Yeast chaperones interact with human prions
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Yeast chaperone protein and mad cow disease
Article highlights or off-site fulltexts 1, 2
Earlier Hsp 104 papers,
GroEL structure/function and human homologes
Is there a human psi?
Hsp 104 at Genbank, SwissProt, Blast, and Proteome
Mammalian homologues of Hsp 104
CJD-Alzheimer similarity?

Chaperone protein could explain mad cow disease

Mon, 8 Dec Reuters World Report
WASHINGTON - A "chaperone" protein helps other proteins change their shapes and could explain how prions cause mad cow disease and related deadly illnesses such as CJD, researchers said on Monday. Researchers at the University of Chicago isolated the protein from yeast, but said it acted on prions from mammals, too.

They said their findings could lead to a treatment for mad cow disease and CJD, which are both incurable and always fatal. Prions are proteins found in the brain, which have been recently linked with bovine spongiform encephalopathy (BSE or mad cow disease) and relatives like CJD. Tests have shown the "bad" prions fold in a certain shape and somehow transmit this to healthy prions. The result is a tangle in the brain and spongelike holes. Such a mechanism for passing on disease has never been seen before, and the prions are not easily destroyed by heat or chemicals.

"These odd diseases have focused attention on what appears to be an entirely new, gene-free mechanism of heredity that increasingly appears to be extraordinarily widespread, now that we know where to look,"said Susan Lindquist, a molecular geneticist at the University of Chicago, who worked on the study.
The University of Chicago team, which reported its findings in the [9 Dec 97] PNAS, was working with chaperone proteins in yeast.
"The yeast and mammalian prion proteins are genetically, structurally and functionally entirely different," Lindquist said in a statement. They were amazed to find the yeast proteins worked with mammal prions. "These chaperones are telling us that, although we haven't found it yet, there is a remarkable underlying biochemical resemblance," Lindquist said

"Although we first became aware of prions because they cause several bizarre neurological diseases, the discovery that something so awesomely similar happens in organisms as different as humans and yeast makes us suspect that there is a fundamental, common biochemical process at work here.".

Although yeasts are very different from animals, scientists often use them for basic research and they have many genes in common. If a yeast and a human do have a gene in common, it indicates the gene is basic to life's fundamental processes.

Chaperone-supervised conversion of prion protein to its protease-resistantİform

Proc. Natl. Acad. Sci. USA Vol. 94, pp. 13938-13943, December 1997
S DebBurman, GJRaymond, BCaughey, and Susan Lindquist
...To gain insight on the conformational transitions of PrP, we tested the ability of several protein chaperones, which supervise the conformational transitions of proteins in diverse ways, to affect conversion of PrPC to its protease-resistant state. None affected conversion in the absence of pre-existing PrPSc. In its presence, only two, GroEL and Hsp104 (heat shock protein 104), significantly affected conversion. Both promoted it, but the reaction characteristics of conversions with the two chaperones were distinct. In contrast, chemical chaperones inhibited conversion.

Griffith (3) first proposed a "protein-only" model to explain the unconventional behavior of the infectious TSE agent... The goal of this study was to assess whether or not molecular chaperones, whose known functions are to alter the conformational states of proteins (14-16), regulate the conversion of PrPC to PrPSc.

To test for chaperone involvement, we used a cell-free assay, wherein metabolically labeled [35-S]PrPC, purified from cultured cells in an acid-treated state, is converted to a conformational state characteristic of PrPSc (17, 18). In this altered state, PrP is aggregated and a specific portion of the molecule is highly resistant to proteolysis. This simple in vitro conversion reaction faithfully recapitulates several salient TSE features.

First, like experimental TSEs, in vitro conversion of PrPC to its protease-resistant form requires pre-existing PrPSc (17-22).

Second, strain-specific PrPSc protease digestion properties, specifically those associated with two mink TSE strains, hyper and drowsy, were precisely propagated from PrPSc to radiolabeled PrPC in this assay (19).

Third, the known in vivo barriers to transmitting TSEs between different species were reflected well in the efficiencies of in vitro conversion (20, 21).

Last, this cell-free assay modeled accurately another in vivo TSE barrier, based on genetic polymorphisms in PrP, which render sheep either highly susceptible, moderately susceptible, or resistant to scrapie (22). Together, these studies provide substantial evidence that in vitro converted, protease-resistant PrP is either authentic PrPSc or has a very similar conformation. However, because neither the putative infectious nature of pure PrPSc protein nor that of the in vitro converted PrP has been demonstrated, we refer to the in vitro converted material operationally as protease-resistant PrP (PrP-res).

Here, we provide the first evidence that molecular chaperones can regulate conformational transitions in PrP. Two protein chaperones, GroEL and Hsp104, promoted in vitro conversion; in contrast, the chemical chaperones, sucrose, trehalose, and dimethyl sulfoxide (DMSO) inhibited it. Importantly, our results with chaperones demonstrate that in vitro converted PrP-res is a bona fide conformationally altered PrP molecule. Chaperones provide new understanding of the nature of PrP intermediates involved in PrP conversion and provide evidence that the conversion process has two steps. We propose that, if chaperone-like molecules supervise PrPSc formation in TSEs in vivo, such molecules will represent important clinical targets to combat this dreaded disease.

Chaperone Proteins: Yeast Hsp40, Hsp70, and Hsp104 [wild type and mutant] were purified as described. Hsp104 promoted the refolding of kinetically trapped, denatured luciferase, but only when Hsp40, Hsp70, and ATP were also present. PrPSc was purified from hamsters infected with 263K strain of scrapie. Cell-Free PrP Conversion: Caughey et al. 1995.

Chaperones alone do not convert Prpc to Prp-res: We first examined the ability of major cellular chaperones GroES (Hsp10), Hsp26, Hsp40, GroEL (Hsp60), Hsp70, Hsp90, and Hsp104, to promote PrPC conversion in the absence of PrPSc. These chaperones were chosen because they employ different mechanisms to affect the conformation and physical state of other proteins (14-16).

GroEL Promotes Conversion in Reactions Nucleated with Untreated PrPSc: Next, we asked whether chaperones influenced PrPC conversion in the presence of PrPSc. To date, efficient in vitro conversion of PrPC to PrP-res has usually required partial chemical denaturation of PrPSc. Untreated and completely denatured PrPSc have little and no converting ability, respectively. We first asked whether chaperones influenced conversion with PrP-res that was not subjected to partial denaturation. Several chaperones produced reproducible, but very small increases in conversion. One, however, facilitated conversion at a high level. With GroEL, typically 25-30%, and occasionally 50-100%, of converted PrPC.

