Biology subjects

Sengupta, S.

Publications and source records attributed to Sengupta, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Hydrogel microneedle patch for low-cost, colorimetric, continuous glucose monitoring

Diabetes is a chronic condition characterized by dysregulated blood glucose levels. Continuous glucose monitors (CGMs) enable minimally invasive, real-time monitoring of glucose levels, but broader access requires devices that are both effective and affordable. Popular CGMs such as Dexcom and FreeStyle Libre have high accuracy and are integrated with mobile applications to display real-time data and alerts but are limited by high cost. We developed a low-cost, electronic-free, microneedle hydrogel patch CGM that utilizes colorimetric detection to continuously monitor glucose levels. The proposed patch penetrates the skin and absorbs interstitial fluid (ISF) containing glucose. This glucose reacts with glucose oxidase (GOx) enzyme embedded in the hydrogel, producing gluconic acid and decreasing the pH of the hydrogel. As glucose levels increase, decreasing pH levels are reported by the loaded pH indicator bromocresol purple (BCP) via a color change from purple to yellow. This system was validated through a combination of in vitro experiments and COMSOL computational modeling to assess performance at physiologically relevant glucose concentrations. The resulting hydrogels demonstrated reversible and dynamic color changes in response to glucose levels ranging from 3.5-10 mmol/L (63-180 mg/dL), supporting their potential for continuous monitoring applications. A quantitative, image-based analysis method enabled estimation of glucose concentration from photographs, demonstrating strong predictive performance (RMSE=8.59 mg/dL, R2=0.94). Although optimization of mechanical strength and diffusion kinetics remain necessary, these findings highlight the promise of integrating colorimetric sensing with microneedle-based sampling for affordable, user-friendly glucose monitoring.

bioengineering↗

bioRxiv2026

dubTAGs enable on-demand stabilization for tunable and reversible control of endogenous protein levels

Precise and rapid control over cellular protein levels is essential to dissect complex biological systems. Chemical genetic approaches such as dTAG, in which a target is fused to a degron tag (FKBP12F36V) and degraded upon small molecule-mediated recruitment of E3 ligases, have enabled rapid and tunable control over protein abundance. However, no analogous tool exists to precisely increase protein levels and actively reverse dTAG-mediated degradation. Here, we developed heterobifunctional small molecules (dubTAGs) that stabilize FKBP12F36V-tagged proteins by recruiting endogenous deubiquitinases. Utilizing stem cell-derived cranial neural crest cells (CNCCs) in which the transcription factors SOX9 or TWIST1 are endogenously tagged with FKBP12F36V, we identified OTUB1- or USP7-recruiting heterobifunctional molecules that demonstrated effective target stabilization and ternary complex formation. We demonstrate that dubTAG-mediated protein stabilization is dependent on deubiquitinase recruitment, target-specific, and can tunably and rapidly reverse dTAG-mediated degradation. We applied dubTAGs to assess how stabilizing endogenous SOX9 impacts chromatin accessibility in CNCCs, finding both monotonic and non-monotonic regulatory element responses that are driven by distinct sequence features. dubTAGs are readily applicable tools for investigating the effects of elevated protein levels and tunably reversing targeted degradation, enabling new approaches to study protein dosage effects in development, disease, and therapeutic discovery.

molecular biology↗

bioRxiv2026

Stochastic Boolean Model of Death Signaling in MCF7:5C Predicts Cell Death Inducers and Inhibitors

Disrupted cell death signaling contributes significantly to the inappropriate survival of cancer cells, enabling them to evade the apoptotic processes that normally eliminate damaged or abnormal cells. To assess the impact of disrupted cell death signaling on cancer cell survival, we developed a continuous-time stochastic Boolean model of apoptosis signaling in MCF7:5C human breast cancer cells that are resistant to estrogen deprivation and undergo apoptosis in response to estrogen. The model was calibrated to replicate the dynamics of key apoptosis regulators in MCF7:5C cells before and after 17{beta}-estradiol (E2) treatment. Subsequently, the calibrated model was used to predict both the single and double perturbations that inhibit cell death in estradiol-treated MCF7:5C cells, and additional single interventions capable of reinducing apoptosis. For example, strains with single-gene deletion of PERK, eIF2, CHOP, or ATF4 enabled E2-treated MCF7:5C cells to evade apoptosis. However, additional inhibition of SRC, PI3K, ESR1, CEBPB, or MAPK8 restored apoptotic responses in these resistant cells. We identified 30 resistant cell strains with double mutations and 506 drug-target combinations capable of inducing four distinct modes of cell death: direct CASP7 activation, extrinsic apoptosis, intrinsic apoptosis, and combined intrinsic/extrinsic apoptosis. Overall, our model successfully explains the drug response behavior of MCF7:5C cells, predicts mechanisms of drug resistance, and suggests treatment strategies to overcome this resistance.

cancer biology↗

bioRxiv2026

GABAergic Circuit Activation Induces a Therapeutically Responsive State for anti-PD-L1 Immunotherapy in Glioblastoma

Glioblastoma (GBM) disrupts cortical excitatory-inhibitory balance and establishes an immunosuppressive microenvironment that limits therapeutic efficacy. Whether restoring inhibitory signaling can restrain glioma progression and improve responsiveness to immune checkpoint blockade remains unknown. Peritumoral parvalbumin-positive (PV+) interneurons were bidirectionally manipulated by chemogenetics in orthotopic GL261 gliomas to assess tumor growth and neurological function. GABAB signaling was pharmacologically activated with baclofen in GL261 and CT-2A models and combined with anti-PD-L1 blockade in GL261. Therapeutic response, survival, tumor rechallenge, and early myeloid remodeling were evaluated. Human GBM single-cell transcriptomic data were analyzed to examine the relationship between GABAergic and PD-L1 intercellular signaling. PV activation transiently restrained glioma growth, reduced tumor proliferation and preserved cortical function, whereas PV+ silencing increased seizure susceptibility and neurological impairment without accelerating tumor growth. Baclofen monotherapy did not affect survival, whereas its combination with anti-PD-L1 immunotherapy induced complete tumor eradication in 66% of GL261-bearing mice, prolonged survival, and conferred durable protection against tumor rechallenge. Combination therapy also altered the proportions of Arg1+ and CD11c+ cells within the intratumoral F4/80+ compartment. Human single-cell analysis revealed a shared myeloid-centered communication axis linking GABAB and PD-L1 signaling. These findings identify GABAergic signaling as a modulator of GBM progression and demonstrate that combining baclofen with anti-PD-L1 induces durable tumor regression, and prolongs survival in the GL261 model, supporting a neuro-immune framework for combining GABAergic modulation with immunotherapy.

cancer biology↗

bioRxiv2026

Network Dynamics and State-Dependent Effects of Electrical Stimulation in Recurrent Excitatory-Inhibitory Populations

Deep Brain Stimulation (DBS) is an established clinical treatment for a variety of neurological disorders, including Parkinsons Disease where it has been shown to reduce motor symptoms as well as disrupt pathological beta oscillations in the basal ganglia. The mechanisms of action of DBS on the collective activity of neuronal circuits is not fully understood. We use a recurrently-connected excitatory-inhbitory network based on the Brunel network architecture that can produce activity in a variety of states. Using a model of DBS that can reproduce observed effects such as antidromic activation, local somatic suppression, and axonal activation, we characterize the effect of stimulation across the entire parameter space of the network. We show that the effects of stimulation are dependent on the baseline state of the network, with the level of beta suppression dependent on the level of inhibition and the external drive. Specifically, networks with higher inhibition and lower drive show greater disruption of beta oscillations. We further show that networks in different states are preferentially sensitive to different frequencies of stimulation, suggesting that alternative protocols to the clinically standard high-frequency stimulation may have therapeutic efficacy.

neuroscience↗

bioRxiv2026

Surface Binding Sites Orchestrate Long-Range Control of Active-Site Dynamics and Glucose Tolerance in β-glucosidase, BglB

