Biology subjects

Newman, J. A.

Publications and source records attributed to Newman, J. A..

15 recordsLinked to original sources

bioRxiv2026

DNA-induced conformational changes in SPRTN relieve its auto-inhibitory effect on protease activity

The DNA-dependent metalloprotease SPRTN has emerged as a key enzyme in the proteolysis of DNA-protein crosslinks (DPCs), thereby protecting us against genome instability, accelerated ageing, and cancer. DNA and ubiquitin chains serve as the primary activator and catalyst of SPRTN proteolysis, respectively, but how they promote SPRTN activation and activity remains incompletely understood. To address this question, we developed a highly sensitive multi-turnover fluorescence resonance energy transfer (FRET) assay to monitor SPRTN proteolysis in real time. We found that the auto-cleaved N-terminal SPRTN fragment, comprising the metalloprotease domain (MPD), zinc-binding domain (ZBD), and basic region (BR), is highly stable, enzymatically active, and retains ubiquitin-dependent activation. Interestingly, the MPD alone exhibits basal intrinsic activity that is independent of both DNA activation and ubiquitin avidity effect. We show that ZBD and MPD together exert steric regulation: ZBD maintains MPD in an autoinhibited state, while MPD largely prevents ZBD from binding DNA. BR, together with DNA, is essential to relieve ZBD-mediated inhibition of MPD. Using a site-trapping approach, we demonstrate that the ZBD-BR-DNA trinity induces an open conformation of the SPRTN N-terminus in cis, thereby releasing autoinhibition. MPD and BR together restrict the DNA-binding stoichiometry of ZBD, enabling SPRTN to function efficiently in proximity to DNA despite its low abundance in vivo. Collectively, our work overturns the long-standing dogma that SPRTN autocleavage inactivates the enzyme and reveals how DNA-induced conformational changes in SPRTN fine-tune its protease activity, providing a prerequisite for subsequent ubiquitin activation and rapid proteolysis of DPCs.

biochemistry↗

bioRxiv2024

Wilting Wildflowers and Bummed-Out Bees: Climate Change Threatens U.S. State Symbols

Species designated as state symbols in the United States carry cultural importance and embody historical heritage. However, they are threatened by climate change and even face the risk of local or global extinction. The responses of these species to climate change have received little attention. In this study, we examine the effects of climate change on 64 state flowers and 68 state insects in the United States by employing correlative species distribution models (SDMs). We select a variety of commonly used SDM algorithms to construct an ensemble forecasting framework aimed at predicting the potential climatic habitats for each species under both historical (1981-2010) and future (2071-2100) climate scenarios (SSP1-2.6 and SSP5-8.5), and how these changes might influence the habitat suitability of flower and insect species within their symbolic states and across the United States. Our results indicate that 30 - 66% of state flowers and 18 - 51% of state insects are projected to experience substantial losses of climatically suitable habitat within their symbolic states. Under the high-emissions scenario (SSP5-8.5), ten state flowers and three state insects are likely to face local extinction by the 2080s. Although most of these species may find suitable habitats in other states, only two are projected to have such areas located adjacent to their current symbolic states, potentially limiting natural dispersal. Nationally, 85% of flower species and 71 - 79% of insect species are expected to shift their suitable habitat both poleward and uphill, with the magnitude of latitudinal and elevational shifts significantly greater under SSP5-8.5 than under SSP1-2.6. These findings highlight the vulnerability of culturally significant species to climate change and underscore the urgency of integrating climate adaptation into conservation planning. Proactive, forward-looking conservation and management strategies may be critical for preserving cultural heritage and maintaining ecosystem resilience.

ecology↗

bioRxiv2024

Molecular insights into the stimulation of SNM1A nuclease activity by CSB during interstrand crosslink processing

