Biology subjects

Ruohola-Baker, H.

Publications and source records attributed to Ruohola-Baker, H..

At least 19 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↗

bioRxiv2026

Designed Minibinders Rewire Receptor Signaling to Enable Functional Human Myogenic Reprogramming

Sarcopenia, loss of muscle mass is a considerable health burden that demands immediate societal attention. Direct myogenic somatic cell reprogramming, a potential muscle regeneration method is constrained by an inability to control the signaling logic that governs cell fate. Here, we show that this barrier can be overcome using AI-designed receptor modulators. Screening de novo minibinders, we identify a synthetic protein cocktail, C6-DPC, that drives efficient human fibroblast-to-muscle transdifferentiation with robust structural and metabolic maturation. C6-DPC reprograms extracellular signaling by activating pro-myogenic FGFR1/2c pathways while suppressing anti-myogenic inputs through ALK1 and TGFBR2; targeted depletion of ALK1 is sufficient to lower the reprogramming barrier. Inflammatory signaling via gp130 emerges as a dominant checkpoint, and its inhibition further enhances conversion. Engineered tissues generate high twitch and tetanic forces in both wild-type and dystrophin-deficient human cells. These findings demonstrate that programmable synthetic ligands can rewrite receptor-level signaling to direct cell fate and enable functional tissue regeneration.

biochemistry↗

bioRxiv2025

Rapamycin Differentially Impacts Germline Stem Cell Quiescence Across Diverse Genetic Backgrounds of Drosophila Melanogaster

In response to ionizing radiation, stem cells enter a state of temporary quiescence, safeguarding the stem cell pool for tissue regeneration. Quiescence requires the inhibition of mTORC1, a kinase complex that promotes growth and suppresses autophagy, and cell cycle re-entry requires the reactivation of mTORC1. The pharmacological inhibition of mTORC1 in quiescent stem cells by rapamycin prevents cell cycle re-entry and subsequent tissue regeneration. It is well established that pharmacological responses can vary across genetic backgrounds, however, the extent to which genetic variation can impact the quiescence response to rapamycin is unclear. Here, we tested the sensitivity of stem cell quiescence to rapamycin in different genetic backgrounds within the Drosophila Genetic Reference Panel. These analyses revealed substantial variation across different genetic backgrounds, indicating that genetic variation can modulate drug-induced effects on stem cell dynamics. Our analyses suggest that mitophagy, rather than DNA damage response, is associated with the persistence of quiescence and delayed tissue regeneration by rapamycin. This work underscores the critical role of genetic background in determining drug efficacy, highlighting important implications for the therapeutic application of rapamycin.

developmental biology↗

bioRxiv2025

Direct cell reprogramming by a designed agonist inducing HER2-FGFR proximity

Growth factor induced receptor dimerization and activation of downstream pathways can modulate cell fate decisions. Here, we investigate the potential of de novo designed synthetic ligands, termed Novokines, to reprogram cell identity by inducing proximity of novel pairs of receptor subunits. We find that a design, H2F, that brings together HER2 (which has no known natural ligand) and the FGF receptor has potent signaling activity. H2F induces robust signaling and reprograms fibroblasts into myogenic cells. Unlike native FGF ligands, H2F selectively activates the MAPK pathway without engaging PLC{gamma}-mediated Ca{superscript 2} signaling. FRET assays confirm H2F-mediated HER2-FGFR proximity, and phosphoproteomic analysis reveals activation of MAPK effectors. H2F-induced ERK phosphorylation is abolished in cells expressing a kinase-dead FGFR1 (K514M) mutant, confirming the requirement for FGFR catalytic activity. H2F treatment significantly increases myofiber formation from adult patient-derived primary myoblasts, demonstrating its capacity to promote myogenic regeneration. Our findings demonstrate that synthetic receptor pairings can rewire signaling outputs to drive regeneration, providing a programmable platform for cell fate engineering.

