A reduced VWA domain-containing proteasomal ubiquitin receptor of Giardia lamblia localizes to the flagellar pore regions in microtubule-dependent manner (original) (raw)
Related papers
Acta Tropica, 2012
Giardia intestinalis is considered an early-branching eukaryote and is therefore a valuable model for studying primordial cellular processes. This work reports the characterization of the ubiquitin-activating enzyme (E1) during growth and different stages of trophozoite differentiation into cysts. We found that in Giardia E1 expression (both at mRNA and protein levels) is regulated during encystation. The enzyme is proteolytically processed mainly into two fragments of 68 kDa (N-terminal) and 47 kDa (C-terminal). This phenomenon has not been described for any other E1. In trophozoites, this enzyme localized at spots within the cytoplasm as detected by using polyclonal antibodies against either E1 Nor C-terminal fragments. This pattern changed during encystation into a diffuse localization throughout the cytoplasm of encysting cells. E1 localizes in mature cysts at cytoplasmic spots and in the cyst wall. Our antisense silencing experiments suggested that E1 is an essential gene for parasite viability. On the other hand, E1 over-expression greatly increased the encystation rate, indicating a relationship between E1 and Giardia differentiation.
The cytoskeleton of Giardia lamblia
International Journal for Parasitology, 2003
Giardia lamblia is a ubiquitous intestinal pathogen of mammals. Evolutionary studies have also defined it as a member of one of the earliest diverging eukaryotic lineages that we are able to cultivate and study in the laboratory. Despite early recognition of its striking structure resembling a half pear endowed with eight flagella and a unique ventral disk, a molecular understanding of the cytoskeleton of Giardia has been slow to emerge. Perhaps most importantly, although the association of Giardia with diarrhoeal disease has been known for several hundred years, little is known of the mechanism by which Giardia exacts such a toll on its host. What is clear, however, is that the flagella and disk are essential for parasite motility and attachment to host intestinal epithelial cells. Because peristaltic flow expels intestinal contents, attachment is necessary for parasites to remain in the small intestine and cause diarrhoea, underscoring the essential role of the cytoskeleton in virulence. This review presents current day knowledge of the cytoskeleton, focusing on its role in motility and attachment. As the advent of new molecular technologies in Giardia sets the stage for a renewed focus on the cytoskeleton and its role in Giardia virulence, we discuss future research directions in cytoskeletal function and regulation. q
PLoS Neglected …, 2010
Background: Giardia passes through two stages during its life cycle, the trophozoite and the cyst. Cyst formation involves the synthesis of cyst wall proteins (CWPs) and the transport of CWPs into encystation-specific vesicles (ESVs). Active vesicular trafficking is essential for encystation, but the molecular machinery driving vesicular trafficking remains unknown. The Rab proteins are involved in the targeting of vesicles to several intracellular compartments through their association with cytoskeletal motor proteins.
Memórias do Instituto Oswaldo Cruz, 2020
BACKGROUND Ubiquitin (Ub) and Ub-like proteins (Ub-L) are critical regulators of complex cellular processes such as the cell cycle, DNA repair, transcription, chromatin remodeling, signal translation, and protein degradation. Giardia intestinalis possesses an experimentally proven Ub-conjugation system; however, a limited number of enzymes involved in this process were identified using basic local alignment search tool (BLAST). This is due to the limitations of BLAST's ability to identify homologous functional regions when similarity between the sequences dips to < 30%. In addition Ub-Ls and their conjugating enzymes have not been fully elucidated in Giardia. OBJETIVE To identify the enzymes involved in the Ub and Ub-Ls conjugation processes using intelligent systems based on the hidden Markov models (HMMs). METHODS We performed an HMM search of functional Pfam domains found in the key enzymes of these pathways in Giardia's proteome. Each open reading frame identified was analysed by sequence homology, domain architecture, and transcription levels. FINDINGS We identified 118 genes, 106 of which corresponded to the ubiquitination process (Ub, E1, E2, E3, and DUB enzymes). The E3 ligase group was the largest group with 82 members; 71 of which harbored a characteristic RING domain. Four Ub-Ls were identified and the conjugation enzymes for NEDD8 and URM1 were described for first time. The 3D model for Ub-Ls displayed the β-grasp fold typical. Furthermore, our sequence analysis for the corresponding activating enzymes detected the essential motifs required for conjugation. MAIN CONCLUSIONS Our findings highlight the complexity of Giardia's Ub-conjugation