Abstract:
Presented herein are polypeptide substrates based on Notch polypeptides, assay methods based on the use of these substrates, and screening methods directed toward identifying inhibitors of γ-secretase activity. The assay methods and the screening methods are adapted for use in high throughput multi-well plate assay apparatuses. In many embodiments the substrate polypeptides are labeled for ease of detection, and/or may bind specific ligands that themselves are labeled. Generally the labels promote high specificity as well as high sensitivity of detection. These features render the assay and screening methods that employ the labeled substrates especially suited for use in high throughput assay formats.

Description:
This application is the §371 U.S. National Stage of International Application No. PCT/US2011/050688, filed 7 Sep. 2011, which claims the benefit of U.S. Provisional Application Ser. No. 61/380,587, filed Sep. 7, 2010, each of which are incorporated by reference herein. 
    
    
     FIELD OF THE INVENTION 
     The present invention includes, but is not limited to, compositions and methods for measuring gamma-secretase (“γ-secretase”) activity and assays for identifying modulators of γ-secretase activity. For instance, this disclosure describes newly designed substrates derived from the Notch cell surface receptor for use in high sensitivity assays for γ-secretase activity. In addition, the γ-secretase substrates identified herein are useful in assays to identify modulators of γ-secretase activity. 
     BACKGROUND OF THE INVENTION 
     γ-Secretase processes a variety of substrates including the amyloid precursor protein (APP) and Notch proteins. γ-Secretase cleaves APP to release Aβ peptides, which are widely considered to play a causative role in Alzheimer disease (AD). 
     The Notch signaling pathway is a highly conserved cell signaling system present in most multicellular organisms (Artavanis-Tsakonas et al., 1999, Science 284 (5415): 770-776). Notch protein family members are present in all metazoans, and mammals possess four different Notch receptors, referred to as NOTCH1, NOTCH2, NOTCH3, and NOTCH4. The Notch receptor is a single-pass transmembrane receptor protein. The ligands that bind Notch proteins and initiate signaling are bound to neighboring cells. Notch is a hetero-oligomer composed of a large extracellular portion, which associates in a calcium-dependent, non-covalent interaction with a smaller piece of the Notch protein composed of a short extracellular region, a single transmembrane-pass, and a small intracellular region (Brou et al., 2000, Mol. Cell. 5 (2): 207-16). 
     In addition to acting on APP, γ-secretase processes Notch and other type I membrane proteins. Notch is also cleaved by multiple proteases. Notch signaling is controlled by ligand binding, which exposes a negative control region that is susceptible to proteolytic cleavage of the receptor. The GenBank Accession Number for human Notch1 cDNA is AF&#39;308602.1, and for the corresponding polypeptide gene product it is AAG33848.1. Human Notch1 has 2556 amino acid residues. In processing Notch1, first, a membrane-proximal cleavage is executed at site S2 (between residues Ala1710 and Val1711 (see Supplementary Figure S1A of van Tetering et al., 2009, J. Biol. Chem., 284:31018-31027) by an ADAM (A Disintegrin And Metalloprotease) metalloprotease, removing the extracellular domain to generate the membrane-anchored Notch extracellular truncation fragment (NEXT). (The ADAM family includes proteins containing disintegrin-like and metalloprotease-like domains. ADAMs are involved in diverse cellular processes such as development, cell-cell interactions and protein ectodomain.) NEXT is subsequently cleaved by γ-secretase at site S3 (see Supplementary Figure S1A of van Tetering et al., 2009, J. Biol. Chem., 284:31018-31027) to generate Notch Intracellular Domain (NICD) which translocates into the nucleus where it modulates expression of target genes that are involved in cell fate decisions during embryogenesis, hematopoeisis, and stem cell differentiation (Chan et al., 1998, Cell, 94, 423-426, Berezovska et al., 1999, Brain Res Mol Brain Res., 69:273-280, and Redmond et al., 2000, Nat. Neurosci., 3:30-40). 
     γ-Secretase is a membrane-bound aspartyl protease consisting of at least four subunits, which are Presenilin (PS, either PSlor PS2), Aph-1, Nicastrin (Nct) and Pen-2. PS is believed to be the catalytic subunit of γ-secretase and mutations in PS1 and PS2 have been linked to Familial Alzheimer&#39;s Disease (FAD) (Sherrington et al., 1995, Nature, 375:754-760, Levy Lahad et al., 1995, Science, 269:973-977). Although the precise pathological mechanism of these FAD mutations is unknown, it has been postulated that they alter the specificity of γ-secretase and lead to an increase in the ratio of Aβ42 to Aβ40 peptide (Borchelt et al., 1996, Neuron, 17:1005-1013, Duff et al., 1996, Nature, 383:710-713, and Bentahir et al., 2006, J. Neurochem., 96:732-742) Aβ42 is more prone to form insoluble aggregates than is Aβ40, so that FAD mutations are deleterious. In addition, FAD mutations also reduce γ-secretase activity for Notch and E-cadherin processing (Song et al., 1999, Proc. Nat. Acad. Sci. USA, 96:6959-6963, Marambaud et al., 2002, EMBO J., 21:1948-1956, and Marambaud et al., 2003, Cell, 114:635-645). Several studies have shown that FAD mutations of PS1 affect Notch cleavage (Bentahir et al., 2006, J. Neurochem., 96:732-742), Song et al., 1999, Proc. Nat. Acad. Sci. USA, 96:6959-6963, Nakajima et al., 2000, J Neurosci Res., 62:311-317, Amtul et al., 2002, Neurobiol Dis., 9:269-273, Chen et al., 2002, J. Biol. Chem., 277:36521-36526). 
     It is presently unclear whether the differential cellular effects of PS1 mutations on Notch1 cleavage are attributed to cellular factors such as the maturation of the γ-secretase complex, trafficking of substrates or modulation of γ-secretase itself. Although a cell-free Notch/γ-secretase assay has been reported using N100Flag substrate (Moehlmann et al., 2002, Proc. Nat. Acad. Sci. USA, 99:8025-8030), it is a western analysis-based assay that is limited in its application for the characterization of γ-secretase since it is labor-intensive, has low throughput and is not quantitative. Moreover, it has been questioned whether N100Flag is processed by γ-secretase (Keller et al., 2006, Biochem., 45:5351-5358). 
     SUMMARY OF THE INVENTION 
     There is a need to determine whether PS1 mutations might directly affect the catalytic activity of γ-secretase. In order to monitor γ-secretase activity for Notch1 cleavage and develop Notch specific γ-secretase inhibitors, there is a need for a robust, specific and sensitive in vitro γ-secretase assay using a substrate based on a Notch protein, such as Notch1. There is clearly a need for reliable Notch-based substrate that is susceptible to facile proteolysis by γ-secretase. There is a need to develop an assay that specifically determines the occurrence and amount of the product of proteolysis of Notch by γ-secretase with high sensitivity. In addition there remains a need to develop an assay of γ-secretase activity on Notch in real time. There remains in addition a need for an efficient assay that can screen candidate compounds for their ability to modulate the activity of PS1 on Notch proteins. The instant disclosure presents a quick and easy in vitro assay of the γ-secretase catalyzed cleavage of Notch1. This novel in vitro γ-secretase assay that detects γ-secretase cleaved Notch1 product will be a valuable means for better characterization of γ-secretase and for identification of modulators of the activity of γ-secretase on Notch receptor family members. 
     In a first aspect this disclosure presents a reagent that specifically binds a product of the cleavage of a Notch receptor protein that is catalyzed by a γ-secretase at the S3 site. In many embodiments the reagent does not bind a Notch receptor protein that has not been cleaved by a γ-secretase. In additional embodiments the reagent includes an antibody. In further embodiments the antibody is a polyclonal antibody. In additional embodiments the antibody is a monoclonal antibody. In further embodiments the antibody is a single chain antibody. In further embodiments the antibody specifically binds VLLSRKRRR (SEQ ID NO:2). In still further embodiments the antibody is elicited by immunizing a host animal with a composition including a peptide containing the sequence VLLSRKRRR (SEQ ID NO:2). Optionally, the sequence VLLSRKRRR (SEQ ID NO:2) may further include a cysteine residue at the carboxy terminal end. 
     In a second aspect this disclosure presents a substrate peptide including at least a portion of a Notch protein wherein the substrate bears a first label, and wherein the amino acid sequence of the substrate is cleavable by the activity of γ-secretase to provide a detectable product peptide that includes the first label. In certain embodiments the substrate peptide includes a Notch substrate, such as, but not limited to, amino acid residues 1733-1812 of human Notch1 protein, which includes a first scissile peptide bond cleavable by γ-secretase. In many embodiments the first scissile peptide bond is the S3 site. In some embodiments the substrate peptide further includes a tag linked to the labeled Notch amino acid sequence or portion thereof wherein the tag specifically binds a target with high affinity; in many embodiments the target is a polysaccharide containing maltose. In still further embodiments the tag is linked to the substrate peptide via a linking amino acid sequence that includes a second scissile peptide bond, wherein the linking amino acid sequence constitutes a target for a specific protease whose action results in removal of the tag from the substrate peptide. In certain embodiments the specific protease is thrombin. In still further embodiments the first label contains a labeling amino acid sequence reactable to attach a first member of a specific binding pair to the labeling amino acid sequence. In still additional embodiments the first member of the specific binding pair includes biotin. (See  FIGS. 1A and 5A ). 
     In particular embodiments the substrate peptide includes, in order from the N-terminus to the C-terminus, a tag that specifically binds a polysaccharide containing maltose, e.g., maltose binding protein, a thrombin target sequence, a Notch1 sequence that includes the first scissile bond corresponding to the S3 site of Notch1 protein, and a labeling amino acid sequence reactable to bind biotin; in certain embodiments the label includes biotin so incorporated. In still a further particular embodiment the substrate peptide includes, in order from the N-terminus to the C-terminus, a labeling amino acid sequence reactable to bind biotin, a Notch1 sequence that includes the first scissile bond, a thrombin target sequence, and the tag that specifically binds a polysaccharide containing maltose. 
     In still a further aspect, this disclosure presents a polynucleotide susceptible of expression of a substrate peptide described in the preceding two paragraphs, when incorporated in a suitable host cell and incubated under appropriate culture conditions. 
     In yet a further aspect, this disclosure includes a method of synthesizing a peptide substrate wherein the polynucleotide described in the preceding paragraph is introduced into a suitable host cell, the host cell is incubated under appropriate culture conditions for a time sufficient to express the peptide substrate, and the peptide substrate is isolated from the cultured cells. In a further aspect, this disclosure presents polypeptides that include a gamma-secretase cleavage site, and is cleaved by a gamma-secretase under suitable conditions. Such polypeptides include a truncated Notch polypeptide that has fewer amino acids than a full length natural Notch cell surface receptor. Such a polypeptide may be referred to herein as a Notch substrate. 
     In a further aspect this disclosure presents a high sensitivity method of assaying the activity of γ-secretase that includes the steps of:
         a) providing composition suspected of containing γ-secretase activity;   b) contacting the composition with a polypeptide substrate for γ-secretase including at least a portion of a Notch protein bound to a detectable label, wherein cleavage of the labeled substrate by γ-secretase provides a detectably labeled product;   c) contacting the labeled product with
           1) a first ligand bearing a first tag wherein the first ligand specifically binds the label, and   2) a second ligand bearing a second tag wherein the second ligand specifically binds the product; and   
           d) determining the presence and/or the amount of the labeled product bound to the first ligand and to the second ligand.
 
In certain embodiments of the method the peptide substrate includes amino acid residues 1733-1812 of human Notch1. In additional embodiments of the assay the detectable label includes biotin. In additional embodiments the first ligand includes an avidin and the first tag contains a detectable first fluorophore, and in further embodiments the second ligand includes a first antibody that a) specifically binds a newly generated chain terminus of the product and that b) is bound to a second antibody bearing a second fluorophore such that fluorescence resonance energy transfer occurs between the first fluorophore and the second fluorophore. In alternative embodiments the first ligand includes an avidin and a photosensitizer that converts triplet oxygen to singlet oxygen, and the second ligand includes a) a first antibody that specifically binds a newly generated chain terminus of the product, b) a second antibody that binds the first antibody, and c) a luminescence emitter excited by a singlet oxygen-dependent chemiluminescence reaction.
       

     In an additional aspect the disclosure presents a high throughput method of assaying the activity of γ-secretase that includes the steps of:
         a) providing a plurality of containers, each container containing a composition suspected of containing γ-secretase activity;   b) adding to each container a polypeptide substrate for γ-secretase including at least a portion of a Notch protein bound to a detectable label, wherein cleavage of the labeled substrate by γ-secretase provides a detectably labeled product to each container;   c) contacting the labeled product with
           1) a first ligand bearing a first tag wherein the first ligand specifically binds the label, and   2) a second ligand bearing a second tag wherein the second ligand specifically binds the product; and   
           d) determining the presence and/or the amount of the labeled product bound to the first ligand and to the second ligand.
 
In various embodiments of this method each container is a well in a multi-well assay plate. In various additional embodiments, a plate contains at least 96 wells, or at least 384 wells, or at least 1536 wells. In certain embodiments of the method the peptide substrate includes amino acid residues 1733-1812 of human Notch1. In additional embodiments of the assay the detectable label includes biotin. In additional embodiments the first ligand includes an avidin and the first tag contains a detectable first fluorophore, and in further embodiments the second ligand includes a first antibody that a) specifically binds a newly generated chain terminus of the product and that b) is bound to a second antibody bearing a second fluorophore such that fluorescence resonance energy transfer occurs between the first fluorophore and the second fluorophore. In alternative embodiments the first ligand includes an avidin and a photosensitizer that converts triplet oxygen to singlet oxygen, and the second ligand includes a) a first antibody that specifically binds a newly generated chain terminus of the product, b) a second antibody that binds the first antibody, and c) a luminescence emitter excited by a singlet oxygen-dependent chemiluminescence reaction.
       

     In an additional aspect a method is disclosed of assaying the activity of γ-secretase in a cell that includes the steps of
         a) providing a cell suspected of containing γ-secretase activity;   b) adding media containing a polypeptide substrate for γ-secretase including at least a portion of a Notch protein bound to a detectable label, wherein cleavage of the labeled substrate by γ-secretase provides a labeled product; and   c) determining the presence of labeled product.
 
In certain embodiments, the determining includes
   c) separating the cells after a suitable incubation period to provide a supernatant; and   d) assaying the supernatant for the labeled product.
 
