Search

Edible insects are high value alternative protein sources, exhibiting high quality nutrient compositions along with potentially health-promoting constituents. Insects are rich in protein (35–61%), lipids (13–33%) and also contain a significant amount of ‘animal’ fiber in form of insoluble chitin. Apart from macronutrients, some insects are also known for their rich minerals and vitamins profiles. However, the nutrient compositions vary with species, diet, developmental stage, sex and the growth environment of insects. Bioavailability and bioaccessibility of insect nutrients are sparsely researched topics, while most of the studies focus on nutrient content and characterization. Similar to any other food, the type of preparation and processing has a significant effect on the amount of bioaccessible nutrients. This article provides an overview of the bioavailability of nutrients present in insects and the factors affecting their variability. Further, the gaps in research studies and scope for future research are also briefly discussed.

Glutamine synthetase (GS), which catalyzes the ATP-dependent synthesis of L-glutamine from L-glutamate and ammonia, is a ubiquitous and conserved enzyme that plays a pivotal role in nitrogen metabolism across all life domains. In vertebrates, GS is highly expressed in astrocytes, where its activity sustains the glutamate-glutamine cycle at glutamatergic synapses and is thus essential for maintaining brain homeostasis. In fact, decreased GS levels or activity have been associated with neurodegenerative diseases, with these alterations attributed to oxidative post-translational modifications of the protein, in particular tyrosine nitration. In this study, we expressed and purified human GS (HsGS) and performed an in-depth analysis of its oxidative inactivation by peroxynitrite (ONOO−) in vitro. We found that ONOO− exposure led to a dose-dependent loss of HsGS activity, the oxidation of cysteine, methionine, and tyrosine residues and also the nitration of tryptophan and tyrosine residues. Peptide mapping by LC-MS/MS through combined H216O/H218O trypsin digestion identified up to 10 tyrosine nitration sites and five types of dityrosine cross-links; these modifications were further scrutinized by structural analysis. Tyrosine residues 171, 185, 269, 283, and 336 were the main nitration targets; however, tyrosine-to-phenylalanine HsGS mutants revealed that their sole nitration was not responsible for enzyme inactivation. In addition, we observed that ONOO− induced HsGS aggregation and activity loss. Thiol oxidation was a key modification to elicit aggregation, as it was also induced by hydrogen peroxide treatment. Taken together, our results indicate that multiple oxidative events at various sites are responsible for the inactivation and aggregation of human GS.

The study of protein aggregates has a long history. While in the first decades until the 80ies of the 20th century only the observation of the presence of such aggregates was reported, later the biochemistry of the formation and the biological effects of theses aggregates were described. This review focusses on the complexity of the biological effects of protein aggregates and its potential role in the aging process.

Aging is a complex phenomenon that has detrimental effects on tissue homeostasis. The skeletal muscle is one of the earliest tissues to be affected and to manifest age-related changes such as functional impairment and the loss of mass. Common to these alterations and to most of tissues during aging is the disruption of the proteostasis network by detrimental changes in the ubiquitin-proteasomal system (UPS) and the autophagy-lysosomal system (ALS). In fact, during aging the accumulation of protein aggregates, a process mainly driven by increased levels of oxidative stress, has been observed, clearly demonstrating UPS and ALS dysregulation. Since the UPS and ALS are the two most important pathways for the removal of misfolded and aggregated proteins and also of damaged organelles, we provide here an overview on the current knowledge regarding the connection between the loss of proteostasis and skeletal muscle functional impairment and also how redox regulation can play a role during aging. Therefore, this review serves for a better understanding of skeletal muscle aging in regard to the loss of proteostasis and how redox regulation can impact its function and maintenance.

The ubiquitin-proteasomal-system (UPS) and the autophagy-lysosomal-system (ALS) are both highly susceptible for disturbances leading to the accumulation of cellular damage. A decline of protein degradation during aging results in the formation of oxidatively damaged and aggregated proteins finally resulting in failure of cellular functionality. Besides protein aggregation in response to oxidative damage, amyloids are a different type of protein aggregates able to distract proteostasis and interfere with cellular functionality. Amyloids are clearly linked to the pathogenesis of age-related degenerative diseases such as Alzheimer's disease. Human amylin is one of the peptides forming fibrils in β-sheet conformation finally leading to amyloid formation. In contrast to rodent amylin, human amylin is prone to form amyloidogenic aggregates, proposed to play a role in the pathogenesis of Type 2 Diabetes by impairing β-cell functionality. Since aggregates such as lipofuscin and β-amyloid are known to impair proteostasis, it is likely to assume similar effects for human amylin. In this review, we focus on the effects of IAPP on UPS and ALS and their role in amylin degradation, since both systems play a crucial role in maintaining proteome balance thereby influencing, at least in part, cellular fate and aging.

