RESUMEN

Throughout its lifecycle, Entamoeba histolytica encounters a variety of stressful conditions. This parasite possesses Heat Shock Response Elements (HSEs) which are crucial for regulating the expression of various genes, aiding in its adaptation and survival. These HSEs are regulated by Heat Shock Transcription Factors (EhHSTFs). Our research has identified seven such factors in the parasite, designated as EhHSTF1 through to EhHSTF7. Significantly, under heat shock conditions and in the presence of the antiamoebic compound emetine, EhHSTF5, EhHSTF6, and EhHSTF7 show overexpression, highlighting their essential role in gene response to these stressors. Currently, only EhHSTF7 has been confirmed to recognize the HSE as a promoter of the EhPgp5 gene (HSE_EhPgp5), leaving the binding potential of the other EhHSTFs to HSEs yet to be explored. Consequently, our study aimed to examine, both in vitro and in silico, the oligomerization, and binding capabilities of the recombinant EhHSTF5 protein (rEhHSTF5) to HSE_EhPgp5. The in vitro results indicate that the oligomerization of rEhHSTF5 is concentration-dependent, with its dimeric conformation showing a higher affinity for HSE_EhPgp5 than its monomeric state. In silico analysis suggests that the alpha 3 α-helix (α3-helix) of the DNA-binding domain (DBD5) of EhHSTF5 is crucial in binding to the major groove of HSE, primarily through hydrogen bonding and salt-bridge interactions. In summary, our results highlight the importance of oligomerization in enhancing the affinity of rEhHSTF5 for HSE_EhPgp5 and demonstrate its ability to specifically recognize structural motifs within HSE_EhPgp5. These insights significantly contribute to our understanding of one of the potential molecular mechanisms employed by this parasite to efficiently respond to various stressors, thereby enabling successful adaptation and survival within its host environment.

Asunto(s)

Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Entamoeba histolytica , Regiones Promotoras Genéticas , Proteínas Protozoarias , Sitios de Unión , Simulación por Computador , Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Respuesta al Choque Térmico/genética , Unión Proteica , Multimerización de Proteína , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/química , Elementos de Respuesta , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo

RESUMEN

The NusG protein family is structurally and functionally conserved in all domains of life. Its members directly bind RNA polymerases and regulate transcription processivity and termination. RfaH, a divergent sub-family in its evolutionary history, is known for displaying distinct features than those in NusG proteins, which allows them to regulate the expression of virulence factors in enterobacteria in a DNA sequence-dependent manner. A striking feature is its structural interconversion between an active fold, which is the canonical NusG three-dimensional structure, and an autoinhibited fold, which is distinctively novel. How this novel fold is encoded within RfaH sequence to encode a metamorphic protein remains elusive. In this work, we used publicly available genomic RfaH protein sequences to construct a complete multiple sequence alignment, which was further augmented with metagenomic sequences and curated by predicting their secondary structure propensities using JPred. Coevolving pairs of residues were calculated from these sequences using plmDCA and GREMLIN, which allowed us to detect the enrichment of key metamorphic contacts after sequence filtering. Finally, we combined our coevolutionary predictions with molecular dynamics to demonstrate that these interactions are sufficient to predict the structures of both native folds, where coevolutionary-derived non-native contacts may play a key role in achieving the compact RfaH novel fold. All in all, emergent coevolutionary signals found within RfaH sequences encode the autoinhibited and active folds of this protein, shedding light on the key interactions responsible for the action of this metamorphic protein.

Asunto(s)

Proteínas de Escherichia coli , Factores de Transcripción , ARN Polimerasas Dirigidas por ADN/química , Proteínas de Escherichia coli/química , Factores de Elongación de Péptidos/química , Factores de Elongación de Péptidos/genética , Factores de Elongación de Péptidos/metabolismo , Transactivadores/química , Factores de Transcripción/química

