Biology subjects

Johnston, B.

Publications and source records attributed to Johnston, B..

6 recordsLinked to original sources

bioRxiv2025

Drug-like antibody design against challenging targets with atomic precision

Computational antibody design has seen rapid progress, with high success rates enabling direct translation to characterization without any high-throughput screening required. In this work, we markedly expand the scope of de novo antibody design by applying our state-of-the-art Chai-2 platform to design drug-like antibodies in full-length monoclonal format. We find that >86% of these full-length mAbs have strong developability profiles on par with therapeutic antibodies. We further show that experimentally determined structures of Chai-2 designs closely match their in silico predictions, demonstrating that Chai-2 produces atomically accurate models of designed antibodies. Building on these foundational capabilities, we showcase two potential applications of Chai-2 against different targets: designing functional antibodies mediating GPCR agonism, and highly specific antibodies selectively binding tumor-specific neoepitopes. Taken together, this work brings new flexibility to modern discovery pipelines, accelerating the path from in silico design to functional validation across both conventional and challenging targets. Beyond reducing the cost and timelines associated with large screening campaigns, in silico design can now open new frontiers for creative, targeted therapeutics that address unmet clinical needs.

molecular biology↗

bioRxiv2025

A Reference Atlas of the Human Dorsal Root Ganglion

Somatosensory perception largely emerges from diverse peripheral sensory neurons whose cell bodies reside in dorsal root ganglia (DRG). Damage or dysfunction of DRG neurons is a major cause of chronic pain and sensory loss. In mice, deep single-cell transcriptomic profiling and genetically defined models have offered important clues into DRG function, but in humans, the cellular and molecular landscape of DRG neurons remains less understood. Here, we constructed a reference cell atlas of the human DRG by profiling transcriptomes of cells and nuclei from 126 donors sampled across cervical, thoracic, and lumbar DRGs. This atlas resolves 22 neuronal subtypes, including known and previously unrecognized subtypes linked to nociception, mechanosensation, thermosensation, and proprioception, as well as 10 types of non-neuronal cells. Cross-species integration, spatial transcriptomics, and microneurography enabled cell-type-specific comparisons of soma size and conduction velocity between species. Human DRG somata are larger across all cell types than their mouse counterparts, and the conduction velocities of human hair follicle innervating A-fibers are faster than in mice, suggesting a functional shift in rapid mechanical detection in humans. This integrated human DRG reference cell atlas provides a resource for exploring new molecular and physiological features of human DRG, which could help identify new strategies for treating chronic pain and other diseases of the peripheral nervous system.

neuroscience↗

bioRxiv2024

MICAL2 Is a Super Enhancer Associated Gene that Promotes Pancreatic Cancer Growth and Metastasis

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest solid cancers and thus identifying more effective therapies is a major unmet need. In this study we characterized the super enhancer (SE) landscape of human PDAC to identify novel, potentially targetable, drivers of the disease. Our analysis revealed that MICAL2 is a super enhancer-associated gene in human PDAC. MICAL2 is a flavin monooxygenase that induces actin depolymerization and indirectly promotes SRF transcription by modulating the availability of serum response factor coactivators myocardin related transcription factors (MRTF-A and MRTF-B). We found that MICAL2 is overexpressed in PDAC and correlates with poor patient prognosis. Transcriptional analysis revealed that MICAL2 upregulates KRAS and EMT signaling pathways, contributing to tumor growth and metastasis. In loss and gain of function experiments in human and mouse PDAC cells, we observed that MICAL2 promotes both ERK1/2 and AKT activation. Consistent with its role in actin depolymerization and KRAS signaling, loss of MICAL2 expression also inhibited macropinocytosis. Through in vitro phenotypic analyses, we show that MICAL2, MRTF-A and MRTF-B influence PDAC cell proliferation, migration and promote cell cycle progression. Importantly, we demonstrate that MICAL2 is essential for in vivo tumor growth and metastasis. Interestingly, we find that MRTF-B, but not MRTF-A, phenocopies MICAL2-driven phenotypes in vivo. This study highlights the multiple ways in which MICAL2 impacts PDAC biology and suggests that its inhibition may impede PDAC progression. Our results provide a foundation for future investigations into the role of MICAL2 in PDAC and its potential as a target for therapeutic intervention.

cancer biology↗

bioRxiv2024

Single-molecule visualization of sequence-specific RNA binding by a designer PPR protein

