Biology subjects

Pramanik, A.

Publications and source records attributed to Pramanik, A..

8 recordsLinked to original sources

bioRxiv2026

P2Y14 receptor agonist impairs, and antagonist improves whole-body glucose homeostasis and liver steatosis

Knockout of the UDP-glucose-activated P2Y14 receptor (P2Y14R) in adipocytes or whole-body has been reported to provide metabolic benefits in obese mice. We hypothesized that selective P2Y14R activation would lead to metabolic impairments, whereas pharmacological antagonism would improve metabolic deficits in diet-induced obese (DIO) mice. Here, we investigated the metabolic effects of a synthetic P2Y14R agonist, UDP-like methylene-bridged MRS2905. Acute P2Y14R activation with MRS2905 triggered a robust and prolonged hyperglycemic effect in lean and obese mice with impaired glucose homeostasis. Moreover, MRS2905 treatment of obese mice lowered fasting plasma insulin and increased glucagon levels, along with upregulation of liver JNK phosphorylation and the expression of rate-limiting gluconeogenic genes. The MRS2905-induced hyperglycemic effect was blunted in whole-body P2Y14R knockout mice compared with wild-type control. In contrast, a potent P2Y14R antagonist mono-ester prodrug (MRS4779) partially reversed the agonist-induced hyperglycemia and restored proper glucose homeostasis after acute treatment. Chronic MRS4779 administration in DIO mice reduced fat mass and improved various metabolic parameters including liver steatosis. Additionally, we examined the roles of P2Y14R in hepatocytes of DIO mice. Here, we report that P2Y14R was upregulated in liver and hepatocytes from obese mice compared to lean mice. Overnight fasting also upregulated hepatic P2Y14R expression. P2Y14R deletion from hepatocytes in DIO mice improved fasting blood glucose level and lipid metabolism without improving glucose homeostasis. These results suggest a novel P2Y14R function in hepatic lipid metabolism, and P2Y14R antagonists may prove useful for the treatment of obesity and obesity-related metabolic disorders.

physiology↗

bioRxiv2026

Leishmania major targets macrophage Syntaxin-2 to impair phagolysosome biogenesis and promote intracellular survival

Macrophages destroy pathogens by engulfing them into phagosomes that mature into degradative phagolysosomes via lysosome fusion. Leishmania parasites subvert this antimicrobial pathway to establish intracellular infection and cause leishmaniasis. We previously identified the SNARE protein syntaxin-2 (Stx2) as a promoter of phagolysosome biogenesis that simultaneously limits particle binding and uptake. Consistent with this dual role, Stx2-depleted macrophages (Stx2-KD) show enhanced binding and internalization of Leishmania major. Stx2-KD macrophages also sustain higher intracellular parasite loads. We find that L. major actively targets macrophage Stx2 by selectively depleting Stx2 from phagosomes through its virulence metalloprotease GP63. Phagosomes containing GP63-deficient L. major retain Stx2 and acquire increased levels of lysosomal hydrolases and v-ATPase, restoring degradative capacity. In BALB/c mice, L. major infection markedly reduces Stx2 in infected tissues in a GP63-dependent manner. Collectively, our findings identify GP63-mediated Stx2 depletion as a key virulence strategy of L. major, positioning the GP63-Stx2 axis as a promising therapeutic target for leishmaniasis.

Cell Biology↗

bioRxiv2026

Multi-lab, Multi-enzyme Study Demonstrates the Versatility of Bacterial Microcompartment Shells as a Modular Platform for Confined Biocatalysis

