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Das, D.

Publications and source records attributed to Das, D..

At least 55 records · Page 3Linked to original sources
bioRxiv2024

Odoribacter splanchnicus mitigates Salmonella-induced gut inflammation and its associated pathogenesis via its secreted bacteriocin

Foodborne pathogens pose a major global health hazard, worsened by drug-resistant strains. Therefore, designing novel therapeutic strategies is crucial. Here, we identified the protective role of gut-commensal Odoribacter splanchnicus (OS) against Salmonella in mice by promoting colonization resistance, preserving gut barrier integrity, and preventing acute inflammation and biofilm formation. In vitro, both OS and their culture supernatant inhibited Salmonella biofilm formation, intracellular proliferation in human intestinal cells, and virulence gene expression. Further, our results depicted that OSs protective role acts over a broad spectrum as it confers protection against Gram-positive, Listeria monocytogenes and Gram-negative, Salmonella Typhimurium. Notably, OS conferred protection even when administered post-infection in mice, highlighting its therapeutic potential. Using several biochemical and proteomics approaches, we characterized key OS-secreted molecules that limit intracellular Salmonella and Listeria replication in human intestinal epithelial cells by regulating key virulence effectors and flagella. Collectively, our study establishes OS as the broad-spectrum protective agent against Salmonella and Listeria infections, with promising therapeutic potential. Author SummaryFoodborne pathogens such as Salmonella and Listeria continue to threaten global health, especially with the rise of antibiotic resistance. In this study, we identify the gut commensal bacterium Odoribacter splanchnicus (OS) as a potent protectant against these pathogens. Using a mouse model, we show that OS enhances colonization resistance, maintains gut barrier integrity, and prevents both inflammation and biofilm formation during infection. Remarkably, OS also limits pathogen proliferation and virulence gene expression in human intestinal cells. These protective effects are mediated by both live OS and its secreted molecules within culture supernatant. Importantly, OS administration post-infection also conferred protection, indicating strong therapeutic potential. Biochemical and proteomic analyses revealed OS-secreted factors that suppress key virulence and motility pathways in pathogens. Our findings establish Odoribacter splanchnicus as a promising broad-spectrum biotherapeutic agent against major foodborne infections. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/609322v4_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@7ea42corg.highwire.dtl.DTLVardef@4c8074org.highwire.dtl.DTLVardef@855350org.highwire.dtl.DTLVardef@120decc_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗
bioRxiv2024

Exact distributions of threshold crossing times of proteins under post-transcriptional regulation by small RNAs

The timings of several cellular events like cell lysis, cell division, or pore formation in endosomes are regulated by the time taken for the relevant proteins to cross a threshold in number or concentration. Since protein synthesis is stochastic, the threshold crossing time is a first passage problem. The exact distributions of these first passage processes have been obtained recently for unregulated and auto-regulated genes. Many proteins are however regulated by post-transcriptional regulation, controlled by small non-coding RNAs (sRNAs). Certain mathematical models of gene expression with post-transcriptional sRNA regulation have been recently exactly mapped to models without sRNA regulation. Utilizing this mapping and the exact distributions, we calculate exact results on fluctuations (full distribution, all cumulants, and characteristic times) of protein threshold crossing times in the presence of sRNA regulation. We derive two interesting predictions from these exact results. We show that the size of the fluctuation of the threshold crossing times have a non-monotonic U-shaped behavior as a function of the rates of binding and unbinding of the sRNA-mRNA complex. Thus there are optimal parameters that minimize noise. Furthermore, the fluctuations in models with sRNA regulation may be higher or lower compared to the model without regulation, depending on the mean protein burst size.

