Biology subjects

Taylor, T.

Publications and source records attributed to Taylor, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Time-Resolved Single-Cell Atlas Reveals Early Endothelial Activation and Stage-Dependent Immune-Stromal Communication in HFpEF

BackgroundHeart failure with preserved ejection fraction (HFpEF) is a heterogeneous syndrome associated with metabolic stress, hypertension, systemic inflammation, and microvascular dysfunction. Early cell-type-specific events and intercellular communication programs that accompany disease onset and progression remain poorly defined. MethodsWe performed a longitudinal study of HFpEF progression in high-fat diet (HFD)+L-NAME mice at control/baseline (0 weeks, 0w/Ctrl), early (1w), intermediate (4w), and established (8w) stages. Metabolic, hemodynamic, exercise, echocardiographic, and single-cardiomyocyte function were assessed. Cardiac non-cardiomyocytes (non-CMs) were profiled by single-cell RNA sequencing (scRNA-seq), with bulk RNA-seq for tissue-level comparison. Endothelial remodeling was assessed in an L-NAME-independent HFD plus mild transverse aortic constriction model (HFD+mTAC) and a published human HFpEF single-nucleus RNA-seq cohort. An endothelial-macrophage adhesion assay tested whether HFpEF-mimic stress promotes endothelial activation and macrophage adhesion. ResultsIn the HFD+L-NAME model, metabolic dysfunction, hypertension, reduced exercise tolerance, abnormal diastolic filling with preserved ejection fraction, and altered cardiomyocyte calcium handling were detected by 1w and persisted through 8w. Bulk RNA-seq showed progressive remodeling, with limited change between 8w and 12w, guiding scRNA-seq timepoint selection. scRNA-seq of 94,848 cardiac non-CMs identified nine major populations with stage-dependent remodeling. Endothelial cells (ECs) were recovered in high proportion and showed an early, pronounced transcriptional response, with inflammatory, adhesion, interferon-response, migratory, and vascular-remodeling programs emerging by 1w. Related EC activation signatures were observed in HFD+mTAC and human HFpEF data. Functionally, HFpEF-mimic stress increased adhesion and chemokine expression in human ECs and enhanced macrophage adhesion. Fibroblast matrix remodeling occurred at later stages, while macrophages progressively shifted toward inflammatory states. CellChat suggested stage-dependent communication remodeling from early endothelial-immune interactions toward later macrophage-fibroblast crosstalk. ConclusionTime-resolved scRNA-seq reveals coordinated, stage-dependent remodeling of the cardiac microvascular and interstitial microenvironment during HFpEF progression. Early endothelial activation emerges before later fibroblast matrix remodeling and inflammatory macrophage remodeling, identifying candidate cell states and signaling pathways for future mechanistic investigation. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIThis study provides a time-resolved single-cell atlas of the cardiac non-cardiomyocyte compartment across baseline, early, intermediate, and established stages of HFpEF progression, rather than a single late-stage snapshot. C_LIO_LIEndothelial cells exhibit early inflammatory, adhesion, interferon-response, and vascular-remodeling programs within the first week of disease, preceding the later predominance of fibroblast matrix remodeling and inflammatory macrophage remodeling. C_LIO_LIThis endothelial activation signature is supported across two mechanistically distinct HFpEF mouse models and aligns with endothelial inflammatory and vascular-remodeling programs in human HFpEF myocardium, supporting its translational relevance. C_LI What Are the Clinical Implications?O_LIEarly endothelial activation may represent a targetable stage of HFpEF pathogenesis that arises before more established structural and fibrotic remodeling. C_LIO_LITherapeutic strategies aimed at limiting endothelial inflammatory activation or endothelial-immune interactions may help attenuate downstream vascular, immune, and stromal remodeling in HFpEF. C_LIO_LIThese findings provide a preclinical foundation for future longitudinal human studies testing whether early endothelial activation can serve as a biomarker, therapeutic target, or disease-staging feature in HFpEF. C_LI

cell biology↗

bioRxiv2026

RADIX: a deep learning framework that maps root barriers across species and reveals genetic and environmental contributions

