Biology subjects

Zhao, Y. T.

Publications and source records attributed to Zhao, Y. T..

6 recordsLinked to original sources

bioRxiv2026

Integrin-independent Tie2 activation using de novo designed proteins

The Angiopoietin-Tie2 pathway is a key regulator of vascular stability, but therapeutic exploitation has been limited by the poor developability of Angiopoietin-1 (Ang1), and there are key unresolved mechanistic questions. Ang1 binds both Tie2 and 5{beta}1 integrin, and the role of the 5{beta}1 interaction in signaling has been unclear. We used RFdiffusion to design a stable, high-affinity Tie2 minibinder that has no affinity for 5{beta}1. The binder is a selective antagonist in monomeric form, and a potent agonist when assembled into an octavalent architecture (H8mb) which drives Tie2 clustering. H8mb signals as potently as Ang1, indicating that integrin engagement is not required for Tie2 activation. However, the duration of signaling is reduced, and internalization of H8mb-Tie2 complexes is more rapid, suggesting that integrin may function as a co-receptor for Ang1 that prolongs signaling by extending the lifetime of the receptor ligand complex on the cell surface. In a mouse model of acute respiratory distress syndrome (ARDS), H8mb markedly improved survival. These results demonstrate that de novo designed receptor binders can enable dissection of co-receptor control of signaling dynamics, and the more stable and manufacturable H8mb provides routes to more developable therapeutic candidates.

biochemistry↗

bioRxiv2022

Top-down design of protein nanomaterials with reinforcement learning

The multisubunit protein assemblies that play critical roles in biology are the result of evolutionary selection for function of the entire assembly, and hence the subunits in structures such as icosahedral viral capsids often fit together with remarkable shape complementarity1,2. In contrast, the large multisubunit assemblies that have been created by de novo protein design, notably the icosahedral nanocages used in a new generation of potent vaccines3-7, have been built by first designing symmetric oligomers with cyclic symmetry and then assembling these into nanocages while keeping the internal structure fixed8-14, which results in more porous structures with less extensive shape matching between the components. Such hierarchical "bottom-up" design approaches have the advantage that one interface can be designed and validated in the context of the cyclic oligomer building block15,16, but the disadvantage that the structural and functional features of the assemblies are limited by the properties of the predesigned building blocks. To overcome this limitation, we set out to develop a "top-down" reinforcement learning based approach to protein nanomaterial design in which both the structures of the subunits and the interactions between them are built up coordinately in the context of the entire assembly. We developed a Monte Carlo tree search (MCTS) method17,18 which assembles protein monomer structures in the context of an overall architecture guided by a loss function which enables specification of any desired overall structural properties such as shape and porosity. We demonstrate the power of the approach by designing hyperstable icosahedral assemblies more compact than any previously observed protein icosahedral structure (designed or naturally occurring), that have very low porosity and are robust to fusion and display of proteins as complex as influenza hemagglutinin. CryoEM structures of two designs are very close to the computational design models. Our top-down reinforcement learning approach should enable the design of a wide variety of complex protein nanomaterials by direct optimization of overall system properties.

synthetic biology↗

bioRxiv2022

Human iPSC Derived Enamel Organoid Guided by Single-Cell Atlas of Human Tooth Development

Tooth enamel secreted by ameloblasts is the hardest material in the human body, acting as a shield protecting the teeth. However, the enamel is gradually damaged or partially lost in over 90% of adults and cannot be regenerated due to a lack of ameloblasts in erupted teeth. Here we use sci-RNA-seq to establish a spatiotemporal single-cell atlas for the developing human tooth and identify regulatory mechanisms controlling the differentiation process of human ameloblasts. We reveal key signaling pathways involved between the support cells and ameloblasts during fetal development and recapitulate those findings in a novel human ameloblast in vitro differentiation from iPSCs. We furthermore develop a mineralizing enamel organ-like 3D organoid system. These studies pave the way for future regenerative dentistry and therapies toward genetic diseases affecting enamel formation.