In certain embodiments assaying the supernatant is carried out by SDS-PAGE and subsequent immunoblotting with an antibody that specifically binds a newly generated chain terminus of the product to identify the presence of the product. In alternative embodiments the determining includes fixing the cell and conducting immunohistochemical identification of the product in the intact cell with an antibody that specifically binds a newly generated chain terminus of the product. In this method the media may further include a detergent. Additionally, in many embodiments the label includes biotin. In still other embodiments assaying for the labeled product includes assaying for a detectable complex containing the product and one or more detectable probes, for example the complex includes a first specific binding member that contains a first detectable probe, wherein the first specific binding member specifically binds the product to form a binary complex. In specific embodiments the first probe includes ruthenium such that the detection is conducted using electrochemiluminescence. In additional embodiments of the assaying further includes
   c) separating the cells after a suitable incubation period to provide a supernatant;   d) contacting the labeled product with
           1) a first ligand bearing a first tag wherein the first ligand specifically binds the label, and   2) a second ligand bearing a second tag wherein the second ligand specifically binds the product; and   
           e) determining the presence and/or the amount of the labeled product bound to the first ligand and to the second ligand.
 
In the latter embodiments, the first ligand includes an avidin and the first tag contains a detectable first fluorophore, and in further embodiments the second ligand includes a first antibody that a) specifically binds a newly generated chain terminus of the product and that b) is bound to a second antibody bearing a second fluorophore such that fluorescence resonance energy transfer occurs between the first fluorophore and the second fluorophore. In still further alternative embodiments the first ligand includes an avidin and a photosensitizer that converts triplet oxygen to singlet oxygen, and the second ligand includes a) a first antibody that specifically binds a newly generated chain terminus of the product, b) a second antibody that binds the first antibody, and c) a luminescence emitter excited by a singlet oxygen-dependent chemiluminescence reaction.
       

     In an additional aspect this disclosure presents a method of screening for a modulator of γ-secretase activity including the steps of:
         a) providing a container containing a composition that includes γ-secretase activity;   b) contacting the composition with a mixture including a candidate compound and a polypeptide substrate for γ-secretase including at least a portion of a Notch protein bound to a detectable label, wherein cleavage of the labeled substrate by γ-secretase provides a labeled product that is detectable; and   c) determining whether the candidate compound modulates formation of the labeled product of the γ-secretase-catalyzed cleavage of the substrate.
 
In certain embodiments of the method the peptide substrate includes amino acid residues 1733-1812 of human Notch1. In many embodiments of the method the label includes biotin. In other embodiments the determining the formation of the labeled product includes assaying for a detectable complex including the product and one or more detectable probes. In certain embodiments the complex contains a first specific binding member that includes a first detectable probe, wherein the first specific binding member specifically binds the product to form a binary complex, and in certain embodiments the first probe includes ruthenium such that the detection is conducted using electrochemiluminescence.
       

     In additional embodiments of the method the determining further includes
         d) contacting the labeled product with
           1) a first ligand bearing a first tag wherein the first ligand specifically binds the label, and   2) a second ligand bearing a second tag wherein the second ligand specifically binds the product; and   
           e) determining the presence and/or the amount of the labeled product bound to the first ligand and to the second ligand.
 
In certain embodiments of this variation the first ligand includes an avidin and the first tag contains a detectable first fluorophore, and in further embodiments the second ligand includes a first antibody that a) specifically binds a newly generated chain terminus of the product and that b) is bound to a second antibody bearing a second fluorophore such that fluorescence resonance energy transfer occurs between the first fluorophore and the second fluorophore. In alternative embodiments the first ligand includes an avidin and a photosensitizer that converts triplet oxygen to singlet oxygen, and the second ligand includes a) a first antibody that specifically binds a newly generated chain terminus of the product, b) a second antibody that binds the first antibody, and c) a luminescence emitter excited by a singlet oxygen-dependent chemiluminescence reaction.
       

     In an additional aspect the disclosure provides a high throughput method of screening for a modulator of γ-secretase activity including the steps of
         a) providing a plurality of containers, each container containing a composition containing γ-secretase activity;   b) adding to each container a composition including a candidate compound and a polypeptide substrate for γ-secretase including at least a portion of a Notch protein bound to a detectable label, wherein cleavage of the labeled substrate by γ-secretase provides a labeled product that is detectable with high sensitivity; and   c) determining whether the candidate compound modulates formation of the labeled product of the γ-secretase-catalyzed cleavage of the substrate.
 
In various embodiments of this screening method each container is a well in a multi-well assay plate; and in particular embodiments of the screen a plate contains at least 96 wells, or at least 384 wells or at least 1536 wells.
       

     In certain embodiments of the method the peptide substrate includes amino acid residues 1733-1812 of human Notch1. In many embodiments of the method the label includes biotin. In other embodiments the determining the formation of the labeled product includes assaying for a detectable complex including the product and one or more detectable probes. In certain embodiments the complex contains a first specific binding member that includes a first detectable probe, wherein the first specific binding member specifically binds the product to form a binary complex, and in certain embodiments the first probe includes ruthenium such that the detection is conducted using electrochemiluminescence. 
     In additional embodiments of the high throughput method of screening the determining further includes
         c) contacting the labeled product with
           1) a first ligand bearing a first tag wherein the first ligand specifically binds the label, and   2) a second ligand bearing a second tag wherein the second ligand specifically binds the product; and   
           d) determining whether the candidate compound modulates the presence and/or the amount of the labeled product bound to the first ligand and to the second ligand.
 
In certain embodiments of this variation the first ligand includes an avidin and the first tag contains a detectable first fluorophore, and in further embodiments the second ligand includes a first antibody that a) specifically binds a newly generated chain terminus of the product and that b) is bound to a second antibody bearing a second fluorophore such that fluorescence resonance energy transfer occurs between the first fluorophore and the second fluorophore. In alternative embodiments the first ligand includes an avidin and a photosensitizer that converts triplet oxygen to singlet oxygen, and the second ligand includes a) a first antibody that specifically binds a newly generated chain terminus of the product, b) a second antibody that binds the first antibody, and c) a luminescence emitter excited by a singlet oxygen-dependent chemiluminescence reaction.
       

     Still a further aspect presented in this disclosure is a method of screening for a modulator of γ-secretase activity in a cell including the steps of
         a) providing a container that contains a cell containing γ-secretase activity;   b) adding to the container media containing a candidate compound and a polypeptide substrate for γ-secretase including at least a portion of a Notch protein bound to a detectable label, wherein cleavage of the labeled substrate by γ-secretase provides a labeled product that is detectable with high sensitivity; and   c) determining whether the candidate compound modulates formation of the labeled product.
 
In certain embodiments, the determining includes
   c) separating the cells after a suitable incubation period to provide a supernatant; and   d) assaying the supernatant to determine whether the candidate compound modulates formation of the labeled product.
 
In certain embodiments assaying the supernatant is carried out by SDS-PAGE and subsequent immunoblotting with an antibody that specifically binds a newly generated chain terminus of the product to identify the presence of the product. In alternative embodiments the determining includes fixing the cell and conducting immunohistochemical identification of the product in the intact cell with an antibody that specifically binds a newly generated chain terminus of the product. In this method the media may further include a detergent. In certain embodiments of the method the peptide substrate includes amino acid residues 1733-1812 of human Notch1. Additionally, in many embodiments the label includes biotin. In still other embodiments assaying for the labeled product includes assaying for a detectable complex containing the product and one or more detectable probes, for example the complex includes a first specific binding member that contains a first detectable probe, wherein the first specific binding member specifically binds the product to form a binary complex. In specific embodiments the first probe includes ruthenium such that the detection is conducted using electrochemiluminescence. In additional embodiments of the assaying further includes
   c) separating the cells after a suitable incubation period to provide a supernatant;   d) contacting the labeled product with
           1) a first ligand bearing a first tag wherein the first ligand specifically binds the label, and   2) a second ligand bearing a second tag wherein the second ligand specifically binds the product; and   
           e) determining whether the candidate compound modulates the presence and/or the amount of the labeled product bound to the first ligand and to the second ligand.
 
In the latter embodiments, the first ligand includes an avidin and the first tag contains a detectable first fluorophore, and in further embodiments the second ligand includes a first antibody that a) specifically binds a newly generated chain terminus of the product and that b) is bound to a second antibody bearing a second fluorophore such that fluorescence resonance energy transfer occurs between the first fluorophore and the second fluorophore. In still further alternative embodiments the first ligand includes an avidin and a photosensitizer that converts triplet oxygen to singlet oxygen, and the second ligand includes a) a first antibody that specifically binds a newly generated chain terminus of the product, b) a second antibody that binds the first antibody, and c) a luminescence emitter excited by a singlet oxygen-dependent chemiluminescence reaction.
       

     In still an additional aspect the disclosure presents a high throughput method of screening for a modulator of γ-secretase activity in a cell that includes the steps of:
         a) providing a plurality of containers, each container containing a cell containing γ-secretase activity;   b) to each container adding media including a candidate compound and a polypeptide substrate for γ-secretase including at least a portion of a Notch protein bound to a detectable label, wherein cleavage of the labeled substrate by γ-secretase provides a labeled product that is detectable with high sensitivity; and   c) determining in each container whether the candidate compound modulates formation of the labeled product of the γ-secretase-catalyzed cleavage of the substrate.
 
In various embodiments of this screening method each container is a well in a multi-well assay plate; in certain embodiments a plate contains at least 96 wells, or at least 384 wells, or at least 1536 wells.
       

    
    
     
       BRIEF DESCRIPTION OF THE DRAWING 
         FIG. 1  Production of biotinylated recombinant N1-Sb1 substrate using plasmid pIAD16-MBP-N-1-Avi. A) Schematic representation of protein expression and purification. 
       B) Coomassie staining of the product. C) LC-MS analysis of the purified product. 
         FIG. 2  Plasmid map of pIAD16 with N1-Sb1 insert. 
         FIG. 3  Nucleotide sequence (SEQ ID NO:4) of MBP-N-1-Sb1 fusion. 
         FIG. 4 . Plasmid sequence (SEQ ID NO:5) of pIAD16 with N1-Sb1 insert. 
         FIG. 5 . In vitro N1-Sb1 γ-secretase assay. A. Schematic representation of in vitro γ-secretase assay and detection using the Amplified Luminescent Proximity Homogeneous Assay. B. Inhibitory potency of L685458 and compound E on γ-secretase activity. C. Kinetic analysis of γ-secretase using N1-Sb1 substrate. 
         FIG. 6  The SM320 antibody specifically recognizes the γ-secretase cleaved Notch product in Western analysis (A) and immunostaining. (B) The ΔE-Notch1 (a Notch construct with deletion of the extracellular portion) or the control vector was transfected into HEK293 cells in the absence and the presence of γ-secretase inhibitor (L-685,458). The cell lysates were analyzed by Western blot with anti-MYC and SM320 antibody (A). The ΔE-Notch1 transfected cells were fixed, permeablized and stained with DAPI (Deep Blue shapes are visible), anti-Myc/anti mouse-Alexa-fluor 594 (red) and SM321/anti-rabbit-Alexa-fluor 488 (green). B. Panel 4. Merged images (Panels 1-3) in the absence of L-685458. Bright yellow to yello-orange spots are seen in the center, on a background of deep blue shapes. Panel 5. SM320 staining in the presence of L-685458. No spots of any color are observed. Panel 6. Merged images for the L-685,458 treated cells. (No green color was detected. Deep red blotches interspersed with black are visible, on a background of faint deep blue shapes.) 
         FIG. 7 . γ-Secretase activity of various PS1 species. A) N1-Sb1 substrate. B) Sb4 substrate. C) N1-Sb1 substrate. D) Sb4 substrate. 
         FIG. 8 . γ-Secretase activity of various PS1 species. A) Signal intensity for various PS1 species as a function of substrate concentration. B) Signal intensity for various PS1 species in the membrane fraction as a function of substrate concentration. C) Inhibition of γ-secretase by Compound E. D) Inhibition of γ-secretase activity by L-685,458. 
         FIG. 9 . Dependence of observed of γ-secretase activity on various experimental parameters in an ALPHA assay. A) Dependence on concentration of SM320 antibody. B) Dependence on concentration of streptavidin donor beads. C) Dependence on concentration of protein A acceptor beads. D) Optimized conditions applied to 384 and 1536 well plates. 
         FIG. 10 . PS1 mutations alter the S2 subpocket conformation of gamma-secretase. (a) Schematic representation of the interactions between of L458 side chains residues (P/P′ positions) with corresponding gamma-secretase subsites (S/S′). (b) Structure of JC8, GY4, and L646, in which a benzophenone was incorporated into the P1′, P1 or P2 site of L458, respectively. Biotin was linked to the probes to facilitate isolation of photolabeled proteins. (c) Photolabeling of PS1 NTF with JC8, GY4 and L646. Membranes isolated from WT, M146L, or E280A PS1 stable cell lines were used for this study. (d) The photolabeled PS1 NTF were quantified by comparing the labeling intensities of GY4 and L646 to the labeling intensity of JC8. (e) Analysis of γ-secretase isolated from M146V or WT PS1 from knock-in mouse brains. Photolabeled PS1 was detected with western blot analysis using anti-PS1 NTF antibody. (f) The analyses of photolabeled PS1 NTF were quantified by comparing the labeling intensities of L646 to JC8. 
         FIG. 11 . PS1 mutants preferred N1-Sb1 substrates with a larger P2 residue. (a) Schematic representation of N1-Sb1 with wild-type P2 residue, cysteine. (b) The relative size differences of the wild-type cysteine residue which was substituted with either alanine, valine or methionine are shown. (c) M146L, E280A and WT PS1 from stable cell lines were used to determine the catalysis of or M N1-Sb1. (d) The catalysis of C and M N1-Sb1 were compared between WT and M146V PS1. (n=3, mean+/−s.d. ***p&lt;0.001, **p&lt;0.01) 
         FIG. 12 . Schematic representation of WT PS1 and FAD PS1 cleavage of APP. (a) Gamma-Secretase cleaves APP at several positions such as gamma-site, for Aβ40 and Aβ42 production, as well as the ε-site to produce Aβ48 and Aβ49 peptides. The sequential cleavage model proposes that gamma-secretase either first cleaves APP at the Aβ49 site followed by Aβ46, Aβ43 and Aβ40 (green dotted line), or gamma-secretase can cleave APP at the Aβ48 site followed by Aβ45 and Aβ42 (red dotted line). (b) A comparison of the relative sizes of the P2 residues during Aβ40, Aβ42/48 or Aβ49 cleavage. Since augmentation of Aβ42/40 and Aβ48/49 ratio are associated with FAD PS1, this suggests that the deeper S2 subpocket of FAD PS1 prefer Aβ42 and Aβ48 cleavages, which have larger P2 residue (Ile) while WT PS1 that has shallower S2 subsite prefers Aβ40 and Aβ49 cleavage that has smaller P2 residue such as Val and Thr. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     As used herein, the term “Notch protein,” and related terms and phrases, relate generally to any member of the Notch family of cell surface receptors present in metazoa. Mammals possess four different Notch receptors, referred to as NOTCH1, NOTCH2, NOTCH3, and NOTCH4. An example of an amino acid sequence of human Notch1 is disclosed in GenBank Acc. No. AAG33848.1 (GI:11275980), and is reproduced below in Table 1. 
     
