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Title:  Methods for identifying factors that control the folding of amyloid proteins of diverse origin
United States Patent: 
7,799,535
Issued: 
September 21, 2010

Inventors:
 Lindquist; Susan (Chicago, IL)
Assignee:
  ARCH Development Corporation (Chicago, IL)
Appl. No.:
 09/207,649
Filed:
 December 8, 1998


 

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Abstract

The present invention provides a yeast cell based system for determining factors that control the folding of amyloid proteins of diverse origins. Further the present invention provides methods of using such a system to screen for reagents that affect amyloid formation, a process that is integral to several devastating human disease including Creutzfeld-Jacob disease (CJD), fatal familial insomnia (FFI), Gertsmann-Straussler-Scheinker (GSS) syndrome, and kuru. The system of the present invention provides a rapid screening system to quickly and cheaply identify reagents that affect the folding and aggregation properties of the target protein.

Description of the Invention

1.0 BACKGROUND OF THE INVENTION

1.1 Field of the Invention

The present invention relates generally to the fields of genetics and cellular biology. More particularly, it concerns a yeast based system for the determination of compounds that affect amyloid formation. The present invention relates to the determination of compounds that affect the amyloid associated with Alzheimer's disease, Transmissible spongiform encephalopathies (TSEs), and several rare human neuropathies: Creutzfeld-Jacob disease (CJD), fatal familial insomnia (FFI), Gertsmann-Straussler-Scheinker (GSS) syndrome, and kuru.

1.2 Description of Related Art

1.2.1 Yeast Prions

Recently, a novel mode of inheritance has been discovered in Saccharomyces cerevisiae (Wickner, 1994; Lindquist, 1997). Phenotypes transmitted by two dominant, cytoplasmically inherited genetic elements, [PSI+] and [URE3], seem to depend upon the inheritance of altered protein structures, rather than altered nucleic acids. The "protein-only" hypothesis for their inheritance led these elements to be called "yeast prions" (Wickner, 1994). The term "prion" was first coined to describe the infectious agent hypothesized to cause mammalian spongiform encephalogathies (TSEs) by a "protein only" mechanism: a normal cellular protein (PrP.sup.C) adopts an altered conformation (PrP.sup.Sc) and interacts with other PrP.sup.C proteins to change their conformation as well (Prusiner, 1996).

The yeast [PSI+] element, the subject of the inventor's work, does not generally kill cells. It reduces the fidelity of ribosome translation termination and thereby suppresses nonsense codons (Lindquist, 1997). 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 (Paushkin et al., 1996; Patino et al., 1996; Paushkin et al., 1997). "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 aggregates too, causing a self-perpetuating loss of function in the termination factor and a heritable change in translational fidelity (Patino et al., 1996; Paushkin et al., 1997).

[PSI+] depends upon the chaperone protein Hsp104. The first known function of Hsp104 was in thermotolerance in yeast, where it increases survival after exposure to extreme temperatures up to 1000-fold (Sanchez and Lindquist, 1990). It does so by promoting the reactivation of proteins that have been damaged by heat and have begun to aggregate (Parsell et al., 1994). 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 (Chernoff et al., 1995). Because the only known function of Hsp104 is to alter the conformational state of other proteins, these observations provide a strong argument that [PSI-1] is indeed based upon 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 (Patino et al., 1996; Chernoff et al., 1995). This is very different from heat-induced aggregates, which remain insoluble in hsp104 deletion strains. Clearly, the relationship between Hsp104 and [PSI-1] is more complex than the relationship between Hsp104 and thermotolerance.

1.2.2 Human Prions

The family of transmissible spongiform encephalopathies (TSEs) include scrapie in sheep, bovine spongiform encephalopathy (BSE) or "mad cow disease" in cattle, and several rare human neuropathies: Creutzfeld-Jacob disease (CJD), fatal familial insomnia (FFI), Gertsmann-Straussler-Scheinker (GSS) syndrome, and kuru (Caughey and Chesebro, 1997; Prusiner, 1996). A central event in TSE pathogenesis is the accumulation in the nervous system of an abnormally-folded version (PrP.sup.Sc) of a normal cellular protein, PrP.sup.C. Griffith first proposed a "protein-only" model to explain the unconventional behavior of the infectious TSE agent (Griffith, 1967). Indeed, the "prion", a term by which the agent is popularly known today, appears to be almost entirely proteinaceous: consisting primarily of PrP.sup.Sc (Caughey and Chesebro, 1997; Prusiner, 1996).

