Pathophysiological importance of aggregated damaged proteins

Free Radical Biology and Medicine 71 (2014) 70–89

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Review Article

Pathophysiological importance of aggregated damaged proteins Annika Höhn, Tobias Jung, Tilman Grune n Department of Nutritional Toxicology, Institute of Nutrition, Friedrich-Schiller-University Jena, 07743 Jena, Germany

art ic l e i nf o

a b s t r a c t

Article history: Received 2 January 2014 Received in revised form 28 February 2014 Accepted 28 February 2014 Available online 12 March 2014

Reactive oxygen species (ROS) are formed continuously in the organism even under physiological conditions. If the level of ROS in cells exceeds the cellular defense capacity, components such as RNA/DNA, lipids, and proteins are damaged and modified, thus affecting the functionality of organelles as well. Proteins are especially prominent targets of various modifications such as oxidation, glycation, or conjugation with products of lipid peroxidation, leading to the alteration of their biological function, nonspecific interactions, and the production of high-molecular-weight protein aggregates. To ensure the maintenance of cellular functions, two proteolytic systems are responsible for the removal of oxidized and modified proteins, especially the proteasome and organelles, mainly the autophagy–lysosomal systems. Furthermore, increased protein oxidation and oxidation-dependent impairment of proteolytic systems lead to an accumulation of oxidized proteins and finally to the formation of nondegradable protein aggregates. Accordingly, the cellular homeostasis cannot be maintained and the cellular metabolism is negatively affected. Here we address the current knowledge of protein aggregation during oxidative stress, aging, and disease. & 2014 Elsevier Inc. All rights reserved.

Keywords: Protein oxidation Protein aggregates Lipofuscin Proteasome Autophagy Free radicals

Contents Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protein oxidation and formation of aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidation-driven protein aggregation and lipofuscin accumulation during aging . . . . . . . . Glycoxidation of proteins and effects of cross-linked AGE–protein aggregates . . . . . . . . . . The proteasomal system and its ability to degrade oxidized, but not aggregated, proteins Autophagic uptake mechanisms and lysosomal degradation of aggregated proteins . . . . . Relevance of proteolytic systems and aggregate formation in several pathologies . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Introduction Abbreviations: Aβ, β-amyloid; AGE, advanced glycation end product (here: proteins modified by AGEs); Atg, autophagy-related gene; CMA, chaperonemediated-autophagy; 3-DG, 3-deoxyglucosone; FIP200, focal adhesion kinase family-interacting protein of 200 kDa; IP3, inositol trisphosphate; JAK, Janus kinase; LC3, microtubule-associated protein light-chain-3; NF-κB, nuclear factor “κ-light-chain enhancer” of activated B cells; PA28, proteasome activator 28 kDa; PA700, proteasome activator 700 kDa; PE, phosphatidylethanolamine; PHF, paired helical filament; polyQ, polyglutamine; RAGE, receptor for advanced glycation end products; ROS, reactive oxygen species; SOD, superoxide dismutase; STAT, signal transducer and activator of transcription; ULK1/2, UNC-51-like kinase 1/2; UPS, ubiquitin–proteasome system; UVRAG, ultraviolet irradiation resistance-associated gene n Corresponding author. Fax: þ49 3641949672. http://dx.doi.org/10.1016/j.freeradbiomed.2014.02.028 0891-5849/& 2014 Elsevier Inc. All rights reserved.

Free radicals are defined as molecules or atoms containing one or more unpaired electrons in an atomic orbital. The presence of an unpaired electron results in common properties shared by radical species. Several radicals are unstable and highly reactive. They can either donate or accept an electron from other molecules, therefore serving as oxidants or reductants. Important oxygencontaining free radicals derived by the partial reduction of oxygen are the superoxide anion radical (Od2
) and the hydroxyl radical (HOd). In addition to radicals, reactive oxygen species (ROS)1 also include a number of nonradical molecular species such as singlet oxygen (1O2) and hydrogen peroxide (H2O2). In biological systems,

A. Höhn et al. / Free Radical Biology and Medicine 71 (2014) 70–89

ROS are often derived from superoxide, which is generated by adding a single electron to molecular oxygen and is only moderately reactive itself. Superoxide can rapidly be converted into hydrogen peroxide, ultimately yielding the highly reactive hydroxyl radical in the presence of reduced iron (Fe2 þ ) or copper (Cu þ ) via the Fenton reaction. In the aerobic metabolism the generation of ROS is an inevitable event of metabolic and energy-transferring processes. ROS are derived either from exogenous sources such as exposure to X-rays, environmental pollutants, ozone, cigarette smoke, drugs/pharmaceuticals, alcohol, pesticides, and industrial solvents, or from essential endogenous metabolic processes (Fig. 1). ROS formation occurs continuously in the cells as a consequence of both enzymatic and nonenzymatic reactions. A number of ROS sources are known in mammalian cells, including the mitochondria (mainly the respiratory chain, but also monoamino oxidase, α-ketoglutarate dehydrogenase, and glycerol phosphate dehydrogenase [1]) and the endoplasmic reticulum (mainly cytochrome P-450 enzymes, endoplasmic reticulum (ER) oxidoreductin 1, and diamine oxidase [2]). In peroxisomes H2O2 is a byproduct of fatty acid β-oxidation [3] and also the synthesis of fatty acids can produce Od2
via acyl-CoA oxidase-catalyzed electron transfer to oxygen [4]. Cytochrome P-450 enzymes, whose function is to dispose of foreign substances, are also able to reduce molecular oxygen to superoxide. Furthermore, H2O2 can be produced by the oxidation of lysine side chains by the enzyme lysyl oxidase, which catalyzes the cross-linking of collagen and elastin fibers in the extracellular matrix [5]. It should be kept in mind that ROS are both physiological intermediates and mediators of oxidative stress. At low levels, ROS participate in several physiological mechanisms such as cellular signaling, gene expression, and pathogen elimination [6]. However, at high levels ROS can cause irreversible and detrimental cellular damage. Yet, cells are not defenseless and complex antioxidant protection mechanisms have evolved to protect cells from ROS, including enzymatic and nonenzymatic systems. Antioxidant enzymes include superoxide dismutases (SODs), catalase, glutathione peroxidases, and peroxiredoxins. But although very important to regulate the levels of ROS in cells, antioxidant compounds and enzymes are not fully effective in preventing oxidative stress and damage. Closely related to ROS are the reactive nitrogen species (RNS). RNS are various nitric oxide (dNO)-derived compounds such as nitroxyl anion (NO
) and nitrosonium cation (NO þ ), higher oxides of nitrogen, S-nitrosothiols, and dinitrosyl iron complexes.

Endoplasmic Reticulum (cytochrome P-450, b5 enzymes, diamine oxidase) Plasma membrane (NADPH oxidases, myeloperoxidase) Cytosol (NOsynthases, lipoxygenases, PGH synthase) Peroxisomes (fatty acid oxidation, Damino acid oxidase, urate oxidase, L-2hydroxyacid oxidase)

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RNS also play an important role in redox biology by acting as important intracellular signaling molecules but are in the same way able to cause cellular damage. dNO per se is not very reactive, but the damaging effect is due to its reaction with Od2
leading to the formation of the highly reactive peroxynitrite (ONOO
). This product is a strong oxidant with a formation rate that has been determined to be three times faster than the scavenging of Od2
by SOD [7]. Oxidative/nitrosative stress represents an imbalance between the formation of reactive oxygen and nitrogen species and their elimination by various reducing or antioxidant systems that destroy reactive intermediates and prevent or repair the resulting damage. In the following the general summarizing terms “ROS” (including ROS and RNS) and “oxidative stress” will be used. The term “oxidative stress” itself was first used by Helmut Sies who described it as “an imbalance between oxidants and antioxidants in favor of the oxidants, potentially leading to damage” [8]; in 2006 this definition was extended to include the “disruption of redox signaling” [9]. Oxidative stress results in direct or indirect ROSmediated cellular damage and has been implicated in carcinogenesis [10], neurodegenerative diseases [11], diabetes [12], atherosclerosis, and aging [10,13]. Targets of ROS include all kinds of molecules in the body; therefore, oxidative stress causes irreversible oxidative damage to DNA bases, lipids, and proteins, interfering with vital cellular functions. Because proteins are most abundant in cells they are major targets for oxidative stress and modifications in cells.

Protein oxidation and formation of aggregates Critical factors determining the degree of protein oxidation are the location of the protein, the protein structure, as well as location and concentration of oxidants and antioxidants. Oxidative protein damage in vivo can impair the functioning of receptors, transport proteins, and enzymes, with effects on various cellular downstream processes. Furthermore, oxidized proteins may be identified as foreign by the immune system triggering antibody formation and possibly autoimmunity [14]. Proteins with modified moieties are able to diffuse or be transported to other cellular parts potentiating the damaging effect [15]. Moreover, secondary damage of other biomolecules can result from protein oxidation as well, e.g., DNA damage as a result of oxidative damage of DNA repair enzymes and histones.

Mitochondria (complex I & III, monoamino oxidase, αketoglutarat dehydrogenase, glycerol phosphate dehydrogenase)

Native Protein

Reactive oxygen species

Environment (UVA/B, pollution, radioactive decay,)

Protein modifications/ oxidized proteins

Fig. 1. Endogenous and exogenous sources of ROS and protein oxidation, according to [1,2,293–296].