       
         
               
             
               
             
           
               
                 TABLE 1  
               
               
                   
               
               
                 (SEQ ID NO: 1). 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                    1 mppllapllc lallpalaar gprcsqpget clnggkceaa ngteacvcgg afvgprcqdp 
               
               
                   
               
               
                   61 npclstpckn agtchvvdrr gvadyacsca lgfsgplclt pldnacltnp crnggtcdll 
               
               
                   
               
               
                  121 tlteykcrcp pgwsgkscqq adpcasnpca nggqclpfea syichcppsf hgptcrqdvn 
               
               
                   
               
               
                  181 ecgqkprlcr hggtchnevg syrcvcrath tgpncerpyv pcspspcqng gtcrptgdvt 
               
               
                   
               
               
                  241 hecaclpgft gqnceenidd cpgnnckngg acvdgvntyn cpcppewtgq yctedvdecq 
               
               
                   
               
               
                  301 lmpnacqngg tchnthggyn cvcvngwtge dcseniddca saacfhgatc hdrvasfyce 
               
               
                   
               
               
                  361 cphgrtgllc hlndacisnp cnegsncdtn pvngkaictc psgytgpacs qdvdecslga 
               
               
                   
               
               
                  421 npcehagkci ntlgsfecqc lqgytgprce idvnecvsnp cqndatcldq igefqcmcmp 
               
               
                   
               
               
                  481 gyegvhcevn tdecasspcl hngrcldkin efqcecptgf tghlcqydvd ecastpckng 
               
               
                   
               
               
                  541 akcldgpnty tcvctegytg thcevdidec dpdpchygsc kdgvatftcl crpgytghhc 
               
               
                   
               
               
                  601 etninecssq pcrlrgtcqd pdnaylcfcl kgttgpncei nlddcasspc dsgtcldkid 
               
               
                   
               
               
                  661 gyecacepgy tgsmcnsnid ecagnpchng gtcedgingf tcrcpegyhd ptclsevnec 
               
               
                   
               
               
                  721 nsnpcvhgac rdslngykcd cdpgwsgtnc dinnnecesn pcvnggtckd mtsgivctcr 
               
               
                   
               
               
                  781 egfsgpncqt ninecasnpc lnkgtciddv agykcncllp ytgatcevvl apcapspcrn 
               
               
                   
               
               
                  841 ggecrqsedy esfscvcpta gakggtcevd inecvlspcr hgascqnthg xyrchcqagy 
               
               
                   
               
               
                  901 sgrncetdid dcrpnpchng gsctdginta fcdclpgfrg tfceedinec asdpcrngan 
               
               
                   
               
               
                  961 ctdcvdsytc tcpagfsgih cenntpdcte sscfnggtcv dginsftclc ppgftgsycq 
               
               
                   
               
               
                 1021 hvvnecdsrp cllggtcqdg rglhrctcpq gytgpncqnl vhwcdsspck nggkcwqtht 
               
               
                   
               
               
                 1081 qyrcecpsgw tglycdvpsv scevaaqrqg vdvarlcqhg glcvdagnth hcrcqagytg 
               
               
                   
               
               
                 1141 sycedlvdec spspcqngat ctdylggysc kcvagyhgvn cseeidecls hpcqnggtcl 
               
               
                   
               
               
                 1201 dlpntykcsc prgtqgvhce invddcnppv dpvsrspkcf nngtcvdqvg gysctcppgf 
               
               
                   
               
               
                 1261 vgercegdvn eclsnpcdar gtqncvqrvn dfhcecragh tgrrcesvin gckgkpckng 
               
               
                   
               
               
                 1321 gtcavasnta rgfickcpag fegatcenda rtcgslrcln ggtcisgprs ptclclgpft 
               
               
                   
               
               
                 1381 gpecqfpass pclggnpcyn qgtceptses pfyrclcpak fngllchild ysfgggagrd 
               
               
                   
               
               
                 1441 ippplieeac elpecqedag nkvcslqcnn hacgwdggdc slnfndpwkn ctqslqcwky 
               
               
                   
               
               
                 1501 fsdghcdsqc nsagclfdgf dcgraeggcn plydqyckdh fsdghcdqgc nsaecewdgl 
               
               
                   
               
               
                 1561 dcaehvperl aagtlvvvvl mppeqlrnss fhflrelsrv lhtnvvfkrd ahgqqmifpy 
               
               
                   
               
               
                 1621 ygreeelrkh pikraaegwa apdallgqvk asllpggseg grrrreldpm dvrgsivyle 
               
               
                   
               
               
                 1681 idnrqcvgas sqcfqsatdv aaflgalasl gslnipykie avqsetvepp ppaqlhfmyv 
               
               
                   
               
               
                 1741 aaaafvllff vgcgvllsrk rrrqhgqlwf pegfkvseas kkkrreplge dsvglkplkn 
               
               
                   
               
               
                 1801 asdgalmddn qnewgdedle tkkfrfeepv vlpdlddqtd hrqwtqqhld aadlrmsama 
               
               
                   
               
               
                 1861 ptppqgevda dcmdvnvrgp dgftplmias csgggletgn seeeedapav isdfiyqgas 
               
               
                   
               
               
                 1921 lhnqtdrtge talhlaarys rsdaakrlle asadaniqdn mgrtplhaav sadaqgvfqi 
               
               
                   
               
               
                 1981 lirnratdld armhdgttpl ilaarlaveg mledlinsha dvnavddlgk salhwaaavn 
               
               
                   
               
               
                 2041 nvdaavvllk ngankdmqnn reetplflaa regsyetakv lldhfanrdi tdhmdrlprd 
               
               
                   
               
               
                 2101 iagermhhdi vrlldeynlv rspqlhgapl ggtptlsppl cspngylgsl kpgvqgkkvr 
               
               
                   
               
               
                 2161 kpsskglacg skeakdlkar rkksqdgkgc lldssgmlsp vdslesphgy lsdvasppll 
               
               
                   
               
               
                 2221 pspfqqspsv plnhlpgmpd thlgighlnv aakpemaalg gggrlafetg pprlshlpva 
               
               
                   
               
               
                 2281 sgtstvlgss sggalnftvg gstslngqce wlsrlqsgmv pnqynplrgs vapgplstqa 
               
               
                   
               
               
                 2341 pslqhgmvgp lhsslaasal sqmmsyqglp strlatqphl vqtqqvqpqn lqmqqqnlqp 
               
               
                   
               
               
                 2401 aniqqqqslq ppppppqphl gvssaasghl grsflsgeps qadvqplgps slavhtilpq 
               
               
                   
               
               
                 2461 espalptslp sslvppvtaa qfltppsqhs ysspvdntps hqlqvpehpf ltpspespdq 
               
               
                   
               
               
                 2521 wssssphsnv sdwsegvssp ptsmqsqiar ipeafk 
               
               
                   
               
             
          
         
       
     
     As used herein the terms “peptide” and “polypeptide” and related terms designate any and all compositions in which a given amino acid residue is linked to a neighboring amino acid residue via a peptide bond. As used herein the term “peptide” is synonymous with “polypeptide”. In this usage the length of the polypeptide is not limited to a specified minimum number of amino acid residues. A polypeptide may be composed of only naturally occurring amino acid residues, or it may include modified, synthetic, or derivatized amino acid residues as well. 
     As used herein, the term “gamma-secretase” or “γ-secretase” refers to any in vivo, ex vivo or in vitro substance containing gamma-secretase activity. Thus, by way of non-limiting example, γ-secretase may be obtained from, or be found in, a live organism (including a human, or a laboratory or veterinary animal such as a mouse, rat, or primate) or a sample therefrom (such as a tissue or body fluid, or extract thereof), a cell (such as a primary cell or cell line, or extract thereof), extracellular medium or matrix or milieu, or isolated protein. Sources of γ-secretase are not limited to naturally occurring gamma-secretase, but may also include engineered and/or synthesized gamma-secretase. A gamma-secretase refers to an enzyme(s) with the ability to cleave at the gamma-secretase site of a gamma-secretase substrate known to have a gamma-secretase cleavage site, e.g., a Notch protein and other gamma-secretase substrates described herein, such as a Notch substrate. As used herein, gamma-secretase includes all recombinant forms, mutations, and other variants of gamma-secretase so long as these maintain a functional capability to catalyze the cleavage of molecules or substrates bearing gamma-secretase cleavage sites. In one embodiment, such a gamma-secretase cleavage site is an S3 site. The identity of Notch S3 cleavage sites are known in the art. 
     As used herein, the term “gamma-secretase substrate” refers to any naturally occurring or synthetic sequence of amino acids (e.g., polypeptides and proteins) including a gamma-secretase cleavage site. Gamma-secretase substrates are know in the art, and non-limiting examples of gamma-secretase substrates include Notch proteins, APP, neuregulin-1, alpha-protocadherin, SCNB2, Tie-1, beta-APP like protein 1, beta-APP like protein 2, nectin-3, nectin-4, alcadein alpha, alcadein gamma, APLP1, APLP2, ApoER2, CD43, CD44, CSF1R, CXCL16, CX3CL1, DCC, Deltal, E-cadherin, EphrinB1, EphrinB2, EphB2, ErbB4, GHR, HLA-A2, IGF1R, IFN-alpha-R2, IL-1R2, IR, IRE1-alpha, Jagged2, L1, LRP, LPR1B, LRP2, LRP6, N-cadherin, Nectin1-alpha, notch, Notch1, Notch2, Notch3, Notch4, NRADD, p75-NTR, PKHD1, Pcdh-alpha-4, Pcdh-gamma-C3, PTP-kappa, PTP-g, PTP-LAR, S or CS1b, SorLA, Sortilin, Syndecan3, Tyrosinase, TYRP1, TYRP2, VEGF-R1, VGSC-beta-2, and VLDLR. 
     Another gamma-secretase substrate is a Notch substrate. As used herein, the term “Notch substrate” refers to any Notch polypeptide that has fewer amino acids than a full length natural Notch cell surface receptor, includes a gamma-secretase cleavage site, and is cleaved by a gamma-secretase under suitable conditions. In some embodiments, the Notch substrate is cleaved at an S3 site. In some embodiments, a Notch substrate further includes at least one amino acid modification. Such a modification may include at least one amino acid substitution, deletion, insertion, or addition. Examples of Notch substrates are shown in Table 2. 
                                 TABLE 2                       Notch1 Peptide    Amino Acid Residues           Substrate   of Notch1 (SEQ ID NO: 1)                           N1Sb1 (NTM2)   1732-1812 (SEQ ID NO: 6)           N1Sb2 (NTM1)   1732-1770 (SEQ ID NO: 7)           N1Sb3   1720-1812 (SEQ ID NO: 9)           N1Sb4   1708-1812 (SEQ ID NO: 10)           N1Sb5   1665-1812 (SEQ ID NO: 11)           N1Sb6   1696-1812 (SEQ ID NO: 12)           N1Sb7   1726-1812 (SEQ ID NO: 13)                        
Other Notch substrates include fragments of Notch substrates disclosed in Table 2. A fragment includes a gamma-secreates cleavage site and can be cleaved by a gamma-secretase under suitable conditions. a fragment may include a deletion of amino acids at the N-terminal end, the C-terminal end, or both. The total number of amino acids deleted may be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 10, at least 12, at least 20, at least 30, at least 40, at least 42, and so on. The total number of amino acids may be deleted from the N-terminal end, the C-terminal end, or the combination of both.
 
