The mechanism of cytokeratin aggresome formation: the role of mutant ubiquitin (UBB +1)

Abstract

Aggresome formation in cells involves the failure of the ubiquitin-proteasome pathway to dispose of proteins destined for degradation by the 26S proteasome. UBB +1 is present in Mallory bodies in alcoholic liver disease and in aggresomes formed in Alzheimer’s desease. The present investigation focuses on the role that UBB +1 plays in cytokeratin aggresome formation in Mallory bodies (MBs) in vitro. Immunoprecipitation with a monoclonal antibody to cytokeratin-8 (CK-8) was used. The immunoprecipitate was incubated for 24 h in the presence of different constituents involved in aggresome formation including ubiquitin, UBB +1, the proteasome inhibitor PS341, an ATP generating energy source, a deubiquitinating enzyme inhibitor, a purified proteasome fraction, and an E 1–3 conjugating enzyme fraction. MB-like protein aggregates formed in the presence of ubiquitin, plus UBB +1 or PS341. These aggregates stained positively for CK-8. UBB +1, and a proteasome subunit Tbp7, as demonstrated on Western blots. A second approach was used to form MBs in vitro in cultured hepatocytes transfected with UBB +1 protein using Chariot. The cells were double stained using CK-8 and ubiquitin antibodies. The two proteins colocalized in MB-like aggregates. The results support the possibility that aggresome formation is a complex multifactor process, which is favored by inhibition of the proteasome and by the presence of UBB +1.