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1.
Nucleic Acids Res ; 49(16): 9280-9293, 2021 09 20.
Artículo en Inglés | MEDLINE | ID: mdl-34387667

RESUMEN

Activator proteins 1 (AP-1) comprise one of the largest families of eukaryotic basic leucine zipper transcription factors. Despite advances in the characterization of AP-1 DNA-binding sites, our ability to predict new binding sites and explain how the proteins achieve different gene expression levels remains limited. Here we address the role of sequence-specific DNA flexibility for stability and specific binding of AP-1 factors, using microsecond-long molecular dynamics simulations. As a model system, we employ yeast AP-1 factor Yap1 binding to three different response elements from two genetic environments. Our data show that Yap1 actively exploits the sequence-specific flexibility of DNA within the response element to form stable protein-DNA complexes. The stability also depends on the four to six flanking nucleotides, adjacent to the response elements. The flanking sequences modulate the conformational adaptability of the response element, making it more shape-efficient to form specific contacts with the protein. Bioinformatics analysis of differential expression of the studied genes supports our conclusions: the stability of Yap1-DNA complexes, modulated by the flanking environment, influences the gene expression levels. Our results provide new insights into mechanisms of protein-DNA recognition and the biological regulation of gene expression levels in eukaryotes.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , ADN/genética , Proteínas de Saccharomyces cerevisiae/genética , Factor de Transcripción AP-1/genética , Factores de Transcripción/genética , Secuencia de Bases/genética , Sitios de Unión/genética , ADN/ultraestructura , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/ultraestructura , Regulación de la Expresión Génica/genética , Sustancias Macromoleculares/ultraestructura , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/ultraestructura , Simulación de Dinámica Molecular , Elementos de Respuesta/genética , Proteínas de Saccharomyces cerevisiae/ultraestructura , Factor de Transcripción AP-1/ultraestructura , Factores de Transcripción/ultraestructura , Proteínas Señalizadoras YAP
2.
Genet Med ; 24(7): 1567-1582, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35482014

RESUMEN

PURPOSE: Diphthamide is a post-translationally modified histidine essential for messenger RNA translation and ribosomal protein synthesis. We present evidence for DPH5 as a novel cause of embryonic lethality and profound neurodevelopmental delays (NDDs). METHODS: Molecular testing was performed using exome or genome sequencing. A targeted Dph5 knockin mouse (C57BL/6Ncrl-Dph5em1Mbp/Mmucd) was created for a DPH5 p.His260Arg homozygous variant identified in 1 family. Adenosine diphosphate-ribosylation assays in DPH5-knockout human and yeast cells and in silico modeling were performed for the identified DPH5 potential pathogenic variants. RESULTS: DPH5 variants p.His260Arg (homozygous), p.Asn110Ser and p.Arg207Ter (heterozygous), and p.Asn174LysfsTer10 (homozygous) were identified in 3 unrelated families with distinct overlapping craniofacial features, profound NDDs, multisystem abnormalities, and miscarriages. Dph5 p.His260Arg homozygous knockin was embryonically lethal with only 1 subviable mouse exhibiting impaired growth, craniofacial dysmorphology, and multisystem dysfunction recapitulating the human phenotype. Adenosine diphosphate-ribosylation assays showed absent to decreased function in DPH5-knockout human and yeast cells. In silico modeling of the variants showed altered DPH5 structure and disruption of its interaction with eEF2. CONCLUSION: We provide strong clinical, biochemical, and functional evidence for DPH5 as a novel cause of embryonic lethality or profound NDDs with multisystem involvement and expand diphthamide-deficiency syndromes and ribosomopathies.


Asunto(s)
Metiltransferasas , Trastornos del Neurodesarrollo , Adenosina Difosfato/metabolismo , Animales , Histidina/análogos & derivados , Histidina/metabolismo , Humanos , Metiltransferasas/genética , Ratones , Ratones Endogámicos C57BL , Trastornos del Neurodesarrollo/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Síndrome
3.
J Chem Inf Model ; 58(7): 1406-1414, 2018 07 23.
Artículo en Inglés | MEDLINE | ID: mdl-29927239