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ROS are described to be able to attack and modify amino acids, peptides, and proteins in different ways (Fig. 2), and reactions of radicals include hydrogen atom abstraction, substitution (elimination or addition of substrates), rearrangement, fragmentation, dimerization, and disproportionation. Reactions of HOd with proteins are able to give rise to carbon-centered radicals by hydrogen atom abstraction from the protein backbone. These radicals react with molecular oxygen and form peroxyl radicals, which can be altered into alkyl peroxide by Od2
. Alternative reactions of carbon-centered radicals induce cross-linked proteins. Because no bond breaking is required, addition reactions are faster than hydrogen atom abstraction and have been reported for amino acids with aromatic rings and sulfur atoms [16]. In general, selectivity and rates of radical attack are well characterized in amino acids, but less clear in peptides and proteins. The susceptibility to oxidative modifications differs between amino acids and some of them, such as histidine, cysteine, methionine, and leucine, as well as the aromatic amino acids tryptophan, tyrosine, and phenylalanine, are more prone to these processes compared to others. Oxidation of leucine results in the formation of several hydroxyleucines [17]. Histidine oxidation leads to the formation of 2-oxohistidine, aspartate, and asparagine, whereas tryptophan oxidation forms N-formylkynurenine, kynurenine, hydroxytryptophan, and further downstream products. Reaction of tyrosine with various oxidants gives rise to tyrosine phenoxyl radical. One possible fate of this tyrosyl radical is dimerization and the formation of dityrosine [18]. This can lead to intra- or intermolecular tyrosine cross-links in proteins [19]. Furthermore, tyrosine oxidation induces several products such as 3,4-dihydroxyphenylalanine, 3-chlorotyrosine owing to reactions with chlorinating species, and 3-nitrotyrosine, which is induced by nitrating species [18]. Phenylalanine is also susceptible to modifications resulting in ortho- and meta-tyrosine and nitrophenylalanine. Methionine residues are also easily oxidized by a large number of species owing to their low redox potential, because the sulfur on the side chain is predisposed for oxidative damage. Methionine can undergo a one-electron oxidation to methionine radical cations or a two-electron oxidation process leading to the

formation of both S- and R-diastereoisomers of methionine sulfoxide, which can be further oxidized to methionine sulfones. The sulfoxide S-isomer is often referred to as MetA and the R-isomer as MetB [20]. It should be noted that the two-electron oxidation to methionine sulfoxides is reversible and that both stereoisomers can be reduced by the action of stereospecific methionine sulfoxide reductases (MSR-A and MSR-B). Both enzymes use thioredoxin (Trx) as a source of reducing equivalents. In the presence of NADPH, the oxidized form of Trx can be converted back to its reduced form by the enzyme Trx reductase. Therefore, the reversible methionine oxidation to sulfoxides can act as a drain for ROS because of its reversibility and further play a role in redox signaling events [21]. Mutations leading to a decrease in MSR activity are associated with a decrease in oxidative stress resistance and life-span reduction, whereas mutations leading to an overproduction of MSR activity result in oxidative stress resistance and life-span extension [22]. It is already known that the level of MSR-A declines with age [23] and in several age-related diseases such as Alzheimer disease [24] and Parkinson disease [25]. In contrast to the two-electron oxidation, the sulfur-centered radical cations arising by one-electron oxidation of methionine are highly unstable and can further complex with oxygen, nitrogen, or other sulfur atoms and react to a series of intermediates and products, of which several represent irreversible posttranslational modifications [26]. Reactions of methionine radical cations are mainly irreversible and ultimately yield carbon-centered and/ or peroxyl radicals representing starting points for chain reactions of protein oxidation. Oxidation of the cysteine thiol group is facile, with a wide range of oxidants leading to thiyl radicals (RSd), which can dimerize to give disulfide (RSSR) or mixed dimers by reaction with numerous other molecules. Reaction with O2 results in peroxyl radicals [27]. Intra- and intermolecular disulfide bonds can be reversed by glutaredoxin-1, the thioredoxin/thioredoxin reductase system, or protein disulfide isomerase, an oxidoreductase of the thioredoxin superfamily [18]. As expected, the concentration of protein –SH groups decreases with age as a consequence of increased levels of mixed disulfides with cysteine and homocysteine [28]. This

Reactive oxygen and nitrogen species Irreversible oxidative modifications

Reversible oxidative modifications

Oxidative modificatione of certain amino acids: • • • • • •

Tryptophan: N-formylkynurenine, kynurenine Histidine: aspartat, asparagine, 2-oxohistidine Phenylalanine: Ortho-/metatyrosine Tyrosine: DOPA, dityrosine, 3-chlorotyrosine, 3-nitrotyrosine Methionine: methionine sulfoxide (reversible) Cysteine: sulfenic (reversible), sulfinic, sulfonic acids, disulfides (reversible) • Proline, arginine, threonine, lysine: protein carbonyls General oxidative protein modifications: • • • • •

Protein carbonyl formation (many amino acids) Glycoxidation HNE-/MDA-adducts Fragmentation Intra-/intermolecular cross-linking (covalent and non-covalent)

Fig. 2. Reversible and irreversible oxidative modifications of proteins and several amino acids.

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age-associated loss has been reported in several tissues and species [29]. Functional consequences of –SH loss include protein misfolding, catalytic inactivation, and decreased antioxidative capacity [29]. Another commonly measured modification of particular significance is the oxidation of proline, arginine, threonine, and lysine residues leading to the formation of carbonyl derivatives [19]. In addition, carbonyl groups can be introduced by secondary reactions with bifunctional aldehydes, such as 4-hydroxy-2nonenal (HNE) or malondialdehyde; both are products of lipid peroxidation [30]. The formation of protein-bound carbonyl groups is more abundant compared to other oxidative protein modifications; therefore, the determination of protein carbonyls has become a widespread method for detection of oxidative protein modifications. Furthermore, carbonyl formation can alter protein conformation. As a result, this can increase protein hydrophobicity, enhancing nonspecific protein–protein interactions and aggregate formation [29,31–33], which compromise cell viability and impair protein turnover [29,33]. In neurological diseases such as Alzheimer and Parkinson disease, aggregates are generally composed of a single protein. In oxidative stress-induced aggregation a multitude of different proteins are involved. In general, if the balance between the generation of oxidatively modified proteins and their repair and/or removal by protein repair and degradation systems is shifted, the final result is accumulation and aggregation of these proteins. Oxidized, misfolded, and unfolded proteins that do not interact with one another under normal conditions tend to form oligomeric complexes driven by covalent and reversible noncovalent (hydrophobic and electrostatic) interactions [34], resulting finally in protein aggregates. Covalent interactions can also be reversible, e.g., the formation of –S–S–, or irreversible, when 2,20 -biphenyl cross-links are formed [32]. These aggregates are independent of the physiological structure of the protein and introduce a toxic factor into the cellular metabolism. It was shown that almost 30% of newly synthesized proteins are misfolded [35]. Under conditions that favor further unfolding, such as oxidative stress, this amount increases. Other reasons—such as mutation of protein-coding genes—also contribute to protein aggregation. Such inherited mutations are also responsible for the Slightly oxidized and unfolded protein; no activity and a substrate for the 20S proteasome

familiar forms of Alzheimer disease, Huntington disease, and Parkinson disease [36–38]. Mutations of members of the heat shock protein family can also disturb the maintenance of the protein pool and trigger aggregation. Molecular chaperones, including the heat shock proteins (Hsp’s), are ubiquitous in cells and are induced during various stresses. Hsp’s are a conserved family of protective proteins, specifically induced in response to environmental stress such as heat shock, oxidative stress, or inflammation [39]. Hsp70 plays a major role in cytoprotection by preventing abnormal folding of newly synthesized proteins and was initially discovered as a protein with increased expression under conditions of stress [40]. However, the main reasons for aggregate formation remain exposure to heat stress, UV irradiation, and, as mentioned above, especially oxidative stress. Because of the complexity of molecular interactions the process of protein aggregation takes place slowly and is not the result of one single exposure to stressors but the result of chronic exposure, sometimes over years [41]. Aggregation might require several steps (Fig. 3): exposure of a native protein in its active form to increasing amounts of oxidants leads to oxidation of more and more amino acid side chains. First, a slight modification such as alteration of –SH groups occurs. These moderately oxidized proteins might not be necessarily inactive depending on the number and location of modified amino acids. As mentioned above, in the case of the sulfur-containing amino acids methionine and cysteine, the emerging modifications can be repaired [42]. Further oxidation is accompanied by increased protein unfolding and hydrophobic amino acids—normally buried in the core of the protein—are exposed to the surface. This unfolded structure of the protein is accompanied by a loss of activity or functionality. In addition, the increased surface hydrophobicity of oxidized proteins is a key factor in their proteolytic recognition by the proteasome [32]. According to the unfolding status, there are different alternatives for the fate of those proteins. These proteins are either degraded by proteolytic systems, mainly the lysosomal and the proteasomal system, or rescued and refolded. Proteolysis has several advantages for the cell, such as reduction of the risk of protein accumulation and aggregation by an efficient removal of oxidized proteins; further, the released intact amino acids can be

Severely oxidized and unfolded protein; susceptible for aggregate formation with other proteins, often hydrophobic surface patterns

Aggregate of proteins, but still (partially?) degradable by proteolytic enzymes; further oxidation can result in covalently cross-linked aggregates, resistant to proteolysis (lipofuscin)

Oxidative stress

Oxidative stress

Oxidative stress

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Native (globular) protein, active and functional

20S proteasome; degrades oxidized proteins preferentially, but is inhibited by lipofuscin

Lipofuscin accumulates in the lysosomes, but can probably be released by lysosomal rupture

Fig. 3. Step-wise protein oxidation and formation of lipofuscin. Lipofuscin formation starts with the ongoing oxidation of proteins. If damage or repair systems fail, oxidized proteins cross-link, become functionally impaired, and change their structure, finally leading to lipofuscin formation. Lipofuscin accumulates mainly in the lysosomal system, but eventually lysosomes are overloaded and might rupture. Lipofuscin is then released into the cytosol and able to inhibit the proteasome.

