Biology subjects

Kang, A.

Publications and source records attributed to Kang, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Design of readers for pathological PTM proteoforms in Alzheimer's disease

Alzheimer's disease (AD) is characterized by heterogeneous Tau phosphorylation and aberrant amyloid-beta (Abeta) processing, yet molecular tools that distinguish closely related molecular states of tau and Abeta with predefined specificity remain difficult to develop. Here, we designed de novo protein readers targeting three disease-associated phospho-Tau regions and N-terminal pyroglutamate-modified Abeta (N3pG-Abeta). Screening 192 designs per target identified 51 modification-selective hits among 768 candidates (6.6%). Representative readers exhibited nanomolar to subnanomolar affinities for modified peptides, discriminated authentic modifications from unmodified or substitution-based controls, and preferentially recognized modified full-length targets. We further programmed local modification specificity by interface redesign, generating pT231 readers conditional on the phosphorylation state of neighboring S235 and S237. A 2.0-angstrom crystal structure of an N3pG-Abeta reader closely matched the computational design model (interface C-alpha RMSD = 0.52 angstrom), and redesign of pT231 readers tuned apparent binding affinities from greater than 1 uM to below 1 nM. In a human 3D neuronal culture with both pathological Abeta and tau, selective readers targeting N3pG-Abeta, Tau pT231, and Tau pT217 recognized disease-associated signals, exhibiting colocalization with related antibodies and much higher signal in the AD line than the control. Tau pT231 readers further quantitatively distinguish AD patients and control human brain lysates in dot-blot assays. Together, these results establish genetically encoded readers with programmable specificity for defined Tau and Abeta modification states, enabling molecular-state-resolved detection in human disease models and biological samples, and lay a foundation for controlled pathological molecular perturbation.

bioengineering↗

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

Complex enzyme active site scaffolding by iteratively detuned catalytic guidance

De novo enzyme design requires sequences whose structures reproduce prescribed catalytic geometries. This remains challenging for active sites that couple multiple catalytic residues, cofactors and substrates. Here we introduce the Catalytic Guidance Engine (CaGE), which optimizes designed sequences to satisfy complex sets of functional constraints by alternating constraint guided structure prediction trajectories with sequence design, gradually fading the strength of the constraint information over cycles to nudge the designed sequence towards satisfying all the constraints. We train RF3 to accept atom-pair distances and other design conditions, and use classifier-free guidance to fade their influence over multiple design cycles. We also develop training-free motif guidance protocols which operate at inference time by directly adjusting selected atoms towards the design target geometry during diffusion sampling. CaGE improves catalytic-motif geometry across a diverse enzyme benchmark and yields sequences that recover complex target active sites in independent AF3 predictions. We experimentally demonstrate the power of CaGE by designing active enzymes with complex catalytic sites: a nonheme-iron design positions four active-site residues around an iron centre and a transition-state ligand, and a PLP-dependent design positions four catalytic residues around a PLP cofactor with multiple functional groups. Active site substitutions and a 2.7[A] crystal structure support the designed mechanisms and structures. Thus, CaGE provides a route to functional de novo enzymes with complex active sites.

bioengineering↗

bioRxiv2026

SEX-DEPENDENT MODULATION OF WHOLE BRAIN cFOS EXPRESSION BY LIGHT AND MELANOPSIN IN THE MOUSE

Light is a fundamental feature of the environment, signaling time of day, weather changes, approaching predators, and other survival cues. The extensive projections from the retina to the brain provide an anatomical substrate for light to adaptively tune circuit function based on changing environmental light. Yet we understand little about the scope and functional impact of light outside of visual and circadian circuits. To address this, we created a whole-brain atlas of light-induced cFos expression in the mouse brain. This approach yielded a strikingly broad pattern of light-driven cFos activation throughout the brain that extended beyond canonical visual circuits, with unexpectedly strong modulation of neuromodulatory centers. These patterns differed starkly in males and females, and were dependent primarily on melanopsin-expressing, intrinsically photosensitive retinal ganglion cells. These findings uncover surprising brainwide patterns of light modulation and are shared as an interactive resource so that they can inspire new, future studies.

