The ubiquitin, proteasome proteolytic pathway is constituted by an ubiquitination enzyme system and a multicatalytic complex, the 26S proteasome. Intracellular proteins to be degraded by this pathway are firstly polyubiquitinated, becoming substrates of 26S proteasome. The present report is a study of this proteolytic pathway in Peripatus acacioi, a threatened of extinguishing  invertebrate of Onychophora phylum, sharing morphological common characteristics with both annelids and  arthropods that is found in Tripui valley, near Ouro Preto, MG, Brazil. Considering that onychophorans have fossil records dating back  to the Cambrian period (more than 400 million years ago), is interesting to identify and characterize the also ancient ubiquitin,proteasome proteolytic pathway in P. Acacioi. For this, the animals  were homogenized  with 1ml of 5mM Tris-HCl pH 8.0, 50 mM leupeptin and 1mM PMSF. The 15,000g, 30 min centrifugation supernatant was passed through a DEAE-cellulose column to obtain Fraction II, an enriched fraction in enzymes belonging to ubiquitin-proteasome proteolytic pathway. 10mg of these proteins were submitted to 10% SDS/PAGE and transferred to a nitrocellulose membrane for Western Blotting employing anti-a1,a2,a3,a5,a6 and a7 proteasome subunits antibodies. These antibodies recognized protein bands ranging from  25 to 30kDa, indicating that proteasome may be present in this invertebrate. After that, we studied if the known proteasome proteolytic activities (chymotrypsin-like, trypsin-like and caspase-like proteolytic activities) were found in Fraction II, using the fluorogenic substrates  SLLVT-MCA; N-Benzoyl-MCA and Suc-Ala-Glu-MCA for detection of each one of the activities, respectively. We found that chymotrypsin-like activity was ten times greater than the other two ones. Classical proteasome inhibitors (MG132, PSI, lactacystin and  epoxomycin) were only partially able to inhibit P. acacioi proteasome activity (46%, 18%, 7% and 36%, respectively). These two results indicate that proteasome exists in Peripatus acacioi and its activities behave distinctly from those found in other organisms where they were studied.
FAPEMIG, FAPESP, IBAMA.