Biology subjects

Cross, S. J.

Publications and source records attributed to Cross, S. J..

11 recordsLinked to original sources

bioRxiv2026

Expanding all-α-helical protein space through rational computational design

De novo protein design is advancing rapidly1,2. This is being driven by AI to generate protein backbones, sequences, and structural models3-7. As a result, de novo designed proteins are becoming larger and more complex8-10, and increasingly explore new protein structures11,12. By contrast, natural proteins have evolved structural and functional complexity by modular combination of recurring protein domains13. Approximately 25% of these natural domains are mostly -helical structures14. Here we show how these can be expanded using rational computational design. Following the domain classification scheme CATH15, we build complex all- de novo proteins hierarchically using sequence-to-structure relationships for helix-helix interactions, systematic rules to connect helices, computational tools to design loops, and in silico evaluation. The pipeline starts with a target architecture of free-standing helices. These are connected into a topology by considering local arrangements of helical bundles using understood sequence-to-structure relationships for helix packing. Single-chain sequences are completed using template- and AI-based methods. Finally, AlphaFold models are assessed to give small numbers of designs for experimental validation. We test 31 designs for 14 different architectures and 25 topologies. 75% of these express as stable, monomeric, water-soluble proteins; and >30% yield X-ray crystal structures matching the designs to atomic accuracy and with new-to-nature structures. Finally, several of the scaffolds are functionalised through one-shot designs to deliver ion, small-molecule and protein binders.

synthetic biology↗

bioRxiv2026

Loss of lmx1ba drives premature osteoarthritis through disruption of skeletal homeostasis

Osteoarthritis is increasingly recognised as a disease of failed integration across the whole joint unit; however, the mechanisms that co-ordinate tissue integrity from development through to adult homeostasis remain largely unresolved. The LIM-homeodomain transcription factor LMX1B is a key determinant of embryonic skeletal patterning, but how it functions to regulate skeletal integrity in the mature skeleton is unknown. Recently, LMX1B was identified as a key driver of osteoarthritis. Here we show that loss of lmx1ba in zebrafish causes premature and progressive severe osteoarthritic pathology in adult spines and jaws, despite largely normal early skeletal patterning, revealing a conserved and continuous requirement for lmx1ba in joint maintenance beyond development. At a cellular level, loss of lmx1ba decouples osteoblast and osteoclast-mediated remodelling leading to bone overgrowth, heterogeneity of bone properties causing increased incidence of spontaneous fractures, and progressive abnormalities in spine morphology. In parallel, we observe degeneration of the intervertebral disc and dysregulation of the proteome and glycosaminoglycans indicative of disrupted extracellular matrix and a breakdown of the coordinated regulation of hard and soft tissue interfaces, which at the organismal level leads to altered joint performance. Notably, degeneration is restricted to mobile joints, and is not observed in cranial sutures, demonstrating a selective requirement for lmx1ba in mechanically active tissues. These changes are consistent with a model of spatially disrupted matrix properties that, under cyclic loading, promote progressive tissue damage. Our findings support a model in which continued expression of LMX1B in adulthood is required to maintain joint structures throughout life.

physiology↗

bioRxiv2025

Biomolecular condensation using de novo designed globular proteins

De novo protein design is advancing rapidly, but many targets remain inaccessible to current AI-based tools. Here we describe de novo designed globular domains that drive biomolecular condensation. Starting from a water-soluble, monomeric protein, we make variants with the same amino-acid composition but different surface-charge distributions: one with large patches of surface charge, and another with a more-homogeneous charge distribution. The individual domains form stable and discrete structures in solution, with the large-patch variant exhibiting more-attractive interprotein interactions. Next, two copies of each variant are joined with disordered linkers to generate dumbbell-like proteins. When expressed in eukaryotic cells, the large-patch variant forms intracellular puncta, whereas that with small patches does not. The assemblies are dynamic, liquid condensates in vitro and in cells. The structured domains facilitate functionalisation: we introduce fluorophore-binding sites to visualise fluorescent condensates directly in cells without a GFP reporter; and we manipulate the condensates using motor proteins.

synthetic biology↗

bioRxiv2025

Individual Staphylococcus aureus SauUSI restriction endonuclease motors fragment methylated DNA

SauUSI is a dimeric, ATP-dependent Type IV restriction enzyme that protects Staphylococcus aureus by cleaving non-self DNA containing 5-methylcytosine or 5-hydroxymethylcytosine. Using biophysical, single-molecule, and nanopore sequencing methods, we show that 5-methylcytosine recognition first induces ATP-driven unidirectional translocation by one helicase-like subunit, displacing its target recognition domain (TRD). The partner TRD can then bind the liberated modified site, stabilizing a growing DNA loop. On symmetrically methylated DNA, both subunits engage in bidirectional loop translocation. Cleavage is triggered by binding a distal methylated site, by preferred DNA sequences, or upon reaching a DNA end. Interactions with other SauUSI dimers or protein roadblocks affect cleavage site distributions but are not required for nuclease activation. While the first cleavages principally generate blunt-ends or one-nucleotide 3' overhangs, multiple binding-translocation cycles by individual enzymes ultimately shred the modified non-self DNA, neutralizing its threat.

