Biology subjects

Bhattacharya, A.

Publications and source records attributed to Bhattacharya, A..

At least 73 records · Page 4Linked to original sources

bioRxiv2022

TBC1D15 potentiates lysosomal regeneration from damaged membranes

Acute lysosomal membrane damage reduces the cellular population of functional lysosomes. However, these damaged lysosomes have a remarkable recovery potential independent of lysosomal biogenesis and remain unaffected in TFEB/TFE3-depleted cells. We combined proximity labelling based proteomics, biochemistry and high-resolution microscopy to unravel a new lysosomal membrane regeneration pathway which is dependent on ATG8, lysosomal membrane protein LIMP2, the Rab7 GAP TBC1D15, and proteins required for autophagic lysosomal reformation (ALR) including Dynamin2, Kinesin5B and Clathrin. Upon lysosomal damage, LIMP2 act as a lysophagy receptor to bind ATG8, which in turn recruits TBC1D15 to damaged membranes. TBC1D15 hydrolyses Rab7-GTP to segregate the damaged lysosomal mass and provides a scaffold to assemble and stabilize the ALR machinery, potentiating the formation of lysosomal tubules and subsequent Dynamin2-dependent scission. TBC1D15-mediated lysosome regeneration was also observed in a cell culture model of oxalate nephropathy.

cell biology↗

bioRxiv2022

Combined promoter-capture Hi-C and Hi-C analysis reveals a fine-tuned regulation of 3D chromatin architecture in colorectal cancer

Hi-C is a widely used method for profiling chromosomal interactions in the 3-dimensional context. Due to limitations on the depth of sequencing, the resolution of most Hi-C datasets is often insufficient for scoring fine-scale interactions. We therefore used promoter-capture Hi-C (PCHi-C) data for mapping these subtle interactions. From multiple colorectal cancer (CRC) studies, we combined PCHi-C with Hi-C datasets to understand the dynamics of chromosomal interactions from cis regulatory elements to topologically associated domain (TAD)-level, enabling detection of fine-scale interactions of disease-associated loci within TADs. Our integrated analyses of PCHi-C and Hi-C datasets from CRC cell lines along with histone modification landscape and transcriptome signatures highlight significant genomic structural instability and their association with tumor-suppressive transcriptional programs. Such analyses also yielded nine dysregulated genes. Transcript profiling revealed a dramatic increase in their expression in CRC cell lines as compared to NT2D1 human embryonic carcinoma cells, supporting the predictions of our bioinformatics analysis. We further report increased occupancy of activation associated histone modifications H3K27ac and H3K4me3 at the promoter regions of the targets analyzed. Our study provides deeper insights into the dynamic 3D genome organization in CRC and identification of affected genes which may serve as potential biomarkers for CRC. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/515643v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@11a9fc8org.highwire.dtl.DTLVardef@f01eb4org.highwire.dtl.DTLVardef@6ff038org.highwire.dtl.DTLVardef@103fc24_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2022

The FIGNL1-interacting protein C1orf112 is synthetic lethal with PICH and mediates RAD51 retention on chromatin.

Joint DNA molecules are natural by-products of DNA replication and repair. Persistent joint molecules give rise to ultrafine DNA bridges (UFBs) in mitosis, which compromise sister chromatid separation. The DNA translocase PICH (ERCC6L) plays a central role in UFB resolution. A genome-wide loss-of-function screen was performed to identify the genetic contexts in which cells become dependent on PICH. In addition to genes involved in DNA condensation, centromere stability and DNA damage repair, we identified the uncharacterized protein C1orf112. We find that C1orf112 interacts with and stabilizes the AAA+ ATPase FIGNL1. Inactivation of either C1orf112 or FIGNL1 resulted in UFB formation, prolonged retention of RAD51 on chromatin, impaired replication fork dynamics, and consequently impaired genome maintenance. Combined, our data reveal that inactivation of C1orf112 and FIGNL1 dysregulates RAD51 dynamics at replication forks, resulting in DNA replication defects, and a dependency on PICH to preserve cell viability.

cancer biology↗

bioRxiv2022

origamiFISH allows universal, label-free, single molecule visualization of DNA origami nanodevices across biological samples