{beta}-Glucosidases are essential for lignocellulosic biomass conversion, yet their industrial utility is limited by inhibition from glucose, the reaction product. Although glucose inhibition has been extensively documented, its molecular basis remains poorly understood. Here, we demonstrate that glucose inhibition in the GH1 {beta}-glucosidase BglB from Paenibacillus polymyxa is mediated by multiple surface-exposed secondary binding sites (SBSs) that function as regulatory elements rather than passive glucose-binding patches. Kinetic analyses revealed a mixed mode of inhibition, with modest activation at low glucose concentrations followed by progressive inhibition at higher concentrations. Glucose association occurred through multiple SBSs in a concentration-dependent manner and induced localized rigidification, particularly at gatekeeper regions surrounding the active-site entrance. These surface interactions propagated through long-range coupling pathways, resulting in structural, energetic, and residue interaction network reorganization that reshaped the catalytic pocket. Functional interrogation of representative SBSs revealed distinct roles for individual sites. Mutation of a gatekeeper-associated SBS reduced local glucose association, increased glucose tolerance by more than 30%, and improved substrate affinity by approximately 38%, whereas disruption of a distal SBS compromised structural integrity and soluble protein production. Together, these findings establish a direct link between surface glucose recognition and active-site regulation, providing, to our knowledge, the first integrated evidence for the existence and functional significance of secondary glucose-binding sites in {beta}-glucosidases. More broadly, this work identifies surface SBSs as promising targets for engineering glucose-tolerant and catalytically robust enzymes for biomass conversion and related biotechnological applications.

biophysics↗

bioRxiv2026

Seizure-related gene 6 (SEZ6) encodes a Cancer stem cell-specific proangiogenic molecule: A novel glioma therapeutic target

Cancer stem-like cells (CSCs) not only initiate tumors but also orchestrate angiogenesis through multiple mechanisms, thereby sustaining vascularization and promoting tumor growth. In glioblastoma (GBM; WHO grade IV glioma), the most aggressive adult brain tumor, glioma stem-like cells (GSCs; glioma CSCs) are the main drivers of tumor progression, drug resistance, and recurrence. An interrogated multi-omic secretome analysis identified Wnt-{beta}-catenin signaling-regulated, membrane-localized Seizure Related 6 Homolog (SEZ6) as a patient-derived GSC-specific proangiogenic molecule. Silencing of SEZ6 inhibited the ability of the GSC secretome to induce angiogenic network formation by brain- and lung-derived endothelial cells, but not GSC growth as neurospheres in vitro. SEZ6 silencing also suppressed patient-derived GSC-initiated glioma tumors, resulting in reduced tumor vasculature in an orthotopic mouse model. We also found that SEZ6 induces TGF{beta}-dependent IL-8 expression in endothelial cells to promote angiogenesis. Coimmunoprecipitation and molecular dynamics simulation experiments determined that the Sushi 3 domain of SEZ6 mediates the interaction with TGF{beta} RII to activate the TGF{beta} pathway. Pharmacological inhibition of BACE1 with an in-house-developed small molecule, or blockade of the SEZ6-TGF{beta} RII interaction using a rationally designed peptide, markedly attenuated SEZ6-driven TGF{beta} signaling, and suppressed angiogenesis. Thus, our findings identify SEZ6 as a novel CSC-specific therapeutic target for GBM.

cancer biology↗

bioRxiv2026

Development of circadian immune regulation in early life

Circadian rhythms temporally regulate immune cell trafficking, abundance, and immune responsiveness in adults, yet how this rhythmic organization emerges during postnatal development remains unknown. Here, we systematically investigated the establishment and development of circadian immune regulation across multiple biological levels. Leukocyte rhythmicity was not present at birth but was gradually established and strengthened during postnatal development in a leukocyte subset-dependent manner. In parallel, promigratory markers displayed dynamic and heterogeneous developmental trajectories, indicating progressive coordination of rhythmic promigratory programs. At the systemic level, peripheral clocks matured and synchronized during development, with tissue- and clock-gene-dependent differences in the acquisition timing, peak phase, and amplitude of circadian oscillations. This multi-level analysis identifies postnatal development as a dynamic period during which circadian immunity is progressively consolidated through leukocyte rhythmicity, regulation of promigratory factors, and peripheral clocks.

