3.3. PA28γ in infection and inflammation

Silencing of the PSME3 gene, as well as application of Nutlin-3, an inhibitor of Mdm2:p53 interaction, resulted in exaggerated stabilization of p53 in HCT116 or A549 cells, respectively [66]. In general, proteasomal contribution to degradation of regulatory proteins has been confirmed by using proteasome inhibitors in an adequate concentration range, thereby stabilizing either the protein of interest (POI) or its ubiquitin conjugates. Ubiquitin conjugates of specific POIs have been demonstrated after transient expression of HA-tagged ubiquitin, HA-pulldown techniques, and consecutive immunoblot analysis. Cellular systems with thermosensitive E1 enzymes were used to demonstrate dispensability of ATP-dependent ubiquitination for some proteasomal substrates. Depletion of subunits crucial for 19S assembly by siRNA silencing and in vitro degradation assays based on purified proteasome complexes either with or without

regulators are alternative approaches to demonstrate UIPP or UPS involvement [1].

DNA double-strand breaks (DSBs) may lead to chromosomal rearrangements, dysfunctional gene expression, or even aneuploidy, if not properly being targeted by the DNA damage response (DDR). The DDR is a tightly regulated nuclear process, organized majorly by posttranslational modification (PTM) signals, implemented by certain stress protein kinases. It has been presumed that DSB mobility and nuclear chromatin dynamics may serve to support DNA repair in nuclear sub-compartments [67]. Major DNA repair pathways are carried out within a highly organized three-dimensional nuclear environment. Homologous recombination (HR) occurs throughout the cell cycle, while nonhomologous end joining (NHEJ) is restricted

Recently, ATM- and PA28γ-dependent recruitment of 20S proteasomes to sites of DSB has been demonstrated by live cell imaging [42]. PA28γ depletion enhanced the focal retention of proteins of the DNA repair machinery (MDC1, 53BP1, RNF8, or BRCA1) at DNA damage sites, whereas early accumulation of focal proteins and initial formation of modified histone γH2AX were not affected. PA28γ silencing moderately reduced the NHEJ pathway, whereas the HR pathway was markedly enhanced. PA28γ seems to assemble proteasomes at the sites of DSB

While varying levels of chromatin compaction result in euchromatin and heterochromatin, nuclear bodies structure interchromatin as well [68]. PA28γ and 20S proteasomes have been found to be physically associated with nuclear structures such as nuclear speckles [40], Cajal bodies (CBs) [41], and PML bodies [69, 70] or have been associated with nuclear survival

Nuclear speckles as subnuclear interchromatin domains are enriched in components of the pre-mRNA splicing machinery. 20S proteasomes and PA28γ co-localize in such NS structures [40]. PSME3 silencing affects NS organization and recruitment of splicing factors of the SR family to transcription sites. Proteasome inhibitors promote the accumulation of SC35 in NS.

3.1. PA28γ in DNA damage response

78 Current Understanding of Apoptosis - Programmed Cell Death

during early stages of the DNA repair pathway [42].

3.2. PA28γ in nuclear dynamics

functions and chromosomal stability [71].

to S and G2 phases.

PA28γ and NF-κB signaling constitute activation loops affecting inflammatory processes [73] and bacterial infection [74]. Since inflammatory states are preceding neoplastic transformation, the molecular link between PA28γ and NF-κB biology might be of relevance in tumor biology. The PSME3 gene is under control of the transcription factor NF-κB. Since inhibitory proteins of NF-κB signaling are substrates of a PA28γ-mediated UIPP, NF-κB-mediated transcription depends on PSME3 expression and vice versa. This positive feedback regulation between PSME3 gene regulation and NF-κB-directed transcriptional regulation has been observed in two different models. First, IκBε degradation prevents the removal of NF-κB from sites of transcription, promoting constitutive activation of inflammatory pathways in the gastrointestinal mucosa. A DSS colitis model demonstrated the requirement of PA28γ for the pathological process. PSME3 knockout resulted in impairment of the DSS colitis pathology [74]. The second target of PA28γ in NF-κB signaling is KLF4, a negative regulator of NF-κB. Targeting of KFL4 by the PA28γ-proteasome releases the negative modulatory effect on NF-κB, resulting in a higher PSME3 expression and further activation of the inflammatory response to counterstrike bacterial infection [73].

Accessing sera of autoimmune and cancer patients, we observed an increased level of extracellularly localized PA28γ in patients suffering from a diverse spectrum of autoimmune diseases. In patients with rheumatoid arthritis, the level correlated with the disease state and responded to treatment with abatacept [49]. Since PA28γ has been associated with sepsis [75] or wound healing [76], development of clinical applications beyond autoimmune or cancer diagnosis is an emerging future perspective.
