Lighting Conditions Influence the Dynamics of Protease Synthesis and Proteasomal Activity in the White Rot Fungus Cerrena unicolor.

Recent transcriptomic and biochemical studies have revealed that light influences the global gene expression profile and metabolism of the white-rot fungus Cerrena unicolor. Here, we aimed to reveal the involvement of proteases and ubiquitin-mediated proteolysis by the 26S proteasome in the response of this fungus to white, red, blue and green lighting conditions and darkness. The changes in the expression profile of C. unicolor genes putatively engaged in proteolysis were found to be unique and specific to the applied wavelength of light. It was also demonstrated that the activity of proteases in the culture fluid and mycelium measured using natural and synthetic substrates was regulated by light and was substrate-dependent. A clear influence of light on protein turnover and the qualitative and quantitative changes in the hydrolytic degradation of proteins catalyzed by various types of proteases was shown. The analysis of activity associated with the 26S proteasome showed a key role of ATP-dependent proteolysis in the initial stages of adaptation of fungal cells to the stress factors. It was suggested that the light-sensing pathways in C. unicolor are cross-linked with stress signaling and secretion of proteases presumably serving as regulatory molecules.

A new model to investigate UVB-induced cellular senescence and pigmentation in melanocytes.

Ultraviolet (UV) light is known to potentially damage human skin and accelerate the skin aging process. Upon UVB exposure, melanocytes execute skin protection by increasing melanin production. Senescent cells, including senescent melanocytes, are known to accumulate in aged skin and contribute to the age-associated decline of tissue function. However, melanocyte senescence is still insufficiently explored. Here we describe a new model to investigate mechanisms of UVB-induced senescence in melanocytes and its role in photoaging. Exposure to mild and repeated doses of UVB directly influenced melanocyte proliferation, morphology and ploidy. We confirmed UVB-induced senescence with increased senescence-associated ß-galactosidase positivity and changed expression of several senescence markers, including p21, p53 and Lamin B1. UVB irradiation impaired proteasome and increased autophagic activity in melanocytes, while expanding intracellular melanin content. In addition, using a co-culture system, we could confirm that senescence-associated secretory phenotype components secreted by senescent fibroblasts modulated melanogenesis. In conclusion, our new model serves as an important tool to explore UVB-induced melanocyte senescence and its involvement in photoaging and skin pigmentation.

Effect of Neoadjuvant Chemoradiation Therapy on Proteasome Pool in Rectal Cancer.

In untreated rectal cancer patients, the chymotrypsin-like activity of proteasomes in tumor tissue was 3-fold higher than that in conventionally normal tissue, which is explained by up-regulation of expression of immunoproteasomes and total pool of proteasomes. After neoadjuvant chemoradiation therapy, expressions of the total pool of proteasomes and immunoproteasomes in the tumor as well as the relative ratios of these indices to those in conventionally normal tissue were smaller by 1.4-3.3 times in comparison with the untreated patients. These changes were paralleled with pronounced (4.5-fold) down-regulation of proteasome activity in the tumor and a 3.7-fold decrease of activity ratio for the proteasomes in tumor and in conventionally normal tissue. The number of immunoproteasome subunits and the chymotrypsin-like activity of proteasomes can be viewed as potential markers to prognosticate effectiveness of neoadjuvant chemoradiation therapy in rectal cancer patients.

The role of the immunoproteasome in interferon-γ-mediated microglial activation.

Microglia regulate the brain microenvironment by sensing damage and neutralizing potentially harmful insults. Disruption of central nervous system (CNS) homeostasis results in transition of microglia to a reactive state characterized by morphological changes and production of cytokines to prevent further damage to CNS tissue. Immunoproteasome levels are elevated in activated microglia in models of stroke, infection and traumatic brain injury, though the exact role of the immunoproteasome in neuropathology remains poorly defined. Using gene expression analysis and native gel electrophoresis we characterize the expression and assembly of the immunoproteasome in microglia following interferon-gamma exposure. Transcriptome analysis suggests that the immunoproteasome regulates multiple features of microglial activation including nitric oxide production and phagocytosis. We show that inhibiting the immunoproteasome attenuates expression of pro-inflammatory cytokines and suppresses interferon-gamma-dependent priming of microglia. These results imply that targeting immunoproteasome function following CNS injury may attenuate select microglial activity to improve the pathophysiology of neurodegenerative conditions or the progress of inflammation-mediated secondary injury following neurotrauma.

