Biology subjects

Rizvi, A.

Publications and source records attributed to Rizvi, A..

14 recordsLinked to original sources

bioRxiv2026

Competing Molecular Interactions Govern the Dynamical Arrest of G3BP1 Condensates

G3BP1 is a central scaffold of stress granules (SGs). Upon cellular stress, G3BP1 forms complex coacervates with translationally repressed mRNAs and recruits multiple RNA-binding proteins to form reversible biomolecular condensates. Persistent SGs are linked to age-dependent dynamical arrest and impaired disassembly. Here, we employ active and passive nanoscale rheology with optical tweezers to show that G3BP1 condensates evolve from being dominantly viscous fluids to dynamically arrested network glasses characterized by nanoscale caging and elastic memory. Integrating atomistic and coarse-grained simulations with experiments, we find that electrostatic interactions between the oppositely charged intrinsically disordered regions drive condensate ageing. RNA modulates these interactions in a length-and structure-dependent manner, delaying dynamic arrest, whereas Caprin-1 binding to the NTF2L domain has little effect. Together, these findings reveal how competing inter-IDR and IDR-RNA interactions govern condensate ageing and material-state transitions. The findings have broader implications for the regulation of SG dynamics in cells.

biophysics↗

bioRxiv2026

Multiscale Physical Effects of CpG Methylation on DNA Mechanics, Nucleosome Wrapping, and Chromatin Condensates

DNA methylation at CpG dinucleotides is a central epigenetic modification linked to transcriptional repression and heterochromatin formation, yet its physical impact on chromatin organization remains incompletely understood. Here, we integrate single-molecule force spectroscopy, FRAP, and nanorheology to quantify how CpG methylation alters chromatin mechanics across scales. Optical tweezer-based measurements show that CpG methylation modestly increases DNA contour and persistence lengths, indicating a slightly extended and stiffer DNA helix. When assembled into nucleosomes, CpG methylation weakens both outer-and inner turn wrapping, reducing unwrapping forces and per-nucleosome unwrapping energy. On a higher-order assembly scale, although CpG methylated and unmethylated chromatin fibers display similar thresholds for salt-induced phase separation, methylated condensates exhibit markedly reduced internal mobility: FRAP reveals slower chromatin exchange, nanorheology shows increased viscoelasticity and longer relaxation times. Finally, CpG methylation enhances HP1-chromatin interactions, lowering the HP1 concentration required for condensation, despite no change in HP1 binding to naked DNA. Together, these results show that CpG methylation remodels chromatin mechanics by tuning DNA rigidity, nucleosome wrapping energetics, and condensate material properties, providing a multiscale physical framework for how DNA methylation contributes to heterochromatin.

biochemistry↗

bioRxiv2025

Organoid Pharmacotyping of Pancreatic Cancer Enables Functional Precision Oncology and Drug Repurposing

PurposePancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest malignancies, with limited benefit from current cytotoxic regimens and poor predictive value of genomics alone. Patient-derived organoids (PDOs) represent a promising platform for functional precision oncology, yet systematic pharmacotyping of genomically annotated PDAC PDOs remains sparse. Experimental DesignWe established a clinically annotated panel of ten treatment-naive PDAC PDOs spanning well-, moderately-, and poorly differentiated tumors. PDOs were evaluated for morphologic and genomic fidelity and screened against 1,813 clinically relevant small molecules in a high-throughput 384-well format. Drug sensitivities were quantified at the compound and drug-family levels and integrated with histologic grade, pathway-level mutational profiles, and available clinical treatment information. ResultsPDOs preserved hallmark tumor features, including glandular organization and subclonal mutational architecture. Pharmacotyping revealed both shared and subtype-specific vulnerabilities. Classical (well/moderately differentiated) PDOs showed enriched mutations in DNA repair, mitotic spindle, and chromatin-regulatory pathways and were preferentially sensitive to topoisomerase inhibitors, microtubule poisons, and HDAC inhibitors. In contrast, basal (poorly differentiated) PDOs displayed coordinated defects in mitochondrial function, vesicle trafficking, and ubiquitin-mediated proteostasis, at the pathway level, that conferred a previously unrecognized vulnerability to cardiac glycosides. Sensitivities to standard PDAC agents were heterogeneous across models, underscoring the limited predictive value of genotype alone and the need for functional drug testing. ConclusionsThis integrated genomic and pharmacologic analysis demonstrates that PDO pharmacotyping identifies biologically grounded, actionable vulnerabilities in PDAC, including novel therapeutic opportunities in basal, chemo-resistant tumors. These findings support PDO-guided functional profiling as a clinically relevant platform for refining drug selection and expanding treatment options for patients with PDAC. SignificancePDAC is dominated by chemoresistance and lacks reliable genomic predictors of therapy response. By integrating high-throughput drug screening with mutation-informed pathway analysis in patient-derived organoids, we identify differentiation-linked therapeutic liabilities, including a previously unrecognized vulnerability to cardiac glycosides in basal PDAC. These results highlight PDO pharmacotyping as a powerful functional complement to genomics for guiding treatment selection in pancreatic cancer.

