CHANDANA BARAT - Academia.edu (original) (raw)

Papers by CHANDANA BARAT

(A) The 23S rRNA of E. coli large ribosomal subuni... more (A) The 23S rRNA of E. coli large ribosomal subunit (PDB: 2I2V) has been displayed (light orange) here with 3′-CCA end of P-site tRNA (PDB: 2J02). The domain V region of 23S rRNA has been shown in grey and the 3′-CCA end of P-site tRNA is represented in red. The nucleotides which have been mutated in this study have also been shown in sticks and are represented in the same colour as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101293#pone-0101293-g004" target="_blank">Fig. 4A</a>. (B) The 23S rRNA of E. coli large ribosomal subunit (PDB: 2I2V) has also been displayed (light orange) here with P-site tRNA (PDB: 2J02) (red colour surface representation). This view is in the same orientation in which Fig. 5A has been shown.</p

Protein & Peptide Letters

Background: Alzheimer’s disease (AD) is characterized by the aggregation of Tau protein and Amylo... more Background: Alzheimer’s disease (AD) is characterized by the aggregation of Tau protein and Amyloid-β peptides (Aβ 1-40 and Aβ 1-42). A loss of ribosomal population is also observed in the neurons in affected regions of AD. Our studies had demonstrated that in vitro aggregation of amyloid forming proteins, Aβ peptides and Tau protein variants (AFPs), in the vicinity of yeast 80S ribosome can induce co-aggregation of ribosomal components. Objective: In this study the ability of minute quantities of AFP-ribosome co-aggregates to seed the aggregation of a large excess of untreated 80S ribosomes was explored. Methods: The AFPs were purified using ion-exchange chromatography. Seeded aggregation of ribosomes in the presence of minute quantities of ribosome-protein co-aggregates or ribosomal components was studied using agarose gel electrophoretic and SDS-PAGE analysis of the pellets and Sucrose Density Gradient centrifugation of the supernatant obtained after centrifugation of the aggrega...

A) Bar diagram shows percent aggregation reduction during BCA II-m refolding by bDV RNA,... more A) Bar diagram shows percent aggregation reduction during BCA II-m refolding by bDV RNA, mDV RNA and bDV RNA mutants U2585C (UC), delG2252 (delG). The turbidities in each case were measured at 320 nm, 12 minutes after return to refolding conditions and turbidity in absence of chaperone was assumed as 100%. B) Comparison of reactivation yields of BCAII-m (0.3 µM) after 30 min of refolding in absence of the chaperone (Self) and in presence of bDV RNA1(R1), bDV RNA1+RNA2 (R1+R2), RNA1+3% Ethanol (R1+EtOH), del G2252 bDV RNA (delG), del G2252+ RNA2 (delG+R2), U2585C (UC), U2585C bDV RNA+RNA2 (UC+R2) and U2585C bDV RNA+3% Ethanol (UC+EtOH). C) Comparison of the reactivation yields of BCAII-m (0.9 µM) after 30 min of refolding in absence of the chaperone (Self), in presence of del G2252 bDV RNA and mDV RNA. BCAII-m reactivation upon addition of RNA 2 portion of bDV at zero minute (mDV+R2 0′and delG+R2 0′) and after ten minutes of initiation of refolding (mDV+R2 10′ and delG+R2 10′) are also shown.</p