biophysics↗
bioRxiv2024

Competitive modulation of Kv1.2 gating by LMAN2 and Slc7a5

Kv1.2 is a prominent ion channel in the CNS, where it regulates neuronal excitability. Kv1.2 structure and function are well understood, but there is less consensus on mechanisms of regulation of Kv1.2 and other potassium channels by auxiliary proteins. We identified novel regulators of Kv1.2 by a mass spectrometry approach. The neutral amino acid transporter Slc7a5 causes a dramatic hyperpolarizing shift of channel activation. In contrast, LMAN2 is a recently identified candidate regulator that has the opposite effect on gating: large depolarizing voltages are required to activate Kv1.2 channels co-expressed with LMAN2. In this study, we characterized the functional interaction between LMAN2 and Slc7a5 on Kv1.2 gating properties and identified key structural elements that underlie sensitivity to each regulator. When LMAN2 and Slc7a5 are expressed together, Kv1.2 activation exhibits a bi-modal voltage-dependence, suggesting two distinct populations of channels regulated either by LMAN2 or Slc7a5, but not both. Using a Kv1.2:1.5 chimeric approach, we identified specific regions between the S1 to S3 segments of the voltage sensing domain (VSD) that are distinct for either Slc7a5 or LMAN2 sensitivity. By replacing either segment with sequence from Kv1.5, modulation by the corresponding regulator was selectively abolished. These results suggest that Slc7a5 and LMAN2 compete for interaction with the Kv1.2 voltage sensor, leading to complex voltage-dependence of channel activity when both regulators are present in the cell.

biophysics↗
bioRxiv2024

Regulation of Kv1.2 redox-sensitive gating by the transmembrane lectin LMAN2

Kv1.2 potassium channels influence excitability and action potential propagation in the nervous system. Unlike closely-related Kv1 channels, Kv1.2 exhibits highly variable voltage-dependence of gating, attributed to regulation by unidentified extrinsic factors. Variable Kv1.2 gating is strongly influenced by the extracellular redox potential, and we demonstrate that Kv1.2 currents in dorsal root ganglion sensory neurons exhibit similar variability and redox sensitivity as observed when the channel is heterologously expressed in cell lines. We used a functional screening approach to test the effects of candidate regulatory proteins on Kv1.2 gating, using patch clamp electrophysiology. Among 52 candidate genes tested, we observed that co-expression with the transmembrane lectin LMAN2 led to a pronounced gating shift of Kv1.2 activation to depolarized voltages in CHO and L(tk-) cell lines, caused by deceleration of activation kinetics. Overexpression of LMAN2 promoted a slow gating mode of Kv1.2 that mimics the functional outcomes of extracellular reducing conditions, and enhanced sensitivity to extracellular reducing agents. In contrast, shRNA-mediated knockdown of endogenous LMAN2 in cell lines reduced Kv1.2 redox sensitivity and gating variability. Kv1.2 sensitivity to LMAN2 is abolished by mutation of neighboring residues F251 and T252 in the intracellular S2-S3 linker, and these also abolish redox-dependent modulation of Kv1.2, suggesting that LMAN2 is an important contributor to the mechanism of redox sensitivity. In conclusion, we identified LMAN2 as a candidate regulatory protein that influences redox-dependent modulation of Kv1.2, and clarified the structural elements of the channel that are required for sensitivity.

biophysics↗
bioRxiv2024

ESRP2-microRNA-122 axis directs the postnatal onset of liver polyploidization and maturation

Hepatocyte polyploidy and maturity are critical to acquiring specialized liver functions. Multiple intra- and extracellular factors influence ploidy, but how they cooperate temporally to steer liver polyploidization and maturation or how post-transcriptional mechanisms integrate into these paradigms is unknown. Here, we identified an important regulatory hierarchy in which postnatal activation of Epithelial-Splicing-Regulatory-Protein-2 (ESRP2) stimulates biogenesis of liver-specific microRNA (miR-122), thereby facilitating polyploidization, maturation, and functional competence of hepatocytes. By determining transcriptome-wide protein-RNA interactions in vivo and integrating them with single-cell and bulk hepatocyte RNA-seq datasets, we delineate an ESRP2-driven RNA processing program that drives sequential replacement of fetal-to-adult transcript isoforms. Specifically, ESRP2 binds the primary miR-122 host gene transcript to promote its processing/biogenesis. Combining constitutive and inducible ESRP2 gain- and loss-of-function mice models with miR-122 rescue experiments, we demonstrate that timed activation of ESRP2 augments miR-122-driven program of cytokinesis failure, ensuring proper onset and extent of hepatocyte polyploidization.

