Proteasomes are located in both the nucleus and cytoplasm of cells, and they play a major role in the ubiquitin-dependent and ubiquitin-independent non-lysosomal pathways of intracellular protein degradation [1, 2]. Proteasomes are also involved in the turnover of various regulatory proteins (e.g., rate-limiting enzymes  and proteins for cell-cycle control  or transcriptional regulation ), antigen processing , cell differentiation , and apoptosis . The 26S proteasome is a multicatalytic enzyme with a highly ordered structure composed of at least 32 different subunits arranged in two subcomplexes: a 20S core and a 19S regulator particle . The 20S-proteasome is composed of four rings of 28 non-identical subunits (two rings are composed of seven alpha subunits, and the other two rings are composed of seven beta subunits). Three of the seven beta subunits have proteolytic sites; the β1, β2, and β5 subunits are associated with caspase-like, trypsin-like, and chymotrypsin-like activities, respectively . These β1, β2, and β5 subunits cleave peptide bonds at post-acidic, −basic, and -hydrophobic amino acid residues, respectively . However, subunits β1, β2, and β5 could be replaced with β1i, β2i, and β5i by interferon-gamma (IFN-γ), and this IFN-γ-inducible proteasome isotype is called the immunoproteasome .
The serum proteasome levels of patients with malignant tumors are elevated because the proteasome is overexpressed in tumor cells. In patients with multiple myeloma, serum proteasome concentrations have been shown to be associated with disease severity and activity : the serum proteasome concentrations were significantly elevated in patients with multiple myeloma compared to controls, in multiple myeloma versus monoclonal gammopathies of undetermined significance (MGUS), and in active versus smoldering multiple myeloma . Similarly, elevated serum proteasome levels were also reported in autoimmune diseases characterized by B-cell abnormality . In the present study, to determine the diagnostic value of the serum proteasome concentration in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), we investigated patients with myeloperoxidase (MPO)-AAV at various stages of the disease.
Patients and controls
We analyzed the cases of 44 patients with MPO-ANCA-associated microscopic polyangiitis (MPA) and renal involvement. The diagnosis of MPA was based on the European Medicines Agency algorithm , and patients with other types of systemic vasculitis (including eosinophlic granuromatosis with polyangiitis, granulomatosis with polyangiitis, and anti-glomerular basement disease) were excluded.
Of the 44 MPO-AAV patients, 30 provided serum samples before the initial treatment, and 30 provided samples during remission; 16 provided samples both before the initial treatment and during remission. The Birmingham Vasculitis Activity Score (BVAS) was used to evaluate patients’ disease activity, and remission was defined as a BVAS of 0. As controls, 14 healthy volunteers and 26 patients with chronic kidney disease (CKD) were investigated. The causes of CKD were nephrosclerosis (n = 10), chronic glomerulonephritis (n = 11), diabetic nephrosclerosis (n = 3), and autosomal dominant polycystic kidney disease (n = 2).
Sample collection and analysis
The serum samples measured by a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Enzo Life Science, Plymouth Meeting, PA, U.S.) in duplicate. In brief, 96-well microtiter plates were coated with a mouse anti-20S-proteasome alpha-6 subunit monoclonal antibody and left overnight at 4 °C, followed by blocking with phosphate-buffered saline (PBS) containing bovine serum albumin for 2 h at room temperature (RT). A serum sample was then added to each well, and the plates were incubated for 1 h at RT. A rabbit anti-20S-proteasome polyclonal antibody was then added to each well, and the plates were incubated for 1 h at RT, followed by incubation with a horseradish-peroxidase-labeled goat anti-rabbit IgG antibody for 1 h at RT. The plates were finally incubated with chromogen (tetramethylbenzidine) and hydrogen peroxide for 30 min at RT and then added with 1 N hydrochloride acid solution.
Between these steps, the plates were washed five times with Tris-buffered saline. The plates were immediately read on a microplate reader (Sunrise Remote®, Tecan Japan, Kanagawa, Japan) set at 450 nm with 540 nm as a reference wavelength. The inter- and intra-assay variations were < 10%.
All statistical analyses were performed using PASW Statistics software, ver. 18 (IBM Japan, Tokyo) for Windows. The data are expressed as means ± standard deviations or as numbers with percentages of the total. The chi-square test with Yates’ continuity correction and Fisher’s exact test were used for differences in categorical variables, and post-hoc comparisons (Bonferroni correction) were performed to detect differences among three groups. The Mann-Whitney U-test was used for two-group comparisons, and we conducted an analysis of variance (ANOVA) to assess differences among three or more groups; post-hoc comparisons were made using the Bonferroni/Dunn test. Correlations were determined using Spearman’s univariate correlation test and a linear regression analysis. The multiple linear regression analysis included the covariates shown to be significantly associated with the serum 20S-proteasome level in the correlation analysis, and the data are expressed as standardized regression coefficients (β). We applied comparative receiver-operating-characteristic (ROC) curves and the area under the curve (AUC) to assess the disease activity accuracy of the the serum 20S-proteasome level and inflammatory variables. P-values were accepted as significant at < 0.05, but in the comparisons of three or more groups, the critical p-value was divided by the number of comparisons being made.