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Metal Metabolism: Transport, Development and Neurodegeneration 1299
Amyloidogenic metal-binding proteins: newinvestigative pathways
Paul Davies, Sarah N. Fontaine, Dima Moualla, Xiaoyan Wang, Josephine A. Wright and David R. Brown
1
Department of Biology and Biochemistry, University of Bath, Bath, BA2 7AY, U.K.
Abstract
Neurodegenerative diseases remain perplexing and problematic for modern research. Those associatedwith amyloidogenic proteins have often been lumped together simply because those proteins aggregate.However, research has identified a more logical reason to group some of these diseases together. Theassociated proteins not only aggregate, but also bind copper. The APP (amyloid precursor protein) bindscopper in an N-terminal region. Binding of copper has been suggested to influence generation of
β
-amyloid from the protein. PrP (prion protein) binds copper, and this appears to be necessary for its normalfunction and might also reduce its probability of conversion into an infectious prion.
α
-Synuclein, a proteinassociated with Parkinson’s disease, also binds copper, but, in this case, it potentially increases the rateat which the protein aggregates. The similarities between these proteins, in terms of metal binding, hasallowed us to investigate them using similar approaches. In the present review, we discuss some of theseapproaches.
Introduction
Two of the most common neurodegenerative disordersare Parkinson’s disease and Alzheimer’s disease. Differentproteins have been associated with each disease. However, itis commonly recognized that the generation of A
β
(amyloid
β
-peptide), especially in the longer form of A
β
-(1–42) is themost likely candidate for the cause [1]. This protein fragmentisderivedfromAPP(amyloidprecursorprotein)bysecretasecleavage [2]. Another protein originally associated with thenon-amyloid component of plaques in Alzheimer’s disease isnow more commonly associated with Parkinson’s disease[3].
α
-Synuclein is the main component of Lewy bodies inboth Parkinson’s disease and other diseases such as DLB(dementia with Lewy bodies). There has only been oneprotein associated with the TSEs (transmissible spongiformencephalopathies). PrP (prion protein) is now accepted asbeing essential for prion diseases or TSEs [4]. In this case,the protein is transformed into an abnormal isoform rich in
β
-sheet structure. All three proteins bind metals.APP has been shown to be a copper-binding protein [5].The copper binding is associated with histidine residuesin the N-terminal domain. Binding of copper preventsdimerization of the protein [6], which potentially reducesthe rate of cleavage by the enzyme BACE1 (
β
-site APP-cleaving enzyme 1), the
β
-secretase that plays a role in thegeneration of A
β
. Additionally, there is evidence that zinccan bind to APP [7]. Once cleaved from the full-lengthprotein, A
β
has also demonstrated copper- and zinc-binding
Key words:
Alzheimer’s disease, amyloid precursor protein, neurodegeneration, Parkinson’sdisease, prion, synuclein, transmissible spongiform encephalopathy (TSE).
Abbreviations used:
A
β
, amyloid
β
-peptide; APP, amyloid precursor protein; APLP, APP-likeprotein; BACE1,
β
-site APP-cleaving enzyme 1; DLB, dementia with Lewy bodies; ITC, isothermaltitration calorimetry; PrP, prion protein; PrP
Sc
, abnormal disease-specific conformation of PrP; TSE,transmissible spongiform encephalopathy.
1
To whom correspondence should be addressed (email bssdrb@bath.ac.uk).
properties that are believed to influence its aggregation [8]. Itis unclear whether the cleavage of APP is necessary for themetal-binding capacity of the A
β
domain.PrP has been shown to bind a number of metals, includingcopper, manganese and zinc [9]. Of these, copper has beenshown repeatedly to be the metal bound to the normalisoform. A variety of functions have been suggested forthis copper binding. These include copper uptake and/orsequestration [10] and possible antioxidant activity [11].Additionally, there is good evidence that the copper bindingcaninhibitproteinconversionintotheabnormalisoform[12]and alter the incubation period of the disease in animals[13]. In contrast, manganese binding is associated withprotein conversion into an abnormal isoform [9]. Proteinpurified from the brains of animals and patients with a priondiseaseshowsthepresenceofmanganese.Therearestructuraldifferences in PrP when it binds either copper or manganese[14], although it can bind both metals at the same time [15].
