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  Polyglutamine repeat sequencesUSPTO Application #: 20060234385Title: Polyglutamine repeat sequences Abstract: Several neurodegenerative diseases result from the aggregation of polyglutamine repeat proteins into insoluble neuronal intranuclear inclusions. The invention provides methods with which to study the processes of these diseases, including methods for solubilizing polypeptides containing a polyglutamine repeat sequence, for storing these polyglutamine polypeptides and inhibit their spontaneous aggregation, for making the aggregates of polyglutamine polypeptides, for assaying the extension of existing polyglutamine aggregates, for determining the ability of a chemical compound to inhibit aggregation, and for inhibiting aggregation of polyglutamine polypeptides. The invention further provides materials with which to study these diseases including a synthetic aggregate that have a capability to recruit additional monomeric polyglutamine polypeptides and chemical compounds that inhibit the formation and/or extension of polyglutamine aggregates. (end of abstract) Agent: Howard Eisenberg, Esq. - Perkasie, PA, US Inventors: Ronald Wetzel, Wen Yang, Valerie Berthelier, Songming Chen USPTO Applicaton #: 20060234385 - Class: 436086000 (USPTO) Related Patent Categories: Chemistry: Analytical And Immunological Testing, Peptide, Protein Or Amino Acid The Patent Description & Claims data below is from USPTO Patent Application 20060234385. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This application is a divisional of U.S. patent application Ser. No. 10/082,673, filed on Feb. 22, 2002. FIELD OF THE INVENTION [0002] The invention relates to the field of diseases that are associated with abnormal aggregation of proteins. Specifically, the invention relates to the field of diseases related to aggregation of intracellular proteins that contain polyglutamine repeat sequences. BACKGROUND OF THE INVENTION [0003] Insoluble aggregates of normally well-behaved proteins are associated with a variety of disease states, including the various forms of amyloidosis, such as Alzheimer's disease, and the prion diseases such as scrapie and bovine spongiform encephalomyelitis, also known as Mad Cow Disease. A family of proteins, called molecular chaperones, exists in cells to overcome the intrinsic propensity of polypeptides to aggregate during the normal folding process. However, under certain circumstances proteins such as the amyloid .beta. (A.beta.) peptide associated with Alzheimer's disease form insoluble protein aggregates in spite of the presence of chaperones. In some amyloid diseases, aggregates appear to be toxic simply by virtue of the effect of their accumulated mass in interfering with normal tissue function. In neurodegenerative diseases like Alzheimer's and Huntington's Diseases, the toxic effect appears to be much more subtle. According to one hypothesis, aggregates become toxic when they clog the cell's normal machinery for clearing aggregates and other superfluous proteins. Another hypothesis holds that aggregate toxicity derives from the ability of aggregates to recruit other essential proteins and in the process deplete the normal environment of their activities. In any case, it would be of considerable value to develop ways of removing these aggregates in a benign way, or preventing their formation, in analogy to the actions of molecular chaperones. [0004] Analogously to the situation in Alzheimer's Disease and A.beta. peptide, there are at least eight inherited neurodegenerative diseases, including Huntington's disease (HD), spinal and bulbar muscular atrophy (SBMA), dentatorubral pallidoluysian atrophy (DRPLA), and spinocerebellar ataxias 1, 2, 3, and 6, that are linked to a particular type of protein aggregate. Although each of these diseases is associated with a different protein, the proteins share the common feature of containing what is referred to as an expanded polyglutamine (poly(Gln)) repeat. These poly(Gln) expansion-related diseases, often referred to as CAG repeat diseases because the glutamine in the poly(Gln) peptide is coded for by the nucleotides CAG, are progressive disorders characterized by motor and/or cognitive impairments and distinctive pathological patterns of neuronal degeneration. The only mutation implicated in these diseases is an expansion of a poly(Gln) sequence in the disease-related protein, generally from a benign length of less than 37 Gln (also referred to as Q.sub.37) to a pathological length of Q.sub.37 or more. [0005] All of these neurodegenerative disorders present a common feature: the aggregation of the poly(Gln) repeat disease-related protein into insoluble neuronal intranuclear inclusions, which has become the neuropathological signature of poly(Gln) disorders. The important role that long poly(Gln) repeats play in poly(Gln)-related disorders has been confirmed in a number of models in which mutant forms of various disease proteins were expressed in transgenic mice, Drosophila, or the nematode Caenorhabditis elegans. [0006] Although these diseases exhibit similar physiological abnormalities, the only common features of disease-related proteins are the poly(Gln) domains. Because of this, the expanded poly(Gln) is believed to be responsible for the pathogenesis. As discussed above, their toxicity is believed to be due to their ability to recruit other critical cellular proteins, via their own poly(Gln) components, into the growing aggregate. The loss of protein activity due to this sequestration appears to be toxic to the cell. [0007] Much about these poly(Gln) diseases remains to be learned. One problem in studying these diseases is that poly(Gln) peptides having a Gln