immunology↗

bioRxiv2026

Network reciprocity reshaped by environmental knowledge and feedback

Cooperative interactions often unfold in environments that are shaped by collective behavior, yet how knowledge about such changing environments feeds back into evolutionary dynamics remains poorly understood. While network reciprocity explains how spatial structure enables clusters of cooperators to emerge and grow under certain conditions, it typically ignores how individuals respond to environmental change. Here, we integrate stochastic environmental feedback with network reciprocity to examine how knowledge about environmental state shapes the evolution of cooperation in structured populations. We compare regimes in which individuals either condition their behavior on the current state or remain unaware of it. Under weak selection, we derive a simple condition showing that cooperation is favored when the benefit-to-cost ratio exceeds a modified classic reciprocity threshold accounting for the effect of environmental transitions and state knowledge. Environmental shifts can either promote or hinder cooperation depending on accessibility and fidelity of state knowledge. Counterintuitively, greater knowledge does not universally enhance cooperation: for certain transition rules, state awareness raises the critical threshold for cooperation, a phenomenon we term a "knowledge curse". Our results reveal that, in an ever-changing environment, cooperation in structured populations emerges from a subtle interplay between environmental feedback and information availability.

animal behavior and cognition↗

bioRxiv2026

Potential of HLA-E-targeting diabodies to induce lysis of HIV-1-infected cells by CD8+ T cells

A long-lived reservoir of cells harboring intact HIV-1 provirus persists throughout decades of antiretroviral therapy and can give rise to rapid viral rebound after treatment interruption. Some cure strategies employ cytotoxic T lymphocytes (CTL) to target this reservoir; however, the applicability and efficacy of immunotherapeutic strategies involving MHC class I-restricted CTL is limited by the polymorphic nature of MHC class I molecules and their downregulation by HIV-1 Nef. The non-polymorphic non-classical class I molecule HLA-E is stably expressed on HIV-1-infected CD4+ T cells and presents a potential universal target. We generated a single-chain diabody RLP-13 that redirects CTLs to target cells presenting a well-characterized peptide derived from Mycobacterium tuberculosis in the context of HLA-E. We verified the affinity and specificity of RLP-13. Through co-culture experiments, we confirmed that RLP-13 mediates polyfunctional, HLA-agnostic CTL responses. Using an HIV-1 reporter construct encoding the target peptide, we demonstrated robust and specific elimination of the HIV-1-expressing cell population. This proof-of-concept study shows that HLA-E antigens are promising immunotherapeutic targets that can bypass the limitations of classical MHC class I antigens - allelic variation and downregulation - and that such bispecific antibodies recognizing HIV-1-derived HLA-E binding epitopes could induce elimination of productively infected cells. SummarySengupta, Bachmann et al. utilize a novel HLA-E-restricted CD3-engaging single-chain diabody to induce antigen-specific polyfunctional CTL-responses that are HLA-type-independent. They further show that such biologics have potential to eliminate HIV-1-infected cells by targeting HLA-E-binding epitopes encoded in the HIV-1 provirus.

microbiology↗

bioRxiv2026

Computational insights into the interaction between Topoisomerase I and Rpc82 subunit of RNA Polymerase III in Saccharomyces cereviseae

The process of DNA transcription leads to the generation of torsional stress, which must be resolved for smooth progression of the transcription machinery. In Saccharomyces cerevisiae, DNA topoisomerase I (Top1), a type IB topoisomerase, plays a critical role in relaxing supercoils and mitigating the topological strain associated with transcription. While several proteins from the transcription machinery have been reported to interact with yeast Top1, detailed characterization and functional relevance of these interactions have remained underexplored. This gap is partly due to the absence of a complete three-dimensional structure of the full-length enzyme, which hinders structure-based computational analyses of its interactome. In this study, we present a template-based model of full-length yeast Top1. Leveraging this model, we investigated its molecular interaction with Rpc82, a key subunit of RNA polymerase III enzyme, responsible for transcribing small non-coding RNAs such as tRNAs and 5S rRNA. Through molecular docking and molecular dynamics simulations, critical residues at the Top1-Rpc82 interface were identified that likely mediate their interaction. Our findings provide new insights into the structural basis of Top1s association with RNA polymerase III and its potential role in regulating Pol III-mediated transcription. The Top1 model developed here offers a valuable framework for future in silico studies aimed at elucidating the broader interactome and regulatory mechanisms of this essential enzyme.

