A degenerate primer allows amplification of part of the 3 '-terminus of three distinct carlavirus species

Sequences of the coat protein amino acids of definitive and tentative species of carlaviruses deposited in GenBank were aligned and a region of seven amino acids (GLGVPTE) was found to be conserved. The corresponding nucleotides were aligned, allowing the design of a degenerate primer that together with an oligo dT anti-sense primer, was effective for the detection of three distinct carlavirus species, two transmitted by aphids and one by whitefly. These primers have the advantage that about 940 nt from the 3'-terminus, comprising part of the CP gene (about 60%), the 11 K gene, and the terminal untranslated region can be amplified for sequencing. The fact that this amino acid sequence is conserved in almost all of the sequenced carlaviruses, allows the prediction that this primer pair will be useful as a diagnostic tool for carlavirus species. (C) 2007 Elsevier B.V. All rights reserved.

Influence of N-Terminus Modifications on the Biological Activity, Membrane Interaction, and Secondary Structure of the Antimicrobial Peptide Hylin-a1

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The N terminus of the human alpha(1D)-adrenergic receptor prevents cell surface expression

We previously reported that truncation of the N-terminal 79 amino acids of alpha(1D)-adrenoceptors (Delta(1-79)alpha(1D)-ARs) greatly increases binding site density. In this study, we determined whether this effect was associated with changes in alpha(1D)-AR subcellular localization. Confocal imaging of green fluorescent protein (GFP)-tagged receptors and sucrose density gradient fractionation suggested that full-length alpha(1D)-ARs were found primarily in intracellular compartments, whereas Delta(1-79)alpha(1D)-ARs were translocated to the plasma membrane. This resulted in a 3- to 4-fold increase in intrinsic activity for stimulation of inositol phosphate formation by norepinephrine. We determined whether this effect was transplantable by creating N-terminal chimeras of alpha(1)-ARs containing the body of one subtype and the N terminus of another (alpha(1A) NT-D, alpha(1B) NT-D, alpha(1D) NT-A, and alpha(1D)NT-B). When expressed in human embryonic kidney 293 cells, radioligand binding revealed that binding densities of alpha(1A)- or alpha(1B)-ARs containing the alpha(1D)-N terminus decreased by 86 to 93%, whereas substitution of alpha(1A)- or alpha(1B)-N termini increased alpha(1D)-AR binding site density by 2- to 3-fold. Confocal microscopy showed that GFP-tagged alpha(1D)NT-B-ARs were found only on the cell surface, whereas GFP-tagged alpha(1B)NT-D-ARs were completely intracellular. Radioligand binding and confocal imaging of GFP-tagged alpha(1D)- and Delta(1-79)alpha(1D)-ARs expressed in rat aortic smooth muscle cells produced similar results, suggesting these effects are generalizable to cell types that endogenously express alpha(1D)-ARs. These findings demonstrate that the N-terminal region of alpha(1D)-ARs contain a transplantable signal that is critical for regulating formation of functional bindings, through regulating cellular localization.

Model peptides mimic the structure and function of the N-terminus of the pore-forming toxin sticholysin II

To investigate the role of the N-terminal region in the lytic mechanism of the pore-forming toxin sticholysin II (St II), we studied the conformational and functional properties of peptides encompassing the first 30 residues of the protein. Peptides containing residues 1-30 (P1-30) and 11-30 (P11-30) were synthesized and their conformational properties were examined in aqueous solution as a function of peptide concentration, pH, ionic strength, and addition of the secondary structure-inducing solvent trifluoroethanol (TFE). CD spectra showed that increasing concentration, pH, and ionic strength led to aggregation of P1-30; as a consequence, the peptide acquired beta-sheet conformation. In contrast, P11-30 exhibited practically no conformational changes under the same conditions, remaining essentially structureless. Moreover, this peptide did not undergo aggregation. These differences clearly point to the modulating effect of the first 10 hydrophobic residues on the peptides aggregation and conformational properties. In TFE both the first ten hydrophobic peptides acquired alpha-helical conformation, albeit to a different extent, P11-30 displayed lower alpha-helical content. P1-30 presented a larger-fraction of residues in alpha-helical conformation in TFE than that found in St II's crystal structure for that portion of the protein. Since TFE mimics the membrane em,, such increase in helical content could also occur upon toxin binding to membranes and represent a step in the mechanism of pore formation. The peptides conformational properties correlated well with their functional behaviour. Thus, P1-30 exhibited much higher hemolytic activity than P11-30. In addition, P11-30 was able to block the toxin's hemolytic activity. The size of pores formed in red blood cells by P 1-30 was estimated by measuring the permeability PEGs of different molecular mass. The pore radius (0.95 +/- 0.01 nm) was very similar to that of the PEGs of different pore formed by the toxin. The results demonstrate that the synthetic peptide P1-30 is a good model of St 11 conformation and function and emphasize the contribution of the toxin's N-terminal region, and, in particular, the hydrophobic residues 1-10 to pore formation. (c) 2005 Wiley Periodicals, Inc.

