Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2001-03-30
2004-03-30
Kemmerer, Elizabeth (Department: 1647)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S007900, C435S007920
Reexamination Certificate
active
06713248
ABSTRACT:
Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
FIELD OF THE INVENTION
The present invention relates generally to the field of plaque amyloid deposits that are the hallmarks of Alzheimer's disease. In particular, the invention relates to an isolated, functionally-active protein that has gamma-secretase activity. Gamma-secretase activity is necessary for amyloid production. The present invention also relates to methods for isolating integral-membrane proteins and protein complexes, including the gamma-secretase protein of the invention, and assays for detecting gamma-secretase activity.
BACKGROUND OF THE INVENTION
Alzheimer's disease is characterized by neuropathological lesions in the brain, marked by extracellular amyloid plaques in the cerebral and limbic cortices and intraneuronal paired helical filaments and neurofibrillary tangles. Commonly, Alzheimer's disease is a disease of the elderly with incidence increasing sharply after 60 years of age. However, early onset of Alzheimer's disease may strike patients only 40-50 years old, and is often associated with Familial Alzheimer's disease (FAD).
The course of both types of Alzheimer's disease appears to be the same. The major proteinaceous component of vascular and plaque amyloid deposits is the A&bgr;-42 peptide which is generated by proteolytic cleavage of &bgr;APP. There is extensive evidence that supports the hypothesis that the A&bgr;-42 peptide plays an essential role in the pathogenesis of Alzheimer's disease. The generation of A&bgr; peptides from the &bgr; amyloid precursor protein (&bgr;APP) involves three different protease activities designated alpha-, beta-, and gamma-secretases, and is altered by mutations in &bgr;APP, and two different presenilins designated PS1 and PS2. To date, nucleotide sequences have been determined for &bgr;APP (Kang, J. et al., 1987
Nature
325:733-736), PS1 (Sherrington, R., et al., 1995
Nature
375:754-760), and PS2 (Levy-Lahad, E., et al., 1995 Science 269:973-977). A candidate nucleotide sequence that may encode the protein having beta-secretase activity (Vassar, R., et al., 1999 Science 286:735-741; U.S. Pat. Nos. 5,744,346 and 5,942,400), and a candidate alpha secretase molecule (Lammich, S., et al., 1999
Proc. Natl. Acad. Sci.
USA 98:3922-3927) have been identified. The isolated sequence for gamma-secretase remains elusive.
The mature &bgr;APP protein is an integral-membrane protein found in the plasma membrane, Golgi apparatus, and endoplasmic reticulum. The &bgr;APP protein resembles a cell-surface receptor having a large extracellular N-terminal domain, a single transmembrane domain, and a small cytoplasmic C-terminal tail (Kang, J., et al., 1987 supra). Splice variants of the &bgr;APP mRNA encode APP polypeptides of 770, 750, and 695 amino acids. All these forms of &bgr;APP include the cleavage region and can give rise to amyloidogenic A&bgr; peptides. In normal cells, &bgr;APP undergoes one of two different sequential cleavage pathways that involve alpha-, beta-, and gamma-secretases (Dovey, H. F., et al., 1993
Neuroreport
4:1039-1042; Selkoe, D. J., et al., 1994
Ann. Rev. of Cell Biol.
10:373-403; Asami-Odaka, A., et al., 1995
Biochemistry
34:10272-10278).
In one cleavage pathway, alpha-secretase cleaves &bgr;APP in the extracellular, membrane/proximal domain (e.g., C-terminus to amino acid residue 687 of the 770 amino acid form of &bgr;APP) to generate a soluble N-terminal fragment (e.g., the alpha-sAPP fragment) and a membrane-bound C-terminal fragment (e.g., the 9 kDa CTF or C83 CTF). Then, gamma-secretase cleaves the membrane-bound CTF, within the membrane-bound domain, to generate the p3 fragment (e.g., the 3 kDa fragment) and a 6 kDa C-terminal fragment.
In another cleavage pathway, beta-secretase cleaves &bgr;APP in the extracellular, membrane-proximal domain (e.g., C-terminal to amino acid residue 671 of the 770 amino acid form of &bgr;APP) to generate a soluble N-terminal fragment (e.g., the 100 kDa NTF or beta-sAPP fragment) and a membrane-bound C-terminal fragment (e.g., the 11 kDa CTF or C 100 CTF). Then, gamma-secretase cleaves the membrane-bound CTF, within the membrane-bound domain, to generate the p6 fragment (e.g., the 6 kDa fragment) and A&bgr; peptide (e.g., the 4 kDa fragment).
