Isolation of functionally active gamma-secretase protein complex and methods for detection of activity and inhibitors thereof

Abstract

The present invention provides an isolated, functionally-active protein that catalyzes cleavage of a gamma-secretase substrate. The functional activity of the isolated protein suggests that the isolated protein includes gamma-secretase. In one embodiment, the isolated gamma-secretase protein is associated with PS1. The present invention also relates to homogeneous methods for monitoring cleavage of β-amyloid precursor protein (βAPP) by gamma-secretase, wherein the steps of of isolating and retrieving cleavage products have been eliminated. Cleavage can be detected by binding a pair of fluorescent adducts to the gamma-cleaved βAPP fragment. Preferably, a first fluorescent adduct binds to the carboxy-terminal end of the gamma-cleaved βAPP fragment, with substantially no cross-reactivity to uncleaved βAPP or to other types of gamma-cleaved βAPP fragments, while a second fluorescent adduct binds to a portion within the amino-terminal region on the gamma-cleaved βAPP fragment. Detection of binding to the gamma-cleaved βAPP fragment is determined by monitoring the fluorescent energy transfer between the adducts.

What is claimed:  
     
         1 . A homogeneous method of detecting cleavage of β-amyloid precursor protein (βAPP) by gamma-secretase, said method comprising detecting binding of a gamma-cleaved βAPP fragment with a pair of fluorescent adducts, wherein a first fluorescent adduct binds specifically to the carboxy-terminal end of the gamma-cleaved βAPP fragment with substantially no cross-reactivity to uncleaved βAPP or to other types of gamma-cleaved βAPP fragments, and wherein a second fluorescent adduct binds to the gamma-cleaved βAPP fragment within an amino acid sequence which corresponds to amino acid sequence 1-31 of β-amyloid peptide (Aβ); and wherein excitation of one of the fluorescent adducts provides a detectable transfer of energy to the other fluorescent adduct.  
     
     
         2 . The method according to  claim 1 , wherein the method is practiced in a fluid sample in the presence of uncleaved βAPP and other types of gamma-cleaved βAPP fragments.  
     
     
         3 . The method according to  claim 2 , wherein the sample comprises membrane fractions having endogenous gamma-secretase and Swedish variant βAPP.  
     
     
         4 . The method according to  claim 2 , wherein the sample comprises solubilized gamma-secretase complex and βAPP.  
     
     
         5 . The method according to  claim 1 , wherein each of the fluorescent adducts separately modifies an antibody.  
     
     
         6 . The method according to  claim 5 , wherein the gamma-cleaved βAPP fragment is Aβ-40.  
     
     
         7 . The method according to  claim 6 , wherein the first fluorescent adduct modifies a first antibody which binds to Aβ-40 at an epitope comprising amino acid residue 40.  
     
     
         8 . The method according to  claim 7 , wherein the second fluorescent adduct modifies a second antibody which binds to Aβ at an epitope comprising amino acid sequence 1-12.  
     
     
         9 . The method according to  claim 1 , wherein excitation of the first fluorescent adduct provides a detectable transfer of energy to the second fluorescent adduct.  
     
     
         10 . The method according to  claim 9 , wherein the first adduct comprises a molecule selected from the group consisting of lanthanide cryptate or chelate, fluorescein, EDANS, salts of N-[6-amino-9-[2-carboxy-phenyl]-4,5-disulfoxy-3H-xanthen-3-ylidene]aminium ion (2-) and salts of 1-(epsilon-carboxypentyl-1′-ethyl-3,3,3′,3′-tetramethylindocarbocyanine-5,5′-disulfonate ion.  
     
     
         11 . The method according to  claim 10 , wherein the first fluorescent adduct comprises a europium cryptate.  
     
     
         12 . The method according to  claim 10 , wherein the second fluorescent adduct comprises xl-APC.  
     
     
         13 . The method according to  claim 12 , wherein the detectable transfer of energy comprises an amplified signal from the second fluorescent adduct.  
     
