Biology subjects

Lyubchenko, Y. L.

Publications and source records attributed to Lyubchenko, Y. L..

17 recordsLinked to original sources

bioRxiv2025

Amyloid β interaction with membranes: Removal of cholesterol from the membranes to catalyze aggregation and amyloid pathology

The interplay between the cholesterol metabolism and assembly of A{beta}42 (the 42- residue form of the amyloid-{beta} peptide) peptides in pathological aggregates is considered as one of the major molecular mechanisms in development of Alzheimers disease (AD). Numerous in vitro studies led to the finding that the high cholesterol levels in membranes accelerate the production of A{beta} aggregates. The molecular mechanisms explaining how cholesterol localized inside the membrane bilayer catalyzes the assembly of A{beta} aggregates above the membrane remain unknown. We addressed this problem by combining different AFM modalities, including imaging and force spectroscopy, with fluorescence spectroscopy. Our combined studies revealed that A{beta}42 was capable of removing cholesterol from the membrane. Importantly, physiologically low concentrations of A{beta}42 demonstrate such ability of monomeric A{beta}42. Extracted cholesterol interacts with A{beta}42 and accelerates its on- membrane aggregation. We propose a model of interaction of A{beta}42 with membranes based on the ability of A{beta}42 to extract cholesterol, which explains several AD associated observations related to cholesterol interplay with A{beta}42 aggregation resulting in the AD onset and progression.

biophysics↗

bioRxiv2024

The Interaction of NF-κB Transcription Factor with Centromeric Chromatin

Centromeric chromatin is a subset of chromatin structure and governs chromosome segregation. The centromere is composed of both CENP-A nucleosomes (CENP-Anuc) and H3 nucleosomes (H3nuc) and is enriched with alpha-satellite (-sat) DNA repeats. These CENP-Anuc have a different structure than H3nuc, decreasing the base pairs (bp) of wrapped DNA from 147 bp for H3nuc to 121 bp for CENP-Anuc. All these factors can contribute to centromere function. We investigated the interaction of H3nuc and CENP-Anuc with NF-{kappa}B, a crucial transcription factor in regulating immune response and inflammation. We utilized Atomic Force Microscopy (AFM) to characterize complexes of both types of nucleosomes with NF-{kappa}B. We found that NF-{kappa}B unravels H3nuc, removing more than 20 bp of DNA, and that NF-{kappa}B binds to the nucleosomal core. Similar results were obtained for the truncated variant of NF-{kappa}B comprised only of the Rel Homology domain and missing the transcription activation domain (TAD), suggesting the RelA TAD is not critical in unraveling H3nuc. By contrast, NF-{kappa}B did not bind to or unravel CENP- Anuc. These findings with different affinities for two types of nucleosomes to NF-{kappa}B may have implications for understanding the mechanisms of gene expression in bulk and centromere chromatin.

biophysics↗

bioRxiv2024

Nanoscale interaction of endonuclease APE-1 with DNA characterized by atomic force microscopy

Apurinic/apyrimidinic endonuclease 1 (APE1) is involved in DNA replication, repair, and transcriptional regulation mechanisms. This multifunctional activity of APE1 should be supported by specific structural properties of APE1 that have not yet been elucidated. Here we applied atomic force microscopy (AFM) to characterize the interactions of APE1 with DNA. Complexes of APE1 with DNA containing G-rich segments were visualized, and analysis of the complexes revealed the affinity of APE1 to G-rich DNA sequences. Furthermore, loops in the DNA-APE1 complexes were visualized, and their yield was as high as 53 %. However, the loops were non-specific, with quantitative analysis revealing the yield of loops bridging two G-rich DNA segments to be 41%. Analysis of protein size in various complexes was performed, and these data showed that loops are formed by APE1 monomer, suggesting that APE1 has two DNA binding sites. The data lead us to a model for the interaction of APE1 with DNA that describes its molecular site search mechanism. The new properties of APE1 in organizing DNA, by bringing two distant sites together, may be important for facilitating the scanning for damage and coordinating repair and transcription.