RESUMEN

Cowpea [Vigna unguiculata (L.) Walp.] is one of the most tolerant legume crops to drought and salt stresses. WRKY transcription factor (TF) family members stand out among plant transcriptional regulators related to abiotic stress tolerance. However, little information is currently available on the expression of the cowpea WRKY gene family (VuWRKY) in response to water deficit. Thus, we analyzed genomic and transcriptomic data from cowpea to identify VuWRKY members and characterize their structure and transcriptional response under root dehydration stress. Ninety-two complete VuWRKY genes were found in the cowpea genome based on their domain characteristics. They were clustered into three groups: I (15 members), II (58), and III (16), while three genes were unclassified. Domain analysis of the encoded proteins identified four major variants of the conserved heptapeptide motif WRKYGQK. In silico analysis of VuWRKY gene promoters identified eight candidate binding motifs of cis-regulatory elements, regulated mainly by six TF families associated with abiotic stress responses. Ninety-seven VuWRKY modulated splicing variants associated with 55 VuWRKY genes were identified via RNA-Seq analysis available at the Cowpea Genomics Consortium (CpGC) database. qPCR analyses showed that 22 genes are induced under root dehydration, with VuWRKY18, 21, and 75 exhibiting the most significant induction levels. Given their central role in activating signal transduction cascades in abiotic stress response, the data provide a foundation for the targeted modification of specific VuWRKY family members to improve drought tolerance in this important climate-resilient legume in the developing world and beyond.

Asunto(s)

Perfilación de la Expresión Génica/métodos , Genómica/métodos , Factores de Transcripción/química , Factores de Transcripción/genética , Vigna/genética , Empalme Alternativo , Secuencias de Aminoácidos , Mapeo Cromosómico , Sequías , Regulación de la Expresión Génica de las Plantas , Familia de Multigenes , Proteínas de Plantas/química , Proteínas de Plantas/genética , Raíces de Plantas/genética , Regiones Promotoras Genéticas , Dominios Proteicos , RNA-Seq , Estrés Fisiológico

RESUMEN

The mixed lineage leukemia 3 or MLL3 is the enzyme in charge of the writing of an epigenetic mark through the methylation of lysine 4 from the N-terminal domain of histone 3 and its deregulation has been related to several cancer lines. An interesting feature of this enzyme comes from its regulation mechanism, which involves its binding to an activating dimer before it can be catalytically functional. Once the trimer is formed, the reaction mechanism proceeds through the deprotonation of the lysine followed by the methyl-transfer reaction. Here we present a detailed exploration of the activation mechanism through a QM/MM approach focusing on both steps of the reaction, aiming to provide new insights into the deprotonation process and the role of the catalytic machinery in the methyl-transfer reaction. Our finding suggests that the source of the activation mechanism comes from conformational restriction mediated by the formation of a network of salt-bridges between MLL3 and one of the activating subunits, which restricts and stabilizes the positioning of several residues relevant for the catalysis. New insights into the deprotonation mechanism of lysine are provided, identifying a valine residue as crucial in the positioning of the water molecule in charge of the process. Finally, a tyrosine residue was found to assist the methyl transfer from SAM to the target lysine.

Asunto(s)

Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Sitios de Unión , Proteínas de Unión al ADN/genética , Epigénesis Genética , Humanos , Lisina/química , Lisina/metabolismo , Simulación de Dinámica Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Multimerización de Proteína , Protones , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Tirosina/química , Tirosina/metabolismo

RESUMEN

Absolute binding free energy calculations with explicit solvent molecular simulations can provide estimates of protein-ligand affinities, and thus reduce the time and costs needed to find new drug candidates. However, these calculations can be complex to implement and perform. Here, we introduce the software BAT.py, a Python tool that invokes the AMBER simulation package to automate the calculation of binding free energies for a protein with a series of ligands. The software supports the attach-pull-release (APR) and double decoupling (DD) binding free energy methods, as well as the simultaneous decoupling-recoupling (SDR) method, a variant of double decoupling that avoids numerical artifacts associated with charged ligands. We report encouraging initial test applications of this software both to re-rank docked poses and to estimate overall binding free energies. We also show that it is practical to carry out these calculations cheaply by using graphical processing units in common machines that can be built for this purpose. The combination of automation and low cost positions this procedure to be applied in a relatively high-throughput mode and thus stands to enable new applications in early-stage drug discovery.

