RATIONALE: Repair mechanisms resulting in alveolar protein degradation in acute respiratory distress syndrome (ARDS) are largely unknown. OBJECTIVES: To test whether the 20S proteasome is present and functional in the alveolar space in patients with ARDS. METHODS: Proteasome antigenic concentration in bronchoalveolar lavage (BAL) supernatants was measured by ELISA in patients with ARDS (n = 64), acute lung injury (ALI) (n = 8), sarcoidosis (n = 13), and in healthy subjects (n = 8). Cleavage of specific fluorogenic substrates (+/-epoxomicin), I(125) albumin degradation rate, and gel filtration were used to quantify and characterize proteasomal activity. The presence of proteasomes was confirmed independently by electron microscopic techniques. MEASUREMENTS AND MAIN RESULTS: Proteasome concentrations in patients with ARDS were markedly increased (1,069 +/- 1,194 ng/ml) in comparison to healthy subjects (60.8 +/- 49.8; P < 0.001), ALI (154 +/- 43; P = 0.006), and sarcoidosis (97.6 +/- 42.2; P = 0.037). All fluorogenic substrates were hydrolyzed (Suc-LLVY-AMC, 3.6 +/- 8.8 pkat/mg; BZ-VGR-AMC, 1.8 +/- 3.1; Suc-LLE-AMC, 1 +/- 1.7) by BAL supernatants of patients with ARDS, with inhibition by epoxomicin (P = 0.0001), and the majority of proteolytic activity was detected in BAL supernatant. Maximum hydrolyzing activity occurred at 660 kD and 20S proteasome was seen microscopically after purification and being released by pneumocytes type II. Proteasomal activity and albumin degradation rate in patients with ARDS were approximately 17-fold lower than in healthy subjects. Proteasomal activity in normal BAL was inhibited by BAL aliquots from patients with ARDS but not by denatured BAL, and returned to normal by purification. CONCLUSIONS: For the first time, we identified extracellular, biologically active 20S proteasome in the alveolar space of patients with ARDS in concentrations much higher than in normal subjects or in those with ALI.
RATIONALE: Repair mechanisms resulting in alveolar protein degradation in acute respiratory distress syndrome (ARDS) are largely unknown. OBJECTIVES: To test whether the 20S proteasome is present and functional in the alveolar space in patients with ARDS. METHODS: Proteasome antigenic concentration in bronchoalveolar lavage (BAL) supernatants was measured by ELISA in patients with ARDS (n = 64), acute lung injury (ALI) (n = 8), sarcoidosis (n = 13), and in healthy subjects (n = 8). Cleavage of specific fluorogenic substrates (+/-epoxomicin), I(125) albumin degradation rate, and gel filtration were used to quantify and characterize proteasomal activity. The presence of proteasomes was confirmed independently by electron microscopic techniques. MEASUREMENTS AND MAIN RESULTS: Proteasome concentrations in patients with ARDS were markedly increased (1,069 +/- 1,194 ng/ml) in comparison to healthy subjects (60.8 +/- 49.8; P < 0.001), ALI (154 +/- 43; P = 0.006), and sarcoidosis (97.6 +/- 42.2; P = 0.037). All fluorogenic substrates were hydrolyzed (Suc-LLVY-AMC, 3.6 +/- 8.8 pkat/mg; BZ-VGR-AMC, 1.8 +/- 3.1; Suc-LLE-AMC, 1 +/- 1.7) by BAL supernatants of patients with ARDS, with inhibition by epoxomicin (P = 0.0001), and the majority of proteolytic activity was detected in BAL supernatant. Maximum hydrolyzing activity occurred at 660 kD and 20S proteasome was seen microscopically after purification and being released by pneumocytes type II. Proteasomal activity and albumin degradation rate in patients with ARDS were approximately 17-fold lower than in healthy subjects. Proteasomal activity in normal BAL was inhibited by BAL aliquots from patients with ARDS but not by denatured BAL, and returned to normal by purification. CONCLUSIONS: For the first time, we identified extracellular, biologically active 20S proteasome in the alveolar space of patients with ARDS in concentrations much higher than in normal subjects or in those with ALI.
Authors: G R Tundo; D Sbardella; A M Santoro; A Coletta; F Oddone; G Grasso; D Milardi; P M Lacal; S Marini; R Purrello; G Graziani; M Coletta Journal: Pharmacol Ther Date: 2020-05-19 Impact factor: 12.310
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