biochemistry↗

bioRxiv2025

High-Throughput De Novo Protein Design Yields Novel Immunomodulatory Agonists

Cytokines regulate cell behavior by bringing together specific receptor subunits to trigger downstream signaling. Designed molecules that bring together non-natural receptor pairs could have novel signaling responses and cell specificities. We present a high-throughput de-novo design approach to create novel cytokines by generating and fusing pairs of computationally designed binders. By combining 33 designed receptor-binding domains, we generated over a thousand potential de novo designed "Novokines", of which 75 activated pSTAT signaling in peripheral blood mononuclear cells. We characterized 18 of these, including new pairings of established common receptors, cross-family pairings such as TrkA-{gamma}common, and a series of pairings with interferon receptor-1 (IFNAR1), revealing that IFNAR1 can function as a versatile common receptor similar to {gamma}common or {beta}common. We identify novokines that drive monocyte proliferation, T cell survival and CD4+ T cell-specific proliferation. Our framework provides a blueprint for expanding the understanding of cytokine signaling and generating novel therapeutic proteins.

synthetic biology↗

bioRxiv2025

Human iPSC-derived salivary gland organoids model diabetic salivary gland dysfunction

Salivary glands are highly susceptible to injury and degeneration. To facilitate studies in human salivary gland disease, we developed a rapid protocol for 3D hiPSC-derived salivary gland organoids that recapitulate human fetal salivary gland gene expression and function, have both ductal and acinar cell types, secretory capacity, and the ability to respond to cholinergic agonism. Oral health issues resulting from diabetes mellitus have been attributed to salivary gland dysfunction, leading to chronic xerostomia and increased dental caries. To study diabetic salivary gland hypofunction, we further developed a diabetic model, demonstrating diabetic hallmarks including FOXO1 nuclear localization, AGE-RAGE signaling, and defective oxidative phosphorylation, which were by treatment with the diabetic drug Metformin. Our model has implications for the development of effective therapeutics against salivary gland dysfunction in diabetes and other metabolic diseases in exocrine tissues.

developmental biology↗

bioRxiv2025

Designed NGF mimetics with reduced nociceptive signatures in neurons

The clinical use of Nerve Growth Factor (NGF) for neuronal regeneration has been hampered by pain sensitization side effects. NGF signals through the receptor tyrosine kinase TrkA and the co-receptor p75NTR; pain sensitization is thought to involve p75NTR. We sought to overcome this limitation by de novo design of a TrkA agonist that does not bind p75NTR. We designed homodimeric TrkA engaging constructs that dimerize TrkA subunits in a variety of geometries, and identified those eliciting the strongest signaling. The resulting designed agonists are able to stimulate transdifferentiated neurons and neuroblastoma cell lines, leading to neurite outgrowth and neuronal differentiation, with considerably reduced transcription of inflammation and pain related genes. These agonists are promising candidates for promoting neuronal regeneration without adverse side effects. HighlightsO_LIDe novo designed TrkA agonists activate MAPK and PI3K-AKT signaling C_LIO_LIRigid fusions allow for highly tunable signaling signatures C_LIO_LITrkA agonists lead to neurite outgrowth in neuroblastoma cells comparable to retinoic acid C_LIO_LIModulation of the TrkA pathway without co-stimulating p75NTR leads to a downregulation of inflammatory and nociceptive signature in neurons. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/648806v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@c48066org.highwire.dtl.DTLVardef@c9c5a7org.highwire.dtl.DTLVardef@cf8ebdorg.highwire.dtl.DTLVardef@a4345a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Designed Soluble Notch Agonist Drives Human Ameloblast Maturation for Tooth Regeneration

Enamel, the hardest material in the human body, is required to protect our living organ, tooth. However, over 90% of adults have lost or damaged enamel and cannot regenerate the protective structure due to lack of enamel producing cells, ameloblasts. iPSC derived mature Ameloblasts (iAM) have promise in future regenerative dentistry. Today it is not known why iAM maturation requires intimate contact with the dentin producing cell type, odontoblast. Here we reveal that one of the critical signaling ligands emanating from odontoblasts for ameloblast maturation is Delta, the ligand for Notch receptor. We showed that our designed, soluble Notch agonist can induce iAM organoid maturation in an unprecedented manner, without interactions with odontoblast layer. This novel maturation procedure enables us to analyze the specific requirements of DLX3 function in ameloblasts, independent of its known function in odontoblasts. We now show that DLX3, the gene associated with Amelogenesis Imperfecta, is required on a cell-autonomous manner in ameloblasts for the expression of Enamelin and MMP20.