cancer biology↗

bioRxiv2025

An image-based CRISPR screen reveals splicing-mediated control of HP1α condensates

Heterochromatin Protein 1 (HP1) is a fundamental component of constitutive heterochromatin, forming subnuclear condensates whose regulation and function remain poorly understood. Here, we present an image-based CRISPR screen targeting nuclear factors that identifies splicing as a pivotal pathway regulating HP1 condensates. We discovered that unspliced intronic RNA modulates HP1 condensates by interacting co-transcriptionally with HP1. By modulating the intron content, RNA processing restricts HP1-RNA interactions at chromatin, thus enabling heterochromatin organization. Disruption of HP1 condensates due to enhanced interactions with unspliced RNA leads to loss of heterochromatin and the activation of stress response protective genes. We propose that RNA is a central component of heterochromatin that modulates HP1 condensates, and that RNA processing enzymes act as a surveillance mechanism for condensates by dynamically regulating the network of multi-valent interactions between RNA and chromatin factors. This model underscores the crosstalk between chromatin organization, transcription, and RNA processing, potentially governing broader nuclear functions.

genetics↗

bioRxiv2024

KipOTIA detoxifies 5-oxoproline and promotes the growth of Clostridioides difficile

Clostridioides difficile is an anaerobic enteric pathogen that disseminates in the environment as a dormant spore. For C. difficile and other sporulating bacteria, the initiation of sporulation is a regulated process that prevents spore formation under favorable growth conditions. In Bacillus subtilis, one such mechanism for preventing sporulation is the Kinase Inhibitory Protein, KipI, which impedes activation of the main sporulation kinase. In addition, KipI functions as part of a complex that detoxifies the intermediate metabolite, 5-oxoproline (OP), a harmful by-product of glutamic acid. In this study, we investigate the orthologous Kip proteins in C. difficile to determine their roles in the regulation of sporulation and metabolism. Using deletion mutants in kipIA and the full kipOTIA operon, we show that unlike in B. subtilis, the Kip proteins have no significant impact on sporulation. However, we found that the kip operon encodes a functional oxoprolinase that facilitates detoxification of OP. Further, our data demonstrate that KipOTIA not only detoxifies OP, but also allows OP to be used as a nutrient source that supports the robust growth of C. difficile, thereby facilitating the conversion of a toxic byproduct of metabolism into an effective energy source.

microbiology↗

bioRxiv2023

Development of a robotic cluster for automated and scalable cell therapy manufacturing

