Publications by authors named "Debashis Mukhopadhyay"

38 Publications

LncRNA Xist, X-chromosome Instability and Alzheimer's Disease.

Curr Alzheimer Res 2020 ;17(6):499-507

Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, Homi Bhabha National Institute, Kolkata 700 064, India.

Neurodegenerative Diseases (NDD) are the major contributors to age-related causes of mental disability on a global scale. Most NDDs, like Alzheimer's Disease (AD), are complex in nature - implying that they are multi-parametric both in terms of heterogeneous clinical outcomes and underlying molecular paradigms. Emerging evidence from high throughput genomic, transcriptomic and small RNA sequencing experiments hint at the roles of long non-coding RNAs (lncRNAs) in AD. X-inactive Specific Transcript (XIST), a component of the Xic, the X-chromosome inactivation centre, is an RNA gene on the X chromosome of the placental mammals indispensable for the X inactivation process. An extensive literature survey shows that aberrations in Xist expression and in some cases, a disruption of the Xchromosome inactivation as a whole play a significant role in AD. Considering the enormous potential of Xist as an endogenous silencing molecule, the idea of using Xist as a non-conventional chromosome silencer to treat diseases harboring chromosomal alterations is also being implemented. Comprehensive knowledge about how Xist could play such a role in AD is still elusive. In this review, we have collated the available knowledge on the possible Xist involvement and deregulation from the perspective of molecular mechanisms governing NDDs with a primary focus on Alzheimer's disease. Possibilities of XIST mediated therapeutic intervention and linkages between XIC and preferential predisposition of females to AD have also been discussed.
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http://dx.doi.org/10.2174/1567205017666200807185624DOI Listing
January 2020

E2F1 activates MFN2 expression by binding to the promoter and decreases mitochondrial fission and mitophagy in HeLa cells.

FEBS J 2019 11 22;286(22):4525-4541. Epub 2019 Jul 22.

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

Mitofusin-2 (MFN2) is primarily involved in mitochondrial fusion and participates in diverse biological processes. Several reports show that MFN2 is a target of different miRNAs; however, the transcriptional regulation of MFN2 has not been extensively studied. To gain insight into the transcriptional regulation of MFN2, we expressed E2F transcription factor 1 (E2F1) exogenously and observed that it increased the endogenous expression of MFN2 by binding to its putative promoter region. Although the levels of E2F1 were shown to vary during the cell cycle, the expression of MFN2 and its regulator SP1 did not change throughout the different phases, suggesting that E2F1 regulates MFN2 in a cell-cycle-independent manner. In the cell-cycle phases, where the expression of E2F1 was reduced, SP1 might act in its place to regulate the expression of MFN2. We showed that E2F1 and SP1 are present as a complex on the promoter of MFN2 during the S-phase as well as in E2F1 overexpressing cells, suggesting that they may regulate the expression of MFN2 synergistically. Furthermore, we found that E2F1 modulated mitochondrial fusion and mitophagy, likely through regulation of MFN2. Bioinformatic analysis revealed that several potential targets of E2F1 are localized in mitochondria and associated with autophagy. Collectively, these data identify the E2F1-MFN2 axis as a regulator of mitochondrial morphology and mitophagy, suggesting a potential therapeutic target for the treatment of mitochondrial disorders.
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http://dx.doi.org/10.1111/febs.14980DOI Listing
November 2019

In vitro degradation of β-amyloid fibrils by microbial keratinase.

Alzheimers Dement (N Y) 2019 16;5:154-163. Epub 2019 May 16.

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

Introduction: Amyloid fibrils are misfolded, protease-resistant forms of normal proteins. They are infectious such as prions or noninfectious such as β-amyloid (Aβ) fibrils causing Alzheimer's disease (AD). Prions and amyloids are structurally similar, possessing cross β-pleated sheet-like structures. As microbial keratinase could degrade prions, we tested keratinase activity on Aβ fibrils.

Methods: Lysozyme treated with urea generates Aβ fibrils demonstrated by immunoblotting with anti-Aβ antibody, high-performance liquid chromatography, and Congo red absorption spectroscopy. Two keratinases, Ker1 and Ker2, were purified from an actinomycete Amycolatopsis sp. MBRL 40 and incubated with Aβ fibrils.

Results: Soluble Ker1 and Ker1 reconstituted on neutral/cationic liposomes degraded Aβ fibrils efficiently. Ker 2 was less potent.

Discussion: Drugs that target AD inhibit acetylcholinesterase or formation of Aβ fibrils and downstream effects. These drugs have side effects and do not benefit globally in cognition. Keratinases are novel molecules for drug development against AD.
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http://dx.doi.org/10.1016/j.trci.2019.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6527806PMC
May 2019

Receptor tyrosine kinases (RTKs) consociate in regulatory clusters in Alzheimer's disease and type 2 diabetes.

Mol Cell Biochem 2019 Sep 1;459(1-2):171-182. Epub 2019 Jun 1.

Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, HBNI, Block-AF, Sector-1, Bidhannagar, Kolkata, WB, 700064, India.

