Discovery of the porcine NGN3 gene and testing its endocrine function in the pig

USDA-ARS?s Scientific Manuscript database

Neurogenin 3 (NGN3) is a member of the basic helix-loop-helix transcription factor family. NGN3 is both necessary and sufficient to drive endocrine differentiation in the developing pancreas in mouse and humans. Until now, the sequence for NGN3 eluded discovery despite completion of the pig genome a...

Selection of a platinum-binding sequence in a loop of a four-helix bundle protein.

PubMed

Yagi, Sota; Akanuma, Satoshi; Kaji, Asumi; Niiro, Hiroya; Akiyama, Hayato; Uchida, Tatsuya; Yamagishi, Akihiko

2018-02-01

Protein-metal hybrids are functional materials with various industrial applications. For example, a redox enzyme immobilized on a platinum electrode is a key component of some biofuel cells and biosensors. To create these hybrid materials, protein molecules are bound to metal surfaces. Here, we report the selection of a novel platinum-binding sequence in a loop of a four-helix bundle protein, the Lac repressor four-helix protein (LARFH), an artificial protein in which four identical α-helices are connected via three identical loops. We created a genetic library in which the Ser-Gly-Gln-Gly-Gly-Ser sequence within the first inter-helical loop of LARFH was semi-randomly mutated. The library was then subjected to selection for platinum-binding affinity by using the T7 phage display method. The majority of the selected variants contained the Tyr-Lys-Arg-Gly-Tyr-Lys (YKRGYK) sequence in their randomized segment. We characterized the platinum-binding properties of mutant LARFH by using quartz crystal microbalance analysis. Mutant LARFH seemed to interact with platinum through its loop containing the YKRGYK sequence, as judged by the estimated exclusive area occupied by a single molecule. Furthermore, a 10-residue peptide containing the YKRGYK sequence bound to platinum with reasonably high affinity and basic side chains in the peptide were crucial in mediating this interaction. In conclusion, we have identified an amino acid sequence, YKRGYK, in the loop of a helix-loop-helix motif that shows high platinum-binding affinity. This sequence could be grafted into loops of other polypeptides as an approach to immobilize proteins on platinum electrodes for use as biosensors among other applications. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Repressed expression of a gene for a basic helix-loop-helix protein causes a white flower phenotype in carnation

PubMed Central

Totsuka, Akane; Okamoto, Emi; Miyahara, Taira; Kouno, Takanobu; Cano, Emilio A.; Sasaki, Nobuhiro; Watanabe, Aiko; Tasaki, Keisuke; Nishihara, Masahiro; Ozeki, Yoshihiro

2018-01-01

In a previous study, two genes responsible for white flower phenotypes in carnation were identified. These genes encoded enzymes involved in anthocyanin synthesis, namely, flavanone 3-hydroxylase (F3H) and dihydroflavonol 4-reductase (DFR), and showed reduced expression in the white flower phenotypes. Here, we identify another candidate gene for white phenotype in carnation flowers using an RNA-seq analysis followed by RT-PCR. This candidate gene encodes a transcriptional regulatory factor of the basic helix-loop-helix (bHLH) type. In the cultivar examined here, both F3H and DFR genes produced active enzyme proteins; however, expression of DFR and of genes for enzymes involved in the downstream anthocyanin synthetic pathway from DFR was repressed in the absence of bHLH expression. Occasionally, flowers of the white flowered cultivar used here have red speckles and stripes on the white petals. We found that expression of bHLH occurred in these red petal segments and induced expression of DFR and the following downstream enzymes. Our results indicate that a member of the bHLH superfamily is another gene involved in anthocyanin synthesis in addition to structural genes encoding enzymes. PMID:29681756

The basic helix-loop-helix transcription factor Nex-1/Math-2 promotes neuronal survival of PC12 cells by modulating the dynamic expression of anti-apoptotic and cell cycle regulators

PubMed Central

Uittenbogaard, Martine; Chiaramello, Anne

2006-01-01

The basic helix-loop-helix transcription factor Nex1/Math-2 belongs to the NeuroD subfamily, which plays a critical role during neuronal differentiation and maintenance of the differentiated state. Previously, we demonstrated that Nex1 is a key regulatory component of the nerve growth factor (NGF) pathway. Further supporting this hypothesis, this study shows that Nex1 has survival-inducing properties similar to NGF, as Nex1-overexpressing PC12 cells survive in the absence of trophic factors. We dissected the molecular mechanism by which Nex1 confers neuroprotection upon serum removal and found that constitutive expression of Nex1 maintained the expression of specific G1 phase cyclin-dependent kinase inhibitors and concomitantly induced a dynamic expression profile of key anti-apoptotic regulators. This study provides the first evidence of the underlying mechanism by which a member of the NeuroD-subfamily promotes an active anti-apoptotic program essential to the survival of neurons. Our results suggest that the survival program may be viewed as an integral component of the intrinsic programming of the differ entiated state. PMID:15659228

Genome-wide identification and analysis of the chicken basic helix-loop-helix factors.

PubMed

Liu, Wu-Yi; Zhao, Chun-Jiang

2010-01-01

Members of the basic helix-loop-helix (bHLH) family of transcription factors play important roles in a wide range of developmental processes. In this study, we conducted a genome-wide survey using the chicken (Gallus gallus) genomic database, and identified 104 bHLH sequences belonging to 42 gene families in an effort to characterize the chicken bHLH transcription factor family. Phylogenetic analyses revealed that chicken has 50, 21, 15, 4, 8, and 3 bHLH members in groups A, B, C, D, E, and F, respectively, while three members belonging to none of these groups were classified as ''orphans". A comparison between chicken and human bHLH repertoires suggested that both organisms have a number of lineage-specific bHLH members in the proteomes. Chromosome distribution patterns and phylogenetic analyses strongly suggest that the bHLH members should have arisen through gene duplication at an early date. Gene Ontology (GO) enrichment statistics showed 51 top GO annotations of biological processes counted in the frequency. The present study deepens our understanding of the chicken bHLH transcription factor family and provides much useful information for further studies using chicken as a model system.

EsxB, a secreted protein from Bacillus anthracis forms two distinct helical bundles

DOE PAGES

Fan, Yao; Tan, Kemin; Chhor, Gekleng; ...

2015-07-03

The EsxB protein from Bacillus anthracis belongs to the WXG100 family, a group of proteins secreted by a specialized secretion system. We have determined the crystal structures of recombinant EsxB and discovered that the small protein (~10 kDa), comprised of a helix-loop-helix (HLH) hairpin, is capable of associating into two different helical bundles. The two basic quaternary assemblies of EsxB are an antiparallel (AP) dimer and a rarely observed bisecting U (BU) dimer. This structural duality of EsxB is believed to originate from the heptad repeat sequence diversity of the first helix of its HLH hairpin, which allows for twomore » alternative helix packing. The flexibility of EsxB and the ability to form alternative helical bundles underscore the possibility that this protein can serve as an adaptor in secretion and can form hetero-oligomeric helix bundle(s) with other secreted members of the WXG100 family, such as EsxW. The highly conserved WXG motif is located within the loop of the HLH hairpin and is mostly buried within the helix bundle suggesting that its role is mainly structural. The exact functions of the motif, including a proposed role as a secretion signal, remain unknown.« less

A genome-wide identification of basic helix-loop-helix motifs in Pediculus humanus corporis (Phthiraptera: Pediculidae).

PubMed

Wang, Xu-Hua; Wang, Yong; Zhang, De-Bao; Liu, A-Ke; Yao, Qin; Chen, Ke-Ping

2014-01-01

Basic helix-loop-helix (bHLH) proteins comprise a large superfamily of transcription factors, which are involved in the regulation of various developmental processes. bHLH family members are widely distributed in various eukaryotes including yeast, fruit fly, zebrafish, mouse, and human. In this study, we identified 55 bHLH motifs encoded in genome sequence of the human body louse, Pediculus humanus corporis (Phthiraptera: Pediculidae). Phylogenetic analyses of the identified P. humanus corporis bHLH (PhcbHLH) motifs revealed that there are 23, 11, 9, 1, 10, and 1 member(s) in groups A, B, C, D, E, and F, respectively. Examination to GenBank annotations of the 55 PhcbHLH members indicated that 29 PhcbHLH proteins were annotated in consistence with our analytical result, 8 were annotated different with our analytical result, 12 were merely annotated as hypothetical protein, and the rest 6 were not deposited in GenBank. A comparison on insect bHLH gene composition revealed that human body louse possibly has more hairy and E(spl) genes than other insect species. Because hairy and E(spl) genes have been found to negatively regulate the differentiation of insect preneural cells, it is suggested that the existence of additional hairy and E(spl) genes in human body louse is probably the consequence of its long period adaptation to the relatively dark and stable environment. These data provide good references for further studies on regulatory functions of bHLH proteins in the growth and development of human body louse. © The Author 2014. Published by Oxford University Press on behalf of the Entomological Society of America.

Tracheophytes Contain Conserved Orthologs of a Basic Helix-Loop-Helix Transcription Factor That Modulate ROOT HAIR SPECIFIC Genes[OPEN

PubMed Central

Cho, Hyun-Min

2017-01-01

ROOT HAIR SPECIFIC (RHS) genes, which contain the root hair-specific cis-element (RHE) in their regulatory regions, function in root hair morphogenesis. Here, we demonstrate that an Arabidopsis thaliana basic helix-loop-helix transcription factor, ROOT HAIR DEFECTVE SIX-LIKE4 (RSL4), directly binds to the RHE in vitro and in vivo, upregulates RHS genes, and stimulates root hair formation in Arabidopsis. Orthologs of RSL4 from a eudicot (poplar [Populus trichocarpa]), a monocot (rice [Oryza sativa]), and a lycophyte (Selaginella moellendorffii) each restored root hair growth in the Arabidopsis rsl4 mutant. In addition, the rice and S. moellendorffii RSL4 orthologs bound to the RHE in in vitro and in vivo assays. The RSL4 orthologous genes contain RHEs in their promoter regions, and RSL4 was able to bind to its own RHEs in vivo and amplify its own expression. This process likely provides a positive feedback loop for sustainable root hair growth. When RSL4 and its orthologs were expressed in cells in non-root-hair positions, they induced ectopic root hair growth, indicating that these genes are sufficient to specify root hair formation. Our results suggest that RSL4 mediates root hair formation by regulating RHS genes and that this mechanism is conserved throughout the tracheophyte (vascular plant) lineage. PMID:28087829

Brassinosteroid-Induced Transcriptional Repression and Dephosphorylation-Dependent Protein Degradation Negatively Regulate BIN2-Interacting AIF2 (a BR Signaling-Negative Regulator) bHLH Transcription Factor.

PubMed

Kim, Yoon; Song, Ji-Hye; Park, Seon-U; Jeong, You-Seung; Kim, Soo-Hwan

2017-02-01

Brassinosteroids (BRs) are plant polyhydroxy-steroids that play important roles in plant growth and development via extensive signal integration through direct interactions between regulatory components of different signaling pathways. Recent studies have shown that diverse helix-loop-helix/basic helix-loop-helix (HLH/bHLH) family proteins are actively involved in control of BR signaling pathways and interact with other signaling pathways. In this study, we show that ATBS1-INTERACTING FACTOR 2 (AIF2), a nuclear-localized atypical bHLH transcription factor, specifically interacts with BRASSINOSTEROID-INSENSITIVE 2 (BIN2) among other BR signaling molecules. Overexpression of AIF2 down-regulated transcript expression of growth-promoting genes, thus resulting in retardation of growth. AIF2 renders plants hyposensitive to BR-induced root growth inhibition, but shows little effects on BR-promoted hypocotyl elongation. Notably, AIF2 was dephosphorylated by BR, and the dephosphorylated AIF2 was subject to proteasome-mediated degradation. AIF2 degradation was greatly induced by BR and ABA, but relatively slightly by other hormones such as auxin, gibberellin, cytokinin and ethylene. Moreover, AIF2 transcription was significantly suppressed by a BRI1/BZR1-mediated BR signaling pathway through a direct binding of BRASSINAZOLE RESISTANT 1 (BZR1) to the BR response element (BRRE) region of the AIF2 promoter. In conclusion, our study suggests that BIN2-driven AIF2 phosphorylation could augment the BIN2/AIF2-mediated negative circuit of BR signaling pathways, and the BR-induced transcriptional repression and protein degradation negatively regulate AIF2 transcription factor, reinforcing the BZR1/BES1-mediated positive BR signaling pathway. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Genetic modification of human B-cell development: B-cell development is inhibited by the dominant negative helix loop helix factor Id3.

PubMed

Jaleco, A C; Stegmann, A P; Heemskerk, M H; Couwenberg, F; Bakker, A Q; Weijer, K; Spits, H

1999-10-15

Transgenic and gene targeted mice have contributed greatly to our understanding of the mechanisms underlying B-cell development. We describe here a model system that allows us to apply molecular genetic techniques to the analysis of human B-cell development. We constructed a retroviral vector with a multiple cloning site connected to a gene encoding green fluorescent protein by an internal ribosomal entry site. Human CD34(+)CD38(-) fetal liver cells, cultured overnight in a combination of stem cell factor and interleukin-7 (IL-7), could be transduced with 30% efficiency. We ligated the gene encoding the dominant negative helix loop helix (HLH) factor Id3 that inhibits many enhancing basic HLH transcription factors into this vector. CD34(+)CD38(-) FL cells were transduced with Id3-IRES-GFP and cultured with the murine stromal cell line S17. In addition, we cultured the transduced cells in a reaggregate culture system with an SV-transformed human fibroblast cell line (SV19). It was observed that overexpression of Id3 inhibited development of B cells in both culture systems. B-cell development was arrested at a stage before expression of the IL-7Ralpha. The development of CD34(+)CD38(-) cells into CD14(+) myeloid cells in the S17 system was not inhibited by overexpression of Id3. Moreover, Id3(+) cells, although inhibited in their B-cell development, were still able to develop into natural killer (NK) cells when cultured in a combination of Flt-3L, IL-7, and IL-15. These findings confirm the essential role of bHLH factors in B-cell development and demonstrate the feasibility of retrovirus-mediated gene transfer as a tool to genetically modify human B-cell development.

Identification of a basic helix-loop-helix-type transcription regulator gene in Aspergillus oryzae by systematically deleting large chromosomal segments.

PubMed

Jin, Feng Jie; Takahashi, Tadashi; Machida, Masayuki; Koyama, Yasuji

2009-09-01

We previously developed two methods (loop-out and replacement-type recombination) for generating large-scale chromosomal deletions that can be applied to more effective chromosomal engineering in Aspergillus oryzae. In this study, the replacement-type method is used to systematically delete large chromosomal DNA segments to identify essential and nonessential regions in chromosome 7 (2.93 Mb), which is the smallest A. oryzae chromosome and contains a large number of nonsyntenic blocks. We constructed 12 mutants harboring deletions that spanned 16- to 150-kb segments of chromosome 7 and scored phenotypic changes in the resulting mutants. Among the deletion mutants, strains designated Delta5 and Delta7 displayed clear phenotypic changes involving growth and conidiation. In particular, the Delta5 mutant exhibited vigorous growth and conidiation, potentially beneficial characteristics for certain industrial applications. Further deletion analysis allowed identification of the AO090011000215 gene as the gene responsible for the Delta5 mutant phenotype. The AO090011000215 gene was predicted to encode a helix-loop-helix binding protein belonging to the bHLH family of transcription factors. These results illustrate the potential of the approach for identifying novel functional genes.

Genome-wide identification and analysis of basic helix-loop-helix domains in dog, Canis lupus familiaris.

PubMed

Wang, Xu-Hua; Wang, Yong; Liu, A-Ke; Liu, Xiao-Ting; Zhou, Yang; Yao, Qin; Chen, Ke-Ping

2015-04-01

The basic helix-loop-helix (bHLH) domain is a highly conserved amino acid motif that defines a group of DNA-binding transcription factors. bHLH proteins play essential regulatory roles in a variety of biological processes in animal, plant, and fungus. The domestic dog, Canis lupus familiaris, is a good model organism for genetic, physiological, and behavioral studies. In this study, we identified 115 putative bHLH genes in the dog genome. Based on a phylogenetic analysis, 51, 26, 14, 4, 12, and 4 dog bHLH genes were assigned to six separate groups (A-F); four bHLH genes were categorized as ''orphans''. Within-group evolutionary relationships inferred from the phylogenetic analysis were consistent with positional conservation, other conserved domains flanking the bHLH motif, and highly conserved intron/exon patterns in other vertebrates. Our analytical results confirmed the GenBank annotations of 89 dog bHLH proteins and provided information that could be used to update the annotations of the remaining 26 dog bHLH proteins. These data will provide good references for further studies on the structures and regulatory functions of bHLH proteins in the growth and development of dogs, which may help in understanding the mechanisms that underlie the physical and behavioral differences between dogs and wolves.

Identification and expression analysis of the apple (Malus × domestica) basic helix-loop-helix transcription factor family.

PubMed

Yang, Jinhua; Gao, Min; Huang, Li; Wang, Yaqiong; van Nocker, Steve; Wan, Ran; Guo, Chunlei; Wang, Xiping; Gao, Hua

2017-02-09

Basic helix-loop-helix (bHLH) proteins, which are characterized by a conserved bHLH domain, comprise one of the largest families of transcription factors in both plants and animals, and have been shown to have a wide range of biological functions. However, there have been very few studies of bHLH proteins from perennial tree species. We describe here the identification and characterization of 175 bHLH transcription factors from apple (Malus × domestica). Phylogenetic analysis of apple bHLH (MdbHLH) genes and their Arabidopsis thaliana (Arabidopsis) orthologs indicated that they can be classified into 23 subgroups. Moreover, integrated synteny analysis suggested that the large-scale expansion of the bHLH transcription factor family occurred before the divergence of apple and Arabidopsis. An analysis of the exon/intron structure and protein domains was conducted to suggest their functional roles. Finally, we observed that MdbHLH subgroup III and IV genes displayed diverse expression profiles in various organs, as well as in response to abiotic stresses and various hormone treatments. Taken together, these data provide new information regarding the composition and diversity of the apple bHLH transcription factor family that will provide a platform for future targeted functional characterization.

An exploration of alternative visualisations of the basic helix-loop-helix protein interaction network

PubMed Central

Holden, Brian J; Pinney, John W; Lovell, Simon C; Amoutzias, Grigoris D; Robertson, David L

2007-01-01

Background Alternative representations of biochemical networks emphasise different aspects of the data and contribute to the understanding of complex biological systems. In this study we present a variety of automated methods for visualisation of a protein-protein interaction network, using the basic helix-loop-helix (bHLH) family of transcription factors as an example. Results Network representations that arrange nodes (proteins) according to either continuous or discrete information are investigated, revealing the existence of protein sub-families and the retention of interactions following gene duplication events. Methods of network visualisation in conjunction with a phylogenetic tree are presented, highlighting the evolutionary relationships between proteins, and clarifying the context of network hubs and interaction clusters. Finally, an optimisation technique is used to create a three-dimensional layout of the phylogenetic tree upon which the protein-protein interactions may be projected. Conclusion We show that by incorporating secondary genomic, functional or phylogenetic information into network visualisation, it is possible to move beyond simple layout algorithms based on network topology towards more biologically meaningful representations. These new visualisations can give structure to complex networks and will greatly help in interpreting their evolutionary origins and functional implications. Three open source software packages (InterView, TVi and OptiMage) implementing our methods are available. PMID:17683601

Origin and Diversification of Basic-Helix-Loop-Helix Proteins in Plants

PubMed Central

Pires, Nuno; Dolan, Liam

2010-01-01

Basic helix-loop-helix (bHLH) proteins are a class of transcription factors found throughout eukaryotic organisms. Classification of the complete sets of bHLH proteins in the sequenced genomes of Arabidopsis thaliana and Oryza sativa (rice) has defined the diversity of these proteins among flowering plants. However, the evolutionary relationships of different plant bHLH groups and the diversity of bHLH proteins in more ancestral groups of plants are currently unknown. In this study, we use whole-genome sequences from nine species of land plants and algae to define the relationships between these proteins in plants. We show that few (less than 5) bHLH proteins are encoded in the genomes of chlorophytes and red algae. In contrast, many bHLH proteins (100–170) are encoded in the genomes of land plants (embryophytes). Phylogenetic analyses suggest that plant bHLH proteins are monophyletic and constitute 26 subfamilies. Twenty of these subfamilies existed in the common ancestors of extant mosses and vascular plants, whereas six further subfamilies evolved among the vascular plants. In addition to the conserved bHLH domains, most subfamilies are characterized by the presence of highly conserved short amino acid motifs. We conclude that much of the diversity of plant bHLH proteins was established in early land plants, over 440 million years ago. PMID:19942615

Highly conserved sequences mediate the dynamic interplay of basic helix-loop-helix proteins regulating retinogenesis.

PubMed

Hernandez, Julio; Matter-Sadzinski, Lidia; Skowronska-Krawczyk, Dorota; Chiodini, Florence; Alliod, Christine; Ballivet, Marc; Matter, Jean-Marc

2007-12-28

The atonal homolog 5 (ATH5) protein is central to the transcriptional network regulating the specification of retinal ganglion cells, and its expression comes under the spatiotemporal control of several basic helix-loop-helix (bHLH) proteins in the course of retina development. Monitoring the in vivo occupancy of the ATH5 promoter by the ATH5, Ngn2, and NeuroM proteins and analyzing the DNA motifs they bind, we show that three evolutionarily conserved E-boxes are required for the bHLH proteins to control the different phases of ATH5 expression. E-box 4 mediates the activity of Ngn2, ATH5, and NeuroM along the pathway leading to the conversion of progenitors into newborn neurons. E-box 1, by mediating the antagonistic effects of Ngn2 and HES1 in proliferating progenitors, controls the expansion of the ATH5 expression domain in early retina. E-box 2 is required for the positive feedback by ATH5 that underlies the up-regulation of ATH5 expression when progenitors are going through their last cell cycle. The combinatorial nature of the regulation of the ATH5 promoter suggests that the bHLH proteins involved have no assigned E-boxes but use a common set at which they either cooperate or compete to finely tune ATH5 expression as development proceeds.

Transcription regulation of the Saccharomyces cerevisiae PIS1 gene by inositol and the pleiotropic regulator, Ume6p.

PubMed

Jani, Niketa M; Lopes, John M

2008-12-01

In Saccharomyces cerevisiae, transcription of most of the phospholipid biosynthetic genes (e.g. INO1, CHO1, CHO2 and OPI3) is repressed by growth in the presence of inositol and choline and derepressed in their absence. This regulation requires the Ino2p and Ino4p activators and the Opi1p repressor. The PIS1 structural gene is required for the synthesis of the essential lipid phosphatidylinositol. Previous reports show that PIS1 expression is uncoupled from inositol/choline regulation, but is regulated by carbon source, hypoxia and zinc. However, in this study we found that the expression of PIS1 is induced twofold by inositol. This regulation did not require Ino2p and Ino4p, although Ino4p was required for full expression. Ino4p is a basic helix-loop-helix protein that requires a binding partner. Curiously, none of the other basic helix-loop-helix proteins affected PIS1 expression. Inositol induction did require another general regulator of phospholipid biosynthesis, Ume6p. Ume6p was found to be a positive regulator of PIS1 gene expression. Ume6p, and several associated factors, were required for inositol-mediated induction and chromatin immunoprecipitation analysis showed that Ume6p directly regulates PIS1 expression. Thus, we demonstrate novel regulation of the PIS1 gene by Ume6p.

The Rice Basic Helix-Loop-Helix Transcription Factor TDR INTERACTING PROTEIN2 Is a Central Switch in Early Anther Development[C][W

PubMed Central

Fu, Zhenzhen; Yu, Jing; Cheng, Xiaowei; Zong, Xu; Xu, Jie; Chen, Mingjiao; Li, Zongyun; Zhang, Dabing; Liang, Wanqi

2014-01-01

In male reproductive development in plants, meristemoid precursor cells possessing transient, stem cell–like features undergo cell divisions and differentiation to produce the anther, the male reproductive organ. The anther contains centrally positioned microsporocytes surrounded by four distinct layers of wall: the epidermis, endothecium, middle layer, and tapetum. Here, we report that the rice (Oryza sativa) basic helix-loop-helix (bHLH) protein TDR INTERACTING PROTEIN2 (TIP2) functions as a crucial switch in the meristemoid transition and differentiation during early anther development. The tip2 mutants display undifferentiated inner three anther wall layers and abort tapetal programmed cell death, causing complete male sterility. TIP2 has two paralogs in rice, TDR and EAT1, which are key regulators of tapetal programmed cell death. We revealed that TIP2 acts upstream of TDR and EAT1 and directly regulates the expression of TDR and EAT1. In addition, TIP2 can interact with TDR, indicating a role of TIP2 in later anther development. Our findings suggest that the bHLH proteins TIP2, TDR, and EAT1 play a central role in regulating differentiation, morphogenesis, and degradation of anther somatic cell layers, highlighting the role of paralogous bHLH proteins in regulating distinct steps of plant cell–type determination. PMID:24755456

The Basic Helix-Loop-Helix Transcription Factor MYC2 Directly Represses PLETHORA Expression during Jasmonate-Mediated Modulation of the Root Stem Cell Niche in Arabidopsis[W][OA

PubMed Central

Chen, Qian; Sun, Jiaqiang; Zhai, Qingzhe; Zhou, Wenkun; Qi, Linlin; Xu, Li; Wang, Bao; Chen, Rong; Jiang, Hongling; Qi, Jing; Li, Xugang; Palme, Klaus; Li, Chuanyou

2011-01-01

The root stem cell niche, which in the Arabidopsis thaliana root meristem is an area of four mitotically inactive quiescent cells (QCs) and the surrounding mitotically active stem cells, is critical for root development and growth. We report here that during jasmonate-induced inhibition of primary root growth, jasmonate reduces root meristem activity and leads to irregular QC division and columella stem cell differentiation. Consistently, jasmonate reduces the expression levels of the AP2-domain transcription factors PLETHORA1 (PLT1) and PLT2, which form a developmentally instructive protein gradient and mediate auxin-induced regulation of stem cell niche maintenance. Not surprisingly, the effects of jasmonate on root stem cell niche maintenance and PLT expression require the functioning of MYC2/JASMONATE INSENSITIVE1, a basic helix-loop-helix transcription factor that involves versatile aspects of jasmonate-regulated gene expression. Gel shift and chromatin immunoprecipitation experiments reveal that MYC2 directly binds the promoters of PLT1 and PLT2 and represses their expression. We propose that MYC2-mediated repression of PLT expression integrates jasmonate action into the auxin pathway in regulating root meristem activity and stem cell niche maintenance. This study illustrates a molecular framework for jasmonate-induced inhibition of root growth through interaction with the growth regulator auxin. PMID:21954460

Genomic organization and chromosomal localization of the gene TCF15 encoding the early mesodermal basic helix-loop-helix factor bHLH-EC2

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

Hidai, H.; Quertermous, E.E.; Quertermous, T.

1995-12-10

bHLH-EC2 is a recently characterized member of a growing family of basic helix-loop-helix transcription factors. This family includes bHLH factors such as twist, which appear to be primarily involved in early mesodermal differentiation, and bHLH factors such as TAL-1, which have been characterized through their association with chromosomal breakpoints associated with T-cell leukemias. To provide for studies aimed at understanding the genetic regulation of bHLH-EC2, we have characterized the organization of this gene and conducted preliminary studies of the transcriptional activity of the upstream promoter region. The mouse bHLH-EC2 gene was found to consist of two exons separated by amore » 5-kb intron, an organization pattern similar to the mouse twist gene. The transcription initiation site was identified by RNase protection assay and primer extension analysis. Linked promoter-reporter gene transfection experiments in cultured cells indicated that while the identified upstream sequence can function to promote transcription, it does not function in a cell-specific fashion. To investigate the possible association of bHLH-EC2 with hematological malignancy, the chromosomal location of this gene in the human was mapped by fluorescence in situ hybridization and assigned to chromosome band 20p13. 16 refs., 3 figs.« less

Genome-wide analysis of basic/helix-loop-helix gene family in peanut and assessment of its roles in pod development.

PubMed

Gao, Chao; Sun, Jianlei; Wang, Chongqi; Dong, Yumei; Xiao, Shouhua; Wang, Xingjun; Jiao, Zigao

2017-01-01

The basic/helix-loop-helix (bHLH) proteins constitute a superfamily of transcription factors that are known to play a range of regulatory roles in eukaryotes. Over the past few decades, many bHLH family genes have been well-characterized in model plants, such as Arabidopsis, rice and tomato. However, the bHLH protein family in peanuts has not yet been systematically identified and characterized. Here, 132 and 129 bHLH proteins were identified from two wild ancestral diploid subgenomes of cultivated tetraploid peanuts, Arachis duranensis (AA) and Arachis ipaensis (BB), respectively. Phylogenetic analysis indicated that these bHLHs could be classified into 19 subfamilies. Distribution mapping results showed that peanut bHLH genes were randomly and unevenly distributed within the 10 AA chromosomes and 10 BB chromosomes. In addition, 120 bHLH gene pairs between the AA-subgenome and BB-subgenome were found to be orthologous and 101 of these pairs were highly syntenic in AA and BB chromosomes. Furthermore, we confirmed that 184 bHLH genes expressed in different tissues, 22 of which exhibited tissue-specific expression. Meanwhile, we identified 61 bHLH genes that may be potentially involved in peanut-specific subterranean. Our comprehensive genomic analysis provides a foundation for future functional dissection and understanding of the regulatory mechanisms of bHLH transcription factors in peanuts.

Inhibition of F1-ATPase Rotational Catalysis by the Carboxyl-terminal Domain of the ϵ Subunit*

PubMed Central

Nakanishi-Matsui, Mayumi; Sekiya, Mizuki; Yano, Shio; Futai, Masamitsu

2014-01-01

Escherichia coli ATP synthase (F0F1) couples catalysis and proton transport through subunit rotation. The ϵ subunit, an endogenous inhibitor, lowers F1-ATPase activity by decreasing the rotation speed and extending the duration of the inhibited state (Sekiya, M., Hosokawa, H., Nakanishi-Matsui, M., Al-Shawi, M. K., Nakamoto, R. K., and Futai, M. (2010) Single molecule behavior of inhibited and active states of Escherichia coli ATP synthase F1 rotation. J. Biol. Chem. 285, 42058–42067). In this study, we constructed a series of ϵ subunits truncated successively from the carboxyl-terminal domain (helix 1/loop 2/helix 2) and examined their effects on rotational catalysis (ATPase activity, average rotation rate, and duration of inhibited state). As expected, the ϵ subunit lacking helix 2 caused about ½-fold reduced inhibition, and that without loop 2/helix 2 or helix 1/loop 2/helix 2 showed a further reduced effect. Substitution of ϵSer108 in loop 2 and ϵTyr114 in helix 2, which possibly interact with the β and γ subunits, respectively, decreased the inhibitory effect. These results suggest that the carboxyl-terminal domain of the ϵ subunit plays a pivotal role in the inhibition of F1 rotation through interaction with other subunits. PMID:25228697

Classification and evolutionary analysis of the basic helix-loop-helix gene family in the green anole lizard, Anolis carolinensis.

PubMed

Liu, Ake; Wang, Yong; Zhang, Debao; Wang, Xuhua; Song, Huifang; Dang, Chunwang; Yao, Qin; Chen, Keping

2013-08-01

Helix-loop-helix (bHLH) proteins play essential regulatory roles in a variety of biological processes. These highly conserved proteins form a large transcription factor superfamily, and are commonly identified in large numbers within animal, plant, and fungal genomes. The bHLH domain has been well studied in many animal species, but has not yet been characterized in non-avian reptiles. In this study, we identified 102 putative bHLH genes in the genome of the green anole lizard, Anolis carolinensis. Based on phylogenetic analysis, these genes were classified into 43 families, with 43, 24, 16, 3, 10, and 3 members assigned into groups A, B, C, D, E, and F, respectively, and 3 members categorized as "orphans". Within-group evolutionary relationships inferred from the phylogenetic analysis were consistent with highly conserved patterns observed for introns and additional domains. Results from phylogenetic analysis of the H/E(spl) family suggest that genome and tandem gene duplications have contributed to this family's expansion. Our classification and evolutionary analysis has provided insights into the evolutionary diversification of animal bHLH genes, and should aid future studies on bHLH protein regulation of key growth and developmental processes.

Aryl hydrocarbon receptor (AHR): "pioneer member" of the basic-helix/loop/helix per-Arnt-sim (bHLH/PAS) family of "sensors" of foreign and endogenous signals.

PubMed

Nebert, Daniel W

2017-07-01

The basic-helix/loop/helix per-Arnt-sim (bHLH/PAS) family comprises many transcription factors, found throughout all three kingdoms of life; bHLH/PAS members "sense" innumerable intracellular and extracellular "signals" - including endogenous compounds, foreign chemicals, gas molecules, redox potential, photons (light), gravity, heat, and osmotic pressure. These signals then initiate downstream signaling pathways involved in responding to that signal. The term "PAS", abbreviation for "per-Arnt-sim" was first coined in 1991. Although the mouse Arnt gene was not identified until 1991, evidence of its co-transcriptional binding partner, aryl hydrocarbon receptor (AHR), was first reported in 1974 as a "sensor" of foreign chemicals, up-regulating cytochrome P450 family 1 (CYP1) and other enzyme activities that usually metabolize the signaling chemical. Within a few years, AHR was proposed also to participate in inflammation. The mouse [Ah] locus was shown (1973-1989) to be relevant to chemical carcinogenesis, mutagenesis, toxicity and teratogenesis, the mouse Ahr gene was cloned in 1992, and the first Ahr(-/-) knockout mouse line was reported in 1995. After thousands of studies from the early 1970s to present day, we now realize that AHR participates in dozens of signaling pathways involved in critical-life processes, affecting virtually every organ and cell-type in the animal, including many invertebrates. Copyright © 2017 Elsevier Ltd. All rights reserved.

Antagonistic Basic Helix-Loop-Helix/bZIP Transcription Factors Form Transcriptional Modules That Integrate Light and Reactive Oxygen Species Signaling in Arabidopsis[W

PubMed Central

Chen, Dongqin; Xu, Gang; Tang, Weijiang; Jing, Yanjun; Ji, Qiang; Fei, Zhangjun; Lin, Rongcheng

2013-01-01

The critical developmental switch from heterotrophic to autotrophic growth of plants involves light signaling transduction and the production of reactive oxygen species (ROS). ROS function as signaling molecules that regulate multiple developmental processes, including cell death. However, the relationship between light and ROS signaling remains unclear. Here, we identify transcriptional modules composed of the basic helix-loop-helix and bZIP transcription factors PHYTOCHROME-INTERACTING FACTOR1 (PIF1), PIF3, ELONGATED HYPOCOTYL5 (HY5), and HY5 HOMOLOGY (HYH) that bridge light and ROS signaling to regulate cell death and photooxidative response. We show that pif mutants release more singlet oxygen and exhibit more extensive cell death than the wild type during Arabidopsis thaliana deetiolation. Genome-wide expression profiling indicates that PIF1 represses numerous ROS and stress-related genes. Molecular and biochemical analyses reveal that PIF1/PIF3 and HY5/HYH physically interact and coordinately regulate the expression of five ROS-responsive genes by directly binding to their promoters. Furthermore, PIF1/PIF3 and HY5/HYH function antagonistically during the seedling greening process. In addition, phytochromes, cryptochromes, and CONSTITUTIVE PHOTOMORPHOGENIC1 act upstream to regulate ROS signaling. Together, this study reveals that the PIF1/PIF3-HY5/HYH transcriptional modules mediate crosstalk between light and ROS signaling and sheds light on a new mechanism by which plants adapt to the light environments. PMID:23645630

Identification of basic/helix-loop-helix transcription factors reveals candidate genes involved in anthocyanin biosynthesis from the strawberry white-flesh mutant.

PubMed

Zhao, Fengli; Li, Gang; Hu, Panpan; Zhao, Xia; Li, Liangjie; Wei, Wei; Feng, Jiayue; Zhou, Houcheng

2018-02-09

As the second largest transcription factor family in plant, the basic helix-loop-helix (bHLH) transcription factor family, characterized by the conserved bHLH domain, plays a central regulatory role in many biological process. However, the bHLH transcription factor family of strawberry has not been systematically identified, especially for the anthocyanin biosynthesis. Here, we identified a total of 113 bHLH transcription factors and described their chromosomal distribution and bioinformatics for the diploid woodland strawberry Fragaria vesca. In addition, transcription profiles of 113 orthologous bHLH genes from various tissues were analyzed for the cultivar 'Benihoppe', its white-flesh mutant 'Xiaobai', and the 'Snow Princess' from their fruit development to the ripening, as well as those under either the ABA or Eth treatment. Both the RT-PCR and qRT-PCR results show that seven selected FabHLH genes (FabHLH17, FabHLH25, FabHLH27, FabHLH29, FabHLH40, FabHLH80, FabHLH98) are responsive to the fruit anthocyanin biosynthesis and hormone signaling according to transcript profiles where three color modes are observed for strawberry's fruit skin and flesh. Further, prediction for the protein interaction network reveals that four bHLHs (FabHLH25, FabHLH29, FabHLH80, FabHLH98) are involved in the fruit anthocyanin biosynthesis and hormone signaling transduction. These bioinformatics and expression profiles provide a good basis for a further investigation of strawberry bHLH genes.

Genome-wide identification, classification, and functional analysis of the basic helix-loop-helix transcription factors in the cattle, Bos Taurus.

PubMed

Li, Fengmei; Liu, Wuyi

2017-06-01

The basic helix-loop-helix (bHLH) transcription factors (TFs) form a huge superfamily and play crucial roles in many essential developmental, genetic, and physiological-biochemical processes of eukaryotes. In total, 109 putative bHLH TFs were identified and categorized successfully in the genomic databases of cattle, Bos Taurus, after removing redundant sequences and merging genetic isoforms. Through phylogenetic analyses, 105 proteins among these bHLH TFs were classified into 44 families with 46, 25, 14, 3, 13, and 4 members in the high-order groups A, B, C, D, E, and F, respectively. The remaining 4 bHLH proteins were sorted out as 'orphans.' Next, these 109 putative bHLH proteins identified were further characterized as significantly enriched in 524 significant Gene Ontology (GO) annotations (corrected P value ≤ 0.05) and 21 significantly enriched pathways (corrected P value ≤ 0.05) that had been mapped by the web server KOBAS 2.0. Furthermore, 95 bHLH proteins were further screened and analyzed together with two uncharacterized proteins in the STRING online database to reconstruct the protein-protein interaction network of cattle bHLH TFs. Ultimately, 89 bHLH proteins were fully mapped in a network with 67 biological process, 13 molecular functions, 5 KEGG pathways, 12 PFAM protein domains, and 25 INTERPRO classified protein domains and features. These results provide much useful information and a good reference for further functional investigations and updated researches on cattle bHLH TFs.

The transcriptional co-repressor TLE3 regulates myogenic differentiation by repressing the activity of the MyoD transcription factor.

PubMed

Kokabu, Shoichiro; Nakatomi, Chihiro; Matsubara, Takuma; Ono, Yusuke; Addison, William N; Lowery, Jonathan W; Urata, Mariko; Hudnall, Aaron M; Hitomi, Suzuro; Nakatomi, Mitsushiro; Sato, Tsuyoshi; Osawa, Kenji; Yoda, Tetsuya; Rosen, Vicki; Jimi, Eijiro

2017-08-04

Satellite cells are skeletal muscle stem cells that provide myonuclei for postnatal muscle growth, maintenance, and repair/regeneration in adults. Normally, satellite cells are mitotically quiescent, but they are activated in response to muscle injury, in which case they proliferate extensively and exhibit up-regulated expression of the transcription factor MyoD, a master regulator of myogenesis. MyoD forms a heterodimer with E proteins through their basic helix-loop-helix domain, binds to E boxes in the genome and thereby activates transcription at muscle-specific promoters. The central role of MyoD in muscle differentiation has increased interest in finding potential MyoD regulators. Here we identified transducin-like enhancer of split (TLE3), one of the Groucho/TLE family members, as a regulator of MyoD function during myogenesis. TLE3 was expressed in activated and proliferative satellite cells in which increased TLE3 levels suppressed myogenic differentiation, and, conversely, reduced TLE3 levels promoted myogenesis with a concomitant increase in proliferation. We found that, via its glutamine- and serine/proline-rich domains, TLE3 interferes with MyoD function by disrupting the association between the basic helix-loop-helix domain of MyoD and E proteins. Our findings indicate that TLE3 participates in skeletal muscle homeostasis by dampening satellite cell differentiation via repression of MyoD transcriptional activity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

A Genome-Wide Identification and Analysis of the Basic Helix-Loop-Helix Transcription Factors in Brown Planthopper, Nilaparvata lugens

PubMed Central

Wan, Pin-Jun; Yuan, San-Yue; Wang, Wei-Xia; Chen, Xu; Lai, Feng-Xiang; Fu, Qiang

2016-01-01

The basic helix-loop-helix (bHLH) transcription factors in insects play essential roles in multiple developmental processes including neurogenesis, sterol metabolism, circadian rhythms, organogenesis and formation of olfactory sensory neurons. The identification and function analysis of bHLH family members of the most destructive insect pest of rice, Nilaparvata lugens, may provide novel tools for pest management. Here, a genome-wide survey for bHLH sequences identified 60 bHLH sequences (NlbHLHs) encoded in the draft genome of N. lugens. Phylogenetic analysis of the bHLH domains successfully classified these genes into 40 bHLH families in group A (25), B (14), C (10), D (1), E (8) and F (2). The number of NlbHLHs with introns is higher than many other insect species, and the average intron length is shorter than those of Acyrthosiphon pisum. High number of ortholog families of NlbHLHs was found suggesting functional conversation for these proteins. Compared to other insect species studied, N. lugens has the highest number of bHLH members. Furthermore, gene duplication events of SREBP, Kn(col), Tap, Delilah, Sim, Ato and Crp were found in N. lugens. In addition, a putative full set of NlbHLH genes is defined and compared with another insect species. Thus, our classification of these NlbHLH members provides a platform for further investigations of bHLH protein functions in the regulation of N. lugens, and of insects in general. PMID:27869716

Human lysozyme possesses novel antimicrobial peptides within its N-terminal domain that target bacterial respiration.

PubMed

Ibrahim, Hisham R; Imazato, Kenta; Ono, Hajime

2011-09-28

Human milk lysozyme is thought to be a key defense factor in protecting the gastrointestinal tract of newborns against bacterial infection. Recently, evidence was found that pepsin, under conditions relevant to the newborn stomach, cleaves chicken lysozyme (cLZ) at specific loops to generate five antimicrobial peptide motifs. This study explores the antimicrobial role of the corresponding peptides of human lysozyme (hLZ), the actual protein in breast milk. Five peptide motifs of hLZ, one helix-loop-helix (HLH), its two helices (H1 and H2), and two helix-sheet motifs, H2-β-strands 1-2 (H2-S12) or H2-β-strands 1-3 (H2-S13), were synthesized and examined for antimicrobial action. The five peptides of hLZ exhibit microbicidal activity to various degrees against several bacterial strains. The HLH peptide and its N-terminal helix (H1) were significantly the most potent bactericidal to Gram-positive and Gram-negative bacteria and the fungus Candida albicans . Outer and inner membrane permeabilization studies, as well as measurements of transmembrane electrochemical potentials, provided evidence that HLH peptide and its N-terminal helix (H1) kill bacteria by crossing the outer membrane of Gram-negative bacteria via self-promoted uptake and are able to dissipate the membrane potential-dependent respiration of Gram-positive bacteria. This finding is the first to describe that hLZ possesses multiple antimicrobial peptide motifs within its N-terminal domain, providing insight into new classes of antibiotic peptides with potential use in the treatment of infectious diseases.

Inhibition of F1-ATPase rotational catalysis by the carboxyl-terminal domain of the ϵ subunit.

PubMed

Nakanishi-Matsui, Mayumi; Sekiya, Mizuki; Yano, Shio; Futai, Masamitsu

2014-10-31

Escherichia coli ATP synthase (F0F1) couples catalysis and proton transport through subunit rotation. The ϵ subunit, an endogenous inhibitor, lowers F1-ATPase activity by decreasing the rotation speed and extending the duration of the inhibited state (Sekiya, M., Hosokawa, H., Nakanishi-Matsui, M., Al-Shawi, M. K., Nakamoto, R. K., and Futai, M. (2010) Single molecule behavior of inhibited and active states of Escherichia coli ATP synthase F1 rotation. J. Biol. Chem. 285, 42058-42067). In this study, we constructed a series of ϵ subunits truncated successively from the carboxyl-terminal domain (helix 1/loop 2/helix 2) and examined their effects on rotational catalysis (ATPase activity, average rotation rate, and duration of inhibited state). As expected, the ϵ subunit lacking helix 2 caused about ½-fold reduced inhibition, and that without loop 2/helix 2 or helix 1/loop 2/helix 2 showed a further reduced effect. Substitution of ϵSer(108) in loop 2 and ϵTyr(114) in helix 2, which possibly interact with the β and γ subunits, respectively, decreased the inhibitory effect. These results suggest that the carboxyl-terminal domain of the ϵ subunit plays a pivotal role in the inhibition of F1 rotation through interaction with other subunits. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Disruption of alpha beta but not of gamma delta T cell development by overexpression of the helix-loop-helix protein Id3 in committed T cell progenitors.

PubMed Central

Blom, B; Heemskerk, M H; Verschuren, M C; van Dongen, J J; Stegmann, A P; Bakker, A Q; Couwenberg, F; Res, P C; Spits, H

1999-01-01

Enforced expression of Id3, which has the capacity to inhibit many basic helix-loop-helix (bHLH) transcription factors, in human CD34(+) hematopoietic progenitor cells that have not undergone T cell receptor (TCR) gene rearrangements inhibits development of the transduced cells into TCRalpha beta and gamma delta cells in a fetal thymic organ culture (FTOC). Here we document that overexpression of Id3, in progenitors that have initiated TCR gene rearrangements (pre-T cells), inhibits development into TCRalpha beta but not into TCRgamma delta T cells. Furthermore, Id3 impedes expression of recombination activating genes and downregulates pre-Talpha mRNA. These observations suggest possible mechanisms by which Id3 overexpression can differentially affect development of pre-T cells into TCRalpha beta and gamma delta cells. We also observed that cell surface CD4(-)CD8(-)CD3(-) cells with rearranged TCR genes developed from Id3-transduced but not from control-transduced pre-T cells in an FTOC. These cells had properties of both natural killer (NK) and pre-T cells. These findings suggest that bHLH factors are required to control T cell development after the T/NK developmental checkpoint. PMID:10329625

Origin and diversification of the basic helix-loop-helix gene family in metazoans: insights from comparative genomics

PubMed Central

Simionato, Elena; Ledent, Valérie; Richards, Gemma; Thomas-Chollier, Morgane; Kerner, Pierre; Coornaert, David; Degnan, Bernard M; Vervoort, Michel

2007-01-01

Background Molecular and genetic analyses conducted in model organisms such as Drosophila and vertebrates, have provided a wealth of information about how networks of transcription factors control the proper development of these species. Much less is known, however, about the evolutionary origin of these elaborated networks and their large-scale evolution. Here we report the first evolutionary analysis of a whole superfamily of transcription factors, the basic helix-loop-helix (bHLH) proteins, at the scale of the whole metazoan kingdom. Results We identified in silico the putative full complement of bHLH genes in the sequenced genomes of 12 different species representative of the main metazoan lineages, including three non-bilaterian metazoans, the cnidarians Nematostella vectensis and Hydra magnipapillata and the demosponge Amphimedon queenslandica. We have performed extensive phylogenetic analyses of the 695 identified bHLHs, which has allowed us to allocate most of these bHLHs to defined evolutionary conserved groups of orthology. Conclusion Three main features in the history of the bHLH gene superfamily can be inferred from these analyses: (i) an initial diversification of the bHLHs has occurred in the pre-Cambrian, prior to metazoan cladogenesis; (ii) a second expansion of the bHLH superfamily occurred early in metazoan evolution before bilaterians and cnidarians diverged; and (iii) the bHLH complement during the evolution of the bilaterians has been remarkably stable. We suggest that these features may be extended to other developmental gene families and reflect a general trend in the evolution of the developmental gene repertoires of metazoans. PMID:17335570

The cold-induced basic helix-loop-helix transcription factor gene MdCIbHLH1 encodes an ICE-like protein in apple

PubMed Central

2012-01-01

Background Plant growth is greatly affected by low temperatures, and the expression of a number of genes is induced by cold stress. Although many genes in the cold signaling pathway have been identified in Arabidopsis, little is known about the transcription factors involved in the cold stress response in apple. Results Here, we show that the apple bHLH (basic helix-loop-helix) gene MdCIbHLH1 (Cold-Induced bHLH1), which encodes an ICE-like protein, was noticeably induced in response to cold stress. The MdCIbHLH1 protein specifically bound to the MYC recognition sequences in the AtCBF3 promoter, and MdCIbHLH1 overexpression enhanced cold tolerance in transgenic Arabidopsis. In addition, the MdCIbHLH1 protein bound to the promoters of MdCBF2 and favorably contributed to cold tolerance in transgenic apple plants by upregulating the expression of MdCBF2 through the CBF (C-repeat-binding factor) pathway. Our findings indicate that MdCIbHLH1 functions in stress tolerance in different species. For example, ectopic MdCIbHLH1 expression conferred enhanced chilling tolerance in transgenic tobacco. Finally, we observed that cold induces the degradation of the MdCIbHLH1 protein in apple and that this degradation was potentially mediated by ubiquitination and sumoylation. Conclusions Based on these findings, MdCIbHLH1 encodes a transcription factor that is important for the cold tolerance response in apple. PMID:22336381

SclR, a basic helix-loop-helix transcription factor, regulates hyphal morphology and promotes sclerotial formation in Aspergillus oryzae.

PubMed

Jin, Feng Jie; Takahashi, Tadashi; Matsushima, Ken-ichiro; Hara, Seiichi; Shinohara, Yasutomo; Maruyama, Jun-ichi; Kitamoto, Katsuhiko; Koyama, Yasuji

2011-07-01

Most known basic-region helix-loop-helix (bHLH) proteins belong to a superfamily of transcription factors often involved in the control of growth and differentiation. Therefore, inappropriate expression of genes encoding bHLH proteins is frequently associated with developmental dysfunction. In our previously reported study, a novel bHLH protein-encoding gene (AO090011000215) of Aspergillus oryzae was identified. The gene-disrupted strain was found to produce dense conidia, but sparse sclerotia, relative to the parent strain. Here, to further analyze its function, we generated an overexpressing strain using the A. oryzae amyB gene promoter. Genetic overexpression led to a large number of initial hyphal aggregations and then the formation of mature sclerotia; it was therefore designated sclR (sclerotium regulator). At the same time, the sclR-overexpressing strain also displayed both delayed and decreased conidiation. Scanning electron microscopy indicated that the aerial hyphae of the sclR-overexpressing strain were extremely branched and intertwined with each other. In the generation of the SclR-enhanced green fluorescent protein (EGFP) expression strain, the SclR-EGFP protein fusion was conditionally detected in the nuclei. In addition, the loss of sclR function led to rapid protein degradation and cell lysis in dextrin-polypeptone-yeast extract liquid medium. Taken together, these observations indicate that SclR plays an important role in hyphal morphology, asexual conidiospore formation, and the promotion of sclerotial production, even retaining normal cell function, at least in submerged liquid culture.

A basic helix-loop-helix transcription factor, PhFBH4, regulates flower senescence by modulating ethylene biosynthesis pathway in petunia.

PubMed

Yin, Jing; Chang, Xiaoxiao; Kasuga, Takao; Bui, Mai; Reid, Michael S; Jiang, Cai-Zhong

2015-01-01

The basic helix-loop-helix (bHLH) transcription factors (TFs) play important roles in regulating multiple biological processes in plants. However, there are few reports about the function of bHLHs in flower senescence. In this study, a bHLH TF, PhFBH4, was found to be dramatically upregulated during flower senescence. Transcription of PhFBH4 is induced by plant hormones and abiotic stress treatments. Silencing of PhFBH4 using virus-induced gene silencing or an antisense approach extended flower longevity, while transgenic petunia flowers with an overexpression construct showed a reduction in flower lifespan. Abundance of transcripts of senescence-related genes (SAG12, SAG29) was significantly changed in petunia PhFBH4 transgenic flowers. Furthermore, silencing or overexpression of PhFBH4 reduced or increased, respectively, transcript abundances of important ethylene biosynthesis-related genes, ACS1 and ACO1, thereby influencing ethylene production. An electrophoretic mobility shift assay showed that the PhFBH4 protein physically interacted with the G-box cis-element in the promoter of ACS1, suggesting that ACS1 was a direct target of the PhFBH4 protein. In addition, ectopic expression of this gene altered plant development including plant height, internode length, and size of leaves and flowers, accompanied by alteration of transcript abundance of the gibberellin biosynthesis-related gene GA2OX3. Our results indicate that PhFBH4 plays an important role in regulating plant growth and development through modulating the ethylene biosynthesis pathway.

Genome-wide analysis of basic helix-loop-helix (bHLH) transcription factors in Brachypodium distachyon.

PubMed

Niu, Xin; Guan, Yuxiang; Chen, Shoukun; Li, Haifeng

2017-08-15

As a superfamily of transcription factors (TFs), the basic helix-loop-helix (bHLH) proteins have been characterized functionally in many plants with a vital role in the regulation of diverse biological processes including growth, development, response to various stresses, and so on. However, no systemic analysis of the bHLH TFs has been reported in Brachypodium distachyon, an emerging model plant in Poaceae. A total of 146 bHLH TFs were identified in the Brachypodium distachyon genome and classified into 24 subfamilies. BdbHLHs in the same subfamily share similar protein motifs and gene structures. Gene duplication events showed a close relationship to rice, maize and sorghum, and segment duplications might play a key role in the expansion of this gene family. The amino acid sequence of the bHLH domains were quite conservative, especially Leu-27 and Leu-54. Based on the predicted binding activities, the BdbHLHs were divided into DNA binding and non-DNA binding types. According to the gene ontology (GO) analysis, BdbHLHs were speculated to function in homodimer or heterodimer manner. By integrating the available high throughput data in public database and results of quantitative RT-PCR, we found the expression profiles of BdbHLHs were different, implying their differentiated functions. One hundred fourty-six BdbHLHs were identified and their conserved domains, sequence features, phylogenetic relationship, chromosomal distribution, GO annotations, gene structures, gene duplication and expression profiles were investigated. Our findings lay a foundation for further evolutionary and functional elucidation of BdbHLH genes.

Characterization of an apple TT2-type R2R3 MYB transcription factor functionally similar to the poplar proanthocyanidin regulator PtMYB134.

PubMed

Gesell, Andreas; Yoshida, Kazuko; Tran, Lan T; Constabel, C Peter

2014-09-01

The apple MdMYB9 gene encodes a positive regulator of proanthocyanidin synthesis that activates anthocyanidin reductase promoters from apple and poplar via interaction with basic helix-loop-helix proteins. The regulation of proanthocyanidins (PAs, condensed tannins) is of great importance in food plants due to the many benefits of PAs in the human diet. Two candidate flavonoid MYB regulators, MdMYB9 and MdMYB11, were cloned from apple (Malus × domestica) based on their similarity to known MYB PA regulators. Transcript accumulation of both MdMYB9 and MdMYB11 was induced by high light and wounding, similar to the poplar (Populus spp) PA regulator PtMYB134. In transient activation assays with various basic helix-loop-helix (bHLH) co-regulators, MdMYB9 activated apple and poplar anthocyanidin reductase (ANR) promoters, while MdMYB11 showed no activity. Potential transcription factor binding elements were found within several ANR promoters, and the importance of the bHLH binding site (E-box) on ANR promoter activation was demonstrated via mutational analysis. The ability of MdMYB9 and PtMYB134 to reciprocally activate ANR promoters from both apple and poplar and to partner with heterologous bHLH co-factors from these plants confirms the high degree of conservation of PA regulatory complexes across species. The similarity in apple and poplar PA regulation suggests that regulatory genes from poplar could be effectively employed for metabolic engineering of the PA pathway in apple.

An auto-inhibitory helix in CTP:phosphocholine cytidylyltransferase hijacks the catalytic residue and constrains a pliable, domain-bridging helix pair

PubMed Central

Ramezanpour, Mohsen; Lee, Jaeyong; Taneva, Svetla G.; Tieleman, D. Peter; Cornell, Rosemary B.

2018-01-01

The activity of CTP:phosphocholine cytidylyltransferase (CCT), a key enzyme in phosphatidylcholine synthesis, is regulated by reversible interactions of a lipid-inducible amphipathic helix (domain M) with membrane phospholipids. When dissociated from membranes, a portion of the M domain functions as an auto-inhibitory (AI) element to suppress catalysis. The AI helix from each subunit binds to a pair of α helices (αE) that extend from the base of the catalytic dimer to create a four-helix bundle. The bound AI helices make intimate contact with loop L2, housing a key catalytic residue, Lys122. The impacts of the AI helix on active-site dynamics and positioning of Lys122 are unknown. Extensive MD simulations with and without the AI helix revealed that backbone carbonyl oxygens at the point of contact between the AI helix and loop L2 can entrap the Lys122 side chain, effectively competing with the substrate, CTP. In silico, removal of the AI helices dramatically increased αE dynamics at a predicted break in the middle of these helices, enabling them to splay apart and forge new contacts with loop L2. In vitro cross-linking confirmed the reorganization of the αE element upon membrane binding of the AI helix. Moreover, when αE bending was prevented by disulfide engineering, CCT activation by membrane binding was thwarted. These findings suggest a novel two-part auto-inhibitory mechanism for CCT involving capture of Lys122 and restraint of the pliable αE helices. We propose that membrane binding enables bending of the αE helices, bringing the active site closer to the membrane surface. PMID:29519816

Convergent evolution of gene networks by single-gene duplications in higher eukaryotes.

PubMed

Amoutzias, Gregory D; Robertson, David L; Oliver, Stephen G; Bornberg-Bauer, Erich

2004-03-01

By combining phylogenetic, proteomic and structural information, we have elucidated the evolutionary driving forces for the gene-regulatory interaction networks of basic helix-loop-helix transcription factors. We infer that recurrent events of single-gene duplication and domain rearrangement repeatedly gave rise to distinct networks with almost identical hub-based topologies, and multiple activators and repressors. We thus provide the first empirical evidence for scale-free protein networks emerging through single-gene duplications, the dominant importance of molecular modularity in the bottom-up construction of complex biological entities, and the convergent evolution of networks.

Novel Structural Components Contribute to the High Thermal Stability of Acyl Carrier Protein from Enterococcus faecalis*

PubMed Central

Park, Young-Guen; Jung, Min-Cheol; Song, Heesang; Jeong, Ki-Woong; Bang, Eunjung; Hwang, Geum-Sook; Kim, Yangmee

2016-01-01

Enterococcus faecalis is a Gram-positive, commensal bacterium that lives in the gastrointestinal tracts of humans and other mammals. It causes severe infections because of high antibiotic resistance. E. faecalis can endure extremes of temperature and pH. Acyl carrier protein (ACP) is a key element in the biosynthesis of fatty acids responsible for acyl group shuttling and delivery. In this study, to understand the origin of high thermal stabilities of E. faecalis ACP (Ef-ACP), its solution structure was investigated for the first time. CD experiments showed that the melting temperature of Ef-ACP is 78.8 °C, which is much higher than that of Escherichia coli ACP (67.2 °C). The overall structure of Ef-ACP shows the common ACP folding pattern consisting of four α-helices (helix I (residues 3–17), helix II (residues 39–53), helix III (residues 60–64), and helix IV (residues 68–78)) connected by three loops. Unique Ef-ACP structural features include a hydrophobic interaction between Phe45 in helix II and Phe18 in the α1α2 loop and a hydrogen bonding between Ser15 in helix I and Ile20 in the α1α2 loop, resulting in its high thermal stability. Phe45-mediated hydrophobic packing may block acyl chain binding subpocket II entry. Furthermore, Ser58 in the α2α3 loop in Ef-ACP, which usually constitutes a proline in other ACPs, exhibited slow conformational exchanges, resulting in the movement of the helix III outside the structure to accommodate a longer acyl chain in the acyl binding cavity. These results might provide insights into the development of antibiotics against pathogenic drug-resistant E. faecalis strains. PMID:26631734

Molecular mechanism of Mg2+-dependent gating in CorA

NASA Astrophysics Data System (ADS)

Dalmas, Olivier; Sompornpisut, Pornthep; Bezanilla, Francisco; Perozo, Eduardo

2014-04-01

CorA is the major transport system responsible for Mg2+ uptake in bacteria and can functionally substitute for its homologue Mrs2p in the yeast inner mitochondrial membrane. Although several CorA crystal structures are available, the molecular mechanism of Mg2+ uptake remains to be established. Here we use electron paramagnetic resonance spectroscopy, electrophysiology and molecular dynamic simulations to show that CorA is regulated by cytoplasmic Mg2+ acting as a ligand and elucidate the basic conformational rearrangements responsible for Mg2+-dependent gating. Mg2+ unbinding at the divalent cation sensor triggers a conformational change that leads to the inward motion of the stalk helix, which propagates to the pore-forming transmembrane helix TM1. Helical tilting and rotation in TM1 generates an iris-like motion that increases the diameter of the permeation pathway, triggering ion conduction. This work establishes the molecular basis of a Mg2+-driven negative feedback loop in CorA as the key physiological event controlling Mg2+ uptake and homeostasis in prokaryotes.

Yeast transcriptional activator INO2 interacts as an Ino2p/Ino4p basic helix-loop-helix heteromeric complex with the inositol/choline-responsive element necessary for expression of phospholipid biosynthetic genes in Saccharomyces cerevisiae.

PubMed Central

Schwank, S; Ebbert, R; Rautenstrauss, K; Schweizer, E; Schüller, H J

1995-01-01

Coordinate transcriptional control of yeast genes involved in phospholipid biosynthesis is mediated by the inositol/choline-responsive element (ICRE) contained in the respective promoter regions. Regulatory genes INO2 and INO4, both encoding basic helix-loop-helix (bHLH) proteins, are necessary for ICRE-dependent gene activation. By the use of size variants and by heterologous expression in E. coli we demonstrate that Ino2p and Ino4p are both necessary and sufficient for the formation of the previously described FAS binding factor 1, Fbf1, interacting with the ICRE. Formation of a heteromeric complex between Ino2p and Ino4p by means of the respective bHLH domains was demonstrated in vivo by the interaction of appropriate two-hybrid constructs and in vitro by Far-Western analyses. Neither Ino2p nor Ino4p binds to the ICRE as a homodimer. When fused to the DNA-binding domain of Gal4p, Ino2p but not Ino4p was able to activate a UASGAL-containing reporter gene even in the absence of the heterologous Fbf1 subunit. By deletion studies, two separate transcriptional activation domains were identified in the N-terminal part of Ino2p. Thus, the bHLH domains of Ino2p and Ino4p constitute the dimerization/DNA-binding module of Fbf1 mediating its interaction with the ICRE, while transcriptional activation is effected exclusively by Ino2p. Images PMID:7862526

The genetics of rhizosheath size in a multiparent mapping population of wheat.

PubMed

Delhaize, Emmanuel; Rathjen, Tina M; Cavanagh, Colin R

2015-08-01

Rhizosheaths comprise soil that adheres to plant roots and, in some species, are indicative of root hair length. In this study, the genetics of rhizosheath size in wheat was investigated by screening the progeny of multiparent advanced generation intercrosses (MAGIC). Two MAGIC populations were screened for rhizosheath size using a high throughput method. One MAGIC population was developed from intercrosses between four parents (4-way) and the other from intercrosses between eight parents (8-way). Transgressive segregation for rhizosheath size was observed in both the 4-way and 8-way MAGIC populations. A quantitative trait loci (QTL) analysis of the 4-way population identified six major loci located on chromosomes 2B, 4D, 5A, 5B, 6A, and 7A together accounting for 42% of the variation in rhizosheath size. Rhizosheath size was strongly correlated with root hair length and was robust across different soil types in the absence of chemical constraints. Rhizosheath size in the MAGIC populations was a reliable surrogate for root hair length and, therefore, the QTL identified probably control root hair elongation. Members of the basic helix-loop-helix family of transcription factors have previously been identified to regulate root hair length in Arabidopsis and rice. Since several wheat members of the basic helix-loop-helix family of genes are located within or near the QTL, these genes are candidates for controlling the long root hair trait. The QTL for rhizosheath size identified in this study provides the opportunity to implement marker-assisted selection to increase root hair length for improved phosphate acquisition in wheat. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

DNA binding specificity of the basic-helix-loop-helix protein MASH-1.

PubMed

Meierhan, D; el-Ariss, C; Neuenschwander, M; Sieber, M; Stackhouse, J F; Allemann, R K

1995-09-05

Despite the high degree of sequence similarity in their basic-helix-loop-helix (BHLH) domains, MASH-1 and MyoD are involved in different biological processes. In order to define possible differences between the DNA binding specificities of these two proteins, we investigated the DNA binding properties of MASH-1 by circular dichroism spectroscopy and by electrophoretic mobility shift assays (EMSA). Upon binding to DNA, the BHLH domain of MASH-1 underwent a conformational change from a mainly unfolded to a largely alpha-helical form, and surprisingly, this change was independent of the specific DNA sequence. The same conformational transition could be induced by the addition of 20% 2,2,2-trifluoroethanol. The apparent dissociation constants (KD) of the complexes of full-length MASH-1 with various oligonucleotides were determined from half-saturation points in EMSAs. MASH-1 bound as a dimer to DNA sequences containing an E-box with high affinity KD = 1.4-4.1 x 10(-14) M2). However, the specificity of DNA binding was low. The dissociation constant for the complex between MASH-1 and the highest affinity E-box sequence (KD = 1.4 x 10(-14) M2) was only a factor of 10 smaller than for completely unrelated DNA sequences (KD = approximately 1 x 10(-13) M2). The DNA binding specificity of MASH-1 was not significantly increased by the formation of an heterodimer with the ubiquitous E12 protein. MASH-1 and MyoD displayed similar binding site preferences, suggesting that their different target gene specificities cannot be explained solely by differential DNA binding. An explanation for these findings is provided on the basis of the known crystal structure of the BHLH domain of MyoD.

Constitutive Overexpression of the Basic Helix-Loop-Helix Nex1/MATH-2 Transcription Factor Promotes Neuronal Differentiation of PC12 Cells and Neurite Regeneration

PubMed Central

Uittenbogaard, Martine; Chiaramello, Anne

2009-01-01

Elucidation of the intricate transcriptional pathways leading to neural differentiation and the establishment of neuronal identity is critical to the understanding and design of therapeutic approaches. Among the important players, the basic helix-loop-helix (bHLH) transcription factors have been found to be pivotal regulators of neurogenesis. In this study, we investigate the role of the bHLH differentiation factor Nex1/MATH-2 in conjunction with the nerve growth factor (NGF) signaling pathway using the rat phenochromocytoma PC12 cell line. We report that the expression of Nex1 protein is induced after 5 hr of NGF treatment and reaches maximal levels at 24 hr, when very few PC12 cells have begun extending neurites and ceased cell division. Furthermore, our study demonstrates that Nex1 has the ability to trigger neuronal differentiation of PC12 cells in the absence of neurotrophic factor. We show that Nex1 plays an important role in neurite outgrowth and has the capacity to regenerate neurite outgrowth in the absence of NGF. These results are corroborated by the fact that Nex1 targets a repertoire of distinct types of genes associated with neuronal differentiation, such as GAP-43, βIII-tubulin, and NeuroD. In addition, our findings show that Nex1 up-regulates the expression of the mitotic inhibitor p21WAF1, thus linking neuronal differentiation to cell cycle withdrawal. Finally, our studies show that overexpression of a Nex1 mutant has the ability to block the execution of NGF-induced differentiation program, suggesting that Nex1 may be an important effector of the NGF signaling pathway. PMID:11782967

A Basic Helix-Loop-Helix Transcription Factor, PtrbHLH, of Poncirus trifoliata Confers Cold Tolerance and Modulates Peroxidase-Mediated Scavenging of Hydrogen Peroxide1[C][W

PubMed Central

Huang, Xiao-San; Wang, Wei; Zhang, Qian; Liu, Ji-Hong

2013-01-01

The basic helix-loop-helix (bHLH) transcription factors are involved in a variety of physiological processes. However, plant bHLHs functioning in cold tolerance and the underlying mechanisms remain poorly understood. Here, we report the identification and functional characterization of PtrbHLH isolated from trifoliate orange (Poncirus trifoliata). The transcript levels of PtrbHLH were up-regulated under various abiotic stresses, particularly cold. PtrbHLH was localized in the nucleus with transactivation activity. Overexpression of PtrbHLH in tobacco (Nicotiana tabacum) or lemon (Citrus limon) conferred enhanced tolerance to cold under chilling or freezing temperatures, whereas down-regulation of PtrbHLH in trifoliate orange by RNA interference (RNAi) resulted in elevated cold sensitivity. A range of stress-responsive genes was up-regulated or down-regulated in the transgenic lemon. Of special note, several peroxidase (POD) genes were induced after cold treatment. Compared with the wild type, POD activity was increased in the overexpression plants but decreased in the RNAi plants, which was inversely correlated with the hydrogen peroxide (H2O2) levels in the tested lines. Treatment of the transgenic tobacco plants with POD inhibitors elevated the H2O2 levels and greatly compromised their cold tolerance, while exogenous replenishment of POD enhanced cold tolerance of the RNAi line. In addition, transgenic tobacco and lemon plants were more tolerant to oxidative stresses. Yeast one-hybrid assay and transient expression analysis demonstrated that PtrbHLH could bind to the E-box elements in the promoter region of a POD gene. Taken together, these results demonstrate that PtrbHLH plays an important role in cold tolerance, at least in part, by positively regulating POD-mediated reactive oxygen species removal. PMID:23624854

Basic helix-loop-helix transcription factor NEUROG1 and schizophrenia: effects on illness susceptibility, MRI brain morphometry and cognitive abilities.

PubMed

Ho, Beng-Choon; Epping, Eric; Wang, Kai; Andreasen, Nancy C; Librant, Amy; Wassink, Thomas H

2008-12-01

Transcription factors, including the basic helix-loop-helix (bHLH) family, regulate numerous genes and play vital roles in controlling gene expression. Consequently, transcription factor mutations can lead to phenotypic pleiotropy, and may be a candidate mechanism underlying the complex genetics and heterogeneous phenotype of schizophrenia. Neurogenin1 (NEUROG1; a.k.a. Ngn1 or Neurod3), a bHLH transcription factor encoded on a known schizophrenia linkage region in 5q31.1, induces glutamatergic and suppresses GABAergic neuronal differentiation during embryonic neurodevelopment. The goal of this study is to investigate NEUROG1 effects on schizophrenia risk and on phenotypic features of schizophrenia. We tested 392 patients with schizophrenia or schizoaffective disorder and 226 healthy normal volunteers for association with NEUROG1. Major alleles on two NEUROG1-associated SNPs (rs2344484-C-allele and rs8192558-G-allele) were significantly more prevalent among patients (p

Structures of bacterial homologues of SWEET transporters in two distinct conformations.

PubMed

Xu, Yan; Tao, Yuyong; Cheung, Lily S; Fan, Chao; Chen, Li-Qing; Xu, Sophia; Perry, Kay; Frommer, Wolf B; Feng, Liang

2014-11-20

SWEETs and their prokaryotic homologues are monosaccharide and disaccharide transporters that are present from Archaea to plants and humans. SWEETs play crucial roles in cellular sugar efflux processes: that is, in phloem loading, pollen nutrition and nectar secretion. Their bacterial homologues, which are called SemiSWEETs, are among the smallest known transporters. Here we show that SemiSWEET molecules, which consist of a triple-helix bundle, form symmetrical, parallel dimers, thereby generating the translocation pathway. Two SemiSWEET isoforms were crystallized, one in an apparently open state and one in an occluded state, indicating that SemiSWEETs and SWEETs are transporters that undergo rocking-type movements during the transport cycle. The topology of the triple-helix bundle is similar yet distinct to that of the basic building block of animal and plant major facilitator superfamily (MFS) transporters (for example, GLUTs and SUTs). This finding indicates two possibilities: that SWEETs and MFS transporters evolved from an ancestral triple-helix bundle or that the triple-helix bundle represents convergent evolution. In SemiSWEETs and SWEETs, two triple-helix bundles are arranged in a parallel configuration to produce the 6- and 6 + 1-transmembrane-helix pores, respectively. In the 12-transmembrane-helix MFS transporters, four triple-helix bundles are arranged into an alternating antiparallel configuration, resulting in a much larger 2 × 2 triple-helix bundle forming the pore. Given the similarity of SemiSWEETs and SWEETs to PQ-loop amino acid transporters and to mitochondrial pyruvate carriers (MPCs), the structures characterized here may also be relevant to other transporters in the MtN3 clan. The insight gained from the structures of these transporters and from the analysis of mutations of conserved residues will improve the understanding of the transport mechanism, as well as allow comparative studies of the different superfamilies involved in sugar transport and the evolution of transporters in general.

Computational modeling of the bHLH domain of the transcription factor TWIST1 and R118C, S144R and K145E mutants

PubMed Central

2012-01-01

Background Human TWIST1 is a highly conserved member of the regulatory basic helix-loop-helix (bHLH) transcription factors. TWIST1 forms homo- or heterodimers with E-box proteins, such as E2A (isoforms E12 and E47), MYOD and HAND2. Haploinsufficiency germ-line mutations of the twist1 gene in humans are the main cause of Saethre-Chotzen syndrome (SCS), which is characterized by limb abnormalities and premature fusion of cranial sutures. Because of the importance of TWIST1 in the regulation of embryonic development and its relationship with SCS, along with the lack of an experimentally solved 3D structure, we performed comparative modeling for the TWIST1 bHLH region arranged into wild-type homodimers and heterodimers with E47. In addition, three mutations that promote DNA binding failure (R118C, S144R and K145E) were studied on the TWIST1 monomer. We also explored the behavior of the mutant forms in aqueous solution using molecular dynamics (MD) simulations, focusing on the structural changes of the wild-type versus mutant dimers. Results The solvent-accessible surface area of the homodimers was smaller on wild-type dimers, which indicates that the cleft between the monomers remained more open on the mutant homodimers. RMSD and RMSF analyses indicated that mutated dimers presented values that were higher than those for the wild-type dimers. For a more careful investigation, the monomer was subdivided into four regions: basic, helix I, loop and helix II. The basic domain presented a higher flexibility in all of the parameters that were analyzed, and the mutant dimer basic domains presented values that were higher than the wild-type dimers. The essential dynamic analysis also indicated a higher collective motion for the basic domain. Conclusions Our results suggest the mutations studied turned the dimers into more unstable structures with a wider cleft, which may be a reason for the loss of DNA binding capacity observed for in vitro circumstances. PMID:22839202

Phage selection of peptide "microantibodies".

PubMed

Fujiwara, Daisuke; Fujii, Ikuo

2013-01-01

A bioactive peptide capable of inhibiting protein-protein interactions has the potential to be a molecular tool for biological studies and a therapeutic by disrupting aberrant interactions involved in diseases. We have developed combinatorial libraries of peptides with helix-loop-helix structure, from which the isolated peptides have the constrained structure to reduce entropy costs in binding, resulting in high binding affinities for target molecules. Previously, we designed a de novo peptide of helix-loop-helix structure that we termed a "microantibody." Using the microantibody as a library scaffold, we have constructed a phage-display library to successfully isolate molecular-targeting peptides against a cytokine receptor (granulocyte colony-stimulating factor receptor), a protein kinase (Aurora-A), and a ganglioside (GM1). Protocols in this article describe a general procedure for the library construction and the library screening.

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

Bae, Brian; Nayak, Dhananjaya; Ray, Ananya

RNA polymerase inhibitors like the CBR class that target the enzyme’s complex catalytic center are attractive leads for new antimicrobials. The catalysis by RNA polymerase involves multiple rearrangements of bridge helix, trigger loop, and active-center side chains that isomerize the triphosphate of bound NTP and two Mg 2+ ions from a preinsertion state to a reactive configuration. CBR inhibitors target a crevice between the N-terminal portion of the bridge helix and a surrounding cap region within which the bridge helix is thought to rearrange during the nucleotide addition cycle. Here, we report crystal structures of CBR inhibitor/Escherichia coli RNA polymerasemore » complexes as well as biochemical tests that establish two distinct effects of the inhibitors on the RNA polymerase catalytic site. One effect involves inhibition of trigger-loop folding via the F loop in the cap, which affects both nucleotide addition and hydrolysis of 3'-terminal dinucleotides in certain backtracked complexes. The second effect is trigger-loop independent, affects only nucleotide addition and pyrophosphorolysis, and may involve inhibition of bridge-helix movements that facilitate reactive triphosphate alignment.« less

PHYTOCHROME INTERACTING FACTOR3 Associates with the Histone Deacetylase HDA15 in Repression of Chlorophyll Biosynthesis and Photosynthesis in Etiolated Arabidopsis Seedlings[W][OA

PubMed Central

Liu, Xuncheng; Chen, Chia-Yang; Wang, Ko-Ching; Luo, Ming; Tai, Ready; Yuan, Lianyu; Zhao, Minglei; Yang, Songguang; Tian, Gang; Cui, Yuhai; Hsieh, Hsu-Liang; Wu, Keqiang

2013-01-01

PHYTOCHROME INTERACTING FACTOR3 (PIF3) is a key basic helix-loop-helix transcription factor of Arabidopsis thaliana that negatively regulates light responses, repressing chlorophyll biosynthesis, photosynthesis, and photomorphogenesis in the dark. However, the mechanism for the PIF3-mediated transcription regulation remains largely unknown. In this study, we found that the REDUCED POTASSIUM DEPENDENCY3/HISTONE DEACETYLASE1-type histone deacetylase HDA15 directly interacted with PIF3 in vivo and in vitro. Genome-wide transcriptome analysis revealed that HDA15 acts mainly as a transcriptional repressor and negatively regulates chlorophyll biosynthesis and photosynthesis gene expression in etiolated seedlings. HDA15 and PIF3 cotarget to the genes involved in chlorophyll biosynthesis and photosynthesis in the dark and repress gene expression by decreasing the acetylation levels and RNA Polymerase II–associated transcription. The binding of HDA15 to the target genes depends on the presence of PIF3. In addition, PIF3 and HDA15 are dissociated from the target genes upon exposure to red light. Taken together, our results indicate that PIF3 associates with HDA15 to repress chlorophyll biosynthetic and photosynthetic genes in etiolated seedlings. PMID:23548744

Loop-to-helix transition in the structure of multidrug regulator AcrR at the entrance of the drug-binding cavity.

PubMed

Manjasetty, Babu A; Halavaty, Andrei S; Luan, Chi-Hao; Osipiuk, Jerzy; Mulligan, Rory; Kwon, Keehwan; Anderson, Wayne F; Joachimiak, Andrzej

2016-04-01

Multidrug transcription regulator AcrR from Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 belongs to the tetracycline repressor family, one of the largest groups of bacterial transcription factors. The crystal structure of dimeric AcrR was determined and refined to 1.56Å resolution. The tertiary and quaternary structures of AcrR are similar to those of its homologs. The multidrug binding site was identified based on structural alignment with homologous proteins and has a di(hydroxyethyl)ether molecule bound. Residues from helices α4 and α7 shape the entry into this binding site. The structure of AcrR reveals that the extended helical conformation of helix α4 is stabilized by the hydrogen bond between Glu67 (helix α4) and Gln130 (helix α7). Based on the structural comparison with the closest homolog structure, the Escherichia coli AcrR, we propose that this hydrogen bond is responsible for control of the loop-to-helix transition within helix α4. This local conformational switch of helix α4 may be a key step in accessing the multidrug binding site and securing ligands at the binding site. Solution small-molecule binding studies suggest that AcrR binds ligands with their core chemical structure resembling the tetracyclic ring of cholesterol. Copyright © 2016. Published by Elsevier Inc.

TRANSPARENT TESTA GLABRA 1-Dependent Regulation of Flavonoid Biosynthesis

PubMed Central

Zhang, Bipei

2017-01-01

The flavonoid composition of various tissues throughout plant development is of biological relevance and particular interest for breeding. Arabidopsis thaliana TRANSPARENT TESTA GLABRA 1 (AtTTG1) is an essential regulator of late structural genes in flavonoid biosynthesis. Here, we provide a review of the regulation of the pathway’s core enzymes through AtTTG1-containing R2R3-MYELOBLASTOSIS-basic HELIX-LOOP-HELIX-WD40 repeat (MBW(AtTTG1)) complexes embedded in an evolutionary context. We present a comprehensive collection of A. thaliana ttg1 mutants and AtTTG1 orthologs. A plethora of MBW(AtTTG1) mechanisms in regulating the five major TTG1-dependent traits is highlighted. PMID:29261137

Structure and function of ameloblastin as an extracellular matrix protein: adhesion, calcium binding, and CD63 interaction in human and mouse.

PubMed

Zhang, Xu; Diekwisch, Thomas G H; Luan, Xianghong

2011-12-01

The functional significance of extracellular matrix proteins in the life of vertebrates is underscored by a high level of sequence variability in tandem with a substantial degree of conservation in terms of cell-cell and cell-matrix adhesion interactions. Many extracellular matrix proteins feature multiple adhesion domains for successful attachment to substrates, such as integrin, CD63, and heparin. Here we have used homology and ab initio modeling algorithms to compare mouse ameloblastin (mAMBN) and human ameloblastin (hABMN) isoforms and to analyze their potential for cell adhesion and interaction with other matrix molecules as well as calcium binding. Sequence comparison between mAMBN and hAMBN revealed a 26-amino-acid deletion in mAMBN, corresponding to a helix-loop-helix frameshift. The human AMBN domain (174Q-201G), homologous to the mAMBN 157E-178I helix-loop-helix region, formed a helix-loop motif with an extended loop, suggesting a higher degree of flexibility of hAMBN compared with mAMBN, as confirmed by molecular dynamics simulation. Heparin-binding domains, CD63-interaction domains, and calcium-binding sites in both hAMBN and mAMBN support the concept of AMBN as an extracellular matrix protein. The high level of conservation between AMBN functional domains related to adhesion and differentiation was remarkable when compared with only 61% amino acid sequence homology. © 2011 Eur J Oral Sci.

