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Background:Natural compounds with potential antioxidant properties have been used in the form of food supplements or extracts with the intent to prevent or treat various diseases. Many of these compounds can activate the cytoprotective Nrf2 pathway. Besides, some of them are known to impact the thyroid gland, often with potential side-effects, but in other instances, with potential utility in the treatment of thyroid disorders.Objective:In view of recent data regarding the multiple roles of Nrf2 in the thyroid, this review summarizes the current bibliography on natural compounds that can have an effect on thyroid gland physiology and pathophysiology, and it discusses the potential implication of the Nrf2 system in the respective mechanisms.Method & Results:Literature searches for articles from 1950 to 2018 were performed in PubMed and Google Scholar using relevant keywords about phytochemicals, Nrf2 and thyroid. Natural substances were categorized into phenolic compounds, sulfur-containing compounds, quinones, terpenoids, or under the general category of plant extracts. For individual compounds in each category, respective data were summarized, as derived from in vitro (cell lines), preclinical (animal models) and clinical studies. The main emerging themes were as follows: phenolic compounds often showed potential to affect the production of thyroid hormones; sulfur-containing compounds impacted the pathogenesis of goiter and the proliferation of thyroid cancer cells; while quinones and terpenoids modified Nrf2 signaling in thyroid cell lines.Conclusion:Natural compounds that modify the activity of the Nrf2 pathway should be evaluated carefully, not only for their potential to be used as therapeutic agents for thyroid disorders, but also for their thyroidal safety when used for the prevention and treatment of non-thyroidal diseases.

The ubiquitin-proteasome system (UPS) is a key player in the maintenance of cellular protein homeostasis (proteostasis). Since proteasome function declines upon aging leading to the acceleration of its progression and the manifestation of age-related pathologies, many attempts have been performed towards proteasome activation as a strategy to promote healthspan and longevity. The marine environment hosts a plethora of organisms that produce a vast array of primary and secondary metabolites, the majority of which are unique, exhibiting a wide spectrum of biological activities. The fact that these biologically important compounds are also present in edible marine organisms has sparked the interest for elucidating their potential health-related applications. In this review, we focus on the antioxidant, anti-aging, anti-aggregation and anti-photoaging properties of various marine constituents. We further discuss representatives of marine compounds classes with regard to their potential (direct or indirect) action on UPS components that could serve as UPS modulators and exert beneficial effects on conditions such as oxidative stress, aging and age-related diseases., Graphical abstract Image 1, Highlights • Proteasome has been implicated in oxidative stress, aging and age-related diseases. • Various marine natural products exert strong antioxidant activities. • Various marine natural products possess anti-aging and anti-aggregation properties. • Various marine natural products can serve as potential proteasome modulators. • Various marine natural products represent promising anti-photoaging agents.

The ubiquitin–proteasome system (UPS) is the main cellular proteolytic system responsible for the degradation of normal and abnormal (e.g. oxidised) proteins. Under catabolic conditions characterised by chronic inflammation, the UPS is activated resulting in proteolysis, muscle wasting and impaired muscle function. Milk proteins provide sulphur-containing amino acid and have been proposed to affect muscle inflammation. However, the response of the UPS to aseptic inflammation and protein supplementation is largely unknown. The aim of this study was to investigate how milk protein supplementation affects UPS activity and skeletal muscle function under conditions of aseptic injury induced by intense, eccentric exercise. In a double-blind, cross-over, repeated measures design, eleven men received either placebo (PLA) or milk protein concentrate (PRO, 4×20 g on exercise day and 20 g/d for the following 8 days), following an acute bout of eccentric exercise (twenty sets of fifteen eccentric contractions at 30°/s) on an isokinetic dynamometer. In each trial, muscle biopsies were obtained from the vastus lateralis muscle at baseline, as well as at 2 and 8 d post exercise, whereas blood samples were collected before exercise and at 6 h, 1 d, 2 d and 8 d post exercise. Muscle strength and soreness were assessed before exercise, 6 h post exercise and then daily for 8 consecutive days. PRO preserved chymotrypsin-like activity and attenuated the decrease of strength, facilitating its recovery. PRO also prevented the increase of NF-κB phosphorylation and HSP70 expression throughout recovery. We conclude that milk PRO supplementation following exercise-induced muscle trauma preserves proteasome activity and attenuates strength decline during the pro-inflammatory phase.

Spinocerebellar ataxia type-1 (SCA1) is caused by an abnormally expanded polyglutamine (polyQ) tract in ataxin-1. These expansions are responsible for protein misfolding and self-assembly into intranuclear inclusion bodies (IIBs) that are somehow linked to neuronal death. However, owing to lack of a suitable cellular model, the downstream consequences of IIB formation are yet to be resolved. Here, we describe a nuclear protein aggregation model of pathogenic human ataxin-1 and characterize IIB effects. Using an inducible Sleeping Beauty transposon system, we overexpressed the ATXN1(Q82) gene in human mesenchymal stem cells that are resistant to the early cytotoxic effects caused by the expression of the mutant protein. We characterized the structure and the protein composition of insoluble polyQ IIBs which gradually occupy the nuclei and are responsible for the generation of reactive oxygen species. In response to their formation, our transcriptome analysis reveals a cerebellum-specific perturbed protein interaction network, primarily affecting protein synthesis. We propose that insoluble polyQ IIBs cause oxidative and nucleolar stress and affect the assembly of the ribosome by capturing or down-regulating essential components. The inducible cell system can be utilized to decipher the cellular consequences of polyQ protein aggregation. Our strategy provides a broadly applicable methodology for studying polyQ diseases.