(A) The secondary structure of the bDV RNA has been shown here. The m... more (A) The secondary structure of the bDV RNA has been shown here. The mutated nucleotides are also shown here using colour code. Nucleotides that are the binding sites of antibiotics blasticidin (b), puromycin (p), josamycin (j) and erythromycin (e) are also shown in the structure. The five specific nucleotides of bDV RNA which interacts with unfolded protein are also indicated by arrows. (B) Reactivation of BCAII in the presence of wild type (wt) and mutants E. coli bDV RNA. The reactivation of BCAII with mutated and wt bDV RNA was represented as bar diagrams. Mutated bDV RNAs are marked as M1 to M9 (M1- A2602C, M2- delA2602, M3- U2585C, M4- delU2585, M5- G2252C, M6- delG2252, M7- A2451G, M8- delA2451, M9- G2553C). BCAII reactivation in absence of bDV RNA is marked as ‘Self’ and in presence of wt bDV RNA is marked as ‘bDV RNA’. (C) Time course of binding of [α- 32P] labelled RNA1 to refolding BCAII. Unfolded BCAII was refolded in presence of [α- 32P] labelled wt RNA1 or mutant RNA1. Aliquots of samples were withdrawn at different time points from the RNA1-refolding protein mix and filtered through nitrocellulose. Percent (%) radioactivity retained (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101293#s2" target="_blank">Materials and Methods</a>) on the filter for wtRNA1 (Δ) and its mutants G2553C (□), A2602C (◊) and U2585C (○) are plotted against time. Time course of RNA2 mediated release of [α- 32P] labelled RNA1 from RNA1-BCAII complex. The [α- 32P] labelled RNA1 (wild type or mutated) was incubated with refolding BCAII for 5 min, to which RNA2 was added. Equal volume of samples were withdrawn at different time points and filtered through nitrocellulose. Percent (%) radioactive (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101293#s2" target="_blank">materials and methods</a>) retained on the filter for wtRNA1 + wtRNA2 (▪), wtRNA1 + G2252CRNA2 (▾), wtRNA1 + delG2252RNA2 (♦), U2585CRNA1 + wtRNA2 (•), G2553CRNA1 + wtRNA2 (▴) are plotted against time.</p

(A) Schematic design of template for in vitro transcription of mRNA. ... more (A) Schematic design of template for in vitro transcription of mRNA. The double stranded DNA was designed such that the ribosome binding site would position the methionine (AUG) and glutamic acid (GAA) codon at P-site and A-site respectively. These sequences are downstream of a T7 promoter sequence that was used for transcription of mRNA by T7 RNA polymerase (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101293#s2" target="_blank">Materials and Methods</a>). (B) Dose dependent inhibition of ribosome's chaperoning ability due to binding of tRNA. The E. coli ribosome programmed with mRNA was allowed to bind to increasing concentrations of Met- tRNA (▪) and total E. coli tRNA (•) in Buffer-P (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101293#pone.0101293.s003" target="_blank">Table S1</a>). Inhibition of chaperoning activity of nonprogrammed ribosome (▴) in presence of increasing concentration of Met-tRNA is also plotted here. Reactivation (%) of BCAII by ribosome-tRNA complex was plotted against its increasing tRNA concentrations. The refolding experiments were repeated at least 3 times and the average values were plotted. The graph was fitted by Boltzmann fit. (C) Effect of presence of both deacylated Met-tRNA and Glu-tRNA on chaperoning ability of programmed ribosome. Bar diagram shows BCAII reactivation by the complexes 2 to 6; programmed ribosome (70S ribosome + mRNA) (2), Glu-tRNA bound programmed ribosome (70S ribosome + mRNA +1.5 µM Glu-tRNA) (3) and Met-tRNA bound programmed ribosome (70S ribosome + mRNA +0.75 µM Met-tRNA) (4). BCAII reactivation by complex 4 upon further addition of 0.75 µM of Glu-tRNA (5) or 70S ribosome + mRNA +1.5 µM Met-tRNA (6) is also shown. (D) Bar diagram represents a comparison of the BCAII reactivation (%) of; Self (1), 70S ribosome + mRNA (2), 70S ribosome + mRNA+1.5 µM Met-tRNA (3), 70S ribosome + mRNA + tetracyclin (4), 70S ribosome + mRNA+ tetracyclin +1.5 µM Met-tRNA (5).</p

Filter binding studies. Refolding of BCAII-m or reduced-denatured lys... more Filter binding studies. Refolding of BCAII-m or reduced-denatured lysozyme was initiated in presence of radiolabeled various domain V RNA, was UV- crosslinked and filtered through nitrocellulose membrane (material and method). A) Time course of interactions of BCAII-m with radiolabeled bDV RNA (-○-), mDV RNA (-▴-), bDV RNA mutants U2585C (-▾-) and delG2252 (-▪-) are shown here. Experiments were repeated thrice and their average values were taken for final data plotting. B) Time course of interactions of reduced-denatured lysozyme and radiolabeled bDV RNA (-•-) and mDV RNA (-▴-) are shown. Size exclusion chromatography. Refolding of FITC labeled BCAII-m or reduced-denatured lysozyme was initiated in presence of 70S ribosome, was UV-crosslinked at 30 second of refolding, and the mix was loaded on Sephacryl S-300 column. The elution of the protein and the ribosome was monitored by fluorescence at 518 nm and absorbance at 260 nm. C) Detection of 70S- BCAII complex. The elution profiles of BCAII-m in presence of ribosome at 30 seconds of refolding (3), reduced denatured lysozyme in presence of ribosome at 30 seconds of refolding (5) are shown. The elution profiles of ribosome (1), native BCAII (2) and native lysozyme (4) are also shown for comparison.</p