neuroscience↗

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

Programmable De Novo Design of Mesoporous Protein Crystal Frameworks

Three-dimensional protein crystals are ordered, porous macroscopic materials with potential applications in catalysis, biosensing, and biomedicine. However, most protein crystals are obtained by empirical screening, providing limited control over the lattice architecture, pore geometry or component composition that determine material function. Here, we present a modular strategy for the programmable design of highly porous, framework-like protein crystals using predefined protein-protein interactions. This strategy yielded over 30 distinct protein crystals, including single-component and multicomponent P213 and I213 lattices that grow to over 100 micrometers in size. Small-angle X-ray scattering and electron microscopy showed close agreement between experimental lattices and computational models. RFdiffusion-guided design generated isomorphous variants with matched lattice parameters, enabling coherent protein crystal alloys, epitaxial core-shell growth and reversible shell assembly. The designed crystals exhibit tunable mesoporous architectures, with limiting apertures of 2-18 nm, and support genetically encoded incorporation of fluorescent protein guests. These results establish a general route to programmable lattice engineering of protein crystals and position them as genetically encoded, compositionally tunable mesoporous materials.

synthetic biology↗

bioRxiv2026

De novo design of autocatalytically forming intra- and intermolecular isopeptide bonds to construct rigid covalent protein assemblies

Isopeptide bonds are amide bonds between amino acid side chains that can form autocatalytically, notably in the pili of Gram-positive bacteria. Here, we design de novo proteins that form both intramolecular and intermolecular isopeptide bonds entirely autocatalytically. We report over 50 designs that form isopeptide bonds, validated by mass spectrometry and 5 crystal structures. We redesign these constructs as split proteins that form a covalent intermolecular isopeptide crosslink when combined. These split designs are orthogonal to the existing isopeptide-based SpyTag/Catcher system, and their formation can be regulated by temperature, providing control over the timing of crosslinking in protein assemblies. We extend these designs to create rigid domain crosslinks that enable the construction of large well ordered symmetric rings of up to 215 kDa that are irreversibly covalently crosslinked by multiple isopeptide bonds into a single molecule. Our results provide insight into the determinants of isopeptide bond formation, considerably expand the set of isopeptide bond crosslinking systems, and establish a framework to construct fully covalent rigid protein assemblies.

biophysics↗

bioRxiv2026

Cytoplasmic DNA Sensing Links LINE-1 Expression to Neuronal Senescence in Alzheimer's Disease

Cellular senescence contributes to neurodegeneration in Alzheimers disease (AD), yet brain-penetrant senotherapeutic strategies remain limited. Here, we identify long interspersed nuclear element 1 (LINE-1) retrotransposons as key regulators of neuronal senescence and the senescence-associated-secretory-phenotype (SASP) in AD. Using transdifferentiated induced neurons (iNs) that preserve donor-specific aging-associated molecular signatures, we show that pharmacological inhibition of LINE-1 with nucleoside reverse transcriptase inhibitors (nRTIs) or antisense oligonucleotides reduces p16 expression, suppresses SASP and interferon-stimulated gene programs, and attenuates paracrine induction of reactive astrogliosis. Spatial transcriptomic analysis of human AD brain tissue further supports that senescent neurons with high LINE-1 expression are localized to inflammatory niches in the brain. Although bulk analysis finds no significant differences in LINE-1 expression between AD and control neurons, long-read single-cell RNA sequencing of iNs identifies a subset of neurons with elevated LINE-1 activity which display transcriptional signatures of neurodegeneration, immune activation, and senescence are enriched in AD relative to controls. RNA velocity analysis indicates that LINE-1 activation precedes the induction of canonical senescence markers, supporting a causal rather than consequential role. Mechanistically, LINE-1-derived cytoplasmic DNA activates the cGAS-STING innate immune pathway in post-mitotic neurons, and inhibition of cGAS phenocopies the effects of LINE-1 suppression. Together, these findings establish a LINE-1/cGAS-STING axis as a driver of neuronal senescence in AD and highlight LINE-1 inhibition as a tractable senomorphic strategy for neurodegenerative disease.

neuroscience↗

bioRxiv2026

Orally available designed miniproteins inhibit enterotoxigenic Bacteroides fragilis pathology by blocking toxin receptor binding

