Biology subjects

Radhakrishnan, R.

Publications and source records attributed to Radhakrishnan, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Single molecule Grb2 dynamics reveal spatiomechanical modulation of RTK signaling across length scales

Extracellular matrix (ECM)-mediated cell adhesion and tension regulate development and disease through crosstalk with Receptor Tyrosine Kinases (RTKs). RTKs stimulate proliferation, survival, and metabolic reprogramming via Ras/ERK and PI3K/Akt signaling. Emerging evidence identify the biophysical and biochemical integration of integrin-mediated adhesions with RTK signaling, however, the molecular mechanisms regulating this phenotype remain poorly understood. Here, we demonstrate that ECM stiffness potentiates ligand-mediated epidermal growth factor receptor (EGFR) signaling at scales ranging from the single adaptor protein, Grb2 upward to multicellular spheroids and tissues. We show that ECM stiffness-driven RTK signaling is mediated by integrin adhesions that reshape the membrane to induce the formation of RTK signaling hotspots distributed globally across the cell membrane. A parsimonious computational model and biochemical membrane perturbations causally implicate membrane curvature and topography in tension-dependent integrin-EGFR crosstalk. The results demonstrate how the mechanical properties of the ECM direct molecular recruitment and confinement of signaling molecules to regulate growth factor-dependent RTK signaling. Given that many RTK-driven solid tumors develop a stiff stroma, the findings could explain why many therapeutic tyrosine kinase inhibitors develop clinical resistance. The findings suggest clarifying how tension regulates transmembrane receptor signaling could identify unique force-dependent molecular regulators towards which new antitumor therapies could be applied.

biophysics↗

bioRxiv2026

Median raphe input to dorsal CA1 shapes VIP interneuron recruitment and novelty-guided spatial memory

Vasoactive intestinal peptide-expressing interneurons (VIP-INs) gate hippocampal inhibition during novel experience, but the long-range signals that engage these cells remain poorly understood. Here we identify median raphe (MnR) projections as a brainstem pathway that tunes dorsal CA1 VIP-IN recruitment through coordinated glutamatergic and serotonergic mechanisms. Anatomical mapping and optogenetic recordings showed that MnR axons innervate multiple VIP-IN subtypes, while transcriptomic and pharmacological analyses revealed fast glutamatergic excitation together with serotonin receptor-dependent modulation of synaptic and intrinsic responsiveness. In vivo calcium imaging showed that novelty preferentially recruited a speed-coupled VIP-IN ensemble, and inhibition of MnR input selectively reduced the magnitude of this response. A hippocampal circuit model linked this pathway to dendritic disinhibition and place-cell recruitment. Behaviorally, inhibition of MnR input preserved exploratory engagement but disrupted the organization of spatial sampling and impaired object-location memory. Thus, MnR input organizes hippocampal disinhibition to support novelty-guided exploration and memory encoding.

neuroscience↗

bioRxiv2026

Mechanistic Insights into MYO1C-Mediated Rhodopsin Trafficking and Rod Photoreceptor Homeostasis.

Rhodopsin trafficking from the photoreceptor inner segment to the outer segment is essential for photoreceptor function, yet the molecular mechanism(s) regulating this process remain incompletely understood. MYO1C is an actin-based motor protein implicated in intracellular cargo trafficking. Here, we investigated its role in rhodopsin trafficking and photoreceptor cell homeostasis. In-silico docking identified a putative interaction between the MYO1C C-terminal region and the C-terminal region of rhodopsin containing the conserved VxPx ciliary trafficking motif. Biochemical studies confirmed that full-length MYO1C interacts with rhodopsin, whereas deletion of the MYO1C C-terminal domain abolished this interaction. Live-cell imaging, ciliary localization, and fluorescence recovery after photobleaching in hTERT-RPE1 cells demonstrated that the MYO1C C-terminal region is required for efficient rhodopsin trafficking, membrane localization, and ciliary targeting. In native murine rod photoreceptors MYO1C localized to both inner and outer segments. Global Myo1c deficiency in mice caused age-dependent rhodopsin mislocalization, apo-opsin accumulation and progressive retinal dysfunction, characterized primarily by reduced scotopic ERG responses and delayed a-wave recovery, beginning at 6-months, while photopic responses were relatively preserved. Rod-specific Myo1c deletion similarly caused progressive scotopic dysfunction and reduced a-wave recovery following light stimulation. In contrast, cone-specific Myo1c deletion preserved photopic function and a-wave recovery. Together, these findings identify MYO1C as an important regulator of rhodopsin trafficking and demonstrate a preferential, cell-autonomous requirement for MYO1C in maintaining rod photoreceptor homeostasis and phototransduction recovery. These findings establish a mechanistic link between MYO1C-dependent rhodopsin trafficking and age-dependent rod photoreceptor cell dysfunction.