Asunto(s)

Descubrimiento de Drogas , Simulación del Acoplamiento Molecular , Proteínas/química , Proteínas/metabolismo , Programas Informáticos , Automatización , Sitios de Unión , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Costos y Análisis de Costo , Descubrimiento de Drogas/economía , Ligandos , Simulación del Acoplamiento Molecular/economía , Simulación de Dinámica Molecular , Estructura Molecular , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Unión Proteica , Conformación Proteica , Programas Informáticos/economía , Solventes/química , Termodinámica , Factores de Transcripción/química , Factores de Transcripción/metabolismo

RESUMEN

Mycobacterium tuberculosis is a pathogen with a unique cell envelope including very long fatty acids, implicated in bacterial resistance and host immune modulation. FasR is a TetR-like transcriptional activator that plays a central role in sensing mycobacterial long-chain fatty acids and regulating lipid biosynthesis. Here we disclose crystal structures of M. tuberculosis FasR in complex with acyl effector ligands and with DNA, uncovering its molecular sensory and switching mechanisms. A long tunnel traverses the entire effector-binding domain, enabling long fatty acyl effectors to bind. Only when the tunnel is entirely occupied, the protein dimer adopts a rigid configuration with its DNA-binding domains in an open state, leading to DNA dissociation. The protein-folding hydrophobic core connects the two domains, and is completed into a continuous spine when the effector binds. Such a transmission spine is conserved in a large number of TetR-like regulators, offering insight into effector-triggered allosteric functional control.

Asunto(s)

Acilcoenzima A/química , Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , Mycobacterium tuberculosis/metabolismo , Factores de Transcripción/química , Acilcoenzima A/metabolismo , Sitio Alostérico , Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Cristalografía por Rayos X , ADN Bacteriano/química , Proteínas de Unión al ADN/metabolismo , Ácidos Grasos/metabolismo , Ligandos , Modelos Moleculares , Conformación Proteica , Factores de Transcripción/metabolismo

RESUMEN

Mechanisms coupling the atypical PKC (aPKC) kinase activity to its subcellular localization are essential for cell polarization. Unlike other members of the PKC family, aPKC has no well-defined plasma membrane (PM) or calcium binding domains, leading to the assumption that its subcellular localization relies exclusively on protein-protein interactions. Here we show that in both Drosophila and mammalian cells, the pseudosubstrate region (PSr) of aPKC acts as a polybasic domain capable of targeting aPKC to the PM via electrostatic binding to PM PI4P and PI(4,5)P2. However, physical interaction between aPKC and Par-6 is required for the PM-targeting of aPKC, likely by allosterically exposing the PSr to bind PM. Binding of Par-6 also inhibits aPKC kinase activity, and such inhibition can be relieved through Par-6 interaction with apical polarity protein Crumbs. Our data suggest a potential mechanism in which allosteric regulation of polybasic PSr by Par-6 couples the control of both aPKC subcellular localization and spatial activation of its kinase activity.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/metabolismo , Membrana Celular/enzimología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Proteínas de la Membrana/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Regulación Alostérica , Animales , Animales Modificados Genéticamente , Membrana Celular/ultraestructura , Polaridad Celular/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Embrión no Mamífero , Células Epiteliales/enzimología , Células Epiteliales/ultraestructura , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Larva/citología , Larva/enzimología , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/química , Fosfatos de Fosfatidilinositol/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteína Quinasa C/química , Proteína Quinasa C/genética , Transducción de Señal , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

Bacteria and archaea accumulate cytoplasmic polyhydroxyalkanoate (PHA) granules under nutrient-limited conditions with excess carbon. The transcriptional regulatory (TR) proteins found on the surface of PHA granules act as repressors as well as activators for the expression of major surface proteins called phasins. Until now, detailed information on the evolutionary relationships between these transcription regulators has not been available. Here, we conducted homology searches and analyzed information available for the domains and protein families of the TR proteins through phylogenetic studies. A total of 282 TR proteins were identified and further classified into four distinct subfamilies based upon the presence of conserved motifs: PHB_acc, TetR-like, AbrB-like, and PadR-like. Depending upon the particular family, the DNA-binding domains were located at either the N- or C-terminus. Our results indicated that TR proteins containing the PHB_acc domain are highly conserved within the bacteria, while other TR proteins are present only within archaea (AbrB-like), gram positive bacteria (PadR-like), or the Pseudomonas genera (TetR-like). The repression domains are charged, hydrophobic, and rich in leucine or glutamine. In phylogenetic analyses, many groups of TR proteins were clustered together according to identical domain architectures showing the independent origins of the TR proteins in the PHA reserve storage system. Further analyses revealed that the TR proteins have experienced multiple gene duplications across prokaryotes. Thus, this study investigated the evolutionary framework of TR proteins and has provided a comprehensive catalog of TR proteins for ongoing studies to characterize the functions of these proteins within diverse organisms.

