Biology subjects

Banerjee, A.

Publications and source records attributed to Banerjee, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

SuBMIT: A Software Toolkit for Facilitating Simulations of Coarse-Grained Structure-Based Models of Biomolecules.

Coarse-grained structure-based models (CG-SBMs; or G[o] models) are simplified potential energy functions of biomolecules or biomolecular complexes that encode their structure. Molecular dynamics simulations of such SBMs have been successfully used to study long time-scale dynamics such as protein and RNA folding, and large conformational transitions of biomolecular complexes. SBMs have several advantages: (1) Their MD simulations are computationally inexpensive, making extensive sampling easily accessible to many researchers. (2) They are easy to modify and can be adapted for the specific biomolecular problem that needs to be investigated. However, the force-fields of SBMs are not usually included in commonly used biomolecular simulation packages resulting in a barrier to their use. Here, we present SuBMIT (Structure Based Models Input Toolkit; https://github.com/sglabncbs/submit), a toolkit for generating coarse-grained SBM input files for performing MD simulations with GROMACS and OpenMM/OpenSMOG. Simulations whose input files can be generated using the different flavors of CG-SBMs present in SuBMIT include the folding and conformational ensembles of proteins with intrinsically disordered regions, 3D-domain-swapping in proteins and the dynamics of RNA-protein assemblies (e.g., simple RNA viruses).

biophysics↗

bioRxiv2026

Generation of Pathway Signatures by Combining Normal Modes with Weighted Ensemble Simulations

Molecular dynamics simulations provide a "computational microscope" by which molecular phenomena can be studied at atomic resolution. However, such simulations are often expensive, usually due to a combination of system size and timescale. Various enhanced sampling methods have been proposed to overcome these challenges. Despite their effectiveness, many suffer from artifacts from energetic biases guiding the simulations, or lack of effective progress coordinates. Proteins normal modes uniquely defined by their 3D fold capture their intrinsic dynamics and could provide unbiased guidance, but how to combine these modes with molecular dynamics to generate continuous, energetically unbiased pathways has been challenging. In this study, we demonstrate that conformations generated along from normal modes using adaptive anisotropic network model provide a physical, intuitive, and generalizable progress coordinate for weighted ensemble simulations, providing a boost in efficiency and a means to generate pathways for any protein system without prior knowledge.

biophysics↗

bioRxiv2026

The transmembrane domain regulates the kinetics of the SARS-CoV-2 spike conformational transition

ABSTRACTThe homotrimeric SARS-CoV-2 spike glycoprotein comprises two subunits: S1, which recognizes host-receptors through its receptor-binding domains (RBDs), and S2, anchored to the viral membrane through its transmembrane domain (TMD), which facilitates the fusion of the viral envelope with the host cell membrane. Upon host-receptor engagement and proteolytic activation, S1 dissociates and triggers a large conformational transition in S2, involving structural rearrangements in the S2 ectomembrane-domains and the TMD. While studies have focused on the ectomembrane-domain dynamics, the TMD has typically been modeled as being in a trimeric state. Here, we use molecular dynamics simulations of a coarse-grained structure-based model (SBM) with an implicit membrane to investigate the role of TMD dynamics in modulating S2 conformational conversion. We first recapitulate previous results from an all-atom SBM with a trimeric TMD and re-emphasize that the extended pre-hairpin intermediate state of S2, which brings the two membranes into apposition, is a byproduct of the prefusion-to-postfusion transition. Next, by introducing dynamics into the TMD, we find a late fusion intermediate structurally similar to a recent cryo-EM structure. A dynamic TMD also makes the conformational transition faster. Simulations including the S1/S2 complex reveal coupled RBD-TMD dynamics: when all three RBDs are in the closed state, they can stabilize the TMD in a trimeric configuration, whereas the opening of a single RBD can trigger a transition to a dynamic TMD. So, the dynamics and the conformational preferences of the TMD can be tuned by the presence and conformation of S1. There is some evidence that the TMDs of class I viral fusion proteins, such as spike, contribute to viral fusion by modulating membrane properties. Our simulations indicate an expanded role for the function of the TMD, where it can directly regulate the kinetics of S2 conformational transitions. SIGNIFICANCE STATEMENTThe SARS-CoV-2 fusion protein, spike, undergoes a large conformational transition, which facilitates the fusion of the viral and host-cell membranes and the delivery of the viral genome into the host cell. Despite extensive studies of the spike conformational conversion, its transmembrane domain (TMD) has largely been viewed as a viral membrane anchor. Using coarse-grained structure-based model simulations, we show that TMD dynamics can modulate the timing of the spike-S2 (fusion subunit) prefusion-to-postfusion conformational conversion. The presence of spike-S1 (receptor-recognition subunit) can suppress TMD dynamics, potentially reducing the rate of spike conformational conversion and viral fusion. Thus, the spike TMD regulates the kinetics of spike-mediated membrane fusion, and TMD-targeting strategies can be an additional avenue for antiviral intervention.

