Biology subjects

Pan, M.

Publications and source records attributed to Pan, M..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

A Cryptic Interfacial Pocket Uncovered in Full CRL4CRBN-IKZF3 Ubiquitylation Complex Enhances IMiD Efficacy

Immunomodulatory imide drugs (IMiDs) redirect the CUL4-RBX1-DDB1-CRBN (CRL4CRBN) ligase to ubiquitylate and degrade disease-linked proteins, but the full picture of how IMiDs glue the neosubstrate within the whole CRL4CRBN complex remains unknown. We determined cryo-electron microscopy structures of eight approved or clinical IMiDs in full CRL4CRBN ubiquitylation complexes at resolutions up to 3.4 [A], and revealed how these structurally distinct IMiDs exploit the structural plasticity of CRL4CRBN to organize conformationally conserved and compact active ubiquitylation assemblies with the neosubstrate IKZF3. Four "next-generation" IMiDs were found to additionally engage a cryptic gluing-driven interfacial (GDI) pocket at the non-degron zinc finger 3 (ZF3) of IKZF3, which contributes to their enhanced efficacy and neosubstrate specificity. The identification of cryptic gluing-driven pockets formed only in full ubiquitylation complexes provides new structure-directed design opportunities for IMiDs to improve therapeutic efficacy.

biochemistry↗

bioRxiv2025

Towards Unmanned Proteomics Data Generation: A Fully Automated Sample-to-Data System for Proteomic Experiments

The {pi}-HuB project requires proteomic measurements of millions of low-input samples that are challenging with current technology. Here, we introduce a pilot {pi}-HuB data factory that features a fully automated sample-to-data system to enable high-throughput digitization of human samples. Our platform, as a proof-of-concept towards unmanned proteomics, was demonstrated to show longitudinal robustness and applied for spatial proteomics and the profiling of plasma and cell lines, thereby serving as the workhorse to drive {pi}-HuB.

biochemistry↗

bioRxiv2025

A lineage-specific heat-induced feedback loop controls HrcA to promote chlamydial fitness under stress

Bacterial stress responses rely on transcriptional regulation by sigma factors and repressors. In Chlamydia, which lacks a dedicated heat shock sigma factor, repressor HrcA limits chaperone gene expression under non-stressed conditions. While HrcA function may be enhanced by the chaperone GroESL in a positive feedback loop, here we identify a heat-induced negative feedback loop mediated by HagF, a protein unique to Chlamydiota and conserved across Chlamydia species. We show that HrcA represses hagF, but heat shock induces hagF expression, leading to HagF accumulation and binding to HrcA. Structural modeling and pulldown assays indicate that HagF blocks HrcA dimerization, impairing DNA binding. This relieves repression of the hrcA-grpE-dnaK operon and promotes secondary differentiation in the developmental cycle, enabling infectious elementary bodies to form under heat stress. Our findings reveal dual feedback regulation that tunes chaperone gene expression and illustrate how transcriptional repression can be modulated in a minimal-genome pathogen.

microbiology↗

bioRxiv2025

Unique Gluing Effect of ASXL1 K351 Monoubiquitination Stimulates PR-DUB-Mediated Nucleosomal H2AK119Ub Deubiquitination

Protein ubiquitination plays a critical role in regulating fundamental biological processes through both proteolytic and nonproteolytic mechanisms. While classically known for its role in targeting proteins for degradation, ubiquitination can also modulate enzymatic activity. However, all of the known mechanisms of such modulation involve spatially constrained mechanisms that take place near the enzyme-substrate interfaces. Here, we report an unprecedented regulatory paradigm mediated by ubiquitination that is located distal to the enzyme-substrate interface but can still activate the polycomb repressive deubiquitinase (PR-DUB) complex. Using cryo-electron microscopy, molecular dynamics (MD) simulations, hydrogen-deuterium exchange mass spectrometry (HDX-MS), and kinetics assays, we determined that the ASXL1 K351 monoubiquitination promotes PR-DUB-mediated nucleosomal H2AK119Ub deubiquitination by stabilizing the catalytic pocket of PR-DUB, causing significant increase in maximal catalytic velocity (Vmax) but almost no change in the substrate binding affinity (Km). Structural analysis revealed that the ubiquitin moiety at ASXL1 K351 bridges the BAP1 and ASXL1 subunits, acting as an intramolecular "glue" that constrains the conformational dynamics of these subunits without altering the nucleosome binding interface. Further MD and HDX-MS results demonstrated the monoubiquitination of ASXL1 K351 enables PR-DUB to effectively maintain a spatial arrangement of the catalytic conformation that closely aligns with substrate cleavage. This study expands our understanding of the mechanisms of ubiquitin function to include intramolecular fastening with a molecular gluing effect and provides a deeper mechanistic understanding of PR-DUB activation in nucleosomal H2AK119Ub removal.