cell biology↗

bioRxiv2026

Percolation-inspired criticality in complement activation: universal scaling and transport-limited complement surface amplification

The collective spatial phenomenon of complement protein opsonization on nanoparticle surfaces is a key component of the immune response to viruses, engineered nanoparticles, and diseased cells. Recent work showed this opsonization follows a sharp, percolation-like transition versus the spacing d between surface-bound attachment sites, leaving two open questions: 1) whether the transition exhibits hallmarks of true criticality, such as diverging susceptibility, and 2) whether it can be distinguished from an alternative first-order cooperative (Hill-type) process producing an equally sharp threshold without true criticality. Here, we resolve both questions using a hierarchical statistical-mechanics treatment spanning stochastic, mean-field, and spatial reaction-diffusion models. The variance of two order parameters, peak complement activity and activation lifetime, diverges near threshold and sharpens systematically with system size, the defining signature of a critical point rather than a smooth cooperative response. Extending the analysis across site spacing and intrinsic kinetic rate constants traces a two-dimensional locus of critical points with consistent critical exponents throughout, establishing a single, robust universality class. The mean-field dynamic exponent for activation lifetime agrees quantitatively with the exact value predicted for the general epidemic process. Finally, a reaction-diffusion model of the nanoparticle surface shows the critical locus is set by a diffusion-limited length scale, establishing complement percolation as a fundamentally transport-limited surface reaction. These results place complement activation within the percolation universality class and identify the physical parameters, diffusion, catalysis, and decay, that govern its critical threshold, with direct implications for rational design of complement-evading nanomaterials, immunology, and evolutionary biology.

biophysics↗

bioRxiv2026

Life inside polymetallic nodules

Deep-sea abyssal plains harbour extensive biodiversity, much of which remains undocumented. Polymetallic nodules in the abyssal plains of the Clarion-Clipperton Zone (CCZ), Central Pacific, harbour diverse meiofaunal communities within their crevices, representing an understudied habitat. Given their high concentrations of nickel, copper, and cobalt, nodules are currently one of the primary targets for the extraction of deep-sea mineral resources. Here, we characterise the physical properties of polymetallic nodules, quantify their meiofaunal crevice communities, and compare them to those from the nearby sediments. We show that nodules contain internal cracks and microfossils, that their crevices harbour meiofaunal communities dominated by nematodes and copepods, and that faunal abundance increases significantly with nodule volume. Nodule-crevice nematode and copepod abundances were equivalent to approximately 10% and 25%, respectively, of those recorded in nearby sediments across the two exploration contract areas in the eastern CCZ. Based on 18S rRNA data, nematode and copepod assemblages recovered from nodule crevices differed from those recovered from nearby sediments collected during the same expedition and within the same exploration contract areas. Our findings, therefore, point to the possibility of nodule-associated crevice meiofauna communities and the consequent risk of biodiversity loss associated with nodule removal through potential future commercial mining.

ecology↗

bioRxiv2026

Coupled Cell-Intrinsic and Microenvironmental Heterogeneity Drives Divergent Trajectories in Castration-Resistant Prostate Cancer