In the last several years, numerous molecules derived from plants and vegetables have been tested for their antioxidant, anti-inflammatory, and anti-aging properties. One of them is sulforaphane (SFN), an isothiocyanate present in cruciferous vegetables. SFN activates the antioxidant and anti-inflammatory responses by inducing Nrf2 pathway and inhibiting NF-κB. It also has an epigenetic effect by inhibiting HDAC and DNA methyltransferases and modifies mitochondrial dynamics. Moreover, SFN preserves proteome homeostasis (proteostasis) by activating the proteasome, which has been shown to lead to increased cellular lifespan and prevent neurodegeneration. In this review, we describe some of the molecular and physical characteristics of SFN, its mechanisms of action, and the effects that SFN treatment induces in order to discuss its relevance as a “miraculous” drug to prevent aging and neurodegeneration.

Engineered bacteria enable the screening of vast molecular libraries for discovering inhibitors of pathogenic protein aggregation., Protein misfolding and aggregation are associated with a many human disorders, including Alzheimer’s and Parkinson’s diseases. Toward increasing the effectiveness of early-stage drug discovery for these conditions, we report a bacterial platform that enables the biosynthesis of molecular libraries with expanded diversities and their direct functional screening for discovering protein aggregation inhibitors. We illustrate this approach by performing, what is to our knowledge, the largest functional screen of small-size molecular entities described to date. We generated a combinatorial library of ~200 million drug-like, cyclic peptides and rapidly screened it for aggregation inhibitors against the amyloid-β peptide (Aβ42), linked to Alzheimer’s disease. Through this procedure, we identified more than 400 macrocyclic compounds that efficiently reduce Aβ42 aggregation and toxicity in vitro and in vivo. Finally, we applied a combination of deep sequencing and mutagenesis analyses to demonstrate how this system can rapidly determine structure-activity relationships and define consensus motifs required for bioactivity.

A progressive loss of protein homeostasis is characteristic of aging and a driver of neurodegeneration. To investigate this process quantitatively, we characterized proteome dynamics during brain aging in the short‐lived vertebrate Nothobranchius furzeri combining transcriptomics and proteomics. We detected a progressive reduction in the correlation between protein and mRNA, mainly due to post‐transcriptional mechanisms that account for over 40% of the age‐regulated proteins. These changes cause a progressive loss of stoichiometry in several protein complexes, including ribosomes, which show impaired assembly/disassembly and are enriched in protein aggregates in old brains. Mechanistically, we show that reduction of proteasome activity is an early event during brain aging and is sufficient to induce proteomic signatures of aging and loss of stoichiometry in vivo. Using longitudinal transcriptomic data, we show that the magnitude of early life decline in proteasome levels is a major risk factor for mortality. Our work defines causative events in the aging process that can be targeted to prevent loss of protein homeostasis and delay the onset of age‐related neurodegeneration., Analyses of proteome dynamics delineate a timeline of molecular events underlying brain aging in the vertebrate Nothobranchius furzeri. Early‐in‐life decline of proteasome activity is associated with loss of stoichiometry of protein complexes and predicts lifespan.

This is a PDF file of an unedited manuscript that has been accepted for publication. The final version of this article is available at [https://doi.org/10.1016/j.mad.2018.12.002].

Signaling networks are an important tool of modern systems biology and drug development. Here, we present a new methodology to qualitatively model signaling networks by combining experimental data and prior knowledge about protein connectivity. Unlike other methods, our approach does not focus solely on selecting which reactions are involved but also on whether a protein is present. This allows the user to model more complicated experiments and incorporate more knowledge into the model. To demonstrate the capabilities of our method we compared the signaling networks of young and replicative senescent human primary HFL-1 fibroblasts, whose differences are expected to be due mainly to changes in the expression of the proteins rather than the reactions involved. The resulting networks indicate that, compared to young cells, aged cells are not as responsive to insulin stimulation and activate pathways that establish and maintain senescence.Author summaryCells have developed a complex network of biochemical reactions to monitor their environment and react to changes. Although multiple pathways, tuned to identify specific stimuli, have been discovered, it is generally understood that the signaling process typically involves multiple pathways and is context depended. Consequently, reconstructing the signaling network utilized by cells at any given moment is not a trivial task. In this article, we report on a novel logic-based method for identifying signaling network by combining experimental data with prior knowledge about the connectivity of the involved proteins. Unlike other methods proposed so far, our method uses data to evaluate the presence or absence of reactions and proteins alike. We reconstructed and compared the signaling network of human primary HFL-1 fibroblasts as they undergo replicative senescence in the presence of 6 different stimuli. The resulting networks indicate that, compared to young cells, senescent cells are not responsive to insulin stimulation and activate pathways that are known to establish and maintain senescence.