Structural DNA nanotechnology enables user-prescribed design of DNA nanostructures (DNs) for biological applications, but how DN design determines their bio-distribution and cellular interactions remain poorly understood. One challenge is that current methods for tracking DN fates in situ, including fluorescent-dye labeling, suffer from low sensitivity and dye-induced artifacts. Here we present origamiFISH, a label-free and universal method for single-molecule fluorescence detection of DNA origami nanostructures in cells and tissues. origamiFISH targets pan-DN scaffold sequences with hybridization chain reaction (HCR) probes to achieve thousand-fold signal amplification. We identify cell-type and shape-specific spatiotemporal uptake patterns within 1 minute of uptake and at picomolar DN concentrations, 10,000x lower than field standards. We additionally optimized compatibility with immunofluorescence and tissue clearing to visualize DN distribution within tissue cryo/vibratome-sections, slice cultures, and whole-mount organoids. Together, origamiFISH enables faithful mapping of DN interactions across subcellular and tissue barriers for guiding the development of DN-based therapeutics.

synthetic biology↗

bioRxiv2022

Amplicon structure creates collateral therapeutic vulnerability in cancer

Although DNA amplifications in cancers frequently harbor passenger genes alongside oncogenes, the functional consequence of such co-amplifications and their impact for therapy remains ill-defined. We discovered that passenger co-amplifications can create amplicon structure-specific collateral vulnerabilities. We present the DEAD-box helicase 1 (DDX1) gene as a bona fide passenger co-amplified with MYCN in cancers. Survival of cancer cells with DDX1 co-amplifications strongly depends on the mammalian target of rapamycin complex 1 (mTORC1). Mechanistically, aberrant DDX1 expression inhibits the tricarboxylic acid cycle through a previously unrecognized interaction with dihydrolipoamide S-succinyltransferase, a component of the alpha-ketoglutarate dehydrogenase complex. Cells expressing aberrant DDX1 levels compensate for the metabolic shift by enhancing mTORC1 activity. Consequently, pharmacological mTORC1 inhibition triggered cell death specifically in cells harboring the DDX1 co-amplification. This work highlights a significant contribution of passenger gene alterations to the therapeutic susceptibility of cancers. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/506647v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@58b27dorg.highwire.dtl.DTLVardef@12e2fccorg.highwire.dtl.DTLVardef@1440f33org.highwire.dtl.DTLVardef@141e933_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2022

Rad51-mediated interhomolog recombination during budding yeast meiosis is promoted by the meiotic recombination checkpoint and the conserved Pif1 helicase

During meiosis, recombination between homologous chromosomes (homologs) generates crossovers that promote proper segregation at the first meiotic division. Recombination is initiated by Spo11-catalyzed double strand breaks (DSBs). 5 end resection of the DSBs creates 3 single strand tails that two recombinases, Rad51 and Dmc1, bind to form presynaptic filaments that search for homology, mediate strand invasion and generate displacement loops (D-loops). D-loop processing then forms crossover and non-crossover recombinants. Meiotic recombination occurs in two temporally distinct phases. During Phase 1, Rad51 is inhibited and Dmc1 mediates the interhomolog recombination that promotes homolog synapsis. In Phase 2, Rad51 becomes active and functions with Rad54 to repair residual DSBs, making increasing use of sister chromatids. The transition from Phase 1 to Phase 2 is controlled by the meiotic recombination checkpoint through the meiosis-specific effector kinase Mek1. This work shows that constitutive activation of Rad51 in Phase 1 results in a subset of DSBs being repaired by a Rad51-mediated interhomolog recombination pathway that is distinct from that of Dmc1. Strand invasion intermediates generated by Rad51 require more time to be processed into recombinants, resulting in a meiotic recombination checkpoint delay in prophase I. Without the checkpoint, Rad51-generated intermediates are more likely to be repaired using a sister chromatid, thereby increasing Meiosis I chromosome nondisjunction. This Rad51 interhomolog recombination pathway is specifically promoted by the conserved 5-3 helicase PIF1 and its paralog, RRM3 and requires Pif1 helicase activity and its interaction with PCNA. This work demonstrates that (1) inhibition of Rad51 during Phase 1 is important to prevent competition with Dmc1 for DSB repair, (2) Rad51-mediated meiotic recombination intermediates are initially processed differently than those made by Dmc1, (3) the meiotic recombination checkpoint provides time during prophase 1 for processing of Rad51-generated recombination intermediates. AUTHOR SUMMARYTo sexually reproduce, cells containing two copies of each chromosome must undergo the specialized cell division of meiosis to sort the chromosomes into gametes containing a single copy of each chromosome. But how do homologous chromosomes know who is who? The answer is by recombination, a process in which double strand breaks on one chromosome are converted to single stranded ends that can search for the complementary sequence on the homolog. In yeast and mammals, this homology search involves binding of single strand ends by two highly conserved recombinases, Rad51 and the meiosis specific Dmc1. Rad51 is used in mitotic cells to repair breaks, primarily using sister chromatids as templates, while Dmc1 functions in meiosis to generate interhomolog crossovers. In budding yeast, Rad51 strand exchange activity is normally inhibited while Dmc1 is active. We show here that when Rad51 and Dmc1 are active at the same time, Rad51 competes with Dmc1 to mediate interhomolog recombination of a subset of double strand breaks. However, because Rad51- generated recombination intermediates take longer to process, there is a need to keep Rad51 inactive while interhomolog recombination is occurring.