Several lines of evidence show that PrP.sup.C is conformationally distinct from PrP.sup.Sc although both molecules derive from the same primary sequence and have no detectable post-translational differences (Caughey and Chesebro, 1997; Prusiner, 1996; Caughey et al., 1991; Pan et al., 1993; Riek et al., 1996). The conversion of PrP.sup.C to PrP.sup.Sc appears to involve direct interactions of PrP.sup.C with pre-existing PrP.sup.Sc (Caughey and Chesebro, 1997; Prusiner, 1996; Kocisko et al., 1994). However, the exact mechanism underlying conversion is not known. Genetic and inhibitor studies have suggested that other cellular factors may influence TSE pathogenesis or serve as regulators of disease (Kenward et al., 1996; Talzelt et al., 1996; Carlson et al., 1988; Caughey et al., 1994; Telling et al., 1995; Edenhofer et al., 1996). None have been conclusively identified; however, cellular osmolytes (sometimes called chemical chaperones; Caughey and Raymond, 1991) and protein chaperones have been frequently speculated to be among them (Kenward et al., 1996; Caughey et al., 1994; Telling et al., 1995; Edenhofer et al., 1996).

2.0 SUMMARY OF THE INVENTION

The chaperone protein Hsp104 controls the genetic behavior of a mysterious yeast prion-like element known as [PSI+]. The chaperone Hsp104 controls the aggregation of Sup35, the protein determinant of [PSI+].

The present invention includes, but is not limited to, the following features:

1) The protein Sup35 forms amyloid-like protein fibers in vitro. This is a property shared by other amyloidogenic proteins that cause human disease.

2) The yeast protein Hsp104 affects the behavior of Sup35 in vitro. It also affects the behavior of PrP (the mammalian prion protein) in vitro in a similar manner and interacts in a specific manner with .beta.-amyloid peptide 1-42 (Alzheimer's disease peptide).

3) When mammalian PrP is expressed in yeast cells, its folding state depends upon the Hsp104 protein. This is the final element that establishes that yeast can provide an excellent model system for studying factors that affect the folding properties of human disease proteins that have an amyloidogenic character.

In important embodiments of the present invention, this yeast system is used in methods of identifying a candidate substance that inhibits the aggregation of an aggregate-prone amyloid protein. Such methods comprise contacting a yeast cell that expresses an aggregate-prone amyloid protein with the candidate substance under conditions effective to allow aggregated amyloid formation, and determining the ability of the candidate substance to inhibit the aggregation of the aggregate-prone amyloid protein.

The term "aggregate-prone amyloid protein" is meant to be any protein that is able to form an amyloid or amyloid-like deposit. Amyloid or amyloid like deposits are generally insoluble fibrillary material. Although many proteins are capable of aggregating at high concentrations, aggregate prone amyloid proteins are able to, and often do, aggregate under physiological conditions, such as inside of a cell. Aggregate-prone amyloid proteins include yeast proteins, such as Sup35 and URE3, and mammalian proteins, such as PrP and .beta.-amyloid polypeptide. The inventors contemplate that a protein of essentially any origin may be used in the present invention.

In some preferred embodiments, the aggregate-prone amyloid protein is a chimeric protein. By "chimeric protein" it is meant that the protein comprises polypeptides that do not naturally occur together in a single protein unit. Preferred chimeric proteins comprises at least the N-terminal domain of Sup35. This domain has been found to form aggregates in yeast and in vitro and is capable of causing, the aggregation of chimeric proteins comprising this domain. Other preferred chimeric proteins include comprises at least an aggregate forming domain of a mammalian amyloid polypeptide, such as at least amino acids 1-42 of the .beta.-amyloid protein or at least the aggregate forming domain of PrP. In an important embodiment, the chimeric protein comprises Sup35 in which the N-terminal domain has been replaced by amino acids 1-42 of .beta.-amyloid protein.

In other embodiments, the chimeric protein comprises at least an aggregate forming domain of an aggregate-prone amyloid protein operably attached to a detectable marker protein. By "operably attached" it is meant that the aggregate forming domain and the marker protein are attached such that the chimeric protein maintains the ability to aggregate and the marker protein maintains the property of allowing detection of aggregation of the chimeric protein. For example, the Sup35 N-terminal domain is operably linked to the green fluorescent protein when this polypeptide is capable of aggregating and the aggregated protein maintains the ability of green fluorescent protein to fluoresce.