The development of chronic, low-grade systemic inflammation in the elderly (inflammaging) has been associated with increased incidence of chronic diseases, geriatric syndromes, and functional impairments. The aim of this study was to examine differences in habitual physical activity (PA), dietary intake patterns, and musculoskeletal performance among community-dwelling elderly men with low and elevated systemic inflammation. Nonsarcopenic older men free of chronic diseases were grouped as ‘low’ (LSI: n = 17; 68.2 ± 2.6 years; hs-CRP: 1 mg/L) systemic inflammation according to their serum levels of high-sensitivity CRP (hs-CRP). All participants were assessed for body composition via Dual Emission X-ray Absorptiometry (DEXA), physical performance using the Short Physical Performance Battery (SPPB) and handgrip strength, daily PA using accelerometry, and daily macro- and micronutrient intake. ESI was characterized by a 2-fold greater hs-CRP value than LSI (p < 0.01). The two groups were comparable in terms of body composition, but LSI displayed higher physical performance (p < 0.05), daily PA (step count/day and time at moderate-to-vigorous PA (MVPA) were greater by 30% and 42%, respectively, p < 0.05), and daily intake of the antioxidant vitamins A (6590.7 vs. 4701.8 IU/day, p < 0.05), C (120.0 vs. 77.3 mg/day, p < 0.05), and E (10.0 vs. 7.5 mg/day, p < 0.05) compared to ESI. Moreover, daily intake of vitamin A was inversely correlated with levels of hs-CRP (r = −0.39, p = 0.035). These results provide evidence that elderly men characterized by low levels of systemic inflammation are more physically active, spend more time in MVPA, and receive higher amounts of antioxidant vitamins compared to those with increased systemic inflammation.

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed., Graphical abstract fx1, Highlights • RONS are chemical mediators and a communication tool. • RONS and disturbed redox balance play a role in a broad range of diseases and aging. • Bacteria and toxins are important stimulators of cellular RONS formation. • Drugs should preserve beneficial redox signaling and inhibit detrimental RONS sources. • Redox drugs may target the origin, identity, location and time of RONS formation.

Protein homeostasis (proteostasis) is one of the nodal points that need to be preserved to retain physiologic cellular/organismal balance. The ubiquitin-proteasome system (UPS) is responsible for the removal of both normal and damaged proteins, with the proteasome being the downstream effector. The proteasome is the major cellular protease with progressive impairment of function during aging and senescence. Despite the documented age-retarding properties of proteasome activation in various cellular models, simultaneous enhancement of the 20S core proteasome content, assembly, and function have never been reported in any multicellular organism. Consequently, the possible effects of the core proteasome modulation on organismal life span are elusive. In this study, we have achieved activation of the 20S proteasome at organismal level. We demonstrate enhancement of proteasome levels, assembly, and activity in the nematode Caenorhabditis elegans, resulting in life span extension and increased resistance to stress. We also provide evidence that the observed life span extension is dependent on the transcriptional activity of Dauer formation abnormal/Forkhead box class O (DAF-16/FOXO), skinhead-1 (SKN-1), and heat shock factor-1 (HSF-1) factors through regulation of downstream longevity genes. We further show that the reported beneficial effects are not ubiquitous but they are dependent on the genetic context. Finally, we provide evidence that proteasome core activation might be a potential strategy to minimize protein homeostasis deficiencies underlying aggregation-related diseases, such as Alzheimer’s disease (AD) or Huntington’s disease (HD). In summary, this is the first report demonstrating that 20S core proteasome up-regulation in terms of both content and activity is feasible in a multicellular eukaryotic organism and that in turn this modulation promotes extension of organismal health span and life span.—Chondrogianni, N., Georgila, K., Kourtis, N., Tavernarakis, N., Gonos, E. S. 20S proteasome activation promotes life span extension andresistance to proteotoxicity in Caenorhabditis elegans.

The advanced glycoxidation end products (AGEs) and lipoxidation end products (ALEs) contribute to the development of diabetic complications and of other pathologies. The review discusses the possibilities of counteracting the formation and stimulating the degradation of these species by pharmaceuticals and natural compounds. The review discusses inhibitors of ALE and AGE formation, cross-link breakers, ALE/AGE elimination by enzymes and proteolytic systems, receptors for advanced glycation end products (RAGEs) and blockade of the ligand–RAGE axis.

