Role of Copper in Prion Disease
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Repeat copper: modularity and universality
Copper binding to octarepeat peptides by electrospray ionization mass spec
Manganese replacement of copper in prion protein function and protease K resistance
Brain copper and cuproenzyme activity: variance with prion protein level not confirmed
Repeat region of prion protein reduces copper: role of tryptophan residues.

Threshold survival of scrapie after ashing at 600C
Sheep: biopsy of the mandibular gland better than tonsil?
Prion missing from complete Drosophila genome
Coexistence of Alzheimer and prion amyloid
Blockers for orphan amyloid diseases

Copper modularity and universality

5 Apr 00 webmaster overview
Copper and TSEs have a long history (see onsite copper article index). The 1967-83 era focused on cuprizone-induced spongiform encephalopathy. While whole-animal copper depletion will have pleiotrophic effects, the uncanny pathological resemblance to prion disease remains noteworthy, though the interpretation of these 16 papers was uncertain then and now. Insufficient copper leads today to far more specific disease scenarios in both ALS and CJD.

The second copper boom began in 1995 with two papers on mammalian and avian repeat region copper binding by MP Hornshaw et al (ie, a full decade had gone by since prion protein sequencing without anyone noticing periodic histidine). Medline now shows 40 papers for 'prion and copper' with 8 from this year alone. However, the experimental dust has still not settled.

Part of the problem is that native protein is rarely used, even though the prion protein is not an integral membrane protein and is readily released from its GPI anchor by phospholipase. Few if any prion researchers are trained in classical protein chemistry, leading to unwarranted whining about low solubility and protein levels (neither remarkable). Denatured protein from E. coli of course lacks the two large glycan side chains, intrinsic metal, and two modified arginines. Some evidence suggests citrulline; partial modification, wrongly read as inessentiality, instead implies functional cycling.

What do binding constants for metals mean in a non-native environment? For copper binding studies, usually synthetic peptides are used, perhaps just a single repeat or two, model peptides only recently include end caps sequences. These flanking regions have been strongly conserved for hundreds of millions of years and cannot comprise a separate domain, so are good to include. Neither loaded or unloaded repeat domain is likely to have an intra-protein binding site on the main globular domain, by a modularity argument.

Nmr and other spectral methods have been frustrated by problems inherent to repeated units and internally repeated glycine in mammals; it is difficult to resolve residues in very similar environments. It may now be possible to crystalize a suitably flanked repeat region in the presence of copper at a favorable pH and determine its xray structure -- the peptide size after all is very small. The alternative is another 40 studies.

On 10 May 98, the webmaster posted the probable repeat structure (a non-alpha proline helix with interior axial copper) and explicitly predicted superoxide dismutase activity (verifiable at a German TSE listserve archive). Since 337,216 university-based visits to those pages have since been recorded, failure to cite this research will henceforth be treated as plagiarism.

That modular structural model is readily adjusted to pH-specific conformations (that is, to 1 or 2 copper atoms bound per repeat pair depending on whether both ring nitrogens coordinate copper), repeat length, residue variation, and repeat unit number via its length, pitch, and diameter. A random unstructured chain - bizarrely based on nmr in EDTA - never made sense for holoprotein and is ruled out by elementary bioinformatics (sequence conservation).

Based on experience with thousands of other proteins, whatever the function of the repeat domain, it is likely very similar in all lineages. This implies that repeat structural and functional proposals viable only for, say, turtle prion, but not bird or mammal prions, are fatally flawed from the get-go by a lack of universality. Turning this around, the divergence in repeat structure (unsurmountable by point mutation) from biphasic proline reptilian hexarepeat to longer monophasic marsupial and placental octarepeats, provides an important constraint (ie, opportunity).

Structure/function proposals must also accommodate repeat phylogenetic divergence within mammals, polymorphisms within species, and mutational types within human (conveniently compiled at 1, 2, 3. That is, abbreviated repeat regions are documented in healthy wildtype lemur and Siberian wild goat; bovids have at least three repeat alleles; many species have repeat unit deletion and insertion alleles; intensively sequenced humans exhibit numerous length variations both admissible and pathological ). Rodents and turtle are noteworthy for slightly variant composition of individual amino acids; marsupial prion, with a 10-10-10-9 pattern PQGGGTNWGQ PHPGGSNWGQ PHPGGSSWGQ PHGGSNWGQ, restrains schemes for placental mammals.

These latter taxa show two types of variability within the first repeat: lengths of 8 or 9 residues appear equally acceptable (the glycine run may be 3 or 4); the glycine preceding the tryptophan may be replaced by threonine (and more rarely serine). Serine also occurs sporadically at this and other positions in other repeats, mainly in rodents, but overall it is safe to infer its hydroxyl group has no role. The terminal repeat also accommodates an extra glycine in many species. A model too rigid to accommodate these length variations and particular substitutions can be safely rejected. On the other hand, other conservative variations have evidently been rejected. The criticality of a polar aromatic residue (tryptophan in mammals, tyrosine in birds/turtle) is especially well-supported, eg phenylalanine is unacceptable (a large number of sequences is at hand). Better conserved than the much-touted histidines, the only explanatory scenario on the table is edge-to-face structural stacking.

The fundamental question is finding a 'universal' 3D structure and function consistent with this observed evolutionary plasticity. It is very clear that neither the exact length of the periodic unit nor the number of repeats nor the exact composition can be critical, within certain ranges. Since birds and mammals had a common ancestor at 310 million years, early amniotes are predicted to lack a repeat region as we know it now but instead have a small proto-repeat generator that gave rise to various repeat lengths in diverging lineages via subsequent replication slippage events. This region may already have had functional copper binding [see below].

This strongly suggests that both structure and function are modular, ie, readily accommodate fewer or more repeat units. As an application of modularity, a published planar ring structure lacks modularity and so is impossible: what bond angles and copper distances would 3x lemur, 4x human, 5x mouse, 6x kudu, and 7x bovine rings have in common? For these relatively new alleles to be fixed, the resulting protein needed to be immediately functional without numerous compensatory secondary mutation.

Only a helix provides the necessary modularity to accommodate different periodicities, the variable numbers of units, and absolutely invariant periodic proline. Modularity implies a translational structural symmetry; the amino acid composition is incompatible with alpha helix or beta sheet. The helical pitch is driven not by periodic hydrogen bonding geometry but by rigid dimensional requirements of copper coordination distances, proline bends, and amino acids per unit. The structure of cap regions must be determined experimentally. (These do not include terminal patches of basic amino acids interdigitated with proline, which are likely in extended conformation, possibly for interaction with membrane phospholipid or heparin sulfates.)

What does functional modularity mean? In the main non-enzymatic scenario, wrongly thought of as copper transport, an unloaded repeat arm binds free copper in the vicinity of the membrane, undergoing a conformational change to a more compact helix; triggering endocytosis of the loaded repeat to interior endosomes where copper is discharged or transferred as key histidines are protonated in a much more acidic cellular compartment (to be safely assimilated into metallothionein etc.) prior to protein recycling to the cell surface (refs 1, 2, 3). This is only incidentally copper transport by potocytosis -- the cell has no shortage of dedicated copper transport systems and mapped copper diseases already; the real function is protection of the synapse and sensitive membrane components from unwanted oxidation generated by copper.

This scenario is modular and universal: give or take a few repeats, functional efficiency is unaffected because a shorter repeat may load faster and recycle more often, whereas a longer repeat recyles less often but brings in more. (Energy efficiency of cycling is of little relevence to mammalian cells -- vast amounts of ATP is wasted in transcribing huge introns.) Individual repeat units that apparently cannot bind copper, as in turtle prion, might still be adaptive in providing extensional length.

Functional modularity in enzymatic scenarios is unprecedented at this small scale size but nonetheless possible. While the geometry and ligands of copper in the proposed repeat helix are those found in copper-zinc superoxide dismutase, many copper proteins that are not really superoxide dismutases nonetheless exhibit this activity (eg, ceruloplasmin, transferrin and ferritin); indeed superoxide turns over extremely rapidly without any enzyme present at all (recall carbonic anhydrase). In other words, this reaction is unusual in that a repeat region or two could carry sufficient structure for catalytic competency (even though the smallest known conventional globular enzyme have some 110 amino acids): the metal is doing all the work, it just needs a small cage.

This scenario is also modular and universal: more repeats simply means more catalytic units per monomer; species with varying repeat lengths could adjust to similar total catalytic activities through regulation of transcription; details such as repeat length don't matter if a copper cage still results. Copper is usually involved in small molecule oxidations involving molecular oxygen and other reactions involving nitric oxide or peroxynitrite present similar considerations.

Functional modularity in purely structural scenarios could also be envisioned though copper is implausible in this context. Here the repeat region extends to bind some component of the extra-cellular matrix; metal binding causes a conformational contraction. Modularity is missing because of length differences; universality is questionable. A variation has repeats binding one-on-one to repeats of another macromolecule. Provided the latter are extensive, modularity strengthens overall binding. Short internal tandem repeats are common enough in proteins but often involve heptapeptide binding of repeats in nuclear DNA, out of the question here.

Recent experimental work on copper, while not producing miracles, has made some definite progress on the role on our understanding of the role of copper in prion protein, but not without some dissenting views. These papers are summarized briefly below .

Copper binding to octarepeat peptides of the prion protein monitored by mass spectrometry

Protein Science (2000), 9: 332-343 (Received July 19, 1999) Free full text
Randy M. Whittal,  Haydn L. Ball, Fred E. Cohen,  Alma L. Burlingame, Stanley B.Prusiner, and Michael A. Baldwin 
Comment (webmaster): The article is well worth reading in the free full text. It has interesting and detailed experimental results, good coverage of earlier related work, and improvements afforded by direct ESI-MS over spectral studies. Highlights include individual binding constants, copper binding upstream of the repeat region (a property expected in the ancestral sequence); a role for the post-repeat conserved residue histidine 96 (not quite conserved in all species); and a conformational shift induced by pH titration of histidine ring nitrogen (See also Miura 1996, 1999; Wong 1999).

Copper binding stoichiometry was markedly pH dependent: four octarepeats bound two Cu2+ ions at pH 6 but four at pH 7.4, where circular dichroism suggested a major structural adjustment. No support was found for Cu+1. Extending C-terminally to include histidine 96 led to a high degree of binding cooperativity. (Dissociation constants for individual copper ions were in the low micromolar range but 100nM for the third and fourth Cu2+ ions at high pH. Cooperativity was also seen by Brown et al., 1997) Copper also bound a peptide preceding the octarepeats and also with a cyclohexyl histidine ring replacement. Significant binding of the first Cu2+ ion in the pH range 4-5 suggested histidine imidazole in a solvent-excluded environment.

Thus flanking regions are important to include; experiments will not be comparable except for the same peptide at the same pH. Note too that hamster has threonine in the first repeat instead of glycine. While the arginines were not covalently modified as found in vivo and ESI-MS not yet brought to bear, an author commented in correspondence that data from J Hope favored citrulline (pK 9.4, the immediate biosynthetic precursor of arginine in the urea cycle), consistent with early mass spec data of a +/- 1 dalton shift. Trypsin can cleave PrPSc at Arg-37, suggesting a significant fraction is unmodified (moot at arg-25, followed by proline). An acid-labile group such as phospho-arginine, lost during HPLC separation of peptides, remains a possibility. Arginine forms a well-known Cu+2 chelate. Note that full length protein deleted for the octameric repeat region does not appear to bind copper (Brown et al, 2000, EMBO J.)

Citrulline is an interesting candidate for the modification, as noted earlier. Intriguingly, it is the byproduct of nitric oxide synthase from an arginine substrate. The observed partial modification could reflect cycling between arginine and citrulline in a scenario where the repeat region itself was the substrate for generating precisely located NO, with endosomal regeneration via argininosuccinate synthase and argininosuccinate lyase. The webmaster previously considered related explanations for prion copper involving the interplay of nitric oxide, superoxide, and peroxynitrite dismutation.