Root anatomical barriers, including the suberized and lignified walls of the endodermis and exodermis, and cortical aerenchyma, regulate water and nutrient transport, gas exchange, and rhizosphere interaction. Their adaptive function places them as an important target for breeding environmentally resilient plant species. Quantifying these structures at high resolution is a manual bottleneck that limits experimental scale. We present RADIX (Root Anatomy Deep- learning Image segmentation across species and platforms), a framework that adapts a large self-supervised vision-transformer foundation encoder (DINOv3), pre-trained on billions of natural images, to root anatomy by fine-tuning its encoder with a dense-prediction-transformer decoder. Transferring these general-purpose vision encoders to a specialized biological domain with a high-quality annotated dataset is what allows RADIX to generalize across species and imaging platforms. We train and evaluate it on the first expert-annotated benchmark of root anatomical structures at scale, comprising 1,695 high-quality fluorescence images spanning 17 monocot and dicot species, six anatomical structures, and three imaging platforms. RADIX segments all six structures at inter-annotator-level accuracy and generalizes to unseen species, genotypes, growth conditions, and an imaging platform from an independent laboratory. A single unified model surpasses monocot- and dicot-specialist models without sacrificing in-group accuracy. Predicted masks yield aerenchyma and suberin/lignin measurements matching expert annotation at [~]1.2 s per image with a single GPU, reducing weeks of manual analysis to minutes. Applying RADIX across genotypes, microbial treatments, and growth systems, we show that these cell type features form a coordinated, multidimensional, and context-dependent system shaped by genetic and environmental factors.

plant biology↗

bioRxiv2025

Disentangling the importance of microbiological and physicochemical properties of Ethiopian field soils for the Striga seed bank and sorghum infestations

Striga hermonthica (Striga) is a parasitic weed that severely affects sorghum yields in sub-Saharan Africa. Recent studies highlighted the soil microbiomes potential to suppress Striga through interference with specific stages in its life cycle. In this study, meta-analysis of 48 Ethiopian field soils revealed that microbial communities and their interactions with soil physico-chemical properties correlated with Striga field occurrence. Striga infestation of sorghum and soil seedbank levels were negatively correlated with clay content and the nutrients potassium, sulfur, calcium, and carbon. Microbiome analyses indicated that fungal communities were more responsive than bacteria to changes in Striga infestation and seedbank levels, with distinct microbial compositions even in soils where Striga was not detected. Specific fungal and bacterial genera showed both positive and negative correlations with Striga measures, but patterns rarely held across taxonomic levels, highlighting the complexity of microbiome-Striga interactions. To begin to validate these correlations, we tested an isolate from the fungal genus Neocosmospora, which negatively correlated with the Striga seedbank, and showed that this isolate promotes Striga seed germination in vitro, suggesting potential for biological control of Striga. The data and analysis methods are integrated and shared in a public Shiny App for broader analysis and continued research on soil-Striga interactions.

plant biology↗

bioRxiv2025

Enhancers mediate euchromatin hopping at chromatin contact points

Enhancer-mediated gene activation involves the recruitment of chromatin modifiers and RNA polymerase to target promoters, but it is unknown if enhancers influence chromatin beyond their target genes. Euchromatin and heterochromatin associated histone modifications separate the genome into opposing nuclear compartments. Whereas heterochromatin marks are known to spread from one modified nucleosome to another, no such ability has been ascribed to euchromatin. Using mono-allelic enhancer deletions, native ChIP-seq, and an engineered interaction between an enhancer and transcriptionally inert DNA, we show that enhancers mediate the acquisition of euchromatin features at distal regions through chromatin looping. We term this phenomenon euchromatin hopping and found it occurring on average [~]270kb bidirectionally from enhancers, redefining our understanding of enhancer-mediated chromatin architecture with implications on enhancer identification using chromatin features. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/674255v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@12c6e9aorg.highwire.dtl.DTLVardef@1165feorg.highwire.dtl.DTLVardef@3f08c8org.highwire.dtl.DTLVardef@598bd3_HPS_FORMAT_FIGEXP M_FIG C_FIG Euchromatin hopping modelFigure showing the proposed euchromatin hopping model. TFs recognize and bind to their binding sites in an active enhancer region. Upon activation enhancers recruit coactivators and RNAPII forming a condensate that supports gene activation. After an abundance of transcriptional machinery and coactivators are recruited, adjacent TF bound sites acquire euchromatin features through physical proximity to the active compartment, we call these regions "bystanders". Upon enhancer deletion, condensate formation is lost and active euchromatin marks are not acquired at the gene promoter or other enhancer chromatin contacts. TFs are displayed in yellow, coactivators in green, RNAPII in pink, and histone modifications in red.