Asunto(s)

Proteínas Arqueales/genética , Proteínas Bacterianas/genética , Secuencia Conservada , Evolución Molecular , Lectinas de Plantas/genética , Polihidroxialcanoatos/biosíntesis , Factores de Transcripción/genética , Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Filogenia , Lectinas de Plantas/metabolismo , Polihidroxialcanoatos/genética , Factores de Transcripción/química , Factores de Transcripción/metabolismo

RESUMEN

The IRF2BP2 (IFN regulatory factor 2 binding protein 2) protein was identified as a nuclear protein that interacts with IFN regulatory factor 2 (IRF-2) and is an IRF-2-dependent transcriptional repressor. IRF2BP2 belongs to the IRF2BP family, which includes IRF2BP1, IRF2BP2, and IRF2BPL (EAP1). Recently, IRF2BP2 has emerged as an important new transcriptional cofactor in different biological systems, acting as a positive and negative regulator of gene expression. IRF2BP2 plays a role in different cellular functions, including apoptosis, survival, and cell differentiation. Additionally, IRF2BP2 may be involved in cancer development. Finally, it has been recently reported that IRF2BP2 may play a role in macrophage regulation and lymphocyte activation, highlighting its function in innate and adaptive immune responses. However, it has become increasingly clear that IRF2BP2 and its isoforms can have specific functions. In this review, we address the possible reasons for these distinct roles of IRF2BP2 and the partner proteins that interact with it. We also discuss the genes regulated by IRF2BP2 during the immune response and in other biological systems.

Asunto(s)

Homeostasis , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Exones/genética , Regulación de la Expresión Génica , Humanos , Inmunidad , Factores de Transcripción/química , Factores de Transcripción/genética

RESUMEN

We have applied the docking methodology to characterize the binding modes of the divalent metal transporter 1 (DMT1) and the zinc transporter 8 (ZIP8) protein channels with: melatonin, some melatonin metabolites, and a few lead complexes of melatonin and its metabolites, in three different coordination modes (mono-coordinated, bi-coordinated and tri-coordinated). Our results show that bi-coordinated and tri-coordinated lead complexes prefer to bind inside the central region of ZIP8. Moreover, the interaction strength is larger compared with that of the free melatonin and melatonin metabolites. On the other hand, the binding modes with DMT1 of such complexes display lower binding energies, compared with the free melatonin and melatonin metabolites. Our results suggest that ZIP8 plays a major role in the translocation of Pb, bi or tri coordinated, when melatonin metabolites are present. Finally, we have characterized the binding modes responsible for the ZIP8 large affinities, found in bi-coordinated and tri-coordinated lead complexes. Our results show that such interactions are greater, because of an increase of the number of hydrogen bonds, the number and intensity of electrostatic interactions, and the interaction overlay degree in each binding mode. Our results give insight into the importance of the ZIP8 channel on lead transport and a possible elimination mechanism in lead detoxification processes. Graphical abstract .

Asunto(s)

Proteínas de Transporte de Catión/metabolismo , Plomo/farmacología , Melatonina/farmacología , Factores de Transcripción/metabolismo , Sitios de Unión , Proteínas de Transporte de Catión/química , Complejos de Coordinación/química , Complejos de Coordinación/farmacología , Humanos , Plomo/química , Melatonina/química , Modelos Moleculares , Simulación del Acoplamiento Molecular , Estructura Molecular , Unión Proteica , Estructura Terciaria de Proteína , Factores de Transcripción/química