Zinc binding, of special interest because of superoxide dismutase, was non-specific at higher Km. However, simultaneous zinc/copper binding was not studied; possibly specific zinc binding takes place after copper is already in place. Manganese, found in another type of SOD, was not studied either. Another factor, difficult to rule out, is a role for an auxillary copper chaperone protein, needed in other situations where holoenzyme formation is not spontaneous. Table 1 of the article helpfully summarizes copper binding data in the literature. Hornshaw et al. provided the original idea and data for copper binding; the lab floats yet another bogus claim for credit based on a commercial copper affinity column used earlier on hexahistidine recombinant protein.

A technical question arises about the validity of using free cupric ion, which does not exist within cells to any extent (less than 1 free ion per cell according to Science 284, 805-808). In the copper protein literature, a chelate to amino acids such as glycine or histidine or denaturating conditions are generally used to mimic the conditions in which copper may bind during protein synthesis . This issue also arises in a previous study (Viles et al. 1999) where the same group did not see copper bound to full length PrP. (Compare to Brown, 1999, J. Neurosci Res. and Brown et al., 1999, Biochem. J.). Circular dichroism measurements were made at 250 M Cu which is not remotely physiological.

How is it possible that noncovalent protein-metal interactions in solution can be maintained during desolvation and transfer to the gas phase allowing non-artefactual mass spectrometry? While this seems too good to be true, the article nicely summarizes supporting literature and explains the underlying physical chemistry:

"We have established that ESI-MS is able to monitor specific interactions between PrP peptides and Cu2+ ions and to distinguish these from weak, seemingly nonspecific associations with other metals. Measuring an equilibrium constant by ESI necessitates some assumptions. The first assumption is that the relative amounts of free and complexed peptide can be measured by assuming equal signal responses for both. The second and perhaps more important is that a measurement of ion current in the gas phase quantitatively represents the solution equilibrium. We know that in the process of desolvation, the charged droplets shrink until they reach the Rayleigh limit and break apart (Kebarle & Tang, 1993)."

"Shrinking droplets are small isolated chambers whose contents become progressively more concentrated, compared with the bulk solution from which they came. As they pass through the interface, the droplets are heated by collisions with hot gas but cooled by evaporation, causing their temperature to fall to 10 C below ambient, and the analyte is completely desolvated within 450 s (Kebarle & Tang, 1993). From this point on the analyte exists in the form of isolated ions that are stable on the timescale of the mass spectrometric separation and detection, thus the sample is effectively "frozen." Ultimately, the validity of this technique for monitoring complex formation is dependent upon competition between the rate of any changes in equilibrium caused by the effects outlined above vs. the rate of evaporation. Only for the fastest solution reactions is this likely to present a problem, and there is increasing acceptance of the use of ESI-MS for such studies of noncovalent interactions. "

The authors conclude that on balance the data support a normal prion repeat function of non-enzymatic copper uptake:

"Endocytosis could then transport PrPC into the cell in endosomes and secondary lysosomes, where lowered pH would cause much of the PrP-bound Cu2+ to be released. Such a mechanism for the sequestration and transport of ions and small molecules into the cell by caveolae has been described as potocytosis." [Anderson et al., Potocytosis: Sequestration and transport of small molecules by caveolae. Science (1992) 255:410-411.]

'Potocytosis' has not really caught on; only 4 articles per year at Medline use it, no examples of ion transport are known and some articles dispute small molecule transport. Indeed, genomic sequencing has established a plethora of more standard metal transport systems. Another 'nutritional' copper uptake system seems redundant; the same could be said for the superoxide dismutase activity, proposed first by the webmaster with experimental support shortly from DR Brown and now 3 other groups.

Consequences of manganese replacement of copper for prion protein function and proteinase resistance

EMBO Journal Vol. 19, pp. 1180-1186, 2000
David R. Brown, Farida Hafiz, Leslie L. Glasssmith, Boon-Seng Wong, Ian M. Jones, Christine Clive and Stephen J. Haswell 
Comment (webmaster): This is another paper in a major series also best read as the full text. Some rather surprising properties of manganese ion are reported, considering this is the 40th paper on prion protein and transition metals. Mn++ can replace copper but this lead to protease K resistance, seen also in astrocytes grown in medium with copper overwhelmed by manganese, ie, seeds may form when the wrong metal is occupying the repeat region binding sites. Brown's work stresses the experimental importance of a chelated copper supply and refolding opportunities, as metal binding otherwise might not attain equilibrium.

Note protease K resistance is not a reliable proxy for infectivity; there may be many unrelated, irrelevent conformational shifts leading to protease resistance; infectivity was not studied here.

While there is no shortage of theories for sporadic TSE already, Brown et al suggest reasonably enough that dietary copper:manganese ratios could explain some fraction of these, supporting a thinly motivated proposal by Purdy. Poorly rationalized neutriceuticals are taken daily by millions of people and livestock also receive unnatural mineral supplementation.

Highlights of the paper:

The prion protein binds copper and has antioxidant activity enhancing the survival of neurones in culture. The ability of the PrP to bind other cations was tested and it was found that only manganese could substitute for copper. Although initially manganese-loaded PrP exhibited similar structure and activity to copper-loaded PrP, after aging, manganese-loaded PrP became proteinase resistant and lost function. It was also found that manganese could be incorporated into PrP expressed by astrocytes and that this PrP was partially proteinase resistant. These results show that it is possible to generate proteinase-resistant PrP from cells and suggest a possible mechanism for the formation of the scrapie isoform of the PrP as generated in sporadic prion disease....

PrPc expression appears to aid resistance to oxidative stress and copper toxicity at least in vitro. PrPc has been shown to influence copper uptake into neurones. Copper taken up in association with PrPc can be utilized at the synapse or can be incorporated in Cu/Zn superoxide dismutase. More importantly, PrPc with copper bound to it can act as a superoxide dismutase, possibly explaining why cells deficient in PrPc expression are sensitive to stress from cell culturing conditions.

Although it has been established that PrPc is a copper-binding protein it is unknown whether the protein can bind other divalent cations. In this report we found that recombinant PrPc will bind manganese and nickel. Of these, manganese appeared to alter PrPc to a proteinase-resistant form that forms fibrils. Furthermore, PrPc expression influences uptake of manganese into cells. Therefore, we speculate that incorporation of manganese into PrPc may be one way in which PrPSc can be formed in vivo.

We have shown previously that full-length PrP binds up to four copper atoms to the octameric repeat region. PrP refolded with copper was demonstrated to have an antioxidant activity like that of superoxide dismutase. It has been suggested from studies with peptides based on the octameric repeat region of PrP (Viles et al., 1999) that the four histidines in the repeat region coordinate four copper atoms, each one associated with two histidines. Alternatively, each histidine could bind one copper atom each. To test this possibility we prepared mutants of mouse PrP with none, one, two or three repeats. The mutants and full-length PrP were expressed in bacteria, extracted from inclusion bodies and refolded in urea in the presence of 5 mM CuSO4. ... The resulting protein was analysed by total X-ray reflection fluorescence (TXRF) spectroscope. The mutant PrP with no octameric repeats bound a residual level of copper. In comparison, the other mutants bound the equivalent of one, two or three atoms of copper depending on how many octameric repeats were left in the protein. These results suggest that the four histidines in the repeat region can bind one copper atom each.

The mutants containing one or no octameric repeats showed no SOD activity. The minimal requirement for superoxide dismutase activity was two octameric repeats. A mutant with three octameric repeats binding three copper atoms had no more activity than a mutant with two. The activity of wild-type PrP (binding four atoms of copper to the octameric repeats) was the highest, and was significantly greater than that of PrP with only two or three repeats.

Recombinant full-length and deleted PrP was refolded as described with other divalent cations including calcium, magnesium, manganese, nickel, iron and zinc at concentrations of 5 mM.... The deletion mutant did not retain any of the divalent cations, indicating that any retention by the full-length protein was a result of binding to the octarepeat region. Of the cations tested only nickel, zinc and manganese were retained by PrP. Of these, zinc was poorly retained. In order to determine whether manganese and nickel could replace copper when in competition, full-length PrP was refolded with either nickel and copper or manganese and copper in equimolar concentrations (5 mM). In these cases copper was preferentially bound in place of nickel but manganese was bound equivalently with copper (i.e. two atoms each per molecule). When the concentration of manganese was decreased during refolding (5 mM copper, 0.5 mM manganese) the amount of manganese binding was reduced to one-tenth that of copper. These results suggest that manganese can substitute equivalently for copper in the holo-form of PrP.

Given that only manganese and nickel substituted for copper in refolded PrP, samples refolded only with these cations were used for assaying superoxide dismutase activity of these proteins. As indicated above, copper-loaded PrP has substantial activity in this assay. Manganese-loaded PrP showed half the activity seen for the same mass of copper-loaded PrP. Manganese-loaded PrP lost no activity after aging, with increased with PK resistance and CD spectrum shifted to higher beta sheet... Astrocytes grown in the presence of manganese or copper were collected and PrP was immunoprecipitated. Native PrP isolated from cells grown in the presence of manganese incorporated manganese and more resistant to PK treatment. Manganese is used in mitochondrial manganese superoxide dismutase. It is possible that PrPc could influence uptake of other cations and influence manganese metabolism.

Stckel et al. (1998) who did not find that recombinant PrP binds manganese; the methods used to incorporate copper or manganese into PrP were possibly inadequate for saturation. Refolding the protein in the presence of cations possibly represents the optimal method for cation incorporation. Furthermore, we have shown that manganese is incorporated into native PrP from cells cultured with manganese in the medium. Therefore, it is possible that manganese can be incorporated into or binds to PrPc in vivo.

The observation that on binding manganese PrPc becomes proteinase resistant is probably the most important finding in the present report. Proteinase resistance, along with fibril formation, is one of the most important characteristics distinguishing PrPSc from PrPc. Ttis is the first report of the de novo formation of proteinase-resistant protein from native PrPc from cells without the interaction with PrPSc. [Note: protease resistance could have many unrelated structural origins. -- webmaster]

An environmental cause for sporadic disease remains a possibility. Recent investigations of scrapie, CJD and chronic wasting disease clusters in Iceland, Slovakia and Colorado, respectively, have indicated that the soil in these regions is low in copper and higher in manganese (Purdy, 2000). The possibility that imbalances in environmental cations entering the food chain may induce conditions favouring the formation of proteinase-resistant PrP is a controversial but intriguing possibility. This idea is supported by the findings presented in this paper.

The finding that incorporation of manganese into PrP makes it proteinase resistant and abolishes its function is a long way from explaining sporadic prion disease. However, a current favourite among hypotheses concerning conversion of PrPc to PrPSc requires the formation of `seeds' or `nuclei' of proteinase-resistant PrP. The formation of sufficient proteinase-resistant PrP to create such a seed is the limiting step in this model of PrPSc formation. Incorporation of manganese in place of copper in the holo-form of PrPc may represent one possible way in which the substance of a `seed' could form.

Copper has differential effect on prion protein with polymorphism of position 129.

Biochem Biophys Res Commun 2000 Mar 24;269(3):726-31 
Wong BS, Clive C, Haswell SJ, Jones IM, Brown DR 
The pathology of human prion diseases is affected by polymorphism at amino acid residue 129 of the prion protein gene. Recombinant mouse prion proteins mimicking either form of the polymorphism were prepared to examine their effect on the conformation and the level of superoxide dismutase (SOD) activity of the prion protein.

Following the binding of copper atoms to prion protein, antibody mapping and CD analysis detected conformational differences between the two forms of protein. However, neither the level of copper binding nor the level of SOD activity associated with this form of prion protein altered with the identity of codon 129. These results suggest that in the holo-metal binding form of the protein, prion structure but not its SOD activity is affected by polymorphism at codon 129.

Comment (webmaster): Hopefully this means human protein as mouse differs at too many other residues for any purpose to be served by changing mouse codon 129. There was no reason to expect an effect on either activity and none was found. This result should be compared to Wadsworth JD et al. Nat Cell Biol 1999 May;1(1):55-9 in which strain-specific prion-protein conformation were determined by metal ions (two strain types were interconverted in vitro by altering their metal-ion occupancy.)

Functional and structural differences between the prion protein from two alleles prnpa and prnpb of mouse.

