The removal of misfolded, ubiquitinated proteins is an essential part of the protein quality control. The ubiquitin‐proteasome system (UPS) and autophagy are two interconnected pathways that mediate the degradation of such proteins. During autophagy, ubiquitinated proteins are clustered in a p62‐dependent manner and are subsequently engulfed by autophagosomes. However, the nature of the protein substrates targeted for autophagy is unclear. Here, we developed a reconstituted system using purified components and show that p62 and ubiquitinated proteins spontaneously coalesce into larger clusters. Efficient cluster formation requires substrates modified with at least two ubiquitin chains longer than three moieties and is based on p62 filaments cross‐linked by the substrates. The reaction is inhibited by free ubiquitin, K48‐, and K63‐linked ubiquitin chains, as well as by the autophagosomal marker LC3B, suggesting a tight cross talk with general proteostasis and autophagosome formation. Our study provides mechanistic insights on how substrates are channeled into autophagy.

No abstract

Worldwide, multiple Fusarium mycotoxins occur as contaminants of cereals with important impacts on human and animal health. The aim of this study was to investigate the effects of the widespread Fusarium secondary metabolite enniatin (ENN), a cyclic hexadepsipeptide, on human cell growth and survival. While short-term exposure (up to 8 h) to ENN at nanomolar concentrations slightly but significantly stimulated cell proliferation, it showed profound apoptosis-inducing effects especially against various human cancer cell types at low micromolar concentrations (already after 24 h of treatment). Several cellular changes indicative for programmed cell death such as cell shrinkage, chromatin condensation, DNA fragmentation, and apoptotic body formation were observed. Correspondingly, the cleavage of poly(ADP-ribosyl)polymerase and the activation of multiple caspases accompanied a distinct loss of mitochondrial membrane potential. To investigate the impact of apoptosis- and cell cycle-regulating proteins on ENN activity, HCT116 cells with homozygously disrupted p53, p21, or bax genes were analyzed. In vitality assays, no significant influences of these proteins on the anticancer activity of ENN were detectable. In contrast, 3H-thymidine incorporation revealed a significantly more efficient block of DNA synthesis in p53 wild-type as compared to knock-out cells. Accordingly, fluorescence-activated cell sorting analysis demonstrated a stronger ENN-induced cell cycle arrest in the G0/G1 phase. Profound ENN-mediated induction of p53 and the p53-downstream cell cycle inhibitor p21 were detectable in p53 wild-type cells by Western blotting. P53-independent p21 induction was also detectable at higher ENN concentrations in p53 (-/-) cells. In contrast, bax activation by ENN was independent of the cellular p53 status. In summary, our results suggest that short-term exposure to very low ENN concentrations, for example, via food intake, might have tumor-promoting functions based on growth stimulation. In contrast, elevated ENN concentrations exert profound p53-dependent cytostatic and p53-independent cytotoxic activities especially against human cancer cells, suggesting a potential quality of ENN as an anticancer drug.