Castration-resistant prostate cancer emerges from coupling between cell-intrinsic heterogeneity and microenvironmental constraints. Mechanistically dissecting this coupling, rather than either factor in isolation, is the central aim of this study. To systematically study the effect of intrinsic and extrinsic spatial axes on disease trajectories, we developed an integrated multiscale framework: a cellular signaling model (MHS) parameterized with TCGA genomic data from control, biochemical recurrence (BR), and treatment-resistant (TR) cohorts, coupled to a spatial agent-based model (ABM). Machine learning surrogates trained on the MHS model identified PTEN, MDM4, and AR as dominant intrinsic drivers, using SHAP-based feature ranking. Clinical validation via Kaplan-Meier and Cox regression across cBioPortal cohorts confirmed these rankings: AR alterations (median OS 20 vs. 86 months), PTEN loss (54 vs. 77 months), and MDM4 amplification (33 vs. 75 months) predicted poor overall survival outcomes independently. At the tissue scale, ABM simulations were run to study the effect of microenvironmental (cell-extrinsic) factors such as physical confinement, androgen uptake kinetics, and adhesion-motility strength, on disease progression. This spatiotemporal analysis revealed that identical genetic alterations produce varied selection outcomes depending on microenvironmental context. Extending this coupling logic to the individual patient level, we parameterized the MHS model using gene expression profiles from 14 patients in the EUREKA1 prospective registry. Patient-specific androgen sensitivity ratios, the ratio of net cell growth under high versus low testosterone, stratified patients by model-predicted androgen dependence without requiring longitudinal PSA observations and showed that PTEN deletion shifts the proliferative response toward androgen independence in a patient-specific magnitude set by the broader expression background. This provides a model-based route from genomic data at diagnosis to personalized prediction of ADT resistance risk. Together, these findings establish that CRPC emergence is an emergent property of the intrinsic-extrinsic coupling, that neither molecular nor spatial analyses in isolation can predict clinical trajectories, and that mechanistic integration of both is required for accurate patient stratification.

systems biology↗

bioRxiv2025

Integrative machine learning predicts activating kinase mutations for precision oncology

Kinases are enzymes that catalyze phosphorylation and play crucial roles in a myriad of cellular regulatory processes and hemostasis. Patient-specific genetic mutations that aberrantly activate kinases can profoundly influence cancer progression and alter drug efficacy. Predicting the impact of such missense mutations across the human kinome on protein function and cellular signaling is therefore a critical step toward personalized targeted therapy. Here, we present Kinome-AI, an integrative machine learning framework that classifies kinase missense mutations as activating or non-activating. Kinome-AI is trained on a rich multi-modal feature set, including residue-level biochemical changes, sequence embeddings from a protein language model, and structural descriptors of kinase-ATP-substrate complexes derived from molecular modeling. Notably, detailed structural features were available for only 21% of mutants; we leverage these as privileged information during training to impute missing structural data for the remaining [~]79. This strategy boosts performance without requiring structural inputs for new (unseen) mutations. The resulting classifier achieves an area under the receiver operating characteristic curve (AUROC) of 0.85 and a balanced accuracy (BACC) of 0.76 across 1,003 mutations spanning 110 different kinases --substantially outperforming existing bioinformatics and general-purpose variant effect predictors. This work provides a robust approach to quantify sequence-structure- function relationships of cancer-driving kinase mutations, paving the way for improved personalized cancer treatment. Significance StatementIn cancer patients, numerous mutations in diverse protein kinases lead to marked differences in disease progression and drug response. Identifying which kinase mutations are activating in individual patients is therefore critical for precision oncology. Drawing inspiration from teacher- student (privileged information) learning, we developed a deep learning framework that integrates structural features from molecular simulations with sequence embeddings from protein language models. This approach enables accurate binary classification of the activation status of kinase mutations. Our study demonstrates how data-driven algorithms can leverage accumulated sequence and structural knowledge of known mutations to predict the effects of novel variants a priori. The model, termed Kinome-AI, shows significant promise for incorporation into personalized cancer therapy decision pipelines.