Alzheimer's disease (AD) and type 2 diabetes (T2D) share the common hallmark of insulin resistance. It is conjectured that receptor tyrosine kinases (RTKs) play definitive roles in the process. To decipher the signaling overlap behind this phenotypic resemblance, the activity status of RTKs is probed in post-mortem AD and T2D tissues and cell models. Activities of only about one-third changed in a similar fashion, whereas about half of them showed opposite outcomes when exposed to contrasting signals akin to AD and T2D. Interestingly, irrespective of disease type, RTKs with enhanced and compromised activities clustered distinctly, indicating separate levels of regulations. Similar regulatory mechanisms within an activity cluster could be inferred, which have potential to impact future therapeutic developments.
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http://dx.doi.org/10.1007/s11010-019-03560-5DOI Listing
September 2019

Altered Levels of Long NcRNAs Meg3 and Neat1 in Cell And Animal Models Of Huntington's Disease.

RNA Biol 2018 26;15(10):1348-1363. Epub 2018 Oct 26.

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

Altered expression levels of protein-coding genes and microRNAs have been implicated in the pathogenesis of Huntington's disease (HD). The involvement of other ncRNAs, especially long ncRNAs (lncRNA), is being realized recently and the related knowledge is still rudimentary. Using small RNA sequencing and PCR arrays we observed perturbations in the levels of 12 ncRNAs in HD mouse brain, eight of which had human homologs. Of these, Meg3, Neat1, and Xist showed a consistent and significant increase in HD cell and animal models. Transient knock-down of Meg3 and Neat1 in cell models of HD led to a significant decrease of aggregates formed by mutant huntingtin and downregulation of the endogenous Tp53 expression. Understanding Meg3 and Neat1 functions in the context of HD pathogenesis is likely to open up new strategies to control the disease.
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http://dx.doi.org/10.1080/15476286.2018.1534524DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6284602PMC
January 2019

Cellular levels of Grb2 and cytoskeleton stability are correlated in a neurodegenerative scenario.

Dis Model Mech 2017 05 30;10(5):655-669. Epub 2017 Mar 30.

Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, Block-AF, Sector-1, Bidhannagar, Kolkata, West Bengal 700064, India

Alzheimer's disease (AD) manifests as neuronal loss. On the premise of Grb2 overexpression in AD mouse brain and brain tissues of AD patients, our study primarily focuses on the stability of cytoskeletal proteins in the context of degenerative AD-like conditions. Two predominant molecular features of AD, extracellular accumulation of β-amyloid oligomers and intracellular elevation of amyloid precursor protein intracellular domain levels, have been used to closely inspect the series of signalling events. In their presence, multiple signalling pathways involving ROCK and PAK1 proteins lead to disassembly of the cytoskeleton, and Grb2 partially counterbalances the cytoskeletal loss. Increased Grb2-NOX4 interactions play a preventive role against cytoskeletal disassembly, in turn blocking the activity of nitrogen oxides and decreasing the expression of slingshot homolog 1 (SSH-1) protein, a potent inducer of cytoskeleton disassembly. This study unravels a unique role of Grb2 in protecting the cytoskeletal architecture in AD-like conditions and presents a potential new strategy for controlling neurodegeneration.
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http://dx.doi.org/10.1242/dmm.027748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5451165PMC
May 2017

Systemic study of a natural feedback loop in Huntington's disease at the onset of neurodegeneration.

Biosystems 2016 Dec 30;150:46-51. Epub 2016 Aug 30.

Biophysics & Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata 700064, India. Electronic address:

Aggregation prone Huntingtin (Htt) protein and its aberrations, causing protein misfolding, have been the prototype of intense research for several decades. Misfolded aggregates or oligomers of different sizes not only deregulate the homeostasis, cellular machinery also counterbalances the effects at least at the initial stages, till the balance tilts towards toxicity and degeneration. In this paper, we combine experimental approaches with system based computational modeling to decipher the molecular mechanisms as well as the hidden dynamics leading to neuronal death in HD. We built an abstracted Boolean gate based electronic circuit that captured the available knowledge and experimental data. We inferred the unknown parameters by simultaneously fitting experimental data generated in both control and perturbed conditions. We demonstrate that, at the initial stages of Htt aggregate formation, individual changes in different protein levels and their interactions in cascade constitute the Grb2-pERK-Foxd3 feedback loop that is sufficient to create Hill-like sensitivity and prevent aggregation to the extent till mutant Htt (mHtt) aggregates become predominant in the cell when they spatially isolate the homeostatic reaction mechanism.
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http://dx.doi.org/10.1016/j.biosystems.2016.08.012DOI Listing
December 2016

Increased expression of ApoA1 after neuronal injury may be beneficial for healing.

Mol Cell Biochem 2017 Jan 13;424(1-2):45-55. Epub 2016 Oct 13.