Thermodynamics of the pseudo-knot in helix 18 of 16S ribosomal RNA.

PubMed

Wojciechowska, Monika; Dudek, Marta; Trylska, Joanna

2018-04-01

A fragment of E. coli 16S rRNA formed by nucleotides 500 to 545 is termed helix 18. Nucleotides 505-507 and 524-526 form a pseudo-knot and its distortion affects ribosome function. Helix 18 isolated from the ribosome context is thus an interesting fragment to investigate the structural properties and folding of RNA with pseudo-knots. With all-atom molecular dynamics simulations, spectroscopic and gel electrophoresis experiments, we investigated thermodynamics of helix 18, with a focus on its pseudo-knot. In solution studies at ambient conditions we observed dimerization of helix 18. We proposed that the loop, containing nucleotides forming the pseudo-knot, interacts with another monomer of helix 18. The native dimer is difficult to break but introducing mutations in the pseudo-knot indeed assured a monomeric form of helix 18. Molecular dynamics simulations at 310 K confirmed the stability of the pseudo-knot but at elevated temperatures this pseudo-knot was the first part of helix 18 to lose the hydrogen bond pattern. To further determine helix 18 stability, we analyzed the interactions of helix 18 with short oligomers complementary to a nucleotide stretch containing the pseudo-knot. The formation of higher-order structures by helix 18 impacts hybridization efficiency of peptide nucleic acid and 2'-O methyl RNA oligomers. © 2018 Wiley Periodicals, Inc.

Regulation of TCF ETS-domain transcription factors by helix-loop-helix motifs.

PubMed

Stinson, Julie; Inoue, Toshiaki; Yates, Paula; Clancy, Anne; Norton, John D; Sharrocks, Andrew D

2003-08-15

DNA binding by the ternary complex factor (TCF) subfamily of ETS-domain transcription factors is tightly regulated by intramolecular and intermolecular interactions. The helix-loop-helix (HLH)-containing Id proteins are trans-acting negative regulators of DNA binding by the TCFs. In the TCF, SAP-2/Net/ERP, intramolecular inhibition of DNA binding is promoted by the cis-acting NID region that also contains an HLH-like motif. The NID also acts as a transcriptional repression domain. Here, we have studied the role of HLH motifs in regulating DNA binding and transcription by the TCF protein SAP-1 and how Cdk-mediated phosphorylation affects the inhibitory activity of the Id proteins towards the TCFs. We demonstrate that the NID region of SAP-1 is an autoinhibitory motif that acts to inhibit DNA binding and also functions as a transcription repression domain. This region can be functionally replaced by fusion of Id proteins to SAP-1, whereby the Id moiety then acts to repress DNA binding in cis. Phosphorylation of the Ids by cyclin-Cdk complexes results in reduction in protein-protein interactions between the Ids and TCFs and relief of their DNA-binding inhibitory activity. In revealing distinct mechanisms through which HLH motifs modulate the activity of TCFs, our results therefore provide further insight into the role of HLH motifs in regulating TCF function and how the inhibitory properties of the trans-acting Id HLH proteins are themselves regulated by phosphorylation.

A single amino acid substitution in IIIf subfamily of basic helix-loop-helix transcription factor AtMYC1 leads to trichome and root hair patterning defects by abolishing its interaction with partner proteins in Arabidopsis.

PubMed

Zhao, Hongtao; Wang, Xiaoxue; Zhu, Dandan; Cui, Sujuan; Li, Xia; Cao, Ying; Ma, Ligeng

2012-04-20

Plant trichomes and root hairs are powerful models for the study of cell fate determination. In Arabidopsis thaliana, trichome and root hair initiation requires a combination of three groups of proteins, including the WD40 repeat protein transparent TESTA GLABRA1 (TTG1), R2R3 repeat MYB protein GLABRA1 (GL1), or werewolf (WER) and the IIIf subfamily of basic helix-loop-helix (bHLH) protein GLABRA3 (GL3) or enhancer of GLABRA3 (EGL3). The bHLH component acts as a docking site for TTG1 and MYB proteins. Here, we isolated a mutant showing defects in trichome and root hair patterning that carried a point mutation (R173H) in AtMYC1 that encodes the fourth member of IIIf bHLH family protein. Genetic analysis revealed partial redundant yet distinct function between AtMYC1 and GL3/EGL3. GLABRA2 (GL2), an important transcription factor involved in trichome and root hair control, was down-regulated in Atmyc1 plants, suggesting the requirement of AtMYC1 for appropriate GL2 transcription. Like its homologs, AtMYC1 formed a complex with TTG1 and MYB proteins but did not dimerized. In addition, the interaction of AtMYC1 with MYB proteins and TTG1 was abrogated by the R173H substitution in Atmyc1-1. We found that this amino acid (Arg) is conserved in the AtMYC1 homologs GL3/EGL3 and that it is essential for their interaction with MYB proteins and for their proper functions. Our findings indicate that AtMYC1 is an important regulator of trichome and root hair initiation, and they reveal a novel amino acid necessary for protein-protein interactions and gene function in IIIf subfamily bHLH transcription factors.

A Single Amino Acid Substitution in IIIf Subfamily of Basic Helix-Loop-Helix Transcription Factor AtMYC1 Leads to Trichome and Root Hair Patterning Defects by Abolishing Its Interaction with Partner Proteins in Arabidopsis*

PubMed Central

Zhao, Hongtao; Wang, Xiaoxue; Zhu, Dandan; Cui, Sujuan; Li, Xia; Cao, Ying; Ma, Ligeng

2012-01-01

Plant trichomes and root hairs are powerful models for the study of cell fate determination. In Arabidopsis thaliana, trichome and root hair initiation requires a combination of three groups of proteins, including the WD40 repeat protein TRANSPARENT TESTA GLABRA1 (TTG1), R2R3 repeat MYB protein GLABRA1 (GL1), or WEREWOLF (WER) and the IIIf subfamily of basic helix-loop-helix (bHLH) protein GLABRA3 (GL3) or ENHANCER OF GLABRA3 (EGL3). The bHLH component acts as a docking site for TTG1 and MYB proteins. Here, we isolated a mutant showing defects in trichome and root hair patterning that carried a point mutation (R173H) in AtMYC1 that encodes the fourth member of IIIf bHLH family protein. Genetic analysis revealed partial redundant yet distinct function between AtMYC1 and GL3/EGL3. GLABRA2 (GL2), an important transcription factor involved in trichome and root hair control, was down-regulated in Atmyc1 plants, suggesting the requirement of AtMYC1 for appropriate GL2 transcription. Like its homologs, AtMYC1 formed a complex with TTG1 and MYB proteins but did not dimerized. In addition, the interaction of AtMYC1 with MYB proteins and TTG1 was abrogated by the R173H substitution in Atmyc1-1. We found that this amino acid (Arg) is conserved in the AtMYC1 homologs GL3/EGL3 and that it is essential for their interaction with MYB proteins and for their proper functions. Our findings indicate that AtMYC1 is an important regulator of trichome and root hair initiation, and they reveal a novel amino acid necessary for protein-protein interactions and gene function in IIIf subfamily bHLH transcription factors. PMID:22334670

The basic helix-loop-helix differentiation factor Nex1/MATH-2 functions as a key activator of the GAP-43 gene

PubMed Central

Uittenbogaard, Martine; Martinka, Debra L.; Chiaramello, Anne

2006-01-01

Nex1/MATH-2 is a neurogenic basic Helix-Loop-Helix (bHLH) transcription factor that belongs to the NeuroD subfamily. Its expression parallels that of the GAP-43 gene and peaks during brain development, when neurite outgrowth and synaptogenesis are highly active. We previously observed a direct correlation between the levels of expression of Nex1 and GAP-43 proteins, which resulted in extensive neurite outgrowth and neuronal differentiation of PC12 cells in the absence of nerve growth factor. Since the GAP-43 gene is a target for bHLH regulation, we investigated whether Nex1 could regulate the activity of the GAP-43 promoter. We found that among the members of the NeuroD subfamily, Nex1 promoted maximal activity of the GAP-43 promoter. The Nex1-mediated activity is restricted to the conserved E1–E2 cluster located near the major transcription start sites. By electrophoretic mobility shift assay and site-directed mutagenesis, we showed that Nex1 binds as homodimers and that the E1 E-box is a high affinity binding site. We further found that Nex1 released the ME1 E-protein-mediated repression in a concentration dependent manner. Thus, the E1–E2 cluster has a dual function: it can mediate activation or repression depending on the interacting bHLH proteins. Finally, a series of N-terminal and C-terminal deletions revealed that Nex1 transcriptional activity is linked to two distinct transactivation domains, TAD1 and TAD2, with TAD1 being unique to Nex1. Together, our results suggest that Nex1 may engage in selective interactions with components of the core transcriptional machinery whose assembly is dictated by the architecture of the GAP-43 promoter and cellular environment. PMID:12562512

De novo design, synthesis and characterisation of MP3, a new catalytic four-helix bundle hemeprotein.

PubMed

Faiella, Marina; Maglio, Ornella; Nastri, Flavia; Lombardi, Angela; Lista, Liliana; Hagen, Wilfred R; Pavone, Vincenzo

2012-12-07

A new artificial metalloenzyme, MP3 (MiniPeroxidase 3), designed by combining the excellent structural properties of four-helix bundle protein scaffolds with the activity of natural peroxidases, was synthesised and characterised. This new hemeprotein model was developed by covalently linking the deuteroporphyrin to two peptide chains of different compositions to obtain an asymmetric helix-loop-helix/heme/helix-loop-helix sandwich arrangement, characterised by 1) a His residue on one chain that acts as an axial ligand to the iron ion; 2) a vacant distal site that is able to accommodate exogenous ligands or substrates; and 3) an Arg residue in the distal site that should assist in hydrogen peroxide activation to give an HRP-like catalytic process. MP3 was synthesised and characterised as its iron complex. CD measurements revealed the high helix-forming propensity of the peptide, confirming the appropriateness of the model procedure; UV/Vis, MCD and EPR experiments gave insights into the coordination geometry and the spin state of the metal. Kinetic experiments showed that Fe(III)-MP3 possesses peroxidase-like activity comparable to R38A-hHRP, highlighting the possibility of mimicking the functional features of natural enzymes. The synergistic application of de novo design methods, synthetic procedures, and spectroscopic characterisation, described herein, demonstrates a method by which to implement and optimise catalytic activity for an enzyme mimetic. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dimerization of the docking/adaptor protein HEF1 via a carboxy-terminal helix-loop-helix domain.

PubMed

Law, S F; Zhang, Y Z; Fashena, S J; Toby, G; Estojak, J; Golemis, E A

1999-10-10

HEF1, p130(Cas), and Efs define a family of multidomain docking proteins which plays a central coordinating role for tyrosine-kinase-based signaling related to cell adhesion. HEF1 function has been specifically implicated in signaling pathways important for cell adhesion and differentiation in lymphoid and epithelial cells. While the SH3 domains and SH2-binding site domains (substrate domains) of HEF1 family proteins are well characterized and binding partners known, to date the highly conserved carboxy-terminal domains of the three proteins have lacked functional definition. In this study, we have determined that the carboxy-terminal domain of HEF1 contains a divergent helix-loop-helix (HLH) motif. This motif mediates HEF1 homodimerization and HEF1 heterodimerization with a recognition specificity similar to that of the transcriptional regulatory HLH proteins Id2, E12, and E47. We had previously demonstrated that the HEF1 carboxy-terminus expressed as a separate domain in yeast reprograms cell division patterns, inducing constitutive pseudohyphal growth. Here we show that pseudohyphal induction by HEF1 requires an intact HLH, further supporting the idea that this motif has an effector activity for HEF1, and implying that HEF1 pseudohyphal activity derives in part from interactions with yeast helix-loop-helix proteins. These combined results provide initial insight into the mode of function of the HEF1 carboxy-terminal domain and suggest that the HEF1 protein may interact with cellular proteins which control differentiation. Copyright 1999 Academic Press.

A curved RNA helix incorporating an internal loop with G·A and A·A non-Watson–Crick base pairing

PubMed Central

Baeyens, Katrien J.; De Bondt, Hendrik L.; Pardi, Arthur; Holbrook, Stephen R.

1996-01-01

The crystal structure of the RNA dodecamer 5′-GGCC(GAAA)GGCC-3′ has been determined from x-ray diffraction data to 2.3-Å resolution. In the crystal, these oligomers form double helices around twofold symmetry axes. Four consecutive non-Watson–Crick base pairs make up an internal loop in the middle of the duplex, including sheared G·A pairs and novel asymmetric A·A pairs. This internal loop sequence produces a significant curvature and narrowing of the double helix. The helix is curved by 34° from end to end and the diameter is narrowed by 24% in the internal loop. A Mn2+ ion is bound directly to the N7 of the first guanine in the Watson–Crick region following the internal loop and the phosphate of the preceding residue. This Mn2+ location corresponds to a metal binding site observed in the hammerhead catalytic RNA. PMID:8917508

Prokaryotic and eukaryotic features observed on the secondary structures of Giardia SSU rRNAs and its phylogenetic implications.

PubMed

Hwang, Ui Wook

2007-04-01

Phylogenetic position of a diplomonad protist Giardia, a principle cause of diarrhea, among eukaryotes has been vigorously debated so far. Through the comparisons of primary and secondary structures of SSU rRNAs of G. intestinalis, G. microti, G. ardeae, and G. muris, I found two major indel regions (a 6-nt indel and a 22-26-nt indel), which correspond to the helix 10 of the V2 region and helices E23-8 to E23-9 of the V4 region, respectively. As generally shown in eukaryotes, G. intestinalis and G. microti have commonly a relatively longer helix 10 (a 7-bp stem and a 4-nt loop), and also the eukaryote-specific helices E23-6 to E23-9. On the other hand, G. muris and G. ardeae have a shorter helix 10: a 2-bp stem and a 6-nt loop in G. ardeae and a 3-bp stem and a 6-nt loop in G. muris. In the V4, they have a single long helix (like the P23-1 helix in prokaryotes) instead of the helices E23-6 to E23-9. Among the four Giardia species, co-appearance of prokaryote- and eukaryote-typical features might be significant evidence to suggest that Giardia (Archezoa) is a living fossil showing an "intermediate stage" during the evolution from prokaryotes to eukaryotes.

Proprioceptor pathway development is dependent on Math1

NASA Technical Reports Server (NTRS)

Bermingham, N. A.; Hassan, B. A.; Wang, V. Y.; Fernandez, M.; Banfi, S.; Bellen, H. J.; Fritzsch, B.; Zoghbi, H. Y.

2001-01-01

The proprioceptive system provides continuous positional information on the limbs and body to the thalamus, cortex, pontine nucleus, and cerebellum. We showed previously that the basic helix-loop-helix transcription factor Math1 is essential for the development of certain components of the proprioceptive pathway, including inner-ear hair cells, cerebellar granule neurons, and the pontine nuclei. Here, we demonstrate that Math1 null embryos lack the D1 interneurons and that these interneurons give rise to a subset of proprioceptor interneurons and the spinocerebellar and cuneocerebellar tracts. We also identify three downstream genes of Math1 (Lh2A, Lh2B, and Barhl1) and establish that Math1 governs the development of multiple components of the proprioceptive pathway.

Interactions between relay helix and Src homology 1 domain helix (SH1) drive the converter domain rotation during the recovery stroke of myosin II

PubMed Central

Baumketner, Andrij

2012-01-01

Myosin motor protein exists in two alternative conformations, pre-recovery state M* and post-recovery state M**, upon ATP binding. The details of the M*-to-M** transition, known as the recovery stroke to reflect its role as the functional opposite of the force-generating power stroke, remain elusive. The defining feature of the post-recovery state is a kink in the relay helix, a key part of the protein involved in force generation. In this paper we determine the interactions that are responsible for the appearance of the kink. We design a series of computational models that contain three other segments, relay loop, converter domain and Src homology 1 domain helix (SH1), with which relay helix interacts, and determine their structure in accurate replica exchange molecular dynamics simulations in explicit solvent. By conducting an exhaustive combinatorial search among different models we find that: 1) the converter domain must be attached to the relay helix during the transition, so it does not interfere with other parts of the protein, 2) the structure of the relay helix is controlled by SH1 helix. The kink is strongly coupled to the position of SH1 helix. It arises as a result of direct interactions between SH1 and the relay helix and leads to a rotation of the C-terminal part of the relay helix which is subsequently transmitted to the converter domain. PMID:22411190

Nature of the Charged-Group Effect on the Stability of the C-Peptide Helix

NASA Astrophysics Data System (ADS)

Shoemaker, Kevin R.; Kim, Peter S.; Brems, David N.; Marqusee, Susan; York, Eunice J.; Chaiken, Irwin M.; Stewart, John M.; Baldwin, Robert L.

1985-04-01

The residues responsible for the pH-dependent stability of the helix formed by the isolated C-peptide (residues 1-13 of ribonuclease A) have been identified by chemical synthesis of analogues and measurement of their helix-forming properties. Each of the residues ionizing between pH 2 and pH 8 has been replaced separately by an uncharged residue. Protonation of Glu-2- is responsible for the sharp decrease in helix stability between pH 5 and pH 2, and deprotonation of His-12+ causes a similar decrease between pH 5 and pH 8. Glu-9- is not needed for helix stability. The results cannot be explained by the Zimm-Bragg model and host-guest data for α -helix formation, which predict that the stability of the C-peptide helix should increase when Glu-2- is protonated or when His-12+ is deprotonated. Moreover, histidine+ is a strong helix-breaker in host-guest studies. In proteins, acidic and basic residues tend to occur at opposite ends of α -helices: acidic residues occur preferentially near the NH2-terminal end and basic residues near the COOH-terminal end. A possible explanation, based on a helix dipole model, has been given [Blagdon, D. E. & Goodman, M. (1975) Biopolymers 14, 241-245]. Our results are consistent with the helix dipole model and they support the suggestion that the distribution of charged residues in protein helices reflects the helix-stabilizing propensity of those residues. Because Glu-9 is not needed for helix stability, a possible Glu-9-\\cdots His-12+ salt bridge does not contribute significantly to helix stability. The role of a possible Glu-2-\\cdots Arg-10+ salt bridge has not yet been evaluated. A charged-group effect on α -helix stability in water has also been observed in a different peptide system [Ihara, S., Ooi, T. & Takahashi, S. (1982) Biopolymers 21, 131-145]: block copolymers containing (Ala)20 and (Glu)20 show partial helix formation at low temperatures, pH 7.5, where the glutamic acid residues are ionized. (Glu)20(Ala)20Phe forms a helix that is markedly more stable than (Ala)20(Glu)20Phe. The results are consistent with a helix dipole model.

MYC2 Differentially Modulates Diverse Jasmonate-Dependent Functions in Arabidopsis[W

PubMed Central

Dombrecht, Bruno; Xue, Gang Ping; Sprague, Susan J.; Kirkegaard, John A.; Ross, John J.; Reid, James B.; Fitt, Gary P.; Sewelam, Nasser; Schenk, Peer M.; Manners, John M.; Kazan, Kemal

2007-01-01

The Arabidopsis thaliana basic helix-loop-helix Leu zipper transcription factor (TF) MYC2/JIN1 differentially regulates jasmonate (JA)-responsive pathogen defense (e.g., PDF1.2) and wound response (e.g., VSP) genes. In this study, genome-wide transcriptional profiling of wild type and mutant myc2/jin1 plants followed by functional analyses has revealed new roles for MYC2 in the modulation of diverse JA functions. We found that MYC2 negatively regulates Trp and Trp-derived secondary metabolism such as indole glucosinolate biosynthesis during JA signaling. Furthermore, MYC2 positively regulates JA-mediated resistance to insect pests, such as Helicoverpa armigera, and tolerance to oxidative stress, possibly via enhanced ascorbate redox cycling and flavonoid biosynthesis. Analyses of MYC2 cis binding elements and expression of MYC2-regulated genes in T-DNA insertion lines of a subset of MYC2–regulated TFs suggested that MYC2 might modulate JA responses via differential regulation of an intermediate spectrum of TFs with activating or repressing roles in JA signaling. MYC2 also negatively regulates its own expression, and this may be one of the mechanisms used in fine-tuning JA signaling. Overall, these results provide new insights into the function of MYC2 and the transcriptional coordination of the JA signaling pathway. PMID:17616737

Structure of the GH1 domain of guanylate kinase-associated protein from Rattus norvegicus

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

Tong, Junsen; Yang, Huiseon; Eom, Soo Hyun

2014-09-12

Graphical abstract: - Highlights: • The crystal structure of GKAP homology domain 1 (GH1) was determined. • GKAP GH1 is a three-helix bundle connected by short flexible loops. • The predicted helix α4 associates weakly with the helix α3, suggesting dynamic nature of the GH1 domain. - Abstract: Guanylate-kinase-associated protein (GKAP) is a scaffolding protein that links NMDA receptor-PSD-95 to Shank–Homer complexes by protein–protein interactions at the synaptic junction. GKAP family proteins are characterized by the presence of a C-terminal conserved GKAP homology domain 1 (GH1) of unknown structure and function. In this study, crystal structure of the GH1 domainmore » of GKAP from Rattus norvegicus was determined in fusion with an N-terminal maltose-binding protein at 2.0 Å resolution. The structure of GKAP GH1 displays a three-helix bundle connected by short flexible loops. The predicted helix α4 which was not visible in the crystal structure associates weakly with the helix α3 suggesting dynamic nature of the GH1 domain. The strict conservation of GH1 domain across GKAP family members and the lack of a catalytic active site required for enzyme activity imply that the GH1 domain might serve as a protein–protein interaction module for the synaptic protein clustering.« less

Local conformational dynamics in alpha-helices measured by fast triplet transfer.

PubMed

Fierz, Beat; Reiner, Andreas; Kiefhaber, Thomas

2009-01-27

Coupling fast triplet-triplet energy transfer (TTET) between xanthone and naphthylalanine to the helix-coil equilibrium in alanine-based peptides allowed the observation of local equilibrium fluctuations in alpha-helices on the nanoseconds to microseconds time scale. The experiments revealed faster helix unfolding in the terminal regions compared with the central parts of the helix with time constants varying from 250 ns to 1.4 micros at 5 degrees C. Local helix formation occurs with a time constant of approximately 400 ns, independent of the position in the helix. Comparing the experimental data with simulations using a kinetic Ising model showed that the experimentally observed dynamics can be explained by a 1-dimensional boundary diffusion with position-independent elementary time constants of approximately 50 ns for the addition and of approximately 65 ns for the removal of an alpha-helical segment. The elementary time constant for helix growth agrees well with previously measured time constants for formation of short loops in unfolded polypeptide chains, suggesting that helix elongation is mainly limited by a conformational search.

GTF2IRD2 is located in the Williams-Beuren syndrome critical region 7q11.23 and encodes a protein with two TFII-I-like helix-loop-helix repeats.

PubMed

Makeyev, Aleksandr V; Erdenechimeg, Lkhamsuren; Mungunsukh, Ognoon; Roth, Jutta J; Enkhmandakh, Badam; Ruddle, Frank H; Bayarsaihan, Dashzeveg

2004-07-27

Williams-Beuren syndrome (also known as Williams syndrome) is caused by a deletion of a 1.55- to 1.84-megabase region from chromosome band 7q11.23. GTF2IRD1 and GTF2I, located within this critical region, encode proteins of the TFII-I family with multiple helix-loop-helix domains known as I repeats. In the present work, we characterize a third member, GTF2IRD2, which has sequence and structural similarity to the GTF2I and GTF2IRD1 paralogs. The ORF encodes a protein with several features characteristic of regulatory factors, including two I repeats, two leucine zippers, and a single Cys-2/His-2 zinc finger. The genomic organization of human, baboon, rat, and mouse genes is well conserved. Our exon-by-exon comparison has revealed that GTF2IRD2 is more closely related to GTF2I than to GTF2IRD1 and apparently is derived from the GTF2I sequence. The comparison of GTF2I and GTF2IRD2 genes revealed two distinct regions of homology, indicating that the helix-loop-helix domain structure of the GTF2IRD2 gene has been generated by two independent genomic duplications. We speculate that GTF2I is derived from GTF2IRD1 as a result of local duplication and the further evolution of its structure was associated with its functional specialization. Comparison of genomic sequences surrounding GTF2IRD2 genes in mice and humans allows refinement of the centromeric breakpoint position of the primate-specific inversion within the Williams-Beuren syndrome critical region.

Control of regulatory T cell and Th17 cell differentiation by inhibitory helix-loop-helix protein Id3

PubMed Central

Maruyama, Takashi; Li, Jun; Vaque, Jose P.; Konkel, Joanne E.; Wang, Weifeng; Zhang, Baojun; Zhang, Pin; Zamarron, Brian; Yu, Dongyang; Wu, Yuntao; Zhuang, Yuan; Gutkind, J. Silvio; Chen, WanJun

2010-01-01

The molecular mechanisms directing Foxp3 gene transcription in CD4+ T cells remain ill defined. We show that deletion of the inhibitory helix-loop-helix (HLH) protein Id3 results in defective Foxp3+ Treg cell generation. We identified two transforming grothw factor-β1 (TGF-β1)-dependent mechanisms that are vital for activation of Foxp3 gene transcription, and are defective in Id3−/− CD4+ T cells. Enhanced binding of the HLH protein E2A to the Foxp3 promoter promoted Foxp3 gene transcription. Id3 was required to relieve inhibition by GATA-3 at the Foxp3 promoter. Further, Id3−/− T cells increased differentiation of Th17 cells in vitro and in a mouse asthma model. A network of factors therefore act in a TGF-β-dependent manner to control Foxp3 expression and inhibit Th17 cell development. PMID:21131965

Thymocyte Maturation Is Regulated by the Activity of the Helix-Loop-Helix Protein, E47

PubMed Central

Bain, Gretchen; Quong, Melanie W.; Soloff, Rachel S.; Hedrick, Stephen M.; Murre, Cornelis

1999-01-01

The E2A proteins, E12 and E47, are required for progression through multiple developmental pathways, including early B and T lymphopoiesis. Here, we provide in vitro and in vivo evidence demonstrating that E47 activity regulates double-positive thymocyte maturation. In the absence of E47 activity, positive selection of both major histocompatibility complex (MHC) class I– and class II–restricted T cell receptors (TCRs) is perturbed. Additionally, development of CD8 lineage T cells in an MHC class I–restricted TCR transgenic background is sensitive to the dosage of E47. Mice deficient for E47 display an increase in production of mature CD4 and CD8 lineage T cells. Furthermore, ectopic expression of an E2A inhibitor helix-loop-helix protein, Id3, promotes the in vitro differentiation of an immature T cell line. These results demonstrate that E2A functions as a regulator of thymocyte positive selection. PMID:10587351

Novel and recurrent non-truncating mutations of the MITF basic domain: genotypic and phenotypic variations in Waardenburg and Tietz syndromes

PubMed Central

Léger, Sandy; Balguerie, Xavier; Goldenberg, Alice; Drouin-Garraud, Valérie; Cabot, Annick; Amstutz-Montadert, Isabelle; Young, Paul; Joly, Pascal; Bodereau, Virginie; Holder-Espinasse, Muriel; Jamieson, Robyn V; Krause, Amanda; Chen, Hongsheng; Baumann, Clarisse; Nunes, Luis; Dollfus, Hélène; Goossens, Michel; Pingault, Véronique

2012-01-01

The microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix leucine zipper transcription factor, which regulates melanocyte development and the biosynthetic melanin pathway. A notable relationship has been described between non-truncating mutations of its basic domain and Tietz syndrome, which is characterized by albinoid-like hypopigmentation of the skin and hair, rather than the patchy depigmentation seen in Waardenburg syndrome, and severe hearing loss. Twelve patients with new or recurrent non-truncating mutations of the MITF basic domain from six families were enrolled in this study. We observed a wide range of phenotypes and some unexpected features. All the patients had blue irides and pigmentation abnormalities that ranged from diffuse hypopigmentation to Waardenburg-like patches. In addition, they showed congenital complete hearing loss, diffuse hypopigmentation of the skin, freckling and ocular abnormalities, more frequently than patients with MITF mutations outside the basic domain. In conclusion, the non-truncating mutations of the basic domain do not always lead to Tietz syndrome but rather to a large range of phenotypes. Sun-exposed freckles are interestingly observed more frequently in Asian populations. This variability argues for the possible interaction with modifier loci. PMID:22258527

Flavonoids: biosynthesis, biological functions, and biotechnological applications

PubMed Central

Falcone Ferreyra, María L.; Rius, Sebastián P.; Casati, Paula

2012-01-01

Flavonoids are widely distributed secondary metabolites with different metabolic functions in plants. The elucidation of the biosynthetic pathways, as well as their regulation by MYB, basic helix-loop-helix (bHLH), and WD40-type transcription factors, has allowed metabolic engineering of plants through the manipulation of the different final products with valuable applications. The present review describes the regulation of flavonoid biosynthesis, as well as the biological functions of flavonoids in plants, such as in defense against UV-B radiation and pathogen infection, nodulation, and pollen fertility. In addition, we discuss different strategies and achievements through the genetic engineering of flavonoid biosynthesis with implication in the industry and the combinatorial biosynthesis in microorganisms by the reconstruction of the pathway to obtain high amounts of specific compounds. PMID:23060891

Genome Wide Identification and Characterization of Apple bHLH Transcription Factors and Expression Analysis in Response to Drought and Salt Stress.

PubMed

Mao, Ke; Dong, Qinglong; Li, Chao; Liu, Changhai; Ma, Fengwang

2017-01-01

The bHLH (basic helix-loop-helix) transcription factor family is the second largest in plants. It occurs in all three eukaryotic kingdoms, and plays important roles in regulating growth and development. However, family members have not previously been studied in apple. Here, we identified 188 MdbHLH proteins in apple "Golden Delicious" ( Malus × domestica Borkh.), which could be classified into 18 groups. We also investigated the gene structures and 12 conserved motifs in these MdbHLH s. Coupled with expression analysis and protein interaction network prediction, we identified several genes that might be responsible for abiotic stress responses. This study provides insight and rich resources for subsequent investigations of such proteins in apple.

Emerging role of Twist1 in fibrotic diseases.

PubMed

Ning, Xiaoxuan; Zhang, Kun; Wu, Qingfeng; Liu, Minna; Sun, Shiren

2018-03-01

Epithelial-mesenchymal transition (EMT) is a pathological process that occurs in a variety of diseases, including organ fibrosis. Twist1, a basic helix-loop-helix transcription factor, is involved in EMT and plays significant roles in various fibrotic diseases. Suppression of the EMT process represents a promising approach for the treatment of fibrotic diseases. In this review, we discuss the roles and the underlying molecular mechanisms of Twist1 in fibrotic diseases, including those affecting kidney, lung, skin, oral submucosa and other tissues. We aim at providing new insight into the pathogenesis of various fibrotic diseases and facilitating the development of novel diagnostic and therapeutic methods for their treatment. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Circadian transcription factor BMAL1 regulates innate immunity against select RNA viruses.

PubMed

Majumdar, Tanmay; Dhar, Jayeeta; Patel, Sonal; Kondratov, Roman; Barik, Sailen

2017-02-01

BMAL1 (brain and muscle ARNT-like protein 1, also known as MOP3 or ARNT3) belongs to the family of the basic helix-loop-helix (bHLH)-PAS domain-containing transcription factors, and is a key component of the molecular oscillator that generates circadian rhythms. Here, we report that BMAL1-deficient cells are significantly more susceptible to infection by two major respiratory viruses of the Paramyxoviridae family, namely RSV and PIV3. Embryonic fibroblasts from Bmal1 -/- mice produced nearly 10-fold more progeny virus than their wild type controls. These results were supported by animal studies whereby pulmonary infection of RSV produced a more severe disease and morbidity in Bmal1 -/- mice. These results show that BMAL1 can regulate cellular innate immunity against specific RNA viruses.

Genome Wide Identification and Characterization of Apple bHLH Transcription Factors and Expression Analysis in Response to Drought and Salt Stress

PubMed Central

Mao, Ke; Dong, Qinglong; Li, Chao; Liu, Changhai; Ma, Fengwang

2017-01-01

The bHLH (basic helix-loop-helix) transcription factor family is the second largest in plants. It occurs in all three eukaryotic kingdoms, and plays important roles in regulating growth and development. However, family members have not previously been studied in apple. Here, we identified 188 MdbHLH proteins in apple “Golden Delicious” (Malus × domestica Borkh.), which could be classified into 18 groups. We also investigated the gene structures and 12 conserved motifs in these MdbHLHs. Coupled with expression analysis and protein interaction network prediction, we identified several genes that might be responsible for abiotic stress responses. This study provides insight and rich resources for subsequent investigations of such proteins in apple. PMID:28443104

STARD4 Membrane Interactions and Sterol Binding

PubMed Central

2016-01-01

The steroidogenic acute regulatory protein-related lipid transfer (START) domain family is defined by a conserved 210-amino acid sequence that folds into an α/β helix-grip structure. Members of this protein family bind a variety of ligands, including cholesterol, phospholipids, sphingolipids, and bile acids, with putative roles in nonvesicular lipid transport, metabolism, and cell signaling. Among the soluble START proteins, STARD4 is expressed in most tissues and has previously been shown to transfer sterol, but the molecular mechanisms of membrane interaction and sterol binding remain unclear. In this work, we use biochemical techniques to characterize regions of STARD4 and determine their role in membrane interaction and sterol binding. Our results show that STARD4 interacts with anionic membranes through a surface-exposed basic patch and that introducing a mutation (L124D) into the Omega-1 (Ω1) loop, which covers the sterol binding pocket, attenuates sterol transfer activity. To gain insight into the attenuating mechanism of the L124D mutation, we conducted structural and biophysical studies of wild-type and L124D STARD4. These studies show that the L124D mutation reduces the conformational flexibility of the protein, resulting in a diminished level of membrane interaction and sterol transfer. These studies also reveal that the C-terminal α-helix, and not the Ω1 loop, partitions into the membrane bilayer. On the basis of these observations, we propose a model of STARD4 membrane interaction and sterol binding and release that requires dynamic movement of both the Ω1 loop and membrane insertion of the C-terminal α-helix. PMID:26168008

Ser298 of MITF, a mutation site in Waardenburg syndrome type 2, is a phosphorylation site with functional significance.

PubMed

Takeda, K; Takemoto, C; Kobayashi, I; Watanabe, A; Nobukuni, Y; Fisher, D E; Tachibana, M

2000-01-01

MITF (microphthalmia-associated transcription factor) is a basic-helix-loop-helix-leucine zipper (bHLHZip) factor which regulates expression of tyrosinase and other melanocytic genes via a CATGTG promoter sequence, and is involved in melanocyte differentiation. Mutations of MITF in mice or humans with Waardenburg syndrome type 2 (WS2) often severely disrupt the bHLHZip domain, suggesting the importance of this structure. Here, we show that Ser298, which locates downstream of the bHLHZip and was previously found to be mutated in individuals with WS2, plays an important role in MITF function. Glycogen synthase kinase 3 (GSK3) was found to phosphorylate Ser298 in vitro, thereby enhancing the binding of MITF to the tyrosinase promoter. The same serine was found to be phosphorylated in vivo, and expression of dominant-negative GSK3beta selectively suppressed the ability of MITF to transactivate the tyrosinase promoter. Moreover, mutation of Ser298, as found in a WS2 family, disabled phos-phorylation of MITF by GSK3beta and impaired MITF function. These findings suggest that the Ser298 is important for MITF function and is phosphorylated probably by GSK3beta.

Ubiquitination-Related MdBT Scaffold Proteins Target a bHLH Transcription Factor for Iron Homeostasis1[OPEN

PubMed Central

Zhao, Qiang; Wang, Qing-Jie; Wang, Xiao-Fei; You, Chun-Xiang

2016-01-01

Iron (Fe) homeostasis is crucial for plant growth and development. A network of basic helix-loop-helix (bHLH) transcription factors positively regulates Fe uptake during iron deficiency. However, their up-regulation or overexpression leads to Fe overload and reactive oxygen species generation, thereby damaging the plants. Here, we found that two BTB/TAZ proteins, MdBT1 and MdBT2, interact with the MbHLH104 protein in apple. In addition, the function of MdBT2 was characterized as a regulator of MdbHLH104 degradation via ubiquitination and the 26S proteasome pathway, thereby controlling the activity of plasma membrane H+-ATPases and the acquisition of iron. Furthermore, MdBT2 interacted with MdCUL3 proteins, which were required for the MdBT2-mediated ubiquitination modification of MdbHLH104 and its degradation. In sum, our findings demonstrate that MdBT proteins interact with MdCUL3 to bridge the formation of the MdBTsMdCUL3 complex, which negatively modulates the degradation of the MdbHLH104 protein in response to changes in Fe status to maintain iron homeostasis in plants. PMID:27660166

Genomic cloning and chromosomal localization of HRY, the human homolog to the Drosophila segmentation gene, hairy

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

Feder, J.N.; Jan, L.Y.; Jan, Y.N.

The Drosophila hairy gene encodes a basic helix- loop-helix protein that functions in at least two steps during Drosophila development: (1) during embryogenesis, when it partakes in the establishment of segments, and (2) during the larval stage, when it functions negatively in determining the pattern of sensory bristles on the adult fly. In the rat, a structurally homologous gene (RHL) behaves as an immediate-early gene in its response to growth factors and can, like that in Drosophila, suppress neuronal differentiation events. Here, the authors report the genomic cloning of the human hairy gene homolog (HRY). The coding region of themore » gene is contained within four exons. The predicted amino acid sequence reveals only four amino acid differences between the human and rat genes. Analysis of the DNA sequence 5[prime] to the coding region reveals a putatitve untranslated exon. To increase the value of the HRY gene as a genetic marker and to assess its potential involvement in genetic disorders, they sublocalized the locus to chromosome 3q28-q29 by fluorescence in situ hybridization. 34 refs., 4 figs., 1 tab.« less

Determination of the force systems produced by different configurations of tear drop orthodontic loops.

PubMed

Thiesen, Guilherme; Shimizu, Roberto Hideo; do Valle, Caio Vinicius Martins; do Valle-Corotti, Karyna Martins; Pereira, Jefferson Ricardo; Conti, Paulo Cesar Rodrigues

2013-03-15

To determine the mechanical characteristics of teardrop loop with and without helix fabricated using different metal alloy compositions (stainless steel and beta-titanium), submitted to different intensities of bends preactivation (0° and 40°), and with different cross-sectional dimension of the wire used to build these loops (0.017 x 0.025-in and 0.019 x 0.025-in). Eighty loops used to close spaces were submitted to mechanical tests. The magnitudes of horizontal force, the moment/force ratio, and the load/deflection ratio produced by the specimens were quantified. Loops were submitted to a total activation of 5.0 mm and the values were registered for each 1.0 mm of activation. For statistic data analysis, a analysis of variance was performed and a Tukey's Multiple Comparison test was used as supplement, considering a 5% level of significance. In general, teardrop loops with helix produced lower magnitudes of horizontal force and load/deflection ratio, and higher moment/force ratio than teardrop loops without helix. Among all analyzed variables, metal alloy composition presented greater influence in the horizontal force and in the load/deflection ratio. The moment/force ratio showed to be more influenced by the preactivation of loops for space closure.

The Basic/Helix-Loop-Helix Protein Family in Gossypium: Reference Genes and Their Evolution during Tetraploidization

PubMed Central

Yan, Qian; Liu, Hou-Sheng; Yao, Dan; Li, Xin; Chen, Han; Dou, Yang; Wang, Yi; Pei, Yan; Xiao, Yue-Hua

2015-01-01

Basic/helix-loop-helix (bHLH) proteins comprise one of the largest transcription factor families and play important roles in diverse cellular and molecular processes. Comprehensive analyses of the composition and evolution of the bHLH family in cotton are essential to elucidate their functions and the molecular basis of cotton development. By searching bHLH homologous genes in sequenced diploid cotton genomes (Gossypium raimondii and G. arboreum), a set of cotton bHLH reference genes containing 289 paralogs were identified and named as GobHLH001-289. Based on their phylogenetic relationships, these cotton bHLH proteins were clustered into 27 subfamilies. Compared to those in Arabidopsis and cacao, cotton bHLH proteins generally increased in number, but unevenly in different subfamilies. To further uncover evolutionary changes of bHLH genes during tetraploidization of cotton, all genes of S5a and S5b subfamilies in upland cotton and its diploid progenitors were cloned and compared, and their transcript profiles were determined in upland cotton. A total of 10 genes of S5a and S5b subfamilies (doubled from A- and D-genome progenitors) maintained in tetraploid cottons. The major sequence changes in upland cotton included a 15-bp in-frame deletion in GhbHLH130D and a long terminal repeat retrotransposon inserted in GhbHLH062A, which eliminated GhbHLH062A expression in various tissues. The S5a and S5b bHLH genes of A and D genomes (except GobHLH062) showed similar transcription patterns in various tissues including roots, stems, leaves, petals, ovules, and fibers, while the A- and D-genome genes of GobHLH110 and GobHLH130 displayed clearly different transcript profiles during fiber development. In total, this study represented a genome-wide analysis of cotton bHLH family, and revealed significant changes in sequence and expression of these genes in tetraploid cottons, which paved the way for further functional analyses of bHLH genes in the cotton genus. PMID:25992947

The Basic/Helix-Loop-Helix Protein Family in Gossypium: Reference Genes and Their Evolution during Tetraploidization.

PubMed

Yan, Qian; Liu, Hou-Sheng; Yao, Dan; Li, Xin; Chen, Han; Dou, Yang; Wang, Yi; Pei, Yan; Xiao, Yue-Hua

2015-01-01

Basic/helix-loop-helix (bHLH) proteins comprise one of the largest transcription factor families and play important roles in diverse cellular and molecular processes. Comprehensive analyses of the composition and evolution of the bHLH family in cotton are essential to elucidate their functions and the molecular basis of cotton development. By searching bHLH homologous genes in sequenced diploid cotton genomes (Gossypium raimondii and G. arboreum), a set of cotton bHLH reference genes containing 289 paralogs were identified and named as GobHLH001-289. Based on their phylogenetic relationships, these cotton bHLH proteins were clustered into 27 subfamilies. Compared to those in Arabidopsis and cacao, cotton bHLH proteins generally increased in number, but unevenly in different subfamilies. To further uncover evolutionary changes of bHLH genes during tetraploidization of cotton, all genes of S5a and S5b subfamilies in upland cotton and its diploid progenitors were cloned and compared, and their transcript profiles were determined in upland cotton. A total of 10 genes of S5a and S5b subfamilies (doubled from A- and D-genome progenitors) maintained in tetraploid cottons. The major sequence changes in upland cotton included a 15-bp in-frame deletion in GhbHLH130D and a long terminal repeat retrotransposon inserted in GhbHLH062A, which eliminated GhbHLH062A expression in various tissues. The S5a and S5b bHLH genes of A and D genomes (except GobHLH062) showed similar transcription patterns in various tissues including roots, stems, leaves, petals, ovules, and fibers, while the A- and D-genome genes of GobHLH110 and GobHLH130 displayed clearly different transcript profiles during fiber development. In total, this study represented a genome-wide analysis of cotton bHLH family, and revealed significant changes in sequence and expression of these genes in tetraploid cottons, which paved the way for further functional analyses of bHLH genes in the cotton genus.

Noncanoncial signal recognition particle RNAs in a major eukaryotic phylum revealed by purification of SRP from the human pathogen Cryptococcus neoformans

PubMed Central

Dumesic, Phillip A.; Rosenblad, Magnus A.; Samuelsson, Tore; Nguyen, Tiffany; Moresco, James J.; Yates, John R.; Madhani, Hiten D.

2015-01-01

Despite conservation of the signal recognition particle (SRP) from bacteria to man, computational approaches have failed to identify SRP components from genomes of many lower eukaryotes, raising the possibility that they have been lost or altered in those lineages. We report purification and analysis of SRP in the human pathogen Cryptococcus neoformans, providing the first description of SRP in basidiomycetous yeast. The C. neoformans SRP RNA displays a predicted structure in which the universally conserved helix 8 contains an unprecedented stem-loop insertion. Guided by this sequence, we computationally identified 152 SRP RNAs throughout the phylum Basidiomycota. This analysis revealed additional helix 8 alterations including single and double stem-loop insertions as well as loop diminutions affecting RNA structural elements that are otherwise conserved from bacteria to man. Strikingly, these SRP RNA features in Basidiomycota are accompanied by phylum-specific alterations in the RNA-binding domain of Srp54, the SRP protein subunit that directly interacts with helix 8. Our findings reveal unexpected fungal SRP diversity and suggest coevolution of the two most conserved SRP features—SRP RNA helix 8 and Srp54—in basidiomycetes. Because members of this phylum include important human and plant pathogens, these noncanonical features provide new targets for antifungal compound development. PMID:26275773

Characterization of the near native conformational states of the SAM domain of Ste11 protein by NMR spectroscopy.

PubMed

Gupta, Sebanti; Bhattacharjya, Surajit

2014-11-01

The sterile alpha motif or SAM domain is one of the most frequently present protein interaction modules with diverse functional attributions. SAM domain of the Ste11 protein of budding yeast plays important roles in mitogen-activated protein kinase cascades. In the current study, urea-induced, at subdenaturing concentrations, structural, and dynamical changes in the Ste11 SAM domain have been investigated by nuclear magnetic resonance spectroscopy. Our study revealed that a number of residues from Helix 1 and Helix 5 of the Ste11 SAM domain display plausible alternate conformational states and largest chemical shift perturbations at low urea concentrations. Amide proton (H/D) exchange experiments indicated that Helix 1, loop, and Helix 5 become more susceptible to solvent exchange with increased concentrations of urea. Notably, Helix 1 and Helix 5 are directly involved in binding interactions of the Ste11 SAM domain. Our data further demonstrate that the existence of alternate conformational states around the regions involved in dimeric interactions in native or near native conditions. © 2014 Wiley Periodicals, Inc.

PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORα (PPARα) AGONISTS DIFFERENTIALLY REGULATE INHIBITOR OF DNA BINDING (ID2) EXPRESSION IN RODENTS AND HUMAN CELLS

EPA Science Inventory

Abstract Inhibitor of DNA binding (Id2) is a member of the helix-loop-helix (HLH) transcription factor family whose members play important roles in cell differentiation and proliferation. Id2 has been linked to the development of cardiovascular diseases since thiazolidinediones,...

Differences in the electrostatic surfaces of the type III secretion needle proteins PrgI, BsaL, and MxiH.

PubMed

Wang, Yu; Ouellette, Andrew N; Egan, Chet W; Rathinavelan, Thenmalarchelvi; Im, Wonpil; De Guzman, Roberto N

2007-08-31

Gram-negative bacteria use a needle-like protein assembly, the type III secretion apparatus, to inject virulence factors into target cells to initiate human disease. The needle is formed by the polymerization of approximately 120 copies of a small acidic protein that is conserved among diverse pathogens. We previously reported the structure of the BsaL needle monomer from Burkholderia pseudomallei by nuclear magnetic resonance (NMR) spectroscopy and others have determined the crystal structure of the Shigella flexneri MxiH needle. Here, we report the NMR structure of the PrgI needle protein of Salmonella typhimurium, a human pathogen associated with food poisoning. PrgI, BsaL, and MxiH form similar two helix bundles, however, the electrostatic surfaces of PrgI differ radically from those of BsaL or MxiH. In BsaL and MxiH, a large negative area is on a face formed by the helix alpha1-alpha2 interface. In PrgI, the major negatively charged surface is not on the "face" but instead is on the "side" of the two-helix bundle, and only residues from helix alpha1 contribute to this negative region. Despite being highly acidic proteins, these molecules contain large basic regions, suggesting that electrostatic contacts are important in needle assembly. Our results also suggest that needle-packing interactions may be different among these bacteria and provide the structural basis for why PrgI and MxiH, despite 63% sequence identity, are not interchangeable in S. typhimurium and S. flexneri.

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

Michael, Alicia K.; Fribourgh, Jennifer L.; Chelliah, Yogarany

The basic helix-loop-helix PAS domain (bHLH-PAS) transcription factor CLOCK:BMAL1 (brain and muscle Arnt-like protein 1) sits at the core of the mammalian circadian transcription/translation feedback loop. Precise control of CLOCK:BMAL1 activity by coactivators and repressors establishes the ~24-h periodicity of gene expression. Formation of a repressive complex, defined by the core clock proteins cryptochrome 1 (CRY1):CLOCK:BMAL1, plays an important role controlling the switch from repression to activation each day. Here in this paper, we show that CRY1 binds directly to the PAS domain core of CLOCK: BMAL1, driven primarily by interaction with the CLOCK PAS-B domain. Integrative modeling and solutionmore » X-ray scattering studies unambiguously position a key loop of the CLOCK PAS-B domain in the secondary pocket of CRY1, analogous to the antenna chromophore-binding pocket of photolyase. CRY1 docks onto the transcription factor alongside the PAS domains, extending above the DNA-binding bHLH domain. Single point mutations at the interface on either CRY1 or CLOCK disrupt formation of the ternary complex, highlighting the importance of this interface for direct regulation of CLOCK:BMAL1 activity by CRY1.« less

Differential regulation of transcription through distinct Suppressor of Hairless DNA binding site architectures during Notch signaling in proneural clusters.

PubMed

Cave, John W; Xia, Li; Caudy, Michael

2011-01-01

In Drosophila melanogaster, achaete (ac) and m8 are model basic helix-loop-helix activator (bHLH A) and repressor genes, respectively, that have the opposite cell expression pattern in proneural clusters during Notch signaling. Previous studies have shown that activation of m8 transcription in specific cells within proneural clusters by Notch signaling is programmed by a "combinatorial" and "architectural" DNA transcription code containing binding sites for the Su(H) and proneural bHLH A proteins. Here we show the novel result that the ac promoter contains a similar combinatorial code of Su(H) and bHLH A binding sites but contains a different Su(H) site architectural code that does not mediate activation during Notch signaling, thus programming a cell expression pattern opposite that of m8 in proneural clusters.

Conformational Preference of ‘CαNN’ Short Peptide Motif towards Recognition of Anions

PubMed Central

Banerjee, Raja

2013-01-01

Among several ‘anion binding motifs’, the recently described ‘CαNN’ motif occurring in the loop regions preceding a helix, is conserved through evolution both in sequence and its conformation. To establish the significance of the conserved sequence and their intrinsic affinity for anions, a series of peptides containing the naturally occurring ‘CαNN’ motif at the N-terminus of a designed helix, have been modeled and studied in a context free system using computational techniques. Appearance of a single interacting site with negative binding free-energy for both the sulfate and phosphate ions, as evidenced in docking experiments, establishes that the ‘CαNN’ segment has an intrinsic affinity for anions. Molecular Dynamics (MD) simulation studies reveal that interaction with anion triggers a conformational switch from non-helical to helical state at the ‘CαNN’ segment, which extends the length of the anchoring-helix by one turn at the N-terminus. Computational experiments substantiate the significance of sequence/structural context and justify the conserved nature of the ‘CαNN’ sequence for anion recognition through “local” interaction. PMID:23516403

High thermodynamic stability of parametrically designed helical bundles

DOE PAGES

Huang, Po -Ssu; Oberdorfer, Gustav; Xu, Chunfu; ...

2014-10-24

Here we describe a procedure for designing proteins with backbones produced by varying the parameters in the Crick coiled coil–generating equations. Combinatorial design calculations identify low-energy sequences for alternative helix supercoil arrangements, and the helices in the lowest-energy arrangements are connected by loop building. We design an antiparallel monomeric untwisted three-helix bundle with 80-residue helices, an antiparallel monomeric right-handed four-helix bundle, and a pentameric parallel left-handed five-helix bundle. The designed proteins are extremely stable (extrapolated ΔG fold > 60 kilocalories per mole), and their crystal structures are close to those of the design models with nearly identical core packing betweenmore » the helices. The approach enables the custom design of hyperstable proteins with fine-tuned geometries for a wide range of applications.« less

Identifying Novel Helix-Loop-Helix Genes in "Caenorhabditis elegans" through a Classroom Demonstration of Functional Genomics

ERIC Educational Resources Information Center

Griffin, Vernetta; McMiller, Tracee; Jones, Erika; Johnson, Casonya M.

2003-01-01

A 14-week, undergraduate-level Genetics and Population Biology course at Morgan State University was modified to include a demonstration of functional genomics in the research laboratory. Students performed a rudimentary sequence analysis of the "Caenorhabditis elegans" genome and further characterized three sequences that were predicted to encode…

A single peptide loop in an alpha-Helix

USDA-ARS?s Scientific Manuscript database

Pitch is not a height but a ratio of rise/run. In an alpha-helix, run can be as the radius (r) from the center of the circle, as a diameter (d) measured across/bisecting a circumference, or as a distance (c) along a circumference; rise in each case can corresponds to same height (h) increase. For ...

Structural insights into the stabilization of MALAT1 noncoding RNA by a bipartite triple helix

PubMed Central

Brown, Jessica A.; Bulkley, David; Wang, Jimin; Valenstein, Max L.; Yario, Therese A.; Steitz, Thomas A.; Steitz, Joan A.

2014-01-01

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a highly-abundant nuclear long noncoding RNA that promotes malignancy. A 3′-stem-loop structure is predicted to confer stability by engaging a downstream A-rich tract in a triple helix, similar to the expression and nuclear retention element (ENE) from the KSHV polyadenylated nuclear RNA. The 3.1-Å resolution crystal structure of the human MALAT1 ENE and A-rich tract reveals a bipartite triple helix containing stacks of five and four U•A-U triples separated by a C+•G-C triplet and C-G doublet, extended by two A-minor interactions. In vivo decay assays indicate that this blunt-ended triple helix, with the 3′ nucleotide in a U•A-U triple, inhibits rapid nuclear RNA decay. Interruption of the triple helix by the C-G doublet induces a “helical reset” that explains why triple-helical stacks longer than six do not occur in nature. PMID:24952594

Structural basis for the appearance of a molten globule state in chimeric molecules derived from lysozyme and alpha-lactalbumin.

PubMed

Joniau, M; Haezebrouck, P; Noyelle, K; Van Dael, H

2001-07-01

The problem as to why alpha-lactalbumin, in the absence of Ca(2+), forms a molten globule intermediate, in contrast to its structural homologue lysozyme, has been addressed by the construction of chimeras of human lysozyme in which either the Ca(2+)-binding loop or a part of helix C of bovine alpha-lactalbumin were transplanted. Previously, we have shown that the introduction of both structural elements together in the lysozyme matrix causes the apo form of the resulting chimera to display molten globule behavior during the course of thermal denaturation. In this article, we demonstrate that this molten globule character is not correlated with the Ca(2+)-binding loop. Also, the Del 101 mutant in which Arg101 was deleted to simulate the alpha-lactalbumin conformation of the connecting loop between helix C and helix D, does not show a stable equilibrium intermediate. Rather, the molten globule character of the chimeras has to be related with a specific part of helix C. More particularly, attention is drawn to the four hydrophobic side-chains I93, V96, I99, and L100, the lysozyme counterparts of which are constituted of less bulky valines and alanine. Our observations are discussed in terms of decreased stability of the native form and increased stability of the intermediate molten globule. Copyright 2001 Wiley-Liss, Inc.

Net (ERP/SAP2) one of the Ras-inducible TCFs, has a novel inhibitory domain with resemblance to the helix-loop-helix motif.

PubMed Central

Maira, S M; Wurtz, J M; Wasylyk, B

1996-01-01

The three ternary complex factors (TCFs), Net (ERP/ SAP-2), ELK-1 and SAP-1, are highly related ets oncogene family members that participate in the response of the cell to Ras and growth signals. Understanding the different roles of these factors will provide insights into how the signals result in coordinate regulation of the cell. We show that Net inhibits transcription under basal conditions, in which SAP-1a is inactive and ELK-1 stimulates. Repression is mediated by the NID, the Net Inhibitory Domain of about 50 amino acids, which autoregulates the Net protein and also inhibits when it is isolated in a heterologous fusion protein. Net is particularly sensitive to Ras activation. Ras activates Net through the C-domain, which is conserved between the three TCFs, and the NID is an efficient inhibitor of Ras activation. The NID, as well as more C-terminal sequences, inhibit DNA binding. Net is more refractory to DNA binding than the other TCFs, possibly due to the presence of multiple inhibitory elements. The NID may adopt a helix-loop-helix (HLH) structure, as evidenced by homology to other HLH motifs, structure predictions, model building and mutagenesis of critical residues. The sequence resemblance with myogenic factors suggested that Net may form complexes with the same partners. Indeed, we found that Net can interact in vivo with the basic HLH factor, E47. We propose that Net is regulated at the level of its latent DNA-binding activity by protein interactions and/or phosphorylation. Net may form complexes with HLH proteins as well as SRF on specific promotor sequences. The identification of the novel inhibitory domain provides a new inroad into exploring the different roles of the ternary complex factors in growth control and transformation. Images PMID:8918463

Net (ERP/SAP2) one of the Ras-inducible TCFs, has a novel inhibitory domain with resemblance to the helix-loop-helix motif.

PubMed

Maira, S M; Wurtz, J M; Wasylyk, B

1996-11-01

The three ternary complex factors (TCFs), Net (ERP/ SAP-2), ELK-1 and SAP-1, are highly related ets oncogene family members that participate in the response of the cell to Ras and growth signals. Understanding the different roles of these factors will provide insights into how the signals result in coordinate regulation of the cell. We show that Net inhibits transcription under basal conditions, in which SAP-1a is inactive and ELK-1 stimulates. Repression is mediated by the NID, the Net Inhibitory Domain of about 50 amino acids, which autoregulates the Net protein and also inhibits when it is isolated in a heterologous fusion protein. Net is particularly sensitive to Ras activation. Ras activates Net through the C-domain, which is conserved between the three TCFs, and the NID is an efficient inhibitor of Ras activation. The NID, as well as more C-terminal sequences, inhibit DNA binding. Net is more refractory to DNA binding than the other TCFs, possibly due to the presence of multiple inhibitory elements. The NID may adopt a helix-loop-helix (HLH) structure, as evidenced by homology to other HLH motifs, structure predictions, model building and mutagenesis of critical residues. The sequence resemblance with myogenic factors suggested that Net may form complexes with the same partners. Indeed, we found that Net can interact in vivo with the basic HLH factor, E47. We propose that Net is regulated at the level of its latent DNA-binding activity by protein interactions and/or phosphorylation. Net may form complexes with HLH proteins as well as SRF on specific promotor sequences. The identification of the novel inhibitory domain provides a new inroad into exploring the different roles of the ternary complex factors in growth control and transformation.

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

Zhang, Bing; Liu, Caini; Qian, Wen

Crystal structure of the SEFIR domain from human IL-17 receptor A provides new insights into IL-17 signaling. Interleukin 17 (IL-17) cytokines play a crucial role in mediating inflammatory and autoimmune diseases. A unique intracellular signaling domain termed SEFIR is found within all IL-17 receptors (IL-17Rs) as well as the key adaptor protein Act1. SEFIR-mediated protein–protein interaction is a crucial step in IL-17 cytokine signaling. Here, the 2.3 Å resolution crystal structure of the SEFIR domain of IL-17RA, the most commonly shared receptor for IL-17 cytokine signaling, is reported. The structure includes the complete SEFIR domain and an additional α-helical C-terminalmore » extension, which pack tightly together to form a compact unit. Structural comparison between the SEFIR domains of IL-17RA and IL-17RB reveals substantial differences in protein topology and folding. The uniquely long insertion between strand βC and helix αC in IL-17RA SEFIR is mostly well ordered, displaying a helix (αCC′{sub ins}) and a flexible loop (CC′). The DD′ loop in the IL-17RA SEFIR structure is much shorter; it rotates nearly 90° with respect to the counterpart in the IL-17RB SEFIR structure and shifts about 12 Å to accommodate the αCC′{sub ins} helix without forming any knots. Helix αC was identified as critical for its interaction with Act1 and IL-17-stimulated gene expression. The data suggest that the heterotypic SEFIR–SEFIR association via helix αC is a conserved and signature mechanism specific for IL-17 signaling. The structure also suggests that the downstream motif of IL-17RA SEFIR together with helix αC could provide a composite ligand-binding surface for recruiting Act1 during IL-17 signaling.« less

Achaete-Scute Homolog 1 Expression Controls Cellular Differentiation of Neuroblastoma

PubMed Central

Kasim, Mumtaz; Heß, Vicky; Scholz, Holger; Persson, Pontus B.; Fähling, Michael

2016-01-01

Neuroblastoma, the major cause of infant cancer deaths, results from fast proliferation of undifferentiated neuroblasts. Treatment of high-risk neuroblastoma includes differentiation with retinoic acid (RA); however, the resistance of many of these tumors to RA-induced differentiation poses a considerable challenge. Human achaete-scute homolog 1 (hASH1) is a proneural basic helix-loop-helix transcription factor essential for neurogenesis and is often upregulated in neuroblastoma. Here, we identified a novel function for hASH1 in regulating the differentiation phenotype of neuroblastoma cells. Global analysis of 986 human neuroblastoma datasets revealed a negative correlation between hASH1 and neuron differentiation that was independent of the N-myc (MYCN) oncogene. Using RA to induce neuron differentiation in two neuroblastoma cell lines displaying high and low levels of hASH1 expression, we confirmed the link between hASH1 expression and the differentiation defective phenotype, which was reversed by silencing hASH1 or by hypoxic preconditioning. We further show that hASH1 suppresses neuronal differentiation by inhibiting transcription at the RA receptor element. Collectively, our data indicate hASH1 to be key for understanding neuroblastoma resistance to differentiation therapy and pave the way for hASH1-targeted therapies for augmenting the response of neuroblastoma to differentiation therapy. PMID:28066180

A WNT/beta-catenin signaling activator, R-spondin, plays positive regulatory roles during skeletal myogenesis.

PubMed

Han, Xiang Hua; Jin, Yong-Ri; Seto, Marianne; Yoon, Jeong Kyo

2011-03-25

R-spondins (RSPOs) are a recently characterized family of secreted proteins that activate WNT/β-catenin signaling. In this study, we investigated the potential roles of the RSPO proteins during myogenic differentiation. Overexpression of the Rspo1 gene or administration of recombinant RSPO2 protein enhanced mRNA and protein expression of a basic helix-loop-helix (bHLH) class myogenic determination factor, MYF5, in both C2C12 myoblasts and primary satellite cells, whereas MYOD or PAX7 expression was not affected. RSPOs also promoted myogenic differentiation and induced hypertrophic myotube formation in C2C12 cells. In addition, Rspo2 and Rspo3 gene knockdown by RNA interference significantly compromised MYF5 expression, myogenic differentiation, and myotube formation. Furthermore, Myf5 expression was reduced in the developing limbs of mouse embryos lacking the Rspo2 gene. Finally, we demonstrated that blocking of WNT/β-catenin signaling by DKK1 or a dominant-negative form of TCF4 reversed MYF5 expression, myogenic differentiation, and hypertrophic myotube formation induced by RSPO2, indicating that RSPO2 exerts its activity through the WNT/β-catenin signaling pathway. Our results provide strong evidence that RSPOs are key positive regulators of skeletal myogenesis acting through the WNT/β-catenin signaling pathway.

A WNT/β-Catenin Signaling Activator, R-spondin, Plays Positive Regulatory Roles during Skeletal Myogenesis*

PubMed Central

Han, Xiang Hua; Jin, Yong-Ri; Seto, Marianne; Yoon, Jeong Kyo

2011-01-01

R-spondins (RSPOs) are a recently characterized family of secreted proteins that activate WNT/β-catenin signaling. In this study, we investigated the potential roles of the RSPO proteins during myogenic differentiation. Overexpression of the Rspo1 gene or administration of recombinant RSPO2 protein enhanced mRNA and protein expression of a basic helix-loop-helix (bHLH) class myogenic determination factor, MYF5, in both C2C12 myoblasts and primary satellite cells, whereas MYOD or PAX7 expression was not affected. RSPOs also promoted myogenic differentiation and induced hypertrophic myotube formation in C2C12 cells. In addition, Rspo2 and Rspo3 gene knockdown by RNA interference significantly compromised MYF5 expression, myogenic differentiation, and myotube formation. Furthermore, Myf5 expression was reduced in the developing limbs of mouse embryos lacking the Rspo2 gene. Finally, we demonstrated that blocking of WNT/β-catenin signaling by DKK1 or a dominant-negative form of TCF4 reversed MYF5 expression, myogenic differentiation, and hypertrophic myotube formation induced by RSPO2, indicating that RSPO2 exerts its activity through the WNT/β-catenin signaling pathway. Our results provide strong evidence that RSPOs are key positive regulators of skeletal myogenesis acting through the WNT/β-catenin signaling pathway. PMID:21252233

A Light-Regulated Genetic Module Was Recruited to Carpel Development in Arabidopsis following a Structural Change to SPATULA[W

PubMed Central

Reymond, Mathieu C.; Brunoud, Géraldine; Chauvet, Aurélie; Martínez-Garcia, Jaime F.; Martin-Magniette, Marie-Laure; Monéger, Françoise; Scutt, Charles P.

2012-01-01

A key innovation of flowering plants is the female reproductive organ, the carpel. Here, we show that a mechanism that regulates carpel margin development in the model flowering plant Arabidopsis thaliana was recruited from light-regulated processes. This recruitment followed the loss from the basic helix-loop-helix transcription factor SPATULA (SPT) of a domain previously responsible for its negative regulation by phytochrome. We propose that the loss of this domain was a prerequisite for the light-independent expression in female reproductive tissues of a genetic module that also promotes shade avoidance responses in vegetative organs. Striking evidence for this proposition is provided by the restoration of wild-type carpel development to spt mutants by low red/far-red light ratios, simulating vegetation shade, which we show to occur via phytochrome B, PHYTOCHROME INTERACTING FACTOR4 (PIF4), and PIF5. Our data illustrate the potential of modular evolutionary events to generate rapid morphological change and thereby provide a molecular basis for neo-Darwinian theories that describe this nongradualist phenomenon. Furthermore, the effects shown here of light quality perception on carpel development lead us to speculate on the potential role of light-regulated mechanisms in plant organs that, like the carpel, form within the shade of surrounding tissues. PMID:22851763

Identification of exosite residues of factor Xa involved in recognition of PAR-2 on endothelial cells.

PubMed

Manithody, Chandrashekhara; Yang, Likui; Rezaie, Alireza R

2012-03-27

Recent results have indicated that factor Xa (FXa) cleaves protease-activated receptor 2 (PAR-2) to elicit protective intracellular signaling responses in endothelial cells. In this study, we investigated the molecular determinants of the specificity of FXa interaction with PAR-2 by monitoring the cleavage of PAR-2 by FXa in endothelial cells transiently transfected with a PAR-2 cleavage reporter construct in which the extracellular domain of the receptor was fused to cDNA encoding for alkaline phosphatase. Comparison of the cleavage efficiency of PAR-2 by a series of FXa mutants containing mutations in different surface loops indicated that the acidic residues of 39-loop (Glu-36, Glu-37, and Glu-39) and the basic residues of 60-loop (Lys-62 and Arg-63), 148-loop (Arg-143, Arg-150, and Arg-154), and 162-helix (Arg-165 and Lys-169) contribute to the specificity of receptor recognition by FXa on endothelial cells. This was evidenced by significantly reduced activity of mutants toward PAR-2 expressed on transfected cells. The extent of loss in the PAR-2 cleavage activity of FXa mutants correlated with the extent of loss in their PAR-2-dependent intracellular signaling activity. Further characterization of FXa mutants indicated that, with the exception of basic residues of 162-helix, which play a role in the recognition specificity of the prothrombinase complex, none of the surface loop residues under study makes a significant contribution to the activity of FXa in the prothrombinase complex. These results provide new insight into mechanisms through which FXa specifically interacts with its macromolecular substrates in the clotting and signaling pathways.

The X-ray structure of Paramecium bursaria Chlorella virus arginine decarboxylase: insight into the structural basis for substrate specificity

PubMed Central

Shah, Rahul; Akella, Radha; Goldsmith, Elizabeth J.; Phillips, Margaret A.

2008-01-01

The group IV pyridoxal-5′-phosphate (PLP)-dependent decarboxylases belong to the β/α barrel structural family, and include enzymes with substrate specificity for a range of basic amino acids. A unique homolog of this family, the Paramecium bursaria Chlorella virus arginine decarboxylase (cvADC), shares about 40% amino acid sequence identity with the eukaryotic ornithine decarboxylases (ODCs). The X-ray structure of cvADC has been solved to 1.95 and 1.8 Å resolution for the free and agmatine (product)-bound enzymes. The global structural differences between cvADC and eukaryotic ODC are minimal (rmsd of 1.2 – 1.4 Å), however, the active site has significant structural rearrangements. The key “specificity element,” is identified as the 310-helix that contains and positions substrate-binding residues such as E296 cvADC (D332 in T. brucei ODC). In comparison to the ODC structures, the 310-helix in cvADC is shifted over 2 Å away from the PLP cofactor, thus accommodating the larger arginine substrate. Within the context of this conserved fold, the protein is designed to be flexible in the positioning and amino acid sequence of the 310-helix, providing a mechanism to evolve different substrate preferences within the family without large structural rearrangements. Also, in the structure, the “K148-loop” (homologous to the “K169-loop” of ODC) is observed in a closed, substrate-bound conformation for the first time. Apparently the K148 loop is a mobile loop, analogous to those observed in triose phosphate isomerase and tryptophan synthetase. In conjunction with prior structural studies these data predict that this loop adopts different conformations throughout the catalytic cycle, and that loop movement may be kinetically linked to the rate-limiting step of product release. PMID:17305368

Formation of the Embryonic Head in the Mouse: Attributes of a Gene Regulatory Network.

PubMed

Tam, Patrick P L; Fossat, Nicolas; Wilkie, Emilie; Loebel, David A F; Ip, Chi Kin; Ramialison, Mirana

2016-01-01

The embryonic head is the first major body part to be constructed during embryogenesis. The allocation and the assembly of the progenitor tissues, which start at gastrulation, are accompanied by the spatiotemporal activity of transcription factors and signaling pathways that drives lineage specification, germ layer formation, and cell/tissue movement. The morphogenesis, regionalization, and patterning of the brain and craniofacial structures rely on the function of LIM-domain, homeodomain, and basic helix-loop-helix transcription factors. These factors constitute the central nodes of a gene regulatory network (GRN) which encompasses and intersects with signaling pathways involved with head formation. It is predicted that the functional output of this "head GRN" impacts on cellular function and cell-cell interactions that are essential for lineage differentiation and tissue modeling, which are key processes underpinning the formation of the head. © 2016 Elsevier Inc. All rights reserved.

The Transcriptional Coregulator LEUNIG_HOMOLOG Inhibits Light-Dependent Seed Germination in Arabidopsis

PubMed Central

Lee, Nayoung; Park, Jeongmoo; Kim, Keunhwa; Choi, Giltsu

2015-01-01

PHYTOCHROME-INTERACTING FACTOR1 (PIF1) is a basic helix-loop-helix transcription factor that inhibits light-dependent seed germination in Arabidopsis thaliana. However, it remains unclear whether PIF1 requires other factors to regulate its direct targets. Here, we demonstrate that LEUNIG_HOMOLOG (LUH), a Groucho family transcriptional corepressor, binds to PIF1 and coregulates its targets. Not only are the transcriptional profiles of the luh and pif1 mutants remarkably similar, more than 80% of the seeds of both genotypes germinate in the dark. We show by chromatin immunoprecipitation that LUH binds a subset of PIF1 targets in a partially PIF1-dependent manner. Unexpectedly, we found LUH binds and coregulates not only PIF1-activated targets but also PIF1-repressed targets. Together, our results indicate LUH functions with PIF1 as a transcriptional coregulator to inhibit seed germination. PMID:26276832

The Clock gene clone and its circadian rhythms in Pelteobagrus vachelli

NASA Astrophysics Data System (ADS)

Qin, Chuanjie; Shao, Ting

2015-05-01

The Clock gene, a key molecule in circadian systems, is widely distributed in the animal kingdom. We isolated a 936-bp partial cDNA sequence of the Clock gene ( Pva-clock) from the darkbarbel catfish Pelteobagrus vachelli that exhibited high identity with Clock genes of other species of fish and animals (65%-88%). The putative domains included a basic helix-loop-helix (bHLH) domain and two period-ARNT-single-minded (PAS) domains, which were also similar to those in other species of fish and animals. Pva-Clock was primarily expressed in the brain, and was detected in all of the peripheral tissues sampled. Additionally, the pattern of Pva-Clock expression over a 24-h period exhibited a circadian rhythm in the brain, liver and intestine, with the acrophase at zeitgeber time 21:35, 23:00, and 23:23, respectively. Our results provide insight into the function of the molecular Clock of P. vachelli.

Structural characterization of the H-NS protein from Xylella fastidiosa and its interaction with DNA.

PubMed

Rosselli-Murai, Luciana K; Sforça, Maurício L; Sassonia, Rogério C; Azzoni, Adriano R; Murai, Marcelo J; de Souza, Anete P; Zeri, Ana C

2012-10-01

The nucleoid-associated protein H-NS is a major component of the bacterial nucleoid involved in DNA compaction and transcription regulation. The NMR solution structure of the Xylella fastidiosa H-NS C-terminal domain (residues 56-134) is presented here and consists of two beta-strands and two alpha helices, with one loop connecting the two beta-strands and a second loop connecting the second beta strand and the first helix. The amide (1)H and (15)N chemical shift signals for a sample of XfH-NS(56-134) were monitored in the course of a titration series with a 14-bp DNA duplex. Most of the residues involved in contacts to DNA are located around the first and second loops and in the first helix at a positively charged side of the protein surface. The overall structure of the Xylella H-NS C-terminal domain differ significantly from Escherichia coli and Salmonella enterica H-NS proteins, even though the DNA binding motif in loop 2 adopt similar conformation, as well as β-strand 2 and loop 1. Interestingly, we have also found that the DNA binding site is expanded to include helix 1, which is not seen in the other structures. Copyright © 2012 Elsevier Inc. All rights reserved.

Formation of a repressive complex in the mammalian circadian clock is mediated by the secondary pocket of CRY1

DOE PAGES

Michael, Alicia K.; Fribourgh, Jennifer L.; Chelliah, Yogarany; ...

2017-01-31

The basic helix-loop-helix PAS domain (bHLH-PAS) transcription factor CLOCK:BMAL1 (brain and muscle Arnt-like protein 1) sits at the core of the mammalian circadian transcription/translation feedback loop. Precise control of CLOCK:BMAL1 activity by coactivators and repressors establishes the ~24-h periodicity of gene expression. Formation of a repressive complex, defined by the core clock proteins cryptochrome 1 (CRY1):CLOCK:BMAL1, plays an important role controlling the switch from repression to activation each day. Here in this paper, we show that CRY1 binds directly to the PAS domain core of CLOCK: BMAL1, driven primarily by interaction with the CLOCK PAS-B domain. Integrative modeling and solutionmore » X-ray scattering studies unambiguously position a key loop of the CLOCK PAS-B domain in the secondary pocket of CRY1, analogous to the antenna chromophore-binding pocket of photolyase. CRY1 docks onto the transcription factor alongside the PAS domains, extending above the DNA-binding bHLH domain. Single point mutations at the interface on either CRY1 or CLOCK disrupt formation of the ternary complex, highlighting the importance of this interface for direct regulation of CLOCK:BMAL1 activity by CRY1.« less

Multisite Light-Induced Phosphorylation of the Transcription Factor PIF3 Is Necessary for Both Its Rapid Degradation and Concomitant Negative Feedback Modulation of Photoreceptor phyB Levels in Arabidopsis[C][W

PubMed Central

Ni, Weimin; Xu, Shou-Ling; Chalkley, Robert J.; Pham, Thao Nguyen D.; Guan, Shenheng; Maltby, Dave A.; Burlingame, Alma L.; Wang, Zhi-Yong; Quail, Peter H.

2013-01-01

Plants constantly monitor informational light signals using sensory photoreceptors, which include the phytochrome (phy) family (phyA to phyE), and adjust their growth and development accordingly. Following light-induced nuclear translocation, photoactivated phy molecules bind to and induce rapid phosphorylation and degradation of phy-interacting basic Helix Loop Helix (bHLH) transcription factors (PIFs), such as PIF3, thereby regulating the expression of target genes. However, the mechanisms underlying the signal-relay process are still not fully understood. Here, using mass spectrometry, we identify multiple, in vivo, light-induced Ser/Thr phosphorylation sites in PIF3. Using transgenic expression of site-directed mutants of PIF3, we provide evidence that a set of these phosphorylation events acts collectively to trigger rapid degradation of the PIF3 protein in response to initial exposure of dark-grown seedlings to light. In addition, we show that phyB-induced PIF3 phosphorylation is also required for the known negative feedback modulation of phyB levels in prolonged light, potentially through codegradation of phyB and PIF3. This mutually regulatory intermolecular transaction thus provides a mechanism with the dual capacity to promote early, graded, or threshold regulation of the primary, PIF3-controlled transcriptional network in response to initial light exposure, and later, to attenuate global sensitivity to the light signal through reductions in photoreceptor levels upon prolonged exposure. PMID:23903316

Basic Tilted Helix Bundle - a new protein fold in human FKBP25/FKBP3 and HectD1.

PubMed

Helander, Sara; Montecchio, Meri; Lemak, Alexander; Farès, Christophe; Almlöf, Jonas; Yi, Yanjun; Yee, Adelinda; Arrowsmith, Cheryl; DhePaganon, Sirano; Sunnerhagen, Maria

2014-04-25

In this paper, we describe the structure of a N-terminal domain motif in nuclear-localized FKBP251-73, a member of the FKBP family, together with the structure of a sequence-related subdomain of the E3 ubiquitin ligase HectD1 that we show belongs to the same fold. This motif adopts a compact 5-helix bundle which we name the Basic Tilted Helix Bundle (BTHB) domain. A positively charged surface patch, structurally centered around the tilted helix H4, is present in both FKBP25 and HectD1 and is conserved in both proteins, suggesting a conserved functional role. We provide detailed comparative analysis of the structures of the two proteins and their sequence similarities, and analysis of the interaction of the proposed FKBP25 binding protein YY1. We suggest that the basic motif in BTHB is involved in the observed DNA binding of FKBP25, and that the function of this domain can be affected by regulatory YY1 binding and/or interactions with adjacent domains. Copyright © 2014 Elsevier Inc. All rights reserved.

TFBSshape: a motif database for DNA shape features of transcription factor binding sites.

PubMed

Yang, Lin; Zhou, Tianyin; Dror, Iris; Mathelier, Anthony; Wasserman, Wyeth W; Gordân, Raluca; Rohs, Remo

2014-01-01

Transcription factor binding sites (TFBSs) are most commonly characterized by the nucleotide preferences at each position of the DNA target. Whereas these sequence motifs are quite accurate descriptions of DNA binding specificities of transcription factors (TFs), proteins recognize DNA as a three-dimensional object. DNA structural features refine the description of TF binding specificities and provide mechanistic insights into protein-DNA recognition. Existing motif databases contain extensive nucleotide sequences identified in binding experiments based on their selection by a TF. To utilize DNA shape information when analysing the DNA binding specificities of TFs, we developed a new tool, the TFBSshape database (available at http://rohslab.cmb.usc.edu/TFBSshape/), for calculating DNA structural features from nucleotide sequences provided by motif databases. The TFBSshape database can be used to generate heat maps and quantitative data for DNA structural features (i.e., minor groove width, roll, propeller twist and helix twist) for 739 TF datasets from 23 different species derived from the motif databases JASPAR and UniPROBE. As demonstrated for the basic helix-loop-helix and homeodomain TF families, our TFBSshape database can be used to compare, qualitatively and quantitatively, the DNA binding specificities of closely related TFs and, thus, uncover differential DNA binding specificities that are not apparent from nucleotide sequence alone.

TFBSshape: a motif database for DNA shape features of transcription factor binding sites

PubMed Central

Yang, Lin; Zhou, Tianyin; Dror, Iris; Mathelier, Anthony; Wasserman, Wyeth W.; Gordân, Raluca; Rohs, Remo

2014-01-01

Transcription factor binding sites (TFBSs) are most commonly characterized by the nucleotide preferences at each position of the DNA target. Whereas these sequence motifs are quite accurate descriptions of DNA binding specificities of transcription factors (TFs), proteins recognize DNA as a three-dimensional object. DNA structural features refine the description of TF binding specificities and provide mechanistic insights into protein–DNA recognition. Existing motif databases contain extensive nucleotide sequences identified in binding experiments based on their selection by a TF. To utilize DNA shape information when analysing the DNA binding specificities of TFs, we developed a new tool, the TFBSshape database (available at http://rohslab.cmb.usc.edu/TFBSshape/), for calculating DNA structural features from nucleotide sequences provided by motif databases. The TFBSshape database can be used to generate heat maps and quantitative data for DNA structural features (i.e., minor groove width, roll, propeller twist and helix twist) for 739 TF datasets from 23 different species derived from the motif databases JASPAR and UniPROBE. As demonstrated for the basic helix-loop-helix and homeodomain TF families, our TFBSshape database can be used to compare, qualitatively and quantitatively, the DNA binding specificities of closely related TFs and, thus, uncover differential DNA binding specificities that are not apparent from nucleotide sequence alone. PMID:24214955

Analysis of eco-innovation with triple helix approach: case-study of biofloc catfish farming in Yogyakarta

NASA Astrophysics Data System (ADS)

Purwadi, D.; Nurlaily, I.

2018-03-01

Concerning environmental into focus of innovation process will expand the number of actor involved. Eco-innovation and triple helix are often frameworks applied to analyse how environmental concern are integrated in innovation process and how different stakeholder groups are having inter relation. Case study from biofloc catfish farming in Yogyakarta is presented to demonstrate a possible approach for researching the success of triple helix frameworks. This case is considered on basic of the result of a survey among farmers, academician and government. The paper concludes the creating of full triple helix encounters problem in practice. It also includes suggestion for further research on fisheries development.