Eur J Biochem 2000 Apr 15;267(8):2452-2459 
Brown DR, Iordanova IK, Wong BS, Venien-Bryan C, Hafiz F, Glasssmith LL, Sy MS, Gambetti
P, Jones IM, Clive C, Haswell SJ
The prion protein is a glycoprotein expressed by neurones and other cells. In its holo-form it binds copper and exhibits superoxide dismutase activity. Studies in mice have led to the description of two distinct alleles. Differences in these alleles are linked to long and short incubation times following infection with scrapie. We studied recombinant mouse protein corresponding to the products of either allele and two intermediates carrying single amino-acid residue substitutions. The different forms of the prion protein exhibited differences in superoxide dismutase (SOD) activity and conformation. Intermediates with single substitutions were less stable than either allelic product. The findings provide insight into the differences between the two alleles and might have consequences for understanding differences in susceptibility to prion disease.

Comment (webmaster): Long incubation period mouse carries two non-conservative amino acid substitutions L108F and T189V relative to rodent consensus sequence (as a result of extreme inbreeding) which are probably deleterious > to normal prion function of the globular domain. However short incubation mouse is itself a messed-up sequence (see rodent alignment, discussion and feature table).

Brain Copper Content and Cuproenzyme Activity Do Not Vary with Prion Protein Expression Level

J Biol Chem, Vol. 275, Issue 11, 7455-7458, March 17, 2000
Darrel J. Waggoner, Bettina Drisaldi,... Jonathan D. Gitlin, and David A. Harris 
Comment (webmaster): This is a strong experimental paper with necessary, but primarily negative, conclusions. Indeed, there was already a surfeit of known proteins -- and corresponding diseases -- adequate to handle mainstream aspects of copper metabolism . The question is how to procede further with decisive positive experiments to elucidate the copper component of normal prion function.

Highlights of the paper:

Although the normal physiological function of PrPC has remained enigmatic, the recent observation that the protein binds copper ions with micromolar affinity suggests a possible role in brain copper metabolism. In this study, we have used mice that express 0, 1, and 10 times the normal level of PrP to assess the effect of PrP expression level on the amount of brain copper and on the properties of two brain cuproenzymes. Using mass spectrometry, we find that the amount of ionic copper in subcellular fractions from brain is similar in all three lines of mice. In addition, the enzymatic activities of Cu-Zn superoxide dismutase and cytochrome c oxidase in brain extracts are similar in these groups of animals, as is the incorporation of radioactive Cu into Cu-Zn superoxide dismutase both in cultured cerebellar neurons and in vivo. Our results differ from those of another set of published studies, and they require a re-evaluation of the role of PrPC in copper metabolism.

A report that there is a 10-15-fold reduction in the content of copper, but not of several other metals, in brain membranes from PrP-null mice compared with wild-type controls suggests that PrPC could be a major copper-binding protein in brain. Loading of Cu-Zn superoxide dismutase (SOD1) are said 50% of normal in brain and cultured cerebellar neurons from PrP-null mice, and that SOD1 activity and copper loading are reported elevated in mice that overexpress PrP. We re-examined this evidence in favor of a role for PrPC in copper metabolism and found no support

Knockout mice from Switzerland were used, meaning that no read-through to doppel could occur. The three different expression levels of PrP came from these, wild-type mice, and Tga20 mice which carry a wild-type PrP transgene and express some 10 times the normal level of PrP. No significant differences among mice of the three genotypes were found in the content of copper, zinc, or iron in any of the brain fractions. The absolute values for total brain copper are close to those reported by other investigators. SOD1 activities in brain lysates fromthe three lines were also similar based on two different spectrophotometric assays. Copper incorporation into SOD1 in cerebellar cultures exhibited no difference in the intensity of this band from mice of the three genotypes; similar results were obtained from the brains of animals given intrathecal injections of 500 Ci of 64Cu.

Our results suggest that PrPC is not the primary carrier responsible for entry of copper into the brain via the blood-brain or blood-CSF barriers or for uptake of the metal into neurons from the extracellular space. If PrPC were an essential component of these global uptake mechanisms, then variations in PrP expression should be directly reflected in the copper content of brain fractions. Our studies also indicate that PrPC does not play a role in the specialized trafficking pathways involved in delivery of copper to SOD1 and COX in brain. Conceivably, PrPC could function in copper trafficking pathways upstream of chaperones such as CCS.

In mammalian cells, copper transported across the plasma membrane is delivered to cuproproteins via specific chaperone molecules, which bind copper and transfer it to the target protein by physical association ). This mechanism is necessary because, due to its extreme toxicity, free copper is kept at negligible concentrations in the cytoplasm (less than 1 ion per cell, Science 284, 805-808) by binding to copper-scavenging proteins such as metallothionein .

One possibility is that copper is an essential cofactor for a previously unidentified enzymatic activity of PrPC such as superoxide dismutase activity. However, cuproenzymes such as SOD1 have much higher affinities for copper than PrPC (femtomolar versus micromolar), and these enzymes, unlike PrPC, usually require partial denaturation for removal of bound copper. In addition, they all contain copper centers characterized by metal bonding to sulfur atoms in methionine or cysteine residues, in addition to nitrogen atoms in histidine residues as for PrPC . [The webmaster disagrees: see Fig 8.19 of Creighton or any pdb entry: the ligands are 6 histidines (one bridging and 1 aspartate.]

A much more likely function for PrPC is to serve as a reversible sink or carrier for copper ions. Consistent with this suggestion, the normal concentration of Cu2+ in plasma and CSF (1-10 M) is similar to the estimated Kd for copper binding to PrPC, and the concentration of the metal in brain tissue is estimated to be even higher (100 M). Most extracellular copper is bound to ceruloplasmin in plasma and CSF, and the remainder is complexed with albumin, peptides, and amino acids. PrPC may function as an endocytic receptor for cellular uptake of copper ions [P Pauly, D Harris J Biol Chem, Vol. 273, Issue 50, 33107-33110], or facilitate some other aspect of copper trafficking such as efflux from the cell or intracellular sequestration of the metal.

The N-Terminal Tandem Repeat Region of Human Prion Protein Reduces Copper: Role of Tryptophan Residues.

