Biology subjects

Nagar, B.

Publications and source records attributed to Nagar, B..

6 recordsLinked to original sources

bioRxiv2026

AMPK-dependent FLCN phosphorylation activates a survival response to hypoxia

Adaptation to low oxygen tensions is required to survive physiological and pathological stress conditions. We discovered an adenosine monophosphate-activated protein kinase (AMPK)-dependent, hypoxia-specific survival pathway through transcription factors (TFEB, TFE3). This pathway is distinct from nutrient sensing and independent of GAP Activity Towards Rags 1 (GATOR1). Mechanistically, AMPK phosphorylates the tumor suppressor folliculin (FLCN) at a highly conserved serine, inhibiting its GAP activity towards the Rag-GTPases, resulting in mechanistic target of rapamycin complex 1 (mTORC1) inhibition, TFE3 activation, resulting in lysosomal and mitochondrial biogenesis essential for hypoxia adaptation. TFEB/3 activity correlates with the hypoxia signature in hypoxic tumor areas. Cancer patients with elevated TFEB/3 activity show poor survival, specifically in hypoxia. These findings identify the AMPK-FLCN-TFE3 axis as a therapeutic target in tumors.

biochemistry↗

bioRxiv2024

Secondary deficiency of neuraminidase 1 contributes to CNS pathology in neurological mucopolysaccharidoses via hypersialylation of brain glycoproteins

Mucopolysaccharidoses (MPS) are lysosomal storage diseases caused by defects in catabolism of glycosaminoglycans. MPS I, II, III and VII are associated with lysosomal accumulation of heparan sulphate and manifest with neurological deterioration. Most of these neurological MPS currently lack effective treatments. Here, we report that, compared to controls, neuraminidase 1 (NEU1) activity is drastically reduced in brain tissues of neurological MPS patients and in mouse models of MPS I, II, IIIA, IIIB and IIIC, but not of other neurological lysosomal disorders not presenting with heparan sulphate storage. We further show that accumulated heparan sulphate disrupts the lysosomal multienzyme complex of NEU1 with cathepsin A (CTSA), {beta}-galactosidase (GLB1) and glucosamine-6-sulfate sulfatase (GALNS) necessary to maintain enzyme activity, and that NEU1 deficiency is linked to partial deficiencies of GLB1 and GALNS in cortical tissues and iPSC-derived cortical neurons of neurological MPS patients. Increased sialylation of N-linked glycans in brain samples of human MPS III patients and MPS IIIC mice implicated insufficient processing of brain N-linked sialylated glycans, except for polysialic acid, which was reduced in the brains of MPS IIIC mice. Correction of NEU1 activity in MPS IIIC mice by lentiviral gene transfer ameliorated previously identified hallmarks of the disease, including memory impairment, behavioural traits, and reduced levels of the excitatory synapse markers VGLUT1 and PSD95. Overexpression of NEU1 also restored levels of VGLUT1-/PSD95-positive puncta in cortical neurons derived from iPSC of an MPS IIIA patient. Together, our data demonstrate that heparan sulphate-induced secondary NEU1 deficiency and aberrant sialylation of glycoproteins implicated in synaptogenesis, memory, and behaviour constitute a novel pathological pathway in neurological MPS spectrum crucially contributing to CNS pathology. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/587986v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@3905c3org.highwire.dtl.DTLVardef@1a9672corg.highwire.dtl.DTLVardef@b4911forg.highwire.dtl.DTLVardef@a3b190_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2019

The mTORC1/S6K/PDCD4/eIF4A axis determines outcome of mitosis.

mTOR is a serine/threonine kinase which acts a master regulator of cell growth and proliferation. Raptor, a scaffolding protein that recruits substrates to mTOR complex 1 (mTORC1), is known to be phosphorylated during mitosis, but the significance of this phosphorylation remains largely unknown. Here we show that raptor expression and mTORC1 activity are dramatically reduced in mitotic arrested cells across a variety of cancer and normal cell lines. Prevention of raptor phosphorylation during mitosis resulted in reactivation of mTORC1 in a rapamycin-sensitive manner. Importantly, expression of a non-phosphorylatable raptor mutant caused a dramatic reduction in cytotoxicity of the spindle poison Taxol. This effect was mediated via degradation of Programmed Cell Death Protein 4 (PDCD4), a tumor suppressor protein that inhibits eIF4A activity and is negatively regulated by the mTORC1/S6K pathway. Moreover, pharmacological inhibition of eIF4A was able to enhance the effects of taxol and restore sensitivity in Taxol resistant cancer cells. These findings indicate that the mTORC1/S6K/PDCD4/eIF4A axis has a pivotal role in death vs. slippage decision during prolonged mitotic arrest and may be exploited to gain a clinical benefit in treating cancers resistant to anti-mitotic drugs.

