Biology subjects

Mackay, J. P.

Publications and source records attributed to Mackay, J. P..

16 recordsLinked to original sources

bioRxiv2025

The structure and composition of native human nucleosomes

Since the first high-resolution structures of recombinantly assembled nucleosomes, efforts have shifted towards understanding chromatin structure in a native context. Most of these efforts have focused on native-like, yet still recombinantly assembled, nucleosomes that contain native DNA sequences. To date, no high-resolution structures of native human nucleosomes have been reported. Here we report the high-resolution cryo-EM structure of native human nucleosomes isolated from HEK293 cells. The HEK293-NCP structure reveals that native human nucleosomes store 145 bp of DNA. Despite the DNA sequence diversity of native nucleosomes, we observe conserved nucleotides that support the idea of a nucleosome positioning code. In addition to these striking features of nucleosomal DNA, we note alternate conformations of several DNA contacting histone residues that hint at dynamics in the HEK293-NCP. To complement the HEK293-NCP structure, we provide a mass spectrometry analysis of histone modifications and variants present in the sample, which demonstrates that a typical HEK293-NCP is composed of canonical histones with N-terminal tails that are methylated at K9, K27 and/or K36 of histone H3. Altogether these findings have implications for biological processes such as chromatin remodelling and transcription and improve our understanding of nucleosome and chromatin structure in a native context.

biochemistry↗

bioRxiv2025

Peptide molecular glues select between BET paralogues by exploiting allosteric sites and conformational dynamics

Achieving selective target inhibition is critical for minimising drug side effects. This can be especially challenging when targeting individual members of protein families with high sequence similarity. A well-recognised example is the Bromodomain and Extraterminal domain (BET) family of proteins. Chemical inhibition of the acetyllysine (AcK)-binding bromodomains (BDs) of BET proteins has shown considerable promise in a range of disease models. However, despite over a decade of medicinal chemistry efforts, it has proven challenging to develop BET BD inhibitors that exhibit high selectivity between BET family paralogues. Cyclic peptides are versatile scaffolds for therapeutic development and often exhibit high selectivity and affinity for their targets. To explore their potential as selective BET BD inhibitors, we have used mRNA display to identify cyclic peptide ligands for the N-terminal BD of BRD2 and BRD4. The structurally diverse cyclic peptides enriched from the selections boast superior selectivity and affinity to previously developed inhibitors. Most strikingly, we isolated cyclic peptides with [~]1000-fold higher affinity for their target BD over the paralogous BDs, far surpassing selectivities reported to date. Our biochemical and structural data suggest that paralogue-selective cyclic peptides act as molecular glues, exploiting both subtle sequence differences at locations far from the AcK-binding pocket and differences in conformational dynamics between BET BD paralogues to achieve this unprecedented level of specificity. This work provides a blueprint for the development of new classes of selective BET inhibitors and, more generally, underscores the potential of exploiting protein dynamics in the design of selective ligands.

biochemistry↗

bioRxiv2025

ZNF512B associates with mitotic spindles, regulates metaphase exit and is crucial for stem cell differentiation

Zinc finger proteins are a large family of DNA-binding factors that play key roles in diverse cellular processes including gene regulation, RNA metabolism and cell cycle control. The zinc finger protein ZNF512B has recently been implicated in chromatin organization and transcriptional repression through its direct interaction with the nucleosome remodeling and deacetylase (NuRD) complex, its DNA-binding ability, and its association with the histone variant H2A.Z. Here, we uncover a previously unrecognized role for ZNF512B that is independent of both its zinc finger domains and NuRD association. We identify ZNF512B as a spindle-associated factor that regulates progression through mitosis, specifically controlling metaphase exit. ZNF512Bs N-terminal internal region, which contains 25 repeats of a six-residue motif predicted to form a {beta}-helix structure, is required and sufficient for its spindle interaction. Elevated ZNF512B levels result in a profound metaphase arrest that is ultimately lethal, a phenotype arising from the combined activity of its spindle-binding and chromatin-tethering functions. Conversely, ZNF512B depletion accelerates stem cell proliferation, impairs differentiation, and upregulates genes linked to cell cycle progression. Our findings position ZNF512B as a multifunctional protein that acts as a transcriptional repressor, a chromatin aggregator and a novel metaphase exit regulator through spindle fiber binding.

molecular biology↗

bioRxiv2025

The BRD4-nucleosome interaction is enhanced modestly and non-selectively by histone acetylation

