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  • Concholepas concholepas our model species for this


    Concholepas concholepas, our model species for this study, is a marine gastropod found along the Chilean coast that has become commercially important [25], [26], [27]. Until now, the main focus of immunology related to C. concholepas is the production of a novel hemocyanin that has been useful for the study of cancer, due to its antitumor properties [28], [29], [30]. However, there are no studies aimed to characterize the genetic component of the immune response of C. concholepas, despite the availability of the EST database generated by 454 pyrosequencing [31]. Recently, we have performed a new 454 pyrosequencing with major genome coverage (unpublished data) and found candidate genes involved in innate immune response. Herein, Ubiquitin-conjugating enzyme E2 (UBE2) gene was partially identified in C. concholepas with a putative SNP variation. The aim of this study was to characterize the immune response of UBE2 gene in the gastropod C. concholepas and to evaluate the association between the putative SNP-UBE2 and changes in gene NECA synthesis of UBE2 in individuals exposed to V. anguillarum.
    Materials and methods
    Results and discussions
    Acknowledgment This research was possible, thanks to funding provided by a FONDEF research grant D09I1065(CONICYT-Chile).
    Introduction Ubiquitylation is an important modification that can tag proteins for proteasomal degradation or alter their properties. The covalent attachment of ubiquitin to target proteins is vital for most cellular processes including for example gene expression, mitosis, protein sorting, and signal transduction. Mitochondria do not contain a ubiquitin proteasome system (UPS), but are nonetheless closely linked to the UPS particularly regarding quality control, dynamics, and apoptosis [1], [2], [3], [4]. Ubiquitylation at the outer mitochondrial membrane is a well-established process for which several E3’s have been identified including MITOL, MULAN and Parkin [5], [6], [7]. There have also been reports of intra-mitochondrial proteins being ubiquitylated [8], though the mechanistic details remain to be clarified. The ubiquitin like (UbL) proteins contain an integrated ubiquitin domain, but cannot themselves be conjugated to other proteins. The ubiquitin domain containing 2 (UBTD2) protein, also termed dendritic cell-derived ubiquitin (ub)-like protein (DC-UbP), is a short UbL protein that is localized to mitochondria and is implicated in apoptosis [9]. Phylogenic analyses presented here revealed that mammals contain a second homolog, UBTD1, which is hitherto unstudied. To gain further insight into UBTD1 function we screened for interacting proteins. A yeast-2-hybrid (Y2H) and affinity capture screen both identified proteins involved in the ubiquitylation pathway. One group of proteins that was identified in both screens was the UBE2D family of E2 ubiquitin conjugating enzymes. Further assays using recombinant proteins showed that UBTD1 and UBE2D form a stoichiometric complex that is stable after purification over several chromatographic steps. Our studies suggest that UBTD1 belongs to an evolutionarily conserved family of proteins that may regulate the activity and/or specificity of E2 ubiquitin conjugating enzymes belonging to the UBE2D family.
    Materials and methods
    Discussion Many UbL proteins have direct links to the UPS, but some are also linked to other cellular processes, making functional predictions difficult [22], [23], [24]. Our findings here directly link UBTD1 to the UPS and identify processes that may be influenced by this protein. UBTD1 is similar in sequence to UBTD2, which has been structurally characterized. The C-terminal region of the UBTD2 protein contains a UbL domain that has been crystallized and shown to assume a ubiquitin fold [9], [16]. Moreover, the N-terminus of UBTD2 (aa 14–141) forms a novel structural fold that acts as a low affinity ubiquitin binding domain [25]. One group of proteins which contain both a UbL and ubiquitin-binding domain are proteasome delivery factors, which are able to bind both polyubiquitylated substrates and the proteasome [26].