Biology subjects

Huang, M.

Publications and source records attributed to Huang, M..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Resolving Heterogeneity in Major Depression: Overcoupling and Undercoupling Subtypes Exhibit Differential Treatment Response and Molecular Pathways

Major depressive disorder (MDD) exhibits significant heterogeneity whose neurobiological mechanisms remain elusive. Alterations in morphological-functional coupling (MFC) have been observed in MDD. This study aims to investigate MDD subtypes based on MFC alterations and their associations with clinical symptoms and molecular basis. We identified two clinically distinct MDD subtypes in multi-center neuroimaging data (Discovery: 828 MDD/776 healthy controls; Validation: 236 MDD/86 healthy controls) by using a semi-supervised machine learning approach based on MFC changes, which were validated and robustly repeated in the validation dataset. Differences among subtypes were then examined in relation to clinical assessments, gene expression patterns, neurotransmitter and cell density, and treatment response. Subtype I (overcoupling) showed elevated MFC in high-order association cortices, linking to synaptic transmission activity and severe symptoms. Subtype II (undercoupling) demonstrated reduced MFC in primary cortices, associated with cell cycle regulation and treatment resistance. Both subtypes shared molecular signatures (cell types: astrocytes and oligodendrocytes; neurotransmitter systems: serotonergic and GABAergic receptors). Crucially, longitudinal data (33 MDD undergoing Escitalopram monotherapy) revealed that undercoupling subtype exhibited better response, implying a potential mechanism of the drug via increasing the MFC. Our work deciphers MFC-driven MDD phenotypes with distinct molecular profiles and differential treatment outcomes, suggesting potential pathways for personalized diagnosis and treatment strategies in MDD, advancing precision psychiatry.

neuroscience↗

bioRxiv2025

An Electromechanical Relay-like Multi-enzymatic System Gates c-di-GMP-Dependent Cell Fate in a Cyanobacterium

Cyclic-di-GMP is a ubiquitous bacterial second messenger that regulates diverse cellular processes. Although many bacteria harbor multiple enzymes for metabolism, the mechanisms underlying coordinated regulation remain unclear. Using the cyanobacterium Anabaena PCC 7120, we generate mutant strains with varying numbers of c-di-GMP metabolic genes deleted, including cdg0 (all degradation genes deleted) and cdgmax (all synthesis genes deleted). We found that cyclic-di-GMP is essential for both cell viability and size regulation. Quantitative analysis identified two critical physiological thresholds: one for maintaining normal cell size and a lower, lethal threshold required for survival. The 16 cyclic-di-GMP metabolic enzymes function as an electromechanical-like dual relay system, where different enzyme groups maintain cyclic-di-GMP homeostasis or activate SOS-like alarm responses when cyclic-di-GMP concentrations drop below the lethal threshold. These regulatory effects are mediated by the cyclic-di-GMP receptor CdgR, depending on the fraction of apo-CdgR form. This dual-threshold system enables dynamic cellular adaptation while preventing lethal consequences, representing a fundamental growth-survival trade-off strategy in living organisms.

microbiology↗

bioRxiv2025

KIF5A binds RNA to orchestrate synaptic mRNA localization and stress granules in ALS

Neuronal health depends on the precise transport and local translation of mRNAs to maintain synaptic function across highly polarized cellular architecture. While kinesin motor proteins are known to mediate mRNA transport, the specificity and direct involvement of individual kinesins as RNA-binding proteins (RBPs) remain unclear. Here, we demonstrate that KIF5A, a neuron-specific kinesin implicated in amyotrophic lateral sclerosis (ALS), functions as an RBP. We show that KIF5A directly binds mRNAs encoding synaptic ribosomal proteins and is required for their synaptic localization and for maintaining normal synaptic composition and function. Additionally, we show ALS-linked KIF5A mutations confer gain-of-function properties, enhancing mRNA binding, increasing synaptic ribosomal protein accumulation, inducing neuronal hyperexcitability, and impairing stress responses. These findings reveal a previously unrecognized mechanism by which mutant KIF5A disrupts synaptic homeostasis. Our work positions a kinesin motor protein as an RBP with critical roles in mRNA transport, local translation, and stress response. HighlightsO_LIKIF5A interacts with mRNA encoding synaptic ribosomal proteins C_LIO_LIKIF5A is required for normal synaptic composition and function C_LIO_LIKIF5A binds to G3BP1 and G3BP1 stress granule associated proteins C_LIO_LIKIF5A mutant ALS patient-derived motor neurons have abnormal synaptic function and stress response C_LI

neuroscience↗

bioRxiv2025

Dosage-sensitive RBFOX2 autoregulation promotes cardiomyocyte differentiation through transcriptome maturation