biophysics↗

bioRxiv2026

Stress Granules Buffers Inflammation by Restricting dsRNA-led Mitochondrial Fragmentation

Stress granules (SGs) are dynamic RNA-RBP condensates that form during stress and inflammation, yet how they modulate inflammatory signalling remains unclear. We uncover a rapid, protective SG-mediated mechanism that preserves mitochondrial integrity. During stress and translation inhibition, mitochondrial fragmentation releases double-stranded RNA (dsRNA), which activates PKR and its downstream effector DRP1, generating a self-amplifying loop of mitochondrial fragmentation and inflammation. We find that released dsRNA nucleates nanoSGs within minutes at ER-mitochondria contact sites--the very sites of mitochondrial division. These nanoSGs grow into macroSGs, effectively sequestering PKR-activating dsRNA from the cytosol. By depleting dsRNA, SGs suppress PKR-DRP1-driven positive-feedback inflammation and maintain mitochondrial integrity and function. Our findings reveal SGs as key guardians of mitochondrial homeostasis and position condensate biology at the centre of chronic mitochondrial-driven inflammation relevant to autoimmunity, ageing, and neurodegenerative disease.

cell biology↗

bioRxiv2026

Examining the impact of the Chlamydia muridarum-induced synthesis of IFN-β during genital tract infection

Chlamydia trachomatis infection of the female genital tract can result in severe reproductive sequelae, including pelvic inflammatory disease, tubal scarring, and infertility. Type I interferons have been implicated in both host defense and immunopathogenesis during chlamydial infection, with conflicting conclusions across experimental systems. However, the specific contributions of individual interferon subtypes remain poorly defined. Here, we examined the role of interferon beta (IFN-{beta}) in regulating epithelial immune responses and intracellular bacterial development during Chlamydia muridarum infection. Using murine oviduct epithelial (OE) cell lines derived from wild-type, IFN{beta}-deficient, and Toll-like receptor 3 (TLR3)-deficient mice, we demonstrate that IFN-{beta} is a critical epithelial-intrinsic mediator of host defense. Loss of IFN-{beta} led to dysregulation of genes associated with inflammation, immune regulation, and fibrosis, altered chlamydial inclusion morphology, enhanced expression of bacterial genes throughout the chlamydial developmental cycle, and increased chlamydial replication. Importantly, exogenous IFN-{beta} restored both immune mediator production and bacterial control during IFN{beta}-deficiency. Parallel analyses revealed that TLR3 deficiency phenocopied IFN-{beta} loss, supporting a TLR3-IFN-{beta} signaling axis that restricts chlamydial growth. Consistent with these in vitro findings, IFN{beta}-deficient mice exhibited enhanced bacterial burden during genital tract infection. Together, these data establish IFN-{beta} as a protective epithelial mediator during chlamydial infection and demonstrate that type I interferon responses are not functionally uniform. Our findings provide a mechanistic framework to reconcile the protective role of IFN-{beta} with reports of reduced pathology in interferon-/{beta} receptor-deficient models and highlight the importance of dissecting individual interferon pathways in chlamydial immunopathogenesis. ImportanceGenital tract infection with Chlamydia trachomatis remains a leading cause of preventable infertility worldwide. Although type I interferons are widely viewed as contributors to chlamydial pathology, most studies have examined global interferon signaling rather than the roles of individual interferon subtypes. In this study, we demonstrate that interferon beta (IFN-{beta}) plays a protective, epithelial-intrinsic role during chlamydial infection by restricting bacterial development and shaping local immune responses. These findings challenge the prevailing view that type I interferons are uniformly detrimental in this setting and reveal that distinct interferon subtypes can exert opposing effects on host defense and disease outcome. By defining a TLR3-IFN-{beta} signaling axis that limits chlamydial replication, this work advances our understanding of epithelial immunity in the female genital tract and has important implications for the design of targeted immunomodulatory strategies to prevent chlamydia-induced reproductive pathology.

