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1.
Nat Commun ; 15(1): 592, 2024 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-38238333

RESUMO

The Arabidopsis thaliana DREB2A transcription factor interacts with the negative regulator RCD1 and the ACID domain of subunit 25 of the transcriptional co-regulator mediator (Med25) to integrate stress signals for gene expression, with elusive molecular interplay. Using biophysical and structural analyses together with high-throughput screening, we reveal a bivalent binding switch in DREB2A containing an ACID-binding motif (ABS) and the known RCD1-binding motif (RIM). The RIM is lacking in a stress-induced DREB2A splice variant with retained transcriptional activity. ABS and RIM bind to separate sites on Med25-ACID, and NMR analyses show a structurally heterogeneous complex deriving from a DREB2A-ABS proline residue populating cis- and trans-isomers with remote impact on the RIM. The cis-isomer stabilizes an α-helix, while the trans-isomer may introduce energetic frustration facilitating rapid exchange between activators and repressors. Thus, DREB2A uses a post-transcriptionally and post-translationally modulated switch for transcriptional regulation.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Isomerismo , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas Nucleares/metabolismo
2.
Cell Rep Methods ; 2(11): 100333, 2022 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-36452862

RESUMO

We apply a computational model, global multi-mutant analysis (GMMA), to inform on effects of most amino acid substitutions from a randomly mutated gene library. Using a high mutation frequency, the method can determine mutations that increase the stability of even very stable proteins for which conventional selection systems have reached their limit. As a demonstration of this, we screened a mutant library of a highly stable and computationally redesigned model protein using an in vivo genetic sensor for folding and assigned a stability effect to 374 of 912 possible single amino acid substitutions. Combining the top 9 substitutions increased the unfolding energy 47 to 69 kJ/mol in a single engineering step. Crystal structures of stabilized variants showed small perturbations in helices 1 and 2, which rendered them closer in structure to the redesign template. This case study illustrates the capability of the method, which is applicable to any screen for protein function.


Assuntos
Dobramento de Proteína , Proteínas , Proteínas/genética , Estabilidade Proteica , Mutação , Substituição de Aminoácidos
3.
Mol Biol Rep ; 49(1): 293-302, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34725746

RESUMO

BACKGROUND: Germin-like proteins (GLPs) are ubiquitous plant proteins, which play significant role in plant responses against various abiotic stresses. However, the potential functions of GLPs in rice (Oryza sativa) against salt and drought stress are still unclear. METHODS AND RESULTS: In this study, transcriptional variation of eight OsGLP genes (OsGLP3-6, OsGLP4-1, OsGLP8-4, OsGLP8-7, OsGLP8-10, OsGLP8-11 and OsGLP8-12) was analyzed in leaves and roots of two economically important Indica rice cultivars, KS282 and Super Basmati, under salt and drought stress at early seedling stage. The relative expression analysis from qRT-PCR indicated the highest increase in expression of OsGLP3-6 in leaves and roots of both rice varieties with a significantly higher expression in KS282. Moreover, relative change in expression of OsGLP8-7, OsGLP8-10 and OsGLP8-11 under salt stress and OsGLP8-7 under drought stress was also commonly higher in leaves and roots of KS282 as compared to Super Basmati. Whereas, OsGLP3-7 and OsGLP8-12 after salt stress and OsGLP8-4 and OsGLP8-12 after drought stress were observed with higher relative expression in roots of Super Basmati than KS282. Importantly, the OsGLP3-6 and OsGLP4-1 from chromosome 3 and 4 respectively showed higher expression in leaves whereas most of the OsGLP genes from chromosome 8 exhibited higher expression in roots. CONCLUSION: Overall, as a result of this comparative analysis, OsGLP genes showed both general and specific expression profiles depending upon a specific rice variety, stress condition as well as tissue type. These results will increase our understanding of role of OsGLP genes in rice crop and provide useful information for the further in-depth research on their regulatory mechanisms in response to these stress conditions.