genetics↗

bioRxiv2022

Intrinsic disorder in CENP-ACse4 tail and its chaperone facilitates synergistic association for kinetochore stabilization

The kinetochore is a complex multiprotein network that assembles at a specialized DNA locus called the centromere to ensure faithful chromosome segregation. The centromere is epigenetically marked by a histone H3 variant - the CenH3. The budding yeast CenH3, called Cse4, consists of an unusually long and disordered N-terminal tail that has a role in kinetochore assembly. Its disordered chaperone, Scm3 is involved in its centromeric deposition as well as in the maintenance of a segregation-competent kinetochore. The dynamics of the Cse4 N-tail and chaperone interaction have not been studied, leaving a gap in our understanding of their roles at the centromere. Previously, we had shown that Scm3 is an intrinsically disordered protein. Here, using NMR and a variety of biophysical and bioinformatics tools, we show that Cse4 N-tail is also disordered, the two proteins interact with each other at multiple sites, and this interaction reduces the disorder in Scm3; the chain opens up relative to the native state ensemble and the conformational exchange is reduced. Interestingly, this interaction between the two intrinsically disordered protein is fairly specific as seen by positive and negative controls, and is majorly driven by electrostatics as both the proteins have multiple acidic and basic regions. The complex retains a fair amount of disorder, which facilitates a synergistic association with the essential inner kinetochore Ctf19-Mcm21-Okp1-Ame1 complex; a model has been suggested to this effect. Given the abundance of intrinsic disorder in the kinetochore proteins, this type of interaction and adaptation may be prevalent in other proteins as well for mediating kinetochore assembly. Thus, the present study, on one hand, provides significant structural and mechanistic insights into the complex and dynamic process of kinetochore assembly, and on the other hand, illustrates a mechanism that intrinsically disordered proteins would adapt to mediate the formation of complex multiprotein networks, in general. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/504061v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@8fe5cforg.highwire.dtl.DTLVardef@138afb1org.highwire.dtl.DTLVardef@96943forg.highwire.dtl.DTLVardef@1deb673_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

The evolution of constitutive bacteriocin production and release

The production of the bacterial anti-competitor toxins, called bacteriocins, is widely described as spite, because the production of the toxins is costly and often requires cell death for release. Given these costs, it reasonable to think that bacteriocin production should be induced by the presence of unrelated competitor strains. Nonetheless, recent evidence in the insect-pathogenic bacterium Xenorhabdus koppenhoeffri showed that bacteriocin production occurs regardless of the presence of unrelated competitors. Could the constitutive production of bacteriocins be favored by natural selection? Here we use a mathematical model to examine this question, both within and between subpopulations. The model assumes that bacteriocin production is constitutive and costly, and that toxin release only occurs during natural cell death. We found that constitutive bacteriocin producers can outcompete non-producer sensitive strains within populations, and that it can spread in a metapopulation provided there is local competition for patches. Hence, the evolution of bacteriocin production does not require the detection of competitors or "suicidal" release of toxins.