The Cockayne Syndrome B (CSB, ERCC6) protein, interacts with the exonuclease SNM1A during transcription-coupled DNA interstrand (ICL) repair, with CSB facilitating localisation of SNM1A to ICL damage. The functional and mechanistic details of this interaction in DNA repair, however, have not been defined. Here, we demonstrate that CSB enhances SNM1A resection through ICLs and identify a specific interaction between the winged-helix domain of CSB and the nuclease core of SNM1A that is crucial for recruitment and enhancement of nuclease degradation. Biochemical and single-molecule studies on DNA containing site-specific ICLs reveal that CSB increases the affinity of SNM1A to damaged DNA substrates and also alters the substrate conformation to enhance ICL processing by SNM1A. Notably, CSB was observed preferentially as a dimer when colocalised with SNM1A at ICLs, constrasting with its monomeric nature observed during repair initiation in classical transcription-coupled nucleotide excision repair. The combined results provide molecular insights into the basis of a direct contribution of CSB to a DNA repair reaction.

biochemistry↗

bioRxiv2024

Structures of the human transcription factor brachyury offer insights into DNA recognition, and identify small molecule binders for the development of degraders for cancer therapy

The transcription factor brachyury is a member of the T-Box family of transcription factors. It is active during embryogenesis and is required for the formation of the posterior mesoderm and the notochord in vertebrates. Aside from its role in embryogenesis, brachyury plays an essential role in tumour growth of the rare chordoma bone cancer and is implicated in other solid tumours. Given that brachyury is minimally expressed in healthy tissues, these findings suggest that brachyury is a potential therapeutic target in cancer. Unfortunately, as a ligandless transcription factor, brachyury has historically been considered undruggable. To investigate direct targeting of brachyury by small molecules, we initially determined the structure of human brachyury both in complex with its cognate DNA and in the absence of DNA. Analysis of these structures provided insights into brachyury DNA binding and the structural context of the G177D variant which is strongly associated with chordoma risk. We used these structures to perform a crystallographic fragment screen of brachyury and identify hotspot regions on numerous pockets on the brachyury surface. Finally, we have performed follow-up chemistry on fragment hits and describe the structure-based progression of a thiazole-containing chemical series. Excitingly, we have produced brachyury binders with low {micro}M potency that can serve as starting point for further medicinal chemistry efforts. These data show that brachyury is ligandable and provides an example of how crystallographic fragment screening may be used to find ligands to target protein classes that are traditionally difficult to address using other approaches.

molecular biology↗

bioRxiv2023

Geographic variation in evolutionary rescue in a predator-prey system under climate change: an example with aphids and ladybird beetles

Under climate change, species can adapt to changing environments through phenotypic plasticity and natural selection, and this kind of evolutionary adaptation can vary geographically. Most species distribution models (SDMs) are built upon the "Niche conservatism" assumption. They often ignore the possibility of "evolutionary rescue" and underestimate species future range limits under climate change. Here, we select aphids and ladybirds as model species and develop an eco-evolutionary model to explore evolutionary rescue in a predator-prey system under climate change. We model the adaptive change of species thermal performance, accounting for biotic interactions of unique life-history trait. Our results show that there is geographic variation in evolutionary rescue for ladybirds (the predator) across different locations in the United States, with ladybirds being more likely to be rescued from extinction in southeastern locations. The possibility of rescue is primarily influenced by the change in seasonality. Our findings also indicate the additive genetic variance of predators has a stronger influence on the phenotype evolution and population dynamics of both prey and predators, compared to the additive genetic variance of the prey. Our research emphasizes the importance of incorporating evolutionary adaptation when predicting species range shift under climate change. The eco-evolutionary model framework can be applied to study the effect of evolution on interacting species population abundance and geographic distribution under climate change.

evolutionary biology↗

bioRxiv2022

Predator-prey interactions in a warming world: the critical role of cold tolerance

Thermal tolerance mismatch within predator-prey systems may have pro-found effects on species population abundances and geographical distributions. To examine the generalized responses of a predator-prey system to climate change, we construct a biologically detailed stage-structured population dynamic model of interactions between ladybird beetles and aphids. We explore the models dynamics across the entire feasible parameter space of mean temperature and seasonality. Within this space, we explore different scenarios of predator and prey thermal tolerance mismatch to gain insight into how these thermal sensitivities affect the interacting species responses to climatic change. Our results indicate a predators cold tolerance has a larger effect on prey abundance than its heat tolerance. Mismatches between the predators and preys thermal tolerances also affect the species response to climate change. We identify three common patterns of species abundance across the feasible parameter space that relate to the type of thermal tolerance mismatches. Our study highlights the importance of understanding the complex interplay between climate change and species interactions.