     Other examples of Notch substrates include amino acid sequence having structural similarity to a reference Notch substrate or fragment thereof. For example, polypeptides having structural similarity to a reference Notch substrate include naturally-occurring allelic variants of a Notch sequence that may exist in the population. A Notch substrate that is structurally similar to the amino acid sequence of a polypeptide described herein has a gamma-secretase cleavage site, and is cleaved by a gamma-secretase under suitable conditions. In one embodiment, a gamma-secretase cleavage site is an S2 cleavage site, and in another embodiment a gamma-secretase cleavage site is an S3 cleavage site. Methods for testing whether a polypeptide is cleaved by a gamma-secretase under suitable conditions are described below. 
     Structural similarity of two polypeptides can be determined by aligning the residues of the two polypeptides (for example, a candidate polypeptide and any appropriate reference polypeptide described herein, such as amino acids 1732-1812 of SEQ ID NO:1 or a fragment thereof) to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order. A reference polypeptide may be a polypeptide described herein. A candidate polypeptide is the polypeptide being compared to the reference polypeptide. A candidate polypeptide may be isolated, for example, from a cell, or can be produced using recombinant techniques, or chemically or enzymatically synthesized. 
     Unless modified as otherwise described herein, a pair-wise comparison analysis of amino acid sequences can be carried out using parameters for polypeptide sequence comparison include the algorithm of Needleman and Wunsch (1970, J. Mol. Biol. 48: 443-453), the comparison matrix BLOSSUM62 from Hentikoff and Hentikoff (1992, Proc. Natl. Acad. Sci. USA, 89:10915-10919), with gap penalty 12 and gap length penalty 4. A program useful with these parameters is publicly available as the “gap” program from Genetics Computer Group, Madison Wis. The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps). 
     In the comparison of two amino acid sequences, structural similarity may be referred to by percent “identity” or may be referred to by percent “similarity.” “Identity” refers to the presence of identical amino acids. “Similarity” refers to the presence of not only identical amino acids but also the presence of conservative substitutions. A conservative substitution for an amino acid in a polypeptide described herein may be selected from other members of the class to which the amino acid belongs. For example, it is known in the art of protein biochemistry that an amino acid belonging to a grouping of amino acids having a particular size or characteristic (such as charge, hydrophobicity and hydrophilicity) can be substituted for another amino acid without altering the activity of a protein, particularly in regions of the protein that are not directly associated with biological activity. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Conservative substitutions include, for example, Lys for Arg and vice versa to maintain a positive charge; Glu for Asp and vice versa to maintain a negative charge; Ser for Thr so that a free —OH is maintained; and Gln for Asn to maintain a free —NH2. 
     Thus, as used herein, a candidate polypeptide useful in the methods described herein includes those with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence similarity to a reference amino acid sequence or fragment thereof. 
     Alternatively, as used herein, a candidate polypeptide useful in the methods described herein includes those with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the reference amino acid sequence or fragment thereof. 
     Also included in the present invention are polynucleotides encoding the polypeptides disclosed herein. As used herein, the term “polynucleotide” refers to a polymeric form of nucleotides of any length, either ribonucleotides, deoxynucleotides, peptide nucleic acids, or a combination thereof, and includes both single-stranded molecules and double-stranded duplexes. A polynucleotide can be obtained directly from a natural source, or can be prepared with the aid of recombinant, enzymatic, or chemical techniques. An example of a polynucleotides encoding SEQ ID NO:1 (a Notch1 polypeptide) is GenBank Accession Number AF308602.1, and the skilled person can readily identify portions of the polynucleotide that encode the Notch substrates disclosed in Table 2. It should be understood that a polynucleotide encoding one of the Notch substrates disclosed in Table 2 is not limited to the appropriate portion of the nucleotide sequence disclosed GenBank Accession Number AF308602.1, but also includes the class of polynucleotides encoding a Notch substrate as a result of the degeneracy of the genetic code. For example, the naturally occurring nucleotide sequence disclosed GenBank Accession Number AF308602.1 is but one member of the class of nucleotide sequences encoding a Notch1 polypeptide having the amino acid sequence SEQ ID NO:1. The class of nucleotide sequences encoding a selected polypeptide sequence is large but finite, and the nucleotide sequence of each member of the class may be readily determined by one skilled in the art by reference to the standard genetic code, wherein different nucleotide triplets (codons) are known to encode the same amino acid. 
     As used herein, the term “isolated,” as it refers to a polypeptide refers to any polypeptide that has been removed or separated from any source, e.g., from a cell that naturally expresses the protein, polypeptide or fragment thereof or that has been engineered to express the protein, polypeptide or fragment thereof. Polypeptides that are produced by recombinant, enzymatic, or chemical techniques are considered to be isolated and purified by definition, since they were never present in a natural environment. 
     The term “contacting” refers to bringing into association, either directly or indirectly, two or more substances or compositions. Contacting may occur in vivo, ex vivo or in vitro. Commonly contacting a first composition with a second composition brings about a transformation in the first composition, the second composition, or both compositions. 
     Conditions that “allow” an event to occur or conditions that are “suitable” for an event to occur, or “suitable” conditions are conditions that do not prevent such events from occurring. Thus, these conditions permit, enhance, facilitate, and/or are conducive to the event. Such conditions, known in the art and described herein, may depend upon, for example, the nature of a polypeptide sequence, temperature, and buffer conditions. These conditions may also depend on what event is desired. 
     As used herein, the term “consists essentially,” with respect to a Notch substrate of the invention, indicates that the sequence may be modified by N-terminal and/or C-terminal additions or deletions that do not cause a substantial decrease in the ability of the gamma-secretase substrate to be cleaved compared to the reference sequence. 
     As used herein, the term “transfection” refers to any of the methods known in the art for introducing DNA into a cell including, but not limited to, the methods of calcium phosphate or calcium chloride mediated transfection, electroporation, and infection with a retroviral vector. 
     As used herein, the terms “fusion protein,” “chimeric protein,” and related terms and phrases, refer to a protein or polypeptide engineered to contain at least two polypeptide regions or domains, each having recognizable structure, function, or similar attribute, and, optionally, a linking peptide to operatively link the two polypeptides into one continuous polypeptide. The at least two polypeptide regions in a fusion protein are derived from different sources, and therefore a fusion protein includes two polypeptide regions not normally joined together in nature. 
     As used herein, the terms “linking sequence” and “linker peptide” refer to one or more amino acid residues joined in peptide bonds that serve to join two polypeptide regions of differing origins in a fusion protein via a peptide bond between the linking sequence and each of the polypeptide regions. 
     As used herein, the terms “tag,” “probe” and “label” refer interchangeably to a moiety bound to a target substance that permits easy detection or assay of the target. A tag, probe or label may include a particular amino acid sequence defining a polypeptide tag, probe or label, or it may include a non-proteinaceous moiety that may be readily detected by a laboratory assay. A given composition or substance, e.g., a polypeptide, may bear one, or more than one, tag, probe or label at the same time. Examples of tags include maltose binding protein, AviTag, the FLAG epitope, biotin, digoxigenin, glutathione dehydrogenase, horse radish peroxidase, and so forth. Additionally a tag, probe or label may include an antibody that specifically binds to a target substance, or to a second antibody. An antibody tag, probe or label may itself further bear a detectable moiety as a tag, probe or label, such as a fluorescent moiety, including a fluorescent moiety that may serve as a fluorescence donor or a fluorescence energy acceptor, or a moiety that responds in a chemiluminescence assay. 
     As used herein, the term “gamma-secretase assay” refers to any assay which may be used to measure the activity of gamma-secretase toward a gamma-secretase substrate. 
     As used herein, the terms “increase,” “increases,” “increased” and “decrease,” “decreases,” and “decreased” in the context of the activity of gamma-secretase refer, in some embodiments, to an increase, or a decrease, respectively: (i) of 0.5%, 1%. 1.5%, 2%, 5%, 10%, 20%, 30%, 40%, 50% or more; or (ii) an increase of 1.5, 2, 3, 4, or 5 fold or more. 
     As used herein, the terms “change,” “changed,” “modulate,” or “modulated,” in the context of the activity of gamma-secretase refer, in some embodiments, to: (i) a positive or a negative change of 0.5%, 1%. 1.5%, 2%, 5%, 10%, 20%, 30%, 40%, 50% or more; or (ii) a positive or a negative change of 1.5, 2, 3, 4, or 5 fold or more. 
     As used herein, the term “compound” and similar terms (such as “substance” and “agent”) refers to any compound being tested for its ability to modulate gamma-secretase activity. As used herein, a compound and similar terms include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, other organic and inorganic compounds (e.g., including heteroorganic and organometallic compounds). A compound may have a molecular weight of less than 10,000 Da, or less than 5,000 Da, or less than 1,000 Da, or less than 500 Da, or less than 100 Da. 
     As used herein, the term “gamma-secretase inhibitor” refers to any molecule, compound, and/or substance capable of reducing and/or eliminating the activity of gamma-secretase. 
     As used herein, the term “small molecule” and analogous terms include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, other organic and inorganic compounds (e.g., including heteroorganic and organometallic compounds) and forms thereof having a molecular weight of less than 10,000 Da, or less than 5,000 Da, or less than 1,000 Da, or less than 500 Da, or less than 100 Da. 
     As used herein, the term “candidate,” when referring to a compound relates to a test compound whose potential activity with respect to a certain property is undergoing assay. Thus the ability of the candidate compound to manifest the property has been unknown or uncharacterized prior to the assay, and becomes apparent upon carrying the assay out. A property of interest in the present disclosure is the ability to modulate gamma-secretase activity. 
     As used herein, the term “therapeutic agent” refers to any compound that is used for the purpose of treating and/or managing a disease or disorder. In one embodiment, an therapeutic agent is a gamma-secretase modulator. Examples of therapeutic agents include, but are not limited to, proteins, compounds, immunoglobulins (e.g., multi-specific Igs, single chain Igs, Ig fragments, polyclonal antibodies and their fragments, monoclonal antibodies and their fragments), peptides (e.g., peptide receptors, selectins), binding proteins, biologics, chemospecific agents, chemotoxic agents (e.g., anti-cancer agents), proliferation-based therapy, radiation, chemotherapy, anti-angiogenic agents, and drugs. 
     As used herein, the term “separating” and similar terms and phrases, when applied to a cell, connote resolving various fractions that may occur in the cell from one another. Frequently a cell is disrupted to disperse its contents into a suspending solvent prior to resolving its fractions. Disruption may be accomplished, for example, by homogenization, extrusion through a high shear device such as a French press, sonication, and so on. The resulting cell-free suspension may then be resolved into fractions as above. In general, as used herein, “separating” includes any disruption of the cell. 
     As used herein, the term “host cell” includes a particular subject cell transformed or transfected with a polynucleotide and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the polynucleotide due to mutations or environmental influences that may occur in succeeding generations or integration of the polynucleotide into the host cell genome. 
     As used herein, the term “isolated,” as it refers to a gamma-secretase inhibitor, means the physical state of a gamma-secretase inhibitor after being separated and/or purified from precursors and other substances found in a synthetic process (e.g., from a reaction mixture) or natural source or combination thereof according to a process or processes described herein or which are well known to the skilled artisan (e.g., chromatography, recrystallization and the like) in sufficient purity to be capable of characterization by standard analytical techniques described herein or well known to the skilled artisan. In a specific embodiment, the gamma-secretase inhibitor is at least 60% pure, at least 65% pure, at least 70% pure, at least 75% pure, at least 80% pure, at least 85% pure, at least 90% pure or at least 99% pure as assessed by techniques known to one of skill in the art. 
     Concentrations, amounts, cell counts, percentages and other numerical values may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly disclosed as the limits of the range but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range were explicitly disclosed. 
     The term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements. 
     The terms “comprises” and variations thereof do not have a limiting meaning where these terms appear in the description and claims. 
     Unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one. 
     Antibodies 
     The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, F ab , F ab′  and F (ab′)2  fragments, and an F ab  expression library. In general, antibody molecules obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgG 1 , IgG 2 , and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species. Any antibody disclosed herein binds “immunospecifically” to its cognate antigen. By immunospecific binding is meant that an antibody raised by challenging a host with a particular immunogen binds to a molecule such as an antigen that includes the immunogenic moiety with a high affinity, and binds with only a weak affinity or not at all to non-immunogen-containing molecules. As used in this definition, high affinity means having a dissociation constant less than 1×10 −6  M, and weak affinity means having a dissociation constant higher than 1×10 −6  M. 
     A Notch substrate described herein or a fragment thereof may be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation. The full-length protein maybe used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens. An antigenic peptide fragment includes at least 6 amino acid residues of the amino acid sequence of the full length protein, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope. In one embodiment the antigenic peptide includes at least 9 amino acid residues, at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. In one embodiment, epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions. 
     In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region of a Notch protein or fragment thereof that is located on the surface of the protein, e.g., a hydrophilic region. A hydrophobicity analysis of the protein sequence will indicate which regions of the polypeptide are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production. As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981 , Proc. Nat. Acad. Sci. USA  78: 3824-3828; Kyte and Doolittle 1982 , J. Mol. Biol.  157: 105-142. Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein. 
     A protein of the present invention or a thereof may be used as an immunogen in the generation of antibodies that immunospecifically bind immunogen. In one embodiment, the polypeptide used as an immunogen is at least 6 consecutive amino acids chosen from VLLSRKRRR (SEQ ID NO:2). In one embodiment the immunogen is VLLSRKRRR (SEQ ID NO:2), and in another embodiment the immunogen is a polypeptide that includes VLLSRKRRR (SEQ ID NO:2). For instance, the immunogen may include VLLSRKRRR (SEQ ID NO:2) and other amino acids that are naturally flank this sequence in wild type Notch polypeptides. In one embodiment, the immunogen may include VLLSRKRRR (SEQ ID NO:2) and other non-native amino acids, and/or other molecules. For instance, the immunogen may include VLLSRKRRR. (SEQ ID NO:2) and a carrier such as, but not limited to, keyhole limpet hemocyanin. In one embodiment antibody binds to an epitope that is exposed, and able to interact with an antiboy, after cleavage by a gamma-secretase at an S2 or S3 site. 
     Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Some of these antibodies are discussed below. 
     1. Polyclonal Antibodies 
     For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative of the foregoing. An appropriate immunogenic preparation may contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein. Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The preparation may further include an adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to, Freund&#39;s (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and  Corynebacterium parvum , or similar immunostimulatory agents. Additional examples of adjuvants which may be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). 
     The polyclonal antibody molecules directed against the immunogenic protein may be isolated from the mammal (e.g., from the blood) and further purified by known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28). 
     2. Monoclonal Antibodies 
     The term “monoclonal antibody” (MAb) or “monoclonal antibody composition,” as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs thus contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it. 
     Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro. 
     The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells. 
     Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which may be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Manassas, Va. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor: J. Jmmunol., 133:3001 (1984); Brodeur et al.: Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63). 
     The culture medium in which the hybridoma cells are cultured may then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). It is an objective, especially important in therapeutic applications of monoclonal antibodies, to identify antibodies having a high degree of specificity and a high binding affinity for the target antigen. 
     After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco&#39;s Modified Eagle&#39;s Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal. 
     The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. 
     The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the invention may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison (1994) Nature 368, 812-13) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide may be substituted for the constant domains of an antibody of the invention, or may be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody. 
     3. F ab  Fragments and Single Chain Antibodies 
     According to the invention, techniques may be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Pat. No. 4,946,778). In addition, methods may be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal F ab  fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F (ab′)2  fragment produced by pepsin digestion of an antibody molecule; (ii) an F ab  fragment generated by reducing the disulfide bridges of an F (ab′)2  fragment; (iii) an F ab  fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F v  fragments. 
     Notch Recombinant Vectors and Host Cells 
     The Notch substrates provided herein may be prepared by standard recombinant DNA techniques or by protein synthetic techniques, e.g., by use of a peptide synthesizer. For example, a polynucleotide encoding a Notch substrate of the invention may be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments may be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which may subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g.,  Current Protocols in Molecular Biology , Ausubel et al., eds., John Wiley &amp; Sons, 1992). 
     The nucleotide sequences encoding Notch substrates may be obtained from any information available to those of skill in the art (e.g., from GenBank, the literature, or by routine cloning). The nucleotide sequence coding for a Notch substrate may be modified, if desired, using approaches known to those of skill in the art, e.g., site-directed mutagenesis, and inserted into an appropriate expression vector, e.g., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. In some instances, a sequence encoding a Notch polypeptide may be truncated in order to remove a specific domain, such as the targeting domain. The techniques for modifying or truncating DNA are well known to those of skill in the art of molecular biology. 
     A variety of host-vector systems may be utilized in the present invention to express the protein-coding sequence. These include, but are not limited to, mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast (e.g.  Pichia ) containing yeast vectors; or bacteria (such as  E. coli ) transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used. 
     Another aspect of the disclosure pertains to vectors, preferably expression vectors, containing a polynucleotide encoding a Notch substrate. As used herein, the term “vector” refers to a polynucleotide capable of transporting another polynucleotide to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. 
     The recombinant expression vectors of the invention include a polynucleotide of the invention in a form suitable for expression of the polynucleotide in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively linked to the polynucleotide sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel (1990) G ENE  E XPRESSION  T ECHNOLOGY :M ETHODS IN  E NZYMOLOGY  185, Academic Press, San Diego, Calif. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention may be introduced into host cells to thereby produce polypeptides such as a Notch substrate. Examples of plasmid vectors that encode a Notch substrate and that is suitably labeled for use in various assay methods disclosed herein include, but are not limited to, those illustrated in  FIGS. 1A  (pIAD16-MBP-N-1-Avi) and  2  (pIAD16 with N1-Sb1 insert). 
     The expression of a Notch substrate of the invention may be controlled by a promoter or enhancer element. Promoters which may be used to control expression of a Notch substrate include, but are not limited to, the SV40 early promoter region (Bemoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980 , Cell  22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981 , Proc. Natl. Acad. Sci. U.S.A.  78:1441-1445), the regulatory sequences of the rnetallothionein gene (Brinster et al., 1982 , Nature  296:39-42), the tetracycline (Tet) promoter (Gossen et al., 1995 , Proc. Nat. Acad. Sci. U.S.A.  89:5547-5551); prokaryotic expression vectors such as the β-lactamase promoter (VIIIa-Kamaroff, et al., 1978 , Proc. Natl. Acad. Sci. U.S.A.  75:3727-3731), or the tac promoter (DeBoer, et al., 1983 , Proc. Natl. Acad. Sci. U.S.A.  80:21-25; see also “Useful proteins from recombinant bacteria,” in  Scientific American,  1980, 242:74-94); plant expression vectors including the nopaline synthetase promoter region (Herrera-Estrella et al.,  Nature  303:209-213) or the cauliflower mosaic virus 35S RNA promoter (Gardner, et al., 1981 , Nucl. Acids Res.  9:2871), and the promoter of the photosynthetic enzyme ribulose biphosphate carboxylase (Herrera-Estrella et al., 1984 , Nature  310:115-120); promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984 , Cell  38:639-646; Omitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985 , Nature  315:115-122), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., 1984 , Cell  38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987 , Mol. Cell. Biol.  7:1436-1444), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986 , Cell  45:485-495), albumin gene control region which is active in liver (Pinkert et al., 1987 , Genes and Devel.  1:268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., 1985 , Mol. Cell. Biol.  5:1639-1648; Hammer et al., 1987 , Science  235:53-58); alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al., 1987, Genes and Devel. 1:161-171), beta-globin gene control region which is active in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94; myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al., 1987 , Cell  48:703-712); myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985 , Nature  314:283-286); neuronal-specific enolase (NSE) which is active in neuronal cells (Morelli et al., 1999 , Gen. Virol.  80:571-83); brain-derived neurotrophic factor (BDNF) gene control region which is active in neuronal cells (Tabuchi et al., 1998 , Biochem. Biophysic. Res. Com.  253:818-823); glial fibrillary acidic protein (GFAP) promoter which is active in astrocytes (Gomes et al., 1999 , Braz. J. Med. Biol. Res.  32(5):619-631; Morelli et al., 1999 , Gen. Virol.  80:571-83) and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986 , Science  234:1372-1378). In one embodiment, the expression of a Notch substrate of the invention is regulated by a constitutive promoter. In another embodiment, the expression is regulated by an inducible promoter. In another embodiment, the expression is regulated by a tissue-specific promoter. 
     The recombinant expression vectors of the invention may be designed for expression of the Notch substrate in prokaryotic or eukaryotic cells. For example, the Notch substrate may be expressed in bacterial cells such as  E. coli , insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel (Goeddel (1990) G ENE  E XPRESSION  T ECHNOLOGY : M ETHODS IN  E NZYMOLOGY  185, Academic Press, San Diego, Calif.). Alternatively, the recombinant expression vector may be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase. 
     In a specific embodiment, a vector is used that includes a promoter operably linked to a Notch substrate encoding polynucleotide, one or more origins of replication and, optionally, one or more selectable markers (e.g., an antibiotic resistance gene). 
     Expression of proteins in prokaryotes is most often carried out in K coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (1) to increase expression of recombinant protein, (2) to increase the solubility of the recombinant protein, and/or (3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include but are not limited to Factor Xa, thrombin and enterokinase. Common fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson (1988)  Gene  67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. 
     Examples of suitable inducible non-fusion  E. coli  expression vectors include pTrc (Amrann et al., (1988)  Gene  69:301-315) and pET 11d (Studier et al. (1990) G ENE  E XPRESSION  T ECHNOLOGY : M ETHODS IN  E NZYMOLOGY  185, Academic Press, San Diego, Calif. 60-89). 
     One strategy to maximize recombinant protein expression in  E. coli  is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, Gottesman (1990) G ENE  E XPRESSION  T ECHNOLOGY : M ETHODS IN  E NZYMOLOGY  185, Academic Press, San Diego, Calif. 119-128. Another strategy is to alter the nucleic acid sequence of the polynucleotide to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in  E. coli  (Wada et al., (1992)  Nucleic Acids Res.  20:2111-2118). Such alteration of nucleic acid sequences of the invention may be carried out by standard DNA synthesis techniques. 
     In another embodiment, the Notch expression vector is a yeast expression vector. Examples of vectors for expression in yeast  S. cerivisae  include pYepSec1 (Baldari, et al., (1987)  EMBO J  6:229-234), pMFa (Kurjan and Herskowitz, (1982)  Cell  30:933-943), pJRY88 (Schultz et al., (1987)  Gene  54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.). 
     Alternatively, the Notch substrate may be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith et al. (1983)  Mol Cell Biol  3:2156-2165) and the pVL series (Lucklow and Summers (1989)  Virology  170:31-39). 
     In yet another embodiment, a polynucleotide of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987)  EMBO J.  6: 187-195). When used in mammalian cells, the expression vector&#39;s control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells. See, e.g., Chapters 16 and 17 of Sambrook et al., M OLECULAR  C LONING : A L ABORATORY  M ANUAL.  2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. 
     In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the polynucleotide preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the polynucleotide). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987)  Genes Dev  1:268-277), lymphoid-specific promoters (Calame and Eaton (1988)  Adv Immunol  43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989)  EMBO J  8:729-733) and immunoglobulins (Banerji et al. (1983)  Cell  33:729-740; Queen and Baltimore (1983)  Cell  33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989)  Proc. Natl. Acad. Sci. USA  86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)  Science  230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss (1990)  Science  249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989)  Genes Dev  3:537-546). 
     Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. 
     A host cell may be any prokaryotic or eukaryotic cell. For example, a Notch substrate may be expressed in bacterial cells such as  E. coli , insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art. 
     Vector DNA may be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign polynucleotides (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells may be found in Sambrook, et al. (2001), Ausubel et al. (2002), and other laboratory manuals. 
     For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. A polynucleotide encoding a selectable marker may be introduced into a host cell on the same vector as that encoding the Notch substrate or may be introduced on a separate vector. Cells stably transfected with the introduced polynucleotide may be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die). 