Assuntos
Mapeamento Cromossômico/métodos , Perfilação da Expressão Gênica/métodos , Glicoproteínas/genética , Oryza/crescimento & desenvolvimento , Secas , Regulação da Expressão Gênica de Plantas , Família Multigênica , Oryza/classificação , Oryza/genética , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/genética , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Reação em Cadeia da Polimerase em Tempo Real , Estresse Salino , Estresse Fisiológico , Distribuição Tecidual
4.
J Phys Chem B ; 125(40): 11141-11149, 2021 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-34592819

RESUMO

Computational protein design has taken big strides in recent years; however, the tools available are still not at a state where a sequence can be designed to fold into a given protein structure at will and with high probability. We have applied here a recent release of Rosetta Design to redesign a set of structurally very similar proteins belonging to the thioredoxin fold. We used a genetic screening tool to estimate solubility/folding of the designed proteins in E. coli and to select the best hits from this for further biochemical characterization. We have previously used this set of template proteins for redesign and found that success was highly dependent on template structure, a trait which was also found in this study. Nevertheless, state-of-the-art design software is now able to predict the best template, most likely due to the introduction of an energy term that reports on stress in covalent bond lengths and angles. The template that led to the greatest fraction of successful designs was the same (a thioredoxin from spinach) as that identified in our previous study. Our previously described redesign of thioredoxin, which also used the spinach protein as a template, however also performed well as a template. In the present study, both of these templates yielded proteins with compact folded structures and enforced the conclusion that any design project must carefully consider different design templates. Fortunately, selecting designs based on energies appears to correctly identify such templates.


Assuntos
Biologia Computacional , Escherichia coli , Escherichia coli/genética , Software , Tiorredoxinas/genética
5.
J Am Chem Soc ; 143(36): 14540-14550, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34473923

RESUMO

Intrinsic disorder (ID) constitutes a new dimension to the protein structure-function relationship. The ability to undergo conformational changes upon binding is a key property of intrinsically disordered proteins and remains challenging to study using conventional methods. A 1994 paper by R. S. Spolar and M. T. Record presented a thermodynamic approach for estimating changes in conformational entropy based on heat capacity changes, allowing quantification of residues folding upon binding. Here, we adapt the method for studies of intrinsically disordered proteins. We integrate additional data to provide a broader experimental foundation for the underlying relations and, based on >500 protein-protein complexes involving disordered proteins, reassess a key relation between polar and nonpolar surface area changes, previously determined using globular protein folding. We demonstrate the improved suitability of the adapted method to studies of the folded αα-hub domain RST from radical-induced cell death 1, whose interactome is characterized by ID. From extensive thermodynamic data, quantifying the conformational entropy changes upon binding, and comparison to the NMR structure, the adapted method improves accuracy for ID-based studies. Furthermore, we apply the method, in conjunction with NMR, to reveal hitherto undetected effects of interaction-motif context. Thus, inclusion of the disordered context of the DREB2A RST-binding motif induces structuring of the binding motif, resulting in major enthalpy-entropy compensation in the interaction interface. This study, also evaluating additional interactions, demonstrates the strength of the ID-adapted Spolar-Record thermodynamic approach for dissection of structural features of ID-based interactions, easily overlooked in traditional studies, and for translation of these into mechanistic knowledge.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Entropia , Proteínas Intrinsicamente Desordenadas/química , Proteínas Nucleares/química , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína , Fatores de Transcrição/química
6.
Sci Rep ; 9(1): 18927, 2019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31831797

RESUMO

Radical-Induced Cell Death1 (RCD1) functions as a cellular hub interacting with intrinsically disordered transcription factor regions, which lack a well-defined three-dimensional structure, to regulate plant stress. Here, we address the molecular evolution of the RCD1-interactome. Using bioinformatics, its history was traced back more than 480 million years to the emergence of land plants with the RCD1-binding short linear motif (SLiM) identified from mosses to flowering plants. SLiM variants were biophysically verified to be functional and to depend on the same RCD1 residues as the DREB2A transcription factor. Based on this, numerous additional members may be assigned to the RCD1-interactome. Conservation was further strengthened by similar intrinsic disorder profiles of the transcription factor homologs. The unique structural plasticity of the RCD1-interactome, with RCD1-binding induced α-helix formation in DREB2A, but not detectable in ANAC046 or ANAC013, is apparently conserved. Thermodynamic analysis also indicated conservation with interchangeability between Arabidopsis and soybean RCD1 and DREB2A, although with fine-tuned co-evolved binding interfaces. Interruption of conservation was observed, as moss DREB2 lacked the SLiM, likely reflecting differences in plant stress responses. This whole-interactome study uncovers principles of the evolution of SLiM:hub-interactions, such as conservation of α-helix propensities, which may be paradigmatic for disorder-based interactomes in eukaryotes.