biophysics↗

bioRxiv2024

Nanoscale Structure, Interactions, and Dynamics of Centromere Nucleosomes

Centromeres are specific segments of chromosomes responsible for the accurate chromosome segregation process. Centromeres are comprised of two types of nucleosomes: canonical nucleosomes containing an octamer of H2A, H2B, H3, and H4 histones, and CENP-A nucleosomes in which H3 is replaced with its analog CENP-A histone. This modification leads to the difference in the nuclear of DNA turns around the histone core, wrapping efficiency. This value is 121 bp of DNA, considerably less than 147 bp found in canonical nucleosomes. We used Atomic Force Microscopy (AFM) to characterize nanoscale features for both types of nucleosomes assembled on the same template, enabling us to evaluate the effect of internucleosomal interaction. We found that CENP-A mononucleosomes have a lower internucleosomal affinity than canonical H3 nucleosomes. We applied time-lapse, high-speed AFM (HS-AFM) to characterize the dynamics of nucleosomes. For both nucleosomes, spontaneous unwrapping of DNA was observed, and this process occurs via a transient state with [~]100 bp DNA wrapped around the core, followed by a rapid dissociation of DNA. The unwrapping process is asymmetric, so when the dissociation starts on one arm, it enlarges the size of the dissociated arm. Additionally, HS-AFM revealed higher stability of CENP-nucleosomes compared with H3 ones, in which dissociation of the histone core occurs prior to the nucleosome dissociation. The histone core of CENP-A nucleosomes remains intact even after the dissociation of DNA.

biophysics↗

bioRxiv2023

Assembly of Synaptic Protein-DNA Complexes: Critical Role of Non-Specific Interactions

The synaptic protein-DNA complexes, formed by specialized proteins that bridge two or more distant sites on DNA, are critically involved in various genetic processes. However, the molecular mechanism by which the protein searches for these sites and how it brings them together is not well understood. Our previous studies directly visualized search pathways used by SfiI, and we identified two pathways, DNA threading and site-bound transfer pathways, specific to the site search process for synaptic DNA-protein systems. To investigate the molecular mechanism behind these site search pathways, we assembled complexes of SfiI with various DNA substrates corresponding to different transient states and measured their stability using a single-molecule fluorescence approach. These assemblies corresponded to specific-specific (synaptic), non-specific-non-specific (non-specific), and specific-non-specific (pre-synaptic) SfiI-DNA states. Unexpectedly, there was an elevated stability in pre-synaptic complexes assembled with specific and non-specific DNA substrates has been found. To explain these surprising observations, a theoretical approach that describes the assembly of these complexes and compares the predictions with the experiment is developed. The theory explains this effect by utilizing entropic arguments, according to which, after the partial dissociation, the non-specific DNA template has multiple possibilities of rebinding, effectively increasing the stability. Such difference in the stabilities of SfiI complexes with specific and non-specific DNA explains the utilization of threading and site-bound transfer pathways in the search process of synaptic protein-DNA complexes discovered in the time-lapse AFM experiments.

biophysics↗

bioRxiv2022

Sequence Dependent Nanoscale Structure of CENP-A Nucleosomes

CENP-A is a histone variant found in high abundance at the centromere. The centromere chromatin similar to the bulk chromatin consist of nucleosomes, but H3 histone of the bulk chromatin nucleosomes (H3 nucleosomes) is replaced with CENP-A histone. Additionally, the centromere comprises tandem repeats of -satellite DNA which CENP-A nucleosomes assemble upon. However, the effect of the DNA sequence on the nucleosome assembly and centromere formation remains poorly understood. Here we investigated the structure of nucleosomes assembled with the CENP-A variant using Atomic Force Microscopy. We assembled both CENP-A nucleosomes and H3 nucleosomes on a DNA substrate containing an -satellite motif and characterized their positioning and wrapping efficiency. We also studied CENP-A nucleosomes on the 601-positioning motif and non-specific DNA to compare their relative positioning and stability. CENP-A nucleosomes assembled on -satellite DNA did not show any positional preference along the substrate, which is similar to H3 nucleosomes and CENP-A nucleosomes on non-specific DNA. The range of nucleosome wrapping efficiency was narrower on -satellite DNA compared with non-specific DNA, suggesting a more stable complex. These findings indicate that DNA sequence and histone composition may be two of many factors required for accurate centromere assembly.