RESUMEN

Eukaryotic diphthine synthase, Dph5, is a promiscuous methyltransferase that catalyzes an extraordinary N, O-tetramethylation of 2-(3-carboxy-3-aminopropyl)-l-histidine (ACP) to yield diphthine methyl ester (DTM). These are intermediates in the biosynthesis of the post-translationally modified histidine residue diphthamide (DTA), a unique and essential residue part of the eukaryotic elongation factor 2 (eEF2). Herein, the promiscuity of Saccharomyces cerevisiae Dph5 has been studied with in silico approaches, including homology modeling to provide the structure of Dph5, protein-protein docking and molecular dynamics to construct the Dph5-eEF2 complex, and quantum mechanics/molecular mechanics (QM/MM) calculations to outline a plausible mechanism. The calculations show that the methylation of ACP follows a typical SN2 mechanism, initiating with a complete methylation (trimethylation) at the N-position, followed by the single O-methylation. For each of the three N-methylation reactions, our calculations support a stepwise mechanism, which first involve proton transfer through a bridging water to a conserved aspartate residue D165, followed by a methyl transfer. Once fully methylated, the trimethyl amino group forms a weak electrostatic interaction with D165, which allows the carboxylate group of diphthine to attain the right orientation for the final methylation step to be accomplished.


Asunto(s)
Histidina/análogos & derivados , Metiltransferasas/química , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteínas de Saccharomyces cerevisiae/química , Ácido Aspártico/química , Vías Biosintéticas , Simulación por Computador , Histidina/química , Metilación , Factor 2 de Elongación Peptídica/química , Unión Proteica , Conformación Proteica , Procesamiento Proteico-Postraduccional , Electricidad Estática
5.
Chemistry ; 22(10): 3292-3295, 2016 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-26781301

RESUMEN

A novel method for the mild photoredox-mediated tandem radical acylarylation and tandem acylation/semipinacol rearrangement has been developed. The synthesis of highly functionalized ketones bearing all-carbon α- or ß-quaternary centers has been achieved using easily available symmetric aromatic carboxylic anhydrides as the acyl radical source. The method allows for a straightforward introduction of the keto functionality and concomitant construction of molecular complexity in a single operation.

6.
NAR Genom Bioinform ; 6(2): lqae058, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38800826

RESUMEN

Antisense oligonucleotides (ASOs) offer ground-breaking possibilities for selective pharmacological intervention for any gene product-related disease. Therapeutic ASOs contain extensive chemical modifications that improve stability to enzymatic cleavage and modulate binding affinity relative to natural RNA/DNA. Molecular dynamics (MD) simulation can provide valuable insights into how such modifications affect ASO conformational sampling and target binding. However, force field parameters for chemically modified nucleic acids (NAs) are still underdeveloped. To bridge this gap, we developed parameters to allow simulations of ASOs with the widely applied phosphorothioate (PS) backbone modification, and validated these in extensive all-atom MD simulations of relevant PS-modified NA systems representing B-DNA, RNA, and DNA/RNA hybrid duplex structures. Compared to the corresponding natural NAs, single PS substitutions had marginal effects on the ordered DNA/RNA duplex, whereas substantial effects of phosphorothioation were observed in single-stranded RNA and B-DNA, corroborated by the experimentally derived structure data. We find that PS-modified NAs shift between high and low twist states, which could affect target recognition and protein interactions for phosphorothioated oligonucleotides. Furthermore, conformational sampling was markedly altered in the PS-modified ssRNA system compared to that of the natural oligonucleotide, indicating sequence-dependent effects on conformational preference that may in turn influence duplex formation.

7.
Sci Rep ; 13(1): 16125, 2023 09 26.
Artículo en Inglés | MEDLINE | ID: mdl-37752333

RESUMEN

Transcription factors (TFs) regulate eukaryotic transcription through selective DNA-binding, can also specifically interact with RNA, which may present another layer of transcriptional control. The mechanisms of the TFs-DNA recognition are often well-characterised, while the details of TFs-RNA complexation are less understood. Here we investigate the dual recognition mechanism of the glucocorticoid receptor (GR), which interacts with similar affinities with consensus DNA and diverse RNA hairpin motifs but discriminates against uniform dsRNA. Using atomic molecular dynamics simulations, we demonstrate that the GR binding to nucleic acids requires a wide and shallow groove pocket. The protein effectively moulds its binding site within DNA major groove, which enables base-specific interactions. Contrary, the GR binding has little effect on the grooves geometry of RNA systems, most notably in uniform dsRNA. Instead, a hairpin motif in RNA yields a wide and shallow major groove pocket, allowing the protein to anchor itself through nonspecific electrostatic contacts with RNA backbone. Addition of a bulge increases RNA hairpin flexibility, which leads to a greater number of GR-RNA contacts and, thus, higher affinity. Thus, the combination of structural motifs defines the GR-RNA selective binding: a recognition mechanism, which may be shared by other zinc finger TFs.