Frailty among elderly people leads to an increased risk for negative health outcomes. To prevent frailty, we need a better understanding of the underlying mechanisms and early detection of individuals at risk. Both may be served by identifying candidate (bio)markers, i.e. biomarkers and markers, for the physical, cognitive, and psychological frailty domains. We used univariate (Rank-ANOVA) and multivariate (elastic net) approaches on the RASIG study population (age range: 35–74 years, n = 2220) of the MARK-AGE study to study up to 331 (bio)markers between individuals with and without frailty for each domain. Biomarkers and markers identified by both approaches were studied further regarding their association with frailty using logistic regression. Univariately, we found lower levels of antioxidants, including β-cryptoxanthin and zeaxanthin, in those who were physically, cognitively or psychologically frail. Additionally, self-reported health was worse in these three frail groups. Multivariately, we observed lower levels of β-cryptoxanthin and zeaxanthin in the cognitively frail. Levels of these carotenoids were inversely associated with the risk of being cognitively frail after adjusting for confounders. Antioxidants and self-reported health are potential (bio)markers to detect persons at risk of becoming frail. The biomarkers identified may indicate the involvement of inflammation in frailty, especially for physical and cognitive frailty.

Aging is accompanied by the accumulation of cellular damage over time in response to stress, lifestyle and environmental factors ultimately leading to age-related diseases and death. Additionally, the number of senescent cells increases with age. Senescence is most likely not a static endpoint, it represents a series of hallmarks including morphological changes, alterations in protein turnover and accumulation of protein aggregates. The importance of protein oxidation and aggregate accumulation in the progression of aging is not yet fully understood and research to what extent the accumulation of oxidized proteins has an effect on senescence and the aging process is still ongoing. To study the mechanisms of aging, the impact of senescence and the role of protein aggregates on the aging process, cell culture models are useful tools. Most notably stress induced premature senescence (SIPS) models have contributed to the identification of mechanisms involved in the aging process and helped unravel the age-related changes in proteolysis and the importance of protein aggregation. Here we review characteristics of replicative and premature senescence, how to induce most frequently used senescence models and gained knowledge on age-related changes in the major proteolytic systems.

Mitochondria have been in the focus of oxidative stress and aging research for decades due to their permanent production of ROS during the oxidative phosphorylation. The hypothesis exists that mitochondria are involved in the formation of lipofuscin, an autofluorescent protein aggregate that accumulates progressively over time in lysosomes of post-mitotic and senescent cells. To investigate the influence and involvement of mitochondria in lipofuscinogenesis, we analyzed lipofuscin amounts as well as the mitochondrial function in young and senescent cells. In addition we used an aging model and Lon protease deficient HeLa cells to investigate the influence of mitochondrial degradation processes on lipofuscin formation.We were able to show that mitophagy is impaired in senescent cells resulting in an increased mitochondrial mass and superoxide formation. In addition, the inhibition of mitochondrial fission leads to increased lipofuscin formation.Moreover, we observed that Lon protease downregulation is linked to a higher lipofuscinogenesis whereas the application of the mitochondrial-targeted antioxidant mitoTEMPO is able to prevent the accumulation of this protein aggregate. Keywords: Lipofuscin, Protein aggregates, Lon protease, Aging, Mitochondria, Oxidative stress

Aging is a complex phenomenon and its impact is becoming more relevant due to the rising life expectancy and because aging itself is the basis for the development of age-related diseases such as cancer, neurodegenerative diseases and type 2 diabetes. Recent years of scientific research have brought up different theories that attempt to explain the aging process. So far, there is no single theory that fully explains all facets of aging. The damage accumulation theory is one of the most accepted theories due to the large body of evidence found over the years. Damage accumulation is thought to be driven, among others, by oxidative stress. This condition results in an excess attack of oxidants on biomolecules, which lead to damage accumulation over time and contribute to the functional involution of cells, tissues and organisms. If oxidative stress persists, cellular senescence is a likely outcome and an important hallmark of aging. Therefore, it becomes crucial to understand how senescent cells function and how they contribute to the aging process. This review will cover cellular senescence features related to the protein pool such as morphological and molecular hallmarks, how oxidative stress promotes protein modifications, how senescent cells cope with them by proteostasis mechanisms, including antioxidant enzymes and proteolytic systems. We will also highlight the nutritional status of senescent cells and aged organisms (including human clinical studies) by exploring trace elements and micronutrients and on their importance to develop strategies that might increase both, life and health span and postpone aging onset., Graphical abstract fx1