molecular biology↗

bioRxiv2024

THE CELLS ARE ALL-RIGHT: Regulation of the Lefty genes by separate enhancers in mouse embryonic stem cells

Enhancers play a critical role in regulating precise gene expression patterns essential for development and cellular identity; however, how gene-enhancer specificity is encoded within the genome is not clearly defined. To investigate how this specificity arises within topologically associated domains (TAD), we performed allele-specific genome editing of sequences surrounding the Lefty1 and Lefty2 paralogs in mouse embryonic stem cells. The Lefty genes arose from a tandem duplication event and these genes interact with each other in chromosome conformation capture assays which place these genes within the same TAD. Despite their physical proximity, we demonstrate that these genes are primarily regulated by separate enhancer elements. Through CRISPR-Cas9 mediated deletions to remove the intervening chromatin between the Lefty genes, we reveal a distance-dependent dosage effect of the Lefty2 enhancer on Lefty1 expression. These findings indicate a potential role for both discrete CTCF bound regions of the genome and chromatin distance in insulating gene expression domains in the Lefty locus in the absence of architectural insulation.

genomics↗

bioRxiv2023

Competition between transcription and loop extrusion modulates promoter and enhancer dynamics

The spatiotemporal configuration of genes with distal regulatory elements, and the impact of chromatin mobility on transcription, remain unclear. Loop extrusion is an attractive model for bringing genetic elements together, but how this functionally interacts with transcription is also largely unknown. We combine live tracking of genomic loci and nascent transcripts with molecular dynamics simulations to assess the 4D arrangement of the Sox2 gene and its enhancer, in response to a battery of perturbations. We find that alterations in chromatin mobility, not promoter-enhancer distance, is more informative about transcriptional status. Active elements display more constrained mobility, consistent with confinement within specialized nuclear sites, and alterations in enhancer mobility distinguish poised from transcribing alleles. Strikingly, we find that whereas loop extrusion and transcription factor-mediated clustering contribute to promoter-enhancer proximity, they have antagonistic effects on chromatin dynamics. This provides an experimental framework for the underappreciated role of chromatin dynamics in genome regulation.

cell biology↗

bioRxiv2022

The soil microbiome reduces Striga infection of sorghum by modulation of host-derived signaling molecules and root development

Sorghum bicolor is one of the most important cereals in the world and a staple crop for smallholder famers in sub-Saharan Africa. However approximately 20% of sorghum yield is annually lost on the African continent due to infestation with the root parasitic weed Striga hermonthica. Existing Striga management strategies often show an inconsistent to low efficacy. Hence, novel and integrated approaches are needed as an alternative strategy. Here, we demonstrate that the soil microbiome suppresses Striga infection in sorghum. We associate this suppression with microbiome-mediated induction of root endodermal suberization and aerenchyma formation, and depletion of haustorium inducing factors (HIFs), root exudate compounds that are critical for the initial stages of Striga infection. We further identify microbial taxa associated with reduced Striga infection with concomitant changes in root cellular anatomy and differentiation as well as HIF degradation. Our study describes novel microbiome-mediated mechanisms of Striga suppression, encompassing repression of haustorium formation and induction of physical barriers in the host root tissue. These findings open new avenues to broaden the effectiveness of Striga management practices.

plant biology↗

bioRxiv2022

Multi-species analysis of inflammatory response elements reveals ancient and lineage-specific contributions of transposable elements to NF-κB binding