developmental biology↗

bioRxiv2024

Improved protein binder design using -pairing targeted RFdiffusion

Despite recent advances in the computational design of protein binders, designing proteins that bind with high affinity to polar protein targets remains an outstanding problem. Here we show that RFdiffusion can be conditioned to efficiently generate protein scaffolds that form geometrically matched extended beta-sheets with target protein edge beta-strands in which polar groups on the target are nearly perfectly complemented with hydrogen bonding groups on the design. We use this approach to design binders against a set of therapeutically relevant polar targets (KIT, PDGFR[a], ALK-2, ALK-3, FCRL5, and NRP1) and find that beta-strand-targeted design yields higher affinities and success rates than unconditioned RFdiffusion. All by all binding experiments show that the designs have affinities ranging from 137 pM to mid nM for their targets and essentially no off target binding despite the sharing of beta-strand interactions, likely reflecting the precise customization of interacting beta-strand geometry and additional designed binder-target interactions. A co-crystal structure of one such design in complex with the KIT receptor is nearly identical to the computational design model confirming the accuracy of the design approach. The ability to robustly generate binders displaying high affinity and specificity to polar interaction surfaces with exposed beta-strands considerably increases the range and capabilities of computational binder design.

biochemistry↗

bioRxiv2024

De Novo Design of Integrin α5β1 Modulating Proteins for Regenerative Medicine

Integrin 5{beta}1 is crucial for cell attachment and migration in development and tissue regeneration, and 5{beta}1 binding proteins could have considerable utility in regenerative medicine and next-generation therapeutics. We use computational protein design to create de novo 5{beta}1-specific modulating miniprotein binders, called NeoNectins, that bind to and stabilize the open state of 5{beta}1. When immobilized onto titanium surfaces and throughout 3D hydrogels, the NeoNectins outperform native fibronectin and RGD peptide in enhancing cell attachment and spreading, and NeoNectin-grafted titanium implants outperformed fibronectin and RGD-grafted implants in animal models in promoting tissue integration and bone growth. NeoNectins should be broadly applicable for tissue engineering and biomedicine. One-Sentence SummaryA de novo-designed fibronectin substitute, NeoNectin, is specific for integrin 5{beta}1 and can be incorporated into biomaterials for regenerative medicine.

bioengineering↗

bioRxiv2024

Two non-coding variants associated with isolated orofacial cleft promote binding of transcriptional repressors FOXE1 or ETS2 and reduce expression of IRF6

Oral facial cleft (OFC) is a multifactorial disorder that can present as a cleft lip with or without cleft palate (CL/P) or a cleft palate only. Genome wide association studies (GWAS) of isolated OFC have identified common single nucleotide polymorphisms (SNPs) at the 1q32/IRF6 locus and many other loci where, like IRF6, the presumed OFC-relevant gene is expressed in embryonic oral epithelium. To identify the functional subset of SNPs at eight such loci we conducted a massively parallel reporter assay in a cell line derived from fetal oral epithelium, revealing SNPs with allele-specific effects on enhancer activity. We filtered these against chromatin-mark evidence of enhancers in relevant cell types or tissues, and then tested a subset in traditional reporter assays, yielding six candidates for functional SNPs in five loci (1q32/IRF6, 3q28/TP63, 6p24.3/TFAP2A, 20q12/MAFB, and 9q22.33/FOXE1). We further tested two SNPs near IRF6 and one near FOXE1 by engineering the genome of induced pluripotent stem cells, differentiating the cells into embryonic oral epithelium, and measuring expression of IRF6 or FOXE1 and binding of transcription factors; the results strongly supported their candidacy. Conditional analyses of a meta-analysis of GWAS suggest that the two functional SNPs near IRF6 account for the majority of risk for CL/P associated with variation at this locus. This study connects genetic variation associated with orofacial cleft to mechanisms of pathogenesis.