evolutionary biology↗

bioRxiv2022

Widespread employment of conserved C. elegans homeobox genes in neuronal identity specification

Homeobox genes are prominent regulators of neuronal identity, but the extent to which their function has been probed in animal nervous systems remains limited. In the nematode Caenorhabditis elegans, each individual neuron class is defined by the expression of unique combinations of homeobox genes, prompting the question of whether each neuron class indeed requires a homeobox gene for its proper identity specification. We present here progress in addressing this question by extending previous mutant analysis of homeobox gene family members and describing multiple examples of homeobox gene function in different parts of the C. elegans nervous system. To probe homeobox function, we make use of a number of reporter gene tools, including a novel multicolor reporter transgene, NeuroPAL, which permits simultaneous monitoring of the execution of multiple differentiation programs throughout the entire nervous system. Using these tools, we add to the previous characterization of homeobox gene function by identifying neuronal differentiation defects for 12 homeobox genes in 20 distinct neuron classes that are mostly unrelated by location, function and lineage history. 10 of these 20 neuron classes had no homeobox gene function ascribed to them before, while in the other 10 neuron classes, we extend the combinatorial code of transcription factors required for specifying terminal differentiation programs. Furthermore, we demonstrate that in a particular lineage, homeotic identity transformations occur upon loss of a homeobox gene and we show that these transformations are the result of changes in homeobox codes. Combining the present with past analysis, 111 of the 118 neuron classes of C. elegans are now known to require a homeobox gene for proper execution of terminal differentiation programs. Such broad deployment indicates that homeobox function in neuronal identity specification may be an ancestral feature of animal nervous systems.

neuroscience↗

bioRxiv2022

Human anelloviruses produced by recombinant expression of synthetic genomes.

Human anelloviruses are acquired universally in infancy, highly prevalent, abundant in blood, and extremely diverse. Their apparent lack of pathogenicity indicates that they are a major component of the commensal human virome. Despite their being extensively intertwined with human biology, these viruses are poorly understood. A major impediment in studying anelloviruses is the lack of an in vitro system for their production and/ or propagation. Here we show that the T cell-derived human cell line MOLT-4 can be transfected with plasmids comprising tandem anellovirus genomes to produce viral particles visualized by electron microscopy. We found that a previously described human anellovirus of the Betatorquevirus genus (LY2), as well as a second Betatorquevirus detected by sequencing DNA extracted from a human retinal pigmental epithelium (nrVL4619), can be synthesized and produced by these means, enabling further molecular virology studies. Southern blot was used to demonstrate replication, and site-directed mutagenesis of the viral genome was performed to show that the production of anellovirus in this cell line is dependent on the expression of certain viral proteins. Finally, experiments performed in mice using purified nrVL4619 particles produced in MOLT-4 cells demonstrated infectivity in vivo in the tissue of origin. These results indicate that anelloviruses can be produced in vitro and manipulated to improve our understanding of this viral family which is ubiquitous in humans and many other mammals. Applications of this work to gene therapy and other therapeutic modalities are currently under investigation. IMPORTANCEAnelloviruses are a major component of the human virome. However, their biology is not well understood mainly due to the lack of an in vitro system for anellovirus production and/or propagation. In this study, we used multiple orthogonal measures to show that two different anelloviruses belonging to the Betatorquevirus genus can be produced in a T-cell-derived human cell line, MOLT-4, via recombinant expression of synthetic genomes. Additionally, we show that anellovirus particles generated in this in vitro system demonstrate infectivity in vivo. Our findings enable new molecular virology studies of this highly prevalent, non-pathogenic, and weakly immunogenic family of viruses, potentially leading to therapeutic applications.

