Impact of Ser17 phosphorylation on the conformational dynamics of the oncoprotein MDM2

MDM2 is an important oncoprotein that downregulates the activity of the tumor suppressor protein p53 via binding of its N-terminal domain to the p53 transactivation domain. The first 24 residues of the MDM2 N-terminal domain form an intrinsically disordered “lid” region that interconverts on a millisecond time scale between “open” and “closed” states in unliganded MDM2. While the former conformational state is expected to facilitate p53 binding, the latter competes in a pseudo-substrate manner with p53 for its binding site. Phosphorylation of serine 17 in the MDM2 lid region is thought to modulate the equilibrium between “open” and “closed” lid states, but contradictory findings on the favored lid conformational state upon phosphorylation have been reported. Here, the nature of the conformational states of MDM2 pSer17 and Ser17Asp variants was addressed by means of enhanced sampling molecular dynamics simulations. Detailed analyses of the computed lid conformational ensembles indicate that both lid variants stabilize a “closed” state, with respect to wild type. Nevertheless, the nature of the closed-state conformational ensembles differs significantly between the pSer17 and Ser17Asp variants. Thus, care should be applied in the interpretation of biochemical experiments that use phosphomimetic variants to model the effects of phosphorylation on the structure and dynamics of this disordered protein region

Unraveling the Differences of the Hydrolytic Activity of Trypanosoma cruzi trans-Sialidase and Trypanosoma rangeli Sialidase: A Quantum Mechanics–Molecular Mechanics Modeling Study

Chagas’ disease, also known as American trypanosomiasis, is a lethal, chronic disease that currently affects more than 10 million people in Central and South America. The trans-sialidase from Trypanosoma cruzi (T. cruzi, TcTS) is a crucial enzyme for the survival of this parasite: sialic acids from the host are transferred to the cell surface glycoproteins of the trypanosome, thereby evading the host’s immune system. On the other hand, the sialidase of T. rangeli (TrSA), which shares 70% sequence identity with TcTS, is a strict hydrolase and shows no trans-sialidase activity. Therefore, TcTS and TrSA represent an excellent framework to understand how different catalytic activities can be achieved with extremely similar structures. By means of combined quantum mechanics–molecular mechanics (QM/MM, SCC-DFTB/Amberff99SB) calculations and umbrella sampling simulations, we investigated the hydrolysis mechanisms of TcTS and TrSA and computed the free energy profiles of these reactions. The results, together with our previous computational investigations, are able to explain the catalytic mechanism of sialidases and describe how subtle differences in the active site make TrSA a strict hydrolase and TcTS a more efficient trans-sialidase.Fil: Bueren Calabuig, Juan A.. University of Florida; Estados UnidosFil: Pierdominici Sottile, Gustavo. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Roitberg, Adrián. University of Florida; Estados Unido

XPF-Dependent DNA Breaks and RNA Polymerase II Arrest Induced by Antitumor DNA Interstrand Crosslinking-Mimetic Alkaloids

SummaryTrabectedin and Zalypsis are two potent anticancer tetrahydroisoquinoline alkaloids that can form a covalent bond with the amino group of a guanine in selected triplets of DNA duplexes and eventually give rise to double-strand breaks. Using well-defined in vitro and in vivo assays, we show that the resulting DNA adducts stimulate, in a concentration-dependent manner, cleavage by the XPF/ERCC1 nuclease on the strand opposite to that bonded by the drug. They also inhibit RNA synthesis by: (1) preventing binding of transcription factors like Sp1 to DNA, and (2) arresting elongating RNA polymerase II at the same nucleotide position regardless of the strand they are located on. Structural models provide a rationale for these findings and highlight the similarity between this type of DNA modification and an interstrand crosslink