The production of commercial autologous cell therapies such as chimeric antigen receptor T cells requires complex manual manufacturing processes. Skilled labor costs and challenges in manufacturing scale-out have contributed to high prices for these products. Here, we present a robotic system that uses industry-standard cell therapy manufacturing equipment to automate the steps involved in cell therapy manufacturing. The robotic cluster consists of a robotic arm and customized modules, allowing the robot to manipulate a variety of standard cell therapy instruments and materials such as incubators, bioreactors, and reagent bags. This system enables existing manual manufacturing processes to be rapidly adapted to robotic manufacturing, without having to adopt a completely new technology platform. Proof-of-concept for the robotic clusters expansion module was demonstrated by activating and expanding human CD8+ T cells. The robotic cultures showed comparable cell yields, viability, and identity to those manually performed. Such modular robotic solutions may support scale-up and scale-out of cell therapies that are developed using classical manual methods in academic laboratories and biotechnology companies.

bioengineering↗

bioRxiv2023

Heterozygous Kmt2d loss diminishes enhancers to render medulloblastoma cells vulnerable to combinatory inhibition of lysine demethylation and oxidative phosphorylation

The histone H3 lysine 4 (H3K4) methyltransferase KMT2D (also called MLL4) is one of the most frequently mutated epigenetic modifiers in medulloblastoma (MB) and many other types of cancer. Notably, heterozygous loss of KMT2D is prevalent in MB and other cancer types. However, what role heterozygous KMT2D loss plays in tumorigenesis has not been well characterized. Here, we show that heterozygous Kmt2d loss highly promotes MB driven by heterozygous loss of the MB suppressor gene Ptch in mice. Heterozygous Kmt2d loss upregulated tumor-promoting programs, including oxidative phosphorylation and G-protein-coupled receptor signaling, in Ptch+/--driven MB genesis. Mechanistically, both downregulation of the transcription-repressive tumor suppressor gene NCOR2 by heterozygous Kmt2d loss and upregulation of the oncogene MycN by heterozygous Ptch loss increased the expression of tumor-promoting genes. Moreover, heterozygous Kmt2d loss extensively diminished enhancer signals (e.g., H3K27ac) and H3K4me3 signature, including those for tumor suppressor genes (e.g., Ncor2). Combinatory pharmacological inhibition of oxidative phosphorylation and the enhancer-decommissioning H3K4 demethylase LSD1 drastically reduced tumorigenicity of MB cells bearing heterozygous Kmt2d loss. These findings reveal the mechanistic basis underlying the MB-promoting effect of heterozygous KMT2D loss, provide a rationale for a therapeutic strategy for treatment of KMT2D-deficient MB, and have mechanistic implications for the molecular pathogenesis of other types of cancer bearing heterozygous KMT2D loss.

cancer biology↗

bioRxiv2023

The predicted acetoin dehydrogenase pathway represses sporulation of Clostridioides difficile

Clostridioides difficile is a major gastrointestinal pathogen that is transmitted as a dormant spore. As an intestinal pathogen, C. difficile must contend with variable environmental conditions, including fluctuations in pH and nutrient availability. Nutrition and pH both influence growth and spore formation, but how pH and nutrition jointly influence sporulation are not known. In this study, we investigated the dual impact of pH and pH-dependent metabolism on C. difficile sporulation. Specifically, we examined the impacts of pH and the metabolite acetoin on C. difficile growth and sporulation. We found that expression of the predicted acetoin dehydrogenase operon, acoRABCL, was pH-dependent and regulated by acetoin. Regulation of the C. difficile aco locus is distinct from other characterized systems and appears to involve a co-transcribed DeoR-family regulator rather than the sigma54-dependent activator. In addition, an acoA null mutant produced significantly more spores and initiated sporulation earlier than the parent strain. However, unlike other Firmicutes, growth and culture density of C. difficile was not increased by acetoin availability or disruption of the aco pathway. Together, these results indicate that acetoin, pH, and the aco pathway play important roles in nutritional repression of sporulation in C. difficile, but acetoin metabolism does not support cell growth as a stationary phase energy source. IMPORTANCEClostridioides difficile, or C. diff, is an anaerobic bacterium that lives within the gut of many mammals and causes infectious diarrhea. C. difficile is able to survive outside of the gut and transmit to new hosts by forming dormant spores. It is known that the pH of the intestine and the nutrients available both affect the growth and sporulation of C. diffiicile, but the specific conditions that result in sporulation in the host are not clear. In this study, we investigated how pH and the metabolite acetoin affect the ability of C. difficile to grow, proliferate, and form spores. We found that a mutant lacking the predicted acetoin metabolism pathway form more spores, but their growth is not impacted. These results show that C. difficile uses acetoin differently than many other species and that acetoin has an important role as an environmental metabolite that influences spore formation.

