3. The PA28γ scaffolding hub in tumor biology

A particular interest of this article is the question, how UPS and UIPP are involved in regulating cell survival pathways intimately linked with tumor biology and therapy resistance (Figure 1). Cyclin-dependent protein kinases (CDKs) are one regulatory implement of proliferative control. As indicated in Figure 1A, the PA28γ-dependent degradation of several CDK inhibitors (CDKIs) has been observed [61]. Surprisingly, expression of thermo-labile E1 enzymes in ts20TG or tsBN75 cells did not impair degradation of p21 [62]. Furthermore, contribution of SCF E3 ligases in p21 degradation could be excluded, since ts41 cells harboring a temperature sensitive mutation in the NEDD8-activating enzyme did not reveal an increased p21 half-life at the restrictive temperature. However, p21Cip1 half-life, and similarly that of lysine-less p16INK4A or p19Arf, was extended in embryonic fibroblasts from PA28γ knockout Proteasome Activator 28γ: Impact on Survival Signaling and Apoptosis http://dx.doi.org/10.5772/intechopen.74731 77

context-dependent cytosolic presence has also been reported. In breast cancer, PA28γ accumulates in nuclei, whereas in pancreatic cancer cells, PA28γ emerges in the cytosol [51], indicating that cellular context generates diverse scenarios with respect to subcellular localization and function of PA28γ. Nuclear-cytosolic shuttling, mediated by posttranslational modification, depends on cell types and conditions [44, 52, 53]. Sumoylation of amino-terminal lysine residues K6/14/12 by PIAS1 (E3) enhances cytosolic localization and increases stability, presumably prerequisites for proteasome association and substrate recognition [44]. Contrarily, acetylation at K195 by CREB-binding protein (CBP) favors assembly of nuclear PA28γ. Deacetylation by sirtuin 1 (SirT1) has been demonstrated in mammalian cells. Since K6 and K14 residues can be acetylated as well, competition with sumoylation cannot be excluded [45]. Furthermore, phosphorylation of PA28γ by protein kinase MEKK3 resulted in a concomitant increase of PA28γ levels in Cos cells [43]. Stress kinases Chk2 and ATM have been reported to phosphorylate PA28γ, thereby affecting protein-protein interactions (PPI) and functions in

Several authors showed the association of PA28γ with tumorigenic pathways and correlation of expression with malignancy and metastasis. In skin carcinogenesis, PA28γ is involved in modulating WNT signaling [54]. The improved survival properties of PA28γ overexpressing cells [46] and the well-documented overexpression in tumors and cancer cell lines raise the question, how PSME3 gene expression is regulated on the transcriptional and translational

PA28γ-mediated mechanisms supporting cell survival in cancer cells are of high relevance for cancer therapy, particularly in the light of profoundly increased levels of PA28γ in cancer cell lines and in tumor tissue. Highly increased steady-state level of PA28γ was observed majorly in epithelial and mesenchymal tumors such as thyroid neoplasm [55]; breast tumors, particularly those with poor prognosis [56–58]; colorectal cancers [48]; hepatocellular carcinoma [59];

A particular interest of this article is the question, how UPS and UIPP are involved in regulating cell survival pathways intimately linked with tumor biology and therapy resistance (Figure 1). Cyclin-dependent protein kinases (CDKs) are one regulatory implement of proliferative control. As indicated in Figure 1A, the PA28γ-dependent degradation of several CDK inhibitors (CDKIs) has been observed [61]. Surprisingly, expression of thermo-labile E1 enzymes in ts20TG or tsBN75 cells did not impair degradation of p21 [62]. Furthermore, contribution of SCF E3 ligases in p21 degradation could be excluded, since ts41 cells harboring a temperature sensitive mutation in the NEDD8-activating enzyme did not reveal an increased p21 half-life at the restrictive temperature. However, p21Cip1 half-life, and similarly that of lysine-less p16INK4A or p19Arf, was extended in embryonic fibroblasts from PA28γ knockout

DNA damage response [42].

level.

2.6. Overexpression of PA28<sup>γ</sup> in cancer

76 Current Understanding of Apoptosis - Programmed Cell Death

and oral squamous cell carcinoma (OSCC) [60].

