemical approaches, we demonstrate that the underlying principle of this functional specificity lies in the ‘sequence-encoded structure’ of the κB-DNA. We show that Dorsal-binding motifs exist in distinct activator and repressor conformations. Molecular dynamics of DNA-Dorsal complexes revealed that repressor κB-motifs typically have A-tract and flexible conformation that facilitates interaction with co-repressors. Deformable structure of repressor motifs, is due to changes in the hydrogen bonding in A:T pair in the ‘A-tract’ core. The sixth nucleotide in the nonameric κB-motif, ‘A’ (A6) in the repressor motifs and ‘T’ (T6) in the activator motifs, is critical to confer this functional specificity as A6 → T6 mutation transformed flexible repressor conformation into a rigid activator conformation. These results highlight that ‘sequence encoded κB DNA-geometry’ regulates gene expression by exerting allosteric effect on binding of Rel proteins which in turn regulates interaction with co-regulators. Further, we identified and characterized putative repressor motifs in Dl-target genes, which can potentially aid in functional annotation of Dorsal gene regulatory network. PMID:21890896

Accurate quantification of microRNA via single strand displacement reaction on DNA origami motif.

PubMed

Zhu, Jie; Feng, Xiaolu; Lou, Jingyu; Li, Weidong; Li, Sheng; Zhu, Hongxin; Yang, Lun; Zhang, Aiping; He, Lin; Li, Can

2013-01-01

DNA origami is an emerging technology that assembles hundreds of staple strands and one single-strand DNA into certain nanopattern. It has been widely used in various fields including detection of biological molecules such as DNA, RNA and proteins. MicroRNAs (miRNAs) play important roles in post-transcriptional gene repression as well as many other biological processes such as cell growth and differentiation. Alterations of miRNAs' expression contribute to many human diseases. However, it is still a challenge to quantitatively detect miRNAs by origami technology. In this study, we developed a novel approach based on streptavidin and quantum dots binding complex (STV-QDs) labeled single strand displacement reaction on DNA origami to quantitatively detect the concentration of miRNAs. We illustrated a linear relationship between the concentration of an exemplary miRNA as miRNA-133 and the STV-QDs hybridization efficiency; the results demonstrated that it is an accurate nano-scale miRNA quantifier motif. In addition, both symmetrical rectangular motif and asymmetrical China-map motif were tested. With significant linearity in both motifs, our experiments suggested that DNA Origami motif with arbitrary shape can be utilized in this method. Since this DNA origami-based method we developed owns the unique advantages of simple, time-and-material-saving, potentially multi-targets testing in one motif and relatively accurate for certain impurity samples as counted directly by atomic force microscopy rather than fluorescence signal detection, it may be widely used in quantification of miRNAs.

Feedback Inhibition Shapes Emergent Computational Properties of Cortical Microcircuit Motifs.

PubMed

Jonke, Zeno; Legenstein, Robert; Habenschuss, Stefan; Maass, Wolfgang

2017-08-30

Cortical microcircuits are very complex networks, but they are composed of a relatively small number of stereotypical motifs. Hence, one strategy for throwing light on the computational function of cortical microcircuits is to analyze emergent computational properties of these stereotypical microcircuit motifs. We are addressing here the question how spike timing-dependent plasticity shapes the computational properties of one motif that has frequently been studied experimentally: interconnected populations of pyramidal cells and parvalbumin-positive inhibitory cells in layer 2/3. Experimental studies suggest that these inhibitory neurons exert some form of divisive inhibition on the pyramidal cells. We show that this data-based form of feedback inhibition, which is softer than that of winner-take-all models that are commonly considered in theoretical analyses, contributes to the emergence of an important computational function through spike timing-dependent plasticity: The capability to disentangle superimposed firing patterns in upstream networks, and to represent their information content through a sparse assembly code. SIGNIFICANCE STATEMENT We analyze emergent computational properties of a ubiquitous cortical microcircuit motif: populations of pyramidal cells that are densely interconnected with inhibitory neurons. Simulations of this model predict that sparse assembly codes emerge in this microcircuit motif under spike timing-dependent plasticity. Furthermore, we show that different assemblies will represent different hidden sources of upstream firing activity. Hence, we propose that spike timing-dependent plasticity enables this microcircuit motif to perform a fundamental computational operation on neural activity patterns. Copyright © 2017 the authors 0270-6474/17/378511-13$15.00/0.

Sulfur-induced structural motifs on copper and gold surfaces

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walen, Holly

The interaction of sulfur with copper and gold surfaces plays a fundamental role in important phenomena that include coarsening of surface nanostructures, and self-assembly of alkanethiols. Here, we identify and analyze unique sulfur-induced structural motifs observed on the low-index surfaces of these two metals. We seek out these structures in an effort to better understand the fundamental interactions between these metals and sulfur that lends to the stability and favorability of metal-sulfur complexes vs. chemisorbed atomic sulfur. The experimental observations presented here—made under identical conditions—together with extensive DFT analyses, allow comparisons and insights into factors that favor the existence ofmore » metal-sulfur complexes, vs. chemisorbed atomic sulfur, on metal terraces. We believe this data will be instrumental in better understanding the complex phenomena occurring between the surfaces of coinage metals and sulfur.« less

Complex lasso: new entangled motifs in proteins

NASA Astrophysics Data System (ADS)

Niemyska, Wanda; Dabrowski-Tumanski, Pawel; Kadlof, Michal; Haglund, Ellinor; Sułkowski, Piotr; Sulkowska, Joanna I.

2016-11-01

We identify new entangled motifs in proteins that we call complex lassos. Lassos arise in proteins with disulfide bridges (or in proteins with amide linkages), when termini of a protein backbone pierce through an auxiliary surface of minimal area, spanned on a covalent loop. We find that as much as 18% of all proteins with disulfide bridges in a non-redundant subset of PDB form complex lassos, and classify them into six distinct geometric classes, one of which resembles supercoiling known from DNA. Based on biological classification of proteins we find that lassos are much more common in viruses, plants and fungi than in other kingdoms of life. We also discuss how changes in the oxidation/reduction potential may affect the function of proteins with lassos. Lassos and associated surfaces of minimal area provide new, interesting and possessing many potential applications geometric characteristics not only of proteins, but also of other biomolecules.

Structural insights into alternative splicing-mediated desensitization of jasmonate signaling.

PubMed

Zhang, Feng; Ke, Jiyuan; Zhang, Li; Chen, Rongzhi; Sugimoto, Koichi; Howe, Gregg A; Xu, H Eric; Zhou, Mingguo; He, Sheng Yang; Melcher, Karsten

2017-02-14

Jasmonate ZIM-domain (JAZ) transcriptional repressors play a key role in regulating jasmonate (JA) signaling in plants. Below a threshold concentration of jasmonoyl isoleucine (JA-Ile), the active form of JA, the C-terminal Jas motif of JAZ proteins binds MYC transcription factors to repress JA signaling. With increasing JA-Ile concentration, the Jas motif binds to JA-Ile and the COI1 subunit of the SCF COI1 E3 ligase, which mediates ubiquitination and proteasomal degradation of JAZ repressors, resulting in derepression of MYC transcription factors. JA signaling subsequently becomes desensitized, in part by feedback induction of JAZ splice variants that lack the C-terminal Jas motif but include an N-terminal cryptic MYC-interaction domain (CMID). The CMID sequence is dissimilar to the Jas motif and is incapable of recruiting SCF COI1 , allowing CMID-containing JAZ splice variants to accumulate in the presence of JA and to re-repress MYC transcription factors as an integral part of reestablishing signal homeostasis. The mechanism by which the CMID represses MYC transcription factors remains elusive. Here we describe the crystal structure of the MYC3-CMID JAZ10 complex. In contrast to the Jas motif, which forms a single continuous helix when bound to MYC3, the CMID adopts a loop-helix-loop-helix architecture with modular interactions with both the Jas-binding groove and the backside of the Jas-interaction domain of MYC3. This clamp-like interaction allows the CMID to bind MYC3 tightly and block access of MED25 (a subunit of the Mediator coactivator complex) to the MYC3 transcriptional activation domain, shedding light on the enigmatic mechanism by which JAZ splice variants desensitize JA signaling.

Application of 1-aminocyclohexane carboxylic acid to protein nanostructure computer design

PubMed Central

Rodríguez-Ropero, Francisco; Zanuy, David; Casanovas, Jordi; Nussinov, Ruth; Alemán, Carlos

2009-01-01

Conformationally restricted amino acids are promising candidates to serve as basic pieces in redesigned protein motifs which constitute the basic modules in synthetic nanoconstructs. Here we study the ability of constrained cyclic amino acid 1-aminocyclohexane-1-carboxylic acid (Ac6c) to stabilize highly regular β-helical motifs excised from naturally occurring proteins. Calculations indicate that the conformational flexibility observed in both the ring and the main chain is significantly higher than that detected for other 1-aminocycloalkane-1-carboxylic acid (Acnc, where n refers to the size of the ring) with smaller cycles. Incorporation of Ac6c into the flexible loops of β-helical motifs indicates that the stability of such excised building blocks as well as the nano-assemblies derived from them is significantly enhanced. Thus, the intrinsic Ac6c tendency to adopt folded conformations combined with the low structural strain of the cyclohexane ring confers the ability to both self-adapt to the β-helix motif and to stabilize the overall structure by absorbing part of its conformational fluctuations. Comparison with other Acnc residues indicates that the ability to adapt to the targeted position improves considerably with the ring size, i.e. when the rigidity introduced by the strain of the ring decreases. PMID:18201062

Novel ATPase activity of the polyprotein intermediate, Viral Protein genome-linked-Nuclear Inclusion-a protease, of Pepper vein banding potyvirus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mathur, Chhavi; Savithri, Handanahal S., E-mail: bchss@biochem.iisc.ernet.in

2012-10-12

Highlights: Black-Right-Pointing-Pointer Pepper vein banding potyvirus VPg harbors Walker motifs. Black-Right-Pointing-Pointer VPg exhibits ATPase activity in the presence of NIa-Pro. Black-Right-Pointing-Pointer Plausible structural and functional interplay between VPg and NIa-Pro. Black-Right-Pointing-Pointer Functional relevance of prolonged presence of VPg-Pro during infection. -- Abstract: Potyviruses temporally regulate their protein function by polyprotein processing. Previous studies have shown that VPg (Viral Protein genome-linked) of Pepper vein banding virus interacts with the NIa-Pro (Nuclear Inclusion-a protease) domain, and modulates the kinetics of the protease. In the present study, we report for the first time that VPg harbors the Walker motifs A and B, andmore » the presence of NIa-Pro, especially in cis (cleavage site (E191A) VPg-Pro mutant), is essential for manifestation of the ATPase activity. Mutation of Lys47 (Walker motif A) and Asp88:Glu89 (Walker motif B) to alanine in E191A VPg-Pro lead to reduced ATPase activity, confirming that this activity was inherent to VPg. We propose that potyviral VPg, established as an intrinsically disordered domain, undergoes plausible structural alterations upon interaction with globular NIa-Pro which induces the ATPase activity.« less

Asymmetrical Macromolecular Complex Formation of Lysophosphatidic Acid Receptor 2 (LPA2) Mediates Gradient Sensing in Fibroblasts*

PubMed Central

Ren, Aixia; Moon, Changsuk; Zhang, Weiqiang; Sinha, Chandrima; Yarlagadda, Sunitha; Arora, Kavisha; Wang, Xusheng; Yue, Junming; Parthasarathi, Kaushik; Heil-Chapdelaine, Rick; Tigyi, Gabor; Naren, Anjaparavanda P.

2014-01-01

Chemotactic migration of fibroblasts toward growth factors relies on their capacity to sense minute extracellular gradients and respond to spatially confined receptor-mediated signals. Currently, mechanisms underlying the gradient sensing of fibroblasts remain poorly understood. Using single-particle tracking methodology, we determined that a lysophosphatidic acid (LPA) gradient induces a spatiotemporally restricted decrease in the mobility of LPA receptor 2 (LPA2) on chemotactic fibroblasts. The onset of decreased LPA2 mobility correlates to the spatial recruitment and coupling to LPA2-interacting proteins that anchor the complex to the cytoskeleton. These localized PDZ motif-mediated macromolecular complexes of LPA2 trigger a Ca2+ puff gradient that governs gradient sensing and directional migration in response to LPA. Disruption of the PDZ motif-mediated assembly of the macromolecular complex of LPA2 disorganizes the gradient of Ca2+ puffs, disrupts gradient sensing, and reduces the directional migration of fibroblasts toward LPA. Our findings illustrate that the asymmetric macromolecular complex formation of chemoattractant receptors mediates gradient sensing and provides a new mechanistic basis for models to describe gradient sensing of fibroblasts. PMID:25542932

Structure of the Human Atg13-Atg101 HORMA Heterodimer: an Interaction Hub within the ULK1 Complex.

PubMed

Qi, Shiqian; Kim, Do Jin; Stjepanovic, Goran; Hurley, James H

2015-10-06

The ULK1 complex, consisting of the ULK1 protein kinase itself, FIP200, Atg13, and Atg101, controls the initiation of autophagy in animals. We determined the structure of the complex of the human Atg13 HORMA (Hop1, Rev7, Mad2) domain in complex with the full-length HORMA domain-only protein Atg101. The two HORMA domains assemble with an architecture conserved in the Mad2 conformational heterodimer and the S. pombe Atg13-Atg101 HORMA complex. The WF finger motif that is essential for function in human Atg101 is sequestered in a hydrophobic pocket, suggesting that the exposure of this motif is regulated. Benzamidine molecules from the crystallization solution mark two hydrophobic pockets that are conserved in, and unique to, animals, and are suggestive of sites that could interact with other proteins. These features suggest that the activity of the animal Atg13-Atg101 subcomplex is regulated and that it is an interaction hub for multiple partners. Copyright © 2015 Elsevier Ltd. All rights reserved.

Characterizing English Poetic Style Using Complex Networks

NASA Astrophysics Data System (ADS)

Roxas-Villanueva, Ranzivelle Marianne; Nambatac, Maelori Krista; Tapang, Giovanni

Complex networks have been proven useful in characterizing written texts. Here, we use networks to probe if there exist a similarity within, and difference across, era as reflected within the poem's structure. In literary history, boundary lines are set to distinguish the change in writing styles through time. We obtain the network parameters and motif frequencies of 845 poems published from 1522 to 1931 and relate this to the writing of the Elizabethan, 17th Century, Augustan, Romantic and Victorian eras. Analysis of the different network parameters shows a significant difference of the Augustan era (1667-1780) with the rest. The network parameters and the convex hull and centroids of the motif frequencies reflect the adjectival sequence pattern of the poems of the Augustan era.

Cloning, 3D modeling and expression analysis of three vacuolar invertase genes from cassava (Manihot Esculenta Crantz).

PubMed

Yao, Yuan; Wu, Xiao-Hui; Geng, Meng-Ting; Li, Rui-Mei; Liu, Jiao; Hu, Xin-Wen; Guo, Jian-Chun

2014-05-15

Vacuolar invertase is one of the key enzymes in sucrose metabolism that irreversibly catalyzes the hydrolysis of sucrose to glucose and fructose in plants. In this research, three vacuolar invertase genes, named MeVINV1-3, and with 653, 660 and 639 amino acids, respectively, were cloned from cassava. The motifs of NDPNG (β-fructosidase motif), RDP and WECVD, which are conserved and essential for catalytic activity in the vacuolar invertase family, were found in MeVINV1 and MeVINV2. Meanwhile, in MeVINV3, instead of NDPNG we found the motif NGPDG, in which the three amino acids GPD are different from those in other vacuolar invertases (DPN) that might result in MeVINV3 being an inactivated protein. The N-terminal leader sequence of MeVINVs contains a signal anchor, which is associated with the sorting of vacuolar invertase to vacuole. The overall predicted 3D structure of the MeVINVs consists of a five bladed β-propeller module at N-terminus domain, and forms a β-sandwich module at the C-terminus domain. The active site of the protein is situated in the β-propeller module. MeVINVs are classified in two subfamilies, α and β groups, in which α group members of MeVINV1 and 2 are highly expressed in reproductive organs and tuber roots (considered as sink organs), while β group members of MeVINV3 are highly expressed in leaves (source organs). All MeVINVs are highly expressed in leaves, while only MeVINV1 and 2 are highly expressed in tubers at cassava tuber maturity stage. Thus, MeVINV1 and 2 play an important role in sucrose unloading and starch accumulation, as well in buffering the pools of sucrose, hexoses and sugar phosphates in leaves, specifically at later stages of plant development.

Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment.

PubMed

Barouch-Bentov, Rina; Neveu, Gregory; Xiao, Fei; Beer, Melanie; Bekerman, Elena; Schor, Stanford; Campbell, Joseph; Boonyaratanakornkit, Jim; Lindenbach, Brett; Lu, Albert; Jacob, Yves; Einav, Shirit

2016-11-01

Enveloped viruses commonly utilize late-domain motifs, sometimes cooperatively with ubiquitin, to hijack the endosomal sorting complex required for transport (ESCRT) machinery for budding at the plasma membrane. However, the mechanisms underlying budding of viruses lacking defined late-domain motifs and budding into intracellular compartments are poorly characterized. Here, we map a network of hepatitis C virus (HCV) protein interactions with the ESCRT machinery using a mammalian-cell-based protein interaction screen and reveal nine novel interactions. We identify HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), an ESCRT-0 complex component, as an important entry point for HCV into the ESCRT pathway and validate its interactions with the HCV nonstructural (NS) proteins NS2 and NS5A in HCV-infected cells. Infectivity assays indicate that HRS is an important factor for efficient HCV assembly. Specifically, by integrating capsid oligomerization assays, biophysical analysis of intracellular viral particles by continuous gradient centrifugations, proteolytic digestion protection, and RNase digestion protection assays, we show that HCV co-opts HRS to mediate a late assembly step, namely, envelopment. In the absence of defined late-domain motifs, K63-linked polyubiquitinated lysine residues in the HCV NS2 protein bind the HRS ubiquitin-interacting motif to facilitate assembly. Finally, ESCRT-III and VPS/VTA1 components are also recruited by HCV proteins to mediate assembly. These data uncover involvement of ESCRT proteins in intracellular budding of a virus lacking defined late-domain motifs and a novel mechanism by which HCV gains entry into the ESCRT network, with potential implications for other viruses. Viruses commonly bud at the plasma membrane by recruiting the host ESCRT machinery via conserved motifs termed late domains. The mechanism by which some viruses, such as HCV, bud intracellularly is, however, poorly characterized. Moreover, whether envelopment of HCV and other viruses lacking defined late domains is ESCRT mediated and, if so, what the entry points into the ESCRT pathway are remain unknown. Here, we report the interaction network of HCV with the ESCRT machinery and a critical role for HRS, an ESCRT-0 complex component, in HCV envelopment. Viral protein ubiquitination was discovered to be a signal for HRS binding and HCV assembly, thereby functionally compensating for the absence of late domains. These findings characterize how a virus lacking defined late domains co-opts ESCRT to bud intracellularly. Since the ESCRT machinery is essential for the life cycle of multiple viruses, better understanding of this virus-host interplay may yield targets for broad-spectrum antiviral therapies. Copyright © 2016 Barouch-Bentov et al.

Coarse-graining and self-dissimilarity of complex networks

NASA Astrophysics Data System (ADS)

Itzkovitz, Shalev; Levitt, Reuven; Kashtan, Nadav; Milo, Ron; Itzkovitz, Michael; Alon, Uri

2005-01-01

Can complex engineered and biological networks be coarse-grained into smaller and more understandable versions in which each node represents an entire pattern in the original network? To address this, we define coarse-graining units as connectivity patterns which can serve as the nodes of a coarse-grained network and present algorithms to detect them. We use this approach to systematically reverse-engineer electronic circuits, forming understandable high-level maps from incomprehensible transistor wiring: first, a coarse-grained version in which each node is a gate made of several transistors is established. Then the coarse-grained network is itself coarse-grained, resulting in a high-level blueprint in which each node is a circuit module made of many gates. We apply our approach also to a mammalian protein signal-transduction network, to find a simplified coarse-grained network with three main signaling channels that resemble multi-layered perceptrons made of cross-interacting MAP-kinase cascades. We find that both biological and electronic networks are “self-dissimilar,” with different network motifs at each level. The present approach may be used to simplify a variety of directed and nondirected, natural and designed networks.

Toxoplasma DJ-1 Regulates Organelle Secretion by a Direct Interaction with Calcium-Dependent Protein Kinase 1

PubMed Central

Child, Matthew A.; Garland, Megan; Foe, Ian; Madzelan, Peter; Treeck, Moritz; van der Linden, Wouter A.; Oresic Bender, Kristina; Weerapana, Eranthie; Wilson, Mark A.; Boothroyd, John C.; Reese, Michael L.

2017-01-01

ABSTRACT Human DJ-1 is a highly conserved and yet functionally enigmatic protein associated with a heritable form of Parkinson’s disease. It has been suggested to be a redox-dependent regulatory scaffold, binding to proteins to modulate their function. Here we present the X-ray crystal structure of the Toxoplasma orthologue Toxoplasma gondii DJ-1 (TgDJ-1) at 2.1-Å resolution and show that it directly associates with calcium-dependent protein kinase 1 (CDPK1). The TgDJ-1 structure identifies an orthologously conserved arginine dyad that acts as a phospho-gatekeeper motif to control complex formation. We determined that the binding of TgDJ-1 to CDPK1 is sensitive to oxidation and calcium, and that this interaction potentiates CDPK1 kinase activity. Finally, we show that genetic deletion of TgDJ-1 results in upregulation of CDPK1 expression and that disruption of the CDPK1/TgDJ-1 complex in vivo prevents normal exocytosis of parasite virulence-associated organelles called micronemes. Overall, our data suggest that TgDJ-1 functions as a noncanonical kinase-regulatory scaffold that integrates multiple intracellular signals to tune microneme exocytosis in T. gondii. PMID:28246362

Readily functionalized AAA-DDD triply hydrogen-bonded motifs.

PubMed

Tong, Feng; Linares-Mendez, Iamnica J; Han, Yi-Fei; Wisner, James A; Wang, Hong-Bo

2018-04-25

Herein we present a new, readily functionalized AAA-DDD hydrogen bond array. A novel AAA monomeric unit (3a-b) was obtained from a two-step synthetic procedure starting with 2-aminonicotinaldehyde via microwave radiation (overall yield of 52-66%). 1H NMR and fluorescence spectroscopy confirmed the complexation event with a calculated association constant of 1.57 × 107 M-1. Likewise, the usefulness of this triple hydrogen bond motif in supramolecular polymerization was demonstrated through viscosity measurements in a crosslinked supramolecular alternating copolymer.

Molecularly Defined Nanostructures Based on a Novel AAA-DDD Triple Hydrogen-Bonding Motif.

PubMed

Papmeyer, Marcus; Vuilleumier, Clément A; Pavan, Giovanni M; Zhurov, Konstantin O; Severin, Kay

2016-01-26

A facile and flexible method for the synthesis of a new AAA-DDD triple hydrogen-bonding motif is described. Polytopic supramolecular building blocks with precisely oriented AAA and DDD groups are thus accessible in few steps. These building blocks were used for the assembly of large macrocycles featuring four AAA-DDD interactions and a macrobicyclic complex with a total of six AAA-DDD interactions. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

ELM server: a new resource for investigating short functional sites in modular eukaryotic proteins

PubMed Central

Puntervoll, Pål; Linding, Rune; Gemünd, Christine; Chabanis-Davidson, Sophie; Mattingsdal, Morten; Cameron, Scott; Martin, David M. A.; Ausiello, Gabriele; Brannetti, Barbara; Costantini, Anna; Ferrè, Fabrizio; Maselli, Vincenza; Via, Allegra; Cesareni, Gianni; Diella, Francesca; Superti-Furga, Giulio; Wyrwicz, Lucjan; Ramu, Chenna; McGuigan, Caroline; Gudavalli, Rambabu; Letunic, Ivica; Bork, Peer; Rychlewski, Leszek; Küster, Bernhard; Helmer-Citterich, Manuela; Hunter, William N.; Aasland, Rein; Gibson, Toby J.

2003-01-01

Multidomain proteins predominate in eukaryotic proteomes. Individual functions assigned to different sequence segments combine to create a complex function for the whole protein. While on-line resources are available for revealing globular domains in sequences, there has hitherto been no comprehensive collection of small functional sites/motifs comparable to the globular domain resources, yet these are as important for the function of multidomain proteins. Short linear peptide motifs are used for cell compartment targeting, protein–protein interaction, regulation by phosphorylation, acetylation, glycosylation and a host of other post-translational modifications. ELM, the Eukaryotic Linear Motif server at http://elm.eu.org/, is a new bioinformatics resource for investigating candidate short non-globular functional motifs in eukaryotic proteins, aiming to fill the void in bioinformatics tools. Sequence comparisons with short motifs are difficult to evaluate because the usual significance assessments are inappropriate. Therefore the server is implemented with several logical filters to eliminate false positives. Current filters are for cell compartment, globular domain clash and taxonomic range. In favourable cases, the filters can reduce the number of retained matches by an order of magnitude or more. PMID:12824381

La-related protein 1 (LARP1) binds the mRNA cap, blocking eIF4F assembly on TOP mRNAs.

PubMed

Lahr, Roni M; Fonseca, Bruno D; Ciotti, Gabrielle E; Al-Ashtal, Hiba A; Jia, Jian-Jun; Niklaus, Marius R; Blagden, Sarah P; Alain, Tommy; Berman, Andrea J

2017-04-07

The 5'terminal oligopyrimidine (5'TOP) motif is a cis -regulatory RNA element located immediately downstream of the 7-methylguanosine [m 7 G] cap of TOP mRNAs, which encode ribosomal proteins and translation factors. In eukaryotes, this motif coordinates the synchronous and stoichiometric expression of the protein components of the translation machinery. La-related protein 1 (LARP1) binds TOP mRNAs, regulating their stability and translation. We present crystal structures of the human LARP1 DM15 region in complex with a 5'TOP motif, a cap analog (m 7 GTP), and a capped cytidine (m 7 GpppC), resolved to 2.6, 1.8 and 1.7 Å, respectively. Our binding, competition, and immunoprecipitation data corroborate and elaborate on the mechanism of 5'TOP motif binding by LARP1. We show that LARP1 directly binds the cap and adjacent 5'TOP motif of TOP mRNAs, effectively impeding access of eIF4E to the cap and preventing eIF4F assembly. Thus, LARP1 is a specialized TOP mRNA cap-binding protein that controls ribosome biogenesis.

Ca2+-Induced Rigidity Change of the Myosin VIIa IQ Motif-Single α Helix Lever Arm Extension.

PubMed

Li, Jianchao; Chen, Yiyun; Deng, Yisong; Unarta, Ilona Christy; Lu, Qing; Huang, Xuhui; Zhang, Mingjie

2017-04-04

Several unconventional myosins contain a highly charged single α helix (SAH) immediately following the calmodulin (CaM) binding IQ motifs, functioning to extend lever arms of these myosins. How such SAH is connected to the IQ motifs and whether the conformation of the IQ motifs-SAH segments are regulated by Ca 2+ fluctuations are not known. Here, we demonstrate by solving its crystal structure that the predicted SAH of myosin VIIa (Myo7a) forms a stable SAH. The structure of Myo7a IQ5-SAH segment in complex with apo-CaM reveals that the SAH sequence can extend the length of the Myo7a lever arm. Although Ca 2+ -CaM remains bound to IQ5-SAH, the Ca 2+ -induced CaM binding mode change softens the conformation of the IQ5-SAH junction, revealing a Ca 2+ -induced lever arm flexibility change for Myo7a. We further demonstrate that the last IQ motif of several other myosins also binds to both apo- and Ca 2+ -CaM, suggesting a common Ca 2+ -induced conformational regulation mechanism. Copyright © 2017 Elsevier Ltd. All rights reserved.

Selective integrin endocytosis is driven by interactions between the integrin α-chain and AP2

PubMed Central

De Franceschi, Nicola; Arjonen, Antti; Elkhatib, Nadia; Denessiouk, Konstantin; Wrobel, Antoni G; Wilson, Thomas A; Pouwels, Jeroen; Montagnac, Guillaume; Owen, David J; Ivaska, Johanna

2016-01-01

Integrins are heterodimeric cell-surface adhesion molecules comprising one of possible 18 α-chains and one of possible 8 β-chains. They control a range of cell functions in a matrix- and ligand-specific manner. Integrins can be internalised by clathrin-mediated endocytosis (CME) through β subunit-based motifs found in all integrin heterodimers. However, whether specific integrin heterodimers can be selectively endocytosed was unknown. Here, we found that a subset of α subunits contain an evolutionarily conserved and functional YxxΦ motif directing integrins to selective internalisation by the most abundant endocytic clathrin adaptor, AP2. We determined the structure of the human integrin α4-tail motif in complex with AP2 C-µ2 subunit and confirmed the interaction by isothermal titration calorimetry. Mutagenesis of the motif impaired selective heterodimer endocytosis and attenuated integrin-mediated cell migration. We propose that integrins evolved to enable selective integrin-receptor turnover in response to changing matrix conditions. PMID:26779610

Selective integrin endocytosis is driven by interactions between the integrin α-chain and AP2.

