Publications by authors named "Abhijit Chakrabarti"

68 Publications

Phospholipid Asymmetry in Biological Membranes: Is the Role of Phosphatidylethanolamine Underappreciated?

J Membr Biol 2021 Apr 19;254(2):127-132. Epub 2021 Jan 19.

Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, HBNI, 1/AF Bidhannagar, Kolkata, 700064, India.

The asymmetric distribution of phospholipids in cell membranes has been the focus of a lot of important research keeping its biological importance in mind. Most of this research is focused on phosphatidylserine (PS) since it is an apoptotic marker, and there is a robust and easy method available its selective quantification. The aim of this commentary is to argue in favour of another highly abundant membrane lipid, phosphatidylethanolamine (PE) almost always associated with PS. PE has one of the smallest headgroups and shows distinctly asymmetric transbilayer distribution. It is a neutral aminophospholipid and capable of a vastly wider range of interactions as seen in its unique ability to act as a molecular chaperone, implicated role in disease biology and its possible role as an anti-cancer target. There are ample evidences to the fact that PE may also bind to Annexin V (ANV), the PS-specific probe, at higher than 10 mol% PE concentrations and absence of Ca ions. An update of the major takeaways from the literature regarding PE asymmetry is also provided.
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http://dx.doi.org/10.1007/s00232-020-00163-wDOI Listing
April 2021

Effects of free soluble iron on thermal aggregation of hemoglobin.

Biophys Chem 2021 Feb 16;269:106527. Epub 2020 Dec 16.

Crystallography & Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata 700064, India; Homi Bhabha National Institute, Mumbai 400094, India. Electronic address:

Aggregation of hemoglobin is implicated in the presentation of diseases like sickle cell disease and thalassemia. Hallmark of the disease being imbalance in the production of globin chains leading to aggregation of excess globin chains and aberrant hemoglobins associated with the disease, broadly categorized as hemoglobinopathy. We have studied thermal aggregation of hemoglobin at 70 °C and pH 6.5 using light scattering, flow cytometry and optical microscopy and tried to investigate effects of few abundant soluble metal ions on such aggregation. Our study indicate that only iron, both in Fe and Fe forms, could inhibit hemoglobin aggregation and the extent of inhibition was 60% in presence of 100 mgL FeCl. Similar effect was not seen in lysozyme aggregation. Metal ions such as, Cu, Zn and Ni also did not have any significant effects on hemoglobin aggregation. Results show this important chaperone like behavior of free iron affecting the kinetics and yield of the aggregation process which could have important consequence in the extent of severity of such hematological diseases.
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http://dx.doi.org/10.1016/j.bpc.2020.106527DOI Listing
February 2021

Multiple Functions of Spectrin: Convergent Effects.

J Membr Biol 2020 12 29;253(6):499-508. Epub 2020 Sep 29.

Crystallography & Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata, 700064, India.

Spectrin is a multifunctional, multi-domain protein most well known in the membrane skeleton of mature human erythrocytes. Here we review the literature on the crosstalk of the chaperone activity of spectrin with its other functionalities. We hypothesize that the chaperone activity is derived from the surface exposed hydrophobic patches present in individual "spectrin-repeat" domains and show a competition between the membrane phospholipid binding functionality and chaperone activity of spectrin. Moreover, we show that post-translational modifications such as glycation which shield these surface exposed hydrophobic patches, reduce the chaperone function. On the other hand, oligomerization which is linked to increase of hydrophobicity is seen to increase it. We note that spectrin seems to prefer haemoglobin as its chaperone client, binding with it preferentially over other denatured proteins. Spectrin is also known to interact with unstable haemoglobin variants with a higher affinity than in the case of normal haemoglobin. We propose that chaperone activity of spectrin could be important in the cellular biochemistry of haemoglobin, particularly in the context of diseases.
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http://dx.doi.org/10.1007/s00232-020-00142-1DOI Listing
December 2020

Chaperone potential of erythroid spectrin: Effects of hemoglobin interaction, macromolecular crowders, phosphorylation and glycation.

Biochim Biophys Acta Proteins Proteom 2019 11 27;1867(11):140267. Epub 2019 Aug 27.

Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064, India; Homi Bhabha National Institute, Mumbai 400094, India. Electronic address:

Spectrin, the major protein component of the erythrocyte membrane skeleton has chaperone like activity and is known to bind membrane phospholipids and hemoglobin. We have probed the chaperone activity of spectrin in presence of hemoglobin and phospholipid SUVs of different compositions to elucidate the effect of phospholipid/hemoglobin binding on chaperone function. It is seen that spectrin displays a preference for hemoglobin over other substrates leading to a decrease in chaperone activity in presence of hemoglobin. A competition is seen to exist between phospholipid binding and chaperone function of spectrin, in a dose dependent manner with the greatest extent of decrease being seen in case of phospholipid vesicles containing aminophospholipids e.g. PS and PE which may have implications in diseases like hereditary spherocytosis where mutation in spectrin is implicated in its detachment from cell membrane. To gain a clearer understanding of the chaperone like activity of spectrin under in-vivo like conditions we have investigated the effect of macromolecular crowders as well as phosphorylation and glycation states on chaperone activity. It is seen that the presence of non-specific, protein and non-protein macromolecular crowders do not appreciably affect chaperone function. Phosphorylation also does not affect the chaperone function unlike glycation which progressively diminishes chaperone activity. We propose a model where chaperone clients adsorb onto spectrin's surface and processes that bind to and occlude these surfaces decrease chaperone activity.
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http://dx.doi.org/10.1016/j.bbapap.2019.140267DOI Listing
November 2019

Localizing the chaperone activity of erythroid spectrin.

Cytoskeleton (Hoboken) 2019 06 19;76(6):383-397. Epub 2019 Aug 19.

Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata, India.

Spectrin, the major protein of the erythrocyte membrane skeleton has canonically been thought to only serve a structural function. We have previously described a novel chaperone-like property of spectrin and also hypothesized that the chaperone activity and binding of a hydrophobic ligand, Prodan are localized in the self-association domain. Here we probe the location and molecular origin of the chaperone activity of multi-domain spectrin using a selection of individual recombinant spectrin domains, which we have characterized using intrinsic tryptophan fluorescence and CD spectroscopy to show their identity to native spectrin. Aggregation assays using insulin, ADH, α- and β-globin as well as enzyme refolding assays using alkaline phosphatase and α-glucosidase show that the chaperone activity is not only localized in the self-association domain but is a generalized property of spectrin domains. This is to our understanding, a unique feature in the case of modular multi-repeat proteins, possibly implicating that the large family of "spectrin-repeat" domain containing proteins may also have chaperone like property. Substrate selectivity of chaperone activity as evidenced by the preferential protection of α- over β-globin chains is seen; which has implications in hemoglobin diseases. Moreover, enzyme-refolding assays also indicate alternate modes of chaperone action. We propose that the molecular origin of chaperone activity resides in the surface exposed hydrophobic patches of the spectrin domains as shown by ANS (1-anilinonaphthalene-8-sulfonic acid) and Prodan (6-propionyl-2[dimethylamino]-naphthalene) binding. We also show that Prodan does indeed have a unique binding site on spectrin located at the self-association domain.
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http://dx.doi.org/10.1002/cm.21556DOI Listing
June 2019

Status of Membrane Asymmetry in Erythrocytes: Role of Spectrin.

Adv Exp Med Biol 2018;1112:3-11

Crystallography & Molecular Biology Division, Saha Institute of Nuclear Physics, HBNI, Kolkata, India.

Spectrin-based proteinaceous membrane skeletal network has been found to be implicated in membrane disorders like hereditary spherocytosis (HS). HS greatly affects eryptosis via loss of membrane asymmetry which is seen to be the case in haemoglobin disorders like thalassemia and sickle cell disease as well. The biological implications of the status of membrane asymmetry are strongly correlated to spectrin interactions with aminophospholipids, e.g. PE and PS. Fluorescence and X-ray reflectivity (XRR) measurements of spectrin interactions with small unilamellar vesicles (SUVs) and cushioned bilayers of phospholipids, respectively, were studied. Both the XRR and fluorescence measurements led to the characterization of spectrin orientation on the surface of lipid bilayer of phosphatidylcholine (PC) and PC/aminophospholipid mixed membrane systems showing formation of a uniform layer of spectrin on top of the mixed phospholipid bilayer. Fluorescence studies show that spectrin interacts with PC and phosphatidylethanolamine (PE)/phosphatidylserine (PS) membranes with binding dissociation constants (K) in the nanomolar range indicating the role of spectrin in the maintenance of the overall membrane asymmetry of erythrocytes.
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http://dx.doi.org/10.1007/978-981-13-3065-0_1DOI Listing
July 2019

Structure-activity relationship of heme and its analogues in membrane damage and inhibition of fusion.

FEBS Lett 2018 07 10;592(14):2458-2465. Epub 2018 Jul 10.

Crystallography& Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata, India.

Under pathological conditions, such as sickle cell disease and malaria, heme concentration increases considerably, and it induces membrane damage. As sickled and normal erythrocytes contain high cholesterol: phospholipid ratio, we investigated the role of lipid composition, chain length, and unsaturation on the partitioning and leakage of hemin in phospholipid vesicles. To establish structure-activity relationship in membrane damage, experiments with two other analogues, protoporphyrin-IX and hematoporphyrin (HP) were also carried out. Hemin and its analogues localize differently in membranes and exhibit distinct roles in partitioning, leakage and fusion. Hemin and HP trigger more leakage in the presence of aminophospholipids, whereas cholesterol buffers the destabilizing effect remarkably. Inhibition of fusion by hemin further suggests its unexplored and important role in membrane trafficking, particularly under diseased conditions.
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http://dx.doi.org/10.1002/1873-3468.13165DOI Listing
July 2018

Effects of GM1 on brain spectrin-aminophospholipid interactions.

Biochim Biophys Acta Biomembr 2019 01 18;1861(1):298-305. Epub 2018 Jun 18.

Crystallography & Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF, Bidhannagar, Kolkata 700064, India; Homi Bhabha National Institute, Mumbai 400094, India. Electronic address:

Spectrin, a major component of the membrane skeletal meshwork of metazoan cells, is implicated to associate with membrane domains and is known to act as a scaffold for stabilization and activation of different signalling modules. We have studied the effect of GM1 (monosialotetrahexosyl ganglioside), a well-known model ganglioside and a signalling moiety, on the interaction of non-erythroid brain spectrin with both saturated and unsaturated aminophospholipids by spectroscopic methods. We observe that GM1 modulates brain spectrin-aminophospholipid interaction to the greatest degree whereas its effect on erythroid spectrin is not as pronounced. Fluorescence quenching studies show that brain spectrin interacts with DMPC/DMPE-based vesicles with a 10-fold increased affinity in presence of very low amounts of 2% and 5% GM1, and the extent of quenching decreases progressively in presence of increasing amounts of GM1. Interaction of brain spectrin with unsaturated membrane systems of DOPC/DOPE weakens in presence GM1. Increase in the mean lifetime of the Trp residues of brain spectrin in presence of GM1 indicates change in the microenvironment of spectrin, without affecting the secondary structure of the protein significantly. Studies on pressure - area isotherm of Langmuir-Blodgett monolayer and Brewster's angle microscopy show that GM1 has an expanding effect on the aminophospholipid monolayers, and ordered regions in DMPC/DMPE mixed monolayers are formed and are stabilized at higher pressure. GM1-induced fluidization of the phospholipid membranes and probable physical contact between bulky sugar head group of GM1 and spectrin, may explain the modulatory role of GM1 on aminophospholipid interactions with nonerythroid brain spectrin.
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http://dx.doi.org/10.1016/j.bbamem.2018.06.011DOI Listing
January 2019

Flow Cytometric Analysis of Protein Aggregates.

Protein Pept Lett 2017 ;24(10):969-973

Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, HBNI, Kolkata. India.

Background: Misfolding of proteins often leads to aggregation. Accumulation of diverse protein aggregates in various cells, tissue and organs is the hallmark of many diseases, such as Alzheimer's disease and Parkinson's disease.

Objectives: The main objective of this study was to present a novel method of characterization of protein aggregates, associated with differential toxicity with different size and composition in vitro using flow cytometry.

Methods: A Beckman Coulter Epics XL flow cytometer with argon ion laser operating at 488 nm was used for flow cytometry analysis. The voltage and the gain settings for individual channels were set at high voltage and gain for the detections of autofluorescence, fluorescence of adsorbed Congo red, forward scattering (FSC) and side scattering (SSC) intensities from the aggregates of proteins and nanoparticles. Each sample was analyzed to characterize and quantify the number of aggregates with a limit of maximum 20,000 events. The flow cytometry data were analyzed using Flowing software version 2.5.1 and Origin 8.0.

Results: Autofluorescence and scattering intensities could distinguish between amyloid and nonamyloid aggregates. Dot plots of both side scattering (SSC) and forward scattering (FSC) intensities also showed characteristic fingerprint of both the types of aggregates when compared with those of well known nanoparticles of oxides of Fe and Cu.

Conclusion: This work reports a novel, simple and robust flow cytometric method of characterization of protein aggregates of different size and composition which would find wider application in characterization of biomolecular aggregates, in general.
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http://dx.doi.org/10.2174/0929866524666170818155030DOI Listing
January 2019

Protein-Protein Interaction Probed by Label-free Second Harmonic Light Scattering: Hemoglobin Adsorption on Spectrin Surface as a Case Study.

J Phys Chem B 2017 08 11;121(33):7797-7802. Epub 2017 Aug 11.

Department of Inorganic and Physical Chemistry, Indian Institute of Science , Bangalore 560 012, India.

In this article, we have studied the binding of different naturally occurring hemoglobin (Hb) variants on erythrocyte skeletal protein, spectrin surface using the label free nondestructive second harmonic light scattering (SHLS) technique in aqueous buffer. Hemoglobin variants like sickle hemoglobin (HbS) and hemoglobin E (HbE) were chosen as they associate with sickle cell disease and HbEβ-thalassemia, respectively, and their interaction with spectrin is compared with normal adult hemoglobin (HbA). The concentration dependent change in the second harmonic light intensity from nanomolar spectrin solution has been measured after addition of small aliquots of hemoglobins. From the second harmonic titration data, the binding constant is calculated using a modified Langmuir adsorption model of hemoglobin binding to the spectrin surface. Interestingly, it is found that the binding constant for HbE (13.8 × 10 M) is 1 order of magnitude higher than that of HbS (1.6 × 10 M) or HbA (2.1 × 10 M) which indicates higher affinity of HbE for spectrin compared to HbA and HbS. The number of the Hb molecules bound to the spectrin surface was estimated to be of the order of hundred's which is determined for the first time.
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http://dx.doi.org/10.1021/acs.jpcb.7b04503DOI Listing
August 2017

Substrate specificity in the context of molecular chaperones.

IUBMB Life 2017 09 27;69(9):647-659. Epub 2017 Jul 27.

Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, HBNI, Kolkata, India.

Molecular chaperones are one of the key players in protein biology and as such their structure and mechanism of action have been extensively studied. However the substrate specificity of molecular chaperones has not been well investigated. This review aims to summarize what is known about the substrate specificity and substrate recognition motifs of chaperones so as to better understand what substrate specificity means in the context of molecular chaperones. Available literature shows that the majority of chaperones have broad substrate range and recognize non-native conformations of proteins depending on recognition of hydrophobic and/or charged patches. Based on these recognition motifs chaperones can select for early, mid or late folding intermediates. Another major contributor to chaperone specificity are the co-chaperones they interact with as well as the sub-cellular location they are expressed in and the inducability of their expression. Some chaperones which have only one or a few known substrates are reported. In their case the mode of recognition seems to be specific structural complementarity between chaperone and substrate. It can be concluded that the vast majority of chaperones do not show a high degree of specificity but recognize elements that signal non-native protein conformation and their substrate range is modulated by the context they function in. However a few chaperones are known that display exquisite specificity of their substrate e.g. mammalian heat shock protein 47 collagen interaction. © 2017 IUBMB Life, 69(9):647-659, 2017.
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http://dx.doi.org/10.1002/iub.1656DOI Listing
September 2017

Enzyme Adsorption on Nanoparticle Surface Probed by Highly Sensitive Second Harmonic Light Scattering.

Methods Enzymol 2017 3;590:33-58. Epub 2017 Apr 3.