molecular biology↗

bioRxiv2022

Sequence Dependent Internucleosomal Interactions Dominate Array Assembly

The organization of the nucleosome array is a critical component of the chromatin assembly into higher order structure as well as its function. Here we investigated the contribution of the DNA sequence and internucleosomal interactions to the organization of the nucleosomal arrays in compact structures using Atomic Force Microscopy. We assembled nucleosomes on DNA substrates allowing for the formation of tetranucleosomes. We found that nucleosomes are capable of forming constructs with the close positioning of nucleosomes with no discernible space between them, even in the case of assembled dinucleosomes. This morphology of the array is in contrast with that observed for arrays assembled with repeats of the nucleosome positioning motifs separated by uniform spacers. Simulated assembly of tetranucleosomes by random placement along the substrates revealed that nucleosome array compaction is promoted by the interaction of the nucleosomes. We developed a theoretical model to account for the role of DNA sequence and internucleosomal interactions in the formation of the nucleosome structures. These findings suggest that, in the chromatin assembly, the affinity of the nucleosomes to the DNA sequence and the strengths of the internucleosomal interactions are the two major factors defining the compactness of the chromatin.

biophysics↗

bioRxiv2021

Restriction of RecG translocation by DNA mispairing

The RecG DNA helicase plays a crucial role in stalled replication fork rescue as the guardian of the bacterial genome. We have recently demonstrated that single-strand DNA binding protein (SSB) promotes binding of RecG to the stalled replication fork by remodeling RecG, enabling the helicase to translocate ahead of the fork. We also hypothesized that mispairing of DNA could limit such translocation of RecG, which plays the role of roadblocks for the fork movement. Here, we used atomic force microscopy (AFM) to directly test this hypothesis and investigate how sensitive RecG translocation is to different types of mispairing. We found that a C-C mismatch at a distance of 30 bp away from the fork position prevents translocation of RecG over this mispairing. A G-bulge placed at the same distance also has a similar roadblock efficiency. However, a C-C mismatch 10 bp away from the fork does not prevent RecG translocation, as 10 bp from fork is within the distance of footprint of RecG on fork DNA. Our findings suggest that retardation of RecG translocation ahead of the replication fork can be a mechanism for the base pairing control for DNA replication machinery.

biophysics↗

bioRxiv2021

Transcription Factor NF-κB Unravels Nucleosomes

NF-{kappa}B is a transcription factor responsible for activating hundreds of genes in mammalian organisms. To accomplish its function, NF-{kappa}B must interact with DNA occupied by nucleosomes, but how this interaction occurs is unclear. Here we used Atomic Force Microscopy to characterize complexes of NF-{kappa}B with nucleosomes assembled on different DNA templates. The assembly of NF-{kappa}B-nucleosome complexes leads to a substantial decrease of DNA wrapping efficiency. Mapping of the nucleosomes did not reveal displacement of under-wrapped nucleosomes from their original position, suggesting that unravelling involves dissociation of one or both flanks of the nucleosomes. We discovered two binding modes of NF-{kappa}B associated with nucleosome unraveling - NF-{kappa}B bound to the nucleosome core and to the DNA flanks and propose models explaining the interaction of NF-{kappa}B with the nucleosome. We speculate that NF-{kappa}B can function as a pioneer factor enhancing its ability to facilitate rapid transcriptional response to cell stress.

biophysics↗

bioRxiv2020

Cholesterol in membranes facilitates aggregation of amyloid beta protein at physiologically low concentrations

