Biology subjects

Zhang, J. Z.

Publications and source records attributed to Zhang, J. Z..

At least 19 recordsLinked to original sources

bioRxiv2026

Vascularized Human Cardiac Organoids Reveal Endothelial-Cardiomyocyte Crosstalk and Mechanisms of Carfilzomib-Induced Cardiotoxicity

Human cardiac organoids (hCOs) provide a powerful platform for modeling heart development and disease, yet their lack of vasculature limits physiological relevance. Here, we generate vascularized human cardiac organoids (vhCOs) by assembling hCOs with blood vessel organoids, enabling the self-organized formation of unified tissues with extensive vascular networks and substantially enhanced cardiomyocyte (CM) maturation. Single-nucleus multiomic profiling reveals CM state resembling the 17-week human fetal heart, while endothelial cells (ECs) acquire cardiac-specific and arterial-like identities. Integrative cell-cell communication analyses and experimental validation identify CM-derived Laminin-2 as a driver of arterial EC specification, while reciprocal EFNB2 signaling from arterial ECs promotes CM maturation, uncovering bidirectional cardiac-vascular crosstalk. Modeling carfilzomib-induced cardiotoxicity in vhCOs reveals ATF4-dependent endoplasmic reticulum (ER) stress and IL8-mediated inflammatory responses that are blunted in non-vascularized hCOs. Pharmacological attenuation of ER stress alleviates carfilzomib-induced cardiotoxicity. Altogether, vhCOs provide a developmentally advanced and physiologically faithful human cardiac model.

cell biology↗

bioRxiv2026

TRU-PE: A Universal, Trackable Prime Editor Toolkit for Robust Single- and Multi-Locus Genome Engineering

Prime editing (PE) holds immense promise for precise genome editing, but its wider adoption is constrained by large payloads and inefficient delivery in recalcitrant cell types. Here, we present TRU-PE (Trackable, Robust, and Universal Prime Editor), a toolkit that overcomes these barriers via a split-vector architecture coupled with fluorescence-guided enrichment. This design ensures stoichiometric component expression, yielding marked improvements in editing efficiency and accelerated workflows compatible with both plasmid and viral delivery. By integrating a polycistronic hCtRNA-sgRNA/pegRNA array, the derivative TRU-MPE system enables simultaneous editing of up to ten genomic loci. Furthermore, it enhances the performance of evolved PE editors, allows flexible vector stoichiometry and inspires novel editing reporters. Leveraging these capabilities, we successfully generated a combinatorial human iPSC-based platform for GATA2 deficiency, which accurately recapitulates genotype-specific hematopoietic defects. TRU-PE thus provides a user-friendly, scalable framework for robust single- and multi-locus genome engineering, accelerating functional genomics and therapeutic development.

genetics↗

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

Bioelectricity generation by Symbiodinium microadriaticum: a symbiont-forming photosynthetic dinoflagellate alga from coral reefs

Climate change is intensifying the phenomenon of coral bleaching, a highly ecologically damaging process that stems from the breakdown in the symbiotic relationship between photosynthetic dinoflagellate algae and Cnidaria (animals). Currently, little is understood about bleaching at the molecular level, since changes in metabolic and redox interactions between the dinoflagellate algae and Cnidaria are challenging to study. Here, we developed electrochemical approaches to measure extracellular electron transfer (EET, a type of bioelectricity) in Symbiodinium microadriaticum, a model species of symbiont-forming dinoflagellate algae, and show how this can be used to assess intracellular bioenergetic fluctuations within the alga. We show that EET is dependent on two major intracellular pathways - photosynthesis and respiration - and reveal that expelled electrons exit the cell via diffusible electron carriers. We confirm that EET activity can be affected by environmental stressors linked to coral bleaching, such as changes in temperature, pH and light intensity. Overall, this is a direct and non-invasive approach to provide quantitative measurements of S. microadriaticums bioenergetics and redox exchanges with the environment in stress conditions. This platform can aid our understanding of the coral-dinoflagellate symbiosis and the molecular mechanisms of bleaching.

microbiology↗

bioRxiv2025

De novo design of miniprotein agonists and antagonists targeting G protein-coupled receptors