Toxigenic bacterial infections in the gut are a significant contributor to the global burden of disease. Advanced tools for protein and live biotherapeutic engineering offer potentially transformative strategies for treating such diseases, while avoiding the collateral effects of traditional antibacterials. Here we used de novo protein design to identify inhibitors of the metzincin family protease Bacteroides fragilis toxin (BFT). These inhibitors, which bind distal to the active site, interfere with toxin-mediated E-cadherin cleavage and downstream proinflammatory signaling by blocking claudin-4 receptor binding. We tested the inhibitors as disulfide-stabilized variants administered directly to the cecum or in drinking water, as well as through in situ secretion by an engineered live biotherapeutic. Across these delivery modalities, the inhibitors successfully neutralized the toxin and effectively prevented BFT-associated gut pathology, including tumor formation. These results highlight the potential of de novo designed proteins as precise, non-antibiotic interventions to mitigate bacterial toxin-driven disease in the gut.

microbiology↗

bioRxiv2026

A Pro-Regenerative Petroleum Jelly-Based, Copper-Doped Bioactive Glass Ointment for Impaired Wound Healing in Metabolic Syndrome

Chronic wound healing is often impaired in conditions such as metabolic syndrome, requiring effective therapeutic interventions to promote tissue regeneration and repair. In this study, we evaluated the wound healing potential of petroleum jelly (P Jelly)-based bioactive glass ointments (PBGCu) with varying copper concentrations (0, 1, and 3 wt%) in both in vitro and in vivo models of wound healing. PBGCu formulations demonstrated high biocompatibility with human dermal fibroblasts (HDF) and human umbilical vein endothelial cells (HUVEC). Additionally, PBGCu ointments exhibited strong antibacterial activity against Staphylococcus aureus, suggesting their utility for the care of chronic wounds. In both metabolic syndrome mouse and pig models, PBGCu3-treated wounds showed significantly faster wound closure, enhanced epithelial regeneration, and increased dermal thickness compared to saline and P Jelly controls. Histological analysis also revealed 50% increased vascularization (p < 0.0001) and a 90% reduction in scar formation (p < 0.0001) in PBGCu3-treated wounds. These findings show that PBGCu formulations, especially at 3 wt% copper concentration, significantly improve wound healing by promoting epithelial regeneration, dermal tissue formation, and vascularization, while also offering antibacterial protection. The sustained Cu2+ ions release from PBGCu ointments provides long-term support for tissue regeneration, positioning this ointment composition as a promising therapeutic tool for chronic wound management. Future studies will focus on elucidating the underlying mechanisms and evaluating the therapeutic efficacy of PBGCu formulations in infected wounds. HighlightsO_LIDeveloped a Petroleum Jelly-based copper-doped bioactive glass ointment (PBGCu) enabling sustained and controlled Cu{superscript 2} ion release. C_LIO_LIPBGCu significantly accelerated wound closure and improved epithelial and dermal tissue regeneration. C_LIO_LIPBGCu enhanced hair follicle regeneration and tissue remodeling in full-thickness wounds. C_LIO_LIValidated therapeutic efficacy in both mouse and pig models that support translational relevance. C_LIO_LIOffers a simple, low-cost, and clinically adaptable topical formulation for metabolic syndrome-related wound complications. C_LI

bioengineering↗

bioRxiv2026

Controlling metal-carbonate phase, form, and function through de novo protein design

Biomineralization enables living systems to construct hybrid materials by controlling the location, orientation, and polymorph of inorganic crystals with proteins and other biomolecules. Despite decades of study, the molecular principles underlying these processes remain difficult to harness in engineered materials, in part because native biomineralization proteins are often intrinsically disordered, heterogeneous, or insoluble. Here we show that de novo designed protein interfaces can be assembled into reconfigurable two-dimensional arrays which template calcite nanocrystals. By fine-tuning RFdiffusion2 on repeat protein scaffolds, we further enable the design of protein architectures which selectively form aragonite, a metastable polymorph of calcium carbonate, in nucleation conditions that otherwise result in a mixture of phases. Extending beyond inorganics found in biological systems, we show that lattice-matched protein designs template cobalt carbonate formation: a flat helical repeat protein interface promotes unconfined growth, whereas soluble D3 cage assemblies yield more homogenous cobalt carbonate nanocrystals confined to the interior of the cage. These protein-cage cobalt carbonate hybrid materials function as electrocatalysts for alkaline water splitting. Our results demonstrate the potential of deep learning-based methods to unlock the structural and functional activity of protein-mineral composites.