In other embodiments, the aggregation of the chimeric protein-leads to loss of function of the marker protein. When the marker protein is an enzyme, aggregation of the marker protein leads to loss of the enzymatic activity of the marker protein. That is to say that the enzymatic marker protein maintains its enzymatic activity when not aggregated, but aggregation leads to the loss of enzymatic activity. Loss of activity may be due to an alteration of the structure of the marker protein or may be due to sequestering of the protein in the aggregates away form the substrate or cofactors. The enzyme may be luciferase, confer drug resistance, or, in preferred embodiments, have the translation termination activity of the Sup35 protein. In other embodiments, the marker protein is a hormone receptor, such as the glucocorticoid receptor.

The inventors contemplate that the aggregation of the aggregate-prone amyloid protein may be detected in a number of ways. Including the methods described above, the aggregate may be detected by staining with Congo Red. In other embodiments, the aggregation is detected by the characteristic protease resistance of the aggregated protein. The ability to detect the protein may be increased by labeling the aggregate-prone amyloid protein. Labels useful in the detection of the aggregate-prone amyloid protein include radioactive isotope labels, such as .sup.35S, fluorophores, such as green fluorescent protein, or chromophores. In some preferred embodiments, aggregation is determined by the presence of a [PSI+] phenotype.

Although overexpression of the chimeric protein comprising the aggregate-prone domain of an aggregate-prone amyloid protein often leads to aggregation of the chimeric protein, the inventor has found that this aggregation is dependent on expression of heat shock proteins, such as Hsp104. Therefore, conditions effective to allow amyloid formation may involve modulating the expression of Hsp104 in the yeast cell. In preferred embodiments, the yeast cell overexpresses Hsp104.

In other embodiments of the present invention, the yeast systems are used to identify candidate substances for therapeutic activity against an amyloidogenic disease in an animal. These methods comprise contacting a yeast cell that expresses an aggregate-prone amyloid protein with said candidate substance under conditions effective to allow amyloid formation and determining the ability of said candidate substance to inhibit aggregation of the aggregate-prone amyloid protein. Thus, the ability to inhibit aggregation is indicative of therapeutic activity.

Amyloidogenic diseases in animals include Alzheimer's disease, scrapie, spongiform encephalopathy in a mammal, kuru, Creutzfeldt-Jakob disease, Gestmann-Strausser-Scheinker disease, or fatal familial insomnia. In preferred embodiments, one would express a protein comprising the aggregate forming domain of the etiological agent of a particular disease in the yeast system to identify therapeutic compounds for that particular disease. Therefore, in determining therapeutic compounds for Alzheimer's disease, one would use a yeast system comprising at least amino acids 1-42 of the .beta.-amyloid protein.

Likewise, in determining therapeutic compounds for scrapie, spongiform encephalopathy in a mammal, kuru, Creutzfeldt-Jakob disease, Gestmann-Strausser-Scheinker disease, or fatal familial insomnia, one would use a yeast system comprising the aggregate-forming domain of PrP. In preferred embodiments, the mammalian encephalopathy is bovine, feline, a mink, deer, elk, a mouse, a hamster, an ape, a monkey, or human.

Although many alternative forms of the PrP gene exist, the inventor contemplates that the expression in the yeast of a gene encoding the form linked to a specific disease is preferred for finding therapeutic agents for that disease. For example, for finding therapeutic agents for scrapie, it is preferred that proteins comprising aggregate forming domain of the goat or sheep PrP protein are expressed in the yeast. Of course, due to similarities between the PrP proteins, and even between the different types amyloid proteins, a therapeutic agent for one amyloidogenic disease may have therapeutic activity for one or more other amyloidogenic diseases.

4.0 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The invention demonstrates that yeast cells provide a system in which the folding of amyloidogenic proteins from diverse organisms is subject to manipulation. The most immediate application is the use of yeast cells to screen for reagents that affect amyloid formation, a process that is integral to several devastating human diseases. Screening for agents that affect these disease factors is very expensive and time consuming in animal models and cultured cells. Yeast will provide a rapid first screening system to quickly and cheaply identify reagents that affect the folding and aggregation properties of the target protein. These can then be screened by conventional methods to determine which are therapeutically applicable.