ecology↗

bioRxiv2021

Allelopathic effects of Epichloë fungal endophytes: experiment and meta-analysis

Host-specific Epichloe spp.endophytic fungal symbionts of pooid grasses that produce herbivore-deterring alkaloids and alter the grass hosts metabolite and protein profiles. Early observations suggested that Epichloe may have negative allelopathic effects on neighbouring plant species, particularly Trifolium spp. clovers, but subsequent allelopathy tests produced variable results. We examined two hypotheses: (1) Epichloe strains differ in allelopathic effect, and (2) Epichloe allelopathy negatively affects other plant species. We performed a greenhouse experiment using root exudates from Lolium perenne L. hosting different E. festucae var. lolii (Latch, M.J. Chr. & Samuels) C.W. Bacon & Schardl strains to compare their allelopathic effects on native legumes and forbs. We then used meta-analysis to examine the evidence to date for allelopathic effects of Epichloe endophytes. We found little difference in effect among E. festucae var. lolii strains and very little evidence for negative allelopathic effects of Epichloe in cool-season grasses across a range of methodologies, target plant species, and response measures. Negative allelopathic effects were detected only for root hair measures, which were from a single study. Positive effects on biomass were found for some experimental subgroups, including legumes. However, the majority of response variables showed no evidence for Epichloe allelopathy. Although there is currently little evidence for negative Epichloe allelopathic effects, our meta-analysis identified several research gaps. Experiments testing the functional belowground effects of Epichloe presence may help to determine its effects on non-host plant performance via plant-soil feedbacks.

ecology↗

bioRxiv2021

Climate change and the complex dynamics of green spruce aphid--spruce plantation interactions

Aphids can have a significant impact on the growth and commercial yield of spruce plantations. Here we develop a mechanistic deterministic mathematical model for the dynamics of the green spruce aphid (Elatobium abietum Walker) growing on Sitka spruce (Picea sitchensis (Bong.) Carr.). These grow in a northern British climate in managed plantations, with planting, thinning and a 60-year rotation. Aphid infestation rarely kills the tree but can reduce growth by up to 55%. We used the Edinburgh Forest Model (efm) to simulate spruce tree growth. The aphid sub-model is described in detail in an appendix. The only environmental variable which impacts immediately on aphid dynamics is air temperature which varies diurnally and seasonally. The efm variables that are directly significant for the aphid are leaf area and phloem nitrogen and carbon. Aphid population predictions include dying out, annual, biennual and other complex patterns, including chaos. Predicted impacts on plantation yield of managed forests can be large and variable, as has been observed; they are also much affected by temperature, CO2 concentration and other climate variables. However increased CO2 concentration appears to ameliorate the severity of the effects of increasing temperatures coupled to worsening aphid infestations on plantation yield.

ecology↗

bioRxiv2021

Costs, Benefits, Parasites and Mutualists: The Use and Abuse of the Mutualism-Parasitism Continuum Concept for Epichloë Fungi

The species comprising the fungal endophyte genus Epichloe are symbionts of cool season grasses. About half the species in this genus are strictly vertically transmitted, and evolutionary theory suggests that these species must be mutualists. Nevertheless, Faeth and Sullivan (e.g. 2003) have argued that such vertically transmitted endophytes are "usually parasitic," and Muller and Krauss (2005) have argued that such vertically transmitted endophytes fall along a mutualism-parasitism continuum. These papers (and others) have caused confusion in the field. We used a systematic review to show that close to half of the published papers in this field incorrectly categorize the interaction. Here, we develop the argument that advantages and disadvantages are not the same things as mutualism and parasitism and that experimental evidence must be interpreted in the context of theory. We argue that, contra Faeth and Sullivan, it is highly unlikely that such strictly vertically transmitted endophytes can be parasites, and that, contra Muller and Krauss, it is inappropriate to apply the continuum concept to strictly vertically transmitted endophytes. We develop a mathematical model to support and illustrate our arguments. We use the model to clarify that parasitism requires some degree of horizontal transmission, and that while it is appropriate to use the continuum concept when referring to the genus as a whole, or to species that possess horizontal transmission, we argue that it is inappropriate to use the concept when referring to species that are strictly vertically transmitted.