PubMed

De Franceschi, Nicola; Arjonen, Antti; Elkhatib, Nadia; Denessiouk, Konstantin; Wrobel, Antoni G; Wilson, Thomas A; Pouwels, Jeroen; Montagnac, Guillaume; Owen, David J; Ivaska, Johanna

2016-02-01

Integrins are heterodimeric cell-surface adhesion molecules comprising one of 18 possible α-chains and one of eight possible β-chains. They control a range of cell functions in a matrix- and ligand-specific manner. Integrins can be internalized by clathrin-mediated endocytosis (CME) through β subunit-based motifs found in all integrin heterodimers. However, whether specific integrin heterodimers can be selectively endocytosed was unknown. Here, we found that a subset of α subunits contain an evolutionarily conserved and functional YxxΦ motif directing integrins to selective internalization by the most abundant endocytic clathrin adaptor, AP2. We determined the structure of the human integrin α4-tail motif in complex with the AP2 C-μ2 subunit and confirmed the interaction by isothermal titration calorimetry. Mutagenesis of the motif impaired selective heterodimer endocytosis and attenuated integrin-mediated cell migration. We propose that integrins evolved to enable selective integrin-receptor turnover in response to changing matrix conditions.

Automatic Network Fingerprinting through Single-Node Motifs

PubMed Central

Echtermeyer, Christoph; da Fontoura Costa, Luciano; Rodrigues, Francisco A.; Kaiser, Marcus

2011-01-01

Complex networks have been characterised by their specific connectivity patterns (network motifs), but their building blocks can also be identified and described by node-motifs—a combination of local network features. One technique to identify single node-motifs has been presented by Costa et al. (L. D. F. Costa, F. A. Rodrigues, C. C. Hilgetag, and M. Kaiser, Europhys. Lett., 87, 1, 2009). Here, we first suggest improvements to the method including how its parameters can be determined automatically. Such automatic routines make high-throughput studies of many networks feasible. Second, the new routines are validated in different network-series. Third, we provide an example of how the method can be used to analyse network time-series. In conclusion, we provide a robust method for systematically discovering and classifying characteristic nodes of a network. In contrast to classical motif analysis, our approach can identify individual components (here: nodes) that are specific to a network. Such special nodes, as hubs before, might be found to play critical roles in real-world networks. PMID:21297963

Structures of minimal catalytic fragments of topoisomerase V reveals conformational changes relevant for DNA binding

PubMed Central

Rajan, Rakhi; Taneja, Bhupesh; Mondragón, Alfonso

2010-01-01

Summary Topoisomerase V is an archaeal type I topoisomerase that is unique among topoisomerases due to presence of both topoisomerase and DNA repair activities in the same protein. It is organized as an N-terminal topoisomerase domain followed by 24 tandem helix hairpin helix (HhH) motifs. Structural studies have shown that the active site is buried by the (HhH) motifs. Here we show that the N-terminal domain can relax DNA in the absence of any HhH motifs and that the HhH motifs are required for stable protein-DNA complex formation. Crystal structures of various topoisomerase V fragments show changes in the relative orientation of the domains mediated by a long bent linker helix, and these movements are essential for the DNA to enter the active site. Phosphate ions bound to the protein near the active site helped model DNA in the topoisomerase domain and shows how topoisomerase V may interact with DNA. PMID:20637419

Roles of the distinct electronic structures of the {Fe(NO)(2)}(9) and {Fe(NO)(2)}(10) dinitrosyliron complexes in modulating nitrite binding modes and nitrite activation pathways.

PubMed

Tsai, Fu-Te; Chen, Pei-Lin; Liaw, Wen-Feng

2010-04-14

Nitrosylation of [PPN](2)[(ONO)(2)Fe(eta(2)-ONO)(2)] [1; PPN = bis(triphenylphosphoranylidene)ammonium] yields the nitrite-containing {Fe(NO)}(7) mononitrosyliron complex (MNIC) [PPN](2)[(NO)Fe(ONO)(3)(eta(2)-ONO)] (2). At 4 K, complex 2 exhibits an S = (3)/(2) axial EPR spectrum with principal g values of g( perpendicular) = 3.971 and g( parallel) = 2.000, suggestive of the {Fe(III)(NO(-))}(7) electronic structure. Addition of 1 equiv of PPh(3) to complex 2 triggers O-atom transfer of the chelating nitrito ligand under mild conditions to yield the {Fe(NO)(2)}(9) dinitrosyliron complex (DNIC) [PPN][(ONO)(2)Fe(NO)(2)] (3). These results demonstrate that both electronic structure [{Fe(III)(NO(-))}(7), S = (3)/(2)] and redox-active ligands ([RS](-) for [(RS)(3)Fe(NO)](-) and [NO(-)] for complex 2) are required for the transformation of {Fe(NO)}(7) MNICs into {Fe(NO)(2)}(9) DNICs. In comparison with the PPh(3)-triggered O-atom abstraction of the chelating nitrito ligand of the {Fe(NO)(2)}(9) DNIC [(1-MeIm)(2)(eta(2)-ONO)Fe(NO)(2)] (5; 1-MeIm = 1-methylimidazole) to generate the {Fe(NO)(2)}(10) DNIC [(1-MeIm)(PPh(3))Fe(NO)(2)] (6), glacial acetic acid protonation of the N-bound nitro ligand in the {Fe(NO)(2)}(10) DNIC [PPN][(eta(1)-NO(2))(PPh(3))Fe(NO)(2)] (7) produced the {Fe(NO)(2)}(9) DNIC [PPN][(OAc)(2)Fe(NO)(2)] (8), nitric oxide, and H(2)O. These results demonstrate that the distinct electronic structures of {Fe(NO)(2)}(9/10) motifs [{Fe(NO)(2)}(9) vs {Fe(NO)(2)}(10)] play crucial roles in modulating nitrite binding modes (O-bound chelating/monodentate nitrito for {Fe(NO)(2)}(9) DNICs vs N-bound nitro as a pi acceptor for {Fe(NO)(2)}(10) DNICs) and regulating nitrite activation pathways (O-atom abstraction by PPh(3) leading to the intermediate with a nitroxyl-coordinated ligand vs protonation accompanied by dehydration leading to the intermediate with a nitrosonium-coordinated ligand). That is, the redox shuttling between the {Fe(NO)(2)}(9) and {Fe(NO)(2)}(10) DNICs modulates the nitrite binding modes and then triggers nitrite activation to generate nitric oxide.

MLL/WDR5 Complex Regulates Kif2A Localization to Ensure Chromosome Congression and Proper Spindle Assembly during Mitosis.

PubMed

Ali, Aamir; Veeranki, Sailaja Naga; Chinchole, Akash; Tyagi, Shweta

2017-06-19

Mixed-lineage leukemia (MLL), along with multisubunit (WDR5, RbBP5, ASH2L, and DPY30) complex catalyzes the trimethylation of H3K4, leading to gene activation. Here, we characterize a chromatin-independent role for MLL during mitosis. MLL and WDR5 localize to the mitotic spindle apparatus, and loss of function of MLL complex by RNAi results in defects in chromosome congression and compromised spindle formation. We report interaction of MLL complex with several kinesin and dynein motors. We further show that the MLL complex associates with Kif2A, a member of the Kinesin-13 family of microtubule depolymerase, and regulates the spindle localization of Kif2A during mitosis. We have identified a conserved WDR5 interaction (Win) motif, so far unique to the MLL family, in Kif2A. The Win motif of Kif2A engages in direct interactions with WDR5 for its spindle localization. Our findings highlight a non-canonical mitotic function of MLL complex, which may have a direct impact on chromosomal stability, frequently compromised in cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

A two-step spin crossover mononuclear iron(II) complex with a [HS-LS-LS] intermediate phase.

PubMed

Bonnet, Sylvestre; Siegler, Maxime A; Costa, José Sánchez; Molnár, Gábor; Bousseksou, Azzedine; Spek, Anthony L; Gamez, Patrick; Reedijk, Jan

2008-11-21

The two-step spin crossover of a new mononuclear iron(ii) complex is studied by magnetic, crystallographic and calorimetric methods revealing two successive first-order phase transitions and an ordered intermediate phase built by the repetition of the unprecedented [HS-LS-LS] motif.

Modeling of DNA local parameters predicts encrypted architectural motifs in Xenopus laevis ribosomal gene promoter.

PubMed

Roux-Rouquie, M; Marilley, M

2000-09-15

We have modeled local DNA sequence parameters to search for DNA architectural motifs involved in transcription regulation and promotion within the Xenopus laevis ribosomal gene promoter and the intergenic spacer (IGS) sequences. The IGS was found to be shaped into distinct topological domains. First, intrinsic bends split the IGS into domains of common but different helical features. Local parameters at inter-domain junctions exhibit a high variability with respect to intrinsic curvature, bendability and thermal stability. Secondly, the repeated sequence blocks of the IGS exhibit right-handed supercoiled structures which could be related to their enhancer properties. Thirdly, the gene promoter presents both inherent curvature and minor groove narrowing which may be viewed as motifs of a structural code for protein recognition and binding. Such pre-existing deformations could simply be remodeled during the binding of the transcription complex. Alternatively, these deformations could pre-shape the promoter in such a way that further remodeling is facilitated. Mutations shown to abolish promoter curvature as well as intrinsic minor groove narrowing, in a variant which maintained full transcriptional activity, bring circumstantial evidence for structurally-preorganized motifs in relation to transcription regulation and promotion. Using well documented X. laevis rDNA regulatory sequences we showed that computer modeling may be of invaluable assistance in assessing encrypted architectural motifs. The evidence of these DNA topological motifs with respect to the concept of structural code is discussed.

Discovery and validation of information theory-based transcription factor and cofactor binding site motifs.

PubMed

Lu, Ruipeng; Mucaki, Eliseos J; Rogan, Peter K

2017-03-17

Data from ChIP-seq experiments can derive the genome-wide binding specificities of transcription factors (TFs) and other regulatory proteins. We analyzed 765 ENCODE ChIP-seq peak datasets of 207 human TFs with a novel motif discovery pipeline based on recursive, thresholded entropy minimization. This approach, while obviating the need to compensate for skewed nucleotide composition, distinguishes true binding motifs from noise, quantifies the strengths of individual binding sites based on computed affinity and detects adjacent cofactor binding sites that coordinate with the targets of primary, immunoprecipitated TFs. We obtained contiguous and bipartite information theory-based position weight matrices (iPWMs) for 93 sequence-specific TFs, discovered 23 cofactor motifs for 127 TFs and revealed six high-confidence novel motifs. The reliability and accuracy of these iPWMs were determined via four independent validation methods, including the detection of experimentally proven binding sites, explanation of effects of characterized SNPs, comparison with previously published motifs and statistical analyses. We also predict previously unreported TF coregulatory interactions (e.g. TF complexes). These iPWMs constitute a powerful tool for predicting the effects of sequence variants in known binding sites, performing mutation analysis on regulatory SNPs and predicting previously unrecognized binding sites and target genes. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Identification, occurrence, and validation of DRE and ABRE Cis-regulatory motifs in the promoter regions of genes of Arabidopsis thaliana.

PubMed

Mishra, Sonal; Shukla, Aparna; Upadhyay, Swati; Sanchita; Sharma, Pooja; Singh, Seema; Phukan, Ujjal J; Meena, Abha; Khan, Feroz; Tripathi, Vineeta; Shukla, Rakesh Kumar; Shrama, Ashok

2014-04-01

Plants posses a complex co-regulatory network which helps them to elicit a response under diverse adverse conditions. We used an in silico approach to identify the genes with both DRE and ABRE motifs in their promoter regions in Arabidopsis thaliana. Our results showed that Arabidopsis contains a set of 2,052 genes with ABRE and DRE motifs in their promoter regions. Approximately 72% or more of the total predicted 2,052 genes had a gap distance of less than 400 bp between DRE and ABRE motifs. For positional orientation of the DRE and ABRE motifs, we found that the DR form (one in direct and the other one in reverse orientation) was more prevalent than other forms. These predicted 2,052 genes include 155 transcription factors. Using microarray data from The Arabidopsis Information Resource (TAIR) database, we present 44 transcription factors out of 155 which are upregulated by more than twofold in response to osmotic stress and ABA treatment. Fifty-one transcripts from the one predicted above were validated using semiquantitative expression analysis to support the microarray data in TAIR. Taken together, we report a set of genes containing both DRE and ABRE motifs in their promoter regions in A. thaliana, which can be useful to understand the role of ABA under osmotic stress condition. © 2013 Institute of Botany, Chinese Academy of Sciences.

The ER Contact Proteins VAPA/B Interact with Multiple Autophagy Proteins to Modulate Autophagosome Biogenesis.

PubMed

Zhao, Yan G; Liu, Nan; Miao, Guangyan; Chen, Yong; Zhao, Hongyu; Zhang, Hong

2018-04-23

The endoplasmic reticulum (ER) is the site of biogenesis of the isolation membrane (IM, autophagosome precursor) and forms extensive contacts with IMs during their expansion into double-membrane autophagosomes. Little is known about the molecular mechanism underlying the formation and/or maintenance of the ER/IM contact. The integral ER proteins VAPA and VAPB (VAPs) participate in establishing ER contacts with multiple membranes by interacting with different tethers. Here, we demonstrate that VAPs also modulate ER/IM contact formation. Depletion of VAPs impairs progression of IMs into autophagosomes. Upon autophagy induction, VAPs are recruited to autophagosome formation sites on the ER, a process mediated by their interactions with FIP200 and PI(3)P. VAPs directly interact with FIP200 and ULK1 through their conserved FFAT motifs and stabilize the ULK1/FIP200 complex at the autophagosome formation sites on the ER. The formation of ULK1 puncta is significantly reduced by VAPA/B depletion. VAPs also interact with WIPI2 and enhance the formation of the WIPI2/FIP200 ER/IM tethering complex. Depletion of VMP1, which increases the ER/IM contact, greatly elevates the interaction of VAPs with these autophagy proteins. The VAPB P56S mutation, which is associated with amyotrophic lateral sclerosis, reduces the ULK1/FIP200 interaction and impairs autophagy at an early step, similar to the effect seen in VAPA/B-depleted cells. Our study reveals that VAPs directly interact with multiple ATG proteins, thereby contributing to ER/IM contact formation for autophagosome biogenesis. Copyright © 2018 Elsevier Ltd. All rights reserved.

Integrative Structure–Function Mapping of the Nucleoporin Nup133 Suggests a Conserved Mechanism for Membrane Anchoring of the Nuclear Pore Complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Seung Joong; Fernandez-Martinez, Javier; Sampathkumar, Parthasarathy

2014-08-19

The nuclear pore complex (NPC) is the sole passageway for the transport of macromolecules across the nuclear envelope. Nup133, a major component in the essential Y-shaped Nup84 complex, is a large scaffold protein of the NPC's outer ring structure. Here, we describe an integrative modeling approach that produces atomic models for multiple states of Saccharomyces cerevisiae (Sc) Nup133, based on the crystal structures of the sequence segments and their homologs, including the related Vanderwaltozyma polyspora (Vp) Nup133 residues 55 to 502 (VpNup133 55–502) determined in this study, small angle X-ray scattering profiles for 18 constructs of ScNup133 and one constructmore » of VpNup133, and 23 negative-stain electron microscopy class averages of ScNup1332–1157. Using our integrative approach, we then computed a multi-state structural model of the full-length ScNup133 and validated it with mutational studies and 45 chemical cross-links determined via mass spectrometry. Finally, the model of ScNup133 allowed us to annotate a potential ArfGAP1 lipid packing sensor (ALPS) motif in Sc and VpNup133 and discuss its potential significance in the context of the whole NPC; we suggest that ALPS motifs are scattered throughout the NPC's scaffold in all eukaryotes and play a major role in the assembly and membrane anchoring of the NPC in the nuclear envelope. Our results are consistent with a common evolutionary origin of Nup133 with membrane coating complexes (the protocoatomer hypothesis); the presence of the ALPS motifs in coatomer-like nucleoporins suggests an ancestral mechanism for membrane recognition present in early membrane coating complexes.« less

Integrative Structure–Function Mapping of the Nucleoporin Nup133 Suggests a Conserved Mechanism for Membrane Anchoring of the Nuclear Pore Complex*

PubMed Central

Kim, Seung Joong; Fernandez-Martinez, Javier; Sampathkumar, Parthasarathy; Martel, Anne; Matsui, Tsutomu; Tsuruta, Hiro; Weiss, Thomas M.; Shi, Yi; Markina-Inarrairaegui, Ane; Bonanno, Jeffery B.; Sauder, J. Michael; Burley, Stephen K.; Chait, Brian T.; Almo, Steven C.; Rout, Michael P.; Sali, Andrej

2014-01-01

The nuclear pore complex (NPC) is the sole passageway for the transport of macromolecules across the nuclear envelope. Nup133, a major component in the essential Y-shaped Nup84 complex, is a large scaffold protein of the NPC's outer ring structure. Here, we describe an integrative modeling approach that produces atomic models for multiple states of Saccharomyces cerevisiae (Sc) Nup133, based on the crystal structures of the sequence segments and their homologs, including the related Vanderwaltozyma polyspora (Vp) Nup133 residues 55 to 502 (VpNup13355–502) determined in this study, small angle X-ray scattering profiles for 18 constructs of ScNup133 and one construct of VpNup133, and 23 negative-stain electron microscopy class averages of ScNup1332–1157. Using our integrative approach, we then computed a multi-state structural model of the full-length ScNup133 and validated it with mutational studies and 45 chemical cross-links determined via mass spectrometry. Finally, the model of ScNup133 allowed us to annotate a potential ArfGAP1 lipid packing sensor (ALPS) motif in Sc and VpNup133 and discuss its potential significance in the context of the whole NPC; we suggest that ALPS motifs are scattered throughout the NPC's scaffold in all eukaryotes and play a major role in the assembly and membrane anchoring of the NPC in the nuclear envelope. Our results are consistent with a common evolutionary origin of Nup133 with membrane coating complexes (the protocoatomer hypothesis); the presence of the ALPS motifs in coatomer-like nucleoporins suggests an ancestral mechanism for membrane recognition present in early membrane coating complexes. PMID:25139911

Switch I-dependent allosteric signaling in a G-protein chaperone-B12 enzyme complex.

PubMed

Campanello, Gregory C; Lofgren, Michael; Yokom, Adam L; Southworth, Daniel R; Banerjee, Ruma

2017-10-27

G-proteins regulate various processes ranging from DNA replication and protein synthesis to cytoskeletal dynamics and cofactor assimilation and serve as models for uncovering strategies deployed for allosteric signal transduction. MeaB is a multifunctional G-protein chaperone, which gates loading of the active 5'-deoxyadenosylcobalamin cofactor onto methylmalonyl-CoA mutase (MCM) and precludes loading of inactive cofactor forms. MeaB also safeguards MCM, which uses radical chemistry, against inactivation and rescues MCM inactivated during catalytic turnover by using the GTP-binding energy to offload inactive cofactor. The conserved switch I and II signaling motifs used by G-proteins are predicted to mediate allosteric regulation in response to nucleotide binding and hydrolysis in MeaB. Herein, we targeted conserved residues in the MeaB switch I motif to interrogate the function of this loop. Unexpectedly, the switch I mutations had only modest effects on GTP binding and on GTPase activity and did not perturb stability of the MCM-MeaB complex. However, these mutations disrupted multiple MeaB chaperone functions, including cofactor editing, loading, and offloading. Hence, although residues in the switch I motif are not essential for catalysis, they are important for allosteric regulation. Furthermore, single-particle EM analysis revealed, for the first time, the overall architecture of the MCM-MeaB complex, which exhibits a 2:1 stoichiometry. These EM studies also demonstrate that the complex exhibits considerable conformational flexibility. In conclusion, the switch I element does not significantly stabilize the MCM-MeaB complex or influence the affinity of MeaB for GTP but is required for transducing signals between MeaB and MCM. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Tetratricopeptide-motif-mediated interaction of FANCG with recombination proteins XRCC3 and BRCA2.

PubMed

Hussain, Shobbir; Wilson, James B; Blom, Eric; Thompson, Larry H; Sung, Patrick; Gordon, Susan M; Kupfer, Gary M; Joenje, Hans; Mathew, Christopher G; Jones, Nigel J

2006-05-10

Fanconi anaemia is an inherited chromosomal instability disorder characterised by cellular sensitivity to DNA interstrand crosslinkers, bone-marrow failure and a high risk of cancer. Eleven FA genes have been identified, one of which, FANCD1, is the breast cancer susceptibility gene BRCA2. At least eight FA proteins form a nuclear core complex required for monoubiquitination of FANCD2. The BRCA2/FANCD1 protein is connected to the FA pathway by interactions with the FANCG and FANCD2 proteins, both of which co-localise with the RAD51 recombinase, which is regulated by BRCA2. These connections raise the question of whether any of the FANC proteins of the core complex might also participate in other complexes involved in homologous recombination repair. We therefore tested known FA proteins for direct interaction with RAD51 and its paralogs XRCC2 and XRCC3. FANCG was found to interact with XRCC3, and this interaction was disrupted by the FA-G patient derived mutation L71P. FANCG was co-immunoprecipitated with both XRCC3 and BRCA2 from extracts of human and hamster cells. The FANCG-XRCC3 and FANCG-BRCA2 interactions did not require the presence of other FA proteins from the core complex, suggesting that FANCG also participates in a DNA repair complex that is downstream and independent of FANCD2 monoubiquitination. Additionally, XRCC3 and BRCA2 proteins co-precipitate in both human and hamster cells and this interaction requires FANCG. The FANCG protein contains multiple tetratricopeptide repeat motifs (TPRs), which function as scaffolds to mediate protein-protein interactions. Mutation of one or more of these motifs disrupted all of the known interactions of FANCG. We propose that FANCG, in addition to stabilising the FA core complex, may have a role in building multiprotein complexes that facilitate homologous recombination repair.

Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily

PubMed Central

Lenoir, Marc; Kufareva, Irina; Abagyan, Ruben; Overduin, Michael

2015-01-01

The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer. PMID:26512702

ANF-RGC gene motif 669WTAPELL675 is vital for blood pressure regulation: Biochemical mechanism

PubMed Central

Duda, Teresa; Pertzev, Alexandre; Sharma, Rameshwar K.

2013-01-01

ANF-RGC is the prototype membrane guanylate cyclase, both the receptor and the signal transducer of the hormones ANF and BNP. After binding them at the extracellular domain it, at its intracellular domain, signals activation of the C-terminal catalytic module and accelerates production of the second messenger, cyclic GMP. This, in turn, controls the physiological processes of blood pressure, cardiovascular function, and fluid secretion, and others: metabolic syndrome, obesity and apoptosis. What is the biochemical mechanism by which this single molecule controls these diverse processes, explicitly of the blood pressure regulation is the subject of the present study. In line with the concept that the structural modules of ANF-RGC are designed to respond to more than one, yet distinctive signals, the study demonstrates the construction of a novel ANF-RGC-In-gene-669WTAPELL675 mouse model. Through this model, the study establishes that 669WTAPELL675 is a vital ANF signal transducer motif of the guanylate cyclase. Its striking physiological features linked with their biochemistry are that (1) it controls the hormonally-dependent cyclic GMP production in the kidney and the adrenal gland; (3) its deletion causes hypertension, and (3) cardiac hypertrophy; and (4) these mice show higher levels of the plasma aldosterone. For the first time, a mere 7-amino acid encoded motif of the mouse gene has been directly linked with the physiological control of the blood pressure regulation, a detailed biochemistry of this linkage has been established and a model for this linkage has been offered. PMID:23464624

Secbase: database module to retrieve secondary structure elements with ligand binding motifs.

PubMed

Koch, Oliver; Cole, Jason; Block, Peter; Klebe, Gerhard

2009-10-01

Secbase is presented as a novel extension module of Relibase. It integrates the information about secondary structure elements into the retrieval facilities of Relibase. The data are accessible via the extended Relibase user interface, and integrated retrieval queries can be addressed using an extended version of Reliscript. The primary information about alpha-helices and beta-sheets is used as provided by the PDB. Furthermore, a uniform classification of all turn families, based on recent clustering methods, and a new helix assignment that is based on this turn classification has been included. Algorithms to analyze the geometric features of helices and beta-strands were also implemented. To demonstrate the performance of the Secbase implementation, some application examples are given. They provide new insights into the involvement of secondary structure elements in ligand binding. A survey of water molecules detected next to the N-terminus of helices is analyzed to show their involvement in ligand binding. Additionally, the parallel oriented NH groups at the alpha-helix N-termini provide special binding motifs to bind particular ligand functional groups with two adjacent oxygen atoms, e.g., as found in negatively charged carboxylate or phosphate groups, respectively. The present study also shows that the specific structure of the first turn of alpha-helices provides a suitable explanation for stabilizing charged structures. The magnitude of the overall helix macrodipole seems to have no or only a minor influence on binding. Furthermore, an overview of the involvement of secondary structure elements with the recognition of some important endogenous ligands such as cofactors shows some distinct preference for particular binding motifs and amino acids.

[Conserved motifs in voltage sensing proteins].

PubMed

Wang, Chang-He; Xie, Zhen-Li; Lv, Jian-Wei; Yu, Zhi-Dan; Shao, Shu-Li

2012-08-25

This paper was aimed to study conserved motifs of voltage sensing proteins (VSPs) and establish a voltage sensing model. All VSPs were collected from the Uniprot database using a comprehensive keyword search followed by manual curation, and the results indicated that there are only two types of known VSPs, voltage gated ion channels and voltage dependent phosphatases. All the VSPs have a common domain of four helical transmembrane segments (TMS, S1-S4), which constitute the voltage sensing module of the VSPs. The S1 segment was shown to be responsible for membrane targeting and insertion of these proteins, while S2-S4 segments, which can sense membrane potential, for protein properties. Conserved motifs/residues and their functional significance of each TMS were identified using profile-to-profile sequence alignments. Conserved motifs in these four segments are strikingly similar for all VSPs, especially, the conserved motif [RK]-X(2)-R-X(2)-R-X(2)-[RK] was presented in all the S4 segments, with positively charged arginine (R) alternating with two hydrophobic or uncharged residues. Movement of these arginines across the membrane electric field is the core mechanism by which the VSPs detect changes in membrane potential. The negatively charged aspartate (D) in the S3 segment is universally conserved in all the VSPs, suggesting that the aspartate residue may be involved in voltage sensing properties of VSPs as well as the electrostatic interactions with the positively charged residues in the S4 segment, which may enhance the thermodynamic stability of the S4 segments in plasma membrane.

Physicochemically Tunable Polyfunctionalized RNA Square Architecture with Fluorogenic and Ribozymatic Properties

PubMed Central

2015-01-01

Recent advances in RNA nanotechnology allow the rational design of various nanoarchitectures. Previous methods utilized conserved angles from natural RNA motifs to form geometries with specific sizes. However, the feasibility of producing RNA architecture with variable sizes using native motifs featuring fixed sizes and angles is limited. It would be advantageous to display RNA nanoparticles of diverse shape and size derived from a given primary sequence. Here, we report an approach to construct RNA nanoparticles with tunable size and stability. Multifunctional RNA squares with a 90° angle were constructed by tuning the 60° angle of the three-way junction (3WJ) motif from the packaging RNA (pRNA) of the bacteriophage phi29 DNA packaging motor. The physicochemical properties and size of the RNA square were also easily tuned by modulating the “core” strand and adjusting the length of the sides of the square via predictable design. Squares of 5, 10, and 20 nm were constructed, each showing diverse thermodynamic and chemical stabilities. Four “arms” extending from the corners of the square were used to incorporate siRNA, ribozyme, and fluorogenic RNA motifs. Unique intramolecular contact using the pre-existing intricacy of the 3WJ avoids relatively weaker intermolecular interactions via kissing loops or sticky ends. Utilizing the 3WJ motif, we have employed a modular design technique to construct variable-size RNA squares with controllable properties and functionalities for diverse and versatile applications with engineering, pharmaceutical, and medical potential. This technique for simple design to finely tune physicochemical properties adds a new angle to RNA nanotechnology. PMID:24971772

Efficient budding of the tacaribe virus matrix protein z requires the nucleoprotein.