microbiology↗

bioRxiv2026

Interferon-β Coordinates Epithelial Immune Networks and Fibrotic Responses During Chlamydia muridarum Infection

Chlamydia trachomatis infection is the most common bacterial sexually transmitted infection worldwide and a leading cause of inflammatory reproductive tract disease and infertility in women. Much of the tissue damage associated with genital chlamydial infection arises from host inflammatory responses rather than direct bacterial cytotoxicity. Epithelial cells lining the female reproductive tract represent the primary host cells infected during chlamydial infection and play key roles in initiating innate immune responses. Among the cytokines produced by infected epithelial cells, type-I interferons have emerged as important regulators of host defense and inflammatory signaling; however, the specific contribution of interferon-{beta} (IFN-{beta}) to epithelial transcriptional responses during chlamydial infection remains incompletely defined. In the present study, we investigated the role of IFN-{beta} in coordinating epithelial immune signaling networks during infection with Chlamydia muridarum. Using wild-type murine oviduct epithelial cells (OE-WT) and IFN-{beta}-deficient epithelial cells (OE-IFN{beta}-KO), we performed pathway-focused RT{superscript 2} Profiler PCR array analyses examining transcriptional responses across four biological pathways: (1) innate and adaptive immune responses, (2) type-I interferon signaling, (3) inflammatory and autoimmune responses, and (4) fibrosis-associated pathways. Infection of OE-WT cells resulted in coordinated induction of cytokines, chemokines, and interferon-stimulated genes associated with antimicrobial defense and immune cell recruitment. In contrast, IFN-{beta} deficiency resulted in widespread dysregulation of these transcriptional programs, including reduced induction of interferon-responsive chemokines such as CCL5 and CXCL10, altered inflammatory cytokine expression, and transcriptional signatures consistent with enhanced tissue remodeling responses. Notably, IFN-{beta} deficiency resulted in increased TNF expression accompanied by reduced IL-6 induction, suggesting disruption of balanced inflammatory signaling networks. Pathway analyses further revealed dysregulated expression of fibrosis-associated genes including Serpine1, Ctgf, and Eng in IFN-{beta}-deficient epithelial cells, indicating potential mechanisms linking interferon signaling to tissue remodeling during infection. Collectively, these findings identify IFN-{beta} as a central regulator of epithelial immune networks during chlamydial infection and suggest that disruption of IFN-{beta} signaling may promote inflammatory and fibrotic pathology within the female reproductive tract. Author SummarySexually transmitted infections caused by Chlamydia trachomatis are a major cause of infertility worldwide. Although antibiotic treatment can eliminate the bacteria, damage to the reproductive tract often results from the bodys own immune response to infection. The epithelial cells lining the reproductive tract are the first cells infected and play an important role in initiating immune responses. In this study, we investigated how a specific immune signaling molecule, interferon-{beta} (IFN-{beta}), regulates the gene expression programs activated in epithelial cells during chlamydial infection. Using pathway-focused gene expression arrays, we found that IFN-{beta} coordinates multiple immune pathways, including interferon signaling, inflammatory cytokine networks, and genes associated with tissue remodeling. When IFN-{beta} was absent, many of these pathways became dysregulated, resulting in altered inflammatory signaling and gene expression patterns linked to fibrosis. These findings suggest that IFN-{beta} functions as a key regulator that helps balance protective immune responses with inflammatory processes that can damage reproductive tissues during infection.

