Copper transport

The plasma copper protein ceruloplasmin (CP) was found to inhibit endothelial nitric-oxide synthase activation in cultured endothelial cells, in line with previous evidence showing that the endothelium-dependent vasorelaxation of the aorta is impaired by physiological concentrations of ceruloplasmin. The data presented here indicate a direct relationship between the extent of inhibition of agonist-triggered endothelial nitric oxide synthase activation and CP-induced enrichment of the copper content of endothelial cells. Copper discharged by CP was mainly localized in the soluble fraction of cells. The subcellular distribution of the metal seems to be of relevance to the inhibitory effect of CP, because it was mimicked by copper chelates, like copper-histidine, able to selectively enrich the cytosolic fraction of cells, but not by copper salts, which preferentially located the metal to the particulate fraction. Ceruloplasmin (CP)1 is a copper-containing glycoprotein that is found in the plasma of all vertebrates, in which it carries approximately 90% of plasma copper (1). Each CP molecule tightly binds six copper atoms to the three different copper binding sites that characterize blue oxidases (2, 3). Nevertheless, as indicated by considerable experimental evidence, it is particularly prone to transfer its copper atoms to tissues (4 -6) delivering copper to intracellular copper proteins (7,8). However, recent studies on aceruloplasminemic patients (9 -11) indicate that this protein has no essential role in copper transport, whereas it plays a primary role in iron homeostasis, possibly through its ferroxidase activity (12).Ceruloplasmin is an acute-phase reactant; its concentration in the plasma increases up to 3-fold during pregnancy and during multiple pathological processes including trauma and inflammation (13). Recent attention has focused on the role that this protein may have in the function of the vascular system in health and disease. CP has been detected in human atherosclerotic lesions (14, 15), and it has been shown to oxidize low density lipoproteins in the presence of vascular cells (16). A copper binding site labile to Chelex treatment has been proposed to be responsible for the oxidative damage to low density lipoproteins (17). On the other hand, we have shown that CP, at physiological concentrations, inhibits the endothelium-dependent relaxation of rabbit aorta induced by agonists and that this effect is not due to a trapping of NO by the copper sites (18).Vasodilation requires a (NO)-cGMP transduction pathway between endothelium and smooth muscle cells (19,20). Endothelial NO synthase (eNOS) is a constitutive enzyme that converts L-arginine into NO and citrulline (21) with a relatively low basal activity (22, 23). After agonist stimulation evoking an increase in the [Ca 2ϩ ] i concentration, Ca 2ϩ -bound calmodulin disrupts the inhibitory eNOS-caveolin-1 interactions (24, 25), thereby allowing conformational changes within eNOS, leading to the activated form that produces NO (26,27). The ago...

Section: Resultsmentioning

confidence: 83%

Inhibition of Endothelial Nitric-oxide Synthase by Ceruloplasmin

Bianchini

Musci

Calabrese

1999

“…This bears a striking resemblance to the Cu 2ϩ binding motif (NH 2 -XXH) at the N terminus of human serum albumin (HSA) (40,41). This site is responsible for Cu 2ϩ transport in blood plasma (42) and has a tight 1.0 pM affinity for Cu 2ϩ , whereas N-terminal tripeptide models possess a subpicomolar affinity (43)(44)(45)(46).…”

mentioning

confidence: 99%

Truncated Amyloid-β(11–40/42) from Alzheimer Disease Binds Cu2+ with a Femtomolar Affinity and Influences Fiber Assembly

Barritt

Viles

2015

Background: N-terminally truncated A␤ (11-40/42) typically constitutes 19% of plaque load in Alzheimer patients. Results: Cu 2ϩ binds to A␤ with a 34-fM dissociation constant and stabilizes very short highly amyloidogenic amyloid rods into longer A␤ fibers. Concussion: The very tight affinity explains the high levels of Cu 2ϩ in amyloid plaques. Significance: Copper, tightly bound to A␤ (11-40/42) , may influence disease pathology.

“…Free Cu 1ϩ or Cu 2ϩ ions are not present in cells or in the serum in mammals, presumably because copper ions can participate in the formation of toxic reactive oxygen species (2,3). The regulation of cellular copper levels involves uptake transporters, Cu-activated ATPases that mediate copper efflux, and several protein-specific chaperones that deliver copper to its intracellular target proteins (4 -8).…”

mentioning

confidence: 99%

O-Linked Glycosylation at Threonine 27 Protects the Copper Transporter hCTR1 from Proteolytic Cleavage in Mammalian Cells

Maryon

Molloy

Kaplan

2007

109

The major human copper uptake protein, hCTR1, has 190 amino acids and a predicted mass of 21 kDa. hCTR1 antibodies recognize multiple bands in SDS-PAGE centered at 35 kDa. Part of this increased mass is due to N-linked glycosylation at Asn-15. We show that in mammalian cells the N15Q mutant protein trafficked to the plasma membrane and mediated copper uptake at 75% of the rate of wild-type hCTR1. We demonstrate that the extracellular amino terminus of hCTR1 also contains O-linked polysaccharides. Glycosidase treatment that removed O-linked sugars reduced the apparent mass of hCTR1 or N15Q mutant protein by 1-2 kDa. Expression of amino-terminal truncations and alanine substitution mutants of hCTR1 in HEK293 and MDCK cells localized the site of O-linked glycosylation to Thr-27. Expression of alanine substitutions at Thr-27 resulted in proteolytic cleavage of hCTR1 on the carboxyl side of the T27A mutations. This cleavage produced a 17-kDa polypeptide missing approximately the first 30 amino acids of hCTR1. Expression of wild-type hCTR1 in mutant Chinese hamster ovary cells that were unable to initiate O-glycosylation also resulted in hCTR1 cleavage to produce the 17-kDa polypeptide. The 17-kDa hCTR1 polypeptide was located in the plasma membrane and mediated copper uptake at about 50% that of the rate of wildtype hCTR1. Thus, O-linked glycosylation at Thr-27 is necessary to prevent proteolytic cleavage that removes half of the extracellular amino terminus of hCTR1 and significantly impairs transport activity of the remaining polypeptide.