Molecular biology of natriuretic peptides in reptiles and birds

The heart produces natriuretic peptides that are critical regulators of blood pressure and renal function. This study examined the molecular evolution of natriuretic peptides in vertebrates and discovered novel forms of the peptides in birds. The research outcomes advanced our knowledge of the importance of these peptides in animal physiology.

Mapping and molecular modeling of S-adenosyl-L-methionine binding sites in N-methyltransferase domains of the multifunctional polypeptide cyclosporin synthetase

We employed a highly specific photoaffinity labeling procedure, using 14C-labeled S-adenosyl-L-methionine (AdoMet) to define the chemical structure of the AdoMet binding centers on cyclosporin synthetase (CySyn). Tryptic digestion of CySyn photolabeled with either [methyl-14C]AdoMet or [carboxyl-14C]AdoMet yielded the sequence H2N-Asn-Asp-Gly-Leu-Glu-Ser-Tyr-Val-Gly-Ile-Glu-Pro-Ser-Arg-COOH (residues 10644-10657), situated within the N-methyltransferase domain of module 8 of CySyn. Radiosequencing detected Glu10654 and Pro10655 as the major sites of derivatization. [carboxyl-14C]AdoMet in addition labeled Tyr10650. Chymotryptic digestion generated the radiolabeled peptide H2N-Ile-Gly-Leu-Glu-Pro-Ser-Gln-Ser-Ala-Val-Gln-Phe-COOH, corresponding to amino acids 2125-2136 of the N-methyltransferase domain of module 2. The radiolabeled amino acids were identified as Glu2128 and Pro2129, which are equivalent in position and function to the modified residues identified with tryptic digestions in module 8. Homology modeling of the N-methyltransferase domains indicates that these regions conserve the consensus topology of the AdoMet binding fold and consensus cofactor interactions seen in structurally characterized AdoMet-dependent methyltransferases. The modified sequence regions correspond to the motif II consensus sequence element, which is involved in directly complexing the adenine and ribose components of AdoMet. We conclude that the AdoMet binding to nonribosomal peptide synthetase N-methyltransferase domains obeys the consensus cofactor interactions seen among most structurally characterized low molecular weight AdoMet-dependent methyltransferases.

Molecular architecture of Aβ fibrils grown in cerebrospinal fluid solution and in a cell culture model of Aβ plaque formation

OBJECTIVES: The detailed structure of brain-derived Aβ amyloid fibrils is unknown. To approach this issue, we investigate the molecular architecture of Aβ(1-40) fibrils grown in either human cerebrospinal fluid solution, in chemically simple phosphate buffer in vitro or extracted from a cell culture model of Aβ amyloid plaque formation. METHODS: We have used hydrogen-deuterium exchange (HX) combined with nuclear magnetic resonance, transmission electron microscopy, seeding experiments both in vitro and in cell culture as well as several other spectroscopic measurements to compare the morphology and residue-specific conformation of these different Aβ fibrils. RESULTS AND CONCLUSIONS: Our data reveal that, despite considerable variations in morphology, the spectroscopic properties and the pattern of slowly exchanging backbone amides are closely similar in the fibrils investigated. This finding implies that a fundamentally conserved molecular architecture of Aβ peptide fold is common to Aβ fibrils.

Molecular and immunological analysis of hen's egg yolk allergens with a focus on YGP42 (Gal d 6)

Allergy to hen's (Gallus domesticus) egg white is one of the most common forms of food allergy. Allergy to hen's yolk also exists however, to a lesser extent when compared to egg white allergy. Two minor allergens from the hen's egg yolk known as α-livetin (Gal d 5) and YGP42 (Gal d 6) were discovered recently. In this study, we investigated whether sensitization to egg white is associated with reactivity to egg yolk as well. Sera obtained from 25 patients with allergy to egg white were tested for specific IgE binding for egg yolk proteins through western immunoblotting. 36% of patients were found with true IgE-sensitization against egg yolk proteins. It was found that most of the IgE reactive yolk proteins were fragments of major precursor proteins of hen; vitellogenin-1 (VTG-1), vitellogenin-2 (VTG-2) and apolipoprotein B (Apo B). The egg yolk allergen Gal d 6 is the C-terminal part of VTG-1 and was found to be allergenic in significant percentage of egg white allergy patients. These results highlight the significance of Gal d 6 as an important allergen of egg yolk. Therefore, the secondary aim of this study involved developing a recombinant version of YGP42 in an Escherichia coli expression system. Recombinant Gal d 6 (rGal d6) was expressed as a fusion peptide with a 6 × His tag and purified using metal chelating resin. The inhibition ELISA results showed that rYGP42 was IgE reactive and was able to inhibit IgE binding to crude egg yolk (CEY) by up to 30%. Traditionally, it was thought that allergy to egg yolk occurred independently from sensitization to egg white. This study underlies the importance of concomitant sensitization to egg yolk proteins in patients allergic to egg white. Evidence reported in this study strongly suggests that egg yolk has potentially undiscovered allergens and therefore warrants further investigation. Furthermore, IgE reactive Gal d 6 presented in this study has the potential to be used in diagnosis and immunotherapy to treat egg allergy.