Haploinsufficiency of the RNA splicing regulator, RBFOX2, is linked to congenital heart disease (CHD), yet its pathogenic mechanisms remain unclear. Here, we demonstrate that RBFOX2 is essential for progressing cardiomyocyte (CM) differentiation by shifting exon usage profiles to more mature patterns in sarcomere, cytoskeletal, and focal adhesion genes, including alpha-actinin-2 (ACTN2). This maturation program is initiated by critical levels of RBFOX2 that facilitate autoregulatory splicing at mutually exclusive exons encoding early and late isoforms with distinct functional roles. In heterozygous CMs, autoregulation is disrupted, which skews isoform ratios and generates a dominant-negative product caused by exon co-inclusion. Finally, we demonstrate that overexpression of ACTN2 rescues heterozygous, but not null, phenotypes by restoring contractility, which triggers a mechanosensing feedback loop involving upregulation of RBFOX2 from the wildtype allele and transcriptome maturation. Our data suggest that decreased RBFOX2 dosage and autoregulation impair CM differentiation, contributing to CHD pathogenesis and heart failure susceptibility.

developmental biology↗

bioRxiv2025

PlantScience.ai: An LLM-Powered Virtual Scientist for Plant Science

The accelerating growth of plant science literature presents a major challenge for researchers seeking to extract accurate, up-to-date knowledge from an increasingly fragmented and domain-specific corpus. General-purpose large language models (LLMs), while powerful, often misinterpret plant science terminology and lack mechanisms for source traceability. We created PlantScience.ai, a virtual plant biology scientist powered by our automated scientific knowledge graph construction pipeline (AutoSKG). PlantScience.ai exhibits expert-level reasoning in plant biology and maintains scholarly rigour in its citations. Through continuous learning, it integrates the latest research, ensuring that its knowledge base remains current and scientifically robust. Apart from providing the answers to the scientific questions, PlantScience.ai can interact with human scientists, follow instructions, and retrieve information with citation awareness, grounding each response in primary sources to ensure accuracy and verifiability. PlantScience.ai marks a pivotal advance toward a collaborative scientific paradigm in which virtual and human plant scientists work synergistically to accelerate discovery while preserving the unique value of human insight. PlantScience.ai is available at https://plantscience.ai.

plant biology↗

bioRxiv2025

NyxBind: enhancing DNN representations via contrastive learning for TFBS prediction

While pretrained genomic language models effectively capture general DNA sequence patterns through masked language modeling, they often struggle to discriminate subtle yet biologically critical differences among transcription factor binding site (TFBS) motifs. Recent studies suggest that contrastive learning can enhance the discriminative power of embeddings by explicitly modeling inter-instance similarities and differences. Building on this insight, we introduce NyxBind, the first TFBS prediction model that applies contrastive learning across multiple TFBS types to enhance regulatory sequence representations. NyxBind better captures discriminative sequence features, enabling more accurate and biologically meaningful TFBS prediction. Extensive evaluations show that NyxBind consistently outperforms alternative models across multiple TFBS classification benchmarks, demonstrating strong robustness and generalizability. Moreover, NyxBind supports both full-parameter and parameter-efficient fine-tuning while maintaining high performance, and supports accurate motif visualization, aligning closely with experimentally validated transcription factor binding profiles. The code are available at https://github.com/ai4nucleome/NyxBind.