microbiology↗

bioRxiv2026

Rational Design of Selective IL-2-based Activators for CAR T Cells Using AlphaFold3 and Physics-Informed Machine Learning

Recombinant human Interleukin-2 (rhIL-2, Aldesleukin) is used in immunotherapy for metastatic melanoma and renal cell carcinoma. Low-dose IL-2 has been investigated for administration after adoptive T cell transfer to enhance CAR T expansion and sustain effector function. However, systemic IL-2 can cause severe toxicities and promote expansion of regulatory T cells (Tregs). Previous attempts at mitigating cytokine-mediated side effects involved isolating CAR T cell signaling from endogenous immune responses by developing IL-2/IL-2R{beta} based selective ligand-receptors systems. Expressing these variant orthogonal (ortho)IL2-R{beta} receptors in CAR T cells and supplying variant orthoIL-2, was shown to dramatically improve selectivity in CAR T cell expansion and anti-tumoral potency in a leukemia mouse model. This study describes the computational design of synthetic orthogonal cytokine receptor-ligand systems based on the scaffolds of the human canonical IL-2 and IL-2R{beta}. Leveraging state-of-the-art AlphaFold3 (AF3) structure prediction capabilities and a physics-informed constrained sequence generator (CSG), the pipeline generates, filters and ranks sets of putative orthoIL-2/orthoIL-2R{beta} mutant designs. Variants displaying minimal predicted off-target interactions and enhanced in target contacts are prioritized for structural modelling. Top designs showed outstanding AF3 structural and interfacial quality metrics ipTM and pTM, with averages between cognate pairs of 0.724{+/-}0.05 and 0.770{+/-}0.042, respectively. All in-silico hits showed ipTM <0.5 for non-cognates, indicating a good likelihood of orthogonality. Additionally, putative hits showed high levels of predicted structural fidelity to wild-type (WT) human IL-2/IL-2R{beta} (PDB: 2ERJ), with an average structural root-mean-square deviation (RMSD) of 0.843{+/-}0.375 [A]. These mutants incorporated 7-26 interfacial mutations derived from multiple interface selection strategies. Altogether, the results support the putative foldability and selective affinity of top-ranking mutants displaying metrics close-to or within experimental reference range. Finally, strengths and limitations are discussed, alongside the experimental implications of coupling a constrained protein design pipeline to the discovery and validation of selective binders based on naturally occurring scaffolds.

bioinformatics↗

bioRxiv2026

Single-molecule spatial genomics reveals the multi-scale organization and plasticity of extrachromosomal DNA in glioblastoma

Extrachromosomal DNA (ecDNA) is a major driver of intratumoral heterogeneity and is associated with poor clinical outcomes across cancers, yet how individual ecDNA molecules are organized and regulated within intact tumors remains unknown. Here, we leveraged single-molecule, multi-modal spatial genomics to resolve the three-dimensional chromatin organization and transcriptional activity of individual EGFR-containing ecDNA molecules in glioblastoma (GBM) cells in vitro, in orthotopic xenografts, and in patient-derived GBM tissue. At the larger scale, we find that distinct GBM molecular and functional states emerge depending on the local cellular environment. EGFR expression was markedly different between GBM subpopulations, and perturbations of EGFR dosage shifted GBM cellular states. ecDNA expression was modulated by multiple mechanisms, including variation in copy number, chromatin organization, DNA sequence, and chromosomal reintegration, which were simultaneously measured within the same cells. At the single-molecule scale, ecDNA adopts a physically expanded chromatin configuration with larger ecDNA molecules having higher transcriptional activity and interaction with active transcriptional machinery. ecDNA regulation was coordinated within cells and across GBM states, and ecDNA copy number, structure, and transcription were spatially organized across the tumor architecture. Co-culturing GBM cells with neurons recapitulated key features of infiltrative regions, including lower EGFR expression, reduced ecDNA copy number, and increased chromosomal reintegration, suggesting a causal role for the microenvironment in shaping ecDNA regulation. Collectively, these findings support a model in which GBM states and ecDNA are linked, plastic, and influenced by microenvironmental contexts, revealing a previously inaccessible layer of genome organization underlying tumor heterogeneity and malignant cell behavior.