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recycled and used for biosynthesis of new proteins. But from an energetic point of view, biosynthesis is highly energy consumptive and repair of proteins should be favored [43]. So, the expression of Hsp70 and Hsp90 is induced upon environmental stress [44]. Hsp70 has a broad substrate range including denatured and newly synthesized proteins and is in general responsible for the refolding process or for shuttling nonrefoldable proteins to protein degradation systems. The main function of Hsp90 is also stabilizing and protecting from aggregation but has a more limited substrate range. The complex of both Hsp’s is responsible for quality control of mature proteins. They are able to interact with unfolded/ misfolded proteins and assist in refolding or degradation after binding of exposed hydrophobic regions of those proteins. Furthermore, small Hsp’s, heat-inducible chaperone-like proteins with a molecular mass between 15 and 40 kDa, are also proposed to prevent insolubilization and irreversible protein aggregation under stress conditions [45–47]. It is not fully understood if this refolding scenario by Hsp’s is also possible for oxidized proteins, because covalent modifications might prevent refolding, but for reversible oxidative modifications refolding is conceivable after repair. Nevertheless, if these oxidative modified proteins are not degraded or rescued, aggregates are formed owing to interactions of the exposed hydrophobic structures for the purpose of minimizing the contact area to the hydrophilic environment. Secondary reactions might also take place, such as ubiquitinylation, AGE formation, and cross-linking, owing to reactions between several radicals of amino acid side chains. Over time more and more proteins tend to bind to the aggregate surface leading to an increase in aggregate size [48]. The occurrence of protein aggregates triggers a multitude of intracellular reactions, including the ability of aggregates to promote cell death [49,50]; furthermore, accumulation and aggregation of oxidized proteins are a key factor in the aging process.

Oxidation-driven protein aggregation and lipofuscin accumulation during aging Aging is a complex process controlled somewhat by genetic factors but is largely influenced by various environmental parameters. The role of several genes involved in stress response (including oxidative stress), and investigations indicating that the overexpression of such genes leads to longevity, combined with observations that increasing concentrations of damaged cellular components are associated with aging, show the importance of maintenance systems in the aging process [51]. Oxidatively modified proteins have been shown to increase during aging, and aggregation of these oxidized proteins mainly appears during the aging process, as measured by the cellular levels of protein carbonyls and dityrosine or by the accumulation of lipofuscin [32,52]. The accumulation of damage is currently assumed to be responsible for the ubiquitous progressive decline in the functional capacity of aging cells and organisms. Although different theories have been proposed to explain the aging process, it is generally agreed that there is a correlation between aging and the accumulation of oxidatively damaged lipids, nucleic acids, and especially proteins. It is believed that the abovementioned aggregated proteins undergo further reactions with cellular metabolites, including aldehydic lipid peroxidation products [53], finally leading to a fluorescent pigment referred to as “lipofuscin” (Fig. 3) [54–58], “age fluorophore,” or “age pigment“ [59,60]. Lipofuscin is accepted to be a highly oxidized material from covalently cross-linked proteins [53,61] and lipids [62,63]. From the fourth decade of life (oligo-)saccharides are also found in this material in human cells [64]. Lipofuscin is one of the most

important factors limiting the life span of a cell and it accumulates intracellularly in a time-dependent manner and is inversely correlated with the remaining lifetime of a cell and thus also of the corresponding organism. The presence of this material has been detected in many different tissues, including heart, liver, kidney, and skin, but it plays a decisive role in postmitotic tissue aging of, e.g., nerve and muscle cells. A steady increase in lipofuscin accumulation is known as an inevitable hallmark of aging, but also some neurodegenerative diseases are linked to increased levels of (specific) protein aggregates. A tissue-specific difference in lipofuscin levels was found in rats, in that old rats showed an increased lipofuscin amount in heart, liver, cerebellum, skeletal muscle, and testis compared to young rats [65,66]. Accumulation of intestinal lipofuscin (and AGEs) has also been used as an effective marker for Caenorhabditis elegans health span [67]. SAMP8 mice (senescence-accelerated mouse prone 8) display early cochlear degeneration accompanied by hearing loss shown to be related to lipofuscin accumulation in spiral ganglion neurons [68]. Lipofuscin, as the final aggregation product, is resistant to degradation by cellular proteolytic systems [69,70], is not exocytosed [71–73], and mainly accumulates in the lysosomal compartment. The currently most widespread hypothesis of lipofuscin formation is the "mitochondrial–lysosomal axis theory of aging" established by Ulf Brunk and Alexei Terman [74]. This theory is supported by the fact that lipofuscin comprises about 30–60% protein, about half of which might be provided by subunit C of the mitochondrial ATP synthase [75]. Moreover, lipofuscin is able to incorporate various transition metals, such as iron, copper, zinc, manganese, and calcium, up to 2 mass% [76]. According to one hypothesis of Brunk and Terman, these transition metals may provide a redox-active surface, able to reduce hydrogen peroxide via the Fenton reaction into the highly reactive hydroxyl radical, which is one of the aspects of its cytotoxic effects [77] and an important factor in further oxidation reactions of the initial aggregate [49]. It was shown in vitro that lipofuscin is able to incorporate and retain redox-active transition metals and this iron-loaded form shows a massively increased radical formation compared to the non-iron-loaded form (Fig. 4A) [49]. Most probably iron incorporation is one of the key factors in lipofuscinogenesis and it was shown that lipofuscin formation is significantly increased by addition of a hydrated iron phosphate complex to the medium of cultured cells, leading to endocytosis and enrichment of low-molecular-weight redox-active iron in the lysosomal system. In contrast, the formation of lipofuscin is significantly restricted by application of the potent iron chelator desferrioxamine [54]. Because of its iron-binding ability and the fact that lipofuscin is a fluorochrome and photosensitizer, this material seems to be able to sensitize lysosomes to blue light, a process that might be important for the development of macular degeneration in aged individuals [54,55]. A2E is one identified fluorophore of lipofuscin that accumulates in retinal cells not only during normal aging, but also in age-related macular degeneration. However, one recently published article by Ablonczy et al. [78] was able to show that there is no correlation between the A2E and the lipofuscin topographies in human retina (peripheral lipofuscin vs macular A2E) and might lead to a reevaluation of several years of doctrine [79]. Another property of lipofuscin is the inhibition of protein degradation by competitively binding the proteasome (Fig. 4B) [80]. As mentioned above, oxidatively modified proteins are normally repaired or degraded by the proteasome or the lysosomal system and in this process replaced by de novo synthesized proteins. The most important process to remove oxidized proteins is degradation by the proteasome (see below). If oxidative damage overstresses the proteasomal ability to degrade substrates,

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Uptake of artificial lipofuscin Caspase-3 activity Apoptosis Cell viability Oxidant formation

75

Intracellular lipofuscin formation Inhibition of macroautophagy via ATG5 knockout

Atg5 Atg12

Fe2+ Fe2+

Fe2+

Degradation with protease K

Proteasome activity Apoptosis, Cell viability

Proteasome activity Apoptosis Cell viability

Atg5 Atg12 Atg16

Increasing cytosolic protein aggregates

Increased cytosolic lipofuscin

Cellular viability Reactive oxygen species

Fig. 4. Consequences of lipofuscin accumulation. One aspect of the lipofuscin cytotoxicity is its ability to incorporate redox-active transition metals, able to catalyze the Fenton reaction. (A) By using artificial lipofuscin-like oxidized, cross-linked protein aggregates (artificial lipofuscin), it was shown that this material is able to promote the formation of free radicals and initiate apoptotic cell death, resulting in a significant loss of cellular viability [49]. (B) Furthermore, protein aggregates are able to inhibit the degradation of oxidized proteins by competitively binding to the proteasome. The proposed mechanism for proteasomal inhibition is binding to exposed peptide structures on the surface of the highly oxidized and covalently cross-linked aggregate in ineffective attempts of degradation. Cellular viability is also affected and significantly reduced [48]. (C) Removal of these exposed peptide structures by using protease K significantly reduces the proteasomal binding sites and the inhibition of the proteasome can be partially reversed. Consequently less proteasomal capacity is detracted in futile attempts of degradation, and cellular viability is largely unaffected compared to the nondegraded material [48]. (D) The Atg12–Atg5 conjugation system is mandatory for the formation of a autophagophore and the uptake of aggregated proteins (and other substrates) into the lysosomal system. Knockdown of Atg5 prevents this uptake. The consequence is an increasing amount of cytosolic protein aggregates and finally cytosolic lipofuscin accumulation combined with a decline in cellular viability and an increased amount of ROS production [85].

oxidatively modified and damaged proteins accumulate within the cell. Furthermore, aggregates act as proteasomal inhibitors and subsequently enhance their own formation by slowing down the degradation of damaged proteins further [48,49,80]. The mechanism of proteasomal inhibition remained dubious for a long time. In 1993, it was proposed that hydrophobic structures might be a possible recognition motif of the proteasome toward the substrates [81]. This was later supported by the fact that proteasomal degradation correlates with the unfolding of RNase A [82]. A possible mechanism for proteasomal inhibition by lipofuscin/ aggregates was suggested: 20S proteasome binds to exposed hydrophobic lipofuscin surface structures yet is unable to degrade these structures completely because of steric/mechanic inhibition. Thus proteolytic capacity is detracted from other potential substrates. Interestingly, the inhibition of the proteasome was partially reversed by removal of potential binding sites for the proteasome using protease K (Fig. 4C) [48]. This effect was shown for isolated as well as intracellular proteasomes. Further, there has also been some speculation about dysfunction of lysosomal proteases due to the accumulation of lipofuscin in the lysosomal system. Macroautophagy is the discussed uptake mechanism for aggregates [83], such as lipofuscin, leading to the introduction of the term “aggrephagy” in 2007 [84]. However, it was still unclear whether the majority of cross-linking steps in the formation of lipofuscin take place in the cytosol or after uptake of lipofuscin precursors via macroautophagy into the lysosomal system. Only recently it was shown that lipofuscin can also be formed under exposure to chronic oxidative stress even when autophagy and, therefore, the uptake of aggregates into the lysosomal system is blocked (Fig. 4D). Elevated levels of cytosolic or rather extralysosomal lipofuscin were detected, accompanied by increased ROS formation and a decrease in cellular viability [85]. Thus,

macroautophagy seems to play a protective role, yet lysosomes are not required components for the formation of lipofuscin or the necessary initial cross-linking steps. Lipofuscin was found to accumulate in a much more rapid process in postmitotic cells of short-lived species compared to long-lived species [86]. Quite recently it was shown that dietary lipofuscin intake by young Drosophila flies results in reduced locomotor performance and accelerated rates of modified and carbonylated protein accumulation in somatic tissues and hemolymph, as well as in a significant reduction of both health and life span, contributing to an aging phenotype in flies [87]. Similar results were found by feeding Drosophila flies with AGEs [87]. Because there is no specific antibody available for lipofuscin, its autofluorescence has become the most important detection instrument by flow cytometry, fluorescence microscopy, or spectroscopy. Furthermore, methods such as Sudan dye black, Fontana–Masson, hematoxylin, Ziehl–Neelson, eosin, or osmium acid staining can be used [88,89]. Because detection of lipofuscin is fast and easy to apply, its formation is a good biomarker correlating with aging.