α
-Synuclein is a more recent addition to this family,although there has been evidence for some time that copperinfluencesitsaggregation.However,morerecentdatasuggestthat it can potentially bind at two different sites in theprotein [16]. Parkinson’s disease is more closely associatedwith changes in iron metabolism than copper. It has alsorecently been suggested that
α
-synuclein can bind iron [17].It is currently unclear whether either metal is associatedwith the protein
in vivo
in the absence of disease or whichmetal is a potential ‘appropriate’ binding partner. However,the conclusion that copper binding could initiate proteinaggregation suggests that the interaction is aberrant.
Metal affinity
Studies of the metal-binding affinities for proteins are nec-essarytoverifythattheywouldbindametalinaphysiological
Biochem. Soc. Trans. (2008) 36, 1299–1303; doi:10.1042/BST0361299
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1300 Biochemical Society Transactions (2008) Volume 36, part 6
context. Many different techniques have been employed toestablishthesevalues,rangingfromNMRtoequilibriumdia-lysis. Our preference has been for ITC (isothermal titrationcalorimetry). This technique allows real-time measurementsthat can give values for both association and dissociationof a metal and stochiometries based on enthalpy changesassociated with binding events. Our research has mostlyfocused on PrP, but current research is determining thesevalues for both
α
-synuclein and APP.Initial studies of metals binding to PrP suggested fairlylow affinity values for copper binding [18,19]. Although theoctameric repeat region was clearly responsible for bindingfour atoms of copper, there was also a suggestion that a fifthsite located in the N-terminus could also bind copper. Laterstudies have shown that this fifth site is associated with twohistidine residues (His
95
and His
110
in mouse) and couldpotentially represent a higher-affinity site [20]. Values for thedissociation range from femtomolar down to micromolar.Studies of copper uptake by cells clearly show that PrP isinvolved in copper entry into cells with
K 
m
values in ananomolar range. This clearly supports a fairly high affinity,possibly in the low nanomolar range [10]. Our own studiesof copper binding suggest that the highest-affinity site has afemtomolar dissociation constant, with the other sites beingmuch lower [21]. Studies of copper-depleted cells indicatethat PrP cannot be purified without at least one copper ionbound, supporting further the notion of a single high-affinitysite [22].Studies of other metals binding to PrP have suggestedpossible binding of zinc, nickel and manganese [9]. Althoughthere is little evidence for zinc and nickel binding to PrP
invivo
, manganese is associated with PrP
Sc
(abnormal disease-specific conformation of PrP) isolated from the brains of patients with CJD (Creutzfeld–Jakob disease) or mice withscrapie[23,24].StructuralstudiesofPrPhaveshownthatbothcopper and manganese can alter the structure of the protein[14]. Binding of manganese to PrP results in a change inconformationtoaformthataggregates,isneurotoxic[25]andhasincreaseproteaseresistance,similartoPrP
Sc
[9].Studiesof affinity of PrP for manganese show that the affinity is muchlower than that of copper. However, this result is expected,as all manganese-binding proteins have low affinities. Thevalues found for PrP are equivalent to those of other knownmanganese-binding proteins [15].Copper and zinc binding to APP has been less wellstudied. The copper-binding domain is found in the N-terminus of APP within amino acid residues 135–175 [5].The copper-binding domain contains the typical His-Xaa-His motif of a type II site similar to that seen for superoxidedismutase and lysyl oxidase copper homoeostasis proteingroups. APP reduces the bound copper to Cu(I), suggestingthat the protein could have a copper reductase activity [26].This reduction leads to oxidation of Cys
144
and Cys
158
,resulting in the formation of a new disulfide bridge [27].The dissociation constant for copper binding to APP hasbeen suggested to be approx. 10 nM [7]. Binding of thecopper to this domain involves the imidazole rings of three histidine residues, supporting the notion that this sitehas higher specificity for copper then other type II sites[28].The metallochemistry of A
