Kausik Chakraborty

High resolution methylome map of rat indicates role of intragenic DNA methylation in identification of coding region.

DNA methylation is crucial for gene regulation and maintenance of genomic stability. Rat has been a key model system in understanding mammalian systemic physiology, however detailed rat methylome remains uncharacterized till date. Here, we present the first high resolution methylome of rat liver generated using Methylated DNA immunoprecipitation and high throughput sequencing (MeDIP-Seq) approach. We observed that within the DNA/RNA repeat elements, simple repeats harbor the highest degree of methylation. Promoter hypomethylation and exon hypermethylation were common features in both RefSeq genes and expressed genes (as evaluated by proteomic approach). We also found that although CpG islands were generally hypomethylated, about 6% of them were methylated and a large proportion (37%) of methylated islands fell within the exons. Notably, we obeserved significant differences in methylation of terminal exons (UTRs); methylation being more pronounced in coding/partially coding exons compared to the non-coding exons. Further, events like alternate exon splicing (cassette exon) and intron retentions were marked by DNA methylation and these regions are retained in the final transcript. Thus, we suggest that DNA methylation could play a crucial role in marking coding regions thereby regulating alternative splicing. Apart from generating the first high resolution methylome map of rat liver tissue, the present study provides several critical insights into methylome organization and extends our understanding of interplay between epigenome, gene expression and genome stability.

Chemical Chaperones Mitigate Experimental Asthma by Attenuating Endoplasmic Reticulum Stress

Endoplasmic reticulum (ER) stress and consequent unfolded protein response (UPR) are important in inflammation but have been poorly explored in asthma. We used a mouse model of allergic airway inflammation (AAI) with features of asthma to understand the role of ER stress and to explore potential therapeutic effects of inhaled chemical chaperones, which are small molecules that can promote protein folding and diminish UPR. UPR markers were initially measured on alternate days during a 7-day daily allergen challenge model. UPR markers increased within 24 hours after the first allergen challenge and peaked by the third challenge, before AAI was fully established (from the fifth challenge onward). Three chemical chaperones-glycerol, trehalose, and trimethylamine-N-oxide (TMAO)-were initially administered during allergen challenge (preventive regimen). TMAO, the most effective of these chemical chaperones and 4-phenylbutyric acid, a chemical chaperone currently in clinical trials, were further tested for potential therapeutic activities after AAI was established (therapeutic regimen). Chemical chaperones showed a dose-dependent reduction in UPR markers, airway inflammation, and remodeling in both regimens. Our results indicate an early and important role of the ER stress pathway in asthma pathogenesis and show therapeutic potential for chemical chaperones.

Classification of Chemical Chaperones Based on Their Effect on Protein Folding Landscapes

Various small molecules present in biological systems can assist protein folding in vitro and are known as chemical chaperones. De novo design of chemical chaperones with higher activity than currently known examples is desirable to ameliorate protein misfolding and aggregation in multiple contexts. However, this development has been hindered by limited knowledge of their activities. It is thought that chemical chaperones are typically poor solvents for a protein backbone and hence facilitate native structure formation. However, it is unknown if different chemical chaperones can act differently to modulate folding energy landscapes. Using a model slow folding protein, double-mutant Maltose-binding protein (DM-MBP), we show that a canonical chemical chaperone, trimethylamine-N-oxide (TMAO), accelerates refolding by decreasing the flexibility of the refolding intermediate (RI). Among a number of small molecules that chaperone DM-MBP folding, proline and serine stabilize the transition state (TS) enthalpically, while trehalose behaves like TMAO and increases the rate of barrier crossing through nonenthalpic processes. We propose a two-group classification of chemical chaperones based upon their thermodynamic effect on RI and TS, which is also supported by single molecule Förster resonance energy transfer (smFRET) studies. Interestingly, for a different test protein, the molecular mechanisms of the two groups of chaperones are not conserved. This provides a glimpse into the complexity of chemical chaperoning activity of osmolytes. Future work would allow us to engineer synergism between the two classes to design more efficient chemical chaperones to ameliorate protein misfolding and aggregation problems.

Converging evidence of mitochondrial dysfunction in a yeast model of homocysteine metabolism imbalance

