Stephan Urs Sixt

Extracellular, circulating proteasomes and ubiquitin — Incidence and relevance

The ubiquitin-proteasome system is the major pathway for intracellular protein degradation and is also deeply involved in the regulation of most basic cellular processes. Its proteolytic core, the 20S proteasome, has found to be attached also to the cell plasma membrane and certain observations are interpreted as to suggest that they may be released into the extracellular medium, e.g. in the alveolar lining fluid, epididymal fluid and possibly during the acrosome reaction. Proteasomes have also been detected in normal human blood plasma and designated circulating proteasomes; these have a comparatively low specific activity, a distinct pattern of subtypes and their exact origin is still enigmatic. In patients suffering from autoimmune diseases, malignant myeloproliferative syndromes, multiple myeloma, acute and chronic lymphatic leukaemia, solid tumour, sepsis or trauma, respectively, the concentration of circulating proteasomes has been found to be elevated, to correlate with the disease state and has even prognostic significance. Similarly, ubiquitin has been discovered as a normal component of human blood and seminal plasma and in ovarian follicular fluid. Increased concentrations were measured in diverse pathological situations, not only in blood plasma but also in cerebrospinal fluid, where it may have neuroprotective effects. As defective spermatozoa are covered with ubiquitin in the epididymal fluid, extracellular ubiquitination is proposed to be a mechanism for quality control in spermatogenesis. Growing evidence exists also for a participation of extracellular proteasomes and ubiquitin in the fertilization process.

ACE I/D but not AGT (-6)A/G polymorphism is a risk factor for mortality in ARDS

The intrapulmonary renin–angiotensin system via tissue concentration of angiotensin II or bradykinin may have multiple effects on pulmonary pathophysiology. Therefore, it was investigated whether the presence of the D allele of the angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism or the A allele of angiotensinogen (AGT) promoter polymorphism (-6)A/G are independent risk factors for 30-day survival in acute respiratory distress syndrome (ARDS) patients. In a prospective study, adults (Germans of Caucasian ethnicity) with ARDS (n = 84) were recruited from the current authors’ intensive care unit and genotyped for the ACE I/D and the AGT (-6)A/G polymorphisms, as were 200 healthy Caucasian controls. Mortality was increased in the ACE DD genotype compared with the I allele, and the ACE I/D polymorphism was an independent prognostic factor for 30-day survival. Patients with a homozygous DD genotype were at highest risk for death (hazard ratio 5.7; 95% confidence interval 1.7–19.2) compared with the II genotype. In contrast, the AGT (-6)A/G polymorphism was neither associated with an increased risk for development of ARDS nor with outcome. In patients with acute respiratory distress syndrome, the angiotensin-converting enzyme insertion/deletion polymorphism but not the angiotensinogen (-6)A/G promoter polymorphism is an independent risk factor with a pronounced effect on 30-day survival.

Alveolar extracellular 20S proteasome in patients with acute respiratory distress syndrome.

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.

The prognostic impact of circulating proteasome concentrations in patients with epithelial ovarian cancer.

BACKGROUND Intracellularly, the ubiquitin-proteasome system participates in crucial functions such as cell cycling, differentiation, proliferation, gene transcription, and apoptosis. However, in malignancies including ovarian cancer increased extracellular concentrations of circulating 20S proteasomes (c-proteasomes) have been detected in blood. We tested the hypothesis that the c-proteasome plasma concentration is a biomarker associated with the clinical course of ovarian cancer patients. METHODS 20S-proteasome venous plasma concentration was measured by ELISA in patients presenting with ovarian cancer before (n=120) and after (n=68) primary treatment, and in healthy volunteers (n=55). The median follow-up time was 19 months. To assess the relation of proteasome expression with c-proteasome concentration, tumor specimens from 27 patients were immunohistochemically stained for 20S proteasome using an antibody directed against the core subunits of the catalytic domain of the 20S proteasome. RESULTS Median c-proteasome concentration was higher (p<0.0001) in untreated ovarian cancer patients (457.5 ng/ml, range: 200-12540 ng/ml) than in healthy controls 290 ng/ml, range: 140-425 ng/ml). Following completion of primary treatment, the median c-proteasome concentration increased (p=0.003) relative to baseline (595 ng/ml, range: 200-20000 ng/ml) and concentrations positively correlated (p=0.031) with residual disease left at primary surgery. Patients with post-treatment c-proteasome concentrations exceeding the cohorts median showed a diminished survival (p=0.045). We found no correlation between c-proteasome concentration and strength of proteasomal staining in tumor specimens. CONCLUSIONS Circulating proteasome concentrations correlate with residual tumor mass and might be a prognostic variable in ovarian cancer following primary therapy.

Angiogenic and Angiostatic Chemokines in Idiopathic Pulmonary Fibrosis and Granulomatous Lung Disease

Background: Angiogenesis-angiostasis balance and leukocyte recruitment are influenced by different concentrations of distinct chemokines. Objective: To investigate the relative contribution of angiogenic and angiostatic CXC chemokines to the pathogenesis of idiopathic pulmonary fibrosis (IPF) and granulomatous lung diseases, we examined the in vitro production of an angiogenic chemokine (IL-8), and 2 angiostatic chemokines (IP-10 and MIG) by alveolar macrophages. Methods: Alveolar macrophages from 16 patients with granulomatous lung diseases [8 with sarcoidosis, 8 with extrinsic allergic alveolitis (EAA)], 16 patients with IPF, and 8 control subjects were cultured for 24 h. IL-8, IL-18, IP-10 and MIG in the culture supernatants were measured by a fluorescent bead-based multiplex technique. Results: In IPF patients, IL-8 was increased and correlated with bronchoalveolar lavage (BAL) neutrophils, whereas the levels of IP-10 and MIG were normal. In sarcoidosis and EAA patients, IL-8, IP-10, and MIG were all increased and IP-10 and MIG correlated with IL-18, a Th1 cytokine, and the percentage and number of BAL lymphocytes. Conclusions: The difference in the expression of CXC chemokines and a Th1 cytokine may contribute to the different immunopathogenesis, clinical course and responsiveness to treatment of these diseases.

