In silico analysis of ubiquitin/ubiquitin-like modifiers and their conjugating enzymes in Entamoeba species

Abstract

Covalent modification of proteins by ubiquitin (Ub) and ubiquitin-like modifiers (Ubls) regulates many cellular functions in eukaryotes. These modifications are likely to be associated with pathogenesis, growth, and development of many protozoan parasites but molecular details about this pathway are unavailable for most protozoa. This study presents an analysis of the Ub pathway in three members of the Entamoeba species. Using bioinformatics tools we have identified all Ub and Ubl genes along with their corresponding activating, conjugating, and ligating enzymes (E1, E2s, and E3s) in three Entamoeba species, Entamoeba histolytica, Entamoeba dispar, and Entamoeba invadens. Phylogenetic trees were established for the identified E2s and RING finger E3s using maximum-likelihood method to infer the relationship among these proteins. In silico co-domain analysis of RING finger E3s implicates these proteins in a variety of functions. Several known and putative regulatory motifs were identified in the upstream regions of RING finger domain containing E3 genes. All E2 and E3 genes were analyzed in genomic context in E. histolytica and E. dispar. Most E2s and E3s were in syntenic positions in the two genomes. Association of these genes with transposable elements (TEs) was compared between E. histolytica and E. dispar. A closer association was found between RING finger E3s with TEs in E. histolytica. In summary, our analyses suggests that the complexity of the Ub pathway in Entamoeba species is close to that observed in higher eukaryotes. This study provides important data for further understanding the role of Ub pathway in the biology of these organisms.

A new gene inventory of the ubiquitin and ubiquitin-like conjugation pathways in Giardia intestinalis

“…Orthologs of HUB1 have been identified from yeasts to humans, in apicomplexan parasites, and in Entamoeba, (deep-branching eukaryote, such as Giardia). (11,18,22) In S. cerevisiae, S. pombe, and humans, HUB1 is mainly involved in alternative splicing of pre-mRNA. (23) Notably, in Giardia, few introns have been reported, and splicing is not essential for the parasite's viability.…”

Structural Determinants of Ubiquitin Conjugation in Entamoeba histolytica

“…EhUbiquitin Is Activated by the E1 Enzyme EhUba1-The E. histolytica genome encodes a single predicted ubiquitin-activating E1 enzyme, as well as predicted activating enzymes for Nedd8, SUMO, and other ubiquitin-like modifiers (15). The putative ubiquitin-activating enzyme EhUba1 (AmoebaDB accession EHI_020270) was cloned from genomic DNA, expressed, and purified from E. coli.…”

“…EhUba1 Engages the E2 Enzyme EhUbc5 and Transfers Activated EhUbiquitin-A number of predicted E2 ubiquitin-conjugating enzymes have been identified within the E. histolytica genome, although none has been functionally assessed (15). We cloned a subset of candidate E2s from the E. histolytica genome and attempted to express and purify each of them from E. coli.…”

“…The E. histolytica genome encodes a large number of putative E3 enzymes including HECT, RING, PHD, and U-box domaincontaining proteins (15). A subset of candidate E3s were cloned from E. histolytica genomic DNA and expressed in E. coli.…”

“…Despite its relatively dissimilar sequence, EhUbiquitin complements deletion of the polyubiquitin gene ubi4 in yeast, suggesting conserved functions in E. histolytica (14). More recent bioinformatic analyses of the E. histolytica genome revealed an extensive family of putative ubiquitin activating, conjugating, and ligating enzymes, as well as parallel systems for other ubiquitin-like modifiers (15). However, functional studies of this putative ubiquitination machinery have not yet emerged.…”