Glycoxidation of proteins and effects of cross-linked AGE–protein aggregates Glucose, as the most abundant of the metabolic sugars in humans, has also been implicated in the process of several pathologies and is closely correlated with oxidative stress by its ability to react nonenzymatically with proteins. Louis Camille Maillard incubated glucose with amino acids and discovered first in 1912 that after a certain time yellowish-brown pigments are formed [90]. These pigments result from a nonenzymatic glycation

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dehydrogenation reactions. The resulting intermediates are also highly reactive. A multitude of heterocyclic compounds, which are known as precursors of melanoidins, are formed by additional bonds and cyclization steps. In the third, final, phase the products of the continuative phase form high-molecular-weight melanoidins by polymerization and cross-linking steps. These melanoidins are called “advanced glycation end products”. In addition to the Maillard reaction other pathways may contribute to the formation of AGEs. For example, the autoxidation of glucose and lipid peroxidation lead to reactive dicarbonyl compounds and finally to the formation of AGEs under terms of oxidative stress [96]. Another well-studied mechanism for the formation of AGEs is the polyol pathway. Here, glucose is converted enzymatically to sorbitol and subsequently to fructose. Metabolites of fructose form α-oxoaldehydes, which react with amino acid side chains to AGEs [97]. In summary, there are at least four different pathways leading to the formation of AGEs: the Maillard reaction, the oxidation of glucose, lipid peroxidation, and the polyol pathway (Fig. 5). In view of these different complex pathways, it is not surprising that the AGEs have enormous structural differences. Furthermore, AGE formation can be divided into nonoxidative and oxidative pathways. If oxidation accompanies glycation, the products are also known as glycoxidation products. A glycoxidative pathway can be accelerated in the presence of transition metals. In this case, the unstable imine reacts in the presence of oxygen to reactive oxoaldehydes [98]. AGEs can form cross-links between proteins and show autofluorescent properties due to their structure, for example, pentosidine [99]. On the other hand, carboxymethyllysine (CML) and pyrraline are nonfluorescent AGEs that do not form cross-links [100]. Some prominent AGEs are shown in [101]. Because of the identification and quantification of AGEs in several foods, the question arises to what extent AGEs of the daily diet are absorbed and metabolized in the body. Clinical relevance is given by the fact that some AGEs such as pyrraline are suspected to have carcinogenic and mutagenic properties [102]. Significant daily consumption of Maillard products in common diets was shown by Henle and Miyata [103], but it is still controversial how

reaction of glucose with amino acids and the browning reaction is named the Maillard reaction after its discoverer. In addition to glucose, galactose and fructose are further starting substances for this reaction and both are between four- and sevenfold more reactive. Glycolytic intermediates are much more reactive compared to glucose, especially the trioses dihydroxyacetone and glyceraldehyde phosphate. Both can spontaneously generate methylglyoxal, a highly glycating agent [91,92]. Owing to their significantly higher proportion of the reactive open-chain form in solution (pH 6.5–7.5), D-ribose and D-fructose are more reactive compared to D-glucose. Maillard products are stable and heterogeneous compounds, which are formed over several steps, and are only partially characterized [93]. Classification of the different steps should not be regarded as dogma and the reaction process goes on transitionfree. In the initial stage, condensation of the amino group with the carbonyl to a glycosylamine takes place. This reaction is reversible and especially occurs at lysine and arginine side chains and at the N-terminal amino groups [94]. The nitrogen of the amino group reacts with the carbonyl group in a nucleophilic attack and forms an unstable imine, which is also called a Schiff base, representing an early glycation product [95]. Afterward the nitrogen is protonated and an immonium ion, which is in equilibrium with an aminocarbenium ion, arises. Deprotonation generates an enaminol, which tautomerizes to a 1-amino-1-desoxyketose, the Amadori product. If the reducing sugar is a ketose, Heyns tautomerization is the analogous reaction to the Amadori rearrangement and the product is a 2-amino-2-deoxyaldose. These Amadori or Heyns products are qualified as intermediate glycation products, but in contrast to the Amadori product formation, during Heyns rearrangement a new asymmetric center is generated. Furthermore, both compounds are colorless in contrast to the continuative and final phase products. During the continuative stage, the early stage products are partially degraded and modified. Reactive intermediates are formed, the so-called desoxyosons, which are able to form shorter α-dicarbonyls via retroaldol cleavage or oxidative induction. α-Dicarbonyl compounds can be subjected to further oxidation, cyclization, isomerization, and

Lipid peroxidation Reactive oxygen species

Maillard reaction

Polyol Pathway

Glucose + Proteins

Sorbitol

Membrane lipids/PUFAs

Details are shown in Fig. 4

Lipidperoxidation

Fructose

Schiff’s Base Details are shown in Fig. 4

Fructose-3- Triose phosphate phosphat

Keton bodies

Amadori product α-Oxoaldehydes

α-Oxoaldehydes Oxidative pathway

Non-oxidative pathway

Formation of Advanced Glycation Endproducts Fig. 5. AGE formation pathways involving the Maillard reaction, polyol pathway, lipid peroxidation, and glucose autoxidation (PUFAs, polyunsaturated fatty acids). Modified according to [297].

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exogenous AGEs may affect the endogenous AGE level. The AGE plasma levels of 90 healthy subjects correlated with the amount of absorbed AGEs. One group was assigned to foods with low AGE content and, as suspected, the AGE plasma levels decreased [104]. These results were confirmed by other studies [105,106]. Yet until today the absorption mechanisms of AGEs in the intestinal tract are not fully understood. Studies report on a peptide transporter, PEPT1, which is able to absorb pyrraline [107]. However, meaningful investigations are still lacking. In addition to bioavailability studies, studies on elimination kinetics have also been carried out in recent years. Förster et al. [108] studied the uptake of pyrraline, CML, pentosidine, and the Amadori product fructoselysine in a human intervention study. Fifty percent of the absorbed amounts of pyrraline and 60% of pentosidine were recovered in the urine over a period of 3 days. Another study examined the recovery of pyrraline while eating a normal Western diet compared to an AGEreduced diet. The reduction in dietary AGE content led to a decline in the renal excretion of pyrraline from 4.8 mg/day (normal diet) to 0.3 mg/day (AGE-reduced diet). Owing to the individual metabolic transit of single AGEs, it is difficult to make a general statement on the potential nutritional risk, yet it can be concluded that a healthy kidney function is essential for the excretion of AGEs. Because AGEs are a heterogeneous group of substances, it is still unclear how AGEs must be processed to undergo renal excretion. Nevertheless, AGEs are formed mainly endogenously and only an estimated 10% are of exogenous origin. The formation in vivo takes place continuously and is part of the normal aging process. Proteins with a long half-life are mainly affected. Under mild physiological conditions (pH 7.4, 371C) the in vivo formation of AGEs might take a prolonged time compared to the partly extreme conditions during food preparation or the conditions used originally by Louis Camille Maillard. This influences the formation of α-dicarbonyl compounds as reactive key components in vivo. Long-chain α-dicarbonyl compounds such as 3-deoxyglucosone (3-DG) and N6-(2,3-dihydroxy-5,6-dioxohexyl)-L-lysinate, as well as short-chain compounds such as glyoxal and methylglyoxal, take part in posttranslational protein modifications. In patients with diabetes mellitus a 3-DG level of 353 nM was found compared to a level of 199 nM measured in healthy subjects [109]. The levels of glyoxal and methylglyoxal in plasma of healthy subjects reach values of 0.2–0.3 mM and increase up to 0.4 and 0.8 mM in poorly controlled diabetic or uremia patients, respectively [110]. Higher concentrations of glyoxal and methylglyoxal were found in patients with chronic renal dysfunction (4 mM) compared to healthy subjects (1 mM) [111]. in vivo, the levels of toxic α-dicarbonyls are around 3 levels of magnitude below the level of blood glucose (4–5 mM). One likely reason is the high reactivity of these compounds and the rapid formation of secondary products [112]. Thus, only about 10% of the glyoxal is present in free form. The vast majority is reversibly bound to cysteine, lysine, and arginine side chains. In addition, an efficient and rapid detoxification is ensured by enzymatic systems, such as the glyoxalase system [113]. Cells overexpressing glyoxalase-I do not show an increase in methylglyoxal concentration after incubation in high-glucose medium and a greater enzymatic conversion of methylglyoxal to its metabolite, suggesting that glyoxalase-I activity is important in hyperglycemia-induced methylglyoxal concentrations [114]. In general, the deleterious effects of AGEs in various tissues are attributed to their chemical, pro-oxidant, and inflammatory actions. The biological effects of AGEs are exerted by two different mechanisms: (1) by direct damage to protein structures (including cross-link and aggregate formation) and extracellular matrix modification and

