Nucleosome Research Articles (Page 1)

Abstract An experimental discharge from the DIII-D tokamak is modeled using the SOLPS-ITER code suite and compared against measurements in the pumped and relatively closed upper divertor. Uncertainties of boundary plasma simulations are identified by attempting to match code inputs to experimental conditions, including iteratively solving transport coefficients to match upstream experimental profiles using varying quantities of core particle flux, different pumping models, and various assumptions of ion thermal transport. Simulated boundary conditions for particle injection at the core interface are shown to be relevant to the plasma solution at the divertor targets, even if upstream transport is modified so that plasma profiles are comparatively similar, although
seperatrix density is not held constant. When upstream plasma profiles are matched to experimental measurements by varying diffusive transport coefficients, using either poloidally symmetric or ballooning structure, the model finds a majority of injected energy being transported radially off the computational domain, in conflict with experimental radiated power measurements and heat flux measurements at the divertor target. Imposing a maximum thermal diffusivity or radially shifting the experimental separatrix location of the fitted profiles to increase power conducted to the targets by increasing the upstream electron temperature does not significantly modify this result. Including a thermalizing plenum volume in the simulation domain is shown to maintain the experimental volumetric pumping rate without knowing the neutral energy distribution incident on the pump duct a priori. By modifying transport parameters to match different assumptions for ion temperature, downstream neutral pressure changes by more than a factor of two, suggesting that attention to ion thermal transport may be a critical parameter for simulations to accurately resolve recycling and neutral transport, particularly in a closed divertor geometry. In addition to quantifying various modeling uncertainties, this work motivates both further experimental study and modeling improvements to improve predictive capabilities.

Structural basis for E3 ubiquitin ligase UHRF1 binding to nucleosome core particle and histone H3 ubiquitination.

The VP7 protein of the African horse sickness virus core particle facilitates binding to Culicoides sonorensis cells in an RGD-independent manner.

The proteasome is a central component of the cellular machinery responsible for degrading misfolded or damaged proteins, thereby maintaining protein homeostasis. Dysregulation of proteasome activity has been implicated in various diseases, including neurodegenerative disorders and cancer. In this article, a new β-cyclodextrin conjugate of suxibuzone (SB-CD) is designed and its proteasome activity on purified human 20S core particle and in differentiated human neuroblastoma SH-SY5Y cells (dSHSY5Y) is investigated. This conjugate enhances the proteolytic activity of the 20S proteasome in a dose-dependent manner, with an increase observed at concentrations as low as 5 µM. The EC50 values for SB-CD are determined to be 0.6 ± 0.1 µM for chymotrypsin-like activity and 1.1 ± 0.3 µM for trypsin-like activity, indicating higher efficacy compared to suxibuzone alone. In dSH-SY5Y cells, a decrease in the accumulation of ubiquitinated proteins is observed, consistent with the activation of the proteasome. High-resolution electrospray ionization mass spectrometry investigations confirmed the internalization of SB-CD in cells and verified the stability of the conjugate in response to cellular protease effects, after incubation for up to 24 h. These promising results suggest that the new conjugate is an effective enhancer of proteasome activity, holding significant promise for therapeutic applications targeting proteasome-related pathologies.

Virus-like particles (VLPs) formed as a result of self-assembly of viral capsid proteins are widely used as a platform for antigen presentation in vaccine development. However, since the inclusion of a foreign peptide into the capsid protein can alter its spatial structure and interfere with VLP assembly, such insertions are usually limited to short peptides. In this study, we have demonstrated the potential of capsid protein (CP) of single-stranded RNA phage PQ465 to present long peptides using green fluorescent protein (GFP) as a model. GFP was genetically linked to either the N- or C-terminus of PQ465 CP. Hybrid proteins were expressed in Escherichia coli and Nicotiana benthamiana plants. Spherical virus-like particles (~35 nm according to transmission electron microscopy) were successfully formed by both N- and C-terminal fusions expressed in E. coli, and by plant-produced CP with GFP fused to the C-terminus. ELISA revealed that GFP in VLPs was accessible for specific antibodies suggesting that it is exposed on the surface of PQ465-GFP particles. VLPs carrying GFP were recognized by anti-CP antibodies with less efficiency than VLPs formed by empty CP, which indicates shielding of the CP core in PQ465-GFP particles. Therefore, PQ465 CP can be used as a chimeric VLP platform for the display of relatively large protein antigens, which can operate in bacterial and plant expression systems.

