A Gopala Krishna

Background: Gold nanoparticles (AuNP) are extensively used as biophysical tools in the area of medicine and technology due to their distinct properties. However, vivid understanding of the consequences of biomolecule-nanomaterial interactions is still lacking. In this context, we explore the affect of conjugation of Gαi1 subunit (of heterotrimeric G-proteins) to AuNP and examine its consequences. We consider two bio-conjugation strategies covalent and non-covalent binding. Results: Affinity of the AuNP to the Gαi1 is 7.58 × 10 12 M-1. AuNP conjugated Gαi1 exhibits altered kinetics of activation, non-covalent bio-conjugates displays retarded kinetics, up to 0.88 fold when GTPγS was used as ligand, of protein activation contrary to covalent conjugates which accelerates it to ~ 5 fold. Conjugation influence intrinsic Gαi1 GTPase function in conflicting modes. Non-covalent conjugation inhibits GTPase function (decrease in activity upto 0.8 fold) whilst covalent conjugation drastically accelerates it (12 fold increase in activity). Altered basal nucleotide uptake in both types of conjugates and GTPase function in non-covalent conjugate are almost comparable except for GTPase property of covalent conjugate. The effect is despite the fact that conjugation does not change global conformation of the protein. Conclusion: These findings provide clear evidence that nanoparticles, in addition to 'passive interaction' with protein (biomolecule), can interact "actively" with biomolecule and modify its function. This concept should be considered while engineering nanoparticle based delivery systems in medicine.

In this article, Figure 1 was published with incorrect image. The corrected Figure 1 is shown here. (Figure presented.) a Representation of crystal structure of APJR-AMG3054 complex (PDB: 5VBL). The modified peptide ligand is depicted in green (sticks) and the receptor is shown in orange (cartoon). b Amino acid sequence of apelin peptide isoforms. Residues of Apelin-36, Apelin-17, Apelin-13, and Pyro-glutamate (Pyr) Apelin-13 are indicated. Grey arrows show the site of cleavage by PCSK3/furin enzyme in the larger isoforms, Apelin-36 and Apelin-17 c Comparison between Apelin-17 and AMG3054. The latter is a modified cyclic peptide (cyclised between Glu10 and Lys13, indicated by dotted lines). The modified residues of AMG3054 are coloured in pink. hARG, homoarginine; CHA, cyclohexylalanine; OIC, Octahydroindole-2-carboxylic Acid; NLE, Norleucine; 4-Cl-PHE, 4-choloro-Phenylalanine The original article has been corrected.

P21 Activated Kinase 1 (PAK1) is an oncogenic serine/threonine kinase known to play a significant role in the regulation of cytoskeleton and cell morphology. Runt-related transcription factor 3 (RUNX3) was initially known for its tumor suppressor function, but recent studies have reported the oncogenic role of RUNX3 in various cancers. Previous findings from our laboratory provided evidence that Threonine 209 phosphorylation of RUNX3 acts as a molecular switch in dictating the tissue-specific dualistic functions of RUNX3 for the first time. Based on these proofs and to explore the translational significance of these findings, we designed a small peptide (RMR) from the protein sequence of RUNX3 flanking the Threonine 209 phosphorylation site. The selection of this specific peptide from multiple possible peptides was based on their binding energies, hydrogen bonding, docking efficiency with the active site of PAK1 and their ability to displace PAK1–RUNX3 interaction in our prediction models. We found that this peptide is stable both in in vitro and in vivo conditions, not toxic to normal cells and inhibits the Threonine 209 phosphorylation in RUNX3 by PAK1. We also tested the efficacy of this peptide to block the RUNX3 Threonine 209 phosphorylation mediated tumorigenic functions in in vitro cell culture models, patient-derived explant (PDE) models and in in vivo tumor xenograft models. These results proved that this peptide has the potential to be developed as an efficient therapeutic molecule for targeting RUNX3 Threonine 209 phosphorylation-dependent tumor phenotypes.

