Characterization of proteolytic activities during intestinal regeneration of Holothuria glaberrima

Gastrointestinal diseases are a major problem worldwide. Each year, thousands of people are affected by diverse pathologies associated, in part, to deficiencies in the mechanisms of intestinal regeneration. Our laboratory has been active in studying the process of intestinal organogenesis using as model organism the sea cucumber Holothuria glaberrima. This organism is an echinoderm with the ability to regenerate its viscera after a process of evisceration.;The first aim of this thesis was to analyze the sequences of three components of the Ubiquitin Proteasome System (UPS) of H. glaberrima. These sequences were Rpn10, a subunit of 19S regulatory particle, beta3, a subunit of 20S proteasome and ubiquitin. Predicted proteins from the mRNA sequences showed high degree of conservation that ranged from 60% (Rpn10) to 98% (Ub-RPL40). This is the first time that UPS components are described in holothurians. These findings provide important information about the evolution and conservation of the proteasome subunit homologues in echinoderms.;Once I demonstrated that these components corresponded to the holothurian homologues, I studied their expression during the regeneration process. Thus, in the second aim I demonstrated by RT-PCR that these genes were up-regulated during intestinal regeneration when compared with non regenerating animals. Additionally, protein expression and localization of alpha subunits of the 20S proteasome and ubiquitinated proteins and/or ubiquitin was shown. These data suggested that if the UPS components are present and are up-regulated in the regenerating intestine, this pathway could be participating in the cellular processes that allow the formation of the new intestine.;The focus of the third aim was to study the function of proteasomes during intestinal regeneration. Initially, I studied the enzymatic activity during intestinal regeneration in order to identify the enzymatic properties. In second place, I studied their function by injecting proteasome and protease inhibitors into regenerating animals. Surprisingly and against the initial hypothesis, my results suggest that the proteasome is not the main enzymatic activity present during intestinal regeneration and point to a role of calpains as the principal mediators of proteolytic activities. In addition, the results suggest that cathepsin and serine proteases might also play some roles in regulating several of the process that take place during intestinal regeneration.;The results provide important information on the proteolytic activities associated with intestinal regeneration of H. glaberrima. At the same time they provide insights into the cellular events where these enzymatic processes might be involved and the outcome of inhibiting their activities.