Evaluating zwitterionic stationary phases for glycan profiling of IgGs from various sources by HPLC-ESI-MS/MS.

Cosmological N-body simulations are among the primary tools for studying structure formation in the Universe. Analyses of these simulations critically depend on accurately identifying and tracking dark matter subhalos over time. In recent years, several new algorithms have been developed to improve the accuracy and consistency of subhalo tracking in cold dark matter (CDM) simulations. These algorithms should be revisited in the context of new physics beyond gravity, which can modify the evolution and final properties of subhalo populations. In this work, we apply the particle-tracking-based subhalo finder Symfind to velocity-dependent self-interacting dark matter (SIDM) simulations with large cross section amplitudes to assess the performance of particle-tracking methods beyond the CDM paradigm. We find that the core-particle-tracking technique, which is key to the success of these algorithms in CDM, does not always yield accurate results in SIDM. In particular, the interplay between dark matter self-interactions and tidal stripping can cause the diffusion of core particles to larger radii, leading particle-tracking-based algorithms to prematurely lose track of SIDM subhalos. For massive core-expansion subhalos and core-collapse subhalos that experience close or repeated pericentric passages, a significant fraction of core particles can be lost, and particle-tracking-based finders such as Symfind offer no clear advantage over traditional methods that rely on identifying phase-space overdensities. On the other hand, for subhalos with large pericentric distances or fewer, more distant passages, Symfind tends to outperform. These differences depend sensitively on the cross section amplitude and turnover velocity of the underlying SIDM model. We therefore recommend a hybrid approach that leverages the strengths of both techniques to produce complete and robust catalogs of core-expansion and core-collapse SIDM subhalos.

A sheet-type sinter-bonding material was developed to form thermally stable and highly heat-conductive joints suitable for wide-bandgap (WBG) semiconductor dies and high-heat-flux devices, and its bonding characteristics were investigated. To enhance the cost-competitiveness of the bonding material, Ag-coated Cu (Cu@Ag) particles were employed as fillers instead of conventional Ag particles. To facilitate accelerated sintering, a bimodal particle size distribution comprising several micron- and submicron-sized particles was adopted by synthesizing and mixing both size ranges. For sheet fabrication, a decomposable resin was used as the essential binder component, which could be removed during the bonding process via thermal decomposition. This approach enabled the formation of a sintered bond line composed entirely of Cu@Ag particles. Thermogravimetric and differential thermal analyses revealed that the decomposition of the resin in the sheet occurred within the temperature range of 290–340 °C. Consequently, sinter-bonding conducted at 350 °C and 370 °C exhibited significantly superior bondability compared to bonding at 330 °C. In particular, sinter-bonding at 350 °C for just 60 s resulted in a highly densified joint microstructure with a low porosity of 7.6% and high shear strength exceeding 25 MPa. The formation of the bond line was initiated by sintering between the outer Ag shells of the adjacent particles. However, with increasing bonding time or temperature, sintering driven by Cu diffusion from the particle cores to the outer Ag shells, particularly in the submicron-sized particles, was progressively enhanced. These results obtained from the fabricated sheet-type materials demonstrate that, even with the use of resin, rapid solid-state sintering between filler particles combined with the removal of resin through decomposition enables the formation of a metallic bond line with excellent thermal conductivity.

Abstract This paper investigates the global particle balance of DIV1D, a 1D scrape-off layer (SOL) model extended with reservoirs for neutrals outside the plasma and for the core particle content. The aim of DIV1D is to capture the global particle balance and SOL solutions as a function of gas puffing. To this end, DIV1D is calibrated on solutions of 1D mapped SOLPS-ITER simulations for a density ramp on the tokamak à configuration variable. In this density ramp (as a function of core ion fluxes and reservoir densities) DIV1D predictions for upstream plasma density, upstream electron temperature, target ion flux and target heat flux align mostly within confidence intervals of mapped 1D SOLPS-ITER solutions. For low upstream densities, the target temperature is overestimated by 2 to 4 eV. Simulating the global particle balance as a function of molecular puffing, DIV1D recovers upstream plasma densities and reservoir atomic densities while the core density sensitivity is overestimated by a factor six, whereas sensitivities of divertor reservoir molecular densities are underestimated by a factor three. Special attention is given to wall association effects outside the plasma and SOL opacity in achieving imperfect but acceptable agreement with mapped SOLPS-ITER solutions.