BRD4 regulates gene transcription in complex eukaryotes, in part through the binding of its tandem bromodomains to acetylated lysine residues found in histones and transcription factors. Despite pharmacological inhibition of these domains showing promise in preclinical studies, clinical trial data have been less encouraging so far. A stronger understanding of BRD4 biochemistry could provide a route to better outcomes. To advance on prior work, which has focused almost entirely on the binding of isolated bromodomains and acetylated peptides, we have sought the preferred nucleosomal binding partner of full-length BRD4. We demonstrate that BRD4 binds with sub-micromolar affinity to both unmodified nucleosomes and to DNA alone. In strong contrast to BRD4-peptide interactions, we also find that the affinity of BRD4 for nucleosomes is increased only 2-4-fold by histone acetylation and that this affinity has little dependence on the acetylation pattern. Despite this modest effect of acetylation, binding of BRD4 to acetyllysine in the nucleosome was more resistant to perturbation by mutation or small-molecule inhibition than BRD4-peptide interactions. Our work helps bridge the gap between cellular and prior in vitro work and provides clues to explain the in vivo chromatin occupancy profile of BRD4 and how it changes upon therapeutic inhibition. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/656505v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1aa532aorg.highwire.dtl.DTLVardef@13a154forg.highwire.dtl.DTLVardef@4d2039org.highwire.dtl.DTLVardef@17e8377_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

ZNF512B binds RBBP4 via a variant NuRD interaction motif and aggregates chromatin in a NuRD complex-independent manner

The evolutionarily conserved histone variant H2A.Z plays a crucial role in various DNA-based processes but the underlying mechanisms by which it acts are not completely understood. Recently, we identified the zinc finger protein ZNF512B as an H2A.Z-, HMG20A- and PWWP2A-associated protein. Here, we report that ZNF512B binds the nucleosome remodeling and deacetylase (NuRD) complex. We discover a conserved amino acid sequence within ZNF512B that resembles the NuRD-interaction motif (NIM) previously identified in FOG-1 and other transcriptional regulators. By solving the crystal structure of this motif bound to the NuRD component RBBP4 and by applying several biochemical assays we demonstrate that this internal NIM is both necessary and sufficient for robust NuRD binding. Transcriptome analyses and reporter assays identify ZNF512B as a repressor of gene expression that can act in both NuRD-dependent and -independent ways. Surprisingly, high levels of ZNF512B expression lead to nuclear protein and chromatin aggregation foci that form independent of the interaction with the NuRD complex but depend on the zinc finger domains of ZNF512B. Our study has implications for diseases in which ZNF512B expression is deregulated, such as cancer and neurodegenerative diseases, and hint at the existence of more proteins as potential NuRD interactors.

molecular biology↗

bioRxiv2022

Discovery and characterization of cyclic peptides selective for the C-terminal bromodomains of BET family proteins

SUMMARYDNA encoded cyclic peptide libraries offer unique opportunities to discover high-potency, high-specificity ligands directed against a target protein. We set out to explore the potential for such libraries to provide ligands that can distinguish between bromodomains from the closely related paralogues of the Bromodomain and ExtraTerminal domain (BET) family of epigenetic regulators. Analysis of peptides isolated from a screen against the C-terminal bromodomain of family member BRD2, together with new peptides discovered in previous screens against the corresponding domain from BRD3 and BRD4, reveals peptides with nanomolar and subnanomolar affinities. X-ray crystal structures of several of these bromodomain-peptide complexes reveal diverse structures and binding modes, which nevertheless display several conserved binding features. A subset of the peptides demonstrates significant paralogue-level specificity, though structural analysis does not reveal clear physicochemical explanations for this specificity. Our data demonstrate the power of cyclic peptides to discriminate between highly similar proteins with high potency and hint that differences in conformational dynamics between BET-family bromodomains might modulate binding affinities amongst family members for particular ligands.

biochemistry↗

bioRxiv2022

Cross-linking mass spectrometry discovers, evaluates, and validates the experimental and predicted structural proteome

Significant recent advances in structural biology, particularly in the field of cryo-electron microscopy, have dramatically expanded our ability to create structural models of proteins and protein complexes. However, many proteins remain refractory to these approaches because of their low abundance, low stability or - in the case of complexes - simply not having yet been analysed. Here, we demonstrate the power of combining cross-linking mass spectrometry (XL-MS) with artificial intelligence-based structure prediction to discover and experimentally substantiate models for protein and protein complex structures at proteome scale. We present the deepest XL-MS dataset to date, describing 28,910 unique residue pairs captured across 4,084 unique human proteins and 2,110 unique protein-protein interactions. We show that integrative models of complexes driven by AlphaFold Multimer and inspired and corroborated by the XL-MS data offer new opportunities to deeply mine the structural proteome and interactome and reveal new mechanisms underlying protein structure and function.

