The mechanism of cytokeratin aggresome formation: the role of mutant ubiquitin (UBB+1).
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).
Bardag-Gorce
F
F
Department of Pathology, Harbor-UCLA Medical Center, Torrance, CA 90509, USA.
Riley
N
N
Nguyen
V
V
Montgomery
R O
RO
French
B A
BA
Li
J
J
van Leeuwen
F W
FW
Lungo
W
W
McPhaul
L W
LW
French
S W
SW
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism* / Q000648:ultrastructure; D007150:Immunohistochemistry; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D007633:Keratins / Q000378:metabolism*; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D009154:Mutation; D010447:Peptide Hydrolases / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D014157:Transcription Factors / Q000378:metabolism; D025801:Ubiquitin / Q000235:genetics* / Q000378:metabolism*
D004268:DNA-Binding Proteins; D014157:Transcription Factors; D025801:Ubiquitin; D007633:Keratins; D000255:Adenosine Triphosphate; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease