RESUMEN
In eukaryotic organisms, genomic DNA associates with histone proteins to form nucleosomes. Nucleosomes provide a basis for genome compaction, epigenetic markup, and mediate interactions of nuclear proteins with their target DNA loci. A negatively charged (acidic) patch located on the H2A-H2B histone dimer is a characteristic feature of the nucleosomal surface. The acidic patch is a common site in the attachment of various chromatin proteins, including viral ones. Acidic patch-binding peptides present perspective compounds that can be used to modulate chromatin functioning by disrupting interactions of nucleosomes with natural proteins or alternatively targeting artificial moieties to the nucleosomes, which may be beneficial for the development of new therapeutics. In this work, we used several computational and experimental techniques to improve our understanding of how peptides may bind to the acidic patch and what are the consequences of their binding. Through extensive analysis of the PDB database, histone sequence analysis, and molecular dynamic simulations, we elucidated common binding patterns and key interactions that stabilize peptide-nucleosome complexes. Through MD simulations and FRET measurements, we characterized changes in nucleosome dynamics conferred by peptide binding. Using fluorescence polarization and gel electrophoresis, we evaluated the affinity and specificity of the LANA1-22 peptide to DNA and nucleosomes. Taken together, our study provides new insights into the different patterns of intermolecular interactions that can be employed by natural and designed peptides to bind to nucleosomes, and the effects of peptide binding on nucleosome dynamics and stability.
Asunto(s)
Histonas , Nucleosomas , Histonas/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Cromatina , ADN/química , Simulación de Dinámica Molecular , Péptidos/metabolismo , Polarización de FluorescenciaRESUMEN
Nucleosomes are the most abundant protein-DNA complexes in eukaryotes that provide compaction of genomic DNA and are implicated in regulation of transcription, DNA replication and repair. The details of DNA positioning on the nucleosome and the DNA conformation can provide key regulatory signals. Hydroxyl-radical footprinting (HRF) of protein-DNA complexes is a chemical technique that probes nucleosome organization in solution with a high precision unattainable by other methods. In this work we propose an integrative modeling method for constructing high-resolution atomistic models of nucleosomes based on HRF experiments. Our method precisely identifies DNA positioning on nucleosome by combining HRF data for both DNA strands with the pseudo-symmetry constraints. We performed high-resolution HRF for Saccharomyces cerevisiae centromeric nucleosome of unknown structure and characterized it using our integrative modeling approach. Our model provides the basis for further understanding the cooperative engagement and interplay between Cse4p protein and the A-tracts important for centromere function.
Asunto(s)
Huella de ADN/métodos , ADN/química , Modelos Moleculares , Nucleosomas/química , Algoritmos , Centrómero/química , Proteínas Cromosómicas no Histona , División del ADN , Proteínas de Unión al ADN , Radical Hidroxilo , Conformación de Ácido Nucleico , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiaeRESUMEN
Twenty-five years have passed since the appearance of the first atomistic model of the nucleosome structure, and since then the number of new structures has gradually increased. With the advent of cryo-microscopy, the rate of accumulation of models has increased significantly. New structures are emerging with different histone variants and a variety of proteins that bind to nucleosomes. At the moment, there are more than four hundred structures containing nucleosomes in the Protein Data Bank. Many of these structures represent similar complexes, others differ in composition, conformation and quality. In this perspective, we investigate the diversity of known nucleosome structures, analyze data and model quality, variations in histone/DNA content of nucleosomes and spectrum of their interactors. We outline those parts of the nucleosome "structurome" that are already explored and those awaiting further exploration.