Notably GroEL not only reduced by 10-fold the quantity of PrPSc required for detectable conversion, but also increased by more than 10-fold the maximal levels of conversion attained, compared with reactions nucleated with the same preparation of untreated PrPSc, but not with GroEL. These effects of GroEL were dose-dependent. GroEL effects require ATP, but not GroES.

Posttranslational PrP modifications modestly affect chaperone-promoted conversions: We used a PrP mutant [cites do not identify -- webmaster] that lacks the GPI anchor and accumulates in mono- and unglycosylated form to determine whether these natural modifications affect chaperone-mediated conversion. Again, of the various chaperones tested, GroEL was the only one that efficiently stimulated conversion in the presence of untreated PrPSc).

With this form of PrP, however, conversion was more efficient (typically 30-40%). Moreover, conversion was also achieved with a combination of Hsp104, Hsp70, and Hsp40, albeit less consistently and less strongly than with GroEL. Similar results were also obtained with unglycosylated PrPC purified from cells cultured with tunicamycin. Therefore, the ability of the chaperones to mediate the conversion of PrPC to PrP-res was modestly facilitated by the absence of N-linked sugars or the GPI anchor.

Conversion Kinetics Reveal a Two-Step Process: We analyzed the kinetics of conversion, monitoring protease resistance and insolubility. In reactions driven with untreated PrPSc and GroEL, protease resistance was acquired at a pace similar to that observed in reactions nucleated with partially denatured PrPSc in the absence of GroEL. Moreover, in both sets of reactions, protease-resistant radioactivity was found only in pelletable material.

Surprisingly, however, when the rate at which PrP became insoluble was examined, the chaperone-driven reaction showed very different kinetics than those driven by partially denatured PrPSc. No pelletable radioactivity was detected at two hours in reactions driven by partially denatured PrPSc. In striking contrast, in chaperone-driven reactions, the conversion of PrP to a pelletable form was virtually complete in 2İhr. This conversion occurred long before PrP converted to its characteristic protease-resistant form. This pelleting of PrPC was almost certainly caused by an association with pre-existing PrPSc, because in parallel reactions with GroEL, but without PrPSc, most PrPC remained soluble.

In reactions nucleated with partially denatured PrPSc, Hsp104 also promotes conversion: Although we did not detect a substantial activity for other chaperones in promoting conversion with untreated PrPSc, another chaperone was effective in reactions seeded with partially denatured PrPSc. For these reactions, a milder denaturant, urea, was used. Strikingly, under these conditions, in addition to GroEL, Hsp104 strongly stimulated conversion. With Hsp104, typically 20-30%, occasionally more than 50% of totalPrPC converted. The stimulatory effects of Hsp104 required partial denaturation of PrPSc, with pretreatments in 3-4 M urea being optimal.

Folded state of PrPSc governs properties of chaperone-promoted conversion: Remarkably, the use of partially denatured PrPSc changed the character of conversions promoted by GroEL as well. These conversions lost ATP-dependence. Moreover, they became refractory to GroES inhibition. Thus, chaperone-mediated conversions are mechanistically distinct in reactions nucleated with partially denatured PrPSc than those nucleated by untreated PrPSc.

In studying the conversion of PrPC to PrP-res, we employed previously characterized chaperones from bacteria and the eukaryotic cytosol because protein chaperones have not yet been identified in compartments where PrPC converts to PrPSc. Of the chaperones we tested, only GroEL and Hsp104 affected conversion... Chaperones provide a strong demonstration of the importance of PrPSc in creating a template for PrPC conversion. The chaperones we tested interact with different folding intermediates, bind them in different ways, and promote conformational changes by distinct mechanisms. Yet none could promote the conversion of acid-treated PrPC to PrP-res in the absence of PrPSc.

With untreated PrPSc, only GroEL stimulated PrP conversions, but with partially denatured PrPSc, GroEL, GroEL/GroES, and Hsp104 were stimulated. Presumably, partial denaturation allows PrPSc to accept PrP in a broader variety of conformational intermediates.

Kinetic analysis with GroEL suggests that conversion is a two-step process with GroEL specifically increasing the rate at which PrPC assumes a pelletable conformation. Because conversion of PrPC to a pelletable state requires PrPSc, this process most likely involves recruitment of a conformational PrP intermediate, generated by the chaperone, into a PrPSc polymer. Once PrP has converted to a pelletable state, conversion to PrP-res follows at a slower pace. Based on these observations, we propose a model:

The ability of chaperones to enhance at least one step in the conversion process may provide an avenue for generating sufficient quantities of PrPSc in vitro to test the "protein-only" hypothesis.

Finally, our observations provide a unifying biochemical connection between mammalian TSEs and PSI+, a genetic element in yeast. The proposed "mammalian prion" determinant PrPSc, and the "yeast prion" determinant Sup35, are functionally unrelated and share no sequence identity. Also, PSI+ produces a heritable change in metabolism rather than a lethal infection. However, both mammalian and yeast prions apparently share a common mode of transmission based on self-propagating changes in protein conformation. Among yeast chaperones, the striking specificity of Hsp104 for PrP conversions, and its known in vivo specificity in regulating PSI+ suggest that conformations of PrP and Sup35 share an underlying biochemical similarity that allows for recognition by particular chaperones and prion-like conformational transitions. In added support of this notion, the accompanying study provides evidence for specific interactions of Hsp104 with PrP and Sup35 proteins with circular dichroism and ATP hydrolysis measurements.

Interactions of the chaperone Hsp104 with yeast Sup35 and mammalianİPrP

Proc. Natl. Acad. Sci. USA Vol. 94, pp. 13932-13937, December 1997 
Eric C. Chirmer and Susan Lindquist
PSI+ is a genetic element in yeast for which a heritable change in phenotype appears to be caused by a heritable change in the conformational state of the Sup35 protein. The inheritance of PSI+ and the physical state of Sup35 in vivo depend on the protein chaperone Hsp104 (heat shock protein 104). Although these observations provide a strong genetic argument in support of the "protein-only" hypothesis, there is, as yet, no direct evidence of an interaction between the two proteins.

We report that when purified Sup35 and Hsp104 are mixed, the circular dichroism spectrum differs from that predicted by the addition of the proteins' individual spectra, and the ATPase activity of Hsp104 is inhibited. Similar results are obtained with two other amyloidogenic substrates, mammalian PrP and beta-amyloid 1-42İpeptide, but not with several control proteins.