Aldose reductase (AR) catalyzes the conversion of aldoses to their corresponding polyols in yeasts and filamentous fungi. ARs have the potential to be exploited for the enzymatic production of xylitol, thus the identification and characterization of ARs from novel strains have gained interest. In this study, we chose the novel yeast Debaryomyces nepalensis as a source for an AR gene. For the first time, here we isolated the AR gene from D. nepalensis (DnAR) that encodes a protein of 320 amino acids with a predicted molecular weight of 36.7 kDa using the RACE technique. It was heterologously expressed in Escherichia coli as a His-tagged fusion protein and purified. The enzyme showed strict NADPH dependence and broad substrate specificity with high catalytic efficiency for arabinose, xylose and 3-nitro benzaldehyde. Remarkably, it was active and stable in the presence of high concentrations of salts (KCl/NaCl), thus exhibiting halotolerance. It showed 75% and 45% activity at 0.5 and 1 M concentration of salts respectively. Enzyme half-lifetime at 1 M KCl and 1 M NaCl was found to be 30 h and 16.5 h respectively. Furthermore, to explore the structural basis of its halotolerance, we built a homology model of DnAR. Surprisingly, we found that the existence of a uniform negative electrostatic potential over the protein surface, which is one of the known mechanisms governing protein halotolerance. Therefore, DnAR could be exploited as a biocatalyst to develop an enzyme based bioprocess for xylitol production from lignocelluloses. Moreover, this is the first report providing the genetic sequence and biochemical characteristics of a halotolerant aldose reductase.

Background: Phospholipid scramblases are a group of four homologous proteins conserved from C. elegans to human. In human, two members of the scramblase family, hPLSCR1 and hPLSCR3 are known to bring about Ca2+ dependent translocation of phosphatidylserine and cardiolipin respectively during apoptotic processes. However, affinities of Ca2+/Mg2+ binding to human scramblases and conformational changes taking place in them remains unknown. Methods: In the present study, we analyzed the Ca2+ and Mg2+ binding to the calcium binding motifs of hPLSCR1-4 and hPLSCR1 by spectroscopic methods and isothermal titration calorimetry. Results: The results in this study show that (i) affinities of the peptides are in the order hPLSCR1 > hPLSCR3 > hPLSCR2 > hPLSCR4 for Ca2+ and in the order hPLSCR1 > hPLSCR2 > hPLSCR3 > hPLSCR4 for Mg2+, (ii) binding of ions brings about conformational change in the secondary structure of the peptides. The affinity of Ca2+ and Mg2+ binding to protein hPLSCR1 was similar to that of the peptide I. A sequence comparison shows the existence of scramblase-like motifs among other protein families. Conclusions: Based on the above results, we hypothesize that the Ca2+ binding motif of hPLSCR1 is a novel type of Ca2+ binding motif. General significance: Our findings will be relevant in understanding the calcium dependent scrambling activity of hPLSCRs and their biological function. © 2009 Elsevier B.V. All rights reserved.

Apelin receptor (APJR) is a class A peptide (apelin) binding G protein-coupled receptor (GPCR) that plays a significant role in regulating blood pressure, cardiac output, and maintenance of fluid homeostasis. It is activated by a wide range of endogenous peptide isoforms of apelin and elabela. The apelin peptide isoforms contain distinct structural features that aid in ligand recognition and activation of the receptor. Site-directed mutagenesis and structure-based studies have revealed the involvement of extracellular and transmembrane regions of the receptor in binding to the peptide isoforms. The structural features of APJR activation of the receptor as well as mediating G-protein and β-arrestin-mediated signaling are delineated by multiple mutagenesis studies. There is increasing evidence that the structural requirements of APJR to activate G-proteins and β-arrestins are different, leading to biased signaling. APJR also responds to mechanical stimuli in a ligand-independent manner. A multitude of studies has focused on developing both peptide and non-peptide agonists and antagonists specific to APJR. Apelin/elabela-activated APJR orchestrates major signaling pathways such as extracellular signal-regulated kinase (ERKs), protein kinase B (PKB/Akt), and p70S. This review focuses on the structural and functional characteristics of apelin, elabela, APJR, and their interactions involved in the binding and activation of the downstream signaling cascade. We also focus on the diverse signaling profile of APJR and its ligands and their involvement in various physiological systems.

Protein aggregation and inclusion body formation have been a key causal phenomenon behind a majority of neurodegenerative disorders. Various approaches aimed at preventing the formation/elimination of protein aggregates are being developed to control these diseases. Molecular chaperones are a class of protein that not only direct the functionally relevant fold of the protein but also perform quality control against stress, misfolding/aggregation. Genes that encode molecular chaperones are induced and expressed in response to extreme stress conditions to “salvage” the cell by the “unfolded protein response” (UPR) signaling pathway. Here we describe in detail the various in vitro and in vivo assays involved in identifying the chaperone activity of proteins using human calnuc as a model protein. Calnuc is a Golgi resident, calcium-binding protein, identified as chaperone protein and is reported to protect the cells against the cytotoxicity caused by amyloidosis and ER stress. Calnuc is also reported to regulate G αi activity and inflammation apart from the role of chaperoning against amyloid proteins.