bioinformatics↗

bioRxiv2025

Pharmacophore-driven antibody discovery on the yeast surface

Protein-small molecule hybrids are structures capable of combining the inhibitory properties of small molecules and the specificities of binding proteins. However, discovery of such synergistic conjugates is a substantial engineering challenge. Here, we describe pharmacophore-driven antibody discovery as a high throughput approach to hybrid discovery. In this approach, we use a yeast display antibody library containing reactive noncanonical amino acids (ncAAs) and further diversify it by conjugating the library to four sulfonamide pharmacophores. Yeast display binding screens with each of the resulting billion-member hybrid collections against bovine carbonic anhydrase (bCA) yielded diverse collections of hybrids. Individual hybrids exhibited double digit nanomolar binding affinities, and frequently exhibited inhibitory properties in solution, despite the fact that the screens were based solely on binding phenotypes. Deep sequencing of sorted populations revealed that enrichments were strongly pharmacophore-dependent. In particular, screens with a potent pharmacophore led to collections of hybrids varying substantially in pharmacophore attachment point and antibody sequence features, while screens with moderate or weak pharmacophores led to collections with much narrower sets of enriched attachment points and antibody sequence features. Identification of the most frequently isolated CDR-H3 sequences and clustering CDR-H3 sequences by similarity within enriched populations provided further evidence for pharmacophore-dependent sorting outcomes. Experimental binding assays in which the pharmacophore warhead used during screening was replaced by another warhead indicated that isolated clones can tolerate alternative pharmacophores, but tend to prefer the warhead used during screening. Overall, these efforts demonstrate the utility of introducing pharmacophores into antibody libraries along with several lines of evidence that screening outcomes are pharmacophore-driven. These findings advance our understanding of hybrid discovery and highlight opportunities to pursue hybrids as research tools and potential therapeutic leads. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/682880v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@11c2a65org.highwire.dtl.DTLVardef@237055org.highwire.dtl.DTLVardef@a3fb4borg.highwire.dtl.DTLVardef@1c71700_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Targeting Endothelial PERK Accelerates Lymphoid Regeneration by Enhancing DLL4-NOTCH3 Signaling at the Pre-B Niche

Delayed immune recovery after hematopoietic stem cell (HSC) transplantation is associated with a poor clinical outcome, yet strategies to enhance lymphocyte regeneration are limited. We studied the role of unfolded protein response (ER stress) in hematopoietic regeneration within the bone marrow (BM) microenvironment. We revealed that PERK activation is a prominent feature of BM endothelium in leukemia patients and is a hallmark response in mouse BM following ionizing irradiation. Ablating endothelial Perk boosted Notch ligand DLL4 expression and promoted DLL4-dependent early HSC and B progenitor regeneration. Single-cell analysis shows that endothelial DLL4 activates NOTCH3 expressed by mesenchymal stroma cells, and that the PERK-DLL4 axis coordinates the regulation of lymphoid commitment and niche cytokine production. NOTCH3 is critical for the upregulation of IL7 following irradiation and for supporting the expansion of lymphoid progenitors in mesenchymal sphere cultures. These findings not only unveil a previously unrecognized ER stress-controlled vascular-stroma signaling mechanism in regenerative hematopoiesis but also highlight PERK blockade as a promising therapeutic strategy to improve immune recovery after myeloablative transplantation. SummaryZou et al unravel that the adaptive ER stress response in bone marrow blood vessels restricts the post-transplant regeneration of immune progenitor cells by attenuating the expression of Notch ligand DLL4. Targeting ER stress sensor PERK can accelerate immune recovery after transplantation by enhancing DLL4-NOTCH3 signaling and IL7 cytokine production.

cell biology↗

bioRxiv2025

Convergent utilization of APN receptor by hedgehog merbecoviruses

Dipeptidyl peptidase-4 (DPP4) and angiotensin-converting enzyme 2 (ACE2) are well-established receptors for merbecoviruses, yet the receptor usage of merbecoviruses of European and Asian hedgehogs (EriCoVs) remains unknown. Here, by testing hedgehog orthologs of known coronavirus receptors, we identify hedgehog aminopeptidase N (APN) as a functional receptor for EriCoVs. Analysis of 94 APN orthologs indicates that EriCoVs have a limited host range, primarily utilizing hedgehog APN and, to a lesser extent, APN from certain felids, shaped by specific determinants at the virus-receptor interface. Cryo-EM reveals an APN-binding mode distinct from those used by alpha- and deltacoronaviruses. Functional assays indicate that hedgehog transmembrane serine protease 2 (TMPRSS2) enhances spike activation and promotes pseudovirus entry. Neutralizing antibodies targeting RBD and APN were developed and could effectively block EriCoV pseudovirus entry and propagation. These findings reveal an unexpected convergent evolution of APN utilization among merbecovirus, establishing a foundation for risk assessment and countermeasure development. Graphic abstractConvergent utilization of hedgehog APN by EriCoVs O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/676546v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1ea334aorg.highwire.dtl.DTLVardef@cd53bforg.highwire.dtl.DTLVardef@352102org.highwire.dtl.DTLVardef@a1eed_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Adiponectin plays a key role in macrophage-driven inflammation of adipose tissue caused by Chronic Intermittent Hypoxia

