Research Paper Volume 11, Issue 16 pp 6134—6152

Beta-amyloid induces apoptosis of neuronal cells by inhibition of the Arg/N-end rule pathway proteolytic activity

Figure 1. Inhibition effect of Aβ42 (10 μM) on the proteolytic activity of the Arg/N-end rule pathway. (A) Diagram of the fDHFR-UbR48-X609-PTPRNf (X = Asp, Arg-Asp) fusion. Co-translational cleavage of the fusion by deubiquitylases produces a test protein X609-PTPRNf and a stable ’reference’ protein fDHFR-UbR48 at the initially equimolar ratio. (B) Degradation of Asp609-PTPRNf in reticulocyte lysate in the presence or absence of Aβ42. Asp609-PTPRNf was expressed in reticulocyte lysate and co-translationally labeled with 35S-Met for 30 min at 30°C in the presence or absence of Aβ42, followed by a chase, immunoprecipitation with anti-flag M2 antibody, SDS-PAGE, and autoradiography. (C) Same as (B) but with Arg-Asp609-PTPRNf fragment. (D) Quantification of (B). The level of Asp609-PTPRNf was normalized on the level of fDHFR-UbR48. The level of Asp609-PTPRNf detected immediately after stopping of protein expression in reticulocyte lysate (0 min chase) was taken as 100%. "% remaining" is the level of non-degraded Asp609-PTPRNf at shown time points after stopping of protein expression. The absence of Aβ42 - dark-gray column; the presence of Aβ42 – light-gray column. (E) Quantification of (C). Each value is the mean ± SD of at least three independent experiments; *p < 0.01, **p < 0.001.