bioinformatics↗

bioRxiv2026

A strategically designed homogeneous antibody-drug conjugate improves safety and therapeutic index in renal cell carcinoma

Renal cell carcinoma (RCC) is the most common form of kidney cancer. It is also one of deadliest cancers, with a 5-year survival rate of less than 20% in advanced cancer patients with distant metastasis. Current treatments rely on targeted therapies, such as sunitinib, which are limited in eHicacy. Recently, antibody drug conjugates (ADCs) have emerged as a promising treatment modality by delivering cytotoxic payloads specifically to cancer cells. Here, we describe the engineering of a novel ADC (LT-025), where the antibody targets the kidney injury molecule (KIM) -1 receptor over-expressed on RCC cells to deliver a toxic maytansinoid (DM1) payload. Unlike prior attempts to engineer a KIM-1 targeted ADC that were limited by a heterogenous product, here we used a microbial transglutaminase (MTGase)-based conjugation strategy, which achieved a site-specific conjugation of the drug-linker to the antibody, resulting in a homogenous ADC with a drug-to-antibody ratio (DAR) of 2. The ADC exhibited prolonged stability, excellent antigen-binding capability, and KIM-1 expression-dependent cellular internalization and cytotoxicity in RCC, including in sunitinib-resistant RCC. Excitingly, LT-025 was well tolerated in vivo, and combining LT-025 and sunitinib exhibited a synergistic antitumor eHicacy in a RCC mouse model. Combining an ADC with a targeted therapeutic could emerge as a paradigm shift in the management of advanced RCC.

cancer biology↗

bioRxiv2025

A yeast model of 5-oxoproline accumulation reveals a general toleration to 5-oxoproline

5-oxoproline (5-OP) or pyroglutamic acid is an intermediate of the degradation arc of the glutathione cycle. It is metabolized into glutamate through the action of the 5-oxoprolinase enzyme, the only enzyme known to act on this metabolite. 5-OP has long been known to be relatively inert with a proposed role as an osomoprotectant. Recent studies on the 5-oxoprolinase enzyme in mammalian cells have, however, shown that knockdown or deletions of the 5-oxoprolinase make mice (and humans) prone to heart failure. This was ascribed to oxidative stress due to a two-fold elevation in 5-OP. To examine the consequences of 5-oxoproline accumulation more rigorously, we have created a yeast model for 5-oxoproline accumulation. We observed retardation of growth only when intracellular levels of 5-OP were increased 12-20 fold over the normal levels. A transcriptomics study was carried out under these conditions. We observed that while a large number of genes were regulated upto 2-fold, there were no single prominent pathways amongst them. Among the key genes upregulated were different efflux pumps. Using knockouts and also overexpression of selected genes, we could observe that many of the upregulated genes were involved in the cellular response to 5-OP accumulation. However, it did not appear that there was any significant oxidative stress response. The study suggests a need to reevaluate previous suppositions of the 5-OP induced oxidative stress response. Instead, we propose an alternative model to explain the possible consequences of 5-oxoprolinase deficiency based on these findings made with the yeast model.

systems biology↗

bioRxiv2025

Automatic Classification of Circulating Blood Cell Clusters based on Multi-channel Flow Cytometry Imaging