PubMed

Groseth, Allison; Wolff, Svenja; Strecker, Thomas; Hoenen, Thomas; Becker, Stephan

2010-04-01

The Z protein has been shown for several arenaviruses to serve as the viral matrix protein. As such, Z provides the principal force for the budding of virus particles and is capable of forming virus-like particles (VLPs) when expressed alone. For most arenaviruses, this activity has been shown to be linked to the presence of proline-rich late-domain motifs in the C terminus; however, for the New World arenavirus Tacaribe virus (TCRV), no such motif exists within Z. It was recently demonstrated that while TCRV Z is still capable of functioning as a matrix protein to induce the formation of VLPs, neither its ASAP motif, which replaces a canonical PT/SAP motif in related viruses, nor its YxxL motif is involved in budding, leading to the suggestion that TCRV uses a novel budding mechanism. Here we show that in comparison to its closest relative, Junin virus (JUNV), TCRV Z buds only weakly when expressed in isolation. While this budding activity is independent of the ASAP or YxxL motif, it is significantly enhanced by coexpression with the nucleoprotein (NP), an effect not seen with JUNV Z. Interestingly, both the ASAP and YxxL motifs of Z appear to be critical for the recruitment of NP into VLPs, as well as for the enhancement of TCRV Z-mediated budding. While it is known that TCRV budding remains dependent on the endosomal sorting complex required for transport, our findings provide further evidence that TCRV uses a budding mechanism distinct from that of other known arenaviruses and suggest an essential role for NP in this process.

Accurate Quantification of microRNA via Single Strand Displacement Reaction on DNA Origami Motif

PubMed Central

Lou, Jingyu; Li, Weidong; Li, Sheng; Zhu, Hongxin; Yang, Lun; Zhang, Aiping; He, Lin; Li, Can

2013-01-01

DNA origami is an emerging technology that assembles hundreds of staple strands and one single-strand DNA into certain nanopattern. It has been widely used in various fields including detection of biological molecules such as DNA, RNA and proteins. MicroRNAs (miRNAs) play important roles in post-transcriptional gene repression as well as many other biological processes such as cell growth and differentiation. Alterations of miRNAs' expression contribute to many human diseases. However, it is still a challenge to quantitatively detect miRNAs by origami technology. In this study, we developed a novel approach based on streptavidin and quantum dots binding complex (STV-QDs) labeled single strand displacement reaction on DNA origami to quantitatively detect the concentration of miRNAs. We illustrated a linear relationship between the concentration of an exemplary miRNA as miRNA-133 and the STV-QDs hybridization efficiency; the results demonstrated that it is an accurate nano-scale miRNA quantifier motif. In addition, both symmetrical rectangular motif and asymmetrical China-map motif were tested. With significant linearity in both motifs, our experiments suggested that DNA Origami motif with arbitrary shape can be utilized in this method. Since this DNA origami-based method we developed owns the unique advantages of simple, time-and-material-saving, potentially multi-targets testing in one motif and relatively accurate for certain impurity samples as counted directly by atomic force microscopy rather than fluorescence signal detection, it may be widely used in quantification of miRNAs. PMID:23990889

SUMOylation target sites at the C terminus protect Axin from ubiquitination and confer protein stability

PubMed Central

Kim, Min Jung; Chia, Ian V.; Costantini, Frank

2008-01-01

Axin is a scaffold protein for the β-catenin destruction complex, and a negative regulator of canonical Wnt signaling. Previous studies implicated the six C-terminal amino acids (C6 motif) in the ability of Axin to activate c-Jun N-terminal kinase, and identified them as a SUMOylation target. Deletion of the C6 motif of mouse Axin in vivo reduced the steady-state protein level, which caused embryonic lethality. Here, we report that this deletion (Axin-ΔC6) causes a reduced half-life in mouse embryonic fibroblasts and an increased susceptibility to ubiquitination in HEK 293T cells. We confirmed the C6 motif as a SUMOylation target in vitro, and found that mutating the C-terminal SUMOylation target residues increased the susceptibility of Axin to polyubiquitination and reduced its steady-state level. Heterologous SUMOylation target sites could replace C6 in providing this protective effect. These findings suggest that SUMOylation of the C6 motif may prevent polyubiquitination, thus increasing the stability of Axin. Although C6 deletion also caused increased association of Axin with Dvl-1, this interaction was not altered by mutating the lysine residues in C6, nor could heterologous SUMOylation motifs replace the C6 motif in this assay. Therefore, some other specific property of the C6 motif seems to reduce the interaction of Axin with Dvl-1.—Kim, M. J., Chia, I. V., Costantini, F. SUMOylation target sites at the C terminus protect Axin from ubiquitination and confer protein stability. PMID:18632848

Identification of cancer-specific motifs in mimotope profiles of serum antibody repertoire.

PubMed

Gerasimov, Ekaterina; Zelikovsky, Alex; Măndoiu, Ion; Ionov, Yurij

2017-06-07

For fighting cancer, earlier detection is crucial. Circulating auto-antibodies produced by the patient's own immune system after exposure to cancer proteins are promising bio-markers for the early detection of cancer. Since an antibody recognizes not the whole antigen but 4-7 critical amino acids within the antigenic determinant (epitope), the whole proteome can be represented by a random peptide phage display library. This opens the possibility to develop an early cancer detection test based on a set of peptide sequences identified by comparing cancer patients' and healthy donors' global peptide profiles of antibody specificities. Due to the enormously large number of peptide sequences contained in global peptide profiles generated by next generation sequencing, the large number of cancer and control sera is required to identify cancer-specific peptides with high degree of statistical significance. To decrease the number of peptides in profiles generated by nextgen sequencing without losing cancer-specific sequences we used for generation of profiles the phage library enriched by panning on the pool of cancer sera. To further decrease the complexity of profiles we used computational methods for transforming a list of peptides constituting the mimotope profiles to the list motifs formed by similar peptide sequences. We have shown that the amino-acid order is meaningful in mimotope motifs since they contain significantly more peptides than motifs among peptides where amino-acids are randomly permuted. Also the single sample motifs significantly differ from motifs in peptides drawn from multiple samples. Finally, multiple cancer-specific motifs have been identified.

A Dbf4p BRCA1 C-Terminal-Like Domain Required for the Response to Replication Fork Arrest in Budding Yeast

PubMed Central

Gabrielse, Carrie; Miller, Charles T.; McConnell, Kristopher H.; DeWard, Aaron; Fox, Catherine A.; Weinreich, Michael

2006-01-01

Dbf4p is an essential regulatory subunit of the Cdc7p kinase required for the initiation of DNA replication. Cdc7p and Dbf4p orthologs have also been shown to function in the response to DNA damage. A previous Dbf4p multiple sequence alignment identified a conserved ∼40-residue N-terminal region with similarity to the BRCA1 C-terminal (BRCT) motif called “motif N.” BRCT motifs encode ∼100-amino-acid domains involved in the DNA damage response. We have identified an expanded and conserved ∼100-residue N-terminal region of Dbf4p that includes motif N but is capable of encoding a single BRCT-like domain. Dbf4p orthologs diverge from the BRCT motif at the C terminus but may encode a similar secondary structure in this region. We have therefore called this the BRCT and DBF4 similarity (BRDF) motif. The principal role of this Dbf4p motif was in the response to replication fork (RF) arrest; however, it was not required for cell cycle progression, activation of Cdc7p kinase activity, or interaction with the origin recognition complex (ORC) postulated to recruit Cdc7p–Dbf4p to origins. Rad53p likely directly phosphorylated Dbf4p in response to RF arrest and Dbf4p was required for Rad53p abundance. Rad53p and Dbf4p therefore cooperated to coordinate a robust cellular response to RF arrest. PMID:16547092

Differential control of the tyrosine kinases Lyn and Syk by the two signaling chains of the high affinity immunoglobulin E receptor.

PubMed

Jouvin, M H; Adamczewski, M; Numerof, R; Letourneur, O; Vallé, A; Kinet, J P

1994-02-25

Nonreceptor tyrosine kinases such as the newly described 70-kDa (ZAP-70/Syk) and Src-related tyrosine kinases are coupled to a variety of receptors, including the antigen receptors on B- and T-cells and the Fc receptors for IgE (Fc epsilon RI) and IgG (Fc gamma RI, Fc gamma RIII/CD16). Various subunits of these receptors contain homologous activation motifs which appear capable of autonomously triggering cell activation. Two forms of this motif are present in the Fc epsilon RI multimeric complex: one in the beta chain and one in the gamma chain. Here we show that each of the two tyrosine kinases known to be involved in Fc epsilon RI signaling is controlled by a distinct motif-containing chain. Lyn associates with the nonactivated beta chain, whereas gamma promotes the activation of Syk. We also show that neither the beta nor the gamma motif alone can account for the full signaling capacity of the entire receptor. We propose that, upon triggering of the tetrameric receptor, Lyn already bound to beta becomes activated and phosphorylates beta and gamma; the phosphorylation of gamma induces the association of Syk with gamma and also the activation of Syk, resulting in the phosphorylation and activation of phospholipase C gamma 1. Cooperative recruitment of specific kinases by the various signaling chains found in this family of antigen receptors could represent a way to achieve the full signaling capacity of the multimeric complexes.

Slide-and-exchange mechanism for rapid and selective transport through the nuclear pore complex.

PubMed

Raveh, Barak; Karp, Jerome M; Sparks, Samuel; Dutta, Kaushik; Rout, Michael P; Sali, Andrej; Cowburn, David

2016-05-03

Nucleocytoplasmic transport is mediated by the interaction of transport factors (TFs) with disordered phenylalanine-glycine (FG) repeats that fill the central channel of the nuclear pore complex (NPC). However, the mechanism by which TFs rapidly diffuse through multiple FG repeats without compromising NPC selectivity is not yet fully understood. In this study, we build on our recent NMR investigations showing that FG repeats are highly dynamic, flexible, and rapidly exchanging among TF interaction sites. We use unbiased long timescale all-atom simulations on the Anton supercomputer, combined with extensive enhanced sampling simulations and NMR experiments, to characterize the thermodynamic and kinetic properties of FG repeats and their interaction with a model transport factor. Both the simulations and experimental data indicate that FG repeats are highly dynamic random coils, lack intrachain interactions, and exhibit significant entropically driven resistance to spatial confinement. We show that the FG motifs reversibly slide in and out of multiple TF interaction sites, transitioning rapidly between a strongly interacting state and a weakly interacting state, rather than undergoing a much slower transition between strongly interacting and completely noninteracting (unbound) states. In the weakly interacting state, FG motifs can be more easily displaced by other competing FG motifs, providing a simple mechanism for rapid exchange of TF/FG motif contacts during transport. This slide-and-exchange mechanism highlights the direct role of the disorder within FG repeats in nucleocytoplasmic transport, and resolves the apparent conflict between the selectivity and speed of transport.

Simultaneous alignment and clustering of peptide data using a Gibbs sampling approach.

PubMed

Andreatta, Massimo; Lund, Ole; Nielsen, Morten

2013-01-01

Proteins recognizing short peptide fragments play a central role in cellular signaling. As a result of high-throughput technologies, peptide-binding protein specificities can be studied using large peptide libraries at dramatically lower cost and time. Interpretation of such large peptide datasets, however, is a complex task, especially when the data contain multiple receptor binding motifs, and/or the motifs are found at different locations within distinct peptides. The algorithm presented in this article, based on Gibbs sampling, identifies multiple specificities in peptide data by performing two essential tasks simultaneously: alignment and clustering of peptide data. We apply the method to de-convolute binding motifs in a panel of peptide datasets with different degrees of complexity spanning from the simplest case of pre-aligned fixed-length peptides to cases of unaligned peptide datasets of variable length. Example applications described in this article include mixtures of binders to different MHC class I and class II alleles, distinct classes of ligands for SH3 domains and sub-specificities of the HLA-A*02:01 molecule. The Gibbs clustering method is available online as a web server at http://www.cbs.dtu.dk/services/GibbsCluster.

A short conserved motif in ALYREF directs cap- and EJC-dependent assembly of export complexes on spliced mRNAs

PubMed Central

Gromadzka, Agnieszka M.; Steckelberg, Anna-Lena; Singh, Kusum K.; Hofmann, Kay; Gehring, Niels H.

2016-01-01

The export of messenger RNAs (mRNAs) is the final of several nuclear posttranscriptional steps of gene expression. The formation of export-competent mRNPs involves the recruitment of export factors that are assumed to facilitate transport of the mature mRNAs. Using in vitro splicing assays, we show that a core set of export factors, including ALYREF, UAP56 and DDX39, readily associate with the spliced RNAs in an EJC (exon junction complex)- and cap-dependent manner. In order to elucidate how ALYREF and other export adaptors mediate mRNA export, we conducted a computational analysis and discovered four short, conserved, linear motifs present in RNA-binding proteins. We show that mutation in one of the new motifs (WxHD) in an unstructured region of ALYREF reduced RNA binding and abolished the interaction with eIF4A3 and CBP80. Additionally, the mutation impaired proper localization to nuclear speckles and export of a spliced reporter mRNA. Our results reveal important details of the orchestrated recruitment of export factors during the formation of export competent mRNPs. PMID:26773052

A unique PDZ domain and arrestin-like fold interaction reveals mechanistic details of endocytic recycling by SNX27-retromer.

PubMed

Gallon, Matthew; Clairfeuille, Thomas; Steinberg, Florian; Mas, Caroline; Ghai, Rajesh; Sessions, Richard B; Teasdale, Rohan D; Collins, Brett M; Cullen, Peter J

2014-09-02

The sorting nexin 27 (SNX27)-retromer complex is a major regulator of endosome-to-plasma membrane recycling of transmembrane cargos that contain a PSD95, Dlg1, zo-1 (PDZ)-binding motif. Here we describe the core interaction in SNX27-retromer assembly and its functional relevance for cargo sorting. Crystal structures and NMR experiments reveal that an exposed β-hairpin in the SNX27 PDZ domain engages a groove in the arrestin-like structure of the vacuolar protein sorting 26A (VPS26A) retromer subunit. The structure establishes how the SNX27 PDZ domain simultaneously binds PDZ-binding motifs and retromer-associated VPS26. Importantly, VPS26A binding increases the affinity of the SNX27 PDZ domain for PDZ- binding motifs by an order of magnitude, revealing cooperativity in cargo selection. With disruption of SNX27 and retromer function linked to synaptic dysfunction and neurodegenerative disease, our work provides the first step, to our knowledge, in the molecular description of this important sorting complex, and more broadly describes a unique interaction between a PDZ domain and an arrestin-like fold.

Dynamic CRM occupancy reflects a temporal map of developmental progression.

PubMed

Wilczyński, Bartek; Furlong, Eileen E M

2010-06-22

Development is driven by tightly coordinated spatio-temporal patterns of gene expression, which are initiated through the action of transcription factors (TFs) binding to cis-regulatory modules (CRMs). Although many studies have investigated how spatial patterns arise, precise temporal control of gene expression is less well understood. Here, we show that dynamic changes in the timing of CRM occupancy is a prevalent feature common to all TFs examined in a developmental ChIP time course to date. CRMs exhibit complex binding patterns that cannot be explained by the sequence motifs or expression of the TFs themselves. The temporal changes in TF binding are highly correlated with dynamic patterns of target gene expression, which in turn reflect transitions in cellular function during different stages of development. Thus, it is not only the timing of a TF's expression, but also its temporal occupancy in refined time windows, which determines temporal gene expression. Systematic measurement of dynamic CRM occupancy may therefore serve as a powerful method to decode dynamic changes in gene expression driving developmental progression.

Kinome-wide Decoding of Network-Attacking Mutations Rewiring Cancer Signaling

PubMed Central

Creixell, Pau; Schoof, Erwin M.; Simpson, Craig D.; Longden, James; Miller, Chad J.; Lou, Hua Jane; Perryman, Lara; Cox, Thomas R.; Zivanovic, Nevena; Palmeri, Antonio; Wesolowska-Andersen, Agata; Helmer-Citterich, Manuela; Ferkinghoff-Borg, Jesper; Itamochi, Hiroaki; Bodenmiller, Bernd; Erler, Janine T.; Turk, Benjamin E.; Linding, Rune

2015-01-01

Summary Cancer cells acquire pathological phenotypes through accumulation of mutations that perturb signaling networks. However, global analysis of these events is currently limited. Here, we identify six types of network-attacking mutations (NAMs), including changes in kinase and SH2 modulation, network rewiring, and the genesis and extinction of phosphorylation sites. We developed a computational platform (ReKINect) to identify NAMs and systematically interpreted the exomes and quantitative (phospho-)proteomes of five ovarian cancer cell lines and the global cancer genome repository. We identified and experimentally validated several NAMs, including PKCγ M501I and PKD1 D665N, which encode specificity switches analogous to the appearance of kinases de novo within the kinome. We discover mutant molecular logic gates, a drift toward phospho-threonine signaling, weakening of phosphorylation motifs, and kinase-inactivating hotspots in cancer. Our method pinpoints functional NAMs, scales with the complexity of cancer genomes and cell signaling, and may enhance our capability to therapeutically target tumor-specific networks. PMID:26388441

Structural analysis of Notch-regulating Rumi reveals basis for pathogenic mutations

DOE PAGES

Yu, Hongjun; Takeuchi, Hideyuki; Takeuchi, Megumi; ...

2016-07-18

We present Rumi O-glucosylates the EGF repeats of a growing list of proteins essential in metazoan development, including Notch. Rumi is essential for Notch signaling, and Rumi dysregulation is linked to several human diseases. Despite Rumi's critical roles, it is unknown how Rumi glucosylates a serine of many but not all EGF repeats. Here we report crystal structures of Drosophila Rumi as binary and ternary complexes with a folded EGF repeat and/or donor substrates. These structures provide insights into the catalytic mechanism and show that Rumi recognizes structural signatures of the EGF motif, the U-shaped consensus sequence, C-X-S-X-(P/A)-C and amore » conserved hydrophobic region. We found that five Rumi mutations identified in cancers and Dowling–Degos disease are clustered around the enzyme active site and adversely affect its activity. In conclusion, our study suggests that loss of Rumi activity may underlie these diseases, and the mechanistic insights may facilitate the development of modulators of Notch signaling.« less

Synthetic Quorum Sensing and Induced Aggregation in Model Microcapsule Colonies with Repressilator Feedback

NASA Astrophysics Data System (ADS)

Shum, Henry; Yashin, Victor; Balazs, Anna

We model a system of synthetic microcapsules that communicate chemically by releasing nanoparticles or signaling molecules. These signaling species bind to receptors on the shells of capsules and modulate the target shell's permeability, thereby controlling nanoparticle release from the target capsule. Using the repressilator regulatory network motif, whereby three species suppress the production of the next in a cyclic fashion, we show that large amplitude oscillations in nanoparticle release can emerge when many capsules are close together. This exemplifies quorum sensing, which is the ability of cells to gauge their population density and collectively initiate a new behavior once a critical density is reached. We present a physically realizable model in which the oscillations exhibited in crowded populations induce aggregation of the microcapsules, mimicking complex biological behavior of the slime mold Dictyostelium discoideum with only simple, synthetic components. We also show that the clusters can be dispersed and reformed repeatedly and controllably by addition of chemical stimuli, demonstrating possible applications in creating reconfigurable or programmable materials.

Structural analysis of Notch-regulating Rumi reveals basis for pathogenic mutations

PubMed Central

Yu, Hongjun; Takeuchi, Hideyuki; Takeuchi, Megumi; Liu, Qun; Kantharia, Joshua; Haltiwanger, Robert S.; Li, Huilin

2016-01-01

Rumi O-glucosylates the EGF repeats of a growing list of proteins essential in metazoan development including Notch. Rumi is essential for Notch signaling, and Rumi dysregulation is linked to several human diseases. Despite Rumi’s critical roles, it is unknown how Rumi glucosylates a serine of many but not all EGF repeats. Here we report crystal structures of Drosophila Rumi as binary or ternary complexes with a folded EGF repeat and/or donor substrates. These structures provide insights into the catalytic mechanism, and show that Rumi recognizes structural signatures of the EGF motif, the U-shaped consensus sequence, C-X-S-X-(P/A)-C and a conserved hydrophobic region. We found that five Rumi mutations identified in cancers and Dowling-Degos disease are clustered around the enzyme active site and adversely affect its activity. Our study suggests that loss of Rumi activity may underlie these diseases, and the mechanistic insights may facilitate the development of modulators of Notch signaling. PMID:27428513

Structural analysis of Notch-regulating Rumi reveals basis for pathogenic mutations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Hongjun; Takeuchi, Hideyuki; Takeuchi, Megumi

We present Rumi O-glucosylates the EGF repeats of a growing list of proteins essential in metazoan development, including Notch. Rumi is essential for Notch signaling, and Rumi dysregulation is linked to several human diseases. Despite Rumi's critical roles, it is unknown how Rumi glucosylates a serine of many but not all EGF repeats. Here we report crystal structures of Drosophila Rumi as binary and ternary complexes with a folded EGF repeat and/or donor substrates. These structures provide insights into the catalytic mechanism and show that Rumi recognizes structural signatures of the EGF motif, the U-shaped consensus sequence, C-X-S-X-(P/A)-C and amore » conserved hydrophobic region. We found that five Rumi mutations identified in cancers and Dowling–Degos disease are clustered around the enzyme active site and adversely affect its activity. In conclusion, our study suggests that loss of Rumi activity may underlie these diseases, and the mechanistic insights may facilitate the development of modulators of Notch signaling.« less

Structural Basis for Sialoglycan Binding by the Streptococcus sanguinis SrpA Adhesin.

PubMed

Bensing, Barbara A; Loukachevitch, Lioudmila V; McCulloch, Kathryn M; Yu, Hai; Vann, Kendra R; Wawrzak, Zdzislaw; Anderson, Spencer; Chen, Xi; Sullam, Paul M; Iverson, T M

2016-04-01

Streptococcus sanguinisis a leading cause of infective endocarditis, a life-threatening infection of the cardiovascular system. An important interaction in the pathogenesis of infective endocarditis is attachment of the organisms to host platelets.S. sanguinisexpresses a serine-rich repeat adhesin, SrpA, similar in sequence to platelet-binding adhesins associated with increased virulence in this disease. In this study, we determined the first crystal structure of the putative binding region of SrpA (SrpABR) both unliganded and in complex with a synthetic disaccharide ligand at 1.8 and 2.0 Å resolution, respectively. We identified a conserved Thr-Arg motif that orients the sialic acid moiety and is required for binding to platelet monolayers. Furthermore, we propose that sequence insertions in closely related family members contribute to the modulation of structural and functional properties, including the quaternary structure, the tertiary structure, and the ligand-binding site. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The structure of a protein primer-polymerase complex in the initiation of genome replication.

PubMed

Ferrer-Orta, Cristina; Arias, Armando; Agudo, Rubén; Pérez-Luque, Rosa; Escarmís, Cristina; Domingo, Esteban; Verdaguer, Nuria

2006-02-22

Picornavirus RNA replication is initiated by the covalent attachment of a UMP molecule to the hydroxyl group of a tyrosine in the terminal protein VPg. This reaction is carried out by the viral RNA-dependent RNA polymerase (3D). Here, we report the X-ray structure of two complexes between foot-and-mouth disease virus 3D, VPg1, the substrate UTP and divalent cations, in the absence and in the presence of an oligoadenylate of 10 residues. In both complexes, VPg fits the RNA binding cleft of the polymerase and projects the key residue Tyr3 into the active site of 3D. This is achieved by multiple interactions with residues of motif F and helix alpha8 of the fingers domain and helix alpha13 of the thumb domain of the polymerase. The complex obtained in the presence of the oligoadenylate showed the product of the VPg uridylylation (VPg-UMP). Two metal ions and the catalytic aspartic acids of the polymerase active site, together with the basic residues of motif F, have been identified as participating in the priming reaction.

Theoretical Study of Oxovanadium(IV) Complexation with Formamidoximate: Implications for the Design of Uranyl-Selective Adsorbents

DOE PAGES

Mehio, Nada; Ivanov, Alexander S.; Ladshaw, Austin P.; ...

2015-11-22

Poly(acrylamidoxime) fibers are the current state of the art adsorbent for mining uranium from seawater. However, the competition between uranyl (UO 2 2+) and vanadium ions poses a challenge to mining on the industrial scale. In this work, we employ density functional theory (DFT) and coupled-cluster methods (CCSD(T)) in the restricted formalism to investigate potential binding motifs of the oxovanadium(IV) ion (VO 2+) with the formamidoximate ligand. Consistent with experimental EXAFS data, the hydrated six-coordinate complex is predicted to be preferred over the hydrated five-coordinate complex. Here, our investigation of formamidoximate-VO 2+ complexes universally identified the most stable binding motifmore » formed by chelating a tautomerically rearranged imino hydroxylamine via the imino nitrogen and hydroxylamine oxygen. The alternative binding motifs for amidoxime chelation via a non-rearranged tautomer and 2 coordination are found to be ~11 kcal/mol less stable. Ultimately, the difference in the most stable VO 2+ and UO 2 2+ binding conformation has important implications for the design of more selective UO 2 2+ ligands.« less

Emergent categorical representation of natural, complex sounds resulting from the early post-natal sound environment

PubMed Central

Bao, Shaowen; Chang, Edward F.; Teng, Ching-Ling; Heiser, Marc A.; Merzenich, Michael M.

2013-01-01

Cortical sensory representations can be reorganized by sensory exposure in an epoch of early development. The adaptive role of this type of plasticity for natural sounds in sensory development is, however, unclear. We have reared rats in a naturalistic, complex acoustic environment and examined their auditory representations. We found that cortical neurons became more selective to spectrotemporal features in the experienced sounds. At the neuronal population level, more neurons were involved in representing the whole set of complex sounds, but fewer neurons actually responded to each individual sound, but with greater magnitudes. A comparison of population-temporal responses to the experienced complex sounds revealed that cortical responses to different renderings of the same song motif were more similar, indicating that the cortical neurons became less sensitive to natural acoustic variations associated with stimulus context and sound renderings. By contrast, cortical responses to sounds of different motifs became more distinctive, suggesting that cortical neurons were tuned to the defining features of the experienced sounds. These effects lead to emergent “categorical” representations of the experienced sounds, which presumably facilitate their recognition. PMID:23747304

Probing the modulated formation of gold nanoparticles-beta-lactoglobulin corona complexes and their applications.

PubMed

Yang, Jiang; Wang, Bo; You, Youngsang; Chang, Woo-Jin; Tang, Ke; Wang, Yi-Cheng; Zhang, Wenzhao; Ding, Feng; Gunasekaran, Sundaram

2017-11-23

Understanding the interactions between proteins and nanoparticles (NPs) along with the underlying structural and dynamic information is of utmost importance to exploit nanotechnology for biomedical applications. Upon adsorption onto a NP surface, proteins form a well-organized layer, termed the corona, that dictates the identity of the NP-protein complex and governs its biological pathways. Given its high biological relevance, in-depth molecular investigations and applications of NPs-protein corona complexes are still scarce, especially since different proteins form unique corona patterns, making identification of the biomolecular motifs at the interface critical. In this work, we provide molecular insights and structural characterizations of the bio-nano interface of a popular food-based protein, namely bovine beta-lactoglobulin (β-LG), with gold nanoparticles (AuNPs) and report on our investigations of the formation of corona complexes by combined molecular simulations and complementary experiments. Two major binding sites in β-LG were identified as being driven by citrate-mediated electrostatic interactions, while the associated binding kinetics and conformational changes in the secondary structures were also characterized. More importantly, the superior stability of the corona led us to further explore its biomedical applications, such as in the smartphone-based point-of-care biosensing of Escherichia coli (E. coli) and in the computed tomography (CT) of the gastrointestinal (GI) tract through oral administration to probe GI tolerance and functions. Considering their biocompatibility, edible nature, and efficient excretion through defecation, AuNPs-β-LG corona complexes have shown promising perspectives for future in vitro and in vivo clinical settings.

A New Scheme to Characterize and Identify Protein Ubiquitination Sites.