Transposable elements (TEs) provide a source of transcription factor binding sites that can rewire conserved gene regulatory networks. NF-{kappa}B is an evolutionary conserved transcription factor complex primarily involved in innate immunity and inflammation. The extent to which TEs have contributed to NF-{kappa}B responses during mammalian evolution is not well established. Here we performed a multi-species analysis of TEs bound by the NF-{kappa}B subunit RELA (also known as p65) in response to the proinflammatory cytokine TNF. By comparing RELA ChIP-seq data from TNF-stimulated primary aortic endothelial cells isolated from human, mouse and cow, we found that 55 TE subfamilies were associated with RELA bound regions. These RELA-bound transposons possess active epigenetic features and reside near TNF-responsive genes. A prominent example of lineage-specific contribution of transposons comes from the bovine SINE subfamilies Bov-tA1/2/3 which collectively contributed over 14,000 RELA bound regions in cow. By comparing RELA binding data across species, we also found several examples of RELA motif-bearing TEs that colonized the genome prior to the divergence of the three species and contributed to species-specific RELA binding. For example, we found human RELA bound MER81 instances were enriched for the interferon gamma pathway and demonstrated that one RELA bound MER81 element can control the TNF-induced expression of Interferon Gamma Receptor 2 (IFNGR2). Using ancestral reconstructions, we found that RELA containing MER81 instances rapidly decayed during early primate evolution (> 50 million years ago (MYA)) before stabilizing since the separation of Old World monkeys (< 50 MYA). Taken together, our results suggest ancient and lineage-specific transposon subfamilies contributed to mammalian NF-{kappa}B regulatory networks.

genomics↗

bioRxiv2022

A CREBZF/Zhangfei isoform activates CHOP during prolonged cellular stress

The basic leucine zipper transcription factor CREBZF (Zhangfei or ZF) was identified through its interaction with Herpes Simplex Virus-1 related cellular protein HCF-1. CREBZF has been implicated in cellular stress responses through its interaction with other proteins, such as CREB3/Luman and ATF4. Here we investigated the production of four CREBZF isoforms, which arise from translational initiation of a downstream AUG at codon 83, and mRNA alternative splicing that adds an IFFFR pentapeptidyl tail to the C-terminus. We found that in addition to transcriptional activation, the short-tailed CREBZF (stZF) isoform was specifically induced by prolonged ER stress treatment. This stZF isoform is a potent transcriptional activator of the pro-apoptotic protein CHOP. Overexpression of stZF activates transcription of CHOP through a CCAAT enhancer binding protein (C/EBP)-ATF site, and promotes apoptosis. We propose that 1) CREBZF is a key component of the Integrated Stress Response (ISR); 2) stZF is essential for the role of CREBZF in inducing CHOP and promoting cell death upon prolonged cellular stress.

molecular biology↗

bioRxiv2022

Transcriptional regulation and chromatin architecture maintenance are decoupled modular functions at the Sox2 locus

How distal regulatory elements control gene transcription and chromatin topology is not clearly defined, yet these processes are closely linked in lineage specification during development. Through allele-specific genome editing and chromatin interaction analyses of the Sox2 locus in mouse embryonic stem cells, we found a striking disconnection between transcriptional control and chromatin architecture. We trace nearly all Sox2 transcriptional activation to a small number of key transcription factor binding sites, whose deletions have no effect on promoter-enhancer interaction frequencies or topological domain organization. Local chromatin architecture maintenance, including at the topologically associating domain (TAD) boundary downstream of the Sox2 enhancer, is widely distributed over multiple transcription factor-bound regions and maintained in a CTCF-independent manner. Furthermore, disruption of promoter-enhancer interactions by ectopic chromatin loop formation has no effect on Sox2 expression. These findings indicate that many transcription factors are involved in modulating chromatin architecture independently of CTCF.

genetics↗

bioRxiv2021

The humanized nanobody RBD-1-2G tolerates the spike N501Y mutation to neutralize SARS-CoV-2

Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants.

synthetic biology↗

bioRxiv2021

Nanopore sequencing methods detect cell-free DNA associated with minimal residual disease and central nervous system infiltration in pediatric Acute Lymphoblastic Leukemia