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(2) by binding to several receptors, such as RAGE (receptor for advanced glycation end products), causing multiple pathological changes in gene expression [115]. The first mechanism involves the ability of glycation to change the polarity, the molecular weight, and especially the threedimensional structure of a protein. The free amino functions of lysine side chains are often of essential importance in the active center of many enzymes and carrier, binding, and structural proteins. They also play an important role in the interactions between proteins such as hormone/hormone receptor, antigen/ antibody, or enzyme/enzyme inhibitor [116]. One can, therefore, assume that the biological function of a protein is influenced by glycation at essential positions. Ribonuclease A loses 50% of its enzymatic activity after 24 h of incubation with glucose in vitro [117], whereas glycation does not affect the activity of transaminases [116]. Apparently the free lysine amino function of ribonuclease A is close to the active site, unlike the corresponding function in transaminases. Hemoglobin A1c is the most widely recognized early glycation product and is also used as an indicator of blood glucose management in patients with diabetes. De Rosa et al. [118] showed that modifications at the N-terminus of hemoglobin have an extensive influence on the structure and biological function of this protein and can cause a disturbed oxygen and carbon dioxide transport in blood. Hyperglycemia increases the glycation process and is especially apparent in insulin-independent tissues such as red peripheral nerve tissue cells, blood cells, eye lens cells, endothelial cells, and kidney cells [119]. Glycation of proteolytic enzymes in diabetes reduces their efficiency, resulting in an increased formation of glycated end products [119]. AGEs have also been implicated in delayed wound healing associated with diabetes, presumably through neurological, vascular, or intermediary metabolic modifications [120]. Glycation of bovine lens proteins is accompanied by opacity of the lens crystalline. Chiou et al. [121] and Monnier and Cerami [122] explained this by conformational changes in the lens proteins as a result of glycation, in which the reactive sulfhydryl group becomes available for dimerization and cross-linking, finally forming lens crystallin aggregates. Other consequences of crosslink formation include sclerosis of renal glomeruli, thickening of the capillary basement membrane, and development of atherosclerosis [123]. Atherosclerosis is thought to involve also trapping of lipoproteins by AGEs formed on the matrix component of vessel walls [124]. Furthermore, AGEs are found in many age-related aggregates such as amyloid plaques and it was shown that plaque formation is accelerated by cross-linking reactions via AGEs [125]. Although a definitive etiology for Alzheimer disease is unknown, oxidative stress has been identified as a main risk factor for this disease. Both aging itself and the presence of AGEs are seen as risk factors because of their roles in pro-oxidant and inflammatory actions as mentioned above. One mechanism to reduce the toxic effect of AGEs is their proteolytic breakdown. It was found that AGEs can be degraded by cathepsins D and L, whereas the proteasome was not able to degrade them [126,127]. Increased amounts of endosomes and lysosomes, as well as increased cathepsin D and L activity, are accompanied by endocytosis of AGEs. After substrate uptake into the lysosomal compartment the acidic pH induces denaturation and exposure of buried hydrophobic areas, making these proteins much more accessible to degradation by cathepsins D and L because both exert substrate specificity against hydrophobic amino acids. Recently, it was found that AGEs can also bind to several receptors on cell surfaces, representing the second mechanism mentioned above. Involved receptors are galectin-3 (AGE-R3), the receptor complex oligosaccharyl transferase 48 (also called p60 or AGE-R1), 80K-H phosphoprotein (also AGE-R2 or called p90)

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[125,128,129], and scavenger receptors such as the macrophage scavenger receptor type A, cluster of differentiation 36, and scavenger receptor type BI [130], as well as RAGE [131]. It is still under investigation which receptor is responsible for the particular cellular reaction. RAGE is highly expressed in the lung at readily measurable levels but increases rapidly at sites of inflammation. It is present either as a membrane-bound or a soluble protein that is upregulated by stress in inflammatory and epithelial cells and involved in the development and maintenance of inflammatory reactions. Signaling pathways of RAGE include the activation of CDC42/Rac [132], Ras-extracellular signal-regulating kinase 1/2 [72], stress-activating kinase/c-Jun-NH2-terminal protein kinase, p38 mitogen-activating protein kinase [133], JAK1/2 [134], and the phosphatidylinositol 3-kinase [135]. Downstream signaling activates members of the STAT (signal transducers and activators of transcription) family [136], C-reactive protein and interleukin-6 (IL-6) [137], cAMP response element binding protein [138], and transcription factors such as NF-κB (nuclear factor “κ-light-chain enhancer” of activated B cells) [139]. NF-κB is a critical factor transducing several pro- or antiapoptotic and inflammatory signals. Importantly, NF-κB activation increases RAGE expression, creating a positive feedback cycle, which converts a transient proinflammatory reaction into a chronic pathophysiological state [140]. The activation of NF-κB induces the expression of adhesion molecules, growth factors (e.g., transforming growth factor-β), and proinflammatory cytokines (such as IL-6 and tumor necrosis factor-α) [141,142]. Furthermore, AGE–RAGE interaction activates NADPH oxidase, increasing intracellular oxidative stress. This sudden increase in oxidative stress by NADPH oxidase in response to AGE–RAGE interaction will again activate NF-κB [143–145]. Thus it is not surprising that RAGE has been implicated in aging, as well as in a number of pathological conditions, including atherosclerosis [146], arthritis, metabolic syndrome, stroke, diabetic complications [147], oncological diseases, chronic inflammation complications [148], and neurodegenerative diseases [133]. Comparison of age-matched control and Alzheimer disease patient brain tissue revealed higher AGE and RAGE expression in the Alzheimer disease brains [149] and there is evidence that RAGE mediates the blood–brain barrier transport of amyloid peptides in certain situations [150]. Of high relevance is the fact that intracellular AGEs are able to inhibit the proteasomal system [151] and activate the immunoproteasome via involvement of RAGE and JAK2/STAT1 signaling [134]. The function of the immunoproteasome in AGE-exposed cells is still unknown but malfunction of the proteasomal system might enhance potentially pathogenic protein aggregation and have a close relationship to the process and acceleration of aging. It is to assume that the permanent presence of AGEs might also be a potential reason for the higher concentration of immunoproteasomes in aged cells [152]. To summarize, aggregated AGE-modified proteins seem to be a major contributor to the initiation and development of diseases by triggering chronic inflammatory responses. Such a response can ultimately influence the development and clinical severity of multiple diseases.

The proteasomal system and its ability to degrade oxidized, but not aggregated, proteins To cope with damaged proteins and to maintain the cellular integrity, a series of enzymes able to recognize and degrade misfolded and oxidatively modified proteins are available, so preventing accumulation, aggregation, and cross-linking of proteins. One of these systems is the proteasomal system, which is essential for normal cell function. The proteasome consists of a central core particle (CP), the 20S proteasome, and numerous

regulatory components, which influence the specificity and activity of the 20S proteasome (Fig. 6) [153]. The 20S proteasome is the functional core of all proteasomal forms. The cylinder-like structure is composed of two α- and two β-rings; the β-rings are centrally located and are completed by the α-rings on the outside. Each ring is composed of seven subunits (α1–α7 and β1–β7). The α-rings bind the regulatory components and support the admission of proteins into the inner chamber of the proteasome. In addition, the α-rings seal the access to the catalytic center in the inactive state. Access to the active site is limited by the N-terminus of the α3-subunit, which interacts with the other α-subunits. Deletion of the α3-subunit N-terminus accordingly leads to permanent access to the catalytic center [154]. At the inner surface of the proteasome, the hydrolysis of proteins is carried out by the β-subunits. Here the activities of the constitutive subunits differ from the inducible ones. The constitutive form of the proteasome includes the enzymatically active β1-, β2-, and β5-subunits. The β1-subunit exhibits a peptidylglutamyl peptide hydrolase, or caspase-like, activity, which is responsible for the cleavage at acidic amino acids. The β2-subunit comprises a trypsin-like activity catalyzing the cleavage at basic amino acids, whereas the β5-subunit includes a chymotrypsin-like activity, which in turn cleaves peptide bonds at neutral and hydrophobic amino acids [153]. Unfolded proteins can reach the inner chamber of the proteasome without the aid of a regulator unit. Oxidation of proteins may induce a conformational change in the protein structure, and hydrophobic amino acids, which are usually hidden in the protein core of a folded protein, are exposed to the protein surface after oxidation. As a result, substrate recognition by the α-subunits of the 20S proteasome occurs [155]. However, the readiness to recognize a substrate depends on the degree of oxidative modification, because hydrophobic surfaces of highly oxidized proteins presumably interact with other proteins to form aggregates as described above and are no longer degradable by the 20S proteasome because of their size and structure [155]. Association of the 19S regulator particle (RP) with the α-rings of the core particle results in the formation of 26S proteasomes with one (RP1CP; 19S–20S) or two (RP2CP; 19S–20S–19S) regulatory particles. Binding of the 19S regulator changes the spectrum of degraded proteins. The regulator recognizes proteins that have been marked in advance for degradation. Identification marking to the intended proteins for degradation is carried out by an ATPdependent transfer of ubiquitin, an 8.5-kDa small peptide molecule, to the substrate, catalyzed by a complex enzyme system [153]. In the first step, the ubiquitin is activated by E1 enzymes using ATP and then transported to E2 enzymes. The E2–ubiquitin complex binds to an E3 enzyme, which transfers the ubiquitin from the E2–ubiquitin complex with its ubiquitin ligase activity onto lysine side chains of target proteins. In repeated cycles more ubiquitin monomers may be added to the already attached ubiquitin. The 19S regulator recognizes the polyubiquitin chain and directs the substrates in an unfolded form to the core proteasome [153]. This regulator consists of some 18 subunits, which can in turn be divided into two subtypes. The base consists of six different AAA-ATPases and other non-ATPase family units [156]. This base binds to the α-subunits of the 20S core unit and causes the ATP-dependent opening of the access to the catalytic site [156]. The upper section of the 19S regulator consists of the non-ATPase units, which are responsible for the binding of ubiquitinated substrates and for the recycling of ubiquitin [154]. In the inducible form of the proteasome, also called immunoproteasome, instead of the constitutively expressed catalytic subunits three inducible subunits are built into the CP. The inducible proteasome is synthetized in response to various stimulants, such as interferons, tumor necrosis factor-α, and lipopolysaccharide [157]. Moreover, induction of immunoproteasomal subtypes can

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Inducible proteasome 20 ± 9 %