Indian Institute of Science, Bangalore, India. Electronic address:

Recent developments in second harmonic light scattering technique and the associated theoretical models have provided a deeper insight of molecular interactions on micro- and nanoparticle surfaces. This technique is extended to probe the thermodynamics of protein adsorption on nanoparticle surface which is crucial for understanding the fate of nanoparticle-based formulations in biomedical applications. A modified Langmuir adsorption model has been applied to extract the thermodynamic parameters from the experimental data. The general applicability of the technique is established by extracting free energy change, association constant, and binding stoichiometry of adsorption of a moderate size protein, alcohol dehydrogenase, and a small size protein, insulin, on gold nanoparticles. The free energy change for the adsorption is found to be of the order of -55kJ/mol, which indicates that the interaction of proteins with the nanoparticle surface involves weak forces. On the other hand, the low value of the free energy change makes the detachment of the protein from the particle surface easier and guarantees reversibility of the binding process. In addition, one gets the binding stoichiometry of the proteins with the nanoparticle surface which opens up the possibility of controlling the payload of the protein- or peptide-based therapeutics in future biomedical applications.
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http://dx.doi.org/10.1016/bs.mie.2017.01.020DOI Listing
August 2017

Cholesterol-Induced Structural Changes in Saturated Phospholipid Model Membranes Revealed through X-ray Scattering Technique.

J Phys Chem B 2017 04 17;121(16):4081-4090. Epub 2017 Apr 17.

Saha Institute of Nuclear Physics, HBNI , 1/AF, Bidhannagar, Kolkata 700064, India.

Lateral and out-of-plane organization of cholesterol and its effect on regulating the physicochemical properties of zwitterionic phospholipid model membranes have been investigated by a pressure-area isotherm study from the Langmuir monolayer, atomic force microscopy (AFM), and X-ray reflectivity (XRR) measurements from supported binary monolayer films. The systematic isotherm studies on the Langmuir monolayer of phospholipids and the subsequent extraction of excess Gibbs free energy (ΔG) revealed the mechanism of cholesterol interaction and the molecular cooperativeness for different arrangements in the phospholipid model membranes. We have found a critical cholesterol molar concentration (χ) up to which the lipid-cholesterol miscibility gradually increases and then further increase in the concentration leads to an inhomogeneous structure formation similar to raft structures. The thickening in the lipid acyl chain and the subsequent lowering of the lipid head group thickness up to χ are also evident from the XRR study. Beyond χ, large-sized domains are observed in the AFM images from the deposited monolayer. χ has also been observed to depend on the phase of the monolayer, in particular, ∼25 molar % in the gel phase and ∼40 molar % in the fluid phase, wherein a regular distribution has been found with the highest separation between the cholesterol molecules. The extracted isothermal compressibility coefficient (C) and ΔG from the monolayer isotherms indicate that the molecular arrangement at χ are the most stable configurations of the monolayer. Our study provides direct evidence into cholesterol-induced evolution in phase behavior and the consequent model on the structure at different phases in the phospholipid Langmuir monolayers.
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http://dx.doi.org/10.1021/acs.jpcb.6b12587DOI Listing
April 2017

Effect of pH on stability, conformation, and chaperone activity of erythroid & non-erythroid spectrin.

Biochim Biophys Acta Proteins Proteom 2017 Jun 2;1865(6):694-702. Epub 2017 Apr 2.

Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, HBNI, Kolkata, India. Electronic address:

Spectrin, a major component of the eukaryotic membrane skeleton, has been shown to have chaperone like activity. Here we investigate the pH induced changes in the structure and stability of erythroid and brain spectrin by spectroscopic methods. We also correlate these changes with modulations of chaperone potential at different pH. We have followed the pH induced structural changes by circular dichroism spectroscopy and intrinsic tryptophan fluorescence. It is seen that lowering the pH from 9 has little effect on structure of the proteins till about pH6. At pH4, there is significant change of the secondary structure of the proteins, along with a 5nm hypsochromic shift of the emission maxima. Below pH4 the proteins undergo acid denaturation. Probing exposed hydrophobic patches on the proteins using protein-bound 8-anilinonaphthalene-1-sulfonate fluorescence demonstrates that there is higher solvent accessibility of hydrophobic surfaces in both forms of spectrin at around pH4. Dynamic light scattering and 90° light scattering studies show that the both forms of spectrin forms oligomers at pH~4. Chemical unfolding data shows that these oligomers are less stable than the tetrameric form. Aggregation studies with BSA show that at pH4, both spectrins exhibit better chaperone activity. This enhancement of chaperone like activity appears to result from an increase in regions of solvent-exposed hydrophobicity and oligomeric state of the spectrins which in turn are induced by moderately acid pH. This may have in-vivo implications in cells facing stress conditions where cytoplasmic pH is lowered.
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http://dx.doi.org/10.1016/j.bbapap.2017.03.012DOI Listing
June 2017

A Possible Role of the Full-Length Nascent Protein in Post-Translational Ribosome Recycling.

PLoS One 2017 18;12(1):e0170333. Epub 2017 Jan 18.

Department of Biophysics, Molecular Biology and Bioinformatics, University College of Science, University of Calcutta, Kolkata, India.

Each cycle of translation initiation in bacterial cell requires free 50S and 30S ribosomal subunits originating from the post-translational dissociation of 70S ribosome from the previous cycle. Literature shows stable dissociation of 70S from model post-termination complexes by the concerted action of Ribosome Recycling Factor (RRF) and Elongation Factor G (EF-G) that interact with the rRNA bridge B2a/B2b joining 50S to 30S. In such experimental models, the role of full-length nascent protein was never considered seriously. We observed relatively slow release of full-length nascent protein from 50Sof post translation ribosome, and in that process, its toe prints on the rRNA in vivo and in in vitro translation with E.coli S30 extract. We reported earlier that a number of chemically unfolded proteins like bovine carbonic anhydrase (BCA), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), lysozyme, ovalbumin etc., when added to free 70Sin lieu of the full length nascent proteins, also interact with identical RNA regions of the 23S rRNA. Interestingly the rRNA nucleotides that slow down release of the C-terminus of full-length unfolded protein were found in close proximity to the B2a/B2b bridge. It indicated a potentially important chemical reaction conserved throughout the evolution. Here we set out to probe that conserved role of unfolded protein conformation in splitting the free or post-termination 70S. How both the RRF-EFG dependent and the plausible nascent protein-EFG dependent ribosome recycling pathways might be relevant in bacteria is discussed here.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0170333PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5242463PMC
August 2017

Localization and dynamics of the anticarcinogenic curcumin with GM and other miceller assemblies.

