Biology subjects

Bhardwaj, G.

Publications and source records attributed to Bhardwaj, G..

13 recordsLinked to original sources

bioRxiv2026

PFKFB3 inhibition restores β-cell responsiveness to acetylcholine in a 3xTg-AD pancreatic tissue-slice mode

The glycolytic regulator PFKFB3 supports dysfunctional {beta}-cell states, but its role in pancreatic Ca2+ signaling remains unclear. We examined responses to PFKFB3 inhibitors (AZ67, AZ26) in pancreatic tissue slices from aged 3xTg-AD mice, a model of Alzheimer's disease that develops impaired glucose tolerance and diabetes as comorbidity. Compared to wild-type controls, {beta}-cells showed weaker glucose-stimulated and acetylcholine-potentiated Ca2+ activity, accompanied by increased nuclear PFKFB3 positivity in insulin-defined islet-cell populations, used here as a PFKFB3-associated stress-state marker. During glucose and acetylcholine stimulation, AZ67 increased {beta}-cell Ca2+ event frequency while maintaining short events. In neighboring acinar cells, PFKFB3 inhibitor exposure was followed by renewed oscillations after acetylcholine-evoked activity had waned. {beta}-cell recruitment was preserved during adrenergic inhibition, and enhanced activity persisted after inhibitor withdrawal, although these recordings were exploratory and residual tissue drug exposure could not be excluded. An effect that survives both drug removal and adrenergic opposition identifies a Ca2+ signaling phenotype in 3xTg-AD {beta}-cells that is acutely inducible yet not transient, and establishes PFKFB3 as a regulator of muscarinic responsiveness in the stressed {beta}-cell.

physiology↗

bioRxiv2026

A scalable recombinant pipeline for disulphide-stapled peptides

Constrained macrocyclic peptides can engage protein surfaces that resist both small molecules and biologics but producing them at library scale still depends on chemical synthesis and macrocyclisation workflows that are slow, specialised and hard to parallelise. We describe a recombinant pipeline for disulphide-stapled cyclic peptides, compatible with 96 well plates, that lowers this barrier. Peptide-encoding sequences are introduced by extension PCR and in vivo assembly (IVA) cloning at about $2 per variant, expressed as small ubiquitin-like modifier (SUMO) fusions secreted to the oxidising periplasm, where the intramolecular disulphide forms spontaneously, and purified by immobilised metal-affinity chromatography (IMAC) with analytical size-exclusion chromatography (SEC). The route from a list of designs to characterised material takes about one week, much of it unattended. Applied to a 60-design library across nine protein targets, the pipeline gave purified products for all 60 designs, at a median total soluble yield of approximately 135 micrograms per 6 mL culture (range 9 to 261) that varied by target. Ellman's assay across 38 designs showed disulphide-formation failure to be sequence-driven and uncorrelated with yield, detecting a failure mode invisible to chromatography. Surface plasmon resonance confirmed target binding and showed that the recombinant SUMO fusion constructs retained affinities comparable to their corresponding peptide-only forms. Complementary to general-purpose recombinant platforms, the pipeline enables higher-throughput disulphide macrocycle screening and is amenable to partially or fully automated workflows.

biochemistry↗

bioRxiv2026

Targeting PFKFB3 Metabolic Gatekeeping of β-Cell Dysfunctional States Enables Durable Glucose Tolerance in Type 2 Diabetes

Progressive {beta}-cell dysfunction underlies type 2 diabetes (T2D), yet existing therapies fail to correct the dysfunctional cellular states that accumulate during disease progression. We show that pharmacological inhibition of PFKFB3 markedly reduced the population of PFKFB3 dysfunctional {beta}-cells while preserving functional islet mass, consistent with selective depletion or phenotypic reprogramming of compromised {beta}-cells. In transgenic diabetic mice expressing human IAPP, PFKFB3 inhibitor AZ67 improved glucose-stimulated insulin secretion and glucose tolerance through a mechanism consistent with direct restoration of {beta}-cell function and distinct from incretin-based therapy. Critically, glycemic improvement persisted after treatment withdrawal, consistent with durable remodeling of islet functional states rather than transient pharmacological suppression. In human islet microtissues under glucotoxic and cytokine stress, AZ67 and its analogue AZ26 improved insulin secretion and proinsulin processing in a stress- and compound-dependent manner while reducing PFKFB3 dysfunctional endocrine populations. Transcriptomic profiling identified suppression of inflammatory programs and activation of cholesterol homeostasis and adaptive metabolic pathways. These findings establish PFKFB3 inhibition as a potential disease-modifying therapeutic strategy for T2D.

