Copper-catalyzed oxidation of the recombinant SHa(29–231) prion protein

113

134

The cellular prion protein is known to be a copper-binding protein. Despite the wide range of studies on the copper binding of PrP, there have been no studies to determine the affinity of the protein on both full-length prion protein and under physiological conditions. We have used two techniques, isothermal titration calorimetry and competitive metal capture analysis, to determine the affinity of copper for wild type mouse PrP and a series of mutants. The cellular prion protein PrP c is a cell surface glycoprotein with large ␣-helical content that is attached to the plasma membrane by a glycosylphosphatidylinositol anchor. This cellular form of the protein binds copper, and it is argued that this copper binding to PrP c is intimately linked to the normal cellular function of the protein either in copper transport, sequestration, or antioxidant activity (1-3). The native cellular protein may undergo a conformational transformation to a proteaseresistant species known as a prion (PrP Sc ) with high ␤-sheet content and a tendency to aggregate. This transformed species is the putative proteinaceous infectious agent of transmissible spongiform encephalopathies or prion diseases (4). Prion diseases are characterized by a metal imbalance in the brain that occurs simultaneously to conversion of PrP c to PrPSc . The metal imbalance is associated with the loss of copper binding from PrP c on conversion to PrP Sc (5, 6). This raises important questions concerning the role of copper and other metals in prion diseases. Loss of protein function as a result of impaired copper binding in diseased PrPSc could have serious implications for disease progression.As a result, knowledge of copper binding to PrP is important in considering both the normal function of PrP c and its influence in prion diseases.Although it is accepted that PrP c is a Cu(II) binding protein, there are many facets of the interaction of Cu(II) and PrP c that are disputed. These include the exact number of binding sites and their relative affinity for Cu(II). Initial studies focused on a fragment of the protein termed the octameric repeat region because it contains four repeats of an octamer. This region contains histidine and so was a likely candidate for the binding site. These early studies (7, 8) suggested Cu(II) binds to this region with a micromolar affinity that was little more specific than histidine on its own. Further studies with larger fragments also suggested that Cu(II) binds PrP c , but again the affinity seen was similarly a low micromolar association (9). A study of the effect of PrP c expression on Cu(II) uptake into cells suggested that the affinity must at least be in the nanomolar range (1). Nanomolar affinities for the octameric repeat region have rarely been suggested (10). Only one publication has suggested that the affinity of Cu(II) for PrP is in the femtomolar range (11). Although such a high affinity implies that PrP is a very specific Cu(II)-binding protein, this finding has never been reproduced. Therefore, there is currently no...

Section: Discussionmentioning

confidence: 99%

“…Direct titration of protein solutions with aqueous Cu(II) salts was avoided because of the tendency of PrP to aggregate under such conditions (18,19) and to prevent the formation of insoluble Cu(II) hydroxide species. These may be avoided by the use of a Cu(II) chelate.…”

Section: Methodsmentioning

confidence: 99%

High Affinity Binding between Copper and Full-length Prion Protein Identified by Two Different Techniques

Thompsett

Abdelraheim

Daniels

et al. 2005

113

134

“…Metal binding to the prion protein is altered in human prion disease (12), with levels of cellular copper affected by scrapie infection (13) and the ability for disease progression in infected mice to be slowed with the use of copper-specific chelation therapy (14). Copper-catalyzed redox damage of PrP (15) is linked to prion disease (16,17), although, the copper binding octarepeat region is not required for prion infectivity and propagation (18). Cu 2ϩ binding has also been noted outside the octarepeats, in the so called 5th site (8, 19 -23).…”

Section: ؉mentioning

confidence: 99%

Deconvoluting the Cu2+ Binding Modes of Full-length Prion Protein

Klewpatinond

Davies

Bowen

et al. 2008

140

Sc , is a proteinaceous infectious particle, devoid of nucleic acids, which is responsible for bovine spongiform encephalopathy in cattle and a number of prion diseases in humans, including Creutzfeldt Jakob Disease (1-3). Normal cellular prion protein (PrP C ) is typically 209 residues long, attached to the cell surface by a glycosylphosphatidylinositol anchor. The C-terminal domain between residues 126 and 231 is mainly ␣-helical (4). In contrast, in the absence of copper ions, the N-terminal domain between residues 23 and 125 is unstructured (5) and exhibits a high degree of main chain flexibility (6). It is this natively unfolded domain that includes octarepeat sequences that bind a number of Cu 2ϩ ions (7-9). Metal imbalance is a feature of prion disease (10) and when isolated from diseased brain, PrP Sc has been found to be occupied with metal (11). Metal binding to the prion protein is altered in human prion disease (12), with levels of cellular copper affected by scrapie infection (13) and the ability for disease progression in infected mice to be slowed with the use of copper-specific chelation therapy (14). Copper-catalyzed redox damage of PrP (15) is linked to prion disease (16, 17), although, the copper binding octarepeat region is not required for prion infectivity and propagation (18). Cu 2ϩ binding has also been noted outside the octarepeats, in the so called 5th site (8, 19 -23). This region is an amyloidogenic, neurotoxic segment of PrP that is essential for prion replication (18, 24 -29). Interestingly, the presence, or absence, of Cu 2ϩ ions may also confer different strains of prion disease (11, 30). PrP C is concentrated at presynaptic membranes in the central nervous system, where Cu 2ϩ is also highly localized (32). The ability of PrP C to bind Cu 2ϩ in vivo and in vitro infers PrP may have a physiological role in copper homeostasis (7, 9). Copper has been shown to promote the endocytosis of PrP C (33, 34), but PrP expression levels do not seem to affect copper delivery (35,36). Copper-induced cleavage of the PrP C main chain has also been reported (37,38) (22), and mass spectrometry (30).There have been numerous Cu 2ϩ binding studies of fragments of PrP centered on two regions within the unstructured N-terminal domain of PrP. These studies include the octarepeat region, residues 58 -91, and a second Cu 2ϩ binding region between the octarepeats and the C-terminal structured domain, between residues 90 and 126. Mammalian PrPs contain a repeating motif of 8 amino acids, typically, four octarepeats between residues 60 and 91 with each repeat containing a histidine residue. It is this unstructured, highly conserved * This work was supported in part by Biotechnology and Biological Sciences Research Council Project grants. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

“…4, E and F (23). Histidine residues in rPrP c are highly susceptible to oxidation (24). Therefore, the additional histidine residues in the octapeptide repeat region of rPrP 8OR may render the mutant protein more susceptible to oxidation.…”

Section: Characterization Of Recombinant Wild Type Prion Protein (Rprmentioning

confidence: 99%

Prion Proteins with Insertion Mutations Have Altered N-terminal Conformation and Increased Ligand Binding Activity and Are More Susceptible to Oxidative Attack

Yin

et al. 2006