Assuntos
Evolução Molecular , Glycine max , Hordeum , Proteínas Nucleares , Mapas de Interação de Proteínas , Proteínas de Soja , Hordeum/química , Hordeum/genética , Hordeum/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estrutura Secundária de Proteína , Proteínas de Soja/química , Proteínas de Soja/genética , Proteínas de Soja/metabolismo , Glycine max/química , Glycine max/genética , Glycine max/metabolismo
7.
Protein Eng Des Sel ; 32(3): 145-151, 2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31553452

RESUMO

While the field of computational protein design has witnessed amazing progression in recent years, folding properties still constitute a significant barrier towards designing new and larger proteins. In order to assess and improve folding properties of designed proteins, we have developed a genetics-based folding assay and selection system based on the essential enzyme, orotate phosphoribosyl transferase from Escherichia coli. This system allows for both screening of candidate designs with good folding properties and genetic selection of improved designs. Thus, we identified single amino acid substitutions in two failed designs that rescued poorly folding and unstable proteins. Furthermore, when these substitutions were transferred into a well-structured design featuring a complex folding profile, the resulting protein exhibited native-like cooperative folding with significantly improved stability. In protein design, a single amino acid can make the difference between folding and misfolding, and this approach provides a useful new platform to identify and improve candidate designs.


Assuntos
Engenharia de Proteínas/métodos , Dobramento de Proteína , Proteínas/química , Proteínas/genética , Sequência de Aminoácidos , Modelos Moleculares , Mutação , Conformação Proteica
8.
Structure ; 26(5): 734-746.e7, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29657132

RESUMO

Communication within cells relies on a few protein nodes called hubs, which organize vast interactomes with many partners. Frequently, hub proteins are intrinsically disordered conferring multi-specificity and dynamic communication. Conversely, folded hub proteins may organize networks using disordered partners. In this work, the structure of the RST domain, a unique folded hub, is solved by nuclear magnetic resonance spectroscopy and small-angle X-ray scattering, and its complex with a region of the transcription factor DREB2A is provided through data-driven HADDOCK modeling and mutagenesis analysis. The RST fold is unique, but similar structures are identified in the PAH (paired amphipathic helix), TAFH (TATA-box-associated factor homology), and NCBD (nuclear coactivator binding domain) domains. We designate them as a group the αα hubs, as they share an αα-hairpin super-secondary motif, which serves as an organizing platform for malleable helices of varying topology. This allows for partner adaptation, exclusion, and selection. Our findings provide valuable insights into structural features enabling signaling fidelity.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Proteínas Nucleares/genética , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Estrutura Secundária de Proteína , Espalhamento a Baixo Ângulo , Difração de Raios X
9.
Biochem J ; 474(15): 2509-2532, 2017 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-28701416

RESUMO

Gene-specific transcription factors (TFs) are key regulatory components of signaling pathways, controlling, for example, cell growth, development, and stress responses. Their biological functions are determined by their molecular structures, as exemplified by their structured DNA-binding domains targeting specific cis-acting elements in genes, and by the significant lack of fixed tertiary structure in their extensive intrinsically disordered regions. Recent research in protein intrinsic disorder (ID) has changed our understanding of transcriptional activation domains from 'negative noodles' to ID regions with function-related, short sequence motifs and molecular recognition features with structural propensities. This review focuses on molecular aspects of TFs, which represent paradigms of ID-related features. Through specific examples, we review how the ID-associated flexibility of TFs enables them to participate in large interactomes, how they use only a few hydrophobic residues, short sequence motifs, prestructured motifs, and coupled folding and binding for their interactions with co-activators, and how their accessibility to post-translational modification affects their interactions. It is furthermore emphasized how classic biochemical concepts like allostery, conformational selection, induced fit, and feedback regulation are undergoing a revival with the appreciation of ID. The review also describes the most recent advances based on computational simulations of ID-based interaction mechanisms and structural analysis of ID in the context of full-length TFs and suggests future directions for research in TF ID.