genomics↗

bioRxiv2026

A room-temperature ⁸⁹Zr⁴⁺ radiolabelling strategy for small extracellular vesicles with enhanced plasma stability for PET Imaging

Both for diagnostic purposes and regenerative medicine, it is essential to develop advanced imaging platforms capable of tracking the biodistribution of small extracellular vesicles (sEVs), as current methods are limited by inadequate resolution and sensitivity. In this study, we introduce a novel labeling strategy utilizing the radioisotope zirconium-89 (89Zr), which boasts a half-life of 78.4 h and is cost-effective to produce. To achieve this, we designed a new chelator tailored for 89Zr4+ that offers enhanced stability compared to the conventional deferoxamine (DFO). This chelator forms a robust complex with 89Zr4+ at room temperature, suitable for sEV labeling for PET imaging applications. The radiolabeling process involved a two-step procedure: first, conjugation of the chelator to the sEVs, and second, radiolabeling with 89Zr4+. The resulting sEV-L1-Zr demonstrated a radiochemical yield of approximately 60% and maintained around 80% stability in plasma over seven days. Importantly, our modifications did not alter the morphology, surface protein composition, internal RNA content, or bioactivity of the sEVs. We successfully visualized sEVs at very low doses in the mouse heart following intravenous injection of sEV-L1-Zr. Additionally, ex vivo experiments using a Langendorff rat heart perfusion model confirmed targeted accumulation of the vesicles in cardiomyocytes as compared to other cells in the heart compartment. This approach provides a promising platform for sensitive and stable in vivo tracking of sEVs, advancing their application in both diagnostic imaging and regenerative therapies.

bioengineering↗

bioRxiv2026

A generalized scaling law to characterize coordinated brain activity

Complex ecological systems obey universal scaling laws such as Taylors law, which links population variance to the sample mean. Whether other complex systems with integrated and segregated functional networks--such as the human brain--follow analogous scaling laws, and how these laws behave under synchronized activity, remains unknown. Progress has been hindered because neural signals at all measurement scales contain both positive and negative values, whereas Taylors law was developed for non-negative ecological data. Here we introduce a generalized spatial scaling law that replaces the sample mean with the root-mean-square (RMS) of detrended activity, extending Taylor-like scaling to signed multivariate time series in neural and other complex systems. Within this framework, we quantify synchrony using a dedicated metric of functional coordination and show analytically, and via numerical simulations using multivariate Poisson, binomial, uniform and gamma distributions, that the scaling exponent is inversely related to synchrony. Applying this approach to human fMRI data from three large lifespan cohorts (N = 840, ages 18-88) reveals distinct age-related trajectories of the scaling exponent, with healthy aging characterized by a substantial synchrony-induced reduction in the exponent. The synchrony-scaling relationship remains stable during resting-state activity but progressively shifts during naturalistic tasks across the lifespan, and is particularly strong in limbic, subcortical, and cerebellar subnetworks, indicating preservation at subnetwork levels. Finally, individuals with ADHD exhibit disrupted synchrony-scaling coupling, highlighting the potential clinical and translational utility of this generalized scaling metric.

neuroscience↗

bioRxiv2025

The synaptic vesicle priming protein Munc13 mediates evoked somatodendritic dopamine release