cancer biology↗

bioRxiv2025

Modeling tumor transport and growth with poroelastic biopolymer networks

The mechanical properties of the extracellular matrix (ECM) regulate tumor growth and invasion in the tumor microenvironment. Models of biopolymer networks have been used to investigate the impact of elasticity and viscoelasticity of ECM on tumor behavior. Under tumor compression, these networks also show poroelastic behavior that is governed by the resistance to water flow through their pores. This work investigates the hypothesis that poroelastic properties regulate tumor growth. Here, alginate hydrogels with tunable ionic and hybrid ionic/covalent crosslinking are used as a model biopolymer system. Hydrogel stiffness, viscoelasticity, and stress relaxation behavior were characterized using stepwise axial compression. Among these properties, we find poroelastic fluid outflow dominates ECM stress relaxation, as the measured water flux was significantly affected under compression. Continuum mechanics-based modeling was developed to formulate and calculate the chemical potential gradients of water (solvent) in the hydrogels under compression. This framework was extended into an advection-diffusion framework to quantify growth factor (solute) distribution under varying strengths of stress and diffusion indexed by the relative strength of convective to diffusive transport, characterized by the Peclet number. An agent-based computational simulation showed that tumor growth was affected by Peclet number. Together, these results highlight the role of the poroelastic properties of ECM on water flux and transport in the tumor microenvironment.

biophysics↗

bioRxiv2025

Rescue of the Stargardt Disease phenotype in Abca4 knockout mice through dietary modulation of the vitamin A receptor RBPR2

Mutations in the ABCA4 gene in Stargardt disease (STGD1) causes accumulation of cytotoxic lipofuscin, resulting in RPE atrophy and photoreceptor dysfunction. One component of lipofuscin is the all-trans-retinal derivative, bisretinoid N-retinylidene-N-retinylethanolamine (A2E). Since ocular A2E biosynthesis relies on circulating all-trans-retinol bound to retinol binding protein 4 (RBP4-ROL), we hypothesized that modulating vitamin A receptors, such as the retinol binding protein receptor 2, RBPR2, which regulate serum RBP4-ROL concentration, should attenuate A2E production. In-silico analysis revealed multiple retinoic acid response element (RARE) binding sites on the murine Rbpr2 gene promotor, which was confirmed in vitro by EMSA and ChIP assays. In vitro luciferase assays showed that Rbpr2 promotor activity was induced by exogenous {beta}-carotene (BC) metabolites. Dietary BC supplementation of Abca4-/- mice, a mouse model for STGD1, increased hepatic all-trans-retinoic acid and 9-cis-retinoic acid production, which induced Rbpr2 mRNA expression. This mechanism decreased serum RBP4 protein levels, fundus autofluorescence (AF) and ocular A2E accumulation, altogether improving photoreceptor and RPE function. Conversely, such a rescue was not observed in either Abca4-/- mice fed a diet devoid of BC or in double knockout Rbpr2-/-;Abca4-/- mice. Thus, there was a significant inverse correlation between dietary BC supplementation and Rbpr2 gene presence in Abca4-/- mice, to that of lipofuscin accumulation in Abca4-/- mice on diets devoid of BC or in Rbpr2-/-;Abca4-/- mice. Our results provide impetus to pursue BC supplemented diets as therapeutic interventions for STGD1 patients with ABCA4 gene mutations and identifies a novel role for the vitamin A receptor RBPR2 in this process.

cell biology↗

bioRxiv2025

MYC and AP-1 oncogenes synergistically bind enhancers to rewire transcription

The transcription factor c-MYC (MYC) is deregulated in about 70% of human cancers. Through de novo motif discovery analysis on published MYC ChIP-seq datasets from cancer cell lines, we found cell-type specific co-enrichment of the TRE motifs (AP-1 binding sites), alongside MYCs canonical EBOX motif. TRE motifs serve as indirect MYC binding sites in synergy with AP-1, predominantly at enhancers rather than promoters. At elevated MYC levels, as seen in cancers, MYC preferentially occupies TRE sites over EBOX sites at enhancers. Integration of ChIP-seq and RNA-seq data revealed TRE enhancer binding sites are frequently associated with MYC-mediated transcriptional repression. Gene Ontology analysis showed that MYC utilizes TRE sites to transcriptionally rewire cells, modulating cancer hallmarks like proliferation, apoptosis, and cell adhesion. These molecular insights into how increased MYC levels alter its DNA binding preference and gene regulation could inform new therapeutic strategies targeting cancer-specific MYC functions and its co-regulators.

