Biology subjects

Joyce, E.

Publications and source records attributed to Joyce, E..

16 recordsLinked to original sources

bioRxiv2026

Programming N-linked glycan composition through de novo protein design

N-linked glycans are ubiquitous post-translational modifications that mediate diverse biological processes, including cell signaling and adhesion, molecular recognition, and host-pathogen interactions. Despite their importance, N-linked glycans have largely remained beyond the reach of biomolecular design due to a lack of methods capable of jointly modeling glycans and protein structure. Here, we develop a general all-atom computational pipeline for creating de novo glycoproteins in which the amino acid sequence encodes both the location and composition of N-linked glycans. The method uses three-dimensional protein structure to tune glycan accessibility to processing enzymes in the eukaryotic secretory pathway. Systematic increases in steric restriction progressively reduced processing, shifting glycans from heterogeneous complex-type structures toward increasingly underprocessed oligomannose-type species resembling those found on many viral glycoproteins. Despite this programmed restriction, the resulting oligomannose glycans remained accessible for biological recognition, inhibiting uropathogenic Escherichia coli adhesion to human bladder cells and binding mannose-binding lectin. These results establish steric control of N-glycan processing as a genetically encodable design principle and extend computational protein design to functional glycoproteins with programmed composition.

biochemistry↗

bioRxiv2026

De novo design of allosterically controlled enzymes

The ability of enzymes to sense signals and respond by changing their structure and activity underlies cellular processes from signaling to metabolic control. While there have been recent advances in the de novo design of enzymes and conformationally switching proteins, combining these to achieve allosteric regulation of enzymatic activity in a fully designed system remains an outstanding challenge. Here, we show that denoising diffusion models enable the design of compact de novo enzymes whose activity can be allosterically activated or repressed by designed protein effectors. Our results establish a general route to allosteric control over a wide range of catalytic and other protein functions.

biochemistry↗

bioRxiv2026

Cell-type-specific parallel pathways in the canonical cortical microcircuit

Information processing in the cortex depends on the integration of bottom-up and top-down signals through recurrent microcircuits spanning layers. Although the canonical microcircuit provides a framework for this integration, how these interactions are implemented at synapse resolution remains unclear. Here, we use large-volume electron microscopy reconstructions of mouse primary visual cortex to map the intralaminar and interlaminar connectivity of intratelencephalic (IT) neurons in layers 2/3 and 5. We find that layer 2/3 IT neurons formed a depth-dependent gradient of recurrent connectivity, with superficial (L2) and deeper (L3) neurons potentially forming two channels associated with top-down and bottom-up processing, respectively. These channels are preserved across layers via cell-type-specific pathways involving distinct L5 IT types, rather than collapsing into a single integrative pool. Moreover, each channel is regulated by a largely separate cohort of inhibitory interneurons, stabilizing recurrent excitation while limiting crosstalk. Together, these results reveal parallel, cell-type-specific processing streams embedded within the canonical circuit.

neuroscience↗

bioRxiv2025

Computational design of cysteine proteases

Despite advances in de novo enzyme design, success has been largely limited to low energy barrier model reactions. Amide bonds such as those linking amino acids along the peptide backbone are stable for hundreds of years in neutral aqueous solution because of the high energy barrier to hydrolysis. Here we describe the de novo design of enzymes which utilize an activated cysteine nucleophile to hydrolyze the polypeptide backbone in a sequence-dependent manner, with a success rate of 13/69=19% and rate enhancements over the background reaction (kcat/kuncat) of up to 3 x 10^7. The designed proteases have folds very different from proteases in nature (TM score < 0.50), and six crystal structures are very close to the design models (Ca RMSDs < 1.2 A), highlighting the capacity for generalization and the accuracy of the design methodology. Experimental and computational analyses suggest that the remaining gap in activity to the most active native cysteine proteases arises from imperfections in active site preorganization and substrate positioning. The designed proteases efficiently cleave their targets in mammalian cells, opening the door to a wide range of synthetic biology applications.