Midbrain dopamine neurons secrete dopamine from their somata and dendrites in addition to their axonal release. Shared and distinct properties have been proposed for somatodendritic and axonal release, but the mechanisms of somatodendritic release remain unclear. We here used mouse genetics, electrophysiology, and imaging to define roles of the synaptic vesicle priming protein Munc13 in somatodendritic release in comparison to axonal secretion. Munc13 ablation in dopamine neurons decreased evoked but not spontaneous somatodendritic dopamine transmission measured as D2 receptor-mediated currents. Imaging with a fluorescent dopamine sensor confirmed this finding and established comparable importance for Munc13 in somatodendritic and axonal secretion. Pharmacological experiments revealed that release from adrenergic terminals contributes to D2 receptor-mediated currents, and their relative contribution was enhanced after Munc13 knockout. Altogether, these data establish important roles for Munc13 in evoked somatodendritic release. These roles are similar to Munc13 functions in axonal dopamine release and at fast synapses. Spontaneous midbrain dopamine release does not necessitate Munc13 in dopamine neurons and may rely on a release pathway that is independent of the prototypical release machinery employed at synapses.

neuroscience↗

bioRxiv2025

Pooled versus structured inhibition drives competitive stimulus interactions across distinct spatial scales of the superior colliculus

Competitive stimulus interactions across the space map in the midbrain superior colliculus (SC) are essential for spatial decision-making. Here, with electrophysiological recordings in intermediate and deep layers of the mouse SC (SCid), we discovered that rules and the spatial profiles of these competitive interactions are different between local, within-RF versus global, across-RF-boundary spatial scales. When one visual stimulus was centered in the receptive field (RF) and a second one was located within the RFs classical inhibitory surround, we found that stimulus interactions followed an averaging rule. This classical surround was spatially restricted, and the strength of inhibition underlying these within-RF interactions decreased with distance from the RF center. By contrast, when the second stimulus was located outside the RF, stimulus interactions followed a divisive rule. Strikingly, this extra-classical inhibitory surround was spatially global, and the strength of competitive inhibition underlying these across-RF-boundary interactions was distance-invariant. Computational modeling revealed that whereas within-RF interactions are well-explained by a divisive normalization-like mechanism driven by pooled inhibition, across-RF-boundary interactions are not, and instead, are well-explained by a winner-take-all-like mechanism driven by structured, donut-like inhibition. Combined, our results offer insights into the mechanistic logic of stimulus competition in the SCid, which critically underlies spatial decision-making in mammals.

neuroscience↗

bioRxiv2025

Retinoic acid coordinates the orderly construction of the mammalian body in the anterior-to-posterior sequence

In vertebrate embryos, anterior structures are formed early during gastrulation, while the posterior body develops subsequently. This temporal anterior-to-posterior developmental sequence is a fundamental aspect of animal development. Recent studies have shown that discrete regulatory modules involving Nodal and Wnt signaling pathways, in conjunction with T-box factors Eomes and Brachyury (Tbxt), underlie the differential specification of anterior and posterior mesoderm. However, the mechanism governing the transition from anterior to posterior mesoderm development remains unclear. Our findings suggest that retinoic acid signaling regulates the anterior-to-posterior developmental transition. Using mouse embryonic stem cell-based gastruloids, we show that the preclusion of retinoic acid signaling drives anterior mesoderm development, whereas the mesoderm specified in the presence of retinoic acid acquires posterior identity. Our observations indicate that retinoic acid signaling modulates the Wnt pathway. We demonstrate that both Eomes and Tbxt are essential for suppressing the posterior fate. The negative regulation of Wnt signaling by these T-box factors is critical, along with the preclusion of retinoic acid signaling, to ensure orderly anterior-posterior developmental progression. We propose that the preclusion of retinoic acid signaling early during gastrulation is crucial to maintain low Wnt signal levels, thereby driving anterior mesoderm specification. Subsequently, the activation of retinoic acid signaling results in high Wnt conditions, facilitating a switch to posterior mesoderm development.