genomics↗

bioRxiv2025

CPSF6 Promotes HIV-1 Preintegration Complex Function

Cleavage and polyadenylation specificity factor 6 (CPSF6) is part of the cellular cleavage factor I mammalian (CFIm) complex that regulates mRNA processing and polyadenylation. CPSF6 also functions as a HIV-1 capsid (CA) binding host factor and promotes viral DNA integration targeting into gene dense regions of the host genome. However, the effects of CPSF6 on the activity of the HIV-1 preintegration complex (PIC) - the machinery that carries out viral DNA integration to establish infection - is unknown. To study CPSF6s role in HIV-1 PIC function, we extracted PICs from cells depleted of CPSF6 or expressing a CPSF6 mutant that cannot bind to CA. These PICs exhibited significantly lower integration activity when compared to the control PICs. Addition of recombinant CPSF6 restored the integration activity of PICs extracted from the mutant cells, suggesting a direct role of CPSF6 in PIC function. To solidify CPSF6s effect on PIC function, we inoculated CPSF6-depleted and CPSF6-mutant cells with HIV-1 particles and measured viral DNA integration into the host genome. A significant reduction in viral integration in these cells was detected and this defect was not a consequence of reduced reverse transcription or nuclear entry. Additionally, mutant viruses deficient in CA-CPSF6 binding showed no integration defect in CPSF6 mutant cells. Finally, sequencing analysis revealed that HIV-1 integration in the CPSF6 mutant cells was significantly redirected from the gene dense regions of the host genome. Collectively, these results suggest that CPSF6-CA interaction regulates PIC function both in vitro and in infected cells. IMPORTANCEHIV-1 infection is dependent on the interaction of the virus with host factors. However, the molecular details of virus-host factor interactions are not fully understood. For instance, HIV-1 capsid provides binding interfaces for several host factors. CPSF6 is one such capsid-binding host factor, whose cellular function is to regulate mRNA processing and polyadenylation. Initial work identified a truncated cytosolic form of CPSF6 that restricted HIV infection by blocking viral nuclear entry. However, it is now established that the full-length CPSF6 primarily promotes integration targeting into gene dense regions of the host genome. Here we report that CPSF6-CA interaction promotes the activity of HIV-1 preintegration complexes (PICs). We also observed that disruption of CPSF6-CA binding in target cells significantly reduced viral integration and directed integration targeting away from gene-dense regions. These findings demonstrate a critical role for the CPSF6-CA interaction in PIC function and integration targeting.

biochemistry↗

bioRxiv2025

Tissue-dependent mechanosensing by cells derived from human tumors

Alterations of the extracellular matrix (ECM), including both mechanical (such as stiffening of the ECM) and chemical (such as variation of adhesion proteins and deposition of hyaluronic acid (HA)) changes, in malignant tissues have been shown to mediate tumor progression. To survey how cells from different tissue types respond to various changes in ECM mechanics and composition, we measured physical characteristics (adherent area, shape, cell stiffness, and cell speed) of 25 cancer and 5 non-tumorigenic cell lines on 7 different substrate conditions. Our results indicate substantial heterogeneity in how cell mechanics changes within and across tissue types in response to mechanosensitive and chemosensitive changes in ECM. The analysis also underscores the role of HA in ECM with some cell lines showing changes in cell mechanics in response to presence of HA in soft substrate that are similar to those observed on stiff substrate. This pan-cancer investigation also highlights the importance of tissue-type and cell line specificity for inferences made based on comparison between physical properties of cancer and normal cells. Lastly, using unsupervised machine learning, we identify phenotypic classes that characterize the physical plasticity, i.e. the distribution of physical feature values attainable, of a particular cell type in response to different ECM-based conditions.