Exploring Flexibility of Progesterone Receptor Ligand Binding Domain Using Molecular Dynamics

PubMed Central

Zheng, Liangzhen; Mu, Yuguang

2016-01-01

Progesterone receptor (PR), a member of nuclear receptor (NR) superfamily, plays a vital role for female reproductive tissue development, differentiation and maintenance. PR ligand, such as progesterone, induces conformation changes in PR ligand binding domain (LBD), thus mediates subsequent gene regulation cascades. PR LBD may adopt different conformations upon an agonist or an antagonist binding. These different conformations would trigger distinct transcription events. Therefore, the dynamics of PR LBD would be of general interest to biologists for a deep understanding of its structure-function relationship. However, no apo-form (non-ligand bound) of PR LBD model has been proposed either by experiments or computational methods so far. In this study, we explored the structural dynamics of PR LBD using molecular dynamics simulations and advanced sampling tools in both ligand-bound and the apo-forms. Resolved by the simulation study, helix 11, helix 12 and loop 895–908 (the loop between these two helices) are quite flexible in antagonistic conformation. Several residues, such as Arg899 and Glu723, could form salt-bridging interaction between helix 11 and helix 3, and are important for the PR LBD dynamics. And we also propose that helix 12 in apo-form PR LBD, not like other NR LBDs, such as human estrogen receptor α (ERα) LBD, may not adopt a totally extended conformation. With the aid of umbrella sampling and metadynamics simulations, several stable conformations of apo-form PR LBD have been sampled, which may work as critical structural models for further large scale virtual screening study to discover novel PR ligands for therapeutic application. PMID:27824891

Reovirus FAST Proteins Drive Pore Formation and Syncytiogenesis Using a Novel Helix-Loop-Helix Fusion-Inducing Lipid Packing Sensor

PubMed Central

Sarker, Muzaddid; de Antueno, Roberto; Langelaan, David N.; Parmar, Hiren B.; Shin, Kyungsoo; Rainey, Jan K.; Duncan, Roy

2015-01-01

Pore formation is the most energy-demanding step during virus-induced membrane fusion, where high curvature of the fusion pore rim increases the spacing between lipid headgroups, exposing the hydrophobic interior of the membrane to water. How protein fusogens breach this thermodynamic barrier to pore formation is unclear. We identified a novel fusion-inducing lipid packing sensor (FLiPS) in the cytosolic endodomain of the baboon reovirus p15 fusion-associated small transmembrane (FAST) protein that is essential for pore formation during cell-cell fusion and syncytiogenesis. NMR spectroscopy and mutational studies indicate the dependence of this FLiPS on a hydrophobic helix-loop-helix structure. Biochemical and biophysical assays reveal the p15 FLiPS preferentially partitions into membranes with high positive curvature, and this partitioning is impeded by bis-ANS, a small molecule that inserts into hydrophobic defects in membranes. Most notably, the p15 FLiPS can be functionally replaced by heterologous amphipathic lipid packing sensors (ALPS) but not by other membrane-interactive amphipathic helices. Furthermore, a previously unrecognized amphipathic helix in the cytosolic domain of the reptilian reovirus p14 FAST protein can functionally replace the p15 FLiPS, and is itself replaceable by a heterologous ALPS motif. Anchored near the cytoplasmic leaflet by the FAST protein transmembrane domain, the FLiPS is perfectly positioned to insert into hydrophobic defects that begin to appear in the highly curved rim of nascent fusion pores, thereby lowering the energy barrier to stable pore formation. PMID:26061049

Helix-length compensation studies reveal the adaptability of the VS ribozyme architecture.

PubMed

Lacroix-Labonté, Julie; Girard, Nicolas; Lemieux, Sébastien; Legault, Pascale

2012-03-01

Compensatory mutations in RNA are generally regarded as those that maintain base pairing, and their identification forms the basis of phylogenetic predictions of RNA secondary structure. However, other types of compensatory mutations can provide higher-order structural and evolutionary information. Here, we present a helix-length compensation study for investigating structure-function relationships in RNA. The approach is demonstrated for stem-loop I and stem-loop V of the Neurospora VS ribozyme, which form a kissing-loop interaction important for substrate recognition. To rapidly characterize the substrate specificity (k(cat)/K(M)) of several substrate/ribozyme pairs, a procedure was established for simultaneous kinetic characterization of multiple substrates. Several active substrate/ribozyme pairs were identified, indicating the presence of limited substrate promiscuity for stem Ib variants and helix-length compensation between stems Ib and V. 3D models of the I/V interaction were generated that are compatible with the kinetic data. These models further illustrate the adaptability of the VS ribozyme architecture for substrate cleavage and provide global structural information on the I/V kissing-loop interaction. By exploring higher-order compensatory mutations in RNA our approach brings a deeper understanding of the adaptability of RNA structure, while opening new avenues for RNA research.

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

Helander, Sara; Montecchio, Meri; Lemak, Alexander

Highlights: • We describe the structure of a novel fold in FKBP25 and HectD. • The new fold is named the Basic Tilted Helix Bundle (BTHB) domain. • A conserved basic surface patch is presented, suggesting a functional role. - Abstract: In this paper, we describe the structure of a N-terminal domain motif in nuclear-localized FKBP25{sub 1–73}, a member of the FKBP family, together with the structure of a sequence-related subdomain of the E3 ubiquitin ligase HectD1 that we show belongs to the same fold. This motif adopts a compact 5-helix bundle which we name the Basic Tilted Helix Bundlemore » (BTHB) domain. A positively charged surface patch, structurally centered around the tilted helix H4, is present in both FKBP25 and HectD1 and is conserved in both proteins, suggesting a conserved functional role. We provide detailed comparative analysis of the structures of the two proteins and their sequence similarities, and analysis of the interaction of the proposed FKBP25 binding protein YY1. We suggest that the basic motif in BTHB is involved in the observed DNA binding of FKBP25, and that the function of this domain can be affected by regulatory YY1 binding and/or interactions with adjacent domains.« less

Segregating neural and mechanosensory fates in the developing ear: patterning, signaling, and transcriptional control

PubMed Central

Raft, Steven; Groves, Andrew K.

2014-01-01

The vertebrate inner ear is composed of multiple sensory receptor epithelia, each of which is specialized for detection of sound, gravity or angular acceleration. Each receptor epithelium contains mechanosensitive hair cells, which are connected to the brainstem by bipolar sensory neurons. Hair cells and their associated neurons are derived from the embryonic rudiment of the inner ear epithelium, but the precise spatial and temporal patterns of their generation, as well as the signals that coordinate these events, have only recently begun to be understood. Gene expression, lineage tracing, and mutant analyses suggest that both neurons and hair cells are generated from a common domain of neural and sensory competence in the embryonic inner ear rudiment. Members of the Shh, Wnt and FGF families, together with retinoic acid signals, regulate transcription factor genes within the inner ear rudiment to establish the axial identity of the ear and regionalize neurogenic activity. Close-range signaling, such as that of the Notch pathway, specifies the fate of sensory regions and individual cell types. We also describe positive and negative interactions between basic helix-loop-helix and SoxB family transcription factors that specify either neuronal or sensory fates in a context-dependent manner. Finally, we review recent work on inner ear development in zebrafish, which demonstrates that the relative timing of neurogenesis and sensory epithelial formation is not phylogenetically constrained. PMID:24902666

Plant proximity perception dynamically modulates hormone levels and sensitivity in Arabidopsis.

PubMed

Bou-Torrent, Jordi; Galstyan, Anahit; Gallemí, Marçal; Cifuentes-Esquivel, Nicolás; Molina-Contreras, Maria José; Salla-Martret, Mercè; Jikumaru, Yusuke; Yamaguchi, Shinjiro; Kamiya, Yuji; Martínez-García, Jaime F

2014-06-01

The shade avoidance syndrome (SAS) refers to a set of plant responses initiated after perception by the phytochromes of light enriched in far-red colour reflected from or filtered by neighbouring plants. These varied responses are aimed at anticipating eventual shading from potential competitor vegetation. In Arabidopsis thaliana, the most obvious SAS response at the seedling stage is the increase in hypocotyl elongation. Here, we describe how plant proximity perception rapidly and temporally alters the levels of not only auxins but also active brassinosteroids and gibberellins. At the same time, shade alters the seedling sensitivity to hormones. Plant proximity perception also involves dramatic changes in gene expression that rapidly result in a new balance between positive and negative factors in a network of interacting basic helix-loop-helix proteins, such as HFR1, PAR1, and BIM and BEE factors. Here, it was shown that several of these factors act as auxin- and BR-responsiveness modulators, which ultimately control the intensity or degree of hypocotyl elongation. It was deduced that, as a consequence of the plant proximity-dependent new, dynamic, and local balance between hormone synthesis and sensitivity (mechanistically resulting from a restructured network of SAS regulators), SAS responses are unleashed and hypocotyls elongate. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Genetic identification and molecular modeling characterization reveal a novel PROM1 mutation in Stargardt4-like macular dystrophy

PubMed Central

Imani, Saber; Cheng, Jingliang; Shasaltaneh, Marzieh Dehghan; Wei, Chunli; Yang, Lisha; Fu, Shangyi; Zou, Hui; Khan, Md. Asaduzzaman; Zhang, Xianqin; Chen, Hanchun; Zhang, Dianzheng; Duan, Chengxia; Lv, Hongbin; Li, Yumei; Chen, Rui; Fu, Junjiang

2018-01-01

Stargardt disease-4 (STGD4) is an autosomal dominant complex, genetically heterogeneous macular degeneration/dystrophy (MD) disorder. In this paper, we used targeted next generation sequencing and multiple molecular dynamics analyses to identify and characterize a disease-causing genetic variant in four generations of a Chinese family with STGD4-like MD. We found a novel heterozygous missense mutation, c.734T>C (p.L245P) in the PROM1 gene. Structurally, this mutation most likely impairs PROM1 protein stability, flexibility, and amino acid interaction network after changing the amino acid residue Leucine into Proline in the basic helix-loop-helix leucine zipper domain. Molecular dynamic simulation and principal component analysis provide compelling evidence that this PROM1 mutation contributes to disease causativeness or susceptibility variants in patients with STGD4-like MD. Thus, this finding defines new approaches in genetic characterization, accurate diagnosis, and prevention of STGD4-like MD. PMID:29416601

Potential for neural regeneration after neurotoxic injury in the adult mammalian retina

NASA Astrophysics Data System (ADS)

Ooto, Sotaro; Akagi, Tadamichi; Kageyama, Ryoichiro; Akita, Joe; Mandai, Michiko; Honda, Yoshihito; Takahashi, Masayo

2004-09-01

It has long been believed that the retina of mature mammals is incapable of regeneration. In this study, using the N-methyl-D-aspartate neurotoxicity model of adult rat retina, we observed that some Müller glial cells were stimulated to proliferate in response to a toxic injury and produce bipolar cells and rod photoreceptors. Although these newly produced neurons were limited in number, retinoic acid treatment promoted the number of regenerated bipolar cells. Moreover, misexpression of basic helix-loop-helix and homeobox genes promoted the induction of amacrine, horizontal, and rod photoreceptor specific phenotypes. These findings demonstrated that retinal neurons regenerated even in adult mammalian retina after toxic injury. Furthermore, we could partially control the fate of the regenerated neurons with extrinsic factors or intrinsic genes. The Müller glial cells constitute a potential source for the regeneration of adult mammalian retina and can be a target for drug delivery and gene therapy in retinal degenerative diseases.

The SCL gene specifies haemangioblast development from early mesoderm.

PubMed

Gering, M; Rodaway, A R; Göttgens, B; Patient, R K; Green, A R

1998-07-15

The SCL gene encodes a basic helix-loop-helix (bHLH) transcription factor that is essential for the development of all haematopoietic lineages. SCL is also expressed in endothelial cells, but its function is not essential for specification of endothelial progenitors and the role of SCL in endothelial development is obscure. We isolated the zebrafish SCL homologue and show that it was co-expressed in early mesoderm with markers of haematopoietic, endothelial and pronephric progenitors. Ectopic expression of SCL mRNA in zebrafish embryos resulted in overproduction of common haematopoietic and endothelial precursors, perturbation of vasculogenesis and concomitant loss of pronephric duct and somitic tissue. Notochord and neural tube formation were unaffected. These results provide the first evidence that SCL specifies formation of haemangioblasts, the proposed common precursor of blood and endothelial lineages. Our data also underline the striking similarities between the role of SCL in haematopoiesis/vasculogenesis and the function of other bHLH proteins in muscle and neural development.

Embryonic expression of zebrafish MiT family genes tfe3b, tfeb, and tfec.

PubMed

Lister, James A; Lane, Brandon M; Nguyen, Anhthu; Lunney, Katherine

2011-11-01

The MiT family comprises four genes in mammals: Mitf, Tfe3, Tfeb, and Tfec, which encode transcription factors of the basic-helix-loop-helix/leucine zipper class. Mitf is well-known for its essential role in the development of melanocytes, however the functions of the other members of this family, and of interactions between them, are less well understood. We have now characterized the complete set of MiT genes from zebrafish, which totals six instead of four. The zebrafish genome contain two mitf (mitfa and mitfb), two tfe3 (tfe3a and tfe3b), and single tfeb and tfec genes; this distribution is shared with other teleosts. We present here the sequence and embryonic expression patterns for the zebrafish tfe3b, tfeb, and tfec genes, and identify a new isoform of tfe3a. These findings will assist in elucidating the roles of the MiT gene family over the course of vertebrate evolution. Copyright © 2011 Wiley-Liss, Inc.

Regulation of the Drosophila Hypoxia-Inducible Factor α Sima by CRM1-Dependent Nuclear Export ▿

PubMed Central

Romero, Nuria M.; Irisarri, Maximiliano; Roth, Peggy; Cauerhff, Ana; Samakovlis, Christos; Wappner, Pablo

2008-01-01

Hypoxia-inducible factor α (HIF-α) proteins are regulated by oxygen levels through several different mechanisms that include protein stability, transcriptional coactivator recruitment, and subcellular localization. It was previously reported that these transcription factors are mainly nuclear in hypoxia and cytoplasmic in normoxia, but so far the molecular basis of this regulation is unclear. We show here that the Drosophila melanogaster HIF-α protein Sima shuttles continuously between the nucleus and the cytoplasm. We identified the relevant nuclear localization signal and two functional nuclear export signals (NESs). These NESs are in the Sima basic helix-loop-helix (bHLH) domain and promote CRM1-dependent nuclear export. Site-directed mutagenesis of either NES provoked Sima nuclear retention and increased transcriptional activity, suggesting that nuclear export contributes to Sima regulation. The identified NESs are conserved and probably functional in the bHLH domains of several bHLH-PAS proteins. We propose that rapid nuclear export of Sima regulates the duration of cellular responses to hypoxia. PMID:18332128

Specification of jaw identity by the Hand2 transcription factor

PubMed Central

Funato, Noriko; Kokubo, Hiroki; Nakamura, Masataka; Yanagisawa, Hiromi; Saga, Yumiko

2016-01-01

Acquisition of the lower jaw (mandible) was evolutionarily important for jawed vertebrates. In humans, syndromic craniofacial malformations often accompany jaw anomalies. The basic helix-loop-helix transcription factor Hand2, which is conserved among jawed vertebrates, is expressed in the neural crest in the mandibular process but not in the maxillary process of the first branchial arch. Here, we provide evidence that Hand2 is sufficient for upper jaw (maxilla)-to-mandible transformation by regulating the expression of homeobox transcription factors in mice. Altered Hand2 expression in the neural crest transformed the maxillae into mandibles with duplicated Meckel’s cartilage, which resulted in an absence of the secondary palate. In Hand2-overexpressing mutants, non-Hox homeobox transcription factors were dysregulated. These results suggest that Hand2 regulates mandibular development through downstream genes of Hand2 and is therefore a major determinant of jaw identity. Hand2 may have influenced the evolutionary acquisition of the mandible and secondary palate. PMID:27329940

Coordinated regulation of Arabidopsis microRNA biogenesis and red light signaling through Dicer-like 1 and phytochrome-interacting factor 4

PubMed Central

Sun, Zhenfei; Li, Min; Zhou, Ying; Guo, Tongtong; Liu, Yin; Zhang, Hui

2018-01-01

Light and microRNAs (miRNAs) are key external and internal signals for plant development, respectively. However, the relationship between the light signaling and miRNA biogenesis pathways remains unknown. Here we found that miRNA processer proteins DCL1 and HYL1 interact with a basic helix-loop-helix (bHLH) transcription factor, phytochrome-interacting factor 4 (PIF4), which mediates the destabilization of DCL1 during dark-to-red-light transition. PIF4 acts as a transcription factor for some miRNA genes and is necessary for the proper accumulation of miRNAs. DCL1, HYL1, and mature miRNAs play roles in the regulation of plant hypocotyl growth. These results uncovered a previously unknown crosstalk between miRNA biogenesis and red light signaling through the PIF4-dependent regulation of miRNA transcription and processing to affect red-light-directed plant photomorphogenesis. PMID:29522510

NPAS1-ARNT and NPAS3-ARNT crystal structures implicate the bHLH-PAS family as multi-ligand binding transcription factors

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

Wu, Dalei; Su, Xiaoyu; Potluri, Nalini

Here, the neuronal PAS domain proteins NPAS1 and NPAS3 are members of the basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) family, and their genetic deficiencies are linked to a variety of human psychiatric disorders including schizophrenia, autism spectrum disorders and bipolar disease. NPAS1 and NPAS3 must each heterodimerize with the aryl hydrocarbon receptor nuclear translocator (ARNT), to form functional transcription complexes capable of DNA binding and gene regulation. Here we examined the crystal structures of multi-domain NPAS1-ARNT and NPAS3-ARNT-DNA complexes, discovering each to contain four putative ligand-binding pockets. Through expanded architectural comparisons between these complexes and HIF-1α-ARNT, HIF-2α-ARNT and CLOCK-BMAL1, we show the widermore » mammalian bHLH-PAS family is capable of multi-ligand-binding and presents as an ideal class of transcription factors for direct targeting by small-molecule drugs.« less

NPAS1-ARNT and NPAS3-ARNT crystal structures implicate the bHLH-PAS family as multi-ligand binding transcription factors

DOE PAGES

Wu, Dalei; Su, Xiaoyu; Potluri, Nalini; ...

2016-10-26

Here, the neuronal PAS domain proteins NPAS1 and NPAS3 are members of the basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) family, and their genetic deficiencies are linked to a variety of human psychiatric disorders including schizophrenia, autism spectrum disorders and bipolar disease. NPAS1 and NPAS3 must each heterodimerize with the aryl hydrocarbon receptor nuclear translocator (ARNT), to form functional transcription complexes capable of DNA binding and gene regulation. Here we examined the crystal structures of multi-domain NPAS1-ARNT and NPAS3-ARNT-DNA complexes, discovering each to contain four putative ligand-binding pockets. Through expanded architectural comparisons between these complexes and HIF-1α-ARNT, HIF-2α-ARNT and CLOCK-BMAL1, we show the widermore » mammalian bHLH-PAS family is capable of multi-ligand-binding and presents as an ideal class of transcription factors for direct targeting by small-molecule drugs.« less

Molecular aspects of flower senescence and strategies to improve flower longevity

PubMed Central

Shibuya, Kenichi

2018-01-01

Flower longevity is one of the most important traits for ornamental plants. Ethylene plays a crucial role in flower senescence in some plant species. In several species that show ethylene-dependent flower senescence, genetic modification targeting genes for ethylene biosynthesis or signaling has improved flower longevity. Although little is known about regulatory mechanisms of petal senescence in flowers that show ethylene-independent senescence, a recent study of Japanese morning glory revealed that a NAC transcription factor, EPHEMERAL1 (EPH1), is a key regulator in ethylene-independent petal senescence. EPH1 is induced in an age-dependent manner irrespective of ethylene signal, and suppression of EPH1 expression dramatically delays petal senescence. In ethylene-dependent petal senescence, comprehensive transcriptome analyses revealed the involvement of transcription factors, a basic helix-loop-helix protein and a homeodomain-leucine zipper protein, in the transcriptional regulation of the ethylene biosynthesis enzymes. This review summarizes molecular aspects of flower senescence and discusses strategies to improve flower longevity by molecular breeding. PMID:29681752

MyoD expression restores defective myogenic differentiation of human mesoangioblasts from inclusion-body myositis muscle.

PubMed

Morosetti, Roberta; Mirabella, Massimiliano; Gliubizzi, Carla; Broccolini, Aldobrando; De Angelis, Luciana; Tagliafico, Enrico; Sampaolesi, Maurilio; Gidaro, Teresa; Papacci, Manuela; Roncaglia, Enrica; Rutella, Sergio; Ferrari, Stefano; Tonali, Pietro Attilio; Ricci, Enzo; Cossu, Giulio

2006-11-07

Inflammatory myopathies (IM) are acquired diseases of skeletal muscle comprising dermatomyositis (DM), polymyositis (PM), and inclusion-body myositis (IBM). Immunosuppressive therapies, usually beneficial for DM and PM, are poorly effective in IBM. We report the isolation and characterization of mesoangioblasts, vessel-associated stem cells, from diagnostic muscle biopsies of IM. The number of cells isolated, proliferation rate and lifespan, markers expression, and ability to differentiate into smooth muscle do not differ among normal and IM mesoangioblasts. At variance with normal, DM and PM mesoangioblasts, cells isolated from IBM, fail to differentiate into skeletal myotubes. These data correlate with lack in connective tissue of IBM muscle of alkaline phosphatase (ALP)-positive cells, conversely dramatically increased in PM and DM. A myogenic inhibitory basic helix-loop-helix factor B3 is highly expressed in IBM mesoangioblasts. Indeed, silencing this gene or overexpressing MyoD rescues the myogenic defect of IBM mesoangioblasts, opening novel cell-based therapeutic strategies for this crippling disorder.

Mechanical stress mediated by both endosperm softening and embryo growth underlies endosperm elimination in Arabidopsis seeds.

PubMed

Fourquin, Chloé; Beauzamy, Léna; Chamot, Sophy; Creff, Audrey; Goodrich, Justin; Boudaoud, Arezki; Ingram, Gwyneth

2016-09-15

Seed development in angiosperms demands the tightly coordinated development of three genetically distinct structures. The embryo is surrounded by the endosperm, which is in turn enclosed within the maternally derived seed coat. In Arabidopsis, final seed size is determined by early expansion of the coenocytic endosperm, which then cellularises and subsequently undergoes developmental programmed cell death, breaking down as the embryo grows. Endosperm breakdown requires the endosperm-specific basic helix-loop-helix transcription factor ZHOUPI. However, to date, the mechanism underlying the Arabidopsis endosperm breakdown process has not been elucidated. Here, we provide evidence that ZHOUPI does not induce the developmental programmed cell death of the endosperm directly. Instead ZHOUPI indirectly triggers cell death by regulating the expression of cell wall-modifying enzymes, thus altering the physical properties of the endosperm to condition a mechanical environment permitting the compression of the cellularised endosperm by the developing embryo. © 2016. Published by The Company of Biologists Ltd.

Nuclear localized protein-1 (Nulp1) increases cell death of human osteosarcoma cells and binds the X-linked inhibitor of apoptosis protein

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

Steen, Hakan; Lindholm, Dan; Minerva Institute for Medical Research, Biomedicum Helsinki, Helsinki

2008-02-08

Nuclear localized protein-1 (Nulp1) is a recently identified gene expressed in mouse and human tissues particularly during embryonic development. Nulp1 belongs to the family of basic helix-loop-helix (bHLH) proteins that are important in development. The precise function of Nulp1 in cells is however not known. We observed that overexpression of Nulp1 induces a large increase in cell death of human osteosarcoma Saos2 cells with DNA fragmentation. In mouse N2A neuroblastoma cells Nulp1 affected cell proliferation and sensitized cells towards death induced by staurosporine. Staining using a novel antibody localized Nulp1 mainly to the cell nucleus and to some extent tomore » the cytoplasm. Nulp1 binds the X-linked inhibitor of apoptosis protein (XIAP) and this interaction was increased during cell death. These results indicate that Nulp1 plays a role in cell death control and may influence tumor growth.« less

Aryl hydrocarbon receptor and intestinal immunity.

PubMed

Lamas, Bruno; Natividad, Jane M; Sokol, Harry

2018-04-07

Aryl hydrocarbon receptor (AhR) is a member of the basic helix-loop-helix-(bHLH) superfamily of transcription factors, which are associated with cellular responses to environmental stimuli, such as xenobiotics and oxygen levels. Unlike other members of bHLH, AhR is the only bHLH transcription factor that is known to be ligand activated. Early AhR studies focused on understanding the role of AhR in mediating the toxicity and carcinogenesis properties of the prototypic ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In recent years, however, it has become apparent that, in addition to its toxicological involvement, AhR is highly receptive to a wide array of endogenous and exogenous ligands, and that its activation leads to a myriad of key host physiological functions. In this study, we review the current understanding of the functions of AhR in the mucosal immune system with a focus on its role in intestinal barrier function and intestinal immune cells, as well as in intestinal homeostasis.

Atoh1-lineal neurons are required for hearing and for the survival of neurons in the spiral ganglion and brainstem accessory auditory nuclei

PubMed Central

Maricich, Stephen M.; Xia, Anping; Mathes, Erin L.; Wang, Vincent Y.; Oghalai, John S.; Fritzsch, Bernd; Zoghbi, Huda Y.

2009-01-01

Atoh1 is a basic helix-loop-helix transcription factor necessary for the specification of inner ear hair cells and central auditory system neurons derived from the rhombic lip. We used the Cre-loxP system and two Cre-driver lines (Egr2Cre and Hoxb1Cre) to delete Atoh1 from different regions of the cochlear nucleus (CN) and accessory auditory nuclei (AAN). Adult Atoh1-conditional knockout mice (Atoh1CKO) are behaviorally deaf, have diminished auditory brainstem evoked responses and disrupted CN and AAN morphology and connectivity. In addition, Egr2; Atoh1CKO mice lose spiral ganglion neurons in the cochlea and AAN neurons during the first 3 days of life, revealing a novel critical period in the development of these neurons. These new mouse models of predominantly central deafness illuminate the importance of the CN for support of a subset of peripheral and central auditory neurons. PMID:19741118

Network theory inspired analysis of time-resolved expression data reveals key players guiding P. patens stem cell development.

PubMed

Busch, Hauke; Boerries, Melanie; Bao, Jie; Hanke, Sebastian T; Hiss, Manuel; Tiko, Theodhor; Rensing, Stefan A

2013-01-01

Transcription factors (TFs) often trigger developmental decisions, yet, their transcripts are often only moderately regulated and thus not easily detected by conventional statistics on expression data. Here we present a method that allows to determine such genes based on trajectory analysis of time-resolved transcriptome data. As a proof of principle, we have analysed apical stem cells of filamentous moss (P. patens) protonemata that develop from leaflets upon their detachment from the plant. By our novel correlation analysis of the post detachment transcriptome kinetics we predict five out of 1,058 TFs to be involved in the signaling leading to the establishment of pluripotency. Among the predicted regulators is the basic helix loop helix TF PpRSL1, which we show to be involved in the establishment of apical stem cells in P. patens. Our methodology is expected to aid analysis of key players of developmental decisions in complex plant and animal systems.

Repression by PRDM13 is critical for generating precision in neuronal identity

PubMed Central

Kollipara, Rahul K; Ma, Zhenzhong; Borromeo, Mark D; Chang, Joshua C

2017-01-01

The mechanisms that activate some genes while silencing others are critical to ensure precision in lineage specification as multipotent progenitors become restricted in cell fate. During neurodevelopment, these mechanisms are required to generate the diversity of neuronal subtypes found in the nervous system. Here we report interactions between basic helix-loop-helix (bHLH) transcriptional activators and the transcriptional repressor PRDM13 that are critical for specifying dorsal spinal cord neurons. PRDM13 inhibits gene expression programs for excitatory neuronal lineages in the dorsal neural tube. Strikingly, PRDM13 also ensures a battery of ventral neural tube specification genes such as Olig1, Olig2 and Prdm12 are excluded dorsally. PRDM13 does this via recruitment to chromatin by multiple neural bHLH factors to restrict gene expression in specific neuronal lineages. Together these findings highlight the function of PRDM13 in repressing the activity of bHLH transcriptional activators that together are required to achieve precise neuronal specification during mouse development. PMID:28850031

Phosphorylation of basic helix-loop-helix transcription factor Twist in development and disease.

PubMed

Xue, Gongda; Hemmings, Brian A

2012-02-01

The transcription factor Twist plays vital roles during embryonic development through regulating/controlling cell migration. However, postnatally, in normal physiological settings, Twist is either not expressed or inactivated. Increasing evidence shows a strong correlation between Twist reactivation and both cancer progression and malignancy, where the transcriptional activities of Twist support cancer cells to disseminate from primary tumours and subsequently establish a secondary tumour growth in distant organs. However, it is largely unclear how this signalling programme is reactivated or what signalling pathways regulate its activity. The present review discusses recent advances in Twist regulation and activity, with a focus on phosphorylation-dependent Twist activity, potential upstream kinases and the contribution of these factors in transducing biological signals from upstream signalling complexes. The recent advances in these areas have shed new light on how phosphorylation-dependent regulation of the Twist proteins promotes or suppresses Twist activity, leading to differential regulation of Twist transcriptional targets and thereby influencing cell fate.

Structural basis for recognition of centromere histone variant CenH3 by the chaperone Scm3

PubMed Central

Zhou, Zheng; Feng, Hanqiao; Zhou, Bing-Rui; Ghirlando, Rodolfo; Hu, Kaifeng; Zwolak, Adam; Miller Jenkins, Lisa M.; Xiao, Hua; Tjandra, Nico; Wu, Carl; Bai, Yawen

2011-01-01

The centromere is a unique chromosomal locus that ensures accurate segregation of chromosomes during cell division by directing the assembly of a multiprotein complex, the kinetochore1. The centromere is marked by a conserved variant of conventional histone H3 termed CenH3 or CENP-A2. A conserved motif of CenH3, the CATD, defined by loop 1 and helix 2 of the histone fold, is necessary and sufficient for specifying centromere functions of CenH33, 4. The structural basis of this specification is of outstanding interest. Yeast Scm3 and human HJURP are conserved nonhistone proteins that interact physically with the (CenH3-H4)2 heterotetramer and are required for the deposition of CenH3 at centromeres in vivo5, 6, 7, 8, 9, 10, 11, 12, 13. Here we have elucidated the structural basis for recognition of budding yeast CenH3 (Cse4) by Scm3. We solved the structure of the Cse4-binding domain (CBD) of Scm3 complexed with Cse4 and H4 in a single chain model. An α-helix and an irregular loop at the conserved N-terminus and a shorter α-helix at the C-terminus of Scm3-CBD wraps around the Cse4-H4 dimer. Four Cse4-specific residues in the N-terminal region of helix 2 are sufficient for specific recognition by conserved and functionally important residues in the N-terminal helix of Scm3 through formation of a hydrophobic cluster. Scm3-CBD induces major conformational changes and sterically occludes DNA binding sites in the structure of Cse4 and H4. These findings have implications for the assembly and architecture of the centromeric nucleosome. PMID:21412236

A conserved phenylalanine as a relay between the α5 helix and the GDP binding region of heterotrimeric Gi protein α subunit.

PubMed

Kaya, Ali I; Lokits, Alyssa D; Gilbert, James A; Iverson, Tina M; Meiler, Jens; Hamm, Heidi E

2014-08-29

G protein activation by G protein-coupled receptors is one of the critical steps for many cellular signal transduction pathways. Previously, we and other groups reported that the α5 helix in the G protein α subunit plays a major role during this activation process. However, the precise signaling pathway between the α5 helix and the guanosine diphosphate (GDP) binding pocket remains elusive. Here, using structural, biochemical, and computational techniques, we probed different residues around the α5 helix for their role in signaling. Our data showed that perturbing the Phe-336 residue disturbs hydrophobic interactions with the β2-β3 strands and α1 helix, leading to high basal nucleotide exchange. However, mutations in β strands β5 and β6 do not perturb G protein activation. We have highlighted critical residues that leverage Phe-336 as a relay. Conformational changes are transmitted starting from Phe-336 via β2-β3/α1 to Switch I and the phosphate binding loop, decreasing the stability of the GDP binding pocket and triggering nucleotide release. When the α1 and α5 helices were cross-linked, inhibiting the receptor-mediated displacement of the C-terminal α5 helix, mutation of Phe-336 still leads to high basal exchange rates. This suggests that unlike receptor-mediated activation, helix 5 rotation and translocation are not necessary for GDP release from the α subunit. Rather, destabilization of the backdoor region of the Gα subunit is sufficient for triggering the activation process. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Conformational Flexibility of a Short Loop near the Active Site of the SARS-3CLpro is Essential to Maintain Catalytic Activity

NASA Astrophysics Data System (ADS)

Li, Chunmei; Teng, Xin; Qi, Yifei; Tang, Bo; Shi, Hailing; Ma, Xiaomin; Lai, Luhua

2016-02-01

The SARS 3C-like proteinase (SARS-3CLpro), which is the main proteinase of the SARS coronavirus, is essential to the virus life cycle. This enzyme has been shown to be active as a dimer in which only one protomer is active. However, it remains unknown how the dimer structure maintains an active monomer conformation. It has been observed that the Ser139-Leu141 loop forms a short 310-helix that disrupts the catalytic machinery in the inactive monomer structure. We have tried to disrupt this helical conformation by mutating L141 to T in the stable inactive monomer G11A/R298A/Q299A. The resulting tetra-mutant G11A/L141T/R298A/Q299A is indeed enzymatically active as a monomer. Molecular dynamics simulations revealed that the L141T mutation disrupts the 310-helix and helps to stabilize the active conformation. The coil-310-helix conformational transition of the Ser139-Leu141 loop serves as an enzyme activity switch. Our study therefore indicates that the dimer structure can stabilize the active conformation but is not a required structure in the evolution of the active enzyme, which can also arise through simple mutations.

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

Manjasetty, Babu A.; Halavaty, Andrei S.; Luan, Chi-Hao

Multidrug transcription regulator AcrR from Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 belongs to the tetracycline repressor family, one of the largest groups of bacterial transcription factors. The crystal structure of dimeric AcrR was determined and refined to 1.56 Å resolution. The tertiary and quaternary structures of AcrR are similar to those of its homologs. The multidrug binding site was identified based on structural alignment with homologous proteins and has a di(hydroxyethyl)ether molecule bound. Residues from helices a4 and a7 shape the entry into this binding site. The structure of AcrR reveals that the extended helical conformation of helixmore » a4 is stabilized by the hydrogen bond between Glu67 (helix a4) and Gln130 (helix a7). Based on the structural comparison with the closest homolog structure, the Escherichia coli AcrR, we propose that this hydrogen bond is responsible for control of the loop-to-helix transition within helix a4. This local conformational switch of helix a4 may be a key step in accessing the multidrug binding site and securing ligands at the binding site. Solution smallmolecule binding studies suggest that AcrR binds ligands with their core chemical structure resembling the tetracyclic ring of cholesterol.« less

The structural coupling between ATPase activation and recovery stroke in the myosin II motor

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

Koppole, Sampath; Smith, Jeremy C; Fischer, S.

2007-07-01

Before the myosin motor head can perform the next power stroke, it undergoes a large conformational transition in which the converter domain, bearing the lever arm, rotates {approx} 65{sup o}. Simultaneous with this 'recovery stroke', myosin activates its ATPase function by closing the Switch-2 loop over the bound ATP. This coupling between the motions of the converter domain and of the 40 {angstrom}-distant Switch-2 loop is essential to avoid unproductive ATP hydrolysis. The coupling mechanism is determined here by finding a series of optimized intermediates between crystallographic end structures of the recovery stroke (Dictyostelium discoideum), yielding movies of the transitionmore » at atomic detail. The successive formation of two hydrogen bonds by the Switch-2 loop is correlated with the successive see-saw motions of the relay and SH1 helices that hold the converter domain. SH1 helix and Switch-2 loop communicate via a highly conserved loop that wedges against the SH1-helix upon Switch-2 closing.« less

The structural coupling between ATPase activation and recovery stroke in the myosin II motor.

PubMed

Koppole, Sampath; Smith, Jeremy C; Fischer, Stefan

2007-07-01

Before the myosin motor head can perform the next power stroke, it undergoes a large conformational transition in which the converter domain, bearing the lever arm, rotates approximately 65 degrees . Simultaneous with this "recovery stroke," myosin activates its ATPase function by closing the Switch-2 loop over the bound ATP. This coupling between the motions of the converter domain and of the 40 A-distant Switch-2 loop is essential to avoid unproductive ATP hydrolysis. The coupling mechanism is determined here by finding a series of optimized intermediates between crystallographic end structures of the recovery stroke (Dictyostelium discoideum), yielding movies of the transition at atomic detail. The successive formation of two hydrogen bonds by the Switch-2 loop is correlated with the successive see-saw motions of the relay and SH1 helices that hold the converter domain. SH1 helix and Switch-2 loop communicate via a highly conserved loop that wedges against the SH1-helix upon Switch-2 closing.

Response of the flat cochlear epithelium to forced expression of Atoh1.

PubMed

Izumikawa, Masahiko; Batts, Shelley A; Miyazawa, Toru; Swiderski, Donald L; Raphael, Yehoash

2008-06-01

Following hair cell elimination in severely traumatized cochleae, differentiated supporting cells are often replaced by a simple epithelium with cuboidal or flat appearance. Atoh1 (previously Math1) is a basic helix-loop-helix transcription factor critical to hair cell differentiation during mammalian embryogenesis. Forced expression of Atoh1 in the differentiated supporting cell population can induce transdifferentiation leading to hair cell regeneration. Here, we examined the outcome of adenovirus mediated over-expression of Atoh1 in the non-sensory cells of the flat epithelium. We determined that seven days after unilateral elimination of hair cells with neomycin, differentiated supporting cells are absent, replaced by a flat epithelium. Nerve processes were also missing from the auditory epithelium, with the exception of infrequent looping nerve processes above the habenula perforata. We then inoculated an adenovirus vector with Atoh1 insert into the scala media of the deafened cochlea. The inoculation resulted in upregulation of Atoh1 in the flat epithelium. However, two months after the inoculation, Atoh1-treated ears did not exhibit clear signs of hair cell regeneration. Combined with previous data on induction of supporting cell to hair cell transdifferentiation by forced expression of Atoh1, these results suggest that the presence of differentiated supporting cells in the organ of Corti is necessary for transdifferentiation to occur.

Basic Helix-Loop-Helix Transcription Factor Bmsage Is Involved in Regulation of fibroin H-chain Gene via Interaction with SGF1 in Bombyx mori

PubMed Central

Li, Qiong-Yan; Hu, Wen-Bo; Zhou, Meng-Ting; Nie, Hong-Yi; Zhang, Yin-Xia; Peng, Zhang-Chuan; Zhao, Ping; Xia, Qing-You

2014-01-01

Silk glands are specialized in the synthesis of several secretory proteins. Expression of genes encoding the silk proteins in Bombyx mori silk glands with strict territorial and developmental specificities is regulated by many transcription factors. In this study, we have characterized B. mori sage, which is closely related to sage in the fruitfly Drosophila melanogaster. It is termed Bmsage; it encodes transcription factor Bmsage, which belongs to the Mesp subfamily, containing a basic helix–loop–helix motif. Bmsage transcripts were detected specifically in the silk glands of B. mori larvae through RT-PCR analysis. Immunoblotting analysis confirmed the Bmsage protein existed exclusively in B. mori middle and posterior silk gland cells. Bmsage has a low level of expression in the 4th instar molting stages, which increases gradually in the 5th instar feeding stages and then declines from the wandering to the pupation stages. Quantitative PCR analysis suggested the expression level of Bmsage in a high silk strain was higher compared to a lower silk strain on day 3 of the larval 5th instar. Furthermore, far western blotting and co-immunoprecipitation assays showed the Bmsage protein interacted with the fork head transcription factor silk gland factor 1 (SGF1). An electrophoretic mobility shift assay showed the complex of Bmsage and SGF1 proteins bound to the A and B elements in the promoter of fibroin H-chain gene(fib-H), respectively. Luciferase reporter gene assays confirmed the complex of Bmsage and SGF1 proteins increased the expression of fib-H. Together, these results suggest Bmsage is involved in the regulation of the expression of fib-H by being together with SGF1 in B. mori PSG cells. PMID:24740008

Arabidopsis MYC Transcription Factors Are the Target of Hormonal Salicylic Acid/Jasmonic Acid Cross Talk in Response to Pieris brassicae Egg Extract1[OPEN

PubMed Central

Schmiesing, André; Gouhier-Darimont, Caroline

2016-01-01

Arabidopsis (Arabidopsis thaliana) plants recognize insect eggs and activate the salicylic acid (SA) pathway. As a consequence, expression of defense genes regulated by the jasmonic acid (JA) pathway is suppressed and larval performance is enhanced. Cross talk between defense signaling pathways is common in plant-pathogen interactions, but the molecular mechanism mediating this phenomenon is poorly understood. Here, we demonstrate that egg-induced SA/JA antagonism works independently of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor ORA59, which controls the ERF branch of the JA pathway. In addition, treatment with egg extract did not enhance expression or stability of JASMONATE ZIM-domain transcriptional repressors, and SA/JA cross talk did not involve JASMONATE ASSOCIATED MYC2-LIKEs, which are negative regulators of the JA pathway. Investigating the stability of MYC2, MYC3, and MYC4, three basic helix-loop-helix transcription factors that additively control jasmonate-related defense responses, we found that egg extract treatment strongly diminished MYC protein levels in an SA-dependent manner. Furthermore, we identified WRKY75 as a novel and essential factor controlling SA/JA cross talk. These data indicate that insect eggs target the MYC branch of the JA pathway and uncover an unexpected modulation of SA/JA antagonism depending on the biological context in which the SA pathway is activated. PMID:26884488

Antagonistic Actions of HLH/bHLH Proteins Are Involved in Grain Length and Weight in Rice

PubMed Central

Heang, Dany; Sassa, Hidenori

2012-01-01

Grain size is a major yield component in rice, and partly controlled by the sizes of the lemma and palea. Molecular mechanisms controlling the sizes of these organs largely remain unknown. In this study, we show that an antagonistic pair of basic helix-loop-helix (bHLH) proteins is involved in determining rice grain length by controlling cell length in the lemma/palea. Overexpression of an atypical bHLH, named POSITIVE REGULATOR OF GRAIN LENGTH 1 (PGL1), in lemma/palea increased grain length and weight in transgenic rice. PGL1 is an atypical non-DNA-binding bHLH and assumed to function as an inhibitor of a typical DNA-binding bHLH through heterodimerization. We identified the interaction partner of PGL1 and named it ANTAGONIST OF PGL1 (APG). PGL1 and APG interacted in vivo and localized in the nucleus. As expected, silencing of APG produced the same phenotype as overexpression of PGL1, suggesting antagonistic roles for the two genes. Transcription of two known grain-length-related genes, GS3 and SRS3, was largely unaffected in the PGL1-overexpressing and APG-silenced plants. Observation of the inner epidermal cells of lemma revealed that are caused by increased cell length. PGL1-APG represents a new grain length and weight-controlling pathway in which APG is a negative regulator whose function is inhibited by PGL1. PMID:22363621

bHLH106 Integrates Functions of Multiple Genes through Their G-Box to Confer Salt Tolerance on Arabidopsis.

PubMed

Ahmad, Aftab; Niwa, Yasuo; Goto, Shingo; Ogawa, Takeshi; Shimizu, Masanori; Suzuki, Akane; Kobayashi, Kyoko; Kobayashi, Hirokazu

2015-01-01

An activation-tagging methodology was applied to dedifferentiated calli of Arabidopsis to identify new genes involved in salt tolerance. This identified salt tolerant callus 8 (stc8) as a gene encoding the basic helix-loop-helix transcription factor bHLH106. bHLH106-knockout (KO) lines were more sensitive to NaCl, KCl, LiCl, ABA, and low temperatures than the wild-type. Back-transformation of the KO line rescued its phenotype, and over-expression (OX) of bHLH106 in differentiated plants exhibited tolerance to NaCl. Green fluorescent protein (GFP) fused with bHLH106 revealed that it was localized to the nucleus. Prepared bHLH106 protein was subjected to electrophoresis mobility shift assays against E-box sequences (5'-CANNTG-3'). The G-box sequence 5'-CACGTG-3' had the strongest interaction with bHLH106. bHLH106-OX lines were transcriptomically analyzed, and resultant up- and down-regulated genes selected on the criterion of presence of a G-box sequence. There were 198 genes positively regulated by bHLH106 and 36 genes negatively regulated; these genes possessed one or more G-box sequences in their promoter regions. Many of these genes are known to be involved in abiotic stress response. It is concluded that bHLH106 locates at a branching point in the abiotic stress response network by interacting directly to the G-box in genes conferring salt tolerance on plants.

The antagonistic basic helix-loop-helix partners BEE and IBH1 contribute to control plant tolerance to abiotic stress.

PubMed

Moreno, Javier E; Moreno-Piovano, Guillermo; Chan, Raquel L

2018-06-01

The bHLH family is composed by canonical and non-canonical transcription factors (TFs) that differ in the presence or absence of their DNA-binding domain, respectively. Since both types of bHLH proteins are able to dimerize, their relative abundance impacts their biological activity. Among this TF family BEE and IBH are canonical and non-canonical bHLHs, respectively and previous reports indicated that BEE2 and IBH1 dimerize. Wondering whether BEE TFs participate in the abiotic stress response and how the dimerization with IBH1 could regulate their role in Arabidopsis, double bee1/bee2 and triple bee1/bee2/bee3 mutants were tested under salinity and drought stresses. The bee1/bee2/bee3 mutant showed an enhanced tolerance whereas the double mutant behaved similar to wild type plants. These results indicated that BEE genes play a role in the stress response and also put in evidence the redundancy within the BEE family. Moreover, ectopic expression of IBH1 on different mutant backgrounds improved plant tolerance to abiotic stress, independently of the background. However, the yield of these transgenic plants was penalized with abortive seeds. Our results suggest that BEE genes are negative regulators of physiological responses to abiotic stress whereas IBH1 is a positive modulator via different pathways, one of them involving BEE TFs. Copyright © 2018 Elsevier B.V. All rights reserved.

Direct Control of SPEECHLESS by PIF4 in the High-Temperature Response of Stomatal Development.

PubMed

Lau, On Sun; Song, Zhuojun; Zhou, Zimin; Davies, Kelli A; Chang, Jessica; Yang, Xin; Wang, Shenqi; Lucyshyn, Doris; Tay, Irene Hui Zhuang; Wigge, Philip A; Bergmann, Dominique C

2018-04-23

Environmental factors shape the phenotypes of multicellular organisms. The production of stomata-the epidermal pores required for gas exchange in plants-is highly plastic and provides a powerful platform to address environmental influence on cell differentiation [1-3]. Rising temperatures are already impacting plant growth, a trend expected to worsen in the near future [4]. High temperature inhibits stomatal production, but the underlying mechanism is not known [5]. Here, we show that elevated temperature suppresses the expression of SPEECHLESS (SPCH), the basic-helix-loop-helix (bHLH) transcription factor that serves as the master regulator of stomatal lineage initiation [6, 7]. Our genetic and expression analyses indicate that the suppression of SPCH and stomatal production is mediated by the bHLH transcription factor PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), a core component of high-temperature signaling [8]. Importantly, we demonstrate that, upon exposure to high temperature, PIF4 accumulates in the stomatal precursors and binds to the promoter of SPCH. In addition, we find SPCH feeds back negatively to the PIF4 gene. We propose a model where warm-temperature-activated PIF4 binds and represses SPCH expression to restrict stomatal production at elevated temperatures. Our work identifies a molecular link connecting high-temperature signaling and stomatal development and reveals a direct mechanism by which production of a specific cell lineage can be controlled by a broadly expressed environmental signaling factor. Copyright © 2018 Elsevier Ltd. All rights reserved.

Arabidopsis MYC Transcription Factors Are the Target of Hormonal Salicylic Acid/Jasmonic Acid Cross Talk in Response to Pieris brassicae Egg Extract.

PubMed

Schmiesing, André; Emonet, Aurélia; Gouhier-Darimont, Caroline; Reymond, Philippe

2016-04-01

Arabidopsis (Arabidopsis thaliana) plants recognize insect eggs and activate the salicylic acid (SA) pathway. As a consequence, expression of defense genes regulated by the jasmonic acid (JA) pathway is suppressed and larval performance is enhanced. Cross talk between defense signaling pathways is common in plant-pathogen interactions, but the molecular mechanism mediating this phenomenon is poorly understood. Here, we demonstrate that egg-induced SA/JA antagonism works independently of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor ORA59, which controls the ERF branch of the JA pathway. In addition, treatment with egg extract did not enhance expression or stability of JASMONATE ZIM-domain transcriptional repressors, and SA/JA cross talk did not involve JASMONATE ASSOCIATED MYC2-LIKEs, which are negative regulators of the JA pathway. Investigating the stability of MYC2, MYC3, and MYC4, three basic helix-loop-helix transcription factors that additively control jasmonate-related defense responses, we found that egg extract treatment strongly diminished MYC protein levels in an SA-dependent manner. Furthermore, we identified WRKY75 as a novel and essential factor controlling SA/JA cross talk. These data indicate that insect eggs target the MYC branch of the JA pathway and uncover an unexpected modulation of SA/JA antagonism depending on the biological context in which the SA pathway is activated. © 2016 American Society of Plant Biologists. All Rights Reserved.

The helix-loop-helix protein id1 controls stem cell proliferation during regenerative neurogenesis in the adult zebrafish telencephalon.

PubMed

Rodriguez Viales, Rebecca; Diotel, Nicolas; Ferg, Marco; Armant, Olivier; Eich, Julia; Alunni, Alessandro; März, Martin; Bally-Cuif, Laure; Rastegar, Sepand; Strähle, Uwe

2015-03-01

The teleost brain has the remarkable ability to generate new neurons and to repair injuries during adult life stages. Maintaining life-long neurogenesis requires careful management of neural stem cell pools. In a genome-wide expression screen for transcription regulators, the id1 gene, encoding a negative regulator of E-proteins, was found to be upregulated in response to injury. id1 expression was mapped to quiescent type I neural stem cells in the adult telencephalic stem cell niche. Gain and loss of id1 function in vivo demonstrated that Id1 promotes stem cell quiescence. The increased id1 expression observed in neural stem cells in response to injury appeared independent of inflammatory signals, suggesting multiple antagonistic pathways in the regulation of reactive neurogenesis. Together, we propose that Id1 acts to maintain the neural stem cell pool by counteracting neurogenesis-promoting signals. © 2014 AlphaMed Press.

Recurrent Mutations in the Basic Domain of TWIST2 Cause Ablepharon Macrostomia and Barber-Say Syndromes.

PubMed

Marchegiani, Shannon; Davis, Taylor; Tessadori, Federico; van Haaften, Gijs; Brancati, Francesco; Hoischen, Alexander; Huang, Haigen; Valkanas, Elise; Pusey, Barbara; Schanze, Denny; Venselaar, Hanka; Vulto-van Silfhout, Anneke T; Wolfe, Lynne A; Tifft, Cynthia J; Zerfas, Patricia M; Zambruno, Giovanna; Kariminejad, Ariana; Sabbagh-Kermani, Farahnaz; Lee, Janice; Tsokos, Maria G; Lee, Chyi-Chia R; Ferraz, Victor; da Silva, Eduarda Morgana; Stevens, Cathy A; Roche, Nathalie; Bartsch, Oliver; Farndon, Peter; Bermejo-Sanchez, Eva; Brooks, Brian P; Maduro, Valerie; Dallapiccola, Bruno; Ramos, Feliciano J; Chung, Hon-Yin Brian; Le Caignec, Cédric; Martins, Fabiana; Jacyk, Witold K; Mazzanti, Laura; Brunner, Han G; Bakkers, Jeroen; Lin, Shuo; Malicdan, May Christine V; Boerkoel, Cornelius F; Gahl, William A; de Vries, Bert B A; van Haelst, Mieke M; Zenker, Martin; Markello, Thomas C

2015-07-02

Ablepharon macrostomia syndrome (AMS) and Barber-Say syndrome (BSS) are rare congenital ectodermal dysplasias characterized by similar clinical features. To establish the genetic basis of AMS and BSS, we performed extensive clinical phenotyping, whole exome and candidate gene sequencing, and functional validations. We identified a recurrent de novo mutation in TWIST2 in seven independent AMS-affected families, as well as another recurrent de novo mutation affecting the same amino acid in ten independent BSS-affected families. Moreover, a genotype-phenotype correlation was observed, because the two syndromes differed based solely upon the nature of the substituting amino acid: a lysine at TWIST2 residue 75 resulted in AMS, whereas a glutamine or alanine yielded BSS. TWIST2 encodes a basic helix-loop-helix transcription factor that regulates the development of mesenchymal tissues. All identified mutations fell in the basic domain of TWIST2 and altered the DNA-binding pattern of Flag-TWIST2 in HeLa cells. Comparison of wild-type and mutant TWIST2 expressed in zebrafish identified abnormal developmental phenotypes and widespread transcriptome changes. Our results suggest that autosomal-dominant TWIST2 mutations cause AMS or BSS by inducing protean effects on the transcription factor's DNA binding. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Insulin stimulates the expression of the SHARP-1 gene via multiple signaling pathways.

PubMed

Takagi, K; Asano, K; Haneishi, A; Ono, M; Komatsu, Y; Yamamoto, T; Tanaka, T; Ueno, H; Ogawa, W; Tomita, K; Noguchi, T; Yamada, K

2014-06-01

The rat enhancer of split- and hairy-related protein-1 (SHARP-1) is a basic helix-loop-helix transcription factor. An issue of whether SHARP-1 is an insulin-inducible transcription factor was examined. Insulin rapidly increased the level of SHARP-1 mRNA both in vivo and in vitro. Then, signaling pathways involved with the increase of SHARP-1 mRNA by insulin were determined in H4IIE rat hepatoma cells. Pretreatments with LY294002, wortmannin, and staurosporine completely blocked the induction effect, suggesting the involvement of both phosphoinositide 3-kinase (PI 3-K) and protein kinase C (PKC) pathways. In fact, overexpression of a dominant negative form of atypical protein kinase C lambda (aPKCλ) significantly decreased the induction of the SHARP-1 mRNA. In addition, inhibitors for the small GTPase Rac or Jun N-terminal kinase (JNK) also blocked the induction of SHARP-1 mRNA by insulin. Overexpression of a dominant negative form of Rac1 prevented the activation by insulin. Furthermore, actinomycin D and cycloheximide completely blocked the induction of SHARP-1 mRNA by insulin. Finally, when a SHARP-1 expression plasmid was transiently transfected with various reporter plasmids into H4IIE cells, the promoter activity of PEPCK reporter plasmid was specifically decreased. Thus, we conclude that insulin induces the SHARP-1 gene expression at the transcription level via a both PI 3-K/aPKCλ/JNK- and a PI 3-K/Rac/JNK-signaling pathway; protein synthesis is required for this induction; and that SHARP-1 is a potential repressor of the PEPCK gene expression. © Georg Thieme Verlag KG Stuttgart · New York.

pH Dependence of a 310-Helix versus a Turn in the M-Loop Region of PDE4: Observations on PDB Entries and an Electronic Structure Study.

PubMed

Usharani, Dandamudi; Srivani, Palakuri; Sastry, G Narahari; Jemmis, Eluvathingal D

2008-06-01

Available X-ray crystal structures of phosphodiesterase 4 (PDE 4) are classified into two groups based on a secondary structure difference of a 310-helix versus a turn in the M-loop region. The only variable that was discernible between these two sets is the pH at the crystallization conditions. Assuming that at lower pH there is a possibility of protonation, thermodynamics of protonation and deprotonation of the aspartic acid, cysteine side chains, and amide bonds are calculated. The models in the gas phase and in the explicit solvent using the ONIOM method are calculated at the B3LYP/6-31+G* and B3LYP/6-31+G*:UFF levels of theory, respectively. The molecular dynamics (MD) simulations are also performed on the M-loop region of a 310-helix and a turn with explicit water for 10 ns under NPT conditions. The isodesmic equations of the various protonation states show that the turn containing structure is thermodynamically more stable when proline or cysteine is protonated. The preference for the turn structure on protonation (pH = 6.5-7.5) is due to an increase in the number of the hydrogen bonding and electrostatic interactions gained by the surrounding environment such as adjacent residues and solvent molecules.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-mediated deregulation of myeloid and sebaceous gland stem/progenitor cell homeostasis.

PubMed

Bock, Karl Walter

2017-06-01

Studies of TCDD toxicity stimulated identification of the responsible aryl hydrocarbon receptor (AHR), a multifunctional, ligand-activated transcription factor of the basic helix-loop-helix/Per-Arnt-Sim family. Accumulating evidence suggests a role of this receptor in homeostasis of stem/progenitor cells, in addition to its known role in xenobiotic metabolism. (1) Regulation of myelopoiesis is complex. As one example, AHR-mediated downregulation of human CD34+ progenitor differentiation to monocytes/macrophages is discussed. (2) Accumulation of TCDD in sebum leads to deregulation of sebocyte differentiation via Blimp1-mediated inhibition of c-Myc signaling and stimulation of Wnt-mediated proliferation of interfollicular epidermis. The resulting sebaceous gland atrophy and formation of dermal cysts may explain the pathogenesis of chloracne, the hallmark of TCDD toxicity. (3) TCDD treatment of confluent liver stem cell-like rat WB-F344 cells leads to release from cell-cell contact inhibition via AHR-mediated crosstalk with multiple signaling pathways. Further work is needed to delineate AHR function in crosstalk with other signaling pathways.

Zfp488 promotes oligodendrocyte differentiation of neural progenitor cells in adult mice after demyelination

PubMed Central

Soundarapandian, Mangala M.; Selvaraj, Vimal; Lo, U-Ging; Golub, Mari S.; Feldman, Daniel H.; Pleasure, David E.; Deng, Wenbin

2011-01-01

Basic helix-loop-helix transcription factors Olig1 and Olig2 critically regulate oligodendrocyte development. Initially identified as a downstream effector of Olig1, an oligodendrocyte-specific zinc finger transcription repressor, Zfp488, cooperates with Olig2 function. Although Zfp488 is required for oligodendrocyte precursor formation and differentiation during embryonic development, its role in oligodendrogenesis of adult neural progenitor cells is not known. In this study, we tested whether Zfp488 could promote an oligodendrogenic fate in adult subventricular zone (SVZ) neural stem/progenitor cells (NSPCs). Using a cuprizone-induced demyelination model in mice, we examined the effect of retrovirus-mediated Zfp488 overexpression in SVZ NSPCs. Our results showed that Zfp488 efficiently promoted the differentiation of the SVZ NSPCs into mature oligodendrocytes in vivo. After cuprizone-induced demyelination injury, Zfp488-transduced mice also showed significant restoration of motor function to levels comparable to control mice. Together, these findings identify a previously unreported role for Zfp488 in adult oligodendrogenesis and functional remyelination after injury. PMID:22355521

An upstream open reading frame represses expression of Lc, a member of the R/B family of maize transcriptional activators

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

Damiani, R.D. Jr.; Wessler, S.R.

1993-09-01

The R/B genes of maize encode a family of basic helix-loop-helix proteins that determine where and when the anthocyanin-pigment pathway will be expressed in the plant. Previous studies showed that allelic diversity among family members reflects differences in gene expression, specifically in transcription initiation. The authors present evidence that the R gene Lc is under translational control. They demonstrate that the 235-nt transcript leader of Lc represses expression 25- to 30-fold in an in vivo assay. Repression is mediated by the presence in cis of a 38-codon upstream open reading frame. Furthermore, the coding capacity of the upstream open readingmore » frame influences the magnitude of repression. It is proposed that translational control does not contribute to tissue specificity but prevents overexpression of the Lc protein. The diversity of promoter and 5' untranslated leader sequences among the R/B genes provides an opportunity to study the coevolution of transcriptional and translational mechanisms of gene regulation. 36 refs., 5 figs.« less

The heterologous expression of a chrysanthemum TCP-P transcription factor CmTCP14 suppresses organ size and delays senescence in Arabidopsis thaliana.

PubMed

Zhang, Ting; Qu, Yixin; Wang, Haibin; Wang, Jingjing; Song, Aiping; Hu, Yueheng; Chen, Sumei; Jiang, Jiafu; Chen, Fadi

2017-06-01

TCP transcription factors are important for plant growth and development, but their activity in chrysanthemum (Chrysanthemum morifolium) has not been thoroughly explored. Here, a chrysanthemum TCP-P sequence, which encodes a protein harboring the conserved basic helix-loop-helix (bHLH) motif, was shown to be related phylogenetically to the Arabidopsis thaliana gene AtTCP14. A yeast-one hybrid assay showed that the encoding protein had no transcriptional activation ability, and a localization experiment indicated that it was localized in the nucleus. Transcription profiling established that the gene was most active in the stem and leaf. Its heterologous expression in A. thaliana down-regulated certain cell cycle-related genes, reduced the size of various organs and increased the chlorophyll and carotenoid contents of the leaf which led to delayed senescence and a prolonged flowering period. Moreover, by screening the cDNA library of chrysanthemum, we found that the CmTCP14 can interact with CmFTL2 and some CmDELLAs. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Artificial ligand binding within the HIF2[alpha] PAS-B domain of the HIF2 transcription factor

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

Scheuermann, Thomas H.; Tomchick, Diana R.; Machius, Mischa

2009-05-12

The hypoxia-inducible factor (HIF) basic helix-loop-helix Per-aryl hydrocarbon receptor nuclear translocator (ARNT)-Sim (bHLH-PAS) transcription factors are master regulators of the conserved molecular mechanism by which metazoans sense and respond to reductions in local oxygen concentrations. In humans, HIF is critically important for the sustained growth and metastasis of solid tumors. Here, we describe crystal structures of the heterodimer formed by the C-terminal PAS domains from the HIF2{alpha} and ARNT subunits of the HIF2 transcription factor, both in the absence and presence of an artificial ligand. Unexpectedly, the HIF2{alpha} PAS-B domain contains a large internal cavity that accommodates ligands identified frommore » a small-molecule screen. Binding one of these ligands to HIF2{alpha} PAS-B modulates the affinity of the HIF2{alpha}:ARNT PAS-B heterodimer in vitro. Given the essential role of PAS domains in forming active HIF heterodimers, these results suggest a presently uncharacterized ligand-mediated mechanism for regulating HIF2 activity in endogenous and clinical settings.« less

Notch pathway signaling in the skin antagonizes Merkel cell development.

PubMed

Logan, Gregory J; Wright, Margaret C; Kubicki, Adam C; Maricich, Stephen M

2018-02-15

Merkel cells are mechanosensitive skin cells derived from the epidermal lineage whose development requires expression of the basic helix-loop-helix transcription factor Atoh1. The genes and pathways involved in regulating Merkel cell development during embryogenesis are poorly understood. Notch pathway signaling antagonizes Atoh1 expression in many developing body regions, so we hypothesized that Notch signaling might inhibit Merkel cell development. We found that conditional, constitutive overexpression of the Notch intracellular domain (NICD) in mouse epidermis significantly decreased Merkel cell numbers in whisker follicles and touch domes of hairy skin. Conversely, conditional deletion of the obligate NICD binding partner RBPj in the epidermis significantly increased Merkel cell numbers in whisker follicles, led to the development of ectopic Merkel cells outside of touch domes in hairy skin epidermis, and altered the distribution of Merkel cells in touch domes. Deletion of the downstream Notch effector gene Hes1 also significantly increased Merkel cell numbers in whisker follicles. Together, these data demonstrate that Notch signaling regulates Merkel cell production and patterning. Copyright © 2018 Elsevier Inc. All rights reserved.

β-Catenin Is Required for Hair-Cell Differentiation in the Cochlea

PubMed Central

Hu, Lingxiang; Jacques, Bonnie E.; Mulvaney, Joanna F.; Dabdoub, Alain

2014-01-01

The development of hair cells in the auditory system can be separated into steps; first, the establishment of progenitors for the sensory epithelium, and second, the differentiation of hair cells. Although the differentiation of hair cells is known to require the expression of basic helix-loop-helix transcription factor, Atoh1, the control of cell proliferation in the region of the developing cochlea that will ultimately become the sensory epithelium and the cues that initiate Atoh1 expression remain obscure. We assessed the role of Wnt/β-catenin in both steps in gain- and loss-of-function models in mice. The canonical Wnt pathway mediator, β-catenin, controls the expression of Atoh1. Knock-out of β-catenin inhibited hair-cell, as well as pillar-cell, differentiation from sensory progenitors but was not required to maintain a hair-cell fate once specified. Constitutive activation of β-catenin expanded sensory progenitors by inducing additional cell division and resulted in the differentiation of extra hair cells. Our data demonstrate that β-catenin plays a role in cell division and differentiation in the cochlear sensory epithelium. PMID:24806673

PRMT1 Is a Novel Regulator of Epithelial-Mesenchymal-Transition in Non-small Cell Lung Cancer*

PubMed Central

Avasarala, Sreedevi; Van Scoyk, Michelle; Karuppusamy Rathinam, Manoj Kumar; Zerayesus, Sereke; Zhao, Xiangmin; Zhang, Wei; Pergande, Melissa R.; Borgia, Jeffrey A.; DeGregori, James; Port, J. David; Winn, Robert A.; Bikkavilli, Rama Kamesh

2015-01-01

Protein arginine methyl transferase 1 (PRMT1) was shown to be up-regulated in cancers and important for cancer cell proliferation. However, the role of PRMT1 in lung cancer progression and metastasis remains incompletely understood. In the present study, we show that PRMT1 is an important regulator of epithelial-mesenchymal transition (EMT), cancer cell migration, and invasion, which are essential processes during cancer progression, and metastasis. Additionally, we have identified Twist1, a basic helix-loop-helix transcription factor and a well-known E-cadherin repressor, as a novel PRMT1 substrate. Taken together, we show that PRMT1 is a novel regulator of EMT and arginine 34 (Arg-34) methylation of Twist1 as a unique “methyl arginine mark” for active E-cadherin repression. Therefore, targeting PRMT1-mediated Twist1 methylation might represent a novel strategy for developing new anti-invasive/anti-metastatic drugs. Moreover, methylated Twist1 (Arg-34), as such, could also emerge as a potential important biomarker for lung cancer. PMID:25847239

The Origin, Development and Molecular Diversity of Rodent Olfactory Bulb Glutamatergic Neurons Distinguished by Expression of Transcription Factor NeuroD1.

PubMed

Roybon, Laurent; Mastracci, Teresa L; Li, Joyce; Stott, Simon R W; Leiter, Andrew B; Sussel, Lori; Brundin, Patrik; Li, Jia-Yi

2015-01-01

Production of olfactory bulb neurons occurs continuously in the rodent brain. Little is known, however, about cellular diversity in the glutamatergic neuron subpopulation. In the central nervous system, the basic helix-loop-helix transcription factor NeuroD1 (ND1) is commonly associated with glutamatergic neuron development. In this study, we utilized ND1 to identify the different subpopulations of olfactory bulb glutamategic neurons and their progenitors, both in the embryo and postnatally. Using knock-in mice, transgenic mice and retroviral transgene delivery, we demonstrate the existence of several different populations of glutamatergic olfactory bulb neurons, the progenitors of which are ND1+ and ND1- lineage-restricted, and are temporally and regionally separated. We show that the first olfactory bulb glutamatergic neurons produced - the mitral cells - can be divided into molecularly diverse subpopulations. Our findings illustrate the complexity of neuronal diversity in the olfactory bulb and that seemingly homogenous neuronal populations can consist of multiple subpopulations with unique molecular signatures of transcription factors and expressing neuronal subtype-specific markers.

Nanohelices from planar polymer self-assembled in carbon nanotubes

PubMed Central

Fu, Hongjin; Xu, Shuqiong; Li, Yunfang

2016-01-01

The polymer possessing with planar structure can be activated and guided to encapsulate the inner space of SWNT and form a helix through van der Waals interaction and the π-π stacking effect between the polymer and the inner surface of SWNT. The SWNT size, the nanostructure and flexibility of polymer chain are all determine the final structures. The basic interaction between the polymer and the nanotubes is investigated, and the condition and mechanism of the helix-forming are explained particularly. Hybrid polymers improve the ability of the helix formation. This study provides scientific basis for fabricating helical polymers encapsulated in SWNTs and eventually on their applications in various areas. PMID:27440493

Helix-Grafted Pleckstrin Homology Domains Suppress HIV-1 Infection of CD4-Positive Cells.

PubMed

Tennyson, Rachel L; Walker, Susanne N; Ikeda, Terumasa; Harris, Reuben S; Kennan, Alan J; McNaughton, Brian R

2016-10-17

The size, functional group diversity and three-dimensional structure of proteins often allow these biomolecules to bind disease-relevant structures that challenge or evade small-molecule discovery. Additionally, folded proteins are often much more stable in biologically relevant environments compared to their peptide counterparts. We recently showed that helix-grafted display-extensive resurfacing and elongation of an existing solvent-exposed helix in a pleckstrin homology (PH) domain-led to a new protein that binds a surrogate of HIV-1 gp41, a validated target for inhibition of HIV-1 entry. Expanding on this work, we prepared a number of human-derived helix-grafted-display PH domains of varied helix length and measured properties relevant to therapeutic and basic research applications. In particular, we showed that some of these new reagents expressed well as recombinant proteins in Escherichia coli, were relatively stable in human serum, bound a mimic of pre-fusogenic HIV-1 gp41 in vitro and in complex biological environments, and significantly lowered the incidence of HIV-1 infection of CD4-positive cells. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structural characterization of CFA/III and Longus type IVb pili from enterotoxigenic Escherichia coli.

PubMed

Kolappan, Subramaniapillai; Roos, Justin; Yuen, Alex S W; Pierce, Owen M; Craig, Lisa

2012-05-01

The type IV pili are helical filaments found on many Gram-negative pathogenic bacteria, with multiple diverse roles in pathogenesis, including microcolony formation, adhesion, and twitching motility. Many pathogenic enterotoxigenic Escherichia coli (ETEC) isolates express one of two type IV pili belonging to the type IVb subclass: CFA/III or Longus. Here we show a direct correlation between CFA/III expression and ETEC aggregation, suggesting that these pili, like the Vibrio cholerae toxin-coregulated pili (TCP), mediate microcolony formation. We report a 1.26-Å resolution crystal structure of CofA, the major pilin subunit from CFA/III. CofA is very similar in structure to V. cholerae TcpA but possesses a 10-amino-acid insertion that replaces part of the α2-helix with an irregular loop containing a 3(10)-helix. Homology modeling suggests a very similar structure for the Longus LngA pilin. A model for the CFA/III pilus filament was generated using the TCP electron microscopy reconstruction as a template. The unique 3(10)-helix insert fits perfectly within the gap between CofA globular domains. This insert, together with differences in surface-exposed residues, produces a filament that is smoother and more negatively charged than TCP. To explore the specificity of the type IV pilus assembly apparatus, CofA was expressed heterologously in V. cholerae by replacing the tcpA gene with that of cofA within the tcp operon. Although CofA was synthesized and processed by V. cholerae, no CFA/III filaments were detected, suggesting that the components of the type IVb pilus assembly system are highly specific to their pilin substrates.

Transmembrane helix prediction: a comparative evaluation and analysis.

PubMed

Cuthbertson, Jonathan M; Doyle, Declan A; Sansom, Mark S P

2005-06-01

The prediction of transmembrane (TM) helices plays an important role in the study of membrane proteins, given the relatively small number (approximately 0.5% of the PDB) of high-resolution structures for such proteins. We used two datasets (one redundant and one non-redundant) of high-resolution structures of membrane proteins to evaluate and analyse TM helix prediction. The redundant (non-redundant) dataset contains structure of 434 (268) TM helices, from 112 (73) polypeptide chains. Of the 434 helices in the dataset, 20 may be classified as 'half-TM' as they are too short to span a lipid bilayer. We compared 13 TM helix prediction methods, evaluating each method using per segment, per residue and termini scores. Four methods consistently performed well: SPLIT4, TMHMM2, HMMTOP2 and TMAP. However, even the best methods were in error by, on average, about two turns of helix at the TM helix termini. The best and worst case predictions for individual proteins were analysed. In particular, the performance of the various methods and of a consensus prediction method, were compared for a number of proteins (e.g. SecY, ClC, KvAP) containing half-TM helices. The difficulties of predicting half-TM helices suggests that current prediction methods successfully embody the two-state model of membrane protein folding, but do not accommodate a third stage in which, e.g., short helices and re-entrant loops fold within a bundle of stable TM helices.

Self-assembled RNA-triple-helix hydrogel scaffold for microRNA modulation in the tumour microenvironment

NASA Astrophysics Data System (ADS)

Conde, João; Oliva, Nuria; Atilano, Mariana; Song, Hyun Seok; Artzi, Natalie

2016-03-01

The therapeutic potential of miRNA (miR) in cancer is limited by the lack of efficient delivery vehicles. Here, we show that a self-assembled dual-colour RNA-triple-helix structure comprising two miRNAs--a miR mimic (tumour suppressor miRNA) and an antagomiR (oncomiR inhibitor)--provides outstanding capability to synergistically abrogate tumours. Conjugation of RNA triple helices to dendrimers allows the formation of stable triplex nanoparticles, which form an RNA-triple-helix adhesive scaffold upon interaction with dextran aldehyde, the latter able to chemically interact and adhere to natural tissue amines in the tumour. We also show that the self-assembled RNA-triple-helix conjugates remain functional in vitro and in vivo, and that they lead to nearly 90% levels of tumour shrinkage two weeks post-gel implantation in a triple-negative breast cancer mouse model. Our findings suggest that the RNA-triple-helix hydrogels can be used as an efficient anticancer platform to locally modulate the expression of endogenous miRs in cancer.

Evidence for an RNA pseudoknot loop-helix interaction essential for efficient -1 ribosomal frameshifting.

PubMed

Liphardt, J; Napthine, S; Kontos, H; Brierley, I

1999-05-07

RNA pseudoknots are structural elements that participate in a variety of biological processes. At -1 ribosomal frameshifting sites, several types of pseudoknot have been identified which differ in their organisation and functionality. The pseudoknot found in infectious bronchitis virus (IBV) is typical of those that possess a long stem 1 of 11-12 bp and a long loop 2 (30-164 nt). A second group of pseudoknots are distinguishable that contain stems of only 5 to 7 bp and shorter loops. The NMR structure of one such pseudoknot, that of mouse mammary tumor virus (MMTV), has revealed that it is kinked at the stem 1-stem 2 junction, and that this kinked conformation is essential for efficient frameshifting. We recently investigated the effect on frameshifting of modulating stem 1 length and stability in IBV-based pseudoknots, and found that a stem 1 with at least 11 bp was needed for efficient frameshifting. Here, we describe the sequence manipulations that are necessary to bypass the requirement for an 11 bp stem 1 and to convert a short non-functional IBV-derived pseudoknot into a highly efficient, kinked frameshifter pseudoknot. Simple insertion of an adenine residue at the stem 1-stem 2 junction (an essential feature of a kinked pseudoknot) was not sufficient to create a functional pseudoknot. An additional change was needed: efficient frameshifting was recovered only when the last nucleotide of loop 2 was changed from a G to an A. The requirement for an A at the end of loop 2 is consistent with a loop-helix contact similar to those described in other RNA tertiary structures. A mutational analysis of both partners of the proposed interaction, the loop 2 terminal adenine residue and two G.C pairs near the top of stem 1, revealed that the interaction was essential for efficient frameshifting. The specific requirement for a 3'-terminal A residue was lost when loop 2 was increased from 8 to 14 nt, suggesting that the loop-helix contact may be required only in those pseudoknots with a short loop 2. Copyright 1999 Academic Press.

NLR-Associating Transcription Factor bHLH84 and Its Paralogs Function Redundantly in Plant Immunity

PubMed Central

Xu, Fang; Kapos, Paul; Cheng, Yu Ti; Li, Meng; Zhang, Yuelin; Li, Xin

2014-01-01

In plants and animals, nucleotide-binding and leucine-rich repeat domain containing (NLR) immune receptors are utilized to detect the presence or activities of pathogen-derived molecules. However, the mechanisms by which NLR proteins induce defense responses remain unclear. Here, we report the characterization of one basic Helix-loop-Helix (bHLH) type transcription factor (TF), bHLH84, identified from a reverse genetic screen. It functions as a transcriptional activator that enhances the autoimmunity of NLR mutant snc1 (suppressor of npr1-1, constitutive 1) and confers enhanced immunity in wild-type backgrounds when overexpressed. Simultaneously knocking out three closely related bHLH paralogs attenuates RPS4-mediated immunity and partially suppresses the autoimmune phenotypes of snc1, while overexpression of the other two close paralogs also renders strong autoimmunity, suggesting functional redundancy in the gene family. Intriguingly, the autoimmunity conferred by bHLH84 overexpression can be largely suppressed by the loss-of-function snc1-r1 mutation, suggesting that SNC1 is required for its proper function. In planta co-immunoprecipitation revealed interactions between not only bHLH84 and SNC1, but also bHLH84 and RPS4, indicating that bHLH84 associates with these NLRs. Together with previous finding that SNC1 associates with repressor TPR1 to repress negative regulators, we hypothesize that nuclear NLR proteins may interact with both transcriptional repressors and activators during immune responses, enabling potentially faster and more robust transcriptional reprogramming upon pathogen recognition. PMID:25144198

The aryl hydrocarbon receptor repressor - More than a simple feedback inhibitor of AhR signaling: Clues for its role in inflammation and cancer.

PubMed

Vogel, Christoph F A; Haarmann-Stemmann, Thomas

2017-02-01

The aryl hydrocarbon receptor repressor (AhRR) was first described as a specific competitive repressor of aryl hydrocarbon receptor (AhR) activity based on its ability to dimerize with the AhR nuclear translocator (ARNT) and through direct competition of AhR/ARNT and AhRR/ARNT complexes for binding to dioxin-responsive elements (DREs). Like AhR, AhRR belongs to the basic Helix-Loop-Helix/Per-ARNT-Sim (bHLH/PAS) protein family but lacks functional ligand-binding and transactivation domains. Transient transfection experiments with ARNT and AhRR mutants examining the inhibitory mechanism of AhRR suggested a more complex mechanism than the simple mechanism of negative feedback through sequestration of ARNT to regulate AhR signaling. Recently, AhRR has been shown to act as a tumor suppressor gene in several types of cancer cells. Furthermore, epidemiological studies have found epigenetic changes and silencing of AhRR associated with exposure to cigarette smoke and cancer development. Additional studies from our laboratories have demonstrated that AhRR represses other signaling pathways including NF-κB and is capable of regulating inflammatory responses. A better understanding of the regulatory mechanisms of AhRR in AhR signaling and adverse outcome pathways leading to deregulated inflammatory responses contributing to tumor promotion and other adverse health effects is expected from future studies. This review article summarizes the characteristics of AhRR as an inhibitor of AhR activity and highlights more recent findings pointing out the role of AhRR in inflammation and tumorigenesis.

An atypical bHLH protein encoded by POSITIVE REGULATOR OF GRAIN LENGTH 2 is involved in controlling grain length and weight of rice through interaction with a typical bHLH protein APG.

PubMed

Heang, Dany; Sassa, Hidenori

2012-06-01

Grain size is an important yield component in rice, however, genes controlling the trait remain poorly understood. Previously, we have shown that an antagonistic pair of basic helix-loop-helix (bHLH) proteins, POSITIVE REGULATOR OF GRAIN LENGTH 1 (PGL1) and ANTAGONIST OF PGL1 (APG), is involved in controlling rice grain length. Here, we report the involvement of another atypical bHLH protein gene, POSITIVE REGULATOR OF GRAIN LENGTH 2 (PGL2), in the regulation of rice grain length. Over-expression of PGL2 in the lemma/palea increased grain length and weight in correlation with the level of transgene expression. Observation of the inner epidermal cells of lemma of PGL2-overexpressing lines revealed that the long grain size is caused by an increase in cell length. PGL2 interacts with a typical bHLH protein APG, a negative regulator of rice grain length and weight, in vitro and in vivo. It was reported that overexpression of BU1 (BRASSINOSTEROID UPREGULATED 1), the closest homolog of PGL2, caused an increase in grain length. However, we detected no interaction between BU1 and APG. These findings suggest that PGL2 and PGL1 redundantly suppress the function of APG by forming heterodimers to positively regulate the rice grain length, while the pathway through which BU1, the closest homolog of PGL2, controls grain length is independent of APG.

An atypical bHLH protein encoded by POSITIVE REGULATOR OF GRAIN LENGTH 2 is involved in controlling grain length and weight of rice through interaction with a typical bHLH protein APG

PubMed Central

Heang, Dany; Sassa, Hidenori

2012-01-01

Grain size is an important yield component in rice, however, genes controlling the trait remain poorly understood. Previously, we have shown that an antagonistic pair of basic helix-loop-helix (bHLH) proteins, POSITIVE REGULATOR OF GRAIN LENGTH 1 (PGL1) and ANTAGONIST OF PGL1 (APG), is involved in controlling rice grain length. Here, we report the involvement of another atypical bHLH protein gene, POSITIVE REGULATOR OF GRAIN LENGTH 2 (PGL2), in the regulation of rice grain length. Over-expression of PGL2 in the lemma/palea increased grain length and weight in correlation with the level of transgene expression. Observation of the inner epidermal cells of lemma of PGL2-overexpressing lines revealed that the long grain size is caused by an increase in cell length. PGL2 interacts with a typical bHLH protein APG, a negative regulator of rice grain length and weight, in vitro and in vivo. It was reported that overexpression of BU1 (BRASSINOSTEROID UPREGULATED 1), the closest homolog of PGL2, caused an increase in grain length. However, we detected no interaction between BU1 and APG. These findings suggest that PGL2 and PGL1 redundantly suppress the function of APG by forming heterodimers to positively regulate the rice grain length, while the pathway through which BU1, the closest homolog of PGL2, controls grain length is independent of APG. PMID:23136524

Id1, Id2 and Id3 are induced in rat melanotrophs of the pituitary gland by dopamine suppression under continuous stress.