Changes in the two main intracellular degradation systems, the Ubiquitin-Proteasome System and the Autophagy-Lysosome pathway (ALP) are widely discussed as a hallmark of the aging process. To follow the age-related behavior of both degradation systems we examined their impact on ferritin, known to be degradable by both. Ferritin H was analyzed in young and senescent human fibroblasts, revealing a higher steady-state level in the senescent cells. By blocking both proteolytic systems, we confirmed that particularly the ALP plays a crucial role in ferritin H turnover. However, an unexpected increase in lysosomal activity in the senescent cells, suggests a dysregulation in the autophagy pathway. To further investigate the impaired ferritin H turnover, confocal microscopic colocalization studies of ferritin H with lysosomal-associated membrane protein 2a (Lamp2a) and monodansylcadaverine (MDC) were performed and clearly revealed the degradation of ferritin by macroautophagy. By induction of autophagy via inhibition of mTOR using rapamycin an increase of ferritin H turnover was obtained in senescent cells, demonstrating a mTOR dependent reduction of autophagy in senescent human fibroblasts.

Overnutrition contributes to insulin resistance, obesity and metabolic stress, initiating a loss of functional beta-cells and diabetes development. Whether these damaging effects are amplified in advanced age is barely investigated. Therefore, New Zealand Obese (NZO) mice, a well-established model for the investigation of human obesity-associated type 2 diabetes, were fed a metabolically challenging diet with a high-fat, carbohydrate restricted period followed by a carbohydrate intervention in young as well as advanced age. Interestingly, while young NZO mice developed massive hyperglycemia in response to carbohydrate feeding, leading to beta-cell dysfunction and cell death, aged counterparts compensated the increased insulin demand by persistent beta-cell function and beta-cell mass expansion. Beta-cell loss in young NZO islets was linked to increased expression of thioredoxin-interacting protein (TXNIP), presumably initiating an apoptosis-signaling cascade via caspase-3 activation. In contrast, islets of aged NZOs exhibited a sustained redox balance without changes in TXNIP expression, associated with higher proliferative potential by cell cycle activation. These findings support the relevance of a maintained proliferative potential and redox homeostasis for preserving islet functionality under metabolic stress, with the peculiarity that this adaptive response emerged with advanced age in diabetes-prone NZO mice., Graphical abstract Image 1, Highlights • Differential expression of redox and cell cycle genes in young and aged islets. • Increased TXNIP expression is associated with the induction of beta-cell apoptosis. • Islets of aged mice maintained redox homeostasis and proliferative potential. • Aging under diet-induced metabolic stress does not amplify beta-cell failure.

Protein carbonyls are widely analysed as a measure of protein oxidation. Several different methods exist for their determination. A previous study had described orders of magnitude variance that existed when protein carbonyls were analysed in a single laboratory by ELISA using different commercial kits. We have further explored the potential causes of variance in carbonyl analysis in a ring study. A soluble protein fraction was prepared from rat liver and exposed to 0, 5 and 15 min of UV irradiation. Lyophilised preparations were distributed to six different laboratories that routinely undertook protein carbonyl analysis across Europe. ELISA and Western blotting techniques detected an increase in protein carbonyl formation between 0 and 5 min of UV irradiation irrespective of method used. After irradiation for 15 min, less oxidation was detected by half of the laboratories than after 5 min irradiation. Three of the four ELISA carbonyl results fell within 95% confidence intervals. Likely errors in calculating absolute carbonyl values may be attributed to differences in standardisation. Out of up to 88 proteins identified as containing carbonyl groups after tryptic cleavage of irradiated and control liver proteins, only seven were common in all three liver preparations. Lysine and arginine residues modified by carbonyls are likely to be resistant to tryptic proteolysis. Use of a cocktail of proteases may increase the recovery of oxidised peptides. In conclusion, standardisation is critical for carbonyl analysis and heavily oxidised proteins may not be effectively analysed by any existing technique., Graphical abstract, Highlights • ELISA of protein carbonyls is sensitive and robust to detect moderate oxidation. • Western blot for protein carbonyls offers a robust semi-quantitative alternative. • Improved standardisation is needed for protein carbonyl analysis methods. • Mass spectrometry is suitable for protein oxidation target discovery. • Fewer carbonyls are detected in heavily-relative to moderately-oxidised proteins.

