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.

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 Normal human fibroblasts undergo a limited number of divisions in culture and progressively reach a state of irreversible growth arrest, a process termed replicative senescence. Senescence has been attributed to a combination of genetic and environmental factors (1). Senescent fibroblasts are viable and fully functional, however, they exhibit several morphologic and biochemical changes as compared with their young/proliferating counterparts (2). We and others (3) have reported loss of proteasome function upon aging of several human tissues as well as in senescent primary cultures. Our previous work in human embryonic fibroblast cultures undergoing replicative senescence has shown that the reduced levels of proteasome activities are accompanied by lower proteasome content due to the down-regulation of its ␤-catalytic subunits (4). The fundamental link between cellular senescence and proteasome function is further supported by our additional studies (5, 6) demonstrating that when the proteasome is partially inhibited in young fibroblasts cultures, an irreversible senescence-like phenotype is triggered. Importantly, reconstitution of the levels of the "down-regulated" ␤-subunits by genetic means in human embryonic fibroblasts, results in enhanced rates of assembled, and functional proteasome and delays senescence by ϳ15% (7).The proteasome is the major cellular non-lysosomal threonine protease, implicated in the removal of normal as well as abnormal, denatured, or otherwise damaged proteins (8). It is a well organized protein complex that contains a proteolytic active 20 S core, composed of 28 subunits, 7 different ␣-and 7 different ␤-subunits, arranged as ␣ 1-7 ␤ 1-7 ␤ 1-7 ␣ 1-7 structure (9). The ␤ 5, ␤ 1 , and ␤ 2 subunits are the catalytic centers of chymotrypsin-like (CT-L), 3 caspase-like/PGPH (peptidylglutamyl-peptide hydrolyzing), and trypsin-like activities, respectively, cleaving peptide bonds after hydrophobic, acidic, and basic residues, respectively (10). The 20 S proteasome is also central to the ATP/ubiquitin-dependent intracellular protein degradation pathway where it represents the proteolytic core of the 26 S complex (11).Although the regulation of human proteasome is not yet entirely elucidated, reports in mice indica...

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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 and resistance to proteotoxicity in Caenorhabditis elegans. FASEB J. 29, 611‐622 (2015). http://www.fasebj.org

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.

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 ␤-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 ␤ 5 subunit. Stable overexpression of the ␤ 5 subunit in WI38/T and HL60 cells resulted in elevated levels of other ␤-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) ␣-type subunits identified to accumulate in vector-transfected cells. In support we have also found low proteasome maturation protein levels in ␤ 5 transfectants, thus revealing an increased rate/level of proteasome assembly in these cells as opposed to vector-transfected cells. Functional studies have shown that ␤ 5 -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 ␤ 5 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.

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 posttranscriptional mechanisms that account for over 40% of the ageregulated 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.

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 ␤ 1 and ␤ 5 subunits in WI38 established cell lines was shown to induce elevated expression levels of ␤ 1 subunit in ␤ 5 transfectants and vice versa.