biophysics↗

bioRxiv2024

Enzyme-Regulated Non-Thermal Fluctuations Enhance Ligand Diffusion and Receptor-Mediated Endocytosis

Active enzymes during catalyzing chemical reactions, have been found to generate significant mechanical fluctuations, which can influence the dynamics of their surroundings. These phenomena open new avenues for controlling mass transport in complex and dynamically inhomogeneous environments through localized chemical reactions. To explore this potential, we studied the uptake of transferrin molecules in retinal pigment epithelium (RPE) cells via clathrin-mediated endocytosis. In the presence of enzyme catalysis in the extracellular matrix, we observed a significant enhancement in the transport of fluorophore-tagged transferrin inside the cells. Fluorescence correlation spectroscopy measurements showed substantial increase in transferrin diffusion in the presence of active fluctuations. This study sheds light on the possibility that enzyme-substrate reactions within the extracellular matrix may induce long-range mechanical influences, facilitating targeted material delivery within intracellular milieu more efficiently than passive diffusion. These insights are expected to contribute to the development of better therapeutic strategies by overcoming limitations imposed by slow molecular diffusion under complex environments.

biophysics↗

bioRxiv2024

Structural and Mechanistic Bases for Resistance of the M66I Capsid Variant to Lenacapavir

Lenacapavir (LEN) is the first in class viral capsid protein (CA) targeting antiretroviral for treating multi-drug-resistant HIV-1 infection. Clinical trials and cell culture experiments have identified resistance associated mutations (RAMs) in the vicinity of the hydrophobic CA pocket targeted by LEN. The M66I substitution conferred by far the highest level of resistance to the inhibitor compared to other RAMs. Here we investigated structural and mechanistic bases for how the M66I change affects LEN binding to CA and viral replication. The high-resolution X-ray structure of the CA(M66I) hexamer revealed that the {beta}-branched side chain of Ile66 induces steric hindrance specifically to LEN thereby markedly reducing the inhibitor binding affinity. By contrast, the M66I substitution did not affect binding of Phe-Gly (FG)-motif-containing cellular cofactors CPSF6, NUP153, or SEC24C, which engage the same hydrophobic pocket of CA. In cell culture the M66I variant did not acquire compensatory mutations or replicate in the presence of LEN. Analysis of viral replication intermediates revealed that HIV-1(M66I CA) predominantly formed correctly matured viral cores, which were more stable than their wildtype counterparts. The mutant cores stably bound to the nuclear envelope but failed to penetrate inside the nucleus. Furthermore, the M66I substitution markedly altered HIV-1 integration targeting. Taken together, our findings elucidate mechanistic insights for how the M66I change confers remarkable resistance to LEN and affects HIV-1 replication. Moreover, our structural findings provide powerful means for future medicinal chemistry efforts to rationally develop second generation inhibitors with a higher barrier to resistance. IMPORTANCELenacapavir (LEN) is a highly potent and long-acting antiretroviral that works by a unique mechanism of targeting the viral capsid protein. The inhibitor is used in combination with other antiretrovirals to treat multi-drug-resistant HIV-1 infection in heavily treatment-experienced adults. Furthermore, LEN is in clinical trials for preexposure prophylaxis (PrEP) with interim results indicating 100 % efficacy to prevent HIV-1 infections. However, one notable shortcoming is a relatively low barrier of viral resistance to LEN. Clinical trials and cell culture experiments identified emergent resistance mutations near the inhibitor binding site on capsid. The M66I variant was the most prevalent capsid substitution identified in patients receiving LEN to treat muti-drug resistant HIV-1 infections. The studies described here elucidate the underlying mechanism by which the M66I substitution confers a marked resistance to the inhibitor. Furthermore, our structural findings will aid future efforts to develop the next generation of capsid inhibitors with enhanced barriers to resistance.

