Conserved and divergent chaperoning effects of Hsp60/10 chaperonins on protein folding landscapes.

The GroEL/ES chaperonin cavity surface charge properties, especially the negative charges, play an important role in its capacity to assist intracavity protein folding. Remarkably, the larger fraction of GroEL/ES negative charges are not conserved among different bacterial species, resulting in a large variation in negative-charge density in the GroEL/ES cavity across prokaryotes. Intriguingly, eukaryotic GroEL/ES homologs have the lowest negative-charge density in the chaperonin cavity. This prompted us to investigate if GroEL's chaperoning mechanism changed during evolution. Using a model in vivo GroEL/ES substrate, we show that the ability of GroEL/ES to buffer entropic traps in the folding pathway of its substrate was partially dependent upon the negative-charge density inside its cavity. While this activity of GroEL/ES was found to be essential for Escherichia coli, it has been perfected in some organisms and diminished in others. However, irrespective of their charges, all the tested homologs retained their ability to regulate polypeptide chain collapse and remove enthalpic traps from folding pathways. The ability of these GroEL/ES homologs to buffer mutational variations in a model substrate correlated with their negative-charge density. Thus, Hsp60/10 chaperonins in different organisms may have changed to accommodate a different spectrum of mutations on their substrates.

Integrating an ER Stress Reporter for Monitoring Genome-Wide UPR-ER in Budding Yeast.

Genetic interaction studies have been instrumental in understanding and organizing cellular pathways. This has been helpful in identifying and arranging genes according to pathways, identifying novel pathways, ascribing gene function, and providing information regarding redundant and antagonistic pathways. Synthetic Genetic Array (SGA) uses growth to identify genome scale gene interaction networks. While this has provided most of the genetic interaction data available, SGA coupled to other reporters have the potential to identify components of pathways that specifically affect the probed reporter. The method described here utilizes SGA principles to understand conserved elements of endoplasmic reticulum (ER) homeostasis in the presence and absence of ER stress. The use of a fluorescent reporter of ER stress allows quantitative measurements and provides a handle to measure the proteostasis capacity of the ER in a high-throughput manner. The integration of such a fluorescent reporter in the background of different mutant/deletion strains is sufficient to identify genetic modules in a high-throughput manner.

Recent advances in understanding the role of proteostasis.

Maintenance of a functional proteome is achieved through the mechanism of proteostasis that involves precise coordination between molecular machineries assisting a protein from its conception to demise. Although each organelle within a cell has its own set of proteostasis machinery, inter-organellar communication and cell non-autonomous signaling bring forth the multidimensional nature of the proteostasis network. Exposure to extrinsic and intrinsic stressors can challenge the proteostasis network, leading to the accumulation of aberrant proteins or a decline in the proteostasis components, as seen during aging and in several diseases. Here, we summarize recent advances in understanding the role of proteostasis and its regulation in aging and disease, including monogenetic and infectious diseases. We highlight some of the emerging as well as unresolved questions in proteostasis that need to be addressed to overcome pathologies associated with damaged proteins and to promote healthy aging.

GROEL/ES Buffers Entropic Traps in Folding Pathway during Evolution of a Model Substrate.

The folding landscape of proteins can change during evolution with the accumulation of mutations that may introduce entropic or enthalpic barriers in the protein folding pathway, making it a possible substrate of molecular chaperones in vivo. Can the nature of such physical barriers of folding dictate the feasibility of chaperone-assistance? To address this, we have simulated the evolutionary step to chaperone-dependence keeping GroEL/ES as the target chaperone and GFP as a model protein in an unbiased screen. We find that the mutation conferring GroEL/ES dependence in vivo and in vitro encode an entropic trap in the folding pathway rescued by the chaperonin. Additionally, GroEL/ES can edit the formation of non-native contacts similar to DnaK/J/E machinery. However, this capability is not utilized by the substrates in vivo. As a consequence, GroEL/ES caters to buffer mutations that predominantly cause entropic traps, despite possessing the capacity to edit both enthalpic and entropic traps in the folding pathway of the substrate protein.

Distinct metabolic states of a cell guide alternate fates of mutational buffering through altered proteostasis.

Metabolic changes alter the cellular milieu; can this also change intracellular protein folding? Since proteostasis can modulate mutational buffering, if change in metabolism has the ability to change protein folding, arguably, it should also alter mutational buffering. Here we find that altered cellular metabolic states in E. coli buffer distinct mutations on model proteins. Buffered-mutants have folding problems in vivo and are differently chaperoned in different metabolic states. Notably, this assistance is dependent upon the metabolites and not on the increase in canonical chaperone machineries. Being able to reconstitute the folding assistance afforded by metabolites in vitro, we propose that changes in metabolite concentrations have the potential to alter protein folding capacity. Collectively, we unravel that the metabolite pools are bona fide members of proteostasis and aid in mutational buffering. Given the plasticity in cellular metabolism, we posit that metabolic alterations may play an important role in cellular proteostasis.