Capsid assembly modulators (CAMs) represent a promising antiviral strategy against hepatitis B virus (HBV), but their effects on pre-formed capsids remain incompletely understood. Here, all-atom molecular dynamics (MD) simulations of intact HBV capsids complexed with prototypical CAM-As (HAP1, HAP18) and CAM-Es (AT130), reveal how structural changes induced by small molecule binding in the interdimer interfaces propagate through the shell lattice to yield global morphological consequences. Each quasi-equivalent interface exhibits a unique response: A sites, located within the pentameric capsomers, are unfilled in these systems and altered marginally by the presence of CAMs in neighboring interfaces. B sites are the most open and “CAM-ready,” suggesting uptake requires minimal conformational perturbation on the local or global level. C sites emerge as hubs of allosteric control and the key drug target, as their occupancy creates local distortion that is broadcast to adjacent sites, driving capsid faceting and – in the case of CAM-As – the destabilization that precedes dissociation in favor of aberrant assembly. D sites, unfilled in these systems, act as structural sinks, absorbing distortions from adjacent interfaces within the hexameric capsomers. The extent of C site adjustment and the nature of D site counterbalance varies with CAM chemotype, highlighting the divergent effects of CAM-As versus CAM-Es. The tensegrity relationship between the four quasi-equivalent interfaces couples them into a global network for strain redistribution that is functionally allosteric, with CAM binding sites displaying signs of both positive and negative cooperativity. These new insights into HBV capsid dynamics clarify how CAMs alter them on the microsecond timescale and suggest that targeting strain redistribution in mature core particles could be leveraged therapeutically.

Sirtuins are a class of NAD-dependent histone deacetylases that regulate important biological pathways in prokaryotes and eukaryotes. This enzyme family comprises seven members, named SIRT1 to SIRT7. Among them, Sirtuin 6 (SIRT6) is a human sirtuin that deacetylates histones and plays a key role in DNA repair, telomere maintenance, carbohydrate and lipid metabolism, and lifespan. SIRT6's structure consists of a zinc finger domain, a Rossmann fold domain containing the NAD+ binding site, and disordered N-terminal and C-terminal (CTD) extensions. The specific role of the CTD in SIRT6's interaction with nucleosomes for histone deacetylation remains unclear. Here, we resort to extended molecular dynamics simulations to uncover the dynamical behavior of the full-length SIRT6 bound to a nucleosome core particle. Our simulations reveal that the CTD preferentially interacts with DNA at the entry/exit near the enzyme's docking site, exhibiting a variety of different binding modes. In specific cases, the CTD contributes to the promotion of DNA unwrapping and enhances H3K27 accessibility to SIRT6's active site, suggesting a pivotal role of this domain for H3K27 deacetylation. This work provides new structural insights into the binding process of the full-length SIRT6 to a nucleosome core particle, shedding light on the conformational behavior and functional role of its CTD. It constitutes an important step toward understanding of SIRT6 deacetylation mechanisms and specificity.

The nucleosome core particle (NCP) regulates genome accessibility through dynamic allosteric communication between histone proteins and DNA. Building on the concept of conditional activity introduced by Lin (2016), we use molecular dynamics simulations and develop an open-source Python library, CONDACT (CONDitional ACTivity), to quantify time-resolved kinetic correlations in nucleosome systems. We analyze long-time simulations of the nucleosome core particle, including two different DNA sequences, the Widom-601 and ASP (alpha-satellite palindromic) sequences. By tracking dihedral angle transitions, we identify residues with high dynamical memory and map inter-residue communication pathways across histone subunits and DNA. Our analysis reveals kinetically connected domains involving post-translational modification sites, oncogenic mutation sites, and DNA contact regions, with dynamic coupling observed over distances up to 7.5 nm. These findings offer new insight into the long-range allosteric behavior of the nucleosome and its potential role in regulating chromatin accessibility. Quantifying this allosteric behavior potentially identifies targetable residues and domains for therapeutic intervention.