Glycoconj J 2017 04 19;34(2):171-179. Epub 2016 Nov 19.

Chemistry Department, University of Calcutta, Kolkata, 700009, India.

Structural transitions involving shape changes play an important role in cellular physiology and enhance the bioavailability of the natural food like curcumin in surfactant aggregates. In this work, we have studied the localization, dynamics and stability of curcumin in various miceller assemblies using a combination of absorbance and fluorescence spectroscopic approaches. The measurements of absorption and fluorescence spectra of curcumin revealed that the nature of interactions of ionic and nonionic surfactants and the glycosphingolipid, GM with curcumin is significantly different with surfactant concentrations. At low concentrations of SDS and the GM the head group of SDS and GM binds to the central β-diketone group of curcumin to form SDS-curcumin or GM-curcumin complexes. At high concentrations, both formed micelles with curcumin completely solubilized inside. Cucurmin is solubilized in the stern layer of SDS micelles. Compared to spherical micelles, rod shaped micelles allow more curcumin to bind through hydrophobic interactions indicated by higher absorption and fluorescence, enhanced partition coefficient and stability. Whereas curcumin associates with GM micelles with lower partition coefficient, solubility and remain closer to aqueous phase decreasing its bioavailability and stability. While cucurmin is solubilized in the palisade layer of deoxycholate and octyl glucopyranoside micelles through the alkyl chains providing more hydrophobic microenvironment to curcumin with enhanced stability and bioavailability. Graphical abstract Schematic diagram of the two different types of detergent micelles and larger GM1 micelles.
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http://dx.doi.org/10.1007/s10719-016-9748-1DOI Listing
April 2017

A FACS Based Case Study on Two HbE-β Thalassaemia Members of a Family, Having Similar Mutational Background.

Scientifica (Cairo) 2016 18;2016:3181937. Epub 2016 Apr 18.

Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064, India.

In this report we have tried to explain the reasons behind the difference in the pattern of transfusion requirement between two members of a family with similar β-globin mutation. The father and younger son both are HbE-β, but the father never had transfusion, whereas the younger son takes transfusion monthly. Mother and the elder son are HbEE without any history of transfusion. β-globin mutations of all family members were determined by ARMS-PCR. These were reconfirmed by direct sequencing of β-globin gene. Father and younger son were found to be Cod 26 (G-A)/IVS 1-5 (G-C), whereas mother and elder son were found to be Cod 26 (G-A)/Cod 26 (G-A). XmnI sequencing also revealed that all members of the family were CC. Then, flow cytometry study of red blood cells (RBCs) was performed to measure the oxidative stress of the RBCs. This study was also done on the light and dense fractions of the RBC population of the father and younger son. It was seen that the younger son suffers severe oxidative stress, which can be explained by his higher transfusion requirement. From our work, we have established the importance of taking oxidative stress of RBCs into consideration to explain the clinical manifestation and progression of haemoglobin related diseases like thalassaemia.
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http://dx.doi.org/10.1155/2016/3181937DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4852363PMC
May 2016

Erythrocyte and platelet proteomics in hematological disorders.

Proteomics Clin Appl 2016 Apr 28;10(4):403-14. Epub 2016 Jan 28.

Biophysics and Structural Genomics Division, Saha institute of Nuclear Physics, Kolkata, India.

Erythrocytes undergo ineffective erythropoesis, hemolysis, and premature eryptosis in sickle cell disease and thalassemia. Abnormal hemoglobin variants associated with hemoglobinopathy lead to vesiculation, membrane instability, and loss of membrane asymmetry with exposal of phosphatidylserine. This potentiates thrombin generation resulting in activation of the coagulation cascade responsible for subclinical phenotypes. Platelet activation also results in the release of microparticles, which express and transfer functional receptors from platelet membrane, playing key roles in vascular reactivity and activation of intracellular signaling pathways. Over the last decade, proteomics had proven to be an important field of research in studies of blood and blood diseases. Blood cells and its fluidic components have been proven to be easy systems for studying differential expressions of proteins in hematological diseases encompassing hemoglobinopathies, different types of anemias, myeloproliferative disorders, and coagulopathies. Proteomic studies of erythrocytes and platelets reported from several groups have highlighted various factors that intersect the signaling networks in these anucleate systems. In this review, we have elaborated on the current scenario of anucleate blood cell proteomes in normal and diseased individuals and the cross-talk between the two major constituent cell types of circulating blood.
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http://dx.doi.org/10.1002/prca.201500080DOI Listing
April 2016

Platelet proteomics in thalassemia: Factors responsible for hypercoagulation.

Proteomics Clin Appl 2016 Mar 14;10(3):239-47. Epub 2015 Dec 14.

Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata, India.

Purpose: Thalassemias can be defined as a group with inherited hemolytic anemia due to differential expressions of α and β globin genes. Hemoglobin E combined with β thalassemia (HbEβ) creates high oxidative stress in platelets producing different degrees of pathophysiological severity. Numerous cases of thalassemia have been reported with thromboembolic complications due to the hypercoagulable state, the mechanism underlying that is not yet well understood.

