Distinct Proteasome Subpopulations in the Alveolar Space of Patients with the Acute Respiratory Distress Syndrome

S. U. Sixt,U. Costabel,A. Kloß,B. Dahlmann,P. R. Jungblut,J. Peters,J. Hakenbeck,M. Adamzik,R. Alami

doi:10.1155/2012/204250

Abstract

There is increasing evidence that proteasomes have a biological role in the extracellular alveolar space, but inflammation could change their composition. We tested whether immunoproteasome protein-containing subpopulations are present in the alveolar space of patients with lung inflammation evoking the acute respiratory distress syndrome (ARDS). Bronchoalveolar lavage (BAL) supernatants and cell pellet lysate from ARDS patients (n = 28) and healthy subjects (n = 10) were analyzed for the presence of immunoproteasome proteins (LMP2 and LMP7) and proteasome subtypes by western blot, chromatographic purification, and 2D-dimensional gelelectrophoresis. In all ARDS patients but not in healthy subjects LMP7 and LMP2 were observed in BAL supernatants. Proteasomes purified from pooled ARDS BAL supernatant showed an altered enzyme activity ratio. Chromatography revealed a distinct pattern with 7 proteasome subtype peaks in BAL supernatant of ARDS patients that differed from healthy subjects. Total proteasome concentration in BAL supernatant was increased in ARDS (971 ng/mL ± 1116 versus 59 ± 25; P < 0.001), and all fluorogenic substrates were hydrolyzed, albeit to a lesser extent, with inhibition by epoxomicin (P = 0.0001). Thus, we identified for the first time immunoproteasome proteins and a distinct proteasomal subtype pattern in the alveolar space of ARDS patients, presumably in response to inflammation.

Highlights

The proteasome is a multicatalytic enzyme complex responsible for the degradation of the vast majority of intracellular proteins [1]
LMP7 and LMP2 were not detected in the Bronchoalveolar lavage (BAL) supernatant (Figures 2(a) and 2(b)) of any healthy subject
The molecular weight of the immunoproteasome positive protein bands in the western blots of the BAL cell pellet lysate from acute respiratory distress syndrome (ARDS) patients was greater than that in their BAL supernatants, suggesting that extracellular immunoproteasome protein-containing proteasomes are assembled from larger intracellular pro-proteins

Summary

Introduction

The proteasome is a multicatalytic enzyme complex responsible for the degradation of the vast majority of intracellular proteins [1]. The 20S proteasome itself is a 660–700 kDa [2, 6] multicatalytic proteinase complex with a cylinder-shaped structure arranged as four axially stacked heptametrical rings composed of seven α subunits (outer rings) and seven β subunits (inner rings), respectively [7]. The α type subunits have highly conserved N-terminal extensions which were proposed to have regulatory and targeting function [38]. Five of the seven β type subunits are synthesized as precursor proteins with N-terminal propeptides that are cleaved off during 20S proteasome biogenesis [13,14,15] that is mediated by accessory proteins like the proteasome maturation protein (POMP) [10]

Methods

Results

Conclusion