Molecular mechanisms of UVB-induced senescence of dermal fibroblasts and its relevance for photoaging of the human skin.

Due to its ability to cross the epidermis and reach the upper dermis where it causes cumulative DNA damage and increased oxidative stress, UVB is considered the most harmful component of sunlight to the skin. The consequences of chronic exposition to UVB are related to photoaging and photocarcinogenesis. There are limitations to the study of human skin aging and for this reason the use of models is required. Human dermal fibroblasts submitted to mild and repeated doses of UVB are considered a versatile model to study UVB effects in the process of skin photoaging, which depends on the accumulation of senescent cells, in particular in the dermis. Here we provide updated information about the current model of UVB-induced senescence with special emphasis on the process of protein quality control.

UVB-Induced Senescence of Human Dermal Fibroblasts Involves Impairment of Proteasome and Enhanced Autophagic Activity.

In the current study, we have extended previous findings aiming at a better understanding of molecular mechanisms underlying UVB-induced senescence of diploid human dermal fibroblasts (HDFs), an experimental model to study the process of photoaging in the skin. We provide evidence that the inhibition of proteasomal degradation of damaged proteins and the activation of autophagosome formation are early events in UVB-induced senescence of HDFs, dependent on UVB-induced accumulation of reactive oxygen species. Our data suggest that autophagy is required for the establishment of the senescent phenotype in UVB-treated HDFs and that inhibition of autophagy is sufficient to change the cell fate from senescence to cell death by apoptosis. Studies in reconstructed skin equivalents revealed that UVB irradiation triggers hallmarks of autophagy induction in the dermal layer. These findings have potential implications for fundamental as well as translational research into skin aging, in particular photoaging.

Radioprotector indralin at early and late manifestations of local radiation injuries.

Experimental model of acute local radiation injuries (LRI) was the degree of radiation skin burns of mouse paws, observed through the 10 day within 1 month after local γ -60Co-irradiation at the doses of 20-45 Gy. For late local radiation injuries with a maximum of over 6 months after exposure to radiation, model was the contracture of animal paws and post-radiation amputation of limbs of the mouse. In the experiments on mice radioprotector indralin (B-190) IP as direct α1-adrenomimetic has a expressed protective effect on reducing acute and late LRI, equal in terms of dose reduction factor (DRF) 1.4 -1.5 that was comparable to their efficacy during radiation injuries of hematopoietic tissues. Indralin fully retain its radioprotective properties (DRF = 1.5-1.7) in the condition of repeated radioprotector administration through one day at total dose of 57 Gy (three times 19 Gy) of fractionated γ -irradiation. The protective effect of indralin improved at parenteral administration in the place of local irradiation. The local topical application of indralin in the ointments or in solution of dimethylsulfoxide has radioprotective effect, equal in the term of DRF to 1.3 -1.5 at acute and late LRI. Indralin also possessed a expressed radioprotective properties (DRF = 1.5) in decrease radiation injuries of salivary glands during local irradiation of head of rats.

Uncoupling phototoxicity-elicited neural dysmorphology and death by insidious function and selective impairment of Ran-binding protein 2 (Ranbp2).