BackgroundThe inflammatory response driven by macrophages in adipose tissue is a key contributor to CIH-induced metabolic disorders. The precise mechanism underlying CIH-induced inflammation in adipose tissue macrophages remains elusive. Adiponectin (Ad) is a highly abundant adipokine secreted by adipocytes that plays a crucial role in insulin sensitivity. MethodsThis study aims to elucidate the pivotal roles of adipocytes and Ad in CIH-induced macrophage inflammation using a co-culture cell approach. The direct co-culture method involved the physical interaction between iBMDM cells and 3T3-L1 adipocytes, while the indirect co-culture method utilized adipocyte-conditioned media as the culture medium for macrophages. Following CIH treatment and Ad intervention, the expression levels of inflammatory factors in the conditioned media of each cell group were quantified. Statistical significance was defined as P < .05. ResultsResults showed CIH did not directly cause the release of inflammatory factors in individually cultured iBMDM cells and 3T3-L1 adipocytes; however, it significantly increased the release of inflammatory factors in co-cultured cells. Ad alleviates the pro-inflammatory effect of adipocytes on macrophages under CIH conditions. ConclusionCIH could disrupt adipocyte function and promote macrophage inflammation through paracrine mechanisms.

cell biology↗

bioRxiv2025

Prokineticin-2 Upregulates GDNF in Astrocytes and Pharmacological Modulation of PK2 Receptors offers Neuroprotection in Experimental Models of Parkinson's Disease

Despite a wealth of preclinical studies establishing neuroprotective and neurorestorative properties of glial cell-line-derived neurotrophic factor (GDNF) in animal models of Parkinsons disease (PD), clinical trials utilizing direct intracranial infusion of GDNF protein, or adeno-associated virus (AAV)-mediated GDNF gene transfer has not achieved the desired efficacy, largely due to challenges in delivery methods. Given GDNFs strong potential for neuroprotection, alternative strategies to elevate its expression by beyond invasive injection or genetic manipulation remain a promising therapeutic avenue for PD. We previously reported that prokineticin signaling provides a compensatory protective response against dopaminergic neuronal degeneration in cell and animal models of PD. Herein, we report a novel finding that PK2 regulates GDNF gene expression in astrocytes, suggesting that PK2 signaling can be harnessed for neuroprotection in PD. Treatment of cultured astrocytes with the PK2 protein, PK2 gene overexpression or prokineticin receptor 1 (PKR1) agonist IS20 significantly induced the GDNF gene expression and the protein secretion, resulting in enhanced dopaminergic cell survival in cell culture models of PD. Importantly, systemic administration of IS20 through intraperitoneal or intranasal routes elevated GDNF levels in the mouse brain, including the nigrostriatal system. Furthermore, IS20 treatment conferred significant neuroprotective effects in both 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced and MitoPark transgenic mouse models of PD. Collectively, our translational findings suggest that pharmacological modulation PK2 signaling may unlock the full clinical benefit of GDNF, offering a novel and non-invasive therapeutic strategy for Parkinsons disease.

pharmacology and toxicology↗

bioRxiv2025

Adaptation of Perceived Animacy from Biological Motion: Evidence for a "Life Motion Detector"

Humans can readily perceive animacy from biological motion (BM) - the distinctive movement patterns of living entities. However, how the human brain extracts animacy information from BM remains largely unclear. The current study investigated this issue using visual adaptation, a non-invasive tool for revealing neural mechanisms underlying the selective processing of specific properties. Results showed that prolonged exposure to intact human walkers, compared to non-BM adaptors, biased the perception of subsequent walking stimuli towards less animate. This adaptation aftereffect persisted following adaptation to local foot motions carrying diagnostic kinematic cues, but was absent after exposure to static body forms, indicating the involvement of neuronal populations encoding animacy from BM based on motion signals. Moreover, adapting to pigeon movements also biased animacy perception for human motions, revealing a shared mechanism for encoding animacy from cross-species BM signals. These results support the existence of an inherent "life detection" system in the human brain, attuned to local foot motions and kinematic features prevalent in vertebrate movements, which may lay a foundation for perceiving life from motion across terrestrial species. Public significance statementsDiscerning whether a moving entity is animate is crucial for survival and for generating appropriate responses. This study explored how the human brain perceives animacy based on a prevalent movement pattern among vertebrates, namely, walking. Leveraging a non-invasive visual adaptation paradigm, five experiments demonstrated that pre-exposure to intact human walking, feet-only walking, and even intact pigeon walking motion sequences - but not static human forms - elicited significant adaptation aftereffects in animacy perception of human walkers. These findings support the existence of neural substrates encoding perceived animacy based on biological motion signals, particularly those carried by local foot motions and cross-species kinematic features. Such mechanisms may be integral to an inherent life detection system in the human brain.

neuroscience↗

bioRxiv2025

IRE1α-XBP1s axis is required for BCG-induced trained immunity against viral infection by inhibiting AHR signaling

Although Bacille Calmette-Guerin (BCG) has been proved to be suitable in inducing trained immunity, mechanism-related issues have not yet been fully resolved. In this study, we found that in vitro BCG training protects both BCG harboring and bystander macrophages against DNA virus and RNA virus infection and demonstrated that BCG harboring cells protect bystanders partly by type I interferon (IFN). We determined aryl hydrocarbon receptor (AHR) as a negative regulator in BCG-induced trained immunity on defense against viral infection in vitro and in vivo and blockade of type I IFN signaling partially impaired the enhanced antiviral effect of AHR deficiency in BCG training. Furthermore, we revealed IRE1-XBP1s signaling as a critical pathway in BCG-induced trained immunity against viral infection in vitro and in mice. Mechanistically, we demonstrated that IRE1-XBP1s pathway influenced BCG-induced trained immunity by limiting AHR mRNA expression and the translocation of AHR protein into the nucleus.

immunology↗

bioRxiv2025

Comparative Temporal Transcriptomic Analysis of SOD1 Mutations in iPSC-Motor Neurons

Mutations in the SOD1 gene are among the most significant genetic contributors to amyotrophic lateral sclerosis (ALS), with different variants linked to varying disease severity. To investigate the molecular mechanisms driving this variability, we conducted RNA sequencing on spinal motor neurons (MNs) differentiated from isogenic human induced pluripotent stem cell (iPSC) lines engineered via CRISPR/Cas9. These lines carried two representative SOD1 heterogenous mutations, D91A and G94A, and were analyzed at Days 10 and 20 of neuronal maturation stage to capture the temporal changes of gene expression. We aim to explore how these mutations affect MN function, identify distinct molecular pathways that may explain the variable severity of ALS, and investigate the role of translation and metabolic dysregulation in disease progression.

bioinformatics↗

bioRxiv2025

In Vivo Evolution of Monoclonal Antibody CR3022 to Achieve Cross-Neutralization of SARS-CoV-2 and Implications for Vaccine Strategies Against SARS-Related Viruses

The epitope that monoclonal CR3022 binds to represents a promising target for broad protection against a wide range of human and zoonotic coronaviruses. We developed a powerful model to evaluate antibody affinity maturation in vivo using immunoglobulin (Ig)-humanized mice that express the predicted germline heavy chain of antibody CR3022. SARS-CoV/SARS-CoV-2 sequential immunization led to the convergent evolution of the germline CR3022 through somatic hypermutation (SHM) that resembled the affinity-matured CR3022 from a human, but now also adapted to key variants and divergent sarbecoviruses. While simple prime-boost strategies drove CR3022-epitope targeting, an intensive vaccination protocol elicited dominant responses to other epitopes. X-ray crystal structures revealed that SARS-CoV-2-neutralizing CR3022-like antibodies exhibit enhanced affinity by increasing polar and electrostatic interactions. Overall, these findings show CR3022-like clones can be readily adapted through SHM to increase breadth and potency to sarbecoviruses by relatively minor shifts in affinity with appropriate vaccination strategies.

immunology↗

bioRxiv2025

DAXX governs the silencing of LINE1 during spermatogenesis in mice

The suppression of transposable elements in germline is critical for safeguarding fertility. Here we identify that the DAXX, a potent transcription repressor and an H3.3 chaperone, is indispensable for transposable elements silencing during spermatogenesis. Male mice with conditional knockout of Daxx in germ cells mediated by Ddx4-cre display delayed meiotic progression, deformed and reduced production of sperms, and an age-dependent decline in fertility. The ablation of DAXX results in activation of evolutionarily young LINE1 and ERVs subfamilies. Mechanistically, DAXX interacts with DMAP1 to maintain DNA methylation of LINE1 in meiotic cells. In summary, this study identifies DAXX as a previously unknown regulator of spermatogenesis and demonstrates that it functions as an epigenetic regulator to maintain the silencing the young LINE1 by DNA methylation.

developmental biology↗

bioRxiv2025

Small tau aggregates exhibit disease-specific molecular profiles across tauopathies

Tauopathies are neurodegenerative diseases marked by pathological tau aggregation. While disease-specific folds of insoluble tau filaments have been established, it remains unclear whether the smaller, earlier species also differ across tauopathies. Here, we characterise these small tau aggregates from post-mortem brain of individuals with Alzheimers disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration, Picks disease, and healthy controls. Using two complementary single-molecule assays, we confirm that small tau aggregates vary in abundance, morphology, and post-translational modifications. AD features specific long, fibrillar-shaped aggregates enriched in phospho-epitopes, while PSP aggregates are shorter, round, and selectively phosphorylated at serine-356, a site we identify as correlating with markers of inflammation and apoptosis. Aggregate properties co-vary with cellular stress signatures and align with disease-specific seeding profiles, suggesting distinct pathological mechanisms. These findings suggest that small tau aggregates are not a shared intermediate, but instead encode disease-specific mechanisms, with potential as both biomarkers and therapeutic targets. Key pointsSmall tau aggregates show disease-specific signatures across tauopathies, differing in abundance, morphology, and post-translational modifications. Tau aggregates in AD show enhanced phosphorylation density and structural heterogeneity, including a distinct population of long fibrillar species detectable in the soluble fraction. Alzheimers disease is characterised by specific long, fibrillar-shaped tau aggregates enriched in disease-relevant phospho-epitopes. PSP features round pSer356-positive aggregates that correlate with apoptotic and inflammatory markers, suggesting a distinct mechanism of toxicity. Isoform-specific biosensor assays reveal divergent seeding behaviour: CBD shows strong 4R seeding, while PSP lacks seeding activity. Features of small aggregates co-vary with distinct patterns of gliosis and cell stress, suggesting disease-specific mechanisms of tau-mediated toxicity.

neuroscience↗

bioRxiv2025

Rules of engagement: determinants of chemokine receptor activation and selectivity by CCL27 and CCL28

The distinct functional roles of chemokines CCL27 and CCL28 in epithelial immunity of skin and mucosal tissues, respectively, are coordinated by their shared receptor, CCR10 and the CCL28-specific receptor, CCR3. In this study, we conducted structure-function studies focused on the N-termini of these two chemokines to identify determinants of receptor activation, internalization and binding specificity. Deletion of two N-terminal residues of CCL27 resulted in a CCR10 antagonist, highlighting the critical roles of these residues in driving receptor pharmacology. Extension with a Phe produced a superagonist by occupying an available subpocket in the receptor binding site. Swapping the N-terminus of CCL28 onto the CCL27 globular domain (NT28-CCL27) also resulted in a superagonist of CCR10, but the opposite swap (NT27-CCL28) showed equivalent or reduced activity compared to WT CCL28, indicating that the CCL28 N-terminus is a stronger driver of CCR10 signaling. The effect of these and other mutations were rationalized by AlphaFold models of the CCR10 complexes. AlphaFold modeling also revealed that the reduced size of the binding pocket, and more basic nature of the N-terminus and extracellular loops of CCR3 compared to CCR10, contribute to its specificity for CCL28 while CCR10 accommodates both ligands. The overall basic nature of CCL28 also contributes to its high affinity for glycosaminoglycans, which is likely important for its retention in mucosal tissues. These data illustrate the modular compositions of these chemokines that has evolved to achieve overlapping but non-redundant functions, and the exploitation of this modular nature to produce engineered chemokines for probing or targeting CCR10 in disease contexts.

pharmacology and toxicology↗