Aims: Proteasomes are constituents of the cellular proteolytic networks that maintain protein homeostasis through regulated proteolysis of normal and abnormal (in any way) proteins. Genetically mediated proteasome activation in multicellular organisms has been shown to promote longevity and to exert protein antiaggregation activity. In this study, we investigate whether compound-mediated proteasome activation is feasible in a multicellular organism and we dissect the effects of such approach in aging and Alzheimer's disease (AD) progression. Results: Feeding of wild-type Caenorhabditis elegans with 18α-glycyrrhetinic acid (18α-GA; a previously shown proteasome activator in cell culture) results in enhanced levels of proteasome activities that lead to a skinhead-1- and proteasome activation-dependent life span extension. The elevated proteasome function confers lower paralysis rates in various AD nematode models accompanied by decreased Aβ deposits, thus ultimately decelerating the progression of AD phenotype. More importantly, similar positive results are also delivered when human and murine cells of nervous origin are subjected to 18α-GA treatment. Innovation: This is the first report of the use of 18α-GA, a diet-derived compound as prolongevity and antiaggregation factor in the context of a multicellular organism. Conclusion: Our results suggest that proteasome activation with downstream positive outcomes on aging and AD, an aggregation-related disease, is feasible in a nongenetic manipulation manner in a multicellular organism. Moreover, they unveil the need for identification of antiaging and antiamyloidogenic compounds among the nutrients found in our normal diet. Antioxid. Redox Signal. 25, 855–869.

Replicative senescence in human fibroblasts is accompanied with alterations of various biological processes, including the impaired function of the proteasome. The proteasome is responsible for the removal of both normal and damaged proteins. Due to its latter function, proteasome is also considered a representative secondary antioxidant cellular mechanism. Nrf2 is a basic transcription factor responsible for the regulation of the cellular antioxidant response that has also been shown to regulate several proteasome subunits in mice. We have established in this study the proteasome-related function of Nrf2 in human fibroblasts undergoing replicative senescence. We demonstrate that Nrf2 has a declined function in senescence, whereas its silencing leads to premature senescence. However, upon its activation by a novel Nrf2 inducer, elevated levels of proteasome activity and content are recorded only in cell lines possessing a functional Nrf2. Moreover, treatment by the Nrf2 inducer results in the enhanced survival of cells following oxidative stress, whereas continuous treatment leads to lifespan extension of human fibroblasts. Importantly the Nrf2-proteasome axis is functional in terminally senescent cultures as these cells retain their responsiveness to the Nrf2 stimuli. In conclusion, these findings open up new directions for future manipulation of the senescence phenotype.

The proteasome is the major cellular proteolytic machinery. It is involved in the regulation of various pathways via the selective degradation of either short-lived normal proteins or damaged proteins permitting the cellular detoxification. Proteasome has impaired function during several biological processes, including aging and diseases; however, it can be activated through overexpression of β5- or β1-subunits, resulting to enhanced survival and extended lifespan. In the current study, we have investigated proteasomal up-regulation via overexpression of hUMP1/POMP protein, the known accessory factor for proteasome assembly in humans. hUMP1/POMP overexpressing fibroblasts have increased levels of functional proteasome and enhanced capacity to cope better and faster with various oxidative stressors. These data highlight hUMP1/POMP role in proteasome assembly and further strengthen the prospect of genetic manipulation of the proteasomal system.

In the version of this Article originally published, in Fig. 1c–e, on the x axes, the lines labelled ‘Aβ42’ and ‘Aβ42(F19S;L34P)’ grouped the data incorrectly; the line labelled Aβ42 should have grouped the data for Random 1–2 and Clones 1–10, and the line labelled Aβ42(F19S;L34P) should have only grouped the data for Random 1–2 on the right end of the plots and blots. These figures have now been corrected in all versions of the Article.