Bacterial microcompartments (BMCs) are proteinaceous organelles that spatially organize metabolic reactions in bacteria and represent an attractive scaffold for pathway engineering. Here, we present a proof-of-concept in vitro study demonstrating a simple, scalable, and modular BMC shell-based platform for enzyme encapsulation using the SpyCatcher-SpyTag (SC-ST) covalent conjugation system. To evaluate the generality of this approach, 16 dehydrogenases were selected, of which 13 were successfully expressed and purified as SC-tagged enzymes in E. coli by five research groups working in parallel. Twelve of these efficiently conjugated to ST-fused BMC-T1 proteins, and addition of urea-solubilized BMC-H triggered rapid self-assembly of HT1 shells, resulting in successful encapsulation of all conjugated enzymes. The only enzyme lacking detectable activity after encapsulation was also inactive in its free SC-fused form, indicating that encapsulation retained enzymatic activity for all tested enzymes. Encapsulation modulated enzymatic activity and kinetic parameters in an enzyme-dependent manner, likely arising from variations in catalytic mechanism, structural flexibility affected by immobilization, and sensitivity to the local microenvironment created by encapsulation. Functional characterization of a subset of encapsulated enzymes revealed enhanced thermal stability up to [~]50 {degrees}C and improved storage stability relative to free SC-fused enzymes. Enzyme-loaded shells could be lyophilized and reconstituted without loss of structural integrity or activity. Finally, we demonstrate co-encapsulation of two enzymes within a single shell and their cooperative function through cofactor recycling. Together, these results establish engineered BMCs as a robust and modular platform for organizing multi-enzyme pathways, enabling rapid assembly, stabilization, and functional integration of enzymes for diverse metabolic engineering applications. HighlightsA single strategy enables encapsulation of 12 diverse dehydrogenases in BMCs. SpyCatcher-SpyTag interactions drive rapid enzyme assembly in BMCs. Encapsulated enzymes are active and show improved thermal stability. The platform enables scalable construction of synthetic metabolic modules. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/712704v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@d34f8dorg.highwire.dtl.DTLVardef@10bba9corg.highwire.dtl.DTLVardef@10868b7org.highwire.dtl.DTLVardef@112cb03_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

EGFR-targeted and MMP-activated membranolytic peptides derived from Polybia paulista MP1 kill cancer cells specifically in vitro and reduce tumour growth in vivo

Membranolytic peptides have demonstrated potential as cancer therapeutics, although targeting to cancer cells and reducing toxicity associated with activity in normal cells remain unmet challenges. We have investigated the membranolytic peptide MP1, from Polybia paulista, in order to assess ways to reduce its non-specific toxicity and thereby improve its characteristics as a cancer therapeutic. Using a panel of human breast cell lines and cell survival assays, we show that C-terminal addition of an EGFR binding sequence, with or without a linking MMP-2 cleavage sequence, generally reduced the efficacy of the peptides relative to wildtype MP1, as determined by increases in IC50 values. Critically, cell lines that show the highest sensitivities to these fusion peptides (MDA-MB-468, MDA-MB-231) expressed the highest EGFR and/or MMP-2 levels, demonstrating that these additions direct the cytotoxic activity to cells expressing these biomarkers. MMP-2 inhibition significantly reduced the cell-killing activity of peptides containing MMP-2 cleavage sites, further demonstrating targeting to this biomarker. Fusion peptides significantly induced apoptosis and reduced survival in EGFR/MMP-2 high cancer cells, while sparing EGFR/MMP-2 low cells in the context of standard tissue culture and 3D-spheroids. Finally, systemic treatment with the EGFR and MMP-2 cleavage fusion significantly reduced tumour size in MDA-MB-468 xenograft models, confirming in vivo efficacy against cancer cells and acceptable systemic toxicity. We present this EGFR-MMP-MP1 peptide as a novel cancer therapeutic for further pre-clinical and clinical development.

cancer biology↗

bioRxiv2025

Determinants of membrane sensitivity to the peptide MP-1 (Polybia paulista)

The membrane-disrupting peptide Mastoparan-1 (MP-1), derived from the wasp Polybia paulista is known to possess antimicrobial properties, and exhibits enhanced activity against a number of cancer cell lines relative to healthy cells. Due to the mechanism of action of MP-1 it is likely that differences in plasma membrane lipid composition arising from cancer associated mutations, such as localisation of phosphatidylserine (PS) lipids to the outer leaflet of the plasma membrane, are involved in driving that enhanced activity. Rapid screening of MP-1 mutants in a combined approach using model membrane and cell-based biological assays, has led to the identification of a number of derivative peptides with enhanced selectivity for cancer-like membrane models and breast cancer cell lines and provided insights into the mechanism of membrane disruption and cell death. Notably, the morphology of the membrane perturbations observed by Atomic Force Microscopy (AFM) and activity in cell model systems can change considerably in response to single-point mutations in the MP-1 sequence, indicating a complex structure-activity relationship.