microbiology↗

bioRxiv2023

Glycine fermentation by C. difficile promotes virulence, spore formation, and is induced by host cathelicidin

The amino acid glycine is enriched in the dysbiotic gut and is suspected to contribute to Clostridioides difficile infection. We hypothesized that the use of glycine as an energy source contributes to colonization of the intestine and pathogenesis of C. difficile. To test this hypothesis, we deleted the glycine reductase genes grdAB, rendering C. difficile unable to ferment glycine, and investigated the impact on growth and pathogenesis. We found that the grd pathway promoted growth, toxin production, sporulation, and pathogenesis of C. difficile in the hamster model of disease. Further, we determined that the grd locus is regulated by host cathelicidin (LL-37) and the cathelicidin-responsive regulator, ClnR, indicating that the host peptide signals to control glycine catabolism. The induction of glycine fermentation by LL-37 demonstrates a direct link between the host immune response and the bacterial reactions of toxin production and spore formation.

microbiology↗

bioRxiv2022

AXL-TBK1 driven nuclear AKT3 promotes metastasis

Epithelial-to-mesenchymal transition (EMT) contributes to tumor cell survival, immune evasion, migration, invasion, and therapy resistance. Across human cancer, tumors that are high grade, poorly differentiated, and have undergone EMT carry a worse prognosis with a higher likelihood of metastasis. AXL, a receptor tyrosine kinase, drives EMT and is implicated in tumor progression, metastasis, and therapy resistance in multiple cancer types including pancreatic cancer and breast cancer. TANK-binding kinase 1 (TBK1) is central to AXL-driven EMT yet, the mechanism of how TBK1 induces EMT remains unclear. Here, we report that AXL activation stimulates TBK1 binding and phosphorylation of AKT3. TBK1 activation of AKT3 drives binding and phosphorylation of slug/snail resulting in protection from proteasomal degradation and translocation of the complex into the nucleus. We show that nuclear translocation of AKT3 is required for AXL-driven EMT and metastasis. Congruently, nuclear AKT3 expression correlates with worse outcome in aggressive breast cancer. To advance AKT3 as a therapeutic target, an AKT3-isoform selective allosteric small molecule inhibitor, BGB214, was developed. BGB214 inhibits AKT3 nuclear translocation, EMT-TF stability, AKT3-mediated invasion of breast cancer cells and reduces tumor initiation in vivo. Our results suggest that AKT3 nuclear activity is an important feature of AXL-driven epithelial plasticity and that selective AKT3 inhibition represents a novel therapeutic avenue for treating aggressive cancer. SignificanceNuclear AKT3 activity is an important feature of AXL-TBK1 driven EMT and metastasis, thus selective AKT3 targeting represents a novel approach to treat aggressive cancer.

cancer biology↗

bioRxiv2021

Synaptojanin1 deficiency upregulates basal level autophagosome formation in astrocytes

Macroautophagy (hereafter, autophagy) dysregulation is implicated in multiple neurological disorders. While the autophagy pathways are heavily investigated in heterologous cells and neurons, how autophagy is regulated in the astrocyte, the most abundant cell type in the mammalian brain, is less understood. Here we report that Synaptojanin1 (Synj1), a neuron enriched lipid phosphatase, is expressed in low levels in astrocytes and represses autophagy at the basal level. Synj1 is encoded by the Synj1 gene, whose missense mutations are linked to Parkinsonism with seizure. While the best-known role of Synj1 is to facilitate synaptic vesicle recycling, recent studies suggest that Synj1 also regulates autophagy. Our previous study using the Synj1 haploinsufficient (Synj1+/-) mouse demonstrated that Synj1 deficiency was associated with an age-dependent autophagy impairment in multiple brain regions. We now use cultured astrocytes from Synj1 deficient mice to investigate its role in astrocyte autophagy. We demonstrate that Synj1 deficient astrocytes exhibit increased LC3 puncta, which is more pronounced when lysosomal acidification is blocked. The increased autophagosome formation is accompanied by reduced autophagy substrate, p62, but an insensitivity to starvation induced autophagy clearance. Moreover, we show, for the first time, that the Parkinsonism associated R839C mutation impacts astrocyte autophagy. The profound impact of this mutation on Synj1s phosphatase functions results in elevated basal level autophagosome formation and clearance that mimics Synj1 deletion. We find that energy sensing molecules, including mTOR and AMPK, are altered in Synj1 deficient astrocytes, which may contribute to the enhanced basal level autophagy.