RESUMEN

Azospirillum brasilense is one of the most studied species of diverse agronomic plants worldwide. The benefits conferred to plants inoculated with Azospirillum have been primarily attributed to its capacity to fix atmospheric nitrogen and synthesize phytohormones, especially indole-3-acetic acid (IAA). The principal pathway for IAA synthesis involves the intermediate metabolite indole pyruvic acid. Successful colonization of plants by Azospirillum species is fundamental to the ability of these bacteria to promote the beneficial effects observed in plants. Biofilm formation is an essential step in this process and involves interactions with the host plant. In this study, the tyrR gene was cloned, and the translated product was observed to exhibit homology to TyrR protein, a NtrC/NifA-type activator. Structural studies of TyrR identified three putative domains, including a domain containing binding sites for aromatic amino acids in the N-terminus, a central AAA+ ATPase domain, and a helix-turn-helix DNA binding motif domain in the C-terminus, which binds DNA sequences in promoter-operator regions. In addition, a bioinformatic analysis of promoter sequences in A. brasilense Sp7 genome revealed that putative promoters encompass one to three TyrR boxes in genes predicted to be regulated by TyrR. To gain insight into the phenotypes regulated by TyrR, a tyrR-deficient strain derived from A. brasilense Sp7, named A. brasilense 2116 and a complemented 2116 strain harboring a plasmid carrying the tyrR gene were constructed. The observed phenotypes indicated that the putative transcriptional regulator TyrR is involved in biofilm production and is responsible for regulating the utilization of D-alanine as carbon source. In addition, TyrR was observed to be absolutely required for transcriptional regulation of the gene dadA encoding a D-amino acid dehydrogenase. The data suggested that TyrR may play a major role in the regulation of genes encoding a glucosyl transferase, essential signaling proteins, and amino acids transporters.

Asunto(s)

Aspergillus , Biopelículas/crecimiento & desarrollo , Proteínas Fúngicas , Factores de Transcripción , Aspergillus/química , Aspergillus/fisiología , D-Aminoácido Oxidasa/biosíntesis , D-Aminoácido Oxidasa/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Secuencias Hélice-Giro-Hélice , Dominios Proteicos , Elementos de Respuesta , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética/fisiología

RESUMEN

Fragaria chiloensis is a strawberry endemic from Chile with attractive white-pink fruit, pleasant aroma and taste. However, this fruit has a limited post-harvest period due to fast softening. Several transcription factors (TFs) are involved in the regulation of fruit ripening, and members of the NAC family have been implicated in cell wall remodeling. FcNAC1 was isolated from F. chiloensis fruit, coding a protein of 332 amino acid residues and displaying a characteristic NAC domain at the N terminus. FcNAC1 protein showed nuclear localization. An increase in transcript level was observed during ripening. A sequence of 1488 bp of FcNAC1 promoter was obtained. In silico analysis identified cis elements able to respond to some hormones and Secondary wall NAC binding elements (SNBE), and responding to auxin and ABA. A structural model of FcNAC1 provided evidence for interaction with DNA sequences containing SNBE, while a dual luciferase assay confirmed the transcriptional activation by FcNAC1 of the promoter of FcPL, a gene involved in cell wall remodeling in F. chiloensis fruit. The results suggest the participation of FcNAC1 during ripening development of strawberry fruit, by regulating pectin metabolism during softening.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Fragaria/fisiología , Proteínas de Plantas/metabolismo , Factores de Transcripción/metabolismo , Ácido Abscísico/metabolismo , Pared Celular/metabolismo , Chile , Clonación Molecular , ADN de Plantas/química , ADN de Plantas/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Frutas/citología , Frutas/metabolismo , Regulación de la Expresión Génica de las Plantas/fisiología , Ácidos Indolacéticos/metabolismo , Simulación de Dinámica Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/aislamiento & purificación , Regiones Promotoras Genéticas/genética , Dominios y Motivos de Interacción de Proteínas , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/aislamiento & purificación

RESUMEN

Flavonoids are widely recognized as natural polydrugs, given their anti-inflammatory, antioxidant, sedative, and antineoplastic activities. Recently, different studies showed that flavonoids have the potential to inhibit bromodomain and extraterminal (BET) bromodomains. Previous reports suggested that flavonoids bind between the Z and A loops of the bromodomain (ZA channel) due to their orientation and interactions with P86, V87, L92, L94, and N140. Herein, a comprehensive characterization of the binding modes of fisetin and the biflavonoid, amentoflavone, is discussed. To this end, both compounds were docked with BET bromodomain 4 (BRD4) using four docking programs. The results were post-processed with protein⁻ligand interaction fingerprints. To gain further insight into the binding mode of the two natural products, the docking results were further analyzed with molecular dynamics simulations. The results showed that amentoflavone makes numerous contacts in the ZA channel, as previously described for flavonoids and kinase inhibitors. It was also found that amentoflavone can potentially make contacts with non-canonical residues for BET inhibition. Most of these contacts were not observed with fisetin. Based on these results, amentoflavone was experimentally tested for BRD4 inhibition, showing activity in the micromolar range. This work may serve as the basis for scaffold optimization and the further characterization of flavonoids as BET inhibitors.