biochemistry↗

bioRxiv2025

A mathematical model of metacarpal subchondral bone adaptation, microdamage, and repair in racehorses

Fractures of the distal limb in Thoroughbred racehorses primarily occur because of accumulation of bone microdamage from high-intensity training. Mathematical models of subchondral bone adaptation of the third metacarpal lateral condyles are capable of approximating existing data for Thoroughbred racehorses in training or at rest. To improve upon previous models, we added a dynamic resorption rate and microdamage accumulation and repair processes. Our ordinary differential equation model simulates the coupled processes of bone adaptation and microdamage accumulation, and is calibrated to data on racehorses in training and rest. Sensitivity analyses of our model suggest that joint loads and distances covered per day are among the most significant parameters for predicting microdamage accumulated during training. We also use the model to compare the impact of incremental increasing training programs as horses enter training from a period of rest and maintenance workloads of horses that are race fit on bone adaptation. We find that high-speed training accounts for the majority of damage to the bone. Furthermore, for horses in race training, the estimated rates of bone repair are unable to offset the rate of damage accumulation under a typical Australian racing campaign, highlighting the need for regular rest from training. Nomenclature O_TBL View this table: org.highwire.dtl.DTLVardef@1d155cborg.highwire.dtl.DTLVardef@57a950org.highwire.dtl.DTLVardef@12cd1b9org.highwire.dtl.DTLVardef@17ce916org.highwire.dtl.DTLVardef@1aaaf66_HPS_FORMAT_FIGEXP M_TBL C_TBL

animal behavior and cognition↗

bioRxiv2025

Multimodal brain cell atlas across the adult macaque lifespan

High-throughput single-cell omics of non-human primate tissues present a remarkable opportunity to study primate brain aging. Here, we introduce a transcriptomic and chromatin accessibility landscape of 1,985,317 cells from eight brain regions of 13 cynomolgus female monkeys spanning adult lifespan including exceptionally old individuals up to 29-years old. This dataset uncovers dynamic molecular changes in critical brain functions such as synaptic communication and axon myelination, exhibiting a high degree of cell type and brain region specificity. We identify the multicellular networks of the pons and medulla as a previously unrecognized hotspot for aging. Furthermore, comparative analyses with human neurodegeneration datasets highlight both shared and distinct mechanisms contributing to aging and disease. In addition, we uncover transcription factors implicated in monkey brain aging and pinpoint aging-regulated loci linked to longevity and neurodegeneration. This spatiotemporal atlas will advance our understanding of primate brain aging and its broader implications for health and disease.

neuroscience↗

bioRxiv2025

Quantitative and large-scale investigation of human TCR-HLA Cross-Reactivity

The interaction between Human leukocyte antigens (HLA) and T cell receptor (TCR) is essential for adaptive immune recognition. While it is known that one TCR can map to multiple HLA alleles, the extent of this cross-reactivity remains poorly understood. Here, we introduce THNet, a TCR-based HLA similarity inference method, and performed a comprehensive analysis of HLA-TCR cross-reactivity. This method is built upon clustering over 47 million TCRs to identify over 9 million significant HLA-TCR pairs. We created similarity networks for both class I and class II HLA alleles, illustrating how peptide cross-presentation contributes to HLA-TCR cross-reactivity. This analysis revealed novel disease-susceptibilities missed by single-HLA enrichment analyses, especially in the Black populations. Finally, we demonstrated that THNet prioritized optimal HLA mismatch candidates for organ transplantation, thereby improving patient survival rates. Our investigation of HLA-TCR cross-reactive network might provide useful insights for autoimmune risk prediction and better transplantation outcomes. One Sentence SummaryWe introduced THNet, a large-scale TCR-based HLA similarity mapping network that uncovers previously unrecognized cross-reactivity patterns across HLA alleles and provides valuable insights into their influence on disease susceptibility and graft rejection.