PubMed

Konishi, H; Ogawa, T; Nakagomi, S; Inoue, K; Tohyama, M; Kiyama, H

2010-09-15

In rats under continuous stress (CS) there is decreased hypothalamic dopaminergic innervation to the intermediate lobe (IL) of the pituitary gland, which causes hyperactivation and subsequent degeneration of melanotrophs in the IL. In this study, we investigated the molecular basis for the changes that occur in melanotrophs during CS. Using microarray analysis, we identified several genes differentially expressed in the IL under CS conditions. Among the genes up-regulated under CS conditions, we focused on the inhibitor of DNA binding/differentiation (Id) family of dominant negative basic helix-loop-helix (bHLH) transcription factors. RT-PCR, Western blotting and in situ hybridization confirmed the significant inductions of Id1, Id2 and Id3 in the IL of CS rats. Administration of the dopamine D2 receptor agonist bromocriptine prevented the inductions of Id1-3 in the IL of CS rats, whereas application of the dopamine D2 antagonist sulpiride induced significant expressions of Id1-3 in the IL of normal rats. Moreover, an in vitro study using primary cultured melanotrophs demonstrated a direct effect on Id1-3 inductions by dopamine suppression. These results suggest that the decreased dopamine levels in the IL during CS induce Id1-3 expressions in melanotrophs. Because Id family members inhibit various bHLH transcription factors, it is conceivable that the induced Id1-3 would cooperatively modulate gene expressions in melanotrophs under CS conditions to induce hormone secretion. (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Structural Basis for Antifreeze Activity of Ice-binding Protein from Arctic Yeast*

PubMed Central

Lee, Jun Hyuck; Park, Ae Kyung; Do, Hackwon; Park, Kyoung Sun; Moh, Sang Hyun; Chi, Young Min; Kim, Hak Jun

2012-01-01

Arctic yeast Leucosporidium sp. produces a glycosylated ice-binding protein (LeIBP) with a molecular mass of ∼25 kDa, which can lower the freezing point below the melting point once it binds to ice. LeIBP is a member of a large class of ice-binding proteins, the structures of which are unknown. Here, we report the crystal structures of non-glycosylated LeIBP and glycosylated LeIBP at 1.57- and 2.43-Å resolution, respectively. Structural analysis of the LeIBPs revealed a dimeric right-handed β-helix fold, which is composed of three parts: a large coiled structural domain, a long helix region (residues 96–115 form a long α-helix that packs along one face of the β-helix), and a C-terminal hydrophobic loop region (243PFVPAPEVV251). Unexpectedly, the C-terminal hydrophobic loop region has an extended conformation pointing away from the body of the coiled structural domain and forms intertwined dimer interactions. In addition, structural analysis of glycosylated LeIBP with sugar moieties attached to Asn185 provides a basis for interpreting previous biochemical analyses as well as the increased stability and secretion of glycosylated LeIBP. We also determined that the aligned Thr/Ser/Ala residues are critical for ice binding within the B face of LeIBP using site-directed mutagenesis. Although LeIBP has a common β-helical fold similar to that of canonical hyperactive antifreeze proteins, the ice-binding site is more complex and does not have a simple ice-binding motif. In conclusion, we could identify the ice-binding site of LeIBP and discuss differences in the ice-binding modes compared with other known antifreeze proteins and ice-binding proteins. PMID:22303017

Analysis of the function of E. coli 23S rRNA helix-loop 69 by mutagenesis

PubMed Central

Liiv, Aivar; Karitkina, Diana; Maiväli, Ülo; Remme, Jaanus

2005-01-01

Background The ribosome is a two-subunit enzyme known to exhibit structural dynamism during protein synthesis. The intersubunit bridges have been proposed to play important roles in decoding, translocation, and the peptidyl transferase reaction; yet the physical nature of their contributions is ill understood. An intriguing intersubunit bridge, B2a, which contains 23S rRNA helix 69 as a major component, has been implicated by proximity in a number of catalytically important regions. In addition to contacting the small ribosomal subunit, helix 69 contacts both the A and P site tRNAs and several translation factors. Results We scanned the loop of helix 69 by mutagenesis and analyzed the mutant ribosomes using a plasmid-borne IPTG-inducible expression system. We assayed the effects of 23S rRNA mutations on cell growth, contribution of mutant ribosomes to cellular polysome pools and the ability of mutant ribosomes to function in cell-free translation. Mutations A1912G, and A1919G have very strong growth phenotypes, are inactive during in vitro protein synthesis, and under-represented in the polysomes. Mutation Ψ1917C has a very strong growth phenotype and leads to a general depletion of the cellular polysome pool. Mutation A1916G, having a modest growth phenotype, is apparently defective in the assembly of the 70S ribosome. Conclusion Mutations A1912G, A1919G, and Ψ1917C of 23S rRNA strongly inhibit translation. Mutation A1916G causes a defect in the 50S subunit or 70S formation. Mutations Ψ1911C, A1913G, C1914A, Ψ1915C, and A1918G lack clear phenotypes. PMID:16053518

NMR structure of the bovine prion protein

PubMed Central

López García, Francisco; Zahn, Ralph; Riek, Roland; Wüthrich, Kurt

2000-01-01

The NMR structures of the recombinant 217-residue polypeptide chain of the mature bovine prion protein, bPrP(23–230), and a C-terminal fragment, bPrP(121–230), include a globular domain extending from residue 125 to residue 227, a short flexible chain end of residues 228–230, and an N-terminal flexibly disordered “tail” comprising 108 residues for the intact protein and 4 residues for bPrP(121–230), respectively. The globular domain contains three α-helices comprising the residues 144–154, 173–194, and 200–226, and a short antiparallel β-sheet comprising the residues 128–131 and 161–164. The best-defined parts of the globular domain are the central portions of the helices 2 and 3, which are linked by the only disulfide bond in bPrP. Significantly increased disorder and mobility is observed for helix 1, the loop 166–172 leading from the β-strand 2 to helix 2, the end of helix 2 and the following loop, and the last turn of helix 3. Although there are characteristic local differences relative to the conformations of the murine and Syrian hamster prion proteins, the bPrP structure is essentially identical to that of the human prion protein. On the other hand, there are differences between bovine and human PrP in the surface distribution of electrostatic charges, which then appears to be the principal structural feature of the “healthy” PrP form that might affect the stringency of the species barrier for transmission of prion diseases between humans and cattle. PMID:10899999

Probing the mechanistic role of the long α-helix in subunit L of respiratory Complex I from Escherichia coli by site-directed mutagenesis

PubMed Central

Belevich, Galina; Knuuti, Juho; Verkhovsky, Michael I; Wikström, Mårten; Verkhovskaya, Marina

2011-01-01

The C-terminus of the NuoL subunit of Complex I includes a long amphipathic α-helix positioned parallel to the membrane, which has been considered to function as a piston in the proton pumping machinery. Here, we have introduced three types of mutations into the nuoL gene to test the piston-like function. First, NuoL was truncated at its C- and N-termini, which resulted in low production of a fragile Complex I with negligible activity. Second, we mutated three partially conserved residues of the amphipathic α-helix: Asp and Lys residues and a Pro were substituted for acidic, basic or neutral residues. All these variants exhibited almost a wild-type phenotype. Third, several substitutions and insertions were made to reduce rigidity of the amphipathic α-helix, and/or to change its geometry. Most insertions/substitutions resulted in a normal growth phenotype, albeit often with reduced stability of Complex I. In contrast, insertion of six to seven amino acids at a site of the long α-helix between NuoL and M resulted in substantial loss of proton pumping efficiency. The implications of these results for the proton pumping mechanism of Complex I are discussed. PMID:22060017

Molecular evolution of pentatricopeptide repeat genes reveals truncation in species lacking an editing target and structural domains under distinct selective pressures.

PubMed

Hayes, Michael L; Giang, Karolyn; Mulligan, R Michael

2012-05-14

Pentatricopeptide repeat (PPR) proteins are required for numerous RNA processing events in plant organelles including C-to-U editing, splicing, stabilization, and cleavage. Fifteen PPR proteins are known to be required for RNA editing at 21 sites in Arabidopsis chloroplasts, and belong to the PLS class of PPR proteins. In this study, we investigate the co-evolution of four PPR genes (CRR4, CRR21, CLB19, and OTP82) and their six editing targets in Brassicaceae species. PPR genes are composed of approximately 10 to 20 tandem repeats and each repeat has two α-helical regions, helix A and helix B, that are separated by short coil regions. Each repeat and structural feature was examined to determine the selective pressures on these regions. All of the PPR genes examined are under strong negative selection. Multiple independent losses of editing site targets are observed for both CRR21 and OTP82. In several species lacking the known editing target for CRR21, PPR genes are truncated near the 17th PPR repeat. The coding sequences of the truncated CRR21 genes are maintained under strong negative selection; however, the 3' UTR sequences beyond the truncation site have substantially diverged. Phylogenetic analyses of four PPR genes show that sequences corresponding to helix A are high compared to helix B sequences. Differential evolutionary selection of helix A versus helix B is observed in both plant and mammalian PPR genes. PPR genes and their cognate editing sites are mutually constrained in evolution. Editing sites are frequently lost by replacement of an edited C with a genomic T. After the loss of an editing site, the PPR genes are observed with three outcomes: first, few changes are detected in some cases; second, the PPR gene is present as a pseudogene; and third, the PPR gene is present but truncated in the C-terminal region. The retention of truncated forms of CRR21 that are maintained under strong negative selection even in the absence of an editing site target suggests that unrecognized function(s) might exist for this PPR protein. PPR gene sequences that encode helix A are under strong selection, and could be involved in RNA substrate recognition.

Crystal Structure of the Minimalist Max-E47 Protein Chimera

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

Ahmadpour, Faraz; Ghirlando, Rodolfo; De Jong, Antonia T.

Max-E47 is a protein chimera generated from the fusion of the DNA-binding basic region of Max and the dimerization region of E47, both members of the basic region/helix-loop-helix (bHLH) superfamily of transcription factors. Like native Max, Max-E47 binds with high affinity and specificity to the E-box site, 5'-CACGTG, both in vivo and in vitro. We have determined the crystal structure of Max-E47 at 1.7 Å resolution, and found that it associates to form a well-structured dimer even in the absence of its cognate DNA. Analytical ultracentrifugation confirms that Max-E47 is dimeric even at low micromolar concentrations, indicating that the Max-E47more » dimer is stable in the absence of DNA. Circular dichroism analysis demonstrates that both non-specific DNA and the E-box site induce similar levels of helical secondary structure in Max-E47. These results suggest that Max-E47 may bind to the E-box following the two-step mechanism proposed for other bHLH proteins. In this mechanism, a rapid step where protein binds to DNA without sequence specificity is followed by a slow step where specific protein:DNA interactions are fine-tuned, leading to sequence-specific recognition. Collectively, these results show that the designed Max-E47 protein chimera behaves both structurally and functionally like its native counterparts.« less

Behavior of Solvent-Exposed Hydrophobic Groove in the Anti-Apoptotic Bcl-XL Protein: Clues for Its Ability to Bind Diverse BH3 Ligands from MD Simulations

PubMed Central

Sankararamakrishnan, Ramasubbu

2013-01-01

Bcl-XL is a member of Bcl-2 family of proteins involved in the regulation of intrinsic pathway of apoptosis. Its overexpression in many human cancers makes it an important target for anti-cancer drugs. Bcl-XL interacts with the BH3 domain of several pro-apoptotic Bcl-2 partners. This helical bundle protein has a pronounced hydrophobic groove which acts as a binding region for the BH3 domains. Eight independent molecular dynamics simulations of the apo/holo forms of Bcl-XL were carried out to investigate the behavior of solvent-exposed hydrophobic groove. The simulations used either a twin-range cut-off or particle mesh Ewald (PME) scheme to treat long-range interactions. Destabilization of the BH3 domain-containing helix H2 was observed in all four twin-range cut-off simulations. Most of the other major helices remained stable. The unwinding of H2 can be related to the ability of Bcl-XL to bind diverse BH3 ligands. The loss of helical character can also be linked to the formation of homo- or hetero-dimers in Bcl-2 proteins. Several experimental studies have suggested that exposure of BH3 domain is a crucial event before they form dimers. Thus unwinding of H2 seems to be functionally very important. The four PME simulations, however, revealed a stable helix H2. It is possible that the H2 unfolding might occur in PME simulations at longer time scales. Hydrophobic residues in the hydrophobic groove are involved in stable interactions among themselves. The solvent accessible surface areas of bulky hydrophobic residues in the groove are significantly buried by the loop LB connecting the helix H2 and subsequent helix. These observations help to understand how the hydrophobic patch in Bcl-XL remains stable in the solvent-exposed state. We suggest that both the destabilization of helix H2 and the conformational heterogeneity of loop LB are important factors for binding of diverse ligands in the hydrophobic groove of Bcl-XL. PMID:23468841

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser

DOE PAGES

Kang, Yanyong; Zhou, X. Edward; Gao, Xiang; ...

2015-07-22

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ~20° rotationmore » between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. In conclusion, this structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.« less

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

PubMed

Kang, Yanyong; Zhou, X Edward; Gao, Xiang; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; Barty, Anton; White, Thomas A; Yefanov, Oleksandr; Han, Gye Won; Xu, Qingping; de Waal, Parker W; Ke, Jiyuan; Tan, M H Eileen; Zhang, Chenghai; Moeller, Arne; West, Graham M; Pascal, Bruce D; Van Eps, Ned; Caro, Lydia N; Vishnivetskiy, Sergey A; Lee, Regina J; Suino-Powell, Kelly M; Gu, Xin; Pal, Kuntal; Ma, Jinming; Zhi, Xiaoyong; Boutet, Sébastien; Williams, Garth J; Messerschmidt, Marc; Gati, Cornelius; Zatsepin, Nadia A; Wang, Dingjie; James, Daniel; Basu, Shibom; Roy-Chowdhury, Shatabdi; Conrad, Chelsie E; Coe, Jesse; Liu, Haiguang; Lisova, Stella; Kupitz, Christopher; Grotjohann, Ingo; Fromme, Raimund; Jiang, Yi; Tan, Minjia; Yang, Huaiyu; Li, Jun; Wang, Meitian; Zheng, Zhong; Li, Dianfan; Howe, Nicole; Zhao, Yingming; Standfuss, Jörg; Diederichs, Kay; Dong, Yuhui; Potter, Clinton S; Carragher, Bridget; Caffrey, Martin; Jiang, Hualiang; Chapman, Henry N; Spence, John C H; Fromme, Petra; Weierstall, Uwe; Ernst, Oliver P; Katritch, Vsevolod; Gurevich, Vsevolod V; Griffin, Patrick R; Hubbell, Wayne L; Stevens, Raymond C; Cherezov, Vadim; Melcher, Karsten; Xu, H Eric

2015-07-30

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser

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

Kang, Yanyong; Zhou, X. Edward; Gao, Xiang

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ~20° rotationmore » between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. In conclusion, this structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.« less

Transcriptome profiling reveals the immune response of goose T cells under selenium stimuli.

PubMed

Cao, Nan; Li, Wanyan; Li, Bingxin; Tian, Yunbo; Xu, Danning

2017-12-01

The goose is an economically important poultry species and a principal natural host of avian viruses. This study aimed to determine the effects of selenium on the immune response of geese. Under selenium stimulation, gene expression profiling was investigated using transcriptome sequencing. The selenoproteins were promoted by selenium stimulation, while the heat shock proteins, interleukin and interferons were mainly down-regulated. After comparison, 2228 differentially expressed genes were primarily involved in immune and environmental response, and infectious disease and genetic information processing related pathways were identified. Specifically, the enzymes of the lysosomes which acted as a safeguard in preventing pathogens were mostly up-regulated and six randomly selected differentially expressed genes were validated by quantitative polymerase chain reaction. In addition, the most proportional increased transcription factor family basic helix-loop-helix (bHLH) located in the 5' flank of selenoprotein P-like protein for selenium metabolism was identified by response to the selenium stimulation in this study. These analyses show that selenium can promote immune function by activating selenoproteins, transcript factors and lysosome pathway related genes, while weakening cytokine content genes in geese. © 2017 Japanese Society of Animal Science.

[Mutation screening of MITF gene in patients with Waardenburg syndrome type 2].

PubMed

Chen, Jing; Yang, Shu-Zhi; Liu, Jun; Han, Bing; Wang, Guo-Jian; Zhang, Xin; Kang, Dong-Yang; Dai, Pu; Young, Wie-Yen; Yuan, Hui-Jun

2008-04-01

Warrgenburg syndrome type 2 (WS2) is the most common autosomal dominantly-inherited syndrome with hearing loss. MITF (microphthalmia associated transcription factor)is a basic-helix-loop-helix-luecine zipper (bHLHZip) factor which regulates expression of tyrosinase, and is involved in melanocyte differentiation. Mutations in MITF associated with WS2 have been identified in some but not all affected families. Here, we report a three-generation Chinese family with a point mutation in the MITF gene causing WS2. The proband exhibits congenital severe sensorineural hearing loss, heterochromia iridis and facial freckles. One of family members manifests sensorineural deafness, and the other patients show premature greying or/and freckles. This mutation, heterozygous deletion c.639delA, creates a stop codon in exon 7 and is predicted to result in a truncated protein lacking normal interaction with its target DNA motif. This mutation is a novel mutation and the third case identified in exon 7 of MITF in WS2. Though there is only one base pair distance between this novel mutation and the other two documented cases and similar amino acids change, significant difference is seen in clinical phenotype, which suggests genetic background may play an important role.

Genetic dissection of endothelial transcriptional activity of zebrafish aryl hydrocarbon receptors (AHRs).

PubMed

Sugden, Wade W; Leonardo-Mendonça, Roberto C; Acuña-Castroviejo, Darío; Siekmann, Arndt F

2017-01-01

The aryl hydrocarbon receptor (AHR) is a basic helix-loop-helix transcription factor conserved across phyla from flies to humans. Activated by a number of endogenous ligands and environmental toxins, studies on AHR function and gene regulation have largely focused on a toxicological perspective relating to aromatic hydrocarbons generated by human activities and the often-deleterious effects of exposure on vertebrates mediated by AHR activation. A growing body of work has highlighted the importance of AHR in physiologic processes, including immune cell differentiation and vascular patterning. Here we dissect the contribution of the 3 zebrafish AHRs, ahr1a, ahr1b and ahr2, to endothelial cyp1a1/b1 gene regulation under physiologic conditions and upon exposure to the AHR ligand Beta-naphthoflavone. We show that in fish multiple AHRs are functional in the vasculature, with vessel-specific differences in the ability of ahr1b to compensate for the loss of ahr2 to maintain AHR signaling. We further provide evidence that AHR can regulate the expression of the chemokine receptor cxcr4a in endothelial cells, a regulatory mechanism that may provide insight into AHR function in the endothelium.

Elicitor-induced transcription factors for metabolic reprogramming of secondary metabolism in Medicago truncatula

PubMed Central

Naoumkina, Marina A; He, XianZhi; Dixon, Richard A

2008-01-01

Background Exposure of Medicago truncatula cell suspension cultures to pathogen or wound signals leads to accumulation of various classes of flavonoid and/or triterpene defense molecules, orchestrated via a complex signalling network in which transcription factors (TFs) are essential components. Results In this study, we analyzed TFs responding to yeast elicitor (YE) or methyl jasmonate (MJ). From 502 differentially expressed TFs, WRKY and AP2/EREBP gene families were over-represented among YE-induced genes whereas Basic Helix-Loop-Helix (bHLH) family members were more over-represented among the MJ-induced genes. Jasmonate ZIM-domain (JAZ) transcriptional regulators were highly induced by MJ treatment. To investigate potential involvement of WRKY TFs in signalling, we expressed four Medicago WRKY genes in tobacco. Levels of soluble and wall bound phenolic compounds and lignin were increased in all cases. WRKY W109669 also induced tobacco endo-1,3-β-glucanase (NtPR2) and enhanced the systemic defense response to tobacco mosaic virus in transgenic tobacco plants. Conclusion These results confirm that Medicago WRKY TFs have broad roles in orchestrating metabolic responses to biotic stress, and that they also represent potentially valuable reagents for engineering metabolic changes that impact pathogen resistance. PMID:19102779

Repression of ESR1 transcription by MYOD potentiates letrozole-resistance in ERα-positive breast cancer cells.

PubMed

Zhang, Qiang; Liu, Xiao-Yan; Li, Shuang; Zhao, Zhao; Li, Juan; Cui, Ming-Ke; Wang, En-Hua

2017-10-21

Transcriptional silencing of estrogen receptor α (ERα) expression is an important etiology contributing to the letrozole-resistance in ERα-positive breast cancer (BCa) cells, but the transcription factors responsible for this transcriptional repression remain largely unidentified. Here we report that the expression of the basic helix-loop-helix myogenic regulatory factor MYOD was abnormally up-regulated in letrozole-resistant BCa tissues and in experimentally-induced letrozole-resistant BCa cells. Overexpression of the exogenous MYOD impaired ERα expression and potentiated letrozole-resistance in letrozole-sensitive MCF7 cells, whereas MYOD knockdown could effectively restore ERα expression and thereby promote letrozole-sensitivity in letrozole-resistant MCF-7/LR cells. Mechanistically, MYOD was shown to be a potent corepressor of ESR1 transcription, and this transcriptional repression was significantly enhanced in the presence of letrozole treatment. Thus, targeted inhibition of MYOD may restore ERα level and lead to resensitization to letrozole-based hormone therapy, providing a novel therapeutic strategy for relapsed ERα-positive BCa patients. Our data also underscore an unexpected chemotherapeutic facet of MYOD, which may operate as a novel regulator of BCa biology. Copyright © 2017 Elsevier Inc. All rights reserved.

A targeted neuroglial reporter line generated by homologous recombination in human embryonic stem cells.

PubMed

Xue, Haipeng; Wu, Sen; Papadeas, Sophia T; Spusta, Steve; Swistowska, Anna Maria; MacArthur, Chad C; Mattson, Mark P; Maragakis, Nicholas J; Capecchi, Mario R; Rao, Mahendra S; Zeng, Xianmin; Liu, Ying

2009-08-01

In this study, we targeted Olig2, a basic helix-loop-helix transcription factor that plays an important role in motoneuron and oligodendrocyte development, in human embryonic stem cell (hESC) line BG01 by homologous recombination. One allele of Olig2 locus was replaced by a green fluorescent protein (GFP) cassette with a targeting efficiency of 5.7%. Targeted clone R-Olig2 (like the other clones) retained pluripotency, typical hESC morphology, and a normal parental karyotype 46,XY. Most importantly, GFP expression recapitulated endogenous Olig2 expression when R-Olig2 was induced by sonic hedgehog and retinoic acid, and GFP-positive cells could be purified by fluorescence-activated cell sorting. Consistent with previous reports on rodents, early GFP-expressing cells appeared biased to a neuronal fate, whereas late GFP-expressing cells appeared biased to an oligodendrocytic fate. This was corroborated by myoblast coculture, transplantation into the rat spinal cords, and whole genome expression profiling. The present work reports an hESC reporter line generated by homologous recombination targeting a neural lineage-specific gene, which can be differentiated and sorted to obtain pure neural progenitor populations.

GTL1 and DF1 regulate root hair growth through transcriptional repression of ROOT HAIR DEFECTIVE 6-LIKE 4 in Arabidopsis

PubMed Central

Breuer, Christian; Kawamura, Ayako; Clark, Natalie M.; Morohashi, Kengo; Busch, Wolfgang; Benfey, Philip N.; Sozzani, Rosangela

2018-01-01

ABSTRACT How plants determine the final size of growing cells is an important, yet unresolved, issue. Root hairs provide an excellent model system with which to study this as their final cell size is remarkably constant under constant environmental conditions. Previous studies have demonstrated that a basic helix-loop helix transcription factor ROOT HAIR DEFECTIVE 6-LIKE 4 (RSL4) promotes root hair growth, but how hair growth is terminated is not known. In this study, we demonstrate that a trihelix transcription factor GT-2-LIKE1 (GTL1) and its homolog DF1 repress root hair growth in Arabidopsis. Our transcriptional data, combined with genome-wide chromatin-binding data, show that GTL1 and DF1 directly bind the RSL4 promoter and regulate its expression to repress root hair growth. Our data further show that GTL1 and RSL4 regulate each other, as well as a set of common downstream genes, many of which have previously been implicated in root hair growth. This study therefore uncovers a core regulatory module that fine-tunes the extent of root hair growth by the orchestrated actions of opposing transcription factors. PMID:29439132

Virulence Factors of Geminivirus Interact with MYC2 to Subvert Plant Resistance and Promote Vector Performance[C][W

PubMed Central

Li, Ran; Weldegergis, Berhane T.; Li, Jie; Jung, Choonkyun; Qu, Jing; Sun, Yanwei; Qian, Hongmei; Tee, ChuanSia; van Loon, Joop J.A.; Dicke, Marcel; Chua, Nam-Hai; Liu, Shu-Sheng

2014-01-01

A pathogen may cause infected plants to promote the performance of its transmitting vector, which accelerates the spread of the pathogen. This positive effect of a pathogen on its vector via their shared host plant is termed indirect mutualism. For example, terpene biosynthesis is suppressed in begomovirus-infected plants, leading to reduced plant resistance and enhanced performance of the whiteflies (Bemisia tabaci) that transmit these viruses. Although begomovirus-whitefly mutualism has been known, the underlying mechanism is still elusive. Here, we identified βC1 of Tomato yellow leaf curl China virus, a monopartite begomovirus, as the viral genetic factor that suppresses plant terpene biosynthesis. βC1 directly interacts with the basic helix-loop-helix transcription factor MYC2 to compromise the activation of MYC2-regulated terpene synthase genes, thereby reducing whitefly resistance. MYC2 associates with the bipartite begomoviral protein BV1, suggesting that MYC2 is an evolutionarily conserved target of begomoviruses for the suppression of terpene-based resistance and the promotion of vector performance. Our findings describe how this viral pathogen regulates host plant metabolism to establish mutualism with its insect vector. PMID:25490915

Organ-specific effects of brassinosteroids on stomatal production coordinate with the action of Too Many Mouths.

PubMed

Wang, Ming; Yang, Kezhen; Le, Jie

2015-03-01

In Arabidopsis, stomatal development initiates after protodermal cells acquire stomatal lineage cell fate. Stomata or their precursors communicate with their neighbor epidermal cells to ensure the "one cell spacing" rule. The signals from EPF/EPFL peptide ligands received by Too Many Mouths (TMM) and ERECTA-family receptors are supposed to be transduced by YODA MAPK cascade. A basic helix-loop-helix transcription factor SPEECHLESS (SPCH) is another key regulator of stomatal cell fate determination and asymmetric entry divisions, and SPCH activity is regulated by YODA MAPK cascade. Brassinosteroid (BR) signaling, one of the most well characterized signal transduction pathways in plants, contributes to the control of stomatal production. But opposite organ-specific effects of BR on stomatal production were reported. Here we confirm that stomatal production in hypocotyls is controlled by BR levels. YODA and CYCD4 are not essential for BR stomata-promoting function. Furthermore, we found that BR could confer tmm hypocotyls clustered stomatal phenotype, indicating that the BR organ-specific effects on stomatal production might coordinate with the TMM organ-specific actions. © 2014 Institute of Botany, Chinese Academy of Sciences.

DAPT mediates atoh1 expression to induce hair cell-like cells.

PubMed

Ren, Hongmiao; Guo, Weiwei; Liu, Wei; Gao, Weiqiang; Xie, Dinghua; Yin, Tuanfang; Yang, Shiming; Ren, Jihao

2016-01-01

Hearing loss is currently an incurable degenerative disease characterized by a paucity of hair cells (HCs), which cannot be spontaneously replaced in mammals. Recent technological advancements in gene therapy and local drug delivery have shed new light for hearing loss. Atoh1, also known as Math1, Hath1, and Cath1, is a proneural basic helix-loop-helix (bHLH) transcription factor that is essential for HC differentiation. At various stages in development, Atoh1 activity is sufficient to drive HC differentiation in the cochlea. Thus, Atoh1 related gene therapy is the most promising option for HC induction. DAPT, an inhibitor of Notch signaling, enhances the expression of Atoh1 indirectly, which in turn promotes the induction of a HC fate. Here, we show that DAPT cooperates with Atoh1 to synergistically promote HC fate in ependymal cells in vitro and promote hair cell regeneration in the cultured basilar membrane (BM) which mimics the microenvironment in vivo. Taken together, our findings demonstrated that DAPT is sufficient to induce HC-like cells via enhancing of the expression of Atoh1 to inhibit the progression of HC apoptosis and to induce new HC formation.

MDL-1, a growth- and tumor-suppressor, slows aging and prevents germline hyperplasia and hypertrophy in C. elegans

PubMed Central

Riesen, Michèle; Feyst, Inna; Rattanavirotkul, Nattaphong; Ezcurra, Marina; Tullet, Jennifer M.A.; Papatheodorou, Irene; Ziehm, Matthias; Au, Catherine; Gilliat, Ann F.; Hellberg, Josephine; Thornton, Janet M.; Gems, David

2014-01-01

In C. elegans, increased lifespan in daf-2 insulin/IGF-1 receptor mutants is accompanied by up-regulation of the MDL-1 Mad basic helix-loop-helix leucine zipper transcription factor. Here we describe the role of mdl-1 in C. elegans germline proliferation and aging. The deletion allele mdl-1(tm311) shortened lifespan, and did so significantly more so in long-lived daf-2 mutants implying that mdl-1(+) contributes to effects of daf-2 on lifespan. mdl-1 mutant hermaphrodites also lay increased numbers of unfertilized oocytes. During aging, unfertilized oocytes in the uterus develop into tumors, whose development was accelerated by mdl-1(tm311). Opposite phenotypes were seen in daf-2 mutants, i.e. mdl-1 and daf-2 mutant germlines are hyperplastic and hypoplastic, respectively. Thus, MDL-1, like its mammalian orthologs, is an inhibitor of cell proliferation and growth that slows progression of an age-related pathology in C. elegans (uterine tumors). In addition, intestine-limited rescue of mdl-1 increased lifespan but not to wild type levels. Thus, mdl-1 likely acts both in the intestine and the germline to influence age-related mortality. PMID:24531613

Drosophila Hey is a target of Notch in asymmetric divisions during embryonic and larval neurogenesis.

PubMed

Monastirioti, Maria; Giagtzoglou, Nikolaos; Koumbanakis, Konstantinos A; Zacharioudaki, Evanthia; Deligiannaki, Myrto; Wech, Irmgard; Almeida, Mara; Preiss, Anette; Bray, Sarah; Delidakis, Christos

2010-01-01

bHLH-O proteins are a subfamily of the basic-helix-loop-helix transcription factors characterized by an 'Orange' protein-protein interaction domain. Typical members are the Hairy/E(spl), or Hes, proteins, well studied in their ability, among others, to suppress neuronal differentiation in both invertebrates and vertebrates. Hes proteins are often effectors of Notch signalling. In vertebrates, another bHLH-O protein group, the Hey proteins, have also been shown to be Notch targets and to interact with Hes. We have studied the single Drosophila Hey orthologue. We show that it is primarily expressed in a subset of newly born neurons, which receive Notch signalling during their birth. Unlike in vertebrates, however, Hey is not expressed in precursor cells and does not block neuronal differentiation. It rather promotes one of two alternative fates that sibling neurons adopt at birth. Although in the majority of cases Hey is a Notch target, it is also expressed independently of Notch in some lineages, most notably the larval mushroom body. The availability of Hey as a Notch readout has allowed us to study Notch signalling during the genesis of secondary neurons in the larval central nervous system.

Linear signaling in the Toll-Dorsal pathway of Drosophila: activated Pelle kinase specifies all threshold outputs of gene expression while the bHLH protein Twist specifies a subset.

PubMed

Stathopoulos, Angelike; Levine, Michael

2002-07-01

Differential activation of the Toll receptor leads to the formation of a broad Dorsal nuclear gradient that specifies at least three patterning thresholds of gene activity along the dorsoventral axis of precellular embryos. We investigate the activities of the Pelle kinase and Twist basic helix-loop-helix (bHLH) transcription factor in transducing Toll signaling. Pelle functions downstream of Toll to release Dorsal from the Cactus inhibitor. Twist is an immediate-early gene that is activated upon entry of Dorsal into nuclei. Transgenes misexpressing Pelle and Twist were introduced into different mutant backgrounds and the patterning activities were visualized using various target genes that respond to different thresholds of Toll-Dorsal signaling. These studies suggest that an anteroposterior gradient of Pelle kinase activity is sufficient to generate all known Toll-Dorsal patterning thresholds and that Twist can function as a gradient morphogen to establish at least two distinct dorsoventral patterning thresholds. We discuss how the Dorsal gradient system can be modified during metazoan evolution and conclude that Dorsal-Twist interactions are distinct from the interplay between Bicoid and Hunchback, which pattern the anteroposterior axis.

Soybean SAT1 (Symbiotic Ammonium Transporter 1) encodes a bHLH transcription factor involved in nodule growth and NH4+ transport.

PubMed

Chiasson, David M; Loughlin, Patrick C; Mazurkiewicz, Danielle; Mohammadidehcheshmeh, Manijeh; Fedorova, Elena E; Okamoto, Mamoru; McLean, Elizabeth; Glass, Anthony D M; Smith, Sally E; Bisseling, Ton; Tyerman, Stephen D; Day, David A; Kaiser, Brent N

2014-04-01

Glycine max symbiotic ammonium transporter 1 was first documented as a putative ammonium (NH4(+)) channel localized to the symbiosome membrane of soybean root nodules. We show that Glycine max symbiotic ammonium transporter 1 is actually a membrane-localized basic helix-loop-helix (bHLH) DNA-binding transcription factor now renamed Glycine max bHLH membrane 1 (GmbHLHm1). In yeast, GmbHLHm1 enters the nucleus and transcriptionally activates a unique plasma membrane NH4(+) channel Saccharomyces cerevisiae ammonium facilitator 1. Ammonium facilitator 1 homologs are present in soybean and other plant species, where they often share chromosomal microsynteny with bHLHm1 loci. GmbHLHm1 is important to the soybean rhizobium symbiosis because loss of activity results in a reduction of nodule fitness and growth. Transcriptional changes in nodules highlight downstream signaling pathways involving circadian clock regulation, nutrient transport, hormone signaling, and cell wall modification. Collectively, these results show that GmbHLHm1 influences nodule development and activity and is linked to a novel mechanism for NH4(+) transport common to both yeast and plants.

Evolution of the Max and Mlx networks in animals.

PubMed

McFerrin, Lisa G; Atchley, William R

2011-01-01

Transcription factors (TFs) are essential for the regulation of gene expression and often form emergent complexes to perform vital roles in cellular processes. In this paper, we focus on the parallel Max and Mlx networks of TFs because of their critical involvement in cell cycle regulation, proliferation, growth, metabolism, and apoptosis. A basic-helix-loop-helix-zipper (bHLHZ) domain mediates the competitive protein dimerization and DNA binding among Max and Mlx network members to form a complex system of cell regulation. To understand the importance of these network interactions, we identified the bHLHZ domain of Max and Mlx network proteins across the animal kingdom and carried out several multivariate statistical analyses. The presence and conservation of Max and Mlx network proteins in animal lineages stemming from the divergence of Metazoa indicate that these networks have ancient and essential functions. Phylogenetic analysis of the bHLHZ domain identified clear relationships among protein families with distinct points of radiation and divergence. Multivariate discriminant analysis further isolated specific amino acid changes within the bHLHZ domain that classify proteins, families, and network configurations. These analyses on Max and Mlx network members provide a model for characterizing the evolution of TFs involved in essential networks.

Helical peptides with three pairs of Asp-Arg and Glu-Arg residues in different orientations and spacings.

PubMed Central

Huyghues-Despointes, B. M.; Scholtz, J. M.; Baldwin, R. L.

1993-01-01

The helix-stabilizing effects of repeating pairs of Asp-Arg and Glu-Arg residues have been characterized using a peptide system of the same design used earlier to study Glu-Lys (Marqusee, S. & Baldwin, R.L., 1987, Proc. Natl. Acad. Sci. USA 84, 8898-8902) and Asp-Lys ion pairs (Marqusee, S. & Baldwin, R.L., 1990, In Protein Folding [Gierasch, L.M. & King, J., Eds.], pp. 85-94, AAAS, Washington, D.C.). The consequences of breaking ion pair and charge-helix dipole interactions by titration to pH 2 have been compared with the results of screening these interactions with NaCl at pH 7.0 and pH 2.5. The four peptides in each set contain three pairs of acidic (A) and basic (B) residues spaced either i, i + 4 or i, i + 3 apart. In one peptide of each kind the pairwise order of residues is AB, with the charges oriented favorably to the helix macrodipole, and in the other peptide the order is BA. The results are as follows: (1) Remarkably, both Asp-Arg and Glu-Arg peptides show the same pattern of helix stabilization at pH 7.0 found earlier for Glu-Lys and Asp-Lys peptides: i + 4 AB > i + 4 BA approximately i + 3 AB > i + 3 BA. (2) The ion pairs and charge-helix dipole interactions cannot be cleanly separated, but the results suggest that both interactions make important contributions to helix stability.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8443591

Effect of polarization on the stability of a helix dimer

NASA Astrophysics Data System (ADS)

Wang, Xing Y.; Zhang, John Z. H.

2011-01-01

Molecular dynamics (MD) simulations have been carried out to study helix-helix interaction using both standard AMBER and polarized force fields. Comparison of the two simulations shows that electrostatic polarization of intra-protein hydrogen bonds plays a significant role in stabilizing the structure of helix dimer. This stabilizing effect is clearly demonstrated by examining the monomer structure, helix crossing angle and stability of backbone hydrogen bonds under AMBER and PPC. Since reliable prediction of protein-protein structure is a significant challenge, the current study should help shed light on the importance of electrostatic polarization of protein in helix-helix interaction and helix bundle structures.

Contributions of the N- and C-terminal helical segments to the lipid-free structure and lipid interaction of apolipoprotein A-I.

PubMed

Tanaka, Masafumi; Dhanasekaran, Padmaja; Nguyen, David; Ohta, Shinya; Lund-Katz, Sissel; Phillips, Michael C; Saito, Hiroyuki

2006-08-29

The tertiary structure of lipid-free apolipoprotein (apo) A-I in the monomeric state comprises two domains: a N-terminal alpha-helix bundle and a less organized C-terminal domain. This study examined how the N- and C-terminal segments of apoA-I (residues 1-43 and 223-243), which contain the most hydrophobic regions in the molecule and are located in opposite structural domains, contribute to the lipid-free conformation and lipid interaction. Measurements of circular dichroism in conjunction with tryptophan and 8-anilino-1-naphthalenesulfonic acid fluorescence data demonstrated that single (L230P) or triple (L230P/L233P/Y236P) proline insertions into the C-terminal alpha helix disrupted the organization of the C-terminal domain without affecting the stability of the N-terminal helix bundle. In contrast, proline insertion into the N terminus (Y18P) disrupted the bundle structure in the N-terminal domain, indicating that the alpha-helical segment in this region is part of the helix bundle. Calorimetric and gel-filtration measurements showed that disruption of the C-terminal alpha helix significantly reduced the enthalpy and free energy of binding of apoA-I to lipids, whereas disruption of the N-terminal alpha helix had only a small effect on lipid binding. Significantly, the presence of the Y18P mutation offset the negative effects of disruption/removal of the C-terminal helical domain on lipid binding, suggesting that the alpha helix around Y18 concealed a potential lipid-binding region in the N-terminal domain, which was exposed by the disruption of the helix-bundle structure. When these results are taken together, they indicate that the alpha-helical segment in the N terminus of apoA-I modulates the lipid-free structure and lipid interaction in concert with the C-terminal domain.

Solution structure of telomere binding domain of AtTRB2 derived from Arabidopsis thaliana

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

Yun, Ji-Hye; Lee, Won Kyung; Kim, Heeyoun

Highlights: • We have determined solution structure of Myb domain of AtTRB2. • The Myb domain of AtTRB2 is located in the N-terminal region. • The Myb domain of AtTRB2 binds to plant telomeric DNA without fourth helix. • Helix 2 and 3 of the Myb domain of AtTRB2 are involved in DNA recognition. • AtTRB2 is a novel protein distinguished from other known plant TBP. - Abstract: Telomere homeostasis is regulated by telomere-associated proteins, and the Myb domain is well conserved for telomere binding. AtTRB2 is a member of the SMH (Single-Myb-Histone)-like family in Arabidopsis thaliana, having an N-terminalmore » Myb domain, which is responsible for DNA binding. The Myb domain of AtTRB2 contains three α-helices and loops for DNA binding, which is unusual given that other plant telomere-binding proteins have an additional fourth helix that is essential for DNA binding. To understand the structural role for telomeric DNA binding of AtTRB2, we determined the solution structure of the Myb domain of AtTRB2 (AtTRB2{sub 1–64}) using nuclear magnetic resonance (NMR) spectroscopy. In addition, the inter-molecular interaction between AtTRB2{sub 1–64} and telomeric DNA has been characterized by the electrophoretic mobility shift assay (EMSA) and NMR titration analyses for both plant (TTTAGGG)n and human (TTAGGG)n telomere sequences. Data revealed that Trp28, Arg29, and Val47 residues located in Helix 2 and Helix 3 are crucial for DNA binding, which are well conserved among other plant telomere binding proteins. We concluded that although AtTRB2 is devoid of the additional fourth helix in the Myb-extension domain, it is able to bind to plant telomeric repeat sequences as well as human telomeric repeat sequences.« less

Direct Ubiquitin Independent Recognition and Degradation of a Folded Protein by the Eukaryotic Proteasomes-Origin of Intrinsic Degradation Signals

PubMed Central

Singh Gautam, Amit Kumar; Balakrishnan, Satish; Venkatraman, Prasanna

2012-01-01

Eukaryotic 26S proteasomes are structurally organized to recognize, unfold and degrade globular proteins. However, all existing model substrates of the 26S proteasome in addition to ubiquitin or adaptor proteins require unstructured regions in the form of fusion tags for efficient degradation. We report for the first time that purified 26S proteasome can directly recognize and degrade apomyoglobin, a globular protein, in the absence of ubiquitin, extrinsic degradation tags or adaptor proteins. Despite a high affinity interaction, absence of a ligand and presence of only helices/loops that follow the degradation signal, apomyoglobin is degraded slowly by the proteasome. A short floppy F-helix exposed upon ligand removal and in conformational equilibrium with a disordered structure is mandatory for recognition and initiation of degradation. Holomyoglobin, in which the helix is buried, is neither recognized nor degraded. Exposure of the floppy F-helix seems to sensitize the proteasome and primes the substrate for degradation. Using peptide panning and competition experiments we speculate that initial encounters through the floppy helix and additional strong interactions with N-terminal helices anchors apomyoglobin to the proteasome. Stabilizing helical structure in the floppy F-helix slows down degradation. Destabilization of adjacent helices accelerates degradation. Unfolding seems to follow the mechanism of helix unraveling rather than global unfolding. Our findings while confirming the requirement for unstructured regions in degradation offers the following new insights: a) origin and identification of an intrinsic degradation signal in the substrate, b) identification of sequences in the native substrate that are likely to be responsible for direct interactions with the proteasome, and c) identification of critical rate limiting steps like exposure of the intrinsic degron and destabilization of an unfolding intermediate that are presumably catalyzed by the ATPases. Apomyoglobin emerges as a new model substrate to further explore the role of ATPases and protein structure in proteasomal degradation PMID:22506054

Functionally Similar WRKY Proteins Regulate Vacuolar Acidification in Petunia and Hair Development in Arabidopsis

PubMed Central

de Vries, Michel

2016-01-01

The WD40 proteins ANTHOCYANIN11 (AN11) from petunia (Petunia hybrida) and TRANSPARENT TESTA GLABRA1 (TTG1) from Arabidopsis thaliana and associated basic helix-loop-helix (bHLH) and MYB transcription factors activate a variety of differentiation processes. In petunia petals, AN11 and the bHLH protein AN1 activate, together with the MYB protein AN2, anthocyanin biosynthesis and, together with the MYB protein PH4, distinct genes, such as PH1 and PH5, that acidify the vacuole. To understand how AN1 and AN11 activate anthocyanin biosynthetic and PH genes independently, we isolated PH3. We found that PH3 is a target gene of the AN11-AN1-PH4 complex and encodes a WRKY protein that can bind to AN11 and is required, in a feed-forward loop, together with AN11-AN1-PH4 for transcription of PH5. PH3 is highly similar to TTG2, which regulates hair development, tannin accumulation, and mucilage production in Arabidopsis. Like PH3, TTG2 can bind to petunia AN11 and the Arabidopsis homolog TTG1, complement ph3 in petunia, and reactivate the PH3 target gene PH5. Our findings show that the specificity of WD40-bHLH-MYB complexes is in part determined by interacting proteins, such as PH3 and TTG2, and reveal an unanticipated similarity in the regulatory circuitry that controls petunia vacuolar acidification and Arabidopsis hair development. PMID:26977085

Non-Periodic Helix TWT Study.

DTIC Science & Technology

1980-05-01

IA-A087 413 LITTON SYSTEMS INC SAN CARLOS CA ELECTRON TUBE DIV F/6 9/1 NON-PERIODIC HELIX TWT STUDY.(U) MAY 80 N00173-76C-014 UNCLASSIFIED ML...IEEEEEEmhEEI U-80 - ’l////////,, 11111 128 25 L1. = *""L 1- 1.8 MICROCOPY RESOLUTION TEST CHART ’IT-, ( (NO-PERIODIC HELIX WTSTUDY0 I EXTENSION OF CONTRACT... Helix Circuit 4 - 2 Non-Periodic Helix Structure 7 3 Unwound Tape for Non-Periodic Helix 8 4 Typical Helix Assembly 10 5 Typical Ladder Assembly 11 6

Autoinhibition of ETV6 DNA Binding Is Established by the Stability of Its Inhibitory Helix

PubMed Central

De, Soumya; Okon, Mark; Graves, Barbara J.; McIntosh, Lawrence P.

2017-01-01

The ETS transcriptional repressor ETV6 (or TEL) is autoinhibited by an α-helix that sterically blocks its DNA-binding ETS domain. The inhibitory helix is marginally stable and unfolds when ETV6 binds to either specific or non-specific DNA. Using NMR spectroscopy, we show that folding of the inhibitory helix requires a buried charge–dipole interaction with helix H1 of the ETS domain. This interaction also contributes directly to autoinhibition by precluding a highly conserved dipole-enhanced hydrogen bond between the phosphodiester backbone of bound DNA and the N terminus of helix H1. To probe further the thermodynamic basis of autoinhibition, ETV6 variants were generated with amino acid substitutions introduced along the solvent exposed surface of the inhibitory helix. These changes were designed to increase the intrinsic helical propensity of the inhibitory helix without perturbing its packing interactions with the ETS domain. NMR-monitored amide hydrogen exchange measurements confirmed that the stability of the folded inhibitory helix increases progressively with added helix-promoting substitutions. This also results in progressively reinforced autoinhibition and decreased DNA-binding affinity. Surprisingly, locking the inhibitory helix onto the ETS domain by a disulfide bridge severely impairs, but does not abolish DNA binding. Weak interactions still occur via an interface displaced from the canonical ETS domain DNA-binding surface. Collectively, these studies establish a direct thermodynamic linkage between inhibitory helix stability and ETV6 autoinhibition, and demonstrate that helix unfolding does not strictly precede DNA binding. Modulating inhibitory helix stability provides a potential route for the in vivo regulation of ETV6 activity. PMID:26920109

Molecular Dynamics Analysis Reveals Structural Insights into Mechanism of Nicotine N-Demethylation Catalyzed by Tobacco Cytochrome P450 Mono-Oxygenase

PubMed Central

Wang, Shan; Yang, Shuo; An, Baiyi; Wang, Shichen; Yin, Yuejia; Lu, Yang; Xu, Ying; Hao, Dongyun

2011-01-01

CYP82E4, a cytochrome P450 monooxygenase, has nicotine N-demethylase (NND) activity, which mediates the bioconversion of nicotine into nornicotine in senescing tobacco leaves. Nornicotine is a precursor of the carcinogen, tobacco-specific nitrosamine. CYP82E3 is an ortholog of CYP82E4 with 95% sequence identity, but it lacks NND activity. A recent site-directed mutagenesis study revealed that a single amino acid substitution, i.e., cysteine to tryptophan at the 330 position in the middle of protein, restores the NND activity of CYP82E3 entirely. However, the same amino acid change caused the loss of the NND activity of CYP82E4. To determine the mechanism of the functional turnover of the two molecules, four 3D structures, i.e., the two molecules and their corresponding cys–trp mutants were modeled. The resulting structures exhibited that the mutation site is far from the active site, which suggests that no direct interaction occurs between the two sites. Simulation studies in different biological scenarios revealed that the mutation introduces a conformation drift with the largest change at the F-G loop. The dynamics trajectories analysis using principal component analysis and covariance analysis suggests that the single amino acid change causes the opening and closing of the transfer channels of the substrates, products, and water by altering the motion of the F-G and B-C loops. The motion of helix I is also correlated with the motion of both the F-G loop and the B-C loop and; the single amino acid mutation resulted in the curvature of helix I. These results suggest that the single amino acid mutation outside the active site region may have indirectly mediated the flexibility of the F-G and B-C loops through helix I, causing a functional turnover of the P450 monooxygenase. PMID:21858078

Insights into the Photoprotective Switch of the Major Light-harvesting Complex II (LHCII)

PubMed Central

Sunku, Kiran; de Groot, Huub. J. M.; Pandit, Anjali

2013-01-01

Light-harvesting antennae of the LHC family form transmembrane three-helix bundles of which two helices are interlocked by conserved arginine-glutamate (Arg-Glu) ion pairs that form ligation sites for chlorophylls. The antenna proteins of photosystem II have an intriguing dual function. In excess light, they can switch their conformation from a light-harvesting into a photoprotective state, in which the excess and harmful excitation energies are safely dissipated as heat. Here we applied magic angle spinning NMR and selective Arg isotope enrichment as a noninvasive method to analyze the Arg structures of the major light-harvesting complex II (LHCII). The conformations of the Arg residues that interlock helix A and B appear to be preserved in the light-harvesting and photoprotective state. Several Arg residues have very downfield-shifted proton NMR responses, indicating that they stabilize the complex by strong hydrogen bonds. For the Arg Cα chemical shifts, differences are observed between LHCII in the active, light-harvesting and in the photoprotective, quenched state. These differences are attributed to a conformational change of the Arg residue in the stromal loop region. We conclude that the interlocked helices of LHCII form a rigid core. Consequently, the LHCII conformational switch does not involve changes in A/B helix tilting but likely involves rearrangements of the loops and helical segments close to the stromal and lumenal ends. PMID:23629658

NMR Studies of the C-Terminus of alpha4 Reveal Possible Mechanism of Its Interaction with MID1 and Protein Phosphatase 2A

PubMed Central

Du, Haijuan; Massiah, Michael A.

2011-01-01

Alpha4 is a regulatory subunit of the protein phosphatase family of enzymes and plays an essential role in regulating the catalytic subunit of PP2A (PP2Ac) within the rapamycin-sensitive signaling pathway. Alpha4 also interacts with MID1, a microtubule-associated ubiquitin E3 ligase that appears to regulate the function of PP2A. The C-terminal region of alpha4 plays a key role in the binding interaction of PP2Ac and MID1. Here we report on the solution structure of a 45-amino acid region derived from the C-terminus of alpha4 (alpha45) that binds tightly to MID1. In aqueous solution, alpha45 has properties of an intrinsically unstructured peptide although chemical shift index and dihedral angle estimation based on chemical shifts of backbone atoms indicate the presence of a transient α-helix. Alpha45 adopts a helix-turn-helix HEAT-like structure in 1% SDS micelles, which may mimic a negatively charged surface for which alpha45 could bind. Alpha45 binds tightly to the Bbox1 domain of MID1 in aqueous solution and adopts a structure consistent with the helix-turn-helix structure observed in 1% SDS. The structure of alpha45 reveals two distinct surfaces, one that can interact with a negatively charged surface, which is present on PP2A, and one that interacts with the Bbox1 domain of MID1. PMID:22194938

A Vitis vinifera basic helix-loop-helix transcription factor enhances plant cell size, vegetative biomass and reproductive yield

DOE PAGES

Lim, Sung Don; Yim, Won Choel; Liu, Degao; ...

2018-04-16

Strategies for improving plant size are critical targets for plant biotechnology to increase vegetative biomass or reproductive yield. To improve biomass production, a codon-optimized helix–loop–helix transcription factor (VvCEB1 opt) from wine grape was overexpressed in Arabidopsis thaliana resulting in significantly increased leaf number, leaf and rosette area, fresh weight and dry weight. Cell size, but typically not cell number, was increased in all tissues resulting in increased vegetative biomass and reproductive organ size, number and seed yield. Ionomic analysis of leaves revealed the VvCEB1 opt-overexpressing plants had significantly elevated, K, S and Mo contents relative to control lines. Increased Kmore » content likely drives increased osmotic potential within cells leading to greater cellular growth and expansion. To understand the mechanistic basis of VvCEB1 opt action, one transgenic line was genotyped using RNA-Seq mRNA expression profiling and revealed a novel transcriptional reprogramming network with significant changes in mRNA abundance for genes with functions in delayed flowering, pathogen–defence responses, iron homeostasis, vesicle-mediated cell wall formation and auxin-mediated signalling and responses. Direct testing of VvCEB1 opt-overexpressing plants showed that they had significantly elevated auxin content and a significantly increased number of lateral leaf primordia within meristems relative to controls, confirming that cell expansion and organ number proliferation were likely an auxin-mediated process. VvCEB1 opt overexpression in Nicotiana sylvestris also showed larger cells, organ size and biomass demonstrating the potential applicability of this innovative strategy for improving plant biomass and reproductive yield in crops.« less

A Vitis vinifera basic helix-loop-helix transcription factor enhances plant cell size, vegetative biomass and reproductive yield

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

Lim, Sung Don; Yim, Won Choel; Liu, Degao

Strategies for improving plant size are critical targets for plant biotechnology to increase vegetative biomass or reproductive yield. To improve biomass production, a codon-optimized helix–loop–helix transcription factor (VvCEB1 opt) from wine grape was overexpressed in Arabidopsis thaliana resulting in significantly increased leaf number, leaf and rosette area, fresh weight and dry weight. Cell size, but typically not cell number, was increased in all tissues resulting in increased vegetative biomass and reproductive organ size, number and seed yield. Ionomic analysis of leaves revealed the VvCEB1 opt-overexpressing plants had significantly elevated, K, S and Mo contents relative to control lines. Increased Kmore » content likely drives increased osmotic potential within cells leading to greater cellular growth and expansion. To understand the mechanistic basis of VvCEB1 opt action, one transgenic line was genotyped using RNA-Seq mRNA expression profiling and revealed a novel transcriptional reprogramming network with significant changes in mRNA abundance for genes with functions in delayed flowering, pathogen–defence responses, iron homeostasis, vesicle-mediated cell wall formation and auxin-mediated signalling and responses. Direct testing of VvCEB1 opt-overexpressing plants showed that they had significantly elevated auxin content and a significantly increased number of lateral leaf primordia within meristems relative to controls, confirming that cell expansion and organ number proliferation were likely an auxin-mediated process. VvCEB1 opt overexpression in Nicotiana sylvestris also showed larger cells, organ size and biomass demonstrating the potential applicability of this innovative strategy for improving plant biomass and reproductive yield in crops.« less

Converting One-Face α-Helix Mimetics into Amphiphilic α-Helix Mimetics as Potent Inhibitors of Protein-Protein Interactions.

PubMed

Lee, Ji Hoon; Oh, Misook; Kim, Hyun Soo; Lee, Huisun; Im, Wonpil; Lim, Hyun-Suk

2016-01-11

Many biologically active α-helical peptides adopt amphiphilic helical structures that contain hydrophobic residues on one side and hydrophilic residues on the other side. Therefore, α-helix mimetics capable of mimicking such amphiphilic helical peptides should possess higher binding affinity and specificity to target proteins. Here we describe an efficient method for generating amphiphilic α-helix mimetics. One-face α-helix mimetics having hydrophobic side chains on one side was readily converted into amphiphilic α-helix mimetics by introducing appropriate charged residues on the opposite side. We also demonstrate that such two-face amphiphilic α-helix mimetics indeed show remarkably improved binding affinity to a target protein, compared to one-face hydrophobic α-helix mimetics. We believe that generating a large combinatorial library of these amphiphilic α-helix mimetics can be valuable for rapid discovery of highly potent and specific modulators of protein-protein interactions.

Computational study of stability of an H-H-type pseudoknot motif.

PubMed

Wang, Jun; Zhao, Yunjie; Wang, Jian; Xiao, Yi

2015-12-01

Motifs in RNA tertiary structures are important to their structural organizations and biological functions. Here we consider an H-H-type pseudoknot (HHpk) motif that consists of two hairpins connected by a junction loop and with kissing interactions between the two hairpin loops. Such a tertiary structural motif is recurrently found in RNA tertiary structures, but is difficult to predict computationally. So it is important to understand the mechanism of its formation and stability. Here we investigate the stability of the HHpk tertiary structure by using an all-atom molecular dynamics simulation. The results indicate that the HHpk tertiary structure is stable. However, it is found that this stability is not due to the helix-helix packing, as is usually expected, but is maintained by the combined action of the kissing hairpin loops and junctions, although the former plays the main role. Stable HHpk motifs may form structural platforms for the molecules to realize their biological functions. These results are useful for understanding the construction principle of RNA tertiary structures and structure prediction.

An Amino Acid Packing Code for α-helical Structure and Protein Design

PubMed Central

Joo, Hyun; Chavan, Archana G.; Phan, Jamie; Day, Ryan; Tsai, Jerry

2012-01-01

This work demonstrates that all packing in α-helices can be simplified to repetitive patterns of a single motif: the knob-socket. Using the precision of Voronoi Polyhedra/Deluaney Tessellations to identify contacts, the knob-socket is a 4 residue tetrahedral motif: a knob residue on one α-helix packs into the 3 residue socket on another α-helix. The principle of the knob-socket model relates the packing between levels of protein structure: the intra-helical packing arrangements within secondary structure that permit inter-helix tertiary packing interactions. Within an α-helix, the 3 residue sockets arrange residues into a uniform packing lattice. Inter-helix packing results from a definable pattern of interdigitated knob-socket motifs between 2 α-helices. Furthermore, the knob-socket model classifies 3 types of sockets: 1) free: favoring only intra-helical packing, 2) filled: favoring inter-helical interactions and 3) non: disfavoring α-helical structure. The amino acid propensities in these 3 socket classes essentially represent an amino acid code for structure in α-helical packing. Using this code, a novel yet straightforward approach for the design of α-helical structure was used to validate the knob-socket model. Unique sequences for 3 peptides were created to produce a predicted amount of α-helical structure: mostly helical, some helical, and no-helix. These 3 peptides were synthesized and helical content assessed using CD spectroscopy. The measured α-helicity of each peptide was consistent with the expected predictions. These results and analysis demonstrate that the knob-socket motif functions as the basic unit of packing and presents an intuitive tool to decipher the rules governing packing in protein structure. PMID:22426125

Evolution of the α-Subunit of Na/K-ATPase from Paramecium to Homo sapiens: Invariance of Transmembrane Helix Topology.

PubMed

Morrill, Gene A; Kostellow, Adele B; Liu, Lijun; Gupta, Raj K; Askari, Amir

2016-05-01

Na/K-ATPase is a key plasma membrane enzyme involved in cell signaling, volume regulation, and maintenance of electrochemical gradients. The α-subunit, central to these functions, belongs to a large family of P-type ATPases. Differences in transmembrane (TM) helix topology, sequence homology, helix-helix contacts, cell signaling, and protein domains of Na/K-ATPase α-subunit were compared in fungi (Beauveria), unicellular organisms (Paramecia), primitive multicellular organisms (Hydra), and vertebrates (Xenopus, Homo sapiens), and correlated with evolution of physiological functions in the α-subunit. All α-subunits are of similar length, with groupings of four and six helices in the N- and C-terminal regions, respectively. Minimal homology was seen for protein domain patterns in Paramecium and Hydra, with high correlation between Hydra and vertebrates. Paramecium α-subunits display extensive disorder, with minimal helix contacts. Increases in helix contacts in Hydra approached vertebrates. Protein motifs known to be associated with membrane lipid rafts and cell signaling reveal significant positional shifts between Paramecium and Hydra vulgaris, indicating that regional membrane fluidity changes occur during evolution. Putative steroid binding sites overlapping TM-3 occurred in all species. Sites associated with G-protein-receptor stimulation occur both in vertebrates and amphibia but not in Hydra or Paramecia. The C-terminus moiety "KETYY," necessary for the Na(+) activation of pump phosphorylation, is not present in unicellular species indicating the absence of classical Na(+)/K(+)-pumps. The basic protein topology evolved earliest, followed by increases in protein domains and ordered helical arrays, correlated with appearance of α-subunit regions known to involve cell signaling, membrane recycling, and ion channel formation.

Molecular dynamics and mutational analysis of the catalytic and translocation cycle of RNA polymerase

PubMed Central

2012-01-01

Background During elongation, multi-subunit RNA polymerases (RNAPs) cycle between phosphodiester bond formation and nucleic acid translocation. In the conformation associated with catalysis, the mobile “trigger loop” of the catalytic subunit closes on the nucleoside triphosphate (NTP) substrate. Closing of the trigger loop is expected to exclude water from the active site, and dehydration may contribute to catalysis and fidelity. In the absence of a NTP substrate in the active site, the trigger loop opens, which may enable translocation. Another notable structural element of the RNAP catalytic center is the “bridge helix” that separates the active site from downstream DNA. The bridge helix may participate in translocation by bending against the RNA/DNA hybrid to induce RNAP forward movement and to vacate the active site for the next NTP loading. The transition between catalytic and translocation conformations of RNAP is not evident from static crystallographic snapshots in which macromolecular motions may be restrained by crystal packing. Results All atom molecular dynamics simulations of Thermus thermophilus (Tt) RNAP reveal flexible hinges, located within the two helices at the base of the trigger loop, and two glycine hinges clustered near the N-terminal end of the bridge helix. As simulation progresses, these hinges adopt distinct conformations in the closed and open trigger loop structures. A number of residues (described as “switch” residues) trade atomic contacts (ion pairs or hydrogen bonds) in response to changes in hinge orientation. In vivo phenotypes and in vitro activities rendered by mutations in the hinge and switch residues in Saccharomyces cerevisiae (Sc) RNAP II support the importance of conformational changes predicted from simulations in catalysis and translocation. During simulation, the elongation complex with an open trigger loop spontaneously translocates forward relative to the elongation complex with a closed trigger loop. Conclusions Switching between catalytic and translocating RNAP forms involves closing and opening of the trigger loop and long-range conformational changes in the atomic contacts of amino acid side chains, some located at a considerable distance from the trigger loop and active site. Trigger loop closing appears to support chemistry and the fidelity of RNA synthesis. Trigger loop opening and limited bridge helix bending appears to promote forward nucleic acid translocation. PMID:22676913

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.

A Conserved Hydrophobic Core in Gαi1 Regulates G Protein Activation and Release from Activated Receptor.

PubMed

Kaya, Ali I; Lokits, Alyssa D; Gilbert, James A; Iverson, T M; Meiler, Jens; Hamm, Heidi E

2016-09-09

G protein-coupled receptor-mediated heterotrimeric G protein activation is a major mode of signal transduction in the cell. Previously, we and other groups reported that the α5 helix of Gαi1, especially the hydrophobic interactions in this region, plays a key role during nucleotide release and G protein activation. To further investigate the effect of this hydrophobic core, we disrupted it in Gαi1 by inserting 4 alanine amino acids into the α5 helix between residues Gln(333) and Phe(334) (Ins4A). This extends the length of the α5 helix without disturbing the β6-α5 loop interactions. This mutant has high basal nucleotide exchange activity yet no receptor-mediated activation of nucleotide exchange. By using structural approaches, we show that this mutant loses critical hydrophobic interactions, leading to significant rearrangements of side chain residues His(57), Phe(189), Phe(191), and Phe(336); it also disturbs the rotation of the α5 helix and the π-π interaction between His(57) and Phe(189) In addition, the insertion mutant abolishes G protein release from the activated receptor after nucleotide binding. Our biochemical and computational data indicate that the interactions between α5, α1, and β2-β3 are not only vital for GDP release during G protein activation, but they are also necessary for proper GTP binding (or GDP rebinding). Thus, our studies suggest that this hydrophobic interface is critical for accurate rearrangement of the α5 helix for G protein release from the receptor after GTP binding. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Stabilizing interactions between aromatic and basic side chains in alpha-helical peptides and proteins. Tyrosine effects on helix circular dichroism.

PubMed

Andrew, Charles D; Bhattacharjee, Samita; Kokkoni, Nicoleta; Hirst, Jonathan D; Jones, Gareth R; Doig, Andrew J

2002-10-30

Here we investigate the structures and energetics of interactions between aromatic (Phe or Tyr) and basic (Lys or Arg) amino acids in alpha-helices. Side chain interaction energies are measured using helical peptides, by quantifying their helicities with circular dichroism at 222 nm and interpreting the results with Lifson-Roig-based helix/coil theory. A difficulty in working with Tyr is that the aromatic ring perturbs the CD spectrum, giving an incorrect helicity. We calculated the effect of Tyr on the CD at 222 nm by deriving the intensities of the bands directly from the electronic and magnetic transition dipole moments through the rotational strengths corresponding to each excited state of the polypeptide. This gives an improved value of the helix preference of Tyr (from 0.48 to 0.35) and a correction to the helicity for the peptides containing Tyr. We find that Phe-Lys, Lys-Phe, Phe-Arg, Arg-Phe, and Tyr-Lys are all stabilizing by -0.10 to -0.18 kcal.mol-1 when placed i, i + 4 on the surface of a helix in aqueous solution, despite the great difference in polarity between these residues. Interactions between these side chains have previously been attributed to cation-pi bonds. A survey of protein structures shows that they are in fact predominantly hydrophobic interactions between the CH2 groups of Lys or Arg and the aromatic rings.

Possible role of region 152–156 in the structural duality of a peptide fragment from sheep prion protein

PubMed Central

Megy, Simon; Bertho, Gildas; Kozin, Sergey A.; Debey, Pascale; Hui Bon Hoa, Gaston; Girault, Jean-Pierre

2004-01-01

The conformational conversion of the nonpathogenic “cellular” prion isoform into a pathogenic “scrapie” protease-resistant isoform is a fundamental event in the onset of transmissible spongiform encephalopathies (TSE). During this pathogenic conversion, helix H1 and its two flanking loops of the normal prion protein are thought to undergo a conformational transition into a β-like structure. A peptide spanning helix H1 and β-strand S2 (residues 142–166 in human numbering) was studied by circular dichroism and nuclear magnetic resonance spectroscopies. This peptide in aqueous solution, in contrast to many prion fragments studied earlier (1) is highly soluble and (2) does not aggregate until the millimolar concentration range, and (3) exhibits an intrinsic propensity to a β-hairpin-like conformation at neutral pH. We found that this peptide can also fold into a helix H1 conformation when dissolved in a TFE/PB mixture. The structures of the peptide calculated by MD showed solvent-dependent internal stabilizing forces of the structures and evidenced a higher mobility of the residues following the end of helix H1. These data suggest that the molecular rearrangement of this peptide in region 152–156, particularly in position 155, could be associated with the pathogenic conversion of the prion protein. PMID:15537751

Localization of functional receptor epitopes on the structure of ciliary neurotrophic factor indicates a conserved, function-related epitope topography among helical cytokines.

PubMed

Panayotatos, N; Radziejewska, E; Acheson, A; Somogyi, R; Thadani, A; Hendrickson, W A; McDonald, N Q

1995-06-09

By rational mutagenesis, receptor-specific functional analysis, and visualization of complex formation in solution, we identified individual amino acid side chains involved specifically in the interaction of ciliary neurotrophic factor (CNTF) with CNTFR alpha and not with the beta-components, gp130 and LIFR. In the crystal structure, the side chains of these residues, which are located in helix A, the AB loop, helix B, and helix D, are surface accessible and are clustered in space, thus constituting an epitope for CNTFR alpha. By the same analysis, a partial epitope for gp130 was also identified on the surface of helix A that faces away from the alpha-epitope. Superposition of the CNTF and growth hormone structures showed that the location of these epitopes on CNTF is analogous to the location of the first and second receptor epitopes on the surface of growth hormone. Further comparison with proposed binding sites for alpha- and beta-receptors on interleukin-6 and leukemia inhibitory factor indicated that this epitope topology is conserved among helical cytokines. In each case, epitope I is utilized by the specificity-conferring component, whereas epitopes II and III are used by accessory components. Thus, in addition to a common fold, helical cytokines share a conserved order of receptor epitopes that is function related.

Teaching helix and problems connected with helix using GeoGebra

NASA Astrophysics Data System (ADS)

Bímová, Daniela

2017-12-01

The contribution presents the dynamic applets created in GeoGebra that show the origin and main properties of a helix and it also presents some constructive problems connected with the helix. There are created the step by step algorithms of some constructions in the chosen applets. Three-dimensional applets include illustrative helix samples and spatial animations that help students better see problems concerning the helix spatially. There is mentioned the website in the contribution on which there is situated GeoGebra book dedicated to the topic "Helix" and containing the mentioned applets. The created applets and materials of the GeoGebra book "Helix" help in teaching and studying the course Constructive Geometry determined for the students of the Faculty of Mechanical Engineering of the Technical University of Liberec.

Optical fiber loops and helices: tools for integrated photonic device characterization and microfluidic trapping

NASA Astrophysics Data System (ADS)

Ren, Yundong; Zhang, Rui; Ti, Chaoyang; Liu, Yuxiang

2016-09-01

Tapered optical fibers can deliver guided light into and carry light out of micro/nanoscale systems with low loss and high spatial resolution, which makes them ideal tools in integrated photonics and microfluidics. Special geometries of tapered fibers are desired for probing monolithic devices in plane as well as optical manipulation of micro particles in fluids. However, for many specially shaped tapered fibers, it remains a challenge to fabricate them in a straightforward, controllable, and repeatable way. In this work, we fabricated and characterized two special geometries of tapered optical fibers, namely fiber loops and helices, that could be switched between one and the other. The fiber loops in this work are distinct from previous ones in terms of their superior mechanical stability and high optical quality factors in air, thanks to a post-annealing process. We experimentally measured an intrinsic optical quality factor of 32,500 and a finesse of 137 from a fiber loop. A fiber helix was used to characterize a monolithic cavity optomechanical device. Moreover, a microfluidic "roller coaster" was demonstrated, where microscale particles in water were optically trapped and transported by a fiber helix. Tapered fiber loops and helices can find various applications ranging from on-the-fly characterization of integrated photonic devices to particle manipulation and sorting in microfluidics.

Structure modulation of helix 69 from Escherichia coli 23S ribosomal RNA by pseudouridylations.

PubMed

Jiang, Jun; Aduri, Raviprasad; Chow, Christine S; SantaLucia, John

2014-04-01

Helix 69 (H69) is a 19-nt stem-loop region from the large subunit ribosomal RNA. Three pseudouridine (Ψ) modifications clustered in H69 are conserved across phylogeny and known to affect ribosome function. To explore the effects of Ψ on the conformations of Escherichia coli H69 in solution, nuclear magnetic resonance spectroscopy was used to reveal the structural differences between H69 with (ΨΨΨ) and without (UUU) Ψ modifications. Comparison of the two structures shows that H69 ΨΨΨ has the following unique features: (i) the loop region is closed by a Watson-Crick base pair between Ψ1911 and A1919, which is potentially reinforced by interactions involving Ψ1911N1H and (ii) Ψ modifications at loop residues 1915 and 1917 promote base stacking from Ψ1915 to A1918. In contrast, the H69 UUU loop region, which lacks Ψ modifications, is less organized. Structure modulation by Ψ leads to alteration in conformational behavior of the 5' half of the H69 loop region, observed as broadening of C1914 non-exchangeable base proton resonances in the H69 ΨΨΨ nuclear magnetic resonance spectra, and plays an important biological role in establishing the ribosomal intersubunit bridge B2a and mediating translational fidelity.

Tissue and cell-type co-expression networks of transcription factors and wood component genes in Populus trichocarpa.

PubMed

Shi, Rui; Wang, Jack P; Lin, Ying-Chung; Li, Quanzi; Sun, Ying-Hsuan; Chen, Hao; Sederoff, Ronald R; Chiang, Vincent L

2017-05-01

Co-expression networks based on transcriptomes of Populus trichocarpa major tissues and specific cell types suggest redundant control of cell wall component biosynthetic genes by transcription factors in wood formation. We analyzed the transcriptomes of five tissues (xylem, phloem, shoot, leaf, and root) and two wood forming cell types (fiber and vessel) of Populus trichocarpa to assemble gene co-expression subnetworks associated with wood formation. We identified 165 transcription factors (TFs) that showed xylem-, fiber-, and vessel-specific expression. Of these 165 TFs, 101 co-expressed (correlation coefficient, r > 0.7) with the 45 secondary cell wall cellulose, hemicellulose, and lignin biosynthetic genes. Each cell wall component gene co-expressed on average with 34 TFs, suggesting redundant control of the cell wall component gene expression. Co-expression analysis showed that the 101 TFs and the 45 cell wall component genes each has two distinct groups (groups 1 and 2), based on their co-expression patterns. The group 1 TFs (44 members) are predominantly xylem and fiber specific, and are all highly positively co-expressed with the group 1 cell wall component genes (30 members), suggesting their roles as major wood formation regulators. Group 1 TFs include a lateral organ boundary domain gene (LBD) that has the highest number of positively correlated cell wall component genes (36) and TFs (47). The group 2 TFs have 57 members, including 14 vessel-specific TFs, and are generally less correlated with the cell wall component genes. An exception is a vessel-specific basic helix-loop-helix (bHLH) gene that negatively correlates with 20 cell wall component genes, and may function as a key transcriptional suppressor. The co-expression networks revealed here suggest a well-structured transcriptional homeostasis for cell wall component biosynthesis during wood formation.

Transcription factors, sucrose, and sucrose metabolic genes interact to regulate potato phenylpropanoid metabolism

PubMed Central

Payyavula, Raja S.; Navarre, Duroy A.

2013-01-01

Much remains unknown about how transcription factors and sugars regulate phenylpropanoid metabolism in tuber crops like potato (Solanum tuberosum). Based on phylogeny and protein similarity to known regulators of phenylpropanoid metabolism, 15 transcription factors were selected and their expression was compared in white, yellow, red, and purple genotypes with contrasting phenolic and anthocyanin profiles. Red and purple genotypes had increased phenylalanine ammonia lyase (PAL) enzyme activity, markedly higher levels of phenylpropanoids, and elevated expression of most phenylpropanoid structural genes, including a novel anthocyanin O-methyltransferase. The transcription factors Anthocyanin1 (StAN1), basic Helix Loop Helix1 (StbHLH1), and StWD40 were more strongly expressed in red and purple potatoes. Expression of 12 other transcription factors was not associated with phenylpropanoid content, except for StMYB12B, which showed a negative relationship. Increased expression of AN1, bHLH1, and WD40 was also associated with environmentally mediated increases in tuber phenylpropanoids. Treatment of potato plantlets with sucrose induced hydroxycinnamic acids, flavonols, anthocyanins, structural genes, AN1, bHLH1, WD40, and genes encoding the sucrose-hydrolysing enzymes SUSY1, SUSY4, and INV2. Transient expression of StAN1 in tobacco leaves induced bHLH1, structural genes, SUSY1, SUSY4, and INV1, and increased phenylpropanoid amounts. StAN1 infiltration into tobacco leaves decreased sucrose and glucose concentrations. In silico promoter analysis revealed the presence of MYB and bHLH regulatory elements on sucrolytic gene promoters and sucrose-responsive elements on the AN1 promoter. These findings reveal an interesting dynamic between AN1, sucrose, and sucrose metabolic genes in modulating potato phenylpropanoids. PMID:24098049

Arabidopsis thaliana G2-LIKE FLAVONOID REGULATOR and BRASSINOSTEROID ENHANCED EXPRESSION1 are low-temperature regulators of flavonoid accumulation.

PubMed

Petridis, Antonios; Döll, Stefanie; Nichelmann, Lars; Bilger, Wolfgang; Mock, Hans-Peter

2016-08-01

Flavonoid synthesis is predominantly regulated at the transcriptional level through the MYB-basic helix-loop-helix (bHLH)-WD40 (MBW) (MYB: transcription factor of the myeloblastosis protein family, WD40: tanscription factor with a short structural motif of 40 amino acids which terminates in an aspartic acid-tryptophan dipeptide) complex, and responds to both environmental and developmental stimuli. Although the developmental regulation of flavonoid accumulation in Arabidopsis thaliana has been examined in great detail, the response of the flavonoid synthesis pathway to abiotic stress (particularly low temperature) remains unclear. A screen of a Dissociation element (Ds) transposon-induced mutation collection identified two lines which exhibited an altered profile of phenylpropanoid accumulation following exposure to low-temperature stress. One of the mutated genes (BRASSINOSTEROID ENHANCED EXPRESSION1 (BEE1)) encoded a brassinosteroid enhanced expression transcription factor, while the other (G2-LIKE FLAVONOID REGULATOR (GFR)) encoded a G2-like flavonoid regulator. Phenylpropanoid-targeted analysis was performed using high-performance LC-MS, and gene expression analysis using quantitative reverse transcription-PCR. In both mutants, the accumulation of quercetins and scopolin was reduced under low-temperature growing conditions, whereas that of anthocyanin was increased. BEE1 and GFR were both shown to negatively regulate anthocyanin accumulation by inhibiting anthocyanin synthesis genes via the suppression of the bHLH (TRANSPARENT TESTA8 (TT8) and GLABROUS3 (GL3)) and/or the MYB (PRODUCTION OF ANTHOCYANIN PIGMENTS2 (PAP2)) components of the MBW complex. Our results provide new insight into the regulatory control of phenylpropanoid metabolism at low temperatures, and reveal that BEE1 and GFR act as important components of the signal transduction chain. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

The Genetic Causes of Nonsyndromic Congenital Retinal Detachment: A Genetic and Phenotypic Study of Pakistani Families.

PubMed

Keser, Vafa; Khan, Ayesha; Siddiqui, Sorath; Lopez, Irma; Ren, Huanan; Qamar, Raheel; Nadaf, Javad; Majewski, Jacek; Chen, Rui; Koenekoop, Robert K

2017-02-01

To evaluate consanguineous pedigrees from Pakistan with a clinical diagnosis of nonsyndromic congenital retinal nonattachment (NCRNA) and identify genes responsible for the disease as currently only one NCRNA gene is known (atonal basic helix-loop-helix transcription factor 7: ATOH7). We implemented a three-step genotyping platform: single nucleotide polymorphism genotyping to identify loss of heterozygosity regions in patients, Retinal Information Network panel screening for mutations in currently known retinal genes. Negative patients were then subjected to whole exome sequencing. We evaluated 21 consanguineous NCRNA pedigrees and identified the causal mutations in known retinal genes in 13 out of our 21 families. We found mutations in ATOH7 in three families. Surprisingly, we then found mutations in familial exudative vitreoretinopathy (FEVR) genes; low-density lipoprotein receptor-related protein 5 mutations (six families), tetraspanin 12 mutations (two families), and NDP mutations (two families). Thus, 62% of the patients were successfully genotyped in our study with seven novel and six previously reported mutations in known retinal genes. Although the clinical diagnosis of all children was NCRNA with severe congenital fibrotic retinal detachments, the molecular diagnosis determined that the disease process was in fact a very severe form of FEVR in 10 families. Because severe congenital retinal detachment has not been previously associated with all the FEVR genes, we have thus expanded the phenotypic spectrum of FEVR, a highly variable retinal detachment phenotype that has clinical overlap with NCRNA. We identified seven novel mutations. We also established for the first time genetic overlap between the Iranian and Pakistani populations. We identified eight NCRNA families that do not harbor mutations in any known retinal genes, suggesting novel causal genes in these families.

The Genetic Causes of Nonsyndromic Congenital Retinal Detachment: A Genetic and Phenotypic Study of Pakistani Families

PubMed Central

Keser, Vafa; Khan, Ayesha; Siddiqui, Sorath; Lopez, Irma; Ren, Huanan; Qamar, Raheel; Nadaf, Javad; Majewski, Jacek; Chen, Rui; Koenekoop, Robert K.

