Rakhi Dasgupta

Hypoglycosylation of dystroglycan due to T192M mutation: a molecular insight behind the fact.

Abnormal glycosylation of dystroglycan (DG), a transmembrane glycoprotein, results in a group of diseases known as dystroglycanopathy. A severe dystroglycanopathy known as the limb girdle disease MDDGC9 [OMIM: 613818] occurs as a result of hypoglycosylation of alpha subunit of DG. Reasons behind this has been traced back to a point mutation (T192M) in DG that leads to weakening of interactions of DG protein with laminin and subsequent loss of signal flow through the DG protein. In this work we have tried to analyze the molecular details of the interactions between DG and laminin1 in order to propose a mechanism about the onset of the disease MDDGC9. We have observed noticeable changes between the modeled structures of wild type and mutant DG proteins. We also have employed molecular docking techniques to study and compare the binding interactions between laminin1 and both the wild type and mutant DG proteins. The docking simulations have revealed that the mutant DG has weaker interactions with laminin1 as compared to the wild type DG. Till date there are no previous reports that deal with the elucidation of the interactions of DG with laminin1 from the molecular level. Our study is therefore the first of its kind which analyzes the differences in binding patterns of laminin1 with both the wild type and mutant DG proteins. Our work would therefore facilitate analysis of the molecular mechanism of the disease MDDGC9. Future work based on our results may be useful for the development of suitable drugs against this disease.

Green synthesized cerium oxide nanoparticle: A prospective drug against oxidative harm

Cerium oxide nanoparticle (CeONP) of size 2-3nm was synthesized by a new, simple and green method at ambient temperature, using cerium nitrate as prime precursor and Aloe vera leaf extract as stabilizing agent. Of the two oxidation states (+3) and (+4) of cerium, it was dominantly present in (+3) state in CeONP and cyclic conversion of Ce(III)O→Ce(IV)O→Ce(III)O by reaction with H2O2 implied uninterrupted antioxidant property of CeONP. Moreover, the higher oxygen defect in the crystal lattice produced particles with higher antioxidant activity. CeONP was found to neutralize the deleterious effects of H2O2 viz., cell death, generation of intracellular reactive oxygen species and loss of connectivity in mouse neural cells. Therefore, CeONP might have potential use in future as an anti-oxidant drug.

GroEL to DnaK chaperone network behind the stability modulation of σ32 at physiological temperature in Escherichia coli

The stability of heat‐shock transcription factor σ32 in Escherichia coli has long been known to be modulated only by its own transcribed chaperone DnaK. Very few reports suggest a role for another heat‐shock chaperone, GroEL, for maintenance of cellular σ32 level. The present study demonstrates in vivo physical association between GroEL and σ32 in E. coli at physiological temperature. This study further reveals that neither DnaK nor GroEL singly can modulate σ32 stability in vivo; there is an ordered network between them, where GroEL acts upstream of DnaK.

Analyses of the presence of mutations in Dystrophin protein to predict their relative influences in the onset of Duchenne Muscular Dystrophy

Muscle plays a vital role in the life of vertebrates like humans. Muscle contraction is the only criterion required for locomotion. Muscle fibers also play a vital role as the provider of mechanical strength and act as a large repository of building blocks for protein synthesis in living beings. Muscles function as per the messages received from the extra-cellular signals. One of the central players responsible for capturing and transmission of extra-cellular signals to maintain the integrity of muscle function is the protein called Dystrophin (Dp). However, the wild type Dp protein accumulates some mutations which lead to a severe disease called Duchenne Muscular Dystrophy (DMD). The disease is so frequent that it is known to affect 1 in 3500 newborns per year. There are a number of reports that identify the mutations leading to DMD. Interestingly, it is also observed that the type of mutations affects the severity of the disease. But the biochemical mechanism of the DMD onset is still obscure. In the present scenario, an attempt has been made to analyze the mutations in the development of the disease. We analyzed the changes in secondary structure, solvent accessibility and stability of the Dp protein associated with the mutations. We tried to correlate the type of mutations with the severity of the disease. So far this is the first report that deals with the analyses of the mutations leading to DMD. This study would therefore be essential to come up with a plausible mechanism of DMD disease onset.

In-silico characterization of Formin Binding Protein 4 Family of proteins

Members of the Formin Binding Protein 4 Family or the FNBP4 were indirectly reported to be associated with many of the biological processes. These proteins possess two WW domains. So far there are practically no reports regarding the characterization and classification of the protein by any means. Keeping in mind the importance of the proteins from this FNBP4 family, we have tried an in silico approach to come up with a comprehensive analysis of the proteins. We have analyzed the proteins by considering their sequence conservation, their phylogenetic distributions among the different organisms. We have also investigated the functional properties of the WW domains in the proteins. Finally, we have made an attempt to elucidate the structural details of the domains and predicted the possible modes of their interactions. Our findings show that FNBP4 is eukaryotic in its distribution and follows a trend of evolution where animal and plant homologues have evolved in an independent manner. While the WW domain is the only common motif present across the FNBP4 family of proteins, there are different classes (mainly two) of WW domains that are found among different FNBP4 proteins. Structure function predictions indicate a possible role of FNBP4 in either protein stabilization control or transcript processing. Our study on FNBP4 may therefore open up new avenues to generate new interest in this highly important but largely unexplored class of proteins. Future studies with proteins from this family may answer many important questions of protein-protein interactions in different biologically important processes.