The inventor has found that Hsp104 controls the behavior of a factor that alters a particular physiological property of yeast cells in a heritable way. This change in physiology was shown to be associated with a heritable change in the aggregation state of a particular protein, Sup35, that is controlled by genetic manipulation of Hsp104. Subsequently, the inventor demonstrated that Sup35 has a very unusual biochemical property that it shares with certain human disease proteins. Specifically it forms amyloid fibers that stain with the dye Congo Red and shows apple green birefringence. Staining with this dye is a common diagnosis for human amyloid diseases (Glover et al., 1997; incorporated herein by reference).

The present invention is based, in part, on the inventor's discovery that, in a purified system in vitro, Hsp104 affects the folding state of the yeast amyloidogenic protein Sup35. Moreover, it also affects the folding state of a mammalian amyloidogenic protein, the prion protein known as PrP. The yeast protein was also shown to interact in a highly specific manner with another mammalian amyloid protein, .beta.-amyloid peptide 1-42 (Alzheimer's disease peptide). The inventor has established that the folding state of the mammalian PrP protein, when expressed in yeast, depends upon the same type of manipulations that the folding of the yeast amyloid Sup35 depends upon. This establishes that yeast provides a surprisingly advantageous and widely applicable system for testing factors that affect the folding and amyloidogenic properties of mammalian disease proteins (Schirmer and Lindquist, 1997; DebBurman et al., 1997).

4.1 Methods of Screening and Selecting Amyloid Formation

The inventor contemplates that the formation of amyloid fibers may be detected by a number of mechanisms. In some embodiments, the aggregation may be detected by its ability to bind Congo Red and show apple green birefringence under polarized light (Baker et al., 1994; Guiroy et al., 1993; Gasset et al., 1992; Tashima et al., 1986; Bockman et al., 1985; Bendheim et al., 1984; Prusiner et al., 1983). However, in other embodiments, the aggregation is detected indirectly. For example, in embodiments comprising the Sup35 aggregation domain (N-terminal domain), the physiologically important C-terminal domain may be sequestered in the cell by the addition of the endogenous Sup35 protein into the aggregation, causing a change in phenotype of the cell. Thus, aggregation may be detected by the presence of the [PSI+] phenotype in the yeast cells. Depending upon how much of the Sup35 comprising protein is expressed and aggregated in the yeast, this phenotype is characterized by an increase in nonsense suppression, lesser aggregation, or cell death, higher aggregation.

Chernoff et al. (1995) used a color test for the [PSI+] phenotype. In this test, a adel-14 strain was used. In this strain, the adel-14 nonsense mutation is suppressed in the presence of the [PSI+] phenotype. This leads to white colored colonies. In the absence of the [PSI+] phenotype, this strain has a red color. This test provides a screen for the [PSI+] phenotype. Therefore, the ability of conditions or compositions to affect the [PSI+] phenotype may be detected by their ability to affect a color change in this [PSI+]/adel-14 strain.

In some preferred embodiments, the [PSI+] phenotype kills the yeast cell. Such cells are particularly useful in screening for the [PSI.sup.+] phenotype. For example, yeast expressing a chimeric protein comprising the .beta.-amyloid peptide (1-42) and the Sup35 C-terminal domain have a [PSI+] phenotype that leads to cell death. The inventor contemplates that such cells are an excellent system for screening candidate compounds for their ability to inhibit .beta.-amyloid aggregation, because only yeast grown in the presence of compounds that inhibit or reverse the [PSI+] phenotype will survive.

The inventor has shown that chimeric proteins comprising an aggregate prone domain have prion properties. For example, in a yeast expressing a chimeric protein comprising the N-terminal domain of Sup35 and GFP, the GFP was shown to aggregate. This same result was seen in a yeast strain expressing a chimeric protein comprising the N-terminal domain of Sup35 and GFP but that lacked expression of the N-terminal domain of the endogenous Sup35. This shows the aggregation of the chimeric protein was independent of the endogenous protein comprising the aggregate prone domain. Furthermore, chimeric proteins comprising GFP may be particularly useful in methods of screening agents that prevent aggregation, as the fluorescence pattern GFP is quickly and easily screened.