Increasing cellular damage during the aging process is considered to be one factor limiting the lifespan of organisms. Besides the DNA and lipids, proteins are frequent targets of non-enzymatic modifications by reactive substances including oxidants and glycating agents. Non-enzymatic protein modifications may alter the protein structure often leading to impaired functionality. Although proteolytic systems ensure the removal of modified proteins, the activity of these proteases was shown to decline during the aging process. The additional age-related increase of reactive compounds as a result of impaired antioxidant systems leads to the accumulation of damaged proteins and the formation of protein aggregates. Both, non-enzymatic modified proteins and protein aggregates impair cellular functions and tissue properties by a variety of mechanisms. This is increasingly important in aging and age-related diseases. In this review, we will give an overview on oxidation and glycation of proteins and the function of modified proteins in aggregate formation. Furthermore, their effects as well as their role in aging and age-related diseases will be highlighted.

Native casein micelles were treated (incubated) with vitamin D2 at different hydrostatical pressures (0.1, 200, 400, and 600MPa) and temperatures (10-50°C) in order to load the vitamin. Pressure induced the release of αs-, β- and κ-casein and calcium from the micelle into the soluble phase. During the pressure stable reassembled micelles were created due to a structural rearrangement of hydrophobic interactions between the hydrophobic domains of the caseins and vitamin D2. Treatment at 600MPa and 50°C increased the loading of vitamin D2 per casein from 2.2±0.2μg/mg (native sample) to 10.4±0.2μg/mg. The reassembled micelles presented an average hydrodynamic diameter of 272±10nm and contained 8.1, 10.3 and 0.8mg of α-, β- and κ-casein, respectively, per 100mg casein.

Here, we review the role of oxidative protein modification as a signal for recognition and degradation of proteins. It was clearly demonstrated that the ATP- and ubiquitin-independent 20S proteasome is playing a key role in the selective removal of oxidized proteins. Furthermore, the current knowledge of the substrate susceptibility on the degradation of oxidized proteins and the role of the immunoproteasome will be highlighted.

Improvements in health care and lifestyle have led to an elevated lifespan and increased focus on age-associated diseases, such as neurodegeneration, cardiovascular disease, frailty and arteriosclerosis. In all these chronic diseases protein, lipid or nucleic acid modifications are involved, including cross-linked and non-degradable aggregates, such as advanced glycation end products (AGEs). Formation of endogenous or uptake of dietary AGEs can lead to further protein modifications and activation of several inflammatory signaling pathways. This review will give an overview of the most prominent AGE-mediated signaling cascades, AGE receptor interactions, prevention of AGE formation and the impact of AGEs during pathophysiological processes.

Ageing is a multidimensional process involving many variables that affect all levels of life. Upon ageing a significant increase of damaged biomolecules, such as accumulated proteins occurs with age pigments being such molecules. The “age pigment” lipofuscin is naturally produced throughout the life of an organism and it is aggregated in post-mitotic cells as ageing progresses resulting in age-dependent degeneration of various cellular systems. Lipofuscin is non-degradable and consists of oxidized, cross-linked proteins, lipids and saccharides. Its accumulation is associated with the proteolytic mechanisms inability to degrade it during ageing. Lipofuscin accumulation promotes ageing as it acts as proteasome inhibitor by directly binding on proteasome complexes. Our aim is to study the effects of lifelong exposure to lipofuscin on organismal lifespan and physiology by exploiting Caenorhabditis elegans. Our preliminary results demonstrated that the nematode lifespan is altered upon treatment with artificial lipofuscin and that the two major cellular degradation mechanisms (proteasome and autophagy) are affected. Detailed analysis of the involved antioxidant mechanisms and metabolic pathways has been performed and additional experimentation will elucidate deeper effects.

