The ubiquitin-proteasome pathway plays an essential role in proteolysis during Trypanosoma cruzi remodeling.
Here, we document for the first time the presence of the 26S proteasome and the ubiquitin pathway in a protozoan parasite that is in an early branch in the eukaryotic lineage. The 26S proteasome of Trypanosoma cruzi epimastigotes was identified as a high molecular weight complex (1400 kDa) with an ATP-dependent chymotrypsin-like activity against the substrate Suc-LLVY-Amc. This activity was inhibited by proteasome inhibitors and showed same electrophorectic migration pattern as yeast 26S proteasome in nondenaturating gels. About 30 proteins in a range of 25-110 kDa were detected in the purified T. cruzi 26S proteasome. Antibodies raised against the AAA family of ATPases from eukaryotic 26S proteasome and the T. cruzi 20S core specifically recognized components of T. cruzi 26S. To confirm the biological role of 26S in this primitive eukaryotic parasite, we analyzed the participation of the ubiquitin (Ub)-proteasome system in protein degradation during the time of parasite remodeling. Protein turnover in trypomastigotes was proteasome and ATP-dependent and was enhanced during the transformation of the parasites into amastigotes. If 20S proteasome activity is inhibited, ubiquitinated proteins accumulate in the parasites. As expected from the profound morphological changes that occur during transformation, cytoskeletal proteins associated with the flagellum are targets of the ubiquitin-proteasome pathway.
de Diego
J L
JL
Michael Heidelberger Division, Pathology Department, New York University School of Medicine, New York, New York 10016, USA. diegoj01@med.nyu.edu
Katz
J M
JM
Marshall
P
P
Gutiérrez
B
B
Manning
J E
JE
Nussenzweig
V
V
González
J
J
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D002460:Cell Line; D002463:Cell Membrane Permeability; D003546:Cysteine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000378:metabolism; D005407:Flagella / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008253:Macaca mulatta; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D046988:Proteasome Endopeptidase Complex; D015800:Protozoan Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D014349:Trypanosoma cruzi / Q000201:enzymology / Q000254:growth & development* / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism / Q000502:physiology*
D015853:Cysteine Proteinase Inhibitors; D003598:Cytoskeletal Proteins; D009097:Multienzyme Complexes; D015800:Protozoan Proteins; D014452:Ubiquitins; C045519:paraflagellar rod proteins, Trypanosoma brucei; C067713:lactacystin; D000255:Adenosine Triphosphate; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D000111:Acetylcysteine