3.6.3. Connecting the p53 activity status and PA28γ biology

The biology of p53, a key regulator of DDR and apoptosis, appeared recently being intimately connected to PA28γ. Our data on UV-C-induced apoptosis in murine fibroblasts overexpressing PA28γ revealed an increase of phosphorylated nuclear p53, paralleled by cytosolic disappearance of p53. Kinetic experiments of apoptosis induction showed that protein levels of pro-apoptotic Bcl-2 family member Bax, cell cycle inhibitor p21, and ubiquitin E3 ligase MDM2 were transcriptionally upregulated in a time-dependent manner, indicating an increased level of transcriptionally active p53 in a pro-apoptotic context.

Furthermore, it was observed in endometrial cancer that mutant p53R248Q promoted the upregulation of the PSME3 gene [91]. That the PSME3 gene locus is under inhibitory transcriptional control has been reported too. Wild-type p53 inhibits the PA28γ-20S UIPP pathway by repressing PSME3 gene transcription, whereas mutant p53 was unable to repress PSME3 transcription [92].

Among other cellular survival functions, the p53 tumor suppressor protein particularly regulates key decision points of DNA repair and apoptosis via affecting transcriptional regulation of pivotal regulators of these two processes. Under normal conditions, the p53 protein is constitutively degraded via Mdm2-mediated ubiquitination and the 30S proteasome (Figure 2D). Under non-apoptotic conditions, PA28γ facilitates cytosolic redistribution of p53 by enhancing its nuclear export via a mechanism involving mono-ubiquitination [53]. Cytosolic p53 can activate MOMP via Bax, a mechanism prevented or balanced by the p53:BclXL and BAX: BclXL interactions [82]. Similarly, a release of cytochrome c into the cytosol, triggering apoptosome formation (Figure 2A), can be antagonized by Cyt c:BclXL [83] or p27:Cyt c interaction (p27, small heat shock protein), while other heat shock proteins counteract via interaction with Apaf-1 (Figure 2A). One key regulator in this picture is BclXL, which responds transcriptionally to p53 activation and is posttranscriptionally downregulated by PARK2, the specific E3 ubiquitin ligase preparing BclXL for UPS-mediated degradation (Figure 2B). Another anti-apoptotic regulator of MOMP, Mcl-1, is similarly targeted for degradation by Mule E3 ubiquitin protein ligase (Figure 2B) [82, 89].

DNA damage-induced ATM protein kinase (Figure 2D) activates p53 by phosphorylating p53 and Mdm2, thereby regulating Mdm2 oligomerization and processivity [93]. As a result of sitespecific phosphorylation (human p53 Ser15P or mouse p53Ser18P), p53 becomes stabilized and translocates into the nucleus. Phosphorylated wild-type p53 tetramers are capable of promotor and coactivator/corepressor binding [94].

of apoptosis due to increasing expression of pro-apoptotic genes, while in cancer cells an anti-

Figure 2. Impact of PA28γ on UIPP- and UPS-mediated regulation of apoptosis. (A) Mitochondrial regulation of apoptosis via MOMP, apoptosome assembly, and caspase activation. (B) Anti-apoptotic role of BclXL and its control via the UPS. (C) Death receptor signaling and extrinsic activation of apoptosis via caspase-8 (CASP-8) depend on the intriguing UPSmediated regulation of the bid protein (reviewed by [89]). CASP-8 cleavage of bid generates amino- and carboxy-terminal peptide fragments (N-tBid; t-bid-C) that act antagonistically. Anti-apoptotic N-tBid still bound to pro-apoptotic tBid-C prevents apoptosis. Noncanonical ubiquitination at Gln and Cys residues initiates UPS-mediated degradation of N-bid. N-bid degradation is required for activation of pro-apoptotic tBid-C. Inactivation of t-bid-C is realized by itch E3 ubiquitin ligase, initiating its UPS-mediated degradation. This prevents mitochondrial outer membrane pore formation and activation of apoptosis. (D) Induction of stress kinases ATM and Chk2 by UV-C irradiation induces phosphorylation of p53, Mdm2, and PA28γ, thereby disconnecting p53 from its constitutive restriction through E3 ubiquitin ligase Mdm2. Phosphorylated nuclear p53 activates transcription of pro-apoptotic Bax, and cytosolic p53 can activate Bax-mediated pore formation directly. It has been observed that PA28γ reduces p53 levels by enhancing Mdm2:p53 interaction [66]. (E) Our model proposes a central role of anti-apoptotic PA28γ-20S UIPP in cancer cells [46]. High levels of PA28γ might be based on altered promotor selectivity of mutant p53, which has been shown to increase PSME3 transcription, whereas wild-type p53 represses PSME3 gene expression (D) [91, 92]. High PA28γ levels and acetylation are favoring heptamer assembly and proteasome activation. UIPP, either based on 20S proteasomes alone or in association with PA28γ, may reduce the level of released cytochrome c or activated caspases (CASP-9; CASP-3). If PA28γ levels are low, restriction of caspases by UIPP is released, and monomeric PA28γ is targeted by effector caspases (CASP-3). Of note, experimental evidences support the proteasomal degradation of effector caspases [46, 95], but the precise role of PA28γ in this process