repeat of more than Q.sub.35 are poorly soluble when transferred directly into denaturing solvents or water. In some circumstances, such as described in Sharma, FEBS Letters, 456:181-185 (1999), the insolubility of long poly(Gln) repeats has presented such an insurmountable problem that studies had to performed on shorter soluble poly(Gln) repeats such as Q.sub.22, even though mutant proteins involved in the spino-cerebellar ataxia type 1 (SCA) are at least Q.sub.40. [0008] One method to increase the solubility of monomeric (non-aggregated) A.beta. peptide, as disclosed in Evans et al., Proc. Natl. Acad. Sci., 92:763-767 (1995), is to dissolve the peptide in a non-volatile disaggregating solvent such as dimethyl sulfoxide (DMSO). This method has the disadvantage in that the DMSO remains as a permanent co-solvent in the final reaction mixture. Therefore, any results obtained in studies of A.beta. peptide dissolved in this way may be biased by the presence of the DMSO. [0009] Use of a volatile disaggregating solvent, trifluoroacetic acid (TFA), to solubilize the A.beta. peptide is disclosed in Jao et al., Amyloid: Int. J. Exp. Clin. Invest., 4:240-252 (1997). Volatile disaggregating solvents have an advantage over non-volatile solvents such as DMSO because they are readily removed from the peptide, and thus do not interfere with studies on the peptide. According to this method, TFA is added to the peptide in a glass container at about a 1:1 ratio (ml TFA:mg peptide). Then the TFA and peptide are sonicated, while adding additional TFA, until the peptide completely dissolves. The TFA is then removed with dry nitrogen gas, leaving a coating of peptide on the walls of the container. Trace amounts of the TFA are removed by adding distilled hexafluoroisopropanol (HFIP), sonicating, and removing the HFIP with dry nitrogen gas. Sequential TFA-HFIP treatment has also been disclosed in Zagorski et al., Meths. Enzymol., 309:189-204 (1999). In this protocol, the role played by HFIP seems to be to simply aid in the removal of TFA, which otherwise will make an aqueous solution of the processed peptide acidic and possibly encourage its reaggregation. In our laboratory, this protocol was determined to be effective at solubilizing and disaggregating peptides in the range of Q.sub.15-Q.sub.35. However, it is poorly effective with peptides greater than Q.sub.35. [0010] The inability to dissolve and disaggregate poly(Gln) of the pathological length of Q.sub.37 or more represents a major obstacle in studying poly(Gln) diseases. A substantial need exists for a method to solubilize and to disaggregate these peptides, and to maintain these peptides in the disaggregated monomeric state. [0011] Another impediment to the study of poly(Gln) aggregation diseases has been the difficulty in making the aggregates in vitro. Typically, as disclosed by Scherzinger et al., Cell, 90:549-558 (1997) and Scherzinger et al., Proc. Natl. Acad. Sci., 96:4604-4609 (1999), such aggregates are made by recombinantly producing a fusion protein (GST-HDex1) containing glutathione S-transferase and exon 1 of the HD (Huntington's Disease). When the fusion protein is cleaved with trypsin, a high molecular weight protein aggregate with a fibrillar or ribbon-like morphology similar to those found in scrapie and in Alzheimer's disease are formed. These recombinantly produced aggregates do not completely correlate with the natural poly(Gln) disease state. [0012] In the disease state, peptides with poly(Gln) repeat lengths as low as Q.sub.15 or Q.sub.20, while of insufficient length to readily spontaneously aggregate, readily add to pre-existing aggregates. As disclosed in Perutz, et al., Proc. Natl. Acad. Sci. USA, 91:5355-5358 (1994), aggregates having such shorter poly(Gln) peptides differ from those seen in Alzheimer's disease. Polyglutamine aggregates made in vitro can adopt a number of morphological forms, each of which may differ in toxic activity. Very little is known for certain about the morphology of the toxic form of polyglutamine aggregates in vivo, but if the recruitment hypothesis is correct, than the aggregates must be especially potent in this activity. Consequently, a substantial need exists for methods to prepare different kinds of poly(Gln) aggregates in vitro in order to identify those that are particularly active in supporting deposition and/or extension of additional polyglutamine peptides. [0013] Given the potential role of poly(Gln) aggregates and poly(Gln) aggregate extension in the pathogenesis of expanded CAG repeat diseases, it is essential to characterize the fundamental aggregation behavior of poly(Gln) sequences. Studies of the aggregation behavior dependence on poly(Gln) repeat length are important to fully understand the correlation between length and disease risk, as well as the rules that control the recruitment of other poly(Gln)-containing peptides and proteins into growing poly(Gln) aggregates. Presently, no assay exists that allows the observation of both the homologous growth of an aggregate as well as the ability of the aggregate to recruit other polyglutarnine peptides into the aggregate via its extension. Such an assay would have use as an assay for poly(Gln) aggregation and recruitment and as a screen for aggregation inhibitors as potential therapeutics. The assay would also be capable of detecting "extension-competent" or "seeding-competent" aggregates in tissue and serum samples that might be crucial for diagnosis and for evaluating the role of poly(Gln) aggregates in the disease mechanism. SUMMARY OF THE INVENTION [0014] In one embodiment, the invention is a method for solubilizing and/or disaggregating a polypeptide. The method of the invention is especially useful for solubilizing