developmental biology↗

bioRxiv2025

Sparse, random sampling is sufficient for central tolerance

Negative selection in the thymus limits autoimmunity by eliminating T cells that react strongly to self. Individual T cells, however, are only exposed to a small fraction of all self peptides during their "training" in the thymus, and it is puzzling how tolerance can be generalized to the remaining "test" self peptides across peripheral tissues in the body. Using a machine learning perspective, we show that such generalization is possible because the immune system satisfies two conditions: first that peptide abundance levels in the human thymus and periphery are highly correlated (i.e., training distribution {approx} test distribution), and second that cross-reactivity allows T cells to effectively learn binding information of similar peptides without explicitly interacting with all of them. Together, we show that sparse, random sampling of only 10% of self peptides in the thymus is sufficient to avoid reactivity to 90% of peripheral self, and we support this result with diverse experimental data. We then validate two predictions by our model; the first is that only 200-250 antigen presenting cells need to be seen by a T cell to ensure its robust selection, and the second relates how peptides missing from the thymus can drive auto-immunity of peripheral tissues. Overall, we provide a plausible answer to a long-standing question underlying adaptive immunity, and we highlight how generalization, a fundamental challenge faced by nearly every learning algorithm, is uniquely tackled by the immune system.

immunology↗

bioRxiv2025

Small molecule activator of phosphatase PP2A remodels scaffold PR65 structural dynamics to promote holoenzyme assembly

Small molecule activators of protein phosphatase 2A (PP2A), hereafter SMAPs, have attracted substantial interest, for their potential to inhibit cancer cell proliferation by targeting PR65, the scaffold subunit of the PP2A heterotrimer. PR65 is a uniquely flexible and stable molecule composed of 15 tandem HEAT (Huntingtin, Elongation factor 3-PP2A-TOR1) repeats. We characterized the binding sites and interactions of two SMAPs ATUX-8385 and DT-061 with PR65 and evaluated effects on PR65 structural dynamics using docking and molecular dynamics simulations. We initiated SMAP-bound PR65 simulations starting from two binding sites: S1, determined by cryo-electron microscopy for DT-061 bound to PP2A, on the inner helices of the HEAT repeats 2 and 3 (2i and 3i); and S2, predicted by docking of ATUX-8385 onto PR65, on 4i and 5i and outer helices 5o and 6o consistent with footprinting experiments. S2 proved to be a stable site for both SMAPs when initiating the simulations at S2. However, neither DT-061 nor ATUX-8385 demonstrated stable binding to S1. DT-061 rapidly dissociated from S1 to settle instead at a neighboring site S4 overlapping with our previously identified S3 for PR65 in extended form, suggesting that binding to S1 may be a 2-step process: an initial binding to PR65 alone, either to S3/S4 or S2, followed by movement to S3/S4, and then an induced relocation to S1 upon complexation with the regulatory and catalytic subunits. Targeted in silico mutagenesis showed that mutations at S2 and S4 destabilized SMAP binding to the PR65 (subunit). Heterotrimeric PP2A simulations showed that S3 and S4 binding were not persistent upon complexation. Together, these results corroborate our findings. Furthermore, preferentially stabilized a relatively extended PR65 conformation that would accommodate, if not promote, the assembly of the catalytic and regulatory subunits to prompt the activation of the trimeric phosphatase.

biophysics↗

bioRxiv2025

Moving Beyond Binary Biomarkers: Machine Learning Model Resolves Concurrent and Molecularly Heterogeneous Mismatch Repair and Homologous Recombination Deficiencies in Prostate Cancer

Current DNA damage repair (DDR) biomarkers employ binary classifications that fail to capture the molecular complexity of tumors with concurrent repair deficiencies. We used genomics analysis to stratify 672 metastatic prostate cancer patients into 11 DDR subgroups, identifying 51 molecular signatures with weighted roles in class identity. We identified a tumor-mutational-burden very-high subset, characterized by 19 mutations/Mb or more, as a molecularly distinct group characterized by preserved genomic integrity and enhanced immunogenicity. Critically, 2.3 percent of tumors exhibited concurrent TMB-High and HRR mutant phenotypes, while 1.5 percent harbored MMR bi-allelic loss without MMRd (mismatch-repair-deficiency) signatures. Clinical validation in 130 patients demonstrated superior immunotherapy responses in tumors with very high TMB levels. We developed CHIMERA DDR, a probabilistic machine learning tool that integrates these 51 genomic features using a nested Random Forest architecture to infer seven clinically relevant DDR subgroups. After negating model overfit concerns, CHIMERA-DDR showed exceptional classification performance (AUCs 0.919-0.999) to accurately detect MMRd and HRR mutant molecular subtypes with or without concurrent DDR deficiencies, resolving admixed phenotypes to enable precision therapeutic stratification beyond binary methods.

