Proteases Shape the Chlamydomonas Secretome: Comparison to Classical Neuropeptide Processing Machinery

The recent identification of catalytically active peptidylglycine α-amidating monooxygenase (PAM) in Chlamydomonas reinhardtii, a unicellular green alga, suggested the presence of a PAM-like gene and peptidergic signaling in the last eukaryotic common ancestor (LECA). We identified prototypical neuropeptide precursors and essential peptide processing enzymes (subtilisin-like prohormone convertases and carboxypeptidase B-like enzymes) in the C. reinhardtii genome. Reasoning that sexual reproduction by C. reinhardtii requires extensive communication between cells, we used mass spectrometry to identify proteins recovered from the soluble secretome of mating gametes, and searched for evidence that the putative peptidergic processing enzymes were functional. After fractionation by SDS-PAGE, signal peptide-containing proteins that remained intact, and those that had been subjected to cleavage, were identified. The C. reinhardtii mating secretome contained multiple matrix metalloproteinases, cysteine endopeptidases, and serine carboxypeptidases, along with one subtilisin-like proteinase. Published transcriptomic studies support a role for these proteases in sexual reproduction. Multiple extracellular matrix proteins (ECM) were identified in the secretome. Several pherophorins, ECM glycoproteins homologous to the Volvox sex-inducing pheromone, were present; most contained typical peptide processing sites, and many had been cleaved, generating stable N- or C-terminal fragments. Our data suggest that subtilisin endoproteases and matrix metalloproteinases similar to those important in vertebrate peptidergic and growth factor signaling play an important role in stage transitions during the life cycle of C. reinhardtii.

Evaluation of Anti-Inflammatory Activity of Pseudananas macrodontes (Morr.) Harms (Bromeliaceae) Fruit Extract in Rats

Several species of the family Bromeliaceae are characterized by the production of proteases in unusual amounts, especially in fruits. Bromelain, an extract rich in cysteine endopeptidases obtained from Ananas comosus L., and a few other proteases have been used as anti-inflammatory agents for some years, but bromelain is still mainly being used as alternative and/or complementary therapy to the treatment with glucocorticoids, nonsteroidal antirheumatics, and immunomodulators. In this study, the anti-inflammatory action of a partially purified extract from Pseudananas macrodontes (Morr.) Harms fruits (PPEPm) is presented, whose main components are cysteine endopeptidases. The effect of PPEPm was assessed in carrageenan-induced and serotonin-induced rat paw edema, as well as in the cotton pellet granuloma model. Doses with equal proteolytic activity of PPEPm and bromelain produced significantly similar anti-inflammatory responses in the acute inflammatory models assayed, supporting the hypothesis that proteolytic activity could be responsible for the anti-inflammatory action. On the contrary, comparable anti-inflammatory effects of PPEPm and bromelain in the chronic inflammatory assay required a much lower proteolytic activity content of PPEPm, which could be due to a differential affinity for the protein target involved in this process.