Experimental Design: We have used 2DE and DIGE coupled with MALDI TOF/TOF-based MS identification and characterization of altered proteins in both splenectomized and nonsplenectomized HbEβ and β thalassemia to investigate the factors responsible for hypercoagulation.

Results: The study revealed elevated levels of chaperones like HSP70, protein disulfide isomerase; oxidative stress proteins like peroxiredoxin2 and superoxide dismutase1 along with high ROS levels. Upregulation of translation initiation factor 5a observed in thalassemia is a novel finding and plays a protective role toward cell survival under oxidative stress.

Conclusions And Clinical Relevance: The altered levels of chaperones and oxidative stress proteins indicate toward regulation of integrin binding and platelet activation under oxidative stress. Altogether, this comparative proteomics study of platelets in thalassemia could provide an insight into better understanding of the pathophysiology of the disease.
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http://dx.doi.org/10.1002/prca.201500049DOI Listing
March 2016

Proteome analysis of the triton-insoluble erythrocyte membrane skeleton.

J Proteomics 2015 Oct 10;128:298-305. Epub 2015 Aug 10.

The Center for Systems and Computational Biology and Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA. Electronic address:

Erythrocyte shape and membrane integrity is imparted by the membrane skeleton, which can be isolated as a Triton X-100 insoluble structure that retains the biconcave shape of intact erythrocytes, indicating isolation of essentially intact membrane skeletons. These erythrocyte "Triton Skeletons" have been studied morphologically and biochemically, but unbiased proteome analysis of this substructure of the membrane has not been reported. In this study, different extraction buffers and in-depth proteome analyses were used to more fully define the protein composition of this functionally critical macromolecular complex. As expected, the major, well-characterized membrane skeleton proteins and their associated membrane anchors were recovered in good yield. But surprisingly, a substantial number of additional proteins that are not considered in erythrocyte membrane skeleton models were recovered in high yields, including myosin-9, lipid raft proteins (stomatin, flotillin1 and 2), multiple chaperone proteins (HSPs, protein disulfide isomerase and calnexin), and several other proteins. These results show that the membrane skeleton is substantially more complex than previous biochemical studies indicated, and it apparently has localized regions with unique protein compositions and functions. This comprehensive catalog of the membrane skeleton should lead to new insights into erythrocyte membrane biology and pathogenic mutations that perturb membrane stability. Biological significance Current models of erythrocyte membranes describe fairly simple homogenous structures that are incomplete. Proteome analysis of the erythrocyte membrane skeleton shows that it is quite complex and includes a substantial number of proteins whose roles and locations in the membrane are not well defined. Further elucidation of interactions involving these proteins and definition of microdomains in the membrane that contain these proteins should yield novel insights into how the membrane skeleton produces the normal biconcave erythrocyte shape and how it is perturbed in pathological conditions that destabilize the membrane.
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http://dx.doi.org/10.1016/j.jprot.2015.08.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4619114PMC
October 2015

Fluorescence study of the effect of cholesterol on spectrin-aminophospholipid interactions.

Eur Biophys J 2015 Dec 17;44(8):635-45. Epub 2015 Jul 17.

Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata, 700064, India.

The ability of the membrane skeletal protein spectrin to interact with phospholipids, and aminophospholipids in particular, in both natural and model membranes, is well documented. The present study involves phospholipid-induced quenching of tryptophan fluorescence to probe spectrin-membrane interactions in the presence and absence of cholesterol. We performed the experiments on small unilamellar vesicles of phospholipids made of DMPC and DMPC/DMPE and of DOPC and DOPC/DOPE with and without cholesterol at two different temperatures, one below at 15 °C and another above, at 50 °C, the main phase transition temperature (T m) of the bulk phospholipid. Results indicate that erythroid and brain spectrin binds DMPC/DMPE membranes by tenfold and 40-fold stronger, respectively, in the presence of 20 % cholesterol, up to which both gel (Lβ) and liquid crystalline (Lα) phases coexists, at 15 °C particularly in DMPC-based membranes containing saturated fatty acyl chains and not in DOPC-based membranes with appreciably lower T m. Time-resolved fluorescence and circular dichroism spectroscopic studies indicated no significant change in the mean lifetime of the tryptophan residues in spectrin and in the secondary structures of the proteins upon binding to the phospholipid SUVs.
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http://dx.doi.org/10.1007/s00249-015-1057-2DOI Listing
December 2015

Hemoglobin interacting proteins and implications of spectrin hemoglobin interaction.

J Proteomics 2015 Oct 30;128:469-75. Epub 2015 Jun 30.

Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064, India. Electronic address:

In this report we have analyzed interacting partners of hemoglobin inside erythrocyte and sought possible implications of hemoglobin-spectrin interaction. Our list of identified cytosolic hemoglobin interacting proteins includes redox regulators like peroxiredoxin-2, Cu-Zn superoxide dismutase, catalase, aldehyde dehydrogenase-1, flavin reductase and chaperones like HSP70, α-hemoglobin stabilizing protein. Others include metabolic enzymes like carbonic anhydrase-1, selenium binding protein-1, purine nucleoside phosphorylase and nucleoside diphosphate kinase. Additionally, various membrane proteins like α and β spectrin, ankyrin, band3, protein4.1, actin and glyceraldehyde 3 phosphate dehydrogenase have been shown to interact with hemoglobin. Our result indicates that major membrane skeleton protein spectrin, that also has a chaperone like activity, helps to fold the unstable alpha-globin chains in vitro. Taken together our results could provide insight into a protein network evolved around hemoglobin molecule inside erythrocyte that may add a new perspective in understanding the hemoglobin function and homeostasis.
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http://dx.doi.org/10.1016/j.jprot.2015.06.014DOI Listing
October 2015

Differential regulation of urine proteins in urothelial neoplasm.