biochemistry↗

bioRxiv2025

De novo design of phospho-tyrosine peptide binders

Phosphorylation on tyrosine is a key step in many signaling pathways. Despite recent progress in de novo design of protein binders, there are no current methods for designing binders that recognize phosphorylated proteins and peptides; this is a challenging problem as phosphate groups are highly charged, and phosphorylation often occurs within unstructured regions. Here we introduce RoseTTAFold Diffusion 2 for Molecular Interfaces (RFD2-MI), a deep generative framework for the design of binders for protein, ligand, and covalently modified protein targets. We demonstrate the power and versatility of this method by designing binders for four critical phosphotyrosine sites on three clinically relevant targets: Cluster of Differentiation 3 (CD3{varepsilon}), Epidermal Growth Factor Receptor (EGFR), Insulin Receptor (INSR) and Signal Transducer and Activator of Transcription 5 (STAT5). Experimental characterization shows that the designs bind their phosphotyrosine containing targets with affinities comparable to native binding sites and have negligible binding to non-phosphorylated targets or phosphopeptides with different sequences. X-ray crystal structures of generated binders to CD3{varepsilon} and EGFR are very close to the design models, demonstrating the accuracy of the design approach. A designed binder to an EGFR intracellular region phosphorylated upon EGF activation co-localizes with the receptor following EGF stimulation in single-particle tracking (SPT) experiments, demonstrating pY specific recognition in living cells. RFD2-MI provides a generalizable all-atom diffusion framework for probing and modulating phosphorylation-dependent signaling, and more generally, for developing research tools and targeted therapeutics against post-translationally modified proteins.

bioengineering↗

bioRxiv2025

De Novo Design of Miniprotein Inhibitors of Bacterial Adhesins

The rise of multidrug-resistant bacterial infections necessitates the discovery of novel antimicrobial strategies. Here, we show that protein design provides a generalizable means of generating new antimicrobials by neutralizing the function of bacterial adhesins, which are virulence factors critical in host-pathogen interactions. We de novo designed high-affinity miniprotein binders to FimH and Abp chaperone usher pili adhesins from uropathogenic Escherichia coli and Acinetobacter baumannii, respectively, which are implicated in mediating both uncomplicated and catheter-associated urinary tract infections (UTI) responsible for significant morbidity worldwide. The designed antagonists have high specificity and stability, disrupt bacterial recognition of host receptors, block biofilm formation, and are effective in treating and preventing murine models of uncomplicated and catheter- associated UTIs in vivo.

microbiology↗

bioRxiv2025

Design of Orthogonal Far-Red, Orange and Green Fluorophore-binding Proteins for Multiplex Imaging

Fluorescent proteins and small molecule dyes have complementary strengths for biological imaging: the former are genetically manipulatable enabling tagging of specific proteins and detection of protein interactions, while the latter have greater photostability and brightness but are difficult to target. To combine these strengths, we used de novo protein design to generate binders to three bright, stable, cell-permeable dyes spanning the visible spectrum: JF657 (far red), JF596 (orange-red) and JF494 (green). For each dye, we obtain nanomolar binders with weak or no binding to the other two dyes; the accuracy of the design approach is confirmed by a crystal structure of one binder which is very close to the design model. Fusion of the JF567, JF596 and JF494 binders to three different targets followed by staining with the three dyes simultaneously enables multiplex imaging. We further expand functionality by incorporating an active site carrying out nucleophilic aromatic substitution to form a covalent linkage with the dye, and developing split versions which reconstitute fluorescence at subcellular locations where both halves are present, enabling both protein-protein interaction detection and chemically induced dimerization with fluorescence reporting. Our designs combine the advantages of fluorescent proteins and small molecule dyes and should be broadly useful for cellular imaging.