Morphological disintegration of neurons is coupled invariably to neural death. In particular, disruption of outer segments of photoreceptor neurons triggers photoreceptor death regardless of the pathological stressors. We show that Ranbp2(-/-)::Tg-Ranbp2(CLDm-HA) mice with mutations in SUMO-binding motif (SBM) of cyclophilin-like domain (CLD) of Ran-binding protein 2 (Ranbp2) expressed in a null Ranbp2 background lack untoward effects in photoreceptors in the absence of light-stress. However, compared to wild type photoreceptors, light-stress elicits profound disintegration of outer segments of Ranbp2(-/-)::Tg-Ranbp2(CLDm-HA) with paradoxical age-dependent resistance of photoreceptors to death and genotype-independent activation of caspases. Ranbp2(-/-)::Tg-Ranbp2(CLDm-HA) exhibit photoreceptor death-independent changes in ubiquitin-proteasome system (UPS), but death-dependent increase of ubiquitin carrier protein 9(ubc9) levels. Hence, insidious functional impairment of SBM of Ranbp2's CLD promotes neuroprotection and uncoupling of photoreceptor degeneration and death against phototoxicity.

NRH:quinone oxidoreductase 2 (NQO2) protein competes with the 20 S proteasome to stabilize transcription factor CCAAT enhancer-binding protein α (C/EBPα), leading to protection against γ radiation-induced myeloproliferative disease.

NRH:quinone oxidoreductase 2 (NQO2) is a flavoprotein that protects cells against radiation and chemical-induced oxidative stress. Disruption of the NQO2 gene in mice leads to γ radiation-induced myeloproliferative diseases. In this report, we showed that the 20 S proteasome and NQO2 both interact with myeloid differentiation factor CCAAT-enhancer-binding protein α (C/EBPα). The interaction of the 20 S proteasome with C/EBPα led to the degradation of C/EBPα. NQO2, in the presence of its cofactor NRH, protected C/EBPα against 20 S degradation. Deletion and site-directed mutagenesis demonstrated that NQO2 and 20 S competed for the same binding region of S(268)GAGAGKAKKSV(279) in C/EBPα. Exposure of mice and HL-60 cells to γ radiation enhanced the levels of NQO2, which led to an increased NQO2 interaction with C/EBPα and decreased 20 S interaction with C/EBPα. NQO2 stabilization of C/EBPα was independent of NQO1, even though both interacted with the same C/EBPα domain. NQO2(-/-) mice, deficient in NQO2, failed to stabilize C/EBPα. This contributed to the development of γ radiation-induced myeloproliferative disease in NQO2(-/-) mice.

Radiation-induced mitotic cell death and glioblastoma radioresistance: a new regulating pathway controlled by integrin-linked kinase, hypoxia-inducible factor 1 alpha and survivin in U87 cells.

We have previously shown that integrin-linked kinase (ILK) regulates U87 glioblastoma cell radioresistance by modulating the main radiation-induced cell death mechanism in solid tumours, the mitotic cell death. To decipher the biological pathways involved in these mechanisms, we constructed a U87 glioblastoma cell model expressing an inducible shRNA directed against ILK (U87shILK). We then demonstrated that silencing ILK enhanced radiation-induced centrosome overduplication, leading to radiation-induced mitotic cell death. In this model, ionising radiations induce hypoxia-inducible factor 1 alpha (HIF-1α) stabilisation which is inhibited by silencing ILK. Moreover, silencing HIF-1α in U87 cells reduced the surviving fraction after 2 Gy irradiation by increasing cell sensitivity to radiation-induced mitotic cell death and centrosome amplification. Because it is known that HIF-1α controls survivin expression, we then looked at the ILK silencing effect on survivin expression. We show that survivin expression is decreased in U87shILK cells. Furthermore, treating U87 cells with the specific survivin suppressor YM155 significantly increased the percentage of giant multinucleated cells, centrosomal overduplication and thus U87 cell radiosensitivity. In consequence, we decipher here a new pathway of glioma radioresistance via the regulation of radiation-induced centrosome duplication and therefore mitotic cell death by ILK, HIF-1α and survivin. This work identifies new targets in glioblastoma with the intention of radiosensitising these highly radioresistant tumours.

A pilot study to investigate the role of the 26S proteasome in radiotherapy resistance and loco-regional recurrence following breast conserving therapy for early breast cancer.

