We have previously shown that iron-containing human lactoferrin (LF) purified from breast milk is able to form both in vitro and in vivo a complex with ceruloplasmin (CP), the copper-containing protein of human plasma. Here we present evidence that the CP-LF complex is dissociated by high concentrations of NaCl, CaCl2, or EDTA, or by decreasing the pH to 4.7. In addition, DNA, bacterial lipopolysaccharide, and heparin can displace CP from its complex with LF. Antibodies to either of the two proteins also cause dissociation of the complex.
A large group of prion-associated proteins was identified in yeast cells using a new approach, comparative analysis of pellet proteins of crude cell lysates in isogenic strains of Saccharomyces cerevisiae differing by their prion composition. Twodimensional (2D) electrophoresis followed by MALDI analysis of the pellet proteins of [PSI + ] and [psi − ] strains after prion elimination by GuHCl and prion transmission by cytoduction permitted identification of ca. 40 proteins whose aggregation state correlated with the change of prion(s) content. Approximately half of these proteins belonged to chaperones and to enzymes of glucose metabolism. Chaperones are known to be involved in prion metabolism and are expected to be present in prioncontaining aggregates, but glucose metabolism enzymes are not predicted to be present. Nevertheless, several recent data suggest that their presence is not incidental. We detected six proteins involved in oxidative stress response and eight in translation. Also notable is a protease. Most of the identified proteins seem to be prion-associated, but we cannot exclude the possibility that several proteins may propagate as prions.
The possibility of obtaining recombinant fibrillogenic fusion proteins such as transthyretin (TTR) and β2-microglobulin (β2M) with a superfolder green fluorescent protein (sfGFP) was studied. According to the literature data, sfGFP is resistant to denaturating influences, does not aggregate during renaturation, possesses improved kinetic characteristics of folding, and folds well when fused to different polypeptides. The corresponding DNA constructs for expression in Escherichia coli were created. It could be shown that during expression of these constructs in E. coli, soluble forms of the fusion proteins are synthesized. Efficient isolation of the fusion proteins was performed with the help of nickel-affinity chromatography. For this purpose a polyhistidine sequence (6-His-tag) was incorporated into the C-terminus of the sfGFP. We could show that the purified fusion proteins contained full-size sequences of the most amyloidogenic TTR variant, TTR(L55P) and β2M, and also sfGFP possessing fluorescent properties. In the course of fibrillogenesis both fusion proteins demonstrated their ability to form fibrils that were clearly detectable by atomic force microscopy. Furthermore, with the help of confocal microscopy we were able to reveal structures (exhibiting fluorescence) that are formed during fibrillogenesis. Thus, the use of sfGFP has made it possible to avoid formation of inclusion bodies (IB) during the synthesis of recombinant fusion proteins and to obtain soluble forms of TTR(L55P) and β2M that are suitable for further studies.
Symmetrical peptide GYDTQAIVENNESTEYG (WT, Wild Type) identical to 35-51 aminoacid residues of human alpha-lactalbumin (HLA) and peptide GYDTQTVVNNNGHTDYG (ID, IDeal symmetry) homologous to beta-domain of mammalian alpha-lactalbumins can form amyloid-like fibrils in conditions required for fibrillogenesis of HLA. The latter peptide can also form fibrils in deionized water. Fibrils formed by these peptides can cause forming of HLA amyloid-like aggregates in physiological conditions. These results provide an evidence for presence of amyloidogenic determinant in beta-domain of alpha-lactalbumin. Thus, symmetry in the primary structure may play the role in fibrillogenesis of proteins.
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