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

ApoA1 is a player in reverse cholesterol transport that initiates multiple cellular pathways on binding to its receptor ABCA1. Its relation to neuronal injury is however unclear. We found ApoA1 to be increasingly abundant at a later time point in the secondary phase of traumatic spinal cord injury. In a cellular injury model of neuroblastoma, ApoA1 showed an initial diminished expression after infliction of injury, which sharply increased thereafter. Subsequently, ApoA1 was shown to alter wound healing dynamics in neuroblastoma injury model. It was observed that an initial lag in scratch wound closure was followed by rapid healing in the ApoA1 treatment group. Activation of ERK pathway and Actin polymerisation by ApoA1 corroborated its role in healing after neuronal injury. We propose that ApoA1 is increasingly expressed and secreted as a delayed response to neuronal injury, and this is a self-protecting mechanism of the injured system.
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http://dx.doi.org/10.1007/s11010-016-2841-8DOI Listing
January 2017

Clinical proteomics of enervated neurons.

Clin Proteomics 2016 5;13:10. Epub 2016 May 5.

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

The dynamic field of neurosciences entails ever increasing search for molecular mechanisms of disease states, especially in the domain of neurodegenerative disorders. The previous century heralded the techniques in proteomics when indexing of the human proteomes relating to various disease conditions became important. Early stage research in certain diseases or pathological conditions requires a more holistic approach of first discovering the proteins of interest for the condition. Despite its limitations, proteomics is one of the most powerful techniques available to us today to dissect the molecular scenario in a particular disease situation. In this review we will discuss about the current clinical research in neurodegenerative disorders that employ proteomics techniques. We will specifically focus on our understanding of Alzheimer's disease, traumatic spinal cord injury and neuromyelitis optica. Discussions will include ongoing worldwide research in these areas, research in India and specifically our laboratory in these domains of neurodegenerative conditions.
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http://dx.doi.org/10.1186/s12014-016-9112-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4857373PMC
May 2016

Possible role of apolipoprotein A1 in healing and cell death after neuronal injury.

Front Biosci (Elite Ed) 2016 06 1;8:460-77. Epub 2016 Jun 1.

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

Limited axonal regeneration after traumatic injuries to the CNS presents a challenge in neuroscience. Investigation of CSF from subjects with spinal cord injury (SCI) has found that the lipid catabolism pathway is implicated in the post injury scenario. Sequestration of the CNS by the blood brain barrier ensures a mechanism of cholesterol metabolism and recycling distinct from that in the peripheral tissues. Apolipoprotein A1, the protein component of high density lipoprotein (HDL), is an abundant protein in the mammalian cerebrospinal fluid. Interaction of ApoA1 with its cellular receptor, ABCA1, gives rise to several signaling events, such as the activation of Cdc42 protein leading to actin polymerisation. Emerging evidences suggest that ApoA1 mediates anti-inflammatory effects and conversely, is negatively regulated by inflammatory cytokines. Collating these findings, added to the clinical evidences of using HDL as a therapeutic target for cardio vascular diseases, we hypothesize that ApoA1 could be useful in neurite outgrowth after mechanical injury by 1) mediating polymerisation of actin and 2) restricting inflammatory responses after injury which are deleterious to healing.
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http://dx.doi.org/10.2741/e780DOI Listing
June 2016

Chaperone-like protein HYPK and its interacting partners augment autophagy.

Eur J Cell Biol 2016 Jun-Jul;95(6-7):182-94. Epub 2016 Apr 1.

Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF, Bidhan Nagar, Kolkata 700 064, India. Electronic address:

To decipher the function(s) of HYPK, a huntingtin (HTT)-interacting protein with chaperone-like activity, we had previously identified 36 novel interacting partners of HYPK. Another 13 proteins were known earlier to be associated with HYPK. On the basis of analysis of the interacting partners of HYPK, it has been shown that HYPK may participate in diverse cellular functions relevant to Huntington's disease. In the present study, we identified additional 5 proteins by co-immunoprecipitation and co-localization. As of now we have 54 primary interactors of HYPK. From the database we collected 1026 unique proteins (secondary interactors) interacting with these 54 primary HYPK interacting partners. We observed that 10 primary and 91 secondary interacting proteins of HYPK are associated with two types of autophagy processes. We next tested the hypothesis that the hub, HYPK, might itself be involved in autophagy. Using mouse striatal STHdh(Q7)/Hdh(Q7) cell lines, we observed that over expression of HYPK significantly increased background cellular autophagy, while knock down of endogenous HYPK decreased the autophagy level as detected by altered LC3I conversion, BECN1 expression, cleavage of GFP from LC3-GFP, ATG5-ATG12 conjugate formation and expression of transcription factors like Tfeb, Srebp2 and Zkscan3. This result shows that HYPK, possibly with its interacting partners, induces autophagy. We further observed that N-terminal mutant HTT reduced the cellular levels of LC3II and BECN1, which could be recovered significantly upon over expression of HYPK in these cells. This result further confirms that HYPK could also be involved in clearing mutant HTT aggregates by augmenting autophagy pathway.
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http://dx.doi.org/10.1016/j.ejcb.2016.03.003DOI Listing
February 2017

Regulation of mitochondrial morphology and cell cycle by microRNA-214 targeting Mitofusin2.

Biochem Biophys Res Commun 2015 Oct 22;465(4):797-802. Epub 2015 Aug 22.

Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF, Bidhan Nagar, Kolkata, 700 064, India; Biomedical Genomics Centre, PG Polyclinic Building (3rd Floor), 5, Suburbun Hospital Road, Kolkata, 700020, India. Electronic address:

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by the increase in CAG repeats beyond 36 at the exon1 of the gene Huntingtin (HTT). Among the various dysfunctions of biological processes in HD, transcription deregulation due to abnormalities in actions of transcription factors has been considered to be one of the important pathological conditions. In addition, deregulation of microRNA (miRNA) expression has been described in HD. Earlier, expression of microRNA-214 (miR-214) has been shown to increase in HD cell models and target HTT gene; the expression of the later being inversely correlated to that of miR-214. In the present communication, we observed that the expressions of several HTT co-expressed genes are modulated by exogenous expression of miR-214 or by its mutant. Among several HTT co-expressed genes, MFN2 was shown to be the direct target of miR-214. Exogenous expression of miR-214, repressed the expression of MFN2, increased the distribution of fragmented mitochondria and altered the distribution of cells in different phases of cell cycle. In summary, we have shown that increased expression of miR-214 observed in HD cell model could target MFN2, altered mitochondrial morphology and deregulated cell cycle. Inhibition of miR-214 could be a possible target of intervention in HD pathogenesis.
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http://dx.doi.org/10.1016/j.bbrc.2015.08.090DOI Listing
October 2015

CSF proteomics of secondary phase spinal cord injury in human subjects: perturbed molecular pathways post injury.

PLoS One 2014 28;9(10):e110885. Epub 2014 Oct 28.

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

Recovery of sensory and motor functions following traumatic spinal cord injury (SCI) is dependent on injury severity. Here we identified 49 proteins from cerebrospinal fluid (CSF) of SCI patients, eight of which were differentially abundant among two severity groups of SCI. It was observed that the abundance profiles of these proteins change over a time period of days to months post SCI. Statistical analysis revealed that these proteins take part in several molecular pathways including DNA repair, protein phosphorylation, tRNA transcription, iron transport, mRNA metabolism, immune response and lipid and ATP catabolism. These pathways reflect a set of mechanisms that the system may adopt to cope up with the assault depending on the injury severity, thus leading to observed physiological responses. Apart from putting forward a picture of the molecular scenario at the injury site in a human study, this finding further delineates consequent pathways and molecules that may be altered by external intervention to restrict neural degeneration.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0110885PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4211693PMC
January 2016

Mutant huntingtin replaces Gab1 and interacts with C-terminal SH3 domain of growth factor receptor binding protein 2 (Grb2).

Neurosci Res 2014 Oct 18;87:77-83. Epub 2014 Jul 18.

Biophysics & Structural Genomics Division, Saha Institute of Nuclear Physics, 1/AF, Bidhannagar, Kolkata 700064, India. Electronic address:

Huntington's disease (HD) is caused due to expansion of CAG repeats in the gene huntingtin (Htt). Adaptor protein Grb2, involved in Ras-MAP kinase pathway, is a known interactor of Htt. Mutant Htt-Grb2 interaction reduces Ras-MAPK signaling in HD models. In normal cells Grb2 forms Grb2-Sos1-Gab1 complex through its N-SH3 and C-SH3 domains respectively, essential for sustained activation of Ras. We found that C-SH3 of Grb2 mediates the interaction with mutant Htt and this interaction being stronger could replace Gab1, with mutant Htt becoming the preferred partner. This would have immense effect on downstream signaling events.
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http://dx.doi.org/10.1016/j.neures.2014.06.009DOI Listing
October 2014

Interaction of Grb2 SH3 domain with UVRAG in an Alzheimer's disease-like scenario.

Biochem Cell Biol 2014 Jun 24;92(3):219-25. Epub 2014 Apr 24.

Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, 1/AF Bidhan Nagar, Kolkata 700 064, India.

Growth factor receptor-bound protein 2 (Grb2) is an adaptor protein which participates in trafficking pathways alongside its role in signaling. Proteins important for actin remodeling and cellular compartmentalization contain SRC Homology 3 (SH3) binding motifs that interact with Grb2. While studying the Grb2-amyloid precursor protein (APP) intracellular domain (AICD) interaction in Alzheimer's disease cell line models, it was seen that Grb2 colocalized to compartments that mature into autophagosomes. The entrapping of AICD in the Grb2 vesicles and its clearance via autophagosomes was a survival contrivance on the part of the cell. Here, we report that Grb2, when in excess, interacts with ultraviolet radiation resistance-associated gene protein (UVRAG) under excess conditions of AICD-Grb2 or Grb2. The N-terminal SH3 domain of Grb2 specifically interacts with UVRAG, unlike the C-terminal SH3 domain. This interaction helps to understand the role of Grb2 in the autophagic maturation of vesicles.
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http://dx.doi.org/10.1139/bcb-2014-0001DOI Listing
June 2014

Grb2 is regulated by foxd3 and has roles in preventing accumulation and aggregation of mutant huntingtin.

PLoS One 2013 8;8(10):e76792. Epub 2013 Oct 8.