J Proteomics 2015 Sep 2;127(Pt A):185-92. Epub 2015 May 2.

Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064, West Bengal, India. Electronic address:

Unlabelled: Urothelial neoplasm of the urinary bladder has a high rate of multifocality and recurrence. To understand this we first need to understand the changes in the molecular level that distinguishes a normal individual from a patient and also a low grade neoplasm from a high grade. In this work we aim to study the urine proteome of Indian patients with urothelial neoplasm categorised on the basis of their p53 immunohistochemistry. The urine samples of pre-operative patients were subjected to two dimensional gel electrophoresis followed by densitometric analysis and spot identification using MALDI mass spectrometry. Our study shows that few proteins such as albumin, alpha 1 antitrypsin, apolipoprotein A1, transferrin, transthyretin, haptoglobin and haemoglobin β chain were upregulated and inter alpha trypsin inhibitor heavy chain was downregulated in the disease samples. Further we have reported that some of these proteins show an association with disease severity. The present study marks the first step in the identification of new diagnostic markers as well as therapeutic targets.

Biological Significance: Bladder carcinoma is the ninth most common cancer worldwide. It has gained attention within both clinicians and cancer biologists because of its recurrence and mortality rate. Identifying the prognostic factors of progression is a challenge, so that high risk patients who may be a candidate for a radical cystectomy may be identified. In this study we have attempted to study the changes observed in the urinary protein levels of urothelial neoplasm patients. The samples were graded based on p53 immunohistochemistry staining. We have reported eight (8) proteins, mostly highly abundant; those have exhibited differential regulation in case of diseased samples. This study is first of its kind that associates the changes in the urinary protein levels to that of the severity of the disease. We believe that the findings can be used as a stepping stone in the development of a noninvasive prognostic tool for the disease. This article is part of a Special Issue entitled: Proteomics in India.
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http://dx.doi.org/10.1016/j.jprot.2015.04.022DOI Listing
September 2015

Organization and dynamics of tryptophan residues in brain spectrin: novel insight into conformational flexibility.

J Fluoresc 2015 May 3;25(3):707-17. Epub 2015 Apr 3.

Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata, 700064, India.

Brain spectrin enjoys overall structural and sequence similarity with erythroid spectrin, but less is known about its function. We utilized the fluorescence properties of tryptophan residues to monitor their organization and dynamics in brain spectrin. Keeping in mind the functional relevance of hydrophobic binding sites in brain spectrin, we monitored the organization and dynamics of brain spectrin bound to PRODAN. Results from red edge excitation shift (REES) indicate that the organization of tryptophans in brain spectrin is maintained to a considerable extent even after denaturation. These results are supported by acrylamide quenching experiments. To the best of our knowledge, these results constitute the first report of the presence of residual structure in urea-denatured brain spectrin. We further show from REES and time-resolved emission spectra that PRODAN bound to brain spectrin is characterized by motional restriction. These results provide useful information on the differences between erythroid spectrin and brain spectrin.
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http://dx.doi.org/10.1007/s10895-015-1556-7DOI Listing
May 2015

Binding of hemin, hematoporphyrin, and protoporphyrin with erythroid spectrin: fluorescence and molecular docking studies.

Eur Biophys J 2015 Apr 4;44(3):171-82. Epub 2015 Mar 4.

Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata, 700064, India.

Free heme has toxic effects, for example lipid peroxidation, DNA damage, and protein aggregation. In severe hemolysis, which occurs during pathological states, for example sickle cell disease, ischemia reperfusion, and malaria, levels of free heme increase inside erythrocytes. The purpose of this study was to investigate whether spectrin, the major erythroid cytoskeleton protein, is involved as an acceptor of free heme. We compared the interactions of three heme derivatives, hemin chloride, hematoporphyrin, and protoporphyrin-IX, with dimeric and tetrameric spectrin. The dissociation constants (K d) for binding to spectrin dimer and tetramer were 0.57 and 1.16 µM respectively. Thermodynamic data associated with this binding revealed the binding to be favored by a positive change in entropy. Although molecular docking studies identified the SH3 domain as the unique binding site of these heme derivatives to erythroid spectrin, experimental results indicated a binding stoichiometry of 1 heme attached to both dimeric and tetrameric spectrin, indicating the common self-associating domain to be the unique binding site. We also noticed heme-induced structural changes in the membrane skeletal protein. Erythroid spectrin could thus act as a potential acceptor of heme, particularly relevant under disease conditions.
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http://dx.doi.org/10.1007/s00249-015-1012-2DOI Listing
April 2015

Peanut protein sensitivity towards trace iron: a novel mode to ebb allergic response.

Food Chem 2015 Jun 29;176:308-13. Epub 2014 Dec 29.

Department of Chemistry, University of Calcutta, 92, APC Road, Kolkata 700 009, India. Electronic address:

Peanut is a rich source of plant protein which is inexpensive and abundant in nature. The peanut proteins however cause hypersensitive immunogenic responses in certain individuals. A minute amount of contamination may cause strong allergic reactions and even death. Many chemical pretreatment procedures have been developed and prescribed earlier for removal of this allergenicity. In the present article we have observed trace level Fe(III) and Cu(II) complexation of the protein fractions of peanut at pH 4.8 using different spectral methods. Consequently we studied the allergic response of Fe(III) complex of the protein fractions using competitive enzyme linked immunosorbent assay (ELISA) technique and found that there were considerable losses in allergenicity of conarachin I and arachin fractions upon complexation. Immunoassay of Cu(II) complex was avoided keeping in view the Cu toxicity in living systems. The results bring up a new strategy towards reduction of allergenicity using an inexpensive and simple methodology.
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http://dx.doi.org/10.1016/j.foodchem.2014.12.081DOI Listing
June 2015

Probing conformational stability and dynamics of erythroid and nonerythroid spectrin: effects of urea and guanidine hydrochloride.

