Gopala Krishna Aradhyam

The repertoire of G protein-coupled receptors in the sea squirt Ciona intestinalis

BackgroundG protein-coupled receptors (GPCRs) constitute a large family of integral transmembrane receptor proteins that play a central role in signal transduction in eukaryotes. The genome of the protochordate Ciona intestinalis has a compact size with an ancestral complement of many diversified gene families of vertebrates and is a good model system for studying protochordate to vertebrate diversification. An analysis of the Ciona repertoire of GPCRs from a comparative genomic perspective provides insight into the evolutionary origins of the GPCR signalling system in vertebrates.ResultsWe have identified 169 gene products in the Ciona genome that code for putative GPCRs. Phylogenetic analyses reveal that Ciona GPCRs have homologous representatives from the five major GRAFS (Glutamate, Rhodopsin, Adhesion, Frizzled and Secretin) families concomitant with other vertebrate GPCR repertoires. Nearly 39% of Ciona GPCRs have unambiguous orthologs of vertebrate GPCR families, as defined for the human, mouse, puffer fish and chicken genomes. The Rhodopsin family accounts for ~68% of the Ciona GPCR repertoire wherein the LGR-like subfamily exhibits a lineage specific gene expansion of a group of receptors that possess a novel domain organisation hitherto unobserved in metazoan genomes.ConclusionComparison of GPCRs in Ciona to that in human reveals a high level of orthology of a protochordate repertoire with that of vertebrate GPCRs. Our studies suggest that the ascidians contain the basic ancestral complement of vertebrate GPCR genes. This is evident at the subfamily level comparisons since Ciona GPCR sequences are significantly analogous to vertebrate GPCR subfamilies even while exhibiting Ciona specific genes. Our analysis provides a framework to perform future experimental and comparative studies to understand the roles of the ancestral chordate versions of GPCRs that predated the divergence of the urochordates and the vertebrates.

Calcium binding studies of peptides of human phospholipid scramblases 1 to 4 suggest that scramblases are new class of calcium binding proteins in the cell

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.

The single C‐terminal helix of human phospholipid scramblase 1 is required for membrane insertion and scrambling activity

Human phospholipid scramblase 1 (hPLSCR1) belongs to the ATP‐independent class of phospholipid translocators which possess a single EF‐hand‐like Ca2+‐binding motif and also a C‐terminal helix (CTH). The CTH domain of hPLSCR1 was believed to be a putative single transmembrane helix at the C‐terminus. Recent homology modeling studies by Bateman et al. predicted that the hydrophobic nature of this helix is due to its packing in the core of the protein domain and proposed that this is not a true transmembrane helix [Bateman A, Finn RD, Sims PJ, Wiedmer T, Biegert A & Johannes S. Bioinformatics 2008, 25, 159]. To determine the exact function of the CTH of hPLSCR1, we deleted the CTH domain and determined: (a) whether CTH plays any role beyond membrane anchorage, (b) the functional consequences of CTH deletion, and (c) any conformational changes associated with CTH in a lipid environment. In vitro reconstitution studies confirm that the predicted CTH is required for membrane insertion and scrambling activity. CTH deletion caused a 50% decrease in binding affinity of Ca2+ for ∆CTH‐hPLSCR1 (Ka = 115 μm) compared with hPLSCR1 (Ka = 249 μm). Far UV‐CD studies revealed that the CTH peptide adopts α‐helicity only in the presence of SDS micelles and negatively charged vesicles, indicating that electrostatic interactions are required for insertion of the peptide. CTH peptide‐quenching studies confirm that the predicted CTH inserts into the membrane and its ability to interact with the membrane depends on the presence of charge interactions. TOXCAT assay revealed that CTH of hPLSCR1 does not oligomerize in the membrane. We conclude that CTH is required for membrane insertion and Ca2+ coordination and also plays an important role in the functional conformation of hPLSCR1.

Chemistry of conjugation to gold nanoparticles affects G-protein activity differently

BackgroundGold 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.ResultsAffinity 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.ConclusionThese 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.

Ion‐binding properties of Calnuc, Ca2+ versus Mg2+– Calnuc adopts additional and unusual Ca2+‐binding sites upon interaction with G‐protein

Calnuc is a novel, highly modular, EF‐hand containing, Ca2+‐binding, Golgi resident protein whose functions are not clear. Using amino acid sequences, we demonstrate that Calnuc is a highly conserved protein among various organisms, from Ciona intestinalis to humans. Maximum homology among all sequences is found in the region that binds to G‐proteins. In humans, it is known to be expressed in a variety of tissues, and it interacts with several important protein partners. Among other proteins, Calnuc is known to interact with heterotrimeric G‐proteins, specifically with the α‐subunit. Herein, we report the structural implications of Ca2+ and Mg2+ binding, and illustrate that Calnuc functions as a downstream effector for G‐protein α‐subunit. Our results show that Ca2+ binds with an affinity of 7 μm and causes structural changes. Although Mg2+ binds to Calnuc with very weak affinity, the structural changes that it causes are further enhanced by Ca2+ binding. Furthermore, isothermal titration calorimetry results show that Calnuc and the G‐protein bind with an affinity of 13 nm. We also predict a probable function for Calnuc, that of maintaining Ca2+ homeostasis in the cell. Using Stains‐all and terbium as Ca2+ mimic probes, we demonstrate that the Ca2+‐binding ability of Calnuc is governed by the activity‐based conformational state of the G‐protein. We propose that Calnuc adopts structural sites similar to the ones seen in proteins such as annexins, c2 domains or chromogrannin A, and therefore binds more calcium ions upon binding to Giα. With the number of organelle‐targeted G‐protein‐coupled receptors increasing, intracellular communication mediated by G‐proteins could become a new paradigm. In this regard, we propose that Calnuc could be involved in the downstream signaling of G‐proteins.