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

Growth factor receptor protein binding protein 2 (Grb2) is known to be associated with intracellular growth and proliferation related signaling cascades. Huntingtin (Htt), a ubiquitously expressed protein, when mutated, forms toxic intracellular aggregates - the hallmark of Huntington's disease (HD). We observed an elevated expression of Grb2 in neuronal cells in animal and cell models of HD. Grb2 overexpression was predominantly regulated by the transcription factor Forkhead Box D3 (Foxd3). Exogenous expression of Grb2 also reduced aggregation of mutant Htt in Neuro2A cells. Grb2 is also known to interact with Htt, depending on epidermal growth factor receptor (EGFR) activation. Grb2- mutant Htt interaction in the contrary, took place in vesicular structures, independent of EGFR activation that eventually merged with autophagosomes and activated the autophagy machinery helping in autophagosome and lysosome fusion. Grb2, with its emerging dual role, holds promise for a survival mechanism for HD.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0076792PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3792889PMC
August 2014

Growth factor receptor-bound protein 2 promotes autophagic removal of amyloid-β protein precursor intracellular domain overload in neuronal cells.

J Alzheimers Dis 2014 ;38(4):881-95

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

The ascertainment of elevated levels of amyloid-β protein precursor intracellular domain (AICD) in Alzheimer's disease (AD) brains and the fact that it contributes to AD-like pathology has geared the search toward a new paradigm. While studying endogenous as well as overexpressed Grb2-AICD interaction in AD cell models, it was found that Grb2 co-localized to compartments along with AICD. We report now that these vesicles form in a clathrin and dynamin independent manner. Both types of vesicles mature into autophagosomes, merge with lysosomes, and relieve the cells of AICD overload. Inhibiting autophagosome formation results in vesicle accumulation. AICD-level is reduced in Grb2 excess condition in Cycloheximide Chase setup. Reduced caspase activity and apoptosis point toward the fact that the cytotoxic effect of AICD is alleviated by its sequestration in autolysosomes. Hence we state that the entrapping of AICD in Grb2 vesicles and its clearance via autophagosomes is a survival contrivance on the part of the cell. This study unravels, for the first time, the roles of Grb2 in autophagy and in handling toxic protein overload in an AD-like scenario.
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http://dx.doi.org/10.3233/JAD-130929DOI Listing
August 2014

Differential expression of neuroblastoma cellular proteome due to AICD overexpression.

J Alzheimers Dis 2014 ;38(4):845-55

Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata, WB, India.

Amyloid-β protein precursor intracellular domain (AICD), which exerts intracellular effects by interacting with proteins involved in a plethora of biological processes, is a key player behind the pathophysiology of Alzheimer's disease (AD). Keeping in mind that overwhelming presence of AICD would mimic AD-like conditions in neuroblastoma cell lines, we hypothesized alteration in the proteomic expression pattern in these cells in the presence of AICD compared to their normal proteome. The rationale behind the study was to distinguish between symptomatic pathophysiological effects as opposed to any artifactual consequence due to protein overload in the cell lines. Using 2D-DIGE analysis and MALDI-MS identifications in neuro2A (mouse) and SHSY5Y (human) cell lines, we have identified several proteins belonging to different functional classes and involved in several biological pathways including protein folding, cytoskeletal dynamics, metabolism, and stress. Many of these were being upregulated or downregulated due to AICD effects and could be correlated directly with AD phenotypes.
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http://dx.doi.org/10.3233/JAD-130695DOI Listing
August 2014

Altered levels of amyloid precursor protein intracellular domain-interacting proteins in Alzheimer disease.

Alzheimer Dis Assoc Disord 2014 Jul-Sep;28(3):283-90

*Structural Genomics Division, Saha Institute of Nuclear Physics †Department of Medicine, Nilratan Sircar Medical College, Kolkata, West Bengal, India.

Background: The amyloid precursor protein intracellular domain (AICD) is an intrinsically unstructured molecule with functional promiscuity that plays an important role in determining the fate of the neurons during its degeneration. Its association with Alzheimer disease (AD) recently played a key role in propelling scientists to choose AICD as a major molecule of interest. Although several studies have been conducted elucidating AICD's participation in inducing neurodegenerative outcomes in AD condition, much remains to be deciphered regarding the linkage of AICD with cellular pathways in the AD scenario.

Results: In the present study, we have pulled down interactors of nonphosphorylated AICD from the cerebrospinal fluid of AD subjects, identified them by matrix assisted laser desorption ionization mass spectrometry, and subsequently studied the differential expression of these interactors in AD and control cases by 2-dimensional difference gel electrophoresis. The study has yielded some AICD-interactors that are differentially expressed in the AD cases and are involved in diverse cellular functions.

Conclusions: This proteomic-based approach highlights the first step in finding the possible cellular pathways engaged in AD pathophysiology on the basis of interaction of one or more of their members with AICD.
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http://dx.doi.org/10.1097/WAD.0000000000000011DOI Listing
April 2015

Novel adaptors of amyloid precursor protein intracellular domain and their functional implications.

