Biology subjects

Chakraborty, D.

Publications and source records attributed to Chakraborty, D..

47 records · Page 3Linked to original sources

bioRxiv2021

On the emergence of orientational order in folded proteins with implications for allostery

The beautiful structures of single and multi-domain proteins are clearly ordered in some fashion but cannot be readily classified using group theory methods that are successfully used to describe periodic crystals. For this reason, protein structures are considered to be aperiodic, and may have evolved this way for functional purposes, especially in instances that require a combination of softness and rigidity within the same molecule. By analyzing the solved protein structures, we show that orientational symmetry is broken in the aperiodic arrangement of the secondary structural elements (SSEs), which we deduce by calculating the nematic order parameter, P2. We find that the folded structures are nematic droplets with a broad distribution of P2. We argue that non-zero values of P2, leads to an arrangement of the SSEs that can resist external stresses forces, which is a requirement for allosteric proteins. Such proteins, which resist mechanical forces in some regions while being flexible in others, transmit signals from one region of the protein to another (action at a distance) in response to binding of ligands (oxygen, ATP or other small molecules).

biophysics↗

bioRxiv2020

Inferring within-flock transmission dynamics of highly pathogenic avian influenza (H5N8) in France, 2020

Following the emergence of highly pathogenic avian influenza (H5N8) in France in early December 2020, we used duck mortality data of the index case to investigate within-flock transmission dynamics. A stochastic epidemic model was adjusted to the daily mortality data and model parameters were estimated using an approximate Bayesian computation sequential Monte Carlo (ABC-SMC) algorithm. Results suggested that the virus was introduced 4 days (95% credible interval: 3 - 5) prior to the day suspicion was reported and that the transmission rate was 3.7 day-1 (95%CI: 2.6 - 5.3). On average, ducks started being infectious 3.1 hours (95%CI: 0.4 - 8.0) after infection and remained infectious for 4.4 days (95%CI: 3.1 - 5.6). Model outputs also suggested that the number of infectious ducks was already 3239 (95%CI: 26 - 3706) the day before suspicion, emphasising the substantial latent threat this virus could pose to other poultry farms and to neighbouring wildlife. These estimations can be applied to upcoming outbreaks and made available to veterinary services within few hours. This study illustrates how mechanistic models can provide rapid relevant insights to contribute to the management of infectious disease outbreaks of farmed animals.

ecology↗

bioRxiv2020

Rapid, field-deployable nucleobase detection and identification using FnCas9

Detection of pathogenic sequences or variants in DNA and RNA through a point-of-care diagnostic approach is valuable for rapid clinical prognosis. In recent times, CRISPR based detection of nucleic acids has provided an economical and quicker alternative to sequencing-based platforms which are often difficult to implement in the field. Here, we present FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that employs a highly accurate enzymatic readout for detecting nucleotide sequences, identifying nucleobase identity and inferring zygosity with precision. We demonstrate that FELUDA output can be adapted to multiple signal detection platforms and can be quickly designed and deployed for versatile applications including rapid diagnosis during infectious disease outbreaks like COVID-19.

molecular biology↗

bioRxiv2020

Differences in the free energies between the excited states of Aβ40 and Aβ42 monomers encode their distinct aggregation propensities

The early events in the aggregation of the intrinsically disordered peptide, A{beta}, involve transitions from the disordered lowest free energy ground state to assembly-competent states. Are the finger-prints of order found in the amyloid fibrils encoded in the conformations that the monomers access at equilibrium? If so, could the enhanced aggregation rate of A{beta}42 compared to A{beta}40 be rationalized from the sparsely populated high free energy states of the monomers? Here, we answer these questions in the affirmative using coarse-grained simulations of the SOP-IDP model of A{beta}40 and A{beta}42. Although both the peptides have practically identical ensemble-averaged properties, characteristic of random coils (RCs), the conformational ensembles of the two monomers exhibit sequence-specific heterogeneity. Hierarchical clustering of conformations reveals that both the peptides populate high free energy aggregation-prone (N*) states, which resemble the monomers in the fibril structure. The free energy gap between the ground (RC) and the N* states of A{beta}42 peptide is smaller than for A{beta}40. By relating the populations of excited states of the two peptides to the fibril formation time scales using an empirical formula, we explain nearly quantitatively the faster aggregation rate of A{beta}42 relative to A{beta}40. The N* concept accounts for fibril polymorphs, leading to the prediction that the less stable N* state of A{beta}42, encoding for the U-bend fibril, should form earlier than the structure with the S-bend topology, which is in accord with the Ostwalds rule rationalizing crystal polymorph formation. Significance StatementAlzheimers disease (AD), a rampant neurodegenerative disorder, is caused by the accumulation of pathological aggregates, primarily composed of the two isoforms A{beta}40 and A{beta}42. Experiments have shown that A{beta}42 is more aggregation-prone compared to A{beta}40. However, the molecular origin of this apparent anomaly remains elusive. Here, we provide a microscopic basis for the different aggregation rates in terms of the distinct populations of high free energy excited fibril-like states (N*) that are encoded in the monomer spectra. The N* theory explains the emergence of fibril polymorphs, and predicts the relative kinetic stabilities of A{beta}42 fibrils using Ostwalds rule of stages. Our work shows that sequence-specific conformational heterogeneity of the monomer ensembles provides important cues for understanding protein aggregation.

biophysics↗

bioRxiv2019

Pharmacological and fasting-induced activation of SIRT1/LXRα signaling alleviates diabetes-induced retinopathy.

In diabetes, the retina, a tissue with unique metabolic needs, demonstrates dysregulation of the intricate balance between nutrient availability and utilization. This results in cholesterol accumulation, pro-inflammatory and pro-apoptotic changes, and consequently neurovascular damage. Sirtuin 1 (SIRT1), a nutrient sensing deacetylase, is downregulated in the diabetic retina. In this study, the effect of SIRT1 stimulation by fasting or by pharmacological activation using SRT1720, was evaluated on retinal cholesterol metabolism, inflammation and neurovascular damage. SIRT1 activation, in retinal endothelial cells (REC) and neuronal retinal progenitor cells (R28), led to Liver X Receptor alpha (LXR) deacetylation and subsequent increased activity, as measured by increased ATP-binding cassette transporter (ABC) A1 and G1 mRNA expression. In turn, increased cholesterol export resulted in decreased REC cholesterol levels. SIRT1 activation also led to decreased inflammation. SIRT1 activation, in vivo, prevented diabetes-induced inflammation and vascular and neural degeneration. Diabetes-induced visual function impairment, as measured by electroretinogram and optokinetic response, was significantly improved as a result of SIRT1 activation. Taken together, activation of SIRT1 signaling is an effective therapeutic strategy that provides a mechanistic link between the advantageous effects associated with fasting regimes and prevention of diabetic retinopathy (DR).

cell biology↗

bioRxiv2019

TUTase mediated site-directed access to clickable chromatin employing CRISPR-dCas9

Locus-specific interrogation of the genome using programmable CRISPR-based technologies is tremendously useful in dissecting the molecular basis of target gene function and modulating its downstream output. Although these tools are widely utilized in recruiting genetically encoded functional proteins, display of small molecules using this technique is not well developed due to inadequate labeling technologies. Here, we report the development of a modular technology, sgRNA-Click (sgR-CLK), which harnesses the power of bioorthogonal click chemistry for remodeling CRISPR to display synthetic molecules on target genes. A terminal uridylyl transferase (TUTase) was repurposed to construct an sgRNA containing multiple minimally invasive bioorthogonal clickable handles, which served as a Trojan horse on CRISPR-dCas9 system to guide synthetic tags site-specifically on chromatin employing copper-catalyzed or strain-promoted click reactions. Our results demonstrate that sgR-CLK could provide a simplified solution for site-directed display of small molecules to study as well as modulate the function of gene targets.

biochemistry↗

bioRxiv2019

Land Use Change Increases Wildlife Parasite Diversity in Anamalai Hills, Western Ghats, India