3. The PA28γ scaffolding hub in tumor biology

Figure 1. Mechanistic diversity of PA28γ-mediated protein turnover. (A) Typical protease complexes and substrates of UIPP. (B) Core components of UPS. Note, that UIPP and UPS are connected by cross talk: 11S and 19S regulators compete for binding to the 20S core protease. Increased assembly of PA28γ complexes is supposed to reduce the relative amount of 30S proteasomes. In cells overexpressing PA28γ, certain proteasomal substrates of PA28γ-20S proteasomes may be recognized by increased turnover, while others may accumulate due to UPS inhibition. The second mode of cross talk takes place, if UIPP degrades E3 enzymes or components required for their activation. As the third mode, PA28γ might increase PPI between E3s (Mdm2) and their substrates (S1, p53), thereby increasing UPS-dependent substrate turnover.

mice [61]. Noteworthy, aforementioned CDKIs had measurable slow rates of proteasomedependent turnover after depletion of PA28γ, indicating that PA28γ-20S proteasome UIPP is more efficient than the PA28γ-independent pathway. Furthermore, inhibition of aminoterminal ubiquitination via acetylation or mutagenesis of internal lysine residues did not impair degradation of p21 [62]. Degradation of the three mentioned CDKIs could be performed by 20S proteasomes, but not by 30S proteasomes in vitro [61].

PA28γ-mediated effects can be assigned mechanistically to distinct categories (Figure 1). Firstly, central to its role in UIPP, PA28γ recruits substrates for degradation by the 20S proteasome. CDKIs revealing features of intrinsically unstructured proteins (IUP) are prototypic for this mode [61].

Secondly, a cross talk between UIPP and UPS is established, since 11S and 19S regulators compete for binding to 20S proteasomes. Interestingly, overexpression of PA28γ correlates with the increase of some UPS substrates such as c-Myc [63], ERα [58], BclXL, or MART-1 [46]. Such stabilization may indicate inhibitory effects on specific E3 enzymes, UIPP degradation of auxiliary UPS factors, or reduction of the active 30S proteasome pool. Perhaps, putative 30S substrates may escape UPS degradation due to activation of DUBs. Thirdly, UPS-mediated degradation might be affected either by degradation of specific E3 enzymes (Smurf1; [64, 65]) or by enhancing the interaction of specific E3s with their substrates, as has recently been shown for Mdm2:p53:PA28γ [66].

Here, PA28γ promotes the interaction of p53 and Mdm2 (Figure 1B), thereby reducing total p53 levels in UV-C-radiated cells via the ubiquitin-dependent proteasomal degradation pathway. 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].

Contributions of UIPP and/or UPS to NS protein dynamics seem to be crucial for nuclear

Proteasome Activator 28γ: Impact on Survival Signaling and Apoptosis

http://dx.doi.org/10.5772/intechopen.74731

79

CBs are sites of assembly of small nuclear ribonucleoproteins and small noncoding RNA traffic [72]. They are specific nuclear targets of the cellular stress response [41]. It has been shown that UV-C irradiation induces a stable association of PA28γ with coilin, the intrinsically disordered marker protein of CBs. The accumulation of PA28γ correlates with the disruption of CBs. Apart from its canonical targeting via Mdm2, the unstructured nature of coilin and its associ-

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

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

Transcription factor c-Myc, a highly unstable protein [63, 77, 78], acts as a heterodimer with Max, recruiting coactivators through CACGTG-binding motifs to promotors of target genes involved in the regulation of cell growth, proliferation, metabolism, and apoptosis. Interestingly, c-Myc appears to be targeted by diverse PTMs and proteolytic mechanisms. UPS-dependent degradation of c-Myc depends on several E3 enzymes, F-Box proteins, and antagonizing pathways [79]. Using gene set enrichment analysis, PA28γ was shown to reprogram energy metabolism via the c-Myc-glycolysis axis, affecting glucose utilization and lactate production in cancer cells. PSME3 gene silencing reduced c-MYC levels, whereas the increased levels of

ation with PA28γ might be the first evidence for coilin targeting via UIPP.

3.3. PA28γ in infection and inflammation

speckle function.

bacterial infection [73].

an emerging future perspective.

3.4. Transcriptional regulation of metabolism

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].