PubMed

Nguyen, Van-Nui; Huang, Kai-Yao; Huang, Chien-Hsun; Lai, K Robert; Lee, Tzong-Yi

2017-01-01

Protein ubiquitination, involving the conjugation of ubiquitin on lysine residue, serves as an important modulator of many cellular functions in eukaryotes. Recent advancements in proteomic technology have stimulated increasing interest in identifying ubiquitination sites. However, most computational tools for predicting ubiquitination sites are focused on small-scale data. With an increasing number of experimentally verified ubiquitination sites, we were motivated to design a predictive model for identifying lysine ubiquitination sites for large-scale proteome dataset. This work assessed not only single features, such as amino acid composition (AAC), amino acid pair composition (AAPC) and evolutionary information, but also the effectiveness of incorporating two or more features into a hybrid approach to model construction. The support vector machine (SVM) was applied to generate the prediction models for ubiquitination site identification. Evaluation by five-fold cross-validation showed that the SVM models learned from the combination of hybrid features delivered a better prediction performance. Additionally, a motif discovery tool, MDDLogo, was adopted to characterize the potential substrate motifs of ubiquitination sites. The SVM models integrating the MDDLogo-identified substrate motifs could yield an average accuracy of 68.70 percent. Furthermore, the independent testing result showed that the MDDLogo-clustered SVM models could provide a promising accuracy (78.50 percent) and perform better than other prediction tools. Two cases have demonstrated the effective prediction of ubiquitination sites with corresponding substrate motifs.

Trithiocarbonates: exploration of a new head group for HDAC inhibitors.

PubMed

Dehmel, Florian; Ciossek, Thomas; Maier, Thomas; Weinbrenner, Steffen; Schmidt, Beate; Zoche, Martin; Beckers, Thomas

2007-09-01

Inhibition of histone deacetylases class I/II enzymes is a new, promising approach for cancer therapy. In the present study, we disclose a new structural class of HDAC inhibitors with the trithiocarbonate motif. A clear structure-activity-relationship was obtained for the cap-linker motif and the putative Zn(2+) complexing head group. Selected analogs display potent inhibition of HDAC enzymatic activity and a cellular potency comparable to that of suberoylanilide hydroxamic acid (SAHA), recently approved for treatment of patients with advanced cutaneous T-cell lymphoma.

Structural insight into RNA recognition motifs: versatile molecular Lego building blocks for biological systems.

PubMed

Muto, Yutaka; Yokoyama, Shigeyuki

2012-01-01

'RNA recognition motifs (RRMs)' are common domain-folds composed of 80-90 amino-acid residues in eukaryotes, and have been identified in many cellular proteins. At first they were known as RNA binding domains. Through discoveries over the past 20 years, however, the RRMs have been shown to exhibit versatile molecular recognition activities and to behave as molecular Lego building blocks to construct biological systems. Novel RNA/protein recognition modes by RRMs are being identified, and more information about the molecular recognition by RRMs is becoming available. These RNA/protein recognition modes are strongly correlated with their biological significance. In this review, we would like to survey the recent progress on these versatile molecular recognition modules. Copyright © 2012 John Wiley & Sons, Ltd.

CO2 directly modulates connexin 26 by formation of carbamate bridges between subunits

PubMed Central

Meigh, Louise; Greenhalgh, Sophie A; Rodgers, Thomas L; Cann, Martin J; Roper, David I; Dale, Nicholas

2013-01-01

Homeostatic regulation of the partial pressure of CO2 (PCO2) is vital for life. Sensing of pH has been proposed as a sufficient proxy for determination of PCO2 and direct CO2-sensing largely discounted. Here we show that connexin 26 (Cx26) hemichannels, causally linked to respiratory chemosensitivity, are directly modulated by CO2. A ‘carbamylation motif’, present in CO2-sensitive connexins (Cx26, Cx30, Cx32) but absent from a CO2-insensitive connexin (Cx31), comprises Lys125 and four further amino acids that orient Lys125 towards Arg104 of the adjacent subunit of the connexin hexamer. Introducing the carbamylation motif into Cx31 created a mutant hemichannel (mCx31) that was opened by increases in PCO2. Mutation of the carbamylation motif in Cx26 and mCx31 destroyed CO2 sensitivity. Course-grained computational modelling of Cx26 demonstrated that the proposed carbamate bridge between Lys125 and Arg104 biases the hemichannel to the open state. Carbamylation of Cx26 introduces a new transduction principle for physiological sensing of CO2. DOI: http://dx.doi.org/10.7554/eLife.01213.001 PMID:24220509

ATtRACT-a database of RNA-binding proteins and associated motifs.

PubMed

Giudice, Girolamo; Sánchez-Cabo, Fátima; Torroja, Carlos; Lara-Pezzi, Enrique

2016-01-01

RNA-binding proteins (RBPs) play a crucial role in key cellular processes, including RNA transport, splicing, polyadenylation and stability. Understanding the interaction between RBPs and RNA is key to improve our knowledge of RNA processing, localization and regulation in a global manner. Despite advances in recent years, a unified non-redundant resource that includes information on experimentally validated motifs, RBPs and integrated tools to exploit this information is lacking. Here, we developed a database named ATtRACT (available athttp://attract.cnic.es) that compiles information on 370 RBPs and 1583 RBP consensus binding motifs, 192 of which are not present in any other database. To populate ATtRACT we (i) extracted and hand-curated experimentally validated data from CISBP-RNA, SpliceAid-F, RBPDB databases, (ii) integrated and updated the unavailable ASD database and (iii) extracted information from Protein-RNA complexes present in Protein Data Bank database through computational analyses. ATtRACT provides also efficient algorithms to search a specific motif and scan one or more RNA sequences at a time. It also allows discoveringde novomotifs enriched in a set of related sequences and compare them with the motifs included in the database.Database URL:http:// attract. cnic. es. © The Author(s) 2016. Published by Oxford University Press.

Identification of sequence-structure RNA binding motifs for SELEX-derived aptamers.

PubMed

Hoinka, Jan; Zotenko, Elena; Friedman, Adam; Sauna, Zuben E; Przytycka, Teresa M

2012-06-15

Systematic Evolution of Ligands by EXponential Enrichment (SELEX) represents a state-of-the-art technology to isolate single-stranded (ribo)nucleic acid fragments, named aptamers, which bind to a molecule (or molecules) of interest via specific structural regions induced by their sequence-dependent fold. This powerful method has applications in designing protein inhibitors, molecular detection systems, therapeutic drugs and antibody replacement among others. However, full understanding and consequently optimal utilization of the process has lagged behind its wide application due to the lack of dedicated computational approaches. At the same time, the combination of SELEX with novel sequencing technologies is beginning to provide the data that will allow the examination of a variety of properties of the selection process. To close this gap we developed, Aptamotif, a computational method for the identification of sequence-structure motifs in SELEX-derived aptamers. To increase the chances of identifying functional motifs, Aptamotif uses an ensemble-based approach. We validated the method using two published aptamer datasets containing experimentally determined motifs of increasing complexity. We were able to recreate the author's findings to a high degree, thus proving the capability of our approach to identify binding motifs in SELEX data. Additionally, using our new experimental dataset, we illustrate the application of Aptamotif to elucidate several properties of the selection process.

La-related protein 1 (LARP1) binds the mRNA cap, blocking eIF4F assembly on TOP mRNAs

PubMed Central

Lahr, Roni M; Fonseca, Bruno D; Ciotti, Gabrielle E; Al-Ashtal, Hiba A; Jia, Jian-Jun; Niklaus, Marius R; Blagden, Sarah P; Alain, Tommy; Berman, Andrea J

2017-01-01

The 5’terminal oligopyrimidine (5’TOP) motif is a cis-regulatory RNA element located immediately downstream of the 7-methylguanosine [m7G] cap of TOP mRNAs, which encode ribosomal proteins and translation factors. In eukaryotes, this motif coordinates the synchronous and stoichiometric expression of the protein components of the translation machinery. La-related protein 1 (LARP1) binds TOP mRNAs, regulating their stability and translation. We present crystal structures of the human LARP1 DM15 region in complex with a 5’TOP motif, a cap analog (m7GTP), and a capped cytidine (m7GpppC), resolved to 2.6, 1.8 and 1.7 Å, respectively. Our binding, competition, and immunoprecipitation data corroborate and elaborate on the mechanism of 5’TOP motif binding by LARP1. We show that LARP1 directly binds the cap and adjacent 5’TOP motif of TOP mRNAs, effectively impeding access of eIF4E to the cap and preventing eIF4F assembly. Thus, LARP1 is a specialized TOP mRNA cap-binding protein that controls ribosome biogenesis. DOI: http://dx.doi.org/10.7554/eLife.24146.001 PMID:28379136

Modeling of DNA local parameters predicts encrypted architectural motifs in Xenopus laevis ribosomal gene promoter

PubMed Central

Roux-Rouquie, Magali; Marilley, Monique

2000-01-01

We have modeled local DNA sequence parameters to search for DNA architectural motifs involved in transcription regulation and promotion within the Xenopus laevis ribosomal gene promoter and the intergenic spacer (IGS) sequences. The IGS was found to be shaped into distinct topological domains. First, intrinsic bends split the IGS into domains of common but different helical features. Local parameters at inter-domain junctions exhibit a high variability with respect to intrinsic curvature, bendability and thermal stability. Secondly, the repeated sequence blocks of the IGS exhibit right-handed supercoiled structures which could be related to their enhancer properties. Thirdly, the gene promoter presents both inherent curvature and minor groove narrowing which may be viewed as motifs of a structural code for protein recognition and binding. Such pre-existing deformations could simply be remodeled during the binding of the transcription complex. Alternatively, these deformations could pre-shape the promoter in such a way that further remodeling is facilitated. Mutations shown to abolish promoter curvature as well as intrinsic minor groove narrowing, in a variant which maintained full transcriptional activity, bring circumstantial evidence for structurally-preorganized motifs in relation to transcription regulation and promotion. Using well documented X.laevis rDNA regulatory sequences we showed that computer modeling may be of invaluable assistance in assessing encrypted architectural motifs. The evidence of these DNA topological motifs with respect to the concept of structural code is discussed. PMID:10982860

Expression, refolding and crystallizations of the Grb2-like (GADS) C-terminal SH3 domain complexed with a SLP-76 motif peptide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Faravelli, Alessandro; Dimasi, Nazzareno, E-mail: ndimasi@gmail.com

Several crystals of the Grb2-like C-terminal SH3 domain in complex with a motif peptide from the SLP-76 protein were obtained and characterized. The Grb2-like adaptor protein GADS is composed of an N-terminal SH3 domain, an SH2 domain, a proline-rich region and a C-terminal SH3 domain. GADS interacts through its C-terminal SH3 domain with the adaptor protein SLP-76, thus recruiting this protein and other associated molecules to the linker for activation of T-cell (LAT) protein. The DNA encoding the C-terminal SH3 domain of GADS (GADS-cSH3) was assembled synthetically using a recursive PCR technique and the protein was overexpressed in Escherichia coli,more » refolded and purified. Several crystals of this domain in complex with the SLP-76 peptide were obtained and characterized.« less

A Luminescent Cocaine Detection Platform Using a Split G-Quadruplex-Selective Iridium(III) Complex and a Three-Way DNA Junction Architecture.

PubMed

Ma, Dik-Lung; Wang, Modi; He, Bingyong; Yang, Chao; Wang, Wanhe; Leung, Chung-Hang

2015-09-02

In this study, a series of 10 in-house cyclometalated iridium(III) complexes bearing different auxiliary ligands were tested for their selectivity toward split G-quadruplex in order to construct a label-free switch-on cocaine detection platform employing a three-way junction architecture and a G-quadruplex motif as a signal output unit. Through two rounds of screening, we discovered that the iridium(III) complex 7 exhibited excellent selectivity toward the intermolecular G-quadruplex motif. A detection limit as low as 30 nM for cocaine can be achieved by this sensing approach with a linear relationship between luminescence intensity and cocaine concentration established from 30 to 300 nM. Furthermore, this sensing approach could detect cocaine in diluted oral fluid. We hope that our simple, signal-on, label-free oligonucleotide-based sensing method for cocaine using a three-way DNA junction architecture could act as a useful platform in bioanalytical research.

A Tyrosine-Based Trafficking Motif of the Tegument Protein pUL71 Is Crucial for Human Cytomegalovirus Secondary Envelopment.

PubMed

Dietz, Andrea N; Villinger, Clarissa; Becker, Stefan; Frick, Manfred; von Einem, Jens

2018-01-01

The human cytomegalovirus (HCMV) tegument protein pUL71 is required for efficient secondary envelopment and accumulates at the Golgi compartment-derived viral assembly complex (vAC) during infection. Analysis of various C-terminally truncated pUL71 proteins fused to enhanced green fluorescent protein (eGFP) identified amino acids 23 to 34 as important determinants for its Golgi complex localization. Sequence analysis and mutational verification revealed the presence of an N-terminal tyrosine-based trafficking motif (YXXΦ) in pUL71. This led us to hypothesize a requirement of the YXXΦ motif for the function of pUL71 in infection. Mutation of both the tyrosine residue and the entire YXXΦ motif resulted in an altered distribution of mutant pUL71 at the plasma membrane and in the cytoplasm during infection. Both YXXΦ mutant viruses exhibited similarly decreased focal growth and reduced virus yields in supernatants. Ultrastructurally, mutant-virus-infected cells exhibited impaired secondary envelopment manifested by accumulations of capsids undergoing an envelopment process. Additionally, clusters of capsid accumulations surrounding the vAC were observed, similar to the ultrastructural phenotype of a UL71-deficient mutant. The importance of endocytosis and thus the YXXΦ motif for targeting pUL71 to the Golgi complex was further demonstrated when clathrin-mediated endocytosis was inhibited either by coexpression of the C-terminal part of cellular AP180 (AP180-C) or by treatment with methyl-β-cyclodextrin. Both conditions resulted in a plasma membrane accumulation of pUL71. Altogether, these data reveal the presence of a functional N-terminal endocytosis motif that is an important determinant for intracellular localization of pUL71 and that is furthermore required for the function of pUL71 during secondary envelopment of HCMV capsids at the vAC. IMPORTANCE Human cytomegalovirus (HCMV) is the leading cause of birth defects among congenital virus infections and can lead to life-threatening infections in immunocompromised hosts. Current antiviral treatments target viral genome replication and are increasingly overcome by viral mutations. Therefore, identifying new targets for antiviral therapy is important for future development of novel treatment options. A detailed molecular understanding of the complex virus morphogenesis will identify potential viral as well as cellular targets for antiviral intervention. Secondary envelopment is an important viral process through which infectious virus particles are generated and which involves the action of several viral proteins, such as tegument protein pUL71. Targeting of pUL71 to the site of secondary envelopment appears to be crucial for its function during this process and is regulated by utilizing host trafficking mechanisms that are commonly exploited by viral glycoproteins. Thus, intracellular trafficking, if targeted, might present a novel target for antiviral therapy. Copyright © 2017 American Society for Microbiology.

Divergent Synthesis of Chondroitin Sulfate Disaccharides and Identification of Sulfate Motifs that Inhibit Triple Negative Breast Cancer

NASA Astrophysics Data System (ADS)

Wei Poh, Zhong; Heng Gan, Chin; Lee, Eric J.; Guo, Suxian; Yip, George W.; Lam, Yulin

2015-09-01

Glycosaminoglycans (GAGs) regulate many important physiological processes. A pertinent issue to address is whether GAGs encode important functional information via introduction of position specific sulfate groups in the GAG structure. However, procurement of pure, homogenous GAG motifs to probe the “sulfation code” is a challenging task due to isolation difficulty and structural complexity. To this end, we devised a versatile synthetic strategy to obtain all the 16 theoretically possible sulfation patterns in the chondroitin sulfate (CS) repeating unit; these include rare but potentially important sulfated motifs which have not been isolated earlier. Biological evaluation indicated that CS sulfation patterns had differing effects for different breast cancer cell types, and the greatest inhibitory effect was observed for the most aggressive, triple negative breast cancer cell line MDA-MB-231.

The Rich Club of the C. elegans Neuronal Connectome

PubMed Central

Vértes, Petra E.; Ahnert, Sebastian E.; Schafer, William R.; Bullmore, Edward T.

2013-01-01

There is increasing interest in topological analysis of brain networks as complex systems, with researchers often using neuroimaging to represent the large-scale organization of nervous systems without precise cellular resolution. Here we used graph theory to investigate the neuronal connectome of the nematode worm Caenorhabditis elegans, which is defined anatomically at a cellular scale as 2287 synaptic connections between 279 neurons. We identified a small number of highly connected neurons as a rich club (N = 11) interconnected with high efficiency and high connection distance. Rich club neurons comprise almost exclusively the interneurons of the locomotor circuits, with known functional importance for coordinated movement. The rich club neurons are connector hubs, with high betweenness centrality, and many intermodular connections to nodes in different modules. On identifying the shortest topological paths (motifs) between pairs of peripheral neurons, the motifs that are found most frequently traverse the rich club. The rich club neurons are born early in development, before visible movement of the animal and before the main phase of developmental elongation of its body. We conclude that the high wiring cost of the globally integrative rich club of neurons in the C. elegans connectome is justified by the adaptive value of coordinated movement of the animal. The economical trade-off between physical cost and behavioral value of rich club organization in a cellular connectome confirms theoretical expectations and recapitulates comparable results from human neuroimaging on much larger scale networks, suggesting that this may be a general and scale-invariant principle of brain network organization. PMID:23575836

Direct AUC optimization of regulatory motifs.

PubMed

Zhu, Lin; Zhang, Hong-Bo; Huang, De-Shuang

2017-07-15

The discovery of transcription factor binding site (TFBS) motifs is essential for untangling the complex mechanism of genetic variation under different developmental and environmental conditions. Among the huge amount of computational approaches for de novo identification of TFBS motifs, discriminative motif learning (DML) methods have been proven to be promising for harnessing the discovery power of accumulated huge amount of high-throughput binding data. However, they have to sacrifice accuracy for speed and could fail to fully utilize the information of the input sequences. We propose a novel algorithm called CDAUC for optimizing DML-learned motifs based on the area under the receiver-operating characteristic curve (AUC) criterion, which has been widely used in the literature to evaluate the significance of extracted motifs. We show that when the considered AUC loss function is optimized in a coordinate-wise manner, the cost function of each resultant sub-problem is a piece-wise constant function, whose optimal value can be found exactly and efficiently. Further, a key step of each iteration of CDAUC can be efficiently solved as a computational geometry problem. Experimental results on real world high-throughput datasets illustrate that CDAUC outperforms competing methods for refining DML motifs, while being one order of magnitude faster. Meanwhile, preliminary results also show that CDAUC may also be useful for improving the interpretability of convolutional kernels generated by the emerging deep learning approaches for predicting TF sequences specificities. CDAUC is available at: https://drive.google.com/drive/folders/0BxOW5MtIZbJjNFpCeHlBVWJHeW8 . dshuang@tongji.edu.cn. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

A Second Las17 Monomeric Actin-Binding Motif Functions in Arp2/3-Dependent Actin Polymerization During Endocytosis

PubMed Central

Feliciano, Daniel; Tolsma, Thomas O.; Farrell, Kristen B.; Aradi, Al; Di Pietro, Santiago M.

2018-01-01

During clathrin-mediated endocytosis (CME), actin assembly provides force to drive vesicle internalization. Members of the Wiskott–Aldrich syndrome protein (WASP) family play a fundamental role stimulating actin assembly. WASP family proteins contain a WH2 motif that binds globular actin (G-actin) and a central-acidic motif that binds the Arp2/3 complex, thus promoting the formation of branched actin filaments. Yeast WASP (Las17) is the strongest of five factors promoting Arp2/3-dependent actin polymerization during CME. It was suggested that this strong activity may be caused by a putative second G-actin-binding motif in Las17. Here, we describe the in vitro and in vivo characterization of such Las17 G-actin-binding motif (LGM) and its dependence on a group of conserved arginine residues. Using the yeast two-hybrid system, GST-pulldown, fluorescence polarization and pyrene-actin polymerization assays, we show that LGM binds G-actin and is necessary for normal Arp2/3-mediated actin polymerization in vitro. Live-cell fluorescence microscopy experiments demonstrate that LGM is required for normal dynamics of actin polymerization during CME. Further, LGM is necessary for normal dynamics of endocytic machinery components that are recruited at early, intermediate and late stages of endocytosis, as well as for optimal endocytosis of native CME cargo. Both in vitro and in vivo experiments show that LGM has relatively lower potency compared to the previously known Las17 G-actin-binding motif, WH2. These results establish a second G-actin-binding motif in Las17 and advance our knowledge on the mechanism of actin assembly during CME. PMID:25615019

Structural basis for genome wide recognition of 5-bp GC motifs by SMAD transcription factors.

PubMed

Martin-Malpartida, Pau; Batet, Marta; Kaczmarska, Zuzanna; Freier, Regina; Gomes, Tiago; Aragón, Eric; Zou, Yilong; Wang, Qiong; Xi, Qiaoran; Ruiz, Lidia; Vea, Angela; Márquez, José A; Massagué, Joan; Macias, Maria J

2017-12-12

Smad transcription factors activated by TGF-β or by BMP receptors form trimeric complexes with Smad4 to target specific genes for cell fate regulation. The CAGAC motif has been considered as the main binding element for Smad2/3/4, whereas Smad1/5/8 have been thought to preferentially bind GC-rich elements. However, chromatin immunoprecipitation analysis in embryonic stem cells showed extensive binding of Smad2/3/4 to GC-rich cis-regulatory elements. Here, we present the structural basis for specific binding of Smad3 and Smad4 to GC-rich motifs in the goosecoid promoter, a nodal-regulated differentiation gene. The structures revealed a 5-bp consensus sequence GGC(GC)|(CG) as the binding site for both TGF-β and BMP-activated Smads and for Smad4. These 5GC motifs are highly represented as clusters in Smad-bound regions genome-wide. Our results provide a basis for understanding the functional adaptability of Smads in different cellular contexts, and their dependence on lineage-determining transcription factors to target specific genes in TGF-β and BMP pathways.

Specific interaction of mutant p53 with regions of matrix attachment region DNA elements (MARs) with a high potential for base-unpairing

PubMed Central

Will, Katrin; Warnecke, Gabriele; Wiesmüller, Lisa; Deppert, Wolfgang

1998-01-01

Mutant, but not wild-type p53 binds with high affinity to a variety of MAR-DNA elements (MARs), suggesting that MAR-binding of mutant p53 relates to the dominant-oncogenic activities proposed for mutant p53. MARs recognized by mutant p53 share AT richness and contain variations of an AATATATTT “DNA-unwinding motif,” which enhances the structural dynamics of chromatin and promotes regional DNA base-unpairing. Mutant p53 specifically interacted with MAR-derived oligonucleotides carrying such unwinding motifs, catalyzing DNA strand separation when this motif was located within a structurally labile sequence environment. Addition of GC-clamps to the respective MAR-oligonucleotides or introducing mutations into the unwinding motif strongly reduced DNA strand separation, but supported the formation of tight complexes between mutant p53 and such oligonucleotides. We conclude that the specific interaction of mutant p53 with regions of MAR-DNA with a high potential for base-unpairing provides the basis for the high-affinity binding of mutant p53 to MAR-DNA. PMID:9811860

Self-assembly of multi-stranded RNA motifs into lattices and tubular structures

DOE PAGES

Stewart, Jaimie Marie; Subramanian, Hari K. K.; Franco, Elisa

2017-02-16

Rational design of nucleic acidmolecules yields selfassembling scaffolds with increasing complexity, size and functionality. It is an open question whether design methods tailored to build DNA nanostructures can be adapted to build RNA nanostructures with comparable features. We demonstrate the formation of RNA lattices and tubular assemblies from double crossover (DX) tiles, a canonical motif in DNA nanotechnology. Tubular structures can exceed 1 m in length, suggesting that this DX motif can produce very robust lattices. Some of these tubes spontaneously form with left-handed chirality. We obtain assemblies by using two methods: a protocol where gel-extracted RNA strands are slowlymore » annealed, and a one-pot transcription and anneal procedure. We then identify the tile nick position as a structural requirement for lattice formation. These results demonstrate that stable RNA structures can be obtained with design tools imported from DNA nanotechnology. These large assemblies could be potentially integrated with a variety of functional RNA motifs for drug or nanoparticle delivery, or for colocalization of cellular components.« less

Paranemic Crossover DNA: There and Back Again.

PubMed

Wang, Xing; Chandrasekaran, Arun Richard; Shen, Zhiyong; Ohayon, Yoel P; Wang, Tong; Kizer, Megan E; Sha, Ruojie; Mao, Chengde; Yan, Hao; Zhang, Xiaoping; Liao, Shiping; Ding, Baoquan; Chakraborty, Banani; Jonoska, Natasha; Niu, Dong; Gu, Hongzhou; Chao, Jie; Gao, Xiang; Li, Yuhang; Ciengshin, Tanashaya; Seeman, Nadrian C

2018-06-18

Over the past 35 years, DNA has been used to produce various nanometer-scale constructs, nanomechanical devices, and walkers. Construction of complex DNA nanostructures relies on the creation of rigid DNA motifs. Paranemic crossover (PX) DNA is one such motif that has played many roles in DNA nanotechnology. Specifically, PX cohesion has been used to connect topologically closed molecules, to assemble a three-dimensional object, and to create two-dimensional DNA crystals. Additionally, a sequence-dependent nanodevice based on conformational change between PX and its topoisomer, JX 2 , has been used in robust nanoscale assembly lines, as a key component in a DNA transducer, and to dictate polymer assembly. Furthermore, the PX motif has recently found a new role directly in basic biology, by possibly serving as the molecular structure for double-stranded DNA homology recognition, a prominent feature of molecular biology and essential for many crucial biological processes. This review discusses the many attributes and usages of PX-DNA-its design, characteristics, applications, and potential biological relevance-and aims to accelerate the understanding of PX-DNA motif in its many roles and manifestations.

Self-assembly of multi-stranded RNA motifs into lattices and tubular structures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stewart, Jaimie Marie; Subramanian, Hari K. K.; Franco, Elisa

Rational design of nucleic acidmolecules yields selfassembling scaffolds with increasing complexity, size and functionality. It is an open question whether design methods tailored to build DNA nanostructures can be adapted to build RNA nanostructures with comparable features. We demonstrate the formation of RNA lattices and tubular assemblies from double crossover (DX) tiles, a canonical motif in DNA nanotechnology. Tubular structures can exceed 1 m in length, suggesting that this DX motif can produce very robust lattices. Some of these tubes spontaneously form with left-handed chirality. We obtain assemblies by using two methods: a protocol where gel-extracted RNA strands are slowlymore » annealed, and a one-pot transcription and anneal procedure. We then identify the tile nick position as a structural requirement for lattice formation. These results demonstrate that stable RNA structures can be obtained with design tools imported from DNA nanotechnology. These large assemblies could be potentially integrated with a variety of functional RNA motifs for drug or nanoparticle delivery, or for colocalization of cellular components.« less

General method to find the attractors of discrete dynamic models of biological systems.

PubMed

Gan, Xiao; Albert, Réka

2018-04-01

Analyzing the long-term behaviors (attractors) of dynamic models of biological networks can provide valuable insight. We propose a general method that can find the attractors of multilevel discrete dynamical systems by extending a method that finds the attractors of a Boolean network model. The previous method is based on finding stable motifs, subgraphs whose nodes' states can stabilize on their own. We extend the framework from binary states to any finite discrete levels by creating a virtual node for each level of a multilevel node, and describing each virtual node with a quasi-Boolean function. We then create an expanded representation of the multilevel network, find multilevel stable motifs and oscillating motifs, and identify attractors by successive network reduction. In this way, we find both fixed point attractors and complex attractors. We implemented an algorithm, which we test and validate on representative synthetic networks and on published multilevel models of biological networks. Despite its primary motivation to analyze biological networks, our motif-based method is general and can be applied to any finite discrete dynamical system.

General method to find the attractors of discrete dynamic models of biological systems

NASA Astrophysics Data System (ADS)

Gan, Xiao; Albert, Réka

2018-04-01

Analyzing the long-term behaviors (attractors) of dynamic models of biological networks can provide valuable insight. We propose a general method that can find the attractors of multilevel discrete dynamical systems by extending a method that finds the attractors of a Boolean network model. The previous method is based on finding stable motifs, subgraphs whose nodes' states can stabilize on their own. We extend the framework from binary states to any finite discrete levels by creating a virtual node for each level of a multilevel node, and describing each virtual node with a quasi-Boolean function. We then create an expanded representation of the multilevel network, find multilevel stable motifs and oscillating motifs, and identify attractors by successive network reduction. In this way, we find both fixed point attractors and complex attractors. We implemented an algorithm, which we test and validate on representative synthetic networks and on published multilevel models of biological networks. Despite its primary motivation to analyze biological networks, our motif-based method is general and can be applied to any finite discrete dynamical system.

