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Prion protein 'binds copper in vivo'
Copper binding not demonstrated: webmaster
...suitability of apo-fragment?
...missing controls
...stoichiometry inconsistencies what about GPI-ceruloplasmin?
...where are those cuprione citations?
Earlier copper chelator studies: cuprizone and scrapie

Comprehensive Dec. 97 review article of copper proteins
Copper in Alzheimer's disease: histidines in APP 135-155
Repeat motifs in copper-binding membrane transporters: Menkes and Wilson disease

"The cellular prion protein binds copper in vivo"

Nature 390, 684 (1997)
D R Brown, K Qin, ..., D Westaway & H Kretzschmar
"The normal cellular form of prion protein is a precursor to the pathogenic protease-resistant forms believed to cause scrapie, BSE and CJD. Its amino terminus contains the octapeptide PHGGGWGQ, which is repeated four times and is among the best-preserved regions of mammalian PrPC.

Here the authors show that the amino-terminal domain of PrPC exhibits five to six sites that bind copper II presented as a glycine chelate. At neutral pH, binding occurs with positive cooperativity, with binding affinity compatible with estimates for extracellular, labile copper.

Two lines of independently derived PrPC gene-ablated mice exhibit severe reductions in the copper content of membrane-enriched brain extracts and similar reductions in synaptosomal and endosome-enriched subcellular fractions. These mice also have altered cellular phenotypes, including a reduction in the activity of copper/zinc superoxide dismutase and altered electrophysiological responses in the presence of excess copper. These findings indicate that PrPC can exist in a Cu-metalloprotein form in vivo."

Copper study commentary

12.19.97  -- webmaster
I have a number of grave reservations concerning the claims and interpretations made in the paper and title. However the low levels of membrane copper in knockout mice are novel and very striking. Normal function of prion protein could indeed, as often suggested, involve copper but that hasn't been shown unequivocably here:

1. Non-native protein: Only a small fragment of prion protein was used, 23-98. Of these 76 residues, only 32 have to do with the repeat region implied by the authors to bind copper. There is no experimental support in this paper to attribute localization to the repeat region. The fragment, produced in E. coli, is unlikely to have correctly modified arginines R25 and R37, and so is an apo-fragment. The effects of these modified arginines on conformation and metal binding properties cannot even be guessed at. The fragment also lacks the puzzling disease-associated residues 102 and 105 that are part of the random coil N-terminus. Although the authors use a GPI-cleaving phospholipase on a primary cerebellar line and mass spectroscopy elsewhere in the paper, no attempt is made to demonstrate released copper-containing native prion nor characterize the modified arginine by mass spec.

2. Controls, active copper species, and stoichiometry: It was not reported whether the purified fragment already contained copper or zinc, either from E. coli or acquired during purification as a contaminant. Dialysis binding studies only measured subtracted copper concentration on the control side, never on the prion side. This is a poor substitute: was the missing copper is bound to the prion fragment, as infered, or lost as precipitate or non-specific binding? Zinc was not considered in the binding studies -- an important specificity control given earlier work demonstrating comparable affinity.

BSA is a garbage collector, not a control; normally, a random polymer of the same amino acid composition as prion fragment would be used. They could simply be looking in part at a weak biuret reaction: copper coordinated with 4 peptide amides; this gives rise to redox cuprous ion. No data addresses the actual species of copper bound; no gel filtration shows copper bound to a correct molecular weight prion species. Not used were a number of readily available reagents that specifically and terminally chelate copper, notably cuprizone (bicyclohexone oxaldihydrazone) for Cu(II) and BCS, neocuproine, or bicinchoninic acid for Cu(I), or the broader-specificity copper chelator, D-penicillamine, for both. Without spectroscopic or chelation support, it is wholly speculative to say Cu(II) is the active species.

