Characterization of proteolytic activities during intestinal regeneration of the sea cucumber, Holothuria glaberrima

Abstract

Proteolysis carried out by different proteases control cellular processes during development and regeneration. Here we investigated the function of the proteasome and other proteases in the process of intestinal regeneration using as a model the sea cucumber Holothuria glaberrima. This echinoderm possesses the ability to regenerate its viscera after a process of evisceration. Enzymatic activity assays showed that intestinal extracts at different stages of regeneration possessed chymotrypsin-like activity. This activity was inhibited by i) MG132, a reversible inhibitor of chymotrypsin and peptidylglutamyl peptidase hydrolase (PGPH) activities of the proteasome, ii) E64d, a permeable inhibitor of cysteine proteases and iii) TPCK, a serine chymotrypsin inhibitor, but not by epoxomicin, an irreversible and potent inhibitor of all enzymatic activities of the proteasome. To elucidate the role which these proteases might play during intestinal regeneration, we carried out in vivo experiments injecting MG132, E64d and TPCK into regenerating animals. The results showed effects on the size of the regenerating intestine, cell proliferation and collagen degradation. These findings suggest that proteolysis by several proteases is important in the regulation of intestinal regeneration in H. glaberrima.

Molecular Aspects of Regeneration Mechanisms in Holothurians

“…In addition to the genes of connective-tissue structural components, holothurians have a wide variety of genes encoding ECM-modifying proteins. Many different serine, cysteine, aspartyl, and metal peptidases and their inhibitors have been identified [ 71 , 94 , 95 , 96 , 97 , 98 ]. In the study of regeneration in holothurians, much attention is paid to MMPs that can degrade collagen [ 96 , 97 , 99 , 100 , 101 ].…”

“…Furthermore, ajMMP-2 and ajMMP-16 presumably regulate the interaction between ECM components and growth factors through site-specific proteolysis of ECM proteins and other biological molecules [ 96 ]. According to Pasten et al [ 94 ], some cysteine proteases, mainly cathepsins, may be involved in collagen degradation in H. glaberrima .…”

Antibiotics Modulate Intestinal Regeneration

“…These structures contain contractile material from dedifferentiated cells. SLSs were detected using rhodamine-labelled phalloidin, as described previously, using the scheme shown in Scheme S1B , adjudicating a 0 to the presence of SLS or muscle fiber from the distal tip of the rudiment, and a 5 when the SLS and fibers were only visible in the mesentery adjacent to the body wall [ 75 ]. At least three animals were used for each experimental condition and at least two sections from each animal were counted and averaged per animal, values are reported as mean ± standard deviation.…”

Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Echinoderms: Structure and Possible Functions

“…A wide variety of proteinases capable of degrading ECM proteins have now been found in echinoderms. They are serine, cysteine, aspartyl, and metal peptidases and their inhibitors [ 35 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 ]. For example, the genome of the sea urchin S. purpuratus contains approximately 240 metalloprotease genes [ 62 ].…”