Assuntos
Eucariotos/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Proteínas Intrinsicamente Desordenadas/química , Cinética , Processamento de Proteína Pós-Traducional , Termodinâmica , Fatores de Transcrição/química
10.
J Biol Chem ; 292(2): 512-527, 2017 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-27881680

RESUMO

Intrinsically disordered protein regions (IDRs) lack a well defined three-dimensional structure but often facilitate key protein functions. Some interactions between IDRs and folded protein domains rely on short linear motifs (SLiMs). These motifs are challenging to identify, but once found they can point to larger networks of interactions, such as with proteins that serve as hubs for essential cellular functions. The stress-associated plant protein radical-induced cell death1 (RCD1) is one such hub, interacting with many transcription factors via their flexible IDRs. To identify the SLiM bound by RCD1, we analyzed the IDRs in three protein partners, DREB2A (dehydration-responsive element-binding protein 2A), ANAC013, and ANAC046, considering parameters such as disorder, context, charges, and pI. Using a combined bioinformatics and experimental approach, we have identified the bipartite RCD1-binding SLiM as (DE)X(1,2)(YF)X(1,4)(DE)L, with essential contributions from conserved aromatic, acidic, and leucine residues. Detailed thermodynamic analysis revealed both favorable and unfavorable contributions from the IDRs surrounding the SLiM to the interactions with RCD1, and the SLiM affinities ranged from low nanomolar to 50 times higher Kd values. Specifically, although the SLiM was surrounded by IDRs, individual intrinsic α-helix propensities varied as shown by CD spectroscopy. NMR spectroscopy further demonstrated that DREB2A underwent coupled folding and binding with α-helix formation upon interaction with RCD1, whereas peptides from ANAC013 and ANAC046 formed different structures or were fuzzy in the complexes. These findings allow us to present a model of the stress-associated RCD1-transcription factor interactome and to contribute to the emerging understanding of the interactions between folded hubs and their intrinsically disordered partners.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/química , Modelos Moleculares , Proteínas Nucleares/química , Dobramento de Proteína , Fatores de Transcrição/química , Motivos de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
11.
J Exp Bot ; 67(17): 5259-73, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27436280

RESUMO

The plant-specific NAC transcription factors have attracted particular attention because of their involvement in stress responses, senescence, and nutrient remobilization. The HvNAC005 gene of barley encodes a protein belonging to subgroup NAC-a6 of the NAC family. This study shows that HvNAC005 is associated with developmental senescence. It was significantly up-regulated following ABA treatment, supported by ABA-responsive elements in its promoter, but it was not up-regulated during dark-induced senescence. The C-termini of proteins closely related to HvNAC005 showed overall high divergence but also contained conserved short motifs. A serine- and leucine-containing central motif was essential for transcriptional activity of the HvNAC005 C-terminus in yeast. Over-expression of HvNAC005 in barley resulted in a strong phenotype with delayed development combined with precocious senescence. The over-expressing plants showed up-regulation of genes involved with secondary metabolism, hormone metabolism, stress, signalling, development, and transport. Up-regulation of senescence markers and hormone metabolism and signalling genes supports a role of HvNAC005 in the cross field of different hormone and signalling pathways. Binding of HvNAC005 to promoter sequences of putative target genes containing the T[G/A]CGT core motif was shown by direct protein-DNA interactions of HvNAC005 with promoters for two of the up-regulated genes. In conclusion, HvNAC005 was shown to be a strong positive regulator of senescence and so is an obvious target for the fine-tuning of gene expression in future attempts to improve nutrient remobilization related to the senescence process in barley.