cell biology↗

bioRxiv2026

Orally available designed miniproteins inhibit enterotoxigenic Bacteroides fragilis pathology by blocking toxin receptor binding

Toxigenic bacterial infections in the gut are a significant contributor to the global burden of disease. Advanced tools for protein and live biotherapeutic engineering offer potentially transformative strategies for treating such diseases, while avoiding the collateral effects of traditional antibacterials. Here we used de novo protein design to identify inhibitors of the metzincin family protease Bacteroides fragilis toxin (BFT). These inhibitors, which bind distal to the active site, interfere with toxin-mediated E-cadherin cleavage and downstream proinflammatory signaling by blocking claudin-4 receptor binding. We tested the inhibitors as disulfide-stabilized variants administered directly to the cecum or in drinking water, as well as through in situ secretion by an engineered live biotherapeutic. Across these delivery modalities, the inhibitors successfully neutralized the toxin and effectively prevented BFT-associated gut pathology, including tumor formation. These results highlight the potential of de novo designed proteins as precise, non-antibiotic interventions to mitigate bacterial toxin-driven disease in the gut.

microbiology↗

bioRxiv2025

Protein Hunter: exploiting structure hallucination within diffusion for protein design

1Interactions between proteins and other biomolecules underlie nearly all biological processes, yet designing such interactions de novo remains challenging. Capturing their specific interactions and co-optimizing sequence and structure are difficult and often require extensive computation. We present Protein Hunter, a fast, fine-tuning-free framework for de novo protein design. Starting from an all-X sequence, we find diffusion-based structure prediction models hallucinate reasonable looking structures that can be further improved through iterative sequence re-design and structure re-prediction. This lightweight strategy achieves high AlphaFold3 in silico success rates across both unconditional and conditional generation tasks, including binders to proteins, cyclic peptides, small molecules, DNA, and RNA. Protein Hunter also supports multi-motif scaffolding and partial redesign, providing a general and efficient platform for de novo protein design across diverse molecular targets.

molecular biology↗

bioRxiv2025

De novo design of high-affinity miniprotein binders targeting Francisella tularensis virulence factor

Francisella tularensis poses considerable public health risk due to its high infectivity and potential for bioterrorism. Francisella-like lipoprotein (Flpp3), a key virulence factor unique to Francisella, plays critical roles in infection and immune evasion, making it a promising target for therapeutic development. However, the lack of well-defined binding pockets and structural information on native interactions has hindered structure-guided ligand discovery against Flpp3. Here, we used a combination of physics-based and deep-learning methods to design high-affinity miniprotein binders targeting two distinct sites on Flpp3. We identified four binders for site I with binding affinities ranging between 24-110 nM. For the second site, an initial binder showed a dissociation constant (KD) of 81 nM, and subsequent site saturation mutagenesis yielded variants with sub-nanomolar affinities. Circular dichroism confirmed the topology of designed miniproteins. The X-ray crystal structure of Flpp3 in complex with a site I binder is nearly identical to the design model (C root-mean-square deviation: 0.9 [A]). These designed miniproteins provide research tools to explore the roles of Flpp3 in tularemia and should enable the development of new therapeutic candidates.

biochemistry↗

bioRxiv2025

TDP-43 directly inhibits RNA accumulation in neurites through modulation of RNA stability

The subcellular localization of hundreds of RNAs to neuronal projections allows neurons to efficiently and rapidly react to spatially restricted external cues. However, for the vast majority of these RNAs, the mechanisms that govern their localization are unknown. Here we demonstrate that the ALS-associated RNA binding protein TDP-43 primarily acts to keep RNAs out of neuronal projections. Using subcellular fractionation and single molecule RNA FISH we find that TDP-43 loss results in the increased neurite accumulation of hundreds of RNAs. These RNAs are highly enriched for known TDP-43 binding sites, suggesting that TDP-43 directly binds them to regulate their localization. We then identified precise regions within RNAs that mediate their TDP-43-dependent localization and interaction with TDP-43 using high-throughput functional assays in cells and high-throughput binding assays in vitro. We found that these regions also mediated TDP-43-dependent RNA instability, identifying the mechanism by which TDP-43 regulates RNA localization. ALS-associated mutations in TDP-43 resulted in similar RNA mislocalization phenotypes as did TDP-43 loss in human iPS-derived motor neurons. These findings establish TDP-43 as a direct negative regulator of RNA abundance in neurites and suggest that mislocalization of specific transcripts may occur in ALS patients.