An elevated level of homocysteine, a thiol amino acid, is associated with various complex disorders. The cellular effects of homocysteine and its precursors S-adenosylhomocysteine (AdoHcy) and S-adenosylmethionine (AdoMet) are, however, poorly understood. We used Saccharomyces cerevisiae as a model to understand the basis of pathogenicity induced by homocysteine and its precursors. Both homocysteine and AdoHcy but not AdoMet inhibited the growth of the str4Δ strain (which lacks the enzyme that converts homocysteine to cystathionine-mimicking vascular cells). Addition of AdoMet abrogated the inhibitory effect of AdoHcy but not that of homocysteine indicating that an increase in the AdoMet/AdoHcy ratio is sufficient to overcome the AdoHcy-mediated growth defect but not that of homocysteine. Also, the transcriptomic profile of AdoHcy and homocysteine showed gross dissimilarity based on gene enrichment analysis. Furthermore, compared with homocysteine, AdoHcy treatment caused a higher level of oxidative stress in the cells. However, unlike a previously reported response in wild type (Kumar, A., John, L., Alam, M. M., Gupta, A., Sharma, G., Pillai, B., and Sengupta, S. (2006) Biochem. J. 396, 61–69), the str4Δ strain did not exhibit an endoplasmic reticulum stress response. This suggests that homocysteine induces varied response depending on the flux of homocysteine metabolism. We also observed altered expression of mitochondrial genes, defective membrane potential, and fragmentation of the mitochondrial network together with the increased expression of fission genes indicating that the imbalance in homocysteine metabolism has a major effect on mitochondrial functions. Furthermore, treatment of cells with homocysteine or AdoHcy resulted in apoptosis as revealed by annexin V staining and TUNEL assay. Cumulatively, our results suggest that elevated levels of homocysteine lead to mitochondrial dysfunction, which could potentially initiate pro-apoptotic pathways, and this could be one of the mechanisms underlying homocysteine-induced pathogenicity.

Cross‐compartment proteostasis regulation during redox imbalance induced ER stress

Imbalance in protein homeostasis in specific subcellular organelles is alleviated through organelle‐specific stress response pathways. As a canonical example of stress activated pathway, accumulation of misfolded proteins in ER activates unfolded protein response (UPR) in almost all eukaryotic organisms. However, very little is known about the involvement of proteins of other organelles that help to maintain the cellular protein homeostasis during ER stress. In this study, using iTRAQ‐based LC–MS approach, we identified organelle enriched proteins that are differentially expressed in yeast (Saccharomyces cerevisiae) during ER stress in the absence of UPR sensor Ire1p. We have identified about 750 proteins from enriched organelle fraction in three independent iTRAQ experiments. Induction of ER stress resulted in the differential expression of 93 proteins in WT strains, 40 of which were found to be dependent on IRE1. Our study reveals a cross‐talk between ER‐ and mitochondrial proteostasis exemplified by an Ire1p‐dependent induction of Hsp60p, a mitochondrial chaperone. Thus, in this study, we show changes in protein levels in various organelles in response to ER stress. A large fraction of these changes were dependent on canonical UPR signalling through Ire1, highlighting the importance of interorganellar cross‐talk during stress.

Cellular responses to proteostasis perturbations reveal non-optimal feedback in chaperone networks

The proteostasis network (PN) comprises a plethora of proteins that are dedicated to aid in protein folding; some with over-lapping functions. Despite this, there are multiple pathophysiological states associated with depletion of chaperones. This is counter-intuitive assuming cells have the ability to re-program transcriptional outputs in accordance with its proteostasic limitations. To this effect, we have used S. cerevisiae to understand the route a cell takes as a response when challenged with different proteostasis impairments. Using 14 single deletion strains of genes of Protein Quality Control (PQC) system, we quantify their proteostasis impairment and the transcriptional response. In most cases cellular response was incapable of restoring proteostasis. The response did not activate proteostasis components or pathways that could complement the function of the missing PQC gene. Over-expression of alternate machineries, could restore part of the proteostasis defect in deletion strains. We posit that epistasis guided synthetic biology approaches may be helpful in realizing the true potential of the cellular chaperone machinery.Protein folding abnormalities are associated with the pathology of many diseases. This is surprising given the plethora of cellular machinery dedicated to aid protein folding. It is though that cellular response to proteotoxicity is generally sufficient, but may be compromised during pathological conditions. We asked if, in a physiological condition, cells have the ability to re-program transcriptional outputs in accordance with proteostasis demands. We have used S. cerevisiae to understand the response of cells when challenged with different proteostasis impairments, by removing one protein quality control (PQC) gene from the system at a time. Using 14 PQC deletions, we investigated the transcriptional response and find the mutants were unable to upregulate pathways that could complement the function of the missing PQC gene. To our surprise, cells have inherently a limited scope of response that is not optimally tuned; with transcriptomic responses being decorrelated with respect to the sign of their epistasis. We conclude that this non-optimality in proteotoxic response may limit the cellular ability to reroute proteins through alternate and productive machineries resulting in pathological states. We posit that epistasis guided synthetic biology approaches may be helpful in realizing the true potential of the cellular chaperone machinery.