Assuntos
Hordeum/crescimento & desenvolvimento , Proteínas de Plantas/fisiologia , Fatores de Transcrição/fisiologia , Envelhecimento/fisiologia , Clonagem Molecular , Ensaio de Desvio de Mobilidade Eletroforética , Hordeum/metabolismo , Hordeum/fisiologia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase em Tempo Real , Saccharomyces cerevisiae , Fatores de Transcrição/metabolismo , Transcriptoma
12.
Plant Signal Behav ; 10(6): e1010967, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26107850

RESUMO

Protein intrinsic disorder (ID), referring to the lack of a fixed tertiary structure, is significant in signaling and transcription. We recently characterized ID in 6 phylogenetically representative Arabidopsis thaliana NAC transcription factors. Their transcription regulatory domains are mostly disordered but contain short, functionally important regions with structure propensities known as molecular recognition features. Here, we analyze for NAC subgroup-specific ID patterns. Some subgroups, such as the VND subgroup implicated in secondary cell wall biosynthesis, and the NAP/SHYG subgroup have highly conserved ID profiles. For the stress-associated ATAF1 subgroup and the CUC/ORE1 subgroup involved in development, only sub clades have similar ID patterns. For similar ID profiles, conserved molecular recognition features and sequence motifs represent likely functional determinants of e.g. transcriptional activation and interactions. Based on our analysis, we suggest that ID profiling of regulatory proteins in general can be used to guide identification of interaction partners of network proteins.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Arabidopsis/química , Filogenia , Estrutura Terciária de Proteína , Fatores de Transcrição/química
13.
Plants (Basel) ; 4(3): 412-48, 2015 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-27135336

RESUMO

Within the last decade, NAC transcription factors have been shown to play essential roles in senescence, which is the focus of this review. Transcriptome analyses associate approximately one third of Arabidopsis NAC genes and many crop NAC genes with senescence, thereby implicating NAC genes as important regulators of the senescence process. The consensus DNA binding site of the NAC domain is used to predict NAC target genes, and protein interaction sites can be predicted for the intrinsically disordered transcription regulatory domains of NAC proteins. The molecular characteristics of these domains determine the interactions in gene regulatory networks. Emerging local NAC-centered gene regulatory networks reveal complex molecular mechanisms of stress- and hormone-regulated senescence and basic physiological steps of the senescence process. For example, through molecular interactions involving the hormone abscisic acid, Arabidopsis NAP promotes chlorophyll degradation, a hallmark of senescence. Furthermore, studies of the functional rice ortholog, OsNAP, suggest that NAC genes can be targeted to obtain specific changes in lifespan control and nutrient remobilization in crop plants. This is also exemplified by the wheat NAM1 genes which promote senescence and increase grain zinc, iron, and protein content. Thus, NAC genes are promising targets for fine-tuning senescence for increased yield and quality.

14.
Biochem J ; 465(2): 281-94, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25348421

RESUMO

Protein ID (intrinsic disorder) plays a significant, yet relatively unexplored role in transcription factors (TFs). In the present paper, analysis of the transcription regulatory domains (TRDs) of six phylogenetically representative, plant-specific NAC [no apical meristem, ATAF (Arabidopsis transcription activation factor), cup-shaped cotyledon] TFs shows that the domains are present in similar average pre-molten or molten globule-like states, but have different patterns of order/disorder and MoRFs (molecular recognition features). ANAC046 (Arabidopsis NAC 046) was selected for further studies because of its simple MoRF pattern and its ability to interact with RCD1 (radical-induced cell death 1). Experiments in yeast and thermodynamic characterization suggest that its single MoRF region is sufficient for both transcriptional activation and interaction with RCD1. The remainder of the large regulatory domain is unlikely to contribute to the interaction, since the domain and truncations thereof have similar affinities for RCD1, which are also similar for ANAC013-RCD1 interactions. However, different enthalpic and entropic contributions to binding were revealed for ANAC046 and ANAC013, suggestive of differences in binding mechanisms. Although substitution of both hydrophobic and acidic residues of the ANAC046 MoRF region abolished binding, substitution of other residues, even with α-helix-breaking proline, was less disruptive. Together, the biophysical analyses suggest that RCD1-ANAC046 complex formation does not involve folding-upon-binding, but rather fuzziness or an unknown structure in ANAC046. We suggest that the ANAC046 regulatory domain functions as an entropic chain with a terminal hot spot interacting with RCD1. RCD1, a cellular hub, may be able to interact with many different TFs by exploiting their ID-based flexibility, as demonstrated for its interactions with ANAC046 and ANAC013.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas Nucleares , Fatores de Transcrição , Ativação Transcricional/fisiologia , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
15.
Nucleic Acids Res ; 42(12): 7681-93, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24914054