     A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, may be used to produce (e.g., express) the Notch substrate. Accordingly, the invention further provides methods for producing the Notch substrate using the host cells of the invention. In one embodiment, the method includes culturing the host cell of invention (into which a recombinant expression vector encoding the Notch substrate has been introduced) in a suitable medium such that the Notch substrate is produced 
     In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters may be elevated in the presence of certain inducers; thus, expression of the genetically engineered gamma-secretase substrates may be controlled. Furthermore, different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, phosphorylation) of proteins. Appropriate cell lines or host systems may be chosen to ensure the desired modification and processing of the foreign protein expressed. For example, expression in a bacterial system will produce an unglycosylated product and expression in yeast will produce a glycosylated product. Eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, NSO, and in particular, neuronal cell lines such as, for example, SK-N-AS, SK-N-FI, SK-N-DZ human neuroblastomas (Sugimoto et al., 1984 , J. Natl. Cancer Inst.  73: 51-57), SK-N-SH human neuroblastoma ( Biochim. Biophys. Acta,  1982, 704: 450-460), Daoy human cerebellar medulloblastoma (He et al., 1992 , Cancer Res.  52: 1144-1148) DBTRG-05MG glioblastoma cells (Kruse et al., 1992 , In Vitro Cell. Dev. Biol.  28A: 609-614), LMR-32 human neuroblastoma ( Cancer Res.,  1970, 30: 2110-2118), 1321N1 human astrocytoma ( Proc. Natl. Acad. Sci. U.S.A.  1977, 74: 4816), MOG-G-CCM human astrocytoma ( Br. J. Cancer  1984, 49: 269), U87MG human glioblastoma-astrocytoma ( Acta Pathol. Microbiol. Scand.  1968, 74: 465-486), A172 human glioblastoma (Olopade et al., 1992 , Cancer Res.  52: 2523-2529), C6 rat glioma cells (Benda et al., 1968, Science 161: 370-371), Neuro-2a mouse neuroblastoma ( Proc. Natl. Acad. Sci. U.S.A.  1970, 65: 129-136), NB41A3 mouse neuroblastoma ( Proc. Natl. Acad. Sci. U.S.A.  1962, 48: 1184-1190), SCP sheep choroid plexus (Bolin et al., 1994 , J. Virol. Methods  48: 211-221), G355-5, PG-4 Cat normal astrocyte (Haapala et al., 1985 , J. Virol.  53: 827-833), Mpf ferret brain (Trowbridge et al., 1982 , In Vitro  18: 952-960), and normal cell lines such as, for example, CTX TNA2 rat normal cortex brain (Radany et al., 1992 , Proc. Natl. Acad. Sci. U.S.A.  89: 6467-6471) such as, for example, CRL7030 and Hs578Bst. Furthermore, different vector/host expression systems may effect processing reactions to different extents. 
     For long-term, high-yield production of gamma-secretase substrates, such as a Notch substrate, stable expression is preferred. For example, cell lines which stably express the gamma-secretase substrate of the invention may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells may be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched medium, and then are switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn may be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express a gamma-secretase substrate of the invention. 
     A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al., 1977 , Cell  11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &amp; Szybalski, 1962 , Proc. Natl. Acad. Sci. U.S.A.  48:2026), and adenine phosphoribosyltransferase (Lowy, et al., 1980 , Cell  22:817) genes may be employed in tk−, hgprt− or aprt− cells, respectively. Also, antimetabolite resistance may be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et al., 1980 , Proc. Natl. Acad. Sci. U.S.A.  77:3567; O&#39;Hare, et al., 1981 , Proc. Natl. Acad. Sci. U.S.A.  78:1527); gpt, which confers resist-once to mycophenolic acid (Mulligan &amp; Berg, 1981 , Proc. Natl. Acad. Sci. U.S.A.  78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., 1981 , J. Mol. Biol.  150:1); and hygro, which confers resistance to hygromycin (Santerre, et al., 1984 , Gene  30:147). 
     Once a gamma-secretase substrate of the invention has been produced by recombinant expression or by chemical synthesis, it may be purified by any method known in the art for purification of a protein, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. 
     Detection and Labeling 
     Generally an analyte, such as a product of cleavage of a Notch substrate by gamma-secretase, may be detected in many ways. Detecting may include any one or more processes that result in the ability to observe the presence and/or the amount of a proteolytic cleavage reaction. Physical, chemical or biological methods may be used to detect and quantify a product. Physical methods include, by way of nonlimiting example, surface plasmon resonance (SPR) detection, using SPR to detect a labeled product to an immobilized probe, or having a probe in a chromatographic medium and detecting binding of a bound product molecule in the chromatographic medium. Physical methods further include a gel electrophoresis or capillary electrophoresis format in which product molecules are resolved from other molecules, and the resolved products are detected. Chemical methods include hybridization methods and formation of specific binding pairs generally in which a product molecule binds to a probe. Biological methods include causing a bound target molecule to exert a biological effect on a cell, and detecting the effect. The present invention discloses examples of biological effects which may be used as a biological assay. In many embodiments, a product or member of a specific binding pair may be labeled as described herein to assist in detection and quantitation. 
     An advantageous way of accomplishing detection is to use a labeled form of a cleavage product molecule, such as a Notch substrate, and to detect the bound label. A label may be a radioisotopic label, such as  125 I,  35 S,  32 P,  14 C, or  3 H, for example, that is detectable by its radioactivity. Alternatively, a label may be selected such that it may be detected using a spectroscopic method, for example. In one instance, a label may be a chromophore, absorbing incident ultraviolet, visible, infrared, microwave or similar electromagnetic radiation. A preferred label is one detectable by luminescence. Generally, luminescence refers to the emission of electromagnetic radiation from a substance or a chemical. The radiation may occur in any region of the electromagnetic spectrum; i.e., the frequency of the emitted radiation may be anywhere in the spectrum. Commonly luminescence occurs in the ultraviolet, visible, or infrared spectral regions. Luminescence includes fluorescence, phosphorescence, and chemiluminescence. Thus a label that fluoresces, or that phosphoresces, or that induces a chemiluminscent reaction, may be employed. Nonlimiting examples of suitable fluorescent labels, or fluorochromes, include a Eu label, a fluorescein label, a rhodamine label, a phycoerythrin label, a phycocyanin label, Cy-3, Cy-5, an allophycocyanin label, an o-phthalaldehyde label, and a fluorescamine label. Luminescent labels afford detection with high sensitivity. A label may furthermore be a magnetic resonance label, such as a stable free radical label detectable by electron paramagnetic resonance, or a nuclear label, detectable by nuclear magnetic resonance. A label may still further be a ligand in a specific ligand-receptor pair; the presence of the ligand is then detected by the secondary binding of an additional ligand-specific receptor, which commonly is itself labeled for detection. Nonlimiting examples of such ligand-receptor pairs include biotin and streptavidin or avidin, a hapten such as digoxigenin or antigen and its specific antibody, and so forth. 
     Detecting, quantitating, including labeling, methods are known generally to workers of skill in fields related to the present invention, including, by way of nonlimiting example, workers of skill in spectroscopy, nucleic acid chemistry, biochemistry, molecular biology and cell biology. Quantitating assesses the quantity, mass, or concentration of a Notch substrate cleavage product, or fragment thereof, that results from the action of a gamma-secreatase. Quantitation includes determining the amount of change in a physical, chemical, or biological property as described herein. For example, the intensity of a signal originating from a label may be used to assess the quantity of the cleaved polypeptide bound to the probe. Any equivalent process yielding a way of detecting the presence and/or the quantity, mass, or concentration of a peptide cleavage product is envisioned to be within the scope of the present invention. 
     Electrochemiluminescence (ECL) Assay of Notch Cleavage Product Peptides 
     In one embodiment, a method for identifying a product of the gamma-secretase catalyzed cleavage of a Notch substrate is an electrochemiluminescence (“ECL”) assay (see Li, et al., 2000 , Proc. Natl. Acad. Sci. USA  97:6138-6143; and Yin, et al., 2007 , J. Biol. Chem.  282:23639-23644). In an ECL assay an analyte to be detected is labeled with a chemiluminescent moiety whose chemiluminescence is redox dependent. A commonly used chemiluminescent moiety is a Ru +2  complex which becomes chemiluminescent, and hence detectable with high sensitivity, upon oxidation to Ru +3 . Alternative electrochemiluminescent (ECL) probes equivalent to Ru +2  complexes are contemplated for use herein. As implemented in various Examples provided herein, an antibody specific for an intended epitope such as one revealed in a gamma-secretase proteolysis product, such as a cleaved Notch substrate, is conjugated to a Ru +2  complex. The moiety so conjugated may be termed “ruthenylated” herein. As applied herein an ECL assay includes: (a) contacting a Notch substrate or a fragment thereof with a composition containing gamma-secretase; (b) incubating the Notch substrate with the gamma-secretase containing composition under suitable conditions for a time period sufficient for gamma-secretase activity to take place; (c) adding a ruthenylated antibody that specifically binds a cleavage product but not the Notch substrate or fragment thereof; and (d) detecting a product bound to the product-specific ruthenylated antibody using ECL. ECL techniques are known in the art and described in, e.g., Yang, et al., 1994 , Bio/Technology  12:193-194; and Khorkova, et al., 1998 , J. Neurosci. Methods  82:159-166 
     In a specific embodiment, the source of gamma-secretase is a cell or cell membrane, e.g., a HeLa cell or other mammalian cell or a constituent membrane thereof, and the incubation of step (b) takes place in the presence of a detergent, e.g., CHAPSO, at a concentration optimized for the assay. 
     In another specific embodiment, the anti-product antibody is one that binds a newly revealed peptide terminus that is created after gamma-secretase mediated cleavage of the Notch substrate, such as an antibody raised using the polypetpide VLLSRKRRR (SEQ ID NO:2). An example of such an antibody is the SM320 antibody described herein. Other antibodies raised against analogous neoepitopes newly revealed upon proteolytic cleavage of a Notch substrate or a variant thereof are also useful for detecting the activity of γ-secretase on the substrate. 
     In another specific embodiment, a Notch substrate of the invention or fragment thereof includes a modification to at least one amino acid, including a conservative amino acid substitution, that preserves the modified substrate as a susceptible to the action of γ-secretase. 
     Homogeneous Time Resolved Fluorescence (HTRF) Assay for Notch Substrate Cleavage 
     In another embodiment, an assay method for detecting and quantifying the gamma-secretase catalyzed proteolysis of a Notch substrate is a Homogeneous Time Resolved Fluorescence (HTRF) assay. HTRF combines a) homogeneous fluorescence detection phase (e.g. carried out in liquid solution) with b) time resolution of signal detection and c) assessment of the distance separating an excitation donor and a fluorescence emitter to eliminate background fluorescence and provide both high sensitivity and high specificity of detection. A long-lived fluorophore, commonly a complex of a rare earth metal ion, such as a cryptate complex of the ion, permits detection to be delayed by time resolution until interfering background fluorescence will already have decayed. Fluorescence resonance energy transfer between specific fluorescence donor and fluorescence acceptor further enhance specificity by restricting ultimate detection of fluorescence to instances of, for example, complex formation between them. As implemented in this disclosure, an HTRF assay includes: (a) contacting a Notch substrate of the invention or a fragment thereof with a source of gamma-secretase, wherein the Notch substrate includes a detectable tag or label; (b) incubating the Notch substrate of the invention with a composition containing, or suspected of containing, gamma-secretase for a time period sufficient for gamma-secretase activity to take place thereby providing a product bearing the detectable tag or label; (c) adding an HTRF detection mixture that includes (i) a first reagent, such as an antibody, that recognizes a gamma-secretase-cleaved peptide resulting from Notch cleavage but does not recognize uncleaved gamma-secretase substrates and (ii) a rare earth metal-labeled second antibody that binds the first antibody, and (iii) a fluorophore-conjugated reagent that binds to the detectable tag or label; (d) incubating the HTRF detection mixture with the product mixture; and (e) measuring the presence and/or amount of the Notch cleavage product or fragment thereof using, generally, Fluorescence Resonance Energy Transfer (“FRET”), or more particularly, Homogeneous Time Resolved Fluorescence (HTRF), by exciting the rare earth metal and detecting fluorescence from the fluorophore of the conjugated reagent. A cognate assay may be performed on a negative control in which either the enzyme or the Notch substrate is omitted. 
     In a specific embodiment, a source of gamma-secretase is a cell or cell membrane, e.g., a HeLa cell or other mammalian cell, or a constituent membrane thereof, and the incubation of step (b) takes place in the presence of a detergent, e.g., CHAPSO, at a concentration optimized for the assay. 
     In a specific embodiment, the antibody that recognizes gamma-secretase-cleaved peptides resulting from cleavage of the Notch substrate or fragment thereof but does recognize uncleaved gamma-secretase substrates is the SM320 antibody. 
     Cell-Based Assays for Cleavage of Notch Substrates 
     In another embodiment, a method for assaying for the proteolytic cleavage of a Notch substrate of the invention by gamma-secretase is a cell-based assay. Such an assay may include: (a) transfecting cells containing, or suspected of containing, gamma-secretase activity with a plasmid containing the nucleotide sequence encoding a Notch substrate of the invention; (b) incubating the cells for a time period sufficient for the expression of the Notch substrate, and sufficient for gamma-secretase activity on the Notch substrate to occur; and (c) detecting a cleaved Notch substrate secreted by the cells, or included in a homogenate prepared from the cells, or in a mounted preparation of the cells on a surface. Detection of secreted product, or of product contained in a homogenate, may be carried out in various embodiments, for example, by Western analysis (SDS-PAGE and immunoblotting using a product-specific antibody), or by ECL, HTRF, chemiluminescence-coupled FRET (see Examples). Detection of product peptides in situ may be carried out by immunohistochemical analysis using an antibody specific for a product of the cleavage reaction. In various embodiments the antibody specific for a product of the cleavage reaction is the novel SM320 antibody disclosed herein. In certain embodiments, more generally, an antibody useful for detection in a cell-based assay specifically binds either the C-terminus or the N-terminus of the product peptide that is exposed after gamma-secretase mediated cleavage of the Notch substrate, such as disclosed herein. 
     In a specific embodiment, the cells contain endogenous gamma-secretase. In another specific embodiment, the cells are HEK293 cells or other mammalian cell. 
     In another embodiment, the level of product peptide resulting from the action of γ-secretase on a Notch substrate of the invention or a fragment thereof is measured by mass spectrometry/surface enhanced laser desorption/ionization time-of-flight analysis (SELDI-TOF). 
     Screens for Gamma-Secretase Modulators 
     ECL Screening Assay for Identifying Modulators of Gamma-Secretase Activity 
     In one embodiment, a method for the identification and/or validation of a gamma-secretase modulator may be an ECL assay, wherein said method includes: (a) contacting a candidate compound which is a potential modulator of gamma-secretase activity with a Notch substrate of the invention and a composition containing gamma-secretase to provide a candidate assay mixture, (b) incubating the candidate assay mixture for a time period sufficient for gamma-secretase activity to take place; (c) adding ruthenylated antibody that specifically binds a cleavage product resulting from the action of the gamma-secretase; and (d) detecting the γ-secretase product by the product-specific ruthenylated antibody using ECL. Using this assay, a candidate compound is identified or validated as a gamma-secretase modulator if the activity of gamma-secretase toward a Notch substrate is altered either positively or negatively relative to the activity of gamma-secretase toward the Notch substrate in the absence of the candidate modulator. 
     In a specific embodiment, the source of gamma-secretase activity is a cell membrane, e.g., a cell membrane from a HeLa cell or other mammalian cell, and the incubation of step (b) takes place in the presence of a detergent, e.g., CHAPSO (0.25%). 
     In another specific embodiment, the antibody binds either the C-terminus or the N-terminus of the Notch substrate of the invention or fragment thereof that is exposed after gamma-secretase mediated cleavage of the substrate, such as the novel SM320 antibody disclosed herein. 
     HTRF Screening Assay for Modulators of Gamma-Secretase Activity 
     In another embodiment, an assay method for the identification and/or validation of a gamma-secretase modulator for proteolytic cleavage of a Notch substrate or fragment thereof by gamma-secretase uses HTRF. This method includes steps of: (a) contacting a candidate compound that is a potential gamma-secretase modulator with a Notch substrate and a composition containing gamma-secretase activity to provide a candidate assay mixture; wherein the Notch substrate includes a detectable tag or label; (b) incubating the candidate assay mixture under suitable conditions for a time period sufficient for gamma-secretase activity to take place; (c) adding an HTRF detection mixture that includes (i) a first antibody that recognizes a gamma-secretase-cleaved peptide resulting from cleavage of the Notch substrate but does not recognize uncleaved Notch substrate, (ii) a rare earth metal-labeled second antibody that binds the first antibody, and (iii) a fluorophore-conjugated reagent that binds to the detectable tag or label; (d) incubating said HTRF detection mixture with the candidate assay mixture; and (e) measuring the cleavage of the Notch substrate of the invention or variant thereof by gamma-secretase using, generally, Fluorescence Resonance Energy Transfer (“FRET”), or more particularly, Homogeneous Time Resolved Fluorescence (HTRF), by exciting the rare earth metal and detecting fluorescence from the fluorophore of the conjugated reagent. A candidate compound is identified or validated as a gamma-secretase modulator if the activity of gamma-secretase toward Notch substrate is modulated either positively or negatively relative to the activity of gamma-secretase toward the Notch substrate in the absence of the candidate gamma-secretase modulator. 
     In a specific embodiment, a source of gamma-secretase is a cell or cell membrane, e.g., a HeLa cell or other mammalian cell or a constituent membrane thereof, and the incubation of step (b) takes place in the presence of a detergent, e.g., CHAPSO, at a concentration optimized for the assay. 
     In a specific embodiment, the antibody that recognizes gamma-secretase-cleaved Notch substrate resulting from cleavage of the Notch substrate but does not recognize uncleaved Notch substrates is the novel SM320 antibody disclosed herein. 
     Cell-Based Assays for Identifying Modulators of Gamma-Secretase Activity 
     In another aspect, an assay method for the identification or validation of a gamma-secretase modulator includes a cell-based assay. This method includes: (a) transfecting cells that harbor gamma-secretase activity with a plasmid containing the nucleotide sequence encoding a Notch substrate; (b) adding a candidate modulator of gamma-secretase activity to provide a candidate assay mixture; (c) incubating said candidate assay mixture for a time period sufficient for gamma-secretase activity to occur; and (d) detecting a cleaved Notch substrate product secreted by the cells, or included in a homogenate prepared from the cells, or in a mounted preparation of the cells on a surface. Detection of secreted product, or of product contained in a homogenate, may be carried out in various embodiments, for example, by Western analysis (SDS-PAGE and immunoblotting using a product-specific antibody), or by ECL, HTRF, chemiluminescence-coupled FRET (see Examples). Detection of product peptides in situ may be carried out by immunohistochemical analysis using an antibody specific for a product of the cleavage reaction. In various embodiments the antibody specific for a product of the cleavage reaction is the novel SM320 antibody disclosed herein. In certain embodiments, more generally, an antibody useful for detection in a cell-based assay specifically binds a gamma-secretase-cleaved peptide resulting from cleavage of the Notch substrate but does not recognize uncleaved Notch substrate. As a result of this assay a modulator of gamma-secretase activity is identified or validated if the activity of gamma-secretase toward the Notch substrate is modulated either positively or negatively relative to the activity of gamma-secretase toward the Notch substrate in the absence of the candidate modulator. 
     In a specific embodiment, a source of gamma-secretase is a cell or cell membrane, e.g., a HeLa cell, HEK293 cell, or other mammalian cell or a constituent membrane thereof, and the incubation of step (b) takes place in the presence of a detergent, e.g., CHAPSO, at a concentration optimized for the assay. 
     In another specific embodiment, an antibody that binds either the C-terminus or the N-terminus of the Notch substrate that is exposed after gamma-secretase mediated cleavage of the substrate, such as the novel SM320 antibody disclosed herein, is added to the cell conditioned media (containing secreted Notch-derived product) and the level of the Notch product peptide secreted by the cells is measured by ECL. 
     In another embodiment, the amount of a Notch product peptide secreted by the cells or contained in a cell homogenate obtained from the candidate assay mixture is measured by Western Blot, using an antibody that specifically binds the Notch product peptide. In a specific embodiment, the antibody binds either the C-terminus or the N-terminus of the Notch substrate of the invention or variant thereof that is exposed after gamma-secretase mediated cleavage of the substrate, such as the novel SM320 antibody disclosed herein 
     In another embodiment, the amount of a Notch product peptide secreted by the cells or contained in a cell homogenate obtained from the candidate assay mixture is measured by mass spectrometry/surface enhanced laser desorption/ionization time-of-flight analysis (SELDI-TOF). 
     Combinatorial Chemical Libraries 
     Assays for gamma-secretase activity, using labeled or detectable Notch substrates, are identified herein. As described herein, and disclosed in several Examples herein, these assays are adaptable for application in high throughput screens of candidate chemical compounds in a quest for modulators of gamma-secretase activity. In many embodiments such assays are implemented in multiwell plates, including 96-well, 384-well, and 1536-well plates. Candidate compounds are provided for these screens from extended chemical libraries. Preparation of chemical libraries are widely known in the field. Combinatorial approaches to introducing framework components as well as peripheral substituents have been developed, including techniques for tagging each synthesis so that intermediates and products are identified throughout the course of the synthesis. Other libraries are prepared from a broad range naturally occurring substances, and still others from assemblages of pharmaceutical agents already known to possess therapeutic effects or therapeutic potential for a broad range of medical indications. Nonlimiting examples of preparation and uses of chemical libraries, including combinatorial chemical libraries, include U.S. Pat. No. 7,083,812, entitled “Chemical library preparation method from natural product”; U.S. Pat. No. 6,936,477, entitled “Complex combinatorial chemical libraries encoded with tags”; U.S. Patent Application Publication 20090005256, entitled “Analysis of Encoded Chemical Libraries”; U.S. Pat. No. 6,800,444, entitled “Complex chemical libraries”; International publication WO/2006/102542, entitled “Diverse Chemical Libraries Bound To Small Particles With Paramagnetic Properties”; U.S. Pat. No. 6,625,546, directed to the direct identification of a chemical compound structure following solid phase synthesis of a chemical compound library; U.S. Pat. No. 6,625,546, directed to methods for using structural identification technology to increase the productivity of solid phase synthesis strategies; and “Designed chemical libraries for hit/lead optimization,” Cooper T and Andrews-Cramer, K, Innovations in Pharmaceutical Technology, June 2000, pp. 46-53. 
     EXAMPLES 
     Experimental Procedures 
     Peptide Synthesis 
     The neoepitope peptide VLLSRKRRRC corresponding to the new N-terminal sequence generated when γ-secretase cleaves human Notch1 at the S3 site (24; residues 1755-1763) was synthesized with an automated solid phase peptide synthesizer (ProteinTech) using Fmoc chemistry. The peptides were cleaved from the resin with Reagent R (90% TFA, 5% thioanisole, 3% EDT, 2% anisole) for two hours and then precipitated with cold ethyl ether. Precipitated peptides were lyophilized and confirmed with HPLC and LC/MS (Agilent). 
     Production of Antibody SM320 Specific for Detecting γ-Secretase Activity on Notch 
     The neoepitope VLLSRKRRRC, which serves as a peptide antigen to detect the Notch cleavage product, was conjugated to maleimide functionalized keyhole limpet hemocyanin (KLH) according to the manufacturer&#39;s instructions (Pierce Chemical Co., Rockford, Ill.). The KLH-conjugated antigen was sent to Covance Inc. for rabbit vaccination. Once serum was collected, two volumes of 60 mM sodium acetate buffer (pH 4.0) was added to the serum. Caprylic acid was added to the serum and the resulting mixture was stirred for 30 minutes at room temperature. The mixture was then centrifuged at 5000 g for 10 minutes and the supernatant was dialyzed into phosphate buffered saline (PBS). Dialzyed supernatant that contains predominantly IgG immunoglobulin was passed through a column of resin (Pierce) derivatized with immobilized VLLSRKRRRC antigen to capture the anti-Notch peptide antibody. After initial characterization of two sera, SM320 and SM321, neoepitope antibodies were purified by affinity chromatography with peptide immobilized resin. SM320 was used for this study. The purified antibody, named SM320, was stored at −80° C. 
     Production of Recombinant Notch1 substrate, N1-Sb1 
     A recombinant synthetic substrate based on human Notch 1 called N1-Sb1 was constructed as diagrammed in  FIG. 1A . First, a fragment of human Notch1 (residues 1733-1812) was subcloned into the PacS vector that contains an AviTag. AviTag, a specific 15 residue peptide sequence (GLNDIFEAQ K IEWHE (SEQ ID NO:3); Avidity, LLC; U.S. Pat. Nos. 5,723,584, 5,874,239 &amp; 5,932,433) may be biotinylated with biotin ligase on the underlined K. This Notch1-AviTag construct was further subcloned into the pIAD16 vector that contains a chimeric maltose binding protein (MBP)-thrombin target sequence to provide a MBP-thrombin site-Notch fragment-AviTag construct. The MBP facilitates protein purification. The resulting MBP-N1-Sb1 plasmid ( FIGS. 2-4 ) was co-transformed into the BL21 (DE3)  E. coli  strain with the pACYC184 plasmid (BirA) that encodes biotin ligase. IPTG (0.1 mM) and 50 μM of biotin were added to the cell culture to induce protein synthesis and biotinylation of the AviTag sequence. Cells were centrifuged at 8000 g for 30 minutes and the pelleted cells were lysed by passage through a French Press (Spectronics Instruments). The cell homogenate was centrifuged at 17,000 g for 30 minutes. The supernatant was affinity purified on an amylose resin column using the AKTAprimer chromatographic system (Amersham Bioscience). The expression of MBP-tagged N1-Sb1 was identified with SDS-PAGE and the mass was confirmed with LC/MS. MBP was removed from N1-Sb1 with thrombin, as demonstrated by SDS-PAGE and LC/MS ( FIG. 1 , panels B and C). The P2 substituted N1-Sb1 substrates were generated with Strategene Site-directed Mutagenesis kit, expressed and purified as described for wild-type N1-Sb1. APP recombinant substrate, Sb4, was produced as described previously (Tian et al., 2010, Nat Struct Mol. Biol., 17:151-158). 
     Cell-Based Notch Cleavage Assay with Western Using ELISA Detection with SM320 Antibody. 
     HEK293 cells were seeded in a 24-well plate and transfected with either truncated Notch1-ΔE construct (N1-ΔE) (gift from Dr. Raphael Kopan, Washington University School of Medicine, St. Louis, Mo.; (see Supplemental  FIG. 1A  of van Tetering et al., 2009, J. Biol. Chem., 284:31018-31027, for a diagram of N1-ΔE)) including cmyc (24), or empty pcDNA3.1 (−) (Invitrogen), using Fugene 6 transfection reagents (Roche). N1-ΔE includes the replete intracellular sequence, the transmembrane domain, and a truncated extracellular portion lacking LNR and the EGF repeat sequences (see Supplemental  FIG. 1A  of van Tetering et al., 2009, J. Biol. Chem., 284:31018-31027). DMSO carrier or 1 μM Compound E were added to the transfected cells, at a final DMSO concentration of 1% (v/v). After 48 hours of treatment, the cells were washed with PBS and lysed with 1×RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.1% (wt/vol) SDS, 1% (vol/vol) Nonidet P-40, and 0.5% (wt/vol) deoxycholic acid). Cell lysates were centrifuged at 13,000×g at 4° C. The cleared supernatant was collected and resolved by SDS-PAGE. The proteins were transferred to PVDF membrane (Millipore) using a semi-dry transfer emit (Bio-Rad) and analyzed by western blot with anti-myc antibody (prepared at Memorial Sloan-Kettering Cancer Center) and SM320 antibody. 
     Cell-Based Notch Cleavage Assay with Immunostaining Using SM320 Antibody. 
     HEK293 cells were seeded in a 4-well chamber slide (Lab-tek) and transfected with N1-ΔE as in the preceding paragraph, using Fugene 6 transfection reagents (Roche Applied Science, Mannheim, Germany). Transfected cells were then treated with either DMSO at a 1% final concentration or 1 μM Compound E. 
                                