RESUMEN
The protein core of the nucleosome is composed of an H3-H4 histone tetramer and two H2A-H2B histone dimers. The tetramer organizes the central 60 DNA bp, while H2A-H2B dimers lock the flanking DNA segments. Being positioned at the sides of the nucleosome, H2A-H2B dimers stabilize the overall structure of the nucleosome and modulate its dynamics, such as DNA unwrapping, sliding, etc. Such modulation at the epigenetic level is achieved through post-translational modifications and the incorporation of histone variants. However, the detailed connection between the sequence of H2A-H2B histones and their structure, dynamics and implications for nucleosome functioning remains elusive. In this work, we present a detailed study of H2A-H2B dimer dynamics in the free form and in the context of nucleosomes via atomistic molecular dynamics simulations (based on X. laevis histones). We supplement simulation results by comparative analysis of information in the structural databases. Particularly, we describe a major dynamical mode corresponding to the bending movement of the longest H2A and H2B α-helices. This overall bending dynamics of the H2A-H2B dimer were found to be modulated by its interactions with DNA, H3-H4 tetramer, the presence of DNA twist-defects with nucleosomal DNA and the amino acid sequence of histones. Taken together, our results shed new light on the dynamical mechanisms of nucleosome functioning, such as nucleosome sliding, DNA-unwrapping and their epigenetic modulation.
Asunto(s)
Histonas , Nucleosomas , Secuencia de Aminoácidos , ADN/metabolismo , Histonas/metabolismo , Simulación de Dinámica MolecularRESUMEN
Nucleosomes are elementary building blocks of chromatin in eukaryotes. They tightly wrap â¼147 DNA base pairs around an octamer of histone proteins. How nucleosome structural dynamics affect genome functioning is not completely clear. Here we report all-atom molecular dynamics simulations of nucleosome core particles at a timescale of 15 microseconds. At this timescale, functional modes of nucleosome dynamics such as spontaneous nucleosomal DNA breathing, unwrapping, twisting, and sliding were observed. We identified atomistic mechanisms of these processes by analyzing the accompanying structural rearrangements of the histone octamer and histone-DNA contacts. Octamer dynamics and plasticity were found to enable DNA unwrapping and sliding. Through multi-scale modeling, we showed that nucleosomal DNA dynamics contribute to significant conformational variability of the chromatin fiber at the supranucleosomal level. Our study further supports mechanistic coupling between fine details of histone dynamics and chromatin functioning, provides a framework for understanding the effects of various chromatin modifications.
Asunto(s)
Cromatina/metabolismo , ADN/metabolismo , Histonas/metabolismo , Nucleosomas/metabolismo , Cromatina/química , ADN/química , Simulación de Dinámica Molecular , Conformación de Ácido NucleicoRESUMEN
The cutaneous delivery route currently accounts for almost 10% of all administered drugs and it is becoming more common. Chemical penetration enhancers (CPEs) increase the transport of drugs across skin layers by different mechanisms that depend on the chemical nature of the penetration enhancers. In our work, we created a chemical penetration enhancer database (CPE-DB) that is, to the best of our knowledge, the first CPE database. We collected information about known enhancers and their derivatives in a single database, and classified and characterized their molecular diversity in terms of scaffold content, key chemical moieties, molecular descriptors, etc. CPE-DB can be used for virtual screening and similarity search to identify new potent and safe enhancers, building quantitative structure-activity relationship (QSAR) and quantitative structure-property relationship (QSPR) models, and other machine-learning (ML) applications for the prediction of biological activity.
RESUMEN
The 70-kDa heat shock proteins (HSP70s) are abundantly present in cancer, providing malignant cells selective advantage by suppressing multiple apoptotic pathways, regulating necrosis, bypassing cellular senescence program, interfering with tumor immunity, promoting angiogenesis and supporting metastasis. This direct involvement of HSP70 in most of the cancer hallmarks explains the phenomenon of cancer "addiction" to HSP70, tightly linking tumor survival and growth to the HSP70 expression. HSP70 operates in different states through its catalytic cycle, suggesting that it can multi-function in malignant cells in any of these states. Clinically, tumor cells intensively release HSP70 in extracellular microenvironment, resulting in diverse outcomes for patient survival. Given its clinical significance, small molecule inhibitors were developed to target different sites of the HSP70 machinery. Furthermore, several HSP70-based immunotherapy approaches were assessed in clinical trials. This review will explore different roles of HSP70 on cancer progression and emphasize the importance of understanding the flexibility of HSP70 nature for future development of anti-cancer therapies.