Advanced glycation end product (AGE)-modified proteins are formed by the nonenzymatic glycation of free amino groups of proteins and, along with lipofuscin (a highly oxidized aggregate of covalently cross-linked proteins, sugars, and lipids), have been found to accumulate during aging and in several age-related diseases. As the in vivo effects of diet-derived AGEs or lipofuscin remain elusive, we sought to study the impact of oral administration of glucose-, fructose-, or ribose-modified albumin or of artificial lipofuscin in a genetically tractable model organism. We report herein that continuous feeding of young Drosophila flies with culture medium enriched in AGEs or in lipofuscin resulted in reduced locomotor performance and in accelerated rates of AGE-modified proteins and carbonylated proteins accumulation in the somatic tissues and hemolymph of flies, as well as in a significant reduction of flies health span and life span. These phenotypic effects were accompanied by reduced proteasome peptidase activities in both the hemolymph and the somatic tissues of flies and higher levels of oxidative stress; furthermore, oral administration of AGEs or lipofuscin in flies triggered an upregulation of the lysosomal cathepsin B, L activities. Finally, RNAi-mediated cathepsin D knockdown reduced flies longevity and significantly augmented the deleterious effects of AGEs and lipofuscin, indicating that lysosomal cathepsins reduce the toxicity of diet-derived AGEs or lipofuscin. Our in vivo studies demonstrate that chronic ingestion of AGEs or lipofuscin disrupts proteostasis and accelerates the functional decline that occurs with normal aging.

Protein carbonylation is an important event in the context of proteostasis because of its frequency, non-enzymatic nature and irreversible effects. The carbonylation of proteins disturbs their function and leads to protein aggregates, which may precede cellular senescence and cell death. Actin, an evolutionarily conserved cytoskeletal protein that is involved in important cellular processes, is one of the proteins most susceptible to carbonylation. Conditions resulting in oxidative stress are likely to lead to its carbonylation, loss of function and aggregate formation. In this review, we summarise actin susceptibility to carbonylation, as verified in cell free extracts, cell lines and animal models, and review its fate through the activation of cell mechanisms aimed at removing damaged proteins. Their insufficient activity may underlie age-related diseases and the ageing process. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.

A simple and sensitive method is presented for the simultaneous quantification (spectrophotometric and spectrofluorimetric) of the main lipid and protein peroxidation products after their initial fractionation: free malondialdehyde (FrMDA), protein-bound malondialdehyde (PrMDA), total hydroperoxides (LOOH), and protein hydroperoxides (PrOOH). FrMDA and PrMDA (released from proteins by alkaline hydrolysis) are measured after the reaction of MDA with thiobarbituric acid (TBA) under acidic conditions, by the specific fluorimetric quantification of the resulting MDA-(TBA)2 adduct chromophore. The measurement of LOOH and PrOOH is based on the reaction of Fe(3+) (resulting from the reaction of LOOH and PrOOH with Fe(2+)) with xylenol orange (XO) and the photometric quantification of the resulting XO-Fe complex. The sensitivity of the assays for FrMDA/PrMDA and LOOH/PrOOH is 20 and 100pmol, respectively. The method was applied successfully on human plasma and can be used for the evaluation of oxidative stress in both basic and clinical research.

The main machinery responsible for cellular protein maintenance is the ubiquitin-proteasomal system, with its core particle the 20S proteasome. The main task of the system is a fast and efficient degradation of proteins not needed anymore in cellular metabolism. For this aim a complex system of regulators evolved, modifying the function of the 20S core proteasome. Here we summarize shortly the structure of the 20S proteasome as well as its associated regulator proteins., Graphical abstract Highlights ► Form and function of the 20S proteasome are described. ► Comparison between Archaeal and eukaryotic 20S proteasome are pointed out. ► Structure and function of proteasomal regulators are highlighted.

Oxidative stress plays a crucial role in the development of the aging process and age dependent diseases. Both are closely connected to disturbances of proteostasis by protein oxidation and an impairment of the proteasomal system. The final consequence is the accumulation of highly cross-linked undegradable aggregates such as lipofuscin. These aggregates of damaged proteins are detrimental to normal cell functions. Here we provide an overview about effect of these aggregates on the proteasomal system, followed by transcription factor activation and loss of cell viability. Furthermore, findings on the mechanism of radical genesis, proteasomal inhibition and the required components of lipofuscin formation were resumed.

The proteasome is responsible for the degradation of polyubiquitinated proteins. Inhibition of the proteasome leads to an accumulation of polyubiquitinated proteins and thus to an impairment of the cellular protein homeostasis. To prevent cellular damage on proteasome inhibition there is an up-regulation of several heat shock proteins (Hsps), including Hsp27, Hsp70, and heme oxygenase-1 (HO-1). It was demonstrated that the induction of classical Hsps, such as Hsp27 and Hsp70, is dependent on a HDAC6-dependent mechanism which releases HSF-1 and induces the expression of newly synthesized Hsps. In this study we demonstrate that the up-regulation of HO-1 on proteasome inhibition is mediated by p38MAPK and Nrf-2. Interestingly we found additional evidence, proving the involvement of HDAC6 in the up-regulation of HO-1. By using RNAi technologies against HDAC6 we demonstrate that there is a lack of the expected induction of HO-1, Nrf-2, and phosphorylated p38 (pp38) after proteasome inhibition. Furthermore, we can show that p38 is acetylated in unstressed cells and is a good substrate for HDAC6-mediated deacetylation. Therefore, we propose that on proteasome inhibition HDAC6 deacetylates p38, allowing the subsequent phosphorylation of p38 and resultant activation of NRF-2. NRF-2 enters the nucleus and functions as a transcription factor for HO-1.

In the current literature, the lysosomal system is considered to be involved in the intracellular formation and accumulation of lipofuscin, a highly oxidized and covalently cross-linked aggregate of proteins that fills the lysosomal volume during aging. In contrast, our experimental results presented here suggest that both the autophagosomes and the lysosomal system are not mandatory for the formation of lipofuscin, since that material accumulates in the cytosolic volume if autophagy or lysosomal activity is inhibited. However, such an inhibition is accompanied by an enhanced toxicity of the formed protein aggregates. Furthermore, it could be proven that macroautophagy is responsible for the uptake of lipofuscin into the lysosomes.

Advanced glycation end product-modified proteins are known for accumulating during aging and in several pathological conditions such as diabetes, renal failure, and neurodegenerative disorders. There is little information about the intracellular fate of endocytosed advanced glycation end products (AGEs) and their influence on proteolytic systems. However, it is known that the lysosomal system is impaired during aging. Therefore, undegraded material may accumulate and play a considerable role in the development of diverse diseases. To investigate if AGEs can be degraded and to test whether they accumulate because of impaired lysosomal proteases we studied the effects of advanced glycation end products on the endosomal–lysosomal system. Five different types of AGEs were generated by bovine serum albumin incubation with glyoxal, methylglyoxal, glucose, fructose, and ribose. The first experiments revealed the uptake of AGEs by the macrophage cell line RAW 264.7. Further investigations demonstrated an increase in cathepsin D and L activity and an increase in mature cathepsins D and L. Increased activities were accompanied by the presence of more lysosomes, measured by staining with LysoTracker blue. To specify the roles of cathepsins D and L we used knockout cells to test the roles of both cathepsins on the toxicity of advanced glycation end products. In summary we conclude that both cathepsins are required for a reduction in advanced glycation end product-induced cytotoxicity.

Experts in the field of carotenoids met at the Hohenheim consensus conference in July 2009 to elucidate the current status of β-carotene research and to summarize the current knowledge with respect to the chemical properties, physiological function, and intake of β-carotene. The experts discussed 17 questions and reached an agreement formulated in a consensus answer in each case. These consensus answers are based on published valid data, which were carefully reviewed by the individual experts and are justified here by background statements. Ascertaining the impact of β-carotene on the total dietary intake of vitamin A is complicated, because the efficiency of conversion of β-carotene to retinol is not a single ratio and different conversion factors have been used in various surveys and following governmental recommendations within different countries. However, a role of β-carotene in fulfilling the recommended intake for vitamin A is apparent from a variety of studies. Thus, besides elucidating the various functions, distribution, and uptake of β-carotene, the consensus conference placed special emphasis on the provitamin A function of β-carotene and the role of β-carotene in the realization of the required/recommended total vitamin A intake in both developed and developing countries. There was consensus that β-carotene is a safe source of vitamin A and that the provitamin A function of β-carotene contributes to vitamin A intake.

Reactive oxygen species (ROS) are formed continuously in the organism even under physiological conditions. Proteins are prominent targets of oxidation reactions. Such oxidized proteins are either degraded or form protein aggregates. Such protein aggregates are accumulating during cellular aging and influence the metabolism in an aged cell. One of the potential adverse reactions of protein aggregates in aged cells is the aggregate-dependent formation of reactive oxygen species. Protein aggregates are able to incorporate metals, especially iron, which is redox active and can only be partially accessed by chelators. We were able to show that such protein aggregates are contributing substantially to the changes in the pro-oxidative status of aged cells. A special role in this formation of protein aggregate-dependent, metal-induced oxidative stress is played by iron containing proteins, which may be included into the protein aggregates. We could show a contribution of both, cytosolic/ferritin and mitochondrial iron sources. While research is often focusing on the pro-oxidant role of mitochondria in aged cells, other sources – as protein aggregates – of oxidants have to be taken into account.