microbiology↗

bioRxiv2024

Mitochondrial genome-encoded mitomiRs regulate cellular plasticity and susceptibility to ferroptosis in triple-negative breast cancer

Ferroptosis is a distinct form of regulated cell death promoted by iron-dependent lipid peroxidation. The metabolic plasticity of cancer cells determines their sensitivity to ferroptosis. Although mitochondrial dysfunction contributes to metabolic reprogramming in cancer cells, its role in ferroptosis remains to be identified. We identified that the mitochondrial genome encodes 13 miRNAs (mitomiRs) that are highly expressed in breast cancer cell lines and patient-derived tumor samples. Expression analysis revealed that mitomiRs are upregulated in basal-like triple-negative breast cancer (TNBC) cells compared to mesenchymal stem-like TNBC cells. Interestingly, 11 out of the 13 mitomiRs bind to the 3'UTR of zinc finger E-box-binding homeobox 1 (ZEB1), a transcription factor, involved in epithelial to mesenchymal transition (EMT) in breast cancer. Using mitomiR-3 mimic, inhibitor and sponges, we confirmed that mitomiR-3 indeed regulate ZEB1 expression in TNBC cells. Increased mesenchymal features in TNBC contributed to vulnerability to pro-ferroptotic metabolic reprogramming sensitizing to cell death in in vitro and in vivo models. Some of the challenges associated with pro-ferroptotic drugs includes lack of cancer cell specificity, low targeting ability, normal tissue toxicity contributing to their limited clinical application as cancer therapeutics. Here, we identified mitomiRs which are highly expressed in TNBC subtypes with low expression in normal breast cells making them an ideal candidate for selective inhibition for targeted therapy. Further, we demonstrated that the inhibition of mitomiRs in triple-negative breast cancer cells promote pro-ferroptotic metabolic reprogramming which can be exploited as novel vulnerability for targeted ferroptotic induction in cancer cells avoiding the normal tissue toxicity. Collectively, our results indicate a novel mechanism of mitochondrial miRNA mediated ferroptosis sensitivity in TNBC subtypes which could be exploited to develop potential miRNA-based therapeutics.

cancer biology↗

bioRxiv2024

A percolation-type criticality threshold controls immune protein coating of surfaces

When a material enters the body, it is immediately attacked by hundreds of proteins, organized into complex networks of binding interactions and reactions. How do such complex systems interact with a material, "deciding" whether to attack? We focus on the "complement" system of [~]40 blood proteins that bind microbes, nanoparticles, and medical devices, initiating inflammation. We show a sharp threshold for complement activation upon varying a fundamental material parameter, the surface density of potential complement attachment points. This sharp threshold manifests at scales spanning single nanoparticles to macroscale pathologies, shown here for diverse engineered and living materials. Computational models show these behaviors arise from a minimal subnetwork of complement, manifesting percolation-type critical transitions in the complement response. This criticality switch explains the "decision" of a complex signaling network to interact with a material, and elucidates the evolution and engineering of materials interacting with the body.

biophysics↗

bioRxiv2024

Coupled Solvent Dynamics and Protein Dynamics Help Drive Functional Differences in Exon-19 Deletion Mutants in the Epidermal Growth Factor Receptor (EGFR/ErbB1/HER1) Kinase Domain

