Autophagy is the process by which organelles and portions of the cytoplasm are degraded in lysosomes. Several different forms of autophagy are known that are distinguishable chiefly by the mode in which cargo is delivered to the lysosome for degradation. Ubiquilin was recently reported to regulate macroautophagy, the form of autophagy in which cytosolic cargo is packaged in a double-membrane structure or autophagosome that fuses with lysosomes for degradation. We confirm here using different morphological and biochemical procedures that ubiquilin is present in autophagosomes in HeLa cells and in brain and liver tissue of mouse. Coimmunoprecipitation studies indicated that ubiquilin binds the autophagosome marker LC3 in a complex and that reduction of ubiquilin expression reduces autophagosome formation, which correlates with a reduction in maturation of LC3-I to the LC3-II form of the protein. We found that ubiquilin is degraded during both macroautophagy and during chaperone-mediated autophagy (CMA), the latter of which involves the active transport of proteins into lysosomes. We discuss the implication of this degradation in mediating cross-talk between macroautophagy and CMA. Finally, we demonstrate that ubiquilin protects cells against starvation-induced cell death propagated by overexpression of mutant Alzheimer's disease PS2N141I protein and green fluorescent protein (GFP)-huntingtin exon-1 fusion protein containing 74 polyglutamines.

Loss of ubiquilin or erasin activates ER stress, increases accumulation of polyubiquitinated proteins, and shortens lifespan in worms.

The transcription factor p53 is under negative regulation by the ubiquitin ligase MDM2 and its close homologue MDM4. In the bound complex between MDM2 and p53, the transactivation domain of p53 adopts an amphipathic helical conformation which optimizes the spatial organization of three key hydrophobic residues (Phe19, Trp23, Leu26) for maximum interactions. The interaction with MDM2 is known to be abrogated by phosphorylation of Ser/Thr residues in the MDM2 N-terminal domain and in the p53 transactivation domain. In the latter, phosphorylation of Thr18 has been attributed to destabilize a key hbond between Thr18 and Asp21. This interaction has been thought to be critical for the formation of the helical conformation of the p53 transactivation domain. Molecular dynamics simulations of the p53 transactivation domain suggest that phosphorylation of either Thr18 or Ser20 does not disrupt its helical structure but does result in reduced affinities for MDM2. While interactions between the Thr18 and Asp21 are indeed broken due to charge-charge repulsions, the peptide has enough inherent flexibility to form alternate patterns of hbonds, resulting in the maintenance of helicity. Electrostatics of MDM2 reveal local anionic patches in the region where Thr18 docks. These suggest that repulsions will arise because the MDM2 surface will force the p53 to bind in a manner that will place the negatively charged phosphorylated Thr18 near this anionic region. A similar, albeit somewhat attenuated pattern of electrostatic modulations, is seen for a model of MDM4 that has been built. Mutants of MDM2 and MDM4 have been designed to attenuate this anionicity and have been computationally demonstrated to enhance the binding of the phosphorylated peptides.

Activin A is a potent growth and differentiation factor whose synthesis and bioactivity are tightly regulated. Both follistatin binding and inhibin subunit heterodimerization block access to the activin receptor and/or receptor activation. We postulated that the activin-␤ C subunit provides another mechanism regulating activin bioactivity. To test our hypothesis, we examined the biological effects of activin C and produced mice that overexpress activin-␤ C . Activin C reduced activin A bioactivity in vitro; in LNCaP cells, activin C abrogated both activin A-induced Smad signaling and growth inhibition, and in L␤T2 cells, activin C antagonized activin A-mediated activity of an follicle-stimulating hormone-␤ promoter. Transgenic mice that overexpress activin-␤C exhibited disease in testis, liver, and prostate. Male infertility was caused by both reduced sperm production and impaired sperm motility. The livers of the transgenic mice were enlarged because of an imbalance between hepatocyte proliferation and apoptosis. Transgenic prostates showed evidence of hypertrophy and epithelial cell hyperplasia. Additionally, there was decreased evidence of nuclear Smad-2 localization in the testis, liver, and prostate, indicating that overexpression of activin-␤ C antagonized Smad signaling in vivo. Underlying the significance of these findings, human testis, liver, and prostate cancers expressed increased activin-␤C immunoreactivity. This study provides evidence that activin-␤ C is an antagonist of activin A and supplies an impetus to examine its role in development and disease.