Self-assembly of multi-stranded RNA motifs into lattices and tubular structures

PubMed Central

Stewart, Jaimie Marie; Subramanian, Hari K. K.

2017-01-01

Abstract Rational design of nucleic acid molecules yields self-assembling scaffolds with increasing complexity, size and functionality. It is an open question whether design methods tailored to build DNA nanostructures can be adapted to build RNA nanostructures with comparable features. Here we demonstrate the formation of RNA lattices and tubular assemblies from double crossover (DX) tiles, a canonical motif in DNA nanotechnology. Tubular structures can exceed 1 μm in length, suggesting that this DX motif can produce very robust lattices. Some of these tubes spontaneously form with left-handed chirality. We obtain assemblies by using two methods: a protocol where gel-extracted RNA strands are slowly annealed, and a one-pot transcription and anneal procedure. We identify the tile nick position as a structural requirement for lattice formation. Our results demonstrate that stable RNA structures can be obtained with design tools imported from DNA nanotechnology. These large assemblies could be potentially integrated with a variety of functional RNA motifs for drug or nanoparticle delivery, or for colocalization of cellular components. PMID:28204562

Evolutionary and biogeographical implications of degraded LAGLIDADG endonuclease functionality and group I intron occurrence in stony corals (Scleractinia) and mushroom corals (Corallimorpharia).

PubMed

Celis, Juan Sebastián; Edgell, David R; Stelbrink, Björn; Wibberg, Daniel; Hauffe, Torsten; Blom, Jochen; Kalinowski, Jörn; Wilke, Thomas

2017-01-01

Group I introns and homing endonuclease genes (HEGs) are mobile genetic elements, capable of invading target sequences in intron-less genomes. LAGLIDADG HEGs are the largest family of endonucleases, playing a key role in the mobility of group I introns in a process known as 'homing'. Group I introns and HEGs are rare in metazoans, and can be mainly found inserted in the COXI gene of some sponges and cnidarians, including stony corals (Scleractinia) and mushroom corals (Corallimorpharia). Vertical and horizontal intron transfer mechanisms have been proposed as explanations for intron occurrence in cnidarians. However, the central role of LAGLIDADG motifs in intron mobility mechanisms remains poorly understood. To resolve questions regarding the evolutionary origin and distribution of group I introns and HEGs in Scleractinia and Corallimorpharia, we examined intron/HEGs sequences within a comprehensive phylogenetic framework. Analyses of LAGLIDADG motif conservation showed a high degree of degradation in complex Scleractinia and Corallimorpharia. Moreover, the two motifs lack the respective acidic residues necessary for metal-ion binding and catalysis, potentially impairing horizontal intron mobility. In contrast, both motifs are highly conserved within robust Scleractinia, indicating a fully functional endonuclease capable of promoting horizontal intron transference. A higher rate of non-synonymous substitutions (Ka) detected in the HEGs of complex Scleractinia and Corallimorpharia suggests degradation of the HEG, whereas lower Ka rates in robust Scleractinia are consistent with a scenario of purifying selection. Molecular-clock analyses and ancestral inference of intron type indicated an earlier intron insertion in complex Scleractinia and Corallimorpharia in comparison to robust Scleractinia. These findings suggest that the lack of horizontal intron transfers in the former two groups is related to an age-dependent degradation of the endonuclease activity. Moreover, they also explain the peculiar geographical patterns of introns in stony and mushroom corals.

Solution Structures of 2 : 1 And 1 : 1 DNA Polymerase - DNA Complexes Probed By Ultracentrifugation And Small-Angle X-Ray Scattering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tang, K.H.; /Ohio State U.; Niebuhr, M.

2009-04-30

We report small-angle X-ray scattering (SAXS) and sedimentation velocity (SV) studies on the enzyme-DNA complexes of rat DNA polymerase {beta} (Pol {beta}) and African swine fever virus DNA polymerase X (ASFV Pol X) with one-nucleotide gapped DNA. The results indicated formation of a 2 : 1 Pol {beta}-DNA complex, whereas only 1 : 1 Pol X-DNA complex was observed. Three-dimensional structural models for the 2 : 1 Pol {beta}-DNA and 1 : 1 Pol X-DNA complexes were generated from the SAXS experimental data to correlate with the functions of the DNA polymerases. The former indicates interactions of the 8 kDamore » 5{prime}-dRP lyase domain of the second Pol {beta} molecule with the active site of the 1 : 1 Pol {beta}-DNA complex, while the latter demonstrates how ASFV Pol X binds DNA in the absence of DNA-binding motif(s). As ASFV Pol X has no 5{prime}-dRP lyase domain, it is reasonable not to form a 2 : 1 complex. Based on the enhanced activities of the 2 : 1 complex and the observation that the 8 kDa domain is not in an optimal configuration for the 5{prime}-dRP lyase reaction in the crystal structures of the closed ternary enzyme-DNA-dNTP complexes, we propose that the asymmetric 2 : 1 Pol {beta}-DNA complex enhances the function of Pol {beta}.« less

Expression and Characterization of a Bifidobacterium adolescentis Beta-Mannanase Carrying Mannan-Binding and Cell Association Motifs

PubMed Central

Kulcinskaja, Evelina; Rosengren, Anna; Ibrahim, Romany; Kolenová, Katarína

2013-01-01

The gene encoding β-mannanase (EC 3.2.1.78) BaMan26A from the bacterium Bifidobacterium adolescentis (living in the human gut) was cloned and the gene product characterized. The enzyme was found to be modular and to contain a putative signal peptide. It possesses a catalytic module of the glycoside hydrolase family 26, a predicted immunoglobulin-like module, and two putative carbohydrate-binding modules (CBMs) of family 23. The enzyme is likely cell attached either by the sortase mechanism (LPXTG motif) or via a C-terminal transmembrane helix. The gene was expressed in Escherichia coli without the native signal peptide or the cell anchor. Two variants were made: one containing all four modules, designated BaMan26A-101K, and one truncated before the CBMs, designated BaMan26A-53K. BaMan26A-101K, which contains the CBMs, showed an affinity to carob galactomannan having a dissociation constant of 0.34 μM (8.8 mg/liter), whereas BaMan26A-53K did not bind, showing that at least one of the putative CBMs of family 23 is mannan binding. For BaMan26A-53K, kcat was determined to be 444 s−1 and Km 21.3 g/liter using carob galactomannan as the substrate at the optimal pH of 5.3. Both of the enzyme variants hydrolyzed konjac glucomannan, as well as carob and guar gum galactomannans to a mixture of oligosaccharides. The dominant product from ivory nut mannan was found to be mannotriose. Mannobiose and mannotetraose were produced to a lesser extent, as shown by high-performance anion-exchange chromatography. Mannobiose was not hydrolyzed, and mannotriose was hydrolyzed at a significantly lower rate than the longer oligosaccharides. PMID:23064345

Evolutionary gradient of predicted nuclear localization signals (NLS)-bearing proteins in genomes of family Planctomycetaceae.

PubMed

Guo, Min; Yang, Ruifu; Huang, Chen; Liao, Qiwen; Fan, Guangyi; Sun, Chenghang; Lee, Simon Ming-Yuen

2017-04-04

The nuclear envelope is considered a key classification marker that distinguishes prokaryotes from eukaryotes. However, this marker does not apply to the family Planctomycetaceae, which has intracellular spaces divided by lipidic intracytoplasmic membranes (ICMs). Nuclear localization signal (NLS), a short stretch of amino acid sequence, destines to transport proteins from cytoplasm into nucleus, and is also associated with the development of nuclear envelope. We attempted to investigate the NLS motifs in Planctomycetaceae genomes to demonstrate the potential molecular transition in the development of intracellular membrane system. In this study, we identified NLS-like motifs that have the same amino acid compositions as experimentally identified NLSs in genomes of 11 representative species of family Planctomycetaceae. A total of 15 NLS types and 170 NLS-bearing proteins were detected in the 11 strains. To determine the molecular transformation, we compared NLS-bearing protein abundances in the 11 representative Planctomycetaceae genomes with them in genomes of 16 taxonomically varied microorganisms: nine bacteria, two archaea and five fungi. In the 27 strains, 29 NLS types and 1101 NLS-bearing proteins were identified, principal component analysis showed a significant transitional gradient from bacteria to Planctomycetaceae to fungi on their NLS-bearing protein abundance profiles. Then, we clustered the 993 non-redundant NLS-bearing proteins into 181 families and annotated their involved metabolic pathways. Afterwards, we aligned the ten types of NLS motifs from the 13 families containing NLS-bearing proteins among bacteria, Planctomycetaceae or fungi, considering their diversity, length and origin. A transition towards increased complexity from non-planctomycete bacteria to Planctomycetaceae to archaea and fungi was detected based on the complexity of the 10 types of NLS-like motifs in the 13 NLS-bearing proteins families. The results of this study reveal that Planctomycetaceae separates slightly from the members of non-planctomycete bacteria but still has substantial differences from fungi, based on the NLS-like motifs and NLS-bearing protein analysis.

The amylase inhibitor montbretin A reveals a new glycosidase inhibition motif.

PubMed

Williams, Leslie K; Zhang, Xiaohua; Caner, Sami; Tysoe, Christina; Nguyen, Nham T; Wicki, Jacqueline; Williams, David E; Coleman, John; McNeill, John H; Yuen, Violet; Andersen, Raymond J; Withers, Stephen G; Brayer, Gary D

2015-09-01

The complex plant flavonol glycoside montbretin A is a potent (Ki = 8 nM) and specific inhibitor of human pancreatic α-amylase with potential as a therapeutic for diabetes and obesity. Controlled degradation studies on montbretin A, coupled with inhibition analyses, identified an essential high-affinity core structure comprising the myricetin and caffeic acid moieties linked via a disaccharide. X-ray structural analyses of the montbretin A-human α-amylase complex confirmed the importance of this core structure and revealed a novel mode of glycosidase inhibition wherein internal π-stacking interactions between the myricetin and caffeic acid organize their ring hydroxyls for optimal hydrogen bonding to the α-amylase catalytic residues D197 and E233. This novel inhibitory motif can be reproduced in a greatly simplified analog, offering potential for new strategies for glycosidase inhibition and therapeutic development.

Identification of new members of the MAPK gene family in plants shows diverse conserved domains and novel activation loop variants.

PubMed

Mohanta, Tapan Kumar; Arora, Pankaj Kumar; Mohanta, Nibedita; Parida, Pratap; Bae, Hanhong

2015-02-06

Mitogen Activated Protein Kinase (MAPK) signaling is of critical importance in plants and other eukaryotic organisms. The MAPK cascade plays an indispensible role in the growth and development of plants, as well as in biotic and abiotic stress responses. The MAPKs are constitute the most downstream module of the three tier MAPK cascade and are phosphorylated by upstream MAP kinase kinases (MAPKK), which are in turn are phosphorylated by MAP kinase kinase kinase (MAPKKK). The MAPKs play pivotal roles in regulation of many cytoplasmic and nuclear substrates, thus regulating several biological processes. A total of 589 MAPKs genes were identified from the genome wide analysis of 40 species. The sequence analysis has revealed the presence of several N- and C-terminal conserved domains. The MAPKs were previously believed to be characterized by the presence of TEY/TDY activation loop motifs. The present study showed that, in addition to presence of activation loop TEY/TDY motifs, MAPKs are also contain MEY, TEM, TQM, TRM, TVY, TSY, TEC and TQY activation loop motifs. Phylogenetic analysis of all predicted MAPKs were clustered into six different groups (group A, B, C, D, E and F), and all predicted MAPKs were assigned with specific names based on their orthology based evolutionary relationships with Arabidopsis or Oryza MAPKs. We conducted global analysis of the MAPK gene family of plants from lower eukaryotes to higher eukaryotes and analyzed their genomic and evolutionary aspects. Our study showed the presence of several new activation loop motifs and diverse conserved domains in MAPKs. Advance study of newly identified activation loop motifs can provide further information regarding the downstream signaling cascade activated in response to a wide array of stress conditions, as well as plant growth and development.

Modular Evolution of DNA-Binding Preference of a Tbrain Transcription Factor Provides a Mechanism for Modifying Gene Regulatory Networks

PubMed Central

Cheatle Jarvela, Alys M.; Brubaker, Lisa; Vedenko, Anastasia; Gupta, Anisha; Armitage, Bruce A.; Bulyk, Martha L.; Hinman, Veronica F.

2014-01-01

Gene regulatory networks (GRNs) describe the progression of transcriptional states that take a single-celled zygote to a multicellular organism. It is well documented that GRNs can evolve extensively through mutations to cis-regulatory modules (CRMs). Transcription factor proteins that bind these CRMs may also evolve to produce novelty. Coding changes are considered to be rarer, however, because transcription factors are multifunctional and hence are more constrained to evolve in ways that will not produce widespread detrimental effects. Recent technological advances have unearthed a surprising variation in DNA-binding abilities, such that individual transcription factors may recognize both a preferred primary motif and an additional secondary motif. This provides a source of modularity in function. Here, we demonstrate that orthologous transcription factors can also evolve a changed preference for a secondary binding motif, thereby offering an unexplored mechanism for GRN evolution. Using protein-binding microarray, surface plasmon resonance, and in vivo reporter assays, we demonstrate an important difference in DNA-binding preference between Tbrain protein orthologs in two species of echinoderms, the sea star, Patiria miniata, and the sea urchin, Strongylocentrotus purpuratus. Although both orthologs recognize the same primary motif, only the sea star Tbr also has a secondary binding motif. Our in vivo assays demonstrate that this difference may allow for greater evolutionary change in timing of regulatory control. This uncovers a layer of transcription factor binding divergence that could exist for many pairs of orthologs. We hypothesize that this divergence provides modularity that allows orthologous transcription factors to evolve novel roles in GRNs through modification of binding to secondary sites. PMID:25016582

Modulation of the multistate folding of designed TPR proteins through intrinsic and extrinsic factors

PubMed Central

Phillips, J J; Javadi, Y; Millership, C; Main, E R G

2012-01-01

Tetratricopeptide repeats (TPRs) are a class of all alpha-helical repeat proteins that are comprised of 34-aa helix-turn-helix motifs. These stack together to form nonglobular structures that are stabilized by short-range interactions from residues close in primary sequence. Unlike globular proteins, they have few, if any, long-range nonlocal stabilizing interactions. Several studies on designed TPR proteins have shown that this modular structure is reflected in their folding, that is, modular multistate folding is observed as opposed to two-state folding. Here we show that TPR multistate folding can be suppressed to approximate two-state folding through modulation of intrinsic stability or extrinsic environmental variables. This modulation was investigated by comparing the thermodynamic unfolding under differing buffer regimes of two distinct series of consensus-designed TPR proteins, which possess different intrinsic stabilities. A total of nine proteins of differing sizes and differing consensus TPR motifs were each thermally and chemically denatured and their unfolding monitored using differential scanning calorimetry (DSC) and CD/fluorescence, respectively. Analyses of both the DSC and chemical denaturation data show that reducing the total stability of each protein and repeat units leads to observable two-state unfolding. These data highlight the intimate link between global and intrinsic repeat stability that governs whether folding proceeds by an observably two-state mechanism, or whether partial unfolding yields stable intermediate structures which retain sufficient stability to be populated at equilibrium. PMID:22170589

Similar modes of interaction enable Trailer Hitch and EDC3 to associate with DCP1 and Me31B in distinct protein complexes.

PubMed

Tritschler, Felix; Eulalio, Ana; Helms, Sigrun; Schmidt, Steffen; Coles, Murray; Weichenrieder, Oliver; Izaurralde, Elisa; Truffault, Vincent

2008-11-01

Trailer Hitch (Tral or LSm15) and enhancer of decapping-3 (EDC3 or LSm16) are conserved eukaryotic members of the (L)Sm (Sm and Like-Sm) protein family. They have a similar domain organization, characterized by an N-terminal LSm domain and a central FDF motif; however, in Tral, the FDF motif is flanked by regions rich in charged residues, whereas in EDC3 the FDF motif is followed by a YjeF_N domain. We show that in Drosophila cells, Tral and EDC3 specifically interact with the decapping activator DCP1 and the DEAD-box helicase Me31B. Nevertheless, only Tral associates with the translational repressor CUP, whereas EDC3 associates with the decapping enzyme DCP2. Like EDC3, Tral interacts with DCP1 and localizes to mRNA processing bodies (P bodies) via the LSm domain. This domain remains monomeric in solution and adopts a divergent Sm fold that lacks the characteristic N-terminal alpha-helix, as determined by nuclear magnetic resonance analyses. Mutational analysis revealed that the structural integrity of the LSm domain is required for Tral both to interact with DCP1 and CUP and to localize to P-bodies. Furthermore, both Tral and EDC3 interact with the C-terminal RecA-like domain of Me31B through their FDF motifs. Together with previous studies, our results show that Tral and EDC3 are structurally related and use a similar mode to associate with common partners in distinct protein complexes.

Structural and energetic study of cation-π-cation interactions in proteins.

PubMed

Pinheiro, Silvana; Soteras, Ignacio; Gelpí, Josep Lluis; Dehez, François; Chipot, Christophe; Luque, F Javier; Curutchet, Carles

2017-04-12

Cation-π interactions of aromatic rings and positively charged groups are among the most important interactions in structural biology. The role and energetic characteristics of these interactions are well established. However, the occurrence of cation-π-cation interactions is an unexpected motif, which raises intriguing questions about its functional role in proteins. We present a statistical analysis of the occurrence, composition and geometrical preferences of cation-π-cation interactions identified in a set of non-redundant protein structures taken from the Protein Data Bank. Our results demonstrate that this structural motif is observed at a small, albeit non-negligible frequency in proteins, and suggest a preference to establish cation-π-cation motifs with Trp, followed by Tyr and Phe. Furthermore, we have found that cation-π-cation interactions tend to be highly conserved, which supports their structural or functional role. Finally, we have performed an energetic analysis of a representative subset of cation-π-cation complexes combining quantum-chemical and continuum solvation calculations. Our results point out that the protein environment can strongly screen the cation-cation repulsion, leading to an attractive interaction in 64% of the complexes analyzed. Together with the high degree of conservation observed, these results suggest a potential stabilizing role in the protein fold, as demonstrated recently for a miniature protein (Craven et al., J. Am. Chem. Soc. 2016, 138, 1543). From a computational point of view, the significant contribution of non-additive three-body terms challenges the suitability of standard additive force fields for describing cation-π-cation motifs in molecular simulations.

Serine Phosphorylation of HIV-1 Vpu and Its Binding to Tetherin Regulates Interaction with Clathrin Adaptors

PubMed Central

Sumner, Jonathan C.; Pickering, Suzanne; Neil, Stuart J. D.

2015-01-01

HIV-1 Vpu prevents incorporation of tetherin (BST2/ CD317) into budding virions and targets it for ESCRT-dependent endosomal degradation via a clathrin-dependent process. This requires a variant acidic dileucine-sorting motif (ExxxLV) in Vpu. Structural studies demonstrate that recombinant Vpu/tetherin fusions can form a ternary complex with the clathrin adaptor AP-1. However, open questions still exist about Vpu’s mechanism of action. Particularly, whether endosomal degradation and the recruitment of the E3 ubiquitin ligase SCFβTRCP1/2 to a conserved phosphorylated binding site, DSGNES, are required for antagonism. Re-evaluation of the phenotype of Vpu phosphorylation mutants and naturally occurring allelic variants reveals that the requirement for the Vpu phosphoserine motif in tetherin antagonism is dissociable from SCFβTRCP1/2 and ESCRT-dependent tetherin degradation. Vpu phospho-mutants phenocopy ExxxLV mutants, and can be rescued by direct clathrin interaction in the absence of SCFβTRCP1/2 recruitment. Moreover, we demonstrate physical interaction between Vpu and AP-1 or AP-2 in cells. This requires Vpu/tetherin transmembrane domain interactions as well as the ExxxLV motif. Importantly, it also requires the Vpu phosphoserine motif and adjacent acidic residues. Taken together these data explain the discordance between the role of SCFβTRCP1/2 and Vpu phosphorylation in tetherin antagonism, and indicate that phosphorylation of Vpu in Vpu/tetherin complexes regulates promiscuous recruitment of adaptors, implicating clathrin-dependent sorting as an essential first step in tetherin antagonism. PMID:26317613

Neurofilament tail phosphorylation: identity of the RT-97 phosphoepitope and regulation in neurons by cross-talk among proline-directed kinases.

PubMed

Veeranna; Lee, Ju-Hyun; Pareek, Tej K; Jaffee, Howard; Boland, Barry; Vinod, K Yaragudri; Amin, Niranjana; Kulkarni, Ashok B; Pant, Harish C; Nixon, Ralph A

2008-10-01

As axons myelinate, establish a stable neurofilament network, and expand in caliber, neurofilament proteins are extensively phosphorylated along their C-terminal tails, which is recognized by the monoclonal antibody, RT-97. Here, we demonstrate in vivo that RT-97 immunoreactivity (IR) is generated by phosphorylation at KSPXK or KSPXXXK motifs and requires flanking lysines at specific positions. extracellular signal regulated kinase 1,2 (ERK1,2) and pERK1,2 levels increase in parallel with phosphorylation at the RT-97 epitope during early postnatal brain development. Purified ERK1,2 generated RT-97 on both KSP motifs on recombinant NF-H tail domain proteins, while cdk5 phosphorylated only KSPXK motifs. RT-97 epitope generation in primary hippocampal neurons was regulated by extensive cross-talk among ERK1,2, c-Jun N-terminal kinase 1,2 (JNK1,2) and cdk5. Inhibition of both ERK1,2 and JNK1,2 completely blocked RT-97 generation. Cdk5 influenced RT-97 generation indirectly by modulating JNK activation. In mice, cdk5 gene deletion did not significantly alter RT-97 IR or ERK1,2 and JNK activation. In mice lacking the cdk5 activator P35, the partial suppression of cdk5 activity increased RT-97 IR by activating ERK1,2. Thus, cdk5 influences RT-97 epitope generation partly by modulating ERKs and JNKs, which are the two principal kinases regulating neurofilament phosphorylation. The regulation of a single target by multiple protein kinases underscores the importance of monitoring other relevant kinases when the activity of a particular one is blocked.

Determinants of the heme-CO vibrational modes in the H-NOX family.

PubMed

Tran, Rosalie; Weinert, Emily E; Boon, Elizabeth M; Mathies, Richard A; Marletta, Michael A

2011-08-02

The Heme Nitric oxide/OXygen binding (H-NOX) family of proteins have important functions in gaseous ligand signaling in organisms from bacteria to humans, including nitric oxide (NO) sensing in mammals, and provide a model system for probing ligand selectivity in hemoproteins. A unique vibrational feature that is ubiquitous throughout the H-NOX family is the presence of a high C-O stretching frequency. To investigate the cause of this spectroscopic characteristic, the Fe-CO and C-O stretching frequencies were probed in the H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX) using resonance Raman (RR) spectroscopy. Four classes of heme pocket mutants were generated to assess the changes in stretching frequency: (i) the distal H-bonding network, (ii) the proximal histidine ligand, (iii) modulation of the heme conformation via Ile-5 and Pro-115, and (iv) the conserved Tyr-Ser-Arg (YxSxR) motif. These mutations revealed important electrostatic interactions that dampen the back-donation of the Fe(II) d(π) electrons into the CO π* orbitals. The most significant change occurred upon disruption of the H-bonds between the strictly conserved YxSxR motif and the heme propionate groups, producing two dominant CO-bound heme conformations. One conformer was structurally similar to Tt H-NOX WT, whereas the other displayed a decrease in ν(C-O) of up to ∼70 cm(-1) relative to the WT protein, with minimal changes in ν(Fe-CO). Taken together, these results show that the electrostatic interactions in the Tt H-NOX binding pocket are primarily responsible for the high ν(C-O) by decreasing the Fe d(π) → CO π* back-donation and suggest that the dominant mechanism by which this family modulates the Fe(II)-CO bond likely involves the YxSxR motif.

A Robust Molecular Network Motif for Period-Doubling Devices.

PubMed

Cuba Samaniego, Christian; Franco, Elisa

2018-01-19

Life is sustained by a variety of cyclic processes such as cell division, muscle contraction, and neuron firing. The periodic signals powering these processes often direct a variety of other downstream systems, which operate at different time scales and must have the capacity to divide or multiply the period of the master clock. Period modulation is also an important challenge in synthetic molecular systems, where slow and fast components may have to be coordinated simultaneously by a single oscillator whose frequency is often difficult to tune. Circuits that can multiply the period of a clock signal (frequency dividers), such as binary counters and flip-flops, are commonly encountered in electronic systems, but design principles to obtain similar devices in biological systems are still unclear. We take inspiration from the architecture of electronic flip-flops, and we propose to build biomolecular period-doubling networks by combining a bistable switch with negative feedback modules that preprocess the circuit inputs. We identify a network motif and we show it can be "realized" using different biomolecular components; two of the realizations we propose rely on transcriptional gene networks and one on nucleic acid strand displacement systems. We examine the capacity of each realization to perform period-doubling by studying how bistability of the motif is affected by the presence of the input; for this purpose, we employ mathematical tools from algebraic geometry that provide us with valuable insights on the input/output behavior as a function of the realization parameters. We show that transcriptional network realizations operate correctly also in a stochastic regime when processing oscillations from the repressilator, a canonical synthetic in vivo oscillator. Finally, we compare the performance of different realizations in a range of realistic parameters via numerical sensitivity analysis of the period-doubling region, computed with respect to the input period and amplitude. Our mathematical and computational analysis suggests that the motif we propose is generally robust with respect to specific implementation details: functionally equivalent circuits can be built as long as the species-interaction topology is respected. This indicates that experimental construction of the circuit is possible with a variety of components within the rapidly expanding libraries available in synthetic biology.

Crystal structure of the tyrosine kinase domain of the hepatocyte growth factor receptor c-Met and its complex with the microbial alkaloid K-252a.

PubMed

Schiering, Nikolaus; Knapp, Stefan; Marconi, Marina; Flocco, Maria M; Cui, Jean; Perego, Rita; Rusconi, Luisa; Cristiani, Cinzia

2003-10-28

The protooncogene c-met codes for the hepatocyte growth factor receptor tyrosine kinase. Binding of its ligand, hepatocyte growth factor/scatter factor, stimulates receptor autophosphorylation, which leads to pleiotropic downstream signaling events in epithelial cells, including cell growth, motility, and invasion. These events are mediated by interaction of cytoplasmic effectors, generally through Src homology 2 (SH2) domains, with two phosphotyrosine-containing sequence motifs in the unique C-terminal tail of c-Met (supersite). There is a strong link between aberrant c-Met activity and oncogenesis, which makes this kinase an important cancer drug target. The furanosylated indolocarbazole K-252a belongs to a family of microbial alkaloids that also includes staurosporine. It was recently shown to be a potent inhibitor of c-Met. Here we report the crystal structures of an unphosphorylated c-Met kinase domain harboring a human cancer mutation and its complex with K-252a at 1.8-A resolution. The structure follows the well established architecture of protein kinases. It adopts a unique, inhibitory conformation of the activation loop, a catalytically noncompetent orientation of helix alphaC, and reveals the complete C-terminal docking site. The first SH2-binding motif (1349YVHV) adopts an extended conformation, whereas the second motif (1356YVNV), a binding site for Grb2-SH2, folds as a type II Beta-turn. The intermediate portion of the supersite (1353NATY) assumes a type I Beta-turn conformation as in an Shc-phosphotyrosine binding domain peptide complex. K-252a is bound in the adenosine pocket with an analogous binding mode to those observed in previously reported structures of protein kinases in complex with staurosporine.

Copper supplementation restores cytochrome c oxidase assembly defect in a mitochondrial disease model of COA6 deficiency.

