Similar to C99, only one of the proteolytic cleavage products of Notch (the C-terminal fragment) has been identified, so that we cannot rule out that the initial -like cleavage of Notch occurs in the middle of the TMD and is followed by a secondary proteolytic event. C99-wild type (wt), most mutants with an altered length of the TMD changed the cleavage site of -secretase, whereas control mutants with mutations outside the TMD did not. Thus, the length of the whole TMD is HNPCC a major determinant for the cleavage site of -secretase. Moreover, the C99-mutants were not only cleaved at one site but at two sites within their TMD. One cleavage site was located around the middle of the TMD, of its actual length regardless. An additional cleavage occurred within the N-terminal half of their TMD and thus at the opposite side of the Notch cleavage site. Regulated intramembrane proteolysis of type I membrane proteins has been shown to play an important UNC0321 role in the pathogenesis of Alzheimer’s disease and in cell differentiation (for a review, see ref. 1). The corresponding proteins involved in these biological processes are the amyloid precursor protein (APP) and Notch, respectively. The intramembrane proteolysis of APP leads to the generation of the amyloid peptide (A), which is deposited in the brains of patients with Alzheimer’s disease (for a review, see ref. 2). To undergo intramembrane proteolysis, APP is first proteolytically processed by one of the proteases termed – and -secretase (for a review, see ref. 3). The -secretase seems to be a member of the ADAM (and divided UNC0321 into three samples with a volume of 1 ml each. A and p3 were immunoprecipitated with antibody W02 (5 g/ml), G2-10 (12.5 g/ml), or G2-11 (17.3 g/ml) and 100 g protein-G Agarose (Boehringer Mannheim), UNC0321 respectively. The immunoprecipitated proteins were separated on 10% Tris/Tricine gels (24). The intensity of the bands was quantified by using a Fuji PhosphorImager (BAS 1000). To determine whether the introduced mutations altered the amount of A generated from C99, A and C99 were immunoprecipitated from the conditioned medium and the cell lysate, respectively, by using antibody W02 (concentration as above). After gel electrophoresis and Western Blotting using antibody W02, the amounts of C99 and A were measured. Each experiment was carried out in three or more independent experiments. For the inhibition of A-generation, the experiments were carried out as described above. The specific -secretase inhibitor {1and test) relative to C99-wt (**, 0.01, ***, 0.001). a, the calculated ratios are 0% because no A42 was detected. In contrast, most mutants with an altered length of the TMD led to a changed ratio of A42/A40 and thus an altered -cleavage site. Both the C-terminal insertion (CTI) and the N-terminal deletion (NT) of two residues led to a similar increase of the A42/A40-ratio (increase: 4.1-fold (CTI), 4.3 fold (NT); A42/A40: 19.6 2.2% (CTI), 20.5 4.7% (NT); 0.001 (CTI), 0.01 (NT)). This increase corresponds to a shift of the -cleavage site in the C-terminal direction. In contrast, the C-terminal deletion of two residues (CT) did not alter the A42/A40-ratio (4.6 1.5%). For C99-NTI and C99-SN, no A42 could be detected so that the calculated ratio A42/A40 was 0%. Both antibodies used for the immunoprecipitation of A40 (G2-10) and A42 (G2-11) also immunoprecipitated p340 and p342 (Fig. ?(Fig.2).2). A comparison with C99-wt revealed that the analyzed mutations altered the ratios p342/p340 to a similar extent as the ratios A42/A40 (data not shown). Mass Spectrometric Analysis of A. To UNC0321 determine exactly how the mutations in the TMD of C99 affect the -cleavage site, A was immunocaptured by using monoclonal antibody 6E10, which binds to the N terminus of A. Subsequently, the immunocaptured A UNC0321 was analyzed by SELDI-TOF mass spectrometry (Fig. ?(Fig.4).4). C99-wt gave a prominent signal for A40 (Fig. ?(Fig.4),4), confirming previous results by.