Factor V Leiden mutation is associated with improved 30-day survival in patients with acute respiratory distress syndrome.

Objective:Activation of coagulation and inflammation are parts of the innate host response to infection that may contribute to organ dysfunction and death when control of these systems is compromised. Thus, functional single nucleotide polymorphisms within candidate genes of the inflammation and coagulation cascade are possible factors which may influence severity and/or mortality in acute respiratory distress syndrome. The aim of this study was to investigate whether the factor V Leiden mutation (Arg506Gln) is associated with altered severity and/or mortality in acute respiratory distress syndrome. Design:Retrospective cohort, genetic association study. Setting:Tertiary care intensive care unit. Patients:Adults (white Germans) with acute respiratory distress syndrome (n = 106). Interventions:Genotyping for the factor V Leiden mutation. Measurements and Main Results:Using Kaplan-Meier estimates to compare outcome, 30-day survival was significantly associated with the factor V Leiden mutation (p = .049). Thirty-day survival rates were 100% for Arg/Gln (n = 7) genotypes but only 58% for Arg/Arg (n = 99) genotypes, respectively. Conclusion:We show for the first time that a heterozygous factor V Leiden genotype is associated with improved 30-day survival in patients with acute respiratory distress syndrome.

Proteasomes in human bronchoalveolar lavage fluid after burn and inhalation injury.

The purpose of this study was to determine whether 26S proteasome is detectable in human bronchoalveolar lavage fluid (BALF) and whether burn and inhalation injury is accompanied by changes in BALF proteasome content or activity. BALF was obtained on hospital admission from 28 patients with burn and inhalation injury (controls: 10 healthy volunteers). Proteasome concentrations were quantified by enzyme-linked immonosorbent assay, and their native molecular mass was assessed by gel filtration. Proteasome peptidase activity was measured using a chymotryptic-like peptide substrate in combination with epoxomicin (specific proteasome inhibitor). BALF protein was increased in patients (P < .001) and correlated positively with the degree of inhalation injury. The 20S/26S proteasomes were detectable in all BALF by enzyme-linked immonosorbent assay. Gel filtration confirmed the presence of intact 20S and 26S proteasome that was stable without soluble ATP/Mg2+. In all BALF chymotryptic-like activity was detectable and could be inhibited with epoxomicin by 60 to 70% (P < .01). Absolute amounts of 20S/26S proteasomes and proteasome activity were increased in patients (P < .001 for all). The relative BALF composition after injury was characterized by increased concentrations of 20S proteasome/mg protein (P = .0034 vs volunteers), decreased concentrations of 26S proteasome/mg protein (P = .041 vs volunteers), and reduced specific proteasome activity (P = .044 vs volunteers). The 26S proteasome per milligram and specific proteasome activity were even further reduced in patients who developed ventilator-associated pneumonia (P = .045 and P = .03 vs patients without ventilator-associated pneumonia). This study supports the novel concept that extracellular proteasomes could play a pathophysiological role in the injured lung and suggests that insufficient proteasome function may increase susceptibility for pulmonary complications.

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

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.

The G-Allele of the PSMA6 -8C>G polymorphism is associated with poor outcome in multiple myeloma independently of circulating proteasome serum levels

Introduction: The proteasome system plays a crucial role in several malignant disorders, especially in multiple myeloma (MM). The G‐allele of a single nucleotide polymorphism (SNP) −8C>G in the gene PSMA6, one of seven α‐subunit genes of the 20S proteasome, was associated with myocardial infarction. Moreover, PSMA6 mRNA expression in human B‐cell lines depended on genotypes. We investigated a potential role of this novel SNP in patients with MM. Methods: PSMA6 genotypes of 116 patients with MM were associated with survival. Circulating proteasome levels (CPL) dependent on −8C>G genotypes of 70 newly diagnosed patients were studied using an anti‐20S proteasome enzyme‐linked immunoabsorbant assay (ELISA). Results: Genotype distribution (69 CC, 44 CG, 3 GG) was compatible with Hardy–Weinberg equilibrium. Kaplan–Meier curves revealed a significant association of PSMA6−8C>G with 5‐yr survival (P = 0.014). Median survival time was 43 months for the GG genotype and 50 months for the CG genotype. It was not reached within follow‐up by the CC genotype (CC 5‐yr survival rate 61.2%). Following hazard ratio (HR) for overall survival was calculated: G‐allele vs. CC genotype: 2.038, 95% CI 1.14–3.65, P = 0.017. In multivariate analysis the G‐allele was an independent prognostic factor (HR 2.1, P = 0.014). CPL were not significantly different between genotypes [mean CPL: CC 284.9 ng/mL vs. 303.3 ng/mL G‐allele carriers (P = 0.709)]. Conclusions: These results suggest the G‐allele of the PSMA6−8C>G polymorphism as a possible survival prognosticator.

[ARDS--pulmonary repair and therapeutic options].

The acute respiratory distress syndrome (ARDS) is characterized by inflammation evoked pulmonary edema, hyaline membranes, diffuse endothelial and epithelial injury, and fibrosis. For survival to occur lung repair is required. This review explores recent advances in the field of fibroproliferation with emphasis on cellular and soluble factors, mechanisms involved in lung repair, and genetic factors which influence severity and survival in ARDS.