Pentatricopeptide repeat (PPR) proteins are a large family of modular RNA-binding proteins that recognize specific ssRNA target sequences. There is significant interest in developing designer PPRs for use in diagnostics or as tools to detect and localize target RNA sequences. However, it is unclear how PPRs search for target sequences within complex transcriptomes and current models to predict PPR binding sites struggle to reconcile the effects that RNA mismatches and secondary structure have on PPR binding. To address this, we determined the structure of a designer PPR (dPPR10) bound to its target sequence and used two- and three-colour single-molecule FRET to interrogate the mechanism of ssRNA binding to individual PPR proteins in real time. We demonstrate that longer RNA sequences were significantly slower to bind (or could not bind at all) and that this is, in part, due to their propensity to form stable secondary structures that sequester the target sequence from dPPR10. Importantly, dPPR10 does not associate with non-target flanking sequences, binding specifically to its target sequence within longer ssRNA species. This data provides evidence that PPRs have limited to no capacity to scan RNA transcripts for target sequences and instead rely on diffusion for cognate searching. The kinetic constraints imposed by random three-dimensional diffusion may explain the long-standing conundrum of why PPR proteins are abundant in organelles, but almost unknown outside them (i.e. in the cytosol and nucleus). These findings will inform improved prediction of PPR binding sites for the development of designer PPRs. SummaryPentatricopeptide repeat proteins (PPR) are a large family of modular RNA-binding proteins, whereby each module can be designed to bind to a specific ssRNA nucleobase and thus any RNA sequence of interest. As such, there is substantial interest in developing designer PPRs for a range of biotechnology applications, including diagnostics or in vivo localisation of RNA species; however, the mechanistic details regarding how PPRs search for and bind to target sequences is unclear. As such, we combined structure-based and single- molecule approaches and determined that PPRs bind only to their target sequences (i.e., they do not associate with non-target RNA sequences) and do not scan longer RNA oligonucleotides for the target sequence. Instead, target searching appears kinetically-constrained by random three-dimensional diffusion, providing an explanation as to why PPRs are found almost exclusively in organelle compartments that typically have smaller transcriptomes. Collectively, this work identifies several key considerations for future designer PPR developments.

biochemistry↗

bioRxiv2022

Ultra-sensitive platelet proteome maps the O-glycosylation landscape and charts the response to thrombin dosage

Platelet activation induces the secretion of proteins that promote platelet aggregation and inflammation. However, detailed analysis of the released platelet proteome is hampered by platelets tendency to pre-activate during their isolation and a lack of sensitive protocols for low abundance releasate analysis. Here we detail the most sensitive analysis to date of the platelet releasate proteome with the detection of >1,300 proteins. Unbiased scanning for post-translational modifications within releasate proteins highlighted O-glycosylation as being a major component. For the first time, we detected O-fucosylation on previously uncharacterised sites including multimerin-1 (MMRN1), a major alpha granule protein that supports platelet adhesion to collagen and is a carrier for platelet factor V. The N-terminal EMI domain of MMRN1, a key site for protein-protein interaction, was O-fucosylated at a conserved threonine within a new consensus sequence. Our data suggest that Protein O-fucosyltransferase 1 (POFUT1) is responsible for this modification. Secretion of MMRN1 was reduced in cells lacking POFUT1, supporting a key role of O-fucosylation in MMRN1 function. By comparing releasates from resting and thrombin-treated platelets, 202 proteins were found to be significantly released after high-dose thrombin stimulation. Complementary quantification of the platelet lysates identified >3,800 proteins, which confirmed the platelet origin of releasate proteins by anti-correlation analysis. Low-dose thrombin treatment yielded a smaller subset of significantly regulated proteins with fewer secretory pathway enzymes. The comprehensive platelet proteome resource provided here (larancelab.com/platelet-proteome) allows identification of novel regulatory mechanisms for drug targeting to address platelet dysfunction and thrombosis. Key PointsO_LIHigh-sensitivity proteome mapping of human platelets identifies O-glycosylation of platelet proteins at key functional sites C_LIO_LIPlatelet O-fucosyltransferase POFUT1 regulates the secretion of adhesive protein multimerin-1 (MMRN1) C_LI

biochemistry↗

bioRxiv2022

CRISPRi-based circuits for genetic computation in plants

Synthetic gene circuits can enable new cellular behaviours by integrating multiple input signals into customisable genetic programs. However, gene circuit development in plants has been limited by a lack of orthogonal and modular parts required for their construction. Here, we present a tool-kit of reversible CRISPRi-based gene circuits for use in plants. First, we created a range of engineered repressible promoters of different strengths and used them as integrators for the construction of NOT and NOR gates in Arabidopsis cells. Next, we determined the optimal processing system to express sgRNAs from RNA Pol II promoters to introduce NOR gate programmability and interface it with host regulatory sequences. Finally, we connected multiple NOR gates together in layered arrangements to create OR, NIMPLY, and AND logic functions. Our CRISPRi circuits are orthogonal, compact, reversible, programmable, and modular, providing a new platform for sophisticated and deliberate spatio-temporal control of gene expression in plants.

synthetic biology↗