After 48 hours of treatment, the cells were washed with PBS, fixed with cold methanol and permeabilized with 0.25% Tween-20. SM320 and anti-myc antibodies were added to detect NICD and full length N1-ΔE, respectively. Alexa Fluor-488 (Invitrogen, San Diego, Calif.) conjugated anti-rabbit and Alexa Fluor-594 (Invitrogen) conjugated anti-mouse antibodies were used as corresponding secondary antibodies. 4′,6-diamidino-2-phenylindole (DAPI) was used to stain cell nuclei. Finally, fluorescent images were captured with a Leica TCS Sp2 AOBS laser scanning confocal microscope (Memorial Sloan-Kettering Cancer Center Molecular Cytology Core Facility). Alternatively, REK 293 cells overexpressing N1-ΔE were treated with DMSO or Compound E, and were then collected through centrifugation and embedded in paraffin. The cell pellets were sliced and mounted on glass slides. The immunodetection of NICD was performed at the Molecular Cytology Core Facility of Memorial Sloan Kettering Cancer Center using Discovery XT processor (Ventana Medical Systems). The cell sections were blocked for 30 minutes in 10% normal goat serum in 0.2% BSA/PBS, followed by incubation for 5 h with 0.5 μg/ml of SM320 and incubation for 60 min with biotinylated goat anti-rabbit IgG (Vector labs, cat#:PK6101; 1:200 dilution). The detection was performed with DAB-MAP kit (Ventana Medical Systems). The slides were scanned and digitized using the Mirax Scanner (Carl Zeiss Microsystems).
 