Η αναπτυξιακή πορεία όλων των πολυκύτταρων οργανισμών περιλαμβάνει διάφορα στάδια, τα οποία χαρακτηρίζονται από μη αντιστρεπτές μεταβολές, στην ανατομία, στη φυσιολογία αλλά και στην προσαρμοστικότητα αυτών, οι οποίες αυξάνουν την πιθανότητα θανάτου. Οι μεταβολές που υφίσταται κάθε οργανισμός μπορεί να είναι απότομες ή και βαθμιαίες. Το φαινόμενο αυτό της αλληλοδιαδοχής διαφόρων αναπτυξιακών σταδίων, με τελικό στάδιο το θάνατο, καλείται γήρανση. Πρόκειται για καθολικό φαινόμενο που εμφανίζεται σε όλους τους οργανισμούς, κατά την εξέλιξη του οποίου, εμφανίζονται διάφορες αλλαγές στη μορφολογία, τη φυσιολογία και τα μοριακά χαρακτηριστικά των δομικών λίθων των οργανισμών, τα κύτταρα. Η αποικοδόμηση και οι πρωτεολυτικοί μηχανισμοί του κυττάρου αποτελούν συστήματα που παίζουν βασικό ρόλο στη διατήρηση της κυτταρικής ομοιοστασίας και που υφίστανται τροποποιήσεις κατά την εξέλιξη του φαινομένου της γήρανσης. Το πρωτεόσωμα αποτελεί τον βασικό κυτταρικό πρωτεολυτικό μηχανισμό. Στα ανθρώπινα κύτταρα, το πρωτεόσωμα αποτελείται από 14 α-ρυθμιστικές υπομονάδες και 14 β-καταλυτικές υπομονάδες σε διάταξη α7β7β7α7. Είναι υπέυθυνο για την αποικοδόμηση τόσο κατεστραμμένων πρωτεϊνών (αποδιαταγμένων, οξειδωμένων κλπ), όσο και φυσιολογικών πρωτεϊνών που πρέπει να πρωτεολυθούν. Παρόλο ότι υπάρχουν πολλά δεδομένα τόσο για τη γήρανση όσο και για το πρωτεόσωμα, δεν είναι πλήρως γνωστό το τι συμβαίνει στον μηχανισμό αυτού κατά τη γήρανση και την εξέλιξή της. Σκοπός της διδακτορικής αυτής διατριβής είναι η μελέτη του βασικού πρωτεολυτικού συστήματος του κυττάρου, του πρωτεοσώματος και η συσχέτιση της έκφρασης και δράσης αυτού με το φαινόμενο της γήρανσης και της μακροβιότητας στον άνθρωπο, in vivo και in vitro. Δείχθηκε πως κατά την in vivo γήρανση, το πρωτεόσωμα υφίσταται αλλαγές τόσο σε επίπεδο πρωτεϊνικής έκφρασης των υπομονάδων του όσο και σε επίπεδο λειτουργικότητας αυτού (μελετώντας τις πρωτεολυτικές του ενεργότητες). Οι μεταβολές αυτές μπορούν να σχετισθούν με την in vivo γήρανση και την εξέλιξη αυτής. Χρησιμοποιώντας το μοντέλο των δοτών διαφορετικών ηλικιών (ανθρώπινοι ινοβλάστες από υγιείς δότες διαφορετικών ηλικιών), έγινε φανερό πως μεταβαίνοντας από κύτταρα δοτών μικρής ηλικίας (18, 28 ετών) σε κύτταρα δοτών μεγάλης ηλικίας (80 ετών), παρατηρείται μείωση των επιπέδων, τόσο RNA όσο και πρωτεϊνικής έκφρασης, των διαφόρων πρωτεοσωμικών υπομονάδων (η μελέτη έγινε με έμφαση στις υπομονάδες του 20S συμπλόκου, τόσο α-ρυθμιστικές όσο και β-καταλυτικές), όσο και των πρωτεοσωμικών ενεργοτήτων αυτού. Οι μειωμένες ενεργότητες του πρωτεοσώματος στους ινοβλάστες ατόμων μεγάλης ηλικίας έχουν άμεσο αντίκτυπο στα παρατηρούμενα επίπεδα οξειδωμένων κυτταρικών πρωτεϊνών, τα οποία παρουσιάζονται αυξημένα στα κύτταρα αυτά. Δεδομένου ότι στο πεδίο της γήρανσης, οι υγιείς αιωνόβιοι αποτελούν χαρακτηριστικά παραδείγματα «επιτυχούς» γήρανσης, συμπεριλήφθησαν στην παραπάνω μελέτη και ινοβλάστες από ανάλογους δότες. Τα επίπεδα RNA και πρωτεϊνικής έκφρασης των διαφόρων πρωτεοσωμικών υπομονάδων αυτών των κυττάρων, βρέθηκαν παρόμοια με αυτά που παρατηρούνται στα κύτταρα δοτών νεαρής ηλικίας (28 ετών) και όχι δοτών μεγαλύτερης ηλικίας (80 ετών). ................................................................

The DNA damage response (DDR) orchestrates the recruitment of repair proteins at sites of damage and arrests cell-cycle progression until completion of repair. Upon irreparable damage, DNA damage foci persist (long-lived foci) and this is believed to induce cellular senescence. The resolution of DNA damage foci has previously been shown to depend on proteasomal degradation and various proteasome subunits have been implicated in the DDR. In this study, we aimed to analyze the possible distinct roles of individual proteasome subunits in the DDR. We show that specific 19S subunits respond to DNA damage by increased protein levels and nuclear translocation. Importantly, two 19S subunits, Rpn7 and Rpn11, colocalize with DNA damage foci over their whole lifespan. Although silencing of Rpn11 does not affect foci stability and lifespan, silencing of Rpn7 promotes faster resolution of DNA damage foci following genotoxic insult. For the first time, we provide evidence that Rpn7 silencing specifically decreases the frequencies of long-lived DNA damage foci without, however, affecting the repair rate of short-lived foci. Therefore, we propose that interaction of Rpn7 with DDR foci in situ mediates the protection of DNA damage foci from premature resolution. We suggest that this interaction is involved in enabling cellular senescence following genotoxic insult.

Solar ultraviolet (UV) A radiation is a well known trigger of signaling responses in human skin fibroblasts. One important consequence of this stress response is the increased expression of matrix metalloproteinase-1 (MMP-1), which causes extracellular protein degradation and thereby contributes to photoaging of human skin. In the present study we identify the proteasome as an integral part of the UVA-induced, intracellular signaling cascade in human dermal fibroblasts. UVA-induced singlet oxygen formation was accompanied by protein oxidation, the cross-linking of oxidized proteins, and an inhibition of the proteasomal system. This proteasomal inhibition subsequently led to an accumulation of c-Jun and phosphorylated c-Jun and activation of activator protein-1, i.e. transcription factors known to control MMP-1 expression. Increased transcription factor activation was also observed if the proteasome was inhibited by cross-linked proteins or lactacystin, indicating a general mechanism. Most importantly, inhibition of the proteasome was of functional relevance for UVA-induced MMP-1 expression, because overexpression of the proteasome or the protein repair enzyme methionine sulfoxide reductase prevented the UVA-induced induction of MMP-1. These studies show that an environmentally relevant stimulus can trigger a signaling pathway, which links intracellular and extracellular protein degradation. They also identify the proteasome as an integral part of the UVA stress response.

