Biology subjects

Yadav, D. K.

Publications and source records attributed to Yadav, D. K..

5 recordsLinked to original sources

bioRxiv2023

O-GlcNAc Signaling Increases Neuron Regeneration Through One-Carbon Metabolism in Caenorhabditis elegans

Cellular metabolism plays an essential role in the regrowth and regeneration of a neuron following physical injury. Yet, our knowledge of the specific metabolic pathways that are beneficial to neuron regeneration remains sparse. Previously, we have shown that modulation of O-linked {beta}-N-acetylglucosamine (O-GlcNAc), a ubiquitous post-translational modification that acts as a cellular nutrient sensor, can significantly enhance in vivo neuron regeneration. Here we define the specific metabolic pathway by which mutation of the O-GlcNAc transferase (ogt-1) increases regenerative outgrowth. Performing in vivo laser axotomy and measuring subsequent regeneration of individual neurons in C. elegans, we find that the ogt-1 mutation increases regeneration by diverting the metabolic flux of enhanced glycolysis towards one carbon metabolism (OCM) and the downstream transsulfuration metabolic pathway (TSP). These effects are abrogated by genetic and/or pharmacological disruption of OCM or the serine synthesis pathway (SSP) that links OCM to glycolysis. Testing downstream branches of this pathway, we find that enhanced regeneration is dependent only on the vitamin B12 independent shunt pathway. These results are further supported by RNA-sequencing that reveals dramatic transcriptional changes, by the ogt-1 mutation, in the genes involved in glycolysis, OCM, TSP and ATP metabolism. Strikingly, the beneficial effects of the ogt-1 mutation can be recapitulated by simple metabolic supplementation of the OCM metabolite methionine in wild-type animals. Taken together, these data unearth the metabolic pathways involved in the increased regenerative capacity of a damaged neuron in ogt-1 animals and highlight the therapeutic possibilities of OCM and its related pathways in the treatment of neuronal injury. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/531166v1_ufig1.gif" ALT="Figure UFIG"> View larger version (19K): org.highwire.dtl.DTLVardef@7da18eorg.highwire.dtl.DTLVardef@1f997d6org.highwire.dtl.DTLVardef@15fa337org.highwire.dtl.DTLVardef@1d476bd_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOAbstarct Figure.C_FLOATNO Metabolic pathways involved in the enhanced neuronal regeneration in ogt-1 animalsThe green highlighted pathway illustrates the metabolic rewiring in ogt-1 mutant animals supporting enhanced axonal regeneration of injured neurons in vivo. C_FIG

neuroscience↗

bioRxiv2022

An efficient method to generate fluorescent amyloid fibrils

Studies on fluorophore-tagged peptides help in elucidating the molecular mechanism of amyloidogenesis including their cellular internalization and crosstalk potential. Despite several advantages, unavoidable difficulties including expensive and tedious synthesis-protocols exist in fluorophore-based tools. Importantly, covalently-tagged fluorophores could introduce structural constraints which may influence the conformation of the monomeric and aggregated forms of protein. To resolve this problem, we describe a robust yet simple method to make fluorescent amyloid fibrils through non-covalent incorporation of fluorophores into amyloid fibrils. We used aggregation protocol in which a small amount of fluorophore is incorporated into the amyloids, and this protocol does not alter the aggregation kinetics and the characteristic {beta}-sheet-conformers of the generated amyloid fibrils. We have successfully prepared fluorescent amyloid fibrils of Insulin, Lysozyme and A{beta}1-42, and the noncovalently incorporated fluorophores remained intact in the amyloid fibrils without leaching, even after serial-dilutions and prolonged-storage. Further, this method enables successful monitoring of cellular-internalization of the fluorescent amyloids into SH-SY5Y and A549 cells, and it also detects FRET-signals during interfibrillar interactions. The findings establish a simple and affordable protocol to prepare fluorescent amyloid structures, which may significantly help amyloid researchers working on both in vitro and animal model systems.

biophysics↗

bioRxiv2020

DNA Binding Induces a cis to trans Switch in Cre Recombinase to Enable Intasome Assembly