bioinformatics↗

bioRxiv2025

Integrating multi-modal transcriptomics identifies cellular subtypes with distinct roles in PDAC progression

BackgroundPancreatic ductal adenocarcinoma (PDAC) features a complex tumor microenvironment (TME) that significantly influences patient outcomes. Understanding the TMEs cellular composition and interactions is crucial for identifying therapeutic targets to improve treatment. MethodsWe performed an integrative analysis combining 88 single-cell RNA sequencing (scRNA-seq) samples with 187,520 cells, 20 Visium spatial transcriptomics (ST-seq) samples with 67,933 spots, and 1383 bulk RNA-seq samples and 2 Xenium high-resolution ST-seq samples with 307,679 cells, to delineate and characterize distinct subpopulations of fibroblasts, macrophages, T/NK cells, and B/plasma cells within the PDAC microenvironment. Correlations among major cell types across 12 PDAC datasets were assessed through gene set variation analysis (GSVA). Kaplan-Meier survival analysis was used to evaluate the prognostic significance of cell subtypes. Furthermore, multiplex immunohistochemistry (mIHC) and the Xenium platform was utilized to validate cellular interactions in human PDAC tissues at both protein and RNA levels. ResultsSix fibroblast subtypes and eight macrophage/monocyte subtypes were identified. POSTN high fibroblasts and SPP1 high macrophages highly infiltrated tumor tissues and were associated with poor prognosis. Most immune cell subtypes mediate adverse prognoses, except for CCL4 high CD8+ T EFF cells and IGHG1 high plasma cells, which are linked to favorable outcomes. ST-seq revealed spatial colocalization of POSTN high Fibro and SPP1 high Macro cells, as well as CCL4 high CD8+ T EFF and IGHG1 high plasma cells. These findings were corroborated by mIHC and validated using Xenium spatial transcriptomics with single-cell resolution, which confirmed the expression and spatial proximity of these markers at both the gene and protein levels. ConclusionThis integrative analysis of the PDAC TME underscores the prognostic importance of POSTN high fibroblasts and SPP1 high macrophages, while also highlighting the protective roles of CCL4 high CD8+ T EFF cells and IGHG1 high plasma cells. These insights could improve PDAC treatment strategies.

cancer biology↗

bioRxiv2025

SCAN-ACT: Adoptive T Cell Therapy Target Discovery Through Single-Cell Transcriptomics

The FDA approval of T cell receptor-engineered T cells (TCR-T) for synovial sarcoma demonstrates the potential for adoptive T cell therapies (ACTs) in solid tumors. However, the paucity of tumor-specific targets without expression in normal tissues remains a major bottleneck, especially in rare cancer subtypes. Here, we present a comprehensive computational pipeline called SCAN-ACT that leverages single cell RNA sequencing and multi-omics data from tumor and normal tissues to nominate and prioritize targets for both chimeric antigen receptor (CAR)- and TCR-T cells. For surface membrane targets, SCAN-ACT proposes target pairs for bispecific Boolean logic-gated CAR T cells. For peptide-MHC targets, SCAN-ACT proposes intracellular peptides bound to a diverse set of human leukocyte antigens. We applied the SCAN-ACT pipeline to soft tissue sarcoma (STS), analyzing 986,749 single cells to identify and prioritize 395 monospecific CAR-T targets, 14,192 bispecific CAR-T targets, and 5,020 peptide-MHC targets for TCR-T cells. Selected targets were validated experimentally by protein expression and for peptide-MHC binding. Proposed targets and target pairs reflected the mesenchymal, neuronal, and hematopoietic ontogeny of STS. This work provides a robust data repository along with a web-based and user-friendly set of analysis tools to accelerate ACT development for solid tumors.

cancer biology↗

bioRxiv2025

Conformation-gated binding drives negative cooperativity in ATP:cob(I)alamin Adenosyltransferase for optimized cobalamin handling