Th9 cytokines curb cervical cancer progression and immune evasion.

Cervical cancer is one of the most common cancers among women in developing countries. Persistent infection with high-risk human papillomavirus (HPV) is the major determinant for the development of cervical cancer. Role of newly discovered T helper 9 (Th9) cells in cervical cancer pathogenesis is yet unfolded. In this study, we observed a huge infiltration of PU.1+ cells and overrepresentation of IL-9R in tissue biopsy specimens of CIN patients in cervical cancer cases. Treatment with Th9 signatory cytokines, IL-9 and IL-21, suppressed proliferation, enhanced apoptosis and stimulated the expression of MHC I and e-cadherin on HeLa cell lines. Th9 thus seems enhance antitumor immune response through T cell cytotoxicity and play crucial role in a controlling malignant cell transformation. Therefore, this study helps in firmer understanding of relevance of Th9 in cervical cancer immunity.

Ncl1-mediated metabolic rewiring critical during metabolic stress.

Nutritional limitation has been vastly studied; however, there is limited knowledge of how cells maintain homeostasis in excess nutrients. In this study, using yeast as a model system, we show that some amino acids are toxic at higher concentrations. With cysteine as a physiologically relevant example, we delineated the pathways/processes that are altered and those that are involved in survival in the presence of elevated levels of this amino acid. Using proteomics and metabolomics approach, we found that cysteine up-regulates proteins involved in amino acid metabolism, alters amino acid levels, and inhibits protein translation-events that are rescued by leucine supplementation. Through a comprehensive genetic screen, we show that leucine-mediated effect depends on a transfer RNA methyltransferase (NCL1), absence of which decouples transcription and translation in the cell, inhibits the conversion of leucine to ketoisocaproate, and leads to tricarboxylic acid cycle block. We therefore propose a role of NCL1 in regulating metabolic homeostasis through translational control.

Dietary restriction improves proteostasis and increases life span through endoplasmic reticulum hormesis.

Unfolded protein response (UPR) of the endoplasmic reticulum (UPRER) helps maintain proteostasis in the cell. The ability to mount an effective UPRER to external stress (iUPRER) decreases with age and is linked to the pathophysiology of multiple age-related disorders. Here, we show that a transient pharmacological ER stress, imposed early in development on Caenorhabditis elegans, enhances proteostasis, prevents iUPRER decline with age, and increases adult life span. Importantly, dietary restriction (DR), that has a conserved positive effect on life span, employs this mechanism of ER hormesis for longevity assurance. We found that only the IRE-1-XBP-1 branch of UPRER is required for the longevity effects, resulting in increased ER-associated degradation (ERAD) gene expression and degradation of ER resident proteins during DR. Further, both ER hormesis and DR protect against polyglutamine aggregation in an IRE-1-dependent manner. We show that the DR-specific FOXA transcription factor PHA-4 transcriptionally regulates the genes required for ER homeostasis and is required for ER preconditioning-induced life span extension. Finally, we show that ER hormesis improves proteostasis and viability in a mammalian cellular model of neurodegenerative disease. Together, our study identifies a mechanism by which DR offers its benefits and opens the possibility of using ER-targeted pharmacological interventions to mimic the prolongevity effects of DR.

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 and maintenance; some with overlapping 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. Here, we have used S. cerevisiae to understand how cells respond to different types of proteostasis impairments. We monitored the proteostasis status and transcriptome of single deletions of fourteen different Protein Quality Control (PQC) genes. In most cases, cellular response did not activate proteostasis components or pathways that could either complement the function of the missing PQC gene or restore proteostasis. Over-expression of alternate machineries could restore part of the proteostasis defect in two representative PQC gene deletion strains. We posit that S. cerevisiae inherently lacks the ability to sense and respond optimally to defects in proteostasis caused due to deletion of specific PQC components.

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.

Proteomic profile of 4-PBA treated human neuronal cells during ER stress.

Perturbations affecting the homoeostasis of endoplasmic reticulum (ER) activate an adaptive signaling known as the unfolded protein response or UPR. Many studies have reported the association between neurological disorders and ER stress. Decreasing ER stress may therefore aid in therapeutic control of neuronal diseases. Sodium 4-phenylbutyrate (4-PBA), a small molecule, has been shown to alleviate ER stress and various neurological diseases, but the mechanistic basis of its action is not well understood. Using an iTRAQ based LC-MS technique we have delineated the effect of 4-PBA on the proteome of human neuroblastoma cells (SK-N-SH) during Tunicamycin-induced ER stress. The proteomic profile of 4-PBA-treated cells revealed that 4-PBA does not alter the cellular proteome to adapt towards ER stress. However, it can alleviate both the toxicity and proteomic alterations, induced by an ER stress inducer. Hence, the therapeutic effect of 4-PBA is primarily due to its ability to resolve ER stress rather than its ability to alter the expression of proteins required for maintaining ER proteostasis. Thus, we posit here that 4-PBA acts as an authentic chemical chaperone by aiding protein folding in the ER.