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Overexpression of the PSME3 gene was accompanied by an increased resistance to apoptosis induction. Even with elevated levels of BclXL and a partially impaired cytochrome c release in PA28γ-overexpressing cells, we observed execution of the caspase-9/caspase-3 activation cascade upon UV-C stimulation [46]. Surprisingly, PA28γ overexpression correlated with significantly

apoptotic scenario prevails.

remains to be investigated in detail.

3.6.4. Feedback regulation: PA28γ inhibits effector caspase activity

In healthy cells, p53wt proteins cooperate with Smad3 in repressing the PMSE3 promotor [92]. Contrarily, tumor cells reveal high levels of p53mut activating the PMSE3 promotor via derepression and/or activation with unknown coactivators. Consistently, elevated PA28γ levels have been detected in several types of cancer and tumor cell lines [92]. These PA28γ-overexpressing cells, hence, reveal higher resistance to apoptotic stimuli and increased proliferation.

In cancer cells, mutant p53 (p53mut) variants reveal alternative promotor selectivity. This might explain how irreparable DNA damage in healthy cells results in transcriptional amplification

3.6.3. Connecting the p53 activity status and PA28γ biology

82 Current Understanding of Apoptosis - Programmed Cell Death

transcription [92].

(Figure 2B) [82, 89].

and coactivator/corepressor binding [94].

increased level of transcriptionally active p53 in a pro-apoptotic context.

The biology of p53, a key regulator of DDR and apoptosis, appeared recently being intimately connected to PA28γ. Our data on UV-C-induced apoptosis in murine fibroblasts overexpressing PA28γ revealed an increase of phosphorylated nuclear p53, paralleled by cytosolic disappearance of p53. Kinetic experiments of apoptosis induction showed that protein levels of pro-apoptotic Bcl-2 family member Bax, cell cycle inhibitor p21, and ubiquitin E3 ligase MDM2 were transcriptionally upregulated in a time-dependent manner, indicating an

Furthermore, it was observed in endometrial cancer that mutant p53R248Q promoted the upregulation of the PSME3 gene [91]. That the PSME3 gene locus is under inhibitory transcriptional control has been reported too. Wild-type p53 inhibits the PA28γ-20S UIPP pathway by repressing PSME3 gene transcription, whereas mutant p53 was unable to repress PSME3

Among other cellular survival functions, the p53 tumor suppressor protein particularly regulates key decision points of DNA repair and apoptosis via affecting transcriptional regulation of pivotal regulators of these two processes. Under normal conditions, the p53 protein is constitutively degraded via Mdm2-mediated ubiquitination and the 30S proteasome (Figure 2D). Under non-apoptotic conditions, PA28γ facilitates cytosolic redistribution of p53 by enhancing its nuclear export via a mechanism involving mono-ubiquitination [53]. Cytosolic p53 can activate MOMP via Bax, a mechanism prevented or balanced by the p53:BclXL and BAX: BclXL interactions [82]. Similarly, a release of cytochrome c into the cytosol, triggering apoptosome formation (Figure 2A), can be antagonized by Cyt c:BclXL [83] or p27:Cyt c interaction (p27, small heat shock protein), while other heat shock proteins counteract via interaction with Apaf-1 (Figure 2A). One key regulator in this picture is BclXL, which responds transcriptionally to p53 activation and is posttranscriptionally downregulated by PARK2, the specific E3 ubiquitin ligase preparing BclXL for UPS-mediated degradation (Figure 2B). Another anti-apoptotic regulator of MOMP, Mcl-1, is similarly targeted for degradation by Mule E3 ubiquitin protein ligase