The 19S regulatory particle (RP) associates with the 20S core particle (CP) to form the 26S proteasome, an evolutionarily conserved holoenzyme that plays key roles in both physiological and pathological processes. Proteasome inhibitors that target the catalytic subunits within the 20S have proven to be valuable research tools and therapeutics for various cancers. Herein we report the discovery of rapaprotin, a 26S proteasome assembly inhibitor from our natural product-inspired hybrid macrocycle rapafucin library. Rapaprotin induces apoptosis in both myeloma and leukemia cell lines. Genome-wide CRISPR-Cas9 screen identified a cytosolic enzyme, prolyl endopeptidase (PREP) that is required for the pro-apoptotic activity of rapaprotin. Further mechanistic studies revealed that rapaprotin acts as a molecular transformer, changing from an inactive cyclic form into an active linear form, rapaprotin-L, upon PREP cleavage, to block 26S proteasome activity. Time-resolved cryogenic electron microscopy (cryo-EM) revealed that rapaprotin-L induces dissociation of the 19S RP from the 26S holoenzyme, which was verified in cells. Furthermore, rapaprotin exhibits a marked synergistic effect with FDA-approved proteasome inhibitors and resensitizes drug-resistant multiple myeloma cells from patients to bortezomib. Taken together, these results suggest that rapaprotin is a new chemical tool to probe the dynamics of the 26S proteasome assembly and a promising anticancer drug lead.

Oct4 is a master regulator of pluripotency. Potential Oct4 interactors have been cataloged but the manner and significance of these interactions are incompletely defined. Oct4 is capable of binding to DNA in multiple configurations, however the relationship between these configurations and cofactor recruitment (and hence transcription output) are unknown. Here, we show that Oct4 interacts with common and unique proteins when bound to DNA in different configurations. A unique protein is Jade1, a component of HBO histone acetyltransferase complexes. Jade1 preferentially associates with Oct4 when bound to M ore palindromic O ctamer- R elated E lement (MORE) DNA sequences that bind Oct4 dimers. Surprisingly, we find that the Oct4 N-terminal activation domain, rather than facilitating Jade1 binding, serves as an autoinhibitory domain that dampens the interaction. ChIP-seq using HBO1, the enzymatic component of the complex, identifies a preference for binding adjacent to Oct4 at MORE sites. Using purified recombinant proteins and nucleosome complexes, we show that the HBO1 complex acetylates histone H3K9 within nucleosomes more efficiently when Oct4 is co-bound to a MORE. An Oct4 mutant with superior MORE binding characteristics also shows superior ability to catalyze H3K9 acetylation. Jade1 knockdown reduces H3K9Ac at regions where Oct4 binds a MORE but not a simple octamer. Cryo-electron microscopy reveals that Oct4 bound to a MORE near the nucleosome entry/exit site partially unwinds DNA from nucleosome core particles, and identifies additional mass associated with the HBO1 complex. These results identify a novel mechanism of transcriptional regulation by Oct4.

The trypanosome vault particle is composed of multiple major vault protein paralogs and harbors vault RNA

Homology-directed DNA repair (HDR) is critical for genome stability and tumor suppression. HDR is initiated by the RAD51 single-stranded (ss)DNA nucleoprotein filament which conducts the homology search and invades a homologous DNA template, creating a displacement-loop (D-loop). The RAD51 filament is assisted in these processes by several proteins. One such protein is RAD51-Associated-Protein 1 (RAD51AP1) which binds DNA and RNA and directly interacts with RAD51. Of note, RAD51AP1 overexpression is associated with poor prognosis in several different cancer types.Here, we show that RAD51AP1 activity is regulated by phosphorylation. RAD51AP1 bearing S277/282A mutations is more proficient in the stimulation of D-loop formation than wild type RAD51AP1 or phosphomimetic RAD51AP1-S277/282D. In EMSAs, RAD51AP1 with S277/282A mutations more avidly binds ssDNA, double-stranded (ds)DNA, and the nucleosome core particle than wild type RAD51AP1 or RAD51AP1-S277/282D. In cells, RAD51AP1-S277/282A confers no rescue of RAD51AP1 deficiency in toxicity tests and DNA replication assays. In contrast, RAD51AP1-S277/282D fully rescues RAD51AP1 deficiency. We provide evidence that RAD51AP1-S277 is a CDK2 target and propose a model in which RAD51AP1-S277/282 phosphorylation ensures RAD51AP1 flexibility for dynamic engagement in consecutive steps of the HDR reaction. Our results provide new mechanistic insights into RAD51AP1 regulation by a CDK.