biochemistry↗

bioRxiv2026

Generative design of programmable asymmetric β-barrel nanopores

Protein nanopores are powerful tools for molecular sensing, sequencing, and separation, but designing pores with programmable function remains challenging. Native homo-oligomeric transmembrane {beta} barrels (TMBs) are used for these applications, but their uniform lumens limit spatial resolution and analyte discrimination. Although monomeric TMBs can be designed using energy-based methods, these approaches remain highly manual and limited to structural design rather than function. Here, we present a generative AI framework for TMB design, with backbone and sequence design models trained on a curated distillation set. We characterized 48 designs spanning 0.7-1.5 nm in diameter, diverse lumen chemistries, and hydrophobic thicknesses. Crystal structures closely match the design models. We demonstrate that our method produces customizable nanopores for ion sensing, DNA translocation, and transport across polymer membranes.

biochemistry↗

bioRxiv2026

Computational design of membrane fusion proteins

The fusion of two distinct biological membranes is an evolutionarily conserved process essential to cellular organization and physiology. Membrane fusion is driven by the refolding of fusogenic proteins into low-energy postfusion states that overcome the energetic barrier to bilayer merger. Here we report a computational method for the design of synthetic fusogens inspired by the architecture of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Using machine learning-guided protein design to extensively remodel backbone geometry and sequence, we generated heterodimeric SNARE-like assemblies that efficiently catalyze cell-cell membrane fusion. These minimal two-component fusogens exhibit substantially higher fusion activity than native multisubunit SNARE complexes. Structural and functional analyses identify the key determinants required for fusogenic activity and reveal a modularity that enables control of fusion through chemically induced heterodimerization. In addition to cell-cell fusion, the synthetic fusogens drive fusion between endoplasmic reticulum and mitochondrial membranes from human cells, demonstrating their potential as tools for programmable manipulation of intracellular membranes. Together, these results establish a general framework for the rational design of synthetic fusogens and expand the toolkit for engineering membrane dynamics in living systems.

biochemistry↗

bioRxiv2026

Dopamine in the ventral and tail of striatum supports global and local evaluation in reward-threat conflict

Survival requires balancing reward seeking and threat avoidance, yet how distinct dopamine systems coordinate to support this remains unclear. Using a naturalistic foraging paradigm in which mice pursue water reward under threat from a monster object, we examined roles of dopamine projections to the ventral striatum (VS) and tail of the striatum (TS). Ablation of VS- projecting dopamine neurons impaired both distal reward pursuit and threat avoidance, with the impairment in threat avoidance paralleling effects of TS dopamine ablation. However, simultaneous recordings revealed different activity rules: VS dopamine tracked radial velocity towards the current goal as animals changed goals (reward or shelter), consistent with a temporal-difference error of spatial value, while TS dopamine encoded proximity and orientation to the threat, reflecting immediate sensory experience. Taken together, VS and TS dopamine evaluates distinct state information for avoidance. VS dopamine facilitates allocentric, goal- directed navigation, while TS dopamine facilitates egocentric, stimulus-driven threat responses.

neuroscience↗

bioRxiv2026

De novo design of a macrocycle induced dimerization system for cellular control

Investigating and manipulating cellular events requires precise control of protein function. To enable control over cellular processes, we set out to design a chemically induced dimerization (CID) system consisting of a de novo designed ligand and protein pair. Here we describe the design of a C2 symmetric membrane permeable macrocyclic peptide and a cognate protein homodimer which binds the macrocycle through a large interface with both chains. The designed homodimer binds the macrocycle with a KD of 36 nM, and the x-ray crystal structure of the protein homodimer-macrocycle complex is very close to the computational design model, with the C2 axis of the macrocycle aligned with the homodimer C2 axis. Transcriptional and split luciferase assays in mammalian cells demonstrates conditional control over both a reporter gene expression and luciferase reconstitution.

