Fig. 4
PA28α overexpression does not increase hippocampal PA28-dependent proteasome activity, but enhances aggregation prevention capacity in the hippocampus. a The PA28-dependent proteasome capacity determined by suc-LLVY-AMC digestion (i.e. β5/β5i chymotrypsin-like activity) under PA28–20S optimizing conditions, with interferon-γ treated MEFs serving as positive control (compare C – FNγ to C + IFNγ). Activity presented is activity inhibited by the proteasome-specific inhibitor epoxomicin (5 µM), which corresponded to 70–98% of total activity (see Methods). Values are mean ± SEM; nPA28αOE = 3 and nWT = 4; differences are not statistically significant (P = 0.72). b Representative blot of K48-linked polyubiquitin western analysis and quantification of K48-linked polyubiquitinated protein signal from western analysis, P = 0.051; Student’s t test). Values are mean ± SEM; nPA28αOE = 6 and nWT = 8. c 20S proteasome capacity (in the presence of 0.02% SDS) in protein extracts made from PA28αOE and WT right hippocampus, values are mean ± SEM; nPA28αOE = 3 and nWT = 4. d Western analysis of the proteasome related markers Rpt2 (19S subunit), β5 (20S) and β5i (20Si) in protein extracts made from PA28αOE and WT left hippocampus. PA28α is induced 13-fold in PA28αOE hippocampus (P < 1E−12; Student’s t test). *Estimated kDa marker placement based on 20 kDa and 37 kDa marker bands. “n.d.” = not included in assay due to limited amount of extract. e Aggregation prevention of heat-sensitive luciferase in the presence of hippocampal protein extracts at 42 °C. Luciferase aggregation prevention capacity was calculated as percentage of non-aggregated luciferase compared to samples without cell extracts. Boiled extracts served as negative control and did not prevent aggregation. Values are mean ± SEM; nPA28αOE = 9 and nWT = 10 (P = 0.036; Student’s t test). f A model of PA28αβ effects on cognitive functions through its role as a chaperone rather than a 20S proteasome activator