Publications by authors named "Ashok K Rout"

Ubiquitin-conjugating enzymes (E2) play a crucial role in the attachment of ubiquitin to proteins. Together with ubiquitin ligases (E3), they catalyze the transfer of ubiquitin (Ub) onto lysines with high chemoselectivity. A subfamily of E2s, including yeast Ubc6 and human Ube2J2, also mediates noncanonical modification of serines, but the structural determinants for this chemical versatility remain unknown.

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Acute myeloid leukaemia (AML) cells interact and modulate components of their surrounding microenvironment into their own benefit. Stromal cells have been shown to support AML survival and progression through various mechanisms. Nonetheless, whether AML cells could establish beneficial metabolic interactions with stromal cells is underexplored.

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Thirty-five peptides selected from functionally-relevant SARS-CoV-2 spike (S), membrane (M), and envelope (E) proteins were suitably modified for immunising MHC class II (MHCII) DNA-genotyped monkeys and matched with HLA-DRβ1* molecules for use in humans. This was aimed at producing the first minimal subunit-based, chemically-synthesised, immunogenic molecules (COLSARSPROT) covering several HLA alleles. They were predicted to cover 48.

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Nuclear magnetic resonance (NMR) has the unique advantage of elucidating the structure and dynamics of biomolecules in solution at physiological temperatures, where they are in constant movement on timescales from picoseconds to milliseconds. Such motions have been shown to be critical for enzyme catalysis, allosteric regulation, and molecular recognition. With NMR being particularly sensitive to these timescales, detailed information about the kinetics can be acquired.

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Chlamydomonas reinhardtii is a single celled alga that undergoes apoptosis in response to UV-C irradiation. UVI31+, a novel UV-inducible DNA endonuclease in C. reinhardtii, which normally localizes near cell wall and pyrenoid regions, gets redistributed into punctate foci within the whole chloroplast, away from the pyrenoid, upon UV-stress.

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Melanoregulin (Mreg) is a small, highly charged, multiply palmitoylated protein present on the membrane of melanosomes. Mreg is implicated in the transfer of melanosomes from melanocytes to keratinocytes, and in promoting the microtubule minus end-directed transport of these organelles. The possible molecular function of Mreg was identified by solving its structure using nuclear magnetic resonance (NMR) spectroscopy.

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Chaperone-mediated autophagy (CMA) is a highly regulated cellular process that mediates the degradation of a selective subset of cytosolic proteins in lysosomes. Increasing CMA activity is one way for a cell to respond to stress, and it leads to enhanced turnover of non-critical cytosolic proteins into sources of energy or clearance of unwanted or damaged proteins from the cytosol. The lysosome-associated membrane protein type 2a (LAMP-2A) together with a complex of chaperones and co-chaperones are key regulators of CMA.

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Article Synopsis
  • EhCaBP1 is a calcium-binding protein from Entamoeba histolytica, consisting of two distinct domains that are both necessary for its stability and functionality.
  • The C-terminal domain has a unique structure, primarily due to the lack of a Phe-Phe interaction commonly found in similar proteins, which led researchers to mutate a specific tyrosine residue to see how it affected the protein's structure and function.
  • The Y81F mutation resulted in a more compact C-terminal structure and altered biological behavior, including changes in membrane localization and actin colocalization, while still allowing the protein to bind actin.
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The mechanism of Ca(2+)-signaling in the protozoan parasite Entamoeba histolytica is yet to be understood as many of the key regulators are still to be identified. E. histolytica encodes a number of multi-EF-hand Ca(2+)-binding proteins (EhCaBPs).

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We have shown that the methodology based on the estimation of root-mean-square deviation (RMSD) between two sets of chemical shifts is very useful to rapidly assign the spectral signatures of (1)H(N), (13)C(α), (13)C(β), (13)C', (1)H(α) and (15)N spins of a given protein in one state from the knowledge of its resonance assignments in a different state, without resorting to routine established procedures (manual and automated). We demonstrate the utility of this methodology to rapidly assign the 3D spectra of a metal-binding protein in its holo-state from the knowledge of its assignments in apo-state, the spectra of a protein in its paramagnetic state from the knowledge of its assignments in diamagnetic state and, finally, the spectra of a mutant protein from the knowledge of the chemical shifts of the corresponding wild-type protein. The underlying assumption of this methodology is that, it is impossible for any two amino acid residues in a given protein to have all the six chemical shifts degenerate and that the protein under consideration does not undergo large conformational changes in going from one conformational state to another.

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The cDNA of UVI31+ was cloned from C. reinhardtii and expressed in E. coli from where the protein was purified to homogeneity.

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We report almost complete sequence specific 1H, 13C and 15N NMR assignments of a 151-residue long calmodulin-like calcium-binding protein from Entamoeba histolytica (EhCaM).

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We propose a methodology that uses GFT (3,2)D CB(CACO)NNH experiment to rapidly collect the data and readily identify six amino acid residue types (Ala, Asn, Asp, Cys, Gly and Ser) in any given protein. Further, the experiment can distinguish the redox state of Cys residues. The proposed experiment in its two forms will have wide range of applications in resonance assignment strategies and structure determination of proteins.

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We present two NMR experiments, (3,2)D HNHA and (3,2)D HNHB, for rapid and accurate measurement of 3J(H N-H alpha) and 3J(N-H beta) coupling constants in polypeptides based on the principle of G-matrix Fourier transform NMR spectroscopy and quantitative J-correlation. These experiments, which facilitate fast acquisition of three-dimensional data with high spectral/digital resolution and chemical shift dispersion, will provide renewed opportunities to utilize them for sequence specific resonance assignments, estimation/characterization of secondary structure with/without prior knowledge of resonance assignments, stereospecific assignment of prochiral groups and 3D structure determination, refinement and validation. Taken together, these experiments have a wide range of applications from structural genomics projects to studying structure and folding in polypeptides.

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