Suramin Affects Coupling of Rhodopsin to Transducin

Suramin, a polysulfonated naphthylurea, is under investigation for the treatment of several cancers. It interferes with signal transduction through G(s), G(i), and G(o), but structural and kinetic aspects of the molecular mechanism are not well understood. Here, we have investigated the influence of suramin on coupling of bovine rhodopsin to G(t), where G-protein activation and receptor structure can be monitored by spectroscopic in vitro assays. G(t) fluorescence changes in response to rhodopsin-catalyzed nucleotide exchange reveal that suramin inhibits G(t) activation by slowing down the rate of complex formation between metarhodopsin-II and G(t). The metarhodopsin-I/-II photoproduct equilibrium, GTPase activity, and nucleotide uptake by G(t) are unaffected. Attenuated total reflection Fourier transform infrared spectroscopy shows that the structure of rhodopsin, metarhodopsin-II, and the metarhodopsin-II G(t) complex is also not altered. Instead, suramin dissociates G(t) from disk membranes in the dark, whereas metarhodopsin-II G(t) complexes are stable. Förster resonance energy transfer suggests a suramin-binding site near Trp(207) on the G(t alpha) subunit (K(d) approximately 0.5 microM). The kinetic analyses and the structural data are consistent with a specific perturbation by suramin of the membrane attachment site on G(t alpha). Disruption of membrane anchoring may contribute to some of the effects of suramin exerted on other G-proteins.

Snail‐mediated Cripto‐1 repression regulates the cell cycle and epithelial–mesenchymal transition‐related gene expression

Transcription factor Snail mediates epithelial to mesenchymal transitions (EMT) by coordinate repression of epithelial markers, facilitating mass cell movement during germ layer formation. Aberrant reprogramming in its signaling pathways causes metastatic cancer. Snails involvement in “fate‐changing” decisions is however not understood. Cripto‐1 shares a common temporal expression pattern with Snail during development. While Cripto‐1 is required for mammary morphogenesis and hematopoietic stem cell renewal, its unregulated expression causes metastatic cancers. Therefore, we suspected that Snail regulates the expression of Cripto‐1 controlling decisions such as motility, transformation and differentiation. We demonstrate that Snail represses Cripto‐1 gene by direct transcriptional interaction and propose a novel mechanism by which it co‐ordinately regulates cell fate decisions during development and could be causal of cancers.

Structural insights into human GPCR protein OA1: a computational perspective

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.

Serine Protease Activity of Calnuc REGULATION BY Zn2+ AND G PROTEINS

Background: Calnuc is a multidomain Ca2+-binding protein with many interacting partners but whose function is still elusive. Results: Calnuc is a serine protease with its active site catalytic triad present in the C-terminal domain. Conclusion: The serine protease activity of calnuc is allosterically regulated by Zn2+-binding and its interaction with G protein α subunit. Significance: Novel proteolytic function of calnuc will have vital implications in its physiological role. The functions of calnuc, a novel Ca2+-binding protein with multiple structural domains and diverse interacting partners, are yet unknown. We demonstrate unknown facets of calnuc, which is a serine protease in which Ser-378 of GXSXG motif, Asp-328 of DTG motif, and His-339 form the “catalytic triad,” locating the enzyme active site in the C-terminal region. Analogous to the active site of Zn2+ carboxypeptidases, calnuc has two high affinity (Kd ∼ 20 nm), well conserved Zn2+-binding sites near its N terminus, although it is inactive as a peptidase. Zn2+ binding allosterically and negatively regulates the serine protease activity of calnuc, inhibition being caused by an “open to close” change in its conformation not seen upon Ca2+ binding. Most strikingly, interaction with G protein α subunit completely inhibits the enzymatic activity of calnuc. We thus illustrate that G proteins and Zn2+ act as two “keys” that control enzymatic activity of calnuc, arresting it in “locked” state. Calnuc, therefore, exists dynamically in two different forms, (i) as a Ca2+-binding protein in Zn2+-bound form and (ii) as a protease in Zn2+-free form, commissioning it to perform multiple functions.

Calnuc: Emerging roles in calcium signaling and human diseases

Calnuc is a recently discovered multidomain protein with EF‐hand calcium binding sites. Several studies have reported various interacting partners for calnuc and, therefore, also different sites of localization in the cell. It interacts with important molecules such as DNA, G protein, COX, and amyloid precursor protein among others in addition to being involved in stress response and trafficking. The immense possibilities (of various functions this protein might be involved in) implicate great future in medicine and physiology. Preliminary studies also implicate the possibility of calnuc being involved in some of the human diseases. These initial observations imply the functions that this protein might be involved in. This review emphasizes the importance of further research on this protein.