Structural and Functional Characterization of Arabidopsis thaliana WW Domain Containing Protein F4JC80

WW domains are the smallest known independently foldable protein structural motifs that are involved in cellular events like protein turnover, splicing, development, and tumor growth control. These motifs bind the polyproline rich ligands. While the WW domains of animal origin are well characterized, the same from plant origin are not well documented yet. Despite the small repertoire of WW proteome of plants (in comparison to animal WW proteome) functional diversity is reported to be equally vivid for plants also. Here, for the first time, we report the structural and functional properties of an Arabidopsis thaliana (At) WW domain containing protein F4JC80 by using homology modeling and docking techniques. Our findings report that the At F4JC80 protein contains two WW domains which bear the standard triple β sheet structure and structurally and functionally resemble Class I WW domains of E3 ubiquitin ligase family but their structural differences impact their polypeptide binding abilities differently.

Understanding the Interaction of Human Formin Binding Protein 4 with Formin FMN1

The proline rich formin homolog 1 (FH1) region of mouse formin FMN1 was initially reported to bind to WW domains and mediate its interaction with formin binding protein 4 (FNBP4) via the WW domain of FNBP4. However further structural, biochemical and functional details about this interaction have never been reported. The nature of the study that first reported this interaction, along with lack of further information, later created doubt about the possibility of this interaction under cellular environment. In this context, this computational study confirms the binding of mouse formin FMN1 FH1 with the 1st WW domain of FNBP4. Combined with our previous reports, this study concludes that only the 1st WW domain of FNBP4 is able to mediate its interaction with formins FH1 regions and its binding is stronger to the PPXXPP motif compared to the PPXP or PPXPP motifs, all of which are found in formin FH1 region.

In vitro holdase activity of E. coli small heat-shock proteins IbpA, IbpB and IbpAB: a biophysical study with some unconventional techniques.

E. coli small heat shock proteins IbpA and IbpB (inclusion body binding proteins A and B) are known to act as holding chaperones on denaturing, aggregate-prone proteins. But, there is no clear understanding about which of the IbpA and IbpB has more holdase activity and how the holdase activity of one was influenced by the presence of the other. This study was conducted to resolve the questions, using some uncommon physical techniques like dynamic light scattering, micro-viscometry and atomic force microscopy in addition to the common techniques of spectrophotometry and spectrofluorimetry. The holdase activity was investigated on the heat-denatured L-lactate dehydrogenase (LDH) of rabbit muscle. LDH was found to be deactivated completely without any aggregation at 52°C and with transient aggregation at 60°C; molecular dynamics simulation also revealed that at 52°C, denaturation occurred only at the active site of LDH. When LDH was allowed to be deactivated in the presence of IbpA, IbpB or (IbpA + IbpB), partial inhibition of i) denaturation at 52°C and ii) aggregation at 60°C were observed. The results further demonstrated that the holdase activity of IbpB was higher than that of IbpA and their combined effect was higher than their individual one.

The Effect of T192M Mutation in Stability of Alpha Dystroglycan: Study with Molecular Dynamics Simulation

Alpha-dystroglycan (α-DG), a cell surface receptor links extracellular matrix with cellular cytoskeleton. Its post translational modification is carried out with number of glycosyltransferases, depending on cell types to make the ligand specific mature α-DG receptor protein. However, T192M mutation in α-DG has been found to cause hypo-glycosylation of the protein disabling its Laminin binding form and thereby triggering the onset of a limb girdle muscular dystrophy affecting early childhood. Here for the first time we exploit the effect of this mutation in protein conformational stability. We have found that this mutation leads to significant changes in secondary structure of the protein as well as in the accessible surface area. All these changes also hamper the crucial disulfide bond that is required to maintain the globular fold at the N terminus of α-DG. This molecular insight will therefore be useful for developing new therapeutic approaches to overcome the disease state.

Evolutionary analysis of prokaryotic heat-shock transcription regulatory protein σ32

Heat-stress to any living cell is known to trigger a universal defense response, called heat-shock response, with rapid induction of tens of different heat-shock proteins. Bacterial heat-shock genes are transcribed by the σ(32)-bound RNA polymerase instead of the normal σ(70)-bound RNA polymerase. In this study, the diversity in sequence, variation in secondary structure and function amongst the different functional regions of the proteobacterial σ(32) family of proteins, and their phylogenetic relationships have been analyzed. Bacterial σ(32) proteins can be subdivided into different functional regions which are referred to as regions 2, 3, and 4. There is a great deal of sequence conservation among the functional regions of proteobacterial σ(32) family of proteins though some mutations are also present in these regions. Region 2 is the most conserved one, while region 4 has comparatively more variable sequences. In the present work, we tried to explore the effects of mutations in these regions. Our study suggests that the sequence diversities due to natural mutations in the different regions of proteobacterial σ(32) family lead to different functions. So far, this study is the first bioinformatic approach towards the understanding of the mechanistic details of σ(32) family of proteins using the protein sequence information only. This study therefore may help in elucidating the hitherto unknown molecular mechanism of the functionalities of σ(32)family of proteins.