Skeletal muscle alterations during aging lead to dysfunctional metabolism, correlating with frailty and early mortality. The loss of proteostasis is a hallmark of aging. Whether proteostasis loss plays a role in muscle aging remains elusive. To address this question we collected muscles, Soleus (SOL, type I) and Extensor digitorum longus (EDL, type II), from young (4 months) and old (25 months) C57BL/6 mice and evaluated the proteasomal system. Initial work showed decreased 26 S activity in old SOL. EDL displayed lower proteasomal activity in both ages compared to any of the SOL ages. Moreover, in order to understand if during aging there is the so-called “fiber switch from fast-to-slow”, we performed western blots against sMHC and fMHC (slow and fast myosin heavy chain, respectively). Preliminary results suggest that young SOL is composed by slow twitch fibers but also contains fast twitch fibers, while young EDL seems to be mostly composed by fast twitch fibers that level down during aging, suggesting the switch. As a conclusion, EDL seems to have less proteasomal activity, however, if this is a contributor or a consequence to the muscle fiber switch during aging still needs further investigation.

Glutamine synthetase (GS) is a key metabolic enzyme that catalyzes the ATP-dependent synthesis of glutamine from glutamate and ammonia. In the CNS, it is mainly located in the cytosol of astrocytes, playing an important role in detoxification of ammonia and excess glutamate. Alterations in GS activity and oxidative posttranslational modifications have been found in vivo. Herein, we studied the effect of oxidants on recombinant hGS function and structure with a combination of experimental and computational studies. Peroxynitrite caused a dose-dependent inactivation of hGS and 3-nitrotyrosine (NT) formation. In addition, higher molecular weight aggregates were formed likely due to 3,3´-dityrosine crosslinks. pH studies suggested that 3,3´-dityrosine formation had a larger impact on GS activity than nitration. MS analysis identified some critical residues modified, namely Tyr 185 and 269. H 2 O 2 -dependent thiol oxidation did not impact on GS activity. Exposure of human astrocytes to SIN-1 led to GS nitration and decreased enzyme activity. Molecular dynamic studies in GS (Issoglio et al. Biochemistry 2016) are being performed in nitrated forms of the enzyme to define with an atomic level of detail how tyrosine oxidative modifications affect GS structure and activity.

Aging is accompanied by an intracellular accumulation of lipofuscin, a hydrophobic yellow-brownish material that accumulates especially in the lysosomal compartment, where it can be neither degraded nor exocytosed from the cell. The intracellular effects of accumulating lipofuscin are still a subject of speculation. In addition to the demonstrated inhibition of the proteasome, it was proposed that lipofuscin is cytotoxic because of its ability to incorporate transition metals such as copper and iron, resulting in a redox-active surface, able to catalyze the Fenton reaction. This characteristic of lipofuscin may contribute to an increased level of radical formation and oxidatively modified cellular components such as proteins, lipids, and RNA/DNA, which has been shown to be extensive in aging cells. In this study for the first time the lipofuscin-mediated formation of oxidants and the role of iron in this process are directly shown in a model of senescent fibroblasts, as well as in vitro with artificial lipofuscin. We demonstrate that this oxidant production is independent of mitochondria and has cytotoxic effects. The ability of lipofuscin to produce oxidants is dependent on the amount of transition metals incorporated. Although the amount of oxidants formed by cellular lipofuscin turned out to be moderate, it is chronic and thus lipofuscin is able to catalyze its own formation.