Cellular Membrane Preparation
 
     HeLa membrane fraction was isolated from HeLa-S3 cells (National Cell Culture Center). The cell pellet was resuspended in Buffer A (50 mM MES, 150 mM KCl, 5 mM CaCl 2 , 5 mM MgCl 2 ) and lysed by passage through a French Press. N2a cells (murine neuroblastoma cells) over-expressing wild-type PS1, or mutants M146L or E280A, were obtained from Dr. Sangram Sisodia (University of Chicago, Chicago, Ill.). These cells were maintained in 50% Dulbecco&#39;s modified Eagle&#39;s medium high glucose, 50% Opti-Mem Reduced Serum Media, and 10% fetal calf serum. Cultured cells were pelleted by centrifugation and resuspended in hypotonic buffer (40 mM Tris, pH 7.4, 10 mM NaCl, 1 mM EDTA, and 0.5 mM DTT) for 20 minutes before being lysed with a dounce homogenizer. Nuclear debris from the HeLa and N2a cells were pelleted at 3000 rpm for 30 minutes. The resulting supernatants were ultracentrifuged at 100,000×g for 1 hour. The pellet that contained total cell membrane was resuspended in Buffer A. Mouse brains overexpressing knock-in wild type or M146V PS1 (gift from Dr. Hui Zheng, Amylin Pharmaceuticals, San Diego, Calif.) were homogenized with dounce homogenizer. The cell lysate was centrifuged at 3000 rpm for 30 minutes. The supernatants were ultracentrifuged at 100,000×g for 1 hour. Protein concentration was determined with the DC Protein Assay Kit according to the manufacturer&#39;s instructions (Bio-Rad, Hercules, Calif.). 
     Development of an In Vitro γ-Secretase Assay with N1-Sb1. 
     An in vitro γ-secretase assay using a novel recombinant APP-derived substrate (termed “Sb4”) is disclosed in co-pending application U.S. Ser. No. 12/776,141. In the present assay based on a Notch-derived substrate, N1-Sb1 was incubated with the cell membrane fraction, prepared as described above, in the presence of 0.25% CHAPSO and 1×PIPES buffer (50 mM PIPES, pH 7.0, 150 mM KCl, 5 mM CaCl 2 , 5 mM MgCl 2 . The reaction was incubated at 37° C. for 2 hours. The Perkin Elmer AlphaScreen™ detection system was employed to assay for Notch cleavage. AlphaScreen (PerkinElmer Life and Analytical Sciences, Shelton, Conn.) is a bead based non-radioactive Amplified Luminescent Proximity Homogeneous Assay (ALPHA). When a biological interaction brings the beads together, a cascade of chemical reactions acts to produce a greatly amplified signal. Specifically, upon laser excitation, a photosensitizer in the “Donor” bead converts ambient (triplet) oxygen to the more reactive singlet state. The singlet state oxygen molecules diffuse into the solvent, and may react with a thioxene derivative in the Acceptor bead if nearby, generating chemiluminescence at 370 nm that further activates fluorophores contained in the same bead. The fluorophores subsequently emit light at 520-620 nm. In the absence of a specific biological interaction, the singlet state oxygen molecules produced by the donor bead go undetected without the close proximity of the acceptor bead. As a result no fluorescence signal at 520-620 nm is produced. 
     In the present system, antibody SM320, Protein A-conjugated acceptor beads, and Streptavidin-conjugated donor beads (PerkinElmer) were added to the reaction at a final concentration of 0.2 μg/ml, 2.5 μg/ml and 5 μg/ml, respectively (diagrammed in  FIG. 5A ). The reaction was incubated in the dark at room-temperature for 4 hours. The ALPHA signal was detected using the Envision plate reader (Perkin Elmer). The reactions were excited at 680 nm and signal was detected at 615 nm. 
     Photolabeling Probes and Photolabeling. 
     Total cell membrane was pre-incubated in the presence of DMSO or 1 μM L458 in PIPES buffer containing 0.25% CHAPSO at 37° C. for 30 minutes. Then, photolabeling probes (JC8, L646 or GY4) (Li et al., 2000, Nature, 405:689-694, Chun et al., 2004, J Org. Chem., 69:7344-7347, Yang et al., 2009, Bioorg Med Chem. Lett., 19:922-925) at 10 nM were added and incubated for an additional 1 hour at 37° C. The reaction mixtures were irradiated at 350 nm for 45 minutes and solubilize with RIPA buffer. Biotinylated proteins in the soluble fraction were captured by Streptavidin resin (Pierce) overnight at 4° C. Bound proteins were eluted by boiling with SDS sample buffer and analyzed by western blotting with anti-PS1 NTF antibodies. 
     Example 1 
     The SM320 Antibody Specifically Recognizes γ-Secretase-Cleaved Notch1 Substrate 
     The SM320 polyclonal antibody directed against the Notch cleavage product epitope (VLLSRKRRRC) was generated as described in Experimental Methods and its specificity determined. The SM320 antibody was purified by conjugating the epitope peptide to an affinity column and capturing the specific antibody, as described. 
     HEK 293 cells were transiently transfected with truncated Notch1-ΔE construct including cmyc (N1-ΔE), a substrate of γ-secretase that is independent of ligand activation, as described in Experimental Methods. Transfected cells were treated with either DMSO vehicle or Compound E, a potent γ-secretase inhibitor. Cell lysates were analyzed by performing SDS-PAGE and immunoblotting the result with anti-myc and SM320 antibody ( FIG. 6A ). The expression of the myc-tagged N1-ΔE protein was confirmed with anti-myc antibody. N1-ΔE was detected only in cells transfected with this construct ( FIG. 6A , upper panel, lanes 1 and 2) but not in the mock transfected cells ( FIG. 6A , upper panel, lane 3). More importantly, SM320 detected a specific band that co-migrates with cleaved Notch IntraCellular Domain (NICD) in N1-ΔE transfected cells ( FIG. 6A , lower panel, lane 1), but not in the Compound E-treated cells in which γ-secretase activity is expected to be blocked ( FIG. 6A , lower panel, lane 2). This western blot analysis confirms that SM320 specifically detects γ-secretase cleaved product, but not the uncleaved substrate. 
     SM320 was further characterized using immunostaining analysis. The transfected cells were fixed and permeabilized. Following this, N1-ΔE and NICD were immunostained with SM320 and anti-myc antibodies, respectively. Using confocal microscopy, it was shown that N1-ΔE protein, as detected by anti-myc antibody, is expressed in cells transfected with the construct ( FIG. 6B , panel 2). SM320 was able to detect NICD only in cells treated with DMSO vehicle ( FIG. 6B , panel 3) but not in cells treated with Compound E ( FIG. 6B , panel 5). Moreover, the NICD staining co-localized with DAPI staining and thus confirming the translocation of NICD into the nucleus upon γ-secretase cleavage ( FIG. 6B , panel 4). Taken together, the western blot and immunostaining analyses established the specificity of antibody SM320 in detecting γ-secretase cleaved Notch product. 
     Example 2 
     Development of a Biotinylated Recombinant Notch1 Substrate and an In Vitro γ-Secretase Assay 
     It has been previously demonstrated that biotinylated recombinant APP substrates are suitable for the development of robust γ-secretase assays (21-23; co-pending application U.S. Ser. No. 12/776,141). In order to develop a similar ELISA-like assay based on Notch, an AviTag and a maltose binding protein (MBP)-thrombin cleavage site tag were appended to a Notch1 protein fragment (residues 1733-1812) coding sequence (see Experimental Procedures). The product is designated MBP-N1-Sb1 ( FIG. 1A ).  E. coli  cells were co-transformed with this plasmid as well as the pACYC184 plasmid, which encodes biotin ligase, in the presence of biotin. Expression of biotin ligase during induction catalyzes the attachment of biotin to the AviTag. The MBP tag was removed from N1-Sb1 by thrombin cleavage and the product was analyzed by SDS-PAGE and LC-MS. The apparent molecular weight of N1-Sb1 in SDS-PAGE was larger than the expected 12 kDa ( FIG. 1B ). However, LC-MS showed two species of N1-Sb1: a minor peak at 12017.5 Da and a major peak at 12246.5 Da ( FIG. 1C ), which match the calculated molecular mass of N1-Sb1 in a non-biotinylated form (12017.7) and biotinylated form (12245.7). Since there is very little non-biotinylated N1-Sb1 (&lt;5%), this suggests a high efficiency of biotin ligation during induction. 
     HeLa membrane was incubated with N1-Sb1 substrate in the presence of 0.25% CHAPSO. The γ-secretase cleavage product (cN1-Sb1) was detected by the PerkinElmer proximity assay (ALPHA) using the SM320 antibody, Protein A-conjugated acceptor-beads (which bind SM320) and streptavidin-conjugated donor-beads (which bind biotin) ( FIG. 5A ). cN1-Sb1 levels in DMSO-treated assay was 10-fold higher than in the presence of the inhibitor L-685,458 (L458; structure shown below)-treated assay. The IC 50  values of L458 and Compound 
                                
E in inhibiting N1-Sb1 cleavage were also determined The values are 0.7 nM and 1 nM, respectively ( FIG. 5B ). Finally, we have used this assay to demonstrate that the apparent Km of N1-Sb1 cleavage by γ-secretase is 0.15±0.03 μM and the Vmax is 38 Unit/μg/min (in arbitrary units;  FIG. 5C ). The above data shows that this in vitro γ-secretase assay is both specific and sensitive, and can be used to assay for γ-secretase cleavage of Notch1.
 