developmental biology↗

bioRxiv2021

Multivalent designed proteins protect against SARS-CoV-2 variants of concern

Escape variants of SARS-CoV-2 are threatening to prolong the COVID-19 pandemic. To address this challenge, we developed multivalent protein-based minibinders as potential prophylactic and therapeutic agents. Homotrimers of single minibinders and fusions of three distinct minibinders were designed to geometrically match the SARS-CoV-2 spike (S) trimer architecture and were optimized by cell-free expression and found to exhibit virtually no measurable dissociation upon binding. Cryo-electron microscopy (cryoEM) showed that these trivalent minibinders engage all three receptor binding domains on a single S trimer. The top candidates neutralize SARS-CoV-2 variants of concern with IC50 values in the low pM range, resist viral escape, and provide protection in highly vulnerable human ACE2-expressing transgenic mice, both prophylactically and therapeutically. Our integrated workflow promises to accelerate the design of mutationally resilient therapeutics for pandemic preparedness. One-Sentence SummaryWe designed, developed, and characterized potent, trivalent miniprotein binders that provide prophylactic and therapeutic protection against emerging SARS-CoV-2 variants of concern.

synthetic biology↗

bioRxiv2020

Designed proteins assemble antibodies into modular nanocages

Antibodies are widely used in biology and medicine, and there has been considerable interest in multivalent antibody formats to increase binding avidity and enhance signaling pathway agonism. However, there are currently no general approaches for forming precisely oriented antibody assemblies with controlled valency. We describe the computational design of two-component nanocages that overcome this limitation by uniting form and function. One structural component is any antibody or Fc fusion and the second is a designed Fc-binding homo-oligomer that drives nanocage assembly. Structures of 8 antibody nanocages determined by electron microscopy spanning dihedral, tetrahedral, octahedral, and icosahedral architectures with 2, 6, 12, and 30 antibodies per nanocage match the corresponding computational models. Antibody nanocages targeting cell-surface receptors enhance signaling compared to free antibodies or Fc-fusions in DR5-mediated apoptosis, Tie2-mediated angiogenesis, CD40 activation, and T cell proliferation; nanocage assembly also increases SARS-CoV-2 pseudovirus neutralization by -SARS-CoV-2 monoclonal antibodies and Fc-ACE2 fusion proteins. We anticipate that the ability to assemble arbitrary antibodies without need for covalent modification into highly ordered assemblies with different geometries and valencies will have broad impact in biology and medicine.

biochemistry↗

bioRxiv2020

F-domain valency determines outcome of signaling through the angiopoietin pathway

Angiopoietin 1 and 2 (Ang1 and Ang2) modulate angiogenesis and vascular homeostasis through engagement of their very similar F-domain modules with the Tie2 receptor tyrosine kinase on endothelial cells. Despite this similarity in the underlying receptor binding interaction, the two angiopoietins have opposite effects: Ang1 induces phosphorylation of protein kinase B (AKT), strengthens cell-cell junctions and enhances endothelial cell survival while Ang2 antagonizes these effects1-4. To investigate the molecular basis for the opposing effects, we examined the protein kinase activation and morphological phenotypes produced by a series of computationally designed protein scaffolds presenting the Ang1 F-domain in a wide range of valencies and geometries. We find two broad phenotypic classes distinguished by the number of presented F-domains: scaffolds presenting 4 F-domains have Ang2 like activity, upregulating pFAK and pERK but not pAKT, and failing to induce cell migration and tube formation, while scaffolds presenting 6 or more F-domains have Ang1 like activity, upregulating pAKT and inducing migration and tube formation. The scaffolds with 8 or more F-domains display superagonist activity, producing stronger phenotypes at lower concentrations than Ang1. When examined in vivo, superagonist icosahedral self-assembling nanoparticles caused significant revascularization in hemorrhagic brains after a controlled cortical impact injury.

biochemistry↗