developmental biology↗
bioRxiv2024

Autoimmunity and clinical pathology amelioration in SLE by Dexamethasone primed Mesenchymal Stem Cell derived conditioned media

This study proposes a novel approach, utilizing cell-free dexamethasone (Dex) primed Whartons jelly mesenchymal stem cells derived conditioned media (DW), offering improved efficacy, simplicity, and alternative medicine for addressing complications associated with systemic lupus erythematosus (SLE). This study explores the immunomodulatory effects of DW treatment on immune cell populations in SLE patients and a pristane-induced lupus (PIL) mouse model. DW induces significant expansion of Tregs, Bregs, suppressing Th17, double-negative T cells, and inflammatory neutrophils through modulating IL-10 and IL-17A production. Comparisons with the standard drug hydroxychloroquine reveal similar effects, suggesting TGF beta; pathway mediation in DWs actions. Compared with the immunosuppressive drug Dex, DW better attenuated autoantibody production, increased anti-inflammatory cytokines and maintained a balanced Th17/Treg ratio. In the preclinical in vivo studies, DW exhibits therapeutic efficacy, reducing mortality, preventing proteinuria, and reversing limb inflammation, seizure and alopecia. Organ specific evaluations using advanced live imaging or histopathological analysis highlighted DWs protective effects on kidneys, liver, lungs, heart, and spleen. This provided insight into the immunomodulatory benefits of DW at various levels and suggested that it could be a potential therapeutic avenue for managing complications related to SLE.

immunology↗
bioRxiv2024

Glucose-regulated circular RNA Rabep1 regulates pancreatic beta-cell growth by modulating miR-335-3p/PTEN axis

HighlightsO_LIIdentified circRNAs expressed in {beta}TC6 cell line C_LIO_LIFirst report identifying glucose-regulated circRNAs in pancreatic {beta}-cell C_LIO_LICircRabep1 regulates {beta}-cell growth by binding to miR-335-3p C_LI Circular RNAs (circRNAs) are a large family of closed-loop RNA molecules emerging as novel regulators of gene expression. Although several circRNAs are known to regulate various biological processes, the functions of most circRNAs expressed in pancreatic {beta}-cells remain to be discovered. Since short-term glucose treatment induces pancreatic {beta}-cell growth and promotes insulin production, we wanted to explore the role of glucose-regulated circRNAs in pancreatic {beta}-cell physiology. Our RNA-seq analysis identified more than 300 differentially expressed circRNAs in high-glucose compared to low-glucose treated {beta}TC6 cells. A subset of differentially expressed and abundant circRNAs was validated by various biochemical methods, including circular RNA Rabep1 (circRabep1). Moreover, the downregulation of circRabep1 in high glucose-treated {beta}TC6 cells suggested a possible function in {beta}-cell physiology. Furthermore, analysis of the circRabep1-miRNA-mRNA regulatory network discovered the association of circRabep1 with miR-335-3p, a suppressor of Pten expression. Importantly, inhibition of miRNA function by miR-335-3p inhibitor results in upregulation of PTEN levels, suppressing {beta}-cell growth and proliferation. Furthermore, silencing circRabep1 decreased PTEN expression by sponging miR-335-3p, promoting cell proliferation. We propose that the downregulation of circRabep1 in high-glucose treated {beta}-cell leads to an increase in {beta}-cell proliferation by suppressing PTEN expression through derepression of miR-335-3p. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=62 SRC="FIGDIR/small/600308v1_ufig1.gif" ALT="Figure 1"> View larger version (10K): org.highwire.dtl.DTLVardef@542b03org.highwire.dtl.DTLVardef@1d3f3faorg.highwire.dtl.DTLVardef@36b476org.highwire.dtl.DTLVardef@181c7b4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Schematic showing the molecular function of glucose-regulated circRabep1 in pancreatic -cell growth by binding to miR-335-3p. C_FIG

molecular biology↗
bioRxiv2024

Targeting MDSC-HTR2B to Improve Immune Checkpoint Inhibitors in Breast to Brain Metastasis