and/or disaggregating a peptide that has a tendency to spontaneously aggregate. Such peptides, and aggregates thereof, are found in certain diseases such as prion disease and Alzheimer's disease, and with CAG repeat (poly(Gln)) diseases. [0015] According to this embodiment of the invention, the peptide is combined with a mixture of trifluoroacetic acid (TFA) and hexafluoroisopropanol (HFIP) and the peptide is permitted to dissolve in the mixture. Utilizing a mixture of these two solvents, rather than alone or sequentially as taught in the prior art, has been unexpectedly discovered to increase the solubility of peptides, even those having long repeating sequences, such as poly(Gln) where Q>35, that are otherwise difficult or impossible to dissolve by presently available methods. This permits the subsequent step of removing the TFA and HFIP and then resolubilizing the monomeric peptides in water so that the peptides will be in a useful form. According to this embodiment of the invention, it is preferred but not essential that the poly(Gln) polypeptide to be solubilized contain charged amino acids flanking the poly(Gln) sequence in order to increase its kinetic solubility in aqueous solution, that is the transient solubility of the polypeptide when initially dissolved. [0016] In another embodiment, the invention is a method for storing monomeric peptides that have a tendency to aggregate. According to this method, solubilized disaggregated peptides are snap frozen at a low temperature where freezing occurs essentially instantaneously and stored at a temperature of below -50.degree. C., preferably about -80.degree. C. or lower. If desired, DMSO may be added to the solution containing the monomeric peptides prior to freezing. The presence of DMSO, although not preferred, may further help to prevent aggregation of the peptides. [0017] In another embodiment, the invention is a method for making an aggregate of peptides that are prone to aggregation, such as peptides containing a glutamine repeat sequence. According to this embodiment, an aggregation-prone peptide, such as a polypeptide having a glutamine repeat sequence, which peptide is in solution, is frozen and incubated in the frozen state at a temperature at which aggregate formation may occur, and then thawed, at which point the aggregates are collected. The method of the invention permits the formation of peptide aggregates that are not otherwise obtainable, or that are impractical to produce, by prior art methods, such as poly(Gln) aggregates of Q.sub.15. If desired, the aggregates thus formed may be further processed by sonication and/or filtration. [0018] In another embodiment, the invention is an vitro-produced aggregate composed of peptides having a glutamine repeat sequence, wherein the aggregate is in the shape of a filament having a diameter of less than 10 nm and a length of less than 100 nm. The aggregate may be produced by the method of the invention for making an aggregate of peptides containing a glutamine repeat sequence. Preferably, there is a multiplicity of aggregates of which the majority of the aggregates in the multiplicity of aggregates are in the shape of a filament having a diameter less than 10 nm and a length of less than 100 nm. [0019] In another embodiment, the invention is an in vitro assay for poly(Gln) aggregate formation and for the extension of existing poly(Gln) aggregates. Present methods of assaying for poly(Gln) aggregate formation depend on measurements of the total mass of an aggregate, and are therefore limited in their ability to provide detailed quantification of heterologous aggregation reactions. The assay of the invention is capable of monitoring the repeat length dependence of heterologous aggregation extension, that is, the ability of a poly(Gln) peptide to assemble into an aggregate of another poly(Gln) peptide. [0020] According to this embodiment, labeled monomeric (non-aggregated) poly(Gln) peptide in solution is added to a support to which are fixed poly(Gln) aggregates so that the monomeric peptide solution contacts the fixed aggregates, and the amount of labeled monomeric peptide that binds to the aggregate is determined, typically by counting the amount of the labeling that remains following removal of any unbound monomeric peptide. Preferably, following the addition of the monomeric peptide to the container, the aggregates and monomeric peptides are incubated for a predetermined amount of time during which time the aggregates and monomeric peptides have an opportunity to bind to one another. Also preferably, at a time following the addition of the monomeric peptide and before the amount of labeling remaining is determined, the container is rinsed to remove any monomeric peptide that does not bind to the aggregates. Preferably, the poly(Gln) aggregates that are fixed to the inner surface of the container are the poly(Gln) aggregates of the invention. [0021] In another embodiment, the invention is a method for determining the ability of a chemical compound to inhibit the expansion of existing poly(Gln) aggregates by either homologous or heterologous extension reactions. According to this embodiment, the assay of the invention is performed wherein a test compound is exposed to either or both of the fixed poly(Gln) aggregates and the monomeric poly(Gln) peptides. In this manner, the ability of the test compound to inhibit the formation of, or the extension of, poly(Gln) aggregates can be determined. Continue reading... Full patent description for Polyglutamine repeat sequences Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Polyglutamine repeat sequences patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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