Asunto(s)
Ácidos Nucleicos , Receptores de Glucocorticoides , Receptores de Glucocorticoides/genética , ADN , ARN Bicatenario , Sitios de Unión
8.
QRB Discov ; 3: e4, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-37529292

RESUMEN

Changing torsional restraints on DNA is essential for the regulation of transcription. Torsional stress, introduced by RNA polymerase, can propagate along chromatin facilitating topological transitions and modulating the specific binding of transcription factors (TFs) to DNA. Despite the importance, the mechanistic details on how torsional stress impacts the TFs-DNA complexation remain scarce. Herein, we address the impact of torsional stress on DNA complexation with homologous human basic helix-loop-helix (BHLH) hetero- and homodimers: MycMax, MadMax and MaxMax. The three TF dimers exhibit specificity towards the same DNA consensus sequence, the E-box response element, while regulating different transcriptional pathways. Using microseconds-long atomistic molecular dynamics simulations together with the torsional restraint that controls DNA total helical twist, we gradually over- and underwind naked and complexed DNA to a maximum of ± 5°/bp step. We observe that the binding of the BHLH dimers results in a similar increase in DNA torsional rigidity. However, under torsional stress the BHLH dimers induce distinct DNA deformations, characterised by changes in DNA grooves geometry and a significant asymmetric DNA bending. Supported by bioinformatics analyses, our data suggest that torsional stress may contribute to the execution of differential transcriptional programs of the homologous TFs by modulating their collaborative interactions.

9.
QRB Discov ; 3: e23, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-37529293

RESUMEN

Selective DNA binding by transcription factors (TFs) is crucial for the correct regulation of DNA transcription. In healthy cells, promoters of active genes are hypomethylated. A single CpG methylation within a TF response element (RE) may change the binding preferences of the protein, thus causing the dysregulation of transcription programs. Here, we investigate a molecular mechanism driving the downregulation of the NDUFA13 gene, due to hypermethylation, which is associated with multiple cancers. Using bioinformatic analyses of breast cancer cell line MCF7, we identify a hypermethylated region containing the binding sites of two TFs dimers, CEBPB and E2F1-DP1, located 130 b.p. from the gene transcription start site. All-atom extended MD simulations of wild type and methylated DNA alone and in complex with either one or both TFs dimers provide mechanistic insights into the cooperative asymmetric binding order of the two dimers; the CEBPB binding should occur first to facilitate the E2F1-DP1-DNA association. The CpG methylation within the E2F1-DP1 RE and the linker decrease the cooperativity effects and renders the E2F1-DP1 binding site less recognizable by the TF dimer. Taken together, the identified CpG methylation site may contribute to the downregulation of the NDUFA13 gene.

10.
Sci Rep ; 10(1): 18795, 2020 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-33139763

RESUMEN

Torsional stress on DNA, introduced by molecular motors, constitutes an important regulatory mechanism of transcriptional control. Torsional stress can modulate specific binding of transcription factors to DNA and introduce local conformational changes that facilitate the opening of promoters and nucleosome remodelling. Using all-atom microsecond scale molecular dynamics simulations together with a torsional restraint that controls the total twist of a DNA fragment, we address the impact of torsional stress on DNA complexation with a human BZIP transcription factor, MafB. We gradually over- and underwind DNA alone and in complex with MafB by 0.5° per dinucleotide step, starting from the relaxed state to a maximum of 5° per dinucleotide step, monitoring the evolution of the protein-DNA contacts at different degrees of torsional strain. Our computations show that MafB changes the DNA sequence-specific response to torsional stress. The dinucleotide steps that are susceptible to absorbing most of the torsional stress become more torsionally rigid, as they are involved in protein-DNA contacts. Also, the protein undergoes substantial conformational changes to follow the stress-induced DNA deformation, but mostly maintains the specific contacts with DNA. This results in a significant asymmetric increase of free energy of DNA twisting transitions, relative to free DNA, where overtwisting is more energetically unfavourable. Our data suggest that specifically bound BZIP factors could act as torsional stress insulators, modulating the propagation of torsional stress along the chromatin fibre, which might promote cooperative binding of collaborative DNA-binding factors.


Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , ADN Superhelicoidal/química , ADN/química , Secuencia de Bases , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/fisiología , Fenómenos Biomecánicos , Cromatina , ADN/genética , Fragmentación del ADN , ADN Superhelicoidal/genética , Humanos , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico
11.
Sci Rep ; 10(1): 17490, 2020 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-33060689

RESUMEN

Inositol-Requiring Enzyme 1α (IRE1α; hereafter IRE1) is a transmembrane kinase/ribonuclease protein related with the unfolded protein response (UPR) signaling. Experimental evidence suggests that IRE1 forms several three dimensional (3D) structural variants: dimers, tetramers and higher order oligomers, where each structural variant can contain different IRE1 conformers in different arrangements. For example, studies have shown that two sets of IRE1 dimers exist; a face-to-face dimer and a back-to-back dimer, with the latter considered the important unit for UPR signaling propagation. However, the structural configuration and mechanistic details of the biologically important IRE1 tetramers are limited. Here, we combine protein-protein docking with molecular dynamics simulations to derive human IRE1 tetramer models and identify a molecular mechanism of IRE1 activation. To validate the derived models of the human IRE1 tetramer, we compare the dynamic behavior of the models with the yeast IRE1 tetramer crystallographic structure. We show that IRE1 tetramer conformational changes could be linked to the initiation of the unconventional splicing of mRNA encoding X-box binding protein-1 (XBP1), which allows for the expression of the transcription factor XBP1s (XBP1 spliced). The derived IRE1 tetrameric models bring new mechanistic insights about the IRE1 molecular activation mechanism by describing the IRE1 tetramers as active protagonists accommodating the XBP1 substrate.


Asunto(s)
Endorribonucleasas/química , Proteínas Serina-Treonina Quinasas/química , Proteína 1 de Unión a la X-Box/química , Biología Computacional , Cristalografía por Rayos X , Humanos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Fosforilación , Análisis de Componente Principal , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Transducción de Señal , Respuesta de Proteína Desplegada
12.
EMBO Mol Med ; 11(6)2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-31040128

RESUMEN

Anterior gradient 2 (AGR2) is a dimeric protein disulfide isomerase family member involved in the regulation of protein quality control in the endoplasmic reticulum (ER). Mouse AGR2 deletion increases intestinal inflammation and promotes the development of inflammatory bowel disease (IBD). Although these biological effects are well established, the underlying molecular mechanisms of AGR2 function toward inflammation remain poorly defined. Here, using a protein-protein interaction screen to identify cellular regulators of AGR2 dimerization, we unveiled specific enhancers, including TMED2, and inhibitors of AGR2 dimerization, that control AGR2 functions. We demonstrate that modulation of AGR2 dimer formation, whether enhancing or inhibiting the process, yields pro-inflammatory phenotypes, through either autophagy-dependent processes or secretion of AGR2, respectively. We also demonstrate that in IBD and specifically in Crohn's disease, the levels of AGR2 dimerization modulators are selectively deregulated, and this correlates with severity of disease. Our study demonstrates that AGR2 dimers act as sensors of ER homeostasis which are disrupted upon ER stress and promote the secretion of AGR2 monomers. The latter might represent systemic alarm signals for pro-inflammatory responses.


Asunto(s)
Estrés del Retículo Endoplásmico , Retículo Endoplásmico/metabolismo , Mucoproteínas/metabolismo , Proteínas Oncogénicas/metabolismo , Multimerización de Proteína , Proteostasis , Animales , Retículo Endoplásmico/genética , Células HEK293 , Humanos , Masculino , Ratones , Mucoproteínas/genética , Proteínas Oncogénicas/genética
13.
Chem Commun (Camb) ; 54(84): 11885-11888, 2018 Oct 18.
Artículo en Inglés | MEDLINE | ID: mdl-30288518

RESUMEN

We describe a potential molecular mechanism explaining how DNA methylation contributes to biological regulation. Using molecular dynamics together with a new torsional restraint, we identify the impact of methylation on DNA response to torsional stress. We observe that, depending on the sequence, DNA methylation hinders overwinding or underwinding molecular transitions.


Asunto(s)
Metilación de ADN , ADN/química , ADN/metabolismo , Fenómenos Biomecánicos , ADN/genética , Modelos Moleculares , Simulación de Dinámica Molecular
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