With a group of peptides that span the PrP protein sequence, those that produced the largest changes in CD spectra also caused the strongest inhibition of ATPase activity in Hsp104. Our observations suggest that previously described genetic interactions between Hsp104 and PSI+ are caused by direct interaction between Hsp104 and Sup35; Sup35 and PrP, the determinants of the yeast and mammalian prions share structural features that lead to a specific interaction with Hsp104; and these interactions couple a change in structure to the ATPase activity of Hsp104.

Phenotypes transmitted by two dominant, cytoplasmically inherited genetic elements, PSI+ and URE3, seem to depend on the inheritance of altered protein structures, rather than altered nucleic acids. The yeast PSI+ element, the subject of our work, does not generally kill cells. It reduces the fidelity of ribosome translation termination and thereby suppresses nonsense codons. This phenotype is thought to result from a change in the state of the translation-termination factor, Sup35, that interferes with its normal function. In psi cells, Sup35 is protease sensitive and is mostly soluble; in PSI+ cells, Sup35 has increased protease resistance and is mostly aggregated. ("Aggregate" is used in a general sense; Sup35 may be polymerized into an amyloid-like structure, or coalesced in a less ordered state.) When pre-existing Sup35 is in the aggregated state, newly made Sup35 also aggregates, causing a self-perpetuating loss of function in the termination factor and a heritable change in translational fidelity.

PSI+ depends on the chaperone protein Hsp104. The first known function of Hsp104 was in thermo-tolerance in yeast, where it increases survival after exposure to extreme temperatures up to 1,000-fold. It does so by promoting the reactivation of proteins that have been damaged by heat and have begun to aggregate. At normal temperatures, Hsp104 overexpression cures cells of PSI+. Sup35 becomes soluble and the fidelity of translation termination is restored. This state is heritable, even when overexpression of Hsp104 ceases. Because the only known function of Hsp104 is to alter the conformational state of other proteins, these observations provide a strong argument that PSI+ is indeed based on a heritable (self-perpetuating) change in the conformational state of Sup35.

Surprisingly, deletions of HSP104 also cure cells of PSI+, and Sup35 is soluble in such cells as well. This is very different from heat-induced aggregates, which remain insoluble in hsp104 deletion strains. Clearly, the relationship between Hsp104 and PSI+ is more complex than the relationship between Hsp104 and thermotolerance.

...We asked whether changes in state could be detected by CD when purified Hsp104 and Sup35 were mixed in vitro. If two proteins do not interact, or if they interact without a substantial change in secondary structure, the CD spectrum of their mixture should equal that predicted from the simple addition of their individual spectra. ...When Hsp104 and Sup35 were mixed, the observed spectrum differed dramatically from the predicted spectrum. Thus, these two proteins interact.

In vivo, the inheritance of PSI+ is associated with the partitioning of Sup35 into aggregates, a change in state that requires Hsp104. In vitro, Sup35 forms highly ordered, amyloid-like fibers after prolonged incubations in the absence of Hsp104 (13).... An increase in Congo red dye binding was detected by the characteristic spectral shift that occurs when this dye binds amyloid proteins. Previous studies identified the N-terminal domain of Sup35 as responsible for the formation of self-seeded amyloid fibrils in vitro.

PrP and Sup35 are unrelated in sequence and in biological function. Nonetheless, the capacity for both proteins to form amyloid-like aggregates suggests an underlying biochemical similarity between them. We asked whether this similarity would extend to shared molecular features in the two proteins that allow recognition by Hsp104. The change in state of mammalian PrP associated with TSEs is characterized by increased beta-sheet content and protease resistance in amino acid segment 90-231.

We also tested another amyloidogenic peptide, beta-amyloid 1-42,İa fragment often found in the neural plaques associated with Alzheimer's disease (11). Again, the ATPase activity of Hsp104 was inhibited. Less inhibition was observed with a less amyloidogenic derivative, beta-amyloid 1-40,İstill less with a peptide containing the same amino acids in the reverse order, and no inhibition was observed with a wide variety of control proteins. Thus, the unexpected inhibitory effects of these three amyloidogenic polypeptides on the ATPase activity of Hsp104 are specific and strongly suggest an underlying biochemical similarity between them.

When Hsp104 was mixed with rPrP, the CD spectrum of the solution differed dramatically from the spectrum predicted by the addition of individual spectra. When rPrP was mixed with several other chaperones, only GroEL (Hsp60) yielded a substantial spectral shift. Other chaperones (Cdc37, Hsp90, Hsp70) yielded spectral shifts with PrP, but they were much smaller.

... The changes in spectra we observe are large, but their nature is unclear. One issue is that the interpretation of CD spectra is based on comparisons with defined structures, and no structures of amyloid fibers have yet been solved [but see recent studies --webmaster] .... The changes we observe are therefore consistent with either a reduction in alpha-helix Because Sup35 has a known capacity to form amyloid-like fibers in vitro, the increase in light scattering and Congo red binding in our experiments suggest that Hsp104 is facilitating a change in the structure of Sup35.

The inhibition of the ATPase activity of Hsp104 was itself surprising. Interactions between other HSP100 proteins and their substrates generally stimulate the chaperone's ATPase activity. At least some of these interactions, however, seem to involve less dramatic structural transitions. For example, ClpA (an Escherichia coli relative of Hsp104) converts the RepA protein from dimers to monomers). Both ClpA and Hsp104 are hexameric proteins with multiple ATP binding sites and, presumably, multiple substrate binding sites. Perhaps the structural transitions of more complex, amyloidogenic substrates involve more coupled or "concerted" work from the chaperone and this inhibits its free-running ATPase activity. [??? -webmaster]

We did not monitor CD spectral shifts with betta-amyloid peptide and Hsp104, but it is striking that this peptide too inhibited the ATPase activity of Hsp104. beta-Amyloid, Sup35, and PrP differ in size and biological function and have unrelated sequences (except for weak homology in a few oligopeptide repeats of Sup35 and PrP). Yet, all share the capacity to assemble into amyloid-like aggregates). The PSI+ genetic trait is linked to the aggregation of Sup35; the pathologies of TSEs and Alzheimer's disease are generally associated with the aggregation of PrP and -amyloid, respectively). Presumably, it is the shared capacity for such conformational transitions that leads to recognition by Hsp104.