Acute Lymphoblastic Leukemia (ALL) patients that are positive for minimal residual disease (MRD) after therapy or have leukemic infiltration into the central nervous system (CNS) are considered high risk and receive intensive chemotherapy regimens. Current methods to diagnose MRD and CNS infiltration rely on detecting leukemic cells in patient samples using pathology, flow cytometry, or next-generation sequencing. However, leukemic blasts may persist in the patient but not be physically present in bone marrow biopsy or biofluid sample, leading to inaccurate or delayed patient diagnosis. We have developed a nanopore sequencing workflow to detect B-ALL-associated cell-free DNA (cfDNA) in blood and cerebrospinal fluid (CSF) samples. Quantitation of B-cell specific VDJ recombination events in cfDNA samples defined B-ALL clonal heterogeneity. This workflow allowed us to track the response of individual B-ALL clones throughout treatment. Detection of cfDNA also predicted the clinical diagnosis of MRD and CNS disease. Importantly, we identified patients diagnosed as CNS negative who had low B-cell derived cell-free DNA levels in their CSF sample that correlated with B-cell clones present in the bone marrow. These data suggest that cfDNA assays may be useful in detecting the presence of ALL in the patient even when blasts are not in the biofluid sample. Nanopore analysis of cell-free DNA is a simple, rapid, and inexpensive assay that can serve as a valuable complement to traditional clinical diagnostic approaches for ALL. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=50 SRC="FIGDIR/small/462067v2_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@1373bd3org.highwire.dtl.DTLVardef@138b2e3org.highwire.dtl.DTLVardef@168661forg.highwire.dtl.DTLVardef@1088626_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2021

Cytolytic memory CD4+ T cell clonotypes are expanded during Plasmodium falciparum infection

Plasmodium falciparum (Pf) malaria causes high rates of morbidity and mortality and lacks a sufficiently effective vaccine. Clinical immunity develops in residents of malaria endemic regions which confers reduced clinical symptoms during infection and protection against severe disease. We hypothesized that understanding the immune mechanisms of clinical immunity could inform vaccine design to improve efficacy. We compared the peripheral blood cellular and humoral immune responses during a mild episode of Pf malaria infection. Participants were classified as either clinically susceptible or clinically protected, based on the number of recurrent clinical infections over an 18-month longitudinal study in a malaria endemic region in Malawi. Susceptible participants had three or more recurrent clinical episodes while clinically immune individuals had one or none. Protected participants exhibited higher plasma immunoglobulin G (IgG) breadth and titers against Pf antigens, and greater antibody (Ab)-dependent Pf opsonization compared to susceptible participants. Using high dimensional mass cytometry (CyTOF), spectral flow cytometry and single-cell transcriptomic analyses, we identified expanded memory CD4+ T cell clones sharing identical T cell receptor clonotypes in the blood of protected participants during malaria infection. These cells express a strong cytolytic T helper 1 effector program with transcripts encoding granzymes (A, B, H, M), granulysin, NKG7 and the Zeb2 master transcriptional regulator of terminally differentiated effector T cells. Memory CD4+ T cells expressing Zeb2+ were CD39hiTIGIThi and expressed multiple chemotactic and checkpoint inhibitory receptors, although the cellular levels of several of these receptors were reduced in protected compared to susceptible individuals. We propose that clonally expanded Zeb2+ cytolytic memory CD4+ Th1 cells could represent essential contributors to clinical immunity against Pf malaria. One Sentence SummaryA population of cytolytic memory CD4+ T cells is clonally expanded in patients with Plasmodium falciparum malaria and has reduced chemotactic and inhibitory receptor expression in patients with naturally acquired clinical malaria immunity.

immunology↗

bioRxiv2021

Characterization of growth and development of sorghum genotypes with differential susceptibility to Striga hermonthica

Variation in strigolactone composition in sorghum root exudates underlies its resistance to the parasitic weed, Striga hermonthica. Root exudates of the Striga susceptible variety Shanqui Red (SQR) contain primarily 5-deoxystrigol, which has a high efficiency of inducing Striga germination. SRN39 roots primarily exude orobanchol, leading to reduced Striga germination and making this variety resistant to Striga. This structural diversity in exuded strigolactones is determined by the polymorphism in the LGS1 (LOW GERMINATION STIMULANT 1) locus. Yet, the effects of the lgs1 mutation as well as the consequences of the vast genetic diversity between SQR and SRN39 have not been addressed in terms of growth and development. Here, we demonstrate additional consequences of LGS1 loss-of-function by phenotypic and molecular characterization. A suite of genes related to metabolism was differentially expressed between SQR and SRN39. Increased levels of gibberellin precursors in SRN39 were accompanied with its slower growth rate and developmental delay and we observed an overall increased SRN39 biomass. The slow-down in growth and differences in transcriptome profiles of SRN39 were strongly associated with plant age. Additionally, analyses of multiple LGS1 loss-of-function genotypes indicated that strigolactone stereochemistry influences root system architecture. In summary, we demonstrate that the consequences of the lgs1 mutation reach further than the changes in strigolactone profile in the root exudate and translate into alterations in growth and development. HighlightSRN39 and Shanqui Red are models for sorghum genotypes that are resistant and susceptible, respectively, to Striga hermonthica. Additional differences in plant growth, development, and hormone abundance should be considered when assessing Striga tolerance.