microbiology↗

bioRxiv2022

Doctoring Direct-to-Consumer Genetic Tests with DNA Spike-Ins

Direct-to-consumer (DTC) genetic testing companies have provided personal genotyping services to millions of customers. Customers mail saliva samples to DTC service providers to have their genotypes analyzed and receive back their raw genetic data. Both consumers and the DTC companies use the results to perform ancestry analyses, relative matching, trait prediction, and estimate predisposition to disease, often relying on genetic databases composed of the data from millions of other DTC-genotyped individuals. While the digital integrity risks to this type of data have been explored, we considered whether data integrity issues could manifest upstream of data generation through physical manipulation of DNA samples themselves, for example by adding synthetic DNA to a saliva sample ("spiked samples") prior to sample processing by a DTC company. Here, we investigated the feasibility of this scenario within the standard DTC genetic testing pipeline. Starting with the purchase of off-the-shelf DTC genetic testing kits, we found that synthetic DNA can be used to precisely manipulate the results of saliva samples genotyped by a popular DTC genetic testing service and that this method can be used to modify arbitrary single nucleotide polymorphisms (SNPs) in multiplex to create customized doctored genetic profiles. This capability has implications for the use of DTC-generated results and the outcomes of their downstream analyses.

genetics↗

bioRxiv2021

Exceptions to the rule: Why does resistance evolution not undermine antibiotic therapy in all bacterial infections?

Antibiotic resistance poses one of the greatest public health challenges of the 21st century. Yet not all pathogens are equally affected by resistance evolution. Why? Here we examine what underlies variation in antibiotic resistance across human bacterial pathogens and the drugs used to treat them. We document the observed prevalence of antibiotic resistance for pathogen x drug combinations across 57 different human bacterial pathogens and 53 antibiotics from 15 drug classes used to treat them. Using AIC-based model selection we analyze 14 different traits of bacteria and antibiotics that are believed to be important in resistance evolution. Using these data, we identify the traits that best explain observed variation in resistance evolution. Our results show that nosocomial pathogens and indirectly transmitted pathogens are significantly associated with increased prevalence of resistance whereas zoonotic pathogens, specifically those with wild animal reservoirs, are associated with reduced prevalence of resistance. We found partial support for associations between drug resistance and gram classification, human microbiome reservoirs, horizontal gene transfer, and documented human-to human transfer. Global drug use, time since drug discovery, mechanism of drug action, and environmental reservoirs did not emerge as statistically robust predictors of drug resistance in our analyses. To the best of our knowledge this work is the first systematic analysis of resistance across such a wide range of human bacterial pathogens, encompassing the vast majority of common bacterial pathogens. Insights from our study may help guide public health policies and future studies on resistance control.

evolutionary biology↗

bioRxiv2021

Functionalizing lipid sponge droplets with DNA

Nucleic acids are among the most versatile molecules for the construction of biomimetic systems because they can serve as information carriers and programmable construction materials. How nucleic acids interact with membranous coacervate compartments such as lipid sponge droplets is not known. Here we systematically characterize the potential of DNA to functionalize lipid sponge droplets and demonstrate a strong size dependence for sequestration into the sponge phase. Double stranded DNA molecules of more than 300 bp are excluded and form a corona on the surface of droplets they are targeted to. Shorter DNA molecules partition efficiently into the lipid sponge phase and can direct DNA-templated reactions to droplets. We demonstrate repeated capture and release of labeled DNA strands by dynamic hybridization and strand displacement reactions that occur inside droplets. Our system opens new opportunities for DNA-encoded functions in lipid sponge droplets such as cargo control and signaling.

synthetic biology↗

bioRxiv2021

Transcriptomic analysis of ribosome biogenesis and pre-rRNA processing during growth stress in Entamoeba histolytica

Ribosome biogenesis, a multi-step process involving the transcription, modification, folding and processing of rRNA is the major consumer of cellular energy. It involves the sequential assembly of ribosomal proteins (RP)s via more than 200 ribogenesis factors. Unlike model organisms where transcription of rRNA and RP genes slows down during stress, in Entamoeba histolytica, pre-rRNA synthesis continues, and unprocessed pre-rRNA accumulates. To gain insight into the vast repertoire of ribosome biogenesis factors and understand the major components playing role during stress we computationally identified the ribosome biogenesis factors in E. histolytica. Of the total [~]279 S. cerevisiae proteins, we could only find 188 proteins in E. histolytica. Some of the proteins missing in E. histolytica were also missing in humans. A number of proteins represented by multiple genes in S. cerevisiae had only a single copy in E. histolytica. It was interesting to note that E. histolytica lacked mitochondrial ribosome biogenesis factors and had far less RNase components as compared to S. cerevisiae. Northern hybridization using probes from different spacer regions depicted the accumulation of unprocessed intermediates during stress. Transcriptomic studies revealed the differential regulation of a number of ribosomal factors both in serum-starved and RRP6KD conditions. The ARB1 protein involved at multiple steps of ribosome biogenesis and NEP1 and TSR3 involved in chemical modification of 18S rRNA previously shown to accumulate pre-rRNA precursors upon downregulation in S. cerevisiae and humans were included. The data reveals the importance of some of the major factors required for regulating pre-rRNA processing during stress. This is the first report on the complete repertoire of ribosome biogenesis factors in E. histolytica.