Letters in Applied Microbiology, 2017

Letters in Applied Microbiology, 2016

The ability of the ribosome to assist in folding of proteins both in vitro and in vivo is well do... more The ability of the ribosome to assist in folding of proteins both in vitro and in vivo is well documented and is a nontranslational function of the ribosome. The interaction of the unfolded protein with the peptidyl transferase centre (PTC) of the bacterial large ribosomal subunit is followed by release of the protein in the folding competent state and rapid dissociation of ribosomal subunits. Our study demonstrates that the PTC‐specific antibiotics, chloramphenicol and blasticidin S inhibit unfolded protein‐mediated subunit dissociation. During post‐termination stage of translation in bacteria, ribosome recycling factor (RRF) is used together with elongation factor G to recycle the 30S and 50S ribosomal subunits for the next round of translation. Ribosome dissociation mediated by RRF and induced at low magnesium concentration was also inhibited by the antibiotics indicating that the PTC antibiotics exert an associative effect on ribosomal subunits. In vivo, the antibiotics can also reduce the ribosomal degradation during carbon starvation, a process requiring ribosome subunit dissociation. This study reveals a new mode of action of the broad‐spectrum antibiotics chloramphenicol and blasticidin.

FEBS Letters

Alzheimer's disease (AD) is characterized by the appearance of neurofibrillary tangles compri... more Alzheimer's disease (AD) is characterized by the appearance of neurofibrillary tangles comprising of the Tau protein and aggregation of amyloid-β peptides (Aβ 1-40 and Aβ 1-42). A concomitant loss of the ribosomal population is also observed in AD-affected neurons. Our studies demonstrate that, similarly to Tau protein aggregation, in vitro aggregation of Aβ peptides in the vicinity of the yeast 80S ribosome can induce co-aggregation of ribosomal components. The RNA-stimulated aggregation of Aβ peptides and the Tau-K18 variant is dependent on the RNA:protein stoichiometric ratio. A similar effect of stoichiometry is also observed on the ribosome-protein co-aggregation process. Polyphenolic inhibitors of amyloid aggregation, such as rosmarinic acid and myricetin, inhibit RNA-stimulated Aβ and Tau-K18 aggregation and can mitigate the co-aggregation of ribosomal components.

Ribosome hibernation is a prominent cellular strategy to modulate protein synthesis during starva... more Ribosome hibernation is a prominent cellular strategy to modulate protein synthesis during starvation and the stationary phase of bacterial cell growth. Translational suppression involves the formation of either factor‐bound inactive 70S monomers or dimeric 100S hibernating ribosomal complexes, the biological significance of which is poorly understood. Here, we demonstrate that the Escherichia coli 70S ribosome associated with stationary phase factors hibernation promoting factor or protein Y or ribosome‐associated inhibitor A and the 100S ribosome isolated from both Gram‐negative and Gram‐positive bacteria are resistant to unfolded protein‐mediated subunit dissociation and subsequent degradation by cellular ribonucleases. Considering that the increase in cellular stress is accompanied by accumulation of unfolded proteins, such resistance of hibernating ribosomes towards dissociation might contribute to their maintenance during the stationary phase. Analysis of existing structures p...

Scientific Reports

The human tau is a microtubule-associated intrinsically unstructured protein that forms intraneur... more The human tau is a microtubule-associated intrinsically unstructured protein that forms intraneuronal cytotoxic deposits in neurodegenerative diseases, like tauopathies. Recent studies indicate that in Alzheimer’s disease, ribosomal dysfunction might be a crucial event in the disease pathology. Our earlier studies had demonstrated that amorphous protein aggregation in the presence of ribosome can lead to sequestration of the ribosomal components. The present study aims at determining the effect of incubation of the full-length tau protein (Ht40) and its microtubule binding 4-repeat domain (K18) on the eukaryotic ribosome. Our in vitro studies show that incubation of Ht40 and the K18 tau variants with isolated non-translating yeast ribosome can induce a loss of ribosome physical integrity resulting in formation of tau-rRNA-ribosomal protein aggregates. Incubation with the tau protein variants also led to a disappearance of the peak indicating the ribosome profile of the HeLa cell lys...