biochemistry↗

bioRxiv2022

The H2A.Z.1/PWWP2A/NuRD-associated protein HMG20A controls early head and heart developmental transcription programs

Abstract/SummarySpecialized chromatin-binding proteins are required for DNA-based processes during development. We recently established PWWP2A as direct histone variant H2A.Z interactor involved in mitosis and cranial-facial development. Here, we identify the H2A.Z/PWWP2A-associated protein HMG20A as part of several chromatin-modifying complexes including NuRD, and show that it localizes to genomic regulatory regions. Hmg20a depletion causes severe head and heart developmental defects in Xenopus laevis. Our data indicate that craniofacial malformations are caused by defects in neural crest cell (NCC) migration and cartilage formation. These developmental defects are pheno-copied in HMG20A-depleted mESCs, which show inefficient differentiation into NCCs and cardiomyocytes (CMs). Accordingly, loss of HMG20A caused striking deregulation of transcription programs involved in epithelial- mesenchymal transition (EMT) and cardiac differentiation, thereby providing insights into the regulatory circuits controlled by HMG20A. Collectively, our findings implicate HMG20A as part of the H2A.Z/PWWP2A/NuRD-axis and reveal it as a key modulator of the intricate developmental transcription programs that guide NCC and cardiomyocyte differentiation.

molecular biology↗

bioRxiv2021

Molecular architecture of nucleosome remodeling and deacetylase sub-complexes by integrative structure determination

The Nucleosome Remodeling and Deacetylase (NuRD) complex is a chromatin-modifying assembly that regulates gene expression and DNA damage repair. Despite its importance, limited structural information describing the complete NuRD complex is available and a detailed understanding of its mechanism is therefore lacking. Drawing on information from SEC-MALLS, DIA-MS, XLMS, negative-stain EM, X-ray crystallography, NMR spectroscopy, secondary structure predictions and homology models, we applied Bayesian integrative structure determination to investigate the molecular architecture of three NuRD sub-complexes: MTA1-HDAC1-RBBP4 (MHR), MTA1N-HDAC1-MBD3GATAD2CC (MHM), and MTA1-HDAC1-RBBP4-MBD3-GATAD2A (NuDe). The integrative structures were corroborated by examining independent crosslinks, cryo-EM maps, biochemical assays, known cancer-associated mutations, and structure predictions from AlphaFold. The robustness of the models was assessed by jack-knifing. Localization of the full-length MBD3, which connects the deacetylase and chromatin remodeling modules in NuRD, has not previously been possible; our models indicate two different locations for MBD3, suggesting a mechanism by which MBD3 in the presence of GATAD2A asymmetrically bridges the two modules in NuRD. Further, our models uncovered three previously unrecognized subunit interfaces in NuDe: HDAC1C-MTA1BAH, MTA1BAH-MBD3MBD, and HDAC160-100-MBD3MBD. Our approach also allowed us to localize regions of unknown structure, such as HDAC1C and MBD3IDR, thereby resulting in the most complete and robustly cross-validated structural characterization of these NuRD sub-complexes so far.

biophysics↗

bioRxiv2021

Altered cortical processing of sensory input in Huntington disease mouse models

Huntington disease (HD), a hereditary neurodegenerative disorder, manifests as progressively impaired movement and cognition. Although early abnormalities of neuronal activity in striatum are well established in HD models, there are fewer in vivo studies of the cortex. Here, we record local field potentials (LFPs) in YAC128 HD model mice versus wild-type mice. In multiple cortical areas, limb sensory stimulation evokes a greater change in LFP power in YAC128 mice. Mesoscopic imaging using voltage-sensitive dyes reveal more extensive spread of evoked sensory signals across the cortical surface in YAC128 mice. YAC128 layer 2/3 sensory cortical neurons ex vivo show increased excitatory events, which could contribute to enhanced sensory responses in vivo. Cortical LFP responses to limb stimulation, visual and auditory input are also significantly increased in zQ175 HD mice. Results presented here extend knowledge of HD beyond ex vivo studies of individual neurons to the intact cortical network.

neuroscience↗

bioRxiv2021

Unique Protein Interaction Networks Define The Chromatin Remodeling Module of The NuRD Complex