Despite criticism of earlier workers, a very familiar copper binding constant was reported: a few micromolar. No real explanation is given why their 1:2 mix of CuSO(4)+glycine [called cupric diglycine] is so different from CuSO(4)+ histidine or better than the CuSO(4) used for decades by copper biochemists: so what if Cu++ is hydrated ['hydrolzed' in paper] in aqueous solution -- what ion isn't, life goes on. Many, many molecular species are in rapid equilbrium. They find 5.6 atoms of copper bound per fragment with cupric diglycine, but only 3.0 with cupric dihistidine: this internal inconsistency deserves comment. That comment would be, as noted by earlier workers, that 1.0 is probably the correct stoichiometry as copper needs 4 ligands. There are no other candidate coordination atoms other than one histidine per repeat (and peptidyl amides).

Blocking suspect histidines with diethylpyrocarbomate (reversible with hydroxylamine) is an easy experiment, as is directed mutagenesis to substitute these histidines. Not discussed are the function of the histidine-displacing glutamine in the first repeat, PQGGGGWGQ, and copper binding by the rather different chicken prion repeat, PHNPGY; the chicken sequence reinforces a role for histidine but not for triglycine or tryptophan indole nitrogen (aromatic tyrosine seems to substitute). Altered numbers of repeats were not studied: the insertions can cause disease, a single deletion seems to be a common functional polymorphism with mild effects.

3. In vivo copper studies and ceruloplasmin: Dramatic loss of copper is noted in wild type neural cell culture after 3 hours of GPI lipase treatment; null mice are steady but had far less to begin with. True, prion proteins are GPI anchored and released by this enzyme, but so might be a couple hundred other GPI proteins, among them the dominant mammalian copper protein, ceruloplasmin, known to markedly increase in neurodegenerative disorders. The copper-release data are simply consistent with less ceruloplasmin expressed in null mice. Released copper is never quantitively associated with prion protein -- until shown otherwise, the default assumption is that miniscule amounts of prion copper are overwhelmed by GPI-ceruloplasmin. Menkes and Wilson disease genes are other neural membrane copper proteins that might be released by phospholipase and variably expressed under experimental conditions used here.

A novel GPl-anchored form of ceruloplasmin expressed by astrocytes.

J Biol Chem 1997 Aug 8;272(32):20185-20190
Patel BN, David S
Ceruloplasmin is a copper-binding protein and the major ferroxidase in plasma. We now provide evidence for a novel membrane-bound form of ceruloplasmin expressed by astrocytes in the mammalian central nervous system. Using a monoclonal antibody, we show that the cell surface antigen recognized by this antibody is ceruloplasmin and that it is directly anchored to the cell surface via a GPI anchor. Our peptide mapping and other immunochemical studies indicate that, except for the GPI anchor, the membrane-bound and secreted plasma forms are similar. We also show that the membrane-bound form of ceruloplasmin has oxidase activity. These studies therefore suggest that the GPI-anchored form of ceruloplasmin may play a role similar to the secreted form in oxidizing ferrous iron. The GPI-anchored form of ceruloplasmin expressed by astrocytes is likely to be the major form of this molecule in the central nervous system because serum ceruloplasmin does not cross the blood-brain barrier. Lack of this form of ceruloplasmin in the CNS could lead to the generation of highly toxic free radicals, which can cause neuronal degeneration as seen in aceruloplasminemia and other neurodegenerative diseases such as Parkinson's and Alzheimer's disease.

Increased regional brain concentrations of ceruloplasmin in neurodegenerative disorders.

Brain Res 1996 Nov 4;738(2):265-274 
Loeffler DA, LeWitt PA,... Kanaley L
Ceruloplasmin, the major plasma anti-oxidant and copper transport protein, is synthesized in several tissues, including the brain. We compared regional brain concentrations of CP and copper between subjects with Alzheimer's disease, Parkinson's disease, Huntington's disease, progressive supranuclear palsy, young adult normal controls, and elderly normal controls. Mean CP concentrations were significantly increased vs. elderly in AD hippocampus, entorhinal cortex, frontal cortex, and putamen. PD hippocampus, frontal, temporal, and parietal cortices, and HD hippocampus, parietal cortex, and substantia nigra. Immunocytochemical staining for CP in AD hippocampus revealed marked staining within neurons, astrocytes, and neuritic plaques. Increased CP concentrations in brain in these disorders may indicate a localized acute phase-type response and/or a compensatory increase to oxidative stress.
4. Missing citations: No paper on prions can be considered authentic without glaring omissions of earlier papers by colleagues. This paper excels: a couple dozen copper-and-prion articles in high-profile journals by well-known researchers such as Gibbs, Marsh, Merz, Kimberlin, and Pattison are overlooked. Earlier articles carry no abstracts at Medline, but cuprizone fortunately is a MESH term, so an effective search is easy.