Normal human fibroblasts undergo a limited number of divisions in culture and progressively they reach a state of irreversible growth arrest, a process termed as replicative senescence. The proteasome is the major cellular proteolytic machinery, the function of which is impaired during replicative senescence. However, the exact causes of its malfunction in these conditions are unknown. Using WI38 fibroblasts as a model for cellular senescence we have observed reduced levels of proteasomal peptidase activities coupled with increased levels of both oxidized and ubiquitinated proteins in senescent cells. We have found the catalytic subunits of the 20 S complex and subunits of the 19 S regulatory complex to be down-regulated in senescent cells. This is accompanied by a decrease in the level of both 20 S and 26 S complexes. Partial inhibition of proteasomes in young cells caused by treatment with specific inhibitors induced a senescence-like phenotype, thus demonstrating the fundamental importance of the proteasome for retaining cellular maintenance and homeostasis. Stable overexpression of β1 and β5 subunits in WI38 established cell lines was shown to induce elevated expression levels of β1 subunit in β5 transfectants and vice versa. Transfectants possess increased proteasome activities and most importantly, increased capacity to cope better with various stresses. In summary these data demonstrate the central role of the proteasome during cellular senescence and survival as well as provide insights toward a better understanding of proteasome regulation.

Normal human fibroblasts undergo replicative senescence due to both genetic and environmental factors. Senescence and aging can be further accelerated by exposure of cells to a variety of oxidative agents that contribute among other effects to the accumulation of damaged proteins. The proteasome, a multicatalytic nonlysosomal protease, has impaired function during aging, while its increased expression delays senescence in human fibroblasts. The aim of this study was to identify natural compounds that enhance proteasome activity and exhibit antiaging properties. We demonstrate that oleuropein, the major constituent of Olea europea leaf extract, olive oil and olives, enhances the proteasome activities in vitro stronger than other known chemical activators, possibly through conformational changes of the proteasome. Moreover, continuous treatment of early passage human embryonic fibroblasts with oleuropein decreases the intracellular levels of reactive oxygen species (ROS), reduces the amount of oxidized proteins through increased proteasome-mediated degradation rates and retains proteasome function during replicative senescence. Importantly, oleuropein-treated cultures exhibit a delay in the appearance of senescence morphology and their life span is extended by approximately 15%. In summary, these data demonstrate the beneficial effect of oleuropein on human fibroblasts undergoing replicative senescence and provide new insights towards enhancement of cellular antioxidant mechanisms by natural compounds that can be easily up-taken through normal diet.

Summary Proteasome-dependent degradation has been extensively investigated and has been shown to play a vital role in the maintenance of cellular homeostasis. Proteasome activity and expression are reduced during aging and replicative senescence. Its activation has been shown to confer lifespan extension in human diploid fibroblasts (HDFs), whereas partial proteasome inhibition triggers an irreversible premature senescent state in young HDFs. As p53 and Rb tumor suppressors regulate both replicative and premature senescence (RS and PS, respectively), in this study we investigated their implication in proteasome inhibition-mediated PS. By taking advantage of a variety of HDFs with defective p53 or/and Rb pathways, we reveal that proteasome activity inhibition to levels normally found in senescent human cells results in immediate growth arrest and/or moderate increase of apoptotic death. These effects are independent of the cellular genetic context. However, in the long term, proteasome inhibition-mediated PS can only be initiated and maintained in the presence of functional p53. More specifically, we demonstrate that following partial proteasome inhibition, senescence is dominant in HDFs with functional p53 and Rb molecules, crisis/death is induced in cells with high p53 levels and defective Rb pathway, whereas stress recovery and restoration of normal cycling occurs in cells that lack functional p53. These data reveal the continuous interplay between the integrity of proteasome function, senescence and cell survival.