The combination of four proteins and their paralogues including MBD2/3, GATAD2A/B, CDK2AP1, and CHD3/4/5, which we refer to as the MGCC module, form the chromatin remodeling module of the Nucleosome Remodeling and Deacetylase (NuRD) complex, a gene repressor complex. Specific paralogues of the MGCC subunits such as MBD2 and CHD4 are amongst the key repressors of adult-stage fetal globin and provide important targets for molecular therapies in beta ({beta})-thalassemia. However, mechanisms by which the MGCC module acquires paralogue-specific function and specificity have not been addressed to date. Understanding the protein-protein interaction (PPI) network of the MGCC subunits is essential in defining underlying mechanisms and developing treatment strategies. Therefore, using pulldown followed by mass spectrometry analysis (PD-MS) we report a proteome-wide interaction network of the MGCC module in a paralogue-specific manner. Our data also demonstrate that the disordered C-terminal region of CHD3/4/5 is a gateway to incorporate remodeling activity into both the ChAHP (CHD4, ADNP, HP1{gamma}) and NuRD complexes in a mutually exclusive manner. We define a short aggregation prone region (APR) within the C-terminal segment of GATAD2B that is essential for the interaction of CHD4 and CDK2AP1 with the NuRD complex. Finally, we also report an association of CDK2AP1 with the Nuclear Receptor Co-Repressor (NCOR) complex. Overall, this study provides insight into the possible mechanisms through which the MGCC module can achieve specificity and diverse biological functions.

biochemistry↗

bioRxiv2020

The Nucleosome Remodeling and Deacetylase complex has an asymmetric, dynamic, and modular architecture

The Nucleosome Remodeling and Deacetylase (NuRD) complex is essential for development in complex animals but has been refractory to biochemical analysis. We present the first integrated analysis of the architecture of the native mammalian NuRD complex, combining quantitative mass spectrometry, covalent cross-linking, protein biochemistry and electron microscopy. NuRD is built around a 2:2:4 pseudo-symmetric deacetylase module comprising MTA, HDAC and RBBP subunits. This module interacts asymmetrically with a remodeling module comprising one copy each of MBD, GATAD2 and CHD subunits. The previously enigmatic GATAD2 controls the asymmetry of the complex and directly recruits the ATP-dependent CHD remodeler. Unexpectedly, the MTA-MBD interaction acts as a point of functional switching. The transcriptional regulator PWWP2A modulates NuRD assembly by competing directly with MBD for binding to the MTA-HDAC-RBBP subcomplex, forming a moonlighting PWWP2A-MTA-HDAC-RBBP complex that likely directs deacetylase activity to PWWP2A target sites. Taken together, our data describe the overall architecture of the intact NuRD complex and reveal aspects of its structural dynamics and functional plasticity.

biochemistry↗

bioRxiv2020

Spontaneous Axonal ER Ca2+ Waves Mediate A Shift From Action Potential-Dependent to Independent Glutamate Release in the YAC128 HD-Model

Action potential-independent (miniature) neurotransmission occurs at all chemical synapses, but remains poorly understood, particularly in pathological contexts. Spontaneous release of Ca2+ from the axonal endoplasmic reticulum (ER) is thought to facilitated miniature neurotransmission, and aberrant ER Ca2+ handling is notably implicated in the progression of Huntingtons disease (HD) and other neurodegenerative diseases. Here, we report elevated glutamate-mediated miniature synaptic event frequencies in YAC128 (HD-model) cortical neurons, which pharmacological experiments suggest is mediated by enhanced spontaneous ER Ca2+ release. Calcium imaging using an axon-localized sensor revealed slow action potential (AP)-independent axonal Ca2+ waves, which were more common in YAC128 cortical neurons. Conversely, spontaneous axonal ER Ca2+ release was associated with reduced AP-dependent axonal Ca2+ events and consequent glutamate release. Together, our results suggest spontaneous release of axonal ER Ca2+ stores oppositely regulates activity-dependent and -independent neurotransmitter release in HD, with potential implications for the fidelity and plasticity of cortical excitatory signaling.

neuroscience↗

bioRxiv2019

Cyclic peptides can engage a single binding pocket through multiple, entirely divergent modes