Asunto(s)

Biflavonoides/química , Biflavonoides/farmacología , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Flavonoides/química , Flavonoides/farmacología , Flavonoles , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica , Dominios Proteicos , Factores de Transcripción/antagonistas & inhibidores

RESUMEN

M. tuberculosis oxidation sense Regulator (MosR) is a transcriptional regulator from Mycobacterium tuberculosis. It senses the environment oxidation and regulates the expression of a secreted oxidoreductase, thus defending the bacilli against oxidative stress from the phagosome. While most of the members of the Multiple antibiotics resistance Regulator (MarR) family are ligand-responsive, MosR may dissociate from its DNA site upon formation of an intrachain disulphide bond. However, the structure of MosR in its oxidized state is not known, and it is not clear how the formation of this disulphide bond would lead to the conformational changes required for dissociation of the DNA. Nonetheless, MosR presents two crystallographically resolved conformations in its reduced state: bound and unbound to DNA. We managed to simulate MosR unbound to the DNA, both in the presence and in the absence of the disulphide bond. Our results indicate that this disulphide bond precludes the N-terminal residues from adopting a conformation that stands in-between the helix α1 and the DNA binding domain (DBD) from the other chain. Once this conformation is achieved in the reduced state, this DBD detaches from the dimerization domain and becomes more flexible, being able to perform motions with higher amplitude and higher degree of collectivity. Only then, MosR may achieve a conformation where its recognition helices fit into the major grooves of its DNA site. The analysis of the collective motions performed by MosR, during the different situations sampled by the molecular dynamics (MDs), was only possible by the method of filtering harmonic modes with specific frequencies. The frequency of the collective motions performed by the DBD of MosR in the reduced state to achieve a DNA-binding conformation is in the range of 20 to 50 MHz, but it may be associated to more sporadic events since it requires the combination of a suitable conformation of the N-terminal residues.

Asunto(s)

Proteínas Bacterianas/metabolismo , ADN/metabolismo , Simulación de Dinámica Molecular , Factores de Transcripción/metabolismo , Proteínas Bacterianas/química , ADN/química , Movimiento (Física) , Mycobacterium tuberculosis , Oxidación-Reducción , Unión Proteica , Conformación Proteica , Factores de Transcripción/química

RESUMEN

BACKGROUND: De novo prediction of Transcription Factor Binding Sites (TFBS) using computational methods is a difficult task and it is an important problem in Bioinformatics. The correct recognition of TFBS plays an important role in understanding the mechanisms of gene regulation and helps to develop new drugs. RESULTS: We here present Memetic Framework for Motif Discovery (MFMD), an algorithm that uses semi-greedy constructive heuristics as a local optimizer. In addition, we used a hybridization of the classic genetic algorithm as a global optimizer to refine the solutions initially found. MFMD can find and classify overrepresented patterns in DNA sequences and predict their respective initial positions. MFMD performance was assessed using ChIP-seq data retrieved from the JASPAR site, promoter sequences extracted from the ABS site, and artificially generated synthetic data. The MFMD was evaluated and compared with well-known approaches in the literature, called MEME and Gibbs Motif Sampler, achieving a higher f-score in the most datasets used in this work. CONCLUSIONS: We have developed an approach for detecting motifs in biopolymers sequences. MFMD is a freely available software that can be promising as an alternative to the development of new tools for de novo motif discovery. Its open-source software can be downloaded at https://github.com/jadermcg/mfmd .