Myeloid Derived Suppressor Cells (MDSCs) support breast cancer growth via immune suppression and non-immunological mechanisms. Although 15% of patients with breast cancer will develop brain metastasis, there is scant understanding of MDSCs contribution within the breast-to-brain metastatic microenvironment. Utilizing co-culture models mimicking a tumor-neuron-immune microenvironment and patient tissue arrays, we identified serotonergic receptor, HTR2B, on MDSCs to upregulate pNF-{kappa}B and suppress T cell proliferation, resulting in enhanced tumor growth. In vivo murine models of metastatic and intracranial breast tumors treated with FDA-approved, anti-psychotic HTR2B antagonist, clozapine, combined with immunotherapy anti-PD-1 demonstrated a significant increase in survival and increased T cell infiltration. Collectively, these findings reveal a previously unknown role of MDSC-HTR2B in breast-to-brain metastasis, suggesting a novel and immediate therapeutic approach using neurological drugs to treat patients with metastatic breast cancer.

cancer biology↗
bioRxiv2024

A scalable CRISPR-Cas9 gene editing system facilitates CRISPR screens in the malaria parasite Plasmodium berghei

Many Plasmodium genes remain uncharacterised due to low genetic tractability. Previous large scale knockout screens have only been able to target about half of the genome in the more genetically tractable rodent malaria parasite Plasmodium berghei. To overcome this limitation, we have developed a scalable CRISPR system called PbHiT, which uses a single cloning step to generate targeting vectors with 100 bp homology arms physically linked to a guide RNA (gRNA) that effectively integrate into the target locus. We show that PbHiT coupled with gRNA sequencing robustly recapitulates known knockout mutant phenotypes in pooled transfections. Furthermore, we provide vector designs and sequences to target the entire P. berghei genome and scale-up vector production using a pooled ligation approach. This work presents for the first time a tool for high-throughput CRISPR screens in Plasmodium for studying the parasites biology at scale.

molecular biology↗
bioRxiv2024

The Candida auris Hog1 MAP kinase is essential for the colonization of murine skin and intradermal persistence

Candida auris, a multidrug-resistant human fungal pathogen, was first identified in 2009 in Japan. Since then, systemic C. auris infections have now been reported in more than 50 countries, with mortality rates of 30-60%. A major contributing factor to its high inter- and intrahospital clonal transmission is that C. auris, unlike most Candida species, displays unique skin tropism and can stay on human skin for a prolonged period. However, the molecular mechanisms responsible for C. auris skin colonization, intradermal persistence, and systemic virulence are poorly understood. Here, we report that C. auris Hog1 mitogen-activated protein kinase (MAPK) is essential for efficient skin colonization, intradermal persistence, as well as systemic virulence. RNA-seq analysis of wildtype parental and hog1{Delta} mutant strains revealed marked down-regulation of genes involved in processes such as cell adhesion, cell-wall rearrangement, and pathogenesis in hog1{Delta} mutant compared to the wildtype parent. Consistent with these data, we found a prominent role for Hog1 in maintaining cell-wall architecture, as the hog1{Delta} mutant demonstrated a significant increase in cell-surface {beta}-glucan exposure and a concomitant reduction in chitin content. Additionally, we observed that Hog1 was required for biofilm formation in vitro and fungal survival when challenged with primary murine macrophages and neutrophils ex vivo. Collectively, these findings have important implications for understanding the C. auris skin adherence mechanisms and penetration of skin epithelial layers preceding bloodstream infections. ImportanceCandida auris is a World Health Organization (WHO) fungal priority pathogen and an urgent public health threat recognized by the Centers for Disease Control and Prevention (CDC). C. auris has a unique ability to colonize human skin. It also persists on abiotic surfaces in healthcare environments for an extended period of time. These attributes facilitate the inter- and intrahospital clonal transmission of C. auris. Therefore, understanding C. auris skin colonization mechanisms are critical for infection control, especially in hospitals and nursing homes. However, despite its profound clinical relevance, the molecular and genetic basis of C. auris skin colonization mechanisms are poorly understood. Herein, we present data on the identification of the Hog1 MAP kinase as a key regulator of C. auris skin colonization. These findings lay foundation for further characterization of unique mechanisms that promote fungal persistence on human skin.