RESUMO

Target gene identification for transcription factors is a prerequisite for the systems wide understanding of organismal behaviour. NAM-ATAF1/2-CUC2 (NAC) transcription factors are amongst the largest transcription factor families in plants, yet limited data exist from unbiased approaches to resolve the DNA-binding preferences of individual members. Here, we present a TF-target gene identification workflow based on the integration of novel protein binding microarray data with gene expression and multi-species promoter sequence conservation to identify the DNA-binding specificities and the gene regulatory networks of 12 NAC transcription factors. Our data offer specific single-base resolution fingerprints for most TFs studied and indicate that NAC DNA-binding specificities might be predicted from their DNA-binding domain's sequence. The developed methodology, including the application of complementary functional genomics filters, makes it possible to translate, for each TF, protein binding microarray data into a set of high-quality target genes. With this approach, we confirm NAC target genes reported from independent in vivo analyses. We emphasize that candidate target gene sets together with the workflow associated with functional modules offer a strong resource to unravel the regulatory potential of NAC genes and that this workflow could be used to study other families of transcription factors.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Fatores de Transcrição/metabolismo , Arabidopsis/metabolismo , Sítios de Ligação , DNA de Plantas/química , DNA de Plantas/metabolismo , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo
16.
J Acoust Soc Am ; 133(5): 2899-909, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23654395

RESUMO

Previous studies have suggested that listeners can identify words spoken by a target talker amidst competing talkers if they are distinguished by their spatial location or vocal characteristics. This "direct" identification of individual words is distinct from an "indirect" identification based on an association with other words (call-signs) that uniquely label the target. The present study assessed listeners' ability to use differences in presentation level between a target and overlapping maskers to identify target words. A new sentence was spoken every 800 ms by an unpredictable talker from an unpredictable location. Listeners reported color and number words in a target sentence distinguished by a unique call-sign. When masker levels were fixed, target words could be identified directly based on their relative level. Speech-reception thresholds (SRTs) were low (-12.9 dB) and were raised by 5 dB when direct identification was disrupted by randomizing masker levels. Thus, direct identification is possible using relative level. The underlying psychometric functions were monotonic even when relative level was a reliable cue. In a further experiment, indirect identification was prevented by removing the unique call-sign cue. SRTs did not change provided that other cues were available to identify target words directly. Thus, direct identification is possible without indirect identification.


Assuntos
Sinais (Psicologia) , Ruído/efeitos adversos , Mascaramento Perceptivo , Detecção de Sinal Psicológico , Percepção da Fala , Estimulação Acústica , Adulto , Limiar Auditivo , Humanos , Localização de Som , Percepção Espacial , Acústica da Fala , Teste do Limiar de Recepção da Fala , Fatores de Tempo , Qualidade da Voz , Adulto Jovem
17.
Int J Mol Sci ; 14(3): 5842-78, 2013 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-23485989

RESUMO

Transcription factors (TFs) are master regulators of abiotic stress responses in plants. This review focuses on TFs from seven major TF families, known to play functional roles in response to abiotic stresses, including drought, high salinity, high osmolarity, temperature extremes and the phytohormone ABA. Although ectopic expression of several TFs has improved abiotic stress tolerance in plants, fine-tuning of TF expression and protein levels remains a challenge to avoid crop yield loss. To further our understanding of TFs in abiotic stress responses, emerging gene regulatory networks based on TFs and their direct targets genes are presented. These revealed components shared between ABA-dependent and independent signaling as well as abiotic and biotic stress signaling. Protein structure analysis suggested that TFs hubs of large interactomes have extended regions with protein intrinsic disorder (ID), referring to their lack of fixed tertiary structures. ID is now an emerging topic in plant science. Furthermore, the importance of the ubiquitin-proteasome protein degradation systems and modification by sumoylation is also apparent from the interactomes. Therefore; TF interaction partners such as E3 ubiquitin ligases and TF regions with ID represent future targets for engineering improved abiotic stress tolerance in crops.

