Figure 1: DJ-1 inhibits the degradation of partially folded proteins by the 20S proteasome.

(a) To examine whether DJ-1 can protect substrates from 20S proteasome proteolysis, we performed a series of time-dependent degradation assays using a range of proteins containing unstructured regions. Intrinsically unstructured proteins such as α-synuclein and the C terminus domain of p53 (Cp53, a.a. 293–393) were used, as well as the mutational variant of NQO1, P187S (NQO1_P187S), which we previously demonstrated to be partially unfolded, and prone to 20S proteasomal degradation³⁶. Calmodulin, which becomes a substrate of the 20S proteasome only upon oxidation (OxCalmodulin), served as a control, demonstrating the specificity of the 20S proteasome towards disordered segments. As an additional control, the proteasome inhibitor MG132 was used. At the indicated time points, aliquots were quenched and evaluated by SDS–PAGE (Cp53, α-synuclein and calmodulin) or western blot (NQO1_P187S; left panel), followed by quantitative image analysis (right panel). In all tested cases, the substrates were stable in the absence of the 20S proteasome; however, after its addition, they were degraded. In the presence of DJ-1, however, there was a marked decrease in the degradation rate of all substrates. (b) DJ-1 was incubated in the presence and absence of purified 20S proteasomes at the indicated time points, and monitored by western blot analysis, using anti-DJ-1 antibody. The results indicate that unlike the 20S substrates, DJ-1 levels remained constant in the presence of the 20S proteasome. (c) Purified 20S proteasomes were analysed for peptidase activity in the presence and absence of DJ-1 or MG132 using the fluorogenic peptide substrate suc-LLVY-AMC. To enhance the peptidase activity, 0.02% SDS was added to the mixture. Error bars in a–c represent the s.d. of three repeats. Molecular weights are indicated in the right of each blot in kDa units.