26S proteasome 15 ± 3 %

20S proteasome 41 ± 5 %

Degradation of oxidized proteins

Generation of antigenic peptides and others

Hybrid proteasome 24 ± 9 %

Degradation of polyubiquitinated proteins

Function still under investigation Fig. 6. Structure, relative amount, and proposed function of various proteasomal complexes. Modified according to [153].

be induced by heat-shock reactions [158], arsenic trioxide [159], and nitric oxide [160]. In this process the β1-, β2-, and β5-subunits of the 20S core are replaced by the respective immunoproteasomal subunits low-molecular-weight protein 2 (or β1i), multicatalytic endopeptidase complex-like 1 (or β2i), and low-molecular-weight protein 7 (or β5i). In addition, the 11S RP (PA28, proteasome activator 28 kDa) binds to the 20S core unit. Together with the inducible β-subunits the 11S regulators build the active inducible proteasome. The 11S regulator is composed of several subunits, which are characterized as α-, β-, or γ-subunits and differ in their cellular localization. In the cytoplasm the heterodimeric 11Sα/β complex is predominant, whereas in the nucleus it is the 11Sγ complex [153]. The heterodimeric 11Sα/β consists of three β- and four α-subunits, which build an annular structure. Increased hydrolysis of peptides was observed by the addition of the 11S to the 20S core unit [161]. Again unfolding of a protein is required to translocate it into the inner core of the inducible proteasome complex [162]. The inducible proteasome is involved in the processing of oligopeptides, which are provided for antigen presentation to the adaptive immune response. However, cells may induce the formation of the inducible proteasome without the necessity of increased antigen processing [163]. It is also known that the constitutive subunits of the proteasomal system are expressed in almost all cells of the organism, whereupon the inducible proteasomal subunits can be found constitutively in high quantities in immune-related cells or tissues and in smaller

quantities in almost any other cell type. In addition, a recent study indicates that the inducible proteasome is responsible for the maintenance of protein homeostasis and cell viability in cytokineinduced oxidative stress [164]. These observations suggest other functions that are not associated with antigen processing. However, the evidence regarding this relationship is still limited. The inducible proteasome also has a clear role in transcription factor processing [165] and in the degradation of oxidized proteins [166]. Although it is often stated that the degradation of oxidatively damaged proteins can occur via a ubiquitin/ATP-dependent (26Sdependent) mechanism [167,168], data showing the degradation of oxidized proteins in an ATP/ubiquitin-dependent pathway are actually not convincing or simply nonexistent. However, most studies have identified a primary role for the 20S proteasome in the degradation of oxidized proteins [166,169–171]. This may be in part because the 20S proteasome is more resistant (compared to the 26S proteasome) to oxidative stress and can maintain its activity even under stress conditions [172,173]. It was further suggested that an increase in oxidative stress leads to disassembly of 26S proteasomes and consequently levels of 20S proteasomes rise [171,174,175]. Oxidative modifications can also impair ubiquitination such as modifications on lysine residues [52] and disable the required designation of proteins for 26S proteasomal degradation. In 2011, Aiken et al. proposed a working model for stressdependent 26S regulation connecting 20S and 26S proteasomal activity [176]. Under normal physiological conditions and in the

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Increasing amount of oxidative modification Degrading-signal strength for the 20S proteasome

Susceptibility to proteasomal degradation

I. Native and functional protein, no need for 20S proteasomal degradation

II. Proteasomal substrate, Slightly oxidized, partially unfolded, no or reduced activity

III. Aggregated and covalently cross-linked proteins, resistant to proteasomes

Fig. 7. Proteolytic susceptibility dependence on oxidative modification. (I) The protein is native and unmodified with no need for proteolytic degradation. (II) With increasing oxidative damage the protein becomes oxidized and the proteolytic susceptibility increases. (III) The protein is severely oxidized, becomes resistant to proteolytic degradation, is even able to inhibit the 20S proteasome, and can be considered as a lipofuscin precursor. According to [153].

absence of oxidative stress, the 26S proteasome is the predominant cellular protein degradation system and responsible for ATPdependent degradation of polyubiquitinated substrates. Under conditions of oxidative stress, however, it has been suggested that proteasomal activity can be initially stimulated for the degradation of mildly oxidized proteins [50]. The underlying mechanisms are still unknown. However, persisting oxidative stress or acute oxidative stress leads to a partial inhibition of 26S activity, stresstriggered 26S disassembly, and consequently an accumulation of ubiquitinated substrates [172,177]. This 26S dissociation liberates 20S complexes and, therefore, increases the cellular capacity for ATP/ubiquitin-independent removal of oxidized proteins. In yeast, 26S proteasome disassembly is regulated by Ecm29, a proteasomeinteracting protein, and Aiken et al. also hypothesized a regulatory mechanism in mammalian cells that is probably more complex because of the quantity of regulatory proteins and proteasomal components. A biphasic susceptibility of substrates for 20S CP-mediated degradation that is dependent on oxidative modification is observed (Fig. 7). At moderate oxidant concentrations, proteolytic susceptibility increases. After a protein reaches the ideal damage level, further oxidation causes a decrease in proteolytic susceptibility, sometimes even below the basal value. This biphasic response seems to be a common behavior of all soluble, globular proteins with defined structures [155]. The origin of the protein seems not to have any importance even in the case of recombinant proteins. Proteins such as oxidized hemoglobin, catalase, aconitase, superoxide dismutase, ferritin, and many more have been used as substrates by several laboratories and revealed similar results. An increase in proteolytic susceptibility should always be seen at an optimal oxidant exposure, although the concentration of the oxidants used differs in experimental setups [81,178,179]. Yet there are some exceptions. Several native-unfolded proteins such as casein, tau [180,181], and α-synuclein [182] are inherently excellent proteasomal substrates and their degradation susceptibility was not increased by mild oxidation. It seems that their proteolytic susceptibility is rather modulated by enzymatic modifications, e.g., phosphorylation [183]. However, in any case, if these proteins become heavily oxidized, these proteins can also form aggregates and cross-links and change into poor substrates for degradation [184].

Autophagic uptake mechanisms and lysosomal degradation of aggregated proteins In addition to the proteasome, several other degradation systems and enzymes exist in mammalian cells. Some of them are present in compartments where the proteasomal system is not expressed, such as the Lon protease in mitochondria and the variety of cathepsins in the lysosomal lumen. Lysosomes are membrane-surrounded intracellular organelles that are equipped with a variety of enzymes. These include hydrolases, proteases, peptidases, lipases, glycosidases, nucleases, phosphatases, and sulfatases [185]. A majority of these enzymes have an optimum pH of 4.5–5.0. This intralysosomal pH range is maintained by protons, which are transported via membrane-bound V-ATPases into the lumen [185]. Lysosomes play a significant role in the degradation of extracellular but also intracellular macromolecules. Extracellular material can be incorporated via pinocytosis, phagocytosis, or receptor-mediated endocytosis. The major lysosomal proteases are the above-mentioned cathepsins (Greek: “kathepsein,” meaning “digest”). Depending on the amino acid located in the active site, the cathepsins can be divided into cysteine cathepsins (cathepsins B, C, F, H, K, L, N, O, S, T, U, W, and X), aspartate cathepsins (cathepsins D and E), and serine cathepsins (cathepsins G and A) [186]. Cathepsins are produced as inactive proenzymes, activated by a lowering of pH value or by other enzymes. The cathepsins B, L, and D are considered the main human lysosomal proteases [187]. Cathepsin B (EC 3.4.22.1) is a cysteine protease and, in addition to its function as an endopeptidase, also possesses carboxypeptidase activity [188]. According to their structural homology to the plant protease papain, cathepsins B and L are counted in the family of papain-like proteases [189]. Cathepsin L (EC 3.4.22.15) is the most important cysteine protease in the lysosomal proteolysis of endocytosed proteins [190]. Cathepsin D (EC 3.4.23.5) belongs to the pepsin family and is classified as an aspartic protease. It has a strong activity as endopeptidase, preferentially cleaving peptide bonds between hydrophobic aromatic amino acids [191]. Cathepsin D is ubiquitously expressed and is primarily involved in the lysosomal degradation of proteins. It is also needed for the proteolytic activation of other proenzymes [107]. Other than the proteasomal system, the lysosomal system is the most important “digestive system” in animal cells, with a

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Autophagosome

Phagophore Lysosome

Degradation hsp70

LAMP-2A

Target Protein Fig. 8. Types of autophagy in mammalian cells. Modified according to [298].

nearly unlimited degradative capacity. Soluble long-lived proteins, extracellular material, large protein aggregates, and even organelles can be targeted to the lysosome in a process called “autophagy” [192]. One of the main functions of lysosomal degradation is to ensure the cellular homeostasis. At a basal level autophagy occurs constitutively in all cell types. The composition of the cytoplasm and the size of the ER are thus regulated by autophagy [193]. Furthermore, autophagy can assume both a protective and a damaging role in different stress situations such as nutrient deficiency or oxidative stress [194]. Energy is provided by degradation of nonessential macromolecules, enabling adaptation and survival in starvation periods. Autophagy may also induce type II apoptosis, and dysregulation of autophagy is implicated in the pathogenesis of several diseases. In mammalian cells, three different types of autophagy can be distinguished: chaperonemediated autophagy (CMA) [195], microautophagy [196], and macroautophagy (Fig. 8) [197]. CMA is a selective and direct elimination system, without building of a vesicle, and is highly selective for a particular group of soluble cytosolic proteins that contain a specific recognition motif, the pentapeptide “KFERQ” or “KFERQ-like motif.” Protein uptake into the lysosomal system occurs via recognition by cytosolic chaperones and receptormediated translocation [198]. First, the substrate binds to the cytosolic heat-shock cognate protein of 70 kDa and its cochaperone and then to the lysosomal-associated membrane protein type 2A. After unfolding, the substrate protein is translocated to the lysosomal lumen with the help of a lysosomal chaperone (lyshsc70). It is assumed that several oxidative protein modifications lead to the formation of a KFERQ-like motif and convert a nonCMA substrate in a CMA substrate, which can be targeted for lysosomal degradation [199]. During microautophagy, small parts of the cytosol and its components are incorporated via invagination of lysosomal membranes followed by vesicle degradation in the lysosomal lumen. After this, breakdown products are liberated in the cytoplasm. This mechanism is only poorly understood. In contrast, macroautophagy remains the best characterized form of autophagy and is often simply referred to as autophagy. This process is responsible for the degradation of larger aggregates and organelles. Macroautophagy involves the formation of a socalled “autophagosome,” a double-membraned vesicle that enwraps cytoplasmic contents or organelles via expansion of an isolation