Deletions in Exon-19 of the epidermal growth factor receptor (EGFR) play a pivotal role in the pathogenesis of non-small cell lung cancer (NSCLC), influencing patient response to tyrosine kinase inhibitors (TKIs). Although these mutations are known to affect treatment efficacy, the precise molecular mechanisms have been unclear. Building upon recent insights from the study [DOI: 10.1038/s41467-022-34398-z], which identified two distinct mutation profiles associated with differential drug sensitivity and clinical outcomes, our research delves into the molecular dynamics that drive these variances. We employed molecular dynamics simulations, enhanced sampling methods, and machine learning to classify Exon-19 deletion mutations into two profiles based on their conformational dynamics. Profile 1 mutations display only localized motions in key subdomains in their fluctuations about the equilibrium state, and a high affinity for ATP and consequent resistance to TKIs, while profile 2 mutations show reduced ATP binding affinity due to delocalized motion characterized by an increased flexibility between the N- and C-lobes of the EGFR kinase domain. This structural flexibility perturbs the ATP binding site, leading to decreased affinity and, heightened sensitivity to TKIs. Our use of the INDirect Umbrella Sampling (INDUS) technique has shed light on the collective solvent dynamics, further elucidating the coupling between long timescale solvent fluctuations and protein conformational dynamics, that likely contributes to the observations in HDX-MS studies. Our free energy analysis, covering timescales relevant to both HDX-MS and ligand interaction, provides a deeper understanding of the relationship between protein and solvent dynamics and their collective impact on drug efficacy in NSCLC with EGFR Exon-19 deletions. Significance StatementEGFR Exon 19 deletion mutations are key drivers in non-small cell lung cancer (NSCLC), yet their drug sensitivity to tyrosine kinase inhibitors (TKIs) varies significantly. This study identifies two mutation profiles: mutations that exhibit high ATP binding affinity and localized conformational motion, driving TKI resistance, and mutations that show reduced ATP affinity due to delocalized structural flexibility, enhancing TKI sensitivity. Using molecular dynamics simulations and free energy sampling techniques, we reveal how solvent fluctuations and protein dynamics collectively affect drug binding and efficacy. These findings provide a mechanistic basis for differential drug sensitivity, informing precision medicine strategies for NSCLC patients with EGFR Exon-19 mutations.

biophysics↗

bioRxiv2024

Interplay between the cyclophilin homology domain of RANBP2 and MX2 regulates HIV-1 capsid dependencies on nucleoporins

Interlinked interactions between the viral capsid (CA), nucleoporins (Nups), the antiviral protein myxovirus resistance 2 (MX2/MXB) influence HIV-1 nuclear entry and the outcome of infection. Although RANBP2/NUP358 has been repeatedly identified as a critical player in HIV-1 nuclear import and MX2 activity, the mechanism by which RANBP2 facilitates HIV-1 infection is not well understood. To explore the interactions between MX2, the viral CA, and RANBP2, we utilized CRISPR-Cas9 to generate cell lines expressing RANBP2 from its endogenous locus but lacking the C-terminal cyclophilin (Cyp) homology domain, and found that both HIV-1 and HIV-2 infection were reduced significantly in RANBP2{Delta}Cyp cells. Importantly, although MX2 still localized to the nuclear pore complex in RANBP2{Delta}Cyp cells, antiviral activity against HIV-1 was decreased. By generating cells expressing specific point mutations in the RANBP2-Cyp domain we determined that the effect of the RANBP2-Cyp domain on MX2 anti-HIV-1 activity is due to direct interactions between RANBP2 and CA. We further determined that CypA and RANBP2-Cyp have similar effects on HIV-1 integration targeting. Finally, we found that the Nup requirements for HIV infection and MX2 activity were altered in cells lacking the RANBP2-Cyp domain. These findings demonstrate that the RANBP2-Cyp domain affects viral infection and MX2 sensitivity by altering CA-specific interactions with cellular factors that affect nuclear import and integration targeting. Significance StatementHIV-1 entry into the nucleus is an essential step in viral replication that involves complex interactions between the viral capsid and multiple cellular proteins, including nucleoporins such as RANBP2. Nups also mediate the function of the antiviral protein MX2, however determining the precise role of Nups in HIV infection has proved challenging due to the complex nature of the nuclear pore and significant pleiotropic effects elicited by Nup depletion. We have used precise gene editing to assess the role of the Cyp domain of RANBP2 in HIV-1 infection and MX2 activity. We find that this domain affects viral infection, nucleoporin requirements, MX2 sensitivity, and integration targeting in a CA-specific manner, providing detailed insights into how RANBP2 contributes to HIV-1 infection.

microbiology↗