     
         14 . The method according to  claim 1 , wherein the other fluorescent adduct comprises a fluorescent quencher molecule.  
     
     
         15 . The method according to  claim 14 , wherein the fluorescent quencher molecule is selected from the group consisting of dabcyl and salts of 9-[2-[[4-carboxy-piperidin-1-yl]sulfonyl]phenyl]-6-(N-methyl-N-phenyl-amino)-3H-xanthen-3-ylidene]-N-methylbenzenaminium ion.  
     
     
         16 . The method according to  claim 15 , wherein each of the fluorescent adducts separately modifies an antibody.  
     
     
         17 . The method according to  claim 16 , wherein the detectable transfer of energy comprises a decrease in fluorescent signal from the fluorescent adduct which is excited.  
     
     
         18 . The method according to claims  13  or  17 , wherein excitation is by laser, xenon flash lamp or deuterium-tungsten lamp.  
     
     
         19 . The method according to  claim 18 , wherein excitation is by laser.  
     
     
         20 . A homogeneous method for determining the presence of β-amyloid peptide (Aβ), said method comprising 
 (1) exposing the sample to a pair of fluorescent adducts, wherein the first fluorescent adduct binds to the carboxy-terminal region of Aβ and the second fluorescent adduct binds to the amino-terminal region of Aβ and at least one fluorescent adduct is substantially free of cross-reactivity to uncleaved βAPP or to other types of gamma-cleaved βAPP fragments; and  
 (2) detecting binding of the pair of fluorescent adducts with Aβ by excitation of one of the fluorescent adducts.  
 
     
     
         21 . The method according to  claim 20 , wherein the first fluorescent adduct binds specifically to the carboxy-terminal end of Aβ with substantially no cross-reactivity to uncleaved βAPP or to other types of gamma-cleaved βAPP fragments.  
     
     
         22 . The method according to  claim 21 , wherein Aβ is Aβ-40.  
     
     
         23 . The method according to  claim 22 , wherein each of the fluorescent adducts separately binds specifically to either the amino- and carboxy-terminal ends of Aβ with substantially no cross-reactivity to uncleaved βAPP or to other types of gamma-cleaved βAPP fragments.  
     
     
         24 . The method according to  claim 21 , wherein excitation is by laser, xenon flash lamp or deuterium-tungsten lamp.  
     
     
         25 . The method according to  claim 24 , wherein excitation is by laser.  
     
     
         26 . A homogeneous method for determining the presence of β-amyloid peptide Aβ-40, said method comprising 
 (1) exposing the sample to a pair of fluorescent adducts, wherein the first fluorescent adduct binds to the carboxy-terminal end of Aβ-40 and the second fluorescent adduct binds to the amino-terminal region of Aβ-40 and the first fluorescent adduct is substantially free of cross-reactivity to uncleaved βAPP or to other types of gamma-cleaved βAPP fragments; and  
 (2) detecting binding of the pair of fluorescent adducts with Aβ-40 by excitation of the first fluorescent adduct.  
 
     
     
         27 . The method according to  claim 26 , wherein the first fluorescent adduct modifies a first antibody which binds to Aβ-40 at an epitope comprising amino acid residue 40.  
     
     
         28 . The method according to  claim 27 , wherein the first fluorescent adduct modifies a europium cryptate.  
     
     
         29 . The method according to  claim 28 , wherein the second fluorescent adduct modifies a second antibody which binds to Aβ-40 at an epitope comprising amino acid sequence 1-12.  
     
     
         30 . The method according to  claim 29 , wherein the second fluorescent adduct comprises xl-APC.  
     
     
         31 . The method according to  claim 30 , wherein the first fluorescent adduct is excited by laser.  
     
     
         32 . A homogeneous method of detecting cleavage of β-amyloid precursor protein (βAPP) by gamma-secretase, said method comprising detecting binding of a 6 kDa fragment with a pair of fluorescent adducts; wherein a first fluorescent adduct binds to the amino-terminal end of the 6 kDa fragment with substantially no cross-reactivity to uncleaved βAPP or to other types of gamma-cleaved βAPP fragments; and wherein a second fluorescent adduct binds to a portion within the carboxy-terminal region of the 6 kDa fragment; and wherein excitation of one of the fluorescent adducts provides a detectable transfer of energy to the other fluorescent adduct.  
     