The biological relevance of the interactions between Hsp104 and PrP is supported by a separate study from our laboratory. DebBurman et al. have found that in the presence of the infectious form of PrP, Hsp104 accelerates the rate at which full-length cellular form, PrPC, assumes the protease resistance pattern that is the hallmark of PrPSc. Of the [several] chaperones tested in that study, only Hsp104 and GroEL accelerated conversion. It is notable therefore that of the several chaperones tested here (Hsp70, Hsp90, Ydj1, Cdc37, GroEL, and Hsp104), only Hsp104 and GroEL produced a strong spectral shift with PrP.

We do not mean to suggest that Hsp104 (or GroEL) homologs regulate the structural transitions of PrP or beta-amyloid in vivo. Rather, we suggest that protein chaperones provide common mechanisms for controlling certain types of conformational switches and, thus, might provide potential avenues for therapeutic intervention. In any case, the strikingly similar and highly specific interactions we observe between Hsp104 and these three very different proteins provides another link between these [conformational disorders].

Earlier HSP 104 papers

Maintenance and inheritance of yeast prions.
Trends Genet 1996 Nov;12(11):467-471
Tuite MF, Lindquist SL
The unusual genetic behaviour of two yeast extrachromosomal elements [PSI] and [URE3] is entirely consistent with a prion-like mechanism of inheritance involving an autocatalytic alteration in the conformation of a normal cellular protein. In the case of both yeast determinants the identity of the underlying cellular prion protein is known. The discovery that the molecular chaperone Hsp104 is essential for the establishment and maintenance of the [PSI] determinant provides an explanation for several aspects of the puzzling genetic behaviour of these determinants. What remains to be explained is whether these determinants represent 'disease states' of yeast or represent the first examples of a unique mechanism for producing a heritable change in phenotype without an underlying change in genotype.
Support for the prion hypothesis for inheritance of a phenotypic trait in yeast.
Science 1996 Aug 2;273(5275):622-626 
Patino MM, Liu JJ, Glover JR, Lindquist S
A cytoplasmically inherited genetic element in yeast, PSI+, was confirmed to be a prionlike aggregate of the cellular protein Sup35 by differential centrifugation analysis and microscopic localization of a Sup35-green fluorescent protein fusion. Aggregation depended on the intracellular concentration and functional state of the chaperone protein Hsp104 in the same manner as did PSI+ inheritance. The amino-terminal and carboxy-terminal domains of Sup35 contributed to the unusual behavior of PSI+. PSI+ altered the conformational state of newly synthesized prion proteins, inducing them to aggregate as well, thus fulfilling a major tenet of the prion hypothesis.
Propagation of the yeast prion-like PSI+ determinant is mediated 
by oligomerization of the SUP35-encod polypeptide chain release factor.
EMBO J 1996 Jun 17;15(12):3127-3134 
Paushkin SV, Kushnirov VV, Smirnov VN, Ter-Avanesyan MD
The Sup35p protein of yeast Saccharomyces cerevisiae is a homologue of the polypeptide chain release factor 3 (eRF3) of higher eukaryotes. It has been suggested that this protein may adopt a specific self-propagating conformation, similar to mammalian prions, giving rise to the PSI+ nonsense suppressor determinant, inherited in a non-Mendelian fashion. Here we present data confirming the prion-like nature of PSI+. We show that Sup35p molecules interact with each other through their N-terminal domains in PSI+, but not [psi-] cells. This interaction is critical for PSI+ propagation, since its disruption leads to a loss of PSI+. Similarly to mammalian prions, in PSI+ cells Sup35p forms high molecular weight aggregates, accumulating most of this protein. The aggregation inhibits Sup35p activity leading to a PSI+ nonsense-suppressor phenotype. N-terminally altered Sup35p molecules are unable to interact with the PSI+ Sup35p isoform, remain soluble and improve the translation termination in PSI+ strains, thus causing an antisuppressor phenotype. The overexpression of Hsp104p chaperone protein partially solubilizes Sup35P aggregates in the PSI+ strain, also causing an antisuppressor phenotype. We propose that Hsp104p plays a role in establishing stable PSI+ inheritance by splitting up Sup35p aggregates and thus ensuring equidistribution of the prion-like Sup35p isoform to daughter cells at cell divisions.
In Vitro Propagation of the Prion-Like State of YeastSup35 Protein.
Science 1997 Jul 18;277(5324):381-383 
Paushkin SV, Kushnirov VV, Smirnov VN, Ter-Avanesyan MD
The yeast cytoplasmically inherited genetic determinant PSI+ is presumed to be a manifestation of the prion-like properties of the Sup35 protein (Sup35p). Here, cell-free conversion of Sup35p from [psi-] cells (Sup35ppsi-) to the prion-like PSI+-specific form (Sup35pPSI+) was observed. The conversion reaction could be repeated for several consecutive cycles, thus modeling in vitro continuous PSI+ propagation. Size fractionation of lysates of PSI+ cells demonstrated that the converting activity was associated solely with Sup35pPSI+ aggregates, which agrees with the nucleation model for PSI+ propagation. Sup35pPSI+ was purified and showed high conversion activity, thus confirming the prion hypothesis for Sup35p.
Role of the chaperone protein Hsp104 in propagation
of the yeast prion-like factor PSI+.
Science 1995 May 12;268(5212):880-884 
Chernoff YO, Lindquist SL, Ono B, Inge-Vechtomov SG, Liebman SW
The yeast non-Mendelian factor PSI+ has been suggested to be a self-modified protein analogous to mammalian prions. Here it is reported that an intermediate amount of the chaperone protein Hsp104 was required for the propagation of the PSI+ factor. Over-production or inactivation of Hsp104 caused the loss of PSI+. These results suggest that chaperone proteins play a role in prion-like phenomena, and that a certain level of chaperone expression can cure cells of prions without affecting viability. This may lead to antiprion treatments that involve the alteration of chaperone amounts or activity.
Genetic and environmental factors affecting the de novo appearance of the PSI+ prion 
Genetics 1997 Oct;147(2):507-519 
Derkatch IL, Bradley ME, Zhou P, Chernoff YO, Liebman SW
It has previously been shown that yeast prion PSI+ is cured by GuHCl, although reports on reversibility of curing were contradictory. Here we show that GuHCl treatment of both PSI+ and [psi-] yeast strains results in two classes of [psi-] derivatives: Pin+, in which PSI+ can be reinduced by Sup35p overproduction, and Pin-, in which overexpression of the complete SUP35 gene does not lead to the PSI+ appearance.