microbiology↗
bioRxiv2024

ATP Prevents Aggregation of Protein via Modulating its Monomeric Conformation

Proteins occurring in significantly high concentrations in cellular environments (over 100 mg/mL) and functioning in crowded cytoplasm, often face the prodigious challenges of aggregation which are the pathological hallmark of aging and are critically responsible for a wide spectrum of rising human diseases. Here we combine a joint-venture of complementary wet-lab experiment and molecular simulation to discern the potential ability of adenosine triphosphate (ATP) as solubilizer of protein aggregates. We show that ATP prevents both condensation of aggregation-prone intrinsically disordered protein A{beta}40 and promotes dissolution of pre-formed aggregates. Computer simulation links ATPs solubilizing role to its ability to modulate proteins structural plasticity by unwinding protein conformation. We show that ATP is positioned as a superior biological solubilizer of protein aggregates over traditional chemical hydrotropes, potentially holding promises in therapeutic interventions in protein-aggregation related diseases. Going beyond its conventional activity as energy currency, the amphiphilic nature of ATP enables its protein-specific interaction that would enhance ATPs efficiency in cellular processes.

biochemistry↗
bioRxiv2023

Reassessing the association of VDR and its polymorphisms with tuberculosis in global populations.

BackgroundVitamin D is a hormone that regulates the calcium homeostasis of the body. Besides this classical function, it is also regarded as an important immunomodulator. Most active Vitamin D actions are mediated through the Vitamin D receptor (VDR), a transcription factor and also a member of the nuclear receptor superfamily. In this study, we explored the phylogeographic attributes of the four most well-known polymorphisms of the VDR gene namely rs7975232 (ApaI), rs731235 (TaqI), rs1544410 (BsmI), rs2228570 (FokI) and also evaluated their association with the incidence of tuberculosis in global populations. This study integrated several in-silico approaches on population databases to evaluate the pattern of distribution, linkage and selection patterns of these SNPs. ResultsThe ancestral alleles of rs7975232, rs731235, and rs1544410 are still present in over 50% frequency in modern human populations. These SNPs also have a very strong linkage disequilibrium among themselves in all population groups but no haplotype blocks are seen in South Asian populations constituting these polymorphisms. The selection results reveal a negative Tajimas D value in West and East Eurasian populations suggesting positive selection in these regions... In correlation studies, we found no association between the incidence of tuberculosis and the allele or genotype frequency of these four SNPs. ConclusionThe four SNPs of VDR behave differently in South Asian populations as compared to West and East Eurasian populations but no significant association was found with the incidence of tuberculosis in global populations.

genomics↗
bioRxiv2023

Discovering Novel Prognostic Biomarkers of Hepatocellular Carcinoma using eXplainable Artificial Intelligence