Webmaster Commentary

19 Jan 98
Basically, in these experiments, cuprizone (a specific copper II chelator) was put in the feed of 4 week old rats, guinea pigs, or hamsters. By 8 weeks, they had developed a severe spongiform encephalopathy. Many experienced people confirmed an uncanny resemblance to the neuropathological changes that occur in scrapie. There must have been a suspicion that copper deficiency was even the cause of scrapie, not unreasonably in view of documented impacts of selenium deficiency etc. on livestock.

However, neither amyloid fibrils nor transmission could not be demonstrated. Interest waned and by the 1980's, cuprizone was used more as just a control. Failure to observe transmission is only as good as conditions used. If it wasn't intra-cerebral with several years incubation in a favorable species, not much is learned from negative results. There are many situations today where fibrils are not seen; sometimes this is simply attributed to size detection thresholds of electron microscopy or preparative dissociation.

It would be quite interesting to repeat some of these one-month experiments using modern methods (null mice, over-producing mice, 15B3 antibody) in view of reported copper binding to prion protein. It would be most provocative if knockout mice were not susceptible to cuprizone encephalopathy.

In one hypothetical motivating scenario, cuprizone passes the blood-brain barrier but is not taken up by neurons. (Cytoplasmic copper levels remain adequate and enzymes such as amine oxidases and superoxide dismutase continue to function.) Cuprizone then competes, with its superior binding constant and higher concentration, for exposed extra-cellular surface copper. Copper-containing GPI-anchored neuroproteins, such as prion protein and ceruloplasmin, lose their copper and ability to function. These now-defective proteins turn over more rapidly and are not processed correctly (because of abnormal configuration due to lost copper), resulting in rogue conformer accumulation. The cell seeks, in futility, to keep normal levels of prion protein on the surface via a feedback mechanism. The bottom line is, cuprizone causes up-regulation and over-production, resulting in spongiform encephalopathy. But not in PrP null mice.

If this came to pass, cuprizone-treated animals would provide a rapid knockout of prion gene function, one that continues to make exportable but non-functional protein. Conventional null mice have had the opportunity to mitigate since fertilization for loss of function, perhaps by inducing compensatory alternative proteins that mask the effect of loss of function.

Commentary (Robert A. LaBudde):

"It would be even more interesting to perform a BSE titer experiment on two cohorts of mice, one with and one without a copper supplement. Perhaps a copper supplement could reduce susceptibility and might be a possible therapy. Obviously copper is toxic, so there are limits to supplementation. "


I should have mentioned that something along these lines was tried already, with a confusing outcome. I recall it was first in 'Clinical and histological recovery from the scrapie-like spongiform encephalopathy produced in mice by feeding them with cuprizone' J Pathol 1973 Mar;109(3):245-250 Sansom BF, Pattison IH, Jebbett JN

Basically, supplemental copper wasn't all that effective in reversing cuprizone. The conditions used baffled me: it seems that they were using a whopping level of 0.5% chelator in the diet but some non-comparable other level of compensatory copper in the drinking water, leaving it unclear whether any or how much copper got through untitrated. And after-onset copper isn't going to fix a sponge. Someone also looked at both of the synthetic precursors used to make cuprizone (as potential contaminants) but they were not toxic.

Another scenario would have copper only addable in the cytoplasm during prion folding and assembly. Sometimes auxillary enzymes and factors are needed with prosthetic groups. (This could be vexatious in reconstitution or binding experiments.) Once stripped off extra-cellularly by chelator, fresh exogenous copper might not be able to go back on. But this is a protein that turns over fairly rapidly and if chelator is overwhelmed, then de novo protein should be ok.

