succinyl-leucyl-leucyl-valyl-tyrosyl-methylcoumarinamide and Disease-Models--Animal

Abstract Deposition of ubiquitinated protein aggregates is a hallmark of neurodegeneration in both acute neural injuries, such as stroke, and chronic conditions, such as Parkinson's disease, but the underlying mechanisms are poorly understood. In the present study, we examined the role of Zn2+ in ischemia-induced impairment of the ubiquitin-proteasome system in the CA1 region of rat hippocampus after transient global ischemia. We found that scavenging endogenous Zn2+ reduced ischemia-induced ubiquitin conjugation and free ubiquitin depletion. Furthermore, exposure to zinc chloride increased ubiquitination and inhibited proteasomal enzyme activity in cultured hippocampal neurons in a concentration- and time-dependent manner. Further studies of the underlying mechanisms showed that Zn(2+)-induced ubiquitination required p38 activation. These findings indicate that alterations in Zn2+ homeostasis impair the protein degradation pathway.

Topics: Actins; Animals; CA1 Region, Hippocampal; Cells, Cultured; Chelating Agents; Coumarins; Disease Models, Animal; Dose-Response Relationship, Drug; Edetic Acid; Embryo, Mammalian; Enzyme Inhibitors; Fluorescent Dyes; Green Fluorescent Proteins; Imidazoles; Ischemia; Leupeptins; Male; Microtubule-Associated Proteins; Neurons; Oligopeptides; p38 Mitogen-Activated Protein Kinases; Proteasome Endopeptidase Complex; Pyrimidines; Rats; Rats, Sprague-Dawley; Statistics, Nonparametric; Time Factors; Transfection; Ubiquitin; Zinc

Proteasomes are multisubunit proteases that exist universally among eukaryotes. They have multiple proteolytic activities and are believed to have important roles in regulating cell cycle, selective intracellular proteolysis, and antigen presentation. Here we have partially purified Leishmania chagasi proteasome. The L. chagasi proteasome rich fraction displayed the typical features of eukaryotic 20S proteasome complexes, being active towards peptidyl substrates with hydrophobic and acidic residues, and sensitive to the proteasome-specific inhibitor lactacystin. We have shown that lactacystin, or its active form clasto-lactacystin beta-lactone, but not E-64, blocks the in vitro growth of L. chagasi promastigotes, demonstrating that the interference with parasite growth is due to the lack of proteasome activity. Furthermore, pre-treatment of L. chagasi promastigotes with lactacystin did not prevent parasite entry in host cells, but markedly restricted its intracellular survival. These results demonstrate that intact parasite proteasome function is required for replication of L. chagasi and for amastigotes survival inside the vertebrate host cell.

Topics: Acetylcysteine; Animals; Coumarins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Disease Models, Animal; Leishmania; Leishmaniasis, Visceral; Leucine; Macrophages, Peritoneal; Male; Mice; Multienzyme Complexes; Oligopeptides; Phenylmethylsulfonyl Fluoride; Proteasome Endopeptidase Complex