Breast conserving therapy is a currently accepted method for managing patients with early stage breast cancer. However, approximately 7% of patients may develop loco-regional tumour recurrence within 5 years. We previously reported that expression of the 26S proteasome may be associated with radio-resistance. Here we aimed to analyse the 26S proteasome in a pilot series of early breast cancers and correlate the findings with loco-regional recurrence. Fourteen patients with early breast cancer who developed loco-regional recurrence within 4 years of completing breast conserving therapy were selected according to strict criteria and compared with those from 14 patients who were disease-free at 10 years. Decreased expression of the 26S proteasome was significantly associated with radio-resistance, manifested as the development of a loco-regional recurrence within 4 years of breast conserving therapy (p = 0.018). This small pilot study provides further suggestion that the 26S proteasome may be associated with response to radiotherapy.

Ikaros is degraded by proteasome-dependent mechanism in the early phase of apoptosis induction.

Ikaros is an important transcription factor involved in the development and differentiation of hematopoietic cells. In this work, we found that chemotherapeutic drugs or ultraviolet radiation (UV) treatment could reduce the expression of full-length Ikaros (IK1) protein in less than 3h in leukemic NB4, Kasumi-1 and Jurkat cells, prior to the activation of caspase-3. Etoposide treatment could not alter the mRNA level of IK1 but it could shorten the half-life of IK1. Co-treatment with the proteasome inhibitor MG132 or epoxomicin but not calpain inhibitor calpeptin inhibited etoposide-induced Ikaros downregulation. Overexpression of IK1 could accelerate etoposide-induced apoptosis in NB4 cells, as evidenced by the increase of Annexin V positive cells and the more early activation of caspase 3. To our knowledge, this is the first report to show that upon chemotherapy drugs or UV treatment, IK1 could be degraded via the proteasome system in the early phase of apoptosis induction. These data might shed new insight on the role of IK1 in apoptosis and the post-translational regulation of IK1.

50 Hz extremely low frequency electromagnetic fields enhance protein carbonyl groups content in cancer cells: effects on proteasomal systems.

Electromagnetic fields are an assessed cause of prolonging free radicals lifespan. This study was carried out to investigate the influence of extremely low frequency electromagnetic fields on protein oxidation and on the 20S proteasome functionality, the complex responsible for the degradation of oxidized proteins. Caco 2 cells were exposed, for 24-72 hours, to 1 mT, 50 Hz electromagnetic fields. The treatment induced a time-dependent increase both in cell growth and in protein oxidation, more evident in the presence of TPA, while no changes in cell viability were detected. Exposing the cells to 50 Hz electromagnetic fields caused a global activation of the 20S proteasome catalytic components, particularly evident at 72 hours exposure and in the presence of TPA. The finding that EGCG, a natural antioxidant compound, counteracted the field-related pro-oxidant effects demonstrates that the increased proteasome activity was due to an enhancement in intracellular free radicals.

Bcl-XL protein is markedly decreased in UVB-irradiated basal cell carcinoma cell lines through proteasome-mediated degradation.

There is considerable evidence that the excessive ultraviolet radiation B (UVB) from sunlight is implicated in skin damage, ultimately inducing the death of keratinocytes. The UVB-induced apoptotic pathways are tightly regulated by the balance between pro-apoptotic and anti-apoptotic molecules. Among them, modulations of Bcl2 family proteins are important to decide the fate of UVB-irradiated cells. If the apoptotic pathway does not work properly, the damaged cells have a chance to transform into a carcinoma, such as basal cell carcinoma or squamous cell carcinoma of the skin. To develop a strategy of inducing apoptosis of skin cancer cells, the current study was performed to investigate the apoptotic pathway, especially focused on Bcl2 family proteins, in curcumin or UVB-treated basal cell carcinoma cell lines. Our data showed that the decreased proliferation rates and apoptotic DNA laddering were clearly observed in UVB irradiation, but not markedly observed in curcumin treatment. The decreased expression of Bcl-XL, which is involved in protection of apoptosis, was also clearly observed in UVB-irradiated cells without markedly changing mRNA levels. However, the expression of Bax or Bcl2 were not markedly changed by UVB-irradiation. The decreased expression of Bcl-XL protein after UVB treatment was partially restored in the presence of MG132, which is an inhibitor of proteasome, implying that the down-regulation of Bcl-XL may be regulated by the proteasome-mediated degradation. Our data demonstrated that the expression of Bcl-XL protein was decreased by proteasome-mediated degradation prior to change of mRNA level in UVB-induced apoptotic basal cell carcinoma cell lines, thereby these results will offer fundamental information to develop a strategy of inducing apoptosis of skin cancer cells.