PLoS One 2015 24;10(1):e0116991. Epub 2015 Jan 24.

Crystallography & Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata, West Bengal, India.

We have studied the conformational stability of the two homologous membrane skeletal proteins, the erythroid and non-erythroid spectrins, in their dimeric and tetrameric forms respectively during unfolding in the presence of urea and guanidine hydrochloride (GuHCl). Fluorescence and circular dichroism (CD) spectroscopy have been used to study the changes of intrinsic tryptophan fluorescence, anisotropy, far UV-CD and extrinsic fluorescence of bound 1-anilinonapthalene-8-sulfonic acid (ANS). Chemical unfolding of both proteins were reversible and could be described as a two state transition. The folded erythroid spectrin and non-erythroid spectrin were directly converted to unfolded monomer without formation of any intermediate. Fluorescence quenching, anisotropy, ANS binding and dynamic light scattering data suggest that in presence of low concentrations of the denaturants (up-to 1M) hydrogen bonding network and van der Waals interaction play a role inducing changes in quaternary as well as tertiary structures without complete dissociation of the subunits. This is the first report of two large worm like, multi-domain proteins obeying twofold rule which is commonly found in small globular proteins. The free energy of stabilization (ΔGuH20) for the dimeric spectrin has been 20 kcal/mol lesser than the tetrameric from.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0116991PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4305312PMC
January 2016

Differential interactions of two local anesthetics with phospholipid membrane and nonerythroid spectrin: Localization in presence of cholesterol and ganglioside, GM1.

Biochim Biophys Acta 2015 Mar 5;1848(3):821-32. Epub 2014 Dec 5.

Chemistry Department, University of Calcutta 92, A. P. C Road, Kolkata 700009, India.

Interactions of two local anesthetics, dibucaine and tetracaine have been studied with phospholipid vesicles containing cholesterol and/or monosialogangliosides (GM1) using fluorescence spectroscopy. The fluorescence intensity of tetracaine showed a marked increase with the increasing molar ratio of the phospholipid to tetracaine, while that of dibucaine showed opposite effects. Steady state anisotropy and the wavelength of maximum emission (λmax) decreased with the increasing phospholipids to tetracaine ratio. The extent of such changes in anisotropy and λmax in the presence and absence of two important components of neuronal membranes, cholesterol and GM1 indicated differential membrane localization of the two local anesthetics. To understand the intercellular mode of action of local anesthetics, we have also studied the interactions of dibucaine and tetracaine with brain spectrin which indicate differential spectrin interactions with similar binding strength. Thermodynamic parameters associated with such binding reveal that binding is favored by entropy. Tetracaine brings about distinct structural changes in spectrin compared to dibucaine, as reflected in the tryptophan mean lifetime and far-UV CD spectra. Tetracaine also exhibits a detergent-like property inducing concentration dependent decrease in spectrin anisotropy, further indicating structural changes in brain spectrin with probable implications in its anesthetic potential.
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http://dx.doi.org/10.1016/j.bbamem.2014.11.022DOI Listing
March 2015

Defects in erythrocyte membrane skeletal architecture.

Adv Exp Med Biol 2015 ;842:41-59

Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata, 700064, India.

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http://dx.doi.org/10.1007/978-3-319-11280-0_4DOI Listing
May 2015

Comparative proteomics and glycoproteomics of plasma proteins in Indian visceral leishmaniasis.

Proteome Sci 2014 22;12(1):48. Epub 2014 Sep 22.

Cancer Biology and Inflammatory Disorder Division, Council of Scientific and Industrial Research-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata, 700 032 India.

Background: Visceral leishmaniasis (VL) is a deadly parasitic diseases caused by Leishmania donovani; it is a major health problem in many countries. A lack of proper understanding of the disease biology, poor diagnostic methods and increasing drug resistance are the main reasons for the growing burden of VL infection. Comparative plasma proteomics are a relatively useful technique that can be used to investigate disease-associated alterations that can help in understanding host responses against pathogens, and might be useful in disease management and diagnosis.

Result: In this study, a comparative proteomics and glycoproteomics approach using 2DE and 2D-DIGE was employed between early diagnosed VL patients of all age groups and healthy endemic and non-endemic controls in order to aid the recognition of disease-associated alterations in host plasma. Comparative proteomics was performed by the depletion of seven highly abundant plasma proteins. Comparative glycoproteomics was performed by the depletion of albumin and IgG, followed by purification of plasma glycoproteins using a multi lectin affinity column. From these two approaches, 39 differentially expressed protein spots were identified and sequenced using MALDI-TOF/TOF mass spectrometry. This revealed ten distinct proteins that appeared in multiple spots, suggesting micro-heterogeneity. Among these proteins, alpha-1-antitrypsin, alpha-1-B glycoprotein and amyloid-A1 precursor were up-regulated, whereas vitamin-D binding protein, apolipoprotein-A-I and transthyretin were down-regulated in VL. Alterations in the levels of these proteins in VL-infected plasma were further confirmed by western blot and ELISA.

Conclusions: These proteins may be involved in the survival of parasites, resisting neutrophil elastase, and in their multiplication in macrophages, potentially maintaining endogenous anti-inflammatory and immunosuppressive conditions. Consequently, the results of this study may help in understanding the host response against L.donovani, which could help in the discovery of new drugs and disease management. Finally, these alterations on protein levels might be beneficial in improving early diagnosis considering those as biomarkers in Indian VL.
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http://dx.doi.org/10.1186/s12953-014-0048-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4179796PMC
October 2014