β
has been investigated in somedetail [29]. A
β
can be rapidly precipitated by Zn
2
+
ions atlow physiological concentrations, and it has been reportedthat other metal ions such as Cu
2
+
and Fe
3
+
, unlike Zn
2
+
,produced a greater aggregation of A
β
under weakly acidicconditions [8]. Such a mildly acidic environment probablyresembles conditions occurring in the brain. The significanceofthese
invitro
studieswithA
β
andmetalionsisemphasizedby other data showing that the homoeostasis of zinc, copperand iron are significantly altered in Alzheimer’s disease brain[30]. The affinities of the Zn
2
+
-binding sites on A
β
-(1–40)were measured as 100 nM and 5
μ
M, indicating that theymay be occupied under physiological conditions [31]. Thehighest affinity Cu
2
+
-binding site on A
β
-(1–42) has ameasured association constant (
 K 
a
) of 10–15 aM [32]. Withsuch strong affinity for Cu
2
+
, A
β
species such as A
β
-(1–42) are likely to bind Cu
2
+
in vivo
. The increase in Cu
2
+
affinity of A
β
-(1–42) over the normal APP is related to APPproteolysis. APP is a membrane-spanning protein, and theCu
2
+
-binding site is probably hidden within the protein,becoming exposed in the proteolysed A
β
fragment. Also,A
β
-(1–42) has a higher
β
-sheet content, and these structuresarefrequentlyfoundinthetertiarystructureofCu
2
+
catalyticsites.Studies of metal affinity for
α
-synuclein are relativelynew. Initial studies of copper binding to
α
-synuclein havetheir origin in the study of its aggregation [33]. Copper-mediated aggregation seemed to be related to interactionof copper with the C-terminus of the protein. This wasshown by limited proteolysis of 
α
-synuclein that cleavedoff part of the C-terminus either at residue 97 or 114. Theshorter fragment showed no response to copper in terms of oligomerization, whereas the 1–114 fragment did produce alimited amount of oligomerization [33]. Despite early studiessuggesting that copper causes aggregation of the proteinvia the C-terminus, a more recent study suggested thatcopper can bind to His
50
. Further binding can occur at theC-terminus, but this is of much lower affinity than theN-terminal site [16]. Binding at the high-affinity site wasshowntobesufficienttodriveoligomerizationoftheprotein.The high-affinity site appears to be a type-II site with squareplanar co-ordination. However, the affinity for this site issuggested to be 0.1
μ
M. That for the second site was shownto be 50
μ
M, which is in line with previous findings [33].Further studies suggest involvement of the N-terminus inbindingatleastonecopperion[34].Metalbindingto
α
-synu-clein has been studied using ITC. The results suggest thatthe protein binds two atoms of copper, but with high-micromolar affinity [17]. The same study also suggested that
α
-synuclein can bind iron with a similar low affinity. Furtherstudy is clearly needed to verify that
α
-synuclein binds thesemetals, but our own studies suggest that they do and withaffinities that would be more likely to suggest metal binding
in vivo
.
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Metal Metabolism: Transport, Development and Neurodegeneration 1301
Figure 1
Cyclic voltammograms recorded at a scan rate of 10mV
·
s
−
1
for the reduction and reoxidation of copper refolded mPrPadsorbed on to a 3 mm diameter boron-doped diamond discelectrode and immersed in 5 mM Mes/Tris buffer solution at pH 7
The broken line indicates the background signal of protein with nocopper bound. The solid red line is the voltammogram recorded forcopper-bound mPrP (murine PrP) at pH 5.5 and the solid green line atpH 7. Our work has demonstrated that copper bound to recombinantPrP is able to undergo reversible redox cycling. The reversible ormidpoint potential for this process is 0.03
±
0.01 V compared with theSCE(saturatedcalomelelectrode)atpH 7.Theproteindemonstrateslittleredox chemistry at pH 5.5 or below, probably as a result of the conditionsfavouringlowcopperoccupancyatsignificantlyreducedaffinity.Logplotsof the data indicate an electron transfer rate-limited process, with themajority of protein adsorbed permanently to the electrode.