Epidermal growth factor receptor vIII expression in U87 glioblastoma cells alters their proteasome composition, function, and response to irradiation.

Little is known about the factors that influence the proteasome structures in cells and their activity, although this could be highly relevant to cancer therapy. We have previously shown that, within minutes, irradiation inhibits substrate degradation by the 26S proteasome in most cell types. Here, we report an exception in U87 glioblastoma cells transduced to express the epidermal growth factor receptor vIII (EGFRvIII) mutant (U87EGFRvIII), which does not respond to irradiation with 26S proteasome inhibition. This was assessed using either a fluorogenic substrate or a reporter gene, the ornithine decarboxylase degron fused to ZsGreen (cODCZsGreen), which targets the protein to the 26S proteasome. To elucidate whether this was due to alterations in proteasome composition, we used quantitative reverse transcription-PCR to quantify the constitutive (X, Y, Z) and inducible 20S subunits (Lmp7, Lmp2, Mecl1), and 11S (PA28alpha and beta) and 19S components (PSMC1 and PSMD4). U87 and U87EGFRvIII significantly differed in expression of proteasome subunits, and in particular immunosubunits. Interestingly, 2 Gy irradiation of U87 increased subunit expression levels by 16% to 324% at 6 hours, with a coincident 30% decrease in levels of the proteasome substrate c-myc, whereas they changed little in U87EGFRvIII. Responses similar to 2 Gy were seen in U87 treated with a proteasome inhibitor, NPI0052, suggesting that proteasome inhibition induced replacement of subunits independent of the means of inhibition. Our data clearly indicate that the composition and function of the 26S proteasome can be changed by expression of the EGFRvIII. How this relates to the increased radioresistance associated with this cell line remains to be established.

Proteasome function is required for DNA damage response and fanconi anemia pathway activation.

Proteasome inhibitors sensitize tumor cells to DNA-damaging agents, including ionizing radiation (IR), and DNA cross-linking agents (melphalan and cisplatin) through unknown mechanisms. The Fanconi anemia pathway is a DNA damage-activated signaling pathway, which regulates cellular resistance to DNA cross-linking agents. Monoubiquitination and nuclear foci formation of FANCD2 are critical steps of the Fanconi anemia pathway. Here, we show that proteasome function is required for the activation of the Fanconi anemia pathway and for DNA damage signaling. Proteasome inhibitors (bortezomib and MG132) and depletion of 19S and 20S proteasome subunits (PSMD4, PSMD14, and PSMB3) inhibited monoubiquitination and/or nuclear foci formation of FANCD2, whereas depletion of DSS1/SHFM1, a subunit of the 19S proteasome that also directly binds to BRCA2, did not inhibit FANCD2 monoubiquitination or foci formation. On the other hand, DNA damage-signaling processes, such as IR-induced foci formation of phosphorylated ATM (phospho-ATM), 53BP1, NBS1, BRCA1, FANCD2, and RAD51, were delayed in the presence of proteasome inhibitors, whereas ATM autophosphorylation and nuclear foci formation of gammaH2AX, MDC1, and RPA were not inhibited. Furthermore, persistence of DNA damage and abrogation of the IR-induced G(1)-S checkpoint resulted from proteasome inhibition. In summary, we showed that the proteasome function is required for monoubiquitination of FANCD2, foci formation of 53BP1, phospho-ATM, NBS1, BRCA1, FANCD2, and RAD51. The dependence of specific DNA damage-signaling steps on the proteasome may explain the sensitization of tumor cells to DNA-damaging chemotherapeutic agents by proteasome inhibitors.

Proteasomes and proteasome activator 200 kDa (PA200) accumulate on chromatin in response to ionizing radiation.

Proteasome activator 200 kDa (PA200) forms nuclear foci after exposure of cells to ionizing radiation and enhances proteasome activity in vitro. Within cells, it is unclear whether PA200 responds to radiation alone or in association with proteasomes. In the present study, we identified three forms of cellular PA200 (termed PA200i, ii and iii) at the mRNA and protein levels. Neither PA200ii nor PA200iii appears to associate with proteasomes. All detectable PA200i is associated with proteasomes, which indicates that PA200i and proteasomes function together within the cell. Consistent with this idea, we find that exposure of cells to radiation leads to an equivalent accumulation of both PA200i and core proteasomes on chromatin. This increase in PA200 and proteasomes on chromatin is not specific to the stage of cell cycle arrest since it occurs in cells that arrest in G(2)/M and cells that arrest in G(1)/S after exposure to radiation. These data provide evidence that PA200 and proteasomes function together within cells and respond to a specific radiation-induced damage independent of the stage of cell cycle arrest.

Proteasome and photoaging: the effects of UV irradiation.

Cellular aging is characterized by the accumulation of oxidatively modified proteins that result, at least in part, from impaired degradation of abnormal proteins. The proteasome is the major intracellular proteolytic system implicated in the removal of abnormal and oxidized proteins. In human epidermal cells, previous studies have evidenced that proteasome function is decreased during aging as well as upon UV irradiation, which is the main component of photoaging. The age-related decline of proteasome activity has been reported to be due to either or both decreased proteasome subunits expression and content, inactivation upon alteration of proteasome subunits, and accumulation of endogenous inhibitors, such as highly oxidized and cross-linked proteins. To gain further insight in the mechanisms that might be implicated in the decreased activity of the proteasome upon photoaging, purified 20S human proteasome has been exposed to UVA- and UVB-irradiation. The effect of such an irradiation on proteasome peptidase activities has been monitored and shown to promote a stimulation or an inhibition of the peptidase activities depending on whether the proteasome is under its latent or a nonphysiological active form. Analysis of the patterns of proteasome subunits by 2D gel electrophoresis has revealed modification for several subunits for UV-irradiated proteasome only in its irreversibly activated form, compared with nonirradiated and irradiated latent forms, indicating that the 20S proteasome is rather resistant to UV irradiation.

Polymerase eta is a short-lived, proteasomally degraded protein that is temporarily stabilized following UV irradiation in Saccharomyces cerevisiae.

Saccharomyces cerevisiae Rad30 is the homolog of human DNA polymerase eta whose inactivation leads to the cancer-prone syndrome xeroderma pigmentosum variant. Both human and yeast polymerase eta are responsible for error-free bypass of UV-induced cis-syn pyrimidine dimers and several other DNA lesions. Here we show, using yeast strains expressing TAP-tagged Rad30, that the level of this protein is post-translationally regulated via ubiquitination and proteasome-mediated degradation. The half-life of Rad30 is 20 min and it increases due to proteasomal defects. Mutations inactivating components of the Skp1/cullin/ F-box (SCF) ubiquitin ligase complex: Skp1 and the F-box protein Ufo1 stabilize Rad30. Our results indicate also that ultraviolet irradiation causes transient stabilization of Rad30, which leads, in turn, to temporary accumulation of this polymerase in the cell. We conclude that proteolysis plays an important role in regulating the cellular abundance of Rad30. These results are the first indication of a role for controlled proteasomal degradation in modulating cellular level of translesion DNA polymerase in eukaryotes.