I haven't been able to get yet 'The effects of cuprizone toxicity on the incubation period of scrapie in mice.' J Comp Pathol 1976 Jul;86(3):489-496. An interesting therapeutic concept though if normal prion function has to do with copper uptake, but would make things worse if the suggested copper detox at synapses is the role. tom

Biochemical changes in cuprizone-induced spongiform encephalopathy.

Neurochem Res 1983 Aug;8(8):1029-1044
Carey EM, Freeman NM

Immunohistochemical demonstration of GFAP in scrapie.

J Comp Pathol 1983 Apr;93(2):251-259 
Mackenzie A
Although little is known about its metabolism, glial fibrillary acidic (GFA) protein has become widely used as a cell-specific, species-non-specific antigenic marker for normal or pathologically altered astroglia. So far there have been few investigations on GFA protein in relation to scrapie and analogous spongiform encephalopathies although there has been a need for an unequivocal method for the discrimination of different types of glia in these diseases. In the present studies, a commercially available antiserum to GFA protein incorporated in a peroxidase-antiperoxidase procedure was applied to paraffin sections of brain and spinal cord from mice affected with scrapie, avirulent Semliki forest virus and cuprizone encephalopathy, and to tissues from healthy and scrapie-affected sheep.

Retinal degeneration during clinical scrapie encephalopathy in hamsters.

J Comp Neurol 1982 Feb 20;205(2):153-160 and Exp Neurol 1982 Dec;78(3):780-785 
Buyukmihci N, Goehring-Harmon F, Marsh RF
Weanling hamsters were inoculated intracerebrally with brain suspensions from normal or scrapie-infected hamsters. A third group of uninoculated animal was fed cuprizone. Histologic and electron microscopic examination of the neural retinas and retinal pigment epithelium was done. At 50 days postinoculation, when scrapie-infected animals began to show clinical signs of encephalopathy, there was a variable degree of photoreceptor degeneration. The neural retinas and retinal pigment epithelium of noninfected and cuprizone-treated animals were normal. We have previously shown that the scrapie agent accumulates in the retina; that together with our present work, we conclude that the scrapie agent is the cause of photoreceptor degeneration in experimentally inoculated hamsters.

The effects of cuprizone toxicity on the incubation period of scrapie in mice.

J Comp Pathol 1976 Jul;86(3):489-496

Profiles of brain glycosidase activity in cuprizone-fed Syrian hamsters and in scrapie-affected mice, rats, Chinese hamsters and Syrian hamsters.

J Comp Pathol 1976 Jan;86(1):135-142 
Kimberlin RH, Millson GC

A comparison of the biochemical changes induced in mouse brain cuprizone toxicity and by scrapie infection.

J Comp Pathol 1974 Apr;84(2):263-270 
Kimberlin RH, Collis SC, Walker CA

Permeability of blood vessels in mice affected with scrapie or fed with cuprizone.

J Comp Pathol 1973 Oct;83(4):461-466 
Kimberlin RH, Millson GC, Bountiff L, Collis SC

Clinical and histological recovery from the scrapie-like spongiform encephalopathy produced in mice by feeding them with cuprizone.

J Pathol 1973 Mar;109(3):245-250 
Sansom BF, Pattison IH, Jebbett JN

Unsuccessful attempts to produce disease with tissues from mice fed on a diet containing cuprizone.

Res Vet Sci 1973 Jan;14(1):128-130 
Pattison IH, Jebbett JN

Brain cell cultures from mice affected with scrapie or fed with cuprizone.

Res Vet Sci 1971 Sep;12(5):478-480 
Pattison IH, Jebbett JN

Clinical and histological observations on cuprizone toxicity and scrapie in mice.

Res Vet Sci 1971 Jul;12(4):378-380 
Pattison IH, Clarke MC, Haig DA, Jebbett JN

Histopathological similarities between scrapie and cuprizone toxicity in mice.

Nature 1971 Mar 12;230(5289):115-117 
Pattison IH, Jebbett JN

Spongiform encephalopathy induced in rats and guinea pigs by cuprizone.