     Example 3 
     PS1 FAD Mutations have Distinct Effects on Notch1 Cleavage 
     The effects of PS1 FAD mutations on Notch cleavage were determined using the new γ-secretase assay described in Example 2. The activities of two PS1 FAD mutations—PS1 M146L and E280A were compared against wild-type PS1 (WT), which has been previously studied (25). Total membrane was isolated from N2a cells that stably express WT, M146L, or E280A mutants. N1-Sb1 was incubated with these membranes and the resulting cN1-Sb1 was detected with SM320. A significant reduction was observed in N1-Sb1 cleavage by both PS1 FAD mutants compared to the wild-type PS1. The M146L and E280A mutants had 40% and 14% activity remaining, respectively, compared to the activity of the wild type ( FIG. 7A ). To compare the effects of these two PS1 FAD mutants on APP cleavage, similar experiments were carried out using the APP substrate Sb4 (25) (see co-pending application U.S. Ser. No. 12/776,141). It was found that M146L and wild-type PS1 have similar activity against Sb4 cleavage as determined by the production of A1340 from Sb4 ( FIG. 7B ). However, PS1 bearing the E280A mutation has 12% activity in the cleavage of Sb4 remaining ( FIG. 7B ). These data suggest that these two PS1 mutations affect the activity of γ-secretase differentially for Notch or Aβ40 cleavage. The E280A PS1 mutant has a significant loss of function with respect to both Notch1 and Aβ40 cleavages while the M146L PS1 mutant has reduced activity against Notch but not Aβ40 cleavage. 
     Mouse brain specimens were obtained that either expressed knock-in wild type PS1 or the M146V PS1 mutation (25, 26). Total membrane from these mouse brains was isolated, and the membrane was incubated with N1-Sb1. The cN1-Sb1 cleavage product was detected with the new in-vitro γ-secretase assay of Example 2. It was shown that the M146V PS1 mutant in mouse brain has less activity against Notch cleavage compare to wild-type PS1, whereas this mutation has no effect on Sb4 cleavage. ( FIGS. 7C and 7D ). 
     Next, the effect of these PS1 mutations on the kinetics of γ-secretase activity for Notch1 cleavage was assessed. The data show that the Km values for N1-Sb1 cleavage by PS1 wild-type, M146L, and E280A FAD mutants are 70, 49, and 50 nM, respectively ( FIG. 8A ). The Vmax values found for PS1 wild-type, M146L, and E280A FAD mutations are 293, 163 and 49 U/μg/min, respectively. Further, N1-Sb1 was titrated in the presence of membrane fractions isolated from PS1 WT and M146V mouse brain. The Km values of the brain membrane data is consistent with the cell-line data. The Km values of WT and M146V PS1 are 60 and 48 nM, respectively; while the Vmax is 52 and 40 U/μg/min, respectively ( FIG. 8B ). These data suggest that M146L, M146V and E280A PS1 mutations reduce the Vmax of PS1 against Notch1 cleavage compared to WT PS1. 
     Further, effects of the FAD mutations on the potencies of γ-secretase inhibitors L458 and Compound E were investigated. It was found that Compound E inhibited wild-type PS1, M146L PS1 and E280A PS1 with similar IC50&#39;s of 0.78 nM, 0.76 nM and 0.71 nM, respectively ( FIG. 8C ); while the IC50&#39;s of L458 inhibition are 0.50 nM, 0.45 nM, and 0.46 nM for wild-type PS1, M146L PS1 and E280A PS1, respectively ( FIG. 8D ). These data suggest that FAD mutations do not alter the potency of γ-secretase inhibitors. 
     Example 4 
     Development of a 384- and 1536 Well Format ALPHA-Based γ-Secretase Assay 
     The 384-well plate assay was optimized by titrating the amount of the SM320 antibody. It was shown that 0.1 ug/ml of SM320 yielded the highest signal ( FIG. 9A ). Next, the concentrations of Protein A conjugated acceptor beads and streptavidin conjugated donor beads were titrated. Although it was found that streptavidin donor beads at 10 ug/ml has higher signal than 5 ug/ml, there is no significant improvement in the signal to noise ratio ( FIG. 9B ). It was also shown that 1.25 ug/ml of protein A acceptor beads yielded optimum signal to noise ratio although higher signal can be obtained when higher bead concentrations are used ( FIG. 9C ). By using 0.1 ug/ml of SM320, 5 ug/ml of streptavidin donor beads and 1.25 ug/ml of protein A acceptor beads in the final 384-well assay conditions, a 25-fold signal to noise ratio was obtained ( FIG. 9D ). 
     Based on the 384-well assay format described above, to the assay was miniaturized to a 1536-well format for automated high-throughput drug screening. Concentrations of SM320, protein A conjugated acceptor beads and streptavidin conjugated donor beads were titrated to minimize reagent usage. It was found that 0.05 ug/ml SM320, 0.5 ug/ml Protein A conjugated acceptor beads and 2.5 ug/ml streptavidin conjugated donor beads yielded optimal 7-fold signal to noise ratio ( FIG. 9D ). The assay was validated by comparing the high and low control signals from full 1536-well plates. The high controls contained 1% final DMSO concentration (v/v), while the low controls contains 250 nM of Compound E also at 1% final DMSO concentration (v/v). The results are in Table 3. 
     
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                   
                 Average 
                 STDEV 
                 Z′ 
                 S/N 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 High Control 
                 2487 
                 198 
                 0.66 
                 7.8 
               
               
                 Low Control  
                 319 
                 51 
               
               
                   
               
               
                 S/N = signal-to-noise. 
               
             
          
         
       
     
     Example 5 
     PS1 Mutations Alter the S2 Subpocket of γ-Secretase 
     Activity-based probes have been widely used to identify and profile various classes of enzymes. L-685,458 (L458), an aspartyl protease transition state mimic that interacts with the active site of γ-secretase ( FIG. 10 a   ), has been a valuable tool to study γ-secretase. Therefore, we have generated various potent photoactivatable probes through “photophore walking”, in which the photoactivatable benzophenone is separately incorporated into different postions along the peptidomimetic core structure of L458. These positions are also known as the P or P′ positions according to Schecter and Berger nomenclature. We intented to apply these L458-based probes to sense the subsite (S and S′) conformational change within the active site of γ-secretase caused by PS1 FAD mutations. The rationale of this strategy is that the efficiency of photolabeling by these photoactivatable probes depends on the orientation of the probes and their proximity to residues within the active site. Conformational changes resulting from PS1 mutations within the active site alter the orientation or distance of contact regions with the probes and could lead to different crosslinking efficiencies. 
     L646, GY4 and JC8, which photolabel the S2, S1, and S1′ subsites within the active site of PS1, respectively, were used for this study ( FIG. 10 b   ). Membranes isolated from N2a cells stably expressing wild-type (WT), M146L, or E280A PS1 were photolabeled with JC8, GY4 or L646 in the presence of 0.25% CHAPSO. The labeling was determined to be specific because incubating the probes in the presence of excess L458 completely prevented labeling. After ITV irradiation, biotinylated proteins were isolated with streptavidin beads and analyzed by western blotting with anti-PS1 NTF antibodies. First, we showed that JC8, GY4 and L646 all photolabeled WT, M146L, and E280A PS1 NTF ( FIG. 10 c   ). To eliminate the effect of various amounts of γ-secretase existing in different cell lines, we normalized each probe with JC8 labeling in the same cell lines. L646 labeled M146L and E280A PS1 NTF with ˜80% less intensity than JC8; while L646 was photoinserted into WT PS1 with the same efficiency as JC8 ( FIG. 10 d   ). This result strongly indicates that the reduced labeling of PS1 mutants by L646 is not due to decreased expression of γ-secretase in the different stable cell lines, but due to conformational changes within the S2 subsites of M146L and E280A PS1. Finally, we showed that GY4 labeled WT, M146L and E280A PS1 NTF with similar efficiencies ( FIGS. 10 c  and 10 d   ). 
     To further investigate the effect of PS1 mutations on the active site in an in vivo setting, we performed the same study using M146V and WT PS1 knock-in mouse brains. The M146V PS1 knock-in mice have been well characterized for memory formation and Aβ production. Membrane isolated from the M146V and WT PS1 knock-in mouse brain was labeled with photoprobes JC8 and L646. Similarly, we found that JC8 and L646 labeled WT PS1 with similar intensities ( FIGS. 10 e  and 10 f   ). However, L646 labeled M146V PS1 with 84% less efficiency than JC8 ( FIGS. 10 e  and 10 f   ). This data also showed that M146V PS1 leads to a similar conformation change at the S2 subsite. Taken together, these studies indicate that PS1 FAD mutations directly influence the shape of the active site within the PS1 γ-secretase complex. However, it is still unclear how this change in the S2 subsite of the gamma-secretase active site affects the interaction and catalysis of substrates such as APP and Notch1. 
     Example 6 
     PS1 Mutants Prefer N1-Sb1 with Larger P2 Residues 
     Finally, we probed the S2 subsite of the γ-secretase active site with mutated P2 Notch1 substrates. The rationale of this study was to investigate whether larger P2 Notch substrate residues can enhance the activity of PS1 FAD mutants. We generated a series of P2 site mutations corresponding to human Notch1 residue 1752 (Cys) that interacts with the S2 subsite within the gamma-secretase active site ( FIG. 11 a   ). We substituted Cys with either Ala, Val, or Met using site-directed mutagenesis ( FIG. 11 b   ). After these substrates were purified, we determined the rate of γ-secretase cleavage from each cell membrane against four substrates ( FIG. 11 c   ). First, the C1752A substrate had significantly reduced reactivity for all three forms of γ-secretase (20-30% remaining) compared with WT substrate. Secondly, C1752V substrate had considerably increased reactivity for M146L PS1 γ-secretase (141.3±17.9%0 (middle panel) and had no effect on WT (left panel) and E280A PS1 (right panel). Thirdly, C1752M substrate had no effect on WT (left panel), but had significantly enhanced activity for M146L (166.4±6.1%) (middle panel) and E280A (185.5±7.4%) PS1 (right panel). Similarly, we showed that C1752M substrate also had no effect on WT PS1 (left panel), but had enhanced activity for M146V knock-in PS1 (127.4±3.7%) (right panel) ( FIG. 11 d   ). Taken together, these data demonstrate that N1-Sb1 with a Met residue at the P2 position is a better substrate for both PS1 mutants, suggesting that Met fits the altered S2 subsites of PS1 mutants better than Cys. On the other hand, N1-Sb1 with a Val at the P2 position is a better substrate for M146L PS1 but not for WT PS1 and E280A PS1, suggesting that the S2 subsite of M146L PS1 is distinguishable from the S2 subsite of E280A PS1. These results indicate that the different PS1 mutations, such as M146L and E280A PS1, can lead to S2 subsite variation, and cause different effects on γ-secretase processing of APP and Notch1. 
     The data presented here demonstrate that the PS1 mutants, M146L and E280A, directly affect γ-secretase activity, which leads to a reduction in the rate of Notch1 cleavage. However, M146L PS1 has no effect on Aβ40 production and E280A PS1 has reduced Aβ40 production. These studies indicate that PS1 mutations could lead to different effects on APP and Notch1 cleavage. Our in vitro assay offers a unique way to characterize the effects of PS1 mutations on Notch1 and APP cleavage, as well as providing a way to address whether PS1 could contribute to AD through altering the processing of APP and Notch1. 
     Furthermore, our Notch1 substrate P2 residue mutagenesis studies demonstrated that both PS1 mutations, M146L and E280A, lead to similar and yet distinguishable S2 subsite alteration. Although both PS1 mutations prefer Met P2 residue over Cys, M146L and E280A exhibited different activities for Val at the P2 position. Moreover, a smaller residue at the P2 position of Notch1 substrate dramatically reduced its reactivity with γ-secretase. Taken together, these multiple photoaffinity labeling and substrate complement studies indicate that both M146L and E280A mutations lead to a deeper and distinct S2 subsite. This conformational change in the active site could be a plausible mechanism on how PS1 mutations affect •-secretase activity for Aβ40 and Aβ42 production. It has been shown through a sequential cleavage mechanism that Aβ40 and Aβ42 peptides are generated from Aβ49 and Aβ48 peptides, respectively ( FIG. 12 a   ). The P2 residues for Aβ40 and Aβ49 cleavages are Val and Thr, respectively ( FIG. 12 b   ), which are relatively smaller residues, while the P2 residues for both Aβ42 and Aβ48 are Ile, a larger residue ( FIG. 12 b   ). A deeper S2 subsite would favor Aβ48 cleavage, leading to an increase in Aβ42. Concurrently, these changes can affect Aβ49 and Aβ40 cleavages ( FIG. 12 a   ). It has been reported that PS1 FAD mutations reduce A1349 production while concomitantly increasing Aβ48 levels, which supports our S2 subsite alteration model ( FIG. 12 a   ). 
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     The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. Supplementary materials referenced in publications (such as supplementary tables, supplementary figures, supplementary materials and methods, and/or supplementary experimental data) are likewise incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims. 
     Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. 
     Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements. 
     All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.