2017-01-01

Purpose To evaluate consanguineous pedigrees from Pakistan with a clinical diagnosis of nonsyndromic congenital retinal nonattachment (NCRNA) and identify genes responsible for the disease as currently only one NCRNA gene is known (atonal basic helix-loop-helix transcription factor 7: ATOH7). Methods We implemented a three-step genotyping platform: single nucleotide polymorphism genotyping to identify loss of heterozygosity regions in patients, Retinal Information Network panel screening for mutations in currently known retinal genes. Negative patients were then subjected to whole exome sequencing. Results We evaluated 21 consanguineous NCRNA pedigrees and identified the causal mutations in known retinal genes in 13 out of our 21 families. We found mutations in ATOH7 in three families. Surprisingly, we then found mutations in familial exudative vitreoretinopathy (FEVR) genes; low-density lipoprotein receptor-related protein 5 mutations (six families), tetraspanin 12 mutations (two families), and NDP mutations (two families). Thus, 62% of the patients were successfully genotyped in our study with seven novel and six previously reported mutations in known retinal genes. Conclusions Although the clinical diagnosis of all children was NCRNA with severe congenital fibrotic retinal detachments, the molecular diagnosis determined that the disease process was in fact a very severe form of FEVR in 10 families. Because severe congenital retinal detachment has not been previously associated with all the FEVR genes, we have thus expanded the phenotypic spectrum of FEVR, a highly variable retinal detachment phenotype that has clinical overlap with NCRNA. We identified seven novel mutations. We also established for the first time genetic overlap between the Iranian and Pakistani populations. We identified eight NCRNA families that do not harbor mutations in any known retinal genes, suggesting novel causal genes in these families. PMID:28192794

The Phenylpropanoid Pathway Is Controlled at Different Branches by a Set of R2R3-MYB C2 Repressors in Grapevine1

PubMed Central

Cavallini, Erika; Matus, José Tomás; Finezzo, Laura; Zenoni, Sara; Loyola, Rodrigo; Guzzo, Flavia; Schlechter, Rudolf; Ageorges, Agnès; Arce-Johnson, Patricio

2015-01-01

Because of the vast range of functions that phenylpropanoids possess, their synthesis requires precise spatiotemporal coordination throughout plant development and in response to the environment. The accumulation of these secondary metabolites is transcriptionally controlled by positive and negative regulators from the MYB and basic helix-loop-helix protein families. We characterized four grapevine (Vitis vinifera) R2R3-MYB proteins from the C2 repressor motif clade, all of which harbor the ethylene response factor-associated amphiphilic repression domain but differ in the presence of an additional TLLLFR repression motif found in the strong flavonoid repressor Arabidopsis (Arabidopsis thaliana) AtMYBL2. Constitutive expression of VvMYB4a and VvMYB4b in petunia (Petunia hybrida) repressed general phenylpropanoid biosynthetic genes and selectively reduced the amount of small-weight phenolic compounds. Conversely, transgenic petunia lines expressing VvMYBC2-L1 and VvMYBC2-L3 showed a severe reduction in petal anthocyanins and seed proanthocyanidins together with a higher pH of crude petal extracts. The distinct function of these regulators was further confirmed by transient expression in tobacco (Nicotiana benthamiana) leaves and grapevine plantlets. Finally, VvMYBC2-L3 was ectopically expressed in grapevine hairy roots, showing a reduction in proanthocyanidin content together with the down-regulation of structural and regulatory genes of the flavonoid pathway as revealed by a transcriptomic analysis. The physiological role of these repressors was inferred by combining the results of the functional analyses and their expression patterns in grapevine during development and in response to ultraviolet B radiation. Our results indicate that VvMYB4a and VvMYB4b may play a key role in negatively regulating the synthesis of small-weight phenolic compounds, whereas VvMYBC2-L1 and VvMYBC2-L3 may additionally fine tune flavonoid levels, balancing the inductive effects of transcriptional activators. PMID:25659381

Thermodynamics of melittin binding to lipid bilayers. Aggregation and pore formation.

PubMed

Klocek, Gabriela; Schulthess, Therese; Shai, Yechiel; Seelig, Joachim

2009-03-31

Lipid membranes act as catalysts for protein folding. Both alpha-helical and beta-sheet structures can be induced by the interaction of peptides or proteins with lipid surfaces. Melittin, the main component of bee venom, is a particularly well-studied example for the membrane-induced random coil-to-alpha-helix transition. Melittin in water adopts essentially a random coil conformation. The cationic amphipathic molecule has a high affinity for neutral and anionic lipid membranes and exhibits approximately 50-65% alpha-helix conformation in the membrane-bound state. At higher melittin concentrations, the peptide forms aggregates or pores in the membrane. In spite of the long-standing interest in melittin-lipid interactions, no systematic thermodynamic study is available. This is probably caused by the complexity of the binding process. Melittin binding to lipid vesicles is fast and occurs within milliseconds, but the binding process involves at least four steps, namely, (i) the electrostatic attraction of the cationic peptide to an anionic membrane surface, (ii) the hydrophobic insertion into the lipid membrane, (iii) the conformational change from random coil to alpha-helix, and (iv) peptide aggregation in the lipid phase. We have combined microelectrophoresis (measurement of the zeta potential), isothermal titration calorimetry, and circular dichroism spectroscopy to provide a thermodynamic analysis of the individual binding steps. We have compared melittin with a synthetic analogue, [D]-V(5,8),I(17),K(21)-melittin, for which alpha-helix formation is suppressed and replaced by beta-structure formation. The comparison reveals that the thermodynamic parameters for the membrane-induced alpha-helix formation of melittin are identical to those observed earlier for other peptides with an enthalpy h(helix) of -0.7 kcal/mol and a free energy g(helix) of -0.2 kcal/mol per peptide residue. These thermodynamic parameters hence appear to be of general validity for lipid-induced membrane folding. As g(helix) is negative, it further follows that helix formation leads to an enhanced membrane binding for the peptides or proteins involved. In this study, melittin binds by approximately 2 orders of magnitude better to the lipid membrane than [D]-V(5,8),I(17),K(21)-melittin which cannot form an alpha-helix. We also found conditions under which the isothermal titration experiment reports only the aggregation process. Melittin aggregation is an entropy-driven process with an endothermic heat of reaction (DeltaH(agg)) of approximately 2 kcal/mol and an aggregation constant of 20-40 M(-1).

Generating structured light with phase helix and intensity helix using reflection-enhanced plasmonic metasurface at 2 μm

NASA Astrophysics Data System (ADS)

Zhao, Yifan; Du, Jing; Zhang, Jinrun; Shen, Li; Wang, Jian

2018-04-01

Mid-infrared (2-20 μm) light has been attracting great attention in many areas of science and technology. Beyond the extended wavelength range from visible and near-infrared to mid-infrared, shaping spatial structures may add opportunities to grooming applications of mid-infrared photonics. Here, we design and fabricate a reflection-enhanced plasmonic metasurface and demonstrate efficient generation of structured light with the phase helix and intensity helix at 2 μm. This work includes two distinct aspects. First, structured light (phase helix, intensity helix) generation at 2 μm, which is far beyond the ability of conventional spatial light modulators, is enabled by the metasurface with sub-wavelength engineered structures. Second, the self-referenced intensity helix against environmental noise is generated without using a spatially separated light. The demonstrations may open up advanced perspectives to structured light applications at 2 μm, such as phase helix for communications and non-communications (imaging, sensing) and intensity helix for enhanced microscopy and advanced metrology.

A Secondary Structural Transition in the C-helix Promotes Gating of Cyclic Nucleotide-regulated Ion Channels*

PubMed Central

Puljung, Michael C.; Zagotta, William N.

2013-01-01

Cyclic nucleotide-regulated ion channels bind second messengers like cAMP to a C-terminal domain, consisting of a β-roll, followed by two α-helices (B- and C-helices). We monitored the cAMP-dependent changes in the structure of the C-helix of a C-terminal fragment of HCN2 channels using transition metal ion FRET between fluorophores on the C-helix and metal ions bound between histidine pairs on the same helix. cAMP induced a change in the dimensions of the C-helix and an increase in the metal binding affinity of the histidine pair. cAMP also caused an increase in the distance between a fluorophore on the C-helix and metal ions bound to the B-helix. Stabilizing the C-helix of intact CNGA1 channels by metal binding to a pair of histidines promoted channel opening. These data suggest that ordering of the C-helix is part of the gating conformational change in cyclic nucleotide-regulated channels. PMID:23525108

A Novel DNA Binding Mechanism for maf Basic Region-Leucine Zipper Factors Inferred from a MafA-DNA Complex Structure and Binding Specificities

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

Lu, Xun; Guanga, Gerald P; Wan, Cheng

2012-11-13

MafA is a proto-oncoprotein and is critical for insulin gene expression in pancreatic β-cells. Maf proteins belong to the AP1 superfamily of basic region-leucine zipper (bZIP) transcription factors. Residues in the basic helix and an ancillary N-terminal domain, the Extended Homology Region (EHR), endow maf proteins with unique DNA binding properties: binding a 13 bp consensus site consisting of a core AP1 site (TGACTCA) flanked by TGC sequences and binding DNA stably as monomers. To further characterize maf DNA binding, we determined the structure of a MafA–DNA complex. MafA forms base-specific hydrogen bonds with the flanking G –5C –4 andmore » central C 0/G 0 bases, but not with the core-TGA bases. However, in vitro binding studies utilizing a pulse–chase electrophoretic mobility shift assay protocol revealed that mutating either the core-TGA or flanking-TGC bases dramatically increases the binding off rate. Comparing the known maf structures, we propose that DNA binding specificity results from positioning the basic helix through unique phosphate contacts. The EHR does not contact DNA directly but stabilizes DNA binding by contacting the basic helix. Collectively, these results suggest a novel multistep DNA binding process involving a conformational change from contacting the core-TGA to contacting the flanking-TGC bases.« less

Crystal structure of the Mus81-Eme1 complex.

PubMed

Chang, Jeong Ho; Kim, Jeong Joo; Choi, Jung Min; Lee, Jung Hoon; Cho, Yunje

2008-04-15

The Mus81-Eme1 complex is a structure-specific endonuclease that plays an important role in rescuing stalled replication forks and resolving the meiotic recombination intermediates in eukaryotes. We have determined the crystal structure of the Mus81-Eme1 complex. Both Mus81 and Eme1 consist of a central nuclease domain, two repeats of the helix-hairpin-helix (HhH) motif at their C-terminal region, and a linker helix. While each domain structure resembles archaeal XPF homologs, the overall structure is significantly different from those due to the structure of a linker helix. We show that a flexible intradomain linker that formed with 36 residues in the nuclease domain of Eme1 is essential for the recognition of DNA. We identified several basic residues lining the outer surface of the active site cleft of Mus81 that are involved in the interaction with a flexible arm of a nicked Holliday junction (HJ). These interactions might contribute to the optimal positioning of the opposite junction across the nick into the catalytic site, which provided the basis for the "nick and counternick" mechanism of Mus81-Eme1 and for the nicked HJ to be the favored in vitro substrate of this enzyme.

The N-Terminal Amphipathic Helix of the Topological Specificity Factor MinE Is Associated with Shaping Membrane Curvature

PubMed Central

Shih, Yu-Ling; Huang, Kai-Fa; Lai, Hsin-Mei; Liao, Jiahn-Haur; Lee, Chai-Siah; Chang, Chiao-Min; Mak, Huey-Ming; Hsieh, Cheng-Wei; Lin, Chu-Chi

2011-01-01

Pole-to-pole oscillations of the Min proteins in Escherichia coli are required for the proper placement of the division septum. Direct interaction of MinE with the cell membrane is critical for the dynamic behavior of the Min system. In vitro, this MinE-membrane interaction led to membrane deformation; however, the underlying mechanism remained unclear. Here we report that MinE-induced membrane deformation involves the formation of an amphipathic helix of MinE2–9, which, together with the adjacent basic residues, function as membrane anchors. Biochemical evidence suggested that the membrane association induces formation of the helix, with the helical face, consisting of A2, L3, and F6, inserted into the membrane. Insertion of this helix into the cell membrane can influence local membrane curvature and lead to drastic changes in membrane topology. Accordingly, MinE showed characteristic features of protein-induced membrane tubulation and lipid clustering in in vitro reconstituted systems. In conclusion, MinE shares common protein signatures with a group of membrane trafficking proteins in eukaryotic cells. These MinE signatures appear to affect membrane curvature. PMID:21738659

Understanding the EF-hand closing pathway using non-biased interatomic potentials.

PubMed

Dupuis, L; Mousseau, Normand

2012-01-21

The EF-hand superfamily of proteins is characterized by the presence of calcium binding helix-loop-helix structures. Many of these proteins undergo considerable motion responsible for a wide range of properties upon binding but the exact mechanism at the root of this motion is not fully understood. Here, we use an unbiased accelerated multiscale simulation scheme, coupled with two force fields - CHARMM-EEF1 and the extended OPEP - to explore in details the closing pathway, from the unbound holo state to the closed apo state, of two EF-hand proteins, the Calmodulin and Troponin C N-terminal nodules. Based on a number of closing simulations for these two sequences, we show that the EF-hand β-scaffold, identified as crucial by Grabarek for the EF-hand opening driven by calcium binding, is also important in closing the EF-hand. We also show the crucial importance of the phenylalanine situated at the end of first EF-hand helix, and identify an intermediate state modulating its behavior, providing a detailed picture of the closing mechanism for these two representatives of EF-hand proteins. © 2012 American Institute of Physics

Conservation of Three-Dimensional Helix-Loop-Helix Structure through the Vertebrate Lineage Reopens the Cold Case of Gonadotropin-Releasing Hormone-Associated Peptide.

PubMed

Pérez Sirkin, Daniela I; Lafont, Anne-Gaëlle; Kamech, Nédia; Somoza, Gustavo M; Vissio, Paula G; Dufour, Sylvie

2017-01-01

GnRH-associated peptide (GAP) is the C-terminal portion of the gonadotropin-releasing hormone (GnRH) preprohormone. Although it was reported in mammals that GAP may act as a prolactin-inhibiting factor and can be co-secreted with GnRH into the hypophyseal portal blood, GAP has been practically out of the research circuit for about 20 years. Comparative studies highlighted the low conservation of GAP primary amino acid sequences among vertebrates, contributing to consider that this peptide only participates in the folding or carrying process of GnRH. Considering that the three-dimensional (3D) structure of a protein may define its function, the aim of this study was to evaluate if GAP sequences and 3D structures are conserved in the vertebrate lineage. GAP sequences from various vertebrates were retrieved from databases. Analysis of primary amino acid sequence identity and similarity, molecular phylogeny, and prediction of 3D structures were performed. Amino acid sequence comparison and phylogeny analyses confirmed the large variation of GAP sequences throughout vertebrate radiation. In contrast, prediction of the 3D structure revealed a striking conservation of the 3D structure of GAP1 (GAP associated with the hypophysiotropic type 1 GnRH), despite low amino acid sequence conservation. This GAP1 peptide presented a typical helix-loop-helix (HLH) structure in all the vertebrate species analyzed. This HLH structure could also be predicted for GAP2 in some but not all vertebrate species and in none of the GAP3 analyzed. These results allowed us to infer that selective pressures have maintained GAP1 HLH structure throughout the vertebrate lineage. The conservation of the HLH motif, known to confer biological activity to various proteins, suggests that GAP1 peptides may exert some hypophysiotropic biological functions across vertebrate radiation.

Conservation of Three-Dimensional Helix-Loop-Helix Structure through the Vertebrate Lineage Reopens the Cold Case of Gonadotropin-Releasing Hormone-Associated Peptide

PubMed Central

Pérez Sirkin, Daniela I.; Lafont, Anne-Gaëlle; Kamech, Nédia; Somoza, Gustavo M.; Vissio, Paula G.; Dufour, Sylvie

2017-01-01

GnRH-associated peptide (GAP) is the C-terminal portion of the gonadotropin-releasing hormone (GnRH) preprohormone. Although it was reported in mammals that GAP may act as a prolactin-inhibiting factor and can be co-secreted with GnRH into the hypophyseal portal blood, GAP has been practically out of the research circuit for about 20 years. Comparative studies highlighted the low conservation of GAP primary amino acid sequences among vertebrates, contributing to consider that this peptide only participates in the folding or carrying process of GnRH. Considering that the three-dimensional (3D) structure of a protein may define its function, the aim of this study was to evaluate if GAP sequences and 3D structures are conserved in the vertebrate lineage. GAP sequences from various vertebrates were retrieved from databases. Analysis of primary amino acid sequence identity and similarity, molecular phylogeny, and prediction of 3D structures were performed. Amino acid sequence comparison and phylogeny analyses confirmed the large variation of GAP sequences throughout vertebrate radiation. In contrast, prediction of the 3D structure revealed a striking conservation of the 3D structure of GAP1 (GAP associated with the hypophysiotropic type 1 GnRH), despite low amino acid sequence conservation. This GAP1 peptide presented a typical helix-loop-helix (HLH) structure in all the vertebrate species analyzed. This HLH structure could also be predicted for GAP2 in some but not all vertebrate species and in none of the GAP3 analyzed. These results allowed us to infer that selective pressures have maintained GAP1 HLH structure throughout the vertebrate lineage. The conservation of the HLH motif, known to confer biological activity to various proteins, suggests that GAP1 peptides may exert some hypophysiotropic biological functions across vertebrate radiation. PMID:28878737

pH-induced conformational changes in human ABO(H) blood group glycosyltransferases confirm the importance of electrostatic interactions in the formation of the semi-closed state.

PubMed

Johal, Asha R; Blackler, Ryan J; Alfaro, Javier A; Schuman, Brock; Borisova, Svetlana; Evans, Stephen V

2014-03-01

The homologous human ABO(H) A and B blood group glycosyltransferases GTA and GTB have two mobile polypeptide loops surrounding their active sites that serve to allow substrate access and product egress and to recognize and sequester substrates for catalysis. Previous studies have established that these enzymes can move from the "open" state to the "semi-closed" then "closed" states in response to addition of a substrate. The contribution of electrostatic interactions to these conformational changes has now been demonstrated by the determination at various pH of the structures of GTA, GTB and the chimeric enzyme ABBA. At near-neutral pH, GTA displays the closed state in which both mobile loops order around the active site, whereas ABBA and GTB display the open state. At low pH, the apparent protonation of the DXD motif in GTA leads to the expulsion of the donor analog to yield the open state, whereas at high pH, both ABBA and GTB form the semi-closed state in which the first mobile loop becomes an ordered α-helix. Step-wise deprotonation of GTB in increments of 0.5 between pH 6.5 and 10.0 shows that helix ordering is gradual, which indicates that the formation of the semi-closed state is dependent on electrostatic forces consistent with the binding of substrate. Spectropolarimetric studies of the corresponding stand-alone peptide in solution reveal no tendency toward helix formation from pH 7.0 to 10.0, which shows that pH-dependent stability is a product of the larger protein environment and underlines the importance of substrate in active site ordering.

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

Ghosh, Tarini Shankar; Chaitanya, S. Krishna; Sankararamakrishnan, Ramasubbu, E-mail: rsankar@iitk.ac.in

New classes of helix–helix interactions in protein structures are reported in which interactions only occur at the terminal regions or between the terminal region of one helix and the middle region of another helix. Helix–helix interactions are important for the structure, stability and function of α-helical proteins. Helices that either cross in the middle or show extensive contacts between each other, such as coiled coils, have been investigated in previous studies. Interactions between two helices can also occur only at the terminal regions or between the terminal region of one helix and the middle region of another helix. Examples ofmore » such helix pairs are found in aquaporin, H{sup +}/Cl{sup −} transporter and Bcl-2 proteins. The frequency of the occurrence of such ‘end-to-end’ (EE) and ‘end-to-middle’ (EM) helix pairs in protein structures is not known. Questions regarding the residue preferences in the interface and the mode of interhelical interactions in such helix pairs also remain unanswered. In this study, high-resolution structures of all-α proteins from the PDB have been systematically analyzed and the helix pairs that interact only in EE or EM fashion have been extracted. EE and EM helix pairs have been categorized into five classes (N–N, N–C, C–C, N–MID and C–MID) depending on the region of interaction. Nearly 13% of 5725 helix pairs belonged to one of the five classes. Analysis of single-residue propensities indicated that hydrophobic and polar residues prefer to occur in the C-terminal and N-terminal regions, respectively. Hydrophobic C-terminal interacting residues and polar N-terminal interacting residues are also highly conserved. A strong correlation exists between some of the residue properties (surface area/volume and length of side chains) and their preferences for occurring in the interface of EE and EM helix pairs. In contrast to interacting non-EE/EM helix pairs, helices in EE and EM pairs are farther apart. In these helix pairs, residues with large surface area/volume and longer side chains are preferred in the interfacial region.« less

Effects of side chains in helix nucleation differ from helix propagation

PubMed Central

Miller, Stephen E.; Watkins, Andrew M.; Kallenbach, Neville R.; Arora, Paramjit S.

2014-01-01

Helix–coil transition theory connects observable properties of the α-helix to an ensemble of microstates and provides a foundation for analyzing secondary structure formation in proteins. Classical models account for cooperative helix formation in terms of an energetically demanding nucleation event (described by the σ constant) followed by a more facile propagation reaction, with corresponding s constants that are sequence dependent. Extensive studies of folding and unfolding in model peptides have led to the determination of the propagation constants for amino acids. However, the role of individual side chains in helix nucleation has not been separately accessible, so the σ constant is treated as independent of sequence. We describe here a synthetic model that allows the assessment of the role of individual amino acids in helix nucleation. Studies with this model lead to the surprising conclusion that widely accepted scales of helical propensity are not predictive of helix nucleation. Residues known to be helix stabilizers or breakers in propagation have only a tenuous relationship to residues that favor or disfavor helix nucleation. PMID:24753597

Structural Basis of the Induced-Fit Mechanism of 1,4-Dihydroxy-2-Naphthoyl Coenzyme A Synthase from the Crotonase Fold Superfamily

PubMed Central

Li, Jie; Li, Yan; Jiang, Ming; Zhou, Jiahai; Guo, Zhihong

2013-01-01

1, 4-Dihydroxy-2-naphthoyl coenzyme A (DHNA-CoA) synthase is a typical crotonase fold enzyme with an implicated role of conformational changes in catalysis. We have identified these conformational changes by determining the structures of its Escherichia coli and Synechocystis sp. PCC6803 orthologues in complex with a product analog. The structural changes include the folding of an active-site loop into a β-hairpin and significant reorientation of a helix at the carboxy terminus. Interestingly, a new interface is formed between the ordered loop and the reoriented helix, both of which also form additional interactions with the coenzyme A moiety of the ligand. Site-directed mutation of the amino acid residues involved in these ligand-induced interactions significantly diminishes the enzyme activity. These results suggest a catalytically essential induced-fit that is likely initiated by the enzyme-ligand interactions at the active site. PMID:23658663

Solution structure and DNA-binding properties of the C-terminal domain of UvrC from E.coli

PubMed Central

Singh, S.; Folkers, G.E.; Bonvin, A.M.J.J.; Boelens, R.; Wechselberger, R.; Niztayev, A.; Kaptein, R.

2002-01-01

The C-terminal domain of the UvrC protein (UvrC CTD) is essential for 5′ incision in the prokaryotic nucleotide excision repair process. We have determined the three-dimensional structure of the UvrC CTD using heteronuclear NMR techniques. The structure shows two helix–hairpin–helix (HhH) motifs connected by a small connector helix. The UvrC CTD is shown to mediate structure-specific DNA binding. The domain binds to a single-stranded–double-stranded junction DNA, with a strong specificity towards looped duplex DNA that contains at least six unpaired bases per loop (‘bubble DNA’). Using chemical shift perturbation experiments, the DNA-binding surface is mapped to the first hairpin region encompassing the conserved glycine–valine–glycine residues followed by lysine–arginine–arginine, a positively charged surface patch and the second hairpin region consisting of glycine–isoleucine–serine. A model for the protein– DNA complex is proposed that accounts for this specificity. PMID:12426397

A parallel implementation of the Wuchty algorithm with additional experimental filters to more thoroughly explore RNA conformational space.

PubMed

Stone, Jonathan W; Bleckley, Samuel; Lavelle, Sean; Schroeder, Susan J

2015-01-01

We present new modifications to the Wuchty algorithm in order to better define and explore possible conformations for an RNA sequence. The new features, including parallelization, energy-independent lonely pair constraints, context-dependent chemical probing constraints, helix filters, and optional multibranch loops, provide useful tools for exploring the landscape of RNA folding. Chemical probing alone may not necessarily define a single unique structure. The helix filters and optional multibranch loops are global constraints on RNA structure that are an especially useful tool for generating models of encapsidated viral RNA for which cryoelectron microscopy or crystallography data may be available. The computations generate a combinatorially complete set of structures near a free energy minimum and thus provide data on the density and diversity of structures near the bottom of a folding funnel for an RNA sequence. The conformational landscapes for some RNA sequences may resemble a low, wide basin rather than a steep funnel that converges to a single structure.

Modification and simulation of Rhizomucor miehei lipase: the influence of surficial electrostatic interaction on enantioselectivity.

PubMed

Xu, Gang; Meng, Xiao; Xu, Lin-Jie; Guo, Li; Wu, Jian-Ping; Yang, Li-Rong

2015-04-01

Surface residues have a significant impact on the enantioselectivity of lipases. But the molecular basis of this has never been explained. In this work, transition state complexes of Rhizomucor miehei lipase (RmL) and (R)- or (S)-n-butyl 2-phenxypropinate were studied using molecular dynamics. According to comparison between B-factor of the two simulated complexes, the β 1-β 2 loop and α 2 helix were considered the enantioselectivity-determining domains of RmL. Interaction analysis of these domains suggested an Asp(61)-Arg(86) electrostatic interaction linking the loop and helix strongly impacting enantioselectivity of RmL. Modification of Arg(86) by 1, 2-cyclohexanedione weakening this interaction decreased the E ratio from 6 to 1, modification by 1-iodo-2, 3-butanedione covalently bonding Asp(61) and Arg(86) strengthening the interaction increased the E ratio to 45. Dynamics simulation and energy calculation of the modified lipases also displayed corresponding decreases or increases of enantioselectivity.

Conformational switching of the pseudokinase domain promotes human MLKL tetramerization and cell death by necroptosis.

PubMed

Petrie, Emma J; Sandow, Jarrod J; Jacobsen, Annette V; Smith, Brian J; Griffin, Michael D W; Lucet, Isabelle S; Dai, Weiwen; Young, Samuel N; Tanzer, Maria C; Wardak, Ahmad; Liang, Lung-Yu; Cowan, Angus D; Hildebrand, Joanne M; Kersten, Wilhelmus J A; Lessene, Guillaume; Silke, John; Czabotar, Peter E; Webb, Andrew I; Murphy, James M

2018-06-21

Necroptotic cell death is mediated by the most terminal known effector of the pathway, MLKL. Precisely how phosphorylation of the MLKL pseudokinase domain activation loop by the upstream kinase, RIPK3, induces unmasking of the N-terminal executioner four-helix bundle (4HB) domain of MLKL, higher-order assemblies, and permeabilization of plasma membranes remains poorly understood. Here, we reveal the existence of a basal monomeric MLKL conformer present in human cells prior to exposure to a necroptotic stimulus. Following activation, toggling within the MLKL pseudokinase domain promotes 4HB domain disengagement from the pseudokinase domain αC helix and pseudocatalytic loop, to enable formation of a necroptosis-inducing tetramer. In contrast to mouse MLKL, substitution of RIPK3 substrate sites in the human MLKL pseudokinase domain completely abrogated necroptotic signaling. Therefore, while the pseudokinase domains of mouse and human MLKL function as molecular switches to control MLKL activation, the underlying mechanism differs between species.

Trigger loop folding determines transcription rate of Escherichia coli’s RNA polymerase

DOE PAGES

Mejia, Yara X.; Nudler, Evgeny; Bustamante, Carlos

2014-12-31

Two components of the RNA polymerase (RNAP) catalytic center, the bridge helix and the trigger loop (TL), have been linked with changes in elongation rate and pausing. Here, single molecule experiments with the WT and two TL-tip mutants of the Escherichia coli enzyme reveal that tip mutations modulate RNAP’s pause-free velocity, identifying TL conformational changes as one of two rate-determining steps in elongation. Consistent with this observation, we find a direct correlation between helix propensity of the modified amino acid and pause-free velocity. Moreover, nucleotide analogs affect transcription rate, suggesting that their binding energy also influences TL folding. A kineticmore » model in which elongation occurs in two steps, TL folding on nucleoside triphosphate (NTP) binding followed by NTP incorporation/pyrophosphate release, quantitatively accounts for these results. The TL plays no role in pause recovery remaining unfolded during a pause. The model suggests a finely tuned mechanism that balances transcription speed and fidelity.« less

A genomewide survey of basic helix–loop–helix factors in Drosophila

PubMed Central

Moore, Adrian W.; Barbel, Sandra; Jan, Lily Yeh; Jan, Yuh Nung

2000-01-01

The basic helix–loop–helix (bHLH) transcription factors play important roles in the specification of tissue type during the development of animals. We have used the information contained in the recently published genomic sequence of Drosophila melanogaster to identify 12 additional bHLH proteins. By sequence analysis we have assigned these proteins to families defined by Atonal, Hairy-Enhancer of Split, Hand, p48, Mesp, MYC/USF, and the bHLH-Per, Arnt, Sim (PAS) domain. In addition, one single protein represents a unique family of bHLH proteins. mRNA in situ analysis demonstrates that the genes encoding these proteins are expressed in several tissue types but are particularly concentrated in the developing nervous system and mesoderm. PMID:10973473

The Role of GH/IGF-I Axis in Muscle Homeostasis During Weightlessness

NASA Technical Reports Server (NTRS)

Schwartz, Robert J.

1997-01-01

Exposure to reduced gravity during space travel profoundly alters the loads placed on bone and muscle. Astronauts suffer significant losses of muscle and bone strength during weightlessness. Exercise as a countermeasure is only partially effective in remedying severe muscle atrophy and bone demineralization. Similar wasting of muscles and bones affects people on Earth during prolonged bed rest or immobilization due to injury. In the absence of weight bearing activity, atrophy occurs primarily in the muscles that act in low power, routine movements and in maintaining posture. Hormonal disfunction could contribute in part to the loss of muscle and bone during spaceflight. Reduced levels of human Growth Hormone (hGH) were found in astronauts during space flight, as well as reduced GH secretory activity was observed from the anterior pituitary in 7-day space flight rats. Growth hormone has been shown to be required for maintenance of muscle mass and bone mineralization, in part by mediating the biosynthesis IGF-I, a small polypeptide growth factor. IGF biosynthesis and secretion plays an important role in potentiating muscle cell differentiation and has been shown to drive the expression of myogenin, a myogenic specific basic helix-loop-helix factor. IGF-I has also been shown to have an important role in potentiating muscle regeneration, repair and adult muscle hypertrophy.

Generation and characterization of Atoh1-Cre knock-in mouse line

PubMed Central

Yang, Hua; Xie, Xiaoling; Deng, Min; Chen, Xiaowei; Gan, Lin

2010-01-01

Summary Atoh1 encodes a basic helix-loop-helix (bHLH) transcription factor required for the development of the inner ear sensory epithelia, the dorsal spinal cord, brainstem, cerebellum, and intestinal secretory cells. In this study to create a genetic tool for the research on gene function in the ear sensory organs, we generated an Atoh1-Cre knock-in mouse line by replacing the entire Atoh1 coding sequences with the Cre coding sequences. Atoh1Cre/+mice were viable, fertile, and displayed no visible defects whereas the Atoh1Cre/Cremice died perinatally. The spatiotemporal activities of Cre recombinase were examined by crossing Atoh1-Cre mice with the R26R-lacZ conditional reporter mice. Atoh1-Cre activities were detected in the developing inner ear, the hindbrain, the spinal cord, and the intestine. In the inner ear, Atoh1-Cre activities were confined to the sensory organs in which lacZ expression is detected in nearly all of the hair cells and in many supporting cells. Thus, Atoh1-Cre mouse line serves as a useful tool for the functional study of genes in the inner ear. In addition, our results demonstrate that Atoh1 is expressed in the common progenitors destined for both hair and supporting cells. PMID:20533400

Identification of Specific DNA Binding Residues in the TCP Family of Transcription Factors in Arabidopsis[W

PubMed Central

Aggarwal, Pooja; Das Gupta, Mainak; Joseph, Agnel Praveen; Chatterjee, Nirmalya; Srinivasan, N.; Nath, Utpal

2010-01-01

The TCP transcription factors control multiple developmental traits in diverse plant species. Members of this family share an ∼60-residue-long TCP domain that binds to DNA. The TCP domain is predicted to form a basic helix-loop-helix (bHLH) structure but shares little sequence similarity with canonical bHLH domain. This classifies the TCP domain as a novel class of DNA binding domain specific to the plant kingdom. Little is known about how the TCP domain interacts with its target DNA. We report biochemical characterization and DNA binding properties of a TCP member in Arabidopsis thaliana, TCP4. We have shown that the 58-residue domain of TCP4 is essential and sufficient for binding to DNA and possesses DNA binding parameters comparable to canonical bHLH proteins. Using a yeast-based random mutagenesis screen and site-directed mutants, we identified the residues important for DNA binding and dimer formation. Mutants defective in binding and dimerization failed to rescue the phenotype of an Arabidopsis line lacking the endogenous TCP4 activity. By combining structure prediction, functional characterization of the mutants, and molecular modeling, we suggest a possible DNA binding mechanism for this class of transcription factors. PMID:20363772

Phylogenetic Analysis and Classification of the Fungal bHLH Domain

PubMed Central

Sailsbery, Joshua K.; Atchley, William R.; Dean, Ralph A.

2012-01-01

The basic Helix-Loop-Helix (bHLH) domain is an essential highly conserved DNA-binding domain found in many transcription factors in all eukaryotic organisms. The bHLH domain has been well studied in the Animal and Plant Kingdoms but has yet to be characterized within Fungi. Herein, we obtained and evaluated the phylogenetic relationship of 490 fungal-specific bHLH containing proteins from 55 whole genome projects composed of 49 Ascomycota and 6 Basidiomycota organisms. We identified 12 major groupings within Fungi (F1–F12); identifying conserved motifs and functions specific to each group. Several classification models were built to distinguish the 12 groups and elucidate the most discerning sites in the domain. Performance testing on these models, for correct group classification, resulted in a maximum sensitivity and specificity of 98.5% and 99.8%, respectively. We identified 12 highly discerning sites and incorporated those into a set of rules (simplified model) to classify sequences into the correct group. Conservation of amino acid sites and phylogenetic analyses established that like plant bHLH proteins, fungal bHLH–containing proteins are most closely related to animal Group B. The models used in these analyses were incorporated into a software package, the source code for which is available at www.fungalgenomics.ncsu.edu. PMID:22114358

Ptf1a determines horizontal and amacrine cell fates during mouse retinal development.

PubMed

Fujitani, Yoshio; Fujitani, Shuko; Luo, Huijun; Qiu, Feng; Burlison, Jared; Long, Qiaoming; Kawaguchi, Yoshiya; Edlund, Helena; MacDonald, Raymond J; Furukawa, Takahisa; Fujikado, Takashi; Magnuson, Mark A; Xiang, Mengqing; Wright, Christopher V E

2006-11-01

The vertebrate neural retina comprises six classes of neurons and one class of glial cells, all derived from a population of multipotent progenitors. There is little information on the molecular mechanisms governing the specification of cell type identity from multipotent progenitors in the developing retina. We report that Ptf1a, a basic-helix-loop-helix (bHLH) transcription factor, is transiently expressed by post-mitotic precursors in the developing mouse retina. Recombination-based lineage tracing analysis in vivo revealed that Ptf1a expression marks retinal precursors with competence to exclusively produce horizontal and amacrine neurons. Inactivation of Ptf1a leads to a fate-switch in these precursors that causes them to adopt a ganglion cell fate. This mis-specification of neurons results in a complete loss of horizontal cells, a profound decrease of amacrine cells and an increase in ganglion cells. Furthermore, we identify Ptf1a as a primary downstream target for Foxn4, a forkhead transcription factor involved in the genesis of horizontal and amacrine neurons. These data, together with the previous findings on Foxn4, provide a model in which the Foxn4-Ptf1a pathway plays a central role in directing the differentiation of retinal progenitors towards horizontal and amacrine cell fates.

Stochastic loss and gain of symmetric divisions in the C. elegans epidermis perturbs robustness of stem cell number

PubMed Central

Katsanos, Dimitris; Koneru, Sneha L.; Mestek Boukhibar, Lamia; Gritti, Nicola; Ghose, Ritobrata; Appleford, Peter J.; Doitsidou, Maria; Woollard, Alison; van Zon, Jeroen S.; Poole, Richard J.

2017-01-01

Biological systems are subject to inherent stochasticity. Nevertheless, development is remarkably robust, ensuring the consistency of key phenotypic traits such as correct cell numbers in a certain tissue. It is currently unclear which genes modulate phenotypic variability, what their relationship is to core components of developmental gene networks, and what is the developmental basis of variable phenotypes. Here, we start addressing these questions using the robust number of Caenorhabditis elegans epidermal stem cells, known as seam cells, as a readout. We employ genetics, cell lineage tracing, and single molecule imaging to show that mutations in lin-22, a Hes-related basic helix-loop-helix (bHLH) transcription factor, increase seam cell number variability. We show that the increase in phenotypic variability is due to stochastic conversion of normally symmetric cell divisions to asymmetric and vice versa during development, which affect the terminal seam cell number in opposing directions. We demonstrate that LIN-22 acts within the epidermal gene network to antagonise the Wnt signalling pathway. However, lin-22 mutants exhibit cell-to-cell variability in Wnt pathway activation, which correlates with and may drive phenotypic variability. Our study demonstrates the feasibility to study phenotypic trait variance in tractable model organisms using unbiased mutagenesis screens. PMID:29108019

Two soybean bHLH factors regulate response to iron deficiency.

PubMed

Li, Lin; Gao, Wenwen; Peng, Qi; Zhou, Bin; Kong, Qihui; Ying, Yinghui; Shou, Huixia

2018-03-25

Iron is an indispensable micronutrient for plant growth and development. Limited bioavailability of Fe in the soil leads to iron deficiency chlorosis in plants and yield loss. In this study, two soybean basic helix-loop-helix transcription factors, GmbHLH57 and GmbHLH300, were identified in response to Fe-deficiency. Both transcription factors are expressed in roots and nodules, and are induced by Fe deficiency; these patterns were confirmed in transgenic hairy roots expressing constructs of the endogenous promoters fused to a GUS reporter gene. Bimolecular fluorescence complementation, yeast two-hybrid and coimmunoprecipitation (co-IP) assays indicated a physical interaction between GmbHLH57 and GmbHLH300. Studies on transgenic soybeans overexpressing GmbHLH57 and GmbHLH300 revealed that overexpression of each transcription factor, alone, results in no change of the responses to Fe deficiency, whereas overexpression of both transcription factors upregulated the downstream Fe uptake genes and increased the Fe content in these transgenic plants. Compared to wild type, these double overexpression transgenic plants were more tolerant to Fe deficiency. Taken together, our findings establish that GmbHLH57 and GmbHLH300 are important transcription factors involved in Fe homeostasis in soybean. © 2018 Institute of Botany, Chinese Academy of Sciences.

Origins and Evolution of Stomatal Development1[OPEN

PubMed Central

2017-01-01

The fossil record suggests stomata-like pores were present on the surfaces of land plants over 400 million years ago. Whether stomata arose once or whether they arose independently across newly evolving land plant lineages has long been a matter of debate. In Arabidopsis, a genetic toolbox has been identified that tightly controls stomatal development and patterning. This includes the basic helix-loop-helix (bHLH) transcription factors SPEECHLESS (SPCH), MUTE, FAMA, and ICE/SCREAMs (SCRMs), which promote stomatal formation. These factors are regulated via a signaling cascade, which includes mobile EPIDERMAL PATTERNING FACTOR (EPF) peptides to enforce stomatal spacing. Mosses and hornworts, the most ancient extant lineages to possess stomata, possess orthologs of these Arabidopsis (Arabidopsis thaliana) stomatal toolbox genes, and manipulation in the model bryophyte Physcomitrella patens has shown that the bHLH and EPF components are also required for moss stomatal development and patterning. This supports an ancient and tightly conserved genetic origin of stomata. Here, we review recent discoveries and, by interrogating newly available plant genomes, we advance the story of stomatal development and patterning across land plant evolution. Furthermore, we identify potential orthologs of the key toolbox genes in a hornwort, further supporting a single ancient genetic origin of stomata in the ancestor to all stomatous land plants. PMID:28356502

Twist functions in vertebral column formation in medaka, Oryzias latipes.

PubMed

Yasutake, Junichi; Inohaya, Keiji; Kudo, Akira

2004-07-01

Medaka twist, a basic helix-loop-helix (bHLH) transcription factor, is expressed in the sclerotome during embryogenesis. We previously established a line of twist-EGFP transgenic medaka, whose EGFP expression is regulated by the twist promoter; therefore, we could observe the behavior of sclerotomal cells in vivo. In the transgenic medaka embryos, EGFP-positive sclerotomal cells migrated dorsally around the notochord and the neural tube, where at a later stage the vertebral column would be formed. This finding strongly suggests that twist-expressing sclerotomal cells participate in vertebral column formation in medaka. To clarify the function of twist gene in the sclerotome, we performed knockdown analysis of twist by using two kinds of morpholino antisense oligonucleotides targeted against twist (MO1 and MO2). Both the MO1 and MO2 morphants exhibited absence of neural arches, which are bilaterally paired, dorsomedially oriented bones on the dorsal aspect of the centrum. In addition, MO2, which blocks translation of only endogenous twist mRNA in the twist-EGFP transgenic medaka, did not affect the migration pattern of EGFP-positive cells, revealing that the migration of sclerotome-derived cells were normal in the absence of twist gene function. These results demonstrate that medaka twist functions in vertebral column formation by regulating the sclerotomal cell differentiation.

GTL1 and DF1 regulate root hair growth through transcriptional repression of ROOT HAIR DEFECTIVE 6-LIKE 4 in Arabidopsis.

PubMed

Shibata, Michitaro; Breuer, Christian; Kawamura, Ayako; Clark, Natalie M; Rymen, Bart; Braidwood, Luke; Morohashi, Kengo; Busch, Wolfgang; Benfey, Philip N; Sozzani, Rosangela; Sugimoto, Keiko

2018-02-08

How plants determine the final size of growing cells is an important, yet unresolved, issue. Root hairs provide an excellent model system with which to study this as their final cell size is remarkably constant under constant environmental conditions. Previous studies have demonstrated that a basic helix-loop helix transcription factor ROOT HAIR DEFECTIVE 6-LIKE 4 (RSL4) promotes root hair growth, but how hair growth is terminated is not known. In this study, we demonstrate that a trihelix transcription factor GT-2-LIKE1 (GTL1) and its homolog DF1 repress root hair growth in Arabidopsis Our transcriptional data, combined with genome-wide chromatin-binding data, show that GTL1 and DF1 directly bind the RSL4 promoter and regulate its expression to repress root hair growth. Our data further show that GTL1 and RSL4 regulate each other, as well as a set of common downstream genes, many of which have previously been implicated in root hair growth. This study therefore uncovers a core regulatory module that fine-tunes the extent of root hair growth by the orchestrated actions of opposing transcription factors. © 2018. Published by The Company of Biologists Ltd.

Development of inner ear afferent connections: forming primary neurons and connecting them to the developing sensory epithelia

NASA Technical Reports Server (NTRS)

Fritzsch, Bernd

2003-01-01

The molecular and cellular origin of the primary neurons of the inner ear, the vestibular and spiral neurons, is reviewed including how they connect to the specific sensory epithelia and what the molecular nature of their survival is. Primary neurons of the ear depend on a single basic Helix-Loop-Helix (bHLH) protein for their formation, neurogenin 1 (ngn1). An immediate downstream gene is the bHLH gene neuronal differentiation (NeuroD). Targeted null mutations of ngn1 results in absence of primary neuron formation; targeted null mutation of NeuroD results in loss of almost all spiral and many vestibular neurons. NeuroD and a later expressed gene, Brn3a, play a role in pathfinding to and within sensory epithelia. The molecular nature of this pathfinding property is unknown. Reduction of hair cells in ngn1 null mutations suggests a clonal relationship with primary neurons. This relationship may play some role in specifying the identity of hair cells and the primary neurons that connect with them. Primary neuron neurites growth to sensory epithelia is initially independent of trophic factors released from developing sensory epithelia, but becomes rapidly dependent on those factors. Null mutations of specific neurotrophic factors lose distinct primary neuron populations which undergo rapid embryonic cell death.

Myrosin Idioblast Cell Fate and Development Are Regulated by the Arabidopsis Transcription Factor FAMA, the Auxin Pathway, and Vesicular Trafficking[W

PubMed Central

Li, Meng; Sack, Fred D.

2014-01-01

Crucifer shoots harbor a glucosinolate-myrosinase system that defends against insect predation. Arabidopsis thaliana myrosinase (thioglucoside glucohydrolase [TGG]) accumulates in stomata and in myrosin idioblasts (MIs). This work reports that the basic helix-loop-helix transcription factor FAMA that is key to stomatal development is also expressed in MIs. The loss of FAMA function abolishes MI fate as well as the expression of the myrosinase genes TGG1 and TGG2. MI cells have previously been reported to be located in the phloem. Instead, we found that MIs arise from the ground meristem rather than provascular tissues and thus are not homologous with phloem. Moreover, MI patterning and morphogenesis are abnormal when the function of the ARF-GEF gene GNOM is lost as well as when auxin efflux and vesicular trafficking are chemically disrupted. Stomata and MI cells constitute part of a wider system that reduces plant predation, the so-called “mustard oil bomb,” in which vacuole breakage in cells harboring myrosinase and glucosinolate yields a brew toxic to many animals, especially insects. This identification of the gene that confers the fate of MIs, as well as stomata, might facilitate the development of strategies for engineering crops to mitigate predation. PMID:25304201

C8orf46 homolog encodes a novel protein Vexin that is required for neurogenesis in Xenopus laevis.

PubMed

Moore, Kathryn B; Logan, Mary A; Aldiri, Issam; Roberts, Jacqueline M; Steele, Michael; Vetter, Monica L

2018-05-01

Neural basic helix-loop helix (bHLH) transcription factors promote progenitor cell differentiation by activation of downstream target genes that coordinate neuronal differentiation. Here we characterize a neural bHLH target gene in Xenopus laevis, vexin (vxn; previously sbt1), that is homologous to human c8orf46 and is conserved across vertebrate species. C8orf46 has been implicated in cancer progression, but its function is unknown. Vxn is transiently expressed in differentiating progenitors in the developing central nervous system (CNS), and is required for neurogenesis in the neural plate and retina. Its function is conserved, since overexpression of either Xenopus or mouse vxn expands primary neurogenesis and promotes early retinal cell differentiation in cooperation with neural bHLH factors. Vxn protein is localized to the cell membrane and the nucleus, but functions in the nucleus to promote neural differentiation. Vxn inhibits cell proliferation, and works with the cyclin-dependent kinase inhibitor p27Xic1 (cdkn1b) to enhance neurogenesis and increase levels of the proneural protein Neurog2. We propose that vxn provides a key link between neural bHLH activity and execution of the neurogenic program. Copyright © 2018 Elsevier Inc. All rights reserved.

Amino-terminal domains of c-myc and N-myc proteins mediate binding to the retinoblastoma gene product

NASA Astrophysics Data System (ADS)

Rustgi, Anil K.; Dyson, Nicholas; Bernards, Rene

1991-08-01

THE proteins encoded by the myc gene family are involved in the control of cell proliferation and differentiation, and aberrant expression of myc proteins has been implicated in the genesis of a variety of neoplasms1. In the carboxyl terminus, myc proteins have two domains that encode a basic domain/helix-loop-helix and a leucine zipper motif, respectively. These motifs are involved both in DNA binding and in protein dimerization2-5. In addition, myc protein family members share several regions of highly conserved amino acids in their amino termini that are essential for transformation6,7. We report here that an N-terminal domain present in both the c-myc and N-myc proteins mediates binding to the retinoblastoma gene product, pRb. We show that the human papilloma virus E7 protein competes with c-myc for binding to pRb, indicating that these proteins share overlapping binding sites on pRb. Furthermore, a mutant Rb protein from a human tumour cell line that carried a 35-amino-acid deletion in its C terminus failed to bind to c-myc. Our results suggest that c-myc and pRb cooperate through direct binding to control cell proliferation.

System analysis identifies distinct and common functional networks governed by transcription factor ASCL1, in glioma and small cell lung cancer.

PubMed

Donakonda, Sainitin; Sinha, Swati; Dighe, Shrinivas Nivrutti; Rao, Manchanahalli R Satyanarayana

2017-07-25

ASCL1 is a basic Helix-Loop-Helix transcription factor (TF), which is involved in various cellular processes like neuronal development and signaling pathways. Transcriptome profiling has shown that ASCL1 overexpression plays an important role in the development of glioma and Small Cell Lung Carcinoma (SCLC), but distinct and common molecular mechanisms regulated by ASCL1 in these cancers are unknown. In order to understand how it drives the cellular functional network in these two tumors, we generated a gene expression profile in a glioma cell line (U87MG) to identify ASCL1 gene targets by an si RNA silencing approach and then compared this with a publicly available dataset of similarly silenced SCLC (NCI-H1618 cells). We constructed TF-TF and gene-gene interactions, as well as protein interaction networks of ASCL1 regulated genes in glioma and SCLC cells. Detailed network analysis uncovered various biological processes governed by ASCL1 target genes in these two tumor cell lines. We find that novel ASCL1 functions related to mitosis and signaling pathways influencing development and tumor growth are affected in both glioma and SCLC cells. In addition, we also observed ASCL1 governed functional networks that are distinct to glioma and SCLC.

Id4 functions downstream of Bmp signaling to restrict TCF function in endocardial cells during atrioventricular valve development.

PubMed

Ahuja, Suchit; Dogra, Deepika; Stainier, Didier Y R; Reischauer, Sven

2016-04-01

The atrioventricular canal (AVC) connects the atrial and ventricular chambers of the heart and its formation is critical for the development of the cardiac valves, chamber septation and formation of the cardiac conduction system. Consequently, problems in AVC formation can lead to congenital defects ranging from cardiac arrhythmia to incomplete cardiac septation. While our knowledge about early heart tube formation is relatively comprehensive, much remains to be investigated about the genes that regulate AVC formation. Here we identify a new role for the basic helix-loop-helix factor Id4 in zebrafish AVC valve development and function. id4 is first expressed in the AVC endocardium and later becomes more highly expressed in the atrial chamber. TALEN induced inactivation of id4 causes retrograde blood flow at the AV canal under heat induced stress conditions, indicating defects in AV valve function. At the molecular level, we found that id4 inactivation causes misexpression of several genes important for AVC and AV valve formation including bmp4 and spp1. We further show that id4 appears to control the number of endocardial cells that contribute to the AV valves by regulating Wnt signaling in the developing AVC endocardium. Copyright © 2016 Elsevier Inc. All rights reserved.

The myogenic repressor gene Holes in muscles is a direct transcriptional target of Twist and Tinman in the Drosophila embryonic mesoderm

PubMed Central

Elwell, Jennifer A.; Lovato, TyAnna L.; Adams, Melanie M.; Baca, Erica M.; Lee, Thai; Cripps, Richard M.

2015-01-01

Understanding the regulatory circuitry controlling myogenesis is critical to understanding developmental mechanisms and developmentally-derived diseases. We analyzed the transcriptional regulation of a Drosophila myogenic repressor gene, Holes in muscles (Him). Previously, Him was shown to inhibit Myocyte enhancer factor-2 (MEF2) activity, and is expressed in myoblasts but not differentiating myotubes. We demonstrate that different phases of Him embryonic expression arise through the actions of different enhancers, and we characterize the enhancer required for its early mesoderm expression. This Him early mesoderm enhancer contains two conserved binding sites for the basic helix-loop-helix regulator Twist, and one binding site for the NK homeodomain protein Tinman. The sites for both proteins are required for enhancer activity in early embryos. Twist and Tinman activate the enhancer in tissue culture assays, and ectopic expression of either factor is sufficient to direct ectopic expression of a Him-lacZ reporter, or of the endogenous Him gene. Moreover, sustained expression of twist expression in the mesoderm up-regulates mesodermal Him expression in late embryos. Our findings provide a model to define mechanistically how Twist can both promotes myogenesis through direct activation of Mef2, and can place a brake on myogenesis, through direct activation of Him. PMID:25704510

OsMYC2 mediates numerous defence-related transcriptional changes via jasmonic acid signalling in rice.

PubMed

Ogawa, Satoshi; Kawahara-Miki, Ryouka; Miyamoto, Koji; Yamane, Hisakazu; Nojiri, Hideaki; Tsujii, Yoshimasa; Okada, Kazunori

2017-05-06

Jasmonic acid (JA) plays central roles in various events in plants, especially defence against pathogens and insects. The basic helix-loop-helix (bHLH) transcription factor MYC2 has attracted attention as a master regulator of JA signalling in dicotyledonous plants. However, how MYC2 functions in monocotyledonous plants, including agriculturally important crops such as cultivated rice, has been poorly understood. To elucidate the comprehensive effects of rice MYC2 (OsMYC2) on the JA-inducible transcriptional modifications, we performed RNA-sequencing by using OsMYC2-knockdown plants (osmyc2RNAi). In osmyc2RNAi, JA-inducible expression of many defence-related genes, for example chitinases and proteinase inhibitors, was compromised. Decrease in JA-dependent activation of the biosynthetic pathways of specialised metabolites, especially defence compounds, was also evident in the osmyc2RNAi line. Furthermore, a substantial change was noted in the expression of distinct types of transcription factors, such as MYB-type factors, likely depicting the importance of OsMYC2 in not only defence responses but also other morphogenetic events. Our findings provide fundamental information to understand the overall functions of MYC2 in JA signalling in monocotyledonous plants, which might yield agricultural benefits. Copyright © 2017 Elsevier Inc. All rights reserved.

TRANSPARENT TESTA GLABRA1 and GLABRA1 Compete for Binding to GLABRA3 in Arabidopsis

PubMed Central

Pesch, Martina; Schultheiß, Ilka; Klopffleisch, Karsten; Clemen, Christoph S.; Hülskamp, Martin

2015-01-01

The MBW (for R2R3MYB, basic helix-loop-helix [bHLH], and WD40) genes comprise an evolutionarily conserved gene cassette that regulates several traits such as (pro)anthocyanin and anthocyanin biosynthesis and epidermal cell differentiation in plants. Trichome differentiation in Arabidopsis (Arabidopsis thaliana) is governed by GLABRA1 (GL1; R2R3MYB), GL3 (bHLH), and TRANSPARENT TESTA GLABRA1 (TTG1; WD40). They are thought to form a trimeric complex that acts as a transcriptional activation complex. We provide evidence that these three MBW proteins form either GL1 GL3 or GL3 TTG1 dimers. The formation of each dimer is counteracted by the respective third protein in yeast three-hybrid assays, pulldown experiments (luminescence-based mammalian interactome), and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer studies. We further show that two target promoters, TRIPTYCHON (TRY) and CAPRICE (CPC), are differentially regulated: GL1 represses the activation of the TRY promoter by GL3 and TTG1, and TTG1 suppresses the activation of the CPC promoter by GL1 and GL3. Our data suggest that the transcriptional activation by the MBW complex involves alternative complex formation and that the two dimers can differentially regulate downstream genes. PMID:25926482

Regulation of the Arabidopsis root vascular initial population by LONESOME HIGHWAY

PubMed Central

Ohashi-Ito, Kyoko; Bergmann, Dominique C.

2011-01-01

Complex organisms consist of a multitude of cell types arranged in precise spatial relation to each other. Arabidopsis roots generally exhibit radial tissue organization; however, within a tissue layer, cells are not identical. Specific vascular cell types are arranged in diametrically opposed longitudinal files that maximize the distance between them and create a bilaterally symmetric (diarch) root. Mutations in the LONESOME HIGHWAY (LHW) gene eliminate bilateral symmetry and reduce the number of cells in the center of the root, resulting in roots with only single and xylem and phloem poles. LHW does not appear to be required for the creation of any specific cell type, but coordinately controls the number of all vascular cell types by regulating the size of the pool of cells from which they arise. We cloned LHW and found that it encodes a protein with weak sequence similarity to basic helix-loop-helix (bHLH) domain proteins. LHW is a transcriptional activator in vitro. In plants, LHW is nuclear localized and is expressed in the root meristems where we hypothesize it acts independently of other known root patterning genes to promote the production of stele cells, but may also indirectly feed into established regulatory networks for the maintenance of the root meristem. PMID:17626058

Effect of the g.-723G-->T polymorphism in the bovine myogenic factor 5 (Myf5) gene promoter region on gene transcript level in the longissimus dorsi muscle and on meat traits of Polish Holstein-Friesian cattle.

PubMed

Robakowska-Hyzorek, Dagmara; Oprzadek, Jolanta; Zelazowska, Beata; Olbromski, Rafał; Zwierzchowski, Lech

2010-06-01

Myogenic factor 5 (Myf5), a product of the Myf5 gene, belongs to the MRF family of basic helix-loop-helix transcription factors that regulate myogenesis. Their roles in muscle growth and development make their genes candidates for molecular markers of meat production in livestock, but nucleotide sequence polymorphism has not been thoroughly studied in MRF genes. We detected four single nucleotide polymorphisms (SNPs) within exon 1 of the Myf5 gene, encoding the NH-terminal transactivation domain of the Myf5 protein. Three of these mutations change the amino acid sequence. The distribution of these SNPs was highly skewed in cattle populations; most of the mutations were found in only a few or even single individuals. Of the nine SNPs found in the promoter region of Myf5, one (transversion g.-723G-->T) was represented by all three genotypes distributed in the cattle populations studied. This polymorphism showed an influence on Myf5 gene expression in the longissimus dorsi muscle and was associated with sirloin weight and fat weight in sirloin in carcasses of Holstein-Friesian cattle.

A Mutation in the bHLH Domain of the SPCH Transcription Factor Uncovers a BR-Dependent Mechanism for Stomatal Development.

PubMed

de Marcos, Alberto; Houbaert, Anaxi; Triviño, Magdalena; Delgado, Dolores; Martín-Trillo, Mar; Russinova, Eugenia; Fenoll, Carmen; Mena, Montaña

2017-06-01

The asymmetric cell divisions necessary for stomatal lineage initiation and progression in Arabidopsis ( Arabidopsis thaliana ) require the function of the basic helix-loop-helix (bHLH) transcription factor SPEECHLESS ( SPCH ). Mutants lacking SPCH do not produce stomata or lineages. Here, we isolated a new spch-5 allele carrying a point mutation in the bHLH domain that displayed normal growth, but had an extremely low number of sometimes clustered stomata in the leaves, whereas the hypocotyls did not have any stomata. In vivo tracking of leaf epidermal cell divisions, combined with marker lines and genetic analysis, showed that the spch-5 leaf phenotype is dosage dependent and results from the decreased ability to initiate and amplify lineages, defects in asymmetric cell fate allocation, and misorientation of asymmetric division planes. Notably, application of brassinosteroids (BRs) partly rescued the stomatal leaf phenotype of spch-5 Transcriptomic analysis combining spch-5 with BR treatments revealed that the expression of a set of SPCH target genes was restored by BRs. Our results also show that BR-dependent stomata formation and expression of some, but not all, SPCH target genes require the integrity of the bHLH domain of SPCH. © 2017 American Society of Plant Biologists. All Rights Reserved.

A Mutation in the bHLH Domain of the SPCH Transcription Factor Uncovers a BR-Dependent Mechanism for Stomatal Development1

PubMed Central

Triviño, Magdalena; Delgado, Dolores; Martín-Trillo, Mar

2017-01-01

The asymmetric cell divisions necessary for stomatal lineage initiation and progression in Arabidopsis (Arabidopsis thaliana) require the function of the basic helix-loop-helix (bHLH) transcription factor SPEECHLESS (SPCH). Mutants lacking SPCH do not produce stomata or lineages. Here, we isolated a new spch-5 allele carrying a point mutation in the bHLH domain that displayed normal growth, but had an extremely low number of sometimes clustered stomata in the leaves, whereas the hypocotyls did not have any stomata. In vivo tracking of leaf epidermal cell divisions, combined with marker lines and genetic analysis, showed that the spch-5 leaf phenotype is dosage dependent and results from the decreased ability to initiate and amplify lineages, defects in asymmetric cell fate allocation, and misorientation of asymmetric division planes. Notably, application of brassinosteroids (BRs) partly rescued the stomatal leaf phenotype of spch-5. Transcriptomic analysis combining spch-5 with BR treatments revealed that the expression of a set of SPCH target genes was restored by BRs. Our results also show that BR-dependent stomata formation and expression of some, but not all, SPCH target genes require the integrity of the bHLH domain of SPCH. PMID:28507175

A molecular framework of light-controlled phytohormone action in Arabidopsis.

PubMed

Zhong, Shangwei; Shi, Hui; Xue, Chang; Wang, Lei; Xi, Yanpeng; Li, Jigang; Quail, Peter H; Deng, Xing Wang; Guo, Hongwei

2012-08-21

Environmental changes strongly affect plant growth and development. Phytohormones, endogenous plant-made small molecules such as ethylene, regulate a wide range of processes throughout the lifetime of plants. The ability of plants to integrate external signals with endogenous regulatory pathways is vital for their survival. Ethylene has been found to suppress hypocotyl elongation in darkness while promoting it in light. How ethylene regulates hypocotyl elongation in such opposite ways is largely unknown. In particular, how light modulates and even reverses the function of ethylene has yet to be characterized. Here we show that the basic-helix-loop-helix transcription factor phytochrome-interacting factor 3 (PIF3) is directly activated by ETHYLENE-INSENSITIVE 3 (EIN3) and is indispensible for ethylene-induced hypocotyl elongation in light. Ethylene via EIN3 concomitantly activates two contrasting pathways: the PIF3-dependent growth-promoting pathway and an ethylene response factor 1 (ERF1)-mediated growth-inhibiting pathway. In the light, growth-promoting PIFs are limiting due to light-dependent destabilization, and thus ethylene stimulates growth under these conditions. In contrast, ERF1 is destabilized, and thus limiting, under dark conditions, explaining why ethylene inhibits growth in the dark. Our findings provide a mechanistic insight into how light modulates internal hormone-regulated plant growth. Copyright © 2012 Elsevier Ltd. All rights reserved.

Ascl1-induced neuronal differentiation of P19 cells requires expression of a specific inhibitor protein of cAMP-dependent protein kinase

PubMed Central

Huang, Holly S.; Turner, David L.; Thompson, Robert C.; Uhler, Michael D.

2011-01-01

cAMP-dependent protein kinase (PKA) plays a critical role in nervous system development by modulating sonic hedgehog and bone morphogenetic protein signaling. In the current studies, P19 embryonic carcinoma cells were neuronally differentiated by expression of the proneural basic helix-loop-helix transcription factor Ascl1. After expression of Ascl1, but prior to expression of neuronal markers such as microtubule associated protein 2 and neuronal β-tubulin, P19 cells demonstrated a large, transient increase in both mRNA and protein for the endogenous protein kinase inhibitor (PKI)β. PKIβ-targeted shRNA constructs both reduced the levels of PKIβ expression and blocked the neuronal differentiation of P19 cells. This inhibition of differentiation was rescued by transfection of a shRNA-resistant expression vector for the PKIβ protein, and this rescue required the PKA-specific inhibitory sequence of the PKIβprotein. PKIβ played a very specific role in the Ascl1-mediated differentiation process since other PKI isoforms were unable to rescue the deficit conferred by shRNA-mediated knockdown of PKIβ. Our results define a novel requirement for PKIβ and its inhibition of PKA during neuronal differentiation of P19 cells. PMID:21623794

Generation of induced neurons by direct reprogramming in the mammalian cochlea.

PubMed

Nishimura, K; Weichert, R M; Liu, W; Davis, R L; Dabdoub, A

2014-09-05

Primary auditory neurons (ANs) in the mammalian cochlea play a critical role in hearing as they transmit auditory information in the form of electrical signals from mechanosensory cochlear hair cells in the inner ear to the brainstem. Their progressive degeneration is associated with disease conditions, excessive noise exposure and aging. Replacement of ANs, which lack the ability to regenerate spontaneously, would have a significant impact on research and advancement in cochlear implants in addition to the amelioration of hearing impairment. The aim of this study was to induce a neuronal phenotype in endogenous non-neural cells in the cochlea, which is the essential organ of hearing. Overexpression of a neurogenic basic helix-loop-helix transcription factor, Ascl1, in the cochlear non-sensory epithelial cells induced neurons at high efficiency at embryonic, postnatal and juvenile stages. Moreover, induced neurons showed typical properties of neuron morphology, gene expression and electrophysiology. Our data indicate that Ascl1 alone or Ascl1 and NeuroD1 is sufficient to reprogram cochlear non-sensory epithelial cells into functional neurons. Generation of neurons from non-neural cells in the cochlea is an important step for the regeneration of ANs in the mature mammalian cochlea. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Myogenin induces higher oxidative capacity in pre-existing mouse muscle fibres after somatic DNA transfer

PubMed Central

Ekmark, Merete; Grønevik, Eirik; Schjerling, Peter; Gundersen, Kristian

2003-01-01

Muscle is a permanent tissue, and in the adult pronounced changes can occur in pre-existing fibres without the formation of new fibres. Thus, the mechanisms responsible for phenotype transformation in the adult might be distinct from mechanisms regulating muscle differentiation during muscle formation and growth. Myogenin is a muscle-specific, basic helix-loop-helix transcription factor that is important during early muscle differentiation. It is also expressed in the adult, where its role is unknown. In this study we have overexpressed myogenin in glycolytic fibres of normal adult mice by electroporation and single-cell intracellular injection of expression vectors. Myogenin had no effects on myosin heavy chain fibre type, but induced a considerable increase in succinate dehydrogenase and NADH dehydrogenase activity, with some type IIb fibres reaching the levels observed histochemically in normal type IIx and IIa fibres. mRNA levels for malate dehydrogenase were similarly altered. The size of the fibres overexpressing myogenin was reduced by 30–50 %. Thus, the transfected fibres acquired a phenotype reminiscent of the phenotype obtained by endurance training in man and other animals, with a higher oxidative capacity and smaller size. We conclude that myogenin can alter pre-existing glycolytic fibres in the intact adult animal. PMID:12598590

The Transcription Factor Atonal homolog 8 Regulates Gata4 and Friend of Gata-2 during Vertebrate Development

PubMed Central

Rawnsley, David R.; Xiao, Jiping; Lee, John S.; Liu, Xi; Mericko-Ishizuka, Patricia; Kumar, Vinayak; He, Jie; Basu, Arindam; Lu, MinMin; Lynn, Francis C.; Pack, Michael; Gasa, Rosa; Kahn, Mark L.

2013-01-01

GATA and Friend of GATA (FOG) form a transcriptional complex that plays a key role in cardiovascular development in both fish and mammals. In the present study we demonstrate that the basic helix-loop-helix transcription factor Atonal homolog 8 (Atoh8) is required for development of the heart in fish but not in mice. Genetic studies reveal that Atoh8 interacts specifically with Gata4 and Fog1 during development of the heart and swim bladder in the fish. Biochemical studies reveal that ATOH8, GATA4, and FOG2 associate in a single complex in vitro. In contrast to fish, ATOH8-deficient mice exhibit normal cardiac development and loss of ATOH8 does not alter cardiac development in Gata4+/− mice. This species difference in the role of ATOH8 is explained in part by LacZ and GFP reporter alleles that reveal restriction of Atoh8 expression to atrial but not ventricular myocardium in the mouse. Our findings identify ATOH8 as a novel regulator of GATA-FOG function that is required for cardiac development in the fish but not the mouse. Whether ATOH8 modulates GATA-FOG function at other sites or in more subtle ways in mammals is not yet known. PMID:23836893

FAMA Is an Essential Component for the Differentiation of Two Distinct Cell Types, Myrosin Cells and Guard Cells, in Arabidopsis[W

PubMed Central

Shirakawa, Makoto; Ueda, Haruko; Nagano, Atsushi J.; Shimada, Tomoo; Kohchi, Takayuki; Hara-Nishimura, Ikuko

2014-01-01

Brassicales plants, including Arabidopsis thaliana, have an ingenious two-compartment defense system, which sequesters myrosinase from the substrate glucosinolate and produces a toxic compound when cells are damaged by herbivores. Myrosinase is stored in vacuoles of idioblast myrosin cells. The molecular mechanism that regulates myrosin cell development remains elusive. Here, we identify the basic helix-loop-helix transcription factor FAMA as an essential component for myrosin cell development along Arabidopsis leaf veins. FAMA is known as a regulator of stomatal development. We detected FAMA expression in myrosin cell precursors in leaf primordia in addition to stomatal lineage cells. FAMA deficiency caused defects in myrosin cell development and in the biosynthesis of myrosinases THIOGLUCOSIDE GLUCOHYDROLASE1 (TGG1) and TGG2. Conversely, ectopic FAMA expression conferred myrosin cell characteristics to hypocotyl and root cells, both of which normally lack myrosin cells. The FAMA interactors ICE1/SCREAM and its closest paralog SCREAM2/ICE2 were essential for myrosin cell development. DNA microarray analysis identified 32 candidate genes involved in myrosin cell development under the control of FAMA. This study provides a common regulatory pathway that determines two distinct cell types in leaves: epidermal guard cells and inner-tissue myrosin cells. PMID:25304202

Functionally Similar WRKY Proteins Regulate Vacuolar Acidification in Petunia and Hair Development in Arabidopsis.

PubMed

Verweij, Walter; Spelt, Cornelis E; Bliek, Mattijs; de Vries, Michel; Wit, Niek; Faraco, Marianna; Koes, Ronald; Quattrocchio, Francesca M

2016-03-01

The WD40 proteins ANTHOCYANIN11 (AN11) from petunia (Petunia hybrida) and TRANSPARENT TESTA GLABRA1 (TTG1) from Arabidopsis thaliana and associated basic helix-loop-helix (bHLH) and MYB transcription factors activate a variety of differentiation processes. In petunia petals, AN11 and the bHLH protein AN1 activate, together with the MYB protein AN2, anthocyanin biosynthesis and, together with the MYB protein PH4, distinct genes, such as PH1 and PH5, that acidify the vacuole. To understand how AN1 and AN11 activate anthocyanin biosynthetic and PH genes independently, we isolated PH3. We found that PH3 is a target gene of the AN11-AN1-PH4 complex and encodes a WRKY protein that can bind to AN11 and is required, in a feed-forward loop, together with AN11-AN1-PH4 for transcription of PH5. PH3 is highly similar to TTG2, which regulates hair development, tannin accumulation, and mucilage production in Arabidopsis. Like PH3, TTG2 can bind to petunia AN11 and the Arabidopsis homolog TTG1, complement ph3 in petunia, and reactivate the PH3 target gene PH5. Our findings show that the specificity of WD40-bHLH-MYB complexes is in part determined by interacting proteins, such as PH3 and TTG2, and reveal an unanticipated similarity in the regulatory circuitry that controls petunia vacuolar acidification and Arabidopsis hair development. © 2016 American Society of Plant Biologists. All rights reserved.

A direct repeat of E-box-like elements is required for cell-autonomous circadian rhythm of clock genes

PubMed Central

Nakahata, Yasukazu; Yoshida, Mayumi; Takano, Atsuko; Soma, Haruhiko; Yamamoto, Takuro; Yasuda, Akio; Nakatsu, Toru; Takumi, Toru

2008-01-01

Background The circadian expression of the mammalian clock genes is based on transcriptional feedback loops. Two basic helix-loop-helix (bHLH) PAS (for Period-Arnt-Sim) domain-containing transcriptional activators, CLOCK and BMAL1, are known to regulate gene expression by interacting with a promoter element termed the E-box (CACGTG). The non-canonical E-boxes or E-box-like sequences have also been reported to be necessary for circadian oscillation. Results We report a new cis-element required for cell-autonomous circadian transcription of clock genes. This new element consists of a canonical E-box or a non-canonical E-box and an E-box-like sequence in tandem with the latter with a short interval, 6 base pairs, between them. We demonstrate that both E-box or E-box-like sequences are needed to generate cell-autonomous oscillation. We also verify that the spacing nucleotides with constant length between these 2 E-elements are crucial for robust oscillation. Furthermore, by in silico analysis we conclude that several clock and clock-controlled genes possess a direct repeat of the E-box-like elements in their promoter region. Conclusion We propose a novel possible mechanism regulated by double E-box-like elements, not to a single E-box, for circadian transcriptional oscillation. The direct repeat of the E-box-like elements identified in this study is the minimal required element for the generation of cell-autonomous transcriptional oscillation of clock and clock-controlled genes. PMID:18177499

NMR structure and conformational dynamics of AtPDFL2.1, a defensin-like peptide from Arabidopsis thaliana.

PubMed

Omidvar, Reza; Xia, Youlin; Porcelli, Fernando; Bohlmann, Holger; Veglia, Gianluigi

2016-12-01

Plant defensins constitute the innate immune response against pathogens such as fungi and bacteria. Typical plant defensins are small, basic peptides that possess a characteristic three-dimensional fold stabilized by three or four disulfide bridges. In addition to known defensin genes, the Arabidopsis genome comprises >300 defensin-like genes coding for small cysteine-rich peptides. One of such genes encodes for AtPDFL2.1, a putative antifungal peptide of 55 amino acids, with six cysteine residues in its primary sequence. To understand the functional role of AtPDFL2.1, we carried out antifungal activity assays and determined its high-resolution three-dimensional structure using multidimensional solution NMR spectroscopy. We found that AtPDFL2.1 displays a strong inhibitory effect against Fusarium graminearum (IC 50 ≈4μM). This peptide folds in the canonical cysteine-stabilized αβ (CSαβ) motif, consisting of one α-helix and one triple-stranded antiparallel β-sheet stabilized by three disulfide bridges and a hydrophobic cluster of residues within its core where the α-helix packs tightly against the β-sheets. Nuclear spin relaxation measurements show that the structure of AtPDFL2.1 is essentially rigid, with the L3 loop located between β-strands 2 and 3 being more flexible and displaying conformational exchange. Interestingly, the dynamic features of loop L3 are conserved among defensins and are probably correlated to the antifungal and receptor binding activities. Copyright © 2016 Elsevier B.V. All rights reserved.

Theory of twisted trunks

NASA Astrophysics Data System (ADS)

Carlqvist, P.; Gahm, G. F.; Kristen, H.

2003-05-01

Using the 2.6 m Nordic Optical Telescope we have observed a large number of elephant trunks in several H II regions. Here, we present a small selection of this material consisting of a few large, well-developed trunks, and some smaller ones. We find that: (i) the well-developed trunks are made up of dark filaments and knots which show evidence of twisted structures, (ii) the trunks are connected with essentially two filamentary legs running in V-shape, and (iii) all trunks have the maximum extinction in their heads. We advance a theory of twisted elephant trunks which is based on the presence of magnetic flux ropes in molecular clouds where hot OB stars are formed. If the rope contains a local condensation it may adopt a V-shape as the H II region around the hot stars expands. If, in addition, the magnetic field in the rope is sufficiently twisted, the rope may form a double helix at the apex of the V. The double helix is identified with the twisted elephant trunks. In order to illustrate the mechanisms behind the double helix we have constructed a mechanical analogy model of the magnetic flux rope in which the rope has been replaced by a bundle of elastic strings loaded by a weight. Experiments with the model clearly show that part of the bundle will transform into a double helix when the twist of the bundle is sufficiently large. We have also worked out a simple theoretical model of a mass-loaded magnetic flux rope. Numerical calculations show that a double helix will indeed form when the twist of the rope exceeds a certain critical limit. Numerical model calculations are applied to both the analogy model experiments and one of the well-developed elephant trunks. On the basis of our model we also suggest a new interpretation of the so called EGGs. The double helix mechanism is quite general, and should be active also in other suitable environments. One such environment may be the shell of supernova remnants. Another example is the expanding bubble outlined by the North Celestial Pole Loop. Based on observations collected at the Nordic Optical Telescope, La Palma, Spain.

Helix coupling

DOEpatents

Ginell, W.S.

1982-03-17

A coupling for connecting helix members in series, which consists of a pair of U-shaped elements, one of which is attached to each helix end with the U sections of the elements interlocked. The coupling is particularly beneficial for interconnecting helical Nitinol elements utilized in thermal actuators or engines. Each coupling half is attached to the associated helix at two points, thereby providing axial load while being easily removed from the helix, and reusable.

Helix coupling

DOEpatents

Ginell, William S.

1989-04-25

A coupling for connecting helix members in series, which consists of a pair of U-shaped elements, one of which is attached to each helix end with the "U" sections of the elements interlocked. The coupling is particularly beneficial for interconnecting helical Nitinol elements utilized in thermal actuators or engines. Each coupling half is attached to the associated helix at two points, thereby providing axial load while being easily removed from the helix, and reusable.

Iterative key-residues interrogation of a phytase with thermostability increasing substitutions identified in directed evolution.

PubMed

Shivange, Amol V; Roccatano, Danilo; Schwaneberg, Ulrich

2016-01-01

Bacterial phytases have attracted industrial interest as animal feed supplement due to their high activity and sufficient thermostability (required for feed pelleting). We devised an approach named KeySIDE,  an iterative Key-residues interrogation of the wild type with Substitutions Identified in Directed Evolution for improving Yersinia mollaretii phytase (Ymphytase) thermostability by combining key beneficial substitutions and elucidating their individual roles. Directed evolution yielded in a discovery of nine positions in Ymphytase and combined iteratively to identify key positions. The "best" combination (M6: T77K, Q154H, G187S, and K289Q) resulted in significantly improved thermal resistance; the residual activity improved from 35 % (wild type) to 89 % (M6) at 58 °C and 20-min incubation. Melting temperature increased by 3 °C in M6 without a loss of specific activity. Molecular dynamics simulation studies revealed reduced flexibility in the loops located next to helices (B, F, and K) which possess substitutions (Helix-B: T77K, Helix-F: G187S, and Helix-K: K289E/Q). Reduced flexibility in the loops might be caused by strengthened hydrogen bonding network (e.g., G187S and K289E/K289Q) and a salt bridge (T77K). Our results demonstrate a promising approach to design phytases in food research, and we hope that the KeySIDE might become an attractive approach for understanding of structure-function relationships of enzymes.

Heterologous expression and solution structure of defensin from lentil Lens culinaris

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

Shenkarev, Zakhar O.; Gizatullina, Albina K.; Moscow Institute of Physics and Technology

2014-08-22

Highlights: • Lentil defensin Lc-def and its {sup 15}N-labeled analog were overexpressed in E. coli. • Lc-def is active against fungi, but does not inhibit growth of G+ and G− bacteria. • Lc-def spatial structure involves triple-stranded β-sheet and α-helix (CSαβ motif). • Lc-def is able to bind to anionic lipid vesicles under low-salt conditions. • NMR data revealed significant μs–ms mobility in the loops 1 and 3 of Lc-def. - Abstract: A new defensin Lc-def, isolated from germinated seeds of the lentil Lens culinaris, has molecular mass 5440.4 Da and consists of 47 amino acid residues. Lc-def and itsmore » {sup 15}N-labeled analog were overexpressed in Escherichia coli. Antimicrobial activity of the recombinant protein was examined, and its spatial structure, dynamics, and interaction with lipid vesicles were studied by NMR spectroscopy. It was shown that Lc-def is active against fungi, but does not inhibit the growth of Gram-positive and Gram-negative bacteria. The peptide is monomeric in aqueous solution and contains one α-helix and triple-stranded β-sheet, which form cysteine-stabilized αβ motif (CSαβ) previously found in other plant defensins. The sterically neighboring loop1 and loop3 protrude from the defensin core and demonstrate significant mobility on the μs–ms timescale. Lc-def does not bind to the zwitterionic lipid (POPC) vesicles but interacts with the partially anionic (POPC/DOPG, 7:3) membranes under low-salt conditions. The Lc-def antifungal activity might be mediated through electrostatic interaction with anionic lipid components of fungal membranes.« less

Expanded turn conformations: characterization and sequence-structure correspondence in alpha-turns with implications in helix folding.

PubMed

Dasgupta, Bhaskar; Pal, Lipika; Basu, Gautam; Chakrabarti, Pinak

2004-05-01

Like the beta-turns, which are characterized by a limiting distance between residues two positions apart (i, i+3), a distance criterion (involving residues at positions i and i+4) is used here to identify alpha-turns from a database of known protein structures. At least 15 classes of alpha-turns have been enumerated based on the location in the phi,psi space of the three central residues (i+1 to i+3)-one of the major being the class AAA, where the residues occupy the conventional helical backbone torsion angles. However, moving towards the C-terminal end of the turn, there is a shift in the phi,psi angles towards more negative phi, such that the electrostatic repulsion between two consecutive carbonyl oxygen atoms is reduced. Except for the last position (i+4), there is not much similarity in residue composition at different positions of hydrogen and non-hydrogen bonded AAA turns. The presence or absence of Pro at i+1 position of alpha- and beta-turns has a bearing on whether the turn is hydrogen-bonded or without a hydrogen bond. In the tertiary structure, alpha-turns are more likely to be found in beta-hairpin loops. The residue composition at the beginning of the hydrogen bonded AAA alpha-turn has similarity with type I beta-turn and N-terminal positions of helices, but the last position matches with the C-terminal capping position of helices, suggesting that the existence of a "helix cap signal" at i+4 position prevents alpha-turns from growing into helices. Our results also provide new insights into alpha-helix nucleation and folding. Copyright 2004 Wiley-Liss, Inc.

Effects of helix and fingertip mutations on the thermostability of xyn11A investigated by molecular dynamics simulations and enzyme activity assays.

PubMed

Sutthibutpong, Thana; Rattanarojpong, Triwit; Khunrae, Pongsak

2017-12-04

Local conformational changes and global unfolding pathways of wildtype xyn11A recombinant and its mutated structures were studied through a series of atomistic molecular dynamics (MD) simulations, along with enzyme activity assays at three incubation temperatures to investigate the effects of mutations at three different sites to the thermostability. The first mutation was to replace an unstable negatively charged residue at a surface beta turn near the active site (D32G) by a hydrophobic residue. The second mutation was to create a disulphide bond (S100C/N147C) establishing a strong connection between an alpha helix and a distal beta hairpin associated with the thermally sensitive Thumb loop, and the third mutation add an extra hydrogen bond (A155S) to the same alpha helix. From the MD simulations performed, MM/PBSA energy calculations of the unfolding energy were in a good agreement with the enzyme activities measured from the experiment, as all mutated structures demonstrated the improved thermostability, especially the S100C/N147C proved to be the most stable mutant both by the simulations and the experiment. Local conformational analysis at the catalytic sites and the xylan access region also suggested that mutated xyn11A structures could accommodate xylan binding. However, the analysis of global unfolding pathways showed that structural disruptions at the beta sheet regions near the N-terminal were still imminent. These findings could provide the insight on the molecular mechanisms underlying the enhanced thermostability due to mutagenesis and changes in the protein unfolding pathways for further protein engineering of the GH11 family xylanase enzymes.

Interactions between the mixotrophic dinoflagellate Takayama helix and common heterotrophic protists.