microbiology↗

bioRxiv2021

EP300 (p300) mediated histone butyrylation is critical for adipogenesis

ObjectiveThe master epigenetic enzyme EP300 (p300) besides having lysine acetyltransferase activity can also catalyse other acylation modifications (propionylation, butyrylation, crotonylation etc.), the physiological implications of which are yet to be established fully. We hypothesized that p300 catalysed histone butyrylation may have a causal relationship with adipogenesis and the consequent obesity. MethodsHistone butyrylation pattern was investigated in 3T3L1 cells upon adipogenesis by immunoblotting and chromatin immunoprecipitation experiments. A small molecule modulator that could specifically inhibit p300 catalysed butyrylation without affecting its canonical acetyltransferase activity was screened from a series of compounds and then administered in differentiating 3T3L1 adipocytes as well as high fat diet-induced and genetically obese mice to validate the importance of butyrylation in adipogenesis. ResultsHistone butyrylation was increased upon adipogenesis both globally and locally in the promoters of pro-adipogenic genes along with an upregulation in the expression of acyl CoA generating enzyme Acss2, knockdown of which led to reduced butyrylation. Treatment of differentiating 3T3L1 cells with the p300 specific butyrylation inhibitor LTK-14A led to abrogation of adipogenesis with reduced expression of pro-adipogenic genes and inhibition of H4K5 butyrylation. LTK-14A administration could also attenuate weight gain in both mice models of obesity by preventing adipocyte hypertrophy via H4K5 butyrylation inhibition. ConclusionOur results indicate that p300 catalysed histone butyrylation may have a causal relationship with the process of adipogenesis. Site specific inhibition of butyrylation could lead to adipogenesis repression and hence this epigenetic modification could be targeted for obesity treatment. HighlightsO_LIHistone butyrylation has been established as a new epigenetic signature in the context of adipogenesis. C_LIO_LITo the best of our knowledge, this is the first report of a selective inhibitor of p300 catalysed histone acylation (butyrylation) without affecting its canonical acetyltransferase activity. C_LIO_LISpecific inhibition of H4K5 butyrylation could be a possible mechanism for inhibiting adipogenesis and hepatic steatosis leading to better control of obesity. C_LIO_LILTK-14A class of molecule could be developed as anti-obesity therapeutics. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=195 HEIGHT=200 SRC="FIGDIR/small/454641v3_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1f29b35org.highwire.dtl.DTLVardef@abc5fcorg.highwire.dtl.DTLVardef@1534388org.highwire.dtl.DTLVardef@176d706_HPS_FORMAT_FIGEXP M_FIG Proposed model for the role of p300-mediated histone butyrylation in adipogenesis: In pre-adipocytes, there exists a basal level of histone acetylation while butyrylation is present to a much lesser extent owing to low stoichiometric levels of butyryl CoA. Induction of adipogenesis causes a simultaneous upregulation of histone acetylation and butyrylation marks leading to increased rate of adipogenesis and concomitant transcriptional activation of pro-adipogeneic genes. Onset of obesity in mice, either due to excess energy intake through high fat diet consumption or increased de novo synthesis of fatty acids due to leptin receptor gene mutation leading to hyperphagic behavior, is accompanied by adipocyte hyperplasia and hypertrophy. Both the organs of adipose tissue and liver were found to have enhanced levels of H4K5 butyrylation during obesity. LTK-14A, a butyrylation specific inhibitor could efficiently prevent the processes of adipogenesis and adipocyte hypertrophy due to inhibition of H4K5 butyrylation in these organs. Thus the compound could attenuate weight gain by selective inhibition of butyrylation without affecting acetylation, thereby highlighting the importance of histone butyrylation in adipogenesis and obesity. C_FIG

molecular biology↗

bioRxiv2021

Expression of fatty acyl-CoA ligase drives one-pot de novo synthesis of membrane-bound vesicles in a cell free transcription-translation system