membrane, called a phagophore. The source of the membrane material is still unclear, but it is believed to involve de novo membrane synthesis and the endoplasmic reticulum is possibly involved [200]. Furthermore, it has been suggested that the Golgi apparatus serves as a source for phagophore formation [201]. Macroautophagy was initially described in mammalian cells but the molecular mechanism has been mostly investigated in yeast. Until today, more than 30 different autophagy-related (Atg) genes and the corresponding Atg proteins have been identified in yeast [202]; for many of them mammalian homologs are known [203]. Atg’s are organized into functional complexes and are involved in each of the following three steps: (1) Initiation: stimulating signals cause mTOR (mammalian target of rapamycin)-dependent activation of the initiation complex and a preautophagosomal structure is formed. (2) Elongation: the isolation membrane grows until it encloses the target material completely and builds the autophagosome. (3) Maturation: the isolated cytoplasm, enclosed by the inner limiting membrane, is delivered to the endo/lysosomal lumen. Both the cytoplasm and the surrounding membrane are degraded by lysosomal hydrolases, and the degradation products are released into the cytoplasm, where they are available for metabolism [204]. The activity of autophagy is under strict control of several signal transduction pathways, which are shifted by various signals [205]. The best characterized and an important regulator of autophagy is the serine/threonine kinase mTOR. In an adequate nutritional state, mTOR is phosphorylated and thus activated, e.g., via kinases of the class I phosphatidylinositol 3-kinase/Akt pathway, which is activated by ligand binding to the insulin receptor [206]. At low cellular energy levels 50 -AMP-activated protein kinase and the eukaryotic initiation factor 2α, which responds to amino acid deficiency [205], inhibit mTOR. Pharmacologically, autophagy can be induced by the mTOR inhibitor rapamycin [207]. In yeast, inactivation of TOR induces dephosphorylation of Atg13, which builds the initiation complex with Atg1 and Atg17 leading to the formation of the preautophagosomal structure (PAS) [208] and is responsible for further recruitment of other Atg proteins [209]. Owing to the lack of knowledge of Atg13 and

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Atg17 homologs, the understanding of mechanisms inducing macroautophagy was limited in higher eukaryotes. Meanwhile, ULK1 (UNC-51-like kinase 1) and ULK2 (UNC-51-like kinase 2) are found as mammalian homologs of Atg1 [210,211]. FIP200 (focal adhesion kinase family-interacting protein of 200 kDa) was identified as having structural and functional characteristics of Atg17 [212]. Further, a mammalian homolog for Atg13 [213] was described. The ULK1 complex controls the first step of initiation and consists of ULK1/2, Atg13, and FIP200, which integrates stress signals from mTOR complex 1, the main regulator of autophagy. The phosphatidylinositol 3-kinase (Vps34 (yeast) or mVps34 (mammals)) is also involved in autophagy initiation as a part of the complex. Furthermore, the proteins Vps15, Atg6 (yeast)/Beclin 1 (mammals), and Atg14 are essential. The molecular function of this complex is not finally revealed but it seems to play a pivotal role during autophagosome formation and further recruitment of Atg proteins (including Atg18, Atg20, Atg21, and Atg24) to the PAS [214,215]. The Beclin 1/Vps24 complex allocates a supplemental level of autophagy control, regulated by the interaction of a series of cofactors [216]. BIF-1/endophilin B1 interacts with Beclin 1 and promotes autophagy via the ultraviolet irradiation resistanceassociated gene (UVRAG) [217]. Binding of activating molecule in Beclin 1-regulated autophagy and Atg14L to the complex (or alternatively UVRAG) stimulates autophagy. Binding of Rubicon (RUN domain and cysteine-rich domain containing Beclin 1-interacting protein) to the complex with UVRAG inhibits autophagy. Binding of apoptosis-related proteins such as BCL-2 or BCL-XL to Beclin 1 also leads to an inhibition of autophagy. During starvation, BCL-2 is phosphorylated by Jun N-terminal kinase 1 and the interaction between Beclin 1 and BCL-2 is disrupted [218]. This mechanism is discussed as also being responsible for the increase in macroautophagy after ER stress or proteasomal inhibition [219]. Inositol and inositol trisphosphate (IP3) levels are able to regulate autophagy independent of mTOR [220]. IP3 downregulates autophagy via binding to IP3 receptors, release of calcium, activation of calpain proteases, and finally increase in cAMP production. For elongation and fusion of the autophagosome with the lysosomal system, secretory and endocytotic transport system components are required, among others SNAREs and Rab proteins. Subsequent acidification using vacuolar H þ -ATPases converts an early autophagosome into a late autophagosome and fusion with lysosomes or late endosomes provides the lysosomal hydrolases for degradation processes. The final vesicle is called the autophagolysosome or autolysosome. Fusion leads to incorporation of the outer membrane of the autophagosome into the lysosomal membrane. The single inner membrane is released into the lysosomal lumen and dismantled [221]. The second elongating step requires two processes similar to ubiquitination: Atg12–Atg5 conjugation and LC3 (microtubule-associated protein light-chain 3) modification [222]. In the first conjugation system (Atg12–Atg5 system) Atg7 activates the ubiquitin-like protein Atg12 and transfers it to Atg10. The formation of this intermediate enables the conjugation of Atg12 to a lysine residue of Atg5 via an isopeptide bond. This reaction appears to be irreversible and a major part of both Atg12 and Atg5 exists in the conjugated form [223]. In the next step the Atg12–Atg5 conjugate forms a noncovalent protein complex with Atg16 on the outside of the nascent autophagosome and has been shown to promote LC3I–PE (phosphatidylethanolamine) formation, analogous to E3 enzymes during ubiquitin-conjugation reactions, and is, therefore, functionally linked to the secondary conjugation system (LC3 modification). In a first step Atg4 eliminates the C-terminal tail of LC3 (in yeast Atg8) to expose a glycine residue (G116). Atg7 activates G116; afterward LC3I is conjugated via a thioester bond with Atg7. LC3I is then transferred to Atg3 and also connected via a thioester bond. Finally, LC3I binds to the

amino group of PE via formation of an amide bond. The LC3–PE complex is called LC3II and is localized owing to its lipid part at the autophagosomal membrane, enabling membrane elongation. LC3II is unconjugated from the phospholipid anchor via Atg4, and the luminal-associated LC3II is degraded. The LC3 system and Atg12–Atg5 complex are both essential for autophagy. Whether the Atg12–Atg5 conjugation system has any additional functions apart from promotion of LC3I–PE conjugation is unclear. However, in yeast [208] and mammalian cells [224,225] autophagosome formation is more heavily impaired by inactivation of the Atg12– Atg5 conjugation system than the Atg8–LC3 system, suggesting additional functions. Interestingly, LC3II is also able to bind to the adapter protein sequestosome 1 (SQSTM1/p62), which mediates recognition and targeting of ubiquitinated protein aggregates for degradation via selective autophagy [226]. Therefore, the lysosomal system via autophagy does play a major role in the fate of intracellular protein aggregates. It seems that ubiquitin coordinates the catabolism of cellular targets by both the UPS and autophagy: substrates recognized by the proteasomal system are thought to be modified by K48-linked polyubiquitin chains, whereas K63-linked chains (revealing a more open conformation than K48 chains) or only monoubiquitinated substrates are preferentially degraded by the autophagosomal pathway [227]. Therefore, despite the use of ubiquitin in both pathways, the various structures of polyubiquitin chains may be adequate to maintain specificity and selectivity of autophagosomal and proteasomal pathways, but owing to incomplete specificity of SQSTM1/p62, there is some overlap. SQSTM1/ p62 itself is an autophagy substrate and able to recruit ubiquitinated proteins via its ubiquitin-associated domain [228]. It is proposed that these complexes are recognized and engulfed by the autophagic machinery [228]. The affinity of p62 for K63-linked monoubiquitin or polyubiquitin chains is higher compared to K48polyubiquitylated proteins [229], but the adaptor molecule is generally able to bind K48-linked ubiquitin, suggesting the possibility of recruiting substrates, which are basically degraded by the proteasome. Therefore, recruitment into autophagosomes seems to be possible under circumstances when the UPS is compromised and acts as an alternative mechanism [229]. The essentiality of the lysosomal system and the variety of functions that can be assigned to autophagy explains the number of diseases in which autophagy deficiencies have been observed.