molecular biology↗

bioRxiv2018

Receptor Mediated Delivery of Cas9-Nanobody Induces Cisplatin Synthetic Dose Sensitivity

The CRISPR/Cas9 system has shown great potential for precisely editing genomic DNA sequences by introducing site-specific DNA cuts that are subsequently repaired by the cell. However, delivery of the CRISPR ribonucleoprotein remains an understudied area and hinders realizing the full potential of the system. We prepared Cas9 ribonucleoprotein complexes chemically conjugated to the 7D12 nanobody and demonstrate receptor-mediated transfection of Cas9 into A549 non-small-cell lung cancer cells via binding to the epithelial growth factor receptor for subsequent cell internalization. We further show that transfection with a Cas9 ribonucleoprotein targeting the BRCA2 gene results in an enhanced sensitivity to the chemotherapeutic drug Cisplatin, and thereby induces a synthetic dose lethality in A549 cells.

biochemistry↗

bioRxiv2018

Efficient Homology Directed Repair by Cas9:DNA Localization and Cationic Polymeric Transfection in Mammalian Cells

Homology directed repair (HDR) induced by site specific DNA double strand breaks (DSB) with CRISPR/Cas9 is a precision gene editing approach that occurs at low frequency in comparison to indel forming non homologous end joining (NHEJ). In order to obtain high HDR percentages in mammalian cells, we engineered Cas9 protein fused to a high-affinity monoavidin domain to deliver biotinylated donor DNA to a DSB site. In addition, we used the cationic polymer, polyethylenimine, to deliver Cas9 RNP-donor DNA complex into the cell. Combining these strategies improved HDR percentages of up to 90% in three tested loci (CXCR4, EMX1, and TLR) in standard HEK293 cells. Our approach offers a cost effective, simple and broadly applicable gene editing method, thereby expanding the CRISPR/Cas9 genome editing toolbox.\n\nSummaryPrecision gene editing occurs at a low percentage in mammalian cells using Cas9. Colocalization of donor with Cas9MAV and PEI delivery raises HDR occurrence.

biochemistry↗

bioRxiv2017

A conserved homo-dimerization interface in human IFIT1 provides insights into IFIT interactome assembly

The Interferon-Induced Proteins with Tetratricopeptide Repeats (IFITs) are a group of potently expressed Interferon Stimulated Genes that mediate antiviral innate immunity. Previous studies have revealed that most IFITs partake in higher order structures, potentially as part of an IFIT interactome that results in viral inhibition. Recent crystal structures of a mutated, monomeric form of IFIT1 revealed the molecular basis of how it recognizes non-self, capped viral mRNAs to selectively inhibit their translation. However, wild-type IFIT1 forms dimers in solution and the role of dimerization was not examined in detail. Here we present a structural and biochemical analysis of wild-type IFIT1 in complex with capped and uncapped RNA. Wild-type IFIT1 forms an antiparallel, elongated dimer that is in stark contrast to the domain-swapped, parallel dimer found in IFIT2. Dimerization takes place through a small, C-terminal interface that is evolutionarily conserved in IFIT1 and IFIT1B proteins. The interface is modular and can be grafted onto IFIT5, which is natively monomeric, to induce dimerization. Mutational analysis of this interface showed that homo-dimerization is not required for full RNA binding or translational inhibition by IFIT1. Sedimentation velocity analytical ultracentrifugation measurements demonstrated a reversible monomer-dimer equilibrium, suggesting that dimerization is of low affinity and could play a role under physiological concentrations, possibly in regulating IFIT interactome assembly. Finally, conformational changes in IFIT1 that occur upon RNA binding provide insight into how RNA enters its binding site in solution.

biochemistry↗