molecular biology↗

bioRxiv2024

Accurate de novo design of high-affinity protein binding macrocycles using deep learning

The development of macrocyclic binders to therapeutic proteins typically relies on large-scale screening methods that are resource-intensive and provide little control over binding mode. Despite considerable progress in physics-based methods for peptide design and deep-learning methods for protein design, there are currently no robust approaches for de novo design of protein-binding macrocycles. Here, we introduce RFpeptides, a denoising diffusion-based pipeline for designing macrocyclic peptide binders against protein targets of interest. We test 20 or fewer designed macrocycles against each of four diverse proteins and obtain medium to high-affinity binders against all selected targets. Designs against MCL1 and MDM2 demonstrate KD between 1-10 M, and the best anti-GABARAP macrocycle binds with a KD of 6 nM and a sub-nanomolar IC50 in vitro. For one of the targets, RbtA, we obtain a high-affinity binder with KD < 10 nM despite starting from the target sequence alone due to the lack of an experimentally determined target structure. X-ray structures determined for macrocycle-bound MCL1, GABARAP, and RbtA complexes match very closely with the computational design models, with three out of the four structures demonstrating Ca RMSD of less than 1.5 [A] to the design models. In contrast to library screening approaches for which determining binding mode can be a major bottleneck, the binding modes of RFpeptides-generated macrocycles are known by design, which should greatly facilitate downstream optimization. RFpeptides thus provides a powerful framework for rapid and custom design of macrocyclic peptides for diagnostic and therapeutic applications.

biochemistry↗

bioRxiv2024

Perilipin 5 Phosphorylation is Dispensable for Upregulation of Hepatic Lipid Metabolism Genes upon Fasting but Required for Insulin Receptor Substrate 2 Expression in Male Mice

ObjectivePerilipin 5 (PLIN5) is a lipid droplet protein highly expressed in cells that actively oxidize fatty acids. Previous in vitro studies have revealed that PLIN5 phosphorylation (p-PLIN5) at serine 155 by PKA is critical for transcriptional regulation of PPARa target genes by which PLIN5 adapt cells for fatty acid oxidation. We aim to determine the extent of p-PLIN5 in vivo and the consequence of impaired PLIN5 phosphorylation in the liver by using a whole-body knock-in of phosphorylation resistant PLIN5 (SA/SA) in mice. MethodsWe measured PLIN5 and p-PLIN5 with mass spectrometry and Phos-tag gels. We assessed serum chemistry in WT and SA/SA mice upon fasting. RNA sequencing and qPCR compared the gene expression in the liver of SA/SA and WT mice after overnight fast. ResultsPlin5 phosphorylation at S155 was increased in the liver LD fraction of fasted mice compared with that of fed mice by mass spectrometry (p<0.05). qPCR of key lipid metabolism genes did not differ between WT and SA/SA liver upon fasting. Male SA/SA mice had a higher fasting blood glucose (p<0.05) without a difference in body weight, serum insulin, or serum lipids. IRS2 was reduced in the liver of fasted male SA/SA mice (p<0.05). ConclusionPLIN5 S155 phosphorylation is dispensable for the upregulation of lipid metabolism genes important for fasting response in vivo. Impaired phosphorylation also had little effect on serum lipids or liver TG. However, SA/SA mice showed decreased IRS2 expression in the liver, which may contribute to glucose intolerance in SA/SA male mice.

molecular biology↗

bioRxiv2024

The RNA binding protein HNRNPA2B1 regulates RNA abundance and motor protein activity in neurites

RNA molecules are localized to subcellular regions through interactions between localization-regulatory cis-elements and trans-acting RNA binding proteins (RBPs). However, the identities of RNAs whose localization is regulated by a specific RBP as well as the impacts of that RNA localization on cell function have generally remained unknown. Here, we demonstrate that the RBP HNRNPA2B1 acts to keep specific RNAs out of neuronal projections. Using subcellular fractionation, high-throughput sequencing, and single molecule RNA FISH, we find that hundreds of RNAs demonstrate markedly increased abundance in neurites in HNRNPA2B1 knockout cells. These RNAs often encode motor proteins and are enriched for known HNRNPA2B1 binding sites and motifs in their 3' UTRs. The speed and processivity of microtubule-based transport is impaired in these cells, specifically in their neurites. HNRNPA2B1 point mutations that increase its cytoplasmic abundance relative to wildtype lead to stronger suppression of RNA mislocalization defects than seen with wildtype HNRNPA2B1. We further find that the subcellular localizations of HNRNPA2B1 target RNAs are sensitive to perturbations of RNA decay machinery, suggesting that it is HNRNPA2B1s known role in regulating RNA stability that may explain these observations. These findings establish HNRNPA2B1 as a negative regulator of neurite RNA abundance and link the subcellular activities of motor proteins with the subcellular abundance of the RNAs that encode them.