PubMed

Ok, Jin Hee; Jeong, Hae Jin; Lim, An Suk; Lee, Kyung Ha

2017-09-01

The phototrophic dinoflagellate Takayama helix that is known to be harmful to abalone larvae has recently been revealed to be mixotrophic. Although mixotrophy elevates the growth rate of T. helix by 79%-185%, its absolute growth rate is still as low as 0.3d -1 . Thus, if the mortality rate of T. helix due to predation is high, this dinoflagellate may not easily prevail. To investigate potential effective protistan grazers on T. helix, feeding by diverse heterotrophic dinoflagellates such as engulfment-feeding Oxyrrhis marina, Gyrodinium dominans, Gyrodinium moestrupii, Polykrikos kofoidii, and Noctiluca scintillans, peduncle-feeding Aduncodinium glandula, Gyrodiniellum shiwhaense, Luciella masanensis, and Pfiesteria piscicida, pallium-feeding Oblea rotunda and Protoperidinium pellucidum, and the naked ciliates Pelagostrobilidium sp. (ca. 40μm in cell length) and Strombidinopsis sp. (ca. 150μm in cell length) on T. helix was explored. Among the tested heterotrophic protists, O. marina, G. dominans, G. moestrupii, A. glandula, L. masanensis, P. kofoidii, P. piscicida, and Strombidinopsis sp. were able to feed on T. helix. The growth rates of all these predators except Strombidinopsis sp. with T. helix prey were lower than those without the prey. The growth rate of Strombidinopsis sp. on T. helix was almost zero although the growth rate of Strombidinopsis sp. with T. helix prey was higher than those without the prey. Moreover, T. helix fed on O. marina and P. pellucidum and lysed the cells of P. kofoidii and G. shiwhaense. With increasing the concentrations of T. helix, the growth rates of O. marina and P. kofoidii decreased, but those of G. dominans and L. masanensis largely did not change. Therefore, reciprocal predation, lysis, no feeding, and the low ingestion rates of the common protists preying on T. helix may result in a low mortality rate due to predation, thereby compensating for this species' low growth rate. Copyright © 2017 Elsevier B.V. All rights reserved.

Impact of ion binding on poly-L-lysine (un)folding energy landscape and kinetics.

PubMed

Xiong, Kan; Asher, Sanford A

2012-06-21

We utilize T-jump UV resonance Raman spectroscopy (UVRR) to study the impact of ion binding on the equilibrium energy landscape and on (un)folding kinetics of poly-L-lysine (PLL). We observe that the relaxation rates of the folded conformations (including π-helix (bulge), pure α-helix, and turns) of PLL are slower than those of short alanine-based peptides. The PLL pure α-helix folding time is similar to that of short alanine-based peptides. We for the first time have directly observed that turn conformations are α-helix and π-helix (bulge) unfolding intermediates. ClO(4)(-) binding to the Lys side chain -NH(3)(+) groups and the peptide backbone slows the α-helix unfolding rate compared to that in pure water, but little impacts the folding rate, resulting in an increased α-helix stability. ClO(4)(-) binding significantly increases the PLL unfolding activation barrier but little impacts the folding barrier. Thus, the PLL folding coordinate(s) differs from the unfolding coordinate(s). The-π helix (bulge) unfolding and folding coordinates do not directly go through the α-helix energy well. Our results clearly demonstrate that PLL (un)folding is not a two-state process.

Mechanical unfolding reveals stable 3-helix intermediates in talin and α-catenin

PubMed Central

2018-01-01

Mechanical stability is a key feature in the regulation of structural scaffolding proteins and their functions. Despite the abundance of α-helical structures among the human proteome and their undisputed importance in health and disease, the fundamental principles of their behavior under mechanical load are poorly understood. Talin and α-catenin are two key molecules in focal adhesions and adherens junctions, respectively. In this study, we used a combination of atomistic steered molecular dynamics (SMD) simulations, polyprotein engineering, and single-molecule atomic force microscopy (smAFM) to investigate unfolding of these proteins. SMD simulations revealed that talin rod α-helix bundles as well as α-catenin α-helix domains unfold through stable 3-helix intermediates. While the 5-helix bundles were found to be mechanically stable, a second stable conformation corresponding to the 3-helix state was revealed. Mechanically weaker 4-helix bundles easily unfolded into a stable 3-helix conformation. The results of smAFM experiments were in agreement with the findings of the computational simulations. The disulfide clamp mutants, designed to protect the stable state, support the 3-helix intermediate model in both experimental and computational setups. As a result, multiple discrete unfolding intermediate states in the talin and α-catenin unfolding pathway were discovered. Better understanding of the mechanical unfolding mechanism of α-helix proteins is a key step towards comprehensive models describing the mechanoregulation of proteins. PMID:29698481

Functional studies of the Ciona intestinalis myogenic regulatory factor reveal conserved features of chordate myogenesis.

PubMed

Izzi, Stephanie A; Colantuono, Bonnie J; Sullivan, Kelly; Khare, Parul; Meedel, Thomas H

2013-04-15

Ci-MRF is the sole myogenic regulatory factor (MRF) of the ascidian Ciona intestinalis, an invertebrate chordate. In order to investigate its properties we developed a simple in vivo assay based on misexpressing Ci-MRF in the notochord of Ciona embryos. We used this assay to examine the roles of three structural motifs that are conserved among MRFs: an alanine-threonine (Ala-Thr) dipeptide of the basic domain that is known in vertebrates as the myogenic code, a cysteine/histidine-rich (C/H) domain found just N-terminal to the basic domain, and a carboxy-terminal amphipathic α-helix referred to as Helix III. We show that the Ala-Thr dipeptide is necessary for normal Ci-MRF function, and that while eliminating the C/H domain or Helix III individually has no demonstrable effect on Ci-MRF, simultaneous loss of both motifs significantly reduces its activity. Our studies also indicate that direct interaction between CiMRF and an essential E-box of Ciona Troponin I is required for the expression of this muscle-specific gene and that multiple classes of MRF-regulated genes exist in Ciona. These findings are consistent with substantial conservation of MRF-directed myogenesis in chordates and demonstrate for the first time that the Ala/Thr dipeptide of the basic domain of an invertebrate MRF behaves as a myogenic code. Copyright © 2013 Elsevier Inc. All rights reserved.

Estimating loop length from CryoEM images at medium resolutions.

PubMed

McKnight, Andrew; Si, Dong; Al Nasr, Kamal; Chernikov, Andrey; Chrisochoides, Nikos; He, Jing

2013-01-01

De novo protein modeling approaches utilize 3-dimensional (3D) images derived from electron cryomicroscopy (CryoEM) experiments. The skeleton connecting two secondary structures such as α-helices represent the loop in the 3D image. The accuracy of the skeleton and of the detected secondary structures are critical in De novo modeling. It is important to measure the length along the skeleton accurately since the length can be used as a constraint in modeling the protein. We have developed a novel computational geometric approach to derive a simplified curve in order to estimate the loop length along the skeleton. The method was tested using fifty simulated density images of helix-loop-helix segments of atomic structures and eighteen experimentally derived density data from Electron Microscopy Data Bank (EMDB). The test using simulated density maps shows that it is possible to estimate within 0.5 Å of the expected length for 48 of the 50 cases. The experiments, involving eighteen experimentally derived CryoEM images, show that twelve cases have error within 2 Å. The tests using both simulated and experimentally derived images show that it is possible for our proposed method to estimate the loop length along the skeleton if the secondary structure elements, such as α-helices, can be detected accurately, and there is a continuous skeleton linking the α-helices.

Structure modulation of helix 69 from Escherichia coli 23S ribosomal RNA by pseudouridylations

PubMed Central

Jiang, Jun; Aduri, Raviprasad; Chow, Christine S.; SantaLucia, John

2014-01-01

Helix 69 (H69) is a 19-nt stem-loop region from the large subunit ribosomal RNA. Three pseudouridine (Ψ) modifications clustered in H69 are conserved across phylogeny and known to affect ribosome function. To explore the effects of Ψ on the conformations of Escherichia coli H69 in solution, nuclear magnetic resonance spectroscopy was used to reveal the structural differences between H69 with (ΨΨΨ) and without (UUU) Ψ modifications. Comparison of the two structures shows that H69 ΨΨΨ has the following unique features: (i) the loop region is closed by a Watson–Crick base pair between Ψ1911 and A1919, which is potentially reinforced by interactions involving Ψ1911N1H and (ii) Ψ modifications at loop residues 1915 and 1917 promote base stacking from Ψ1915 to A1918. In contrast, the H69 UUU loop region, which lacks Ψ modifications, is less organized. Structure modulation by Ψ leads to alteration in conformational behavior of the 5' half of the H69 loop region, observed as broadening of C1914 non-exchangeable base proton resonances in the H69 ΨΨΨ nuclear magnetic resonance spectra, and plays an important biological role in establishing the ribosomal intersubunit bridge B2a and mediating translational fidelity. PMID:24371282

Conformational and dynamics changes induced by bile acids binding to chicken liver bile acid binding protein.

PubMed

Eberini, Ivano; Guerini Rocco, Alessandro; Ientile, Anna Rita; Baptista, António M; Gianazza, Elisabetta; Tomaselli, Simona; Molinari, Henriette; Ragona, Laura

2008-06-01

The correlation between protein motions and function is a central problem in protein science. Several studies have demonstrated that ligand binding and protein dynamics are strongly correlated in intracellular lipid binding proteins (iLBPs), in which the high degree of flexibility, principally occurring at the level of helix-II, CD, and EF loops (the so-called portal area), is significantly reduced upon ligand binding. We have recently investigated by NMR the dynamic properties of a member of the iLBP family, chicken liver bile acid binding protein (cL-BABP), in its apo and holo form, as a complex with two bile salts molecules. Binding was found to be regulated by a dynamic process and a conformational rearrangement was associated with this event. We report here the results of molecular dynamics (MD) simulations performed on apo and holo cL-BABP with the aim of further characterizing the protein regions involved in motion propagation and of evaluating the main molecular interactions stabilizing bound ligands. Upon binding, the root mean square fluctuation values substantially decrease for CD and EF loops while increase for the helix-loop-helix region, thus indicating that the portal area is the region mostly affected by complex formation. These results nicely correlate with backbone dynamics data derived from NMR experiments. Essential dynamics analysis of the MD trajectories indicates that the major concerted motions involve the three contiguous structural elements of the portal area, which however are dynamically coupled in different ways whether in the presence or in the absence of the ligands. Motions of the EF loop and of the helical region are part of the essential space of both apo and holo-BABP and sample a much wider conformational space in the apo form. Together with NMR results, these data support the view that, in the apo protein, the flexible EF loop visits many conformational states including those typical of the holo state and that the ligand acts stabilizing one of these pre-existing conformations. The present results, in agreement with data reported for other iLBPs, sharpen our knowledge on the binding mechanism for this protein family. (c) 2008 Wiley-Liss, Inc.

Inhibition of Bacterial Rna Polymerase by Streptolydigin: Stabilization of A Straight-Bridge-Helix Active-Center Conformation

PubMed Central

Tuske, Steven; Sarafianos, Stefan G.; Wang, Xinyue; Hudson, Brian; Sineva, Elena; Mukhopadhyay, Jayanta; Birktoft, Jens J.; Leroy, Olivier; Ismail, Sajida; Clark, Arthur D.; Dharia, Chhaya; Napoli, Andrew; Laptenko, Oleg; Lee, Jookyung; Borukhov, Sergei; Ebright, Richard H.; Arnold, Eddy

2009-01-01

We define the target, mechanism, and structural basis of inhibition of bacterial RNA polymerase (RNAP) by the tetramic-acid antibiotic streptolydigin (Stl). Stl binds to a site adjacent to, but not overlapping, the RNAP active center and stabilizes an RNAP-active-center conformational state with a straight bridge helix. The results provide direct support for the proposals that alternative straight-bridge-helix and bent-bridge-helix RNAP-active-center conformations exist, and that cycling between straight-bridge-helix and bent-bridge-helix RNAP-active-center conformations is required for RNAP function. The results set bounds on models for RNAP function and suggest strategies for design of novel antibacterial agents. PMID:16122422

Designing cooperatively folded abiotic uni- and multimolecular helix bundles

NASA Astrophysics Data System (ADS)

de, Soumen; Chi, Bo; Granier, Thierry; Qi, Ting; Maurizot, Victor; Huc, Ivan

2018-01-01

Abiotic foldamers, that is foldamers that have backbones chemically remote from peptidic and nucleotidic skeletons, may give access to shapes and functions different to those of peptides and nucleotides. However, design methodologies towards abiotic tertiary and quaternary structures are yet to be developed. Here we report rationally designed interactional patterns to guide the folding and assembly of abiotic helix bundles. Computational design facilitated the introduction of hydrogen-bonding functionalities at defined locations on the aromatic amide backbones that promote cooperative folding into helix-turn-helix motifs in organic solvents. The hydrogen-bond-directed aggregation of helices not linked by a turn unit produced several thermodynamically and kinetically stable homochiral dimeric and trimeric bundles with structures that are distinct from the designed helix-turn-helix. Relative helix orientation within the bundles may be changed from parallel to tilted on subtle solvent variations. Altogether, these results prefigure the richness and uniqueness of abiotic tertiary structure behaviour.

Peptide mimics of the M13 coat protein transmembrane segment. Retention of helix-helix interaction motifs.

PubMed

Wang, C; Deber, C M

2000-05-26

Sequence-specific noncovalent helix-helix interactions between transmembrane (TM) segments in proteins are investigated by incorporating selected TM sequences into synthetic peptides using the construct CKKK-TM-KKK. The peptides are of suitable hydrophobicity for spontaneous membrane insertion, whereas formation of an N-terminal S-S bond can bring pairs of TM helices into proximity and promote their parallel orientation. Using the propensity of the protein to undergo thermally induced alpha-helix --> beta-sheet transitions as a parameter for helix stability, we compared the wild type and mutant (V29A and V31A) bacteriophage M13 coat proteins with their corresponding TM peptide constructs (M13 residues 24-42). Our results demonstrated that the relevant helix-helix tertiary contacts found in the intact proteins persist in the peptide mimics. Molecular dynamics simulations support the tight "two in-two out" dimerization motif for V31A consistent with mutagenesis data. The overall results reinforce the notion of TM segments as autonomous folding domains and suggest that the generic peptide construct provides a viable reductionist system for membrane protein structural and computational analysis.

Three-dimensional structure of the human immunodeficiency virus type 1 matrix protein.

PubMed

Massiah, M A; Starich, M R; Paschall, C; Summers, M F; Christensen, A M; Sundquist, W I

1994-11-25

The HIV-1 matrix protein forms an icosahedral shell associated with the inner membrane of the mature virus. Genetic analyses have indicated that the protein performs important functions throughout the viral life-cycle, including anchoring the transmembrane envelope protein on the surface of the virus, assisting in viral penetration, transporting the proviral integration complex across the nuclear envelope, and localizing the assembling virion to the cell membrane. We now report the three-dimensional structure of recombinant HIV-1 matrix protein, determined at high resolution by nuclear magnetic resonance (NMR) methods. The HIV-1 matrix protein is the first retroviral matrix protein to be characterized structurally and only the fourth HIV-1 protein of known structure. NMR signal assignments required recently developed triple-resonance (1H, 13C, 15N) NMR methodologies because signals for 91% of 132 assigned H alpha protons and 74% of the 129 assignable backbone amide protons resonate within chemical shift ranges of 0.8 p.p.m. and 1 p.p.m., respectively. A total of 636 nuclear Overhauser effect-derived distance restraints were employed for distance geometry-based structure calculations, affording an average of 13.0 NMR-derived distance restraints per residue for the experimentally constrained amino acids. An ensemble of 25 refined distance geometry structures with penalties (sum of the squares of the distance violations) of 0.32 A2 or less and individual distance violations under 0.06 A was generated; best-fit superposition of ordered backbone heavy atoms relative to mean atom positions afforded root-mean-square deviations of 0.50 (+/- 0.08) A. The folded HIV-1 matrix protein structure is composed of five alpha-helices, a short 3(10) helical stretch, and a three-strand mixed beta-sheet. Helices I to III and the 3(10) helix pack about a central helix (IV) to form a compact globular domain that is capped by the beta-sheet. The C-terminal helix (helix V) projects away from the beta-sheet to expose carboxyl-terminal residues essential for early steps in the HIV-1 infectious cycle. Basic residues implicated in membrane binding and nuclear localization functions cluster about an extruded cationic loop that connects beta-strands 1 and 2. The structure suggests that both membrane binding and nuclear localization may be mediated by complex tertiary structures rather than simple linear determinants.

NHLH2: At the intersection of obesity and fertility

PubMed Central

Good, Deborah J.; Braun, Thomas

2013-01-01

Nescient helix loop helix 2 (NSCL2/NHLH2) is a neuronal transcription factor originally thought to be involved in neuronal development and childhood neuroblastomas. Accumulating evidence has since identified roles for NHLH2 in adult phenotypes of obesity and fertility. Here, we summarize these findings, and attempt to link genotype with phenotype in mouse models and humans. In particular, NHLH2 (Nhlh2 in mice) is one of only two genes that are genetically linked to physical activity levels. Nhlh2 also controls obesity and fertility, with strong sexual dimorphism displayed for both phenotypes by Nhlh2 mutant animals. We propose that Nhlh2 might function as a molecular sensor in different adult hypothalamic neurons to regulate energy balance, leading to normal body weight and reproduction. PMID:23684566

Informative priors based on transcription factor structural class improve de novo motif discovery.

PubMed

Narlikar, Leelavati; Gordân, Raluca; Ohler, Uwe; Hartemink, Alexander J

2006-07-15

An important problem in molecular biology is to identify the locations at which a transcription factor (TF) binds to DNA, given a set of DNA sequences believed to be bound by that TF. In previous work, we showed that information in the DNA sequence of a binding site is sufficient to predict the structural class of the TF that binds it. In particular, this suggests that we can predict which locations in any DNA sequence are more likely to be bound by certain classes of TFs than others. Here, we argue that traditional methods for de novo motif finding can be significantly improved by adopting an informative prior probability that a TF binding site occurs at each sequence location. To demonstrate the utility of such an approach, we present priority, a powerful new de novo motif finding algorithm. Using data from TRANSFAC, we train three classifiers to recognize binding sites of basic leucine zipper, forkhead, and basic helix loop helix TFs. These classifiers are used to equip priority with three class-specific priors, in addition to a default prior to handle TFs of other classes. We apply priority and a number of popular motif finding programs to sets of yeast intergenic regions that are reported by ChIP-chip to be bound by particular TFs. priority identifies motifs the other methods fail to identify, and correctly predicts the structural class of the TF recognizing the identified binding sites. Supplementary material and code can be found at http://www.cs.duke.edu/~amink/.

Curated collection of yeast transcription factor DNA binding specificity data reveals novel structural and gene regulatory insights

PubMed Central

2011-01-01

Background Transcription factors (TFs) play a central role in regulating gene expression by interacting with cis-regulatory DNA elements associated with their target genes. Recent surveys have examined the DNA binding specificities of most Saccharomyces cerevisiae TFs, but a comprehensive evaluation of their data has been lacking. Results We analyzed in vitro and in vivo TF-DNA binding data reported in previous large-scale studies to generate a comprehensive, curated resource of DNA binding specificity data for all characterized S. cerevisiae TFs. Our collection comprises DNA binding site motifs and comprehensive in vitro DNA binding specificity data for all possible 8-bp sequences. Investigation of the DNA binding specificities within the basic leucine zipper (bZIP) and VHT1 regulator (VHR) TF families revealed unexpected plasticity in TF-DNA recognition: intriguingly, the VHR TFs, newly characterized by protein binding microarrays in this study, recognize bZIP-like DNA motifs, while the bZIP TF Hac1 recognizes a motif highly similar to the canonical E-box motif of basic helix-loop-helix (bHLH) TFs. We identified several TFs with distinct primary and secondary motifs, which might be associated with different regulatory functions. Finally, integrated analysis of in vivo TF binding data with protein binding microarray data lends further support for indirect DNA binding in vivo by sequence-specific TFs. Conclusions The comprehensive data in this curated collection allow for more accurate analyses of regulatory TF-DNA interactions, in-depth structural studies of TF-DNA specificity determinants, and future experimental investigations of the TFs' predicted target genes and regulatory roles. PMID:22189060

OVERVIEW OF HELIX HOUSE NO. 2 (S87), WITH ANTENNA TOWERS, ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

OVERVIEW OF HELIX HOUSE NO. 2 (S-87), WITH ANTENNA TOWERS, HELIX HOUSE NO. 1 (S-3) AND TRANSMITTER BLDG. (S-2) AT REAR, LOOKING WEST SOUTHWEST. - Naval Computer & Telecommunications Area Master Station, Eastern Pacific, Radio Transmitter Facility Lualualei, Helix House No. 2, Base of Radio Antenna Structure No. 427, Makaha, Honolulu County, HI

Assignment of the sup 1 H and sup 15 N NMR spectra of Rhodobacter capsulatus ferrocytochrome c sub 2

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

Gooley, P.R.; Caffrey, M.S.; Cusanovich, M.A.

1990-03-06

The peptide resonances of the {sup 1}H and {sup 15}N nuclear magnetic resonance spectra of ferrocytochrome c{sub 2} from Rhodobacter capsulatus are sequentially assigned by a combination of 2D {sup 1}H-{sup 1}H and {sup 1}H-{sup 15}N spectroscopy, the latter performed on {sup 15}N-enriched protein. Short-range nuclear Overhauser effect (NOE) data show {alpha}-helices from residues 3-17, 55-65, 69-88, and 103-115. Within the latter two {alpha}-helices, there are three single 3{sub 10} turns, 70-72, 76-78, and 107-109. In addition {alpha}H-NH{sub i+1} and {alpha}H-NH{sub i+2} NOEs indicate that the N-terminal helix (3-17) is distorted. Compared to horse or tuna cytochrome c and cytochromemore » c{sub 2} of Rhodospirillium rubrum, there is a 6-residue insertion at residues 23-29 in R. capsulatus cytochrome c{sub 2}. The NOE data show that this insertion forms a loop, probably an {Omega} loop. {sup 1}H-{sup 15}N heteronuclear multiple quantum correlation experiments are used to follow NH exchange over a period of 40 h. As the 2D spectra are acquired in short time periods (30 min), rates for intermediate exchanging protons can be measured. Comparison of the NH exchange data for the N-terminal helix of cytochrome c{sub 2} of R. capsulatus with the highly homologous horse heart cytochrome c shows that this helix is less stable in cytochrome c{sub 2}.« less

Characterization of the biomechanical properties of T4 pili expressed by Streptococcus pneumoniae--a comparison between helix-like and open coil-like pili.

PubMed

Castelain, Mickaël; Koutris, Efstratios; Andersson, Magnus; Wiklund, Krister; Björnham, Oscar; Schedin, Staffan; Axner, Ove

2009-07-13

Bacterial adhesion organelles, known as fimbria or pili, are expressed by gram-positive as well as gram-negative bacteria families. These appendages play a key role in the first steps of the invasion and infection processes, and they therefore provide bacteria with pathogenic abilities. To improve the knowledge of pili-mediated bacterial adhesion to host cells and how these pili behave under the presence of an external force, we first characterize, using force measuring optical tweezers, open coil-like T4 pili expressed by gram-positive Streptococcus pneumoniae with respect to their biomechanical properties. It is shown that their elongation behavior can be well described by the worm-like chain model and that they possess a large degree of flexibility. Their properties are then compared with those of helix-like pili expressed by gram-negative uropathogenic Escherichia coli (UPEC), which have different pili architecture. The differences suggest that these two types of pili have distinctly dissimilar mechanisms to adhere and sustain external forces. Helix-like pili expressed by UPEC bacteria adhere to host cells by single adhesins located at the distal end of the pili while their helix-like structures act as shock absorbers to dampen the irregularly shear forces induced by urine flow and to increase the cooperativity of the pili ensemble, whereas open coil-like pili expressed by S. pneumoniae adhere to cells by a multitude of adhesins distributed along the pili. It is hypothesized that these two types of pili represent different strategies of adhering to host cells in the presence of external forces. When exposed to significant forces, bacteria expressing helix-like pili remain attached by distributing the external force among a multitude of pili, whereas bacteria expressing open coil-like pili sustain large forces primarily by their multitude of binding adhesins which presumably detach sequentially.

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.

Helix-packing motifs in membrane proteins.

PubMed

Walters, R F S; DeGrado, W F

2006-09-12

The fold of a helical membrane protein is largely determined by interactions between membrane-imbedded helices. To elucidate recurring helix-helix interaction motifs, we dissected the crystallographic structures of membrane proteins into a library of interacting helical pairs. The pairs were clustered according to their three-dimensional similarity (rmsd

Folding of a helix is critically stabilized by polarization of backbone hydrogen bonds: study in explicit water.

PubMed

Duan, Li L; Gao, Ya; Mei, Ye; Zhang, Qing G; Tang, Bo; Zhang, John Z H

2012-03-15

Multiple single-trajectory molecular dynamics (MD) simulation at room temperature (300 K) in explicit water was carried out to study the folding dynamics of an α-helix (PDB 2I9M ) using a polarized charge scheme that includes electronic polarization of backbone hydrogen bonds. Starting from an extended conformation, the 17-residue peptide was successfully folded into the native structure (α-helix) between 80 and 130 ns with a root-mean-square deviation of ~1.0 Å. Analysis of the time-dependent trajectories revealed that helix formation of the peptide started at the terminals and progressed toward the center of the peptide. For comparison, MD trajectories generated under various versions of standard AMBER force fields failed to show any significant or stable helix formation in our simulation. Our result shows clear evidence that the electronic polarization of backbone hydrogen bonds energetically stabilizes the helix formation and is critical to the stable folding of the short helix structure. © 2012 American Chemical Society

Structure of LacY with an α-substituted galactoside: Connecting the binding site to the protonation site

PubMed Central

Kumar, Hemant; Finer-Moore, Janet S.; Kaback, H. Ronald; Stroud, Robert M.

2015-01-01

The X-ray crystal structure of a conformationally constrained mutant of the Escherichia coli lactose permease (the LacY double-Trp mutant Gly-46→Trp/Gly-262→Trp) with bound p-nitrophenyl-α-d-galactopyranoside (α-NPG), a high-affinity lactose analog, is described. With the exception of Glu-126 (helix IV), side chains Trp-151 (helix V), Glu-269 (helix VIII), Arg-144 (helix V), His-322 (helix X), and Asn-272 (helix VIII) interact directly with the galactopyranosyl ring of α-NPG to provide specificity, as indicated by biochemical studies and shown directly by X-ray crystallography. In contrast, Phe-20, Met-23, and Phe-27 (helix I) are within van der Waals distance of the benzyl moiety of the analog and thereby increase binding affinity nonspecifically. Thus, the specificity of LacY for sugar is determined solely by side-chain interactions with the galactopyranosyl ring, whereas affinity is increased by nonspecific hydrophobic interactions with the anomeric substituent. PMID:26157133

Structural basis of sterol recognition and nonvesicular transport by lipid transfer proteins anchored at membrane contact sites.

PubMed

Tong, Junsen; Manik, Mohammad Kawsar; Im, Young Jun

2018-01-30

Membrane contact sites (MCSs) in eukaryotic cells are hotspots for lipid exchange, which is essential for many biological functions, including regulation of membrane properties and protein trafficking. Lipid transfer proteins anchored at membrane contact sites (LAMs) contain sterol-specific lipid transfer domains [StARkin domain (SD)] and multiple targeting modules to specific membrane organelles. Elucidating the structural mechanisms of targeting and ligand recognition by LAMs is important for understanding the interorganelle communication and exchange at MCSs. Here, we determined the crystal structures of the yeast Lam6 pleckstrin homology (PH)-like domain and the SDs of Lam2 and Lam4 in the apo form and in complex with ergosterol. The Lam6 PH-like domain displays a unique PH domain fold with a conserved N-terminal α-helix. The Lam6 PH-like domain lacks the basic surface for phosphoinositide binding, but contains hydrophobic patches on its surface, which are critical for targeting to endoplasmic reticulum (ER)-mitochondrial contacts. Structures of the LAM SDs display a helix-grip fold with a hydrophobic cavity and a flexible Ω1-loop as a lid. Ergosterol is bound to the pocket in a head-down orientation, with its hydrophobic acyl group located in the tunnel entrance. The Ω1-loop in an open conformation is essential for ergosterol binding by direct hydrophobic interaction. Structural comparison suggested that the sterol binding mode of the Lam2 SD2 is likely conserved among the sterol transfer proteins of the StARkin superfamily. Structural models of full-length Lam2 correlated with the sterol transport function at the membrane contact sites.

A Proposed Model for Protein Crystal Nucleation and Growth

NASA Technical Reports Server (NTRS)

Pusey, Marc; Curreri, Peter A. (Technical Monitor)

2002-01-01

How does one take a molecule, strongly asymmetric in both shape and charge distribution, and assemble it into a crystal? We propose a model for the nucleation and crystal growth process for tetragonal lysozyme, based upon fluorescence, light, neutron, and X-ray scattering data, size exclusion chromatography experiments, dialysis kinetics, AFM, and modeling of growth rate data, from this and other laboratories. The first species formed is postulated to be a 'head to side' dimer. Through repeating associations involving the same intermolecular interactions this grows to a 4(sub 3) helix structure, that in turn serves as the basic unit for nucleation and subsequent crystal growth. High salt attenuates surface charges while promoting hydrophobic interactions. Symmetry facilitates subsequent helix-helix self-association. Assembly stability is enhanced when a four helix structure is obtained, with each bound to two neighbors. Only two unique interactions are required. The first are those for helix formation, where the dominant interaction is the intermolecular bridging anion. The second is the anti-parallel side-by-side helix-helix interaction, guided by alternating pairs of symmetry related salt bridges along each side. At this stage all eight unique positions of the P4(sub3)2(sub 1),2(sub 1) unit cell are filled. The process is one of a) attenuating the most strongly interacting groups, such that b) the molecules begin to self-associate in defined patterns, so that c) symmetry is obtained, which d) propagates as a growing crystal. Simple and conceptually obvious in hindsight, this tells much about what we are empirically doing when we crystallize macromolecules. By adjusting the growth parameters we are empirically balancing the intermolecular interactions, preferentially attenuating the dominant strong (for lysozyme the charged groups) while strengthening the lesser strong (hydrophobic) interactions. In the general case for proteins the lack of a singularly defined association pathway may lead to formation of multiple species, i.e., amorphous precipitation. Weak interactions, such as hydrogen bonds, are promiscuous, serving to strengthen rather than define specific interactions. Participation in an interaction sequesters that surface from subsequent interactions, and we expect the strongest bonds to form first. This model, its basis, how it fits into the currently understood osmotic second virial coefficient approach to crystallization, and what it suggests will be discussed.

Lipid functions in cytochrome bc complexes: an odd evolutionary transition in a membrane protein structure

PubMed Central

Hasan, S. Saif; Cramer, William A.

2012-01-01

Lipid-binding sites and properties were compared in the hetero-oligomeric cytochrome (cyt) b6f and the yeast bc1 complexes that function, respectively, in photosynthetic and respiratory electron transport. Seven lipid-binding sites in the monomeric unit of the dimeric cyanobacterial b6f complex overlap four sites in the Chlamydomonas reinhardtii algal b6f complex and four in the yeast bc1 complex. The proposed lipid functions include: (i) interfacial–interhelix mediation between (a) the two 8-subunit monomers of the dimeric complex, (b) between the core domain (cyt b, subunit IV) and the six trans membrane helices of the peripheral domain (cyt f, iron–sulphur protein (ISP), and four small subunits in the boundary ‘picket fence’); (ii) stabilization of the ISP domain-swapped trans-membrane helix; (iii) neutralization of basic residues in the single helix of cyt f and of the ISP; (iv) a ‘latch’ to photosystem I provided by the β-carotene chain protruding through the ‘picket fence’; (v) presence of a lipid and chlorophyll a chlorin ring in b6f in place of the eighth helix in the bc1 cyt b polypeptide. The question is posed of the function of the lipid substitution in relation to the evolutionary change between the eight and seven helix structures of the cyt b polypeptide. On the basis of the known n-side activation of light harvesting complex II (LHCII) kinase by the p-side level of plastoquinol, one possibility is that the change was directed by the selective advantage of p- to n-side trans membrane signalling functions in b6f, with the lipid either mediating this function or substituting for the trans membrane helix of a signalling protein lost in crystallization. PMID:23148267

Assessment and Innovation: One Darn Thing Leads to Another

ERIC Educational Resources Information Center

Rutz, Carol; Lauer-Glebov, Jacqulyn

2005-01-01

Using recent experience at Carleton College in Minnesota as a case history, the authors offer a model for assessment that provides more flexibility than the well-known assessment feedback loop, which assumes a linear progression within a hierarchical administrative structure. The proposed model is based on a double helix, with values and feedback…

Chirality-specific lift forces of helix under shear flows: Helix perpendicular to shear plane.

PubMed

Zhang, Qi-Yi

2017-02-01

Chiral objects in shear flow experience a chirality-specific lift force. Shear flows past helices in a low Reynolds number regime were studied using slender-body theory. The chirality-specific lift forces in the vorticity direction experienced by helices are dominated by a set of helix geometry parameters: helix radius, pitch length, number of turns, and helix phase angle. Its analytical formula is given. The chirality-specific forces are the physical reasons for the chiral separation of helices in shear flow. Our results are well supported by the latest experimental observations. © 2016 Wiley Periodicals, Inc.

Introduction of potential helix-capping residues into an engineered helical protein.

PubMed

Parker, M H; Hefford, M A

1998-08-01

MB-1 is an engineered protein that was designed to incorporate high percentages of four amino acid residues and to fold into a four-alpha-helix bundle motif. Mutations were made in the putative loop I and III regions of this protein with the aim of increasing the stability of the helix ends. Four variants, MB-3, MB-5, MB-11 and MB-13, have replacements intended to promote formation of an 'N-capping box'. The loop I and III sequences of MB-3 (both GDLST) and MB-11 (GGDST) were designed to cause alphaL C-terminal 'capping' motifs to form in helices I and III. MB-5 has a sequence, GPDST, that places proline in a favourable position for forming beta-turns, whereas MB-13 (GLDST) has the potential to form Schellman C-capping motifs. Size-exclusion chromatography suggested that MB-1, MB-3, MB-5, MB-11 and MB-13 all form dimers, or possibly trimers. Free energies for the unfolding of each of these variants were determined by urea denaturation, with the loss of secondary structure followed by CD spectroscopy. Assuming an equilibrium between folded dimer and unfolded monomer, MB-13 had the highest apparent stability (40.5 kJ/mol, with +/-2.5 kJ/mol 95% confidence limits), followed by MB-11 (39.3+/-5.9 kJ/mol), MB-3 (36.4+/-1.7 kJ/mol), MB-5 (34.7+/-2.1 kJ/mol) and MB-1 (29.3+/-1.3 kJ/mol); the same relative stabilities of the variants were found when a folded trimer to unfolded monomer model was used to calculate stabilities. All of the variants were relatively unstable for dimeric proteins, but were significantly more stable than MB-1. These findings suggest that it might be possible to increase the stability of a protein for which the three-dimensional structure is unknown by placing amino acid residues in positions that have the potential to form helix- and turn-stabilizing motifs.

Transcriptional deregulation of oncogenic myocyte enhancer factor 2C in T-cell acute lymphoblastic leukemia.

PubMed

Nagel, Stefan; Venturini, Letizia; Meyer, Corinna; Kaufmann, Maren; Scherr, Michaela; Drexler, Hans G; Macleod, Roderick A F

2011-02-01

Myocyte enhancer factor 2C (MEF2C) encodes a transcription factor which is ectopically expressed in T-cell acute lymphoblastic leukemia (T-ALL) cell lines, deregulated directly by ectopically expressed homeodomain protein NKX2-5 or by loss of promoter regions via del(5)(q14). Here, we analyzed the MEF2C 5'-region, thus identifying potential regulatory binding sites for GFI1B, basic helix-loop-helix proteins, STAT5, and HOXA9/HOXA10. Chromatin immunoprecipitation and overexpression analyses demonstrated direct activation by GFI1B and LYL1 and inhibition by STAT5. HOXA9/HOXA10 activated expression of NMYC which in turn mediated MEF2C repression, indicating an indirect mode of regulation via NMYC interactor (NMI) and STAT5. Lacking comma: Chromosomal deletion of the STAT5 binding site in LOUCY cells reduced protein levels of STAT5 in some MEF2C-positve T-ALL cell lines, and the presence of inhibitory IL7-JAK-STAT5 signaling highlighted the repressive impact of this factor in MEF2C regulation. Taken together, our results indicate that the expression of MEF2C in T-ALL cells is principally deregulated via activating leukemic transcription factors GFI1B or NKX2-5 and by escaping inhibitory developmental STAT5 signaling.

A Conserved Network of Transcriptional Activators and Repressors Regulates Anthocyanin Pigmentation in Eudicots[C][W][OPEN

PubMed Central

Albert, Nick W.; Davies, Kevin M.; Lewis, David H.; Zhang, Huaibi; Montefiori, Mirco; Brendolise, Cyril; Boase, Murray R.; Ngo, Hanh; Jameson, Paula E.; Schwinn, Kathy E.

2014-01-01

Plants require sophisticated regulatory mechanisms to ensure the degree of anthocyanin pigmentation is appropriate to myriad developmental and environmental signals. Central to this process are the activity of MYB-bHLH-WD repeat (MBW) complexes that regulate the transcription of anthocyanin genes. In this study, the gene regulatory network that regulates anthocyanin synthesis in petunia (Petunia hybrida) has been characterized. Genetic and molecular evidence show that the R2R3-MYB, MYB27, is an anthocyanin repressor that functions as part of the MBW complex and represses transcription through its C-terminal EAR motif. MYB27 targets both the anthocyanin pathway genes and basic-helix-loop-helix (bHLH) ANTHOCYANIN1 (AN1), itself an essential component of the MBW activation complex for pigmentation. Other features of the regulatory network identified include inhibition of AN1 activity by the competitive R3-MYB repressor MYBx and the activation of AN1, MYB27, and MYBx by the MBW activation complex, providing for both reinforcement and feedback regulation. We also demonstrate the intercellular movement of the WDR protein (AN11) and R3-repressor (MYBx), which may facilitate anthocyanin pigment pattern formation. The fundamental features of this regulatory network in the Asterid model of petunia are similar to those in the Rosid model of Arabidopsis thaliana and are thus likely to be widespread in the Eudicots. PMID:24642943

MYC2 Orchestrates a Hierarchical Transcriptional Cascade That Regulates Jasmonate-Mediated Plant Immunity in Tomato.

PubMed

Du, Minmin; Zhao, Jiuhai; Tzeng, David T W; Liu, Yuanyuan; Deng, Lei; Yang, Tianxia; Zhai, Qingzhe; Wu, Fangming; Huang, Zhuo; Zhou, Ming; Wang, Qiaomei; Chen, Qian; Zhong, Silin; Li, Chang-Bao; Li, Chuanyou

2017-08-01

The hormone jasmonate (JA), which functions in plant immunity, regulates resistance to pathogen infection and insect attack through triggering genome-wide transcriptional reprogramming in plants. We show that the basic helix-loop-helix transcription factor (TF) MYC2 in tomato ( Solanum lycopersicum ) acts downstream of the JA receptor to orchestrate JA-mediated activation of both the wounding and pathogen responses. Using chromatin immunoprecipitation sequencing (ChIP-seq) coupled with RNA sequencing (RNA-seq) assays, we identified 655 MYC2-targeted JA-responsive genes. These genes are highly enriched in Gene Ontology categories related to TFs and the early response to JA, indicating that MYC2 functions at a high hierarchical level to regulate JA-mediated gene transcription. We also identified a group of MYC2-targeted TFs (MTFs) that may directly regulate the JA-induced transcription of late defense genes. Our findings suggest that MYC2 and its downstream MTFs form a hierarchical transcriptional cascade during JA-mediated plant immunity that initiates and amplifies transcriptional output. As proof of concept, we showed that during plant resistance to the necrotrophic pathogen Botrytis cinerea , MYC2 and the MTF JA2-Like form a transcription module that preferentially regulates wounding-responsive genes, whereas MYC2 and the MTF ETHYLENE RESPONSE FACTOR.C3 form a transcription module that preferentially regulates pathogen-responsive genes. © 2017 American Society of Plant Biologists. All rights reserved.

MYC2 Orchestrates a Hierarchical Transcriptional Cascade That Regulates Jasmonate-Mediated Plant Immunity in Tomato[OPEN

PubMed Central

Liu, Yuanyuan; Deng, Lei; Wu, Fangming; Huang, Zhuo; Zhou, Ming; Chen, Qian; Zhong, Silin

2017-01-01

The hormone jasmonate (JA), which functions in plant immunity, regulates resistance to pathogen infection and insect attack through triggering genome-wide transcriptional reprogramming in plants. We show that the basic helix-loop-helix transcription factor (TF) MYC2 in tomato (Solanum lycopersicum) acts downstream of the JA receptor to orchestrate JA-mediated activation of both the wounding and pathogen responses. Using chromatin immunoprecipitation sequencing (ChIP-seq) coupled with RNA sequencing (RNA-seq) assays, we identified 655 MYC2-targeted JA-responsive genes. These genes are highly enriched in Gene Ontology categories related to TFs and the early response to JA, indicating that MYC2 functions at a high hierarchical level to regulate JA-mediated gene transcription. We also identified a group of MYC2-targeted TFs (MTFs) that may directly regulate the JA-induced transcription of late defense genes. Our findings suggest that MYC2 and its downstream MTFs form a hierarchical transcriptional cascade during JA-mediated plant immunity that initiates and amplifies transcriptional output. As proof of concept, we showed that during plant resistance to the necrotrophic pathogen Botrytis cinerea, MYC2 and the MTF JA2-Like form a transcription module that preferentially regulates wounding-responsive genes, whereas MYC2 and the MTF ETHYLENE RESPONSE FACTOR.C3 form a transcription module that preferentially regulates pathogen-responsive genes. PMID:28733419

CD26-mediated regulation of periostin expression contributes to migration and invasion of malignant pleural mesothelioma cells

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

Komiya, Eriko; Ohnuma, Kei, E-mail: kohnuma@juntendo.ac.jp; Yamazaki, Hiroto

Highlights: • CD26-expressing MPM cells upregulate production of periostin. • The intracytoplasmic region of CD26 mediates the upregulation of periostin. • CD26 expression leads to nuclear translocation of Twist1 via phosphorylation of Src. • Secreted periostin enhances migration and invasion of MPM cells. - Abstract: Malignant pleural mesothelioma (MPM) is an aggressive malignancy arising from mesothelial lining of pleura. It is generally associated with a history of asbestos exposure and has a very poor prognosis, partly due to the lack of a precise understanding of the molecular mechanisms associated with its malignant behavior. In the present study, we expanded onmore » our previous studies on the enhanced motility and increased CD26 expression in MPM cells, with a particular focus on integrin adhesion molecules. We found that expression of CD26 upregulates periostin secretion by MPM cells, leading to enhanced MPM cell migratory and invasive activity. Moreover, we showed that upregulation of periostin expression results from the nuclear translocation of the basic helix-loop-helix transcription factor Twist1, a process that is mediated by CD26-associated activation of Src phosphorylation. While providing new and profound insights into the molecular mechanisms involved in MPM biology, these findings may also lead to the development of novel therapeutic strategies for MPM.« less

Heat Shock Protein 83 (Hsp83) Facilitates Methoprene-tolerant (Met) Nuclear Import to Modulate Juvenile Hormone Signaling*

PubMed Central

He, Qianyu; Wen, Di; Jia, Qiangqiang; Cui, Chunlai; Wang, Jian; Palli, Subba R.; Li, Sheng

2014-01-01

Juvenile hormone (JH) receptors, methoprene-tolerant (Met) and Germ-cell expressed (Gce), transduce JH signals to induce Kr-h1 expression in Drosophila. Dual luciferase assay identified a 120-bp JH response region (JHRR) in the Kr-h1α promoter. Both in vitro and in vivo experiments revealed that Met and Gce transduce JH signals to induce Kr-h1 expression through the JHRR. DNA affinity purification identified chaperone protein Hsp83 as one of the proteins bound to the JHRR in the presence of JH. Interestingly, Hsp83 physically interacts with PAS-B and basic helix-loop-helix domains of Met, and JH induces Met-Hsp83 interaction. As determined by immunohistochemistry, Met is mainly distributed in the cytoplasm of fat body cells of the larval when the JH titer is low and JH induces Met nuclear import. Hsp83 was accumulated in the cytoplasm area adjunct to the nucleus in the presence of JH and Met/Gce. Loss-of-function of Hsp83 attenuated JH binding and JH-induced nuclear import of Met, resulting in a decrease in the JHRR-driven reporter activity leading to reduction of Kr-h1 expression. These data show that Hsp83 facilitates the JH-induced nuclear import of Met that induces Kr-h1 expression through the JHRR. PMID:25122763

Pod-1/Capsulin shows a sex- and stage-dependent expression pattern in the mouse gonad development and represses expression of Ad4BP/SF-1.

PubMed

Tamura, M; Kanno, Y; Chuma, S; Saito, T; Nakatsuji, N

2001-04-01

Mammalian sex-determination and differentiation are controlled by several genes, such as Sry, Sox-9, Dax-1 and Mullerian inhibiting substance (MIS), but their upstream and downstream genes are largely unknown. Ad4BP/SF-1, encoding a zinc finger transcription factor, plays important roles in gonadogenesis. Disruption of this gene caused disappearance of the urogenital system including the gonad. Ad4BP/SF-1, however, is also involved in the sex differentiation of the gonad at later stages, such as the regulation of steroid hormones and MIS. Pod-1/Capsulin, a member of basic helix-loop-helix transcription factors, is expressed in a pattern closely related but mostly complimentary to that of the Ad4BP/SF-1 expression in the developing gonad. In the co-transfection experiment using cultured cells, overexpression of Pod-1/Capsulin repressed expression of a reporter gene that carried the upstream regulatory region of the Ad4BP/SF-1 gene. Furthermore, forced expression of Pod-1/Capsulin repressed expression of Ad4BP/SF-1 in the Leydig cell-derived I-10 cells. These results suggest that Pod-1/Capsulin may play important roles in the development and sex differentiation of the mammalian gonad via transcriptional regulation of Ad4BP/SF-1.

Overexpression of EcbHLH57 Transcription Factor from Eleusine coracana L. in Tobacco Confers Tolerance to Salt, Oxidative and Drought Stress

PubMed Central

Nataraja, Karaba N.; Udayakumar, M.

2015-01-01

Basic helix-loop-helix (bHLH) transcription factors constitute one of the largest families in plants and are known to be involved in various developmental processes and stress tolerance. We report the characterization of a stress responsive bHLH transcription factor from stress adapted species finger millet which is homologous to OsbHLH57 and designated as EcbHLH57. The full length sequence of EcbHLH57 consisted of 256 amino acids with a conserved bHLH domain followed by leucine repeats. In finger millet, EcbHLH57 transcripts were induced by ABA, NaCl, PEG, methyl viologen (MV) treatments and drought stress. Overexpression of EcbHLH57 in tobacco significantly increased the tolerance to salinity and drought stress with improved root growth. Transgenic plants showed higher photosynthetic rate and stomatal conductance under drought stress that resulted in higher biomass. Under long-term salinity stress, the transgenic plants accumulated higher seed weight/pod and pod number. The transgenic plants were also tolerant to oxidative stress and showed less accumulation of H202 and MDA levels. The overexpression of EcbHLH57 enhanced the expression of stress responsive genes such as LEA14, rd29A, rd29B, SOD, APX, ADH1, HSP70 and also PP2C and hence improved tolerance to diverse stresses. PMID:26366726

bHLH-O proteins balance the self-renewal and differentiation of Drosophila neural stem cells by regulating Earmuff expression.

PubMed

Li, Xiaosu; Chen, Rui; Zhu, Sijun

2017-11-15

Balancing self-renewal and differentiation of stem cells requires differential expression of self-renewing factors in two daughter cells generated from the asymmetric division of the stem cells. In Drosophila type II neural stem cell (or neuroblast, NB) lineages, the expression of the basic helix-loop-helix-Orange (bHLH-O) family proteins, including Deadpan (Dpn) and E(spl) proteins, is required for maintaining the self-renewal and identity of type II NBs, whereas the absence of these self-renewing factors is essential for the differentiation of intermediate neural progenitors (INPs) generated from type II NBs. Here, we demonstrate that Dpn maintains type II NBs by suppressing the expression of Earmuff (Erm). We provide evidence that Dpn and E(spl) proteins suppress Erm by directly binding to C-sites and N-boxes in the cis-regulatory region of erm. Conversely, the absence of bHLH-O proteins in INPs allows activation of erm and Erm-mediated maturation of INPs. Our results further suggest that Pointed P1 (PntP1) mediates the dedifferentiation of INPs resulting from the loss of Erm or overexpression of Dpn or E(spl) proteins. Taken together, these findings reveal mechanisms underlying the regulation of the maintenance of type II NBs and differentiation of INPs through the differential expression of bHLH-O family proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

Neurog1 Genetic Inducible Fate Mapping (GIFM) Reveals the Existence of Complex Spatiotemporal Cyto-Architectures in the Developing Cerebellum.

PubMed

Obana, Edwin A; Lundell, Travis G; Yi, Kevin J; Radomski, Kryslaine L; Zhou, Qiong; Doughty, Martin L

2015-06-01

Neurog1 is a pro-neural basic helix-loop-helix (bHLH) transcription factor expressed in progenitor cells located in the ventricular zone and subsequently the presumptive white matter tracts of the developing mouse cerebellum. We used genetic inducible fate mapping (GIFM) with a transgenic Neurog1-CreER allele to characterize the contributions of Neurog1 lineages to cerebellar circuit formation in mice. GIFM reveals Neurog1-expressing progenitors are fate-mapped to become Purkinje cells and all GABAergic interneuron cell types of the cerebellar cortex but not glia. The spatiotemporal sequence of GIFM is unique to each neuronal cell type. GIFM on embryonic days (E) 10.5 to E12.5 labels Purkinje cells with different medial-lateral settling patterns depending on the day of tamoxifen delivery. GIFM on E11.5 to P7 labels interneurons and the timing of tamoxifen administration correlates with the final inside-to-outside resting position of GABAergic interneurons in the cerebellar cortex. Proliferative status and long-term BrdU retention of GIFM lineages reveals Purkinje cells express Neurog1 around the time they become post-mitotic. In contrast, GIFM labels mitotic and post-mitotic interneurons. Neurog1-CreER GIFM reveals a correlation between the timing of Neurog1 expression and the spatial organization of GABAergic neurons in the cerebellar cortex with possible implications for cerebellar circuit assembly.

ABA Suppresses Root Hair Growth via the OBP4 Transcriptional Regulator1[OPEN

PubMed Central

Kawamura, Ayako; Schäfer, Sabine; Breuer, Christian; Shibata, Michitaro; Mitsuda, Nobutaka; Ohme-Takagi, Masaru; Matsui, Minami

2017-01-01

Plants modify organ growth and tune morphogenesis in response to various endogenous and environmental cues. At the cellular level, organ growth is often adjusted by alterations in cell growth, but the molecular mechanisms underlying this control remain poorly understood. In this study, we identify the DNA BINDING WITH ONE FINGER (DOF)-type transcription regulator OBF BINDING PROTEIN4 (OBP4) as a repressor of cell growth. Ectopic expression of OBP4 in Arabidopsis (Arabidopsis thaliana) inhibits cell growth, resulting in severe dwarfism and the repression of genes involved in the regulation of water transport, root hair development, and stress responses. Among the basic helix-loop-helix transcription factors known to control root hair growth, OBP4 binds the ROOT HAIR DEFECTIVE6-LIKE2 (RSL2) promoter to repress its expression. The accumulation of OBP4 proteins is detected in expanding root epidermal cells, and its expression level is increased by the application of abscisic acid (ABA) at concentrations sufficient to inhibit root hair growth. ABA-dependent induction of OBP4 is associated with the reduced expression of RSL2. Furthermore, ectopic expression of OBP4 or loss of RSL2 function results in ABA-insensitive root hair growth. Taken together, our results suggest that OBP4-mediated transcriptional repression of RSL2 contributes to the ABA-dependent inhibition of root hair growth in Arabidopsis. PMID:28167701

The Birth of a Black Rice Gene and Its Local Spread by Introgression

PubMed Central

Oikawa, Tetsuo; Maeda, Hiroaki; Oguchi, Taichi; Yamaguchi, Takuya; Tanabe, Noriko; Ebana, Kaworu; Yano, Masahiro; Izawa, Takeshi

2015-01-01

The origin and spread of novel agronomic traits during crop domestication are complex events in plant evolution. Wild rice (Oryza rufipogon) has red grains due to the accumulation of proanthocyanidins, whereas most cultivated rice (Oryza sativa) varieties have white grains induced by a defective allele in the Rc basic helix-loop-helix (bHLH) gene. Although the events surrounding the origin and spread of black rice traits remain unknown, varieties with black grains due to anthocyanin accumulation are distributed in various locations throughout Asia. Here, we show that the black grain trait originated from ectopic expression of the Kala4 bHLH gene due to rearrangement in the promoter region. Both the Rc and Kala4 genes activate upstream flavonol biosynthesis genes, such as chalcone synthase and dihydroflavonol-4-reductase, and downstream genes, such as leucoanthocyanidin reductase and leucoanthocyanidin dioxygenase, to produce the respective specific pigments. Genome analysis of 21 black rice varieties as well as red- and white-grained landraces demonstrated that black rice arose in tropical japonica and its subsequent spread to the indica subspecies can be attributed to the causal alleles of Kala4. The relatively small size of genomic fragments of tropical japonica origin in some indica varieties indicates that refined introgression must have occurred by natural crossbreeding in the course of evolution of the black trait in rice. PMID:26362607

The Birth of a Black Rice Gene and Its Local Spread by Introgression.

PubMed

Oikawa, Tetsuo; Maeda, Hiroaki; Oguchi, Taichi; Yamaguchi, Takuya; Tanabe, Noriko; Ebana, Kaworu; Yano, Masahiro; Ebitani, Takeshi; Izawa, Takeshi

2015-09-01

The origin and spread of novel agronomic traits during crop domestication are complex events in plant evolution. Wild rice (Oryza rufipogon) has red grains due to the accumulation of proanthocyanidins, whereas most cultivated rice (Oryza sativa) varieties have white grains induced by a defective allele in the Rc basic helix-loop-helix (bHLH) gene. Although the events surrounding the origin and spread of black rice traits remain unknown, varieties with black grains due to anthocyanin accumulation are distributed in various locations throughout Asia. Here, we show that the black grain trait originated from ectopic expression of the Kala4 bHLH gene due to rearrangement in the promoter region. Both the Rc and Kala4 genes activate upstream flavonol biosynthesis genes, such as chalcone synthase and dihydroflavonol-4-reductase, and downstream genes, such as leucoanthocyanidin reductase and leucoanthocyanidin dioxygenase, to produce the respective specific pigments. Genome analysis of 21 black rice varieties as well as red- and white-grained landraces demonstrated that black rice arose in tropical japonica and its subsequent spread to the indica subspecies can be attributed to the causal alleles of Kala4. The relatively small size of genomic fragments of tropical japonica origin in some indica varieties indicates that refined introgression must have occurred by natural crossbreeding in the course of evolution of the black trait in rice. © 2015 American Society of Plant Biologists. All rights reserved.

Control of lysosomal biogenesis and Notch-dependent tissue patterning by components of the TFEB-V-ATPase axis in Drosophila melanogaster.

PubMed

Tognon, Emiliana; Kobia, Francis; Busi, Ilaria; Fumagalli, Arianna; De Masi, Federico; Vaccari, Thomas

2016-01-01

In vertebrates, TFEB (transcription factor EB) and MITF (microphthalmia-associated transcription factor) family of basic Helix-Loop-Helix (bHLH) transcription factors regulates both lysosomal function and organ development. However, it is not clear whether these 2 processes are interconnected. Here, we show that Mitf, the single TFEB and MITF ortholog in Drosophila, controls expression of vacuolar-type H(+)-ATPase pump (V-ATPase) subunits. Remarkably, we also find that expression of Vha16-1 and Vha13, encoding 2 key components of V-ATPase, is patterned in the wing imaginal disc. In particular, Vha16-1 expression follows differentiation of proneural regions of the disc. These regions, which will form sensory organs in the adult, appear to possess a distinctive endolysosomal compartment and Notch (N) localization. Modulation of Mitf activity in the disc in vivo alters endolysosomal function and disrupts proneural patterning. Similar to our findings in Drosophila, in human breast epithelial cells we observe that impairment of the Vha16-1 human ortholog ATP6V0C changes the size and function of the endolysosomal compartment and that depletion of TFEB reduces ligand-independent N signaling activity. Our data suggest that lysosomal-associated functions regulated by the TFEB-V-ATPase axis might play a conserved role in shaping cell fate.

Mxi1 is a repressor of the c-Myc promoter and reverses activation by USF.

PubMed

Lee, T C; Ziff, E B

1999-01-08

The basic region/helix-loop-helix/leucine zipper (B-HLH-LZ) oncoprotein c-Myc is abundant in proliferating cells and forms heterodimers with Max protein that bind to E-box sites in DNA and stimulate genes required for proliferation. A second B-HLH-LZ protein, Mxi1, is induced during terminal differentiation, and forms heterodimers with Max that also bind E-boxes but tether the mSin3 transcriptional repressor protein along with histone deacetylase thereby antagonizing Myc-dependent activation. We show that Mxi1 also antagonizes Myc by a second pathway, repression of transcription from the major c-myc promoter, P2. Repression was independent of Mxi1 binding to mSin3 but dependent on the Mxi1 LZ and COOH-terminal sequences, including putative casein kinase II phosphorylation sites. Repression targeted elements of the myc P2 promoter core (-35/+10), where it reversed transactivation by the constitutive transcription factor, USF. We show that Zn2+ induction of a stably transfected, metallothionein promoter-regulated mxi1 gene blocked the ability of serum to induce transcription of the endogenous c-myc gene and cell entry into S phase. Thus, induction of Mxi1 in terminally differentiating cells may block Myc function by repressing the c-myc gene P2 promoter, as well as by antagonizing Myc-dependent transactivation through E-boxes.

Helix formation and stability in membranes.

PubMed

McKay, Matthew J; Afrose, Fahmida; Koeppe, Roger E; Greathouse, Denise V

2018-02-13

In this article we review current understanding of basic principles for the folding of membrane proteins, focusing on the more abundant alpha-helical class. Membrane proteins, vital to many biological functions and implicated in numerous diseases, fold into their active conformations in the complex environment of the cell bilayer membrane. While many membrane proteins rely on the translocon and chaperone proteins to fold correctly, others can achieve their functional form in the absence of any translation apparatus or other aides. Nevertheless, the spontaneous folding process is not well understood at the molecular level. Recent findings suggest that helix fraying and loop formation may be important for overall structure, dynamics and regulation of function. Several types of membrane helices with ionizable amino acids change their topology with pH. Additionally we note that some peptides, including many that are rich in arginine, and a particular analogue of gramicidin, are able passively to translocate across cell membranes. The findings indicate that a final protein structure in a lipid-bilayer membrane is sequence-based, with lipids contributing to stability and regulation. While much progress has been made toward understanding the folding process for alpha-helical membrane proteins, it remains a work in progress. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo. Copyright © 2018 Elsevier B.V. All rights reserved.

Comparison of Genome-Wide Binding of MyoD in Normal Human Myogenic Cells and Rhabdomyosarcomas Identifies Regional and Local Suppression of Promyogenic Transcription Factors

PubMed Central

MacQuarrie, Kyle L.; Yao, Zizhen; Fong, Abraham P.; Diede, Scott J.; Rudzinski, Erin R.; Hawkins, Douglas S.

2013-01-01

Rhabdomyosarcoma is a pediatric tumor of skeletal muscle that expresses the myogenic basic helix-loop-helix protein MyoD but fails to undergo terminal differentiation. Prior work has determined that DNA binding by MyoD occurs in the tumor cells, but myogenic targets fail to activate. Using MyoD chromatin immunoprecipitation coupled to high-throughput sequencing and gene expression analysis in both primary human muscle cells and RD rhabdomyosarcoma cells, we demonstrate that MyoD binds in a similar genome-wide pattern in both tumor and normal cells but binds poorly at a subset of myogenic genes that fail to activate in the tumor cells. Binding differences are found both across genomic regions and locally at specific sites that are associated with binding motifs for RUNX1, MEF2C, JDP2, and NFIC. These factors are expressed at lower levels in RD cells than muscle cells and rescue myogenesis when expressed in RD cells. MEF2C is located in a genomic region that exhibits poor MyoD binding in RD cells, whereas JDP2 exhibits local DNA hypermethylation in its promoter in both RD cells and primary tumor samples. These results demonstrate that regional and local silencing of differentiation factors contributes to the differentiation defect in rhabdomyosarcomas. PMID:23230269

The myogenic repressor gene Holes in muscles is a direct transcriptional target of Twist and Tinman in the Drosophila embryonic mesoderm.

PubMed

Elwell, Jennifer A; Lovato, TyAnna L; Adams, Melanie M; Baca, Erica M; Lee, Thai; Cripps, Richard M

2015-04-15

Understanding the regulatory circuitry controlling myogenesis is critical to understanding developmental mechanisms and developmentally-derived diseases. We analyzed the transcriptional regulation of a Drosophila myogenic repressor gene, Holes in muscles (Him). Previously, Him was shown to inhibit Myocyte enhancer factor-2 (MEF2) activity, and is expressed in myoblasts but not differentiating myotubes. We demonstrate that different phases of Him embryonic expression arises through the actions of different enhancers, and we characterize the enhancer required for its early mesoderm expression. This Him early mesoderm enhancer contains two conserved binding sites for the basic helix-loop-helix regulator Twist, and one binding site for the NK homeodomain protein Tinman. The sites for both proteins are required for enhancer activity in early embryos. Twist and Tinman activate the enhancer in tissue culture assays, and ectopic expression of either factor is sufficient to direct ectopic expression of a Him-lacZ reporter, or of the endogenous Him gene. Moreover, sustained expression of twist in the mesoderm up-regulates mesodermal Him expression in late embryos. Our findings provide a model to define mechanistically how Twist can both promotes myogenesis through direct activation of Mef2, and can place a brake on myogenesis, through direct activation of Him. Copyright © 2015 Elsevier Inc. All rights reserved.

Multiple zebrafish atoh1 genes specify a diversity of neuronal types in the zebrafish cerebellum.

PubMed

Kidwell, Chelsea U; Su, Chen-Ying; Hibi, Masahiko; Moens, Cecilia B

2018-06-01

A single Atoh1 basic-helix-loop-helix transcription factor specifies multiple neuron types in the mammalian cerebellum and anterior hindbrain. The zebrafish genome encodes three paralagous atoh1 genes whose functions in cerebellum and anterior hindbrain development we explore here. With use of a transgenic reporter, we report that zebrafish atoh1c-expressing cells are organized in two distinct domains that are separated both by space and developmental time. An early isthmic expression domain gives rise to an extracerebellar population in rhombomere 1 and an upper rhombic lip domain gives rise to granule cell progenitors that migrate to populate all four granule cell territories of the fish cerebellum. Using genetic mutants we find that of the three zebrafish atoh1 paralogs, atoh1c and atoh1a are required for the full complement of granule neurons. Surprisingly, the two genes are expressed in non-overlapping granule cell progenitor populations, indicating that fish use duplicate atoh1 genes to generate granule cell diversity that is not detected in mammals. Finally, live imaging of granule cell migration in wildtype and atoh1c mutant embryos reveals that while atoh1c is not required for granule cell specification per se, it is required for granule cells to delaminate and migrate away from the rhombic lip. Copyright © 2018 Elsevier Inc. All rights reserved.

A Novel Soybean ERF Transcription Factor, GmERF113, Increases Resistance to Phytophthora sojae Infection in Soybean

PubMed Central

Zhao, Yuanling; Chang, Xin; Qi, Dongyue; Dong, Lidong; Wang, Guangjin; Fan, Sujie; Jiang, Liangyu; Cheng, Qun; Chen, Xi; Han, Dan; Xu, Pengfei; Zhang, Shuzhen

2017-01-01

Phytophthora root and stem rot of soybean caused by the oomycete Phytophthora sojae, is a destructive disease worldwide. Ethylene response factors (ERFs) play important roles in regulating plant biotic and abiotic stress tolerance. In this study, a new ERF gene, GmERF113, was isolated from the highly resistant soybean ‘Suinong 10.’ Sequence analysis suggested that the protein encoded by GmERF113 contained a conserved AP2/ERF domain of 58 amino acid and belonged to the B-4 subgroup of the ERF subfamily. Expression of GmERF113 was significantly induced by P. sojae, ethylene, and methyl jasmonate. GmERF113 protein localized to the nucleus when transiently expressed in Arabidopsis protoplasts, could bind to the GCC-box, and acted as a transcription activator. In addition, a region of the full-length GmERF113, GmERF113-II, interacted with a basic helix-loop-helix transcription factor (GmbHLH) in yeast cells. Full-length GmERF113 also interacted with GmbHLH in planta. GmERF113-overexpressing transgenic plants in susceptible cultivar ‘Dongnong 50’ soybean exhibited increased resistance to P. sojae and positively regulated the expression of the pathogenesis-related genes, PR1 and PR10-1. These results indicate that GmERF113 may play a crucial role in the defense of soybean against P. sojae infection. PMID:28326092

Sumoylation of the Basic Helix-Loop-Helix Transcription Factor Sharp-1 Regulates Recruitment of the Histone Methyltransferase G9a and Function in Myogenesis*

PubMed Central

Wang, Yaju; Shankar, Shilpa Rani; Kher, Devaki; Ling, Belinda Mei Tze; Taneja, Reshma

2013-01-01

Sumoylation is an important post-translational modification that alters the activity of many transcription factors. However, the mechanisms that link sumoylation to alterations in chromatin structure, which culminate in tissue specific gene expression, are not fully understood. In this study, we demonstrate that SUMO modification of the transcription factor Sharp-1 is required for its full transcriptional repression activity and function as an inhibitor of skeletal muscle differentiation. Sharp-1 is modified by sumoylation at two conserved lysine residues 240 and 255. Mutation of these SUMO acceptor sites in Sharp-1 does not impact its subcellular localization but attenuates its ability to act as a transcriptional repressor and inhibit myogenic differentiation. Consistently, co-expression of the SUMO protease SENP1 with wild type Sharp-1 abrogates Sharp-1-dependent inhibition of myogenesis. Interestingly, sumoylation acts as a signal for recruitment of the co-repressor G9a. Thus, enrichment of G9a, and histone H3 lysine 9 dimethylation (H3K9me2), a signature of G9a activity, is dramatically reduced at muscle promoters in cells expressing sumoylation-defective Sharp-1. Our findings demonstrate how sumoylation of Sharp-1 exerts an impact on chromatin structure and transcriptional repression of muscle gene expression through recruitment of G9a. PMID:23637228

Conserved regulatory mechanism controls the development of cells with rooting functions in land plants.

PubMed

Tam, Thomas Ho Yuen; Catarino, Bruno; Dolan, Liam

2015-07-21

Land plants develop filamentous cells-root hairs, rhizoids, and caulonemata-at the interface with the soil. Members of the group XI basic helix-loop-helix (bHLH) transcription factors encoded by LOTUS JAPONICUS ROOTHAIRLESS1-LIKE (LRL) genes positively regulate the development of root hairs in the angiosperms Lotus japonicus, Arabidopsis thaliana, and rice (Oryza sativa). Here we show that auxin promotes rhizoid and caulonema development by positively regulating the expression of PpLRL1 and PpLRL2, the two LRL genes in the Physcomitrella patens genome. Although the group VIII bHLH proteins, AtROOT HAIR DEFECTIVE6 and AtROOT HAIR DEFECTIVE SIX-LIKE1, promote root-hair development by positively regulating the expression of AtLRL3 in A. thaliana, LRL genes promote rhizoid development independently of PpROOT HAIR DEFECTIVE SIX-LIKE1 and PpROOT HAIR DEFECITVE SIX-LIKE2 (PpRSL1 and PpRSL2) gene function in P. patens. Together, these data demonstrate that both LRL and RSL genes are components of an ancient auxin-regulated gene network that controls the development of tip-growing cells with rooting functions among most extant land plants. Although this network has diverged in the moss and the angiosperm lineages, our data demonstrate that the core network acted in the last common ancestor of the mosses and angiosperms that existed sometime before 420 million years ago.

Scleraxis is a transcriptional activator that regulates the expression of Tenomodulin, a marker of mature tenocytes and ligamentocytes.

PubMed

Shukunami, Chisa; Takimoto, Aki; Nishizaki, Yuriko; Yoshimoto, Yuki; Tanaka, Seima; Miura, Shigenori; Watanabe, Hitomi; Sakuma, Tetsushi; Yamamoto, Takashi; Kondoh, Gen; Hiraki, Yuji

2018-02-16

Tenomodulin (Tnmd) is a type II transmembrane glycoprotein predominantly expressed in tendons and ligaments. We found that scleraxis (Scx), a member of the Twist-family of basic helix-loop-helix transcription factors, is a transcriptional activator of Tnmd expression in tenocytes. During embryonic development, Scx expression preceded that of Tnmd. Tnmd expression was nearly absent in tendons and ligaments of Scx-deficient mice generated by transcription activator-like effector nucleases-mediated gene disruption. Tnmd mRNA levels were dramatically decreased during serial passages of rat tenocytes. Scx silencing by small interfering RNA significantly suppressed endogenous Tnmd mRNA levels in tenocytes. Mouse Tnmd contains five E-box sites in the ~1-kb 5'-flanking region. A 174-base pair genomic fragment containing a TATA box drives transcription in tenocytes. Enhancer activity was increased in the upstream region (-1030 to -295) of Tnmd in tenocytes, but not in NIH3T3 and C3H10T1/2 cells. Preferential binding of both Scx and Twist1 as a heterodimer with E12 or E47 to CAGATG or CATCTG and transactivation of the 5'-flanking region were confirmed by electrophoresis mobility shift and dual luciferase assays, respectively. Scx directly transactivates Tnmd via these E-boxes to positively regulate tenocyte differentiation and maturation.

CCAR1 is required for Ngn3-mediated endocrine differentiation

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

Lu, Chung-Kuang; Lai, Yi-Chyi; Lin, Yung-Fu

2012-02-10

Highlights: Black-Right-Pointing-Pointer We identify CCAR1 to directly interact with Ngn3. Black-Right-Pointing-Pointer CCAR1 is co-localized with Ngn3 in the nucleus. Black-Right-Pointing-Pointer CCAR1 cooperates with Ngn3 in activating NeuroD expression. Black-Right-Pointing-Pointer CCAR1 is required for Ngn3-mediated PANC-1 transdifferentiation. -- Abstract: Neurogenin3 (Ngn3) is a basic helix-loop-helix transcription factor that specifies pancreatic endocrine cell fates during pancreas development. It can also initiate a transdifferentiation program when expressed in pancreatic exocrine and ductal cells. However, how Ngn3 initiates a transcriptional cascade to achieve endocrine differentiation is still poorly understood. Here, we show that cell cycle and apoptosis regulator 1 (CCAR1), which is a transcriptionalmore » coactivator for nuclear receptors, also interacts with Ngn3. The association between Ngn3 and CCAR1 was verified by pull-down assays and co-immunoprecipitation analyses. Using gene reporter assays, we found that CCAR1 is essential for Ngn3 to activate the expression of the reporter genes containing the NeuroD promoter. Moreover, down-regulation of endogenous CCAR1 in the PANC-1 pancreatic ductal cell line inhibits the transdifferentiation program initiated by Ngn3. CCAR1 is, therefore, a novel partner of Ngn3 in mediating endocrine differentiation.« less

Functional Profiling Identifies Genes Involved in Organ-Specific Branches of the PIF3 Regulatory Network in Arabidopsis[C][W

PubMed Central

Sentandreu, Maria; Martín, Guiomar; González-Schain, Nahuel; Leivar, Pablo; Soy, Judit; Tepperman, James M.; Quail, Peter H.; Monte, Elena

2011-01-01

The phytochrome (phy)-interacting basic helix-loop-helix transcription factors (PIFs) constitutively sustain the etiolated state of dark-germinated seedlings by actively repressing deetiolation in darkness. This action is rapidly reversed upon light exposure by phy-induced proteolytic degradation of the PIFs. Here, we combined a microarray-based approach with a functional profiling strategy and identified four PIF3-regulated genes misexpressed in the dark (MIDAs) that are novel regulators of seedling deetiolation. We provide evidence that each one of these four MIDA genes regulates a specific facet of etiolation (hook maintenance, cotyledon appression, or hypocotyl elongation), indicating that there is branching in the signaling that PIF3 relays. Furthermore, combining inferred MIDA gene function from mutant analyses with their expression profiles in response to light-induced degradation of PIF3 provides evidence consistent with a model where the action of the PIF3/MIDA regulatory network enables an initial fast response to the light and subsequently prevents an overresponse to the initial light trigger, thus optimizing the seedling deetiolation process. Collectively, the data suggest that at least part of the phy/PIF system acts through these four MIDAs to initiate and optimize seedling deetiolation, and that this mechanism might allow the implementation of spatial (i.e., organ-specific) and temporal responses during the photomorphogenic program. PMID:22108407

Extension of helix II of an HIV-1-directed hammerhead ribozyme with long antisense flanks does not alter kinetic parameters in vitro but causes loss of the inhibitory potential in living cells.

PubMed Central

Homann, M; Tabler, M; Tzortzakaki, S; Sczakiel, G

1994-01-01

When designed to cleave a target RNA in trans, the hammerhead ribozyme contains two antisense flanks which form helix I and helix III by pairing with the complementary target RNA. The sequences forming helix II are contained on the ribozyme strand and represent a major structural component of the hammerhead structure. In the case of an inhibitory 429 nucleotides long trans-ribozyme (2as-Rz12) which was directed against the 5'-leader/gag region of the human immunodeficiency virus type 1 (HIV-1), helix II was not pre-formed in the single-stranded molecule. Thus, major structural changes are necessary before cleavage can occur. To study whether pre-formation of helix II in the non-paired 2as-Rz12 RNA could influence the observed cleavage rate in vitro and its inhibitory activity on HIV-1 replication, we extended the 4 base pair helix II of 2as-Rz12 to 6, 10, 21, and 22 base pairs respectively. Limited RNase cleavage reactions performed in vitro at 37 degrees C and at physiological ion strength indicated that a helix II of the hammerhead domain was pre-formed when its length was at least six base pairs. This modification neither affected the association rate with target RNA nor the cleavage rate in vitro. In contrast to this, extension of helix II led to a significantly decreased inhibition of HIV-1 replication in human cells. Together with the finding of others that shortening of helix II to less than two base pairs reduces the catalytic activity in vitro, this observation indicates that the length of helix II in the naturally occurring RNAs with a hammerhead domain is already close or identical to the optimal length for catalytic activity in vitro and in vivo. Images PMID:7524030

Solution properties of the archaeal CRISPR DNA repeat-binding homeodomain protein Cbp2

PubMed Central

Kenchappa, Chandra S.; Heidarsson, Pétur O.; Kragelund, Birthe B.; Garrett, Roger A.; Poulsen, Flemming M.

2013-01-01

Clustered regularly interspaced short palindromic repeats (CRISPR) form the basis of diverse adaptive immune systems directed primarily against invading genetic elements of archaea and bacteria. Cbp1 of the crenarchaeal thermoacidophilic order Sulfolobales, carrying three imperfect repeats, binds specifically to CRISPR DNA repeats and has been implicated in facilitating production of long transcripts from CRISPR loci. Here, a second related class of CRISPR DNA repeat-binding protein, denoted Cbp2, is characterized that contains two imperfect repeats and is found amongst members of the crenarchaeal thermoneutrophilic order Desulfurococcales. DNA repeat-binding properties of the Hyperthermus butylicus protein Cbp2Hb were characterized and its three-dimensional structure was determined by NMR spectroscopy. The two repeats generate helix-turn-helix structures separated by a basic linker that is implicated in facilitating high affinity DNA binding of Cbp2 by tethering the two domains. Structural studies on mutant proteins provide support for Cys7 and Cys28 enhancing high thermal stability of Cbp2Hb through disulphide bridge formation. Consistent with their proposed CRISPR transcriptional regulatory role, Cbp2Hb and, by inference, other Cbp1 and Cbp2 proteins are closely related in structure to homeodomain proteins with linked helix-turn-helix (HTH) domains, in particular the paired domain Pax and Myb family proteins that are involved in eukaryal transcriptional regulation. PMID:23325851

Ligatoxin B, a new cytotoxic protein with a novel helix-turn-helix DNA-binding domain from the mistletoe Phoradendron liga.

PubMed Central

Li, Shi-Sheng; Gullbo, Joachim; Lindholm, Petra; Larsson, Rolf; Thunberg, Eva; Samuelsson, Gunnar; Bohlin, Lars; Claeson, Per

2002-01-01

A new basic protein, designated ligatoxin B, containing 46 amino acid residues has been isolated from the mistletoe Phoradendron liga (Gill.) Eichl. (Viscaceae). The protein's primary structure, determined unambiguously using a combination of automated Edman degradation, trypsin enzymic digestion, and tandem MS analysis, was 1-KSCCPSTTAR-NIYNTCRLTG-ASRSVCASLS-GCKIISGSTC-DSGWNH-46. Ligatoxin B exhibited in vitro cytotoxic activities on the human lymphoma cell line U-937-GTB and the primary multidrug-resistant renal adenocarcinoma cell line ACHN, with IC50 values of 1.8 microM and 3.2 microM respectively. Sequence alignment with other thionins identified a new member of the class 3 thionins, ligatoxin B, which is similar to the earlier described ligatoxin A. As predicted by the method of homology modelling, ligatoxin B shares a three-dimensional structure with the viscotoxins and purothionins and so may have the same mode of cytotoxic action. The novel similarities observed by structural comparison of the helix-turn-helix (HTH) motifs of the thionins, including ligatoxin B, and the HTH DNA-binding proteins, led us to propose the working hypothesis that thionins represent a new group of DNA-binding proteins. This working hypothesis could be useful in further dissecting the molecular mechanisms of thionin cytotoxicity and of thionin opposition to multidrug resistance, and useful in clarifying the physiological function of thionins in plants. PMID:12049612

Structures of the transmembrane helices of the G-protein coupled receptor, rhodopsin.

PubMed

Katragadda, M; Chopra, A; Bennett, M; Alderfer, J L; Yeagle, P L; Albert, A D

2001-07-01

An hypothesis is tested that individual peptides corresponding to the transmembrane helices of the membrane protein, rhodopsin, would form helices in solution similar to those in the native protein. Peptides containing the sequences of helices 1, 4 and 5 of rhodopsin were synthesized. Two peptides, with overlapping sequences at their termini, were synthesized to cover each of the helices. The peptides from helix 1 and helix 4 were helical throughout most of their length. The N- and C-termini of all the peptides were disordered and proline caused opening of the helical structure in both helix 1 and helix 4. The peptides from helix 5 were helical in the middle segment of each peptide, with larger disordered regions in the N- and C-termini than for helices 1 and 4. These observations show that there is a strong helical propensity in the amino acid sequences corresponding to the transmembrane domain of this G-protein coupled receptor. In the case of the peptides from helix 4, it was possible to superimpose the structures of the overlapping sequences to produce a construct covering the whole of the sequence of helix 4 of rhodopsin. As similar superposition for the peptides from helix 1 also produced a construct, but somewhat less successfully because of the disordering in the region of sequence overlap. This latter problem was more severe for helix 5 and therefore a single peptide was synthesized for the entire sequence of this helix, and its structure determined. It proved to be helical throughout. Comparison of all these structures with the recent crystal structure of rhodopsin revealed that the peptide structures mimicked the structures seen in the whole protein. Thus similar studies of peptides may provide useful information on the secondary structure of other transmembrane proteins built around helical bundles.

Conformational and dynamics simulation study of antimicrobial peptide hedistin-heterogeneity of its helix-turn-helix motif.

PubMed

Xu, Guohua; Wu, Min; Wang, Lin; Zhang, Xu; Cao, Shufen; Liu, Maili; Cui, Yanfang

2009-12-01

Hedistin is an antimicrobial peptide isolated from the coelomocytes of Nereis diversicolor, possessing activity against a large spectrum of bacteria including the methicillin resistant Staphylococcus aureus and Vibrio alginolyticus. The three-dimensional structure of hedistin in both aqueous solution and deuterated dodecylphosphocholine (DPC) micelles was examined using circular dichroism (CD) and nuclear magnetic resonance (NMR) techniques. And, the early events of the antibacterial process of hedistin were simulated using palmitoyl-oleoyl-phophatidylcholine (POPC) lipid bilayers and molecular dynamics (MD) simulation methods. Hedistin lacks secondary structure in aqueous solution, however, in DPC micelles, it features with a heterogeneous helix-turn-helix moiety and exhibits obvious amphipathic nature. The turn region (residues Val9-Thr12) in the moiety is a four-residue hinge, lying in between the first N-terminal alpha-helix (residues Leu5-Lys8) and the second alpha-helix (residues Val13-Ala17) regions and causing an approximately 120 degrees angle between the axes of the two helices. The segmental and nonlinear nature of hedistin structure is referred to as the heterogeneity of its helix-turn-helix motif which was found to be corresponding to a kind of discrete dynamics behavior, herein coined as its dynamical heterogeneity, at the early stage (0-50 ns) of the MD simulations. That is, the first helix segment, prior to (at 310 K) or following (at 363 K) the second helix, binds to the lipid head-group region and subsequently permeates into the hydrophobic lipid tail region, and the hinge is the last portion entering the lipid environment. This result implies that hedistin may adopt a "carpet" model action when disrupting bacterial membrane.