Cyclic peptide display screening techniques can identify drug leads and biological probes with exceptional affinity and specificity. To date, however, the structural and functional diversity encoded in such peptide libraries remains unexplored. We have used the Random nonstandard Peptide Integrated Discovery (RaPID) system to develop cyclic peptide inhibitors of several acetyllysine-binding bromodomains from the Bromodomain and Extra-Terminal domain (BET) family of epigenetic regulators. These peptides have very high affinities for their targets and exhibit extraordinary selectivity (up to 106-fold), making them the highest-affinity and most specific BET-binding molecules discovered to date. Crystal structures of 13 distinct peptide-bromodomain complexes, which all target the acetyllysine-binding pocket, reveal remarkable diversity in both peptide structure and binding mode, and include both -helical and {beta}-sheet type structures. The peptides can exhibit a high degree of structural pre-organization and bivalent binding of two BDs by one peptide was common, flagging the potential for a new direction in inhibitor design that could bring stronger discrimination between BET-family paralogues. Our data demonstrate for the first time the enormous potential held in these libraries to provide a wide array of modes against a single target, maximizing the opportunity to attain high potency and specificity ligands to a wide variety of proteins.

biochemistry↗

bioRxiv2019

Reversible Reorganization Of Basolateral Amygdala Dendrites in Male Rats Accompanies Stress Resilience

Endogenous neuropeptide Y (NPY) and corticotrophin-releasing factor (CRF) modulate the responses of the Basolateral amygdala (BLA) to stress, and are associated respectively with the development of stress resilience and vulnerability. We characterized the persistent effects of repeated NPY and CRF treatment on the structure and function of BLA principal neurons (PN) in a novel organotypic slice culture (OTC) model of rat BLA, and examined the contributions of specific NPY receptor subtypes to these neural and behavioral effects. In BLA principal neurons within the OTCs, repeated NPY treatment caused persistent attenuation of excitatory input and induced dendritic hypotrophy via Y5 receptors; conversely, CRF increased excitatory input and induced hypertrophy of BLA PNs. Repeated treatment of OTCs with NPY followed by an identical treatment with CRF, or vice versa inhibited or reversed all structural changes in OTCs. These structural responses to NPY or CRF required calcineurin or CaMKII, respectively. Finally, repeated intra-BLA injections of NPY or a Y5 receptor agonist increased social interaction and recapitulated structural changes in BLA neurons seen in OTCs, while a Y5 receptor antagonist prevented NPYs effects both on behavior and on structure. These results implicate the Y5 receptor in the long-term, anxiolytic-like effects of NPY in the BLA, consistent with an intrinsic role in stress buffering, and highlight a remarkable mechanism by which BLA neurons may adapt to different levels of stress. Moreover, BLA OTCs offer a robust model to study mechanisms associated with resilience and vulnerability to stress in BLA.\n\nSignificance StatementWithin the basolateral amygdala (BLA), Neuropeptide Y (NPY) is associated with buffering the neural stress response induced by CRF, and promoting stress resilience. We used a novel organotypic slice culture (OTC) model of BLA, complemented with in vivo studies, to examine the cellular mechanisms associated with the actions of NPY. In OTCs, repeated NPY treatment reduces the complexity of the dendritic extent of anxiogenic BLA principal neurons, making them less excitable. NPY, via activation of Y5 receptors, additionally inhibits and reverses the increases in dendritic extent and excitability induced by the stress hormone, corticotropin releasing factor (CRF). This NPY-mediated neuroplasticity indicates that resilience or vulnerability to stress may thus involve neuropeptide-mediated dendritic remodeling in BLA PNs.

neuroscience↗

bioRxiv2019

A heme-binding protein produced by Haemophilus haemolyticus inhibits non-typeable Haemophilus influenzae

Many commensal bacteria and opportunistic pathogens scavenge heme from their environment. Pathogens and host are engaged in an arms race to control access to heme, but similar conflicts between bacterial species that might regulate pathogen colonisation are largely unknown. We show here that a commensal bacterium, Haemophilus haemolyticus, makes hemophilin, a heme-binding protein that not only allows the bacterium to effectively scavenge heme for its own growth, but also inhibits co-culture of the opportunistic pathogen, non-typeable Haemophilus influenzae (NTHi), by heme starvation. Knockout of the hemophilin gene abrogates the ability of H. haemolyticus to inhibit NTHi and an x-ray crystal structure shows that hemophilin has a previously unreported heme-binding structure. The bound heme molecule is deeply buried and the heme iron atom is coordinated through a single histidine side chain. Biochemical characterization shows that this arrangement allows heme to be captured in the ferrous or ferric state, and with small ferrous or ferric heme-ligands bound, suggesting hemophilin could function over in a wide range of physiological conditions. Our data raise the possibility that competition for heme between commensal and pathogenic bacteria can influence bacterial colonisation, and therefore disease likelihood, and suggest that strains of H. haemolyticus that overproduce hemophilin might have therapeutic uses in reducing colonisation and subsequent opportunistic infection by NTHi.

biochemistry↗