Exp Mol Pathol 1969 Jun;10(3):274-287 
Pattison IH, Jebbett JN

Status spongiosus of CNS and hepatic changes induced by cuprizone.

Am J Pathol 1969 Feb;54(2):307-325
Carlton WW Suzuki K, Kikkawa Y

Studies on the induction of hydrocephalus and spongy degeneration by cuprizone

Life Sci 1967 Jan 1;6(1):11-19 
Carlton WW

Infection-specific particle from the unconventional slow virus diseases.

Science 1984 Jul 27;225(4660):437-440
Merz PA, Rohwer RG, Kascsak R, Wisniewski HM, Somerville RA, Gibbs CJ Jr, Gajdusek DC
Scrapie-associated fibrils, first observed in brains of scrapie-infected mice, were also observed in scrapie-infected
hamsters and monkeys, in humans with CJD, and in kuru-infected monkeys...These fibrils are also found in preclinical scrapie and in the spleens of scrapie-infected mice...[cuprizone shows up in Medline as a Mesh term for this abstract]

The extended environment of copper metal centers in protein structures

Proc. Natl. Acad. Sci. USA Vol. 94, pp. 14225-14230, December 1997
Samuel Karlin, Zhan-Yang Zhu, and Kenneth D. Karlin
Both histidine ligands of type I (blue) copper ions exclusively attach the N1 (proximal) nitrogen of the histidine imidazole ring to the metal, whereas histidine ligands for all mononuclear iron ions and nearly all type II copper ions are ligated via the N2 (distal) nitrogen. Multinuclear copper centers are coordinated predominantly by histidine N2. Explanations in terms of steric differences between N1 and N2 are considered.

The second-shell composition favors polar residues, except for type 1 copper where the second shell generally contains multiple methionine residues, which are elements of a statistically significant histidine-cysteine-methionine cluster, half solvent-accessible residues, facilitating electron transfer. Mononuclear copper atoms are never found with acidic carboxylate ligands.

Foremost ligands of Cu, Fe, Mn, and Zn ions include an imidazole nitrogen(s) of histidine , carboxylates, sulfur (both cys and met), water, and carbonyl oxygens. Fe ions are predominantly ligated by multiple H residues in conjunction with one or two acidic residues and occasionally Y. The primary ligands of Mn2+ are often only acidic residues. The principal ligands coordinating Zn2+ ions consist of combinations of H, D, E, C, and sometimes Y, N, S, and T. Copper coordination rarely includes unibidentates (same amino acid providing two bonds, eg, aspartic acid). Coordination patterns and metal-ligand bond lengths reflect the flexibility of the coordination geometry, steric constraints, stability in the coordination process, alternative electrostatics, and redox potential.

The bonding of the trinuclear copper complexes of ceruloplasmin, ascorbate oxidase, and the binuclear copper ions of hemocyanin are ligated in all but one contact by histidines via the N2 geometry. Steric differences between N1 and N2 may be decisive. The N2-metal interaction has the histidine main-chain displaced radially away from the metal site, whereas for the N1 contact the tautomeric geometry is displaced mostly in a tangential direction. In particular, two histidine N1 ligation contacts would sandwich the metal ion between them and thereby provide a more stable metal-ligand coordination and proximity to the backbone and second-shell environment. This may be a favorable situation for an electron transfer center such as in type I Cu. On the other hand, N2 ligand contacts afford more space to allow for metal-substrate catalytic interactions such as for O2 transport, O2 detoxification, or substrate oxidation occurring at mononuclear Fe and type II Cu sites. Zinc metalloproteins, including an abundance of hydrolases and transcription factors, do not engage in redox activity. Notably, there are to date no examples of metalloproteins having three or more histidine ligands that involve more than one N1 contact for any metal site.

The familiar type I Cu2+ ion ligands consist of H, C, H, M. For type I Cu2+ ions, the polar residues of the second shell include generally at least one N (asparagine), often part of an N-S or N-T pair, and one or more acidic residues.