Functional and Topological Studies with Trp-Containing Analogs of the Peptide StII(1-30) Derived From the N-Terminus of the Pore Forming Toxin Sticholysin II: Contribution to Understand its Orientation in Membrane

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Studies on Transformations of H-Phosphonates into DNA Analogues Containing P-S or P-C Bonds

In this thesis, mechanistic and synthetic studies on transformations of H-phosphonates into DNA analogues containing P-S or P-C bonds are described. Configurational stability of dinucleoside H-phosphonates and the stereochemical course of their sulfurisation in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) were investigated. In light of these studies, the reported stereoselective sulfurisation of dinucleoside H-phosphonates and benzoylphosphonates in the presence of DBU was proved to be incorrect. Efficient protocols for the synthesis of new nucleotide analogues with non-ionic C-phosphonate internucleotide linkages were developed. The synthesis of dinucleoside 2-pyridylphosphonates was successfully performed by a DBU-promoted reaction of H-phosphonate diesters with N-methoxypyridinium salts. The thio analogues, 2-pyridyl- and 4-pyridyl phosphonothioate diesters, could be obtained by modifying the reactions developed for their oxo counterparts. Dinucleoside 3-pyridylphosphonates were prepared via a palladium(0)-catalysed cross coupling strategy that could be extended also to the synthesis of nucleotide analogues with metal-complexing properties, i.e. terpyridyl- and bipyridylphosphonate derivatives. Oligonucleotides modified with pyridylphosphonate internucleotide linkages have been prepared and preliminary studies on their hybridisation properties and resistance towards enzymatic degradation were performed. Finally, nucleotidic units for the incorporation of pyridylphosphonate groups at the 5’-terminus of oligonucleotides were designed. Condensations of such units with a suitably protected nucleoside afforded after oxidation the expected dinucleoside (3’-5’)-phosphates with pyridylphosphonate monoester functions at the 5’-ends.

Structural and functional analysis of the hepatitis C virus non-structural protein 5A

Das Hepatitis C Virus (HCV) ist ein umhülltes RNA Virus aus der Familie der Flaviviridae. Sein Genom kodiert für ein ca. 3000 Aminosäuren langes Polyprotein, welches co- und posttranslational in seine funktionellen Einheiten gespalten wird. Eines dieser viralen Proteine ist NS5A. Es handelt sich hierbei um ein stark phosphoryliertes Protein, das eine amphipatische α-Helix im Amino-Terminus trägt, welche für die Membran-Assoziation von NS5A verantwortlich ist. Welche Rolle die Phosphorylierung für die Funktion des Proteins spielt, bzw. welche Funktion NS5A überhaupt ausübt, ist zur Zeit noch unklar. Beobachtungen lassen Vermutungen über eine Funktion von NS5A bei der Resistenz infizierter Zellen gegenüber Interferon-alpha zu. Weiterhin wird vermutet, das NS5A als Komponente des membranständigen HCV Replikasekomplexes an der RNA Replikation beteiligt ist. Das Ziel dieser Doktorarbeit war es, die Funktion von NS5A für die RNA Replikation zu untersuchen. Zu diesem Zweck wurde eine Serie von Phosphorylierungsstellen-Mutanten generiert, die auf Ihre Replikationsfähigkeit und den Phosphorylierungsstatus hin untersucht wurden. Wir fanden, dass bestimmte Serin-Substitutionen im Zentrum von NS5A zu einer gesteigerten RNA Replikation führten, bei gleichzeitig reduzierter NS5A Hyperphosphorylierung. Weiterhin studierten wir den Einfluß von Mutationen in der Amino-terminalen amphipatischen α-Helix von NS5A auf die RNA-Replikation, sowie Phosphorylierung und subzelluläre Lokalisation des Proteins. Wir fanden, dass geringfügige strukturelle Veränderungen der amphipatischen Helix zu einer veränderten subzellulären Lokalisation von NS5A führten, was mit einer reduzierten oder komplett inhibierten RNA Replikation einherging. Zudem interferierten die strukturellen Veränderungen mit der Hyperphosphorylierung des Proteins, was den Schluß nahe legt, dass die amphipatische Helix eine wichtige strukturelle Komponente des Proteins darstellt, die für die korrekte Faltung und Phosphorylierung des Proteins essentiell ist. Als weitere Aspekte wurden die Trans-Komplementationsfähigkeit der verschiedenen viralen Komponenten des HCV Replikasekomplexes untersucht, sowie zelluläre Interaktionspartner von NS5A identifiziert. Zusammenfassend zeigen die Ergebnisse dieser Doktorarbeit, dass NS5A eine wichtige Rolle bei der RNA-Replikation spielt. Diese Funktion wird wahrscheinlich über den Phosphorylierungszustand des Proteins reguliert.

