BACKGROUND: Proteasomes are 'proteolytic machineries' implicated in many cellular functions, including protein turnover, inflammatory response and immunosurveillance. They exist in various forms sharing the same catalytic core - the 20S proteasome. This core consists of 28 subunits codified by 14 different genes, 3 of which - beta 1, beta 2 and beta 5 - are catalytically active and show peptidyl-glutamyl peptide hydrolyzing (PGPH), trypsin-like and chymo-trypsin-like activities, respectively. Under IFN- delta and TNF- alfa stimuli, the 3 active constitutive subunits are replaced by the corresponding ones - i.e., LMP2, MECL-1, LMP7 - known as inducible subunits, thus resulting in the constitution of the 'immunoproteasome' that is specifically implicated in MHC class I-presented peptide generation. This process is enhanced when the proteasome is associated with the polymeric protein 11S regulator/PA28 made up of 4 alfa and 3 beta subunits. METHODS: The 20S proteasome was purified from post mortem specimens of human kidney cortex by chromatographic and ultracentrifugation techniques. It was then characterized on the basis of (i) multicatalytic activity evaluated using specific fluorogenic peptides, (ii) electrophoretic mobility on non-denaturating polyacrylamide gels followed by in-gel visualization by fluorogenic peptide overlaying and Coomassie blue staining and (iii) subunit composition as ascertained by SDS-PAGE and 2-dimensional electrophoresis followed by silver staining or Western immunoblotting using specific antibodies against the proteasome subunits. The 20S proteasome was also studied for its association with the 11S regulator by Western immunoblotting using an antibody to the regulator alfa subuniT. RESULTS: T he purified proteasome was shown to have PGPH, trypsin-like and chymotrypsin-like activities. Furthermore, it incorporated the inducible subunits and was associated with the 11S regulator. CONCLUSIONS:The features we observed make renal cells susceptible to an over-expression of inflammatory response to immunological challenges.
Structural and functional properties of proteasomes purified from the human kidney
BALDOVINO, Simone;PICCININI, Marco;RINAUDO, Maria Teresa;SENA, Luigi Massimino;ROCCATELLO, Dario
2006-01-01
Abstract
BACKGROUND: Proteasomes are 'proteolytic machineries' implicated in many cellular functions, including protein turnover, inflammatory response and immunosurveillance. They exist in various forms sharing the same catalytic core - the 20S proteasome. This core consists of 28 subunits codified by 14 different genes, 3 of which - beta 1, beta 2 and beta 5 - are catalytically active and show peptidyl-glutamyl peptide hydrolyzing (PGPH), trypsin-like and chymo-trypsin-like activities, respectively. Under IFN- delta and TNF- alfa stimuli, the 3 active constitutive subunits are replaced by the corresponding ones - i.e., LMP2, MECL-1, LMP7 - known as inducible subunits, thus resulting in the constitution of the 'immunoproteasome' that is specifically implicated in MHC class I-presented peptide generation. This process is enhanced when the proteasome is associated with the polymeric protein 11S regulator/PA28 made up of 4 alfa and 3 beta subunits. METHODS: The 20S proteasome was purified from post mortem specimens of human kidney cortex by chromatographic and ultracentrifugation techniques. It was then characterized on the basis of (i) multicatalytic activity evaluated using specific fluorogenic peptides, (ii) electrophoretic mobility on non-denaturating polyacrylamide gels followed by in-gel visualization by fluorogenic peptide overlaying and Coomassie blue staining and (iii) subunit composition as ascertained by SDS-PAGE and 2-dimensional electrophoresis followed by silver staining or Western immunoblotting using specific antibodies against the proteasome subunits. The 20S proteasome was also studied for its association with the 11S regulator by Western immunoblotting using an antibody to the regulator alfa subuniT. RESULTS: T he purified proteasome was shown to have PGPH, trypsin-like and chymotrypsin-like activities. Furthermore, it incorporated the inducible subunits and was associated with the 11S regulator. CONCLUSIONS:The features we observed make renal cells susceptible to an over-expression of inflammatory response to immunological challenges.
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