Asunto(s)
Proteínas HSP70 de Choque Térmico/inmunología , Inmunoterapia/métodos , Neoplasias/inmunología , Apoptosis , Proliferación Celular , HumanosRESUMEN
Phytoplankton is the base of the marine food chain as well as oxygen and carbon cycles and thus plays a global role in climate and ecology. Nucleocytoplasmic Large DNA Viruses that infect phytoplankton organisms and regulate the phytoplankton dynamics encompass genes of rhodopsins of two distinct families. Here, we present a functional and structural characterization of two proteins of viral rhodopsin group 1, OLPVR1 and VirChR1. Functional analysis of VirChR1 shows that it is a highly selective, Na+/K+-conducting channel and, in contrast to known cation channelrhodopsins, it is impermeable to Ca2+ ions. We show that, upon illumination, VirChR1 is able to drive neural firing. The 1.4 Å resolution structure of OLPVR1 reveals remarkable differences from the known channelrhodopsins and a unique ion-conducting pathway. Thus, viral rhodopsins 1 represent a unique, large group of light-gated channels (viral channelrhodopsins, VirChR1s). In nature, VirChR1s likely mediate phototaxis of algae enhancing the host anabolic processes to support virus reproduction, and therefore, might play a major role in global phytoplankton dynamics. Moreover, VirChR1s have unique potential for optogenetics as they lack possibly noxious Ca2+ permeability.
Asunto(s)
Fitoplancton/virología , Rodopsina/química , Rodopsina/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Animales , Calcio/metabolismo , Cationes , Células Cultivadas , Channelrhodopsins/metabolismo , Células HEK293 , Humanos , Activación del Canal Iónico , Luz , Neuronas/metabolismo , Filogenia , Conformación Proteica , Ratas Wistar , Rodopsina/genética , Relación Estructura-Actividad , Proteínas Virales/genética , Difracción de Rayos XRESUMEN
Nucleosomes are fundamental units of chromatin compaction, which organize â¼200 DNA base pairs using an octamer of histone proteins. Their ubiquitous presence in the cell nucleus since the first eukaryotes compelled the chromatin machinery to coevolve and learn how to exploit various modes of nucleosome dynamics and sense differences in nucleosome composition. Alterations to histone or DNA sequences, post-translational modifications (PTM) of histones, recruitment of chromatin proteins modulate nucleosome dyn amics and provide epigenetic regulation to the DNA processing pathways (transcription, replication, repair, etc.). Our understanding of this complex interplay between nucleosome composition, dynamics and functioning is constantly evolving through new insights and discoveries. In this review, we highlight recent contributions to the field while attempting to organize them in a unified framework.
Asunto(s)
Nucleosomas/metabolismo , ADN/genética , ADN/metabolismo , Histonas/genética , Histonas/metabolismo , Modelos Moleculares , Nucleosomas/química , Nucleosomas/genéticaRESUMEN
Hydroxyl-radical footprinting (HRF) is a powerful method for probing structures of nucleic acid-protein complexes with single-nucleotide resolution in solution. To tap the full quantitative potential of HRF, we describe a protocol, hydroxyl-radical footprinting interpretation for DNA (HYDROID), to quantify HRF data and integrate them with atomistic structural models. The stages of the HYDROID protocol are extraction of the lane profiles from gel images, quantification of the DNA cleavage frequency at each nucleotide and theoretical estimation of the DNA cleavage frequency from atomistic structural models, followed by comparison of experimental and theoretical results. Example scripts for each step of HRF data analysis and interpretation are provided for several nucleosome systems; they can be easily adapted to analyze user data. As input, HYDROID requires polyacrylamide gel electrophoresis (PAGE) images of HRF products and optionally can use a molecular model of the DNA-protein complex. The HYDROID protocol can be used to quantify HRF over DNA regions of up to 100 nucleotides per gel image. In addition, it can be applied to the analysis of RNA-protein complexes and free RNA or DNA molecules in solution. Compared with other methods reported to date, HYDROID is unique in its ability to simultaneously integrate HRF data with the analysis of atomistic structural models. HYDROID is freely available. The complete protocol takes ~3 h. Users should be familiar with the command-line interface, the Python scripting language and Protein Data Bank (PDB) file formats. A graphical user interface (GUI) with basic functionality (HYDROID_GUI) is also available.
