Proteasome Activator 28γ: Impact on Survival Signaling and Apoptosis

This chapter aims to explain the functional impact of the proteasome activator (PA)28γ protein in cellular survival. The mechanistic complexity of this proteasome activator, encoded by the PSME3 gene and overexpressed in tumors in correlation with the degree of severity, is attracting growing attention. Taking anti-apoptotic properties of PA28γ into account, a simple view might explain therapy resistance of tumors by the presence of high concentrations of this proteasome regulator. A more sophisticated approach would consider functional parameters such as subcellular distribution, competition with other proteasome regulators, and factors affecting heptamer assembly, proteasome binding, and activation. Recently, PA28γ has been attributed as a proteasomal recognin, particularly for intrinsically unstructured proteins (IUPs), targeted by ubiquitin-independent proteasomal protein (UIPP) degradation. Other reports demonstrated inhibitory or stimulatory effects of PA28γ on turnover of substrates of the ubiquitin- and ATP-dependent proteasome system (UPS). Since the understanding of functional implications of PA28γ on diverse signaling processes has grown exponentially and the orchestration of proteolytic systems within apoptosis is fairly complex, this article summarizes the recent developments in PA28γ biology with emphasis on cell survival signaling pathways such as DNA repair and apoptosis

Untersuchungen zur strukturellen und funktionellen Plastizität des 20S-Proteasoms der Maus und seiner Modulierung durch den Proteasomaktivator PA28

Die Studie beinhaltet eine biochemisch-molekularbiologische Analyse des 20S-Proteasoms und seiner Aktivierung durch Proteine der PA28-Familie. Das 20S-Proteasom ist die zentrale Epitop-prozessierende cytosolisch-nukleäre Protease des MHC-Klasse-I-Antigenpräsentationsweges. In Mikroglia, wie auch in anderen Zellen, unterliegt das Proteasom einer Interferon-g-(IFN-g)-vermittelten strukturellen Plastizität, d.h. einer Substitution der Untereinheiten der Aktiven Zentren. Durch diesen Austauschmechanismus werden proteolytische Schnittpräferenzen modifiziert, was für die Hierarchie von cytotoxischen T-Zellantworten von Bedeutung ist. Lipopolysaccharide (LPS) bewirken in Mikroglia ebenfalls Veränderungen der proteasomalen Zusammensetzung des 20S-Komplexes und seines PA700-Aktivators. Dies ist ein Hinweis auf die Rolle des Proteasoms auch bei der Prozessierung von Antigenen des endolysosomalen Antigenpräsentationsweges. Die Modulation der Zusammensetzung des 20S-Proteasoms in Mikroglia durch IFN-g und LPS ist ein weiterer Beleg für die Rolle der Mikroglia bei der zellulären Immunantwort im Zentralnervensystem. Funktionen des Proteasoms in der MHC-Klasse-I-Antigenpräsentation werden durch den Proteasomaktivator PA28 optimiert. Die PA28-Proteinfamilie besteht aus den Proteinen PA28a, PA28b und PA28g. Diese Studie trägt - basierend auf kinetischen Modellierungen, Mutagenese- und Protein-Protein-Interaktionsstudien und Untersuchungen zur MHC-Klasse-I-Antigen-präsentation in PA28-Transfektanten - zur funktionellen Neubewertung dieser drei Proteine bei. Die drei PA28-Proteine sind autonome Aktivatoren des Proteasoms. Heteromere PA28ab-Komplexe verursachen eine stärkere Aktivierung des Proteasoms als die homomeren PA28a-oder PA28b-Komplexe. Das PA28g-Protein ist in vitro ein schwacher Aktivator verschiedener proteasomaler Peptidaseaktivitäten, der dennoch in unserem in vivo-Modell eine verbesserte MHC-Klasse-I-Antigenpräsentation bewirkt. Kinetische Argumente sprechen für Funktionen der PA28a und PA28b-Proteine als Translokasen für Peptidsubstate und -produkte des Proteasoms. Anhand des HBx-Proteins des Hepatitis B-Virus wird die Möglichkeit illustriert, das vorgestellte Modell zur Analyse viraler Faktoren anzuwenden, die mit der Aktivierung des Proteasoms durch PA28 interferieren.The 20S proteasome is the proteolytic core complex of the main cytoplasmic and nuclear protein degradation system. One of the numerous functions assigned to the 20S proteasome is the generation of antigenic peptides from intracellular proteins. MHC class I surface presentation of antigenic peptides is one key event of the cellular immune response. The presented study was aimed on the biochemical and molecular-biological analysis of the 20S proteasome and its activation by regulatory proteins of the PA28 family. In the brain, microglial cells are the major antigen presenting cells and they respond sensitive to pathologic events. Cultured mouse microglia was used as a model to study the correlation between microglial activation parameters and structural plasticity of the 20S/26S proteasome. In response to interferon-g , constitutive active site subunits were replaced by inducible subunits as described for other cellular systems. These replacements result in altered proteolytic cleavage preferences, indicating that activated microglia adapts its proteasomal subunit composition to the requirements of an optimized MHC class I epitope processing. Since lipopolysaccharide (LPS), a glycolipid of bacterial pathogens, also alters proteasome subunit composition of the 20S proteasome and its activator PA700, a function of microglial proteasome in processing of antigens of the endolysosomal pathway has been postulated. The modulation of the 20S proteasome subunit composition indicates that microglia is part of the cellular immune response in the central nervous system. The proteasome activator PA28 has been reported to optimize MHC class I antigen presentation. The PA28 protein family is composed of three proteins: PA28a, PA28b and PA28b. Based on kinetic modelling approaches, mutagenesis of activator proteins, protein-protein interaction studies and investigation of MHC class I antigen presentation in PA28-transfected cell lines a evaluation of functions of these three proteins has been performed. In vitro investigations revealed that the recombinant PA28 proteins are activators of peptidase activities of the proteasome. Heteromeric PA28ab complexes are stronger activators than homomeric PA28a o PA28b complexes. Recombinant PA28g is a weak activator of several peptidase activities. Nevertheless, in PA28g transfected B8-fibroblasts preliminary results indicate an improved MHC class I presentation. Based on kinetic evidence, a model has been presented indicating that PA28a and PA28b proteins act as peptide translocases, performing the import of substrates or the export of products into the catalytic chamber of the 20S proteasome. Finally, as examplified with the HBx protein of Hepatitis B virus, the opportunity is presented to use an in vitro reconstitution system of proteasomal activation to examine viral proteins interfering with proteasomal activation

Increased proteasome activator 28 gamma (PA28γ) levels are unspecific but correlate with disease activity in rheumatoid arthritis

Background PA28γ (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, PA28γ is overexpressed in pathological tissue of various cancers affecting e. g. breast, bowl and thyroids. Furthermore, anti-PA28γ autoantibodies have been linked to several autoimmune disorders. The aim of this study was to develop and evaluate a novel and sensitive PA28γ sandwich ELISA for the quantification of PA28γ serum levels in patients with cancer and autoimmune diseases for diagnostic and prognostic purposes. Methods PA28γ-specific polyclonal antibodies and recombinant His-tagged PA28γ were purified and used to develop a sandwich ELISA for the detection of circulating PA28γ. With this new assay, PA28γ serum levels of patients with various cancers, rheumatoid arthritis (RA), Sjögren’s syndrome (SS), adult-onset Still’s disease (AOSD) and different connective- tissue diseases (CTD) were compared with healthy control subjects. Anti-PA28γ autoantibodies were additionally confirmed using a newly developed microbead assay. Results The developed PA28γ sandwich ELISA showed a high specificity with a detection limit of 3 ng/ml. A significant up-regulation of circulating PA28γ 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-PA28γ autoantibody levels with circulating PA28γ protein levels. Furthermore, PA28γ 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). Conclusion The application of PA28γ 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 PA28γ is a useful marker which should be included in studies related to novel treatments, e.g. abatacep