genetics↗

bioRxiv2023

Modulation of FGF pathway signaling and vascular differentiation using designed oligomeric assemblies

Growth factors and cytokines signal by binding to the extracellular domains of their receptors and drive association and transphosphorylation of the receptor intracellular tyrosine kinase domains, initiating downstream signaling cascades. To enable systematic exploration of how receptor valency and geometry affects signaling outcomes, we designed cyclic homo-oligomers with up to 8 subunits using repeat protein building blocks that can be modularly extended. By incorporating a de novo designed fibroblast growth-factor receptor (FGFR) binding module into these scaffolds, we generated a series of synthetic signaling ligands that exhibit potent valency- and geometry-dependent Ca2+ release and MAPK pathway activation. The high specificity of the designed agonists reveal distinct roles for two FGFR splice variants in driving endothelial and mesenchymal cell fates during early vascular development. The ability to incorporate receptor binding domains and repeat extensions in a modular fashion makes our designed scaffolds broadly useful for probing and manipulating cellular signaling pathways. HighlightsO_LIDe novo designed cyclic oligomers with tunable geometric properties C_LIO_LICyclic, homo-oligomeric FGFR binding modules induce geometry- and valency-dependent activity of isoform-specific FGF signaling C_LIO_LIModulation of FGFR isoform activity controls bifurcation of endothelial and mesenchymal fate during vascular development C_LIO_LIC-isoform activation favors arterial endothelial cell formation while B-isoform induces pericyte differentiation C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/532666v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@151f6eforg.highwire.dtl.DTLVardef@2ae877org.highwire.dtl.DTLVardef@103e0d1org.highwire.dtl.DTLVardef@19cd695_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

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

Robust de novo design of protein binding proteins from target structural information alone

The design of proteins that bind to a specific site on the surface of a target protein using no information other than the three-dimensional structure of the target remains an outstanding challenge. We describe a general solution to this problem which starts with a broad exploration of the very large space of possible binding modes and interactions, and then intensifies the search in the most promising regions. We demonstrate its very broad applicability by de novo design of binding proteins to 12 diverse protein targets with very different shapes and surface properties. Biophysical characterization shows that the binders, which are all smaller than 65 amino acids, are hyperstable and bind their targets with nanomolar to picomolar affinities. We succeeded in solving crystal structures of four of the binder-target complexes, and all four are very close to the corresponding computational design models. Experimental data on nearly half a million computational designs and hundreds of thousands of point mutants provide detailed feedback on the strengths and limitations of the method and of our current understanding of protein-protein interactions, and should guide improvement of both. Our approach now enables targeted design of binders to sites of interest on a wide variety of proteins for therapeutic and diagnostic applications.

synthetic 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↗

bioRxiv2021

Stem cell quiescence requires PRC2/PRC1-mediated mitochondrial checkpoint

Both normal and tumorous stem cells can arrest cell division, avoid apoptosis, and then regenerate lost daughter cells following acute genotoxic insult. This protective, reversible proliferative arrest, known as "quiescence," is still poorly understood. Here, we show that mTOR-regulated mitophagy is required for radiation insult-induced quiescence in Drosophila germline stem cells (GSCs). In GSCs, depletion of mito-fission (Drp1) or mitophagy (Pink1 and Parkin) eliminates entry into quiescence, while depletion of mitochondrial biogenesis (PGC1) or fusion (Mfn2) eliminates exit from quiescence. We also find that mitophagy-dependent quiescence is under epigenetic control; knockdown of Jarid2 (PRC2) or Pc or Sce (PRC1) stabilizes the mitochondria and locks GSCs out of quiescence, while knockdown of PRC2-specific demethylase, Utx, prevents re-accumulation of the mitochondria and locks GSCs in quiescence. These data suggest that mitochondrial number coordinates reversible quiescence. We further identify that the mechanism of quiescence in both GSCs and human induced pluripotent stem cells (iPSCs) relies on mitophagy to deplete the mitochondrial pool of CycE and limit cell cycle progression. This alternative method of G1/S regulation may present new opportunities for therapeutic purposes.

biochemistry↗

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↗