microbiology↗

bioRxiv2023

Five inhibitory receptors display distinct vesicular distributions in T cells

T cells can express multiple inhibitory receptors. Upon induction of T cell exhaustion in response to persistent antigen, prominently in the anti-tumor immune response, many are expressed simultaneously. Key inhibitory receptors are CTLA-4, PD-1, LAG3, TIM3 and TIGIT, as investigated here. These receptors are important as central therapeutic targets in cancer immunotherapy. Inhibitory receptors are not constitutively expressed on the cell surface, but substantial fractions reside in intracellular vesicular structures. It remains unresolved to which extent the subcellular localization of different inhibitory receptors is distinct. Using quantitative imaging of subcellular distributions and plasma membrane insertion as complemented by proximity proteomics and a biochemical analysis of the association of the inhibitory receptors with trafficking adaptors, the subcellular distributions of the five inhibitory receptors were discrete. The distribution of CTLA-4 was most distinct with preferential association with lysosomal-derived vesicles and the sorting nexin 1/2/5/6 transport machinery. With a lack of evidence for the existence of specific vesicle subtypes to explain divergent inhibitory receptor distributions, we suggest that such distributions are driven by divergent trafficking through an overlapping joint set of vesicular structures. This extensive characterization of the subcellular localization of five inhibitory receptors in relation to each other lays the foundation for the molecular investigation of their trafficking and its therapeutic exploitation.

immunology↗

bioRxiv2023

ModularImageAnalysis (MIA): Assembly of modularised image and object analysis workflows in ImageJ

ModularImageAnalysis (MIA) is an ImageJ plugin providing a code-free graphical environment in which complex automated analysis workflows can be constructed and distributed. The near 200 included modules cover all stages of a typical analysis workflow, from image loading through image processing, object detection, extraction of measurements, measurement-based filtering, visualisation, and data exporting. MIA provides out-of-the-box compatibility with many advanced image processing plugins for ImageJ including Bio-Formats, DeepImageJ, MorphoLibJ and TrackMate, allowing these tools and their outputs to be directly incorporated into analysis workflows. By default, modules support spatially calibrated 5D images, meaning measurements can be acquired in both pixel and calibrated units. A hierarchical object relationship model allows for both parent-child (one-to-many) and partner (many-to-many) relationships to be established. These relationships underpin MIAs ability to track objects through time, represent complex spatial relationships (e.g. skeletons) and measure object distributions (e.g. count puncta per cell). MIA features dual graphical interfaces: the "editing view" offers access to the full list of modules and parameters in the workflow, while the simplified "processing view" can be configured to display only a focused subset of controls. All workflows are batch-enabled by default, with image files within a specified folder being processed automatically and exported to a single spreadsheet. Beyond the included modules, functionality can be extended both internally, through integration with the ImageJ scripting interface, and externally, by developing third-party Java modules that extend the core MIA framework. Here we describe the design and functionality of MIA in the context of a series of real-world example analyses.

bioinformatics↗

bioRxiv2023

Assembling membraneless organelles from de novo designed proteins

Recent advances in de novo protein design have delivered a diversity of discrete de novo protein structures and complexes. A new challenge for the field is to use these designs directly in cells to intervene in biological process and augment natural systems. The bottom-up design of self-assembled objects like microcompartments and membraneless organelles is one such challenge, which also presents opportunities for chemical and synthetic biology. Here, we describe the design of genetically encoded polypeptides that form membraneless organelles in Escherichia coli (E. coli). To do this, we combine de novo -helical sequences, intrinsically disordered linkers, and client proteins in single-polypeptide constructs. We tailor the properties of the helical regions to shift protein assembly from diffusion-limited assemblies to dynamic condensates. The designs are characterised in cells and in vitro using biophysical and soft-matter physics methods. Finally, we use the designed polypeptide to co-compartmentalise a functional enzyme pair in E. coli.

synthetic biology↗

bioRxiv2022

Multiomic Approach Characterises the Neuroprotective Role of Retromer in Regulating Lysosomal Health