Differential strengths of molecular determinants guide environment specific mutational fates

Organisms maintain competitive fitness in the face of environmental challenges through molecular evolution. However, it remains largely unknown how different biophysical factors constrain molecular evolution in a given environment. Here, using deep mutational scanning, we quantified empirical fitness of >2000 single site mutants of the Gentamicin-resistant gene (GmR) in Escherichia coli, in a representative set of physical (non-native temperatures) and chemical (small molecule supplements) environments. From this, we could infer how different biophysical parameters of the mutations constrain molecular function in different environments. We find ligand binding, and protein stability to be the best predictors of mutants’ fitness, but their relative predictive power differs across environments. While protein folding emerges as the strongest predictor at minimal antibiotic concentration, ligand binding becomes a stronger predictor of mutant fitness at higher concentration. Remarkably, strengths of environment-specific selection pressures were largely predictable from the degree of mutational perturbation of protein folding and ligand binding. By identifying structural constraints that act as determinants of fitness, our study thus provides coarse mechanistic insights into the environment specific accessibility of mutational fates.

The transcriptome of proteostasis perturbations reveal non-optimal response in chaperone networks

The proteostasis network (PN) comprises a plethora of proteins that are dedicated to aid in protein folding; some with over-lapping functions. Despite this, there are multiple pathophysiological states associated with depletion of chaperones. This is counter-intuitive assuming cells have the ability to re-program transcriptional outputs in accordance with its proteostasic limitations. To this effect, we have used S. cerevisiae to understand the route a cell takes as a response when challenged with different proteostasis impairments. Using 14 single deletion strains of genes of Protein Quality Control (PQC) system, we quantify their proteostasis impairment and the transcriptional response. In most cases cellular response was incapable of restoring proteostasis. The response did not activate proteostasis components or pathways that could complement the function of the missing PQC gene. Over-expression of alternate machineries, could restore part of the proteostasis defect in deletion strains. We posit that epistasis guided synthetic biology approaches may be helpful in realizing the true potential of the cellular chaperone machinery.Protein folding abnormalities are associated with the pathology of many diseases. This is surprising given the plethora of cellular machinery dedicated to aid protein folding. It is though that cellular response to proteotoxicity is generally sufficient, but may be compromised during pathological conditions. We asked if, in a physiological condition, cells have the ability to re-program transcriptional outputs in accordance with proteostasis demands. We have used S. cerevisiae to understand the response of cells when challenged with different proteostasis impairments, by removing one protein quality control (PQC) gene from the system at a time. Using 14 PQC deletions, we investigated the transcriptional response and find the mutants were unable to upregulate pathways that could complement the function of the missing PQC gene. To our surprise, cells have inherently a limited scope of response that is not optimally tuned; with transcriptomic responses being decorrelated with respect to the sign of their epistasis. We conclude that this non-optimality in proteotoxic response may limit the cellular ability to reroute proteins through alternate and productive machineries resulting in pathological states. We posit that epistasis guided synthetic biology approaches may be helpful in realizing the true potential of the cellular chaperone machinery.

Chaperone Network Based On Cellular Response Reveals The Non-Optimal Response To Perturbed Proteostasis

The proteostasis network (PN) comprises a plethora of proteins that are dedicated to aid in protein folding; some with over-lapping functions. Despite this, there are multiple pathophysiological states associated with depletion of chaperones. This is counter-intuitive assuming cells have the ability to re-program transcriptional outputs in accordance with its proteostasic limitations. To this effect, we have used S. cerevisiae to understand the route a cell takes as a response when challenged with different proteostasis impairments. Using 14 single deletion strains of genes of Protein Quality Control (PQC) system, we quantify their proteostasis impairment and the transcriptional response. In most cases cellular response was incapable of restoring proteostasis. The response did not activate proteostasis components or pathways that could complement the function of the missing PQC gene. Over-expression of alternate machineries, could restore part of the proteostasis defect in deletion strains. We posit that epistasis guided synthetic biology approaches may be helpful in realizing the true potential of the cellular chaperone machinery.Protein folding abnormalities are associated with the pathology of many diseases. This is surprising given the plethora of cellular machinery dedicated to aid protein folding. It is though that cellular response to proteotoxicity is generally sufficient, but may be compromised during pathological conditions. We asked if, in a physiological condition, cells have the ability to re-program transcriptional outputs in accordance with proteostasis demands. We have used S. cerevisiae to understand the response of cells when challenged with different proteostasis impairments, by removing one protein quality control (PQC) gene from the system at a time. Using 14 PQC deletions, we investigated the transcriptional response and find the mutants were unable to upregulate pathways that could complement the function of the missing PQC gene. To our surprise, cells have inherently a limited scope of response that is not optimally tuned; with transcriptomic responses being decorrelated with respect to the sign of their epistasis. We conclude that this non-optimality in proteotoxic response may limit the cellular ability to reroute proteins through alternate and productive machineries resulting in pathological states. We posit that epistasis guided synthetic biology approaches may be helpful in realizing the true potential of the cellular chaperone machinery.