Mutant presenilin 1 polypeptides

Details Mutant presenilin 1 polypeptides Mutant presenilin 1 polypeptides

2001-06-29

2004-02-03

Kemmerer, Elizabeth (Department: 1647)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C530S300000, C514S002600, C514S012200, C514S183000, C424S094100, C435S069100, C435S252300, C435S320100, C435S325000

Reexamination Certificate

active

06686449

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides mutant presenilin 1 and presenilin 2 polypeptides and polynucleotides encoding the polypeptides and methods for their production by recombinant and PCR techniques are disclosed. Methods for utilizing the mutant polypeptides in cell based and in-vitro assays for inhibitors of activity are also disclosed.
BACKGROUND OF THE INVENTION
Alzheimer's disease was originally thought to be a rare disorder primarily affecting only people under the age of 65. It is now recognized as the most common form of dementia, and alone is responsible for about 50% of all dementias; an additional 15-20% of dementias have combined Alzheimer's and vascular pathology.
The prevalence of the Alzheimer's is directly related to age. It can occur in the fourth decade of life but is extraordinarily rare at this age. The prevalence then increases logarithmically with each succeeding decade. Over the age of 85 at least one person in four is afflicted. Because persons over the 85 form the rapidly growing portion of the population Alzheimer's disease represents a major health problem. Zigmond, et.: Fundamental Neuroscience, Academic Press, 1999.
Alzheimer's disease is thought to be initiated by the deposition of amyloid plaque in cortex and hippocampus. The material deposited in plaque is proteinaceous. It consists primarily of the amyloid &bgr;-peptide (A&bgr;), a peptide of 39-43 amino acids which is derived from a larger precursor, the amyloid peptide precursor (APP), through the action of specific proteases. APP is a large, type-I transmembrane protein of 695-770 amino acids that is expressed by a variety of cell types including neurons, glia and somatic cells. The cleavage of A&bgr; from APP is accomplished by the action of two proteolytic activities commonly denoted as beta-secretase (Asp2) and gamma-secretase. Processing at the &ggr;-secretase site is somehow dependent on presenilin-1 (as it does not occur in PS1 null embryonic neurons grown in culture, DeStrooper et al., 1997), but the protease responsible has not been identified. Deletion of the PS1 gene in mice greatly reduces gamma secretase activity. With less than 5% of the APP made by the cell processed through the amyloidogenic pathway to A&bgr;. DeStrooper (1998); Qian (1998).
A causative role for A&bgr; peptide in Alzheimer's disease is supported by genetic studies of familial, early-onset Alzheimer's disease in which inheritance follows an autosomal dominant mode of transmission. In such patients, genetic forms of Alzheimer's disease have been associated with mutations in the APP gene (Groate et al., 1991; Mullan et al. 1992), and two related genes, presenilin-1 (PS-1; Sherrington et al., 1995) and presenilin-2 (PS-2; Levy-Lahad et al., 1995; Rogaev et al., 1995). Mutations in all three genes alter production of the A&bgr; peptide in specific ways.
PS1 and PS2 mutations subtly increase the production of A&bgr;
1-42
peptide as compared to the A&bgr;
1-40
peptide (e.g., Citron et al., 1997), Mehta et al. (1998), Murayama et al. (1999), Xia et al. (1997). A&bgr;
1-42
is generally recognized as being more toxic to cells than A&bgr;
1-40.
Because PS1 and PS2 are intimately involved with the processing of APP both genes are attractive targets for drug screening in which aberrant APP processing is a causative or exacerbating factor. It has been postulated that both presenilin 1 and presenilin 2 have some intrinsic protease activity but this activity is so weak that designing a method of screening test agents which inhibit the intrinsic activity is problematic. The invention provides mutant presenilin 1 and presenilin 2 with enhanced proteolytic activities suitable for high throughput screening.
Literature Cited
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Nature
360, 372-374 (1992).
2. Citron, M.; Westaway, D.; Xia, W.; Carlson, G.; Diehl, T.; Levesque, G.; Johnson-Wood, K.; Lee, M.; Seubert, P.; Davis, A.; Kholodenko, D.; Motter, R.; Sherrington, R.; Perry, B.; Yao, H.; Strome, R.; Lieberburg, I.; Rommens, J.; Kim. S.; Schenk, D.; Fraser, P.; St George Hyslop, P.; Selkoe, D. J. : Mutant presenilins of Alzheimer's disease increase production of 42-residue amyloid beta-protein in both transfected cells and transgenic mice. Nature Med. 3: 67-72, 1997.
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Nat Genet.
1, 345-347 (1992).
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Fundamental Neuroscience, Academic Press, 1999.
15. Li Y M, Xu M, Lai M T, Huang Q, Castro J L, DiMuzio-Mower

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