molecular biology↗

bioRxiv2023

Cyclic peptide structure prediction and design using AlphaFold

Deep learning networks offer considerable opportunities for accurate structure prediction and design of biomolecules. While cyclic peptides have gained significant traction as a therapeutic modality, developing deep learning methods for designing such peptides has been slow, mostly due to the small number of available structures for molecules in this size range. Here, we report approaches to modify the AlphaFold network for accurate structure prediction and design of cyclic peptides. Our results show this approach can accurately predict the structures of native cyclic peptides from a single sequence, with 36 out of 49 cases predicted with high confidence (pLDDT > 0.85) matching the native structure with root mean squared deviation (RMSD) less than 1.5 [A]. Further extending our approach, we describe computational methods for designing sequences of peptide backbones generated by other backbone sampling methods and for de novo design of new macrocyclic peptides. We extensively sampled the structural diversity of cyclic peptides between 7-13 amino acids, and identified around 10,000 unique design candidates predicted to fold into the designed structures with high confidence. X-ray crystal structures for seven sequences with diverse sizes and structures designed by our approach match very closely with the design models (root mean squared deviation < 1.0 [A]), highlighting the atomic level accuracy in our approach. The computational methods and scaffolds developed here provide the basis for custom-designing peptides for targeted therapeutic applications.

biochemistry↗

bioRxiv2021

Pre-metastatic niche drives breast cancer invasion by modulating MSC homing and CAF differentiation

The extent to which cancer-associated alterations in extracellular matrix stiffness influences the crosstalk between cancer cells and mesenchymal stem cells (MSCs) remains unclear. By analyzing multiple singlecell RNA sequencing datasets, we establish the existence of a cell sub-population co-expressing MSC and cancer associated fibroblast (CAF) markers in highly aggressive triple-negative breast cancers in primary tumor, secondary sites, and in circulatory tumor cell clusters. Using hydrogels of varying stiffness corresponding to different stages of cancer progression, we show that on pre-metastatic stroma mimetic 2 kPa gels, MDA-MB-231 breast cancer cell secreted conditioned media drives efficient MSC chemotaxis and induces stable CAF differentiation in a TGF{beta}/contractility-dependent manner. In addition to enhancing cancer cell proliferation, 2 kPa CAFs maximally boost local invasion and confer resistance to flow-induced shear stresses. Together, our results suggest that homing of MSCs at the pre-metastatic stage and their differentiation into CAFs actively drives breast cancer invasion and metastasis.

cancer biology↗

bioRxiv2020

Perilipin2 down-regulation in beta cells impairs insulin secretion under nutritional stress and damages mitochondria

Perilipin 2 (PLIN2) is the lipid droplet (LD) protein in {beta} cells that increases under nutritional stress. Down-regulation of PLIN2 is often sufficient to reduce LD accumulation. To determine whether PLIN2 positively or negatively affects {beta} cell function under nutritional stress, PLIN2 was down-regulated in mouse {beta} cells, INS1 cells, and human islet cells. {beta} cell specific deletion of PLIN2 in mice on a high fat diet reduced glucose-stimulated insulin secretion (GSIS) in vivo and in vitro. Down-regulation of PLIN2 in INS1 cells blunted GSIS after 24 h incubation with 0.2 mM palmitic acids. Down-regulation of PLIN2 in human pseudoislets cultured at 5.6 mM glucose impaired both phases of GSIS, indicating that PLIN2 is critical for GSIS. Down-regulation of PLIN2 decreased specific OXPHOS proteins in all three models and reduced oxygen consumption rates in INS1 cells and mouse islets. Moreover, we found that PLIN2 deficient INS1 cells increased the distribution of a fluorescent oleic acid analog to mitochondria and showed signs of mitochondrial stress as indicated by susceptibility to fragmentation and alterations of acyl-carnitines and glucose metabolites. Collectively, PLIN2 in {beta} cells have an important role in preserving insulin secretion, {beta} cell metabolism and mitochondrial function under nutritional stress.

molecular biology↗