Molecular basis of RNA guanine-7 methyltransferase (RNMT) activation by RAM

Maturation and translation of mRNA in eukaryotes requires the addition of the 7-methylguanosine cap. In vertebrates, the cap methyltransferase, RNA guanine-7 methyltransferase (RNMT), has an activating subunit, RNMT-Activating Miniprotein (RAM). Here we report the first crystal structure of the human RNMT in complex with the activation domain of RAM. A relatively unstructured and negatively charged RAM binds to a positively charged surface groove on RNMT, distal to the active site. This results in stabilisation of a RNMT lobe structure which co-evolved with RAM and is required for RAM binding. Structure-guided mutagenesis and molecular dynamics simulations reveal that RAM stabilises the structure and positioning of the RNMT lobe and the adjacent α-helix hinge, resulting in optimal positioning of helix A which contacts substrates in the active site. Using biophysical and biochemical approaches, we observe that RAM increases the recruitment of the methyl donor, AdoMet (S-adenosyl methionine), to RNMT. Thus we report the mechanism by which RAM allosterically activates RNMT, allowing it to function as a molecular rheostat for mRNA cap methylation

Next-generation sequencing in bone marrow failure syndromes and isolated cytopenias: experience of the spanish network on bone marrow failure sundromes

© 2021 the Author(s).Inherited bone marrow failure syndromes (IBMFSs) are a group of congenital rare diseases characterized by bone marrow failure, congenital anomalies, high genetic heterogeneity, and predisposition to cancer. Appropriate treatment and cancer surveillance ideally depend on the identification of the mutated gene. A next-generation sequencing (NGS) panel of genes could be 1 initial genetic screening test to be carried out in a comprehensive study of IBMFSs, allowing molecular detection in affected patients. We designed 2 NGS panels of IBMFS genes: version 1 included 129 genes and version 2 involved 145 genes. The cohort included a total of 204 patients with suspected IBMFSs without molecular diagnosis. Capture-based targeted sequencing covered > 99% of the target regions of 145 genes, with more than 20 independent reads. No differences were seen between the 2 versions of the panel. The NGS tool allowed a total of 91 patients to be diagnosed, with an overall molecular diagnostic rate of 44%. Among the 167 patients with classified IBMFSs, 81 patients (48%) were diagnosed. Unclassified IBMFSs involved a total of 37 patients, of whom 9 patients (24%) were diagnosed. The preexisting diagnosis of 6 clinically classified patients (6%) was amended, implying a change of therapy for some of them. Our NGS IBMFS gene panel assay is a useful tool in the molecular diagnosis of IBMFSs and a reasonable option as the first tier genetic test in these disorders

Temperature-induced melting of double-stranded DNA in the absence and presence of covalently bonded antitumour drugs: insight from molecular dynamics simulations

The difference in melting temperature of a double-stranded (ds) DNA molecule in the absence and presence of bound ligands can provide experimental information about the stabilization brought about by ligand binding. By simulating the dynamic behaviour of a duplex of sequence 5′-d(TAATAACGGATTATT)·5′-d(AATAATCCGTTATTA) in 0.1 M NaCl aqueous solution at 400 K, we have characterized in atomic detail its complete thermal denaturation profile in <200 ns. A striking asymmetry was observed on both sides of the central CGG triplet and the strand separation process was shown to be strongly affected by bonding in the minor groove of the prototypical interstrand crosslinker mitomycin C or the monofunctional tetrahydroisoquinolines trabectedin (Yondelis®), Zalypsis® and PM01183®. Progressive helix unzipping was clearly interspersed with some reannealing events, which were most noticeable in the oligonucleotides containing the monoadducts, which maintained an average of 6 bp in the central region at the end of the simulations. These significant differences attest to the demonstrated ability of these drugs to stabilize dsDNA, stall replication and transcription forks, and recruit DNA repair proteins. This stabilization, quantified here in terms of undisrupted base pairs, supports the view that these monoadducts can functionally mimic a DNA interstrand crosslink

"Bioinformática con Ñ v1.0": a collaborative project of young Spanish scientists to write a complete book about Bioinformatics

Here we present a project aiming to provide specialized educational bibliography on Bioinformatics for Spanish speakers. The idea of writing a book in Spanish language covering the most important topics in the field of Bioinformatics was born in the XIth Spanish Symposium on Bioinformatics in Barcelona two years ago. Different scientists have been involved in the project, from senior scientists to PhD students from different countries. The book intends to be the beginning of an open project, where all the chapters are susceptible of being updated and new topics can be incorporated in future versions. Current book version can be accessed online at http://goo.gl/UYG0o7.Peer Reviewe