genomics↗

bioRxiv2025

CryoEM of ATP-driven dynamics and itraconazole binding in a fungal ABC pump

Azole resistance in Candida species is often caused by the overexpression of Cdr1. Despite its clinical relevance, the structural basis for its ATP-driven efflux pump function remains elusive. We present four high-resolution cryo-EM structures for Candida glabrata Cdr1 under active turnover conditions in the absence and presence of ATP-Mg{superscript 2}, itraconazole, and vanadate. Additional transient cryo-EM structures were unveiled by 3D variability analysis offering a detailed view of the step-by-step transitions triggered by ATP-hydrolysis. The motion cascade starts with a 4 [A] piston-like retraction of the C-helix from the {gamma}-phosphate/vanadate of the hydrolyzed ATP. This causes the nearby transmembrane helix-1 (TMH-1) to open the drug-binding site via lateral displacement and unwinding of the inner-leaflet region of TMH-2. A reverse squeeze-and-push motion of TMH-2 possibly drives substrate extrusion. High resolution structures also reveal how itraconazole adapts its shape to fit into the drug-binding site. Our findings provide a dynamic structural framework for Cdr1-mediated azole resistance and the conserved chemo-mechanical cycle of ABC proteins, including non-membranous members.

biochemistry↗

bioRxiv2025

dCas9 targeted proteome profiling reveals p300-mediated reciprocal regulation of SMAD and SP1 as a driver of GM2 synthase transcription in renal cell carcinoma

Glycolipids constitute an important component of the plasma membrane based on both abundance as well as function. Gangliosides, being a class of structurally diverse and functionally varied glycolipids, can act both as a receptor as well as a ligand and therefore is established as a crucial player in several normal cellular processes. In certain diseases, and in particular cancer, select gangliosides are over-expressed often leading to disease manifestation. GM2-synthase, the enzyme responsible for the formation of a pro-tumorigenic ganglioside, GM2 is well reported to be over-expressed across various cancer tissues and cell lines. This over-expression of GM2-synthase has been linked with increased migration, invasion and epithelial to mesenchymal transition (1) as well as induction of a local and systemic host immune suppression in cancer. Despite only a handful of studies demonstrating an epigenetic regulation underlying the transcriptional regulation of GM2-synthase (B4GalNT1) gene, the detailed mechanism still remains unclear. Here we identified the total proteome associated with the GM2-synthase promoter through a two-step CRISPR-dCas9 based proteome profiling approach by categorizing all the identified proteins leading to a detailed elucidation of the molecular drivers behind GM2-synthase transcription. While the previous study identified an acetylation-dependent de-repression of the transcription factor SP1 causing GM2-synthase activation, the underlying molecular mechanism driving its activation wasnt clear. This study demonstrated that the histone acetyl transferase (2), p300 acts as a pivotal factor which on one hand cause acetylation-mediated degradation of SP1, and on the other hand activates SMAD2/4 to have a direct positive impact on GM2-synthase gene transcription. We identified p300 to have an activator role in GM2-synthase gene transcription through knock out, knock down and over-expression experiments. Furthermore, SP1 degradation, SMAD activation and their DNA binding patterns show the reciprocal role of p300 on SP1 and SMAD complexes. Altogether we have identified SMAD 2/4 as an activator complex, p300 as a positive regulator and uncovered a critical p300-SMAD-SP1 regulatory axis in GM2-synthase transcriptional regulation.

molecular biology↗