Proteins with type II Cu sites include galactose oxidase and two copper amine oxidase structures from Escherichia coli and pea seedling. An essential organic molecule in these copper amine oxidases is the modified tyrosine cofactor TPQ (2,4,5-trihydroxyphenylalanine quinone). The copper ion in chain A is tightly coordinated by H-524, H-526, H-689, and the TPQ residue.

Galactose oxidase functions in catalysis of the stereospecific oxidation of a broad range of primary alcohol substrates. The active site is the mononuclear copper cofactor. Apart from two H, the other ligands are two Y residues and an exogenous acetate ion. Almost all the blue copper motifs are embedded in {CHM} clusters usually carrying at least two methionine and at least three histidine residues. The homotrimer nitrite reductase features a {HED} cluster about the type I copper.

Azurin , thought to transfer electrons from cytochrome c-551 to cytochrome oxidase, contains a highly significant {CHM} cluster embodying residues of both chains (from chain B: six M, three H, two C, two Y, three G, P, F, S, and D and from chain A: four M, three H, C, S, and F) and covering the Cu sites of both chains. The large number of methionine residues and aromatic residues in the cluster with many glycine fillers is striking. Azurin is of all beta-structure class, yet most of the second shell are residues of coil elements. Pseudoazurin and plastocyanin contain a significant {CHM} cluster enveloping the blue copper ion. Among type II copper sites galactose oxidase contains a highly significant {CHY} cluster about the copper ion.

Ligands in known copper proteins [from table]: HCHM, HHH H20, HCHM, HCHM,HCHM, HCHM, HCH, HCHM; YYHH, HHH TPQ, HHH H20.

For zinc, a second paper shows the principal ligands coordinating Zn ions comprise combinations of H (histidine), acidic (D and/or E), and C (cysteine) residues, water ligands, and sometimes the residues Y, N, S, and T. The ligand composition and geometry suggest six natural classes (Table 1): class I, ligand group involving at least three histidine residues, which share an elongated zinc binding motif HEXXHXXGXXH (E and G are not ligands); class II, ligand arrays that feature a proximal histidine pair HXH and a third histidine rather distant in the sequence; class III, combinations of H and C ligands; class IV, two separated H ligands, an acidic unibidentate plus a bound water molecule; class V, predominantly acidic ligands; and class VI, other ligand compositions.

A ligand group of three H abiding by the sequence motif HX3HX5H invariably invokes N2 contacts. A ligand group containing three H residues in the arrangement HXH and a distant H adopts for the proximal histidines the conformation N2, whereas the distant ligand acquires about equally the N2 or N1 conformation, with the exception of the metallo--lactamase structure. A ligand group including precisely two H, HXmH, bonds to the metal via the N2 contact. (iv) A ligand group of two H, from HXmH, adopts about equally the tautomeric alternatives: both N2, both N1, or mixed with one N2 and one N1. (v) The ligand group with a single H generally favors ligation via N1.

Class I are characterized as mononuclear zinc proteins having a histidine ligand group following the motif HEX2HX2GX2H . Several of these structures have an additional Y ligand. Class II: These are examples of zinc metals involving three histidine ligands with two in the sequence order HXH and the third H more than 20 positions away. Class IV: These are mononuclear zinc ion proteins coordinated by two histidines, one acidic unibidentate residue, and a bound water.

The amyloid precursor protein of Alzheimer's disease in the reduction of copper(II) to copper.

Science 1996 Mar 8;271(5254):1406-1409 
Multhaup G, Schlicksupp A, Hesse L, Beher D, Ruppert T, Masters CL, Beyreuther K
The transition metal ion copper(II) has a critical role in chronic neurologic diseases. The amyloid precursor protein (APP) of Alzheimer's disease or a synthetic peptide representing its copper-binding site reduced bound copper(II) to copper(I). This copper ion-mediated redox reaction led to disulfide bond formation in APP, which indicated that free sulfhydryl groups of APP were involved. Neither superoxide nor hydrogen peroxide had an effect on the kinetics of copper(II) reduction. The reduction of copper(II) to copper(I) by APP involves an electron-transfer reaction and could enhance the production of hydroxyl radicals, which could then attack nearby sites. Thus, copper-mediated toxicity may contribute to neurodegeneration in Alzheimer's disease.

