Herein, we demonstrate that the ubiquitin-associated (UBA) domain of sequestosome 1/p62 displays a preference for binding K63-polyubiquitinated substrates. Furthermore, the UBA domain of p62 was necessary for aggregate sequestration and cell survival. However, the inhibition of proteasome function compromised survival in cells with aggregates. Mutational analysis of the UBA domain reveals that the conserved hydrophobic patch MGF as well as the conserved leucine in helix 2 are necessary for binding polyubiquitinated proteins and for sequestration-aggregate formation. We report that p62 interacts with the proteasome by pull-down assay, coimmunoprecipitation, and colocalization. Depletion of p62 levels results in an inhibition of ubiquitin proteasome-mediated degradation and an accumulation of ubiquitinated proteins. Altogether, our results support the hypothesis that p62 may act as a critical ubiquitin chain-targeting factor that shuttles substrates for proteasomal degradation.Sequestosome 1/p62 is a cellular protein which was initially identified as a phosphotyrosine-independent ligand of the Src homology 2 (SH2) domain of p56 lck (27). The protein has been cloned by two independent groups as a cointeracting protein of the atypical protein kinase C (aPKC) and is also named ZIP for PKC-interacting protein (47, 50). p62 has been shown to bind ubiquitin noncovalently (56), and its overexpression results in large cytoplasmic aggregates (47). We have recently determined that p62 possesses sequence homology with other proteins possessing a ubiquitin-associated (UBA) domain at their C terminus, amino acids 386 to 434 (16), in p62. Interestingly, p62 contains several structural motifs which suggest that it might participate in the formation of multimeric signaling complexes. These domains include an acidic interaction domain (AID/ORCA/PC/PB1) that binds the aPKC, a ZZ finger, a binding site for the RING finger protein TRAF6, two PEST sequences, and the UBA domain (16).Ubiquitin is a small polypeptide of 76 amino acids that can be covalently attached to other proteins. Monoubiquitination serves as a novel endocytosis signal (19), whereas polyubiquitin chains target substrates for degradation by the proteasome (45). p62 has been shown to interact in a noncovalent fashion with polyubiquitin chains (5, 53), which is consistent with reports demonstrating that proteins possessing UBA domains are more likely to bind polyubiquitin chains over monoubiquitin (59). Conjugation of ubiquitin to substrate proteins requires three enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). Initially, E1 activates ubiquitin by forming a high-energy thioester intermediate with the C-terminal glycine using ATP. The activated ubiquitin is sequentially transferred to E2 and then to E3 which catalyzes isopeptide bond formation between the activated C-terminal glycine of ubiquitin and an ε-amino group of a lysine residue in the substrate (45). However, only HECT-type E3s can form thioester bon...

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Conjugates of curcumin to two differently sized poly(ethylene glycol) molecules were synthesized in an attempt to overcome the low aqueous solubility of this natural product with cytotoxic activity against some human cancer cell lines. The soluble conjugates exhibited enhanced cytotoxicity as compared to that of the parent drug. Synthesis, analyses of the rate of drug release, and cytotoxicity studies are herein reported. The water-soluble conjugates may provide information useful for the development of injectable curcumin conjugates.