ecology↗

bioRxiv2021

A FOXN1 mutation competitively displaces wild-type FOXN1 from higher order nuclear condensates to cause immunodeficiency

The transcription factor FOXN1 is a master regulator of thymic epithelial cell development and function. Here we demonstrate that FOXN1 expression is differentially regulated during organogenesis and participates in multi-molecular nuclear condensates essential for the factors transcriptional activity. FOXN1s C-terminal sequence regulates the diffusion velocity within these aggregates and modulates the binding to proximal gene regulatory regions. These dynamics are significantly altered in a patient with a mutant FOXN1 which is modified in its C-terminal sequence. This mutant is transcriptionally inactive and acts as a dominant negative factor displacing wild-type FOXN1 from condensates and causing athymia and severe lymphopenia in heterozygotes. Expression of the mutated mouse ortholog, selectively impairs mouse thymic epithelial cell (TEC) differentiation revealing a gene dose dependency for individual TEC subtypes. We have therefore identified the cause for a primary immunodeficiency disease and determined the mechanism by which this FOXN1 gain-of-function mutant mediates its dominant negative effect.

immunology↗

bioRxiv2021

Structure, Mechanism and Crystallographic fragment screening of the SARS-CoV-2 NSP13 helicase

The global COVID-19 pandemic is caused by the SARS-CoV-2 virus and has infected over 100 million and caused over 2 million fatalities worldwide at the point of writing. There is currently a lack of effective drugs to treat people infected with SARS-CoV-2. The SARS-CoV-2 Non-structural protein 13 (NSP13) is a superfamily1B helicase that has been identified as a possible target for anti-viral drugs due to its high sequence conservation and essential role in viral replication. In this study we present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and the non-hydrolysable ATP analogue (AMP-PNP). Comparisons of these structures reveal details of global and local conformational changes that are induced by nucleotide binding and hydrolysis and provide insights into the helicase mechanism and possible modes of inhibition. Structural analysis reveals two pockets on NSP13 that are classified as "druggable" and include one of the most conserved sites in the entire SARS-CoV-2 proteome. To identify possible starting points for anti-viral drug development we have performed a crystallographic fragment screen against SARS-CoV-2 NSP13 helicase. The fragment screen reveals 65 fragment hits across 52 datasets, with hot spots in pockets predicted to be of functional importance, including the druggable nucleotide and nucleic acid binding sites, opening the way to structure guided development of novel antiviral agents.

biochemistry↗

bioRxiv2021

Structural and mechanistic insights into the Artemis endonuclease and strategies for its inhibition

Artemis (DCLRE1C) is an endonuclease that plays a key role in development of B- and T-lymphocytes and in DNA double-strand break repair by non-homologous end-joining (NHEJ). Artemis is phosphorylated by DNA-PKcs and acts to open DNA hairpin intermediates generated during V(D)J and class-switch recombination. Consistently, Artemis deficiency leads to radiosensitive congenital severe immune deficiency (RS-SCID). Artemis belongs to a structural superfamily of nucleases that contain conserved metallo-{beta}-lactamase (MBL) and {beta}-CASP (CPSF-Artemis-SNM1-Pso2) domains. Here, we present crystal structures of the catalytic domain of wild type and variant forms of Artemis that cause RS-SCID Omenn syndrome. The truncated catalytic domain of the Artemis is a constitutively active enzyme that with similar activity to a phosphorylated full-length protein. Our structures help explain the basis of the predominantly endonucleolytic activity of Artemis, which contrast with the predominantly exonuclease activity of the closely related SNM1A and SNM1B nucleases. The structures also reveal a second metal binding site in its {beta}-CASP domain that is unique to Artemis. By combining our structural data that from a recently reported structure we were able model the interaction of Artemis with DNA substrates. Moreover, co-crystal structures with inhibitors indicate the potential for structure-guided development of inhibitors.