Biochem Biophys Res Commun 2000 Mar 16;269(2):491-495 
Ruiz FH, Silva E, Inestrosa NC
Toward its N-terminal region, PrP bears an octapeptide repeat which has been shown to bind copper. We found that a human synthetic peptide corresponding to the four repeats of Pro-His-Gly-Gly-Gly-Trp-Gly-Gln has the ability to reduce copper. A mutant peptide lacking tryptophan displayed only 24% of the wild-type copper-reducing activity. Experiments performed in a nitrogen atmosphere confirmed that O(2) is not involved in the reaction. Our results indicated that cell surface PrP, besides its ability to bind copper, bears the capacity to reduce copper in vitro. The potential physiological role of copper reduction by PrP is discussed.

Comment (webmaster): It is not clear what to make of this observation. If the authors relied on bathocuproine disulfonic acid to measure Cu+1, this may not have been specific. What accounts for the residual copper-reducing activity? A comparable reaction seems unlikely in avian prion, which has tyrosine in this position, so an in vivo role lacks universality. Even if non-specific, oxidation of tryptophan raises questiongs about interpreting binding of copper quenching rp fluorescence emission, shifting the emission spectrum to shorter wavelengths, and induced changes in the near-UV CD spectrum [Biochemistry 1998 May 19;37(20):7185-93].

Copper-oxidized low density lipoprotein contained kynurenine residues in place of tryptophan. Tryptophan and tyrosine residues in proteins may be posttranslationally modified to form enzyme cofactors such as covalently cross-linked tryptophan tryptophylquinone (TTQ), the cofactor of methylamine dehydrogenase (MADH), which incorporates two oxygen atoms into one of the indole rings to form a quinone. TTQ mediates electron transfer from substrate to a copper protein called amicyanin.

The octapeptide repeat region of prion protein binds Cu(II) in the redox-inactive state.

Biochem Biophys Res Commun 2000 Jan 7;267(1):398-402
Shiraishi N, Ohta Y, Nishikimi M 
The octapeptide repeat region of human prion protein is known to bind four Cu(II) ions per molecule. A peptide representing this region was tested for inhibitory effects on copper-catalyzed oxidation of l-ascorbate or glutathione and on generation of OH(*) during the former reaction. The result indicated that the catalytic activity of the first Cu(II) ion bound to an Octa(4) molecule was completely suppressed.

The valence state of the copper under reducing conditions was Cu(II), as determined by a newly developed method using bathocuproinedisulfonate under acidic conditions. Furthermore, it was shown that Escherichia coli cells expressing the octapeptide repeat region were significantly resistant to copper treatment compared with control cells. The results taken together indicate that prion protein can function to sequester copper ions in the redox-inactive state, rendering copper-induced generation of reactive oxygen species impossible.

Comment (webmaster): This result suggests either physical sequestration of copper or its exclusionary coordination. Hydroxyl radicals are important to consider, as are the plethora of new related oxygen and nitrogen singlet states and reactive non-specific oxidants.

Alzheimer and prion amyloid

JAMA 5 Apr 2000 G. Leuba et al.
Familial Alzheimer disease (AD) with early onset has been linked to 3 different genes with an autosomal dominant mode of inheritance: -amyloid, protein precursor, and the presenilins 1 and 2, representing not more than 50% of all cases of early-onset AD cases.

Thus, the genetic defect remains unexplained in at least half of the families with histories of early onset of AD. We have recently described such a Swiss family whose members presented with a standard clinical and neuropathologic profile of AD.

In particular, severe neurofibrillary tangle degeneration was present in the hippocampus and in several cortical areas, together with a large amount of beta-amyloid deposits and senile plaques. However, known mutations have not been found, either in the beta-amyloid precursor protein or in the presenilin 1 and 2 genes.2 We now report that the brains of 5 deceased members of this family, from 2 generations, present a coexisting beta-amyloid and prion protein (PrP) pathology

....Coexistence of Creutzfeldt-Jakob disease (CJD) and AD in some patients has been described but appears mainly related to age in patients proven to have CJD. However, since the individuals in the Swiss family died over a long interval and were all similarly affected, it is unlikely that CJD is purely coincidental.

...The data presented herein support the existence of a possible new subtype of familial early-onset AD with a concomitant -amyloid and prion brain pathology, together with a massive neurofibrillary tangle degeneration. Although all known mutations have been excluded in the coding regions of the AD genes, numerous candidate chromosome sites, either in the AD genes outside the coding regions or in other genes including PrP, must be considered.

Comment (webmaster): Coexistence of prion and AD amyloid has an intriguing history. The autosomal dominant mutation here could be of interest in terms of common ground to certain aspects of pathology.

However, this group goes to far with dribble-down of data in LPUs (least-publishable-units). In the first letter they did a quick check of known coding mutations of 3 easy genes. In the second meagre letter [above], they stained a brain section with two antibodies. In the third, they will look at non-coding AD mutations. In the fourth, they will look for coding prion mutations. In the fifth , they will stain for still other amyloids (recall inclusion body myosititis). In the sixth, they will roughly map the autosomal dominant. In the seventh, they will look at the human genome project and specualte about which gene. In the eigth, they will sequence a candidate gene in the region.

Most likely, they have done all of this already but want to dribble it out in as many small papers as possible.

Prion missing from complete Drosophila genome

Thu, 23 Mar 2000 Science
This issue of Science is largely allocated to the newly completed drosphila genome. Some 58 neurological disease genes with orthologues in fly, nematode, and/or yeast -- a lot of familiar amyloidoses are there (AD, HD, ALS, SCA1,2,6,7) but prion disease.

There were nine other neurological disease genes also drawing a blanks. However, the apparent absence of a prion gene orthologue might be a matter of not having a good idea what the prion gene looked like back then. Prion sequence from fish would enable a much better probe than what is currently available.

Human Disease Genes

Studies in model organisms have provided important insights into our understanding of genes and pathways that are involved in a variety of human diseases. In order to estimate the extent to which different types of human disease genes are found in flies, worms, and yeast, we compiled a set of 289 genes that are mutated, altered, amplified, or deleted in a diverse set of human diseases and searched for similar genes in D. melanogaster, C. elegans, and S. cerevisiae, as described in the legend to Fig. 1. Of these 289 human genes, 177 (61%) appear to have an ortholog in Drosophila (Fig. 1).

