Purified 20 kDA presenilin 2 C-terminal fragment and methods of screening for compounds that inhibit proteolysis of presenilin 2

#1. Field of the Invention

#The present invention relates, in general, to presenilin 2 proteolytic fragments. In particular, the present invention relates to a purified 20 kDa presenilin 2 C-terminal fragment (PS2-CTF); purified nucleic acid molecules coding for the 20 kDa PS2-CTF protein; cells containing the nucleic acid molecules; non-human organisms containing the nucleic acid molecule; antibodies having specific binding affinity to the 20 kDa PS2-CTF; hybridomas containing the antibodies; methods of detecting 20 kDa PS2-CTF in a sample; diagnostic kits; methods for screening compounds that inhibit proteolytic processing of presenilin 2 in a cell, isolated compounds that inhibit proteolytic processing of presenilin 2 in a cell, and methods of inhibiting apoptotic cell death by preventing proteolytic cleavage of presenilin 2 at a cleavage site which generates a 20 kDa C-terminal fragment.

#2. Related Art

#FIG. 1. Inducible expression and endoproteolytic cleavage of PS2. Detection of C- and N-terminal PS2-derived cellular fragments. Western blot analysis of SDS lysates prepared from stably transformed, PS2-inducible H4 human neuroglioma cells grown in the presence (1 μg/ml) or absence of tetracycline for 48 h. PS2-inducible lines were established using the tetracycline-repressible transactivator (Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992)). The presence of tetracycline in the culture medium suppresses PS2 expression, while its withdrawal results in induction of PS2 expression. H4 neuroglioma founder cells were stably transfected with constructs encoding full-length PS2 fused with FLAG epitope peptide in C terminus (clone: WF26; FIG. 1A) or in N terminus (clone: 5WF11; FIG. 1B). Full-length PS2 with an apparent molecular mass of 53-55 kDa (54K PS2) and a high molecular mass form of PS2 (HMW-PS2) were detected in Western blot analysis using monoclonal anti-FLAG antibody, M2. In addition, 20-kDa C-terminal (PS2-CTF: FIG. 1A) and 34-kDa-terminal (PS2-NTF: FIG. 1B) fragments were also detected only in the induced (-tetracycline) lanes.

#FIG. 2. Detergent solubility of C- and N-terminal PS2 fragments. Cells expressing either C-terminal or N-terminal epitope-tagged PS2 were induced for 48 h, extracted as described herein. Equal amounts of proteins (FIG. 2A) were analyzed by Western blotting using anti-FLAG antibody, and equal volumes (FIG. 2B) were normalized for total cellular proteins using the αPS2Loop. While 34-kDa-terminal fragments (NTF) were only detectable in detergent-extractable cellular pools, the 20-kDa C-terminal fragment (CTF) was highly concentrated in the detergent-resistant cellular fraction.

#FIG. 3. Time course of PS2 induction. Inducible PS2 cells (clone WF2) were grown in the absence of tetracycline and harvested at timed intervals up to 72 h. Equal amounts of proteins (30 μg) from detergent-soluble (FIG. 3A) and detergent-resistant fractions (FIG. 3B) were analyzed by Western blotting to detect the 54K PS2, HMW-PS2, and 20K CTF. The 54K PS2 and HMW-PS2 were extractable by the detergent treatment.

#For purposes of clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the following subsections: I. Purified Presenilin 2 Proteolytic Fragments. II. An Antibody Having Binding Affinity to a Presenilin 2 Proteolytic Fragment and a Hybridoma Containing the Antibody. III. A Method of Detecting a Presenilin 2 Proteolytic Fragment or Antibody in a Sample. IV. A Diagnostic Kit Comprising a Presenilin 2 Proteolytic Fragment or Antibody. V. Isolated Nucleic Acid Molecules Coding for a Presenilin 2 Proteolytic Fragment. VI. DNA Constructs Comprising a Nucleic Acid Molecule Encoding a Presenilin 2 Proteolytic Fragment and Cells Containing These Constructs. VII. Methods of Screening for Compounds that Inhibit Proteolysis of Presenilin 2 VIII. Diagnostic Screening I. Purified Presenilin 2 Proteolytic Fragments

#Mutations in the presenilin genes, PS1 and PS2, cause a major portion of early onset familial Alzheimer's disease (FAD). The biological roles of the presenilins and how their pathological mutations confer FAD are unknown. The processing and degradation pathways of PS2 are described herein. For regulated expression of PS2, inducible cell lines expressing PS2 under the tight control of the tetracycline-responsive transactivator were established. Western blot analysis revealed that PS2 was detected as an ˜53-55-kDa polypeptide (54-kDa PS2) as well as a high molecular mass form (HMW-PS2). Using a stably transfected, inducible cell system, it was found that PS2 is proteolytically cleaved into two stable cellular polypeptides including an ˜20-kDa C-terminal fragment and an ˜34-kDa N-terminal fragment. PS2 is polyubiquitinated in vivo, and the degradation of PS2 is inhibited by proteasome inhibitors, N-acetyl-L-leucinal-L-norleucinal and lactacystin. These studies suggest that PS2 normally undergoes endoproteolytic cleavage and is degraded via the proteasome pathway.

#Both PS1 and PS2 were alternatively cleaved at sites distal to their normal cleavage sites when apoptosis was induced in untransfected H4 human neuroglioma cells, or when these genes were overexpressed. Alternative cleavage of PS1 and PS2 could be blocked by treatment with either zVAD (a broad spectrum caspase inhibitor) or zDEVD (CPP32-like protease inhibitor), indicating that the enzyme responsible is a CPP32-like protease. In PS2, this alternative cleavage site was identified as DSYDS (a.a. 326-330)(SEQ ID NO: 1). In H4 cells overexpressing PS2 containing the N141I FAD mutation, the ratio of apoptotic:normal cleavage fragments was significantly elevated compared to wild-type PS2-expressing cells. Thus, apoptosis-associated endoproteolysis of the presenilins mediated by a CPP32-like protease plays a role in the pathogenesis of FAD.