The amino acid sequence of the gamma-secretase cleavage region is known (Duffy, C. L., et al., 1988
Brain Res.
474:100-111; Castano, E. M. and Frangione, B. 1988
Lab. Invest.
58:122-132). Gamma-secretase cleaves at variable sites within the cleavage region (Haass, C. and Selkoe, D. J. 1993
Cell
75:1039-1042) to generate a population of A&bgr; peptides having heterogeneous C-terminal ends. In normal patients, the A&bgr; peptide is found in two predominant forms, the majority A&bgr;-40 form and the minority A&bgr;-42 form each having a distinct COOH-terminus. Patients with the most common form of FAD show an increase in the amount of the 42 form. The A&bgr;-40 form is not associated with early deposits of amyloid plaques. In contrast, the A&bgr;-42 form accumulates early and predominantly in the parenchymal plaques and there is strong evidence that A&bgr;-42 plays a major role in amyloid plaque deposits in FAD patients (Roher, A. E., et al., 1993
Proc. Natl. Acad. Sci. USA
90:10836; Iwatasubo, T., et al., 1994
Neuron
13:45; Yamaguchi, H., et al., 1995
Amyloid Int. J. Clin. Invest.
2:7-16; Mann, D. M., et al., 1996
Am. J. Pathol.
148:1257).
It has been generally thought that the same gamma-secretase enzyme generates the −40 and −42 forms. To date, this question remains unsettled because researchers in the field have reported conflicting results. For example, two research groups have independently reported in vitro results which suggest certain protease inhibitors selectively decrease the levels of A&bgr;-42 and concluded that A&bgr;-40 and 42 are generated by two different gamma-secretases (Citron, M., et al., 1996
Proc. Nat. Acad. Sci.
USA 93:13170-13175; Klafki, H. -W., et al., 1996
J. Biol. Chem.
271:28655-28659). A third research group has compared the relative ability of a series of protease inhibitors to inhibit secretion of A&bgr;-40 and 42 peptides and reached the opposing conclusion that the A&bgr;-40 and -42 peptides are generated by a single protease (Durkin, J. T. et al., 1999
Journal of Biological Chemistry
274:20499-20504).
The A&bgr;-40 and -42 forms are secreted constitutively in a wide variety of cells/tissues, and are found as soluble forms in biological fluids (Seubert, P., et al., 1992
Nature
359:325 375; Shoji, M., et al., 1992
Science
258:126-129) thus allowing extensive analysis of both forms of the A&bgr; peptide in FAD patients. Some FAD patients have elevated levels of the A&bgr;-42 peptide in their serum (Scheuner, D., et al., 1996
Nat. Med.
2:864-870). It is known that mutations in the &bgr;APP, PS1 or PS2 gene, found in FAD patients, alter cleavage of the &bgr;APP protein to increase the relative amount of the AD-42 peptide (Tomita, T. et al., 1997
Proc. Natl. Acad. Sci. USA
94:2025-2030; Duff, K., et al., 1996
Nature
383:710-713; Borchelt, D., et al., 1996
Neuron
17:1005-1013; Citron, M., et al., 1997
Nat. Med.
3:67-72).
Point mutations of the &bgr;APP gene are linked to a relatively small number of FAD pedigrees such as &bgr;APP-London, &bgr;APP-Flemish, and &bgr;APP-Swedish (Goate, A. M., et al., 1991
Nature
349:704-706; Chartier-Harlin, M. -C., et al., 1991
Nature
353:844-846; Murrell, J., et al., 1991
Science
254:97-99; Karlinsky, H., et al., 1992
Neurology
42:1445-1453; Mullan, M., et al., 1992
Nature Genetics
1:345-347). Point mutations of the PS2 gene are also linked to a minority of FAD cases (Levy-Lahad, E., et al., 1995
Science
269:973-977; Rogaev, E. I., et al., 1995
Nature
376:775-778). The majority of FAD cases are cause
Pak Roger Hochoon
Roberts Susan B.
Bristol--Myers Squibb Company
Buchholz Briana
Kemmerer Elizabeth
Lamerdin John A.
Nichols Christopher James
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