system, which is drastically different from that previously reported, and provides evidence for the presence of NEDDylation and URMylation enzymes in the genome and transcriptome of G. intestinalis. Key words: ubiquitin-ubiquitin-like protein-ubiquitin ligase-deubiquitinating enzymes-Giardia-hidden Markov models Ubiquitin (Ub) and ubiquitin-like modifiers (Ub-Ls) are small proteins that covalently attach to protein substrates and regulate various cellular processes such as the cell cycle, endocytosis, signaling pathways, intracellular trafficking, DNA repair and transcription, among others. (1) Ub and Ub-Ls share two common features: a β-grasp fold composed of a five-stranded β-sheet and a C-terminal diglycine motif (GG) used for conjugation to target proteins. Currently, the Ub-L family includes 10 members: small ubiquitin modifier (SUMO), neural precursor cell expressed developmentally downregulated 8 (NEDD8) or Related to Ubiquitin 1 (RUB 1) in yeast, Ubiquitin-Related Modifier-1 (URM1), Ubiquitinfold Modifier 1 (UFM1), autophagy-related proteins 8 and 12 (ATG8 and ATG12), interferon-stimulated gene 15 (ISG15), human leukocyte antigen (HLA)-F adjacent transcript 10 (FAT10), fan ubiquitin-like protein 1 (FUB1), and histone mono-ubiquitination 1 (HUB1). (2) The first step in the Ub-conjugation cascade is activation, which is mediated by the E1 protein (UBA1 in the budding yeast). Further, Ub is transferred to a Ub
Mining the Giardia genome and proteome for conserved and unique basal body proteins
Giardia lamblia is a flagellated protozoan parasite and a major cause of diarrhoea in humans. Its microtubular cytoskeleton mediates trophozoite motility, attachment and cytokinesis, and is characterised by an attachment disk and eight flagella that are each nucleated in a basal body. To date, only 10 giardial basal body proteins have been identified, including universal signalling proteins that are important for regulating mitosis or differentiation. In this study, we have exploited bioinformatics and proteomic approaches to identify new Giardia basal body proteins and confocal microscopy to confirm their localisation in interphase trophozoites. This approach identified 75 homologs of conserved basal body proteins in the genome including 65 not previously known to be associated with Giardia basal bodies. Thirteen proteins were confirmed to co-localise with centrin to the Giardia basal bodies. We also demonstrate that most basal body proteins localise to additional cytoskeletal structures in interphase trophozoites. This might help to explain the roles of the four pairs of flagella and Giardia-specific organelles in motility and differentiation. A deeper understanding of the composition of the Giardia basal bodies will contribute insights into the complex signalling pathways that regulate its unique cytoskeleton and the biological divergence of these conserved organelles.
Experimental Parasitology, 2002
To investigate the complexity of the endomembrane transport system in the early diverging eukaryote, Giardia lamblia, we characterized homologues of the GTP-binding proteins, Rab1 and Rab2, involved in regulating vesicular trafficking between the endoplasmic reticulum and Golgi in higher eukaryotes, and GDI, which plays a key role in the cycling of Rab proteins. G. lamblia Rab1, 2.1, and GDI sequences largely resemble yeast and mammalian homologues, are transcribed as 0.66-, 0.62-, and 1.4-kb messages, respectively, and are expressed during growth and encystation. Western analyses detected an abundant Rab/GDI complex at approximately 80 kDa, and free GDI (60 kDa) in both trophozoites and encysting cells. Immunoelectron microscopy with antibody to Rab1 localized Rab with ER, encystation secretory vesicles, and lysosome-like peripheral vesicles. GDI associated with these structures, and with small vesicles found throughout the cytoplasm, consistent with GDI's key role in Rab cycling between organelles within the cell.
Proteomic analysis of the ventral disc of Giardia lamblia
BMC Research Notes, 2012
Background Giardia lamblia is a multiflagellated protozoan that inhabits the small intestine of vertebrates, causing giardiasis. To colonize the small intestine, the trophozoites form of the parasite remains attached to intestinal epithelial cells by means of cytoskeletal elements that form a structure known as the ventral disc. Previous studies have shown that the ventral disc is made of tubulin and giardins. Results To obtain further information on the composition of the ventral disc, we developed a new protocol and evaluated the purity of the isolation by transmission electron microscopy. Using 1D- and 2D-PAGE and mass spectrometry, we identified proteins with functions associated with the disc. In addition to finding tubulin and giardin, proteins known to be associated with the ventral disc, we also identified proteins annotated in the Giardia genome, but whose function was previously unknown. Conclusions The isolation of the ventral disc shown in this work, compared to previous...