However, in both Pin+ and Pin- derivatives PSI+ is reinduced by overproduction of a short Sup35p N-terminal fragment, thus, in principle, PSI+ curing remains reversible in both cases. Neither suppression nor growth inhibition caused by SUP35 overexpression in Pin+ [psi-] derivatives are observed in Pin- [psi-] derivatives.

Genetic analyses show that the Pin+ phenotype is determined by a non-Mendelian factor, which, unlike the PSI+ prion, is independent of the Sup35p N-terminal domain. A Pin- [psi-] derivative was also generated by transient inactivation of the heat shock protein, Hsp104, while PSI+ curing by Hsp104 overproduction resulted exclusively in Pin+ [psi-] derivatives. We hypothesize that in addition to the PSI+ prion-determining domain in the Sup35p N-terminus, there is another self-propagating conformational determinant in the C-proximal part of Sup35p and that this second prion is responsible for the Pin+ phenotype.

Is there a human [psi]?

C R Acad Sci III 1996 Jun;319(6):487-492 
Jean-Jean O, Le Goff X, Philippe M
The yeast Sup35p protein which is responsible for the [psi] phenotype, is a GTP-binding protein involved in translation termination. It was suggested recently that the [psi] determinant has prion-like properties that were localized in the 114 N-terminal amino acids of the protein. In this study, we show that the 5' end of the human SUP35 gene open reading frame is longer than previously reported by 138 codons. This N-terminal sequence presents similarities with the N-terminus of S. cerevisiae Sup35p protein, involved in [psi] maintenance. By transfection of human cells and Western blotting, we demonstrate that translation is initiated at the first AUG encountered at the 5' end of the human SUP35 gene. The longest form of the protein, which contains the N-terminal extension, is the major form of Sup35p protein in non transfected cells. Moreover, an analog of the long form of Sup35p protein is found in various mouse tissues. We suggest that the protein encoded by SUP35 gene could have, at least in human, the properties described for the yeast [psi] element.
Structure and functional similarity of yeast Sup35p
and Ure2p proteins to mammalian prions
Mol Biol (Mosk) 1995 Jul;29(4):750-755[Article in Russian]  
Kushnirov VV, Ter-Avanesian MD, Smirnov VN
The results of studies of yeast cytoplasmically-inherited determinants [psi] and [URE3] are summarized. The existence of tandem amino acid repeats in N-terminal regions of yeast Sup35p protein and in prions of higher eukaryotes is shown. The prion-like properties of yeast Sup35p and Ure2p proteins and a role of tandem amino acid repeats localized in N-terminal region of Sup35p protein in inheritance of [psi] determinants is discussed. The suggestion is made that the PSI+ status of yeast cell depends on the specific conformation of the N-terminal domain of Sup35p and that this protein can induce the specific conformational state of its N-terminal domain on newly synthesized Sup35p molecules via protein-protein interaction, thus representing a molecular basis of inheritance of PSI+ determinant. Other proteins containing amino acid repeats of similar type are considered and the suggestion is made that some of these proteins may show prion-like behavior.
Prion-inducing domain 2-114 of yeast Sup35 protein
transforms in vitro into amyloid-like filaments.
Proc Natl Acad Sci U S A 1997 Jun 24;94(13):6618-6622
King CY, Tittmann P, Gross H, Gebert R, Aebi M, Wuthrich K
The yeast non-Mendelian genetic factor [PSI], which enhances the efficiency of tRNA-mediated nonsense suppression in Saccharomyces cerevisiae, is thought to be an abnormal cellular isoform of the Sup35 protein. Genetic studies have established that the N-terminal part of the Sup35 protein is sufficient for the genesis as well as the maintenance of [PSI]. Here we demonstrate that the N-terminal polypeptide fragment consisting of residues 2-114 of Sup35p, Sup35pN, spontaneously aggregates to form thin filaments in vitro. The filaments show a beta-sheet-type circular dichroism spectrum, exhibit increased protease resistance, and show amyloid-like optical properties. It is further shown that filament growth in freshly prepared Sup35pN solutions can be induced by seeding with a dilute suspension of preformed filaments. These results suggest that the abnormal cellular isoform of Sup35p is an amyloid-like aggregate and further indicate that seeding might be responsible for the maintenance of the [PSI] element in vivo.

Hsp 104 at Genbank

ACCESSION   M67479 or P31539
 Hsp104 is a highly conserved protein with two essential nucleotide-binding sites
Nature 353 (6341), 270-273 (1991)
Parsell DA, Sanchez Y, Stitzel JD, Lindquist S
In Saccharomyces cerevisiae, heat-shock protein hsp104 is vital for tolerance to heat, ethanol and other stresses. The mammalian hsp110 protein is nucleolar and redistributes with growth state, nutritional conditions and heat shock. The relationships between hsp110, hsp104 and the high molecular mass heat-shock proteins of other organisms were unknown. We report here that hsp104 is a member of the highly conserved ClpA/ClpB protein family first identified in Escherichia coli and that additional heat-inducible members of this family are present in Schizosaccharomyces pombe and in mammals. Mutagenesis of two putative nucleotide-binding sites in hsp104 indicates that both are essential for function in thermotolerance.
908 amino acids, mw 102,008, cyclic hexamer, N-acetylated, 2 N-glc sites
Region 212..219 nucleotide-binding motif A (P-loop) 
Region 280..284 nucleotide-binding motif B
Region 614..621 nucleotide-binding motif A (P-loop)
Region 682..686 nucleotide-binding motif B 
domain  16..411  i
domain 541..731 ii
n_bind 212..219 ATP
n_bind 614..621 ATP

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      661 gyvgydeggf ltnqlqykpy svllfdevek ahpdvltvml qmlddgrits gqgktidcsn
      721 civimtsnlg aefinsqqgs kiqestknlv mgavrqhfrp eflnrissiv ifnklsrkai
      781 hkivdirlke ieerfeqndk hyklnltqea kdflakygys ddmgarplnr liqneilnkl
      841 alrilkneik dketvnvvlk kgksrdenvp eeaeeclevl pnheatigad tlgdddneds
      901 meidddld

Yeast chromosomal location and neighbors:

Yeast 1997 Feb;13(2):183-188
The analysis of a 32 kb DNA fragment from cosmid 2G12 on the left arm of chromosome XII identifies 14 open reading frames (ORFs) numbered L0948 to L1325, a new tRNA for proline, a delta remnant and two putative ARS. Six ORFs have been previously identified: HSP104, SSA2, SPA2, KNS1, DPS1/APS and SDC25. Three putative ORFs have significant homology with known proteins: L0968 is a new member of the very large 'seripauperins' family, comprising at least 20 yeast members; L1313 is a new ABC transporter highly homologous to the yeast cadmium resistance protein Ycf1p and to the human multidrug resistance protein hMRP1; the C-terminal part of L1325 present in our sequence is very homologous to the fruit fly abdominal segment formation protein Pumilio. Finally, two ORFs, L1201 and L1205, have weak homology with two yeast hypothetical proteins of unknown function identified by the yeast systematic sequencing genome.