Summary Homeostasis is a key feature of cellular lifespan. Mainte- nance of cellular homeostasis influences the rate of aging and is determined by several factors, including efficient proteolysis of damaged proteins. Protein degradation is predominately cata- lyzed by the proteasome. Specifically, the proteasome is respon- sible for cell clearance of abnormal, denatured or in general damaged proteins as well as for the regulated degradation of short-lived proteins. As proteasome has an impaired function during aging, emphasis has been given recently in identifying ways of its activation. A number of studies have shown that the proteasome can be activated by genetic manipulations as well as by factors that affect its conformation and stability. Impor- tantly the developed proteasome activated cell lines exhibit an extended lifespan. This review article discusses in details the various factors that are involved in proteasome biosynthesis and assembly and how they contribute to its activation. Finally as few natural compounds have been identified having protea- some activation properties, we discuss the advantages of this novel antiaging strategy. 2008 IUBMB IUBMB Life, 60(10): 651-655, 2008 Protein degradation is predominately catalyzed by the protea- some. The proteasome is responsible for cell clearance of abnormal, denatured or in general damaged proteins as well as, for the regulated degradation of short-lived proteins (2, 3). The 20S proteasome, a 700 kDa multisubunit enzyme complex, is a barrel-shape stack of four heptameric rings localized in both cytoplasm and nucleus. The two outer a-subunits rings (a1-7) embrace two central head-to-head oriented rings containing b-subunits (b1-7). The internal chamber that is composed by b-subunits hosts the proteolytic active sites. Three of the b-sub- units, b1, b2, and b5, are responsible for the proteasome hydro- lyzing activities that cleave peptide bonds on the carboxyl site of acidic (peptidylglutamylpeptide hydrolyzing activity, PGPH), basic (trypsin-like activity, T-L), and hydrophobic (chymotryp- sin-like activity, CT-L) amino acids, respectively (3). Capping of each side of 20S particle by 19S regulatory com- plexes gives rise to 26S proteasome that is responsible for the ATP/ubiquitin-dependent protein degradation. The 19S particle is composed of two subcomplexes, namely the lid and the base. The lid covers the base and it is involved in the recognition and ubiquitin chain processing of substrates before their transloca- tion and degradation. The base is consisted of six ATPase subu- nits involved in the unfolding and further translocation of the substrate (4). The target protein is first labeled via covalent attachment of multiple ubiquitin molecules before the degrada- tion by the 26S complex. Three steps are involved in the conju- gation of ubiquitin to the substrate. Ubiquitin is activated by E1, the ubiquitin-activating enzyme, and then transferred by an E2 enzyme, the ubiquitin-carrier protein or ubiquitin-conjugat- ing enzyme, to a member of the ubiquitin-protein ligase family, E3, to which the target protein is specifically bound. This enzyme catalyzes the repeated conjugation of ubiquitin to the substrate prior of the 26S complex-mediated degradation (2). Proteasome has also been implicated in antigen presentation process and immune response, mainly through immunoprotea- somes. In this proteasome type, the constitutively expressed b1, b2, and b5 subunits are substituted during de novo proteasome biosynthesis by b1i, b2i, and b5i subunits respectively. As a result, although these particles digest proteins at rates similar to

We have investigated immunoproteasomes in human fibroblasts during replicative senescence. Unlike levels of constitutive proteasome catalytic subunits and 26S proteasome regulatory subunits, levels of immunosubunits did not decrease dramatically in senescent cells. However, the induction of immunosubunits by interferon-gamma (IFN-gamma) was lost in senescent cells. In contrast, levels of the 11S proteasome regulator, PA28, were increased by IFN-gamma even in senescent cells, and both immunosubunits and PA28 increased with the reversible growth arrest in confluent cell cultures. The results highlight differences in the mechanisms of regulation of immunoproteasomes compared to constitutive proteasomes and in the irreversible growth arrest of senescent cells compared to reversible contact-induced growth arrest.

The proteasome is the major cellular proteolytic machinery responsible for the degradation of both normal and damaged proteins. Proteasomes play a fundamental role in retaining cellular homeostasis. Alterations of proteasome function have been recorded in various biological phenomena including aging. We have recently shown that the decrease in proteasome activity in senescent human fibroblasts relates to the down-regulation of beta-type subunits. In this study we have followed our preliminary observation by developing and further characterizing a number of different human cell lines overexpressing the beta5 subunit. Stable overexpression of the beta5 subunit in WI38/T and HL60 cells resulted in elevated levels of other beta-type subunits and increased levels of all three proteasome activities. Immunoprecipitation experiments have shown increased levels of assembled proteasomes in stable clones. Analysis by gel filtration has revealed that the recorded higher level of proteasome assembly is directly linked to the efficient integration of "free" (not integrated) alpha-type subunits identified to accumulate in vector-transfected cells. In support we have also found low proteasome maturation protein levels in beta5 transfectants, thus revealing an increased rate/level of proteasome assembly in these cells as opposed to vector-transfected cells. Functional studies have shown that beta5-overexpressing cell lines confer enhanced survival following treatment with various oxidants. Moreover, we demonstrate that this increased rate of survival is due to higher degradation rates following oxidative stress. Finally, because oxidation is considered to be a major factor that contributes to aging and senescence, we have overexpressed the beta5 subunit in primary IMR90 human fibroblasts and observed a delay of senescence by 4-5 population doublings. In summary, these data demonstrate the phenotypic effects following genetic up-regulation of the proteasome and provide insights toward a better understanding of proteasome regulation.