Mechanism of Ca(v)1.2 channel modulation by the amino terminus of cardiac beta2-subunits

L-type calcium channels are composed of a pore, alpha1c (Ca(V)1.2), and accessory beta- and alpha2delta-subunits. The beta-subunit core structure was recently resolved at high resolution, providing important information on many functional aspects of channel modulation. In this study we reveal differential novel effects of five beta2-subunits isoforms expressed in human heart (beta(2a-e)) on the single L-type calcium channel current. These splice variants differ only by amino-terminal length and amino acid composition. Single-channel modulation by beta2-subunit isoforms was investigated in HEK293 cells expressing the recombinant L-type ion conducting pore. All beta2-subunits increased open probability, availability, and peak current with a highly consistent rank order (beta2a approximately = beta2b > beta2e approximately = beta2c > beta2d). We show graded modulation of some transition rates within and between deep-closed and inactivated states. The extent of modulation correlates strongly with the length of amino-terminal domains. Two mutant beta2-subunits that imitate the natural span related to length confirm this conclusion. The data show that the length of amino termini is a relevant physiological mechanism for channel closure and inactivation, and that natural alternative splicing exploits this principle for modulation of the gating properties of calcium channels.

Fatty acylated proteins and the protein kinase C signaling pathway

Numerous proteins in intracellular signaling pathways are known to be covalently modified by long chain fatty acids. The objective of this project was to identify potentially novel components of the protein kinase C signaling pathway by virtue of their fatty acylation. A 64 kDa palmitoylated protein (p64) was identified that became deacylated following stimulation of quiescent cells with serum, FGF, or PDBu, suggesting that stimulus-dependent deacylation might alter interactions between p64 and other membrane/cytoskeletal components. A myristoylated protein of 68 kDa observed during these studies was identified as the "80K" PKC substrate. This protein was acylated cotranslationally with myristate through an amide linkage. The majority of the 80K protein was tightly associated with the plasma membrane, with approximately 20% in the cytosol. Although phosphorylation of the membrane-bound and soluble forms of the protein was increased 6-fold in response to PDBu, no changes in the subcellular distribution or myristoylation of the protein were observed. A cDNA encoding the murine form of this protein was cloned, and its deduced amino acid sequence revealed the presence of an N-terminal myristoylation consensus and five potential sites for phosphorylation by PKC. A mutant in which the N-terminal glycine residue was changed to alanine was no longer a substrate for NMT and consequently lost its membrane-binding potential. However, its ability to be phosphorylated in response to purified growth factors and phorbol esters was unimpaired. These results indicate that the myristoylated N-terminus of the 80K protein is required for its association with the plasma membrane, and that the cytoplasmic form of the protein can be phosphorylated independently of the membrane-bound form. Mutants of PKC were constructed in which the regulatory domain was removed and replaced by the N-terminus of the 80K or Al proteins. Unexpectedly, both the myristoylated and nonmyristoylated fusion proteins were tightly associated with the nuclear envelope. Further deletion analyses mapped nuclear targeting signals to the hinge region and a portion of the catalytic domain of PKC, explaining the ability of PKC to be translocated to the nucleus in response to certain stimuli. ^

Experimental dissection and characterization of the functional domains in cardiac troponin C