The mitochondrial heat shock protein Hsp78 is a member of the Hsp104/Clp family with unknown function... One induced protein, Hsp104, contributes to both thermotolerance and ethanol tolerance, while others are anti-oxidant enzymes. Heat and ethanol stress cause similar changes to plasma membrane protein composition, reducing the levels of plasma membrane H(+)-ATPase protein and inducing the plasma membrane-associated Hsp30...Hsp104 and Hsp70 proteins have different, but related, functions in protecting cells from the toxic effects of high temperatures...homologue to the bacterial ClpB gene (homologs: L0948, YLL026w)...

A number of ATP-binding proteins that are are thought to protect cells from extreme stress by controlling the aggregation of denaturation of vital cellular structures have been shown [1,2] to be evolutionary related. The size of these proteins range from 84 Kd (clpA) to slightly more than 100 Kd (HSP104). They all share two conserved regions of about 200 amino acids that each contains an ATP-binding site. These proteins are listed below.

- Escherichia coli clpA, which acts as the regulatory subunit of the ATP- dependent protease clp.
- Rhodopseudomonas blastica clpA homolog.
- Escherichia coli heat shock protein clpB and homologs in other bacteria.
- Bacillus subtilis protein mecB.
- Yeast heat shock protein 104 (gene HSP104), which is vital for tolerance to heat, ethanol and other stresses.
- Neurospora heat shock protein hsp98.
- Yeast mitochondrial heat shock protein 78 (gene HSP78) [3].
- CD4A and CD4b, two highly related tomato proteins that seem to be located in the chloroplast.
- Trypanosoma brucei protein clp.
- Porphyra purpurea chloroplast encoded clpC.

Yeast heat shock proteins overview

Offsite: hotlist of yeast proteins in the heat shock category or see awesome Proteome review and bibliography of HSP104 properties. See also yeast-against-mammal Blast server.
35 proteins of yeast genome place in heat shock category:

 DDR2İİStress protein induced by DNA damage, heat shock, osmotic shock and oxidative stress.

 DDR48İİStress protein induced by heat shock, DNA damage, or osmotic stress.

 FPR2İİ rapamycin-binding protein of the endoplasmic reticulum, homolog of human FKBP13.

 HSC82İİChaperonin homologous to E. coli HtpG and mammalian HSP90.

 HSP104İİHeat shock protein required for thermotolerance, important for reactivation of mRNA splicing after heat shock.

 HSP12İİHeat shock protein of 12kDa, induced by heat, osmostress, oxidative stress and stationary phase.

 HSP150İİSecreted O-glycosylated protein required for tolerance to heat shock, member of Pir1/Hsp150p/Pir3 family of proteins with
 variable number of tandem internal repeats.

 HSP26İHeat shock protein 26kD, expressed during entry to stationary phase and induced by osmostress.

 HSP30İİHeat shock protein located in cell membrane, expressed during entry to stationary phase.

 HSP42İİHeat shock protein with similarity to Hsp26p, involved in restoration of the cytoskeleton during mild stress.

 HSP60İİMitochondrial chaperonin that cooperates with Hsp10p, homolog of E. coli GroEL.

 HSP78İİHeat shock protein of ClpB family of ATP-dependent proteases, mitochondrial.

 HSP82İİHeat-inducible chaperonin homologous to E. coli HtpG and mammalian HSP90.

 KAR2İİHSP70 family member of the ER lumen, required for protein translocation across the ER membrane and for nuclear fusion.

 MDJ1İİHomolog of E. coli DnaJ protein, involved in mitochondrial biogenesis and protein folding.

 PGM2İİPhosphoglucomutase, major isozyme, interconverts Glc-1-P and Glc-6-P.

 SIS1İİHomolog of E. coli DnaJ, required for initiation of translation.

 SOD2İİManganese superoxide dismutase, mitochondrial.

 SSA1İİHeat shock protein of HSP70 family, cytoplasmic.

 SSA2İİHeat shock protein of HSP70 family, cytoplasmic.

 SSA3İİHeat shock protein of HSP70 family, cytoplasmic.

 SSA4İİHeat shock protein of HSP70 family, cytoplasmic.

 SSB1İİHeat shock protein of HSP70 family involved in the translational apparatus.

 SSB2İİHeat shock protein of HSP70 family, cytoplasmic.

 SSC1İİMitochondrial heat shock protein of HSP70 family, plays a chaperonin role in receiving and folding of protein chains during import.

 SSE1İİHeat shock protein of the HSP70 family, multicopy suppressor of mutants with hyperactivated ras/cAMP pathway.

 SSE2İİHeat shock protein of the HSP70 family, present at low level at 23 deg but greatly induced after shift to 37 deg.

 STI1WİİStress-induced protein required for optimal growth at high and low temperature, has tetratricopeptide (TPR) repeats.

 TPS2İTrehalose-6-phosphate phosphatase, converts trehalose-6-phosphate to trehalose.

 UBI4İİUbiquitin, mature protein is cleaved from polyubiquitin (Ubi4p) or from fusions with ribosomal proteins 

 YDJ1İİHomolog of E. coli DnaJ, involved in protein import into mitochondria and ER.

 CAT5İİProtein necessary for depression of gluconeogenic enzymes and required for coenzyme Q (ubiquinone) biosynthesis.

 CPR1İİCyclophilin (peptidylprolyl isomerase) of the cytosol, plays a role in the stress response.

 CPR2İİCyclophilin (peptidylprolyl isomerase), ER or secreted isoform, plays a role in the stress response.

 CTT1İİCatalase T (cytosolic).