The inventor contemplates that, because chimeric proteins comprising an aggregate prone domain take on prion-like properties in yeast, such proteins are useful in developing screens or selections for the presence of aggregation. When a chimeric protein comprising an aggregate prone domain, such as the N-terminal domain of Sup35, and another polypeptide, such as luciferase or the glucocorticoid receptor, is expressed in yeast under conditions that lead to aggregation, aggregation of the chimeric protein leads to changes in the activities of the other polypeptide. Therefore, in yeast cells comprising the Sup35 aggregate prone domain and luciferase, the presence of aggregation can be detected by the loss of luciferase activity in the cells. In other preferred embodiments, the chimeric protein comprises an aggregate prone domain and a drug-resistance marker. In such embodiments, aggregation leads to antibiotic sensitivity.

4.2 Amyloid Diseases

In an important embodiment, the present invention is a screen for compounds that are therapeutic for amyloid diseases. The inventors contemplate that, by using polypeptides comprising the etiological agent of the amyloid disease, the methods of the present invention may be used to find therapeutic compounds for essentially any amyloid disease. A number of amyloid diseases occur in mammals and are discussed herein.

A number of neurodegenerative diseases in mammals have been linked to the aggregation of the product of the PrP gene (prion protein). Such diseases include scrapie in sheep and goats, mad cow disease (bovine spongiform encephalopathy), transmissible mink encephalopathy, chronic wating disease in captive mule deer and elk, feline spongiform encephalopathy, and prion diseases of other animals including mice, hamsters, nyala, greater kudu, eland, gembok, arabian oryx. Of course, prion diseases are also seen in apes, monkeys, and humans.

In humans, as in many animals, prion diseases can be sporadic, inherited, or may be brought on by inoculation with infectious prion particles. Common names of prion diseases in humans are kuru, Creutzfeld-Jakob disease (CJD), Gerstmann-Straussler-Scheinker (GSS), and fatal familial insomnia (FFI). The classification of human prion diseases is based on clinical and neuropathological findings (Prusiner, 1996; incorporated herein by reference).

Prion diseases resulting from the horizontal transmission of infectious prions are iatrogenic CJD and kuru. Inherited forms GSS, fanilial CJD, and FFI have all been associated with oneor more mutations in the protein coding region of the PrP gene (Bertoni et al., 1992; Dlouhy et al., 1992; Doh-ura et al., 1989; Gabizon et al., 1993; Goldfarb et al., 1990; Goldfarb et al., 1991; Goldfarb et al., 1992; Goldgaber et al., 1989; Hsiao et al., 1989; Kitamoto et al., 1993a; Kitamoto et al., 1993b; Medori et al., 1992; Petersen et al., 1992; Poulter et al., 1992). Sporadic forms of prion disease in humans comprise most cases of CJD and some cases of GSS (Masters et al., 1978).

Regardless of the origin of the prion diseases, all have been associated with abnormal folding of the cellular protein PrP.sup.c into a protease resistant form, Pa.sup.sc, that aggregates (Oesch et al., 1985; Bolton et al., 1982; McKinley et al., 1982; Bolton et al., 1984; Prusiner et al., 1984; Bolton et al., 1987).

Another amyloid disease in humans is Alzheimer's disease (AD). One of the key events in AD is the deposition of amyloid as insoluble fibrous masses (amyloidogenesis) resulting in extracellularneuritic plaques and deposits around the walls of cerebral blood vessels (WO 96/39834; incorporated herein by reference). The main component of amyloid is a 4.1-4.3 kDa peptide, called .beta.-amyloid, that is part of a much longer amyloid precursor protein APP (Muller-Hill and Beyreuther, 1989). Peptides containing the sequence 1-40 or 1-42 of .beta.-amyloid and shorter derivatives can form amyloid-like fibrils in the absence of other protein (Pike et al., 1993).

The inventors have shown that proteins comprising PrP and .beta.-amyloid polypeptides are capable of forming aggregates in a yeast based system. Thus, this system provides a mechanism of testing compounds for their ability to inhibit the aggregation of these polypeptides in an inducible, yeast-based system. Such compounds may be used as amyloid disease therapeutic compounds.
 

Claim 1 of 30 Claims

1. A method of identifying a candidate substance that inhibits the aggregation of a mammalian aggregate-prone amyloid protein in a yeast cell, comprising: (a) contacting a yeast cell that expresses a chimeric protein comprising a mammalian aggregate-prone amyloid protein with said candidate substance under conditions effective to allow aggregated amyloid formation in the yeast cell, wherein the chimeric protein comprises at least an aggregate forming domain of .beta.-amyloid; and (b) determining the ability of said candidate substance to inhibit the aggregation of the aggregate-prone amyloid protein in the yeast cell.
 

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