Mechanistic understanding of DNA recombination in the Cre-loxP system has largely been guided by crystallographic structures of tetrameric synaptic complexes. Those studies have suggested a role for protein conformational dynamics that has not been well characterized at the atomic level. We used solution NMR to discover the link between intrinsic flexibility and function in Cre recombinase. TROSY-NMR spectra show the N-terminal and C-terminal catalytic domains (CreNTD, CreCat) to be structurally independent. Amide 15N relaxation measurements of the CreCat domain reveal fast time scale dynamics in most regions that exhibit conformational differences in active and inactive Cre protomers in crystallographic tetramers. However, the C-terminal helix N, implicated in assembly of synaptic complexes and regulation of DNA cleavage activity via trans protein-protein interactions, is unexpectedly rigid in free Cre. Chemical shift perturbations and intra- and inter-molecular paramagnetic relaxation enhancement (PRE) NMR data reveal an alternative auto-inhibitory conformation for the N region of free Cre, wherein it packs in cis over the protein DNA binding surface and active site. Moreover, binding to loxP DNA induces a conformational change that dislodge the C-terminus, resulting in a cis to trans switch that is likely to enable protein-protein interactions required for assembly of recombinogenic Cre intasomes. These findings necessitate a re-examination of the mechanisms by which this widely-utilized gene editing tool selects target sites, avoids spurious DNA cleavage activity, and controls DNA recombination efficiency. SIGNIFICANCE STATEMENTThe Cre-loxP system is a widely used gene editing tool that has enabled transformative advances in immunology, neuroscience and cardiovascular research. Still, off-target activities confound research results and present obstacles to biomedical applications. Overcoming those limitations requires understanding the steps leading to assembly of recombination complexes, intasomes. We measured the magnetic properties of nitrogen nuclei in the backbone of the enzyme to correlate its intrinsic dynamics with its function in DNA recognition and cleavage. Remarkably, we found that in the absence of DNA the C-terminus of Cre appears to block the DNA binding surface and active site of the enzyme. Binding to loxP DNA induces a conformational switch that would enable the intermolecular protein-protein interactions required for assembly of recombinogenic Cre intasomes.

biophysics↗

bioRxiv2018

Stress-responsive and metabolic gene regulation are altered in low S-adenosylmethionine

S-adenosylmethionine (SAM) is the methyl donor that modifies proteins such as histones, nucleic acids and produces phosphatidylcholine. Thus variations in SAM levels could affect processes from lipogenesis to epigenetic gene regulation. SAM is hypothesized to link metabolism and chromatin modification, however, its role in acute gene regulation is poorly understood. We recently found that Caenorhabditis elegans with reduced SAM had deficiencies in bacterial-induced H3K4 trimethylation at selected pathogen-response genes, decreasing their expression and limiting survival on the pathogen Pseudomonas aeruginosa. This led us to the hypothesis that SAM may be generally required stress-responsive transcription. Here we show that C. elegans with low SAM fail to activate genome-wide transcriptional programs when exposed to bacterial or xenotoxic stress. However, heat shock responses were unaffected. We also investigated the role of two H3K4 methyltransferases that use SAM, set-2/SET1, and set-16/MLL and found that set-2/SET1 has a specific requirement in bacterial stress responses, whereas set-16/MLL was required for survival in all three stresses. These results define a role for SAM and H3K4 methyltransferases in the acute genome-wide remodeling of gene expression in response to stress. Finally, we find that the ability to modify metabolic gene expression correlates with enhanced survival in stress conditions.

molecular biology↗

bioRxiv2018

Single-nucleotide and Copy-number variance related to severity of Hypospadias

The genetic association of Hypospadias-risk studies has been conducted in Caucasians, Chinese-Han populations and few in Indian populations. Although no comprehensive approach has been followed to assess genetic involvement in the severity of the disorder. The study evaluated to establish the correlation between genotyped SNPs/CNVs and Hypospadias-severity by an association in a total 30 SNPs in genes related to sex hormone-biosynthesis and metabolism; embryonic-development and Phospholipase-D-signalling pathways on 138 surgery-confirmed hypospadias-cases from North-India (84 Penile and 28 cases of Penoscrotal-Hypospadias compared against 31 cases of Glanular+Coronal), and analyzed and identified copy number variants (CNVs) in four Familial samples (18 members) and three paired-sporadic cases (6 samples) using array-based comparative-genomic-hybridization and validated in 32 Hypospadias samples by TaqMan assay. Based on Odds Ratio at 95% CI, Z Statistic and Significance Levels, STS gene-rs17268974 was associated with Penile-Hypospadias and 9-SNPs (seven-SNPs (rs5934740; rs5934842; rs5934913; rs6639811; rs3923341; rs17268974; rs5934937) of STS gene; rs7562326-SRD5A2 and rs1877031-STARD3 were associated with Penoscrotal-Hypospadias. On aggregate analysis with p <0.001, we identified homozygous-loss of Ch7:q34 (PRSS3P2, PRSS2). On validation in previously CNV-characterized and new (32-hypospadias-cases), we identified PRSS3P2-loss in most of the grade 3 and 4 hypospadias. Hence, Grade 1 and 2 (coronal and granular) show no-PRSS3P2-loss and no-association with SNPs in STS; SRD5A2; STARD3-gene but Grade 3 and 4 (Penile and Penoscrotal) show PRSS3P2-loss accompanied with the association of SNPs in STS; SRD5A2; STARD3. Hence, homozygous-loss of PRSS3P2 accompanied with the association of STS; SRD5A2; STARD3 may link to the severity of the disease.

genomics↗