Hepatocellular carcinoma (HCC) remains a global health challenge with high mortality rates, largely due to late diagnosis and suboptimal efficacy of current therapies. With the imperative need for more reliable, non-invasive diagnostic tools and novel therapeutic strategies, this study focuses on the discovery and application of novel genetic biomarkers for HCC using explainable artificial intelligence (XAI). Despite advances in HCC research, current biomarkers like Alpha-fetoprotein (AFP) exhibit limitations in sensitivity and specificity, necessitating a shift towards more precise and reliable markers. This paper presents an innovative XAI framework to identify and validate key genetic biomarkers for HCC prognosis. Our methodology involved analyzing clinical and gene expression data to identify potential biomarkers with prognostic significance. The study utilized robust AI models validated against extensive gene expression datasets, demonstrating not only the predictive accuracy but also the clinical relevance of the identified biomarkers through explainable metrics. The findings highlight the importance of biomarkers such as TOP3B, SSBP3, and COX7A2L, which were consistently influential across multiple models, suggesting their role in improving the predictive accuracy for HCC prognosis beyond AFP. Notably, the study also emphasizes the relevance of these biomarkers to the Hispanic population, aligning with the larger goal of demographic-specific research. The application of XAI in biomarker discovery represents a significant advancement in HCC research, offering a more nuanced understanding of the disease and laying the groundwork for improved diagnostic and therapeutic strategies.

cancer biology↗
bioRxiv2023

Subchronic exposure to ambient PM2.5 impairs novelty recognition and spatial memory

Air pollution remains a great challenge for public health, with the detrimental effects of air pollution on cardiovascular, rhinosinusitis, and pulmonary health increasingly well understood. Recent epidemiological associations point to the adverse effects of air pollution on cognitive decline and neurodegenerative diseases. Mouse models of subchronic exposure to PM2.5 (ambient air particulate matter < 2.5 {micro}m) provide an opportunity to demonstrate the causality of target diseases. Here, we subchronically exposed mice to concentrated ambient PM2.5 for 7 weeks (5 days/week; 8h/day) and assessed its effect on behavior using standard tests measuring cognition or anxiety-like behaviors. Average daily PM2.5 concentration was 200 {micro}g/m3 in the PM2.5 group and 10 {micro}g/m3 in the filtered air group. The novel object recognition (NOR) test was used to assess the effect of PM2.5 exposure on recognition memory. The increase in exploration time for a novel object versus a familiarized object was lower for PM2.5-exposed mice (42% increase) compared to the filtered air (FA) control group (110% increase). In addition, the calculated discrimination index for novel object recognition was significantly higher in FA mice (67 %) compared to PM2.5 exposed mice (57.3%). The object location test (OLT) was used to examine the effect of PM2.5 exposure on spatial memory. In contrast to the FA-exposed control mice, the PM2.5 exposed mice exhibited no significant increase in their exploration time between novel location versus familiarized location indicating their deficit in spatial memory. Furthermore, the discrimination index for novel location was significantly higher in FA mice (62.6%) compared to PM2.5 exposed mice (51%). Overall, our results demonstrate that subchronic exposure to higher levels of PM2.5 in mice causes impairment of novelty recognition and spatial memory.

pharmacology and toxicology↗
bioRxiv2023

SARS-CoV-2 infection triggers pro-atherogenic inflammatory 1 responses in human coronary vessels

COVID-19 patients present higher risk for myocardial infarction (MI), acute coronary syndrome, and stroke for up to 1 year after SARS-CoV-2 infection. While the systemic inflammatory response to SARS-CoV-2 infection likely contributes to this increased cardiovascular risk, whether SARS-CoV-2 directly infects the coronary vasculature and attendant atherosclerotic plaques to locally promote inflammation remains unknown. Here, we report that SARS-CoV-2 viral RNA (vRNA) is detectable and replicates in coronary atherosclerotic lesions taken at autopsy from patients with severe COVID-19. SARS-CoV-2 localizes to plaque macrophages and shows a stronger tropism for arterial lesions compared to corresponding perivascular fat, correlating with the degree of macrophage infiltration. In vitro infection of human primary macrophages highlights that SARS-CoV-2 entry is increased in cholesterol-loaded macrophages (foam cells) and is dependent, in part, on neuropilin-1 (NRP-1). Furthermore, although viral replication is abortive, SARS-CoV-2 induces a robust inflammatory response that includes interleukins IL-6 and IL-1{beta}, key cytokines known to trigger ischemic cardiovascular events. SARS-CoV-2 infection of human atherosclerotic vascular explants recapitulates the immune response seen in cultured macrophages, including pro-atherogenic cytokine secretion. Collectively, our data establish that SARS-CoV-2 infects macrophages in coronary atherosclerotic lesions, resulting in plaque inflammation that may promote acute CV complications and long-term risk for CV events.