DNA damage-induced ATM protein kinase (Figure 2D) activates p53 by phosphorylating p53 and Mdm2, thereby regulating Mdm2 oligomerization and processivity [93]. As a result of sitespecific phosphorylation (human p53 Ser15P or mouse p53Ser18P), p53 becomes stabilized and translocates into the nucleus. Phosphorylated wild-type p53 tetramers are capable of promotor

In healthy cells, p53wt proteins cooperate with Smad3 in repressing the PMSE3 promotor [92]. Contrarily, tumor cells reveal high levels of p53mut activating the PMSE3 promotor via derepression and/or activation with unknown coactivators. Consistently, elevated PA28γ levels have been detected in several types of cancer and tumor cell lines [92]. These PA28γ-overexpressing

In cancer cells, mutant p53 (p53mut) variants reveal alternative promotor selectivity. This might explain how irreparable DNA damage in healthy cells results in transcriptional amplification

cells, hence, reveal higher resistance to apoptotic stimuli and increased proliferation.

Figure 2. Impact of PA28γ on UIPP- and UPS-mediated regulation of apoptosis. (A) Mitochondrial regulation of apoptosis via MOMP, apoptosome assembly, and caspase activation. (B) Anti-apoptotic role of BclXL and its control via the UPS. (C) Death receptor signaling and extrinsic activation of apoptosis via caspase-8 (CASP-8) depend on the intriguing UPSmediated regulation of the bid protein (reviewed by [89]). CASP-8 cleavage of bid generates amino- and carboxy-terminal peptide fragments (N-tBid; t-bid-C) that act antagonistically. Anti-apoptotic N-tBid still bound to pro-apoptotic tBid-C prevents apoptosis. Noncanonical ubiquitination at Gln and Cys residues initiates UPS-mediated degradation of N-bid. N-bid degradation is required for activation of pro-apoptotic tBid-C. Inactivation of t-bid-C is realized by itch E3 ubiquitin ligase, initiating its UPS-mediated degradation. This prevents mitochondrial outer membrane pore formation and activation of apoptosis. (D) Induction of stress kinases ATM and Chk2 by UV-C irradiation induces phosphorylation of p53, Mdm2, and PA28γ, thereby disconnecting p53 from its constitutive restriction through E3 ubiquitin ligase Mdm2. Phosphorylated nuclear p53 activates transcription of pro-apoptotic Bax, and cytosolic p53 can activate Bax-mediated pore formation directly. It has been observed that PA28γ reduces p53 levels by enhancing Mdm2:p53 interaction [66]. (E) Our model proposes a central role of anti-apoptotic PA28γ-20S UIPP in cancer cells [46]. High levels of PA28γ might be based on altered promotor selectivity of mutant p53, which has been shown to increase PSME3 transcription, whereas wild-type p53 represses PSME3 gene expression (D) [91, 92]. High PA28γ levels and acetylation are favoring heptamer assembly and proteasome activation. UIPP, either based on 20S proteasomes alone or in association with PA28γ, may reduce the level of released cytochrome c or activated caspases (CASP-9; CASP-3). If PA28γ levels are low, restriction of caspases by UIPP is released, and monomeric PA28γ is targeted by effector caspases (CASP-3). Of note, experimental evidences support the proteasomal degradation of effector caspases [46, 95], but the precise role of PA28γ in this process remains to be investigated in detail.

of apoptosis due to increasing expression of pro-apoptotic genes, while in cancer cells an antiapoptotic scenario prevails.

#### 3.6.4. Feedback regulation: PA28γ inhibits effector caspase activity

Overexpression of the PSME3 gene was accompanied by an increased resistance to apoptosis induction. Even with elevated levels of BclXL and a partially impaired cytochrome c release in PA28γ-overexpressing cells, we observed execution of the caspase-9/caspase-3 activation cascade upon UV-C stimulation [46]. Surprisingly, PA28γ overexpression correlated with significantly decreased active effector caspase levels. Consistently and vice versa, PMSE3 miRNA-mediated PA28γ downregulation was accompanied by increased sensitivity to butyrate-triggered apoptosis of HT-29 cells and increased levels of active caspase-3/caspase-7 [46]. Reduced caspase activities were not due to transcriptional but posttranslational regulation.

