Extracellular proteasome in the human alveolar space: a new housekeeping enzyme?

Abstract

We hypothesized that 20S proteasome is present and functional in the extracellular alveolar space in humans. Proteasomal activity was measured in bronchoalveolar lavage (BAL) supernatant from eight humans using specific proteasomal fluorogenic substrates and I 125 -albumin with and without specific proteasome inhibitors. Furthermore, gelfiltration, Western blot technique, and mass spectrometry were applied for proteasome characterization. All proteasomal fluorogenic substrates were hydrolyzed by BAL supernatant, with hydrolysis inhibited by epoxomicin (P ϭ 0.024) and other proteasome inhibitors as well. E64, a lysosomal inhibitor, did not inhibit enzyme activity. The majority of proteolytic activity was detected in BAL supernatant rather than in the cell pellet. No correlation was found between proteasomal hydrolysis in BAL supernatant and lactate dehydrogenase activity, the total cell count in the cell pellet, and the fraction of avital cells in the cell pellet, ruling out cell lysis as a major source of proteasomal activity. Gelfiltration revealed hydrolyzing activity in the supernatant at 660 kDa and proteasome core proteins after analysis by ESI-QqTOF mass spectrometry. Furthermore, Western blots using a polyclonal antibody against proteasomal ␣-/␤-subunits detected proteasomal proteins in the typical 20-to 30-kDa range in BAL supernatant. Incubation of BAL supernatant with I 125 -albumin showed a high mean cleavage rate (101.8 g/ml ϫ h lavage Ϯ 46 SD) that was inhibited by epoxomicin (P ϭ 0.013) and was ATP and ubiquitin independent. We identified for the first time extracellular, biologically active, ATP-and ubiquitin-independent 20S proteasome in the human alveolar space, with a high albumin cleavage rate. Possibly, the proteasome assists in maintenance of a low intraalveolar oncotic pressure and/or alveolar protein degradation. albumin; bronchoalveolar lavage; circulating proteasome; fluorogenic peptides; alveolar protein degradation; lung proteins THE UBIQUITIN/PROTEASOME SYSTEM is a major pathway for selective intracellular non-lysosomal protein degradation in eukaryotic cells and plays an important role in numerous processes (3, 13, 53). The 20S proteasome is a multicatalytic proteinase complex with a cylinder-shaped structure arranged as four axially stacked heptameric rings composed of seven ␣-subunits (outer rings) and seven ␤-subunits (inner rings) (36), respectively. Its catalytic sites are exclusively associated with the ␤-subunits (40, 56) and are ATP and ubiquitin independent. The caspase-like activity of the ␤ 1 -subunit cleaves after acidic residues, the trypsin-like activity of the ␤ 2 -subunit cleaves after basic residues, and the chymotrypsin-like activity of the ␤ 5 -subunit cleaves after hydrophobic residues. Together, these three enzyme activities allow the proteasome to cleave many different substrates into diverse products. Within the cell, the 20S proteasome is associated with large ATP-and ubiquitindependent 19S regulatory cap-like complexes, together yielding a 26S complex. It is ...

Proteasomal Regulation of Pulmonary Fibrosis

“…In a subsequent study, BALF from patients with ARDS was found to have increased proteasomal proteins compared with BALF from control subjects, but proteasomal activity was significantly decreased compared with healthy control subjects. In addition, ARDS BALF inhibited proteasome activity when mixed with BAL from healthy patients, suggesting the presence of a soluble inhibitor (41).…”

Bladder cancer detection using a peptide substrate of the 20S proteasome

“…Moreover, the 20S proteasome can be released from the cell. Sixt et al [12] identified an extracellular and biologically active 20S proteasome in the human alveolar space. More importantly, Dutaund et al [13] applied an ELISA technique to detect the 20S proteasome in human serum.…”

Ubiquitination and Proteolysis in Acute Lung Injury

“…Finally, BAL supernatant successfully cleaved albumin in an ATP-and ubiquitin-independent manner. 81 These data point to the presence and biologic activity of proteasome in the alveolar space; furthermore, they imply a unique, ubiquitin-independent role for extracellular proteasome activity. The previous investigation could have profound implications on human pulmonary diseases, particularly those that involve an alveolar fi lling process that alters intraalveolar protein makeup or oncotic pressure.…”

“…A small study illustrates the presence and activity of proteasome in the alveolar space. 81 All three proteasome activities were identifi ed in a cell-free supernatant of BAL fl uid and were inhibited by epoxomicin. Cell lysis was also ruled out as a potential source of alveolar proteasome.…”