Senescent human fibroblasts exhibit several genetic and biochemical differences as compared to their young counterparts including abnormalities of the main proteolytic mechanism, namely the proteasome. Specifically, we and others have shown that there is an impaired function of the proteasome, as senescent cells have reduced proteolytic activities and less proteasome content. In a complementary work we have recently shown that inhibition of the proteasome by a specific inhibitor induces a senescence-like phenotype in young WI38 fibroblasts [Chondrogianni et al. (2003) J Biol Chem 278: 28026-28037]. In this study we tested whether the induction of a senescence-like phenotype following treatment with proteasome inhibitors is a common feature of primary human fibroblasts. A comparative biochemical analysis, after employing three different human fibroblasts cell lines (IMR90, MRC5 and WI38 cells), as well as two proteasome inhibitors (epoxomicin and MG132), has shown that proteasome inhibition results in the appearance of a senescence-like phenotype in all cell lines used. Proteasome inhibitors treated cells were irreversibly stopped dividing, exhibited positive staining to beta-galactosidase as well as reduced CT-L and PGPH activities. In summary, these data reveal the fundamental role of the proteasome in the progression of replicative senescence and open new dimensions towards a better understanding of protein degradation.

Heavy metals like CrVI, CdII, PbII and SnII have many applications in industry. They also represent a group of labour pollutants, as they are involved in several physiological disorders, such as carcinogenesis and various tissue dysfunctions. However, limited knowledge exists regarding their effects on ageing. In the current work we provide evidence that workers chronically exposed to CrVI have considerably reduced serum levels of the biomarker of senescence and cell survival, Apolipoprotein J/Clusterin (ApoJ/CLU). Moreover, we have found that both the degree and the time of exposure to CrVI associate negatively with ApoJ/CLU serum levels. To further examine whether CrVI directly affects cellular senescence we treated for 10 weeks two adult skin fibroblasts cultures as well as embryonic fibroblasts with a range of CrVI concentrations that approximate the values recorded in the blood circulation of exposed workers. Cellular treatment with a CrVI concentration that approximates the highest concentration in the blood was extremely toxic and nearly all cells died immediately after the first treatment. Interestingly, continuous treatment with a 10-fold lower CrVI concentration resulted in the induction of premature senescence. More specifically, treated cells were growth arrested, acquired an irregular shape, were positive to β-galactosidase staining, accumulated oxidized proteins and over-expressed the cyclin-dependent kinase inhibitor p21 and ApoJ/CLU. Similar treatments with three additional labour pollutants resulted in the induction of premature senescence by CdII, but not by SnII or PbII. In summary, our results indicate that exposure to CrVI induces alterations of senescence biomarkers both in vivo and in vitro. They also provide new valuable tools for monitoring CrVI cytotoxic effects in vivo as well as for re-evaluating the maximum permissive values of some labour pollutants, like CrVI and CdII.

Normal human fibroblasts undergo a limited number of divisions in culture and progressively they reach a state of irreversible growth arrest, a process termed as replicative senescence. The proteasome is the major cellular proteolytic machinery, the function of which is impaired during replicative senescence. However, the exact causes of its malfunction in these conditions are unknown. Using WI38 fibroblasts as a model for cellular senescence we have observed reduced levels of proteasomal peptidase activities coupled with increased levels of both oxidized and ubiquitinated proteins in senescent cells. We have found the catalytic subunits of the 20 S complex and subunits of the 19 S regulatory complex to be down-regulated in senescent cells. This is accompanied by a decrease in the level of both 20 S and 26 S complexes. Partial inhibition of proteasomes in young cells caused by treatment with specific inhibitors induced a senescence-like phenotype, thus demonstrating the fundamental importance of the proteasome for retaining cellular maintenance and homeostasis. Stable overexpression of beta1 and beta5 subunits in WI38 established cell lines was shown to induce elevated expression levels of beta1 subunit in beta5 transfectants and vice versa. Transfectants possess increased proteasome activities and most importantly, increased capacity to cope better with various stresses. In summary these data demonstrate the central role of the proteasome during cellular senescence and survival as well as provide insights toward a better understanding of proteasome regulation.

Background Pollen is a bee-product known for its medical properties from ancient times. In our days is increasingly used as health food supplement and especially as a tonic primarily with appeal to the elderly to ameliorate the effects of ageing. In order to evaluate the chemical composition and the biological activity of Greek pollen which has never been studied before, one sample with identified botanical origin from sixteen different common plant taxa of Greece has been evaluated. Results Three different extracts of the studied sample of Greek pollen, have been tested, in whether could induce proteasome activities in human fibroblasts. The water extract was found to induce a highly proteasome activity, showing interesting antioxidant properties. Due to this activity the aqueous extract was further subjected to chemical analysis and seven flavonoids have been isolated and identified by modern spectral means. From the methanolic extract, sugars, lipid acids, phenolic acids and their esters have been also identified, which mainly participate to the biosynthetic pathway of pollen phenolics. The total phenolics were estimated with the Folin-Ciocalteau reagent and the total antioxidant activity was determined by the DPPH method while the extracts and the isolated compounds were also tested for their antimicrobial activity by the dilution technique. Conclusions The Greek pollen is rich in flavonoids and phenolic acids which indicate the observed free radical scavenging activity, the effects of pollen on human fibroblasts and the interesting antimicrobial profile.