G protein-coupled receptors (GPCRs) play key roles in physiology and are central targets for drug discovery and development, but the design of protein agonists and antagonists has been challenging as GPCRs are integral membrane proteins and conformationally dynamic. Here we describe computational de novo design methods and a high throughput "receptor diversion" microscopy-based screen for generating GPCR binding miniproteins with high affinity, potency and selectivity, and the use of these methods to generate agonists for MRGPRX1, NK1R and CCR5, as well as antagonists for CXCR4, CCR5, OXTR, GLP1R, GIPR, GCGR, PTH1R and CGRPR.. Cryo-electron microscopy data reveals atomic-level agreement between designed and experimentally determined structures for CGRPR- and CXCR4-bound antagonists and MRGPRX1-bound agonists. Our de novo design and screening approach opens new frontiers in GPCR drug discovery and development.

bioengineering↗

bioRxiv2025

Dissecting a biological electron transport network with electrochemistry

Photosynthetic membranes contain complex networks of redox proteins and molecules which direct electrons along various energy-to-chemical interconversion reactions important for sustaining life on Earth. Analysing and disentangling the mechanisms, regulation and interdependencies of these electron transfer pathways is extremely difficult, owing to the large number of interacting components in the native membrane environment. Whilst electrochemistry is well established for studying electron transfer in purified proteins, it has proven difficult to directly wire into proteins within their native membrane environments, and even harder to probe on a systems-level the electron transfer networks they are entangled within. Here, we show how photosynthetic membranes from cyanobacteria can be directly wired to electrodes to access their complex electron transfer networks. Measurements of native membranes with structured electrodes revealed distinctive electrochemical signatures, enabling analysis from the scale of individual proteins to entire biochemical pathways, as well as their interplay. This includes measurements of overlapping photosynthetic and respiratory pathways, the redox activities of membrane-bound quinones, along with validation using in operando spectroscopic measurements. Importantly, we further demonstrated direct extraction of electrons from native membrane-bound Photosystem I at -600 mV, which is [~]1 V more negative than from purified photosystems. This finding opens up opportunities for biotechnologies for solar electricity, fuel and chemical generation. We foresee this electrochemical method being adapted to analyze other photosynthetic and non-photosynthetic membranes, as well as aiding the development of new biocatalytic, biohybrid and biomimetic systems.

biochemistry↗

bioRxiv2025

Bayesian phylodynamic inference of population dynamics with dormancy

Many organisms employ reversible dormancy, or seedbank, in response to environmental fluctuations. This life-history strategy alters fundamental eco-evolutionary forces, leading to distinct patterns of genetic diversity. Two models of dormancy have been proposed based on the average duration of dormancy relative to coalescent timescales: weak seedbank, induced by scheduled seasonality (e.g., plants, invertebrates), and strong seedbank, where individuals stochastically switch between active and dormant states (e.g., bacteria, fungi). The weak seedbank coalescent is statistically equivalent to the Kingman coalescent with a scaled mutation rate, allowing the use of existing inference methods. In contrast, the strong seedbank coalescent differs fundamentally, as only active lineages can coalesce, while dormant lineages cannot. Additionally, dormant individuals typically mutate at a slower rate than active ones. Consequently, despite the significant role of dormancy in the eco-evolutionary dynamics of many organisms, no methods currently exist for inferring population dynamics involving dormancy and associated parameters. We present a Bayesian framework for jointly inferring a latent genealogy, seedbank parameters, and evolutionary parameters from molecular sequence data under the strong seedbank coalescent. We derive the exact probability density of genealogies sampled under the strong seedbank coalescent, characterize the corresponding likelihood function, and present efficient computational algorithms for its evaluation based on our theoretical framework. We develop a tailored Markov chain Monte Carlo sampler and implement our inference framework as a package SeedbankTree within BEAST2. Our work provides both a theoretical foundation and practical inference framework for studying the population genetic and genealogical impacts of dormancy.

evolutionary biology↗

bioRxiv2024

Admixed populations in the neighbor-joining algorithm: a geometric analysis with five taxa

