The next step of neurogenesis in the context of Alzheimer’s disease

Molecular Biology Reports - Among different pathological mechanisms, neuronal loss and neurogenesis impairment in the hippocampus play important roles in cognitive decline in Alzheimer’s...     

Structural and functional insights of Wilson disease copper-transporting ATPase

Wilson disease is an autosomal recessive disorder of copper metabolism. The gene for this disorder has been cloned and identified to encode a copper-transporting ATPase (ATP7B), a member of a large family of cation transporters, the P-type ATPases. In addition to the core elements common to all P-type ATPases, the Wilson copper-transporting ATPase has a large cytoplasmic N-terminus comprised six heavy metal associated (HMA) domains, each of which contains the copper-binding sequence motif GMT/HCXXC. Extensive studies addressing the functional, regulatory, and structural aspects of heavy metal transport by heavy metal transporters in general, have offered great insights into copper transport by Wilson copper-transporting ATPase. The findings from these studies have been used together with homology modeling of the Wilson disease copper-transporting ATPases based on the X-ray structure of the sarcoplasmic reticulum (SR) calcium-ATPase, to present a hypothetical model of the mechanism of copper transport by copper-transporting ATPases.     

Copper(I) interaction with model peptides of WD6 and TM6 domains of Wilson ATPase: regulatory and mechanistic implications

With the aim to investigate the mechanism of Cu(I) transport by Wilson ATPase (ATP7B), we have studied the interaction of the peptides 2K10p (CH(3)CO-Lys-Gly-Met-Thr-Cys-Ala-Ser-Cys-Val-His-Asn-Lys-CONH(2)), and 2K8p (CH(3)CO-Lys-Leu-Cys-Ile-Ala-Cys-Pro-Cys-Ser-Lys-CONH(2)), part of the sixth metal binding domain (WD6) and the sixth transmembrane segment (TM6) of Wilson ATPase, respectively, by means of CD, NMR spectroscopy and homology modeling. In addition, the interaction of Cu(I) with the 2K8p mutants 1s (CH(3)CO-Lys-Leu-Ser-Ile-Ala-Cys-Pro-Cys-Ser-Lys-CONH(2)), 2s (CH(3)CO-Lys-Leu-Cys-Ile-Ala-Ser-Pro-Cys-Ser-Lys-CONH(2)) and 3s (CH(3)CO-Lys-Leu-Cys-Ile-Ala-Cys-Pro-Ser-Ser-Lys-CONH(2)), containing two cysteines in various positions, have been studied with the same methods, in order to understand the role of each cysteine in copper binding. Our studies show that the three cysteine thiolates present in the 2K8p peptide sequence act mainly as bridging ligands for Cu(I) binding, and dithiothreitol acts as an important ligand in Cu(I) ligation by 2K10p and the 2K8p mutants. Formation of oligomeric species has been evidenced for all peptides except 2s. Shift of the equilibrium between the various oligomeric species has been accomplished by reducing the Cu(I):peptide ratio. Significant shifts of proline protons upon interaction with Cu(I) have been observed for all proline containing peptides implying a possible role of proline in facilitating Cu(I) binding. These findings have been further discussed with respect to the molecular basis of copper trafficking and intermolecular interactions.     

Identification of the "missing domain" of the rat copper-transporting ATPase, atp7b: insight into the structural and metal binding characteristics of its N-terminal copper-binding domain

Wilson disease is an autosomal disorder of copper transport caused by mutations in the ATP7B gene encoding a copper-transporting P-type ATPase. The Long Evans Cinnamon (LEC) rat is an established animal model for Wilson disease. We have used structural homology modelling of the N-terminal copper-binding region of the rat atp7b protein (rCBD) to reveal the presence of a domain, the fourth domain (rD4), which was previously thought to be missing from rCBD. Although the CXXC motif is absent from rD4, the overall fold is preserved. Using a wide range of techniques, rCBD is shown to undergo metal-induced secondary and tertiary structural changes similar to WCBD. Competition 65Zn(II)-blot experiments with rCBD demonstrate a binding cooperativity unique to Cu(I). Far-UV circular dichroism (CD) spectra suggest significant secondary structural transformation occurring when 2-3 molar equivalents of Cu(I) is added. Near-UV CD spectra, which indicate tertiary structural transformations, show a proportional decrease in rCBD disulfide bonds upon the incremental addition of Cu(I), and a maximum 5:1 Cu(I) to protein ratio. The similarity of these results to those obtained for the Wilson disease N-terminal copper-binding region (WCBD), which has six copper-binding domains, suggests that the metal-dependent conformational changes observed in both proteins may be largely determined by the protein-protein interactions taking place between the heavy metal-associated (HMA) domains, and remain largely unaffected by the absence of one of the six CXXC copper-binding sites.     

Polysialic acid is released by human umbilical vein endothelial cells (HUVEC) in vitro

Background

Sialic acids represent common terminal residues on numerous mammalian glycoconjugates, thereby influencing e.g. lumen formation in developing blood vessels. Interestingly, besides monosialylated also polysialylated glycoconjugates are produced by endothelial cells. Polysialic acid (polySia) is formed in several organs during embryonal and postnatal development influencing, for instance, cell migration processes. Furthermore, the function of cytokines like basic fibroblast growth factor (bFGF) is modulated by polySia.

Results

In this study, we demonstrated that human umbilical vein endothelial cells (HUVEC) also secrete polysialylated glycoconjugates. Furthermore, an interaction between polySia and vascular endothelial growth factor (VEGF) was observed. VEGF modulates like bFGF the migration of HUVEC. Since both growth factors interact with polySia, we examined, if polySia modulates the migration of HUVEC. To this end scratch assays were performed showing that the migration of HUVEC is stimulated, when polySia was degraded.

Conclusions

Since polySia can interact with bFGF as well as VEGF and the degradation of polySia resulted in an increased cell migration capacity in the applied scratch assay, we propose that polySia may trap these growth factors influencing their biological activity. Thus, polySia might also contribute to the fine regulation of physiological processes in endothelial cells.