biochemistry↗

bioRxiv2020

Crystal Structure of the Werner's Syndrome Helicase

Werner syndrome helicase (WRN) plays important roles in multiple pathways of DNA repair and the maintenance of genome integrity. Recently, loss of WRN was identified as a strong synthetic lethal interaction for microsatellite instable (MSI) cancers making WRN a promising drug target. Yet, structural information for the helicase domain is lacking, which prevents structure-based design of drug molecules. In this study, we show that ATP binding and hydrolysis in the helicase domain are required for genome integrity and viability of MSI cancer cells. We then determined the crystal structure of an ADP bound form of the WRN helicase core at 2.2 [A] resolution. The structure features an atypical mode of nucleotide binding with extensive contacts formed by motif VI, which in turn defines the relative positioning of the two RecA like domains. The structure features a novel additional {beta}-hairpin in the second RecA and an unusual helical hairpin in the Zn2+ binding domain, and modelling DNA substrates based on existing RecQ DNA complexes suggests roles for these features in the binding of alternative DNA structures. We have further analysed possible interfaces formed from the interactions between the HRDC domain and the helicase core by NMR. Together, this study will facilitate the structure-based design of inhibitors against WRN helicase.

biochemistry↗

bioRxiv2018

The structural basis for forkhead box family specificity revealed by the crystal structure of human FOXN1 in complex with DNA

SignificanceFOXN1 is a transcription factor that is essential for the development of the thymus and the production of T-lymphocytes. It is a member of a large family of transcription factors which recognize DNA sequences through the conserved Forkhead (FH) domain. FOXN1 recognizes a DNA sequence that is different from the common consensus binding sequence of FH domains, although key binding residues are identical. We present crystal structures of the FH domain of FOXN1, free and DNA-bound, which shed light on the different binding specificities; the structure also revelas the basis of the immunocompromised nude mutation, as well as a preferential binding to non-methylated CpG motifs.\n\nAbstractFOXN1 is a member of the forkhead box (FOX) family of transcription factors, and plays an important role in thymic epithelial cell differentiation and function. FOXN1 mutations in humans and mice give rise to the \"nude\" phenotype which is marked by athymia. FOXN1 belongs to a subset of the FOX family that recognize an alternate consensus sequence (GACGC), which is different from the more widely-recognized canonical sequence consensus RYAAAYA. Here, we present the structure of FOXN1 in complex with DNA at 1.6 [A] resolution, in which the DNA sequence is recognised by a mixture of direct and water-mediated contacts provided by residues in an a-helix inserted in the DNA major groove (the recognition helix). Comparisons with other FOX family structures reveal that the canonical and alternate DNA sequences are bound in two distinct modes, with partially different registers for the protein DNA contacts. We identify a single alternate rotamer within the recognition helix itself as an important determinant of DNA specificity, and indicate sequence features in the recognition helix that could be used to predict the specificity of other FOX family members. Finally we demonstrate that FOXN1 has a significantly reduced affinity for DNA containing 5-methylcytosine, which may have implications for the role of FOXN1 in thymic senescence.

biochemistry↗

bioRxiv2018

A Ubiquitin-Binding Domain that Does Not Bind Ubiquitin

Ubiquitylation, the post-translational linkage of ubiquitin moieties to lysines in target proteins, helps regulate a myriad of biological processes. Ubiquitin, and sometimes ubiquitin-homology domains, are recognized by ubiquitin-binding domains, including CUE domains. CUE domains are thus generally thought to function exclusively by mediating interactions with ubiquitylated proteins. The chromatin remodeler, SMARCAD1, interacts with KAP1, a transcriptional corepressor. We show that the SMARCAD1-KAP1 interaction is direct and involves the first SMARCAD1 CUE domain (CUE1) and the RBCC domain of KAP1. A structural model of the minimal KAP1 RBCC-SMARCAD1 CUE1 complex based on X-ray crystallography analysis is presented. Remarkably, the CUE1 domain, which resembles a canonical CUE domain, recognizes 2 clusters of exposed hydrophobic residues on KAP1, but these are presented in the context of a coiled-coil domain, not in a structure resembling ubiquitin. Together, these data challenge the well-established dogma that CUE domains exclusively recognize the ubiquitin-fold.

biochemistry↗