Oxidative Homeostasis Regulates the Response to Reductive Endoplasmic Reticulum Stress through Translation Control.

Reductive stress leads to the loss of disulfide bond formation and induces the unfolded protein response of the endoplasmic reticulum (UPR(ER)), necessary to regain proteostasis in the compartment. Here we show that peroxide accumulation during reductive stress attenuates UPR(ER) amplitude by altering translation without any discernible effect on transcription. Through a comprehensive genetic screen in Saccharomyces cerevisiae, we identify modulators of reductive stress-induced UPR(ER) and demonstrate that oxidative quality control (OQC) genes modulate this cellular response in the presence of chronic but not acute reductive stress. Using a combination of microarray and relative quantitative proteomics, we uncover a non-canonical translation attenuation mechanism that acts in a bipartite manner to selectively downregulate highly expressed proteins, decoupling the cell's transcriptional and translational response during reductive ER stress. Finally, we demonstrate that PERK, a canonical translation attenuator in higher eukaryotes, helps in bypassing a ROS-dependent, non-canonical mode of translation attenuation.

Selective inhibition of miR-21 by phage display screened peptide.

miRNAs are nodal regulators of gene expression and deregulation of miRNAs is causally associated with different diseases, including cancer. Modulation of miRNA expression is thus of therapeutic importance. Small molecules are currently being explored for their potential to downregulate miRNAs. Peptides have shown to have better potency and selectivity toward their targets but their potential in targeting and modulating miRNAs remain unexplored. Herein, using phage display we found a very selective peptide against pre-miR-21. Interestingly, the peptide has the potential to downregulate miR-21, by binding to pre-miR-21 and hindering Dicer processing. It is selective towards miR-21 inside the cell. By antagonising miR-21 function, the peptide is able to increase the expression of its target proteins and thereby increase apoptosis and suppress cell proliferation, invasion and migration. This peptide can further be explored for its anti-cancer activity in vivo and may be even extended to clinical studies.

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.

In silico studies of the properties of water hydrating a small protein.

Atomistic molecular dynamics simulation of an aqueous solution of the small protein HP-36 has been carried out with explicit solvent at room temperature. Efforts have been made to explore the influence of the protein on the relative packing and ordering of water molecules around its secondary structures, namely, three α-helices. The calculations reveal that the inhomogeneous water ordering and density distributions around the helices are correlated with their relative hydrophobicity. Importantly, we have identified the existence of a narrow relatively dehydrated region containing randomly organized "quasi-free" water molecules beyond the first layer of "bound" waters at the protein surface. These water molecules with relatively weaker binding energies form the transition state separating the "bound" and "free" water molecules at the interface. Further, increased contribution of solid-like caging motions of water molecules around the protein is found to be responsible for reduced fluidity of the hydration layer. Interestingly, we notice that the hydration layer of helix-3 is more fluidic with relatively higher entropy as compared to the hydration layers of the other two helical segments. Such characteristics of helix-3 hydration layer correlate well with the activity of HP-36, as helix-3 contains the active site of the protein.

How do eubacterial organisms manage aggregation-prone proteome?

Eubacterial genomes vary considerably in their nucleotide composition. The percentage of genetic material constituted by guanosine and cytosine (GC) nucleotides ranges from 20% to 70%.  It has been posited that GC-poor organisms are more dependent on protein folding machinery. Previous studies have ascribed this to the accumulation of mildly deleterious mutations in these organisms due to population bottlenecks. This phenomenon has been supported by protein folding simulations, which showed that proteins encoded by GC-poor organisms are more prone to aggregation than proteins encoded by GC-rich organisms. To test this proposition using a genome-wide approach, we classified different eubacterial proteomes in terms of their aggregation propensity and chaperone-dependence using multiple machine learning models. In contrast to the expected decrease in protein aggregation with an increase in GC richness, we found that the aggregation propensity of proteomes increases with GC content. A similar and even more significant correlation was obtained with the GroEL-dependence of proteomes: GC-poor proteomes have evolved to be less dependent on GroEL than GC-rich proteomes. We thus propose that a decrease in eubacterial GC content may have been selected in organisms facing proteostasis problems.

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.

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.

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.