Author details

Ralf Stohwasser

References

bbcan.2008.05.004

jcs.067405

09.001

12.012

bbamcr.2013.07.007

Address all correspondence to: ralf.stohwasser@b-tu.de

DOI: 10.2174/1386207320666170710124746

Brandenburg Technical University Cottbus-Senftenberg, Senftenberg, Germany

[1] Jariel-Encontre I, Bossis G, Piechaczyk M. Ubiquitin-independent degradation of proteins by the proteasome. Biochimica et Biophysica Acta. 2008;1786:153-177. DOI: 10.1016/j.

Proteasome Activator 28γ: Impact on Survival Signaling and Apoptosis

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

85

[2] Mao I, Liu J, Li X, Luo H. REGgamma, a proteasome activator and beyond? Cellular and Molecular Life Sciences. 2008;65:3971-3980. DOI: 10.1007/s00018-008-8291-z.10.1242/

[3] Ao N, Chen Q, Liu G. The small molecules targeting ubiquitin-proteasome system for cancer therapy. Combinatorial Chemistry & High Throughput Screening. 2017;20:403-413.

[4] Ciechanover A. Intracellular protein degradation: From a vague idea thru the lysosome and the ubiquitin-proteasome system and onto human diseases and drug targeting. Best Practice & Research. Clinical Haematology. 2017;30:341-355. DOI: 10.1016/j.beha.2017.

[5] Ciechanover A, Stanhill A. The complexity of recognition of ubiquitinated substrates by the 26S proteasome. Biochimica et Biophysica Acta. 2014;1843:86-96. DOI: 10.1016/j.

[6] Erales J, Coffino P. Ubiquitin-independent proteasomal degradation. Biochimica et

[7] Qiu GZ, Sun W, Jin MZ, Lin J, Lu PG, Jin WL. The bad seed gardener: Deubiquitinases in the cancer stem-cell signaling network and therapeutic resistance. Pharmacology & Ther-

[8] Kwon YT, Ciechanover A. The ubiquitin code in the ubiquitin-proteasome system and autophagy. Trends in Biochemical Sciences. 2017;42:873-886. DOI: 10.1016/j.tibs.2017.09.002

[9] Tanaka K. The proteasome: Overview of structure and functions. Proceedings of the Japan

[10] Dahlmann B. Mammalian proteasome subtypes: Their diversity in structure and function. Archives of Biochemistry and Biophysics. 2016;591:132-140. DOI: 10.1016/j.abb.2015.

Biophysica Acta. 2014;1843:216-221. DOI: 10.1016/j.bbamcr.2013.05.008

apeutics. 2017;172:127-138. DOI: 10.1016/j.pharmthera.2016.12.003

Academy. Series B, Physical and Biological Sciences. 2009;85:12-36

The anti-apoptotic impact of high PA28γ levels could not solely be explained by enhanced PA28γ-mediated degradation of pro-apoptotic p53 as suggested by others [66]. Our current findings support a model (Figure 2A and E), where high PA28γ levels inhibit effector caspase-3/caspase-7, while at low PA28γ levels, caspase-3/caspase-7 activity and PA28γ turnover are increased. As a reasonable explanation, the high level of PA28γ favors heptamer stability, whereas at low concentrations, monomeric PA28γ may be more susceptible to cleavage by activated effector caspases [46, 47]. Furthermore, we found that proteasome inhibition stabilized active caspase-3 levels, indicating that the PA28γ-dependent degradation of caspase-3/ caspase-7 is indeed proteasome-dependent [46]. Since the RING domain of IAP proteins ubiquitinates caspase-3/caspase-7 [95], the canonical UPS is certainly involved in regulating effector caspase activity. If PA28γ conducts restriction of caspase-3/caspase-7 activity through enhancement of physical interaction between IAPs and caspases, or directly via the PA28γ-20S proteasome UIPP route, has to be clarified in the future.