plant biology↗

bioRxiv2020

Improved production of SARS-CoV-2 spike receptor-binding domain (RBD) for serology assays

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a commonly used antigen for serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Different versions of the RBD protein have been developed and utilized in assays, with higher sensitivity attributed to particular forms of the protein. To improve the yield of these high-sensitivity forms of RBD and support the increased demand for this antigen in serology assays, we investigated several protein expression variables including DNA elements such as promoters and signal peptides, cell culture expression parameters, and purification processes. Through this investigation, we developed a simplified and robust purification strategy that consistently resulted in high levels of the high-sensitivity form of RBD and demonstrated that a carboxyterminal tag is responsible for the increased sensitivity in the ELISA. These improved reagents and processes produce high-quality proteins which are functional in serology assays and can be used to investigate seropositivity to SARS-CoV-2 infection. Highlights: O_LIImproved yields of SARS-CoV-2 spike RBD through modification of DNA constructs and purification parameters C_LIO_LITwo versions of RBD show different sensitivity in serology assays C_LIO_LIYields of greater than 50 mg/l obtained under optimal conditions C_LIO_LIMagnetic bead purification technology improves throughput of protein production C_LI

biochemistry↗

bioRxiv2020

REGN-COV2 antibody cocktail prevents and treats SARS-CoV-2 infection in rhesus macaques and hamsters

An urgent global quest for effective therapies to prevent and treat COVID-19 disease is ongoing. We previously described REGN-COV2, a cocktail of two potent neutralizing antibodies (REGN10987+REGN10933) targeting non-overlapping epitopes on the SARS-CoV-2 spike protein. In this report, we evaluate the in vivo efficacy of this antibody cocktail in both rhesus macaques and golden hamsters and demonstrate that REGN-COV-2 can greatly reduce virus load in lower and upper airway and decrease virus induced pathological sequalae when administered prophylactically or therapeutically. Our results provide evidence of the therapeutic potential of this antibody cocktail.

microbiology↗

bioRxiv2020

KRAS interaction with RAF1 RAS-binding domain and cysteine-rich domain provides insights into RAS-mediated RAF activation

A vital first step of RAF activation involves binding to active RAS, resulting in the recruitment of RAF to the plasma membrane. To understand the molecular details of RAS-RAF interaction, we solved crystal structures of wild-type and oncogenic mutants of KRAS complexed with the RAS-binding domain (RBD) and the membrane-interacting cysteine-rich domain (CRD) from the N-terminal regulatory region of RAF1. Our structures revealed that RBD and CRD interact with each other to form one structural entity in which both RBD and CRD interact extensively with KRAS. Mutation at the KRAS-CRD interface resulted in a significant reduction in RAF1 activation despite only a modest decrease in binding affinity. Combining our structures and published data, we provide a model of RAS-RAF complexation at the membrane, and molecular insights into RAS-RAF interaction during the process of RAS-mediated RAF activation.

biophysics↗

bioRxiv2020

RAS-inhibiting biologics identify and probe druggable pockets including an SII-α3 allosteric site.

RAS mutations are the most common oncogenic drivers across human cancers, but there remains a paucity of clinically-validated pharmacological inhibitors of RAS, as druggable pockets have proven difficult to identify. We have identified two RAS-binding Affimer proteins, K3 and K6, that inhibit nucleotide exchange and downstream signalling pathways with distinct isoform and mutant profiles. Affimer K6 is the first biologic to bind in the SI/SII pocket, whilst Affimer K3 is the first non-covalent inhibitor of the SII region, revealing a novel RAS conformer with a large, druggable SII/3 pocket. This work demonstrates the potential of using biologics with small interface surfaces to select novel druggable conformations in conjunction with pharmacophore identification for hard-to-drug proteins.

molecular biology↗