immunology↗
bioRxiv2023

BRD4 isoforms have distinct roles in tumor progression and metastasis in embryonal rhabdomyosarcoma

BRD4, a bromodomain and extraterminal (BET) protein, is deregulated in multiple cancers and has emerged as a promising drug target. However, the function of the two main BRD4 isoforms (BRD4-L and BRD4-S) has not been analyzed in parallel in most cancers. This complicates determining therapeutic efficacy of pan-BET inhibitors. In this study, using functional and transcriptomic analysis, we show that BRD-L and BRD4-S isoforms play distinct roles in embryonal rhabdomyosarcoma. BRD4-L has an oncogenic role and inhibits myogenic differentiation, at least in part, by activating myostatin expression. Depletion of BRD4-L in vivo impairs tumor progression but does not impact metastasis. On the other hand, depletion of BRD4-S has no significant impact on tumor growth, but strikingly promotes metastasis in vivo. Interestingly, BRD4-S loss results in the enrichment of BRD4-L and RNA Polymerase II at integrin gene promoters resulting in their activation. Our work unveils isoform-specific functions of BRD4 and demonstrates that BRD4-S functions as a gatekeeper to constrain the full oncogenic potential of BRD4-L.

cancer biology↗
bioRxiv2023

A sucrose non-fermenting-1-related protein kinase 1 gene from wheat, TaSnRK1α regulates starch biosynthesis by modulating AGPase activity

Major portion of wheat grain consist of carbohydrate, mainly starch. The proportion of amylose and amylopectin in starch greatly influence the end product quality. Advancement in understanding starch biosynthesis pathway and modulating key genes has enabled the genetic modification of crops resulting in enhanced starch quality. However, the regulation of starch biosynthesis genes still remains unexplored. So, to expand the limited knowledge, here, we characterized a Ser/Thr kinase, SnRK1 in wheat and determined its role in regulating starch biosynthesis. SnRK1 is an evolutionary conserved protein kinase and share homology to yeast SNF1. Yeast complementation assay suggest TaSnRK1 restore growth defect and promotes glycogen accumulation. Domain analysis and complementation assay with truncated proteins suggest the importance of ATP-binding and UBA domain in TaSnRK1 activity. Sub-cellular localization identified nuclear and cytoplasmic localization of TaSnRK1 in tobacco leaves. Further, heterologous over-expression (O/E) of TaSnRK1 in Arabidopsis not only led to increase in starch content but also enlarges the starch granules. TaSnRK1 was found to restore starch accumulation in Arabidopsis kin10. Remarkably, TaSnRK1 O/E increase the AGPase activity suggesting the direct regulation of rate limiting enzyme AGPase involved in starch biosynthesis. Furthermore, in vitro and in vivo interaction assay reveal that TaSnRK1 interacts with AGPase large sub-unit. Overall, our findings indicate that TaSnRK1 plays role in starch biosynthesis by regulating AGPase activity. HighlightsO_LITaSnRK1 is Ser/Thr kinase in wheat and show dual localization in nucleus and cytoplasm. C_LIO_LIOverexpression of TaSnRK1 increases starch content and enlarges starch granules in Arabidopsis. C_LIO_LITaSnRK1 enhances AGPase activity thereby regulating starch biosynthesis. C_LIO_LITaSnRK1 directly interact with AGPase large subunit in vivo and in vitro. C_LI

molecular biology↗