PubMed

Ghosh, Alok; Trivedi, Prachi P; Timbalia, Shrishiv A; Griffin, Aaron T; Rahn, Jennifer J; Chan, Sherine S L; Gohil, Vishal M

2014-07-01

Mitochondrial respiratory chain biogenesis is orchestrated by hundreds of assembly factors, many of which are yet to be discovered. Using an integrative approach based on clues from evolutionary history, protein localization and human genetics, we have identified a conserved mitochondrial protein, C1orf31/COA6, and shown its requirement for respiratory complex IV biogenesis in yeast, zebrafish and human cells. A recent next-generation sequencing study reported potential pathogenic mutations within the evolutionarily conserved Cx₉CxnCx₁₀C motif of COA6, implicating it in mitochondrial disease biology. Using yeast coa6Δ cells, we show that conserved residues in the motif, including the residue mutated in a patient with mitochondrial disease, are essential for COA6 function, thus confirming the pathogenicity of the patient mutation. Furthermore, we show that zebrafish embryos with zfcoa6 knockdown display reduced heart rate and cardiac developmental defects, recapitulating the observed pathology in the human mitochondrial disease patient who died of neonatal hypertrophic cardiomyopathy. The specific requirement of Coa6 for respiratory complex IV biogenesis, its intramitochondrial localization and the presence of the Cx₉CxnCx₁₀C motif suggested a role in mitochondrial copper metabolism. In support of this, we show that exogenous copper supplementation completely rescues respiratory and complex IV assembly defects in yeast coa6Δ cells. Taken together, our results establish an evolutionarily conserved role of Coa6 in complex IV assembly and support a causal role of the COA6 mutation in the human mitochondrial disease patient. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The methyltransferase NSD3 has chromatin-binding motifs, PHD5-C5HCH, that are distinct from other NSD (nuclear receptor SET domain) family members in their histone H3 recognition.

PubMed

He, Chao; Li, Fudong; Zhang, Jiahai; Wu, Jihui; Shi, Yunyu

2013-02-15

The NSD (nuclear receptor SET domain-containing) family members, consisting of NSD1, NSD2 (MMSET/WHSC1), and NSD3 (WHSC1L1), are SET domain-containing methyltransferases and aberrant expression of each member has been implicated in multiple diseases. They have specific mono- and dimethylase activities for H3K36, whereas play nonredundant roles during development. Aside from the well characterized catalytic SET domain, NSD proteins have multiple potential chromatin-binding motifs that are clinically relevant, including the fifth plant homeodomain (PHD5) and the adjacent Cys-His-rich domain (C5HCH) located at the C terminus. Herein, we report the crystal structures of the PHD5-C5HCH module of NSD3, in the free state and in complex with H3(1-7) (H3 residues 1-7), H3(1-15) (H3 residues 1-15), and H3(1-15)K9me3 (H3 residues 1-15 with trimethylation on K9) peptides. These structures reveal that the PHD5 and C5HCH domains fold into a novel integrated PHD-PHD-like structural module with H3 peptide bound only on the surface of PHD5 and provide the molecular basis for the recognition of unmodified H3K4 and trimethylated H3K9 by NSD3 PHD5. Structural studies and binding assays show that differences exist in histone binding specificity of the PHD5 domain between three members of the NSD family. For NSD2, the PHD5-C5HCH:H3 N terminus interaction is largely conserved, although with a stronger preference for unmethylated H3K9 (H3K9me0) than trimethylated H3K9 (H3K9me3), and NSD1 PHD5-C5HCH does not bind to H3 peptides. Our results shed light on how NSD proteins that mediate H3K36 methylation are localized to specific genomic sites and provide implications for the mechanism of functional diversity of NSD proteins.

Phosphoproteomic Analysis of Protein Kinase C Signaling in Saccharomyces cerevisiae Reveals Slt2 Mitogen-activated Protein Kinase (MAPK)-dependent Phosphorylation of Eisosome Core Components*

PubMed Central

Mascaraque, Victoria; Hernáez, María Luisa; Jiménez-Sánchez, María; Hansen, Rasmus; Gil, Concha; Martín, Humberto; Cid, Víctor J.; Molina, María

2013-01-01

The cell wall integrity (CWI) pathway of the model organism Saccharomyces cerevisiae has been thoroughly studied as a paradigm of the mitogen-activated protein kinase (MAPK) pathway. It consists of a classic MAPK module comprising the Bck1 MAPK kinase kinase, two redundant MAPK kinases (Mkk1 and Mkk2), and the Slt2 MAPK. This module is activated under a variety of stimuli related to cell wall homeostasis by Pkc1, the only member of the protein kinase C family in budding yeast. Quantitative phosphoproteomics based on stable isotope labeling of amino acids in cell culture is a powerful tool for globally studying protein phosphorylation. Here we report an analysis of the yeast phosphoproteome upon overexpression of a PKC1 hyperactive allele that specifically activates CWI MAPK signaling in the absence of external stimuli. We found 82 phosphopeptides originating from 43 proteins that showed enhanced phosphorylation in these conditions. The MAPK S/T-P target motif was significantly overrepresented in these phosphopeptides. Hyperphosphorylated proteins provide putative novel targets of the Pkc1–cell wall integrity pathway involved in diverse functions such as the control of gene expression, protein synthesis, cytoskeleton maintenance, DNA repair, and metabolism. Remarkably, five components of the plasma-membrane-associated protein complex known as eisosomes were found among the up-regulated proteins. We show here that Pkc1-induced phosphorylation of the eisosome core components Pil1 and Lsp1 was not exerted directly by Pkc1, but involved signaling through the Slt2 MAPK module. PMID:23221999

Genome-wide inference of regulatory networks in Streptomyces coelicolor.

PubMed

Castro-Melchor, Marlene; Charaniya, Salim; Karypis, George; Takano, Eriko; Hu, Wei-Shou

2010-10-18

The onset of antibiotics production in Streptomyces species is co-ordinated with differentiation events. An understanding of the genetic circuits that regulate these coupled biological phenomena is essential to discover and engineer the pharmacologically important natural products made by these species. The availability of genomic tools and access to a large warehouse of transcriptome data for the model organism, Streptomyces coelicolor, provides incentive to decipher the intricacies of the regulatory cascades and develop biologically meaningful hypotheses. In this study, more than 500 samples of genome-wide temporal transcriptome data, comprising wild-type and more than 25 regulatory gene mutants of Streptomyces coelicolor probed across multiple stress and medium conditions, were investigated. Information based on transcript and functional similarity was used to update a previously-predicted whole-genome operon map and further applied to predict transcriptional networks constituting modules enriched in diverse functions such as secondary metabolism, and sigma factor. The predicted network displays a scale-free architecture with a small-world property observed in many biological networks. The networks were further investigated to identify functionally-relevant modules that exhibit functional coherence and a consensus motif in the promoter elements indicative of DNA-binding elements. Despite the enormous experimental as well as computational challenges, a systems approach for integrating diverse genome-scale datasets to elucidate complex regulatory networks is beginning to emerge. We present an integrated analysis of transcriptome data and genomic features to refine a whole-genome operon map and to construct regulatory networks at the cistron level in Streptomyces coelicolor. The functionally-relevant modules identified in this study pose as potential targets for further studies and verification.

Genomic agonism and phenotypic antagonism between estrogen and progesterone receptors in breast cancer

PubMed Central

Singhal, Hari; Greene, Marianne E.; Tarulli, Gerard; Zarnke, Allison L.; Bourgo, Ryan J.; Laine, Muriel; Chang, Ya-Fang; Ma, Shihong; Dembo, Anna G.; Raj, Ganesh V.; Hickey, Theresa E.; Tilley, Wayne D.; Greene, Geoffrey L.

2016-01-01

The functional role of progesterone receptor (PR) and its impact on estrogen signaling in breast cancer remain controversial. In primary ER+ (estrogen receptor–positive)/PR+ human tumors, we report that PR reprograms estrogen signaling as a genomic agonist and a phenotypic antagonist. In isolation, estrogen and progestin act as genomic agonists by regulating the expression of common target genes in similar directions, but at different levels. Similarly, in isolation, progestin is also a weak phenotypic agonist of estrogen action. However, in the presence of both hormones, progestin behaves as a phenotypic estrogen antagonist. PR remodels nucleosomes to noncompetitively redirect ER genomic binding to distal enhancers enriched for BRCA1 binding motifs and sites that link PR and ER/PR complexes. When both hormones are present, progestin modulates estrogen action, such that responsive transcriptomes, cellular processes, and ER/PR recruitment to genomic sites correlate with those observed with PR alone, but not ER alone. Despite this overall correlation, the transcriptome patterns modulated by dual treatment are sufficiently different from individual treatments, such that antagonism of oncogenic processes is both predicted and observed. Combination therapies using the selective PR modulator/antagonist (SPRM) CDB4124 in combination with tamoxifen elicited 70% cytotoxic tumor regression of T47D tumor xenografts, whereas individual therapies inhibited tumor growth without net regression. Our findings demonstrate that PR redirects ER chromatin binding to antagonize estrogen signaling and that SPRMs can potentiate responses to antiestrogens, suggesting that cotargeting of ER and PR in ER+/PR+ breast cancers should be explored. PMID:27386569

Herpes Simplex Virus 1 (HSV-1) and HSV-2 Mediate Species-Specific Modulations of Programmed Necrosis through the Viral Ribonucleotide Reductase Large Subunit R1

PubMed Central

Yu, Xiaoliang; Li, Yun; Chen, Qin; Su, Chenhe; Zhang, Zili; Yang, Chengkui; Hu, Zhilin; Hou, Jue; Zhou, Jinying; Gong, Ling; Jiang, Xuejun

2015-01-01

ABSTRACT Receptor-interacting protein kinase 3 (RIP3) and its substrate mixed-lineage kinase domain-like protein (MLKL) are core regulators of programmed necrosis. The elimination of pathogen-infected cells by programmed necrosis acts as an important host defense mechanism. Here, we report that human herpes simplex virus 1 (HSV-1) and HSV-2 had opposite impacts on programmed necrosis in human cells versus their impacts in mouse cells. Similar to HSV-1, HSV-2 infection triggered programmed necrosis in mouse cells. However, neither HSV-1 nor HSV-2 infection was able to induce programmed necrosis in human cells. Moreover, HSV-1 or HSV-2 infection in human cells blocked tumor necrosis factor (TNF)-induced necrosis by preventing the induction of an RIP1/RIP3 necrosome. The HSV ribonucleotide reductase large subunit R1 was sufficient to suppress TNF-induced necrosis, and its RIP homotypic interaction motif (RHIM) domain was required to disrupt the RIP1/RIP3 complex in human cells. Therefore, this study provides evidence that HSV has likely evolved strategies to evade the host defense mechanism of programmed necrosis in human cells. IMPORTANCE This study demonstrated that infection with HSV-1 and HSV-2 blocked TNF-induced necrosis in human cells while these viruses directly activated programmed necrosis in mouse cells. Expression of HSV R1 suppressed TNF-induced necrosis of human cells. The RHIM domain of R1 was essential for its association with human RIP3 and RIP1, leading to disruption of the RIP1/RIP3 complex. This study provides new insights into the species-specific modulation of programmed necrosis by HSV. PMID:26559832

Herpes Simplex Virus 1 (HSV-1) and HSV-2 Mediate Species-Specific Modulations of Programmed Necrosis through the Viral Ribonucleotide Reductase Large Subunit R1.

PubMed

Yu, Xiaoliang; Li, Yun; Chen, Qin; Su, Chenhe; Zhang, Zili; Yang, Chengkui; Hu, Zhilin; Hou, Jue; Zhou, Jinying; Gong, Ling; Jiang, Xuejun; Zheng, Chunfu; He, Sudan

2016-01-15

Receptor-interacting protein kinase 3 (RIP3) and its substrate mixed-lineage kinase domain-like protein (MLKL) are core regulators of programmed necrosis. The elimination of pathogen-infected cells by programmed necrosis acts as an important host defense mechanism. Here, we report that human herpes simplex virus 1 (HSV-1) and HSV-2 had opposite impacts on programmed necrosis in human cells versus their impacts in mouse cells. Similar to HSV-1, HSV-2 infection triggered programmed necrosis in mouse cells. However, neither HSV-1 nor HSV-2 infection was able to induce programmed necrosis in human cells. Moreover, HSV-1 or HSV-2 infection in human cells blocked tumor necrosis factor (TNF)-induced necrosis by preventing the induction of an RIP1/RIP3 necrosome. The HSV ribonucleotide reductase large subunit R1 was sufficient to suppress TNF-induced necrosis, and its RIP homotypic interaction motif (RHIM) domain was required to disrupt the RIP1/RIP3 complex in human cells. Therefore, this study provides evidence that HSV has likely evolved strategies to evade the host defense mechanism of programmed necrosis in human cells. This study demonstrated that infection with HSV-1 and HSV-2 blocked TNF-induced necrosis in human cells while these viruses directly activated programmed necrosis in mouse cells. Expression of HSV R1 suppressed TNF-induced necrosis of human cells. The RHIM domain of R1 was essential for its association with human RIP3 and RIP1, leading to disruption of the RIP1/RIP3 complex. This study provides new insights into the species-specific modulation of programmed necrosis by HSV. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

TRIM25 enhances cell growth and cell survival by modulating p53 signals via interaction with G3BP2 in prostate cancer.

PubMed

Takayama, Ken-Ichi; Suzuki, Takashi; Tanaka, Tomoaki; Fujimura, Tetsuya; Takahashi, Satoru; Urano, Tomohiko; Ikeda, Kazuhiro; Inoue, Satoshi

2018-04-01

Prostate cancer growth is promoted by the gene regulatory action of androgen receptor (AR) and its downstream signals. The aberrant dysfunction of tumor suppressor p53 has an important role in the prognosis of cancer. We previously found that androgen treatments translocate p53 to the cytoplasm. The mechanism of this translocation depends on sumoylation of p53 by complex of SUMO E3 ligase RanBP2 with androgen-induced GTPase-activating protein-binding protein 2 (G3BP2). Here, we identified tripartite motif-containing protein 25 (TRIM25)/estrogen-responsive finger protein (Efp) as a novel interacting partner of G3BP2 protein complex. Then, we demonstrated that TRIM25 knockdown resulted in p53 downstream activation for cell cycle inhibition and apoptosis induction in LNCaP and 22Rv1 cells. In contrast, overexpression of TRIM25 promoted prostate cancer cell proliferation and inhibited apoptosis by docetaxel treatment in LNCaP cells. We observed that p53 activity was reduced by mechanism of G3BP2-mediated nuclear export in TRIM25-overexpressing prostate cancer cells. We also found TRIM25 is important for G3BP2/RanBP2-mediated p53 modification. Clinically, we newly demonstrated that TRIM25 is a prognostic factor for prostate cancer patients. Expression of TRIM25 is significantly associated with cytoplasmic p53 expression and G3BP2. Moreover, TRIM25 knockdown results in reduced tumor growth and increased p53 activity in the mouse xenograft model of prostate cancer. Thus, our findings show that overexpression of TRIM25 promoted prostate cancer cell proliferation and cell survival by modulating p53 nuclear export mechanism with G3BP2 interaction.

Characterization of ultrasonic vocalizations of Fragile X mice.

PubMed

Belagodu, Amogh P; Johnson, Aaron M; Galvez, Roberto

2016-09-01

Fragile X Syndrome (FXS) is the leading form of inherited intellectual disability. It is caused by the transcriptional silencing of FMR1, the gene which codes for the Fragile X Mental Retardation Protein (FMRP). Patients who have FXS exhibit numerous behavioral and cognitive impairments, such as attention-deficit/hyperactivity disorder, obsessive compulsive disorder, and autistic-like behaviors. In addition to these behavioral abnormalities, FXS patients have also been shown to exhibit various deficits in communication such as abnormal sentence structures, increased utterances, repetition of sounds and words, and reduced articulation. These deficits can dramatically hinder communication for FXS patients, exacerbating learning and cognition impairments while decreasing their quality of life. To examine the biological underpinnings of these communication abnormalities, studies have used a mouse model of the Fragile X Syndrome; however, these vocalization studies have resulted in inconsistent findings that often do not correlate with abnormalities observed in FXS patients. Interestingly, a detailed examination of frequency modulated vocalizations that are believed to be a better assessment of rodent communication has never been conducted. The following study used courtship separation to conduct a detailed examination of frequency modulated ultrasonic vocalizations (USV) in FXS mice. Our analyses of frequency modulated USVs demonstrated that adult FXS mice exhibited longer phrases and more motifs. Phrases are vocalizations consisting of multiple frequency modulated ultrasonic vocalizations, while motifs are repeated frequency modulated USV patterns. Fragile X mice had a higher proportion of "u" syllables in all USVs and phrases while their wildtype counterparts preferred isolated "h" syllables. Although the specific importance of these syllables towards communication deficits still needs to be evaluated, these findings in production of USVs are consistent with the repetitive and perseverative speech patterns observed in FXS patients. This study demonstrates that FXS mice can be used to study the underlying biological mechanism(s) mediating FXS vocalization abnormalities. Copyright © 2016 Elsevier B.V. All rights reserved.

Tsg101 regulates PI(4,5)P2/Ca2+ signaling for HIV-1 Gag assembly

PubMed Central

Ehrlich, Lorna S.; Medina, Gisselle N.; Photiadis, Sara; Whittredge, Paul B.; Watanabe, Susan; Taraska, Justin W.; Carter, Carol A.

2014-01-01

Our previous studies identified the 1,4,5-inositol trisphosphate receptor (IP3R), a channel mediating release of Ca2+ from ER stores, as a cellular factor differentially associated with HIV-1 Gag that might facilitate ESCRT function in virus budding. Channel opening requires activation that is initiated by binding of 1,4,5-triphosphate (IP3), a product of phospholipase C (PLC)-mediated PI(4,5)P2 hydrolysis. The store emptying that follows stimulates store refilling which requires intact PI(4,5)P2. Raising cytosolic Ca2+ promotes viral particle production and our studies indicate that IP3R and the ER Ca2+ store are the physiological providers of Ca2+ for Gag assembly and release. Here, we show that Gag modulates ER store gating and refilling. Cells expressing Gag exhibited a higher cytosolic Ca2+ level originating from the ER store than control cells, suggesting that Gag induced release of store Ca2+. This property required the PTAP motif in Gag that recruits Tsg101, an ESCRT-1 component. Consistent with cytosolic Ca2+ elevation, Gag accumulation at the plasma membrane was found to require continuous IP3R activation. Like other IP3R channel modulators, Gag was detected in physical proximity to the ER and to endogenous IP3R, as indicated respectively by total internal reflection fluorescence (TIRF) and immunoelectron microscopy (IEM) or indirect immunofluorescence. Reciprocal co-immunoprecipitation suggested that Gag and IP3R proximity is favored when the PTAP motif in Gag is intact. Gag expression was also accompanied by increased PI(4,5)P2 accumulation at the plasma membrane, a condition favoring store refilling capacity. Supporting this notion, Gag particle production was impervious to treatment with 2-aminoethoxydiphenyl borate, an inhibitor of a refilling coupling interaction. In contrast, particle production by a Gag mutant lacking the PTAP motif was reduced. We conclude that a functional PTAP L domain, and by inference Tsg101 binding, confers Gag with an ability to modulate both ER store Ca2+ release and ER store refilling. PMID:24904548

Cohesin regulates tissue-specific expression by stabilizing highly occupied cis-regulatory modules

PubMed Central

Faure, Andre J.; Schmidt, Dominic; Watt, Stephen; Schwalie, Petra C.; Wilson, Michael D.; Xu, Huiling; Ramsay, Robert G.; Odom, Duncan T.; Flicek, Paul

2012-01-01

The cohesin protein complex contributes to transcriptional regulation in a CTCF-independent manner by colocalizing with master regulators at tissue-specific loci. The regulation of transcription involves the concerted action of multiple transcription factors (TFs) and cohesin's role in this context of combinatorial TF binding remains unexplored. To investigate cohesin-non-CTCF (CNC) binding events in vivo we mapped cohesin and CTCF, as well as a collection of tissue-specific and ubiquitous transcriptional regulators using ChIP-seq in primary mouse liver. We observe a positive correlation between the number of distinct TFs bound and the presence of CNC sites. In contrast to regions of the genome where cohesin and CTCF colocalize, CNC sites coincide with the binding of master regulators and enhancer-markers and are significantly associated with liver-specific expressed genes. We also show that cohesin presence partially explains the commonly observed discrepancy between TF motif score and ChIP signal. Evidence from these statistical analyses in wild-type cells, and comparisons to maps of TF binding in Rad21-cohesin haploinsufficient mouse liver, suggests that cohesin helps to stabilize large protein–DNA complexes. Finally, we observe that the presence of mirrored CTCF binding events at promoters and their nearby cohesin-bound enhancers is associated with elevated expression levels. PMID:22780989

DIBS: a repository of disordered binding sites mediating interactions with ordered proteins.

PubMed

Schad, Eva; Fichó, Erzsébet; Pancsa, Rita; Simon, István; Dosztányi, Zsuzsanna; Mészáros, Bálint

2018-02-01

Intrinsically Disordered Proteins (IDPs) mediate crucial protein-protein interactions, most notably in signaling and regulation. As their importance is increasingly recognized, the detailed analyses of specific IDP interactions opened up new opportunities for therapeutic targeting. Yet, large scale information about IDP-mediated interactions in structural and functional details are lacking, hindering the understanding of the mechanisms underlying this distinct binding mode. Here, we present DIBS, the first comprehensive, curated collection of complexes between IDPs and ordered proteins. DIBS not only describes by far the highest number of cases, it also provides the dissociation constants of their interactions, as well as the description of potential post-translational modifications modulating the binding strength and linear motifs involved in the binding. Together with the wide range of structural and functional annotations, DIBS will provide the cornerstone for structural and functional studies of IDP complexes. DIBS is freely accessible at http://dibs.enzim.ttk.mta.hu/. The DIBS application is hosted by Apache web server and was implemented in PHP. To enrich querying features and to enhance backend performance a MySQL database was also created. dosztanyi@caesar.elte.hu or bmeszaros@caesar.elte.hu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press.

Crystal structure of dimeric cardiac L-type calcium channel regulatory domains bridged by Ca[superscript 2+]·calmodulins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fallon, Jennifer L.; Baker, Mariah R.; Xiong, Liangwen

2009-11-10

Voltage-dependent calcium channels (Ca(V)) open in response to changes in membrane potential, but their activity is modulated by Ca(2+) binding to calmodulin (CaM). Structural studies of this family of channels have focused on CaM bound to the IQ motif; however, the minimal differences between structures cannot adequately describe CaM's role in the regulation of these channels. We report a unique crystal structure of a 77-residue fragment of the Ca(V)1.2 alpha(1) subunit carboxyl terminus, which includes a tandem of the pre-IQ and IQ domains, in complex with Ca(2+).CaM in 2 distinct binding modes. The structure of the Ca(V)1.2 fragment is anmore » unusual dimer of 2 coiled-coiled pre-IQ regions bridged by 2 Ca(2+).CaMs interacting with the pre-IQ regions and a canonical Ca(V)1-IQ-Ca(2+).CaM complex. Native Ca(V)1.2 channels are shown to be a mixture of monomers/dimers and a point mutation in the pre-IQ region predicted to abolish the coiled-coil structure significantly reduces Ca(2+)-dependent inactivation of heterologously expressed Ca(V)1.2 channels.« less

Discovery of a Kelch-like ECH-associated protein 1-inhibitory tetrapeptide and its structural characterization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sogabe, Satoshi; Sakamoto, Kotaro; Kamada, Yusuke

Keap1 constitutively binds to the transcription factor Nrf2 to promote its degradation, resulting in negative modulation of genes involved in cellular protection against oxidative stress. Keap1 is increasingly recognized as an attractive target for treating diseases involving oxidative stress, including cancer, atherosclerosis, diabetes, arthritis, and neurodegeneration. We used phage-display peptide screening to identify a tetrapeptide showing moderate binding affinity, which inhibits the interaction between Nrf2 and Keap1. The tetrapeptide does not include an ETGE motif, which is a commonly found consensus sequence in known peptidic inhibitors. In addition to affinity parameters, IC{sub 50}, K{sub D}, and thermodynamic parameters, the crystalmore » structure of the complex was determined to elucidate the binding conformation. The binding interactions resemble those of known small-molecule inhibitors as opposed to those of substrates and peptidic inhibitors. Although the tetrapeptide's affinity is not very high, our results may help facilitate the designing of small-molecule inhibitors during lead generation in drug discovery. - Highlights: • Keap1 inhibitory tetrapeptide with moderate affinity was discovered. • Crystal structure of the complex showed the unique binding mode. • Structural information gives a valuable insight for design of therapeutic compounds.« less

Complexity of the 5' Untranslated Region of EIF4A3, a Critical Factor for Craniofacial and Neural Development.

PubMed

Hsia, Gabriella S P; Musso, Camila M; Alvizi, Lucas; Brito, Luciano A; Kobayashi, Gerson S; Pavanello, Rita C M; Zatz, Mayana; Gardham, Alice; Wakeling, Emma; Zechi-Ceide, Roseli M; Bertola, Debora; Passos-Bueno, Maria Rita

2018-01-01

Repeats in coding and non-coding regions have increasingly been associated with many human genetic disorders, such as Richieri-Costa-Pereira syndrome (RCPS). RCPS, mostly characterized by midline cleft mandible, Robin sequence and limb defects, is an autosomal-recessive acrofacial dysostosis mainly reported in Brazilian patients. This disorder is caused by decreased levels of EIF4A3 , mostly due to an increased number of repeats at the EIF4A3 5'UTR. EIF4A3 5'UTR alleles are CG-rich and vary in size and organization of three types of motifs. An exclusive allelic pattern was identified among affected individuals, in which the CGCA-motif is the most prevalent, herein referred as "disease-associated CGCA-20nt motif." The origin of the pathogenic alleles containing the disease-associated motif, as well as the functional effects of the 5'UTR motifs on EIF4A3 expression, to date, are entirely unknown. Here, we characterized 43 different EIF4A3 5'UTR alleles in a cohort of 380 unaffected individuals. We identified eight heterozygous unaffected individuals harboring the disease-associated CGCA-20nt motif and our haplotype analyses indicate that there are more than one haplotype associated with RCPS. The combined analysis of number, motif organization and haplotypic diversity, as well as the observation of two apparently distinct haplotypes associated with the disease-associated CGCA-20nt motif, suggest that the RCPS alleles might have arisen from independent unequal crossing-over events between ancient alleles at least twice. Moreover, we have shown that the number and sequence of motifs in the 5'UTR region is associated with EIF4A3 repression, which is not mediated by CpG methylation. In conclusion, this study has shown that the large number of repeats in EIF4A3 does not represent a dynamic mutation and RCPS can arise in any population harboring alleles with the CGCA-20nt motif. We also provided further evidence that EIF4A3 5'UTR is a regulatory region and the size and sequence type of the repeats at 5'UTR may contribute to clinical variability in RCPS.

Identification of structural motifs as tunneling two-level systems in amorphous alumina at low temperatures

NASA Astrophysics Data System (ADS)

Paz, Alejandro Pérez; Lebedeva, Irina V.; Tokatly, Ilya V.; Rubio, Angel

2014-12-01

One of the most accepted models that describe the anomalous thermal behavior of amorphous materials at temperatures below 1 K relies on the quantum mechanical tunneling of atoms between two nearly equivalent potential energy wells forming a two-level system (TLS). Indirect evidence for TLSs is widely available. However, the atomistic structure of these TLSs remains an unsolved topic in the physics of amorphous materials. Here, using classical molecular dynamics, we found several hitherto unknown bistable structural motifs that may be key to understanding the anomalous thermal properties of amorphous alumina at low temperatures. We show through free energy profiles that the complex potential energy surface can be reduced to canonical TLSs. The tunnel splitting predicted from instanton theory, the number density, dipole moment, and coupling to external strain of the discovered motifs are consistent with experiments.

Weak interactions involving organic fluorine: analysis of structural motifs in Flunazirine and Haloperidol

NASA Astrophysics Data System (ADS)

Prasanna, M. D.; Row, T. N. Guru

2001-05-01

The crystal structure of Flunazirine, an anticonvulsant drug, is analyzed in terms of intermolecular interactions involving fluorine. The structure displays motifs formed by only weak interactions C-H⋯F and C-H⋯π. The motifs thus generated show cavities, which could serve as hosts for complexation. The structure of Flunazirine displays cavities formed by C-H⋯F and C-H⋯π interactions. Haloperidol, an antipsychotic drug, shows F⋯F interactions in the crystalline lattice in lieu of Cl⋯Cl interactions. However, strong O-H⋯N interactions dominate packing. The salient features of the two structures in terms of intermolecular interactions reveal, even though organic fluorine has lower tendency to engage in hydrogen bonding and F⋯F interactions, these interactions could play a significant role in the design of molecular assemblies via crystal engineering.

NK cell activation: distinct stimulatory pathways counterbalancing inhibitory signals.