Critical domain interactions for type A RNase P RNA catalysis with and without the specificity domain

PubMed Central

Mao, Guanzhong; Srivastava, Abhishek S.; Wu, Shiying; Kosek, David; Lindell, Magnus

2018-01-01

The natural trans-acting ribozyme RNase P RNA (RPR) is composed of two domains in which the catalytic (C-) domain mediates cleavage of various substrates. The C-domain alone, after removal of the second specificity (S-) domain, catalyzes this reaction as well, albeit with reduced efficiency. Here we provide experimental evidence indicating that efficient cleavage mediated by the Escherichia coli C-domain (Eco CP RPR) with and without the C5 protein likely depends on an interaction referred to as the "P6-mimic". Moreover, the P18 helix connects the C- and S-domains between its loop and the P8 helix in the S-domain (the P8/ P18-interaction). In contrast to the "P6-mimic", the presence of P18 does not contribute to the catalytic performance by the C-domain lacking the S-domain in cleavage of an all ribo model hairpin loop substrate while deletion or disruption of the P8/ P18-interaction in full-size RPR lowers the catalytic efficiency in cleavage of the same model hairpin loop substrate in keeping with previously reported data using precursor tRNAs. Consistent with that P18 is not required for cleavage mediated by the C-domain we show that the archaeal Pyrococcus furiosus RPR C-domain, which lacks the P18 helix, is catalytically active in trans without the S-domain and any protein. Our data also suggest that the S-domain has a larger impact on catalysis for E. coli RPR compared to P. furiosus RPR. Finally, we provide data indicating that the absence of the S-domain and P18, or the P8/ P18-interaction in full-length RPR influences the charge distribution near the cleavage site in the RPR-substrate complex to a small but reproducible extent. PMID:29509761

Structure of a protein G helix variant suggests the importance of helix propensity and helix dipole interactions in protein design.

PubMed Central

Strop, P.; Marinescu, A. M.; Mayo, S. L.

2000-01-01

Six helix surface positions of protein G (Gbeta1) were redesigned using a computational protein design algorithm, resulting in the five fold mutant Gbeta1m2. Gbeta1m2 is well folded with a circular dichroism spectrum nearly identical to that of Gbeta1, and a melting temperature of 91 degrees C, approximately 6 degrees C higher than that of Gbeta1. The crystal structure of Gbeta1m2 was solved to 2.0 A resolution by molecular replacement. The absence of hydrogen bond or salt bridge interactions between the designed residues in Gbeta1m2 suggests that the increased stability of Gbeta1m2 is due to increased helix propensity and more favorable helix dipole interactions. PMID:10933505

Equilibrium shift in solution: molecular shape recognition and precipitation of a synthetic double helix using helicene-grafted silica nanoparticles.

PubMed

Miyagawa, Masamichi; Ichinose, Wataru; Yamaguchi, Masahiko

2014-01-27

Chiral silica nanoparticles (70 nm) grafted with (P)-helicene recognized the molecular shape of double helix and random coil (P)-ethynylhelicene oligomers in solution. A mixture of the (P)-nanoparticles and double helix precipitated much faster than a mixture of the (P)-nanoparticles and random coil, and the precipitate contained only the double helix. The mixture of the (P)-nanoparticles and (P)-ethynylhelicene pentamer reversibly dispersed in trifluoromethylbenzene upon heating at 70 °C and precipitated upon cooling at 25 °C. When a 10:90 equilibrium mixture of the double helix and random coil in solution was treated with the (P)-nanoparticles, the double helix was precipitated in 53% yield and was accompanied by equilibrium shift. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Position-dependent interactions between cysteine residues and the helix dipole

PubMed Central

Miranda, JJ L.

2003-01-01

A protein model was developed for studying the interaction between cysteine residues and the helix dipole. Site-directed mutagenesis was used to introduce cysteine residues at the N-terminus of helix H in recombinant sperm whale myoglobin. Based on the difference in thiol pKa between folded proteins and an unfolded peptide, the energy of interaction between the thiolate and the helix dipole was determined. Thiolates at the N1 and N2 positions of the helix were stabilized by 0.3 kcal/mole and 0.7 kcal/mole, respectively. A thiolate at the Ncap position was stabilized by 2.8 kcal/mole, and may involve a hydrogen bond. In context with other studies, an experimentally observed helix dipole effect may be defined in terms of two distinct components. A charge-dipole component involves electrostatic interactions with peptide bond dipoles in the first two turns of the helix and affects residues at all positions of the terminus; a hydrogen bond component involves one or more backbone amide groups and is only possible at the capping position due to conformational restraints elsewhere. The nature and magnitude of the helix dipole effect is, therefore, position-dependent. Results from this model system were used to interpret cysteine reactivity in rodent hemoglobins and the thioredoxin family. PMID:12493830

Dynamics and unfolding pathway of chimeric azurin variants: insights from molecular dynamics simulation.

PubMed

Evoli, Stefania; Guzzi, Rita; Rizzuti, Bruno

2013-10-01

The spectroscopic, thermal, and functional properties of blue copper proteins can be modulated by mutations in the metal binding loop. Molecular dynamics simulation was used to compare the conformational properties of azurin and two chimeric variants, which were obtained by inserting into the azurin scaffold the copper binding loop of amicyanin and plastocyanin, respectively. Simulations at room temperature show that the proteins retain their overall structure and exhibit concerted motions among specific inner regions, as revealed by principal component analysis. Molecular dynamics at high temperature indicates that the first events in the unfolding pathway are structurally similar in the three proteins and unfolding starts from the region of the α-helix that is far from the metal binding loop. The results provide details of the denaturation process that are consistent with experimental data and in close agreement with other computational approaches, suggesting a distinct mechanism of unfolding of azurin and its chimeric variants. Moreover, differences observed in the dynamics of specific regions in the three proteins correlate with their thermal behavior, contributing to the determination of the basic factors that influence the stability.

Mode selection in swirling jet experiments: a linear stability analysis

NASA Astrophysics Data System (ADS)

Gallaire, François; Chomaz, Jean-Marc

2003-11-01

The primary goal of the study is to identify the selection mechanism responsible for the appearance of a double-helix structure in the pre-breakdown stage of so-called screened swirling jets for which the circulation vanishes away from the jet. The family of basic flows under consideration combines the azimuthal velocity profiles of Carton & McWilliams (1989) and the axial velocity profiles of Monkewitz (1988). This model satisfactorily represents the nozzle exit velocity distributions measured in the swirling jet experiment of Billant et al. (1998). Temporal and absolute/convective instability properties are directly retrieved from numerical simulations of the linear impulse response for different swirl parameter settings. A large range of negative helical modes, winding with the basic flow, are destabilized as swirl is increased, and their characteristics for large azimuthal wavenumbers are shown to agree with the asymptotic analysis of Leibovich & Stewartson (1983). However, the temporal study fails to yield a clear selection principle. The absolute/convective instability regions are mapped out in the plane of the external axial flow and swirl parameters. The absolutely unstable domain is enhanced by rotation and it remains open for arbitrarily large swirl. The swirling jet with zero external axial flow is found to first become absolutely unstable to a mode of azimuthal wavenumber m {=} {-}2, winding with the jet. It is suggested that this selection mechanism accounts for the experimental observation of a double-helix structure.

Exploring the membrane fusion mechanism through force-induced disassembly of HIV-1 six-helix bundle

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

Gao, Kai; Beijing Key Laboratory of Noncoding RNA, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101; University of Chinese Academy of Sciences, Beijing 100049

Enveloped virus, such as HIV-1, employs membrane fusion mechanism to invade into host cell. HIV-1 gp41 ectodomain uses six-helix bundle configuration to accomplish this process. Using molecular dynamic simulations, we confirmed the stability of this six-helix bundle by showing high occupancy of hydrogen bonds and hydrophobic interactions. Key residues and interactions important for the bundle integration were characterized by force-induced unfolding simulations of six-helix bundle, exhibiting the collapse order of these groups of interactions. Moreover, our results in some way concerted with a previous theory that the formation of coiled-coil choose a route which involved cooperative interactions between the N-terminalmore » and C-terminal helix. -- Highlights: •Unfolding of HIV-1 gp41 six-helix bundle is studied by molecular dynamics simulations. •Specific interactions responsible for the stability of HIV-1 envelope post-fusion conformation were identified. •The gp41 six-helix bundle transition inducing membrane fusion might be a cooperative process of the three subunits.« less

Kinase Regulation by Hydrophobic Spine Assembly in Cancer

PubMed Central

Ahuja, Lalima G.; Meharena, Hiruy S.; Kannan, Natarajan; Kornev, Alexandr P.

2014-01-01

A new model of kinase regulation based on the assembly of hydrophobic spines has been proposed. Changes in their positions can explain the mechanism of kinase activation. Here, we examined mutations in human cancer for clues about the regulation of the hydrophobic spines by focusing initially on mutations to Phe. We identified a selected number of Phe mutations in a small group of kinases that included BRAF, ABL1, and the epidermal growth factor receptor. Testing some of these mutations in BRAF, we found that one of the mutations impaired ATP binding and catalytic activity but promoted noncatalytic allosteric functions. Other Phe mutations functioned to promote constitutive catalytic activity. One of these mutations revealed a previously underappreciated hydrophobic surface that functions to position the dynamic regulatory αC-helix. This supports the key role of the C-helix as a signal integration motif for coordinating multiple elements of the kinase to create an active conformation. The importance of the hydrophobic space around the αC-helix was further tested by studying a V600F mutant, which was constitutively active in the absence of the negative charge that is associated with the common V600E mutation. Many hydrophobic mutations strategically localized along the C-helix can thus drive kinase activation. PMID:25348715

A Comprehensive Review of Travelling-Wave Tube Technology

DTIC Science & Technology

1979-11-01

drivers for crossed-field amplifiers. Coupled-cavity TWT interaction is similar to helix TWT interaction in one respect, that is, power flows along the...resulting in a similar improvement in tube efficiency. Also, an improvement in efficiency can be obtained in helix TWTs by increasing the circuit period...Impedance. Based on theory and measurement the helix is far superior to any other type of propagating structure. The low power helix TWT is still the

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

Li, Chunhua; Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan 45108; Lv, Dashuai

Riboswitches are noncoding mRNA segments that can regulate the gene expression via altering their structures in response to specific metabolite binding. We proposed a coarse-grained Gaussian network model (GNM) to examine the unfolding and folding dynamics of adenosine deaminase (add) A-riboswitch upon the adenine dissociation, in which the RNA is modeled by a nucleotide chain with interaction networks formed by connecting adjoining atomic contacts. It was shown that the adenine binding is critical to the folding of the add A-riboswitch while the removal of the ligand can result in drastic increase of the thermodynamic fluctuations especially in the junction regionsmore » between helix domains. Under the assumption that the native contacts with the highest thermodynamic fluctuations break first, the iterative GNM simulations showed that the unfolding process of the adenine-free add A-riboswitch starts with the denature of the terminal helix stem, followed by the loops and junctions involving ligand binding pocket, and then the central helix domains. Despite the simplified coarse-grained modeling, the unfolding dynamics and pathways are shown in close agreement with the results from atomic-level MD simulations and the NMR and single-molecule force spectroscopy experiments. Overall, the study demonstrates a new avenue to investigate the binding and folding dynamics of add A-riboswitch molecule which can be readily extended for other RNA molecules.« less

Open and Lys–His Hexacoordinated Closed Structures of a Globin with Swapped Proximal and Distal Sites

PubMed Central

Teh, Aik-Hong; Saito, Jennifer A.; Najimudin, Nazalan; Alam, Maqsudul

2015-01-01

Globins are haem-binding proteins with a conserved fold made up of α-helices and can possess diverse properties. A putative globin-coupled sensor from Methylacidiphilum infernorum, HGbRL, contains an N-terminal globin domain whose open and closed structures reveal an untypical dimeric architecture. Helices E and F fuse into an elongated helix, resulting in a novel site-swapped globin fold made up of helices A–E, hence the distal site, from one subunit and helices F–H, the proximal site, from another. The open structure possesses a large cavity binding an imidazole molecule, while the closed structure forms a unique Lys–His hexacoordinated species, with the first turn of helix E unravelling to allow Lys52(E10) to bind to the haem. Ligand binding induces reorganization of loop CE, which is stabilized in the closed form, and helix E, triggering a large conformational movement in the open form. These provide a mechanical insight into how a signal may be relayed between the globin domain and the C-terminal domain of HGbRL, a Roadblock/LC7 domain. Comparison with HGbI, a closely related globin, further underlines the high degree of structural versatility that the globin fold is capable of, enabling it to perform a diversity of functions. PMID:26094577

NMR structure and localization of a large fragment of the SARS-CoV fusion protein: Implications in viral cell fusion.

PubMed

Mahajan, Mukesh; Chatterjee, Deepak; Bhuvaneswari, Kannaian; Pillay, Shubhadra; Bhattacharjya, Surajit

2018-02-01

The lethal Coronaviruses (CoVs), Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV) and most recently Middle East Respiratory Syndrome Coronavirus, (MERS-CoV) are serious human health hazard. A successful viral infection requires fusion between virus and host cells carried out by the surface spike glycoprotein or S protein of CoV. Current models propose that the S2 subunit of S protein assembled into a hexameric helical bundle exposing hydrophobic fusogenic peptides or fusion peptides (FPs) for membrane insertion. The N-terminus of S2 subunit of SARS-CoV reported to be active in cell fusion whereby FPs have been identified. Atomic-resolution structure of FPs derived either in model membranes or in membrane mimic environment would glean insights toward viral cell fusion mechanism. Here, we have solved 3D structure, dynamics and micelle localization of a 64-residue long fusion peptide or LFP in DPC detergent micelles by NMR methods. Micelle bound structure of LFP is elucidated by the presence of discretely folded helical and intervening loops. The C-terminus region, residues F42-Y62, displays a long hydrophobic helix, whereas the N-terminus is defined by a short amphipathic helix, residues R4-Q12. The intervening residues of LFP assume stretches of loops and helical turns. The N-terminal helix is sustained by close aromatic and aliphatic sidechain packing interactions at the non-polar face. 15 N{ 1 H}NOE studies indicated dynamical motion, at ps-ns timescale, of the helices of LFP in DPC micelles. PRE NMR showed that insertion of several regions of LFP into DPC micelle core. Together, the current study provides insights toward fusion mechanism of SARS-CoV. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Structure of the Ebola virus envelope protein MPER/TM domain and its interaction with the fusion loop explains their fusion activity.

PubMed

Lee, Jinwoo; Nyenhuis, David A; Nelson, Elizabeth A; Cafiso, David S; White, Judith M; Tamm, Lukas K

2017-09-19

Ebolavirus (EBOV), an enveloped filamentous RNA virus causing severe hemorrhagic fever, enters cells by macropinocytosis and membrane fusion in a late endosomal compartment. Fusion is mediated by the EBOV envelope glycoprotein GP, which consists of subunits GP1 and GP2. GP1 binds to cellular receptors, including Niemann-Pick C1 (NPC1) protein, and GP2 is responsible for low pH-induced membrane fusion. Proteolytic cleavage and NPC1 binding at endosomal pH lead to conformational rearrangements of GP2 that include exposing the hydrophobic fusion loop (FL) for insertion into the cellular target membrane and forming a six-helix bundle structure. Although major portions of the GP2 structure have been solved in pre- and postfusion states and although current models place the transmembrane (TM) and FL domains of GP2 in close proximity at critical steps of membrane fusion, their structures in membrane environments, and especially interactions between them, have not yet been characterized. Here, we present the structure of the membrane proximal external region (MPER) connected to the TM domain: i.e., the missing parts of the EBOV GP2 structure. The structure, solved by solution NMR and EPR spectroscopy in membrane-mimetic environments, consists of a helix-turn-helix architecture that is independent of pH. Moreover, the MPER region is shown to interact in the membrane interface with the previously determined structure of the EBOV FL through several critical aromatic residues. Mutation of aromatic and neighboring residues in both binding partners decreases fusion and viral entry, highlighting the functional importance of the MPER/TM-FL interaction in EBOV entry and fusion.

Conformational changes associated with L16P and T118M mutations in the membrane-embedded PMP22 protein, consequential in CMT-1A.

PubMed

Bello, Martiniano; Torres, Mixtli J; Méndez-Tenorio, Alfonso; Correa-Basurto, José

2017-10-01

Peripheral myelin protein 22 (PMP22) resides in the plasma membrane and is required for myelin formation in the peripheral nervous system. Excess PMP22 mutants accumulate in the endoplasmic reticulum (ER) resulting in the inherited neuropathies of Charcot-Marie-Tooth disease. However, there was no evidence of the structure of PMP22 or how mutations affect its folding. Therefore, in this study, we combined bioinformatics and homology modeling approaches to obtain three-dimensional native and mutated PMP22 models and its anchoring to a POPC membrane, submitted to .5-μs MD simulations, to determine how the L16P and T118M mutations affect the conformational behavior of PMP22. In addition, we investigated the ability of the native and mutated species to accumulate in the ER, via interaction with RER1, by combining protein-protein docking and MD simulations, taking the conformations that were most representative of the native and mutated PMP22 systems and RER1 conformations. Principal component analysis over MD simulations revealed that L16P and T118M mutations resulted in increased structural instability compared to the native form, which is consistent with previous experimental findings of increased structural fluctuations along a loop connecting transmembrane α-helix1 and α-helix2. Docking and MD simulations coupled with the MMGBSA approach allowed the identification that the binding interface for the PMP22-RER1 complex takes place through transmembrane α-helix1 and α-helix2, with higher effective binding free energy values between the mutated PMP22 systems and RER1 than for the native PMP22, mainly through van der Waals interactions.

Structural investigation of a C-terminal EphA2 receptor mutant: Does mutation affect the structure and interaction properties of the Sam domain?

PubMed

Mercurio, Flavia A; Costantini, Susan; Di Natale, Concetta; Pirone, Luciano; Guariniello, Stefano; Scognamiglio, Pasqualina L; Marasco, Daniela; Pedone, Emilia M; Leone, Marilisa

2017-09-01

Ephrin A2 receptor (EphA2) plays a key role in cancer, it is up-regulated in several types of tumors and the process of ligand-induced receptor endocytosis, followed by degradation, is considered as a potential path to diminish tumor malignancy. Protein modulators of this mechanism are recruited at the cytosolic Sterile alpha motif (Sam) domain of EphA2 (EphA2-Sam) through heterotypic Sam-Sam associations. These interactions engage the C-terminal helix of EphA2 and close loop regions (the so called End Helix side). In addition, several studies report on destabilizing mutations in EphA2 related to cataract formation and located in/or close to the Sam domain. Herein, we analyzed from a structural point of view, one of these mutants characterized by the insertion of a novel 39 residue long polypeptide at the C-terminus of EphA2-Sam. A 3D structural model was built by computational methods and revealed partial disorder in the acquired C-terminal tail and a few residues participating in an α-helix and two short β-strands. We investigated by CD and NMR studies the conformational properties in solution of two peptides encompassing the whole C-terminal tail and its predicted helical region, respectively. NMR binding experiments demonstrated that these peptides do not interact relevantly with either EphA2-Sam or its interactor Ship2-Sam. Molecular dynamics (MD) simulations further indicated that the EphA2 mutant could be represented only through a conformational ensemble and that the C-terminal tail should not largely wrap the EphA2-Sam End-Helix interface and affect binding to other Sam domains. Copyright © 2017 Elsevier B.V. All rights reserved.

Overlapping Binding Sites of Structurally Different Antiarrhythmics Flecainide and Propafenone in the Subunit Interface of Potassium Channel Kv2.1*

PubMed Central

Madeja, Michael; Steffen, Wibke; Mesic, Ivana; Garic, Bojan; Zhorov, Boris S.

2010-01-01

Kv2.1 channels, which are expressed in brain, heart, pancreas, and other organs and tissues, are important targets for drug design. Flecainide and propafenone are known to block Kv2.1 channels more potently than other Kv channels. Here, we sought to explore structural determinants of this selectivity. We demonstrated that flecainide reduced the K+ currents through Kv2.1 channels expressed in Xenopus laevis oocytes in a voltage- and time-dependent manner. By systematically exchanging various segments of Kv2.1 with those from Kv1.2, we determined flecainide-sensing residues in the P-helix and inner helix S6. These residues are not exposed to the inner pore, a conventional binding region of open channel blockers. The flecainide-sensing residues also contribute to propafenone binding, suggesting overlapping receptors for the drugs. Indeed, propafenone and flecainide compete for binding in Kv2.1. We further used Monte Carlo-energy minimizations to map the receptors of the drugs. Flecainide docking in the Kv1.2-based homology model of Kv2.1 predicts the ligand ammonium group in the central cavity and the benzamide moiety in a niche between S6 and the P-helix. Propafenone also binds in the niche. Its carbonyl group accepts an H-bond from the P-helix, the amino group donates an H-bond to the P-loop turn, whereas the propyl group protrudes in the pore and blocks the access to the selectivity filter. Thus, besides the binding region in the central cavity, certain K+ channel ligands can expand in the subunit interface whose residues are less conserved between K+ channels and hence may be targets for design of highly desirable subtype-specific K+ channel drugs. PMID:20709754

The structure of the ends of α-helices in globular proteins: effect of additional hydrogen bonds and implications for helix formation.

PubMed

Leader, David P; Milner-White, E James

2011-03-01

We prepared a set of about 2000 α-helices from a relational database of high-resolution three-dimensional structures of globular proteins, and identified additional main chain i ← i+3 hydrogen bonds at the ends of the helices (i.e., where the hydrogen bonding potential is not fulfilled by canonical i ← i+4 hydrogen bonds). About one-third of α-helices have such additional hydrogen bonds at the N-terminus, and more than half do so at the C-terminus. Although many of these additional hydrogen bonds at the C-terminus are associated with Schellman loops, the majority are not. We compared the dihedral angles at the termini of α-helices having or lacking the additional hydrogen bonds. Significant differences were found, especially at the C-terminus, where the dihedral angles at positions C2 and C1 in the absence of additional hydrogen bonds deviate substantially from those occurring within the α-helix. Using a novel approach we show how the structure of the C-terminus of the α-helix can emerge from that of constituent overlapping α-turns and β-turns, which individually show a variation in dihedral angles at different positions. We have also considered the direction of propagation of the α-helix using this approach. If one assumes that helices start as a single α-turn and grow by successive addition of further α-turns, the paths for growth in the N → C and C → N directions differ in a way that suggests that extension in the C → N direction is favored. Copyright © 2010 Wiley-Liss, Inc.

Structures and mechanism of dipeptidyl peptidases 8 and 9, important players in cellular homeostasis and cancer.

PubMed

Ross, Breyan; Krapp, Stephan; Augustin, Martin; Kierfersauer, Reiner; Arciniega, Marcelino; Geiss-Friedlander, Ruth; Huber, Robert

2018-02-13

Dipeptidyl peptidases 8 and 9 are intracellular N-terminal dipeptidyl peptidases (preferentially postproline) associated with pathophysiological roles in immune response and cancer biology. While the DPP family member DPP4 is extensively characterized in molecular terms as a validated therapeutic target of type II diabetes, experimental 3D structures and ligand-/substrate-binding modes of DPP8 and DPP9 have not been reported. In this study we describe crystal and molecular structures of human DPP8 (2.5 Å) and DPP9 (3.0 Å) unliganded and complexed with a noncanonical substrate and a small molecule inhibitor, respectively. Similar to DPP4, DPP8 and DPP9 molecules consist of one β-propeller and α/β hydrolase domain, forming a functional homodimer. However, they differ extensively in the ligand binding site structure. In intriguing contrast to DPP4, where liganded and unliganded forms are closely similar, ligand binding to DPP8/9 induces an extensive rearrangement at the active site through a disorder-order transition of a 26-residue loop segment, which partially folds into an α-helix (R-helix), including R160/133, a key residue for substrate binding. As vestiges of this helix are also seen in one of the copies of the unliganded form, conformational selection may contributes to ligand binding. Molecular dynamics simulations support increased flexibility of the R-helix in the unliganded state. Consistently, enzyme kinetics assays reveal a cooperative allosteric mechanism. DPP8 and DPP9 are closely similar and display few opportunities for targeted ligand design. However, extensive differences from DPP4 provide multiple cues for specific inhibitor design and development of the DPP family members as therapeutic targets or antitargets.

The visible extinction peaks of Ag nanohelixes: A periodic effective dipole model

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

Zhang, Z.-Y.; Zhao, Y.-P.

2011-02-21

Using the discrete dipole approximation method, two visible extinction peaks are found for Ag nanohelixes. Both of them redshift periodically in an approximate half pitch with the helix height and redshift linearly with the helix diameter and pitch height. At the two absorbance peaks, an integer number of E-field maxima occur along the helix. These field maxima could be treated as results of collective electron oscillations by periodic effective dipoles within a half pitch along the helix. The wavelengths of the absorbance peaks are found to scale with the effective dipole length, which is consistent with the periodic structure ofmore » the helix.« less

Compressive response and helix formation of a semiflexible polymer confined in a nanochannel

NASA Astrophysics Data System (ADS)

Hayase, Yumino; Sakaue, Takahiro; Nakanishi, Hiizu

2017-05-01

Configurations of a single semiflexible polymer is studied when it is pushed into a nanochannel in the case where the polymer persistence length lp is much longer than the channel diameter D :lp/D ≫1 . Using numerical simulations, we show that the polymer undergoes a sequence of recurring structural transitions upon longitudinal compression: random deflection along the channel, a helix going around the channel wall, double-fold random deflection, double-fold helix, etc. We find that the helix transition can be understood as buckling of deflection segments, and the initial helix formation takes place at very small compression with no appreciable weak compression regime of the random deflection polymer.

RNA secondary structure prediction using soft computing.

PubMed

Ray, Shubhra Sankar; Pal, Sankar K

2013-01-01

Prediction of RNA structure is invaluable in creating new drugs and understanding genetic diseases. Several deterministic algorithms and soft computing-based techniques have been developed for more than a decade to determine the structure from a known RNA sequence. Soft computing gained importance with the need to get approximate solutions for RNA sequences by considering the issues related with kinetic effects, cotranscriptional folding, and estimation of certain energy parameters. A brief description of some of the soft computing-based techniques, developed for RNA secondary structure prediction, is presented along with their relevance. The basic concepts of RNA and its different structural elements like helix, bulge, hairpin loop, internal loop, and multiloop are described. These are followed by different methodologies, employing genetic algorithms, artificial neural networks, and fuzzy logic. The role of various metaheuristics, like simulated annealing, particle swarm optimization, ant colony optimization, and tabu search is also discussed. A relative comparison among different techniques, in predicting 12 known RNA secondary structures, is presented, as an example. Future challenging issues are then mentioned.

13-Helix folding of a β/γ-peptide manifold designed from a "minimal-constraint" blueprint.

PubMed

Grison, Claire M; Robin, Sylvie; Aitken, David J

2016-06-14

A bottom-up design rationale was adopted to devise β/γ-peptide foldamer manifolds which would adopt preferred 13-helix conformations, relying on minimal steric imposition brought by the constituent amino acid residues. In this way, a well-defined 13-helix conformer was revealed for short oligomers of trans-2-aminocyclobutanecarboxylic acid and γ(4)-amino acids in alternation, which gave good topological superposition upon an α-helix motif.

Effect of Hedera helix on lung histopathology in chronic asthma.

PubMed

Hocaoglu, Arzu Babayigit; Karaman, Ozkan; Erge, Duygu Olmez; Erbil, Guven; Yilmaz, Osman; Kivcak, Bijen; Bagriyanik, H Alper; Uzuner, Nevin

2012-12-01

Hedera helix is widely used to treat bronchial asthma for many years. However, effects of this herb on lung histopathology is still far from clear. We aimed to determine the effect of oral administration of Hedera helix on lung histopathology in a murine model of chronic asthma.BALB/c mice were divided into four groups; I (Placebo), II (Hedera helix), III (Dexamethasone) and IV (Control). All mice except controls were sensitized and challenged with ovalbumin. Then, mice in group I received saline, group II 100 mg/kg Hedera helix and group III 1 mg/kg dexamethasone via orogastic gavage once daily for one week. Airway histopathology was evaluated by using light and electron microscopy in all groups.Goblet cell numbers and thicknesses of basement membrane were found significantly lower in group II, but there was no statistically significant difference in terms of number of mast cells, thicknesses of epithelium and subepithelial smooth muscle layers between group I and II. When Hedera helix and dexamethasone groups were compared with each other, thickness of epithelium, subepithelial muscle layers, number of mast cells and goblet cells of group III were significantly ameliorated when compared with the group II. Although Hedera helix administration reduced only goblet cell counts and the thicknesses of basement membrane in the asthmatic airways, dexamethasone ameliorated all histopathologic parameters except thickness of basement membrane better than Hedera helix.

Probing sequence dependence of folding pathway of α-helix bundle proteins through free energy landscape analysis.

PubMed

Shao, Qiang

2014-06-05

A comparative study on the folding of multiple three-α-helix bundle proteins including α3D, α3W, and the B domain of protein A (BdpA) is presented. The use of integrated-tempering-sampling molecular dynamics simulations achieves reversible folding and unfolding events in individual short trajectories, which thus provides an efficient approach to sufficiently sample the configuration space of protein and delineate the folding pathway of α-helix bundle. The detailed free energy landscape analyses indicate that the folding mechanism of α-helix bundle is not uniform but sequence dependent. A simple model is then proposed to predict folding mechanism of α-helix bundle on the basis of amino acid composition: α-helical proteins containing higher percentage of hydrophobic residues than charged ones fold via nucleation-condensation mechanism (e.g., α3D and BdpA) whereas proteins having opposite tendency in amino acid composition more likely fold via the framework mechanism (e.g., α3W). The model is tested on various α-helix bundle proteins, and the predicted mechanism is similar to the most approved one for each protein. In addition, the common features in the folding pathway of α-helix bundle protein are also deduced. In summary, the present study provides comprehensive, atomic-level picture of the folding of α-helix bundle proteins.

Kinks, loops, and protein folding, with protein A as an example

PubMed Central

Krokhotin, Andrey; Liwo, Adam; Maisuradze, Gia G.; Niemi, Antti J.; Scheraga, Harold A.

2014-01-01

The dynamics and energetics of formation of loops in the 46-residue N-terminal fragment of the B-domain of staphylococcal protein A has been studied. Numerical simulations have been performed using coarse-grained molecular dynamics with the united-residue (UNRES) force field. The results have been analyzed in terms of a kink (heteroclinic standing wave solution) of a generalized discrete nonlinear Schrödinger (DNLS) equation. In the case of proteins, the DNLS equation arises from a Cα-trace-based energy function. Three individual kink profiles were identified in the experimental three-α-helix structure of protein A, in the range of the Glu16-Asn29, Leu20-Asn29, and Gln33-Asn44 residues, respectively; these correspond to two loops in the native structure. UNRES simulations were started from the full right-handed α-helix to obtain a clear picture of kink formation, which would otherwise be blurred by helix formation. All three kinks emerged during coarse-grained simulations. It was found that the formation of each is accompanied by a local free energy increase; this is expressed as the change of UNRES energy which has the physical sense of the potential of mean force of a polypeptide chain. The increase is about 7 kcal/mol. This value can thus be considered as the free energy barrier to kink formation in full α-helical segments of polypeptide chains. During the simulations, the kinks emerge, disappear, propagate, and annihilate each other many times. It was found that the formation of a kink is initiated by an abrupt change in the orientation of a pair of consecutive side chains in the loop region. This resembles the formation of a Bloch wall along a spin chain, where the Cα backbone corresponds to the chain, and the amino acid side chains are interpreted as the spin variables. This observation suggests that nearest-neighbor side chain–side chain interactions are responsible for initiation of loop formation. It was also found that the individual kinks are reflected as clear peaks in the principal modes of the analyzed trajectory of protein A, the shapes of which resemble the directional derivatives of the kinks along the chain. These observations suggest that the kinks of the DNLS equation determine the functionally important motions of proteins. PMID:24437917

Elevated temperature triggers human respiratory syncytial virus F protein six-helix bundle formation

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

Yunus, Abdul S.; Jackson, Trent P.; Crisafi, Katherine

2010-01-20

Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infection in infants, immunocompromised patients, and the elderly. The RSV fusion (F) protein mediates fusion of the viral envelope with the target cell membrane during virus entry and is a primary target for antiviral drug and vaccine development. The F protein contains two heptad repeat regions, HR1 and HR2. Peptides corresponding to these regions form a six-helix bundle structure that is thought to play a critical role in membrane fusion. However, characterization of six-helix bundle formation in native RSV F protein has been hindered by themore » fact that a trigger for F protein conformational change has yet to be identified. Here we demonstrate that RSV F protein on the surface of infected cells undergoes a conformational change following exposure to elevated temperature, resulting in the formation of the six-helix bundle structure. We first generated and characterized six-helix bundle-specific antibodies raised against recombinant peptides modeling the RSV F protein six-helix bundle structure. We then used these antibodies as probes to monitor RSV F protein six-helix bundle formation in response to a diverse array of potential triggers of conformational changes. We found that exposure of 'membrane-anchored' RSV F protein to elevated temperature (45-55 deg. C) was sufficient to trigger six-helix bundle formation. Antibody binding to the six-helix bundle conformation was detected by both flow cytometry and cell-surface immunoprecipitation of the RSV F protein. None of the other treatments, including interaction with a number of potential receptors, resulted in significant binding by six-helix bundle-specific antibodies. We conclude that native, untriggered RSV F protein exists in a metastable state that can be converted in vitro to the more stable, fusogenic six-helix bundle conformation by an increase in thermal energy. These findings help to better define the mechanism of RSV F-mediated membrane fusion and have important implications for the identification of therapeutic strategies and vaccines targeting RSV F protein conformational changes.« less

An allosterically regulated reversible mechanical molecular switch: A de novo protein maquette functions as a redox/ionic strength sensor coupling chemical binding energy or charge interactions to conformational change

NASA Astrophysics Data System (ADS)

Grosset, Anne Marie

2000-10-01

Switch-like structural rearrangements of subunits due to charge-interactions are common in the basic biological action of proteins that couple and transfer chemical and ionic signals, sensing and regulation, mechanical force and electrochemical free energy. A simple synthetic protein model (maquette) has been designed to better understand the engineering of natural switches. Basic thermodynamic principles define the two key elements required for biological or chemical function of a switch. First, there must be two well-defined states. In this case, the two conformational states must have an energetic difference (DeltaDeltaG°) that is spanned by the applied driving force. Second, there must be an external stimulus, which preferentially interacts with one of the two states. The external stimulus provides the driving force that shifts the equilibrium from the first state to the second state (≥10:1 shifting towards ≤1:10). The energetic difference between the states must be the same order of magnitude as the driving force. In this synthetic protein, the two conformational states correspond to parallel (syn) and antiparallel (anti) assembly of the two identical helix-ss-helix subunits that bind heme close to the di-sulfide loop region. Charge interactions between two ferric hemes bound to histidines provide a driving force on the order of 2 kcal/mol (corresponding in the syn-topology to the 75--100 mV split in the heme redox potentials, or the 25--80 times weaker binding for the second ferric heme). The tetra-alpha-helix bundle has been modified to have a DeltaG around 1.8--2.5 kcal/mol (a 50--80 fold difference in the anti/syn ratio). Therefore, oxidation and reduction of the heme, or the binding of a second charged ferric heme can reversibly switch between syn- and anti-topologies, providing a sensitive detector of redox state or heme concentration. External solution conditions (e.g. ionic composition) can act on the protein remotely from the primary internal switch action and confer a secondary level of allosteric regulation. Bifunctional ligands can link subunits to shift topology. Scanning redox potentiometry can monitor the kinetics of topological change. Point amino acid substitutions and computer repacking of the hydrophobic core can modulate both the kinetics and the energetics.

Model for an RNA tertiary interaction from the structure of an intermolecular complex between a GAAA tetraloop and an RNA helix.

PubMed

Pley, H W; Flaherty, K M; McKay, D B

1994-11-03

In large structured RNAs, RNA hairpins in which the strands of the duplex stem are connected by a tetraloop of the consensus sequence 5'-GNRA (where N is any nucleotide, and R is either G or A) are unusually frequent. In group I introns there is a covariation in sequence between nucleotides in the third and fourth positions of the loop with specific distant base pairs in putative RNA duplex stems: GNAA loops correlate with successive 5'-C-C.G-C base pairs in stems, whereas GNGA loops correlate with 5'-C-U.G-A. This has led to the suggestion that GNRA tetraloops may be involved in specific long-range tertiary interactions, with each A in position 3 or 4 of the loop interacting with a C-G base pair in the duplex, and G in position 3 interacting with a U-A base pair. This idea is supported experimentally for the GAAA loop of the P5b extension of the group I intron of Tetrahymena thermophila and the L9 GUGA terminal loop of the td intron of bacteriophage T4 (ref. 4). NMR has revealed the overall structure of the tetraloop for 12-nucleotide hairpins with GCAA and GAAA loops and models have been proposed for the interaction of GNRA tetraloops with base pairs in the minor groove of A-form RNA. Here we describe the crystal structure of an intermolecular complex between a GAAA tetraloop and an RNA helix. The interactions we observe correlate with the specificity of GNRA tetraloops inferred from phylogenetic studies, suggesting that this complex is a legitimate model for intramolecular tertiary interactions mediated by GNRA tetraloops in large structured RNAs.

Myotonic Dystrophy Type 1 RNA Crystal Structures Reveal Heterogeneous 1×1 Nucleotide UU Internal Loop Conformations⊥

PubMed Central

Kumar, Amit; Park, HaJeung; Fang, Pengfei; Parkesh, Raman; Guo, Min; Nettles, Kendall W.; Disney, Matthew D.

2011-01-01

RNA internal loops often display a variety of conformations in solution. Herein, we visualize conformational heterogeneity in the context of the 5′CUG/3′GUC repeat motif present in the RNA that causes myotonic dystrophy type 1 (DM1). Specifically, two crystal structures are disclosed of a model DM1 triplet repeating construct, 5′r(UUGGGC(CUG)3GUCC)2, refined to 2.20 Å and 1.52 Å resolution. Here, differences in orientation of the 5′ dangling UU end between the two structures induce changes in the backbone groove width, which reveals that non-canonical 1×1 nucleotide UU internal loops can display an ensemble of pairing conformations. In the 2.20 Å structure, CUGa, the 5′UU forms one hydrogen-bonded pairs with a 5′UU of a neighboring helix in the unit cell to form a pseudo-infinite helix. The central 1×1 nucleotide UU internal loop has no hydrogen bonds, while the terminal 1×1 nucleotide UU internal loops each form a one hydrogen-bonded pair. In the 1.52 Å structure, CUGb, the 5′ UU dangling end is tucked into the major groove of the duplex. While the canonical paired bases show no change in base pairing, in CUGb the terminal 1×1 nucleotide UU internal loops form now two hydrogen-bonded pairs. Thus, the shift in major groove induced by the 5′UU dangling end alters non-canonical base patterns. Collectively, these structures indicate that 1×1 nucleotide UU internal loops in DM1 may sample multiple conformations in vivo. This observation has implications for the recognition of this RNA, and other repeating transcripts, by protein and small molecule ligands. PMID:21988728

Myotonic dystrophy type 1 RNA crystal structures reveal heterogeneous 1 × 1 nucleotide UU internal loop conformations.

PubMed

Kumar, Amit; Park, HaJeung; Fang, Pengfei; Parkesh, Raman; Guo, Min; Nettles, Kendall W; Disney, Matthew D

2011-11-15

RNA internal loops often display a variety of conformations in solution. Herein, we visualize conformational heterogeneity in the context of the 5'CUG/3'GUC repeat motif present in the RNA that causes myotonic dystrophy type 1 (DM1). Specifically, two crystal structures of a model DM1 triplet repeating construct, 5'r[UUGGGC(CUG)(3)GUCC](2), refined to 2.20 and 1.52 Å resolution are disclosed. Here, differences in the orientation of the 5' dangling UU end between the two structures induce changes in the backbone groove width, which reveals that noncanonical 1 × 1 nucleotide UU internal loops can display an ensemble of pairing conformations. In the 2.20 Å structure, CUGa, the 5' UU forms a one hydrogen-bonded pair with a 5' UU of a neighboring helix in the unit cell to form a pseudoinfinite helix. The central 1 × 1 nucleotide UU internal loop has no hydrogen bonds, while the terminal 1 × 1 nucleotide UU internal loops each form a one-hydrogen bond pair. In the 1.52 Å structure, CUGb, the 5' UU dangling end is tucked into the major groove of the duplex. While the canonically paired bases show no change in base pairing, in CUGb the terminal 1 × 1 nucleotide UU internal loops now form two hydrogen-bonded pairs. Thus, the shift in the major groove induced by the 5' UU dangling end alters noncanonical base patterns. Collectively, these structures indicate that 1 × 1 nucleotide UU internal loops in DM1 may sample multiple conformations in vivo. This observation has implications for the recognition of this RNA, and other repeating transcripts, by protein and small molecule ligands.

Myotonic Dystrophy Type 1 RNA Crystal Structures Reveal Heterogeneous 1 × 1 Nucleotide UU Internal Loop Conformations

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

Kumar, Amit; Park, HaJeung; Fang, Pengfei

2012-03-27

RNA internal loops often display a variety of conformations in solution. Herein, we visualize conformational heterogeneity in the context of the 5'CUG/3'GUC repeat motif present in the RNA that causes myotonic dystrophy type 1 (DM1). Specifically, two crystal structures of a model DM1 triplet repeating construct, 5'r[{und UU}GGGC(C{und U}G){sub 3}GUCC]{sub 2}, refined to 2.20 and 1.52 {angstrom} resolution are disclosed. Here, differences in the orientation of the 5' dangling UU end between the two structures induce changes in the backbone groove width, which reveals that noncanonical 1 x 1 nucleotide UU internal loops can display an ensemble of pairing conformations.more » In the 2.20 {angstrom} structure, CUGa, the 5' UU forms a one hydrogen-bonded pair with a 5' UU of a neighboring helix in the unit cell to form a pseudoinfinite helix. The central 1 x 1 nucleotide UU internal loop has no hydrogen bonds, while the terminal 1 x 1 nucleotide UU internal loops each form a one-hydrogen bond pair. In the 1.52 {angstrom} structure, CUGb, the 5' UU dangling end is tucked into the major groove of the duplex. While the canonically paired bases show no change in base pairing, in CUGb the terminal 1 x 1 nucleotide UU internal loops now form two hydrogen-bonded pairs. Thus, the shift in the major groove induced by the 5' UU dangling end alters noncanonical base patterns. Collectively, these structures indicate that 1 x 1 nucleotide UU internal loops in DM1 may sample multiple conformations in vivo. This observation has implications for the recognition of this RNA, and other repeating transcripts, by protein and small molecule ligands.« less

Rosalind Franklin's X-ray photo of DNA as an undergraduate optical diffraction experiment

NASA Astrophysics Data System (ADS)

Thompson, J.; Braun, G.; Tierney, D.; Wessels, L.; Schmitzer, H.; Rossa, B.; Wagner, H. P.; Dultz, W.

2018-02-01

Rosalind Franklin's X-ray diffraction patterns of DNA molecules rendered the important clue that DNA has the structure of a double helix. The most famous X-ray photograph, Photo 51, is still printed in most Biology textbooks. We suggest two optical experiments for undergraduates that make this historic achievement comprehensible for students by using macromodels of DNA and visible light to recreate a diffraction pattern similar to Photo 51. In these macromodels, we replace the double helix both mathematically and experimentally with its two-dimensional (flat) projection and explain why this is permissible. Basic optical concepts are used to infer certain well-known characteristics of DNA from the diffraction pattern.

A TWT upgrade to study wave-particle interactions in plasma

NASA Astrophysics Data System (ADS)

Doveil, Fabrice; Caetano de Sousa, Meirielen; Guyomarc'h, Didier; Kahli, Aissa; Elskens, Yves

2015-11-01

Beside industrial applications, Traveling Wave Tubes (TWT) are useful to mimic and study wave-particle interaction in plasma. We upgraded a TWT, whose slow wave structure is a 4 m long helix (diameter 3.4 cm, pitch 1 mm) of Be-Cu wire (diameter 0.6 mm) wrapped in insulating tape. The helix is inserted in a vacuum glass tube. At one end, an electron gun produces a beam propagating along the helix, radially confined by a constant axial magnetic field. Movable probes, capacitively coupled to the helix through the glass tube, launch and monitor waves generated by an arbitrary waveform generator at a few tens of MHz. At the other end of the helix, a trochoidal analyzer allows to reconstruct the electron distribution functions of the beam after its self-consistent interaction with the waves. Linear properties of the new device will be reported. The measured coupling coefficients of each probe with the helix are used to reconstruct the growth and saturation of a launched wave as it interacts with the electron beam. J-B. Faure and V. Long are thanked for their efficient help in designing and using a new way to build the helix.

Characterization of the Interactions between Calmodulin and Death Receptor 5 in Triple-negative and Estrogen Receptor-positive Breast Cancer Cells

PubMed Central

Fancy, Romone M.; Wang, Lingyun; Zeng, Qinghua; Wang, Hong; Zhou, Tong; Buchsbaum, Donald J.; Song, Yuhua

2016-01-01

Activation of death receptor-5 (DR5) leads to the formation of death inducing signaling complex (DISC) for apoptotic signaling. Targeting DR5 to induce breast cancer apoptosis is a promising strategy to circumvent drug resistance and present a target for breast cancer treatment. Calmodulin (CaM) has been shown to regulate DR5-mediated apoptotic signaling, however, its mechanism remains unknown. In this study, we characterized CaM and DR5 interactions in breast cancer cells with integrated experimental and computational approaches. Results show that CaM directly binds to DR5 in a calcium dependent manner in breast cancer cells. The direct interaction of CaM with DR5 is localized at DR5 death domain. We have predicted and verified the CaM-binding site in DR5 being 354WEPLMRKLGL363 that is located at the α2 helix and the loop between α2 helix and α3 helix of DR5 DD. The residues of Trp-354, Arg-359, Glu-355, Leu-363, and Glu-367 in DR5 death domain that are important for DR5 recruitment of FADD and caspase-8 for DISC formation to signal apoptosis also play an important role for CaM-DR5 binding. The changed electrostatic potential distribution in the CaM-binding site in DR5 DD by the point mutations of W354A, E355K, R359A, L363N, or E367K in DR5 DD could directly contribute to the experimentally observed decreased CaM-DR5 binding by the point mutations of the key residues in DR5 DD. Results from this study provide a key step for the further investigation of the role of CaM-DR5 binding in DR5-mediated DISC formation for apoptosis in breast cancer cells. PMID:27129269

NGC 7293, the Helix Nebula

NASA Image and Video Library

2012-05-16

NGC 7293, better known as the Helix nebula, displays its ultraviolet glow courtesy of NASA GALEX. The Helix is the nearest example of a planetary nebula, which is the eventual fate of a star, like our own Sun, as it approaches the end of its life.

Quadrifilar Helix Antenna for Enhanced Air-to-Ground Communications

DTIC Science & Technology

2016-05-01

ARL-TR-7679 ● MAY 2016 US Army Research Laboratory Quadrifilar Helix Antenna for Enhanced Air-to- Ground Communications by...Research Laboratory Quadrifilar Helix Antenna for Enhanced Air-to- Ground Communications by Steven D Keller, William O Coburn, Theodore K Anthony...

Experimental and Theoretical Investigation of Innovative Broadband Microwave Devices

DTIC Science & Technology

1991-05-01

over conventional devices such as the helix TWT . The TSA is also a broad band device; however, the bandwidth of the configuration chosen ultimately...by the TSA over the helix TWT in regard to bandwidth as well. Although the high gain in the TSA confers the advantage of being relatively less...prebuncher for the output helix stage. Thus. the device is essentially a helix TWT im (k) =45C with enhanced bunching due to the two-stream interac-Re() 2+C

Interplay of secondary structures and side-chain contacts in the denatured state of BBA1

NASA Astrophysics Data System (ADS)

Wen, Edward Z.; Luo, Ray

2004-08-01

The denatured state of a miniprotein BBA1 is studied under the native condition with the AMBER/Poisson-Boltzmann energy model and with the self-guided enhanced sampling technique. Forty independent trajectories are collected to sample the highly diversified denatured structures. Our simulation data show that the denatured BBA1 contains high percentage of native helix and native turn, but low percentage of native hairpin. Conditional population analysis indicates that the native helix formation and the native hairpin formation are not cooperative in the denatured state. Side-chain analysis shows that the native hydrophobic contacts are more preferred than the non-native hydrophobic contacts in the denatured BBA1. In contrast, the salt-bridge contacts are more or less nonspecific even if their populations are higher than those of hydrophobic contacts. Analysis of the trajectories shows that the native helix mostly initiates near the N terminus and propagates to the C terminus, and mostly forms from 310-helix/turn to α helix. The same analysis shows that the native turn is important but not necessary in its formation in the denatured BBA1. In addition, the formations of the two strands in the native hairpin are rather asymmetric, demonstrating the likely influence of the protein environment. Energetic analysis shows that the native helix formation is largely driven by electrostatic interactions in denatured BBA1. Further, the native helix formation is associated with the breakup of non-native salt-bridge contacts and the accumulation of native salt-bridge contacts. However, the native hydrophobic contacts only show a small increase upon the native helix formation while the non-native hydrophobic contacts stay essentially the same, different from the evolution of hydrophobic contacts observed in an isolated helix folding.

Government and Governance of Regional Triple Helix Interactions

ERIC Educational Resources Information Center

Danson, Mike; Todeva, Emanuela

2016-01-01

This conceptual paper contributes to the discussion of the role of regional government and regional Triple Helix constellations driving economic development and growth within regional boundaries. The impact of regionalism and subsidiarity on regional Triple Helix constellations, and the questions of governmentality, governance and institutional…

The swimming of a perfect deforming helix

NASA Astrophysics Data System (ADS)

Koens, Lyndon; Zhang, Hang; Mourran, Ahmed; Lauga, Eric

2017-11-01

Many bacteria rotate helical flagellar filaments in order to swim. When at rest or rotated counter-clockwise these flagella are left handed helices but they undergo polymorphic transformations to right-handed helices when the motor is reversed. These helical deformations themselves can generate motion, with for example Rhodobacter sphaeroides using the polymorphic transformation of the flagellum to generate rotation, or Spiroplasma propagating a change of helix handedness across its body's length to generate forward motion. Recent experiments reported on an artificial helical microswimmer generating motion without a propagating change in handedness. Made of a temperature sensitive gel, these swimmers moved by changing the dimensions of the helix in a non-reciprocal way. Inspired by these results and helix's ubiquitous presence in the bacterial world, we investigate how a deforming helix moves within a viscous fluid. Maintaining a single handedness along its entire length, we discuss how a perfect deforming helix can create a non-reciprocal swimming stroke, identify its principle directions of motion, and calculate the swimming kinematics asymptotically.

The helix bundle: A reversible lipid binding motif

PubMed Central

Narayanaswami, Vasanthy; Kiss, Robert S.; Weers, Paul M.M.

2009-01-01

Apolipoproteins are the protein components of lipoproteins that have the innate ability to inter convert between a lipid-free and a lipid-bound form in a facile manner, a remarkable property conferred by the helix bundle motif. Composed of a series of four or five amphipathic α-helices that fold to form a helix bundle, this motif allows the en face orientation of the hydrophobic faces of the α-helices in the protein interior in the lipid-free state. A conformational switch then permits helix-helix interactions to be substituted by helix-lipid interactions upon lipid binding interaction. This review compares the apolipoprotein high resolution structures and the factors that trigger this switch in insect apolipophorin III and the mammalian apolipoproteins, apolipoprotein E and apolipoprotein A-I, pointing out the commonalities and key differences in the mode of lipid interaction. Further insights into the lipid bound conformation of apolipoproteins are required to fully understand their functional role under physiological conditions. PMID:19770066

Osmolyte effects on helix formation in peptides and the stability of coiled-coils

PubMed Central

Celinski, Scott A.; Scholtz, J. Martin

2002-01-01

The ability of several naturally occurring substances known as osmolytes to induce helix formation in an alanine-based peptide have been investigated. As predicted by the osmophobic effect hypothesis, the osmolytes studies here do induce helix formation. Trimethylamine-N-oxide (TMAO) is the best structure-inducing osmolytes investigated here, but it is not as effective in promoting helix formation as the common cosolvent trifluoroethanol (TFE). We also provide a semiquantitative study of the ability of TMAO to induce helix formation and urea, which acts as a helix (and protein) denaturant. We find that on a molar basis, these agents are exactly counteractive as structure inducing and unfolding agents. Finally, we extend the investigations to the effects of urea and TMAO on the stability of a dimeric coiled-coil peptide and find identical results. Together these results support the tenets of the osmophobic hypothesis and highlight the importance of the polypeptide backbone in protein folding and stability. PMID:12142459

Cytotoxic Helix-Rich Oligomer Formation by Melittin and Pancreatic Polypeptide

PubMed Central

Singh, Pradeep K.; Ghosh, Dhiman; Tewari, Debanjan; Mohite, Ganesh M.; Carvalho, Edmund; Jha, Narendra Nath; Jacob, Reeba S.; Sahay, Shruti; Banerjee, Rinti; Bera, Amal K.; Maji, Samir K.

2015-01-01

Conversion of amyloid fibrils by many peptides/proteins involves cytotoxic helix-rich oligomers. However, their toxicity and biophysical studies remain largely unknown due to their highly dynamic nature. To address this, we chose two helical peptides (melittin, Mel and pancreatic polypeptide, PP) and studied their aggregation and toxicity. Mel converted its random coil structure to oligomeric helical structure upon binding to heparin; however, PP remained as helix after oligomerization. Interestingly, similar to Parkinson’s associated α-synuclein (AS) oligomers, Mel and PP also showed tinctorial properties, higher hydrophobic surface exposure, cellular toxicity and membrane pore formation after oligomerization in the presence of heparin. We suggest that helix-rich oligomers with exposed hydrophobic surface are highly cytotoxic to cells irrespective of their disease association. Moreover as Mel and PP (in the presence of heparin) instantly self-assemble into stable helix-rich amyloidogenic oligomers; they could be represented as models for understanding the biophysical and cytotoxic properties of helix-rich intermediates in detail. PMID:25803428

Apex Dips of Experimental Flux Ropes: Helix or Cusp?

NASA Astrophysics Data System (ADS)

Wongwaitayakornkul, Pakorn; Haw, Magnus A.; Li, Hui; Li, Shengtai; Bellan, Paul M.

2017-10-01

We present a new theory for the presence of apex dips in certain experimental flux ropes. Previously such dips were thought to be projections of a helical loop axis generated by the kink instability. However, new evidence from experiments and simulations suggest that the feature is a 2D cusp rather than a 3D helix. The proposed mechanism for cusp formation is a density pileup region generated by nonlinear interaction of neutral gas cones emitted from fast-gas nozzles. The results indicate that density perturbations can result in large distortions of an erupting flux rope, even in the absence of significant pressure or gravitational forces. The density pileup at the apex also suppresses the m = 1 kink mode by acting as a stationary node. Consequently, more accurate density profiles should be considered when attempting to model the stability and shape of solar and astrophysical flux ropes.

Apex Dips of Experimental Flux Ropes: Helix or Cusp?

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

Wongwaitayakornkul, Pakorn; Haw, Magnus A.; Bellan, Paul M.

We present a new theory for the presence of apex dips in certain experimental flux ropes. Previously such dips were thought to be projections of a helical loop axis generated by the kink instability. However, new evidence from experiments and simulations suggest that the feature is a 2D cusp rather than a 3D helix. The proposed mechanism for cusp formation is a density pileup region generated by nonlinear interaction of neutral gas cones emitted from fast-gas nozzles. The results indicate that density perturbations can result in large distortions of an erupting flux rope, even in the absence of significant pressuremore » or gravitational forces. The density pileup at the apex also suppresses the m = 1 kink mode by acting as a stationary node. Consequently, more accurate density profiles should be considered when attempting to model the stability and shape of solar and astrophysical flux ropes.« less

Sequence analysis of RNase MRP RNA reveals its origination from eukaryotic RNase P RNA

PubMed Central

Zhu, Yanglong; Stribinskis, Vilius; Ramos, Kenneth S.; Li, Yong

2006-01-01

RNase MRP is a eukaryote-specific endoribonuclease that generates RNA primers for mitochondrial DNA replication and processes precursor rRNA. RNase P is a ubiquitous endoribonuclease that cleaves precursor tRNA transcripts to produce their mature 5′ termini. We found extensive sequence homology of catalytic domains and specificity domains between their RNA subunits in many organisms. In Candida glabrata, the internal loop of helix P3 is 100% conserved between MRP and P RNAs. The helix P8 of MRP RNA from microsporidia Encephalitozoon cuniculi is identical to that of P RNA. Sequence homology can be widely spread over the whole molecule of MRP RNA and P RNA, such as those from Dictyostelium discoideum. These conserved nucleotides between the MRP and P RNAs strongly support the hypothesis that the MRP RNA is derived from the P RNA molecule in early eukaryote evolution. PMID:16540690

Impaired Thermogenesis and a Molecular Signature for Brown Adipose Tissue in Id2 Null Mice

PubMed Central

Zhou, Peng; Robles-Murguia, Maricela; Mathew, Deepa; Duffield, Giles E.

2016-01-01

Inhibitor of DNA binding 2 (ID2) is a helix-loop-helix transcriptional repressor rhythmically expressed in many adult tissues. Our previous studies have demonstrated that Id2 null mice have sex-specific elevated glucose uptake in brown adipose tissue (BAT). Here we further explored the role of Id2 in the regulation of core body temperature over the circadian cycle and the impact of Id2 deficiency on genes involved in insulin signaling and adipogenesis in BAT. We discovered a reduced core body temperature in Id2−/− mice. Moreover, in Id2−/− BAT, 30 genes including Irs1, PPARs, and PGC-1s were identified as differentially expressed in a sex-specific pattern. These data provide valuable insights into the impact of Id2 deficiency on energy homeostasis of mice in a sex-specific manner. PMID:27144179

Carboxylation of cytosine (5caC) in the CG dinucleotide in the E-box motif (CGCAG|GTG) increases binding of the Tcf3|Ascl1 helix-loop-helix heterodimer 10-fold.

PubMed

Golla, Jaya Prakash; Zhao, Jianfei; Mann, Ishminder K; Sayeed, Syed K; Mandal, Ajeet; Rose, Robert B; Vinson, Charles

2014-06-27

Three oxidative products of 5-methylcytosine (5mC) occur in mammalian genomes. We evaluated if these cytosine modifications in a CG dinucleotide altered DNA binding of four B-HLH homodimers and three heterodimers to the E-Box motif CGCAG|GTG. We examined 25 DNA probes containing all combinations of cytosine in a CG dinucleotide and none changed binding except for carboxylation of cytosine (5caC) in the strand CGCAG|GTG. 5caC enhanced binding of all examined B-HLH homodimers and heterodimers, particularly the Tcf3|Ascl1 heterodimer which increased binding ~10-fold. These results highlight a potential function of the oxidative products of 5mC, changing the DNA binding of sequence-specific transcription factors. Published by Elsevier Inc.

Evidence for a Role of the Transcriptional Regulator Maid in Tumorigenesis and Aging

PubMed Central

Fujisawa, Koichi; Terai, Shuji; Matsumoto, Toshihiko; Takami, Taro; Yamamoto, Naoki; Nishina, Hiroshi; Furutani-Seiki, Makoto; Sakaida, Isao

2015-01-01

Maid is a helix-loop-helix protein that is involved in cell proliferation. In order to further elucidate its physiological functions, we studied Maid activity in two small fish model systems. We found that Maid expression was greatest in zebrafish liver and that it increased following partial hepatectomy. Maid levels were also high in hepatic preneoplastic foci induced by treatment of zebrafish with diethylnitrosamine (DEN), but low in hepatocellular carcinomas (HCC), mixed tumors, and cholangiocarcinomas developing in these animals. In DEN-treated transgenic medaka overexpressing Maid, hepatic BrdU uptake and proliferation were reduced. After successive breedings, Maid transgenic medaka exhibited decreased movement and a higher incidence of abnormal spine curvature, possibly due to the senescence of spinal cord cells. Taken together, our results suggest that Maid levels can influence the progression of liver cancer. In conclusion, we found that Maid is important regulator of hepatocarconogenesis and aging. PMID:26107180

Mouse interleukin-2 structure-function studies: substitutions in the first alpha-helix can specifically inactivate p70 receptor binding and mutations in the fifth alpha-helix can specifically inactivate p55 receptor binding.

PubMed Central

Zurawski, S M; Zurawski, G

1989-01-01

The function of two alpha-helical regions of mouse interleukin-2 were analyzed by saturation substitution analysis. The functional parts of the first alpha-helix (A) was defined as residues 31-39 by the observation that proline substitutions within this region inactivate the protein. Four residues within alpha-helix A, Leu31, Asp34, Leu35 and Leu38, were found to be crucial for biological activity. Structural modeling suggested that these four residues are clustered on one face of alpha-helix A. Residues 31 and 35 had to remain hydrophobic for the molecule to be functional. At residue 38 there was a preference for hydrophobic side chain residues, while at residue 34 some small side chain residues as well as acidic or amide side chain residues were functionally acceptable. Inactivating changes at residue 34 had no effect upon the ability of the protein to interact with the p55 receptor. Disruption of the fifth alpha-helix (E), which had little effect upon biological activity, resulted in an inability of the protein to interact with the p55 receptor. Mutagenesis of the alpha-helix E region demonstrated that alpha-helicity and the nature of the side chain residues in this region were unimportant for biological activity. The region immediately proximal to alpha-helix E was important only for the single intramolecular disulfide linkage. PMID:2583124

A Cytosolic Amphiphilic α-Helix Controls the Activity of the Bile Acid-sensitive Ion Channel (BASIC)*

PubMed Central

Schmidt, Axel; Löhrer, Daniel; Alsop, Richard J.; Lenzig, Pia; Oslender-Bujotzek, Adrienne; Wirtz, Monika; Rheinstädter, Maikel C.; Gründer, Stefan; Wiemuth, Dominik

2016-01-01

The bile acid-sensitive ion channel (BASIC) is a member of the degenerin/epithelial Na+ channel (Deg/ENaC) family of ion channels. It is mainly found in bile duct epithelial cells, the intestinal tract, and the cerebellum and is activated by alterations of its membrane environment. Bile acids, one class of putative physiological activators, exert their effect by changing membrane properties, leading to an opening of the channel. The physiological function of BASIC, however, is unknown. Deg/ENaC channels are characterized by a trimeric subunit composition. Each subunit is composed of two transmembrane segments, which are linked by a large extracellular domain. The termini of the channels protrude into the cytosol. Many Deg/ENaC channels contain regulatory domains and sequence motifs within their cytosolic domains. In this study, we show that BASIC contains an amphiphilic α-helical structure within its N-terminal domain. This α-helix binds to the cytosolic face of the plasma membrane and stabilizes a closed state. Truncation of this domain renders the channel hyperactive. Collectively, we identify a cytoplasmic domain, unique to BASIC, that controls channel activity via membrane interaction. PMID:27679529

A Cytosolic Amphiphilic α-Helix Controls the Activity of the Bile Acid-sensitive Ion Channel (BASIC).

PubMed

Schmidt, Axel; Löhrer, Daniel; Alsop, Richard J; Lenzig, Pia; Oslender-Bujotzek, Adrienne; Wirtz, Monika; Rheinstädter, Maikel C; Gründer, Stefan; Wiemuth, Dominik

2016-11-18

The bile acid-sensitive ion channel (BASIC) is a member of the degenerin/epithelial Na + channel (Deg/ENaC) family of ion channels. It is mainly found in bile duct epithelial cells, the intestinal tract, and the cerebellum and is activated by alterations of its membrane environment. Bile acids, one class of putative physiological activators, exert their effect by changing membrane properties, leading to an opening of the channel. The physiological function of BASIC, however, is unknown. Deg/ENaC channels are characterized by a trimeric subunit composition. Each subunit is composed of two transmembrane segments, which are linked by a large extracellular domain. The termini of the channels protrude into the cytosol. Many Deg/ENaC channels contain regulatory domains and sequence motifs within their cytosolic domains. In this study, we show that BASIC contains an amphiphilic α-helical structure within its N-terminal domain. This α-helix binds to the cytosolic face of the plasma membrane and stabilizes a closed state. Truncation of this domain renders the channel hyperactive. Collectively, we identify a cytoplasmic domain, unique to BASIC, that controls channel activity via membrane interaction. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

VIEW OF EAST ELEVATION OF HELIX HOUSE NO. 2 (S87), ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

VIEW OF EAST ELEVATION OF HELIX HOUSE NO. 2 (S-87), LOOKING WEST (without scale stick). - Naval Computer & Telecommunications Area Master Station, Eastern Pacific, Radio Transmitter Facility Lualualei, Helix House No. 2, Base of Radio Antenna Structure No. 427, Makaha, Honolulu County, HI

VIEW OF EAST ELEVATION OF HELIX HOUSE NO. 2 (S87), ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

VIEW OF EAST ELEVATION OF HELIX HOUSE NO. 2 (S-87), LOOKING WEST (with scale stick). - Naval Computer & Telecommunications Area Master Station, Eastern Pacific, Radio Transmitter Facility Lualualei, Helix House No. 2, Base of Radio Antenna Structure No. 427, Makaha, Honolulu County, HI

VIEW OF HELIX HOUSE NO. 2 (S87), WITH ANTENNA TOWER ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

VIEW OF HELIX HOUSE NO. 2 (S-87), WITH ANTENNA TOWER CABLE SUPPORT IN FOREGROUND, LOOKING SOUTHEAST. - Naval Computer & Telecommunications Area Master Station, Eastern Pacific, Radio Transmitter Facility Lualualei, Helix House No. 2, Base of Radio Antenna Structure No. 427, Makaha, Honolulu County, HI

Folding thermodynamics of pseudoknotted chain conformations

PubMed Central

Kopeikin, Zoia; Chen, Shi-Jie

2008-01-01

We develop a statistical mechanical framework for the folding thermodynamics of pseudoknotted structures. As applications of the theory, we investigate the folding stability and the free energy landscapes for both the thermal and the mechanical unfolding of pseudoknotted chains. For the mechanical unfolding process, we predict the force-extension curves, from which we can obtain the information about structural transitions in the unfolding process. In general, a pseudoknotted structure unfolds through multiple structural transitions. The interplay between the helix stems and the loops plays an important role in the folding stability of pseudoknots. For instance, variations in loop sizes can lead to the destabilization of some intermediate states and change the (equilibrium) folding pathways (e.g., two helix stems unfold either cooperatively or sequentially). In both thermal and mechanical unfolding, depending on the nucleotide sequence, misfolded intermediate states can emerge in the folding process. In addition, thermal and mechanical unfoldings often have different (equilibrium) pathways. For example, for certain sequences, the misfolded intermediates, which generally have longer tails, can fold, unfold, and refold again in the pulling process, which means that these intermediates can switch between two different average end-end extensions. PMID:16674261

Peptide Fragments of Odin-Sam1: Conformational Analysis and Interaction Studies with EphA2-Sam.

PubMed

Mercurio, Flavia A; Di Natale, Concetta; Pirone, Luciano; Scognamiglio, Pasqualina L; Marasco, Daniela; Pedone, Emilia M; Saviano, Michele; Leone, Marilisa

2015-07-27

Odin is a protein belonging to the ANKS family, and has two tandem Sam domains. The first, Odin-Sam1, binds to the Sam domain of the EphA2 receptor (EphA2-Sam); this interaction could be crucial for the regulation of receptor endocytosis and might have an impact on cancer. Odin-Sam1 associates with EphA2-Sam by adopting a "mid-loop/end-helix" model. In this study three peptide sequences, encompassing the mid-loop interacting portion of Odin-Sam1 and its C-terminal α5 helix, were designed. Their conformational properties were analyzed by CD and NMR. In addition, their abilities to interact with EphA2-Sam were investigated by SPR studies. The peptides adopt a predominantly disordered state in aqueous buffer, but a higher helical content is evident in the presence of the cosolvent trifluoroethanol. Dissociation constants towards EphA2-Sam were in the high micromolar range. The structural findings suggest further routes for the design of potential anti-cancer therapeutics as inhibitors of EphA2-Sam heterotypic interactions. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

VIEW OF SOUTH ELEVATION OF HELIX HOUSE NO. 2 (S87) ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

VIEW OF SOUTH ELEVATION OF HELIX HOUSE NO. 2 (S-87) SHOWING MAIN ENTRY DOOR, LOOKING NORTH NORTHWEST. - Naval Computer & Telecommunications Area Master Station, Eastern Pacific, Radio Transmitter Facility Lualualei, Helix House No. 2, Base of Radio Antenna Structure No. 427, Makaha, Honolulu County, HI

Low Cost Expendable TWT Amplifier for ECM.

DTIC Science & Technology

1976-08-01

Figure Tit le Page 4 1 43 Proposed Low Cost TWT 90 44 “T” Shaped Tapered Helix 91 45 Coupled Helix Input 92 46 SImulated Helix by L—C Circuit 94 47...asylTuietric mode even if the helices are syninetrical in phase and amplitude at the input. In a broadband TWT , this l eads to power holes in the f...The 1-Shaped helix was fi rst descri bed by L. Winslow , as an alternative to notching support rods in high power TWT ’ s to Increase interactive

Intramolecular triple helix as a model for regular polyribonucleotide (CAA)(n).

PubMed

Efimov, Alexander V; Spirin, Alexander S

2009-10-09

The regular (CAA)(n) polyribonucleotide, as well as the omega leader sequence containing (CAA)-rich core, have recently been shown to form cooperatively melted and compact structures. In this report, we propose a structural model for the (CAA)(n) sequence in which the polyribonucleotide chain is folded upon itself, so that it forms an intramolecular triple helix. The triple helix is stabilized by hydrogen bonding between bases thus forming coplanar triads, and by stacking interactions between the base triads. A distinctive feature of the proposed triple helix is that it does not contain the canonical double-helix elements. The difference from the known triple helices is that Watson-Crick hydrogen bond pairings do not take place in the interactions between the bases within the base triads.

Magneto-chiral anisotropy of the free electron on a helix

NASA Astrophysics Data System (ADS)

Krstić, V.; Rikken, G. L. J. A.

2002-09-01

The free electron on a helix as a simple model of a molecular chiral conductor is considered. In the presence of a static magnetic field parallel to the helix axis, it is found that both ballistic and diffusive electrical transport along the helix show a conductivity that depends linearly on the current and the magnetic field and on the handedness of the helix. These results agree qualitatively with the recently experimentally observed electrical magneto-chiral anisotropy in carbon nanotubes [V. Krstić, S. Roth, M. Burghard, K. Kern, G.L.J.A. Rikken, Max-Planck-Institut für Festkörperforschung, Stuttgart, Germany; Grenoble High Magnetic Field Laboratory, MPI-CNRS, Grenoble, France; Laboratoire National des Champs Magnétiques Pulsés, CNRS/INSA/UPS, UMS 5462, Toulouse, France].

An α-Helix-Mimicking 12,13-Helix: Designed α/β/γ-Foldamers as Selective Inhibitors of Protein-Protein Interactions.

PubMed

Grison, Claire M; Miles, Jennifer A; Robin, Sylvie; Wilson, Andrew J; Aitken, David J

2016-09-05

A major current challenge in bioorganic chemistry is the identification of effective mimics of protein secondary structures that act as inhibitors of protein-protein interactions (PPIs). In this work, trans-2-aminocyclobutanecarboxylic acid (tACBC) was used as the key β-amino acid component in the design of α/β/γ-peptides to structurally mimic a native α-helix. Suitably functionalized α/β/γ-peptides assume an α-helix-mimicking 12,13-helix conformation in solution, exhibit enhanced proteolytic stability in comparison to the wild-type α-peptide parent sequence from which they are derived, and act as selective inhibitors of the p53/hDM2 interaction. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Anderson transition in a multiply-twisted helix.

PubMed

Ugajin, R

2001-06-01

We investigated the Anderson transition in a multiply-twisted helix in which a helical chain of components, i.e., atoms or nanoclusters, is twisted to produce a doubly-twisted helix, which itself can be twisted to produce a triply-twisted helix, and so on, in which there are couplings between adjacent rounds of helices. As the strength of the on-site random potentials increases, an Anderson transition occurs, suggesting that the number of dimensions is 3 for electrons running along the multiply-twisted helix when the couplings between adjacent rounds are strong enough. If the couplings are weakened, the dimensionality becomes less, resulting in localization of electrons. The effect of random connections between adjacent rounds of helices and random magnetic fields that thread the structure is analyzed using the spectral statistics of a quantum particle.

Design and characterization of an engineered gp41 protein from human immunodeficiency virus-1 as a tool for drug discovery

NASA Astrophysics Data System (ADS)

Stewart, Kent D.; Steffy, Kevin; Harris, Kevin; Harlan, John E.; Stoll, Vincent S.; Huth, Jeffrey R.; Walter, Karl A.; Gramling-Evans, Emily; Mendoza, Renaldo R.; Severin, Jean M.; Richardson, Paul L.; Barrett, Leo W.; Matayoshi, Edmund D.; Swift, Kerry M.; Betz, Stephen F.; Muchmore, Steve W.; Kempf, Dale J.; Molla, Akhter

2007-01-01

Two new proteins of approximately 70 amino acids in length, corresponding to an unnaturally-linked N- and C-helix of the ectodomain of the gp41 protein from the human immunodeficiency virus (HIV) type 1, were designed and characterized. A designed tripeptide links the C-terminus of the C-helix with the N-terminus of the N-helix in a circular permutation so that the C-helix precedes the N-helix in sequence. In addition to the artificial peptide linkage, the C-helix is truncated at its N-terminus to expose a region of the N-helix known as the "Trp-Trp-Ile" binding pocket. Sedimentation, crystallographic, and nuclear magnetic resonance studies confirmed that the protein had the desired trimeric structure with an unoccupied binding site. Spectroscopic and centrifugation studies demonstrated that the engineered protein had ligand binding characteristics similar to previously reported constructs. Unlike previous constructs which expose additional, shallow, non-conserved, and undesired binding pockets, only the single deep and conserved Trp-Trp-Ile pocket is exposed in the proteins of this study. This engineered version of gp41 protein will be potentially useful in research programs aimed at discovery of new drugs for therapy of HIV-infection in humans.

The purple cauliflower arises from activation of a MYB transcription factor.

PubMed

Chiu, Li-Wei; Zhou, Xiangjun; Burke, Sarah; Wu, Xianli; Prior, Ronald L; Li, Li

2010-11-01

Anthocyanins are responsible for the color of many flowers, fruits, and vegetables. An interesting and unique Purple (Pr) gene mutation in cauliflower (Brassica oleracea var botrytis) confers an abnormal pattern of anthocyanin accumulation, giving the striking mutant phenotype of intense purple color in curds and a few other tissues. To unravel the nature of the Pr mutation in cauliflower, we isolated the Pr gene via a combination of candidate gene analysis and fine mapping. Pr encoded a R2R3 MYB transcription factor that exhibited tissue-specific expression, consistent with an abnormal anthocyanin accumulation pattern in the mutant. Transgenic Arabidopsis (Arabidopsis thaliana) and cauliflower plants expressing the Pr-D allele recapitulated the mutant phenotype, confirming the isolation of the Pr gene. Up-regulation of Pr specifically activated a basic helix-loop-helix transcription factor and a subset of anthocyanin structural genes encoding flavonoid 3'-hydroxylase, dihydroflavonol 4-reductase, and leucoanthocyanidin dioxygenase to confer ectopic accumulation of pigments in the purple cauliflower. Our results indicate that the genetic variation including a Harbinger DNA transposon insertion in the upstream regulatory region of the Pr-D allele is responsible for the up-regulation of the Pr gene in inducing phenotypic change in the plant. The successful isolation of Pr provides important information on the regulatory control of anthocyanin biosynthesis in Brassica vegetables, and offers a genetic resource for development of new varieties with enhanced health-promoting properties and visual appeal.

The Purple Cauliflower Arises from Activation of a MYB Transcription Factor1[W][OA

PubMed Central

Chiu, Li-Wei; Zhou, Xiangjun; Burke, Sarah; Wu, Xianli; Prior, Ronald L.; Li, Li

2010-01-01

Anthocyanins are responsible for the color of many flowers, fruits, and vegetables. An interesting and unique Purple (Pr) gene mutation in cauliflower (Brassica oleracea var botrytis) confers an abnormal pattern of anthocyanin accumulation, giving the striking mutant phenotype of intense purple color in curds and a few other tissues. To unravel the nature of the Pr mutation in cauliflower, we isolated the Pr gene via a combination of candidate gene analysis and fine mapping. Pr encoded a R2R3 MYB transcription factor that exhibited tissue-specific expression, consistent with an abnormal anthocyanin accumulation pattern in the mutant. Transgenic Arabidopsis (Arabidopsis thaliana) and cauliflower plants expressing the Pr-D allele recapitulated the mutant phenotype, confirming the isolation of the Pr gene. Up-regulation of Pr specifically activated a basic helix-loop-helix transcription factor and a subset of anthocyanin structural genes encoding flavonoid 3’-hydroxylase, dihydroflavonol 4-reductase, and leucoanthocyanidin dioxygenase to confer ectopic accumulation of pigments in the purple cauliflower. Our results indicate that the genetic variation including a Harbinger DNA transposon insertion in the upstream regulatory region of the Pr-D allele is responsible for the up-regulation of the Pr gene in inducing phenotypic change in the plant. The successful isolation of Pr provides important information on the regulatory control of anthocyanin biosynthesis in Brassica vegetables, and offers a genetic resource for development of new varieties with enhanced health-promoting properties and visual appeal. PMID:20855520

Cloning of cDNA encoding the nuclear form of chicken sterol response element binding protein-2 (SREBP-2), chromosomal localization, and tissue expression of chicken SREBP-1 and -2 genes.

PubMed

Assaf, S; Hazard, D; Pitel, F; Morisson, M; Alizadeh, M; Gondret, F; Diot, C; Vignal, A; Douaire, M; Lagarrigue, S

2003-01-01

Sterol regulatory element binding protein-1 and -2 (SREBP-1 and -2) are key transcription factors involved in the biosynthesis of cholesterol and fatty adds. The SREBP have mainly been studied in rodents in which lipogenesis is regulated in both liver and adipose tissue. There is, however, a paucity of information on birds, in which lipogenesis occurs essentially in the liver as in humans. As a prelude to the investigation of the role of SREBP in lipid metabolism regulation in chicken, we sequenced the cDNA, encoding the mature nuclear form of chicken SREBP-2 protein, mapped SREBP-1 and -2 genes and studied their tissue expressions. The predicted chicken SREBP-2 amino acid sequence shows a 77 to 79% identity with human, mouse, and hamster homologues, with a nearly perfect conservation in all the important functional motifs, basic, helix-loop-helix, and leucine zipper (bHLH-Zip) region as well as cleavage sites. As in the human genome, SREBP-1 and SREBP-2 chicken genes are located on two separate chromosomes, respectively microchromosome 14 and macrochromosome 1. Tissue expression data show that SREBP-1 and SREBP-2 are expressed in a wide variety of tissues in chicken. However, unlike SREBP-2, SREBP-1 is expressed preferentially in the liver and uropygial gland, suggesting an important role of SREBP-1 in the regulation of lipogenesis in avian species.

Bhlhb5 is Required for the Subtype Development of Retinal Amacrine and Bipolar Cells in Mice

PubMed Central

Huang, Liang; Hu, Fang; Feng, Liang; Luo, Xiong-Jian; Liang, Guoqing; Zeng, Xiang-Yun; Yi, Jing-Lin; Gan, Lin

2014-01-01

Background BHLHB5, an OLIG-related basic helix-loop-helix transcription factor, is required for the development of a subset of gamma-amino butyric acid–releasing (GABAergic) amacrine cells and OFF-cone bipolar (CB) cells in mouse retinas. In order to determine BHLHB5’s functional mechanism in retinogenesis, we used the Cre-loxP recombination system to genetically trace the lineage of BHLHB5+ cells in normal and Bhlhb5-null retinas. The Bhlhb5-Cre knock-in allele was used to activate the constitutive expression of a GFP reporter in the Bhlhb5-expressing cells, and the cell fates of Bhlhb5-lineage cells were identified by using specific cell markers and were compared between normal and Bhlhb5-null retinas. Results In addition to GABAergic amacrine and OFF-CB cells, Bhlhb5 lineage cells give rise to ganglion, glycinergic amacrine, rod bipolar, ON-bipolar, and rod photoreceptor cells during normal retinal development. Targeted deletion of Bhlhb5 resulted in the loss of GABAergic amacrine, glycinergic amacrine, dopaminergic amacrine, and Type 2 OFF-CB cells. Furthermore, in the absence of BHLHB5, a portion of Bhlhb5 lineage cells switch their fate and differentiate into cholinergic amacrine cells. Conclusions Our data reveal a broad expression pattern of Bhlhb5 throughout retinogenesis and demonstrate the cell-autonomous as well as non-cell-autonomous role of Bhlhb5 in the specification of amacrine and bipolar subtypes. PMID:24123365

Shade Promotes Phototropism through Phytochrome B-Controlled Auxin Production.

PubMed

Goyal, Anupama; Karayekov, Elizabeth; Galvão, Vinicius Costa; Ren, Hong; Casal, Jorge J; Fankhauser, Christian

2016-12-19

Phototropism is an asymmetric growth response enabling plants to optimally position their organs. In flowering plants, the phototropin (phot) blue light receptors are essential to detect light gradients. In etiolated seedlings, the phototropic response is enhanced by the red/far-red (R/FR)-sensing phytochromes (phy) with a predominant function of phyA. In this study, we analyzed the influence of the phytochromes on phototropism in green (de-etiolated) Arabidopsis seedlings. Our experiments in the laboratory and outdoors revealed that, in open environments (high R/FR ratio), phyB inhibits phototropism. In contrast, under foliar shade, where access to direct sunlight becomes important, the phototropic response was strong. phyB modulates phototropism, depending on the R/FR ratio, by controlling the activity of three basic-helix-loop-helix (bHLH) transcription factors of the PHYTOCHROME INTERACTING FACTORs (PIFs) family. Promotion of phototropism depends on PIF-mediated induction of several members of the YUCCA gene family, leading to auxin production in the cotyledons. Our study identifies PIFs and YUCCAs as novel molecular players promoting phototropism in photoautotrophic, but not etiolated, seedlings. Moreover, our findings reveal fundamental differences in the phytochrome-phototropism crosstalk in etiolated versus green seedlings. We propose that in natural conditions where the light environment is not homogeneous, the uncovered phytochrome-phototropin co-action is important for plants to adapt their growth strategy to optimize photosynthetic light capture. Copyright © 2016 Elsevier Ltd. All rights reserved.

An anther development F-box (ADF) protein regulated by tapetum degeneration retardation (TDR) controls rice anther development.