Threshold survival of scrapie after ashing at 600C

14 Mar 2000 PNAS
Paul Brown, Edward H. Rau, Bruce K. Johnson, Alfred E. Bacote, Clarence J. Gibbs Jr., and D. Carleton Gajdusek
The infectious agents responsible for transmissible spongiform encephalopathy (TSE) are notoriously resistant to most physical and chemical methods used for inactivating pathogens, including heat. It has long been recognized, for example, that boiling is ineffective and that higher temperatures are most efficient when combined with steam under pressure (i.e., autoclaving). As a means of decontamination, dry heat is used only at the extremely high temperatures achieved during incineration, usually in excess of 600C. It has been assumed, without proof, that incineration totally inactivates the agents of TSE, whether of human or animal origin. It also has been assumed that the replication of these agents is a strictly biological process (1), although the notion of a "virus" nucleant of an inorganic molecular cast of the infectious -pleated peptide also has been advanced (2). In this paper, we address these issues by means of dry heat inactivation studies.

One-gram samples from a pool of crude brain tissue from hamsters infected with the 263K strain of hamster-adapted scrapie agent were placed in covered quartz-glass crucibles and exposed for either 5 or 15 min to dry heat at temperatures ranging from 150C to 1,000C. Residual infectivity in the treated samples was assayed by the intracerebral inoculation of dilution series into healthy weanling hamsters, which were observed for 10 months; disease transmissions were verified by Western blot testing for proteinase-resistant protein in brains from clinically positive hamsters.

... Exposure to 600C completely ashed the brain samples, which, when reconstituted with saline to their original weights, transmitted disease to 5 of 35 inoculated hamsters. No transmissions occurred after exposure to 1,000C. These results suggest that an inorganic molecular template with a decomposition point near 600C is capable of nucleating the biological replication of the scrapie agent.

...To minimize the lag times for heating and recooling specimens, the oven was heated to approximately 100C above the desired temperature, and once the specimens had reached temperature, the platform was repeatedly moved in and out of the oven such that the thermocouple temperature was maintained within 10C of the chosen test temperature. Upon completion of heating at the test temperature for either 5 or 15 min, the crucibles were placed in dry ice. This procedure resulted in a heating-up period of 1-2 min (depending on the test temperature) and a cooling-down period of less than 30 sec....

...Different agent strains show different degrees of heat resistance, and the hamster-adapted 263K strain of scrapie used in the present experiments ranks among the most resistant strains studied in comparative testing.

A small fraction of infectivity in wet brain tissue can withstand autoclaving at 134C, or exposure to dry heat at 200C, and if the tissue has been freeze-dried under partial vacuum, some survival is detectable after exposure to 360C . Formaldehyde fixation increases resistance to autoclave inactivation. ...

The survival of infectivity at 600C, even at the very low levels observed in this study, suggests that the agent may not be fully inactivated in the residual ash from the first chamber, and ash formed in this chamber usually is removed without being exposed to the 1,000C temperature used to treat the gaseous and particulate emissions that enter the second chamber before discharge to the atmosphere.

There is also no assurance that any combustion emissions containing the agent that enter the secondary chamber would be inactivated because the residence time there is at most only a few seconds. Moreover, we assayed only the ashed residue for infectivity and not the combustion emissions. Further experiments are in progress, including chemical, immunological, and electron microscopic studies, to assess inactivation of the agent in ash and emissions under conditions simulating those of medical waste incinerators.

.. [Extreme thermophile ] temperatures refer to conditions under which growth and replication can occur, and not resistance to thermal destruction, which is an entirely different matter. The upper thermal stability limit for biological macromolecules in vitro is 150-160C ; denaturation of proteins and nucleic acids occurs at temperatures well below their decomposition temperatures because of thermal disruption of hydrogen and hydrophobic noncovalent bonds . The best-studied examples of microbial heat resistance are found among bacterial spores, which typically are used to test the efficiency of autoclaves and incinerators: wet spores of Bacillus subtilis and Serratia marcescens have been shown to resist brief exposures to temperatures between 270C and 340C, and some viability of dry spores persists after exposure to 370C .

Exposure to 600C or 1,000C produced a flaming tissue combustion that lasted several seconds and yielded a residue of glowing black ash that had lost 98-99% of its initial weight. Under these conditions it is difficult to imagine the survival of any replicative (or even nonreplicative) biological structure composed of proteins or other organic compounds. Yet, combustion is a series of pyrolysis and oxidation reactions that proceed rapidly but incompletely.

At the comparatively low combustion temperature of 600C and with limited oxygen penetration into the crucibles, it is remotely possible that some of the organic molecules originally present might have escaped destruction. Incomplete combustion of organic compounds also would be likely to introduce pyrolysis products and elemental carbon into the combustion residues, which probably was responsible for their black color. Carbon has been reported to partially protect TSE infectivity at autoclave temperatures...

Comment (webmaster): This data, while falling within a rarely studied experimental regime, will do nothing to calm concerned communities in England downwind of BSE incinerators burning warehouses of rendered animals. Medical waste, while much smaller in volume, is also of some concern; more importantly, it is certain that conventional hospital procedures (or even forseeable improvements) can promise much in the way of sterilization.

Sheep: biopsy of the mandibular gland better than tonsil?

Annual Agricultural Research Forum (UCD), March 14-15th,2000
At the Annual Agricultural Research Forum (UCD), March 14-15th, 2000, Melinda Flavin et al (Dept. of Animal Husbandry, Vet College and Vet Research Lab, Abbotstown, Dublin) presented new findings on the diagnosis of scrapie in sheep.

"The anatomical distribution of PrPSc in the peripheral lymphoid tissues of sheep naturally affected with scrapie".

Proceedings of the Agric Res Forum, Univ Coll Dublin, March 14-15th, pp 115-116.(2000)
Flavin M, Roche AM, Monks E, Church A, McElroy M, Sayers R, Roche JF & Sweeney T
Flavin et al examined 9 peripheral lymphoid tissues from 32 sheep with a clinical diagnosis of scrapie. The tissues were the prescapular, prefemoral, mesenteric, retropharyngeal, bronchial and mandibular glands and the tonsil, spleen and ileum.

They compared two methods of marking PrPSc (ELISA and Immunohistochemistry) in the lymphoid follicles of those tissues. ELISA marked PrPSc in 25/30 sheep examined. Immunohistochemistry identified PrPSc in a high percentage of the follicles in the the tonsil and mandibular glands of 100% of clinically affected sheep examined.