PubMed

Bakker, A B; Wu, J; Phillips, J H; Lanier, L L

2000-01-01

A delicate balance between positive and negative signals regulates NK cell effector function. Activation of NK cells may be initiated by the triggering of multiple adhesion or costimulatory molecules, and can be counterbalanced by inhibitory signals induced by receptors for MHC class I. A common pathway of inhibitory signaling is provided by immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the cytoplasmic domains of these receptors which mediate the recruitment of SH2 domain-bearing tyrosine phosphate-1 (SHP-1). In contrast to the extensive progress that has been made regarding the negative regulation of NK cell function, our knowledge of the signals that activate NK cells is still poor. Recent studies of the activating receptor complexes have shed new light on the induction of NK cell effector function. Several NK receptors using novel adaptors with immunoreceptor tyrosine-based activation motifs (ITAMs) and with PI 3-kinase recruiting motifs have been implicated in NK cell stimulation.

Orthorhombic Zr2Co11 phase revisited

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, X. -Z.; Zhang, W. Y.; Sellmyer, D. J.

2014-10-01

The structure of the orthorhombic Zr2Co11 phase was revisited in the present work. Selected-area electron diffraction (SAED) and high-resolution electron microscopy (HREM) techniques were used to investigate the structure. They show the orthorhombic Zr2Co11 phase has a 1-D incommensurate modulated structure. The structure can be approximately described as a B-centered orthorhombic lattice. The lattice parameters of the orthorhombic Zr2Co11 phase have been determined by a tilt series of SAED patterns. A hexagonal network with a modulation wave has been observed in the HREM image and the hexagonal motif is considered as the basic structural unit.

Programming Recognition Arrays through Double Chalcogen-Bonding Interactions.

PubMed

Biot, Nicolas; Bonifazi, Davide

2018-04-11

In this work, we have programmed and synthesized a recognition motif constructed around a chalcogenazolo-pyridine scaffold (CGP) that, through the formation of frontal double chalcogen-bonding interactions, associates into dimeric EX-type complexes. The reliability of the double chalcogen-bonding interaction has been shown at the solid-state by X-ray analysis, depicting the strongest recognition persistence for a Te-congener. The high recognition fidelity, chemical and thermal stability and easy derivatization at the 2-position makes CGP a convenient motif for constructing supramolecular architectures through programmed chalcogen-bonding interactions. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Energetics and Structure Prediction of the Network of Homo- and Hetero-Oligomers Formed by the Transmembrane Domains of the ErbB Receptor Family of Proteins

DTIC Science & Technology

2005-06-01

acid residue motif, Small-x-x-Large-G/A, consist- complexes is critical to understanding the signal ing of a small residue (Gly, Ala , Ser, Thr, or Pro...in transduction process. Recent structures of the the zero position, a large aliphatic residue ( Ala , Val, ligand-binding domains of the erbB...receptors Leu, or Ile) in position 3, followed by Gly or Ala in have begun to provide insight into the mechanisms position four.’ This motif was identified

Web server to identify similarity of amino acid motifs to compounds (SAAMCO).

PubMed

Casey, Fergal P; Davey, Norman E; Baran, Ivan; Varekova, Radka Svobodova; Shields, Denis C

2008-07-01

Protein-protein interactions are fundamental in mediating biological processes including metabolism, cell growth, and signaling. To be able to selectively inhibit or induce protein activity or complex formation is a key feature in controlling disease. For those situations in which protein-protein interactions derive substantial affinity from short linear peptide sequences, or motifs, we can develop search algorithms for peptidomimetic compounds that resemble the short peptide's structure but are not compromised by poor pharmacological properties. SAAMCO is a Web service ( http://bioware.ucd.ie/ approximately saamco) that facilitates the screening of motifs with known structures against bioactive compound databases. It is built on an algorithm that defines compound similarity based on the presence of appropriate amino acid side chain fragments and a favorable Root Mean Squared Deviation (RMSD) between compound and motif structure. The methodology is efficient as the available compound databases are preprocessed and fast regular expression searches filter potential matches before time-intensive 3D superposition is performed. The required input information is minimal, and the compound databases have been selected to maximize the availability of information on biological activity. "Hits" are accompanied with a visualization window and links to source database entries. Motif matching can be defined on partial or full similarity which will increase or reduce respectively the number of potential mimetic compounds. The Web server provides the functionality for rapid screening of known or putative interaction motifs against prepared compound libraries using a novel search algorithm. The tabulated results can be analyzed by linking to appropriate databases and by visualization.

Translation Control of Swarming Proficiency in Bacillus subtilis by 5-Amino-pentanolylated Elongation Factor P.

PubMed

Rajkovic, Andrei; Hummels, Katherine R; Witzky, Anne; Erickson, Sarah; Gafken, Philip R; Whitelegge, Julian P; Faull, Kym F; Kearns, Daniel B; Ibba, Michael

2016-05-20

Elongation factor P (EF-P) accelerates diprolyl synthesis and requires a posttranslational modification to maintain proteostasis. Two phylogenetically distinct EF-P modification pathways have been described and are encoded in the majority of Gram-negative bacteria, but neither is present in Gram-positive bacteria. Prior work suggested that the EF-P-encoding gene (efp) primarily supports Bacillus subtilis swarming differentiation, whereas EF-P in Gram-negative bacteria has a more global housekeeping role, prompting our investigation to determine whether EF-P is modified and how it impacts gene expression in motile cells. We identified a 5-aminopentanol moiety attached to Lys(32) of B. subtilis EF-P that is required for swarming motility. A fluorescent in vivo B. subtilis reporter system identified peptide motifs whose efficient synthesis was most dependent on 5-aminopentanol EF-P. Examination of the B. subtilis genome sequence showed that these EF-P-dependent peptide motifs were represented in flagellar genes. Taken together, these data show that, in B. subtilis, a previously uncharacterized posttranslational modification of EF-P can modulate the synthesis of specific diprolyl motifs present in proteins required for swarming motility. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Interactions of U24 from Roseolovirus with WW domains: canonical vs noncanonical.

PubMed

Sang, Yurou; Zhang, Rui; Creagh, A Louise; Haynes, Charles A; Straus, Suzana K

2017-06-01

U24 is a C-terminal membrane-anchored protein found in both human herpes virus type 6 and 7 (HHV-6 and HHV-7), with an N-terminal segment that is rich in prolines (PPxY motif in both HHV-6A and 7; PxxP motif in HHV-6A). Previous work has shown that U24 interacts strongly with Nedd4 WW domains, in particular, hNedd4L-WW3*. It was also shown that this interaction depends strongly on the nature of the amino acids that are upstream from the PY motif in U24. In this contribution, data was obtained from pull-downs, isothermal titration calorimetry, and NMR to further determine what modulates U24:WW domain interactions. Specifically, 3 non-canonical WW domains from human Smad ubiquitination regulatory factor (Smurf), namely hSmurf2-WW2, hSmurf2-WW3, and a tandem construct hSmurf2-WW2 + 3, were studied. Overall, the interactions between U24 and these Smurf WW domains were found to be weaker than those in U24:Nedd4 WW domain pairs, suggesting that U24 function is tightly linked to specific E3 ubiqitin ligases.

Supramolecular Hydrogels Based on Minimalist Amphiphilic Squaramide-Squaramates for Controlled Release of Zwitterionic Biomolecules.

PubMed

López, Carlos; Ximenis, Marta; Orvay, Francisca; Rotger, Carmen; Costa, Antonio

2017-06-01

Supramolecular hydrogels with tunable properties have innovative applications in biomedicine, catalysis, and materials chemistry. Minimalist low-molecular-weight hydrogelators based on squaramide and squaramic acid motifs have been designed. This approach benefits from the high acidity of squaramic acids and the aromaticity of squaramides. Moreover, substituents on the aryl ring tune the π density of the arylsquaramide motif. Thus, materials featuring distinct thermal and mechanical properties have been successfully prepared. The hydrogel (G'≈400 Pa, G''≈57 Pa; at 1.0 % w/v; 1 Hz) obtained from 4-nitrophenylsquaramide motif 1 is thermoreversible (T=57 °C at 0.2 % w/v), thixotropic, self-healable, and undergoes irreversible shrinking in response to saline stress. Furthermore, the hydrogel is injectable and can be loaded with substantial amounts (5:1 excess molar ratio) of zwitterionic biomolecules, such as l-carnitine, γ-aminobutyric acid (GABA), or d,l-Ala-d,l-Ala, without any loss of structural integrity. Then, the release of these molecules can be modulated by saline solutions. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

β-Catenin recognizes a specific RNA motif in the cyclooxygenase-2 mRNA 3′-UTR and interacts with HuR in colon cancer cells

PubMed Central

Kim, Inae; Kwak, Hoyun; Lee, Hee Kyu; Hyun, Soonsil; Jeong, Sunjoo

2012-01-01

RNA-binding proteins regulate multiple steps of RNA metabolism through both dynamic and combined binding. In addition to its crucial roles in cell adhesion and Wnt-activated transcription in cancer cells, β-catenin regulates RNA alternative splicing and stability possibly by binding to target RNA in cells. An RNA aptamer was selected for specific binding to β-catenin to address RNA recognition by β-catenin more specifically. Here, we characterized the structural properties of the RNA aptamer as a model and identified a β-catenin RNA motif. Similar RNA motif was found in cellular RNA, Cyclooxygenase-2 (COX-2) mRNA 3′-untranslated region (3′-UTR). More significantly, the C-terminal domain of β-catenin interacted with HuR and the Armadillo repeat domain associated with RNA to form the RNA–β-catenin–HuR complex in vitro and in cells. Furthermore, the tertiary RNA–protein complex was predominantly found in the cytoplasm of colon cancer cells; thus, it might be related to COX-2 protein level and cancer progression. Taken together, the β-catenin RNA aptamer was valuable for deducing the cellular RNA aptamer and identifying novel and oncogenic RNA–protein networks in colon cancer cells. PMID:22544606

Auxiliary KChIP4a Suppresses A-type K+ Current through Endoplasmic Reticulum (ER) Retention and Promoting Closed-state Inactivation of Kv4 Channels*

PubMed Central

Tang, Yi-Quan; Liang, Ping; Zhou, Jingheng; Lu, Yanxin; Lei, Lei; Bian, Xiling; Wang, KeWei

2013-01-01

In the brain and heart, auxiliary Kv channel-interacting proteins (KChIPs) co-assemble with pore-forming Kv4 α-subunits to form a native K+ channel complex and regulate the expression and gating properties of Kv4 currents. Among the KChIP1–4 members, KChIP4a exhibits a unique N terminus that is known to suppress Kv4 function, but the underlying mechanism of Kv4 inhibition remains unknown. Using a combination of confocal imaging, surface biotinylation, and electrophysiological recordings, we identified a novel endoplasmic reticulum (ER) retention motif, consisting of six hydrophobic and aliphatic residues, 12–17 (LIVIVL), within the KChIP4a N-terminal KID, that functions to reduce surface expression of Kv4-KChIP complexes. This ER retention capacity is transferable and depends on its flanking location. In addition, adjacent to the ER retention motif, the residues 19–21 (VKL motif) directly promote closed-state inactivation of Kv4.3, thus leading to an inhibition of channel current. Taken together, our findings demonstrate that KChIP4a suppresses A-type Kv4 current via ER retention and enhancement of Kv4 closed-state inactivation. PMID:23576435

A short conserved motif in ALYREF directs cap- and EJC-dependent assembly of export complexes on spliced mRNAs.

PubMed

Gromadzka, Agnieszka M; Steckelberg, Anna-Lena; Singh, Kusum K; Hofmann, Kay; Gehring, Niels H

2016-03-18

The export of messenger RNAs (mRNAs) is the final of several nuclear posttranscriptional steps of gene expression. The formation of export-competent mRNPs involves the recruitment of export factors that are assumed to facilitate transport of the mature mRNAs. Using in vitro splicing assays, we show that a core set of export factors, including ALYREF, UAP56 and DDX39, readily associate with the spliced RNAs in an EJC (exon junction complex)- and cap-dependent manner. In order to elucidate how ALYREF and other export adaptors mediate mRNA export, we conducted a computational analysis and discovered four short, conserved, linear motifs present in RNA-binding proteins. We show that mutation in one of the new motifs (WxHD) in an unstructured region of ALYREF reduced RNA binding and abolished the interaction with eIF4A3 and CBP80. Additionally, the mutation impaired proper localization to nuclear speckles and export of a spliced reporter mRNA. Our results reveal important details of the orchestrated recruitment of export factors during the formation of export competent mRNPs. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Improved modeling of side-chain--base interactions and plasticity in protein--DNA interface design.

PubMed

Thyme, Summer B; Baker, David; Bradley, Philip

2012-06-08

Combinatorial sequence optimization for protein design requires libraries of discrete side-chain conformations. The discreteness of these libraries is problematic, particularly for long, polar side chains, since favorable interactions can be missed. Previously, an approach to loop remodeling where protein backbone movement is directed by side-chain rotamers predicted to form interactions previously observed in native complexes (termed "motifs") was described. Here, we show how such motif libraries can be incorporated into combinatorial sequence optimization protocols and improve native complex recapitulation. Guided by the motif rotamer searches, we made improvements to the underlying energy function, increasing recapitulation of native interactions. To further test the methods, we carried out a comprehensive experimental scan of amino acid preferences in the I-AniI protein-DNA interface and found that many positions tolerated multiple amino acids. This sequence plasticity is not observed in the computational results because of the fixed-backbone approximation of the model. We improved modeling of this diversity by introducing DNA flexibility and reducing the convergence of the simulated annealing algorithm that drives the design process. In addition to serving as a benchmark, this extensive experimental data set provides insight into the types of interactions essential to maintain the function of this potential gene therapy reagent. Published by Elsevier Ltd.

Regulation of HTLV-1 Gag budding by Vps4A, Vps4B, and AIP1/Alix

PubMed Central

Urata, Shuzo; Yokosawa, Hideyoshi; Yasuda, Jiro

2007-01-01

Background HTLV-1 Gag protein is a matrix protein that contains the PTAP and PPPY sequences as L-domain motifs and which can be released from mammalian cells in the form of virus-like particles (VLPs). The cellular factors Tsg101 and Nedd4.1 interact with PTAP and PPPY, respectively, within the HTLV-1 Gag polyprotein. Tsg101 forms a complex with Vps28 and Vps37 (ESCRT-I complex) and plays an important role in the class E Vps pathway, which mediates protein sorting and invagination of vesicles into multivesicular bodies. Nedd4.1 is an E3 ubiquitin ligase that binds to the PPPY motif through its WW motif, but its function is still unknown. In the present study, to investigate the mechanism of HTLV-1 budding in detail, we analyzed HTLV-1 budding using dominant negative (DN) forms of the class E proteins. Results Here, we report that DN forms of Vps4A, Vps4B, and AIP1 inhibit HTLV-1 budding. Conclusion These findings suggest that HTLV-1 budding utilizes the MVB pathway and that these class E proteins may be targets for prevention of mother-to-infant vertical transmission of the virus. PMID:17601348

Auxiliary KChIP4a suppresses A-type K+ current through endoplasmic reticulum (ER) retention and promoting closed-state inactivation of Kv4 channels.

PubMed

Tang, Yi-Quan; Liang, Ping; Zhou, Jingheng; Lu, Yanxin; Lei, Lei; Bian, Xiling; Wang, KeWei

2013-05-24

In the brain and heart, auxiliary Kv channel-interacting proteins (KChIPs) co-assemble with pore-forming Kv4 α-subunits to form a native K(+) channel complex and regulate the expression and gating properties of Kv4 currents. Among the KChIP1-4 members, KChIP4a exhibits a unique N terminus that is known to suppress Kv4 function, but the underlying mechanism of Kv4 inhibition remains unknown. Using a combination of confocal imaging, surface biotinylation, and electrophysiological recordings, we identified a novel endoplasmic reticulum (ER) retention motif, consisting of six hydrophobic and aliphatic residues, 12-17 (LIVIVL), within the KChIP4a N-terminal KID, that functions to reduce surface expression of Kv4-KChIP complexes. This ER retention capacity is transferable and depends on its flanking location. In addition, adjacent to the ER retention motif, the residues 19-21 (VKL motif) directly promote closed-state inactivation of Kv4.3, thus leading to an inhibition of channel current. Taken together, our findings demonstrate that KChIP4a suppresses A-type Kv4 current via ER retention and enhancement of Kv4 closed-state inactivation.

AMP-acetyl CoA synthetase from Leishmania donovani: identification and functional analysis of 'PX4GK' motif.

PubMed

Soumya, Neelagiri; Kumar, I Sravan; Shivaprasad, S; Gorakh, Landage Nitin; Dinesh, Neeradi; Swamy, Kayala Kambagiri; Singh, Sushma

2015-04-01

An adenosine monophosphate forming acetyl CoA synthetase (AceCS) which is the key enzyme involved in the conversion of acetate to acetyl CoA has been identified from Leishmania donovani for the first time. Sequence analysis of L. donovani AceCS (LdAceCS) revealed the presence of a 'PX4GK' motif which is highly conserved throughout organisms with higher sequence identity (96%) to lower sequence identity (38%). A ∼ 77 kDa heterologous protein with C-terminal 6X His-tag was expressed in Escherichia coli. Expression of LdAceCS in promastigotes was confirmed by western blot and RT-PCR analysis. Immunolocalization studies revealed that it is a cytosolic protein. We also report the kinetic characterization of recombinant LdAceCS with acetate, adenosine 5'-triphosphate, coenzyme A and propionate as substrates. Site directed mutagenesis of residues in conserved PX4GK motif of LdAceCS was performed to gain insight into its potential role in substrate binding, catalysis and its role in maintaining structural integrity of the protein. P646A, G651A and K652R exhibited more than 90% loss in activity signifying its indispensible role in the enzyme activity. Substitution of other residues in this motif resulted in altered substrate specificity and catalysis. However, none of them had any role in modulation of the secondary structure of the protein except G651A mutant. Copyright © 2015 Elsevier B.V. All rights reserved.

Unusual sugar specificity of banana lectin from Musa paradisiaca and its probable evolutionary origin. Crystallographic and modelling studies.

PubMed

Singh, D D; Saikrishnan, K; Kumar, Prashant; Surolia, A; Sekar, K; Vijayan, M

2005-10-01

The crystal structure of a complex of methyl-alpha-D-mannoside with banana lectin from Musa paradisiaca reveals two primary binding sites in the lectin, unlike in other lectins with beta-prism I fold which essentially consists of three Greek key motifs. It has been suggested that the fold evolved through successive gene duplication and fusion of an ancestral Greek key motif. In other lectins, all from dicots, the primary binding site exists on one of the three motifs in the three-fold symmetric molecule. Banana is a monocot, and the three motifs have not diverged enough to obliterate sequence similarity among them. Two Greek key motifs in it carry one primary binding site each. A common secondary binding site exists on the third Greek key. Modelling shows that both the primary sites can support 1-2, 1-3, and 1-6 linked mannosides with the second residue interacting in each case primarily with the secondary binding site. Modelling also readily leads to a bound branched mannopentose with the nonreducing ends of the two branches anchored at the two primary binding sites, providing a structural explanation for the lectin's specificity for branched alpha-mannans. A comparison of the dimeric banana lectin with other beta-prism I fold lectins, provides interesting insights into the variability in their quaternary structure.

Cellular microRNAs up-regulate transcription via interaction with promoter TATA-box motifs.

PubMed

Zhang, Yijun; Fan, Miaomiao; Zhang, Xue; Huang, Feng; Wu, Kang; Zhang, Junsong; Liu, Jun; Huang, Zhuoqiong; Luo, Haihua; Tao, Liang; Zhang, Hui

2014-12-01

The TATA box represents one of the most prevalent core promoters where the pre-initiation complexes (PICs) for gene transcription are assembled. This assembly is crucial for transcription initiation and well regulated. Here we show that some cellular microRNAs (miRNAs) are associated with RNA polymerase II (Pol II) and TATA box-binding protein (TBP) in human peripheral blood mononuclear cells (PBMCs). Among them, let-7i sequence specifically binds to the TATA-box motif of interleukin-2 (IL-2) gene and elevates IL-2 mRNA and protein production in CD4(+) T-lymphocytes in vitro and in vivo. Through direct interaction with the TATA-box motif, let-7i facilitates the PIC assembly and transcription initiation of IL-2 promoter. Several other cellular miRNAs, such as mir-138, mir-92a or mir-181d, also enhance the promoter activities via binding to the TATA-box motifs of insulin, calcitonin or c-myc, respectively. In agreement with the finding that an HIV-1-encoded miRNA could enhance viral replication through targeting the viral promoter TATA-box motif, our data demonstrate that the interaction with core transcription machinery is a novel mechanism for miRNAs to regulate gene expression. © 2014 Zhang et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

FF483–484 motif of human Polη mediates its interaction with the POLD2 subunit of Polδ and contributes to DNA damage tolerance

PubMed Central

Baldeck, Nadège; Janel-Bintz, Régine; Wagner, Jérome; Tissier, Agnès; Fuchs, Robert P.; Burkovics, Peter; Haracska, Lajos; Despras, Emmanuelle; Bichara, Marc; Chatton, Bruno; Cordonnier, Agnès M.

2015-01-01

Switching between replicative and translesion synthesis (TLS) DNA polymerases are crucial events for the completion of genomic DNA synthesis when the replication machinery encounters lesions in the DNA template. In eukaryotes, the translesional DNA polymerase η (Polη) plays a central role for accurate bypass of cyclobutane pyrimidine dimers, the predominant DNA lesions induced by ultraviolet irradiation. Polη deficiency is responsible for a variant form of the Xeroderma pigmentosum (XPV) syndrome, characterized by a predisposition to skin cancer. Here, we show that the FF483–484 amino acids in the human Polη (designated F1 motif) are necessary for the interaction of this TLS polymerase with POLD2, the B subunit of the replicative DNA polymerase δ, both in vitro and in vivo. Mutating this motif impairs Polη function in the bypass of both an N-2-acetylaminofluorene adduct and a TT-CPD lesion in cellular extracts. By complementing XPV cells with different forms of Polη, we show that the F1 motif contributes to the progression of DNA synthesis and to the cell survival after UV irradiation. We propose that the integrity of the F1 motif of Polη, necessary for the Polη/POLD2 interaction, is required for the establishment of an efficient TLS complex. PMID:25662213

Roles of conserved proline and glycosyltransferase motifs of EmbC in biosynthesis of lipoarabinomannan.

PubMed

Berg, Stefan; Starbuck, James; Torrelles, Jordi B; Vissa, Varalakshmi D; Crick, Dean C; Chatterjee, Delphi; Brennan, Patrick J

2005-02-18

D-Arabinans, composed of D-arabinofuranose (D-Araf), dominate the structure of mycobacterial cell walls in two settings, as part of lipoarabinomannan (LAM) and arabinogalactan, each with markedly different structures and functions. Little is known of the complexity of their biosynthesis. beta-D-Arabinofuranosyl-1-monophosphoryldecaprenol is the only known sugar donor. EmbA, EmbB, and EmbC, products of the paralogous genes embA, embB, and embC, the sites of resistance to the anti-tuberculosis drug ethambutol (EMB), are the only known implicated enzymes. EmbA and -B apparently contribute to the synthesis of arabinogalactan, whereas EmbC is reserved for the synthesis of LAM. The Emb proteins show no overall similarity to any known proteins beyond Mycobacterium and related genera. However, functional motifs, equivalent to a proline-rich motif of several bacterial polysaccharide co-polymerases and a superfamily of glycosyltransferases, were found. Site-directed mutagenesis in glycosyltransferase superfamily C resulted in complete ablation of LAM synthesis. Point mutations in three amino acids of the proline motif of EmbC resulted in marked reduction of LAM-arabinan synthesis and accumulation of an unknown intermediate and of the known precursor lipomannan. Yet the pattern of the differently linked d-Araf units observed in wild type LAM-arabinan was largely retained in the proline motif mutants. The results allow for the presentation of a unique model of arabinan synthesis.

Identification of sequence–structure RNA binding motifs for SELEX-derived aptamers

PubMed Central

Hoinka, Jan; Zotenko, Elena; Friedman, Adam; Sauna, Zuben E.; Przytycka, Teresa M.

2012-01-01

Motivation: Systematic Evolution of Ligands by EXponential Enrichment (SELEX) represents a state-of-the-art technology to isolate single-stranded (ribo)nucleic acid fragments, named aptamers, which bind to a molecule (or molecules) of interest via specific structural regions induced by their sequence-dependent fold. This powerful method has applications in designing protein inhibitors, molecular detection systems, therapeutic drugs and antibody replacement among others. However, full understanding and consequently optimal utilization of the process has lagged behind its wide application due to the lack of dedicated computational approaches. At the same time, the combination of SELEX with novel sequencing technologies is beginning to provide the data that will allow the examination of a variety of properties of the selection process. Results: To close this gap we developed, Aptamotif, a computational method for the identification of sequence–structure motifs in SELEX-derived aptamers. To increase the chances of identifying functional motifs, Aptamotif uses an ensemble-based approach. We validated the method using two published aptamer datasets containing experimentally determined motifs of increasing complexity. We were able to recreate the author's findings to a high degree, thus proving the capability of our approach to identify binding motifs in SELEX data. Additionally, using our new experimental dataset, we illustrate the application of Aptamotif to elucidate several properties of the selection process. Contact: przytyck@ncbi.nlm.nih.gov, Zuben.Sauna@fda.hhs.gov PMID:22689764

Structural and functional analysis of the GABARAP interaction motif (GIM)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rogov, Vladimir V.; Stolz, Alexandra; Ravichandran, Arvind C.

Through the canonical LC3 interaction motif (LIR), [W/F/Y]–X 1–X 2[I/L/V], protein complexes are recruited to autophagosomes to perform their functions as either autophagy adaptors or receptors. How these adaptors/receptors selectively interact with either LC3 or GABARAP families remains unclear. Herein, we determine the range of selectivity of 30 known core LIR motifs towards individual LC3s and GABARAPs. From these, we define a GABARAP Interaction Motif (GIM) sequence ([W/F]–[V/I]–X 2–V) that the adaptor protein PLEKHM1 tightly conforms to. Using biophysical and structural approaches, we show that the PLEKHM1–LIR is indeed 11–fold more specific for GABARAP than LC3B. Selective mutation of themore » X 1 and X 2 positions either completely abolished the interaction with all LC3 and GABARAPs or increased PLEKHM1–GIM selectivity 20–fold towards LC3B. Finally, we show that conversion of p62/SQSTM1, FUNDC1 and FIP200 LIRs into our newly defined GIM, by introducing two valine residues, enhances their interaction with endogenous GABARAP over LC3B. In conclusion, the identification of a GABARAP–specific interaction motif will aid the identification and characterization of the expanding array of autophagy receptor and adaptor proteins and their in vivo functions.« less

Structural and functional analysis of the GABARAP interaction motif (GIM)

DOE PAGES

Rogov, Vladimir V.; Stolz, Alexandra; Ravichandran, Arvind C.; ...

2017-06-27

Through the canonical LC3 interaction motif (LIR), [W/F/Y]–X 1–X 2[I/L/V], protein complexes are recruited to autophagosomes to perform their functions as either autophagy adaptors or receptors. How these adaptors/receptors selectively interact with either LC3 or GABARAP families remains unclear. Herein, we determine the range of selectivity of 30 known core LIR motifs towards individual LC3s and GABARAPs. From these, we define a GABARAP Interaction Motif (GIM) sequence ([W/F]–[V/I]–X 2–V) that the adaptor protein PLEKHM1 tightly conforms to. Using biophysical and structural approaches, we show that the PLEKHM1–LIR is indeed 11–fold more specific for GABARAP than LC3B. Selective mutation of themore » X 1 and X 2 positions either completely abolished the interaction with all LC3 and GABARAPs or increased PLEKHM1–GIM selectivity 20–fold towards LC3B. Finally, we show that conversion of p62/SQSTM1, FUNDC1 and FIP200 LIRs into our newly defined GIM, by introducing two valine residues, enhances their interaction with endogenous GABARAP over LC3B. In conclusion, the identification of a GABARAP–specific interaction motif will aid the identification and characterization of the expanding array of autophagy receptor and adaptor proteins and their in vivo functions.« less

Function of multiple Lis-Homology domain/WD-40 repeat-containing proteins in feed-forward transcriptional repression by silencing mediator for retinoic and thyroid receptor/nuclear receptor corepressor complexes.

