Anterior gradient 2 ( AGR 2) is a dimeric protein disulfide isomerase family member involved in the regulation of protein quality control in the endoplasmic reticulum ( ER ). Mouse AGR 2 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 AGR 2 function toward inflammation remain poorly defined. Here, using a protein–protein interaction screen to identify cellular regulators of AGR 2 dimerization, we unveiled specific enhancers, including TMED 2, and inhibitors of AGR 2 dimerization, that control AGR 2 functions. We demonstrate that modulation of AGR 2 dimer formation, whether enhancing or inhibiting the process, yields pro‐inflammatory phenotypes, through either autophagy‐dependent processes or secretion of AGR 2, respectively. We also demonstrate that in IBD and specifically in Crohn's disease, the levels of AGR 2 dimerization modulators are selectively deregulated, and this correlates with severity of disease. Our study demonstrates that AGR 2 dimers act as sensors of ER homeostasis which are disrupted upon ER stress and promote the secretion of AGR 2 monomers. The latter might represent systemic alarm signals for pro‐inflammatory responses.
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.
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 β-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.
We explain, with atomic-level detail, how DNA methylation may contribute to the regulation of biological processes. We show that DNA sequence environment modulates the impact of methylation on the physical properties of the double helix.
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.
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 S2 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.
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 remodeling. Using all-atom microsecond scale molecular dynamics simulations together with a torsional restraint that controls the total helical twist of a DNA fragment, we addressed 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 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 absorb most of the torsional stress become more torsionally rigid, as they are involved in the protein-DNA contacts. Also, the protein undergoes substantial conformational changes to follow the stressinduced 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 unfavorable. Our data suggest that MafB could act as a torsional stress insulator, modulating the propagation of torsional stress along the chromatin fiber, which might promote cooperative binding of other transcription factors.Torsional restraints on DNA, referred to as DNA supercoiling, constantly change during the life of the cell, and regulate transcriptional control on many levels. 1-5 DNA supercoiling represents a sum of writhe and twist -the two interchangeable variables. DNA writhing generally dominates supercoiling changes on a larger scale through the formation of loops and knots, while DNA twisting occurs when shorter DNA fragments, up to ~100 base pairs (b.p.), experience changes in torsional restraints. The net state of genomic DNA is neutral, but regions of positive and negative supercoiling can exist locally, created by RNA polymerases that expose DNA to torsional stress. 3,5 This introduces DNA undertwisting (negative supercoiling) upstream and overtwisting (positive supercoiling) downstream of a transcribed gene.Torsional stress can propagate along DNA, modulating transcription of near-located genes 1,5 by altering the stability of nucleosomes and other protein-DNA complexes, 3,4,6,7 changing the accessibility of the genetic code. The ranges and speeds of torsional stress propagation depend on the underlying nucleotide sequence. 1 Computational experiments confirm: DNA responds to torsional stress in a heterogeneous and sequencedependent manner. 8,9 Certain dinucleotide steps, mainly pyrimidine-purine (YpR) but also purine-purine (RpR), in specific sequence environments, absorb a large part of DNA over-and undertwisting, while the rest of the molecule preserves its ...
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.
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