biochemistry↗

bioRxiv2025

Hebb's Vision: The Structural Underpinnings of Hebbian Assemblies

1In 1949, Donald Hebb proposed that groups of neurons that activate stereotypically form the organizational building blocks of perception, cognition, and behavior. Finding the structural underpinning of such assemblies has been technically challenging, due to a lack of large-scale structure-activity maps. Here, we analyze this relation using a novel dataset that links in vivo optical physiology to connectivity using postmortem elec-tron microscopy (EM). From the fluorescence traces, we extract neural assemblies from higher-order correlations in neural activity. Physiologically, we show that these assemblies exhibit properties consistent with Hebbs theory, including more reliable responses to repeated natural movie inputs than size-matched random ensembles and superior decoding of visual stimuli. Structurally, we find that neurons that participate in assemblies are significantly more integrated into the structural network than those that do not. Contrary to Hebbs original prediction, we do not observe a marked increase in the strength of monosynaptic excitatory connections between cells participating in the same assembly. However, we find significantly stronger indirect feed-forward inhibitory connections targeting cells in other assemblies. These results show that assemblies can be useful components of perception, and, surprisingly, they are delineated by mutual inhibition.

neuroscience↗

bioRxiv2025

Inhibition of ice recrystallization with designed twistless helical repeat proteins

Given the repetitive structure of crystalline ice, its unsurprising that highly active ice-binding proteins, often with beta-roll structures, also have repeating motifs. Here, we introduce a de novo designed family of ice-binding twistless alpha-helical repeat (iTHR) proteins. Each iTHR protein comprises two planar layers of parallel alpha-helices connected by loops--a structural topology not seen in native ice-binding proteins. The ice-binding helices feature an ordered array of TXXXAXXXAXX motifs, spaced to match the pyramidal {201} and secondary prism plane {110} ice lattice facets, and engineered with a 98.2{degrees} turn angle per residue to orient threonines uniformly towards the ice surface. iTHR proteins show high solubility, thermostability, and produce varied ice crystal morphologies depending on their intended target facet. Crucially, iTHRs exhibit ice recrystallization inhibition (IRI) at critical concentrations comparable to those of many native globular ice-binding proteins. Extensive site-specific mutagenesis shows that ice-binding activity in iTHR proteins is robust, remaining largely unaffected by changes in chemical composition. Variation in the repeat number reveals a non-monotonic relationship to IRI activity. X-ray crystal structures of two designs confirm the intended orientation of threonines, uniformly pointing toward the ice surface. The iTHR family provides a versatile platform to systematically investigate the complex structure-activity relationships underlying protein-ice interactions.

biophysics↗

bioRxiv2025

Massively parallel assessment of designed protein solution properties using mass spectrometry and peptide barcoding

Library screening and selection methods can determine the binding activities of individual members of large protein libraries given a physical link between protein and nucleotide sequence, which enables identification of functional molecules by DNA sequencing. However, the solution properties of individual protein molecules cannot be probed using such approaches because they are completely altered by DNA attachment. Mass spectrometry enables parallel evaluation of protein properties amenable to physical fractionation such as solubility and oligomeric state, but current approaches are limited to libraries of 1,000 or fewer proteins. Here, we improved mass spectrometry barcoding by co-synthesizing proteins with barcodes optimized to be highly multiplexable and minimally perturbative, scaling to libraries of >5,000 proteins. We use these barcodes together with mass spectrometry to assay the solution behavior of libraries of de novo-designed monomeric scaffolds, oligomers, binding proteins and nanocages, rapidly identifying design failure modes and successes.

biochemistry↗

bioRxiv2024

A new phylogenetic framework for the genus Kalanchoe (Crassulaceae) and implications for infrageneric classification