Electrochemistry
As metal-binding proteins, APP, A
β
, PrP and
α
-synucleinare linked to redox-active metals that could potentiallyhave damaging effects on cells or other proteins. Thereforestudying the redox chemistry of the metal–protein complexcould give valuable insight into potential disease mechanismsinduced by these proteins as a result of metal binding.PrPhasbeensuggestedtobeanantioxidant[10].Althoughother researchers have suggested that this is the case, theirevidence has been based on experiments using high-affinitychelators that are likely to strip the protein of any boundcopper [35,36]. Strong new evidence supports the notion thatPrPcanactasanantioxidant.Studieswithcyclicvoltammetryshow that, when copper is bound to the protein, it can fullycycle between reductive and oxidative potentials (Figure 1)and has a midpoint potential close to 0 V [15]. This clearlyindicated that the electrochemical properties of PrP areequivalent to those of other known antioxidants. In contrast,manganese bound to PrP is predominantly oxidized, whichsuggests a non-cyclable behaviour [15].APP can reduce Cu(II) to Cu(I) in a cell-free system,potentially leading to increased oxidative stress in neurons[26]. The domain that contributes to such activities is thecopper-bindingdomain[5]residingbetweenresidues135and158ofAPP,aregionthatshowsstronghomologywithAPLP(APP-like protein) 2, but not to APLP1. Potentially, APP–Cu(I) complexes are involved that reduce hydrogen peroxideto form an APP–Cu(II)–hydroxyl radical intermediate [27].APP residues 135–158 consisting of cysteine and copper-co-ordinating histidine residues can modulate copper-mediatedlipid peroxidation and neurotoxicity in culture of APP-knockout (APP0/0) and wild-type neurons [37]. Wild-typeneurons were found to be more susceptible than APP0/0neurons to physiological concentrations of copper, but notother metals.A recent study has examined the electrochemistry of the A
β
–copper complex [38]. It reveals that Cu(II) co-ordinates with A
β
in a 1:1 ratio. Independently of themethionine residue, the oxidation state of the copper centrein the complex is 2+. The data suggest that the presumedreductionreactionofftheCu(II)centretoCu(I)cannotoccur
in vitro
[38]. The midpoint potential for the reduction of thecopper centre was determined to be 0.08 V (compared withAg/AgCl) [38]. This group’s result suggests that Met
35
can beoxidized by the Cu(II) centre. The authors suggest that thecopper–A
β
complex could interfere with electron transportof the mitochondria, thus altering cell viability [38].Binding of metals to
α
-synuclein is a relatively newdiscovery. Therefore there has been no significant study of the electrochemistry resulting from this interaction. It hasbeen established that copper can cause the oxidation of theprotein,andthisisassociatedwithincreasedaggregation[39],but further work is still needed.
Aggregation
PrP, synuclein and A
β
are influenced by metals when itcomes to their aggregation (Figure 2). Polymerization studiesusually rely on recombinant protein or peptides, and theproducts of the reactions are analysed with fluorimetricassay such the thioflavin T assay, and other techniquessuch as electron microscopy, atomic force microscopy orvarious kinds of spectroscopy. In some cases, the presenceof metals accelerates the aggregation process, as is knownfor
α
-synuclein [40]. In the case of prion disease, copper hasbeenshowntoinhibitthepotentialoftheproteintoaggregate[12], although the picture is confusing as, in some cases, non-specific effects will accelerate aggregation [41]. Binding of manganese to PrP generates a form of protein that can actas a seed and catalyse aggregation [15]. However, manganeseitself has little effect on this form of aggregation.
Infection
Of the three diseases discussed in the present article, onlyprion disease can be associated with an ‘infection’. Thisusually means experimental transmission of prion diseaseeither from one animal to another or in cell culture. Justas metals play a role in the potential of the protein toaggregate, they also influence formation of the infectiousagent. Copper increases prion infectivity in animal models[42], and chelators can alter the incubation period [13].Understanding how metals influence this process has beenpoorly explored and remains an interesting prospect forfuture study.
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