The Neighbor-Joining (NJ) algorithm is a widely used method for constructing phylogenetic trees from genetic distances. While NJ is known to perform well with tree-like data, its behavior under admixture remains understudied. In this work, we present a geometric framework for analyzing the NJ algorithm under a linear admixture model. We focus on three key properties related to clustering order, distance, and topological path length in the resulting NJ trees involving five taxa. Our approach leverages polyhedral geometry to define NJ cones, which correspond to distinct cherry-picking orders and partition the space of dissimilarity vectors. We project dissimilarity vectors with admixture into a lower-dimensional space without admixture, defining polyhedral regions induced by NJ cones that satisfy specified properties. We compute the exact probabilities that these properties hold by directly calculating the volumes of the induced NJ cones and compare them with Monte Carlo integration and standard NJ simulation methods. Our results show that the property on clustering order is always satisfied, while the other properties are highly probable but depend on the admixture fraction. We also prove that certain induced NJ cones have zero volume, indicating that the corresponding NJ tree topologies are infeasible under admixture. We have implemented our methods as a publicly available module NeighborJoining within Macaulay2, providing an efficient tool for analyzing NJ cones and their properties. This work provides new insights into the geometric structure inherent to the NJ algorithm in the presence of admixture, identifying the conditions under which admixture influences the resulting phylogenetic trees.

evolutionary biology↗

bioRxiv2024

De novo design of Ras isoform selective binders

The proto-oncogene Ras which governs diverse intracellular pathways has four major isoforms (KRAS4A, KRAS4B, HRAS, and NRAS) with substantial sequence homology and similar in vitro biochemistry. There is considerable interest in investigating the roles of these independently as their association with different cancers vary, but there are few Ras isoform-specific binding reagents as the only significant sequence differences are in their disordered and highly charged C-termini which have been difficult to elicit antibodies against. To overcome this limitation, we use deep learning-based methods to de novo design Ras isoform-specific binders (RIBs) for all major Ras isoforms that specifically target the Ras C-terminus. The RIBs bind to their target Ras isoforms both in vitro and in cells with remarkable specificity, disrupting their membrane localization and inhibiting Ras activity, and should contribute to dissecting the distinct roles of Ras isoforms in biology and disease.

bioengineering↗

bioRxiv2024

Manganese transporter SLC30A10 and iron transporters SLC40A1 and SLC11A2 impact dietary manganese absorption

SLC30A10 deficiency is a disease of severe manganese excess attributed to loss of SLC30A10-dependent manganese excretion via the gastrointestinal tract. Patients develop dystonia, cirrhosis, and polycythemia. They are treated with chelators but also respond to oral iron, suggesting that iron can outcompete manganese for absorption in this disease. Here we explore the latter observation. Intriguingly, manganese absorption is increased in Slc30a10-deficient mice despite manganese excess. Studies of multiple mouse models indicate that increased dietary manganese absorption reflects two processes: loss of manganese export from enterocytes into the gastrointestinal tract lumen by SLC30A10, and increased absorption of dietary manganese by iron transporters SLC11A2 (DMT1) and SLC40A1 (ferroportin). Our work demonstrates that aberrant absorption contributes prominently to SLC30A10 deficiency and expands our understanding of biological interactions between iron and manganese. Based on these results, we propose a reconsideration of the role of iron transporters in manganese homeostasis is warranted.

genetics↗

bioRxiv2023

Single-cell signaling analysis reveals that Major Vault Protein facilitates RasG12C inhibitor resistance

Recently developed covalent inhibitors for RasG12C provide the first pharmacological tools to target mutant Ras-driven cancers. However, the rapid development of resistance to current clinical Ras G12C inhibitors is common. Presumably, a subpopulation of RasG12C-expressing cells adapt their signaling to evade these inhibitors and the mechanisms for this phenomenon are unclear due to the lack of tools that can measure signaling with single-cell resolution. Here, we utilized recently developed Ras sensors to profile the environment of active Ras and to measure the activity of endogenous Ras in order to pair structure (Ras signalosome) to function (Ras activity), respectively, at a single-cell level. With this approach, we identified a subpopulation of KRasG12C cells treated with RasG12C-GDP inhibitors underwent oncogenic signaling and metabolic changes driven by WT Ras at the golgi and mutant Ras at the mitochondria, respectively. Our Ras sensors identified Major Vault Protein (MVP) as a mediator of Ras activation at both compartments by scaffolding Ras signaling pathway components and metabolite channels. We found that recently developed RasG12C-GTP inhibitors also led to MVP-mediated WT Ras signaling at the golgi, demonstrating that this a general mechanism RasG12C inhibitor resistance. Overall, single-cell analysis of structure-function relationships enabled the discovery of a RasG12C inhibitor-resistant subpopulation driven by MVP, providing insight into the complex and heterogenous rewiring occurring during drug resistance in cancer.