The formation of amyloid {beta} (1-42) (A{beta}42) oligomers is considered to be a critical step in the development of Alzheimers disease (AD). However, the mechanism underlying this process at physiologically low concentrations of A{beta}42 remains unclear. We have previously shown that oligomers assemble at such low A{beta}42 monomer concentrations in vitro on phospholipid membranes. We hypothesized that membrane composition is the factor controlling the aggregation process. Accumulation of cholesterol in membranes is associated with AD development, suggesting that insertion of cholesterol into membranes may initiate the A{beta}42 aggregation, regardless of a low monomer concentration. We used atomic force microscopy (AFM) to directly visualize the aggregation process of A{beta}42 on the surface of a lipid bilayer containing cholesterol. Time-lapse AFM imaging unambiguously demonstrates that cholesterol in the lipid bilayer significantly enhances the aggregation process of A{beta}42 at nanomolar monomer concentration. Quantitative analysis of the AFM data shows that both the number of A{beta}42 oligomers and their sizes grow when cholesterol is present. Importantly, the aggregation process is dynamic, so the aggregates assembled on the membrane can dissociate from the bilayer surface into the bulk solution. Computational modeling demonstrated that the lipid bilayer containing cholesterol had an elevated affinity to A{beta}42. Moreover, monomers adopted the aggregation-prone conformations present in amyloid fibrils. The low energy barriers between these conformations facilitate the transition between monomer states and is another factor promoting the self-association of the monomers.

biophysics↗

bioRxiv2020

High-Speed AFM directly visualizes conformational dynamics of the HIV-Vif protein complex

Viral infectivity factor (Vif) is a protein that is essential for the replication of the HIV-1 virus. The key function of Vif is to disrupt the antiviral activity of APOBEC3 proteins, which mutate viral nucleic acids. Inside the cell, Vif binds to the host cell proteins Elongin-C, Elongin-B, and CBF-{beta}, forming a four-protein complex called VCBC. The structure of VCBC in complex with the Cullin5 (Cul5) protein has been solved by X-ray crystallography, and recently, using molecular dynamic (MD) simulations, the dynamics of VCBC and VCBC-Cul5 complexes were characterized. Here, we applied time-lapse high-speed atomic force microscopy (HS-AFM) to visualize the conformational changes of the VCBC complex. We determined the three most favorable conformations of the VCBC complex, which we identified as triangle, dumbbell, and globular structures. In addition, we characterized the dynamics of each of these structures. While our data show a very dynamic behavior for all these structures, we found the triangle and dumbbell structures to be the most dynamic. These findings provide insight into the structure and dynamics of the VCBC complex and support further research into the improvement of HIV treatment, as Vif is essential for virus survival in the cell.

biophysics↗

bioRxiv2020

AFM characterization of the interaction of PriA helicase with stalled DNA replication forks

In bacteria, the restart of stalled DNA replication forks requires the PriA DNA helicase. PriA recognizes and remodels abandoned DNA replication forks performing the DNA unwinding in 3 to 5-direction and facilitates loading of the DnaB helicase onto the DNA to restart replication. The single stranded DNA binding protein (SSB) is typically present at the abandoned forks, but there is gap in the knowledge on the interaction between SSB and PriA protein. Here, we used atomic force microscopy (AFM) to visualize the interaction of PriA with DNA substrates in the absence or presence of SSB. Results show that in the absence of SSB, PriA binds preferentially to a fork substrate with a gap in the leading strand. Preferential binding occurs only on forked DNA structures as 5- and 3-tailed duplexes were bound equally well. Furthermore, in the absence of SSB, PriA bound exclusively to the fork regions of substrates. In contrast, fork bound SSB loads PriA onto the duplex DNA arms of forks. When the fork has a gap in the leading strand, PriA localizes to both the parental and lagging strand arms. When the gap is present in the lagging strand, PriA is loaded preferentially onto the leading strand arm of the fork. Remodeling of PriA requires a functional C-terminal domain of SSB.

biophysics↗

bioRxiv2019

Two C-terminal sequence variations determine differential neurotoxicity between human and mouse α-synuclein

-Synuclein (aSyn) aggregation is thought to play a central role in neurodegenerative disorders termed synucleinopathies, including Parkinsons disease (PD). Mouse aSyn contains a threonine residue at position 53 that mimics the human familial PD substitution A53T, yet in contrast to A53T patients, mice show no evidence of aSyn neuropathology even after aging. Here we studied the neurotoxicity of human A53T, mouse aSyn, and various human-mouse chimeras in cellular and in vivo models as well as their biochemical properties relevant to aSyn pathobiology. We report that mouse aSyn is less neurotoxic than the human A53T variant as a result of inhibitory effects of two C-terminal amino acid substitutions on membrane-induced aSyn aggregation and aSyn-mediated vesicle permeabilization. Our findings highlight the importance of membrane-induced self-assembly in aSyn neurotoxicity and suggest that inhibiting this process by targeting the C-terminal domain could slow neurodegeneration in PD and other synucleinopathy disorders.