Oxidative stress is an inevitable process in the nucleus, especially in antitumor chemotherapy, and adaptation by defense mechanisms seems to be one element in the development of long-term resistance to many chemotherapeutic drugs. In this study, a potential chromatin repair mechanism during oxidative stress was investigated in HT22 cells. The 20S proteasome has been shown to be largely responsible for the degradation of oxidatively modified histone proteins in the nucleus. Poly(ADP-ribosyl)ation reactions also play an important role in DNA repair as a consequence of oxidative damage and single-strand breaks. Such a reaction may occur also with the 20S proteasome-with a known increase in enzymatic activity-and also with histones-reducing their proteolytic susceptibility as shown for the first time here. After hydrogen peroxide treatment of HT22 cells, degradation of the model peptide substrate suc-LLVY-MCA and degradation of oxidized histones by nuclear proteasome increased. During the removal of protein carbonyls, single-strand breaks and 8-hydroxy-2'-deoxyguanosine, proteasome, and poly(ADP-ribose) polymerase-1 enzymes were shown to play tightly interacting roles. Our results following the repair of oxidative damage show the proteolytic activation of proteasome concerning poly(ADP-ribosyl)ation together with a decline in poly(ADP-ribosyl)ation of oxidized histones, leading to a selective recognition of oxidatively modified histones. (C) 2009 Elsevier Inc. All rights reserved.

Infrared radiation is a substantial part of the solar energy output reaching the earth surface. Therefore, exposure of humans to infrared radiation is common. However, whether and how infrared (IR) or infrared A acts on human skin cells is still under debate. Recently the generation of reactive oxygen species by water-filtered infrared A (wIRA) irradiation was postulated. wIRA shows a spectral distribution similar to that of solar irradiation at the earth's surface. Thus, the need for protection of human skin from both solar- and artificially generated infrared A irradiation was concluded. Here we demonstrate that in human dermal fibroblasts this reactive oxygen species generation is dependent on heat formation by infrared A and can be reproduced by thermal exposure. On the other hand wIRA irradiation had no detectable effect if the temperature in the cells was kept constant, even if irradiance exceeded the extraterrestrial solar irradiance in the IR range by a factor of about 4 and the maximum at noontime in the tropics by a factor up to about 6. This could be demonstrated by the measurement of oxidant formation using H(2)DCFDA and the determination of protein carbonyls. In additional experiments we could show that during thermal exposure the mitochondria contribute significantly to oxidant production. Further experiments revealed that the major absorbance of infrared is due to absorption of the energy by cellular water.

Several studies have demonstrated that proteasome activity decreases whereas protein oxidation increases with aging in various tissues. However, no studies are available correlating both parameters directly comparing different tissues of one organism. Therefore, we determined whether there is an age-related change in proteasome activity and protein oxidation in heart, lung, liver, kidney and skeletal muscle samples of 6-, 10-, 18- and 26-month-old rats. There was a significant age-related increase in protein carbonyls at 18 and 26 months compared to young rats. Thereby, protein carbonyl formation was rather due to a general than a specific protein carbonylation as shown by immunblot studies. The highest increase in protein carbonyl formation was found in liver, lung and kidney samples. Proteasome activity decreased significantly with age in lung and liver samples. Proteasome activity in liver and lung decreased by factor five compared to young rats. Strong correlations between proteasome activity and protein oxidation were found in liver and lung, whereas in other tissues only a trend was found. These results demonstrate that the increase in protein oxidation and the decline in proteasome activity are correlating. Further studies are needed to determine the mechanisms which cause organ-specific aging-rates and their consequences.

Cellular reactions to oxidative stress always include a response in the protein turnover. Therefore, cellular handling of proteins is important to observe. In this method review, radioactive labeling of proteins in vitro and in intact cells is described. The use of techniques based on the radioactive quantification of amino acids is much more selective and reliable than other nonradioactive methods for studying the protein turnover of both long- and short-lived proteins. Variations of such measurements allow one to measure protein synthesis, protein degradation, formation of insoluble proteins, and, perhaps, the turnover of individual proteins.

Oxidized proteins are recognized and degraded preferentially by the proteasome. This is true for numerous proteins including calmodulin (CaM). The degradation of CaM was investigated in a human fibroblast cell line under conditions of oxidative stress. Low molecular CaM fragments or peptides were found under such conditions. In in vitro experiments it was investigated whether this CaM breakdown product formation is induced by protein oxidation or is due to a limited proteolysis-derived degradation by the 20S proteasome. Native unoxidized CaM was not degraded by 20S proteasome, oxidized CaM was degraded in a time- and H2O2 concentration-dependent manner. Peptides of similar molecular weight were detected in isolated calmodulin as in oxidatively stressed fibroblasts. The peptides were identified using isolated calmodulin. Therefore, in oxidatively stressed fibroblasts and in vitro CaM is forming oxidation-driven fragments and proteasomal cleavage peptides of approximately 30 amino acids which undergo a slow or no degradation.