biophysics↗

bioRxiv2024

A chaotrope-based approach for rapid in vitro assembly and loading of bacterial microcompartment shells

Bacterial microcompartments (BMCs) are proteinaceous organelles that self-assemble into selectively permeable shells that encapsulate enzymatic cargo. BMCs enhance catalytic pathways by reducing crosstalk among metabolites, preventing harmful intermediates from leaking into the cytosol, and increasing reaction efficiency via enzyme colocalization. The intrinsic properties of BMCs make them attractive for biotechnological engineering. However, in vivo expression methods for shell synthesis have significant drawbacks that limit the potential design space for these nanocompartments. Here we describe the development of a new, efficient, and rapid method for in vitro assembly of BMC shells from their protein building blocks. Our method enables large-scale construction of BMC shells by utilizing urea as a chaotropic agent to control self-assembly, and provides an approach for encapsulation of both biotic and abiotic cargo under a broad range of reaction conditions. We demonstrate an enhanced level of control over the assembly of BMC shells in vitro and expand the design parameter space for engineering BMC systems with specialized and enhanced catalytic properties.

synthetic biology↗

bioRxiv2024

Optimized LC-MS/MS method for Doxorubicin quantification: validating drug uptake and tumor reduction in zebrafish xenograft model of breast cancer

Doxorubicin, a potent chemotherapeutic drug, is widely used against various cancers, notably breast cancer. While its efficacy is well-documented, precise dosage determination in experimental models remains challenging. Zebrafish xenografts of various cancers confirm doxorubicins anti-cancerous effect; however, since doxorubicin treatment of zebrafish larva is done by adding doxorubicin to fish water, the precise chemotherapeutic dosage for zebrafish larva remains unknown. In this study, we provide a liquid chromatography tandem mass-spectrometry (LC-MS/MS) method for quantifying doxorubicin uptake in zebrafish larvae and thus provide a direct estimate of doses required for the therapeutic effect. Alongside quantification, we measured the therapeutic effect of doxorubicin in zebrafish larvae xenografted with triple negative breast cancer cell line, MDA-MB-231. LD50 value of doxorubicin was first determined by incubating 3-days post fertilization (dpf) larvae with different doses of doxorubicin for 72 h. Doxorubicin was quantified both from zebrafish larval homogenate and treatment solution. Analysis was performed by selected-reaction monitoring (SRM) scans in positive ionization mode. LD50 value for 72 h calculated to be 35.95 mg/L. As expected, doxorubicin-treated xenografts exhibited a significant reduction in tumor growth. The range of limit of detection (LOD) and limit of quantification (LOQ) for doxorubicin were 2 and 5 g/L respectively. Intra- and inter-day accuracy was within the range of 82-114%. Overall, in this study we describe a reliable method for quantifying doxorubicin in zebrafish larvae. Our study facilitates precise dosage estimation, enhancing the relevance of zebrafish xenograft model in cancer research and potentially improving translational applications of chemotherapeutic treatments.

cancer biology↗

bioRxiv2019

Renal Localization of Plasmodium vivax and Zoonotic Monkey Parasite Plasmodium knowlesi in Malaria Associated Acute Kidney Injury

Acute kidney injury (AKI) is a frequent presentation in Plasmodium vivax (P. vivax) associated severe malaria. However, tissue presence of P. vivax parasites in the kidney has never been demonstrated. In the present study, we show that renal biopsies from malaria associated AKI patients harbor the human malaria parasite P. vivax and, in several cases, the zoonotic monkey malaria parasite P. knowlesi in addition to P. vivax. Presence of microvascular injury in a majority of the malaria associated AKI cases suggests vascular involvement of P. vivax and P. knowlesi. This study implicates Plasmodium species as a possible pathogenic mechanism for AKI, and suggests host renal tissue interaction of P. vivax and P. knowlesi.

microbiology↗