18.
J Biol Chem ; 288(6): 4424-35, 2013 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-23275343

RESUMO

Gentamicin is an aminoglycoside widely used in treatments of, in particular, enterococcal, mycobacterial, and severe Gram-negative bacterial infections. Large doses of gentamicin cause nephrotoxicity and ototoxicity, entering the cell via the receptor megalin. Until now, no structural information has been available to describe the interaction with gentamicin in atomic detail, and neither have any three-dimensional structures of domains from the human megalin receptor been solved. To address this gap in our knowledge, we have solved the NMR structure of the 10th complement type repeat of human megalin and investigated its interaction with gentamicin. Using NMR titration data in HADDOCK, we have generated a three-dimensional model describing the complex between megalin and gentamicin. Gentamicin binds to megalin with low affinity and exploits the common ligand binding motif previously described (Jensen, G. A., Andersen, O. M., Bonvin, A. M., Bjerrum-Bohr, I., Etzerodt, M., Thogersen, H. C., O'Shea, C., Poulsen, F. M., and Kragelund, B. B. (2006) J. Mol. Biol. 362, 700-716) utilizing the indole side chain of Trp-1126 and the negatively charged residues Asp-1129, Asp-1131, and Asp-1133. Binding to megalin is highly similar to gentamicin binding to calreticulin. We discuss the impact of this novel insight for the future structure-based design of gentamicin antagonists.


Assuntos
Gentamicinas/química , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Simulação de Acoplamento Molecular , Motivos de Aminoácidos , Gentamicinas/metabolismo , Humanos , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Estrutura Terciária de Proteína , Sequências Repetitivas de Aminoácidos , Relação Estrutura-Atividade
19.
Structure ; 20(2): 270-82, 2012 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-22325776

RESUMO

The prolactin receptor (PRLR) is activated by binding of prolactin in a 2:1 complex, but the activation mechanism is poorly understood. PRLR has a conserved WSXWS motif generic to cytokine class I receptors. We have determined the nuclear magnetic resonance solution structure of the membrane proximal domain of the human PRLR and find that the tryptophans of the motif adopt a T-stack conformation in the unbound state. By contrast, in the hormone bound state, a Trp/Arg-ladder is formed. The conformational change is hormone-dependent and influences the receptor-receptor dimerization site 3. In the constitutively active, breast cancer-related receptor mutant PRLR(I146L), we observed a stabilization of the dimeric state and a change in the dynamics of the motif. Here we demonstrate a structural link between the WSXWS motif, hormone binding, and receptor dimerization and propose it as a general mechanism for class 1 receptor activation.


Assuntos
Receptores de Citocinas/química , Receptores da Prolactina/química , Motivos de Aminoácidos , Sítios de Ligação , Dicroísmo Circular , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Tamanho da Partícula , Prolactina/química , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Titulometria
20.
J Mol Recognit ; 24(4): 533-47, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-20842635

RESUMO

The cytokine hormone prolactin has a vast number of diverse functions. Unfortunately, it also exhibits tumor growth promoting properties, which makes the development of prolactin receptor antagonists a priority. Prolactin binds to its cognate receptor with much lower affinity at low pH than at physiological pH and since the extracellular environment around solid tumors often is acidic, it is desirable to develop antagonists that have improved binding affinity at low pH. The pK(a) value of a histidine side chain is ∼6.8 making histidine residues obvious candidates for examination. From evaluation of known molecular structures of human prolactin, of the prolactin receptor and of different complexes of the two, three histidine residues in the hormone-receptor binding site 1 were selected for mutational studies. We analyzed 10 variants by circular dichroism spectroscopy, affinity and thermodynamic characterization of receptor binding by isothermal titration calorimetry combined with in vitro bioactivity in living cells. Histidine residue 27 was recognized as a central hot spot for pH sensitivity and conservative substitutions at this site resulted in strong receptor binding at low pH. Pure antagonists were developed earlier and the histidine mutations were introduced within such background. The antagonistic properties were maintained and the high affinity at low pH conserved. The implications of these findings may open new areas of research in the field of prolactin cancer biology.


Assuntos
Prolactina/metabolismo , Receptores da Prolactina/antagonistas & inibidores , Receptores da Prolactina/metabolismo , Calorimetria , Dicroísmo Circular , Histidina/química , Histidina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Prolactina/genética , Ligação Proteica , Conformação Proteica , Receptores da Prolactina/química , Receptores da Prolactina/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinâmica
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