Anthropogenic landscape change such as land use change and habitat fragmentation are known to alter wildlife diversity. Since host and parasite diversities are strongly connected, landscape changes are also likely to change wildlife parasite diversity with implication for wildlife health. However, research linking anthropogenic landscape change and wildlife parasite diversity is limited, especially comparing effects of land use change and habitat fragmentation, which often cooccur but may affect parasite diversity substantially differently. Here, we assessed how anthropogenic land use change (presence of plantation, livestock foraging and human settlement) and habitat fragmentation may change the gastrointestinal parasite diversity of wild mammalian host species (n=23) in Anamalai hills, India. We found that presence of plantations, and potentially livestock, significantly increased parasite diversity due possibly to spillover of parasites from livestock to wildlife. However, effect of habitat fragmentation on parasite diversity was not significant. Together, our results showed how human activities may increase wildlife parasite diversity within human-dominated landscape and highlighted the complex pattern of parasite diversity distribution as a result of cooccurrence of multiple anthropogenic landscape changes.

ecology↗

bioRxiv2019

Francisella novicida Cas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing

Genome editing using the CRISPR Cas9 system has been used to manipulate eukaryotic DNA and make precise heritable changes. Although the widely used Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene-editing applications across different platforms, concerns remain, regarding their putative off targeting at multiple loci across the genome. Here we report that Francisella novicida Cas9 (FnCas9) shows a very high specificity of binding to its intended targets and negligible binding to off-target loci. The specificity is determined by its minimal binding affinity with DNA when mismatches to the target sgRNA are present in the sgRNA:DNA heteroduplex. FnCas9 produces staggered cleavage, higher HDR rates and very low non-specific genome editing compared to SpCas9. We demonstrate FnCas9 mediated correction of the sickle cell mutation in patient derived iPSCs and propose that it can be used for precise therapeutic genome editing for a wide variety of genetic disorders.\n\nSIGNIFICANCE STATEMENTTherapeutic genome editing has been significantly accentuated by the advent of CRISPR based gene correction. However, genomic off-targeting has been a major setback for clinical translation. Although high fidelity versions of Cas9 have been rationally designed, they recognize and bind to off-targets. In this study, we characterize a naturally occurring Cas9 from Francisella novicida (FnCas9) that shows negligible binding affinity to off targets differing by one or more mismatches, rendering it highly specific in target recognition and editing. We show that FnCas9 can direct both HDR and NHEJ mediated DNA repair, generates higher rate of HDR and negligible off-target editing. Finally we show its potential in therapeutic genome editing by correcting the sickle cell anemia mutation in patient derived iPSCs.

molecular biology↗

bioRxiv2019

Enhanced autophagic-lysosomal activity and increased BAG3-mediated selective macroautophagy as adaptive response of neuronal cells to chronic oxidative stress

Oxidative stress and a disturbed cellular protein homeostasis (proteostasis) belong to the most important hallmarks of aging and of neurodegenerative disorders. The proteasomal and autophagic-lysosomal degradation pathways are key measures to maintain proteostasis. Here, we report that hippocampal cells selected for full adaptation and resistance to oxidative stress induced by hydrogen peroxide (oxidative stress-resistant cells, OxSR cells) showed a massive increase in the expression of components of the cellular autophagic-lysosomal network and a significantly higher overall autophagic activity. A comparative expression analysis revealed that distinct key regulators of autophagy are upregulated in OxSR cells. The observed adaptive autophagic response was found to be independent of the upstream autophagy regulator mTOR but is accompanied by a significant upregulation of further downstream components of the canonical autophagy network such as Beclin1, WIPI1 and the transmembrane ATG9 proteins. Interestingly, the expression of the HSP70 co-chaperone BAG3, mediator of BAG3-mediated selective macroautophagy and highly relevant for the clearance of aggregated proteins in cells was found to be increased in OxSR cells that were consequently able to effectively overcome proteotoxic stress. Overexpression of BAG3 in oxidative stress-sensitive HT22 wildtype cells partly established the vesicular phenotype and the enhanced autophagic flux seen in OxSR cells suggesting that BAG3 takes over a key part in the adaptation process. A full proteome analysis demonstrated additional changes in the expression of mitochondrial proteins, metabolic enzymes and different pathway regulators in OxSR cells as consequence of the adaptation to oxidative stress in addition to autophagy-related proteins. Taken together, this analysis revealed a wide variety of pathways and players that act as adaptive response to chronic redox stress in neuronal cells.