Vitamin B12 (cobalamin) is a high-value yet scarce cofactor required for various metabolic processes, making its efficient handling important for maintaining metabolic homeostasis. While the involvement of ATP:cob(I)alamin adenosyltransferases (MMAB) in the synthesis, delivery, and repair of 5-deoxyadenosylcobalamin (AdoCbl) is well established, the kinetic mechanisms that regulate this process, particularly its negative cooperativity, remain poorly understood. Understanding these mechanisms is key to clarifying how MMAB efficiently uses AdoCbl, prevents resource wastage, and supports bacterial survival in nutrient-limited environments. Using single-molecule relative fluorescence (SRF) spectroscopy, we found that conformation-gated binding is the driving force behind MMABs preference for AdoCbl over hydroxocobalamin and is the underlying mechanism for negative cooperativity. This mechanism significantly slows down the binding of the second equivalent of AdoCbl, favoring the singly bound state. Our findings indicate that MMAB predominantly binds a single AdoCbl, optimizing the AdoCbl loading to methylmalonyl-CoA mutase. Additionally, our SRF approach also serves as a tool to explore other cofactor interactions, such as those between riboswitches and cobalamin derivatives, to provide insights into regulatory mechanisms of cobalamin sensing and gene regulation, which are crucial for bacterial adaptation to changing nutrient conditions. Significance StatementMMAB is important for B12-dependent propionate metabolism in bacteria. Our findings reveal that conformation-driven binding mechanism underlines the negative cooperativity of MMAB, as it favors the binding of the first AdoCbl while limiting further binding. The larger kon for the first site, combined with similar unbinding rates for both sites, could provide a solution for optimizing cobalamin handling and minimize unnecessary waste. Our single-molecule fluorescence approach offers a powerful tool for investigating other dynamic cofactor interactions, providing new insights into regulatory mechanisms in bacterial metabolism.

biophysics↗

bioRxiv2024

Chlamydia plasmid-encoded protein Pgp2 is a replication initiator with a unique beta-hairpin necessary for iteron-binding and plasmid replication

The virulence plasmid of the obligate intracellular bacterium Chlamydia encodes eight proteins. Among these, Pgp3 is crucial for pathogenicity, and Pgp4 functions as a transcriptional regulator of both plasmid and chromosomal genes. The remaining proteins, Pgp1, Pgp5, Pgp6, Pgp7, and Pgp8, are predicted to play various roles in plasmid replication or maintenance based on their amino acid sequences. However, the function of Pgp2 remains unknown, even though it is required for transformation. In this study, we utilized AlphaFold to predict the 3-dimensional (3-D) structure of C. trachomatis Pgp2. Despite a lack of apparent sequence homology, the AlphaFold structure exhibited high similarity to experimentally determined structures of several plasmid replication initiators. Notably, Pgp2 features a unique {beta}-hairpin motif near the DNA-binding domain, absent in other plasmid replication initiators with overall 3-D structures similar to Pgp2. This {beta}-hairpin motif was also present in AlphaFold models of Pgp2s across all 13 Chlamydia species. To assess its significance, we engineered a plasmid lacking the 11 amino acids constituting the {beta}-hairpin motif in C. trachomatis Pgp2. Although this deletion did not alter the overall structure of Pgp2, the mutated plasmid failed to transform plasmid-free C. trachomatis. These findings reveal that Pgp2 is a plasmid replication initiator, with the {beta}-hairpin motif playing a critical role in binding to its cognate iteron sequences in the replication origin of the chlamydial plasmid.

microbiology↗

bioRxiv2024

Medicinal chemistry of benzoisoxazole-4,7-dione analogues identifies the ribosomal small subunit as a target for specific mycobacterial translational fidelity

