title: Characterisation of 20S Proteasome in Tritrichomonas foetus and Its Role during the Cell Cycle and Transformation into Endoflagellar Form.
abstract: Proteasomes are intracellular complexes that control selective protein degradation in organisms ranging from Archaea to higher eukaryotes. These structures have multiple proteolytic activities that are required for cell differentiation, replication and maintaining cellular homeostasis. Here, we document the presence of the 20S proteasome in the protist parasite Tritrichomonas foetus. Complementary techniques, such as a combination of whole genome sequencing technologies, bioinformatics algorithms, cell fractionation and biochemistry and microscopy approaches were used to characterise the 20S proteasome of T. foetus. The 14 homologues of the typical eukaryotic proteasome subunits were identified in the T. foetus genome. Alignment analyses showed that the main regulatory and catalytic domains of the proteasome were conserved in the predicted amino acid sequences from T. foetus-proteasome subunits. Immunofluorescence assays using an anti-proteasome antibody revealed a labelling distributed throughout the cytosol as punctate cytoplasmic structures and in the perinuclear region. Electron microscopy of a T. foetus-proteasome-enriched fraction confirmed the presence of particles that resembled the typical eukaryotic 20S proteasome. Fluorogenic assays using specific peptidyl substrates detected presence of the three typical peptidase activities of eukaryotic proteasomes in T. foetus. As expected, these peptidase activities were inhibited by lactacystin, a well-known specific proteasome inhibitor, and were not affected by inhibitors of serine or cysteine proteases. During the transformation of T. foetus to endoflagellar form (EFF), also known as pseudocyst, we observed correlations between the EFF formation rates, increases in the proteasome activities and reduced levels of ubiquitin-protein conjugates. The growth, cell cycle and EFF transformation of T. foetus were inhibited after treatment with lactacystin in a dose-dependent manner. Lactacystin treatment also resulted in an accumulation of ubiquitinated proteins and caused increase in the amount of endoplasmic reticulum membranes in the parasite. Taken together, our results suggest that the ubiquitin-proteasome pathway is required for cell cycle and EFF transformation in T. foetus.
authors.affiliation: Programa de Pós-graduação em Ciências Morfológicas, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil; Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil; Fiocruz, Centro de Pesquisa Aggeu Magalhães, Departamento de Microbiologia, Laboratório de Microbiologia e Biologia Celular, Recife, PE, Brazil.
authors.forename: Antonio
authors.initials: A
authors.lastname: Pereira-Neves
authors.affiliation: Laboratório Nacional de Computação Cientifica (LNCC/MCT), Petrópolis, RJ, Brazil.
authors.forename: Luiz
authors.initials: L
authors.lastname: Gonzaga
authors.affiliation: Fiocruz, Instituto Oswaldo Cruz, Rio de Janeiro, RJ, Brazil.
authors.forename: Rubem F S
authors.initials: RF
authors.lastname: Menna-Barreto
authors.affiliation: Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil; UNIGRANRIO- Universidade do Grande Rio, Duque de Caxias, RJ, Brazil; Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
authors.forename: Marlene
authors.initials: M
authors.lastname: Benchimol
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002453:Cell Cycle; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D005407:Flagella / Q000378:metabolism / Q000648:ultrastructure; D008018:Life Cycle Stages / Q000187:drug effects; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010802:Phylogeny; D046988:Proteasome Endopeptidase Complex / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D017386:Sequence Homology, Amino Acid; D033761:Spores, Protozoan / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D016835:Tritrichomonas foetus / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*
chemical_list: D015853:Cysteine Proteinase Inhibitors; D021122:Protein Subunits; D015800:Protozoan Proteins; C067713:lactacystin; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine