Accumulation of polyubiquitylated proteins in response to Ala-Ala-Phe-chloromethylketone is independent of the inhibition of Tripeptidyl peptidase II.
In the present study we have addressed the issue of proteasome independent cytosolic protein degradation. Tripeptidyl peptidase II (TPPII) has been suggested to compensate for a reduced proteasome activity, partly based on evidence using the inhibitor Ala-Ala-Phe-chloromethylketone (AAF-cmk). Here we show that AAF-cmk induces the formation of polyubiquitin-containing accumulations in osteosarcoma and Burkitt's lymphoma cell lines. These accumulations meet many of the landmarks of the aggresomes that form after proteasome inhibition. Using a combination of experiments with chemical inhibitors and interference of gene expression, we show that TPPII inhibition is not responsible for these accumulations. Our evidence suggests that the relevant target(s) is/are in the ubiquitin-proteasome pathway, most likely upstream the proteasome. We obtained evidence supporting this model by inhibition of Hsp90, which also acts upstream the proteasome. Although our data suggest that Hsp90 is not a target of AAF-cmk, its inhibition resulted in accumulations similar to those obtained with AAF-cmk. Therefore, our results question the proposed role for TPPII as a prominent alternative to the proteasome in cellular proteolysis.
Villasevil
Eugenia M
EM
Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas/Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain.
Guil
Sara
S
López-Ferreras
Lorena
L
Sánchez
Carlos
C
Del Val
Margarita
M
Antón
Luis C
LC
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology*; D000626:Aminopeptidases / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000502:physiology; D045744:Cell Line, Tumor; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D009842:Oligopeptides / Q000494:pharmacology; D025821:Polyubiquitin / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism / Q000502:physiology; D061988:Proteasome Inhibitors; D055503:Protein Multimerization / Q000187:drug effects*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D012697:Serine Endopeptidases / Q000378:metabolism / Q000502:physiology; D057149:Ubiquitinated Proteins / Q000378:metabolism*; D056811:Unfolded Protein Response / Q000187:drug effects
D000590:Amino Acid Chloromethyl Ketones; D004791:Enzyme Inhibitors; D009842:Oligopeptides; D061988:Proteasome Inhibitors; D057149:Ubiquitinated Proteins; C055604:alanyl-alanyl-phenylalanine chloromethyl ketone; D025821:Polyubiquitin; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C048001:tripeptidyl-peptidase 2; D012697:Serine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C078846:epoxomicin