ABSTRACT Proteasomes degrade a multitude of protein substrates in the cytosol and nucleus, and thereby are essential for many aspects of cellular function. Because the proteolytic sites are sequestered in a closed barrel-shaped structure, activators are required to facilitate substrate access. Structural and biochemical studies of two activator families, 11S and Blm10, have provided insights to proteasome activation mechanisms, although the biological functions of these factors remain obscure. Recent advances have improved our understanding of the third activator family, including the 19S activator, which targets polyubiquitylated proteins for degradation. Here we present a structural perspective on how proteasomes are activated and how substrates are delivered to the proteolytic sites.
SourceAvailable from: Stefan Rödiger[Show abstract] [Hide abstract]
ABSTRACT: PA28gamma (also known as Ki, REG gamma, PMSE3), a member of the ubiquitin-and ATP-independent proteasome activator family 11S, has been proved to show proteasome-dependent and -independent effects on several proteins including tumor suppressor p53, cyclin-dependent kinase inhibitor p21 and steroid receptor co-activator 3 (SCR-3). Interestingly, PA28gamma is overexpressed in pathological tissue of various cancers affecting e. g. breast, bowl and thyroids. Furthermore, anti-PA28gamma autoantibodies have been linked to several autoimmune disorders. The aim of this study was to develop and evaluate a novel and sensitive PA28gamma sandwich ELISA for the quantification of PA28gamma serum levels in patients with cancer and autoimmune diseases for diagnostic and prognostic purposes. PA28gamma-specific polyclonal antibodies and recombinant His-tagged PA28gamma were purified and used to develop a sandwich ELISA for the detection of circulating PA28gamma. With this new assay, PA28gamma serum levels of patients with various cancers, rheumatoid arthritis (RA), Sjogren's syndrome (SS), adult-onset Still's disease (AOSD) and different connective-tissue diseases (CTD) were compared with healthy control subjects. Anti-PA28gamma autoantibodies were additionally confirmed using a newly developed microbead assay. The developed PA28gamma sandwich ELISA showed a high specificity with a detection limit of 3 ng/ml. A significant up-regulation of circulating PA28gamma was detected in the sera of patients with cancer, RA, SS and CTD. A significant correlation was observed dependent on age as well as anti-PA28gamma autoantibody levels with circulating PA28gamma protein levels. Furthermore, PA28gamma serum levels showed a correlation with disease activity in patients with RA under treatment with the T-cell directed biological compound abatacept according to disease activity score 28 (DAS28) and erythrocyte sedimentation rate (ESR). The application of PA28gamma as a novel biomarker for diagnostic purposes of a specific disease is limited, since elevated levels were observed in different disorders. However, the correlation with disease activity in patients with RA suggests a prognostic value, which needs to be addressed by further studies. Therefore our results show that PA28gamma is a useful marker which should be included in studies related to novel treatments, e.g. abatacept.BMC Musculoskeletal Disorders 12/2014; 15(1):414. DOI:10.1186/1471-2474-15-414 · 1.90 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: In eukaryotic cells, intracellular protein breakdown is mainly performed by the ubiquitin-proteasome system. Proteasomes are supramolecular protein complexes formed by the association of multiple sub-complexes and interacting proteins. Therefore, they exhibit a very high heterogeneity whose function is still not well understood. Here, using a newly developed method based on the combination of affinity purification and protein correlation profiling associated with high-resolution mass spectrometry, we comprehensively characterized proteasome heterogeneity and identified previously unknown preferential associations within proteasome sub-complexes. In particular, we showed for the first time that the two main proteasome subtypes, standard proteasome and immunoproteasome, interact with a different subset of important regulators. This trend was observed in very diverse human cell types and was confirmed by changing the relative proportions of both 20S proteasome forms using interferon-γ. The new method developed here constitutes an innovative and powerful strategy that could be broadly applied for unraveling the dynamic and heterogeneous nature of other biologically relevant supramolecular protein complexes. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.Molecular Systems Biology 01/2015; 11(1). DOI:10.15252/msb.20145497 · 14.10 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Evading apoptosis is a cancer hallmark that remains a serious obstacle in current treatment approaches. While proteasome inhibitors (PIs) have transformed management of multiple myeloma (MM), drug resistance emerges through induction of the aggresome+autophagy pathway as a compensatory protein clearance mechanism. Genome-wide profiling identified miRs differentially expressed in bortezomib-resistant myeloma cells compared to drug-naïve cells. The effect of individual miRs on proteasomal degradation of short-lived fluorescent reporter proteins was then determined in live cells. MiR-29b was significantly reduced in bortezomib-resistant cells as well as in cells resistant to second-generation PIs carfilzomib and ixazomib. Luciferase reporter assays demonstrated that miR-29b targeted PSME4 which encodes the proteasome activator PA200. Synthetically-engineered miR-29b replacements impaired the growth of myeloma cells, patient tumor cells and xenotransplants. MiR-29b replacements also decreased PA200 association with proteasomes, reduced the proteasome's peptidase activity and inhibited ornithine decarboxylase turnover, a proteasome substrate degraded through ubiquitin-independent mechanisms. Immunofluorescence studies revealed that miR-29b replacements enhanced the bortezomib-induced accumulation of ubiquitinated proteins but did not aggresome or autophagosome formation. Taken together, our study identifies miR-29b replacements as the first-in-class miR-based PIs that also disrupt the autophagy pathway and highlights their potential to synergistically enhance the anti-myeloma effect of bortezomib.Leukemia accepted article preview online, 19 September 2014. doi:10.1038/leu.2014.279.Leukemia 09/2014; DOI:10.1038/leu.2014.279 · 9.38 Impact Factor