Another neurological disorder, familial amyotrophic lateral sclerosis (FALS), supports our view that AD and FALS may be linked through a common mechanism. In FALS, SOD-Cu(I) complexes are affected by hydrogen peroxide and free radicals are produced. In AD, the reduction of Cu(II) to Cu(I) by APP involves an electron-transfer reaction and could also lead to a production of hydroxyl radicals. Thus, copper-mediated toxicity of APP-Cu(II)/(I) complexes may contribute to neurodegeneration in AD.

The beta A4 amyloid precursor protein binding to copper.

FEBS Lett 1994 Jul 25;349(1):109-116 
Hesse L, Beher D, Masters CL, Multhaup G
Previously it has been shown that the extracellular domain of transmembrane beta A4 amyloid precursor protein (APP) includes binding sites for zinc(II) and for molecules of the extracellular matrix such as collagen, laminin and the heparin sulfate chains of proteoglycans (HSPGs).

Here we report that APP also binds copper ions. A copper type II binding site was located within residues 135-155 of the cysteine-rich domain of APP 695 which is present in all eight APP splice isoforms known so far. The two essential histidines in the type II copper binding site of APP are conserved in the related protein APLP2. Copper(II) binding is shown to inhibit homophilic APP binding. The identification of a copper(II) binding site in APP suggests that APP and APLP2 may be involved in electron transfer and radical reactions.


     Site            108 "copper (His) (type 2)
     Site            110 'copper (His) (type 2)

     Site            147 "copper (His) (type 2)
     Site            149 "copper (His) (type 2)
     Site            151 "copper (His) (type 2)

     Site            388 "copper (His) (type 2)
     Site            390 "copper (His) (type 2)

Copper, iron, and zinc imbalances in severely degenerated brain regions in Alzheimer's disease: possible relation to oxidative stress.

J Neurol Sci 1996 Nov;143(1-2):137-142 
Deibel MA, Ehmann WD, Markesbery WR
Copper (Cu), iron (Fe), and zinc (Zn) levels in five different brain regions (amygdala, hippocampus, inferior parietal lobule, superior and middle temporal gyri, and cerebellum) were determined by instrumental neutron activation analysis (INAA) in samples from Alzheimer's disease (AD) patients and age-matched control subjects. A significant decrease in Cu, and significant increases in Zn and Fe were found in AD hippocampus and amygdala, areas showing severe histopathologic alterations in AD. None of these elements were significantly imbalanced in the cerebellum which is minimally affected in AD.

S2P metalloprotease cleaves APP

This gene encodes an unusual type of polytopic membrane protein that contains a consensus metalloprotease metal binding site of the HEXXH type... This site has been defined more extensively as abxHEbbHbc in which a is most commonly valine or threonine; b is an uncharged residue; c is hydrophobic; and x can be any amino acid except proline. The corresponding sequence in human and hamster S2P is GVVHEIGHGI, which matches the extended consensus precisely except that the a residue is glycine instead of valine or threonine. these coordinating residues can be located at widely varying distances from the HEXXH. The coordinating residues also vary. Histidine, tyrosine, and glutamic acid residues are among the most common.

The only protein that has been shown definitively to undergo cleavage within a transmembrane segment is the amyloid precursor protein (APP) (Selkoe, 1996 ). Intramembranous cleavage of APP is carried out by proteases called secretases that can cleave at either of two sites to generate peptides of 40 or 42 amino acids. In the brain, either of these peptides can form aggregates that lead to the toxic amyloid deposits of Alzheimer's disease, but the 42 aa peptide is by far the more potent. Recent evidence indicates that the protease that gives rise to the 40 aa amyloid peptide is inhibited by calpain inhibitors , which would not be expected to inhibit a metalloprotease such as S2P. The nature of the protease that creates the 42 aa peptide is not yet established, and it remains possible that this protease is S2P or one of its relatives.