The FEBS journal, Nov 1, 2017

The ability of the ribosome to assist in the folding of proteins both in vitro and in vivo is wel... more The ability of the ribosome to assist in the folding of proteins both in vitro and in vivo is well documented. The interaction of an unfolded protein with the peptidyltransferase center of the bacterial large ribosomal subunit is followed by release of the protein in a folding-competent state and rapid dissociation of ribosome into its subunits. Our studies demonstrate that the 50S subunit-associated antiassociation ability of an unfolded protein might contribute significantly to its ability to mediate energy-independent and stable dissociation of the ribosome into its subunits. The stoichiometry of the protein present with respect to the ribosome is an important factor in determining whether the ribosome has a chaperoning effect on protein folding or if the protein acts as a 50S subunit antiassociation factor. Sustained interaction of the protein with the ribosome at higher protein concentrations and the hindrance in the formation of the central intersubunit bridge B2a could underl...

Scientific reports, Jan 7, 2017

An understanding of the mechanisms underlying protein aggregation and cytotoxicity of the protein... more An understanding of the mechanisms underlying protein aggregation and cytotoxicity of the protein aggregates is crucial in the prevention of several diseases in humans. Ribosome, the cellular protein synthesis machine is capable of acting as a protein folding modulator. The peptidyltransferase center residing in the domain V of large ribosomal subunit 23S rRNA is the centre for the protein folding ability of the ribosome and is also the cellular target of several antiprion compounds. Our in vitro studies unexpectedly reveal that the partial unfolding or aggregation of lysozyme under reducing conditions in presence of the ribosome can induce aggregation of ribosomal components. Electrostatic interactions complemented by specific rRNA-protein interaction drive the ribosome-protein aggregation process. Under similar conditions the rRNA, especially the large subunit rRNA and in vitro transcribed RNA corresponding to domain V of 23S rRNA (bDV RNA) stimulates lysozyme aggregation leading ...

PLoS ONE, 2014

Background: The ribosome, which acts as a platform for mRNA encoded polypeptide synthesis, is als... more Background: The ribosome, which acts as a platform for mRNA encoded polypeptide synthesis, is also capable of assisting in folding of polypeptide chains. The peptidyl transferase center (PTC) that catalyzes peptide bond formation resides in the domain V of the 23S rRNA of the bacterial ribosome. Proper positioning of the 39-CCA ends of the A-and P-site tRNAs via specific interactions with the nucleotides of the PTC are crucial for peptidyl transferase activity. This RNA domain is also the center for ribosomal chaperoning activity. The unfolded polypeptide chains interact with the specific nucleotides of the PTC and are released in a folding competent form. In vitro transcribed RNA corresponding to this domain (bDV RNA) also displays chaperoning activity. Results: The present study explores the effects of tRNAs, antibiotics that are A-and P-site PTC substrate analogs (puromycin and blasticidin) and macrolide antibiotics (erythromycin and josamycin) on the chaperoning ability of the E. coli ribosome and bDV RNA. Our studies using mRNA programmed ribosomes show that a tRNA positioned at the P-site effectively inhibits the ribosome's chaperoning function. We also show that the antibiotic blasticidin (that mimics the interaction between 39-CCA end of P/P-site tRNA with the PTC) is more effective in inhibiting ribosome and bDV RNA chaperoning ability than either puromycin or the macrolide antibiotics. Mutational studies of the bDV RNA could identify the nucleotides U2585 and G2252 (both of which interact with P-site tRNA) to be important for its chaperoning ability. Conclusion: Both protein synthesis and their proper folding are crucial for maintenance of a functional cellular proteome. The PTC of the ribosome is attributed with both these abilities. The silencing of the chaperoning ability of the ribosome in the presence of P-site bound tRNA might be a way to segregate these two important functions.