Asunto(s)

Algoritmos , Factores de Transcripción/metabolismo , Secuencia de Bases , Sitios de Unión , Internet , Factores de Transcripción/química , Factores de Transcripción/genética , Interfaz Usuario-Computador

RESUMEN

The Neurospora crassa NIT-2 transcription factor belongs to the GATA transcription factor family and plays a fundamental role in the regulation of nitrogen metabolism. Because NIT-2 acts by accessing DNA inside the nucleus, understanding the nuclear import process of NIT-2 is necessary to characterize its function. Thus, in the present study, NIT-2 nuclear transport was investigated using a combination of biochemical, cellular, and biophysical methods. A complemented strain that produced an sfGFP-NIT-2 fusion protein was constructed, and nuclear localization assessments were made under conditions that favored protein translocation to the nucleus. Nuclear translocation was also investigated using HeLa cells, which showed that the putative NIT-2 nuclear localization sequence (NLS; 915TISSKRQRRHSKS927) was recognized by importin-α and that subsequent transport occurred via the classical import pathway. The interaction between the N. crassa importin-α (NcImpα) and the NIT-2 NLS was quantified with calorimetric assays, leading to the observation that the peptide bound to two sites with different affinities, which is typical of a monopartite NLS sequence. The crystal structure of the NcImpα/NIT-2 NLS complex was solved and revealed that the NIT-2 peptide binds to NcImpα with the major NLS-binding site playing a primary role. This result contrasts other recent studies that suggested a major role for the minor NLS-binding site in importin-α from the α2 family, indicating that both sites can be used for different cargo proteins according to specific metabolic requirements.

Asunto(s)

Transporte Activo de Núcleo Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Neurospora crassa/metabolismo , Factores de Transcripción/metabolismo , alfa Carioferinas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión/fisiología , Células Cultivadas , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Células HeLa , Humanos , Neurospora crassa/genética , Estructura Secundaria de Proteína , Esporas Fúngicas , Factores de Transcripción/química , Factores de Transcripción/genética , Difracción de Rayos X , alfa Carioferinas/química , alfa Carioferinas/genética

RESUMEN

The zinc fingers proteins (ZNF) are the largest family of DNA binding proteins and can act as transcriptional factors in eukaryotes. ZNF are implicated in activation in response to environmental stimulus by biometals such as Zn2+. Many of these proteins have the classical C2H2 zinc finger motifs (C2H2-ZNFm) of approximately 30 amino acids, where a Zn2+ ion is coordinated by two cysteine and two histidine residues. Trichomonas vaginalis is a protozoan parasite than responds to environmental changes including Zn2+. Until now has not been described any ZNF that could be involved in the regulation of genic expression of T. vaginalis. Here, we characterized in silico and experimentally an annoted ZNF (TvZNF1) from T. vaginalis and isolated the gene, tvznf1 encoding it. TvZNF1 have eight C2H2-ZNFm with residues that maybe involved in the structural stability of DNA binding motifs. In this work we confirmed the Zn2+ upregulation expression of tvznf1 gene. Recombinant TvZNF1 was able to bind to specific DNA sequences according to EMSA assay. Additionally, we demonstrated that recombinant TvZNF1 bind to MRE signature in vitro, which strongly suggests its role in transcriptional regulation, similar to the one observed for mammalian MTF-1. This result suggested a conserved mechanism of genic regulation mediated by ZNFs in T. vaginalis.

Asunto(s)

Dedos de Zinc CYS2-HIS2 , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Trichomonas vaginalis/genética , Sitios de Unión , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Estructura Secundaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Elementos de Respuesta , Factores de Transcripción/genética , Trichomonas vaginalis/química , Trichomonas vaginalis/metabolismo , Zinc/metabolismo