A central dogma of molecular biology is the "speed-accuracy trade-off," where ribosomes must slow down to ensure accurate protein synthesis. In mycobacteria, a high basal level of mistranslation at glutamine and asparagine codons, caused by an indirect tRNA aminoacylation pathway, promotes tolerance to the antibiotic rifampicin. While pharmacologically increasing translational fidelity is a promising strategy to combat antibiotic tolerance, the underlying mechanisms remain poorly understood. Here, we screened 9,000 synthetic compounds and identified benzo[d]isoxazole-4,7-diones as a novel chemical class that reduces mycobacterial mistranslation. Medicinal chemistry optimization yielded a lead compound, 9787, with superior potency in decreasing mistranslation and reversing rifampicin tolerance. Using competitive chemical proteomics, we identified the 30S ribosomal protein S5 (RpS5) as the specific cellular target. Remarkably, compound 9787 enhances translational fidelity at concentrations that do not measurably impact the overall rate of protein synthesis. Our findings challenge the universality of the speed-accuracy trade-off, demonstrating that fidelity can be improved independently of translation speed. This work reveals that the ribosomal small subunit is a druggable target for modulating translational quality control and introduces a new strategy for combating antibiotic-tolerant bacteria without the associated fitness cost of slowed translation. ImportanceA fundamental principle in molecular biology holds that ribosomes face a trade-off between translation speed and accuracy: going faster means making more errors, while maintaining high fidelity requires slowing down. This study challenges that paradigm by identifying a small molecule that increases translational accuracy in mycobacteria without affecting the rate of protein synthesis. The compound targets ribosomal protein S5 and specifically reduce errors arising from physiologically mischarged tRNAs - a quality control problem distinct from the well-studied codon*anticodon mismatches. This form of mistranslation contributes to antibiotic tolerance in tuberculosis, making it a potential therapeutic target. Our findings reveal unexpected flexibility in how ribosomes maintain translation quality and suggest that pharmacologically increasing fidelity without the fitness cost of slowed protein synthesis may be an attractive strategy for combating antibiotic-tolerant bacteria.

microbiology↗

bioRxiv2024

Bioinspired Bimetallic Ions Functionalized MOF SAzyme Nanocomposites for Synergistic Ferroptosis/Cuproptosis-Enhanced Immune Checkpoint Therapy

Immune checkpoint blockade (ICB) generates sustained responses in immunogenic cancers, but its effectiveness is limited in tumors lacking immune activity. Here, we construct a bioinspired bimetallic ions functionalized nanoscale metal-organic framework (NMOF) single-atom nanozyme (SAzyme) loaded with doxorubicin (Dox) (NMOF-Fe/Cu-Dox nanocomposite) to effectively trigger anti-tumor immune responses while addressing the immunosuppressive tumor microenvironment (TME). The NMOF-Fe/Cu-Dox nanocomposite has been demonstrated to efficiently reverse the TME by generating reactive oxygen species and oxidizing glutathione. Camouflaging NMOF-Fe/Cu-Dox nanocomposites with bioinspired cancer cell membrane (NMOF-Fe/Cu-Dox@M) enables its navigation to the tumor region through homologous targeting. The highly efficient uptaken by cancer cells selectively induced synergistic ferroptosis and cuproptosis in these cells. Furthermore, in vitro and in vivo experiments demonstrate that the ferroptosis in cancer cells can polarize tumor-associated macrophages (TAMs) towards anti-tumoral M1 phenotype and significantly diminish pro-tumoral M2 phenotype. We find that NMOF-Fe/Cu-Dox@M could induce the ferroptosis of M2 macrophages, while no effect to M1 macrophages. In addition, a significant increase of anti-tumor infiltrating CD8+ T cells, while a remarkable decrease of CD4+ regulatory T cells were observed. These findings suggested that NMOF-Fe/Cu-Dox@M could efficiently modulate TME, enhance tumoricidal immunity and elevate the therapeutic efficiency of ICB. Moreover, the combination of NMOF-Fe/Cu-Dox@M with PD-1 effectively eradicated hepatocellular carcinoma cells in vivo, outperforming the use of either NMOF-Fe/Cu-Dox@M or PD-1 alone. In summary, our study presents a therapeutic strategy that leverages coordinated ferroptosis and cuproptosis with therapeutic efficiency of ICB, underscoring the promise of combined chemoimmunotherapy.