     
         33 . The method according to  claim 32 , wherein each of the fluorescent adducts separately modifies an antibody.  
     
     
         34 . The method according to  claim 33 , wherein one of the fluorescent adducts comprises a molecule selected from the group consisting of lanthanide cryptate or chelate, fluorescein, EDANS, salts of N-[6-amino-9-[2-carboxy-phenyl]-4,5-disulfoxy-3H-xanthen-3-ylidene]aminium ion (2-) and salts of 1-(epsilon-carboxypentyl-1′-ethyl-3,3,3′,3′-tetramethylindocarbocyanine-5,5′-disulfonate ion.  
     
     
         35 . The method according to  claim 34 , wherein the other fluorescent adduct comprises a molecule selected from the group consisting of cross-linked allophycocyanins (“xl-APC”), coumarin, rhodamine, tetramethylrhodamine and salts of 1-(epsilon-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethylindodicarbocyanine-5,5′-disulfonate ion.  
     
     
         36 . The method according to  claim 34 , wherein the other fluorescent adduct comprises a fluorescent quencher molecule selected from the group consisting of dabcyl and salts of 9-[2-[[4-carboxy-piperidin-1-yl]sulfonyl]phenyl]-6-(N-methyl-N-phenyl-amino)-3H-xanthen-3-ylidene]-N-methylbenzenaminium ion.  
     
     
         37 . A homogeneous method of detecting cleavage of β-amyloid precursor protein (βAPP) by gamma-secretase, comprising the steps of 
 (1) binding a first fluorescent adduct to a 6 kDa fragment and a second fluorescent adduct to either a β-amyloid peptide (Aβ) or a p3 fragment, wherein at least one of the fluorescent adducts has substantially no cross-reactivity to other portions of uncleared βAPP, and wherein each fluorescent adduct separately comprises either a donor molecule or an acceptor molecule; and,  
 (2) exciting the donor molecule by laser, xenon flash lamp or deuterium-tungsten lamp; and  
 (3) detecting a substantially decreased transfer of energy to the acceptor molecule.  
 
     
     
         38 . The homogeneous method according to  claim 37 , wherein the donor molecule is selected from the group consisting of lanthanide cryptate or chelate, fluorescein, EDANS, salts of N-[6-amino-9-[2-carboxy-phenyl]-4,5-disulfoxy-3H-xanthen-3-ylidene]aminium ion (2-) and salts of 1-(epsilon-carboxypentyl-1′-ethyl-3,3,3′,3′-tetramethylindocarbocyanine-S, 5′-disulfonate ion.  
     
     
         39 . The method according to  claim 38 , wherein the acceptor molecule is selected from the group consisting of cross-linked allophycocyanins (“xl-APC”), coumarin, rhodamine, tetramethylrhodamine and salts of 1-(epsilon-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethylindodicarbocyanine-5,5′-disulfonate ion.  
     
     
         40 . The method according to  claim 39 , wherein the step of detecting a substantially decreased transfer of energy comprises detecting little or no amplified signal from the acceptor molecule.  
     
     
         41 . The method according to  claim 38 , wherein the acceptor molecule is a fluorescent quencher molecule selected from the group consisting of dabcyl and salts of 9-[2-[[4-carboxy-piperidin-1-yl]sulfonyl]phenyl]-6-(N-methyl-N-phenyl-amino)-3H-xanthen-3-ylidene]-N-methylbenzenaminium ion.  
     
     
         42 . The method according to  claim 41 , wherein the step of detecting a substantially decreased transfer of energy comprises detecting an unchanged fluorescent signal from the donor molecule.  
     