Circulating blood cell clusters (CCCs) containing red blood cells (RBCs), white blood cells (WBCs), and platelets are significant biomarkers linked to conditions like thrombosis, infection, and inflammation. Flow cytometry, paired with fluorescence staining, is commonly used to analyze these cell clusters, revealing cell morphology and protein profiles. While computational approaches based on machine learning have advanced the automatic analysis of single-cell flow cytometry images, there is a lack of effort to build tools to automatically analyze images containing CCCs. Unlike single cells, cell clusters often exhibit irregular shapes and sizes. In addition, these cell clusters often consist of heterogeneous cell types, which require multi-channel staining to identify the specific cell types within the clusters. This study introduces a new computational framework for analyzing CCC images and identifying cell types within clusters. Our framework uses a two-step analysis strategy. First, it categorizes images into cell cluster and non-cluster groups by fine-tuning the You Only Look Once(YOLOv11) model, which outperforms traditional convolutional neural networks (CNNs), Vision Transformers (ViT). Then, it identifies cell types by overlaying cluster contours with regions from multi-channel fluorescence stains, enhancing accuracy despite cell debris and staining artifacts. This approach achieved over 95% accuracy in both cluster classification and phenotype identification. In summary, our automated framework effectively analyzes CCC images from flow cytometry, leveraging both bright-field and fluorescence data. Initially tested on blood cells, it holds potential for broader applications, such as analyzing immune and tumor cell clusters, supporting cellular research across various diseases.

bioinformatics↗

bioRxiv2025

A TCR-mimic bispecific antibody reduces HIV-1 provirus and delays viral rebound in HLA-matched humanized mice

Bispecific antibodies that reroute cytotoxic effectors toward infected cells are promising HIV-1 cure agents, yet existing formats bind Env and are limited by antigenic variation and Env down-regulation. We engineered a TCR-mimic single-chain diabody, HI12, that recognizes a conserved Pol-derived peptide presented by HLA-A*02:01 and evaluated its effect in HLA-matched, HIV-infected humanized mice. When administered during early antiretroviral therapy (ART), HI12 was well tolerated, activated HIV-specific CD8+ T cells and accelerated plasma virus decay. Treatment produced four-to six-fold reductions in intact and total proviral DNA within lymph-node and splenic CD4+ T cells, indicating substantive reservoir clearance. After ART interruption, HI12-treated animals showed a significant delay in viral rebound compared with controls, linking reservoir reduction to improved post-therapy control. These findings provide the first in-vivo evidence that a peptide-HLA-directed bispecific antibody can both shrink the intact HIV reservoir and defer viral recrudescence, supporting further development of TCR-mimic bispecific antibodies for cure strategies. Significance StatementAn HIV-1 cure strategy will require novel therapeutics to facilitate immune-mediated elimination of infected cells and reduction of blood and tissue reservoirs. Here, we demonstrate that an TCR-mimic bispecific antibody that recognizes a conserved Pol epitope can promote CD8+ T cell-mediated clearance of infected cells in HLA-matched, HIV-infected humanized mice. The bispecific antibody therapy reduced HIV-1 proviral DNA in lymph node and splenic tissues and improved post-therapy viral control. This study highlights the potential of developing novel TCR-mimic bispecific antibodies in HIV cure-directed strategies.

immunology↗

bioRxiv2025

Purine nucleotides are competitive inhibitors of apo-GOT1

The malate-aspartate shuttle (MAS) plays a key role in cellular metabolism by transferring electrons from cytosolic NADH into the mitochondrial matrix, thereby supporting oxidative phosphorylation, in addition to the citric acid cycle and amino acid metabolism. Here, we sought to identify allosteric regulatory metabolites of the MAS enzymes cytosolic glutamic-oxaloacetic transaminase 1 (GOT1) and mitochondrial GOT2. Using the Atavistik Metabolite Proprietary Screening platform, we identified several structurally similar metabolite hits-- most notably deoxyadenosine monophosphate (dAMP) and deoxyguanosine monophosphate (dGMP)--as candidate interactors with GOT1. Follow-up thermal shift assays revealed that dAMP and dGMP destabilize GOT1 in the absence of its cofactor, pyridoxal 5-phosphate (PLP), but have no destabilizing effect when PLP is present. Crystallographic analysis confirmed that dAMP and dGMP bind in the PLP pocket of GOT1, suggesting competitive binding. Together, these results indicate that nucleotide metabolites can interact with GOT1, offering potential insights into MAS regulation and therapeutic intervention strategies.

