26S proteasome is a 2,5 MDa ATP-dependent macromolecular complex that regulates protein degradation in eukaryotic cells degrading proteins after their ubiquitination (1). Proteasome is also involved in antigen presentation since peptides produced during this process are presented on cellular surface in association with MHC class-I (2). In this work we aim to perform a comparative study of proteasomal enzymatic activities in species of veterinary interest to better understand the regulation of antigen presentation in farm animals, to improve knowledge of antigen processing in animals and to compare it with human. Proteasomal peptidase activities were measured in eight different species after a partial purification through differential centrifugation of 26S constitutive proteasome (from muscle) and 26S immunoproteasome (from spleen) using specific fluorogenic peptides: 100µM Suc-LLVY-amc (chymotrypsin-like activity), 100µM Z-YVAD-amc (caspase-like activity), 100µM Bz-VGR-amc (trypsin-like activity). Western blot analysis were also performed using primary antibody anti α3, α5 and α6 no catalytic subunits. Results of spectrofluorimetric measurements show that proteasomal enzimatic activity is overall conserved in the animal species examined despite some species-specific differences, since the ratio between peptidase activities are maintained, both for constitutive and immuno 26S proteasomes. A substantial conservation of the proteasomal active sites in animals of veterinary interest is further demonstrated by a similar sensitivity of the β5 subunit (chymotrypsin-like peptidase activity) to three of the most specific inhibitors currently available. Further western blot analysis on three different α subunits confirm how the proteasomal structure is highly conserved in the species examined. To clarify in more detail how the proteasome system works in farm animals we plan to test the sensitivity of the other active sites to proteasomal inhibitors and to study degradation products of model substrates. REFERENCES: 1. GLICKMAN MH AND CIECHANOVER A., “THE UBIQUITIN-PROTEASOME PROTEOLYTIC PATHWAY: DESTRUCTION FOR THE SAKE OF CONSTRUCTION”. PHYSIOL REV. 2002 APR;82(2):373-428. 2. GOLDBERG AL., “FUNCTIONS OF THE PROTEASOME: FROM PROTEIN DEGRADATION AND IMMUNE SURVEILLANCE TO CANCER THERAPY” . BIOCHEM SOC TRANS. 2007 FEB;35(PT 1):12-7.
Comparative study of proteasomal enzymatic activities in farm animals to better understand biochemical mechanisms of antigen processing
RAULE, MARY;CERRUTI, Fulvia;CASCIO, Paolo
2014-01-01
Abstract
26S proteasome is a 2,5 MDa ATP-dependent macromolecular complex that regulates protein degradation in eukaryotic cells degrading proteins after their ubiquitination (1). Proteasome is also involved in antigen presentation since peptides produced during this process are presented on cellular surface in association with MHC class-I (2). In this work we aim to perform a comparative study of proteasomal enzymatic activities in species of veterinary interest to better understand the regulation of antigen presentation in farm animals, to improve knowledge of antigen processing in animals and to compare it with human. Proteasomal peptidase activities were measured in eight different species after a partial purification through differential centrifugation of 26S constitutive proteasome (from muscle) and 26S immunoproteasome (from spleen) using specific fluorogenic peptides: 100µM Suc-LLVY-amc (chymotrypsin-like activity), 100µM Z-YVAD-amc (caspase-like activity), 100µM Bz-VGR-amc (trypsin-like activity). Western blot analysis were also performed using primary antibody anti α3, α5 and α6 no catalytic subunits. Results of spectrofluorimetric measurements show that proteasomal enzimatic activity is overall conserved in the animal species examined despite some species-specific differences, since the ratio between peptidase activities are maintained, both for constitutive and immuno 26S proteasomes. A substantial conservation of the proteasomal active sites in animals of veterinary interest is further demonstrated by a similar sensitivity of the β5 subunit (chymotrypsin-like peptidase activity) to three of the most specific inhibitors currently available. Further western blot analysis on three different α subunits confirm how the proteasomal structure is highly conserved in the species examined. To clarify in more detail how the proteasome system works in farm animals we plan to test the sensitivity of the other active sites to proteasomal inhibitors and to study degradation products of model substrates. REFERENCES: 1. GLICKMAN MH AND CIECHANOVER A., “THE UBIQUITIN-PROTEASOME PROTEOLYTIC PATHWAY: DESTRUCTION FOR THE SAKE OF CONSTRUCTION”. PHYSIOL REV. 2002 APR;82(2):373-428. 2. GOLDBERG AL., “FUNCTIONS OF THE PROTEASOME: FROM PROTEIN DEGRADATION AND IMMUNE SURVEILLANCE TO CANCER THERAPY” . BIOCHEM SOC TRANS. 2007 FEB;35(PT 1):12-7.
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