     
         43 . A homogeneous method of screening for inhibitors of gamma-secretase cleavage in β-amyloid precursor protein (βAPP), said method comprising the steps of 
 (1) adding a test compound to a sample comprising gamma-secretase and βAPP;  
 (2) then adding a pair of fluorescent adducts to the sample, wherein a first fluorescent adduct has binding specificity to the carboxy-terminal end of a gamma-cleaved βAPP fragment with substantially no cross-reactivity to uncleaved βAPP or to other types of gamma-cleaved βAPP fragments, and a second fluorescent adduct has binding specificity to the gamma-cleaved βAPP within an amino acid sequence corresponding to 1-31 of β-amyloid peptide (Aβ), and wherein each fluorescent adduct separately comprises either a donor molecule or an acceptor molecule; and  
 (3) detecting a substantially decreased transfer of fluorescent energy between the fluorescent adducts after excitation of the donor molecule.  
 
     
     
         44 . The method according to  claim 43 , wherein the donor molecule is selected from the group consisting of lanthanide cryptate or chelate, fluorescein, EDANS, salts of N-[6-amino-9-[2-carboxy-phenyl]-4,5-disulfoxy-3H-xanthen-3-ylidene]aminium ion (2-) and salts of 1-(epsilon-carboxypentyl-1′-ethyl-3,3,3′,3′-tetramethylindocarbocyanine-5,5′-disulfonate ion.  
     
     
         45 . The method according to  claim 44 , wherein the acceptor molecule is selected from the group consisting of cross-linked allophycocyanins (“xl-APC”), coumarin, rhodamine, tetramethylrhodamine and salts of 1-(epsilon-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethylindodicarbocyanine-5,5′-disulfonate ion.  
     
     
         46 . The method according to  claim 45 , wherein the step of detecting a substantially decreased transfer of energy comprises detecting little or no amplified signal from the acceptor molecule.  
     
     
         47 . The method according to  claim 44 , wherein the acceptor molecule is a fluorescent quencher molecule selected from the group consisting of dabcyl and salts of 9-[2-[[4-carboxy-piperidin-1-yl]sulfonyl]phenyl]-6-(N-methyl-N-phenyl-amino)-3H-xanthen-3-ylidene]-N-methylbenzenaminium ion.  
     
     
         48 . The method according to  claim 47 , wherein the step of detecting a substantially decreased transfer of energy comprises detecting an unchanged fluorescent signal from the donor molecule.  
     
     
         49 . A homogeneous method of screening for inhibitors of gamma-secretase cleavage in β-amyloid precursor protein (βAPP), said method comprising the steps of 
 (1) adding a test compound to a sample comprising gamma-secretase and βAPP;  
 2) then binding a pair of fluorescent adducts to uncleaved βAPP; wherein a first fluorescent adduct binds to a portion within amino acid sequence 722-770 of uncleaved βAPP, a second fluorescent adduct binds to a portion within amino acid sequence 671-702 of uncleaved βAPP, and at least one of the fluorescent adducts has substantially no cross-reactivity to other portions of uncleaved βAPP, and wherein each fluorescent adduct separately comprises either a donor molecule or an acceptor molecule; and,  
 (3) detecting a transfer of energy between the fluorescent adducts after excitation of the donor molecule.  
 
     
     
         50 . The method according to  claim 49 , wherein the donor molecule is selected from the group consisting of lanthanide cryptate or chelate, fluorescein, EDANS, salts of N-[6-amino-9-[2-carboxy-phenyl]-4,5-disulfoxy-3H-xanthen-3-ylidene]aminium ion (2-) and salts of 1-(epsilon-carboxypentyl-1′-ethyl-3,3,3′,3′-tetramethylindocarbocyanine-5,5′-disulfonate ion.  
     
     
         51 . The method according to  claim 50 , wherein the acceptor molecule is selected from the group consisting of cross-linked allophycocyanins (“xl-APC”), coumarin, rhodamine, tetramethylrhodamine and salts of 1-(epsilon-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethylindodicarbocyanine-5,5′-disulfonate ion.  
     
     
         52 . The method according to  claim 51 , wherein the step of detecting a transfer of energy comprises detecting an amplified signal from the acceptor molecule.  
     
     
         53 . The method according to  claim 52 , wherein the acceptor molecule is a fluorescent quencher molecule selected from the group consisting of dabcyl and salts of 9-[2-[[4-carboxy-piperidin-1-yl]sulfonyl]phenyl]-6-(N-methyl-N-phenyl-amino)-3H-xanthen-3-ylidene]-N-methylbenzenaminium ion.  
     
     
         54 . The method according to  claim 53 , wherein the step of detecting a transfer of energy comprises detecting a decrease of fluorescent signal from the donor molecule.  
     
     
         55 . An isolated protein having gamma-secretase activity.  
     
     
         56 . An isolated protein comprising gamma-secretase.  
     
     
         57 . The isolated protein of  claim 56 , wherein the gamma-secretase recognizes and cleaves a substrate having a gamma secretase cleavage site.  
     
     
         58 . The isolated protein of  claim 57 , wherein cleavage of the substrate by the gamma secretase at the gamma-secretase cleavage site generates a β-amyloid peptide (Aβ) and a 6 kDa fragment.  
     
     
         59 . The isolated protein of  claim 56  which is a protein complex comprising gamma secretase and PS1.  
     
     
         60 . A membrane fragment comprising gamma-secretase.  
     
     
         61 . A method for isolating gamma-secretase from a sample by isolating gamma secretase complexed with PS1.  
     
     
         62 . The method of  claim 61 , wherein isolating gamma-secretase complexed with PS1 comprises contacting the sample with an agent that recognizes and binds PS1 so that an agent/PS1/gamma secretase complex forms thereby isolating the molecule having gamma-secretase activity.  
     
     
         63 . A molecule having gamma-secretase activity isolated by the method of  claim 61 .  
     
     
         64 . The method of  claim 62 , wherein the agent that recognizes and binds PS1 comprises an anti-PS1 antibody.  
     
     
         65 . A method for isolating a protein complex having gamma-secretase activity from a sample, comprising: 
 a) contacting the sample with a molecule that recognizes and binds PS1 so that a molecule/PS1 complex forms; and    b) removing the molecule/PS1 complex from the sample, thereby isolating the protein complex having gamma secretase activity.    
     
     
         66 . A protein complex having gamma-secretase activity isolated by the method of  claim 65 .  
     
     
         67 . The method of  claim 65 , wherein the molecule that recognizes and binds PS1 comprises an anti-PS1 antibody.  
     
     
         68 . The method of  claim 65 , wherein the protein complex comprises gamma secretase and PS1.  
     
     
         69 . A protein complex isolated by the method of  claim 65 .  
     
     
         70 . A method for isolating a protein complex comprising gamma secretase and PS1, comprising: 
 a. solubilizing a gamma-secretase positive cell thereby resulting in a mixture of a protein complex comprising gamma-secretase and PS1 and other cell components; and    b. contacting the mixture with a molecule that recognizes and binds PS1 so that a molecule/PS1 complex forms; and    c. removing the complex from the other cell components thereby isolating a protein complex comprising gamma secretase and PS1.    
     
     
         71 . A protein complex comprising gamma secretase and Ps1 isolated by the method of  claim 70 .  
     
     
         72 . The method of  claim 70 , wherein the molecule that recognizes and binds PS1 is an anti-PS1 antibody.  
     
     
         73 . The method of  claim 70 , wherein in step (a) the gamma-secretase positive cell is solubilized in a solution comprising N-[3[(dimethylamino)propyl]3,7,12-trihydroxy(3a, 5b, 7a, 12a)cholan-2-amide].  
     
     
         74 . An isolated functionally-active substrate which is cleaved by gamma-secretase.  
     
     
         75 . The functionally-active substrate of  claim 74  comprising βAPP.  
     
     
         76 . A method for cleaving a functionally-active substrate comprising incubating the functionally-active substrate with a molecule having gamma-secretase activity under conditions so that the molecule having gamma-secretase activity cleaves the functionally-active substrate thereby producing cleavage products.  
     