cell biology↗

bioRxiv2019

IRSp53 shapes the plasma membrane and controls polarized transport at the nascent lumen during epithelial morphogenesis

Establishment of apical-basal cell polarity is necessary for generation of luminal and tubular structures during epithelial morphogenesis. Molecules acting at the membrane/ actin interface are expected to be crucial in governing these processes. Here, we show that the I-BAR-containing IRSp53 protein is restricted to the luminal side of epithelial cells of various glandular organs, and is specifically enriched in renal tubules in human, mice, and zebrafish. Using three-dimensional cultures of renal MDCK and intestinal Caco-2 cysts, we show that IRSp53 is recruited early after the first cell division along the forming apical lumen, and is essential for formation of a single lumen and for positioning of the polarity determinants aPKC and podocalyxin. Molecularly, IRSp53 directly binds to and controls localization of the inactive form of the small GTPase RAB35, a tethering factor for apical determinants. The interaction of IRSp53 with the actin capping protein EPS8 is critical for restricting IRSp53 localization. Correlative light and electron microscopy shows that IRSp53 loss perturbs the shape and continuity of the opposing apical membrane during the initial phase of lumenogenesis, which leads to preservation of multiple cytoplasmic bridges that interrupt the continuity of the nascent lumen. At the organism level, genetic removal of IRSp53 results in abnormal renal tubulogenesis, with defects in tubular polarity and architectural organization in both IRSp53 zebrafish mutant lines and IRSp53-KO murine models. Thus, IRSp53 acts as a platform for spatiotemporal regulation of assembly of the multi-protein complexes that shape the luminal membrane during the early steps of epithelial lumen morphogenesis.

developmental biology↗

bioRxiv2018

Structural basis of hypoxic gene regulation by the Rv0081 transcription factor of Mycobacterium tuberculosis

The transcription factor Rv0081 of M. tuberculosis controls the hypoxic gene expression and acts as a regulatory hub in the latent phase of tuberculosis infection. We report here the crystal structure of Rv0081 at 3.3 [A] resolution revealing that it belongs to the well-known ArsR/SmtB family proteins. ArsR/SmtB family transcriptional repressors exert gene regulation by reversible metal binding. Hypoxia in general is sensed by bacterial transcriptional regulators via metals or Cys-mediated thiol switches. Oxygen sensing typically leads to transcriptional repressor changing its conformational state with altered DNA-binding property under different oxygen levels. Surprisingly Rv0081 neither has a metal binding domain nor does it possess Cys residues suggesting an alternate mechanism of gene regulation. Our structural analysis identified Ser 48, Ser 49, Ser 52 and Gln 53 as potential residues of Rv0081 involved in DNA binding. We probed DNA-binding of Rv0081 with electrophoretic mobility shift assay (EMSA) as well as surface plasmon resonance (SPR), where the Alanine mutants of these residues showed diminished DNA binding. Similarly, Aspartate mutants of these Ser residues was shown to fail to bind to DNA. Since, phosphorylation of various regulatory proteins is one of the important controlling mechanisms, we expected the role of Ser-phosphorylation of Rv0081 in hypoxic condition. Probing Rv0081 with anti-phosphoserine antibodies in M. tuberculosis cell lysate showed marked enhancement in the phosphorylation of Rv0081 protein under hypoxia. Overall, our structural and biochemical analysis provides the molecular basis for the regulation of Rv0081 in the latent phase of tuberculosis infection.\n\nIMPORTANCETuberculosis is one of the deadliest infectious diseases caused by the bacterium Mycobacterium tuberculosis. In about 90% of the infected people, M. tuberculosis exists in a dormant or a latent stage which can be reactivated in favorable conditions. Hypoxia (low oxygen pressure) is one of causes of dormancy. Understanding hypoxic gene regulation in M. tuberculosis is therefore an important step to understand latency. Rv0081 is a transcriptional regulator of genes expressed during hypoxia. In order to understand the mechanism by which Rv00081 regulates gene expression during hypoxia, we have solved the crystal structure of Rv0081 and identified amino acid residues which are critical in its transcriptional regulator activity. The crystal structure is suggestive of mechanism of gene regulation under hypoxia.

