The poly(A)-binding protein (PABP) recognizes the 3' mRNA poly(A) tail and plays an essential role in eukaryotic translation initiation and mRNA stabilization/degradation. PABP is a modular protein, with four N-terminal RNA-binding domains and an extensive C terminus. The C-terminal region of PABP is essential for normal growth in yeast and has been implicated in mediating PABP homo-oligomerization and protein-protein interactions. A small, proteolytically stable, highly conserved domain has been identified within this C-terminal segment. Remarkably, this domain is also present in the Hyperplastic discs protein (HYD) family of ubiquitin ligases. To better understand the function of this conserved region, an x-ray structure of the PABP-like segment of the human HYD protein has been determined at 1.04-Å resolution. The conserved domain adopts a novel fold resembling a right-handed supercoil of four alpha-helices. Sequence profile searches and comparative protein structure modeling identified a small ORF from the Arabidopsis thaliana genome that encodes a structurally similar but distantly related PABP/HYD domain. Phylogenetic analysis of the experimentally determined (HYD) and homology modeled (PABP) protein surfaces revealed a conserved feature that may be responsible for binding to a PABP interacting protein, Paip1, and other shared interaction partners (Deo, 2001).
Use of the differential display technique to isolate progestin-regulated genes in T-47D human breast cancer cells led to identification of a novel gene, EDD. The cDNA sequence contains a 2799 amino acid open reading frame sharing 40% identity with the predicted 2894 amino acid product of the Drosophila melanogaster tumor suppressor gene hyperplastic discs, while the carboxy-terminal 889 amino acids show 96% identity to a rat 100 kDa HECT domain protein. EDD mRNA is progestin-induced in T-47D cells and is highly abundant in testes and expressed at moderately high levels in other tissues, suggesting a broad role for EDD. Anti-EDD antibodies immunoprecipitated an approximately 300 kDa protein from T-47D cell lysates. HECT family proteins function as E3 ubiquitin-protein ligases, targeting specific proteins for ubiquitin-mediated proteolysis. EDD is likely to function as an E3 since in vitro translated protein binda ubiquitin reversibly through a conserved HECT domain cysteine residue. EDD was localized by FISH to chromosome 8q22, a locus disrupted in a variety of cancers. Given the homology between EDD and the Hyperplastic discs protein, which is required for control of imaginal disc growth in Drosophila, EDD potentially has a role in regulation of cell proliferation or differentiation (Callaghan, 2002).
The ubiquitin-protein ligase EDD encodes an ortholog of the hyperplastic discs tumor suppressor gene, which has a critical role in Drosophila development. Frequent allelic imbalance at the EDD chromosomal locus in human cancers suggests a role in tumorigenesis. In addition to a HECT (homologous to E6-AP carboxyl terminus) domain, the EDD protein contains a UBR1 zinc finger motif and ubiquitin-associated domain, each of which indicates involvement in ubiquitinylation pathways. This study shows that EDD interacts with importin alpha 5 through consensus basic nuclear localization signals and is localized in cell nuclei. EDD also binds progesterone receptor (PR) and potentiates progestin-mediated gene transactivation. This activity is comparable with that of the coactivator SRC-1, but, in contrast, the interaction between EDD and PR does not appear to involve an LXXLL receptor-binding motif. EDD also binds calcium- and integrin-binding protein/DNA-dependent protein kinase-interacting protein, a potential target of ubiquitin-mediated proteolysis, and an altered association is found between EDD and calcium- and integrin-binding protein/DNA-dependent protein kinase-interacting protein in response to DNA damage. The data presented here demonstrate a role for EDD in PR signaling but also suggest a link to cancer through DNA damage response pathways (Henderson, 2002).
The human homolog of the regulator of cell proliferation hyperplastic discs in Drosophila, designated hHYD, is a HECT-domain ubiquitin ligase. hHYD provides a ubiquitin system for a cellular response to DNA damage. A yeast two-hybrid screen has shown that DNA topoisomerase IIbeta-binding protein 1 (TopBP1) interacts with hHYD. Endogenous hHYD binds the BRCA1 C-terminus domains of TopBP1 that are highlighted in DNA damage checkpoint proteins and cell cycle regulators. Using an in vitro reconstitution, specific E2 (ubiquitin-conjugating) enzymes (human UbcH4, UbcH5B, and UbcH5C) transfer ubiquitin molecules to hHYD, leading to the ubiquitination of TopBP1. TopBP1 is usually ubiquitinated and degraded by the proteosome, whereas X-irradiation diminishes the ubiquitination of TopBP1, probably via phosphorylation, resulting in the stable colocalization of up-regulated TopBP1 with gamma-H2AX nuclear foci in DNA breaks. These results demonstrate that hHYD coordinates TopBP1 in the DNA damage response (Honda, 2002).
Conjugation of ubiquitin to proteins is activated during spermatogenesis. Ubiquitination is mediated by ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (UBCs or E2s), and ubiquitin protein ligases (E3s). Since activated ubiquitination is UBC4 dependent, Rat100, a UBC4-dependent E3 expressed in the testis, was characterized. Analysis of expressed sequence tag sequences and immunoblotting have shown that Rat100 is actually a 300-kDa protein expressed mainly in the brain and testis and is similar to the human E3 identified by differential display (EDD) protein and the Drosophila hyperplastic discs gene, mutants of which cause a defect in spermatogenesis. Rat100 is induced during postnatal development of the rat testis, peaking at d 25. It is localized only in germ cells and is highly expressed in spermatocytes, moderately in round and slightly in elongating spermatids. In contrast to UBC4 whose removal from a testis extract abrogates much of the conjugation activity, immmunodepletion of Rat100 from the extracts has little effect. Rat100 therefore has a limited subset of substrates, some of which appear associated with the E3, since the immunoprecipitate containing Rat100 supports incorporation of (125)I-ubiquitin into high molecular weight proteins. Thus, Rat100 is the homolog of human EDD and likely of Drosophila Hyperplastic discs. This homology suggests that induction of this E3 results in ubiquitination of specific substrates, some of which are important in male germ cell development (Oughtred, 2002).
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