bioengineering↗

bioRxiv2024

Quantifiable Blood TCR Repertoire Components Associated with Immune Aging

T cell senescence results in decayed adaptive immune protection in older individuals, with decreased or increased abundance of certain T cell phenotypic subpopulations. However, no study has linked aging to the dynamic changes of T cell clones. Through a newly develop computational framework, Repertoire Functional Units (RFU), we investigated over 6,500 TCR repertoire sequencing samples from multiple human cohorts. Our analysis identified age-associated RFUs repeatedly and consistently across different cohorts. Quantification of RFU decreases with aging revealed accelerated loss under immunosuppressive conditions. Systematic analysis of age-associated RFUs in clinical samples manifested a potential link between these RFUs and improved clinical outcomes during acute viral infections, such as lower ICU admission and reduced risk of developing complications. Finally, our investigation of bone-marrow transplantation patients indicated a secondary expansion of the age-associated clones upon receiving stem cells from younger donors. Together, our results suggest the existence of certain clones or a TCR clock that could reflect the immune functions in aging populations.

immunology↗

bioRxiv2024

Association study of Plasmodium falciparum Acetyl-CoA Synthetase Mutations S868G and V949I with antimalarial drugs

Plasmodium falciparum acetyl-CoA synthetase (PfACAS) protein is an important source of acetyl-CoA. We detected the mutations S868G and V949I in PfACAS by whole-genome sequencing analysis in some recrudescent parasites after antimalarial treatment with artesunate and dihydroartemisinin-piperaquine, suggesting that they may confer drug resistance. Using CRISPR/Cas9 technology, we engineered parasite lines carrying the PfACAS S868G and V949I mutations in two genetic backgrounds and evaluated their susceptibility to antimalarial drugs in vitro. The results demonstrated that PfACAS S868G and V949I mutations alone or in combination were not enough to provide resistance to antimalarial drugs.

microbiology↗

bioRxiv2024

Energy-based Modelling of Single Actin Filament Polymerisation Using Bond Graphs

Bond graphs provide an energy-based methodology for modelling complex systems in a hierarchical fashion; at the moment, the method allows biological system with both chemical and electrical subsystems to be modelled. Herein, the bond graph approach is extended to include chemomechanical transduction thus extending the range of biological systems to be modelled. Actin filament polymerisation and force generation is used as an example of chemo-mechanical transduction and it is shown that the TF (transformer) bond graph component provides a practical, and conceptually simple, alternative to the Brownian ratchet approach of Peskin, Odell, Oster & Mogilner. Furthermore it is shown that the bond graph approach leads to the same equation as the Brownian ratchet approach in the simplest case. The approach is illustrated by showing that flexibility and non-normal incidence can be modelled by simply adding additional bond graph components and that compliance leads to non-convexity of the force-velocity curve. Energy flows are fundamental to life; for this reason, the energy based approach is utilised to investigate the power transmission by the actin filament and its corresponding efficiency. The bond graph model is fitted to experimental data by adjusting model physical parameters.

systems biology↗

bioRxiv2024

Energy-based Analysis of Biochemical Oscillators Using Bond Graphs and Linear Control Theory.

The bond graph approach has been recognised as a useful conceptual basis for understanding the behaviour of living entities modelled as a system with hierarchical interacting parts exchanging energy. One such behaviour is oscillation, which underpins many essential biological functions. In this paper, energy-based modelling of biochemical systems using the bond graph approach is combined with classical feedback control theory to give a novel approach to the analysis, and potentially synthesis, of biochemical oscillators. It is shown that oscillation is dependent on the interplay between active and passive feedback and this interplay is formalised using classical frequency-response analysis of feedback systems. In particular, the phase margin is suggested as a simple scalar indicator of the presence or absence of oscillations; it is shown how this indicator can be used to investigate the effect of both the structure and parameters of biochemical system on oscillation. It follows that the combination of classical feedback control theory and the bond graph approach to systems biology gives a novel analysis and design methodology for biochemical oscillators. The approach is illustrated using an introductory example similar to the Goodwin oscillator, the Selkov model of Glycolytic Oscillations and the Repressilator.

systems biology↗

bioRxiv2024

Acetyl-CoA Synthetase Mutations S868G and V949I Do Not Confer Resistance to Antimalarial Drugs in vitro in Plasmodium falciparum.

microbiology↗