Genomics Proteomics Bioinformatics 2012 Aug 25;10(4):208-16. Epub 2012 Jul 25.

Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata, West Bengal, India.

Amyloid precursor protein intracellular domain (AICD) is one of the potential candidates in deciphering the complexity of Alzheimer's disease. It plays important roles in determining cell fate and neurodegeneration through its interactions with several adaptors. The presence or absence of phosphorylation at specific sites determines the choice of partners. In this study, we identified 20 novel AICD-interacting proteins by in vitro pull down experiments followed by 2D gel electrophoresis and MALDI-MS analysis. The identified proteins can be grouped into different functional classes including molecular chaperones, structural proteins, signaling and transport molecules, adaptors, motor proteins and apoptosis determinants. Interactions of nine proteins were further validated either by colocalization using confocal imaging or by co-immunoprecipitation followed by immunoblotting. The cellular functions of most of the proteins can be correlated with AD. Hence, illustration of their interactions with AICD may shed some light on the disease pathophysiology.
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http://dx.doi.org/10.1016/j.gpb.2012.07.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5054717PMC
August 2012

Biophysical studies with AICD-47 reveal unique binding behavior characteristic of an unfolded domain.

Biochem Biophys Res Commun 2012 Aug 22;425(2):201-6. Epub 2012 Jul 22.

Structural Genomics Division, Saha Institute of Nuclear Physics, 1/AF Bidhan Nagar, Kolkata 700 064, WB, India.

APP intracellular C-terminal domain (AICD-47), generated upon γ-secretase cleavage of amyloid precursor's protein (APP), bears the signature of a classical intrinsically unstructured domain (IUD). Comparing the recent crystal structures of AICD-47 peptides bound to its different adaptors such as protein-tyrosine-binding domain-2 (PTB2) of Fe65 and Src homology 2 (SH2) domain of growth factor receptor binding protein 2 (Grb2), the "conformational switching" of AICD-47 becomes evident. In order to understand different binding processes undertaken by this flexible molecule, upon recognizing different interfaces resulting in different 3D conformations, spectroscopic and calorimetric studies have been done. CD spectroscopy has revealed an overall random coil like structure in different pHs while TFE (2'-2'-2'-trifluoro ethanol) and HFIP (Hexa fluoro isopropanol) induced α-helicity to a certain extent. Binding of Tyr phosphorylated AICD-47 ((P)AICD-47) to Grb2-SH2 domain was carried out by a favorable enthalpic change (ΔH=-197.5±6.2 kcal mole(-1) at 25 °C) and an unfavorable entropic contribution (ΔS=-631 cal mol(-1) deg(-1) at 25 °C). Alternative conformation of AICD-47 in different biological contexts is another remarkable feature of IUDs which presumably has definitive roles in regulating Alzheimer's disease phenotype.
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http://dx.doi.org/10.1016/j.bbrc.2012.07.067DOI Listing
August 2012

The N-terminal SH3 domain of Grb2 is required for endosomal localization of AβPP.

J Alzheimers Dis 2012 ;32(2):479-93

Structural Genomics Division, Saha Institute of Nuclear Physics, West Bengal, Kolkata, India.

Based on the observations that Grb2 overexpression altered the trafficking route of amyloid-β protein precursor (AβPP) by inhibiting its release via exosomal vesicles, and subsequently increased its endogenous level, in the present study we aimed to elucidate the mechanism of traffic impairment and the role of different Grb2 domains in this process. We found that the N-SH3 domain of Grb2 was involved in the protein vesicular localization. The C-SH3 domain could also form very small puncta, but were not characteristic Grb2 containing vesicles. Vesicles containing the N-SH3-SH2 domain had a mixed population of early and late endosomes but C-SH3-SH2 domain containing vesicles were of early endosomal type. The N-SH3 domain therefore seems to be involved in the maturation of early endosomes to late endosomes. Almost all the features shown by overexpression of full-length type Grb2, for example, entrapment of endogenous AβPP in vesicles, affecting the turnover of AβPP in terms of decrease in exosomal release and increase in endogenous concentration of the protein, could be reproduced by the N-SH3-SH2 domain and, to a very limited extent, by the C-SH3-SH2 domain. The middle SH2 domain alone did not show any involvement in AβPP trafficking. By mutational analysis of both N and C terminal SH3 domains, attempts were made to elucidate the molecular basis of this functional anomaly.
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http://dx.doi.org/10.3233/JAD-2012-120388DOI Listing
April 2013

"Patch"-ing up the neurons: revival or enervation?

Vitam Horm 2012 ;88:439-59

Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata, West Bengal, India.

Since its identification Patched1 (Ptch1) has gained importance for playing a cardinal role in developmental patterning through Hedgehog (Hh) pathway, acting as a transmembrane receptor. Involvement of this protein in diverse aspects of the neuronal system, from development to regeneration and protection, including uncontrolled proliferation in oncogenic perspectives, makes it an intriguing candidate for investigation in neurobiology. Stem cell population of adult nervous system is also found to be regulated by Ptch1. Though not elaborately studied, research in this field for the past one decade has suggested a new spectrum of Ptch1 function through an alternative route independent of Hh. In this chapter, the available knowledge about Ptch1 in neuronal system is critically reviewed and further functional insights about this protein are evaluated.
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http://dx.doi.org/10.1016/B978-0-12-394622-5.00019-5DOI Listing
August 2012

Functional implications of the conformational switch in AICD peptide upon binding to Grb2-SH2 domain.