neuroscience↗

bioRxiv2019

Spontaneous Self-assembly of Amyloid β (1-40) into Dimers

The self-assembly and fibrillation of amyloid {beta} (A{beta}) proteins is the neuropathological hallmark of Alzheimers disease. However, the molecular mechanism of how disordered monomers assemble into aggregates remains largely unknown. In this work, we characterize the assembly of A{beta} (1-40) monomers into dimers using long-time molecular dynamics simulations. Upon interaction, the monomers undergo conformational transitions, accompanied by change of the structure, leading to the formation of a stable dimer. The dimers are primarily stabilized by interactions in the N-terminal region (residues 5-12), in the central hydrophobic region (residues 16-23), and in the C-terminal region (residues 30-40); with inter-peptide interactions focused around the N- and C- termini. The dimers do not contain long {beta}-strands that are usually found in fibrils.

biophysics↗

bioRxiv2018

A molecular model of the surface-assisted protein aggregation process

The importance of cell surfaces in the self-assembly of proteins is widely accepted. One biologically significant event is the assembly of amyloidogenic proteins into aggregates, which leads to neurodegenerative disorders like Alzheimers and Parkinsons. The interaction of amyloidogenic proteins with cellular membranes appears to dramatically facilitate the aggregation process. Recent findings indicate that, in the presence of surfaces, aggregation occurs at physiologically low concentrations, suggesting interaction with surfaces plays a critical role in the disease-prone aggregation process. However, the molecular mechanisms behind on-surface aggregation remain unclear. Here we provide a theoretical model that offers a molecular explanation. According to this model, monomers transiently immobilized to surfaces increase the local monomer protein concentration and thus work as nuclei to dramatically accelerate the entire aggregation process. This theory was verified by experimental studies, using mica surfaces, to examine the aggregation kinetics of amyloidogenic-synuclein protein (-Syn) and non-amyloidogenic cytosine deaminase APOBEC3G (A3G).

biophysics↗

bioRxiv2018

Phospholipid membranes promote the early stage assembly of α-synuclein aggregates

Development of Parkinsons disease is associated with spontaneous self-assembly of -synuclein (-syn). Efforts aimed at understanding this process have produced little clarity and the mechanism remains elusive. We report a novel effect of phospholipid bilayers on the catalysis of -syn aggregation from monomers. We directly visualized -syn aggregation on supported lipid bilayers using time-lapse atomic force microscopy. We discovered that -syn assemble in aggregates on bilayer surfaces even at the nanomolar concentration of monomers in solution. The efficiency of the aggregation process depends on the membrane composition, being highest for a negatively charged bilayer. Furthermore, assembled aggregates can dissociate from the surface, suggesting that on-surface aggregation can be a mechanism by which pathological aggregates are produced. Computational modeling revealed that interaction of -syn with bilayer surface changes the protein conformation and its affinity to assemble into dimers, and these properties depend on the bilayer composition. A model of the membrane-mediated aggregation triggering the assembly of neurotoxic aggregates is proposed.

neuroscience↗

bioRxiv2017

Nanoscale Dynamics of Centromere Nucleosomes and the Critical Roles of CENP-A

In the absence of a functioning centromere, chromosome segregation becomes aberrant, leading to an increased rate of aneuploidy. The highly specific recognition of centromeres by kinetochores suggests that specific structural characteristics define this region, however, the structural details and mechanism underlying this recognition remains a matter of intense investigation. To address this, High speed atomic force microscopy was used for direct visualization of the spontaneous dynamics of CENP-A nucleosomes at the sub-second time scale. We report that CENP-A nucleosomes change conformation spontaneously and reversibly, utilizing two major pathways: unwrapping, and looping of the DNA; enabling core transfer between neighboring DNA substrates. Along with these nucleosome dynamics we observed that CENP-A stabilizes the histone core against dissociating to histone subunits, unique from H3 cores which are only capable of such plasticity in the presence of remodeling factors. These findings have implications for the dynamics and integrity of nucleosomes at the centromere.

biophysics↗