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The associations of circulating 20S proteasomes (c20S) with clinical and serologic disease indices in patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) are unknown. We present the initial report that c20S levels are elevated in MCTD and correlate with clinically relevant changes in disease activity in SLE and MCTD.

The ubiquitin-proteasome pathway plays major roles in all aspects of biology and contributes to various disease processes. Due to the lack of assays that permit proteasome quantification in crude cell extracts, its concentrations in health and disease states as well as the relationship between free 20S core particles (20S) and 26S proteasomes (26S) that consist of 20S singly or doubly capped with 19S regulator complexes (19S) are still largely unknown. Thus, we established a 20S ELISA for the detection of total 20S, and developed a specific 26S ELISA. The latter utilizes the ATP/Mg2+ requirement for 26S stability and shows no cross-reactivity with 20S. Both ELISAs demonstrate intra- and inter-assay variations between 4.9% and 9.4% and recoveries of 105%-109%. Initial application showed that maintenance of the physiological ATP concentration is essential for accurate 26S assessment. Measurements in erythrocyte and peripheral blood mononuclear cell (PBMNC) extracts revealed that the concentrations of 20S were 15-fold and of 26S 130-fold higher in PBMNCs, and suggested that the 26S is the physiological relevant form in PBMNCs (molar ratio 20S/26S 1.1+/-0.4), whereas free 20S is predominant in erythrocytes (molar ratio 20S/26S: 11.5+/-4.0). During storage of packed red blood cell units spontaneous 26S assembly was detectable while specific 26S enzyme activities decreased, indicating that these assays are useful to assess the dynamic interplay between the 20S and 19S. During 26S assay development we further observed that solid phase affinity immobilization (SPAI) of 26S enables quantification of its dissociation into 20S and 19S. Utilizing the SPAI-26S method in combination with the non-hydrolyzable analogue ATP[beta,gamma-NH] and Mg2+ depletion, we provided evidence that ATP binding without hydrolysis via a high affinity binding site (Kd 4-6 microM) as well as ATP binding with hydrolysis via a low affinity binding site that is virtually not saturable under physiological conditions is required to fully stabilize the 26S. Application of these immunological techniques is expected to facilitate proteasome analyses, and may help to better understand its roles in health and disease processes.

Background: Recent data suggest that ubiquitin (Ub) is systemically released after trauma, has pleiotropic effects on host defense mechanisms, and that Ub administration reduces fluid shifts into tissues during inflammation. Ub release after burns (B) has not been studied and its association with injury severity and outcome after blunt trauma (T) is unknown. Thus, we evaluated Ubs association with injury severity and outcomes after B and T., Methods: Injury severity was assessed with the Injury Severity Score (ISS) in T and burn size (% total body surface area, %TBSA) in B. A total of 129 T (ISS: 26 +/- 13) and 55 B (46% +/- 18% TBSA) were observed for sepsis/multiple organ failure (MOF) and survival. In B, sequential organ failure assessment scores were documented daily. Fifty volunteers served as controls (C) Ub serum levels were measured on day 0 (admission), 1, 3, 5, and 7 by enzyme-linked immunosorbent assay. Data were analyzed using bivariate or partial correlation analyses, t test, and analysis of variance with Tukey post-hoc test for multiple comparisons (two-tailed p < 0.05)., Results: Ub was significantly elevated in patients. Peak levels (ng/mL) were detectable on day 0 (C: 118 +/- 76; T: 359 +/- 205; B: 573 +/- 331) and increased with increased ISS, %TBSA, and presence of inhalation injury. In T, Ub normalized by day 3, but remained elevated in B. In B, Ub correlated significantly negative with sequential organ failure assessment scores (r: -0.143; p = 0.0147), sepsis/MOF development (r: -0.363; p = 0.001), and survival (r: -0.231; p = 0.009). Compared with B who recovered uneventfully, Ub levels were significantly lower on days 1 to 7 and on days 5/7 in B who developed sepsis/MOF or died, respectively., Conclusion: Ub concentrations reflect the extent of tissue damage. Along with Ubs previously described anti- inflammatory properties, this study suggests that its systemic release is protective, that burn patients who develop sepsis/MOF have a relative Ub deficiency and that Ub could play an important role during the physiologic response to burn injury.

Recent observations suggest that the ubiquitin-proteasome system (UPS) contributes to the pathophysiology of myocardial ischemia-reperfusion injury. Since its regulation during cold ischemia-reperfusion is unknown, we evaluated the cardiac UPS in a model of heart transplantation in mice. Cardiac ubiquitylation rates and ubiquitin-protein conjugates increased after 3h of cold ischemia (CI) and normalized post-transplant. 20S proteasome content and proteasome peptidase activities were unchanged after CI. 4h/24h post-transplant 20S proteasome concentrations decreased and chymotryptic-like but not tryptic-like proteasome peptidase activity was inactivated. Epoxomicin sensitivity of the proteasome increased 5.7-fold during CI and normalized 4h/24h post-transplant. This was accompanied by the disappearance of a 13.5 kDa-ubiquitin-conjugate during CI that could be attenuated by addition of epoxomicin to the preservation fluid. We conclude that substrate specificity of the proteasome changes during cold ischemia and that proteasome inhibition preserves the physiological ubiquitin-protein conjugate pool during organ preservation. Reduced proteasome activity during reperfusion is caused by a decrease in proteasome content and enzyme inhibition.