J Mol Biol 2011 Nov 4;414(2):217-30. Epub 2011 Oct 4.

Structural Genomics Division, Saha Institute of Nuclear Physics, 1/AF Bidhan Nagar, WB, Kolkata 700 064, India.

It has been hypothesized previously that synergistic effect of both amyloid precursor protein intracellular C-terminal domain (AICD) and Aβ aggregation could contribute to Alzheimer's disease pathogenesis. Structural studies of AICD have found no stable globular fold over a broad range of pH. Present work is based on the premises that a conformational switch involving the flipping of C-terminal helix of AICD would be essential for effective binding with the Src homology 2 (SH2) domain of growth factor receptor binding protein-2 (Grb2) and subsequent initiation of Grb2-mediated endo-lysosomal pathway. High-resolution crystal structures of Grb2-SH2 domain bound to AICD peptides reveal a unique mode of binding where the peptides assume a noncanonical conformation that is unlike other structures of AICD peptides bound to protein-tyrosine-binding domains or that of its free state; rather, a flipping of the C-terminal helix of AICD is evident. The involvement of different AICD residues in Grb2-SH2 interaction is further elucidated through fluorescence-based assays. Our results reveal the significance of a specific interaction of the two molecules to optimize the rapid transport of AICD inside endosomal vesicles presumably to reduce the cytotoxic load.
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http://dx.doi.org/10.1016/j.jmb.2011.09.046DOI Listing
November 2011

Intrinsically unstructured proteins and neurodegenerative diseases: conformational promiscuity at its best.

IUBMB Life 2011 Jul;63(7):478-88

Structural Genomics Division, Saha Institute of Nuclear Physics, 1/AF Bidhan Nagar, Kolkata, WB, India.

Neurodegenerative diseases are complex, multifactorial disorders where misfolding of proteins cause aberrant protein-protein interactions. They are not usually characterized by specific mutations especially for nonfamilial disease types. Most of the causative proteins, however, are intrinsically unstructured (IUP), loss of whose fine balance could play pivotal role in these processes. Very fast conformational switch of these IUPs between different functional forms, so as to choose different interaction partners and different functional niches within the cell, is the basic premise on which these proteins maintain their interaction network. We are working on the hypothesis that even small perturbations in conformation leads to disruption of the network and to the disease phenotype. Based on a comprehensive data search, the evidence was obtained for the role of IUPs in neurodegenerative disorders, and their mode of action through conformational promiscuity is elaborated through three case studies.
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http://dx.doi.org/10.1002/iub.498DOI Listing
July 2011

AICD Overexpression in Neuro 2A Cells Regulates Expression of PTCH1 and TRPC5.

Int J Alzheimers Dis 2010 Aug 15;2011. Epub 2010 Aug 15.

Structural Genomics Section, Saha Institute of Nuclear Physics, 1/AF Bidhan Nagar, WB, Kolkata 700 064, India.

Amyloid precursor protein (APP), implicated in Alzheimer's disease, is a transmembrane protein of undetermined function. APP is cleaved by gamma-secretase that releases the APP intracellular domain (AICD) in the cytoplasm. In vitro and in vivo studies have implicated the role of AICD in cell signaling and transcriptional regulation of Gsk3β, KAI1, BACE1, EGFR, and other proteins. In this study, by overexpressing AICD in mouse neuroblastoma cell lines, we have demonstrated the alteration in the expressions of two proteins, patched homolog 1 (PTCH1), a receptor for sonic hedgehog signaling, and transient receptor potential cation channel subfamily C member 5 (TRPC5), a component of receptor-activated nonselective calcium permeant cation channel. Our results indicate the possibility of regulation by AICD in developmental processes as well as in the maintenance of calcium homeostasis at the transcription level.
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http://dx.doi.org/10.4061/2011/239453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2935165PMC
August 2010

A large, systematic molecular-genetic study of G6PD in Indian populations identifies a new non-synonymous variant and supports recent positive selection.

Infect Genet Evol 2010 Dec 14;10(8):1228-36. Epub 2010 Aug 14.

Human Genetics Unit, Indian Statistical Institute, Kolkata, India.