Poly(ADP-ribose) polymerases are critical enzymes contributing to regulation of numerous cellular processes, including DNA repair and chromatin remodelling. Within the PARP family, PARP1 and PARP2 primarily facilitate PARylation in the nucleus, particularly responding to genotoxic stress. The activity of PARPs is influenced by the nature of DNA damage and multiple protein partners, with HPF1 being the important one. Forming a joint active site with PARP1/PARP2, HPF1 contributes to histone PARylation and subsequent chromatin relaxation during genotoxic stress events. This study elucidates interrelation between the presence and location of a one-nucleotide gap within the nucleosome core particle (NCP) and PARP activities in automodification and heteromodification of histones. Utilizing a combination of classical biochemical methods with fluorescence-based technique and a single-molecule mass photometry approach, we have shown that the NCP architecture impacts the efficiency and pattern of histone ADP-ribosylation and binding to the histones-associated damaged DNA more significantly for PARP2 than for PARP1. Analysis based on existing studies of HPF1-dependent ADP-ribosylome and NCP structural dynamics allows to suggest that the DNA damage location and the conformational flexibility of histone tails modulated by post-translational modifications are crucial for delineating the distinct roles of PARP1 and PARP2 during genotoxic stress responses.

DNA-protein cross-links (DPCs) are cytotoxic lesions whose study in cells is complicated by the lack of exogenous agents that produce them selectively over DNA-DNA interstrand cross-links (ICLs). The synthesis and reactivity of a chimeric bis-electrophile (MEBAC) that is comprised of a highly reactive alkylating agent and a lysine selective o-ethynyl benzaldehyde is described. DPC formation in nucleosome core particles (NCPs) by MEBAC is >40-times greater than that of ICLs. Cell viability experiments and the single cell Comet assay are consistent with NCP reactivity. Compared to a nitrogen mustard (mechlorethamine, MCE) MEBAC produces higher DPC yields and lower ICL yields in NCPs and in cells at comparable cytotoxicity. Cell viability experiments show that while DPCs from MEBAC are repaired by the metalloprotease SPRTN and the proteasome, only the former repairs such lesions produced by MCE. The inability of the proteasome to repair DPCs in MCE-treated cells likely contributes to the cytotoxicity of the nitrogen mustard. Proteomic analysis identifies several cysteine-rich E3 ligases involved in ubiquitination that are cross-linked to DNA in MCE-treated but not MEBAC-treated cells and suggests a chemical basis for why DPCs produced by the nitrogen mustard are not repaired by the proteasome. This investigation reveals a previously unknown source of nitrogen mustard cytotoxicity and indicates that MEBAC and molecules like it will be useful tools for studying DPCs in cells.

Solution-based investigation of the physical nature of nucleosomes has its roots in X-ray and neutron scattering experiments, including those that provided the initial observation that DNA wraps around core histones. In this study, we performed a comprehensive small-angle scattering study to compare canonical nucleosomes with variant centromeric nucleosomes harboring the histone variant, CENP-A. We used nucleosome core particles (NCPs) assembled on an artificial positioning sequence (Widom 601) and compared these to those assembled on a natural α- satellite DNA cloned from human centromeres. We establish the native solution properties of octameric H3 and CENP-A NCPs using analytical ultracentrifugation (AUC), small-angle X-ray scattering (SAXS), and contrast variation small-angle neutron scattering (CV-SANS). Using high-pressure SAXS (HP-SAXS), we discovered that both histone identity and DNA sequence have an impact on the stability of octameric nucleosomes in solution under high pressure (300 MPa), with evidence of reversible unwrapping in these experimental conditions. Both canonical nucleosomes harboring conventional histone H3 and their centromeric counterparts harboring CENP-A have a substantial increase in their radius of gyration, but this increase is much less prominent for centromeric nucleosomes. More broadly for chromosome-related research, we note that as HP-SAXS methodologies expand in their utility, we anticipate this will provide a powerful solution-based approach to study nucleosomes and higher- order chromatin complexes.

Poly[ADP-ribose] polymerase 1 (PARP1) detects DNA strand breaks that occur in duplex DNA and chromatin. We employed correlative optical tweezers and fluorescence microscopy to quantify how single molecules of PARP1 identify single-strand breaks (i.e., nicks), undamaged nucleosome core particles (NCP) and NCPs containing DNA nicks. Fluorescently-tagged PARP1 or PARP2 from nuclear extracts bound nicks with nanomolar affinity but did not engage undamaged dsDNA regions. In contrast, PARP1 avidly bound undamaged NCPs, and partial NCP unwrapping induced by DNA tension significantly increased the on rate and affinity. Catalytically dead PARP1 or EB-47 inhibition greatly increased PARP1 affinity to DNA nicks and undamaged NCP, implicating a mechanism where PARP1 reverse allostery regulates PARP1 retention to undamaged chromatin. We also monitored ADP-ribosylation in real time upon PARP1 binding undamaged or nicked NCPs. These results provide key mechanistic insights into domain allostery and how pharmacological intervention alters PARP1 binding dynamics for therapeutic impacts.