Based on their findings, they propose that in-vivo field screening of sheep for PrPSc be based on biopsy of the mandibular gland, as it is easier biopsy that gland than the tonsil.

Exacerbated Spongiform Lesions in the Cerebral Cortex in Japanese Sheep, in an Outbreak of Scrapie during 1984-1987.

Jpn J Infect Dis 1999 Dec;52(6):242-244 
Mizoi S, Yoshino T, Momotani E, Kubosaki A, Nakamura Y, Onodera T
The present study dealt with the pathology of natural scrapie in Japanese Suffolk sheep in a certain selected area. Vacuolations in the cytoplasm of neurons were conspicuous. They were particularly evident in many areas of the medulla and pons, extending into and through pedunculus cerebri and thalamus to the septal area and olfactory tubercle. Proliferation of astrocytes was also easily observed with glial fibrillary acidic protein staining. Neural vacuolations in the cerebral cortex were observed in 73% of the cases.

Abnormal prion protein deposits were seen in all cases observed by hydrolytic autoclaving, and subsequent peroxidase and anti-peroxidase immunostaining. Abnormal prion protein staining was the most conspicuous in the polymorphic layers of the hippocampus.

Familial amyloid polyneuropathy blocker

Lancet 2000; 355: 1161 - 1168 Xavier Bosch
Modified NSAIDs for orphan amyloid diseases? Compounds based on non-steroidal anti-inflammatory drugs (NSAIDs) may become tools to treat familial amyloid polyneuropathy, according to US researchers.

Familial amyloid polyneuropathy is one of several uncommon disorders that involve the deposition of amyloid plaques. So far, the pharmaceutical and biotechnology industries have tended to ignore these "orphan" amyloid diseases and have focused mainly on the development of drugs to treat Alzheimer's disease.

Familial amyloid polyneuropathy is caused by the irregular build-up of the protein transthyretin into amyloid fibrils. Transthyretin deposition in peripheral nerves and the heart can lead to death from the disorder within 10 years of diagnosis. Transthyretin normally functions as a tetramer, but only the individual monomers can associate to form fibrils. In familial amyloid polyneuropathy, mutations of transthyretin cause tetramers to dissociate more easily, thereby increasing the tendency to form fibrils.

By means of X-ray crystallography, Thomas Klabunde (Texas A&M University, College Station, TX, USA) and co-workers have shown that the NSAIDs diclofenac and flurbiprofen can bind transthyretin, stabilise the tetramer, and strongly inhibit the formation of transthyretin amyloid fibrils in vitro. The scientists have also designed a series of transthyretin inhibitors derived from NSAIDs but with no anti-inflammatory activity. These new compounds are expected to be highly potent, and to have a high gastric tolerability (Nat Struct Biol 2000; 7: 312-21 ).

"The designed fibril formation inhibitors . . . appear to be excellent candidates for a small molecule therapeutic approach against transthyretin-associated amyloidosis", assert Klabunde and colleagues.

An accompanying editorial states that "the millions of dollars pouring into [Alzheimer's disease] research will hopefully also aid and encourage work on the less well-known amyloid diseases. Their markets may be smaller . . . but they are just as large in terms of devastation to the individual".

Amyloid and Non-amyloid Forms of 5q31-linked Corneal Dystrophy

J Biol Chem, Vol. 275, Issue 15, 11465-11469, April 14, 2000
Elena Korvatska,..., and Daniel F. Schordere
Mutations in kerato-epithelin are responsible for a group of hereditary cornea-specific deposition diseases, 5q31-linked corneal dystrophies. These conditions are characterized by progressive accumulation of protein deposits of different ultrastructure. Herein, we studied the corneas with mutations at kerato-epithelin residue Arg-124 resulting in amyloid (R124C), non-amyloid (R124L), and a mixed pattern of deposition (R124H). We found that aggregated kerato-epithelin comprised all types of pathological deposits. Each mutation was associated with characteristic changes of protein turnover in corneal tissue. Amyloidogenesis in R124C corneas was accompanied by the accumulation of N-terminal kerato-epithelin fragments, whereby species of 44 kDa were the major constituents of amyloid fibrils.... It appears that substitutions at the same residue (Arg-124) result in cornea-specific deposition of kerato-epithelin via distinct aggregation pathways each involving altered turnover of the protein in corneal tissue.

Inhibition of Polyglutamine Protein Aggregation and Cell Death by Novel Peptides Identified by Phage Display Screening

J Biol Chem, Vol. 275, Issue 14, 10437-10442, April 7, 2000
Yoshitaka Nagai, Timothy Tucker,..., Warren J. Strittmatter, and James R. Burke
Proteins with expanded polyglutamine domains cause eight inherited neurodegenerative diseases, including Huntington's, but the molecular mechanism(s) responsible for neuronal degeneration are not yet established. Expanded polyglutamine domain proteins possess properties that distinguish them from the same proteins with shorter glutamine repeats. Unlike proteins with short polyglutamine domains, proteins with expanded polyglutamine domains display unique protein interactions, form intracellular aggregates, and adopt a novel conformation that can be recognized by monoclonal antibodies. Any of these polyglutamine length-dependent properties could be responsible for the pathogenic effects of expanded polyglutamine proteins.

To identify peptides that interfere with pathogenic polyglutamine interactions, we screened a combinatorial peptide library expressed on M13 phage pIII protein to identify peptides that preferentially bind pathologic-length polyglutamine domains. We identified six tryptophan-rich peptides that preferentially bind pathologic-length polyglutamine domain proteins. Polyglutamine-binding peptide 1 (QBP1) potently inhibits polyglutamine protein aggregation in an in vitro assay, while a scrambled sequence has no effect on aggregation. QBP1 and a tandem repeat of QBP1 also inhibit aggregation of polyglutamine-yellow fluorescent fusion protein in transfected COS-7 cells. Expression of QBP1 potently inhibits polyglutamine-induced cell death. Selective inhibition of pathologic interactions of expanded polyglutamine domains with themselves or other proteins may be a useful strategy for preventing disease onset or for slowing progression of the polyglutamine repeat diseases.

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