Retromer controls cellular homeostasis through regulating integral membrane protein sorting and transport and by controlling late-stage maturation of the endo-lysosomal network. Retromer dysfunction, which is linked to neurodegenerative disorders including Parkinsons and Alzheimers diseases, manifests in complex cellular phenotypes, though the precise nature of this dysfunction, and its relation to neurodegeneration, remain unclear. Here, we perform the first integrated multiomics approach to provide precise insight into the impact of Retromer dysfunction on endo-lysosomal health and homeostasis within a human neuroglioma cell model. We quantify profound changes to the lysosomal proteome, indicative of broad lysosomal dysfunction and inefficient autophagic lysosome reformation, coupled with a reconfigured cell surface proteome and secretome reflective of increased lysosomal exocytosis. Through this global proteomic approach and parallel transcriptomic analysis, we provide an unprecedented integrated view of Retromer function in regulating lysosomal homeostasis and emphasise its role in neuroprotection.

neuroscience↗

bioRxiv2022

Characterizing the polarization continuum of macrophage subtypes M1, M2a and M2c

Macrophages are vital components of the inflammatory response and exhibit phenotypical plasticity through active conversion between pro- and anti-inflammatory cell subtypes, a feature which can be reproduced in ex vivo culture. We employed a multifaceted approach utilizing proteomics, flow cytometry, activity assays and livecell microscopy imaging to characterize four cultured macrophage subtypes: unstimulated MO, classically activated M1, alternatively activated M2a, and deactivated M2c macrophages. Whole cell proteomics identified a total of 5435 proteins, with >50% of these proteins exhibiting significant alterations in abundance between the different subtypes. This confirms that four distinct macrophage subtypes are induced from the same originating donor material through stimulation with specific cytokines. Additional surfaceome analysis revealed that M2c macrophages significantly upregulate pro-inflammatory markers compared to the MO baseline and thus appear to be activated or primed to activate, similar to M1. Surface protein expression provided further subtype characterization, in particular distinguishing between the M2a and M2c macrophages. We next explored the re-polarization capabilities of macrophages using dexamethasone, an anti-inflammatory glucocorticoid known to induce macrophage polarization towards the M2c de-activated phenotype. We show that activated M1 macrophages treated with dexamethasone for 48-hours upregulate the levels of CD163 and CD206, markers synonymous with a phenotypical shift from M1 to M2c yet retain key surface markers and display the functional phenotype of M1 macrophages. The observed repolarization of M1 pro-inflammatory macrophages provides a potential mechanism through which dexamethasone treatment improves COVID-19 prognosis and constitutes evidence of partial repolarization along the macrophage continuum. These proteomic and functional ex vivo macrophage datasets provide a valuable resource for studying macrophage polarity and the impact of dexamethasone on macrophage phenotype and function.

immunology↗

bioRxiv2021

ENDO-Pore: High-throughput linked-end mapping of single DNA cleavage events using nanopore sequencing

Mapping the precise position of DNA cleavage events plays a key role in determining the mechanism and function of endonucleases. ENDO-Pore is a high-throughput nanopore-based method that allows the time resolved mapping single molecule DNA cleavage events in vitro. Following linearisation of a circular DNA substrate by the endonuclease, a resistance cassette is ligated recording the position of the cleavage event. A library of single cleavage events is constructed and subjected to rolling circle amplification to generate concatemers. These are sequenced and used to produce accurate consensus sequences. To identify the cleavage site(s), we developed CSI (Cleavage Site Investigator). CSI recognizes the ends of the cassette ligated into the cleaved substrate and triangulates the position of the dsDNA break. We firstly benchmarked ENDO-Pore using Type II restriction endonucleases. Secondly, we analysed the effect of crRNA length on the cleavage pattern of CRISPR Cas12a. Finally, we mapped the time-resolved DNA cleavage by the Type ISP restriction endonuclease LlaGI that introduces random double-strand breaks into its DNA substrates.

molecular biology↗

bioRxiv2021

The adenosine 2a receptor and TIM3 directly inhibit killing of tumor cells by cytotoxic T lymphocytes through interference with cytoskeletal polarization

Tumors generate an immune-suppressive environment that prevents effective killing of tumor cells by CD8+ cytotoxic T cells (CTL). It remains largely unclear upon which cell type and at which stage of the anti-tumor response mediators of suppression act. We have combined an in vivo tumor model with a matching in vitro reconstruction of the tumor microenvironment based on tumor spheroids to identify suppressors of anti-tumor immunity that directly act on interaction between CTL and tumor cells and to determine mechanisms of action. An adenosine 2a receptor antagonist, as enhanced by blockade of TIM3, slowed tumor growth in vivo. Engagement of the adenosine 2a receptor and TIM3 reduced tumor cell killing in spheroids, impaired CTL cytoskeletal polarization ex vivo and in vitro and inhibited CTL infiltration into tumors and spheroids. With this focus on CTL killing, blocking A2aR and TIM3 complements therapies that enhance T cell priming, e.g. anti-PD1 and anti-CTLA-4.

immunology↗