During proteasomal stress, cells can alleviate the accumulation of polyubiquitinated proteins by targeting them to perinuclear aggresomes for autophagic degradation, but the mechanism underlying the activation of this compensatory pathway remains unclear. Here we report that PINK1-s, a short form of Parkinson disease (PD)-related protein kinase PINK1 (PTEN induced putative kinase 1), is a major regulator of aggresome formation. PINK1-s is extremely unstable due to its recognition by the N-end rule pathway, and tends to accumulate in the cytosol during proteasomal stress. Overexpression of PINK1-s induces aggresome formation in cells with normal proteasomal activities, while loss of PINK1-s function leads to a significant decrease in the efficiency of aggresome formation induced by proteasomal inhibition. PINK1-s exerts its effect through phosphorylation of the ubiquitin-binding protein SQSTM1 (sequestosome 1) and increasing its ability to sequester polyubiquitinated proteins into aggresomes. These findings pinpoint PINK1-s as a sensor of proteasomal activities that transduces the proteasomal impairment signal to the aggresome formation machinery.

Aggresome formation is a major strategy to enable cells to cope with proteasomal stress. Misfolded proteins are assembled into micro-aggregates and transported to the microtubule organizing center (MTOC) to form perinuclear aggresomes before their degradation through autophagy. So far, multiple factors have been identified as the activators of micro-aggregate formation, but much less is known about the regulatory mechanisms of their transport. Here, we report that proteasomal stress leads to the activation of p38 MAPK family members. Two of them, p38γ (MAPK12) and p38δ (MAPK13), are dispensable for micro-aggregate formation but are required for their targeting to the MTOC. Interestingly, p38δ promotes micro-aggregate transport by phosphorylating SQSTM1, a major scaffold protein that assembles soluble ubiquitylated proteins into micro-aggregates. Expression of the phospho-mimetic mutant of SQSTM1 in p38δ-knockout cells completely rescued their aggresome formation defects and enhanced their resistance to proteasomal stress to wild-type levels. This study reveals p38δ-mediated SQSTM1 phosphorylation as a critical signal for the targeting of micro-aggregates to the MTOC and provides direct evidence for the survival advantages associated with aggresome formation in cells under proteasomal stress.

Ribosome-inactivating proteins (RIPs) are a family of enzymes that depurinate rRNA and inhibit protein biosynthesis. Here we report the purification, apoptosis-inducing activity, and polyethylene glycol (PEG) modification of RIP from the bitter melon seeds. The protein has a homogenous N-terminal sequence of NAsp- Val-Ser-Phe-Arg. Moreover, the RIP displayed strong apoptosis-inducing activity and suppressed cancer cell growth. This might be attributed to the activation of caspases-3. To make it available for in vivo application, the immunogenicity of RIP was reduced by chemical modification with 20 kDa (mPEG)(2)-Lys-NHS. The inhibition activity of both PEGylated and non-PEGylated RIP against cancer cells was much stronger than against normal cells, and the antigenicity of PEGylated RIP was reduced significantly. Our results suggested that the PEGylated RIP might be potentially developed as anti-cancer drug.