molecular biology↗

bioRxiv2025

Phospholipids that plug the pores of cholesteryl ester transfer protein control its lipid transfer dynamics

The mechanism by which cholesteryl ester transfer protein (CETP), a key drug target in cardiovascular disease, moves lipids between lipoproteins, has remained elusive. CETP is the primary protein that regulates plasma circulating levels of lipids (within high, low, and very low-density lipoproteins. The protein, in addition to its terminal openings, has two additional openings, plugged by two phospholipids (PLs). Small molecule inhibitors targeting CETP tunnel displace PL during CETP inhibition. Here, using steered molecular dynamics simulations followed by in-vitro mutagenesis we show that CETP-bound PLs are indispensable in establishing the optimal architecture of CETP tunnel. Our structural and functional analyses revealed that lipid traversal through CETPs central tunnel is facilitated through hydrophobic interaction-mediated diffusion. PLs are critical in synchronizing domain movements of CETP while accelerating triglyceride traversal through the tunnel and their activity regulated through salt-bridge interactions. Most notably, we show for the first time that phospholipids (PLs) bound within CETPs tunnel accelerate lipid movement through a novel "gliding" mechanism, representing the first known example of a lipid regulating the movement of another. We identified conserved phenylalanine (Phe) flaps that act as a regulatory valve, opening and closing concertedly to prevent lipid backflow in the absence of an active motor. This study provides in-depth understanding of the mechanism of lipid exchange by CETP guided, and accentuated by its interaction with PLs. The conservation of these critical structural elements across the BPI/LBP family suggests that this mechanism is broadly applicable, expanding our understanding of lipid transport in this clinically significant protein class. Significance StatementThe mechanism of lipid transport by the cholesteryl ester transfer protein (CETP), a major drug target, has long been a scientific mystery. Neutral lipid exchange through CETP is dependent on the hydrophobic characteristics of the tunnel that has been exploited during inhibitor design. We provide a detailed, molecular-level explanation, showing for the first time that phospholipids (PLs), previously thought to be static plugs, are indispensable for lipid transfer. They actively accelerate lipid movement through a novel gliding mechanism, the first documented instance of a lipid regulating another. This discovery identifies new therapeutic targets for CETP inhibition. Our findings also suggest that this fundamental mechanism is conserved across a large protein family, with broad implications for understanding lipid transport and function.

biophysics↗

bioRxiv2025

Macrophage signaling and function are regulated by distinct sterol biochemistries

Membranes require continuous reorganization of lipid components, including sterols, to dynamically alter their rigidity to deform and bend during scission events which occur during fundamental cellular functions such as endocytosis. While diseases of cholesterol biosynthesis result in reduced cellular cholesterol and accumulation of precursor sterols, limited studies have addressed the intracellular consequences of disease-associated sterol changes on the ability of eukaryotic cellular membranes to function and signal normally. Here, we utilized bone marrow-derived macrophages (BMDMs) to investigate how altered sterol content impacts macrophage signaling and membrane function. Through pharmacological inhibition of cholesterol biosynthetic enzymes, reduced cholesterol and increased levels of disease-associated sterol intermediates coincided with reduced expression of cell surface proteins and impaired macropinocytosis. Macropinocytic activity was sensitive to both reduced plasma membrane cholesterol and sterols containing functional groups substituted for the C3 hydroxyl group. Transcriptomic analyses of cholesterol-inhibited BMDMs revealed alterations in immune and chemokine signaling pathways. Decreased cholesterol was also associated with dysregulated vesicular sorting pathways and elevated expression of endosomal/lysosomal markers. Disrupted endosome expression and impaired macropinocytosis was also observed in BMDMs from mouse models of the cholesterol biosynthesis disorder Smith-Lemli-Opitz syndrome (SLOS). Our findings detail an important connection between sterol imbalance, membrane dynamics, and immune cell function.

cell biology↗