Background and AimsKalanchoe is a diverse genus in the Crassulaceae, with a centre of diversity in Madagascar and sub-Saharan Africa. The genus is known for its popularity in horticulture, its use as a model system for research on CAM photosynthesis and vegetative reproduction, its high invasive potential, and its use in traditional medicine. The genus-rank circumscription and infrageneric classification of Kalanchoe has been the subject of debate for centuries, especially regarding the status and rank of what is now treated as K. subg. Bryophyllum and K. subg. Kitchingia. We aim to generate a densely sampled phylogeny of Kalanchoe s.l. and evaluate the current infrageneric classification system. MethodsWe inferred a phylogenetic tree for Kalanchoe using a ddRAD sequencing approach, covering 70% of taxa and four out of five subgenera currently recognised in the genus. Key ResultsWe recovered four well-supported clades, partially corresponding to the current subgeneric classification. Kalanchoe subg. Calophygia resolves as sister to the rest of the genus. The relationships among the three remaining clades, however, receive less support. The predominantly mainland African K. subg. Kalanchoe forms a strongly supported clade that resolves as sister to K. subg. Bryophyllum. These two clades are together sister to a clade containing mainly species from K. subg. Kitchingia and K. sect. Pubescentes. ConclusionsThe current subgeneric classification of Kalanchoe is partially backed up by our phylogenetic tree but requires further refinement. The tree topology suggests a Malagasy origin of the genus and one dispersal event to the African mainland, with subsequent dispersal from continental Africa to the Arabian Peninsula and Southeast Asia. The formation of bulbils on the leaf margin is restricted to a larger clade within K. subg. Bryophyllum and thus only evolved once. Our tree provides a framework for further taxonomic, evolutionary, and physiological research on the genus.

evolutionary biology↗

bioRxiv2024

Target-conditioned diffusion generates potent TNFR superfamily antagonists and agonists

Despite progress in designing protein binding proteins, the shape matching of designs to targets is lower than in many native protein complexes, and design efforts have failed for TNF receptor (TNFR1) and other protein targets with relatively flat and polar surfaces. We hypothesized that free diffusion from random noise could generate shape-matched binders for challenging targets, and tested this on TNFR1. We obtain designs with low picomolar affinity whose specificity can be completely switched to other family members using partial diffusion. Designs function as antagonists or as superagonists when presented at higher valency for OX40 and 4-1BB. The ability to design high-affinity and specificity antagonists and agonists for pharmacologically important targets in silico presages a new era in which binders are made by computation rather than immunization or random screening approaches.

biochemistry↗

bioRxiv2023

The Synaptic Architecture of Layer 5 Thick Tufted Excitatory Neurons in the Visual Cortex of Mice

The neocortex is one of the most critical structures that makes us human, and it is involved in a variety of cognitive functions from perception to sensory integration and motor control. Composed of repeated modules, or microcircuits, the neocortex relies on distinct cell types as its fundamental building blocks. Despite significant progress in characterizing these cell types 1-5, an understanding of the complete synaptic partners associated with individual excitatory cell types remain elusive. Here, we investigate the connectivity of arguably the most well recognized and studied excitatory neuron in the neocortex: the thick tufted layer 5 pyramidal cell 6-10 also known as extra telencephalic (ET) 11 neurons. Although the synaptic interactions of ET neurons have been extensively explored, a comprehensive characterization of their local connectivity remains lacking. To address this knowledge gap, we leveraged a 1 mm3 electron microscopic (EM) dataset. We found that ET neurons primarily establish connections with inhibitory cells in their immediate vicinity. However, when they extend their axons to other cortical regions, they tend to connect more with excitatory cells. We also find that the inhibitory cells targeted by ET neurons are a specific group of cell types, and they preferentially inhibit ET cells. Finally, we observed that the most common excitatory targets of ET neurons are layer 5 IT neurons and layer 6 pyramidal cells, whereas synapses with other ET neurons are not as common. These findings challenge current views of the connectivity of ET neurons and suggest a circuit design that involves local competition among ET neurons and collaboration with other types of excitatory cells. Our results also highlight a specific circuit pattern where a subclass of excitatory cells forms a network with specific inhibitory cell types, offering a framework for exploring the connectivity of other types of excitatory cells.