biochemistry↗

bioRxiv2026

RUNX1 and YY1 modulate neuronal fate and energy metabolism in Alzheimer's disease

Loss of neuronal identity and metabolic dysfunction are features of Alzheimers disease (AD), yet the upstream-acting molecular drivers remain incompletely understood. By integrating multi-omics data from patient-derived induced neurons (iNs) and AD post-mortem human brains, we discovered that AD neurons express two master transcription factors (TFs), RUNX1 and YY1. While these TFs are primarily expressed during development where they play fundamental roles in cell fate determination and cellular bioenergetics, respectively, they can be reactivated in adult neurons in response to stress. To understand their functional role in AD neurons, we overexpressed RUNX1 or YY1 in aged iNs and found that the expression of each TF was sufficient to recapitulate two AD-associated features. Specifically, RUNX1 overexpression caused loss of neuronal fate, whereas YY1 overexpression regulated gene regulatory programs associated with metabolic dysfunction. Conversely, downregulation of either TF, in AD iNs, reinstated gene regulatory programs associated with a healthy mature neuronal phenotype. Together, these findings identify two transcriptional master regulators of the AD neuronal phenotype and establish a mechanistic foundation for further studying their role in the pathogenesis of AD and as putative therapeutical targets for the treatment of AD and age-associated neurodegeneration.

neuroscience↗

bioRxiv2026

AI-Guided CRISPR Screen Accelerates Discovery of New Drug Targets

Psoriasis affects over 125 million people worldwide, yet the mechanistic understanding of keratinocyte-driven inflammation remains incomplete, limiting therapeutic innovation beyond costly systemic biologics that are prone to side effects. Here, we performed the first genome-wide CRISPR knockout screen in primary human adult epidermal keratinocytes to systematically identify regulators of IL-17 receptor A (IL17RA), a central node in psoriatic inflammation. To prioritize therapeutically tractable targets from over 19,000 screened genes, we integrated a large language model - VirtualCRISPR - trained on functional genomics data, identifying arachidonate 5-lipoxygenase (ALOX5) and oxytocin receptor (OXTR) as high-confidence novel hits with minimal prior association with psoriasis. Multi-omics validation revealed that ALOX5 and OXTR regulate IL17RA expression through distinct signaling pathways - ALOX5 through lipid mediators that stabilize the receptor at the cell surface, and OXTR through calcium signaling that reprograms cellular metabolism. Topical delivery of their inhibitors Zileuton (ALOX5) and Cligosiban (OXTR) exhibited therapeutic efficacy comparable to systemic anti-IL17RA antibody in the imiquimod-induced psoriasis model, suppressing pathogenic Th17/Tc17 responses, polarizing macrophages toward anti-inflammatory phenotypes, and normalizing epidermal hyperproliferation. Proteomic profiling in human 3D organotypic skin and murine models confirmed on-target pharmacology and revealed convergent suppression of neutrophil-keratinocyte inflammatory circuits. The use of VirtualCRISPR significantly shortened the timescale from screen to the identification of druggable hits with robust validation, and this work establishes a blueprint for integrating AI-driven target prioritization with functional genomics to accelerate therapeutic discovery.

genomics↗

bioRxiv2025

De novo design of protein nanoparticles with integrated functional motifs

Computational design of self-assembling proteins has long relied on pre-existing structures and sequences, fundamentally limiting control over their structural and functional properties. Recent machine learning-based methods have transformed our ability to design functional small de novo proteins and oligomers, yet methods to design large de novo protein assemblies with structures tailored to specific applications are still underexplored. Here, we develop a generalizable method for designing de novo symmetric protein complexes that incorporate target functional motifs into their structures. We report 34 new protein nanoparticles that form on-target assemblies with cubic point group symmetries. The nanoparticles exhibit a wide variety of backbones that were designed with atom-level accuracy, as evidenced by several cryo-EM and crystal structures that reveal minimal deviations from the design models. We use the method to generate a de novo antigen-tailored nanoparticle vaccine that elicits robust immune responses in mice. These results establish a generalizable approach that can be used to design functional self-assembling protein complexes with structures tailored to specific applications.

biochemistry↗