Mammalian homologues of Hsp 104

Iidentification of a mammalian member of the Clp/HSP104 family
Gene 1995 Jan 23;152(2):157-163 
Perier F, Radeke CM, Raab-Graham KF, Vandenberg CA
A cDNA encoding a novel mammalian member of the Clp/HSP104 family was isolated from a mouse macrophage-like cell line; the full-length version of this cDNA, termed SKD3, encodes a putative 76-kDa protein of 677 amino acids. The deduced aa sequence of the SKD3 polypeptide contains four ankyrin-like repeats in the N-terminal domain and a single ATP-binding consensus site in the C-terminal domain [residues 351-358]. The 378-aa C-terminal domain of SKD3 has 57-64% similarity (30-40% identity) with members of the Clp/HSP104 family, including the ClpA regulatory subunit of the Clp protease and S. cerevisiae heat-shock protein 104. Northern analysis showed that the 2.3-kb SKD3 transcript is present in a wide variety of tissues, is abundant in mouse heart, skeletal muscle and kidney, and is most abundant in testis. Members of the Clp/HSP104 family have been identified previously from bacteria, yeast and chloroplasts, and are ATPases regulating Clp protease activity and specificity, or mediating cellular responses involved in thermotolerance. SKD3 is the first member of this protein family identified in a higher eukaryote.
677aa;76003mw SwissProt Q60649
mmlsavlrrttpaprlflglikspslqsrggaygrgvvtgdrgepqrlraaawvrpgass
tfvpgrgaatwgrrgerteipyltaassergpspeetlpgqdswngvpnktglgmwalam
alvvqcysknpsnkdaalmeaarannvqevrrllsegadvnarhklgwtalmvasishne
svvqvllaagadpnlgdefssvyktaneqgvhslevlvtreddfnnrlnhrasfkgctal
hyavladdysivkelldrganplqrnemghtpldyaregevmkllktsetkymekqrkre
aeerrrfpleqrlkehiigqesaiatvgaairrkengwydeehplvflflgssgigktel
akqtakymhkdakkgfirldmsefqerhevakfigsppgyigheeggqltkklkqcpnav
vlfdevdkahpdvltimlqlfdegrltdgkgktidckdaifimtsnvasdeiaqhalqlr
qealemsrnriaenlgdvqmsdkitisknfkenvirpilkahfrrdeflgrineivyflp
fchseliqlvnkelnfwakrakqrhnitllwdrevadvlvdgynvhygarsikheverrv
vnqlaaayeqdllpggctlritvedsdkhllkspelpspqaekrpptlrleiidkdsktr
kldiqaplhpekvcyti
Ankyrins attach integral membrane proteins to cyto-skeletal elements; they bind to the erythrocyte membrane, to Na-K atpase, to the lymphocyte membrane, and to the cytoskeletal proteins fodrin, tubulin, vimentin and desmin. Erythrocyte ankyrins also link spectrin (beta chain) to the cytoplasmic domain of the erythrocytes anion exchange protein; they retain most or all of these binding functions. Tissue specificity: plasma membrane of neurons as well as glial cells throughout the brain. This motif consists of approximately 33 amino acids containing a highly conserved central hydrophobic alpha helix and is abundant in a wide variety of proteins, particularly those participating in the protein-protein or protein-membrane interactions involved in signal transduction, regulation of the cell cycle and control of transcription. The binding domain of ankyrin itself consists of 24 tandem repeats of a 33-amino acid helix-turn-helix motif that is present on a wide variety of otherwise unrelated proteins.

Alignment of SKD3 to yeast Hsp104

Sum P(4) = 6.1e-49 Identities = 54/100 (54%), Positives = 74/100 (74%)
SKD3:     371 DAKKGFIRLDMSEFQERHEVAKFIGSPPGYIGHEEGGQLTKKLKQCPNAVVLFDEVDKAH 430
              D +   IR DMSEFQE+H V++ IG+PPGY+  E GGQLT+ +++ P AVVLFDE +KAH
Hsp104:   560 DDESNVIRFDMSEFQEKHTVSRLIGAPPGYVLSESGGQLTEAVRRKPYAVVLFDEFEKAH 619

SKD3:     431 PDVLTIMLQLFDEGRLTDGKGKTIDCKDAIFIMTSNVASD 470
              PDV  ++LQ+ DEG+LTD  G  +D ++ I +MTSN+  D
Hsp104:   620 PDVSKLLLQVLDEGKLTDSLGHHVDFRNTIIVMTSNIGQD 659

Sum P(4) = 6.1e-49 Identities = 27/109 (24%), Positives = 52/109 (47%)

SKD3:     527 EFLGRINEIVYFLPFCHSELIQLVNKELNFWAKRAKQRHNITLLWDREVADVLVDGYNVH 586
              EF+ RI++I+ F       L  +V+  +     R  ++     L D     +   GY+  
Hsp104:   692 EFINRIDDILVFNRLSKKVLRSIVDIRIAEIQDRLAEKRMKIDLTDEAKDWLTDKGYDQL 751

SKD3:     587 YGARSIKHEVERRVVNQLAAAYEQDLLPGGCTLRITVEDSDKHLLKSPE 635
              YGAR +   + R+++N +A    +  +  G T+R+ V+D+   +L + E
Hsp104:   752 YGARPLNRLIHRQILNSMATFLLKGQIRNGETVRVVVKDTKLVVLPNHE 800