neuroscience↗

bioRxiv2020

HybPhaser: a workflow for the detection and phasing of hybrids in target capture datasets

Premise of the studyHybrids contain divergent alleles that can confound phylogenetic analyses but can provide insights into reticulated evolution when identified and phased. We developed a workflow to detect hybrids in target capture datasets and phase reads into parental lineages using a similarity and phylogenetic framework. MethodsWe used Angiosperms353 target capture data for Nepenthes including known hybrids to test the novel workflow. Reference mapping was used to assess heterozygous sites across the dataset, detect hybrid accessions and paralogous genes. Hybrid samples were phased by mapping reads to multiple references and sorting reads according to similarity. Phased accessions were included in the phylogenetic framework. ResultsAll known Nepenthes hybrids and nine more samples had high levels of heterozygous sites, reads associated with multiple divergent clades, and were phased into accessions resembling divergent haplotypes. Phylogenetic analysis including phased accessions increased clade support and confirmed parental lineages of hybrids. DiscussionHybPhaser provides a novel approach to detect and phase hybrids in target capture datasets, which can provide insights into reticulations by revealing origins of hybrids and reduce conflicting signal leading to more robust phylogenetic analyses.

evolutionary biology↗

bioRxiv2019

Cohesin promotes interactions across domain boundaries to regulate gene expression

The mammalian genome can be segmented into thousands of topologically associated domains (TADs) based on chromosome conformation capture studies, such as Hi-C. TADs have been proposed to act as insulated neighborhoods, spatially sequestering and insulating the enclosed genes and regulatory elements through chromatin looping and self-association. Recent results indicate that inter-TAD interactions can also occur, suggesting boundaries may be semi-permissible. However, the nature, extent, and function, if any, of these inter-TAD interactions remains unclear. Here, we combine super-and high-resolution microscopy with Oligopaint technology to precisely quantify the interaction frequency within and between neighboring domains in human cells. We find that intermingling across domain boundaries is a widespread feature of the human genome, with varying levels of interactions across different loci that correlate with their differing boundary strengths by Hi-C. Moreover, we find that cohesin depletion, which is known to abolish TADs at the population-average level, does not induce ectopic interactions but instead reduces both intra- and inter-domain interactions to a similar extent. Reduced chromatin intermixing due to cohesin loss affects domain incorporation and transcriptional bursting frequencies of genes close to architectural boundaries, potentially explaining the gene expression changes observed in the cohesinopathy Cornelia de Lange syndrome. Together, our results provide a mechanistic explanation for stochastic domain intermingling, arguing that cohesin partially bypasses boundaries to promote alternating incorporation of boundary-proximal genes into neighboring regulatory domains.

cell biology↗

bioRxiv2019

Oncogenic Notch promotes long-range regulatory interactions within hyperconnected 3D cliques

Chromatin loops enable transcription factor-bound distal enhancers to interact with their target promoters to regulate transcriptional programs. Although developmental transcription factors, such as active forms of Notch, can directly stimulate transcription by activating enhancers, the effect of their oncogenic subversion on the 3-dimensional (3D) organization of the cancer genome is largely undetermined. By mapping chromatin looping genome-wide in Notch-dependent triple-negative breast cancer and B-cell lymphoma, we show that far beyond the well-characterized role of Notch as an activator of distal enhancers, Notch regulates its direct target genes through establishing new long-range regulatory interactions. Moreover, a large fraction of Notch-promoted regulatory loops forms highly interacting enhancer and promoter spatial clusters, termed "3D cliques". Loss-and gain-of-function experiments show that Notch preferentially targets hyperconnected 3D cliques that regulate the expression of crucial proto-oncogenes. Our observations suggest that oncogenic hijacking of developmental transcription factors can dysregulate transcription through widespread effects on the spatial organization of cancer genomes.

cancer biology↗