     
         77 . A method for detecting gamma-secretase activity in a molecule of interest by determining whether the molecule can cleave a substrate in accordance with the method of  claim 76 .  
     
     
         78 . The method of  claim 76 , wherein the functionally-active substrate comprising βAPP.  
     
     
         79 . The method of  claim 76 , wherein the functionally-active substrate and the molecule having gamma-secretase activity are incubated in a solution comprising N-[3[(dimethylamino)propyl]3,7,12-trihydroxy(3a, 5b, 7a, 12a)cholan-2-amide].  
     
     
         80 . A method for isolating a functionally-active substrate, comprising: 
 a) generating a substrate comprising a gamma-secretase cleavage sequence;    b) inserting the substrate into a microsomal membrane fragment to generate a functionally-active substrate; and    c) isolating the microsomal membrane fragment which includes the functionally-active substrate.    
     
     
         81 . A functionally-active substrate generated by the method of  claim 80 .  
     
     
         82 . The method of  claim 80 , wherein the substrate comprises βAPP.  
     
     
         83 . The method of  claim 80 , wherein the substrate comprises the amino acid sequence as described in SEQ ID NO.: 2 or 4.  
     
     
         84 . The method of  claim 80 , wherein the functionally-active substrate includes a detectable label.  
     
     
         85 . The method of  claim 80 , wherein the functionally-active substrate is solubilized from the microsomal membrane fragment with a solution comprising N-[3[(dimethylamino)propyl]3,7,12-trihydroxy(3a, 5b, 7a, 12a)cholan-2-amide].  
     
     
         86 . The method of  claim 80  further comprising: 
 a) solubilizing the functionally-active substrate from the microsomal membrane fragment; and  
 b) isolating the functionally-active substrate.  
 
     
     
         87 . A method for identifying an agent of interest that inhibits gamma-secretase activity in a sample comprising: 
 a) contacting the sample and the agent of interest with a functionally-active substrate; and    b) detecting whether a cleavage product of the functionally-active substrate is generated in the sample, the lack of the cleavage product in the sample being indicative that the agent inhibits gamma-secretase activity in the sample.    
     
     
         88 . The method of  claim 87 , wherein the cleavage product is detected with an antibody that recognizes and binds to the N-terminal end of the cleavage product.  
     
     
         89 . The method of  claim 87 , wherein the cleavage product is detected with an antibody that recognizes and binds to the C-terminal end of the cleavage product.  
     
     
         90 . The method of  claim 87 , wherein the cleavage product is detected with a pair of fluorescent adducts wherein a first fluorescent adduct binds to the N-terminal end of the cleavage product and a second fluorescent adduct binds to the C-terminal end of the cleavage product, and wherein excitation of one of the fluorescent adducts provides a detectable transfer of energy to the other fluorescent adduct.  
     
     
         91 . The method according to  claim 87  which comprises contacting a plurality of substantially identical samples each separately with a different agent of interest.  
     
     
         92 . The method of  claim 87 , wherein the plurality of samples comprises more than about 10 4  samples.  
     
     
         93 . The method of  claim 87 , wherein the plurality of samples comprises more than about 10 5  samples.  
     
     
         94 . The method of  claim 87 , wherein the plurality of samples comprises more than about 10 6  samples.  
     
     
         95 . The method of  claim 87 , wherein the plurality of substantially identical samples are each contacted essentially simultaneously with a different agent of interest.  
     
     
         96 . A method for isolating an integral membrane protein or protein complex comprising: 
 a) solubilizing a cell with a solution comprising N-[3[(dimethylamino)propyl]3,7,12-trihydroxy(3a, 5b, 7a, 12a)cholan-2-amide] thereby obtaining a mixture having the integral membrane protein or protein complex and other cell components; and    b) isolating the integral membrane protein or protein complex.    
     
     
         97 . An integral membrane protein or protein complex isolated by the method of  claim 96.