Repeat motifs in copper-binding membrane transporters

PNAS 23 Dec 97
P-type ATPases form a large family of cation-transporting membrane proteins, with more than 50 members identified to date . A recently identified subfamily of putative soft metal P-type ATPases has been implicated in metal homeostasis. Representative members of this subfamily include bacterial enzymes such as CadA, CopA and CopB, CtaA, and eukaryotic Cu(I)-transporting ATPases such as the Menkes and Wilson disease-associated proteins.

The most striking feature of soft metal P-type ATPases is the presence of 1-6 motifs, GXXCXXC or (M/H)XXMDH(S/G)XM, at the N terminus of the molecule that are putative metal-binding domains. Differences in the number of times this metal-binding motif is repeated provide the basis for the enormous variation in mass of these proteins.

In addition, members of this group have either CPC or CPH in a putative transmembrane helix that might be part of the cation channel.Recently peptides derived from Menkes and Wilson proteins have been demonstrated to bind Cu(I). In addition, members of this group have either CPC or CPH in a putative transmembrane helix that might be part of the cation channel.

This is the first demonstration of Zn(II) transport by a P-type ATPase. Considering the importance of zinc homeostasis, we would predict the existence of a homolog of ZntA for zinc metabolism in humans, with inheritable metabolic diseases perhaps as severe as the Menkes and Wilson diseases.

The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene.

Nat Genet 1993 Dec;5(4):327-337 
Bull PC, Thomas GR, Rommens JM, Forbes JR, Cox DW
Wilson disease is an autosomal recessive disorder of copper transport, resulting in copper accumulation and toxicity to the liver and brain. The gene has been mapped to chromosome 13. On yeast artificial chromosomes from this region we have identified a sequence, similar to that coding for the proposed copper binding regions of the putative ATPase gene defective in Menkes disease. We show that this sequence forms part of a P-type ATPase gene that is very similar to MNK, with six putative metal binding regions similar to those found in prokaryotic heavy metal transporters.

Isolation of a candidate gene for Menkes disease and evidence that it encodes a copper-transporting ATPase.

Nat Genet 1993 Jan;3(1):7-13 
Vulpe C, Levinson B, Whitney S, Packman S, Gitschier J
Menkes disease is an X-linked disorder of copper transport characterized by progressive neurological degeneration and death in early childhood. We have isolated a candidate gene for Menkes disease and find qualitative or quantitative abnormalities in the mRNA in sixteen of twenty-one Menkes patients. Four patient showed rearrangements of the Menkes gene. The gene codes for a 1,500 amino acid protein, predicted to be a P-type cation-transporting ATPase. The gene product is most similar to a bacterial copper-transporting ATPase and additionally contains six putative metal-binding motifs at the N-terminus.

N-terminal Domains of Human Copper-transporting ATPases: One Copper Per Metal-binding Repeat

JBC Vol 272, Number 30,  July 25, 1997 pp. 18939-18944
Svetlana Lutsenko, Matthew J. Cooper  , Conrad T. Gilliam and Jack H. Kaplan
N-terminal domains of the Wilson's and Menkes disease proteins (N-WND and N-MNK) were overexpressed in a soluble form in Escherichia coli as fusions with maltose-binding protein, purified, and their metal-binding properties were characterized. Both N-MNK and N-WND bind copper specifically as indicated by the results of metal-chelate chromatography, direct copper-binding measurements, and chemical modification of Cys residues in the presence of different heavy metals.

N-MNK and N-WND bind copper in vivo with stoichiometry of 5-6 nmol of copper/nmol of protein. Copper released from the copper-N-MNK and copper-N-WND complexes reacts with the Cu(I)-selective chelator bicinchoninic acid in the absence of reducing agents. This suggests that in proteins, it is bound in reduced Cu(I) form, in agreement with the spectroscopic properties of the copper-bound domains.

Copper bound to the domains in vivo or in vitro specifically protects the N-MNK and N-WND against labeling with the cysteine-directed probe; this indicates that Cys residues in the repetitive motifs GMTCXXCXXXIE are involved in coordination of copper.

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