PubMed

Li, Li; Li, Yixing; Song, Shufeng; Deng, Huafeng; Li, Na; Fu, Xiqin; Chen, Guanghui; Yuan, Longping

2015-01-01

In this study, we reported that a F-box protein, OsADF, as one of the direct targets of TDR , plays a critical role in rice tapetum cell development and pollen formation. The tapetum, the innermost sporophytic tissue of anther, plays an important supportive role in male reproduction in flowering plants. After meiosis, tapetal cells undergo programmed cell death (PCD) and provide nutrients for pollen development. Previously we showed that tapetum degeneration retardation (TDR), a basic helix-loop-helix transcription factor, can trigger tapetal PCD and control pollen wall development during anther development. However, the comprehensive regulatory network of TDR remains to be investigated. In this study, we cloned and characterized a panicle-specific expression F-box protein, anther development F-box (OsADF). By qRT-PCR and RNA in situ hybridization, we further confirmed that OsADF expressed specially in tapetal cells from stage 9 to stage 12 during anther development. In consistent with this specific expression pattern, the RNAi transgenic lines of OsADF exhibited abnormal tapetal degeneration and aborted microspores development, which eventually grew pollens with reduced fertility. Furthermore, we demonstrated that the TDR, a key regulator in controlling rice anther development, could regulate directly the expression of OsADF by binding to E-box motifs of its promoter. Therefore, this work highlighted the possible regulatory role of TDR, which regulates tapetal cell development and pollen formation via triggering the possible ADF-mediated proteolysis pathway.

ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE1 (ADPG1), ADPG2, and QUARTET2 Are Polygalacturonases Required for Cell Separation during Reproductive Development in Arabidopsis[W

PubMed Central

Ogawa, Mikihiro; Kay, Pippa; Wilson, Sarah; Swain, Stephen M.

2009-01-01

Cell separation is thought to involve degradation of pectin by several hydrolytic enzymes, particularly polygalacturonase (PG). Here, we characterize an activation tagging line with reduced growth and male sterility caused by increased expression of a PG encoded by QUARTET2 (QRT2). QRT2 is essential for pollen grain separation and is part of a small family of three closely related endo-PGs in the Arabidopsis thaliana proteome, including ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE1 (ADPG1) and ADPG2. Functional assays and complementation experiments confirm that ADPG1, ADPG2, and QRT2 are PGs. Genetic analysis demonstrates that ADPG1 and ADPG2 are essential for silique dehiscence. In addition, ADPG2 and QRT2 contribute to floral organ abscission, while all three genes contribute to anther dehiscence. Expression analysis is consistent with the observed mutant phenotypes. INDEHISCENT (IND) encodes a putative basic helix-loop-helix required for silique dehiscence, and we demonstrate that the closely related HECATE3 (HEC3) gene is required for normal seed abscission and show that IND and HEC3 are required for normal expression of ADPG1 in the silique dehiscence zone and seed abscission zone, respectively. We also show that jasmonic acid and ethylene act together with abscisic acid to regulate floral organ abscission, in part by promoting QRT2 expression. These results demonstrate that multiple cell separation events, including both abscission and dehiscence, require closely related PG genes. PMID:19168715

Regulation of BDNF chromatin status and promoter accessibility in a neural correlate of associative learning

PubMed Central

Ambigapathy, Ganesh; Zheng, Zhaoqing; Keifer, Joyce

2015-01-01

Brain-derived neurotrophic factor (BDNF) gene expression critically controls learning and its aberrant regulation is implicated in Alzheimer's disease and a host of neurodevelopmental disorders. The BDNF gene is target of known DNA regulatory mechanisms but details of its activity-dependent regulation are not fully characterized. We performed a comprehensive analysis of the epigenetic regulation of the turtle BDNF gene (tBDNF) during a neural correlate of associative learning using an in vitro model of eye blink classical conditioning. Shortly after conditioning onset, the results from ChIP-qPCR show conditioning-dependent increases in methyl-CpG-binding protein 2 (MeCP2) and repressor basic helix-loop-helix binding protein 2 (BHLHB2) binding to tBDNF promoter II that corresponds with transcriptional repression. In contrast, enhanced binding of ten-eleven translocation protein 1 (Tet1), extracellular signal-regulated kinase 1/2 (ERK1/2), and cAMP response element-binding protein (CREB) to promoter III corresponds with transcriptional activation. These actions are accompanied by rapid modifications in histone methylation and phosphorylation status of RNA polymerase II (RNAP II). Significantly, these remarkably coordinated changes in epigenetic factors for two alternatively regulated tBDNF promoters during conditioning are controlled by Tet1 and ERK1/2. Our findings indicate that Tet1 and ERK1/2 are critical partners that, through complementary functions, control learning-dependent tBDNF promoter accessibility required for rapid transcription and acquisition of classical conditioning. PMID:26336984

The E2A splice variant E47 regulates the differentiation of projection neurons via p57(KIP2) during cortical development.

PubMed

Pfurr, Sabrina; Chu, Yu-Hsuan; Bohrer, Christian; Greulich, Franziska; Beattie, Robert; Mammadzada, Könül; Hils, Miriam; Arnold, Sebastian J; Taylor, Verdon; Schachtrup, Kristina; Uhlenhaut, N Henriette; Schachtrup, Christian

2017-11-01

During corticogenesis, distinct classes of neurons are born from progenitor cells located in the ventricular and subventricular zones, from where they migrate towards the pial surface to assemble into highly organized layer-specific circuits. However, the precise and coordinated transcriptional network activity defining neuronal identity is still not understood. Here, we show that genetic depletion of the basic helix-loop-helix (bHLH) transcription factor E2A splice variant E47 increased the number of Tbr1-positive deep layer and Satb2-positive upper layer neurons at E14.5, while depletion of the alternatively spliced E12 variant did not affect layer-specific neurogenesis. While ChIP-Seq identified a big overlap for E12- and E47-specific binding sites in embryonic NSCs, including sites at the cyclin-dependent kinase inhibitor (CDKI) Cdkn1c gene locus, RNA-Seq revealed a unique transcriptional regulation by each splice variant. E47 activated the expression of the CDKI Cdkn1c through binding to a distal enhancer. Finally, overexpression of E47 in embryonic NSCs in vitro impaired neurite outgrowth, and overexpression of E47 in vivo by in utero electroporation disturbed proper layer-specific neurogenesis and upregulated p57(KIP2) expression. Overall, this study identifies E2A target genes in embryonic NSCs and demonstrates that E47 regulates neuronal differentiation via p57(KIP2). © 2017. Published by The Company of Biologists Ltd.

Scaffold protein enigma homolog 1 overcomes the repression of myogenesis activation by inhibitor of DNA binding 2

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

Nakatani, Miyuki; Ito, Jumpei; Japan Society for the Promotion of Science, Tokyo, 102-0083

Enigma Homolog 1 (ENH1) is a scaffold protein for signaling proteins and transcription factors. Previously, we reported that ENH1 overexpression promotes the differentiation of C2C12 myoblasts. However, the molecular mechanism underlying the role of ENH1 in the C2C12 cells differentiation remains elusive. ENH1 was shown to inhibit the proliferation of neuroblastoma cells by sequestering Inhibitor of DNA binding protein 2 (Id2) in the cytosol. Id2 is a repressor of basic Helix-Loop-Helix transcription factors activity and prevents myogenesis. Here, we found that ENH1 overcome the Id2 repression of C2C12 cells myogenic differentiation and that ENH1 overexpression promotes mice satellite cells activation, the firstmore » step toward myogenic differentiation. In addition, we show that ENH1 interacted with Id2 in C2C12 cells and mice satellite cells. Collectively, our results suggest that ENH1 plays an important role in the activation of myogenesis through the repression of Id2 activity. -- Highlights: •Enigma Homolog 1 (ENH1) is a scaffold protein. •ENH1 binds to inhibitor of DNA binding 2 (Id2) in myoblasts. •ENH1 overexpression overcomes the Id2's repression of myogenesis. •The Id2-ENH1 complex play an important role in the activation of myogenesis.« less

Can the 'neuron theory' be complemented by a universal mechanism for generic neuronal differentiation.

PubMed

Ernsberger, Uwe

2015-01-01

With the establishment of the 'neuron theory' at the turn of the twentieth century, this remarkably powerful term was introduced to name a breathtaking diversity of cells unified by a characteristic structural compartmentalization and unique information processing and propagating features. At the beginning of the twenty-first century, developmental, stem cell and reprogramming studies converged to suggest a common mechanism involved in the generation of possibly all vertebrate, and at least a significant number of invertebrate, neurons. Sox and, in particular, SoxB and SoxC proteins as well as basic helix-loop-helix proteins play major roles, even though their precise contributions to progenitor programming, proliferation and differentiation are not fully resolved. In addition to neuronal development, these transcription factors also regulate sensory receptor and endocrine cell development, thus specifying a range of cells with regulatory and communicative functions. To what extent microRNAs contribute to the diversification of these cell types is an upcoming question. Understanding the transcriptional and post-transcriptional regulation of genes coding for cell type-specific cytoskeletal and motor proteins as well as synaptic and ion channel proteins, which mark differences but also similarities between the three communicator cell types, will provide a key to the comprehension of their diversification and the signature of 'generic neuronal' differentiation. Apart from the general scientific significance of a putative universal core instruction for neuronal development, the impact of this line of research for cell replacement therapy and brain tumor treatment will be of considerable interest.

A credit-card library approach for disrupting protein-protein interactions.

PubMed

Xu, Yang; Shi, Jin; Yamamoto, Noboru; Moss, Jason A; Vogt, Peter K; Janda, Kim D

2006-04-15

Protein-protein interfaces are prominent in many therapeutically important targets. Using small organic molecules to disrupt protein-protein interactions is a current challenge in chemical biology. An important example of protein-protein interactions is provided by the Myc protein, which is frequently deregulated in human cancers. Myc belongs to the family of basic helix-loop-helix leucine zipper (bHLH-ZIP) transcription factors. It is biologically active only as heterodimer with the bHLH-ZIP protein Max. Herein, we report a new strategy for the disruption of protein-protein interactions that has been corroborated through the design and synthesis of a small parallel library composed of 'credit-card' compounds. These compounds are derived from a planar, aromatic scaffold and functionalized with four points of diversity. From a 285 membered library, several hits were obtained that disrupted the c-Myc-Max interaction and cellular functions of c-Myc. The IC50 values determined for this small focused library for the disruption of Myc-Max dimerization are quite potent, especially since small molecule antagonists of protein-protein interactions are notoriously difficult to find. Furthermore, several of the compounds were active at the cellular level as shown by their biological effects on Myc action in chicken embryo fibroblast assays. In light of our findings, this approach is considered a valuable addition to the armamentarium of new molecules being developed to interact with protein-protein interfaces. Finally, this strategy for disrupting protein-protein interactions should prove applicable to other families of proteins.

ThMYC4E, candidate Blue aleurone 1 gene controlling the associated trait in Triticum aestivum

PubMed Central

Chen, Wenjie; Zhang, Bo; Wang, Daowen; Liu, Dengcai; Zhang, Huaigang

2017-01-01

Blue aleurone is a useful and interesting trait in common wheat that was derived from related species. Here, transcriptomes of blue and white aleurone were compared for isolating Blue aleurone 1 (Ba1) transferred from Thinopyrum ponticum. In the genes involved in anthocyanin biosynthesis, only a basic helix-loop-helix (bHLH) transcription factor, ThMYC4E, had a higher transcript level in blue aleurone phenotype, and was homologous to the genes on chromosome 4 of Triticum aestivum. ThMYC4E carried the characteristic domains (bHLH-MYC_N, HLH and ACT-like) of a bHLH transcription factor, and clustered with genes regulating anthocyanin biosynthesis upon phylogenetic analysis. The over-expression of ThMYC4E regulated anthocyanin biosynthesis with the coexpression of the MYB transcription factor ZmC1 from maize. ThMYC4E existed in the genomes of the addition, substitution and near isogenic lines with the blue aleurone trait derived from Th. ponticum, and could not be detected in any germplasm of T. urartu, T. monococcum, T. turgidum, Aegilops tauschii or T. aestivum, with white aleurone. These results suggested that ThMYC4E was candidate Ba1 gene controlling the blue aleurone trait in T. aestivum genotypes carrying Th. ponticum introgression. The ThMYC4E isolation aids in better understanding the genetic mechanisms of the blue aleurone trait and in its more effective use during wheat breeding. PMID:28704468

The bHLH Transcription Factor bHLH104 Interacts with IAA-LEUCINE RESISTANT3 and Modulates Iron Homeostasis in Arabidopsis

PubMed Central

Zhang, Jie; Liu, Bing; Li, Mengshu; Feng, Dongru; Jin, Honglei; Wang, Peng; Liu, Jun; Xiong, Feng; Wang, Jinfa; Wang, Hong-Bin

2015-01-01

Iron (Fe) is an indispensable micronutrient for plant growth and development. The regulation of Fe homeostasis in plants is complex and involves a number of transcription factors. Here, we demonstrate that a basic helix-loop-helix (bHLH) transcription factor, bHLH104, belonging to the IVc subgroup of bHLH family, acts as a key component positively regulating Fe deficiency responses. Knockout of bHLH104 in Arabidopsis thaliana greatly reduced tolerance to Fe deficiency, whereas overexpression of bHLH104 had the opposite effect and led to accumulation of excess Fe in soil-grown conditions. The activation of Fe deficiency-inducible genes was substantially suppressed by loss of bHLH104. Further investigation showed that bHLH104 interacted with another IVc subgroup bHLH protein, IAA-LEUCINE RESISTANT3 (ILR3), which also plays an important role in Fe homeostasis. Moreover, bHLH104 and ILR3 could bind directly to the promoters of Ib subgroup bHLH genes and POPEYE (PYE) functioning in the regulation of Fe deficiency responses. Interestingly, genetic analysis showed that loss of bHLH104 could decrease the tolerance to Fe deficiency conferred by the lesion of BRUTUS, which encodes an E3 ligase and interacts with bHLH104. Collectively, our data support that bHLH104 and ILR3 play pivotal roles in the regulation of Fe deficiency responses via targeting Ib subgroup bHLH genes and PYE expression. PMID:25794933

Members of an R2R3-MYB transcription factor family in Petunia are developmentally and environmentally regulated to control complex floral and vegetative pigmentation patterning.

PubMed

Albert, Nick W; Lewis, David H; Zhang, Huaibi; Schwinn, Kathy E; Jameson, Paula E; Davies, Kevin M

2011-03-01

We present an investigation of anthocyanin regulation over the entire petunia plant, determining the mechanisms governing complex floral pigmentation patterning and environmentally induced vegetative anthocyanin synthesis. DEEP PURPLE (DPL) and PURPLE HAZE (PHZ) encode members of the R2R3-MYB transcription factor family that regulate anthocyanin synthesis in petunia, and control anthocyanin production in vegetative tissues and contribute to floral pigmentation. In addition to these two MYB factors, the basic helix-loop-helix (bHLH) factor ANTHOCYANIN1 (AN1) and WD-repeat protein AN11, are also essential for vegetative pigmentation. The induction of anthocyanins in vegetative tissues by high light was tightly correlated to the induction of transcripts for PHZ and AN1. Interestingly, transcripts for PhMYB27, a putative R2R3-MYB active repressor, were highly expressed during non-inductive shade conditions and repressed during high light. The competitive inhibitor PhMYBx (R3-MYB) was expressed under high light, which may provide feedback repression. In floral tissues DPL regulates vein-associated anthocyanin pigmentation in the flower tube, while PHZ determines light-induced anthocyanin accumulation on exposed petal surfaces (bud-blush). A model is presented suggesting how complex floral and vegetative pigmentation patterns are derived in petunia in terms of MYB, bHLH and WDR co-regulators. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Rhythmic expression of DEC2 protein in vitro and in vivo.

PubMed

Sato, Fuyuki; Muragaki, Yasuteru; Kawamoto, Takeshi; Fujimoto, Katsumi; Kato, Yukio; Zhang, Yanping

2016-06-01

Basic helix-loop-helix (bHLH) transcription factor DEC2 (bHLHE41/Sharp1) is one of the clock genes that show a circadian rhythm in various tissues. DEC2 regulates differentiation, sleep length, tumor cell invasion and apoptosis. Although studies have been conducted on the rhythmic expression of DEC2 mRNA in various tissues, the precise molecular mechanism of DEC2 expression is poorly understood. In the present study, we examined whether DEC2 protein had a rhythmic expression. Western blot analysis for DEC2 protein revealed a rhythmic expression in mouse liver, lung and muscle and in MCF-7 and U2OS cells. In addition, AMP-activated protein kinase (AMPK) activity (phosphorylation of AMPK) in mouse embryonic fibroblasts (MEFs) exhibited a rhythmic expression under the condition of medium change or glucose-depleted medium. However, the rhythmic expression of DEC2 in MEF gradually decreased in time under these conditions. The medium change affected the levels of DEC2 protein and phosphorylation of AMPK. In addition, the levels of DEC2 protein showed a rhythmic expression in vivo and in MCF-7 and U2OS cells. The results showed that the phosphorylation of AMPK immunoreactivity was strongly detected in the liver and lung of DEC2 knockout mice compared with that of wild-type mice. These results may provide new insights into rhythmic expression and the regulation between DEC2 protein and AMPK activity.

Transcriptome-wide analysis of jasmonate-treated BY-2 cells reveals new transcriptional regulators associated with alkaloid formation in tobacco.

PubMed

Yang, Yuping; Yan, Pengcheng; Yi, Che; Li, Wenzheng; Chai, Yuhui; Fei, Lingling; Gao, Ping; Zhao, Heping; Wang, Yingdian; Timko, Michael P; Wang, Bingwu; Han, Shengcheng

2017-08-01

Jasmonates (JAs) are well-known regulators of stress, defence, and secondary metabolism in plants, with JA perception triggering extensive transcriptional reprogramming, including both activation and/or repression of entire metabolic pathways. We performed RNA sequencing based transcriptomic profiling of tobacco BY-2 cells before and after treatment with methyl jasmonate (MeJA) to identify novel transcriptional regulators associated with alkaloid formation. A total of 107,140 unigenes were obtained through de novo assembly, and at least 33,213 transcripts (31%) encode proteins, in which 3419 transcription factors (TFs) were identified, representing 72 gene families, as well as 840 transcriptional regulators (TRs) distributed among 19 gene families. After MeJA treatment BY-2 cells, 7260 differentially expressed transcripts were characterised, which include 4443 MeJA-upregulated and 2817 MeJA-downregulated genes. Of these, 227 TFs/TRs in 36 families were specifically upregulated, and 102 TFs/TRs in 38 families were downregulated in MeJA-treated BY-2 cells. We further showed that the expression of 12 ethylene response factors and four basic helix-loop-helix factors increased at the transcriptional level after MeJA treatment in BY-2 cells and displayed specific expression patterns in nic mutants with or without MeJA treatments. Our data provide a catalogue of transcripts of tobacco BY-2 cells and benefit future study of JA-modulated regulation of secondary metabolism in tobacco. Copyright © 2017 Elsevier GmbH. All rights reserved.

Program Specificity for Ptf1a in Pancreas versus Neural Tube Development Correlates with Distinct Collaborating Cofactors and Chromatin Accessibility

PubMed Central

Meredith, David M.; Borromeo, Mark D.; Deering, Tye G.; Casey, Bradford H.; Savage, Trisha K.; Mayer, Paul R.; Hoang, Chinh; Tung, Kuang-Chi; Kumar, Manonmani; Shen, Chengcheng; Swift, Galvin H.

2013-01-01

The lineage-specific basic helix-loop-helix transcription factor Ptf1a is a critical driver for development of both the pancreas and nervous system. How one transcription factor controls diverse programs of gene expression is a fundamental question in developmental biology. To uncover molecular strategies for the program-specific functions of Ptf1a, we identified bound genomic regions in vivo during development of both tissues. Most regions bound by Ptf1a are specific to each tissue, lie near genes needed for proper formation of each tissue, and coincide with regions of open chromatin. The specificity of Ptf1a binding is encoded in the DNA surrounding the Ptf1a-bound sites, because these regions are sufficient to direct tissue-restricted reporter expression in transgenic mice. Fox and Sox factors were identified as potential lineage-specific modifiers of Ptf1a binding, since binding motifs for these factors are enriched in Ptf1a-bound regions in pancreas and neural tube, respectively. Of the Fox factors expressed during pancreatic development, Foxa2 plays a major role. Indeed, Ptf1a and Foxa2 colocalize in embryonic pancreatic chromatin and can act synergistically in cell transfection assays. Together, these findings indicate that lineage-specific chromatin landscapes likely constrain the DNA binding of Ptf1a, and they identify Fox and Sox gene families as part of this process. PMID:23754747

Endosulfan inhibiting the meiosis process via depressing expressions of regulatory factors and causing cell cycle arrest in spermatogenic cells.

PubMed

Guo, Fang-Zi; Zhang, Lian-Shuang; Wei, Jia-Liu; Ren, Li-Hua; Zhang, Jin; Jing, Li; Yang, Man; Wang, Ji; Sun, Zhi-Wei; Zhou, Xian-Qing

2016-10-01

Endosulfan is a persistent organic pollutant and widely used in agriculture as a pesticide. It is present in air, water, and soil worldwide; therefore, it is a health risk affecting especially the reproductive system. The aim of this study was to evaluate the toxicity of endosulfan in the reproductive system. To investigate the effect of endosulfan on meiosis process, 32 rats were divided into four groups, treated with 0, 1, 5, and 10 mg/kg/day endosulfan, respectively, and sacrificed after the 21 days of treatments. Results show that endosulfan caused the reductions in sperm concentration and motility rate, which resulted into an increased in sperm abnormality rate; further, endosulfan induced downregulation of spermatogenesis- and oogenesis-specific basic helix-loop-helix transcription factor (Sohlh1) which controls the switch on meiosis in mammals, as well cyclin A1, cyclin-dependent kinases 1 (CDK1), and cyclin-dependent kinases 2 (CDK2). In vitro, endosulfan induced G2/M phase arrest in the spermatogenic cell cycle and caused proliferation inhibition. Moreover, endosulfan induced oxidative stress and DNA damage in vivo and vitro. The results suggested that endosulfan could inhibit the start of meiosis by downregulating the expression of Sohlh1 and induce G2/M phase arrest of cell cycle by decreasing the expression of cyclin A1, CDK1, and CDK2 via oxidative damage, which inhibits the meiosis process, and therefore decrease the amount of sperm.

Enhancing Flower Color through Simultaneous Expression of the B-peru and mPAP1 Transcription Factors under Control of a Flower-Specific Promoter

PubMed Central

Kim, Da-Hye; Park, Sangkyu; Lee, Jong-Yeol; Ha, Sun-Hwa; Lim, Sun-Hyung

2018-01-01

Flower color is a main target for flower breeding. A transgenic approach for flower color modification requires a transgene and a flower-specific promoter. Here, we expressed the B-peru gene encoding a basic helix loop helix (bHLH) transcription factor (TF) together with the mPAP1 gene encoding an R2R3 MYB TF to enhance flower color in tobacco (Nicotiana tabacum L.), using the tobacco anthocyanidin synthase (ANS) promoter (PANS) to drive flower-specific expression. The transgenic tobacco plants grew normally and produced either dark pink (PANSBP_DP) or dark red (PANSBP_DR) flowers. Quantitative real time polymerase chain reaction (qPCR) revealed that the expression of five structural genes in the flavonoid biosynthetic pathway increased significantly in both PANSBP_DP and PANSBP_DR lines, compared with the non-transformed (NT) control. Interestingly, the expression of two regulatory genes constituting the active MYB-bHLH-WD40 repeat (WDR) (MBW) complex decreased significantly in the PANSBP_DR plants but not in the PANSBP_DP plants. Total flavonol and anthocyanin abundance correlated with flower color, with an increase of 1.6–43.2 fold in the PANSBP_DP plants and 2.0–124.2 fold in the PANSBP_DR plants. Our results indicate that combinatorial expression of B-peru and mPAP1 genes under control of the ANS promoter can be a useful strategy for intensifying flower color without growth retardation. PMID:29361688

The transcription factor MYB115 contributes to the regulation of proanthocyanidin biosynthesis and enhances fungal resistance in poplar.

PubMed

Wang, Lijun; Ran, Lingyu; Hou, Yisu; Tian, Qiaoyan; Li, Chaofeng; Liu, Rui; Fan, Di; Luo, Keming

2017-07-01

Proanthocyanidins (PAs) are major defense phenolic compounds in the leaves of poplar (Populus spp.) in response to abiotic and biotic stresses. Transcriptional regulation of PA biosynthetic genes by the MYB-basic helix-loop-helix (bHLH)-WD40 complexes in poplar is not still fully understood. Here, an Arabidopsis TT2-like gene MYB115 was isolated from Populus tomentosa and characterized by various molecular, genetic and biochemical approaches. MYB115 restored PA productions in the seed coat of the Arabidopsis tt2 mutant. Overexpression of MYB115 in poplar activated expression of PA biosynthetic genes, resulting in a significant increase in PA concentrations. By contrast, the CRISPR/Cas9-generated myb115 mutant exhibited reduced PA content and decreased expression of PA biosynthetic genes. MYB115 directly activated the promoters of PA-specific structural genes. MYB115 interacted with poplar TT8. Coexpression of MYB115, TT8 and poplar TTG1 significantly enhanced the expression of ANR1 and LAR3. Additionally, transgenic plants overexpressing MYB115 had increased resistance to the fungal pathogen Dothiorella gregaria, whereas myb115 mutant exhibited greater sensitivity compared with wild-type plants. Our data provide insight into the regulatory mechanisms controlling PA biosynthesis by MYB115 in poplar, which could be effectively employed for metabolic engineering of PAs to improve resistance to fungal pathogens. No claim to original Chinese Government works New Phytologist © 2017 New Phytologist Trust.

Expression profiling upon Nex1/MATH-2-mediated neuritogenesis in PC12 cells and its implication in regeneration

PubMed Central

Uittenbogaard, Martine; Chiaramello, Anne

2006-01-01

The expression of Nex1 peaks during brain development when neurite outgrowth and synaptogenesis are highly active. We previously showed that Nex1 is a critical effector of the nerve growth factor (NGF) pathway and its overexpression results in spontaneous neuritogenesis. Furthermore, the PC12-Nex1 cells exhibit accelerated neurite extension upon NGF exposure, and have the capacity to regenerate neurites in the absence of NGF. In this study, we identify the repertoire of genes targeted by Nex1 to unravel the molecular mecha nisms by which Nex1 promotes differentiation and regeneration. Our transcriptional analysis reveals that Nex1 modulates a wide spectrum of genes with diverse functions, many of them being key downstream regulators of the NGF pathway, and critical to neuritogenesis, such as microtubules, microtubule-associated proteins (MAPs) and intermediate filaments. We also provide the first evidence that a basic helix-loop-helix (bHLH) protein stimulates the expression of the cyclin-dependent kinase (CDK) inhibitors belonging to the INK4 family, which plays a role in promoting cell-cycle arrest. Finally, we show a dramatic synergistic effect between Nex1 and cAMP, resulting in an impressive regeneration of an elaborate and dense neurite network. Thus, Nex1 has endowed the PC12-Nex1 cells with a distinct combination of gene products that takes part in the complex regulation of neuritogenesis and regeneration. PMID:15584910

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

Mehmood, Rashid; Yasuhara, Noriko; Oe, Souichi

The transition from undifferentiated pluripotent cells to terminally differentiated neurons is coordinated by a repertoire of transcription factors. NeuroD1 is a type II basic helix loop helix (bHLH) transcription factor that plays critical roles in neuronal differentiation and maintenance in the central nervous system. Its dimerization with E47, a type I bHLH transcription factor, leads to the transcriptional regulation of target genes. Mounting evidence suggests that regulating the localization of transcription factors contributes to the regulation of their activity during development as defects in their localization underlie a variety of developmental disorders. In this study, we attempted to understand themore » nuclear import mannerisms of NeuroD1 and E47. We found that the nuclear import of NeuroD1 and E47 is energy-dependent and involves the Ran-mediated pathway. Herein, we demonstrate that NeuroD1 and E47 can dimerize inside the cytoplasm before their nuclear import. Moreover, this dimerization promotes nuclear import as the nuclear accumulation of NeuroD1 was enhanced in the presence of E47 in an in vitro nuclear import assay, and NLS-deficient NeuroD1 was successfully imported into the nucleus upon E47 overexpression. NeuroD1 also had a similar effect on the nuclear accumulation of NLS-deficient E47. These findings suggest a novel role for dimerization that may promote, at least partially, the nuclear import of transcription factors allowing them to function efficiently in the nucleus.« less

Functional importance of evolutionally conserved Tbx6 binding sites in the presomitic mesoderm-specific enhancer of Mesp2.

PubMed

Yasuhiko, Yukuto; Kitajima, Satoshi; Takahashi, Yu; Oginuma, Masayuki; Kagiwada, Harumi; Kanno, Jun; Saga, Yumiko

2008-11-01

The T-box transcription factor Tbx6 controls the expression of Mesp2, which encodes a basic helix-loop-helix transcription factor that has crucial roles in somitogenesis. In cultured cells, Tbx6 binding to the Mesp2 enhancer region is essential for the activation of Mesp2 by Notch signaling. However, it is not known whether this binding is required in vivo. Here we report that an Mesp2 enhancer knockout mouse bearing mutations in two crucial Tbx6 binding sites does not express Mesp2 in the presomitic mesoderm. This absence leads to impaired skeletal segmentation identical to that reported for Mesp2-null mice, indicating that these Tbx6 binding sites are indispensable for Mesp2 expression. T-box binding to the consensus sequences in the Mesp2 upstream region was confirmed by chromatin immunoprecipitation assays. Further enhancer analyses indicated that the number and spatial organization of the T-box binding sites are critical for initiating Mesp2 transcription via Notch signaling. We also generated a knock-in mouse in which the endogenous Mesp2 enhancer was replaced by the core enhancer of medaka mespb, an ortholog of mouse Mesp2. The homozygous enhancer knock-in mouse was viable and showed normal skeletal segmentation, indicating that the medaka mespb enhancer functionally replaced the mouse Mesp2 enhancer. These results demonstrate that there is significant evolutionary conservation of Mesp regulatory mechanisms between fish and mice.

Bhlhb5 is required for the subtype development of retinal amacrine and bipolar cells in mice.

PubMed

Huang, Liang; Hu, Fang; Feng, Liang; Luo, Xiong-Jian; Liang, Guoqing; Zeng, Xiang-Yun; Yi, Jing-Lin; Gan, Lin

2014-02-01

BHLHB5, an OLIG-related basic helix-loop-helix transcription factor, is required for the development of a subset of gamma-amino butyric acid-releasing (GABAergic) amacrine cells and OFF-cone bipolar (CB) cells in mouse retinas. In order to determine BHLHB5's functional mechanism in retinogenesis, we used the Cre-loxP recombination system to genetically trace the lineage of BHLHB5+ cells in normal and Bhlhb5-null retinas. The Bhlhb5-Cre knock-in allele was used to activate the constitutive expression of a GFP reporter in the Bhlhb5-expressing cells, and the cell fates of Bhlhb5-lineage cells were identified by using specific cell markers and were compared between normal and Bhlhb5-null retinas. In addition to GABAergic amacrine and OFF-CB cells, Bhlhb5 lineage cells give rise to ganglion, glycinergic amacrine, rod bipolar, ON-bipolar, and rod photoreceptor cells during normal retinal development. Targeted deletion of Bhlhb5 resulted in the loss of GABAergic amacrine, glycinergic amacrine, dopaminergic amacrine, and Type 2 OFF-CB cells. Furthermore, in the absence of BHLHB5, a portion of Bhlhb5 lineage cells switch their fate and differentiate into cholinergic amacrine cells. Our data reveal a broad expression pattern of Bhlhb5 throughout retinogenesis and demonstrate the cell-autonomous as well as non-cell-autonomous role of Bhlhb5 in the specification of amacrine and bipolar subtypes. Copyright © 2013 Wiley Periodicals, Inc.

Disruption of neurogenesis and cortical development in transgenic mice misexpressing Olig2, a gene in the Down syndrome critical region.

PubMed

Liu, Wei; Zhou, Hui; Liu, Lei; Zhao, Chuntao; Deng, Yaqi; Chen, Lina; Wu, Laiman; Mandrycky, Nicole; McNabb, Christopher T; Peng, Yuanbo; Fuchs, Perry N; Lu, Jie; Sheen, Volney; Qiu, Mengsheng; Mao, Meng; Lu, Q Richard

2015-05-01

The basic helix-loop-helix (bHLH) transcription factor Olig2 is crucial for mammalian central nervous system development. Human ortholog OLIG2 is located in the Down syndrome critical region in trisomy 21. To investigate the effect of Olig2 misexpression on brain development, we generated a developmentally regulated Olig2-overexpressing transgenic line with a Cre/loxP system. The transgenic mice with Olig2 misexpression in cortical neural stem/progenitor cells exhibited microcephaly, cortical dyslamination, hippocampus malformation, and profound motor deficits. Ectopic misexpression of Olig2 impaired cortical progenitor proliferation and caused precocious cell cycle exit. Massive neuronal cell death was detected in the developing cortex of Olig2-misexpressing mice. In addition, Olig2 misexpression led to a significant downregulation of neuronal specification factors including Ngn1, Ngn2 and Pax6, and a defect in cortical neurogenesis. Chromatin-immunoprecipitation and sequencing (ChIP-Seq) analysis indicates that Olig2 directly targets the promoter and/or enhancer regions of Nfatc4, Dscr1/Rcan1 and Dyrk1a, the critical neurogenic genes that contribute to Down syndrome phenotypes, and inhibits their expression. Together, our study suggests that Olig2 misexpression in neural stem cells elicits neurogenesis defects and neuronal cell death, which may contribute to developmental disorders including Down syndrome, where OLIG2 is triplicated on chromosomal 21. Copyright © 2015 Elsevier Inc. All rights reserved.

Hey1- and p53-dependent TrkC proapoptotic activity controls neuroblastoma growth.

PubMed

Ménard, Marie; Costechareyre, Clélia; Ichim, Gabriel; Blachier, Jonathan; Neves, David; Jarrosson-Wuilleme, Loraine; Depping, Reinhard; Koster, Jan; Saintigny, Pierre; Mehlen, Patrick; Tauszig-Delamasure, Servane

2018-05-01

The neurotrophin-3 (NT-3) receptor tropomyosin receptor kinase C (TrkC/NTRK3) has been described as a dependence receptor and, as such, triggers apoptosis in the absence of its ligand NT-3. This proapoptotic activity has been proposed to confer a tumor suppressor activity to this classic tyrosine kinase receptor (RTK). By investigating interacting partners that might facilitate TrkC-induced cell death, we have identified the basic helix-loop-helix (bHLH) transcription factor Hey1 and importin-α3 (karyopherin alpha 4 [KPNA4]) as direct interactors of TrkC intracellular domain, and we show that Hey1 is required for TrkC-induced apoptosis. We propose here that the cleaved proapoptotic portion of TrkC intracellular domain (called TrkC killer-fragment [TrkC-KF]) is translocated to the nucleus by importins and interacts there with Hey1. We also demonstrate that Hey1 and TrkC-KF transcriptionally silence mouse double minute 2 homolog (MDM2), thus contributing to p53 stabilization. p53 transcriptionally regulates the expression of TrkC-KF cytoplasmic and mitochondrial interactors cofactor of breast cancer 1 (COBRA1) and B cell lymphoma 2-associated X (BAX), which will subsequently trigger the intrinsic pathway of apoptosis. Of interest, TrkC was proposed to constrain tumor progression in neuroblastoma (NB), and we demonstrate in an avian model that TrkC tumor suppressor activity requires Hey1 and p53.

Characterization of sequences in human TWIST required for nuclear localization

PubMed Central

Singh, Shalini; Gramolini, Anthony O

2009-01-01

Background Twist is a transcription factor that plays an important role in proliferation and tumorigenesis. Twist is a nuclear protein that regulates a variety of cellular functions controlled by protein-protein interactions and gene transcription events. The focus of this study was to characterize putative nuclear localization signals (NLSs) 37RKRR40 and 73KRGKK77 in the human TWIST (H-TWIST) protein. Results Using site-specific mutagenesis and immunofluorescences, we observed that altered TWISTNLS1 K38R, TWISTNLS2 K73R and K77R constructs inhibit nuclear accumulation of H-TWIST in mammalian cells, while TWISTNLS2 K76R expression was un-affected and retained to the nucleus. Subsequently, co-transfection of TWIST mutants K38R, K73R and K77R with E12 formed heterodimers and restored nuclear localization despite the NLSs mutations. Using a yeast-two-hybrid assay, we identified a novel TWIST-interacting candidate TCF-4, a basic helix-loop-helix transcription factor. The interaction of TWIST with TCF-4 confirmed using NLS rescue assays, where nuclear expression of mutant TWISTNLS1 with co-transfixed TCF-4 was observed. The interaction of TWIST with TCF-4 was also seen using standard immunoprecipitation assays. Conclusion Our study demonstrates the presence of two putative NLS motifs in H-TWIST and suggests that these NLS sequences are functional. Furthermore, we identified and confirmed the interaction of TWIST with a novel protein candidate TCF-4. PMID:19534813

Early stages in the development of human T, natural killer and thymic dendritic cells.

PubMed

Spits, H; Blom, B; Jaleco, A C; Weijer, K; Verschuren, M C; van Dongen, J J; Heemskerk, M H; Res, P C

1998-10-01

T-cell development is initiated when CD34+ pluripotent stem cells or their immediate progeny leave the bone marrow to migrate to the thymus. Upon arrival in the thymus the stem cell progeny is not yet committed to the T-cell lineage as it has the capability to develop into T, natural killer (NK) and dendritic cells (DC). Primitive hematopoietic progenitor cells in the human thymus express CD34 and lack CD1a. When these progenitor cells develop into T cells they traverse a number of checkpoints. One early checkpoint is the induction of T-cell commitment, which correlates with appearance of CD1a and involves the loss of capacity to develop into NK cells and DC and the initiation of T-cell receptor (TCR) gene rearrangements. Basic helix-loop-helix transcription factors play a role in induction of T-cell commitment. CD1a+CD34+ cells develop into CD4+CD8 alpha+ beta+ cells by upregulating first CD4, followed by CD8 alpha and then CD8 beta. Selection for productive TCR beta gene rearrangements (beta selection) likely occurs in the CD4+CD8 alpha+ beta- and CD4+CD8 alpha+ beta+ populations. Although the T and NK-cell lineages are closely related to each other, NK cells can develop independently of the thymus. The fetal thymus is most likely one site of NK-cell development.

β-Catenin Up-regulates Atoh1 Expression in Neural Progenitor Cells by Interaction with an Atoh1 3′ Enhancer*

PubMed Central

Shi, Fuxin; Cheng, Yen-fu; Wang, Xiaohui L.; Edge, Albert S. B.

2010-01-01

Atoh1, a basic helix-loop-helix transcription factor, plays a critical role in the differentiation of several epithelial and neural cell types. We found that β-catenin, the key mediator of the canonical Wnt pathway, increased expression of Atoh1 in mouse neuroblastoma cells and neural progenitor cells, and baseline Atoh1 expression was decreased by siRNA directed at β-catenin. The up-regulation of Atoh1 was caused by an interaction of β-catenin with the Atoh1 enhancer that could be demonstrated by chromatin immunoprecipitation. We found that two putative Tcf-Lef sites in the 3′ enhancer of the Atoh1 gene displayed an affinity for β-catenin and were critical for the activation of Atoh1 transcription because mutation of either site decreased expression of a reporter gene downstream of the enhancer. Tcf-Lef co-activators were found in the complex that bound to these sites in the DNA together with β-catenin. Inhibition of Notch signaling, which has previously been shown to induce bHLH transcription factor expression, increased β-catenin expression in progenitor cells of the nervous system. Because this could be a mechanism for up-regulation of Atoh1 after inhibition of Notch, we tested whether siRNA to β-catenin prevented the increase in Atoh1 and found that β-catenin expression was required for increased expression of Atoh1 after Notch inhibition. PMID:19864427

Identification of Candidate Genes Underlying an Iron Efficiency Quantitative Trait Locus in Soybean1

PubMed Central

Peiffer, Gregory A.; King, Keith E.; Severin, Andrew J.; May, Gregory D.; Cianzio, Silvia R.; Lin, Shun Fu; Lauter, Nicholas C.; Shoemaker, Randy C.

2012-01-01

Prevalent on calcareous soils in the United States and abroad, iron deficiency is among the most common and severe nutritional stresses in plants. In soybean (Glycine max) commercial plantings, the identification and use of iron-efficient genotypes has proven to be the best form of managing this soil-related plant stress. Previous studies conducted in soybean identified a significant iron efficiency quantitative trait locus (QTL) explaining more than 70% of the phenotypic variation for the trait. In this research, we identified candidate genes underlying this QTL through molecular breeding, mapping, and transcriptome sequencing. Introgression mapping was performed using two related near-isogenic lines in which a region located on soybean chromosome 3 required for iron efficiency was identified. The region corresponds to the previously reported iron efficiency QTL. The location was further confirmed through QTL mapping conducted in this study. Transcriptome sequencing and quantitative real-time-polymerase chain reaction identified two genes encoding transcription factors within the region that were significantly induced in soybean roots under iron stress. The two induced transcription factors were identified as homologs of the subgroup lb basic helix-loop-helix (bHLH) genes that are known to regulate the strategy I response in Arabidopsis (Arabidopsis thaliana). Resequencing of these differentially expressed genes unveiled a significant deletion within a predicted dimerization domain. We hypothesize that this deletion disrupts the Fe-DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT)/bHLH heterodimer that has been shown to induce known iron acquisition genes. PMID:22319075

Site-directed mutagenesis of the hinge peptide from the hemagglutinin protein: enhancement of the pH-responsive conformational change.

PubMed

Casali, Monica; Banta, Scott; Zambonelli, Carlo; Megeed, Zaki; Yarmush, Martin L

2008-06-01

Environmentally responsive proteins and peptides are increasingly finding utility in various engineered systems due to their ability to respond to the presentation of external stimuli. A classic example of this behavior is the influenza hemagglutinin (HA) fusion protein. At neutral pH, HA exists in a non-fusogenic state, but upon exposure to low pH, the conformation of the structure changes to expose a fusogenic peptide. During this structural change, massive rearrangements occur in a subunit of HA (HA2). Crystallography data has shown that a loop of 28 amino acids (residues 54-81) undergoes a dramatic transition from a random coil to an alpha-helix. This segment connects to two flanking helical regions (short and long) to form a long, continuous helix. Here, we report the results of site-directed mutagenesis study on LOOP-36 to further understand the mechanism of this important stimulus-responsive peptide. The conformational transition of a bacterially expressed LOOP-36 was found to be less dramatic than has been previously reported. The systematic mutation of glutamate and histidine residues in the peptide to glutamines (glutamine scanning) did not impact the conformational behavior of the peptide, but the substitution of the glycine residue at position 22 with alanine resulted in significant pH-responsive behavior. Therefore this mutant stimulus-responsive peptide may be more valuable for future protein engineering and bionanotechnology efforts.

Structure-function characterization and optimization of a plant-derived antibacterial peptide.

PubMed

Suarez, Mougli; Haenni, Marisa; Canarelli, Stéphane; Fisch, Florian; Chodanowski, Pierre; Servis, Catherine; Michielin, Olivier; Freitag, Ruth; Moreillon, Philippe; Mermod, Nicolas

2005-09-01

Crushed seeds of the Moringa oleifera tree have been used traditionally as natural flocculants to clarify drinking water. We previously showed that one of the seed peptides mediates both the sedimentation of suspended particles such as bacterial cells and a direct bactericidal activity, raising the possibility that the two activities might be related. In this study, the conformational modeling of the peptide was coupled to a functional analysis of synthetic derivatives. This indicated that partly overlapping structural determinants mediate the sedimentation and antibacterial activities. Sedimentation requires a positively charged, glutamine-rich portion of the peptide that aggregates bacterial cells. The bactericidal activity was localized to a sequence prone to form a helix-loop-helix structural motif. Amino acid substitution showed that the bactericidal activity requires hydrophobic proline residues within the protruding loop. Vital dye staining indicated that treatment with peptides containing this motif results in bacterial membrane damage. Assembly of multiple copies of this structural motif into a branched peptide enhanced antibacterial activity, since low concentrations effectively kill bacteria such as Pseudomonas aeruginosa and Streptococcus pyogenes without displaying a toxic effect on human red blood cells. This study thus identifies a synthetic peptide with potent antibacterial activity against specific human pathogens. It also suggests partly distinct molecular mechanisms for each activity. Sedimentation may result from coupled flocculation and coagulation effects, while the bactericidal activity would require bacterial membrane destabilization by a hydrophobic loop.

Structure-Function Characterization and Optimization of a Plant-Derived Antibacterial Peptide

PubMed Central

Suarez, Mougli; Haenni, Marisa; Canarelli, Stéphane; Fisch, Florian; Chodanowski, Pierre; Servis, Catherine; Michielin, Olivier; Freitag, Ruth; Moreillon, Philippe; Mermod, Nicolas

2005-01-01

Crushed seeds of the Moringa oleifera tree have been used traditionally as natural flocculants to clarify drinking water. We previously showed that one of the seed peptides mediates both the sedimentation of suspended particles such as bacterial cells and a direct bactericidal activity, raising the possibility that the two activities might be related. In this study, the conformational modeling of the peptide was coupled to a functional analysis of synthetic derivatives. This indicated that partly overlapping structural determinants mediate the sedimentation and antibacterial activities. Sedimentation requires a positively charged, glutamine-rich portion of the peptide that aggregates bacterial cells. The bactericidal activity was localized to a sequence prone to form a helix-loop-helix structural motif. Amino acid substitution showed that the bactericidal activity requires hydrophobic proline residues within the protruding loop. Vital dye staining indicated that treatment with peptides containing this motif results in bacterial membrane damage. Assembly of multiple copies of this structural motif into a branched peptide enhanced antibacterial activity, since low concentrations effectively kill bacteria such as Pseudomonas aeruginosa and Streptococcus pyogenes without displaying a toxic effect on human red blood cells. This study thus identifies a synthetic peptide with potent antibacterial activity against specific human pathogens. It also suggests partly distinct molecular mechanisms for each activity. Sedimentation may result from coupled flocculation and coagulation effects, while the bactericidal activity would require bacterial membrane destabilization by a hydrophobic loop. PMID:16127062

Conformational dynamics of the inner pore helix of voltage-gated potassium channels

NASA Astrophysics Data System (ADS)

Choe, Seungho; Grabe, Michael

2009-06-01

Voltage-gated potassium (Kv) channels control the electrical excitability of neurons and muscles. Despite this key role, how these channels open and close or gate is not fully understood. Gating is usually attributed to the bending and straightening of pore-lining helices at glycine and proline residues. In this work we focused on the role of proline in the Pro-Val-Pro (PVP) motif of the inner S6 helix in the Kv1.2 channel. We started by developing a simple hinged-rod model to fully explore the configurational space of bent helices and we related these configurations to the degree of pore opening. We then carried out fully atomistic simulations of the S6 helices and compared these simulations to the hinged-rod model. Both methods suggest that Kv1 channels are not tightly closed when the inner helices are straight, unlike what is seen in the non-PVP containing channels KcsA and KirBac. These results invite the possibility that the S6 helices may be kinked when Kv1 channels are closed. Our simulations indicate that the wild-type helix adopts multiple spatially distinct configurations, which is consistent with its role in adopting a closed state and an open state. The two most dominant configurational basins correspond to a 6 Å movement of the helix tail accompanied by the PVP region undergoing a local α-helix to 310-helix transition. We explored how single point mutations affect the propensity of the S6 helix to adopt particular configurations. Interestingly, mutating the first proline, P405 (P473 in Shaker), to alanine completely removed the bistable nature of the S6 helix possibly explaining why this mutation compromises the channel. Next, we considered four other mutations in the area known to affect channel gating and we saw similarly dramatic changes to the helix's dynamics and range of motion. Our results suggest a possible mechanism of helix pore closure and they suggest differences in the closed state of glycine-only channels, like KcsA, and PVP containing channels.

Kevlar: Transitioning Helix for Research to Practice

DTIC Science & Technology

2016-03-01

entropy randomization techniques, automated program repairs leveraging highly-optimized virtual machine technology, and developing a novel framework...attacker from exploiting residual vulnerabilities in a wide variety of classes. Helix/Kevlar uses novel, fine-grained, high- entropy diversification...the Air Force, and IARPA). Salient features of Helix/Kevlar include developing high- entropy randomization techniques, automated program repairs

Triple Helix Systems: An Analytical Framework for Innovation Policy and Practice in the Knowledge Society

ERIC Educational Resources Information Center

Ranga, Marina; Etzkowitz, Henry

2013-01-01

This paper introduces the concept of Triple Helix systems as an analytical construct that synthesizes the key features of university--industry--government (Triple Helix) interactions into an "innovation system" format, defined according to systems theory as a set of components, relationships and functions. Among the components of Triple…

"Special Issue": Regional Dimensions of the Triple Helix Model

ERIC Educational Resources Information Center

Todeva, Emanuela; Danson, Mike

2016-01-01

This paper introduces the rationale for the special issue and its contributions, which bridge the literature on regional development and the Triple Helix model. The concept of the Triple Helix at the sub-national, and specifically regional, level is established and examined, with special regard to regional economic development founded on…

Regional Dimensions of the Triple Helix Model: Setting the Context

ERIC Educational Resources Information Center

Todeva, Emanuela; Danson, Mike

2016-01-01

This paper introduces the rationale for the special issue and its contributions, which bridge the literature on regional development and the Triple Helix model. The concept of the Triple Helix at the sub-national, and specifically regional, level is established and examined, with special regard to regional economic development founded on…

IFITM3 requires an amphipathic helix for antiviral activity.

PubMed

Chesarino, Nicholas M; Compton, Alex A; McMichael, Temet M; Kenney, Adam D; Zhang, Lizhi; Soewarna, Victoria; Davis, Matthew; Schwartz, Olivier; Yount, Jacob S

2017-10-01

Interferon-induced transmembrane protein 3 (IFITM3) is a cellular factor that blocks virus fusion with cell membranes. IFITM3 has been suggested to alter membrane curvature and fluidity, though its exact mechanism of action is unclear. Using a bioinformatic approach, we predict IFITM3 secondary structures and identify a highly conserved, short amphipathic helix within a hydrophobic region of IFITM3 previously thought to be a transmembrane domain. Consistent with the known ability of amphipathic helices to alter membrane properties, we show that this helix and its amphipathicity are required for the IFITM3-dependent inhibition of influenza virus, Zika virus, vesicular stomatitis virus, Ebola virus, and human immunodeficiency virus infections. The homologous amphipathic helix within IFITM1 is also required for the inhibition of infection, indicating that IFITM proteins possess a conserved mechanism of antiviral action. We further demonstrate that the amphipathic helix of IFITM3 is required to block influenza virus hemagglutinin-mediated membrane fusion. Overall, our results provide evidence that IFITM proteins utilize an amphipathic helix for inhibiting virus fusion. © 2017 The Authors.

The Effect of C-Terminal Helix on the Stability of FF Domain Studied by Molecular Dynamics Simulation

PubMed Central

Zhao, Liling; Cao, Zanxia; Wang, Jihua

2012-01-01

To investigate the effect of C-terminal helix on the stability of the FF domain, we studied the native domain FF3-71 from human HYPA/FBP11 and the truncated version FF3-60 with C-terminal helix being deleted by molecular dynamics simulations with GROMACS package and GROMOS 43A1 force field. The results indicated that the structures of truncated version FF3-60 were evident different from those of native partner FF3-71. Compared with FF3-71, the FF3-60 lost some native contacts and exhibited some similar structural characters to those of intermediate state. The C-terminal helix played a major role in stabilizing the FF3-71 domain. To a certain degree, the FF domain had a tendency to form an intermediate state without the C-terminal helix. In our knowledge, this was the first study to examine the role of C-terminal helix of FF domain in detail by molecular dynamics simulations, which was useful to understand the three-state folding mechanism of the small FF domain. PMID:22408419

Approach to the unfolding and folding dynamics of add A-riboswitch upon adenine dissociation using a coarse-grained elastic network model

NASA Astrophysics Data System (ADS)

Li, Chunhua; Lv, Dashuai; Zhang, Lei; Yang, Feng; Wang, Cunxin; Su, Jiguo; Zhang, Yang

2016-07-01

Riboswitches are noncoding mRNA segments that can regulate the gene expression via altering their structures in response to specific metabolite binding. We proposed a coarse-grained Gaussian network model (GNM) to examine the unfolding and folding dynamics of adenosine deaminase (add) A-riboswitch upon the adenine dissociation, in which the RNA is modeled by a nucleotide chain with interaction networks formed by connecting adjoining atomic contacts. It was shown that the adenine binding is critical to the folding of the add A-riboswitch while the removal of the ligand can result in drastic increase of the thermodynamic fluctuations especially in the junction regions between helix domains. Under the assumption that the native contacts with the highest thermodynamic fluctuations break first, the iterative GNM simulations showed that the unfolding process of the adenine-free add A-riboswitch starts with the denature of the terminal helix stem, followed by the loops and junctions involving ligand binding pocket, and then the central helix domains. Despite the simplified coarse-grained modeling, the unfolding dynamics and pathways are shown in close agreement with the results from atomic-level MD simulations and the NMR and single-molecule force spectroscopy experiments. Overall, the study demonstrates a new avenue to investigate the binding and folding dynamics of add A-riboswitch molecule which can be readily extended for other RNA molecules.

When proteome meets genome: the alpha helix and the beta strand of proteins are eschewed by mRNA splice junctions and may define the minimal indivisible modules of protein architecture.

PubMed

Barik, Sailen

2004-09-01

The significance of the intron-exon structure of genes is a mystery. As eukaryotic proteins are made up of modular functional domains, each exon was suspected to encode some form of module; however, the definition of a module remained vague. Comparison of pre-mRNA splice junctions with the three-dimensional architecture of its protein product from different eukaryotes revealed that the junctions were far less likely to occur inside the alpha-helices and beta-strands of proteins than within the more flexible linker regions ('turns' and 'loops') connecting them. The splice junctions were equally distributed in the different types of linkers and throughout the linker sequence, although a slight preference for the central region of the linker was observed. The avoidance of the alpha-helix and the beta-strand by splice junctions suggests the existence of a selection pressure against their disruption, perhaps underscoring the investment made by nature in building these intricate secondary structures. A corollary is that the helix and the strand are the smallest integral architectural units of a protein and represent the minimal modules in the evolution of protein structure. These results should find use in comparative genomics, designing of cloning strategies, and in the mutual verification of genome sequences with protein structures.

Data on diverse roles of helix perturbations in membrane proteins.

PubMed

Shelar, Ashish; Bansal, Manju

2016-12-01

The various structural variations observed in TM helices of membrane proteins have been deconstructed into 9 distinct types of helix perturbations. These perturbations are defined by the deviation of TM helices from the predominantly observed linear α-helical conformation, to form 3 10 - and π-helices, as well as adopting curved and kinked geometries. The data presented here supplements the article 'Helix perturbations in Membrane Proteins Assist in Inter-helical Interactions and Optimal Helix Positioning in the Bilayer' (A. Shelar, M. Bansal, 2016) [1]. This data provides strong evidence for the role of various helix perturbations in influencing backbone torsion angles of helices, mediating inter-helical interactions, oligomer formation and accommodation of hydrophobic residues within the bilayer. The methodology used for creation of various datasets of membrane protein families (Sodium/Calcium exchanger and Heme Copper Oxidase) has also been mentioned.

The CDRH helix. A phase I clinical trial.

PubMed

Shimm, D S; Cetas, T C; Hynynen, K H; Buechler, D N; Anhalt, D P; Sykes, H F; Cassady, J R

1989-04-01

Seventeen patients have been given regional hyperthermia treatments using the Center for Devices and Radiologic Health (CDRH) Helix, a resonant helical coil unit. Most of these patients had large, clinically advanced tumors, whose mean volume exceeded 1000 cc. Mean maximum, minimum, and average temperatures were 40.6, 38.6, and 39.6 degrees C, respectively, for all sites combined. The pelvic heating capabilities of the CDRH Helix and the BSD-1000 annular phased array were compared, and generally were equivalent. Although the Helix could be used in a wider variety of locations, and was more comfortable and easier to use than the BSD-1000 annular phased array, neither device was particularly effective in generating clinically useful temperatures; the Helix is currently under investigation for use in regional-systemic hyperthermia in combination with antineoplastic drugs and biologic response modifiers.

Theory of helix traveling wave tubes with dielectric and vane loading

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

Freund, H.P.; Zaidman, E.G.; Antonsen, T.M. Jr.

1996-08-01

A time-dependent nonlinear analysis of a helix traveling wave tube (TWT) is presented for a configuration where an electron beam propagates through a sheath helix surrounded by a conducting wall. The effects of dielectric and vane loading are included in the formulation as is efficiency enhancement by tapering the helix pitch. Dielectric loading is described under the assumption that the gap between the helix and the wall is uniformly filled by a dielectric material. The vane-loading model describes the insertion of an arbitrary number of vanes running the length of the helix, and the polarization of the field between themore » vanes is assumed to be an azimuthally symmetric transverse-electric mode. The field is represented as a superposition of azimuthally symmetric waves in a vacuum sheath helix. An overall explicit sinusoidal variation of the form exp({ital ikz}{minus}{ital i}{omega}{ital t}) is assumed (where {omega} denotes the angular frequency corresponding to the wave number {ital k} in the vacuum sheath helix), and the polarization and radial variation of each wave is determined by the boundary conditions in a vacuum sheath helix. The propagation of each wave {ital in} {ital vacuo} as well as the interaction of each wave with the electron beam is included by allowing the amplitudes of the waves to vary in {ital z} and {ital t}. A dynamical equation for the field amplitudes is derived analogously to Poynting{close_quote}s equation, and solved in conjunction with the three-dimensional Lorentz force equations for an ensemble of electrons. Electron beams with a both a continuous and emission-gated pulse format are analyzed, and the model is compared with linear theory of the interaction as well as with the performance of a TWTs operated at the Naval Research Laboratory and at Northrop{endash}Grumman Corporation. {copyright} {ital 1996 American Institute of Physics.}« less

Complex between triple helix of collagen and double helix of DNA in aqueous solution.

PubMed

Mrevlishvili, George M; Svintradze, David V

2005-06-01

We demonstrate in this paper that one example of a biologically important and molecular self-assembling complex system is a collagen-DNA ordered aggregate which spontaneously forms in aqueous solutions. Interaction between the collagen and the DNA leads to destruction of the hydration shell of the triple helix and stabilization of the double helix structure. From a molecular biology point of view this nano-scale self-assembling superstructure could increase the stability of DNA against the nucleases during collagen diseases and the growth of collagen fibrills in the presence of DNA.

Rotational strength of dye-helix complexes as studied by a potential model theory

NASA Astrophysics Data System (ADS)

Kamiya, Mamoru

1988-03-01

The fundamental features of the induced optical activity in dye-helix complexes are clarified by the trap potential model. The effect of the potential depth on the induced rotational strength is explained in terms of the relative magnitudes of the wave-phase and helix-phase variations in the path of an electron moving along a restricted helix segment just like an exciton trapped around a dye intercalation site. The potential parameters have been optimized so as to reproduce the ionic strength effect upon the rotational strengths induced in proflavine-DNA intercalation complexes.

Empirical parameterization of a model for predicting peptide helix/coil equilibrium populations.

PubMed Central

Andersen, N. H.; Tong, H.

1997-01-01

A modification of the Lifson-Roig formulation of helix/coil transitions is presented; it (1) incorporates end-capping and coulombic (salt bridges, hydrogen bonding, and side-chain interactions with charged termini and the helix dipole) effects, (2) helix-stabilizing hydrophobic clustering, (3) allows for different inherent termination probabilities of individual residues, and (4) differentiates helix elongation in the first versus subsequent turns of a helix. Each residue is characterized by six parameters governing helix formation. The formulation of the conditional probability of helix initiation and termination that we developed is essentially the same as one presented previously (Shalongo W, Stellwagen, E. 1995. Protein Sci 4:1161-1166) and nearly the mathematical equivalent of the new capping formulation incorporated in the model presented by Rohl et al. (1996. Protein Sci 5:2623-2637). Side-chain/side-chain interactions are, in most cases, incorporated as context dependent modifications of propagation rather than nucleation parameters. An alternative procedure for converting [theta]221 values to experimental fractional helicities () is presented. Tests of the program predictions suggest this method may have some advantages both for designed peptides and for the analysis of secondary structure preferences that could drive the formation of molten-globule intermediates on protein folding pathways. The model predicts the fractional helicity of 385 peptides with a root-mean-square deviation (RMSD) of 0.050 and locates (with precise definition of the termini in many cases) helices in proteins as well as competing methods. The propagation and nucleation parameters were derived from NMR data and from the CD data for a 79 peptide "learning set" for which an excellent fit resulted (RMSD = 0.0295). The current set of parameter corrections for capping boxes, helix dipole interactions, and side-chain/side-chain interactions (coulombic, hydrogen bonding and hydrophobic clustering), although still under development provide a significant improvement in both helix/coil equilibrium prediction for peptides and helix location in protein sequences. This is clearly evident in the rms deviations between CD measures and calculated values of fractional helicity for different classes of peptides before and after applying the corrections: for peptides lacking capping boxes and i/i + 3 and i/i + 4 side-chain/side-chain interactions RMSD = 0.044 (n = 164) versus RMSD = 0.054 (0.172 without the corrections, n = 221) for peptides that required context-dependent corrections of the parameters. If we restrict the analysis to N-acylated peptides with helix stabilizing side-chain/side-chain interactions (including N-capping boxes), the degree to which our corrections account for the stabilizing interaction can be judged from the change in helicity underestimation, (calc-CD): -0.15 +/- 0.10, which is reduced to -0.018 +/- 0.048 (n = 191) upon applying the corrections. PMID:9300492

Environmental Public Health Tracking: Health and Environment Linked for Information Exchange-Atlanta (HEXIX-Atlanta: A cooperative Program Between CDC and NASA for Development of an Environmental Public Health Tracking Network in the Atlanta Metropolitan Area

NASA Technical Reports Server (NTRS)

Quattrochi, Dale A.; Niskar, Amanda Sue

2005-01-01

The Centers for Disease Control and Prevention (CDC) is coordinating HELIX- Atlanta to provide information regarding the five-county Metropolitan Atlanta Area (Clayton, Cobb, DeKalb, Fulton, and Gwinett) via a network of integrated environmental monitoring and public health data systems so that all sectors can take action to prevent and control environmentally related health effects. The HELIX-Atlanta Network is a tool to access interoperable information systems with optional information technology linkage functionality driven by scientific rationale. HELIX-Atlanta is a collaborative effort with local, state, federal, and academic partners, including the NASA Marshall Space Flight Center. The HELIX-Atlanta Partners identified the following HELIX-Atlanta initial focus areas: childhood lead poisoning, short-latency cancers, developmental disabilities, birth defects, vital records, respiratory health, age of housing, remote sensing data, and environmental monitoring, HELIX-Atlanta Partners identified and evaluated information systems containing information on the above focus areas. The information system evaluations resulted in recommendations for what resources would be needed to interoperate selected information systems in compliance with the CDC Public Health Information Network (PHIN). This presentation will discuss the collaborative process of building a network that links health and environment data for information exchange, including NASA remote sensing data, for use in HELIX-Atlanta.

A directional nucleation-zipping mechanism for triple helix formation

PubMed Central

Alberti, Patrizia; Arimondo, Paola B.; Mergny, Jean-Louis; Garestier, Thérèse; Hélène, Claude; Sun, Jian-Sheng

2002-01-01

A detailed kinetic study of triple helix formation was performed by surface plasmon resonance. Three systems were investigated involving 15mer pyrimidine oligonucleotides as third strands. Rate constants and activation energies were validated by comparison with thermodynamic values calculated from UV-melting analysis. Replacement of a T·A base pair by a C·G pair at either the 5′ or the 3′ end of the target sequence allowed us to assess mismatch effects and to delineate the mechanism of triple helix formation. Our data show that the association rate constant is governed by the sequence of base triplets on the 5′ side of the triplex (referred to as the 5′ side of the target oligopurine strand) and provides evidence that the reaction pathway for triple helix formation in the pyrimidine motif proceeds from the 5′ end to the 3′ end of the triplex according to the nucleation-zipping model. It seems that this is a general feature for all triple helices formation, probably due to the right-handedness of the DNA double helix that provides a stronger base stacking at the 5′ than at the 3′ duplex–triplex junction. Understanding the mechanism of triple helix formation is not only of fundamental interest, but may also help in designing better triple helix-forming oligonucleotides for gene targeting and control of gene expression. PMID:12490709

Sequence specific peptidomimetic molecules inhibitors of a protein-protein interaction at the helix 1 level of c-Myc.

PubMed

Nieddu, Erika; Melchiori, A; Pescarolo, M P; Bagnasco, L; Biasotti, B; Licheri, B; Malacarne, D; Tortolina, L; Castagnino, N; Pasa, S; Cimoli, G; Avignolo, C; Ponassi, R; Balbi, C; Patrone, E; D'arrigo, C; Barboro, P; Vasile, F; Orecchia, P; Carnemolla, B; Damonte, G; Millo, E; Palomba, D; Fassina, G; Mazzei, M; Parodi, S

2005-04-01

Our work is focused in the broad area of strategies and efforts to inhibit protein-protein interactions. The possible strategies in this field are definitely much more varied than in the case of ATP-pocket inhibitors. In our previous work (10), we reported that a retro-inverso (RI) form of Helix1 (H1) of c-Myc, linked to an RI-internalization sequence arising from the third alpha-helix of Antennapedia (Int) was endowed with an antiproliferative and proapoptotic activity toward the cancer cell lines MCF-7 and HCT-116. The activity apparently was dependent upon the presence of the Myc motif. In this work, by ala-scan mapping of the H1 portion of our molecules with D-aa, we found two amino acids necessary for antiproliferative activity: D-Lys in 4 and D-Arg in 5 (numbers refer to L-forms). In the natural hetero-dimer, these two side chains project to the outside of the four alpha-helix bundle. Moreover, we were able to obtain three peptides more active than the original lead. They strongly reduced cell proliferation and survival (RI-Int-VV-H1-E2A,S6A,F8A; RI-Int-VV-H1-S6A,F8A,R11A; RI-Int-VV-H1-S6A,F8A,Q13A): after 8 days at 10 muM total cell number was approximately 1% of the number of cells initially seeded. In these more potent molecules, the ablated side chains project to the inside in the corresponding natural four alpha-helix bundle. In the present work, we also investigated the behavior of our molecules at the biochemical level. Using both a circular dichroism (CD) and a fluorescence anisotropy approach, we noted that side chains projecting at the interior of the four alpha-helix bundle are needed for inducing the partial unfolding of Myc-H2, without an opening of the leucine zipper. Side chains projecting at the outside are not required for this biochemical effect. However, antiproliferative activity had the opposite requirements: side chains projecting at the outside of the bundle were essential, and, on the contrary, ablation of one side chain at a time projecting at the inside increased rather than decreased biological activity. We conclude that our active molecules probably interfere at the level of a protein-protein interaction between Myc-Max and a third protein of the transcription complex. Finally, CD and nuclear magnetic resonance (NMR) data, plus dynamic simulations, suggest a prevalent random coil conformation of the H1 portion of our molecules, at least in diluted solutions. The introduction of a kink (substitution with proline in positions 5 or 7) led to an important reduction of biological activity. We have also synthesized a longer peptido-mimetic molecule (RI-Int-H1-S6A,F8A-loop-H2) with the intent of obtaining a wider zone of interaction and a stronger interference at the level of the higher-order structure (enhanceosome). RI-Int-H1-S6A,F8A-loop-H2 was less active rather than more active in respect to RI-Int-VV-H1-S6A,F8A, apparently because it has a clear bent to form a beta-sheet (CD and NMR data).

Probing the Non-Native H Helix Translocation in Apomyoglobin Folding Intermediates

PubMed Central

2015-01-01

Apomyoglobin folds via sequential helical intermediates that are formed by rapid collapse of the A, B, G, and H helix regions. An equilibrium molten globule with a similar structure is formed near pH 4. Previous studies suggested that the folding intermediates are kinetically trapped states in which folding is impeded by non-native packing of the G and H helices. Fluorescence spectra of mutant proteins in which cysteine residues were introduced at several positions in the G and H helices show differential quenching of W14 fluorescence, providing direct evidence of translocation of the H helix relative to helices A and G in both the kinetic and equilibrium intermediates. Förster resonance energy transfer measurements show that a 5-({2-[(acetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid acceptor coupled to K140C (helix H) is closer to Trp14 (helix A) in the equilibrium molten globule than in the native state, by a distance that is consistent with sliding of the H helix in an N-terminal direction by approximately one helical turn. Formation of an S108C–L135C disulfide prevents H helix translocation in the equilibrium molten globule by locking the G and H helices into their native register. By enforcing nativelike packing of the A, G, and H helices, the disulfide resolves local energetic frustration and facilitates transient docking of the E helix region onto the hydrophobic core but has only a small effect on the refolding rate. The apomyoglobin folding landscape is highly rugged, with several energetic bottlenecks that frustrate folding; relief of any one of the major identified bottlenecks is insufficient to speed progression to the transition state. PMID:24857522

Molecular dynamics simulations of fluoropolymers in the solid state

NASA Astrophysics Data System (ADS)

Holt, David Bryan

1998-10-01

Molecular mechanics and dynamics simulations have been utilized to address the behavior of helix reversal defects in fluoropolymers. The results of the simulations confirm that helix reversals do form and migrate in PTFE crystals. The most important defect structure is a helix reversal band: two helix reversals which bracker a small chain segment (typically 6-7 backbone atoms) having the opposite helical sense from the parent molecule. Small reversal bands had velocities ranging between 100 m/s (low temperature)-250 m/s (high temperature). The size of this reversal band defect is dependent upon the helical conformation and is equal to approximately half of the helical repeat unit in the low and intermediate temperature phases. In the high temperature phase where intermolecular effects are diminished, a wider distribution of reversal band sizes was observed during the simulations. A mechanism is identified by which significant reorientation of a chain segment about the molecular axis can occur when it is bracketed by two helix reversal bands. Simulations with a model containing a perfluoromethyl (PFM) group at low temperature showed that the presence of the PFM group significantly restricts chain mobility locally. However, a significant reduction in the helix reversal defect density was observed on neighboring chains as well. During simulations in which a shear deformation was applied to the models with and without a PFM group, an increase in reversal defect density was observed. However, the helix reversal density in the sheared model containing the PFM branch was less than that in the model without a PFM branch under no shear. These data implicate helix reversal defects and associated chain segment motions in the mechanical behavior of fluoropolymer materials.

MacA is a second cytochrome c peroxidase of Geobacter sulfurreducens.

PubMed

Seidel, Julian; Hoffmann, Maren; Ellis, Katie E; Seidel, Antonia; Spatzal, Thomas; Gerhardt, Stefan; Elliott, Sean J; Einsle, Oliver

2012-04-03

The metal-reducing δ-proteobacterium Geobacter sulfurreducens produces a large number of c-type cytochromes, many of which have been implicated in the transfer of electrons to insoluble metal oxides. Among these, the dihemic MacA was assigned a central role. Here we have produced G. sulfurreducens MacA by recombinant expression in Escherichia coli and have solved its three-dimensional structure in three different oxidation states. Sequence comparisons group MacA into the family of diheme cytochrome c peroxidases, and the protein indeed showed hydrogen peroxide reductase activity with ABTS(-2) as an electron donor. The observed K(M) was 38.5 ± 3.7 μM H(2)O(2) and v(max) was 0.78 ± 0.03 μmol of H(2)O(2)·min(-1)·mg(-1), resulting in a turnover number k(cat) = 0.46 · s(-1). In contrast, no Fe(III) reductase activity was observed. MacA was found to display electrochemical properties similar to other bacterial diheme peroxidases, in addition to the ability to electrochemically mediate electron transfer to the soluble cytochrome PpcA. Differences in activity between CcpA and MacA can be rationalized with structural variations in one of the three loop regions, loop 2, that undergoes conformational changes during reductive activation of the enzyme. This loop is adjacent to the active site heme and forms an open loop structure rather than a more rigid helix as in CcpA. For the activation of the protein, the loop has to displace the distal ligand to the active site heme, H93, in loop 1. A H93G variant showed an unexpected formation of a helix in loop 2 and disorder in loop 1, while a M297H variant that altered the properties of the electron transfer heme abolished reductive activation.

MacA is a Second Cytochrome c Peroxidase of Geobacter sulfurreducens

PubMed Central

Seidel, Julian; Hoffmann, Maren; Ellis, Katie E.; Seidel, Antonia; Spatzal, Thomas; Gerhardt, Stefan; Elliott, Sean J.

2012-01-01

The metal-reducing δ-proteobacterium Geobacter sulfurreducens produces a large number of c-type cytochromes, many of which have been implicated in the transfer of electrons to insoluble metal oxides. Among these, the dihemic MacA was assigned a central role. Here we have produced G. sulfurreducens MacA by recombinant expression in Escherichia coli and have solved its three-dimensional structure in three different oxidation states. Sequence comparisons group MacA into the family of diheme cytochrome c peroxidases, and the protein indeed showed hydrogen peroxide reductase activity with ABTS2– as an electron donor. The observed KM was 38.5 ± 3.7 μM H2O2 and vmax was 0.78 ± 0.03 μmol H2O2·min–1·mg–1, resulting in a turnover number kcat = 0.46 · s–1. In contrast, no Fe(III) reductase activity was observed. MacA was found to display similar electrochemical properties to other bacterial diheme peroxidases, in additional to the ability to electrochemically mediate electron transfer to the soluble cytochrome PpcA. Differences in activity between CcpA and MacA can be rationalized with structural variations in one of the three loop regions, loop 2, that undergo conformational changes during reductive activation of the enzyme. This loop is adjacent to the active site heme and forms an open loop structure rather than a more rigid helix as in CcpA. For the activation of the protein the loop has to displace the distal ligand to the active site heme, H93, in loop 1. A H93G variant showed an unexpected formation of a helix in loop 2 and disorder in loop 1, while a M297H variant that altered the properties of the electron transfer heme abolished reductive activation. PMID:22417533

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

Jones, J.P.; Bangs, A.L.; Butler, P.L.

Hetero Helix is a programming environment which simulates shared memory on a heterogeneous network of distributed-memory computers. The machines in the network may vary with respect to their native operating systems and internal representation of numbers. Hetero Helix presents a simple programming model to developers, and also considers the needs of designers, system integrators, and maintainers. The key software technology underlying Hetero Helix is the use of a compiler'' which analyzes the data structures in shared memory and automatically generates code which translates data representations from the format native to each machine into a common format, and vice versa. Themore » design of Hetero Helix was motivated in particular by the requirements of robotics applications. Hetero Helix has been used successfully in an integration effort involving 27 CPUs in a heterogeneous network and a body of software totaling roughly 100,00 lines of code. 25 refs., 6 figs.« less

Crystal Structure of the Passenger Domain of the Escherichia coli Autotransporter EspP

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

Khan, Shekeb; Mian, Hira S.; Sandercock, Linda E.

2013-03-07

Autotransporters represent a large superfamily of known and putative virulence factors produced by Gram-negative bacteria. They consist of an N-terminal 'passenger domain' responsible for the specific effector functions of the molecule and a C-terminal '{beta}-domain' responsible for translocation of the passenger across the bacterial outer membrane. Here, we present the 2.5-{angstrom} crystal structure of the passenger domain of the extracellular serine protease EspP, produced by the pathogen Escherichia coli O157:H7 and a member of the serine protease autotransporters of Enterobacteriaceae (SPATEs). Like the previously structurally characterized SPATE passenger domains, the EspP passenger domain contains an extended right-handed parallel {beta}-helix precededmore » by an N-terminal globular domain housing the catalytic function of the protease. Of note, however, is the absence of a second globular domain protruding from this {beta}-helix. We describe the structure of the EspP passenger domain in the context of previous results and provide an alternative hypothesis for the function of the {beta}-helix within SPATEs.« less

Antimicrobial Effects of Helix D-derived Peptides of Human Antithrombin III*

PubMed Central

Papareddy, Praveen; Kalle, Martina; Bhongir, Ravi K. V.; Mörgelin, Matthias; Malmsten, Martin; Schmidtchen, Artur

2014-01-01

Antithrombin III (ATIII) is a key antiproteinase involved in blood coagulation. Previous investigations have shown that ATIII is degraded by Staphylococcus aureus V8 protease, leading to release of heparin binding fragments derived from its D helix. As heparin binding and antimicrobial activity of peptides frequently overlap, we here set out to explore possible antibacterial effects of intact and degraded ATIII. In contrast to intact ATIII, the results showed that extensive degradation of the molecule yielded fragments with antimicrobial activity. Correspondingly, the heparin-binding, helix d-derived, peptide FFFAKLNCRLYRKANKSSKLV (FFF21) of human ATIII, was found to be antimicrobial against particularly the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. Fluorescence microscopy and electron microscopy studies demonstrated that FFF21 binds to and permeabilizes bacterial membranes. Analogously, FFF21 was found to induce membrane leakage of model anionic liposomes. In vivo, FFF21 significantly reduced P. aeruginosa infection in mice. Additionally, FFF21 displayed anti-endotoxic effects in vitro. Taken together, our results suggest novel roles for ATIII-derived peptide fragments in host defense. PMID:25202017

Antimicrobial effects of helix D-derived peptides of human antithrombin III.

PubMed

Papareddy, Praveen; Kalle, Martina; Bhongir, Ravi K V; Mörgelin, Matthias; Malmsten, Martin; Schmidtchen, Artur

2014-10-24

Antithrombin III (ATIII) is a key antiproteinase involved in blood coagulation. Previous investigations have shown that ATIII is degraded by Staphylococcus aureus V8 protease, leading to release of heparin binding fragments derived from its D helix. As heparin binding and antimicrobial activity of peptides frequently overlap, we here set out to explore possible antibacterial effects of intact and degraded ATIII. In contrast to intact ATIII, the results showed that extensive degradation of the molecule yielded fragments with antimicrobial activity. Correspondingly, the heparin-binding, helix D-derived, peptide FFFAKLNCRLYRKANKSSKLV (FFF21) of human ATIII, was found to be antimicrobial against particularly the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. Fluorescence microscopy and electron microscopy studies demonstrated that FFF21 binds to and permeabilizes bacterial membranes. Analogously, FFF21 was found to induce membrane leakage of model anionic liposomes. In vivo, FFF21 significantly reduced P. aeruginosa infection in mice. Additionally, FFF21 displayed anti-endotoxic effects in vitro. Taken together, our results suggest novel roles for ATIII-derived peptide fragments in host defense. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Changes in the myosin secondary structure and shrimp surimi gel strength induced by dense phase carbon dioxide.

PubMed

Guo, Minghui; Liu, Shucheng; Ismail, Marliya; Farid, Mohammed M; Ji, Hongwu; Mao, Weijie; Gao, Jing; Li, Chengyong

2017-07-15

Dense phase carbon dioxide (DPCD) could induce protein conformation changes. Myosin and shrimp surimi from Litopenaeus vannamei were treated with DPCD at 5-25MPa and 40-60°C for 20min. Myosin secondary structure was investigated by circular dichroism and shrimp surimi gel strength was determined using textural analysis to develop correlations between them. DPCD had a greater effect on secondary structure and gel strength than heating. With increasing pressure and temperature, the α-helix content of DPCD-treated myosin decreased, while the β-sheet, β-turn and random coil contents increased, and the shrimp surimi gel strength increased. The α-helix content was negatively correlated with gel strength, while the β-sheet, β-turn and random coil contents were positively correlated with gel strength. Therefore, when DPCD induced myosin to form a gel, the α-helix of myosin was unfolded and gradually converted to a β-sheet. Such transformations led to protein-protein interactions and cross-linking, which formed a three-dimensional network to enhance the gel strength. Copyright © 2017 Elsevier Ltd. All rights reserved.

A look at the effect of sequence complexity on pressure destabilisation of DNA polymers.

PubMed

Rayan, Gamal; Macgregor, Robert B

2015-04-01

Our previous studies on the helix-coil transition of double-stranded DNA polymers have demonstrated that molar volume change (ΔV) accompanying the thermally-induced transition can be positive or negative depending on the experimental conditions, that the pressure-induced transition is more cooperative than the heat-induced transition [Rayan and Macgregor, J Phys Chem B2005, 109, 15558-15565], and that the pressure-induced transition does not occur in the absence of water [Rayan and Macgregor, Biophys Chem, 2009, 144, 62-66]. Additionally, we have shown that ΔV values obtained by pressure-dependent techniques differ from those obtained by ambient pressure techniques such as PPC [Rayan et al. J Phys Chem B2009, 113, 1738-1742] thus shedding light on the effects of pressure on DNA polymers. Herein, we examine the effect of sequence complexity, and hence cooperativity on pressure destabilisation of DNA polymers. Working with Clostridium perfringes DNA under conditions such that the estimated ΔV of the helix-coil transition corresponds to -1.78 mL/mol (base pair) at atmospheric pressure, we do not observe the pressure-induced helix-coil transition of this DNA polymer, whereas synthetic copolymers poly[d(A-T)] and poly[d(I-C)] undergo cooperative pressure-induced transitions at similar ΔV values. We hypothesise that the reason for the lack of pressure-induced helix-coil transition of C. perfringens DNA under these experimental conditions lies in its sequence complexity. Copyright © 2015 Elsevier B.V. All rights reserved.

Sequence diagrams and the presentation of structural and evolutionary relationships among proteins.

PubMed

Thomas, B R

1975-01-01

Protein sequences mapped on two-dimensional diagrams show characteristic patterns that should be of value in visualising sequence information and in distinguishing simpler structures. A convenient map form for comparative purposes is the alpha-helix diagram with aminoacid distribution analogous to the surface of an alpha-helix oriented so that an alpha-helix structure corresponds on the diagram to a vertical band 3.6 residues wide. The sequence diagram for an alpha-keratin, high-sulphur protein suggests a new form of polypeptide helix based on a repeating unit of five which may be an important component of alpha-keratin fibres.