Despite the central importance of lipid membranes in cellular organization, it is challenging to reconstitute their de novo formation from minimal chemical and biological elements. Here we describe a chemoenzymatic route to membrane-forming non-canonical phospholipids in which cysteine-modified lysolipids undergo spontaneous coupling with fatty acyl-CoA thioesters generated enzymatically by a fatty acyl-CoA ligase. Due to the high efficiency of the reaction, we were able to optimize phospholipid membrane formation in a cell-free transcription-translation (TX-TL) system. Combining DNA encoding for the fatty acyl-CoA ligase with suitable lipid precursors, enabled spontaneous one-pot de novo synthesis of membrane-bound vesicles. Non-canonical sphingolipid synthesis was also possible by using a cysteine-modified lysosphingomyelin as a precursor. When the sphingomyelin-interacting protein lysenin is co-expressed alongside the acyl CoA ligase, the in situ assembled membranes were spontaneously modified with protein. Our strategy of coupling gene expression with membrane lipid synthesis in a one-pot fashion could facilitate the generation of proteoliposomes and brings us closer to the bottom-up generation of synthetic cells using recombinant synthetic biology platforms.

synthetic biology↗

bioRxiv2021

Bacterial hopanoids: a newly identified potent inducer of monocyte to macrophage differentiation

Monocyte to macrophage differentiation is an extremely essential cellular and immunological process aimed to combat the assault of foreign pathogens as well as this process has immense importance in the maintenance of physiological homeostasis. Differentiated macrophage, serving as specialized phagocytes is an indispensable component of innate immunity. Besides this, being a well-documented antigen-presenting cell (APC) macrophages also function as a key regulator of both humoral and cell-mediated immunity: the two integral components of adaptive immunity. Bacterial hopanoids are a primitive analogous of eukaryotic sterols that are established as a prominent immunomodulator. In this study, we have demonstrated the isolation protocol of neutral lipid fractions from different Gram-negative bacteria, and adopting several analytical approaches we proposed that the extracted lipid fractions may contain hopanoid as an active component. We have delved deeper into the biological effect of these probable hopanoids like compounds. Here, by considering the several structural and functional attributes like altered phenotype, expression of macrophage-specific markers, increased intracellular organelles, acquirement of enhanced phagocytic and inflammatory property, induction of autophagy, etc we have established hopanoids as a potential inducer of monocyte to macrophage differentiation. Thus, our study has unraveled a new immunomodulator present in Gram-negative bacteria and would undoubtedly help to understand the intricacies of host-pathogen interaction in a better and conclusive manner.

cell biology↗

bioRxiv2021

The Fragile X Mental Retardation Protein protects the lung from xenobiotic stress by facilitating the Integrated Stress Response

Stress response pathways protect the lung from the damaging effects of environmental toxicants. Here we investigate the role of the Fragile X Mental Retardation Protein (FMRP), a multifunctional protein implicated in stress responses, in the lung. We report that FMRP is expressed in murine and human lungs, in the airways and more broadly. Analysis of airway stress responses in mice and in a murine cell line ex vivo, using the well-established Naphthalene (Nap) injury model, reveals that FMRP-deficient cells exhibit increased expression of markers of oxidative and genotoxic stress and increased cell death. We find that FMRP-deficient cells fail to actuate the Integrated Stress Response Pathway (ISR) and upregulate the transcription factor ATF4. Knockdown of ATF4 expression phenocopies the loss of FMRP. We extend our analysis of the role of FMRP to human bronchial BEAS-2B cells, using a 9, 10-Phenanthrenequinone air pollutant model, to find that FMRP-deficient BEAS-2B also fail to actuate the ISR and exhibit greater susceptibility. Taken together, our data suggest that FMRP has a conserved role in protecting the airways by facilitating the ISR.

cell biology↗