cancer biology↗

bioRxiv2023

De novo designed Hsp70 activator dissolves intracellular condensates

Protein quality control (PQC) is carried out in part by the chaperone Hsp70, in concert with adapters of the J-domain protein (JDP) family. The JDPs, also called Hsp40s, are thought to recruit Hsp70 into complexes with specific client proteins. However, the molecular principles regulating this process are not well understood. We describe the de novo design of a set of Hsp70 binding proteins that either inhibited or stimulated Hsp70s ATPase activity; a stimulating design promoted the refolding of denatured luciferase in vitro, similar to native JDPs. Targeting of this design to intracellular condensates resulted in their nearly complete dissolution. The designs inform our understanding of chaperone structure-function relationships and provide a general and modular way to target PQC systems to condensates and other cellular targets.

biochemistry↗

bioRxiv2023

Designed Endocytosis-Triggering Proteins mediate Targeted Degradation and amplify signaling

Endocytosis and lysosomal trafficking of cell surface receptors can be triggered by interaction with endogenous ligands. Therapeutic approaches such as LYTAC1,2 and KineTAC3, have taken advantage of this to target specific proteins for degradation by fusing modified native ligands to target binding proteins. While powerful, these approaches can be limited by possible competition with the endogenous ligand(s), the requirement in some cases for chemical modification that limits genetic encodability and can complicate manufacturing, and more generally, there may not be natural ligands which stimulate endocytosis through a given receptor. Here we describe general protein design approaches for designing endocytosis triggering binding proteins (EndoTags) that overcome these challenges. We present EndoTags for the IGF-2R, ASGPR, Sortillin, and Transferrin receptors, and show that fusing these tags to proteins which bind to soluble or transmembrane protein leads to lysosomal trafficking and target degradation; as these receptors have different tissue distributions, the different EndoTags could enable targeting of degradation to different tissues. The modularity and genetic encodability of EndoTags enables AND gate control for higher specificity targeted degradation, and the localized secretion of degraders from engineered cells. The tunability and modularity of our genetically encodable EndoTags should contribute to deciphering the relationship between receptor engagement and cellular trafficking, and they have considerable therapeutic potential as targeted degradation inducers, signaling activators for endocytosis-dependent pathways, and cellular uptake inducers for targeted antibody drug and RNA conjugates.

synthetic biology↗

bioRxiv2023

Computational design of non-porous, pH-responsive antibody nanoparticles

Programming protein nanomaterials to respond to changes in environmental conditions is a current challenge for protein design and important for targeted delivery of biologics. We describe the design of octahedral non-porous nanoparticles with the three symmetry axes (four-fold, three-fold, and two-fold) occupied by three distinct protein homooligomers: a de novo designed tetramer, an antibody of interest, and a designed trimer programmed to disassemble below a tunable pH transition point. The nanoparticles assemble cooperatively from independently purified components, and a cryo-EM density map reveals that the structure is very close to the computational design model. The designed nanoparticles can package a variety of molecular payloads, are endocytosed following antibody-mediated targeting of cell surface receptors, and undergo tunable pH-dependent disassembly at pH values ranging between to 5.9-6.7. To our knowledge, these are the first designed nanoparticles with more than two structural components and with finely tunable environmental sensitivity, and they provide new routes to antibody-directed targeted delivery.

bioengineering↗

bioRxiv2023

Hypoxia-inducible factor 2 is a key determinant of manganese excess and polycythemia in SLC30A10 deficiency