PubMed

Choi, Hyo-Kyoung; Choi, Kyung-Chul; Kang, Hee-Bum; Kim, Han-Cheon; Lee, Yoo-Hyun; Haam, Seungjoo; Park, Hyoung-Gi; Yoon, Ho-Geun

2008-05-01

Lis-homology (LisH) motifs are involved in protein dimerization, and the discovery of the conserved N-terminal LisH domain in transducin beta-like protein 1 and its receptor (TBL1 and TBLR1) led us to examine the role of this domain in transcriptional repression. Here we show that multiple beta-transducin (WD-40) repeat-containing proteins interact to form oligomers in solution and that oligomerization depends on the presence of the LisH domain in each protein. Repression of transcription, as assayed using Gal4 fusion proteins, also depended on the presence of the LisH domain, suggesting that oligomerization is a prerequisite for efficient transcriptional repression. Furthermore, we show that the LisH domain is responsible for the binding to the hypoacetylated histone H4 tail and for stable chromatin targeting by the nuclear receptor corepressor complex. Mutations in conserved residues in the LisH motif of TBL1 and TBLR1 block histone binding, oligomerization, and transcriptional repression, supporting the functional importance of the LisH motif in transcriptional repression. Our results indicate that another WD-40 protein, TBL3, also preferentially binds to the N-terminal domain of TBL1 and TBLR1, and forms oligomers with other WD-40 proteins. Finally, we observed that the WD-40 proteins RbAp46 and RbAp48 of the sin3A corepressor complex failed to dimerize. We also found the specific interaction UbcH/E2 with TBL1, but not RbAp46/48. Altogether, our results thus indicate that the presence of multiple LisH/WD-40 repeat containing proteins is exclusive to nuclear receptor corepressor/ silencing mediator for retinoic and thyroid receptor complexes compared with other class 1 histone deacetylase-containing corepessor complexes.

Isolation and Characterization of FORMATE/NI(CYCLAM)^{2+} Complexes with Cryogenic Ion Vibrational Predissociation

NASA Astrophysics Data System (ADS)

Wolk, Arron B.; Fournier, Joseph A.; Wolke, Conrad T.; Johnson, Mark A.

2013-06-01

Transition metal-based organometallic catalysts are a promising means of converting CO_{2} to transportable fuels. Ni(cyclam)^{2+}(cyclam = 1,4,8,11-tetraazacyclotetradecane), a Ni^{II} complex ligated by four nitrogen centers, has shown promise as a catalyst selective for CO_{2} reduction in aqueous solutions. The cyclam ligand has four NH hydrogen bond donors that can adopt five conformations, each offering distinct binding motifs for coordination of CO_{2} close to the metal center. To probe the ligand conformation and the role of hydrogen bonding in adduct binding, we extract Ni(cyclam)^{2+} complexes with the formate anion and some of its analogs from solution using electrospray ionization, and characterize their structures using cryogenic ion vibrational predissociation spectroscopy. Using the signature vibrational features of the embedded carboxylate anion and the NH groups as reporters, we compare the binding motifs of oxalate, benzoate, and formate anions to the Ni(cyclam)^{2+} framework. Finally, we comment on possible routes to generate the singly charged Ni(cyclam)^{+} complex, a key intermediate that has been invoked in the catalytic CO_{2} reduction cycle, but has never been isolated through ion processing techniques.

Identification of Nucleic Acid Binding Sites on Translin-Associated Factor X (TRAX) Protein

PubMed Central

Gupta, Gagan Deep; Kumar, Vinay

2012-01-01

Translin and TRAX proteins play roles in very important cellular processes such as DNA recombination, spatial and temporal expression of mRNA, and in siRNA processing. Translin forms a homomeric nucleic acid binding complex and binds to ssDNA and RNA. However, a mutant translin construct that forms homomeric complex lacking nucleic acid binding activity is able to form fully active heteromeric translin-TRAX complex when co-expressed with TRAX. A substantial progress has been made in identifying translin sites that mediate its binding activity, while TRAX was thought not to bind DNA or RNA on its own. We here for the first time demonstrate nucleic acid binding to TRAX by crosslinking radiolabeled ssDNA to heteromeric translin-TRAX complex using UV-laser. The TRAX and translin, photochemically crosslinked with ssDNA, were individually detected on SDS-PAGE. We mutated two motifs in TRAX and translin, designated B2 and B3, to help define the nucleic acid binding sites in the TRAX sequence. The most pronounced effect was observed in the mutants of B3 motif that impaired nucleic acid binding activity of the heteromeric complexes. We suggest that both translin and TRAX are binding competent and contribute to the nucleic acid binding activity. PMID:22427937

BamA POTRA Domain Interacts with a Native Lipid Membrane Surface.

PubMed

Fleming, Patrick J; Patel, Dhilon S; Wu, Emilia L; Qi, Yifei; Yeom, Min Sun; Sousa, Marcelo Carlos; Fleming, Karen G; Im, Wonpil

2016-06-21

The outer membrane of Gram-negative bacteria is an asymmetric membrane with lipopolysaccharides on the external leaflet and phospholipids on the periplasmic leaflet. This outer membrane contains mainly β-barrel transmembrane proteins and lipidated periplasmic proteins (lipoproteins). The multisubunit protein β-barrel assembly machine (BAM) catalyzes the insertion and folding of the β-barrel proteins into this membrane. In Escherichia coli, the BAM complex consists of five subunits, a core transmembrane β-barrel with a long periplasmic domain (BamA) and four lipoproteins (BamB/C/D/E). The BamA periplasmic domain is composed of five globular subdomains in tandem called POTRA motifs that are key to BAM complex formation and interaction with the substrate β-barrel proteins. The BAM complex is believed to undergo conformational cycling while facilitating insertion of client proteins into the outer membrane. Reports describing variable conformations and dynamics of the periplasmic POTRA domain have been published. Therefore, elucidation of the conformational dynamics of the POTRA domain in full-length BamA is important to understand the function of this molecular complex. Using molecular dynamics simulations, we present evidence that the conformational flexibility of the POTRA domain is modulated by binding to the periplasmic surface of a native lipid membrane. Furthermore, membrane binding of the POTRA domain is compatible with both BamB and BamD binding, suggesting that conformational selection of different POTRA domain conformations may be involved in the mechanism of BAM-facilitated insertion of outer membrane β-barrel proteins. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Mammalian Fe-S proteins: definition of a consensus motif recognized by the co-chaperone HSC20.

PubMed

Maio, N; Rouault, T A

2016-10-01

Iron-sulfur (Fe-S) clusters are inorganic cofactors that are fundamental to several biological processes in all three kingdoms of life. In most organisms, Fe-S clusters are initially assembled on a scaffold protein, ISCU, and subsequently transferred to target proteins or to intermediate carriers by a dedicated chaperone/co-chaperone system. The delivery of assembled Fe-S clusters to recipient proteins is a crucial step in the biogenesis of Fe-S proteins, and, in mammals, it relies on the activity of a multiprotein transfer complex that contains the chaperone HSPA9, the co-chaperone HSC20 and the scaffold ISCU. How the transfer complex efficiently engages recipient Fe-S target proteins involves specific protein interactions that are not fully understood. This mini review focuses on recent insights into the molecular mechanism of amino acid motif recognition and discrimination by the co-chaperone HSC20, which guides Fe-S cluster delivery.

Ankyrin repeats of ANKRA2 recognize a PxLPxL motif on the 3M syndrome protein CCDC8.

PubMed

Nie, Jianyun; Xu, Chao; Jin, Jing; Aka, Juliette A; Tempel, Wolfram; Nguyen, Vivian; You, Linya; Weist, Ryan; Min, Jinrong; Pawson, Tony; Yang, Xiang-Jiao

2015-04-07

Peptide motifs are often used for protein-protein interactions. We have recently demonstrated that ankyrin repeats of ANKRA2 and the paralogous bare lymphocyte syndrome transcription factor RFXANK recognize PxLPxL/I motifs shared by megalin, three histone deacetylases, and RFX5. We show here that that CCDC8 is a major partner of ANKRA2 but not RFXANK in cells. The CCDC8 gene is mutated in 3M syndrome, a short-stature disorder with additional facial and skeletal abnormalities. Two other genes mutated in this syndrome encode CUL7 and OBSL1. While CUL7 is a ubiquitin ligase and OBSL1 associates with the cytoskeleton, little is known about CCDC8. Binding and structural analyses reveal that the ankyrin repeats of ANKRA2 recognize a PxLPxL motif at the C-terminal region of CCDC8. The N-terminal part interacts with OBSL1 to form a CUL7 ligase complex. These results link ANKRA2 unexpectedly to 3M syndrome and suggest novel regulatory mechanisms for histone deacetylases and RFX7. Copyright © 2015 Elsevier Ltd. All rights reserved.

A novel swarm intelligence algorithm for finding DNA motifs.

PubMed

Lei, Chengwei; Ruan, Jianhua

2009-01-01

Discovering DNA motifs from co-expressed or co-regulated genes is an important step towards deciphering complex gene regulatory networks and understanding gene functions. Despite significant improvement in the last decade, it still remains one of the most challenging problems in computational molecular biology. In this work, we propose a novel motif finding algorithm that finds consensus patterns using a population-based stochastic optimisation technique called Particle Swarm Optimisation (PSO), which has been shown to be effective in optimising difficult multidimensional problems in continuous domains. We propose to use a word dissimilarity graph to remap the neighborhood structure of the solution space of DNA motifs, and propose a modification of the naive PSO algorithm to accommodate discrete variables. In order to improve efficiency, we also propose several strategies for escaping from local optima and for automatically determining the termination criteria. Experimental results on simulated challenge problems show that our method is both more efficient and more accurate than several existing algorithms. Applications to several sets of real promoter sequences also show that our approach is able to detect known transcription factor binding sites, and outperforms two of the most popular existing algorithms.

A peptide affinity column for the identification of integrin alpha IIb-binding proteins.

PubMed

Daxecker, Heide; Raab, Markus; Bernard, Elise; Devocelle, Marc; Treumann, Achim; Moran, Niamh

2008-03-01

To understand the regulation of integrin alpha(IIb)beta(3), a critical platelet adhesion molecule, we have developed a peptide affinity chromatography method using the known integrin regulatory motif, LAMWKVGFFKR. Using standard Fmoc chemistry, this peptide was synthesized onto a Toyopearl AF-Amino-650 M resin on a 6-aminohexanoic acid (Ahx) linker. Peptide density was controlled by acetylation of 83% of the Ahx amino groups. Four recombinant human proteins (CIB1, PP1, ICln and RN181), previously identified as binding to this integrin regulatory motif, were specifically retained by the column containing the integrin peptide but not by a column presenting an irrelevant peptide. Hemoglobin, creatine kinase, bovine serum albumin, fibrinogen and alpha-tubulin failed to bind under the chosen conditions. Immunodetection methods confirmed the binding of endogenous platelet proteins, including CIB1, PP1, ICln RN181, AUP-1 and beta3-integrin, from a detergent-free platelet lysate. Thus, we describe a reproducible method that facilitates the reliable extraction of specific integrin-binding proteins from complex biological matrices. This methodology may enable the sensitive and specific identification of proteins that interact with linear, membrane-proximal peptide motifs such as the integrin regulatory motif LAMWKVGFFKR.

A sequence-specific transcription activator motif and powerful synthetic variants that bind Mediator using a fuzzy protein interface.

PubMed

Warfield, Linda; Tuttle, Lisa M; Pacheco, Derek; Klevit, Rachel E; Hahn, Steven

2014-08-26

Although many transcription activators contact the same set of coactivator complexes, the mechanism and specificity of these interactions have been unclear. For example, do intrinsically disordered transcription activation domains (ADs) use sequence-specific motifs, or do ADs of seemingly different sequence have common properties that encode activation function? We find that the central activation domain (cAD) of the yeast activator Gcn4 functions through a short, conserved sequence-specific motif. Optimizing the residues surrounding this short motif by inserting additional hydrophobic residues creates very powerful ADs that bind the Mediator subunit Gal11/Med15 with high affinity via a "fuzzy" protein interface. In contrast to Gcn4, the activity of these synthetic ADs is not strongly dependent on any one residue of the AD, and this redundancy is similar to that of some natural ADs in which few if any sequence-specific residues have been identified. The additional hydrophobic residues in the synthetic ADs likely allow multiple faces of the AD helix to interact with the Gal11 activator-binding domain, effectively forming a fuzzier interface than that of the wild-type cAD.

Alteration of the carbohydrate-binding specificity of a C-type lectin CEL-I mutant with an EPN carbohydrate-binding motif.

PubMed

Hatakeyama, Tomomitsu; Ishimine, Tomohiro; Baba, Tomohiro; Kimura, Masanari; Unno, Hideaki; Goda, Shuichiro

2013-07-01

CEL-I is a Gal/GalNAc-specific C-type lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-recognition domains (CRDs) with the carbohydrate-recognition motif QPD (Gln-Pro- Asp), which is generally known to exist in galactose-specific C-type CRDs. In the present study, a mutant CEL-I with EPN (Glu-Pro-Asn) motif, which is thought to be responsible for the carbohydrate-recognition of mannose-specific Ctype CRDs, was produced in Escherichia coli, and its effects on the carbohydrate-binding specificity were examined using polyamidoamine dendrimer (PD) conjugated with carbohydrates. Although wild-type CEL-I effectively formed complexes with N-acetylgalactosamine (GalNAc)-PD but not with mannose-PD, the mutant CEL-I showed relatively weak but definite affinity for mannose-PD. These results indicated that the QPD and EPN motifs play a significant role in the carbohydrate-recognition mechanism of CEL-I, especially in the discrimination of galactose and mannose. Additional mutations in the recombinant CEL-I binding site may further increase its specificity for mannose, and should provide insights into designing novel carbohydrate-recognition proteins.

The inhibition of cAMP-dependent protein kinase by full-length hepatitis C virus NS3/4A complex is due to ATP hydrolysis.

PubMed

Aoubala, M; Holt, J; Clegg, R A; Rowlands, D J; Harris, M

2001-07-01

Hepatitis C virus (HCV) is an important cause of chronic liver disease, but the molecular mechanisms of viral pathogenesis remain to be established. The HCV non-structural protein NS3 complexes with NS4A and has three enzymatic activities: a proteinase and a helicase/NTPase. Recently, catalytically inactive NS3 fragments containing an arginine-rich motif have been reported to interact with, and inhibit, the catalytic subunit of cAMP-dependent protein kinase (PKA C-subunit). Here we demonstrate that full-length, catalytically active NS3/4A, purified from recombinant baculovirus-infected insect cells, is also able to inhibit PKA C-subunit in vitro. This inhibition was abrogated by mutation of either the arginine-rich motif or the conserved helicase motif II, both of which also abolished NTPase activity. As PKA C-subunit inhibition was also enhanced by poly(U) (an activator of NS3 NTPase activity), we hypothesized that PKA C-subunit inhibition could be due to NS3/4A-mediated ATP hydrolysis. This was confirmed by experiments in which a constant ATP concentration was maintained by addition of an ATP regeneration system--under these conditions PKA C-subunit inhibition was not observed. Interestingly, the mutations also abrogated the ability of wild-type NS3/4A to inhibit the PKA-regulated transcription factor CREB in transiently transfected hepatoma cells. Our data are thus not consistent with the previously proposed model in which the arginine-rich motif of NS3 was suggested to act as a pseudosubstrate inhibitor of PKA C-subunit. However, in vivo effects of NS3/4A suggest that ATPase activity may play a role in viral pathology in the infected liver.

Apparent loss-of-function mutant GPCRs revealed as constitutively desensitized receptors.

PubMed

Wilbanks, Alyson M; Laporte, Stéphane A; Bohn, Laura M; Barak, Larry S; Caron, Marc G

2002-10-08

The DRY motif is a triplet amino acid sequence (aspartic acid, arginine, and tyrosine) that is highly conserved in G protein-coupled receptors (GPCRs). Recently, we have shown that a molecular determinant for nephrogenic diabetes insipidus, the vasopressin receptor with a substitution at the DRY motif arginine (V2R R137H), is a constitutively desensitized receptor that is unable to couple to G proteins due to its constitutive association with beta-arrestin [Barak, L. S. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 93-98]. Additionally, the mutant receptors are localized in endocytic vesicles, identical to wild-type receptors stimulated with agonist. In this study, we asked whether the constitutively desensitized phenotype observed in the V2R R137H represents a general paradigm that may be extended to other GPCRs. We show that arginine substitutions in the DRY motifs of the alpha(1B) adrenergic receptor (alpha(1B)-AR) and angiotensin II type 1A receptor (AT(1A)R) result in receptors that are uncoupled from G proteins, associated with beta-arrestins, and found localized in endocytic vesicles rather than at the plasma membrane in the absence of agonists. The localization of the alpha(1B)-ARs and AT(1A)Rs with arginine substitutions can be restored to the plasma membrane by either using selective antagonists or preventing the endocytosis of the beta-arrestin-receptor complexes. These results indicate that the arginine residue of the DRY motif is essential for preserving the localization of the inactive receptor complex. Furthermore, constitutive desensitization may underlie some loss-of-function receptor phenotypes and represent an unappreciated mechanism of hormonal resistance.

PEBP1, a RAF kinase inhibitory protein, negatively regulates starvation-induced autophagy by direct interaction with LC3.

PubMed

Noh, Hae Sook; Hah, Young-Sool; Zada, Sahib; Ha, Ji Hye; Sim, Gyujin; Hwang, Jin Seok; Lai, Trang Huyen; Nguyen, Huynh Quoc; Park, Jae-Yong; Kim, Hyun Joon; Byun, June-Ho; Hahm, Jong Ryeal; Kang, Kee Ryeon; Kim, Deok Ryong

2016-11-01

Autophagy plays a critical role in maintaining cell homeostasis in response to various stressors through protein conjugation and activation of lysosome-dependent degradation. MAP1LC3B/LC3B (microtubule- associated protein 1 light chain 3 β) is conjugated with phosphatidylethanolamine (PE) in the membranes and regulates initiation of autophagy through interaction with many autophagy-related proteins possessing an LC3-interacting region (LIR) motif, which is composed of 2 hydrophobic amino acids (tryptophan and leucine) separated by 2 non-conserved amino acids (WXXL). In this study, we identified a new putative LIR motif in PEBP1/RKIP (phosphatidylethanolamine binding protein 1) that was originally isolated as a PE-binding protein and also a cellular inhibitor of MAPK/ERK signaling. PEBP1 was specifically bound to PE-unconjugated LC3 in cells, and mutation (WXXL mutated to AXXA) of this LIR motif disrupted its interaction with LC3 proteins. Interestingly, overexpression of PEBP1 significantly inhibited starvation-induced autophagy by activating the AKT and MTORC1 (mechanistic target of rapamycin [serine/threonine kinase] complex 1) signaling pathway and consequently suppressing the ULK1 (unc-51 like autophagy activating kinase 1) activity. In contrast, ablation of PEBP1 expression dramatically promoted the autophagic process under starvation conditions. Furthermore, PEBP1 lacking the LIR motif highly stimulated starvation-induced autophagy through the AKT-MTORC1-dependent pathway. PEBP1 phosphorylation at Ser153 caused dissociation of LC3 from the PEBP1-LC3 complex for autophagy induction. PEBP1-dependent suppression of autophagy was not associated with the MAPK pathway. These findings suggest that PEBP1 can act as a negative mediator in autophagy through stimulation of the AKT-MTORC1 pathway and direct interaction with LC3.

Toxic and nontoxic components of botulinum neurotoxin complex are evolved from a common ancestral zinc protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Inui, Ken; Japan Society for the Promotion of Science, 1-8 Chiyoda-ku, Tokyo 102-8472; Sagane, Yoshimasa

2012-03-16

Highlights: Black-Right-Pointing-Pointer BoNT and NTNHA proteins share a similar protein architecture. Black-Right-Pointing-Pointer NTNHA and BoNT were both identified as zinc-binding proteins. Black-Right-Pointing-Pointer NTNHA does not have a classical HEXXH zinc-coordinating motif similar to that found in all serotypes of BoNT. Black-Right-Pointing-Pointer Homology modeling implied probable key residues involved in zinc coordination. -- Abstract: Zinc atoms play an essential role in a number of enzymes. Botulinum neurotoxin (BoNT), the most potent toxin known in nature, is a zinc-dependent endopeptidase. Here we identify the nontoxic nonhemagglutinin (NTNHA), one of the BoNT-complex constituents, as a zinc-binding protein, along with BoNT. A protein structuremore » classification database search indicated that BoNT and NTNHA share a similar domain architecture, comprising a zinc-dependent metalloproteinase-like, BoNT coiled-coil motif and concanavalin A-like domains. Inductively coupled plasma-mass spectrometry analysis demonstrated that every single NTNHA molecule contains a single zinc atom. This is the first demonstration of a zinc atom in this protein, as far as we know. However, the NTNHA molecule does not possess any known zinc-coordinating motif, whereas all BoNT serotypes possess the classical HEXXH motif. Homology modeling of the NTNHA structure implied that a consensus K-C-L-I-K-X{sub 35}-D sequence common among all NTNHA serotype molecules appears to coordinate a single zinc atom. These findings lead us to propose that NTNHA and BoNT may have evolved distinct functional specializations following their branching out from a common ancestral zinc protein.« less

Structural and Histone Binding Ability Characterizations of Human PWWP Domains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Hong; Zeng, Hong; Lam, Robert

2013-09-25

The PWWP domain was first identified as a structural motif of 100-130 amino acids in the WHSC1 protein and predicted to be a protein-protein interaction domain. It belongs to the Tudor domain 'Royal Family', which consists of Tudor, chromodomain, MBT and PWWP domains. While Tudor, chromodomain and MBT domains have long been known to bind methylated histones, PWWP was shown to exhibit histone binding ability only until recently. The PWWP domain has been shown to be a DNA binding domain, but sequence analysis and previous structural studies show that the PWWP domain exhibits significant similarity to other 'Royal Family' members,more » implying that the PWWP domain has the potential to bind histones. In order to further explore the function of the PWWP domain, we used the protein family approach to determine the crystal structures of the PWWP domains from seven different human proteins. Our fluorescence polarization binding studies show that PWWP domains have weak histone binding ability, which is also confirmed by our NMR titration experiments. Furthermore, we determined the crystal structures of the BRPF1 PWWP domain in complex with H3K36me3, and HDGF2 PWWP domain in complex with H3K79me3 and H4K20me3. PWWP proteins constitute a new family of methyl lysine histone binders. The PWWP domain consists of three motifs: a canonical {beta}-barrel core, an insertion motif between the second and third {beta}-strands and a C-terminal {alpha}-helix bundle. Both the canonical {beta}-barrel core and the insertion motif are directly involved in histone binding. The PWWP domain has been previously shown to be a DNA binding domain. Therefore, the PWWP domain exhibits dual functions: binding both DNA and methyllysine histones.« less

Disabling Cas9 by an anti-CRISPR DNA mimic.

PubMed

Shin, Jiyung; Jiang, Fuguo; Liu, Jun-Jie; Bray, Nicolas L; Rauch, Benjamin J; Baik, Seung Hyun; Nogales, Eva; Bondy-Denomy, Joseph; Corn, Jacob E; Doudna, Jennifer A

2017-07-01

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. However, the mechanism of CRISPR-Cas9 inhibition is not known, and the potential applications for Cas9 inhibitor proteins in mammalian cells have not been fully established. We show that the anti-CRISPR protein AcrIIA4 binds only to assembled Cas9-single-guide RNA (sgRNA) complexes and not to Cas9 protein alone. A 3.9 Å resolution cryo-electron microscopy structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with a 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif. Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on preformed Cas9-sgRNA-DNA complexes. Timed delivery of AcrIIA4 into human cells as either protein or expression plasmid allows on-target Cas9-mediated gene editing while reducing off-target edits. These results provide a mechanistic understanding of AcrIIA4 function and demonstrate that inhibitors can modulate the extent and outcomes of Cas9-mediated gene editing.

The cellular transcription factor CREB corresponds to activating transcription factor 47 (ATF-47) and forms complexes with a group of polypeptides related to ATF-43.

PubMed

Hurst, H C; Masson, N; Jones, N C; Lee, K A

1990-12-01

Promoter elements containing the sequence motif CGTCA are important for a variety of inducible responses at the transcriptional level. Multiple cellular factors specifically bind to these elements and are encoded by a multigene family. Among these factors, polypeptides termed activating transcription factor 43 (ATF-43) and ATF-47 have been purified from HeLa cells and a factor referred to as cyclic AMP response element-binding protein (CREB) has been isolated from PC12 cells and rat brain. We demonstrated that CREB and ATF-47 are identical and that CREB and ATF-43 form protein-protein complexes. We also found that the cis requirements for stable DNA binding by ATF-43 and CREB are different. Using antibodies to ATF-43 we have identified a group of polypeptides (ATF-43) in the size range from 40 to 43 kDa. ATF-43 polypeptides are related by their reactivity with anti-ATF-43, DNA-binding specificity, complex formation with CREB, heat stability, and phosphorylation by protein kinase A. Certain cell types vary in their ATF-43 complement, suggesting that CREB activity is modulated in a cell-type-specific manner through interaction with ATF-43. ATF-43 polypeptides do not appear simply to correspond to the gene products of the ATF multigene family, suggesting that the size of the ATF family at the protein level is even larger than predicted from cDNA-cloning studies.

Identification of an Amino Acid Domain Encoded by the Capsid Protein Gene of Porcine Circovirus Type 2 that Modulates Viral Protein Distribution During Replication

USDA-ARS?s Scientific Manuscript database

Previous work showed that distinct amino acid motifs are encoded by the Rep, Cap and ORF3 genes of two subgroups of porcine circoviruses (PCV), PCV2a and PCV2b. At a specific location of the gene, a certain amino acid residue or sequence is preferred. Specifically, two amino acid domains located in ...

A viral, transporter associated with antigen processing (TAP)-independent, high affinity ligand with alternative interactions endogenously presented by the nonclassical human leukocyte antigen E class I molecule.

PubMed

Lorente, Elena; Infantes, Susana; Abia, David; Barnea, Eilon; Beer, Ilan; García, Ruth; Lasala, Fátima; Jiménez, Mercedes; Mir, Carmen; Morreale, Antonio; Admon, Arie; López, Daniel

2012-10-12

The transporter associated with antigen processing (TAP) enables the flow of viral peptides generated in the cytosol by the proteasome and other proteases to the endoplasmic reticulum, where they complex with nascent human leukocyte antigen (HLA) class I. Later, these peptide-HLA class I complexes can be recognized by CD8(+) lymphocytes. Cancerous cells and infected cells in which TAP is blocked, as well as individuals with unusable TAP complexes, are able to present peptides on HLA class I by generating them through TAP-independent processing pathways. Here, we identify a physiologically processed HLA-E ligand derived from the D8L protein in TAP-deficient vaccinia virus-infected cells. This natural high affinity HLA-E class I ligand uses alternative interactions to the anchor motifs previously described to be presented on nonclassical HLA class I molecules. This octameric peptide was also presented on HLA-Cw1 with similar binding affinity on both classical and nonclassical class I molecules. In addition, this viral peptide inhibits HLA-E-mediated cytolysis by natural killer cells. Comparison between the amino acid sequences of the presenting HLA-E and HLA-Cw1 alleles revealed a shared structural motif in both HLA class molecules, which could be related to their observed similar cross-reactivity affinities. This motif consists of several residues located on the floor of the peptide-binding site. These data expand the role of HLA-E as an antigen-presenting molecule.

ATM activation and its recruitment to damaged DNA require binding to the C terminus of Nbs1.

PubMed

You, Zhongsheng; Chahwan, Charly; Bailis, Julie; Hunter, Tony; Russell, Paul

2005-07-01

ATM has a central role in controlling the cellular responses to DNA damage. It and other phosphoinositide 3-kinase-related kinases (PIKKs) have giant helical HEAT repeat domains in their amino-terminal regions. The functions of these domains in PIKKs are not well understood. ATM activation in response to DNA damage appears to be regulated by the Mre11-Rad50-Nbs1 (MRN) complex, although the exact functional relationship between the MRN complex and ATM is uncertain. Here we show that two pairs of HEAT repeats in fission yeast ATM (Tel1) interact with an FXF/Y motif at the C terminus of Nbs1. This interaction resembles nucleoporin FXFG motif binding to HEAT repeats in importin-beta. Budding yeast Nbs1 (Xrs2) appears to have two FXF/Y motifs that interact with Tel1 (ATM). In Xenopus egg extracts, the C terminus of Nbs1 recruits ATM to damaged DNA, where it is subsequently autophosphorylated. This interaction is essential for ATM activation. A C-terminal 147-amino-acid fragment of Nbs1 that has the Mre11- and ATM-binding domains can restore ATM activation in an Nbs1-depleted extract. We conclude that an interaction between specific HEAT repeats in ATM and the C-terminal FXF/Y domain of Nbs1 is essential for ATM activation. We propose that conformational changes in the MRN complex that occur upon binding to damaged DNA are transmitted through the FXF/Y-HEAT interface to activate ATM. This interaction also retains active ATM at sites of DNA damage.