Human ocular albinism type 1 protein (OA1) - a member of the G-protein coupled receptor (GPCR) superfamily - is an integral membrane glycoprotein expressed exclusively by intracellular organelles known as melanocytes, and is responsible for the proper biogenesis of melanosomes. Mutations in the Oa1 gene are responsible for the disease ocular albinism. Despite its clinical importance, there is a lack of in-depth understanding of its structure and mechanism of activation due to the absence of a crystal structure. In the present study, homology modeling was applied to predicting OA1 structure following thorough sequence analysis and secondary structure predictions. The predicted model had the signature residues and motifs expected of GPCRs, and was used for carrying out molecular docking studies with an endogenous ligand, L-DOPA and an antagonist, dopamine; the results agreed quite well with the available experimental data. Finally, three sets of explicit molecular dynamics simulations were carried out in lipid bilayer, the results of which not only confirmed the stability of the predicted model, but also helped witness some differences in structural features such as rotamer toggle switch, helical tilts and hydrogen bonding pattern that helped distinguish between the agonist- and antagonist-bound receptor forms. In place of the typical "D/ERY"- motif-mediated "ionic lock", a hydrogen bond mediated by the "DAY" motif was observed that could be used to distinguish the agonist and antagonist bound forms of OA1. In the absence of a crystal structure, this study helped to shed some light on the structural features of OA1, and its behavior in the presence of an agonist and an antagonist, which might be helpful in the future drug discovery process for ocular albinism. © Springer-Verlag 2011.

Phospholipid scramblases are a group of homologous proteins that are conserved in all eukaryotic organisms. They are believed to be involved in destroying plasma membrane phospholipid asymmetry at critical cellular events like cell activation, injury and apoptosis. However, a detailed mechanism of phospholipid scrambling still awaits a proper understanding. The most studied member of this family, phospholipid scramblase 1 (PLSCR1) (a 37 kDa protein), is involved in rapid Ca2+ dependent transbilayer redistribution of plasma membrane phospholipids. Recently the function of PLSCR1 as a phospholipids translocator has been challenged and evidences suggest that PLSCR1 acts as signaling molecule. It has been shown to be involved in protein phosphorylation and as a potential activator of genes in response to interferon and other cytokines. Interferon induced rapid biosynthesis of PLSCR1 targets some of the protein into the nucleus, where it binds to the promoter region of inositol 1,4,5-triphosphate (IP3) receptor type 1 (IP3R1) gene and induces its expression. Palmitoylation of PLSCR1 acts as a switch, controlling its localization either to the PM or inside the nucleus. In the present review, we discuss the current understanding of PLSCR1 in relation to its trafficking, localization and signaling functions. © 2007 Elsevier Inc. All rights reserved.

The characteristic features of cancer cells are aberrant (acidic) intracellular pH and elevated levels of phosphatidylserine. The primary focus of cancer research is concentrated on the discovery of biomarkers directed towards early diagnosis and therapy. It has been observed that azoxymethane-treated mice demonstrate an increased expression of calnuc (a multi-domain, Ca2+- and DNA-binding protein) in their colon, suggesting it to be a good biomarker of carcinogenesis. We show that culture supernatants from tumor cells have significantly higher amounts of secreted calnuc compared to non-tumor cells, selectively packaged into exosomes. Exosomal calnuc is causal for epithelial-mesenchymal transition and atypical migration in non-tumor cells, which are key events in tumorigenesis and metastasis. In vitro studies reveal a significant affinity for calnuc towards phosphatidylserine, specifically to its C-terminal region, leading to the formation of 'molten globule' conformation. Similar structural changes are observed at acidic pH (pH 4), which demonstrates the role of the acidic microenvironment in causing the molten globule conformation and membrane interaction. On a precise note, we propose that the molten globule structure of calnuc caused by aberrant conditions in cancer cells to be the causative mechanism underlying its exosome-mediated secretion, thereby driving metastasis.