Manganese is an essential yet potentially toxic metal. Initially reported in 2012, mutations in SLC30A10 are the first known inherited cause of manganese excess. SLC30A10 is an apical membrane transport protein that exports manganese from hepatocytes into bile and from enterocytes into the lumen of the gastrointestinal tract. SLC30A10 deficiency results in impaired gastrointestinal manganese excretion, leading to severe manganese excess, neurologic deficits, liver cirrhosis, polycythemia, and erythropoietin excess. Neurologic and liver disease are attributed to manganese toxicity. Polycythemia is attributed to erythropoietin excess, but the basis of erythropoietin excess in SLC30A10 deficiency has yet to be established. Here we demonstrate that erythropoietin expression is increased in liver but decreased in kidneys in Slc30a10-deficient mice. Using pharmacologic and genetic approaches, we show that liver expression of hypoxia-inducible factor 2 (Hif2), a transcription factor that mediates the cellular response to hypoxia, is essential for erythropoietin excess and polycythemia in Slc30a10-deficient mice, while hypoxia-inducible factor 1 (HIF1) plays no discernible role. RNA-seq analysis determined that Slc30a10-deficient livers exhibit aberrant expression of a large number of genes, most of which align with cell cycle and metabolic processes, while hepatic Hif2 deficiency attenuates differential expression of half of these genes in mutant mice. One such gene downregulated in Slc30a10-deficient mice in a Hif2-dependent manner is hepcidin, a hormonal inhibitor of dietary iron absorption. Our analyses indicate that hepcidin downregulation serves to increase iron absorption to meet the demands of erythropoiesis driven by erythropoietin excess. Finally, we also observed that hepatic Hif2 deficiency attenuates tissue manganese excess, although the underlying cause of this observation is not clear at this time. Overall, our results indicate that HIF2 is a key determinant of pathophysiology in SLC30A10 deficiency. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=148 HEIGHT=200 SRC="FIGDIR/small/529270v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@c25fc8org.highwire.dtl.DTLVardef@11e70feorg.highwire.dtl.DTLVardef@18c5518org.highwire.dtl.DTLVardef@26b61f_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2022

Designed sensors reveal normal and oncogenic Ras signaling in endomembranes and condensates

Genetically encoded biosensors have accelerated biological discovery, however many important targets such as active Ras (Ras-GTP) are difficult to sense as strategies to match a sensors sensitivity to the physiological range of target are lacking. Here, we use computational protein design to generate and optimize intracellular sensors of Ras activity (LOCKR-based Sensor for Ras activity: Ras-LOCKR-S) and proximity labelers of the signaling environment of Ras (LOCKR-based, Ras activity-dependent Proximity Labeler: Ras-LOCKR-PL). We demonstrate that our tools can measure endogenous Ras activity and environment at subcellular resolution. We illustrate the application of these tools by using them to identify Ras effectors, notably Src-Associated in Mitosis 68 kDa protein (SAM68), enriched in oncogenic EML4-Alk granules. Localizing these sensors to these granules revealed that SAM68 enhances Ras activity specifically at the granules, and SAM68 inhibition sensitizes EML4-Alk-driven cancer cells to existing drug therapies, suggesting a possible therapeutic strategy.

cell biology↗

bioRxiv2021

A modular toolset for electrogenetics

Synthetic biology research and its industrial applications rely on the deterministic spatiotemporal control of gene expression. Recently, electrochemical control of gene expression has been demonstrated in electrogenetic systems (redox-responsive promoters used alongside redox inducers and an electrode), allowing for the direct integration of electronics with complex biological processes for a variety of new applications. However, the use of electrogenetic systems is limited by poor activity, tunability and standardisation. Here, we have developed a variety of genetic and electrochemical tools that facilitate the design and vastly improve the performance of electrogenetic systems. We developed a strong, unidirectional, redox-responsive promoter before deriving a mutant promoter library with a spectrum of strengths. We then constructed genetic circuits with these parts and demonstrated their activation by multiple classes of redox molecules. Finally, we demonstrated electrochemical activation of gene expression in aerobic conditions utilising a novel, modular bioelectrochemical device. This toolset provides researchers with all the elements needed to design and build optimised electrogenetic systems for specific applications.

synthetic biology↗

bioRxiv2021

Detection of antibodies neutralizing historical and emerging SARS-CoV-2 strains using a thermodynamically coupled de novo biosensor system

With global vaccination efforts against SARS-CoV-2 underway, there is a need for rapid quantification methods for neutralizing antibodies elicited by vaccination and characterization of their strain dependence. Here, we describe a designed protein biosensor that enables sensitive and rapid detection of neutralizing antibodies against wild type and variant SARS-CoV-2 in serum samples. More generally, our thermodynamic coupling approach can better distinguish sample to sample differences in analyte binding affinity and abundance than traditional competition based assays.

molecular biology↗