Malaria has been endemic in India. G6PD deficiency is known to confer resistance to malaria. Many G6PD deficiency variants, some of which are India-specific, are known to occur in high frequencies in India. This is the first systematic molecular-genetic study in multiple populations from India drawn from diverse ethnic, socio-cultural and geographical backgrounds. Resequencing of the G6PD gene was carried out in 80 males and then the polymorphic variants were genotyped in 400 individuals of both genders, drawn from 10 ethnic groups of India. Our study has identified one new exonic variant (M159I; exon-5), occurring in multiple populations, that is predicted to result in G6PD deficiency. A strong geographical sub-structuring of known G6PD variants has also been established. We have compared all available data from public-domain resources with those generated in this study to identify the nature and extent of natural selection. Our results (a) provide indication of weak negative selection, and (b) reveal signals of recent positive selection for the G6PD Orissa and G6PD Coimbra mutation bearing haplotypes. These inferences have been interpreted in the light of malarial protection to the populations that have been long exposed to plasmodium infection.
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http://dx.doi.org/10.1016/j.meegid.2010.08.003DOI Listing
December 2010

Grb2-mediated alteration in the trafficking of AbetaPP: insights from Grb2-AICD interaction.

J Alzheimers Dis 2010 ;20(1):275-92

Structural Genomics Section, Saha Institute of Nuclear Physics, Kolkata, India.

The amyloid-beta protein precursor (AbetaPP) is processed by various proteases located along the endosomal lysosomal pathway and any alteration in its trafficking would be important in the pathogenesis of Alzheimer's disease (AD). Our current study is based on the clinical evidence that an AbetaPP intracellular domain (AICD) "adaptor" protein, growth factor receptor protein binding protein 2 (Grb2), gets concentrated in neuronal cell bodies in AD patients. Here we show that both endogenous and exogenously transfected Grb2 interact with AbetaPP in Neuro 2A cells. Endogenous Grb2 partially co-localizes to late endosomal compartments along with AbetaPP and AICD. Increase in the concentration of Grb2 confines it in enlarged late endosomes leading to more sequestration of AbetaPP and AICD within these compartments. This confinement of AbetaPP due to Grb2 overexpression affects its turnover by inhibiting its release via exosomal vesicles. As a consequence, the level of intracellular AbetaPP and AICD increases. The effect of Grb2 overexpression has been verified by knocking down Grb2 as well as by overexpressing Grb2 in Grb2 knocked down cells. Having established the Grb2-mediated trafficking of AICD and its impairment, the significance of its consequence has now become apparent in the downstream events of AD pathogenesis.
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http://dx.doi.org/10.3233/JAD-2010-1371DOI Listing
July 2010

The role of intrinsically unstructured proteins in neurodegenerative diseases.

PLoS One 2009 15;4(5):e5566. Epub 2009 May 15.

Structural Genomics Section, Saha Institute of Nuclear Physics, Kolkata, India.

The number and importance of intrinsically disordered proteins (IUP), known to be involved in various human disorders, are growing rapidly. To test for the generalized implications of intrinsic disorders in proteins involved in Neurodegenerative diseases, disorder prediction tools have been applied to three datasets comprising of proteins involved in Huntington Disease (HD), Parkinson's disease (PD), Alzheimer's disease (AD). Results show, in general, proteins in disease datasets possess significantly enhanced intrinsic unstructuredness. Most of these disordered proteins in the disease datasets are found to be involved in neuronal activities, signal transduction, apoptosis, intracellular traffic, cell differentiation etc. Also these proteins are found to have more number of interactors and hence as the proportion of disorderedness (i.e., the length of the unfolded stretch) increased, the size of the interaction network simultaneously increased. All these observations reflect that, "Moonlighting" i.e. the contextual acquisition of different structural conformations (transient), eventually may allow these disordered proteins to act as network "hubs" and thus they may have crucial influences in the pathogenecity of neurodegenerative diseases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0005566PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2679209PMC
August 2009

Bacterial exotoxins downregulate cathelicidin (hCAP-18/LL-37) and human beta-defensin 1 (HBD-1) expression in the intestinal epithelial cells.

Cell Microbiol 2008 Dec 20;10(12):2520-37. Epub 2008 Aug 20.

National Institute of Cholera and Enteric Diseases, Kolkata, India.

Cathelicidin (hCAP-18/LL-37) and beta-defensin 1 (HBD-1) are human antimicrobial peptides (AMPs) with high basal expression levels, which form the first line of host defence against infections over the epithelial surfaces. The antimicrobial functions owe to their direct microbicidal effects as well as the immunomodulatory role. Pathogenic microorganisms have developed multiple modalities including transcriptional repression to combat this arm of the host immune response. The precise mechanisms and the pathogen-derived molecules responsible for transcriptional downregulation remain unknown. Here, we have shown that enteric pathogens suppress LL-37 and HBD-1 expression in the intestinal epithelial cells (IECs) with Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) exerting the most dramatic effects. Cholera toxin (CT) and labile toxin (LT), the major virulence proteins of V. cholerae and ETEC, respectively, are predominantly responsible for these effects, both in vitro and in vivo. CT transcriptionally downregulates the AMPs by activating several intracellular signalling pathways involving protein kinase A (PKA), ERK MAPKinase and Cox-2 downstream of cAMP accumulation and inducible cAMP early repressor (ICER) may mediate this role of CT, at least in part. This is the first report to show transcriptional repression of the AMPs through the activation of cellular signal transduction pathways by well-known virulence proteins of pathogenic microorganisms.
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http://dx.doi.org/10.1111/j.1462-5822.2008.01227.xDOI Listing
December 2008