cell biology↗

bioRxiv2018

Sequence effects on size, shape, and structural heterogeneity in Intrinsically Disordered Proteins

Intrinsically disordered proteins (IDPs) lack well-defined three-dimensional structures, thus challenging the archetypal notion of structure-function relationships. Determining the ensemble of conformations that IDPs explore under physiological conditions is the first step towards understanding their diverse cellular functions. Here, we quantitatively characterize the structural features of IDPs as a function of sequence and length using coarse-grained simulations. For diverse IDP sequences, with the number of residues (NT) ranging from 24 to 441, our simulations not only reproduce the radii of gyration (Rg) obtained from experiments, but also predict the full scattering intensity profiles in very good agreement with Small Angle X-ray Scattering experiments. The Rg values are well-described by the standard Flory scaling law, [Formula], with v {approx} 0.588, making it tempting to assert that IDPs behave as polymers in a good solvent. However, clustering analysis reveals that the menagerie of structures explored by IDPs is diverse, with the extent of heterogeneity being highly sequence-dependent, even though ensemble-averaged properties, such as the dependence of Rg on chain length, may suggest synthetic polymer-like behavior in a good solvent. For example, we show that for the highly charged Prothymosin-, a substantial fraction of conformations is highly compact. Even if the sequence compositions are similar, as is the case for -Synuclein and a truncated construct from the Tau protein, there are substantial differences in the conformational heterogeneity. Taken together, these observations imply that metrics based on net charge or related quantities alone, cannot be used to anticipate the phases of IDPs, either in isolation or in complex with partner IDPs or RNA. Our work sets the stage for probing the interactions of IDPs with each other, with folded protein domains, or with partner RNAs, which are critical for describing the structures of stress granules and biomolecular condensates with important cellular functions. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/427476v2_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@1b50bd9org.highwire.dtl.DTLVardef@11cf542org.highwire.dtl.DTLVardef@29ebb5org.highwire.dtl.DTLVardef@31e965_HPS_FORMAT_FIGEXP M_FIG Graphical TOC Entry C_FIG

biophysics↗

bioRxiv2018

Sequence-dependent Three Interaction Site (TIS) Model for Single and Double-stranded DNA

We develop a robust coarse-grained model for single and double stranded DNA by representing each nucleotide by three interaction sites (TIS) located at the centers of mass of sugar, phosphate, and base. The resulting TIS model includes base-stacking, hydrogen bond, and electrostatic interactions as well as bond-stretching and bond angle potentials that account for the polymeric nature of DNA. The choices of force constants for stretching and the bending potentials were guided by a Boltzmann inversion procedure using a large representative set of DNA structures extracted from the Protein Data Bank. Some of the parameters in the stacking interactions were calculated using a learning procedure, which ensured that the experimentally measured melting temperatures of dimers are faithfully reproduced. Without any further adjustments, the calculations based on the TIS model reproduces the experimentally measured salt and sequence dependence of the size of single stranded DNA (ssDNA), as well as the persistence lengths of poly(dA) and poly(dT) chains. Interestingly, upon application of mechanical force the extension of poly(dA) exhibits a plateau, which we trace to the formation of stacked helical domains. In contrast, the force-extension curve (FEC) of poly(dT) is entropic in origin, and could be described by a standard polymer model. We also show that the persistence length of double stranded DNA, formed from two complementary ssDNAs with one hundred and thirty base pairs, is consistent with the prediction based on the worm-like chain. The persistence length, which decreases with increasing salt concentration, is in accord with the Odijk-Skolnick-Fixman theory intended for stiff polyelectrolyte chains near the rod limit. The range of applications, which did not require adjusting any parameter after the initial construction based solely on PDB structures and melting profiles of dimers, attests to the transferability and robustness of the TIS model for ssDNA and dsDNA.

biophysics↗