RESUMEN

AtHB1 is an Arabidopsis (Arabidopsis thaliana) homeodomain-leucine zipper transcription factor that participates in hypocotyl elongation under short-day conditions. Here, we show that its expression is posttranscriptionally regulated by an upstream open reading frame (uORF) located in its 5' untranslated region. This uORF encodes a highly conserved peptide (CPuORF) that is present in varied monocot and dicot species. The Arabidopsis uORF and its maize (Zea mays) homolog repressed the translation of the main open reading frame in cis, independent of the sequence of the latter. Published ribosome footprinting results and the analysis of a frame-shifted uORF, in which the repression capability was lost, indicated that the uORF causes ribosome stalling. The regulation exerted by the CPuORF was tissue specific and did not act in the absence of light. Moreover, a photosynthetic signal is needed for the CPuORF action, since plants with uncoupled chloroplasts did not show uORF-dependent repression. Plants transformed with the native AtHB1 promoter driving AtHB1 expression did not show differential phenotypes, whereas those transformed with a construct in which the uORF was mutated exhibited serrated leaves, compact rosettes, and, most significantly, short nondehiscent anthers and siliques containing fewer or no seeds. Thus, we propose that the uncontrolled expression of AtHB1 is deleterious for the plant and, hence, finely repressed by a translational mechanism.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Sistemas de Lectura Abierta/genética , Especificidad de Órganos/genética , Factores de Transcripción/metabolismo , Regiones no Traducidas 5'/genética , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Codón/genética , Secuencia Conservada/genética , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Homocigoto , Mutación/genética , Especificidad de Órganos/efectos de la radiación , Fenotipo , Biosíntesis de Proteínas , Ribosomas/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Zea mays/genética

RESUMEN

Bromodomains, protein domains involved in epigenetic regulation, are able to bind small molecules with high affinity. In the present study, we report free energy calculations for the binding of seven ligands to the first BRD4 bromodomain, using the attach-pull-release (APR) method to compute the reversible work of removing the ligands from the binding site and then allowing the protein to relax conformationally. We test three different water models, TIP3P, TIP4PEw, and SPC/E, as well as the GAFF and GAFF2 parameter sets for the ligands. Our simulations show that the apo crystal structure of BRD4 is only metastable, with a structural transition happening in the absence of the ligand typically after 20 ns of simulation. We compute the free energy change for this transition with a separate APR calculation on the free protein and include its contribution to the ligand binding free energies, which generally causes an underestimation of the affinities. By testing different water models and ligand parameters, we are also able to assess their influence in our results and determine which one produces the best agreement with the experimental data. Both free energies associated with the conformational change and ligand binding are affected by the choice of water model, with the two sets of ligand parameters affecting their binding free energies to a lesser degree. Across all six combinations of water model and ligand potential function, the Pearson correlation coefficients between calculated and experimental binding free energies range from 0.55 to 0.83, and the root-mean-square errors range from 1.4-3.2 kcal/mol. The current protocol also yields encouraging preliminary results when used to assess the relative stability of ligand poses generated by docking or other methods, as illustrated for two different ligands. Our method takes advantage of the high performance provided by graphics processing units and can readily be applied to other ligands as well as other protein systems.

Asunto(s)

Ligandos , Proteínas Nucleares/química , Factores de Transcripción/química , Sitios de Unión , Proteínas de Ciclo Celular , Humanos , Simulación de Dinámica Molecular , Proteínas Nucleares/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Termodinámica , Factores de Transcripción/metabolismo , Agua/química , Agua/metabolismo

RESUMEN

Plants are sessile organisms. This intriguing nature provokes the question of how they survive despite the continual perturbations caused by their constantly changing environment. The large amount of knowledge accumulated to date demonstrates the fascinating dynamic and plastic mechanisms, which underpin the diverse strategies selected in plants in response to the fluctuating environment. This phenotypic plasticity requires an efficient integration of external cues to their growth and developmental programs that can only be achieved through the dynamic and interactive coordination of various signaling networks. Given the versatility of intrinsic structural disorder within proteins, this feature appears as one of the leading characters of such complex functional circuits, critical for plant adaptation and survival in their wild habitats. In this review, we present information of those intrinsically disordered proteins (IDPs) from plants for which their high level of predicted structural disorder has been correlated with a particular function, or where there is experimental evidence linking this structural feature with its protein function. Using examples of plant IDPs involved in the control of cell cycle, metabolism, hormonal signaling and regulation of gene expression, development and responses to stress, we demonstrate the critical importance of IDPs throughout the life of the plant.

Asunto(s)

Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Criptocromos/química , Criptocromos/metabolismo , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Histona Desacetilasas/química , Histona Desacetilasas/metabolismo , Proteínas Intrínsecamente Desordenadas/química , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Desarrollo de la Planta , Proteínas de Plantas/química , Transducción de Señal , Estrés Fisiológico , Factores de Transcripción/química , Factores de Transcripción/metabolismo