GroEL structure and function

GroEL is from E.coli. Yeast homologue is mitochrodrial chaperonin Hsp 60, with Hsp10 playing role of GroES and an ATPase inhibitor. SwissProt P19882:
sequence: 572 aa, 60751 mw 
     mlrssvvrsr atlrpllrra ysshkelkfg vegrasllkg vetlaeavaa tlgpkgrnvl
     ieqpfgppki tkdgvtvaks ivlkdkfenm gakllqevas ktneaagdgt tsatvlgrai
     ftesvknvaa gcnpmdlrrg sqvavekvie flsankkeit tseeiaqvat isangdshvg
     kllasamekv gkegvitire grtledelev tegmrfdrgf ispyfitdpk sskvefekpl
     lllsekkiss iqdilpalei snqsrrplli iaedvdgeal aacilnklrg qvkvcavkap
     gfgdnrknti gdiavltggt vfteeldlkp eqctienlgs cdsitvtked tvilngsgpk
     eaiqerieqi kgsiditttn syekeklqer laklsggvav irvggaseve vgekkdrydd
     alnatraave egilpgggta lvkasrvlde vvvdnfdqkl gvdiirkait rpakqiiena
     geegsviigk lideygddfa kgydasksey tdmlatgiid pfkvvrsglv dasgvaslla
     ttevaivdap eppaaagagg mpggmpgmpg mm
In mice, the homologue is P19226 and humans P10809; these are both called Hsp 60 as well.
Prion protein PrPc interacts with molecular chaperones of the Hsp60 family.
J Virol 1996 Jul;70(7):4724-4728 
Edenhofer F, Rieger R, Famulok M, Wendler W, Weiss S, Winnacker EL
... Here we employ a Saccharomyces cerevisiae two-hybrid screen to search for proteins which interact specifically with the Syrian golden hamster prion protein. Screening of a HeLa cDNA library identified Hsp60, a cellular chaperone as a major interactor for PrPc. The specificity of the interaction was confirmed in vitro for the recombinant proteins PrPc23-231 and rPrP27-30 fused to glutathione S-transferase with recombinant human Hsp60 as well as the bacterial GroEL. The interaction site for recombinant Hsp60 and GroEL proteins was mapped between amino acids 180 and 210 of the prion protein by screening with a set of recombinant PrPc fragments. The binding of Hsp60 and GroEL occurs within a region which contains parts of the putative alpha-helical domains H3 and H4 of the prion protein.
The crystal structure of the asymmetric GroEL-GroES-(ADP)7 chaperonin complex.
Nature 1997 Aug 21;388(6644):741-750 Brookhaven pdb: 1AON
Xu Z, Horwich AL, Sigler PB
Chaperonins assist protein folding with the consumption of ATP. They exist as multi-subunit protein assemblies comprising rings of subunits stacked back to back. In Escherichia coli, asymmetric intermediates of GroEL are formed with the co-chaperonin GroES and nucleotides bound only to one of the seven-subunit rings (the cis ring) and not to the opposing ring (the trans ring).

The structure of the GroEL-GroES-(ADP)7 complex reveals how large en bloc movements of the cis ring's intermediate and apical domains enable bound GroES to stabilize a folding chamber with ADP confined to the cis ring. Elevation and twist of the apical domains double the volume of the central cavity and bury hydrophobic peptide-binding residues in the interface with GroES, as well as between GroEL subunits, leaving a hydrophilic cavity lining that is conducive to protein folding. An inward tilt of the cis equatorial domain causes an outward tilt in the trans ring that opposes the binding of a second GroES. When combined with new functional results, this negative allosteric mechanism suggests a model for an ATP-driven folding cycle that requires a double toroid.

Protein folding. Folding with a two-stroke motor.
Nature 1997 Aug 21;388(6644):720-721 
Lorimer G
Protein folding. The difference with prokaryotes.
Nature 1997 Jul 24;388(6640):329 
Gething MJ

Distinct actions of cis and trans ATP within the double ring of the chaperonin GroEL.
Nature 1997 Aug 21;388(6644):792-798 
Rye HS, Burston SG, Fenton WA, Beechem JM, Xu Z, Sigler PB, Horwich AL
The chaperonin GroEL is a double-ring structure with a central cavity in each ring that provides an environment for the efficient folding of proteins when capped by the co-chaperone GroES in the presence of adenine nucleotides. Productive folding of the substrate rhodanese has been observed in cis ternary complexes, where GroES and polypeptide are bound to the same ring, formed with either ATP, ADP or non-hydrolysable ATP analogues, suggesting that the specific requirement for ATP is confined to an action in the trans ring that evicts GroES and polypeptide from the cis side.

We show here, however, that for the folding of malate dehydrogenase and Rubisco there is also an absolute requirement for ATP in the cis ring, as ADP and AMP-PNP are unable to promote folding. We investigated the specific roles of binding and hydrolysis of ATP in the cis and trans rings using mutant forms of GroEL that bind ATP but are defective in its hydrolysis. Binding of ATP and GroES in cis initiated productive folding inside a highly stable GroEL-ATP-GroES complex. To discharge GroES and polypeptide, ATP hydrolysis in the cis ring was required to form a GroEL-ADP-GroES complex with decreased stability, priming the cis complex for release by ATP binding (without hydrolysis) in the trans ring. These observations offer an explanation of why GroEL functions as a double-ring complex.

X-Priority: 3 X-MSMail-Priority: Normal X-MimeOLE: Produced By Microsoft MimeOLE V4.71.1712.3 X-Sender: 0241932072-0001@t-online.de Approved-By: "Roland Heynkes @ T-Online" Date: Mon, 15 Dec 1997 17:40:24 +0100 Reply-To: Bovine Spongiform Encephalopathy Sender: Bovine Spongiform Encephalopathy From: "Roland Heynkes @ T-Online" Subject: CJD-Alzheimer similarity? onference abstract may be interesting also with respect to CJD therapy:

CJD-Alzheimer similarity?

Conference Abstract
Pappolla,M.A.; Soto,C.; Bozner,P.; Frangione,B.; Ghiso,J.
Journal of Neuropathology and Experimental Neurology 1996; 55(N5): 15
Melatonin prevents formation of beta-sheets and fibrils of beta-amyloid a new therapy for AD?
It is generally postulated that the progressive neurodegeneration observed in sporadic A1zheimer's disease (AD) is a consequence, at least in part, of the neurotoxic properties of the beta-amyloid protein (Abeta). Previous studies have shown an association between aggregation and the secondary structure of Abeta, and its ability to promote neurodegeneration.

In addition, these mentioned characteristics of Abeta appear to determine its increased resistance to proteolysis and impaired clearance (key elements for ainyloid accumulation). We report that melatonin, a pineal hormone recently found to protect cells against Abeta toxicity (Pappolla et al. J Neurosci, in press), markedly inhibited the progressive formation of beta-sheet structures in Abeta, as well as its polymerisation into fibrils.

We have used circular dichroism (CD) and electron microscopy to monitor the gradual development of beta-sheets and of fibrillogenesis, respectively, of solutions containing Abeta(l-40). As expected, the content in beta-sheet conformation of the Abeta(l-40) peptide incubated alone increased with time, reaching a maximum value after 24 hr at 37 C.

This structure (76% beta-sheet and 16% random coil) strikingly changed by the addition of melatonin to sister tubes. In this case, there was an increase of the random conformation (67%) while the original values of beta-sheet dramatically diminished, reaching 30.7% after 24 hr incubation under identical conditions. Ultrastructural examination (2 day incubation) paralleled the conformational changes with virtually no fibrils detected in the tubes containing melatonin at various molarity ratios above 1:25 (melatonin:Abeta).

In contrast, a profuse number of amyloid fibrils were easily detected in the absence of melatonin. Remarkably, these effects were not observed in control preparations containing the melatonin analog 5-hydroxy-N-acetyl-tryptamine. These findings suggest a novel therapeutic approach for AD.

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