biophysics↗

bioRxiv2017

The miR-96/RARγ signaling axis governs androgen signaling and PCa progression

Expression levels of retinoic acid receptor gamma (NR1B3/RARG, encodes RAR{gamma}), are commonly reduced in prostate cancer (PCa). Therefore we sought to establish the cellular and gene regulatory consequences of reduced RAR{gamma} expression, and determine RAR{gamma} regulatory mechanisms. RARG shRNA approaches in non-malignant (RWPE-1 and HPr1-AR) and malignant (LNCaP) prostate models revealed that reducing RAR{gamma} levels, rather than adding exogenous retinoid ligand, had the greatest impact on prostate cell viability and gene expression. ChIP-Seq defined the RAR{gamma} cistrome which was significantly enriched at active enhancers associated with AR binding sites. Reflecting a significant genomic role for RAR{gamma} to regulate androgen signaling, RAR{gamma} knockdown in HPr1-AR cells significantly regulated the magnitude of the AR transcriptome. RAR{gamma} down-regulation was explained by increased miR-96 in PCa cell and mouse models, and TCGA PCa cohorts. Biochemical approaches confirmed that miR-96 directly regulated RAR{gamma} expression and function Capture of the miR-96 targetome by biotin-miR96 identified that RAR{gamma} and a number of RAR{gamma} interacting co-factors including TACC1 were all targeted by miR-96, and expression of these genes were prominently altered, positively and negatively, in the TCGA-PRAD cohort. Differential gene expression analyses between tumors in the TCGA-PRAD cohort with lower quartile expression levels of RARG and TACC1 and upper quartile miR-96, compared to the reverse, identified a gene network including several RAR{gamma} target genes (e.g. SOX15) that significantly associated with worse disease free survival (hazard ratio 2.23, 95% CI 1.58 to 2.88, p=0.015). In summary, miR-96 targets a RAR{gamma} network to govern AR signaling, PCa progression and disease outcome.\n\nConflict of interestThe authors certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.\n\nFUNDINGLESC acknowledges support, in part, of Roswell Park Comprehensive Cancer Center-University of Pittsburg Cancer Institute Ovarian Cancer Specialized Program of Research Excellence National Institutes of Health [P50CA159981-01A1].\n\nMDL acknowledges support of Molecular Pharmacology and Experimental Therapeutics NRSA T32 program [T32CA009072] held at Roswell Park Comprehensive Cancer Center.\n\nMJC and DJS acknowledges support in part from the Prostate program of the Department of Defense Congressionally Directed Medical Research Programs [W81XWH-14-1-0608, W81XWH-11-2-0033] and the National Cancer Institute (NCI) grant P30CA016056 involving the use of Roswell Park Comprehensive Cancer Centers Genomic Shared Resource.\n\nMJC, GL, AR, HW and PvdB acknowledges support from the European Union-United States Atlantis Program [P116J090011].\n\nMJC and LESC acknowledge support from the National Cancer Institute (NCI) grant P30CA016056 involving the use of OSUCCC The James, CCSG P30CA016058

cancer biology↗