Contraction of vertebrate cardiac muscle is regulated by the binding of Ca$\sp{2+}$ to the troponin C (cTnC) subunit of the troponin complex. In this study, we have used site-directed mutagenesis and a variety of assay techniques to explore the functional roles of regions in cTnC, including Ca$\sp{2+}$/Mg$\sp{2+}$-binding sites III and IV, the functionally inactive site I, the N-terminal helix, the N-terminal hydrophobic pocket and the two cysteine residues with regard to their ability to form disulfide bonds. Conversion of the first Ca$\sp{2+}$ ligand from Asp to Ala inactivated sites III and IV and decreased the apparent affinity of cTnC for the thin filament. Conversion of the second ligand from Asn to Ala also inactivated these sites in the free protein but Ca$\sp{2+}$-binding was recovered upon association with troponin I and troponin T. The Ca$\sp{2+}$-concentrations required for tight thin filament-binding by proteins containing second-ligand mutations were significantly greater than that required for the wild-type protein. Mutation of site I such that the primary sequence was that of an active site with the first Ca$\sp{2+}$ ligand changed from Asp to Ala resulted in a 70% decrease in maximal Ca$\sp{2\sp+}$ dependent ATPase activity in both cardiac and fast skeletal myofibrils. Thus, the primary sequence of the inactive site I in cTnC is functionally important. Major changes in the sequence of the N-terminus had little effect on the ability of cTnC to recover maximal activity but deletion of the first nine residues resulted in a 60 to 80% decrease in maximal activity with only a minor decrease in the pCa$\sb{50}$ of activation, suggesting that the N-terminal helix must be present but that a specific sequence is not required. The formation of an inter- or intramolecular disulfide bonds caused the exposure of hydrophobic surfaces on cTnC and rendered the protein Ca$\sp{2+}$ independent. Finally, elution patterns from a hydrophobic interactions column suggest that cTnC undergoes a significant change in hydrophobicity upon Ca$\sp{2+}$ binding, the majority of which is caused by site II. These latter data show an interesting correlation between exposure of hydrophobic surfaces on and activation of cTnC. Overall, these results represent significant progress toward the elucidation of the functional roles of a variety of structural regions in cTnC. ^

Identification and characterization of a conserved family of protein serine/threonine phosphatases homologous to Drosophila retinal degeneration C (rdgC)

The Drosophila retinal degeneration C (rdgC) gene encodes an unusual protein serine/threonine phosphatase in that it contains at least two EF-hand motifs at its carboxy terminus. By a combination of large-scale sequencing of human retina cDNA clones and searches of expressed sequence tag and genomic DNA databases, we have identified two sequences in mammals [Protein Phosphatase with EF-hands-1 and 2 (PPEF-1 and PPEF-2)] and one in Caenorhabditis elegans (PPEF) that closely resemble rdgC. In the adult, PPEF-2 is expressed specifically in retinal rod photoreceptors and the pineal. In the retina, several isoforms of PPEF-2 are predicted to arise from differential splicing. The isoform that most closely resembles rdgC is localized to rod inner segments. Together with the recently described localization of PPEF-1 transcripts to primary somatosensory neurons and inner ear cells in the developing mouse, these data suggest that the PPEF family of protein serine/threonine phosphatases plays a specific and conserved role in diverse sensory neurons.

The primary fibrin polymerization pocket: Three-dimensional structure of a 30-kDa C-terminal γ chain fragment complexed with the peptide Gly-Pro-Arg-Pro

After vascular injury, a cascade of serine protease activations leads to the conversion of the soluble fibrinogen molecule into fibrin. The fibrin monomers then polymerize spontaneously and noncovalently to form a fibrin gel. The primary interaction of this polymerization reaction is between the newly exposed N-terminal Gly-Pro-Arg sequence of the α chain of one fibrin molecule and the C-terminal region of a γ chain of an adjacent fibrin(ogen) molecule. In this report, the polymerization pocket has been identified by determining the crystal structure of a 30-kDa C-terminal fragment of the fibrin(ogen) γ chain complexed with the peptide Gly-Pro-Arg-Pro. This peptide mimics the N terminus of the α chain of fibrin. The conformational change in the protein upon binding the peptide is subtle, with electrostatic interactions primarily mediating the association. This is consistent with biophysical experiments carried out over the last 50 years on this fundamental polymerization reaction.

The N terminus of the Drosophila Numb protein directs membrane association and actin-dependent asymmetric localization

Drosophila Numb is a membrane associated protein of 557 amino acids (aa) that localizes asymmetrically into a cortical crescent in mitotic neural precursor cells and segregates into one of the daughter cells, where it is required for correct cell fate specification. We demonstrate here that asymmetric localization but not membrane localization of Numb in Drosophila embryos is inhibited by latrunculin A, an inhibitor of actin assembly. We also show that deletion of either the first 41 aa or aa 41–118 of Numb eliminates both localization to the cell membrane and asymmetric localization during mitosis, whereas C-terminal deletions or deletions of central portions of Numb do not affect its subcellular localization. Fusion of the first 76 or the first 119 aa of Numb to β-galactosidase results in a fusion protein that localizes to the cell membrane, but fails to localize asymmetrically during mitosis. In contrast, a fusion protein containing the first 227 aa of Numb and β-galactosidase localizes asymmetrically during mitosis and segregates into the same daughter cell as the endogenous Numb protein, demonstrating that the first 227 aa of the Numb protein are sufficient for asymmetric localization.