Asunto(s)
Huella de ADN/métodos , ADN/química , Radical Hidroxilo/química , Huella de Proteína/métodos , Proteínas/química , Programas Informáticos , ADN/metabolismo , División del ADN , Huella de ADN/estadística & datos numéricos , Electroforesis en Gel de Poliacrilamida/estadística & datos numéricos , Humanos , Modelos Moleculares , Nucleosomas/química , Nucleosomas/metabolismo , Huella de Proteína/estadística & datos numéricos , Proteínas/metabolismo , SolucionesRESUMEN
We present here raw trajectories of molecular dynamics simulations for nucleosome with linker DNA strands as well as minimalistic nucleosome core particle model. The simulations were done in explicit solvent using CHARMM36 force field. We used this data in the research article Shaytan et al., 2016 [1]. The trajectory files are supplemented by TCL scripts providing advanced visualization capabilities.
RESUMEN
An octamer of histone proteins wraps about 200bp of DNA into two superhelical turns to form nucleosomes found in chromatin. Although the static structure of the nucleosomal core particle has been solved, details of the dynamic interactions between histones and DNA remain elusive. We performed extensively long unconstrained, all-atom microsecond molecular dynamics simulations of nucleosomes including linker DNA segments and full-length histones in explicit solvent. For the first time, we were able to identify and characterize the rearrangements in nucleosomes on a microsecond timescale including the coupling between the conformation of the histone tails and the DNA geometry. We found that certain histone tail conformations promoted DNA bulging near its entry/exit sites, resulting in the formation of twist defects within the DNA. This led to a reorganization of histone-DNA interactions, suggestive of the formation of initial nucleosome sliding intermediates. We characterized the dynamics of the histone tails upon their condensation on the core and linker DNA and showed that tails may adopt conformationally constrained positions due to the insertion of "anchoring" lysines and arginines into the DNA minor grooves. Potentially, these phenomena affect the accessibility of post-translationally modified histone residues that serve as important sites for epigenetic marks (e.g., at H3K9, H3K27, H4K16), suggesting that interactions of the histone tails with the core and linker DNA modulate the processes of histone tail modifications and binding of the effector proteins. We discuss the implications of the observed results on the nucleosome function and compare our results to different experimental studies.
Asunto(s)
Histonas/química , Histonas/metabolismo , Conformación de Ácido Nucleico , Nucleosomas/química , Nucleosomas/metabolismo , Conformación Proteica , Animales , Humanos , Cinética , Modelos Moleculares , Simulación de Dinámica Molecular , Xenopus laevisRESUMEN
DNA accessibility to regulatory proteins is substantially influenced by nucleosome structure and dynamics. The facilitates chromatin transcription (FACT) complex increases the accessibility of nucleosomal DNA, but the mechanism and extent of its nucleosome reorganization activity are unknown. Here we determined the effects of FACT from the yeast Saccharomyces cerevisiae on single nucleosomes by using single-particle Förster resonance energy transfer (spFRET) microscopy. FACT binding results in dramatic ATP-independent, symmetrical and reversible DNA uncoiling that affects at least 70% of the DNA within a nucleosome, occurs without apparent loss of histones and proceeds via an 'all-or-none' mechanism. A mutated version of FACT is defective in uncoiling, and a histone mutation that suppresses phenotypes caused by this FACT mutation in vivo restores the uncoiling activity in vitro. Thus, FACT-dependent nucleosome unfolding modulates the accessibility of nucleosomal DNA, and this activity is an important function of FACT in vivo.