Relevance of proteolytic systems and aggregate formation in several pathologies The relationship between the proteolytic systems and protein aggregates has been questioned for some time and several studies suggest that the decline in proteasomal and lysosomal proteolytic activity correlates with the accumulation of damaged and aggregated proteins, common features accompanying aging [54,230–233]. In lysosomes, the accumulation of lipofuscin interferes with their ability to fuse with autophagosomes and degrade their cargo, thus initializing a vicious cycle leading to a progressive decline in autophagosome degradation. Also severe defects of proteasome-mediated proteolysis were observed during aging owing to decreased expression and assembly of proteasomal subunits [234] and increased formation of protein aggregates inhibiting proteasomal function [48,50]. Furthermore, the failure of the proteasomal system is involved in several pathologies. Proteolysis defects have been reported for several processes such as cardiovascular diseases (e.g., atherosclerosis), immune system-associated diseases (e.g., rheumatoid arthritis), muscle-related diseases (e.g., muscular dystrophy), and cancer. Furthermore, malfunction of the proteasomal system as a consequence of protein aggregation is also a key process for most

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neurodegenerative diseases, such as Alzheimer disease, Parkinson disease, Huntington disease, and Friedreich ataxia, characterized by a selective loss of neurons in disease-specific regions of the brain. These aggregates consist of unfolded, insoluble, mostly polyubiquitinated polypeptides that fail to be targeted and degraded by the proteasome [235]. These observations have led to the assumption that the UPS plays a direct role in their formation. In fact, the presence of ubiquitinated proteins in these aggregates reflects unsuccessful proteasomal degradation attempts. However, it remains still unknown whether ubiquitinated proteins aggregate or whether the aggregated proteins are ubiquitinated in a secondstep reaction. In addition, advancing impairment of chaperones may lead to an intracellular accumulation of incorrectly folded proteins, which cannot be processed efficiently by an aged UPS. In these cases, proteins such as α-synuclein in Parkinson disease and tau in Alzheimer disease become more prone to aggregation and form inclusions. Because neurons are postmitotic and terminally differentiated cells, they are particularly affected by such changes in cellular metabolism. As mentioned before, intracellular aggregates/inclusions are formed when the capacity of the proteasomal system becomes overwhelmed [236]. In the case of familial inherited neurodegenerative diseases, this may occur owing to the presence of a mutant protein. The missense mutation A53T in α-synuclein in some families with Parkinson disease leads to protein misfolding and subsequent loss of function [237]. The expansion of amino acid repeats, as observed in polyglutamine (polyQ) disorders such as Huntington disease causes a rapid aggregation of these proteins into inclusions. These aggregates are composed of mutant huntingtin protein (mHtt), which contains a prolonged polyQ stretch (N-terminal) [238]. Huntington disease is a progressive neurodegenerative disorder, characterized by cognitive decline, psychotic symptoms, and motoric dysfunctions [239]. During disease progression, severe neuronal loss occurs in hippocampus, spinal cord, thalamus, and other brain regions [240,241]. To date, no prevention or treatment for Huntington disease is known. It was shown that small aggregates especially of oligomeric, fibrillary mHtt cause cellular toxicity [242–244]. However, the role of the proteasome in Huntington disease remains contradictory. PolyQ aggregates were not able to inhibit proteasomal function in vitro, whereas fibrillary huntingtin protein from mouse brain was [245–247]. Using short-lived proteins containing polyQ that are rapidly targeted for proteasomal degradation, it was shown that these polyQ proteins were efficiently degraded when targeted to the proteasome unless these proteins were aggregated [248,249], suggesting that proteasomes are capable of digesting polyQ sequences. On the other hand, it was reported that the UPS is impaired in Huntington disease, which may be the underlying cause of the neurotoxicity. Purified mammalian 26S proteasome was able to cleave only within the flanking sequences or after the first glutamine of a polyQ peptide, whereas the remaining polyQ stretch was released [250]. The remaining polyQ peptide is much longer than peptides normally released by the proteasome; perhaps it is unable to diffuse out of the proteasomal gate, thereby clogging the proteasome, resulting in proteasomal impairment. Supporting evidence was given by FRET experiments showing a stable interaction between the proteasomal subunit β1i and mHtt. In this case the fluorophore was on the outside of the proteasome and not in the inner proteolytic chamber and may thus reflect proteasome binding to mHtt aggregates [251]. In general it is believed that proteasomes make a significant contribution to the disease course, but so far it is unknown whether proteasomes can efficiently degrade nuclear mHtt fragments; generate toxic, aggregation-prone polyQ peptides; or become clogged and impaired by the polyQ fragments [252]. It is known that heat shock proteins can diminish aggregation in polyQ models; two

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members of the Hsp40 family (DNAJB8 and DNAJB6) are especially auspicious candidates [253,254]. The most common neurodegenerative disease is Alzheimer disease; its patients are about 60% of all cases of various existing neurodegenerative diseases. Symptoms are malfunctions of both the short-term memory and the sense of direction; later the longterm memory is affected. Also an impaired motor coordination can occur, caused by affected motor neurons. Physical manifestations are brain atrophy and loss of neurons and synapses, mostly found in the temporal, frontal, and parietal cortex, as well as in hippocampus and amygdala. Also both an extracellular accumulation of β-amyloid (Aβ) aggregates and an intracellular accumulation of hyperphosphorylated tau protein occur [255]. Involvement of the UPS in the pathology of Alzheimer disease is evidenced [256] because Aβ plaques and hyperphosphorylated tau filaments are both found polyubiquitinated but not degraded. Further, it has been shown that a mutant form of ubiquitin [257] is involved, perhaps contributing to the neurotoxicity of Aβ [258]. Also the ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), a deubiquitinating protein [259], is found oxidatively modified and downregulated in early disease stages [260]. Aβ protein is able to inhibit (ubiquitin-dependent) proteasomal degradation [261], by binding to the inner chamber of the proteasome as shown by electron microscopy [262]. In 2000, Shringarpure et al. [263] demonstrated that the inhibitory effect of the peptide depends on the crosslinking status of Aβ. Hyperphosphorylated tau proteins also tend to form larger structures such as paired helical filaments (PHFs) [264] and neurofibrillary tangles [265]. PHFs are able to bind and inhibit the proteasome [184]. Furthermore, hyperphosphorylated tau has been shown to be resistant to proteasomal degradation [183] and is furthermore able to form cross-linked aggregates in the presence of HNE, which are able to inhibit the proteasome [266]. The very fact of proteasomal inhibition seems to be adequate to induce neurodegeneration, playing a major role in neuronal damage related to Alzheimer disease [184]. Parkinson disease is the second most common neurodegenerative disorder after Alzheimer disease. About 1% of the population 465 years of age is affected, and 95% of all patients suffer from the late-onset sporadic form. The early-onset form is mostly found in the hereditary form of Parkinson disease [267]. Clinical symptoms are resting tremor, rigidity, and bradykinesia [267]. The most important histological marker is the cytosolic formation of Lewy bodies formed mainly by α-synuclein [268], in a polyubiquitinated state. A missense mutation of α-synuclein is responsible for some early-onset forms of familial Parkinson disease. Another protein that forms aggregates in Parkinson disease is parkin [269], normally functioning as an E3 ligase of the UPS [270]. Mutations in the encoding gene are a common cause of the hereditary form of Parkinson disease. Finally, another protein mainly found attached to aggregates is UCH-L1. This deubiquitinating enzyme is exclusively found in neurons and hydrolyzes polyubiquitin sequences into monomers [271]. Aside from protein aggregation, oxidative stress seems to be one of the main actors in Parkinson disease, even if it is still unclear whether the increased formation of ROS is the initiating or just a resulting event of cellular changes due to the disease pathology [267]; furthermore, a reduction in complex I of the mitochondrial respiratory chain in cells of the substantia nigra was shown, pointing in the direction of a mitochondrial involvement in Parkinson disease [272]. Consequences are decreased levels of cellular ATP, influencing ubiquitination and protein degradation via the UPS, de novo protein synthesis, and the energy supply of the cellular antioxidative systems; also increased ROS formation aggravates the functioning of the cell [273]. In the dopaminergic neurons of the substantia nigra the amount of proteasomal α-subunits, but not β-subunits, has been shown to be decreased in comparison to an age-matched

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control group [274]. This may cause a decrease in the available level of functional proteasomes and thus a reduction of overall proteasomal activity. In Parkinson disease it was shown that the expression of both of the regulator caps PA700 and PA28 was reduced [275]. By treating rats with the proteasome inhibitor lactacystin a quick progression of the somatic symptoms of Parkinson disease and an increased loss of dopaminergic neurons were shown; at the same time the formation of α-synuclein aggregates was induced [276]. This result suggests an important role for proteasomal impairment in the pathology and progression of this disease. Several findings in recent years have helped to strengthen a connection between macroautophagy and neurodegenerative disorders. Beclin 1 is an essential component of the initiation complex and cellular levels of Beclin 1 have often been correlated with autophagic activity. It was shown that heterozygous deletion of Beclin 1 leads to neurodegeneration [277]. In contrast, the increases in Beclin 1 described in neurodegenerative diseases often reflect neuronal upregulation of macroautophagy as a response to the accumulation of pathogenic proteins [278]. Interestingly, a paradoxical decline in macroautophagy-mediated degradation, despite the correct formation and clearance of autophagosomes, has been identified in Huntington disease models [279]. A marked decrease in cargo content was detected, conveying the impression of empty autophagosomes possibly due to failure of cargo recognition. Alterations in the clearance of autophagosomes have become a widespread topic for an increasing amount of neurodegenerative disorders. An increased amount of autophagic vacuoles was not associated with increased autophagic flux. Defects may be caused by the inability to mobilize autophagosomes from their formation site toward lysosomal or endosomal compartments, decreased autophagosome–lysosome/ endosome fusion, or decreased lysosomal proteolysis [280–282]. Another connection between lysosomal dysfunction and neurodegeneration was found in lysosomal storage disorders (LSDs). The majority of these disorders are characterized by severe neurodegenerative phenotypes. In many LSDs, a deficit or malfunction of specific lysosomal enzymes leads to impaired autophagic turnover of proteins, causing particularly extensive pathology in the brain [283]. For example, loss-of-function mutations of cathepsin D cause neuronal ceroid lipofuscinoses (NCLs; also known as Batten disease), a severe neurodegenerative disorder associated with congenital mental retardation, or a juvenile neurodegenerative NCL subtype associated with dementia [284]. The identification of specific mechanisms in the various neuronal pathologies is an important consideration for the future development of therapeutic interventions and special attention has to be paid to the interplay between macroautophagy and the UPS. Cells respond to acute proteasome inhibition by upregulating macroautophagy [285], whereas prolonged chronic inhibition of the proteasome constitutively upregulates macroautophagy, but further activation of macroautophagy in response to stress fails [286]. This suggests that autophagy may be activated as a compensatory mechanism in consequence of insufficient proteasomal degradation, eventually able to facilitate the accumulation of protein aggregates in neurodegeneration. Aggregates formed by some pathogenic proteins have proven amenable to degradation by macroautophagy [287]. Huntingtin and α-synuclein aggregates, caused by overexpression of mutant forms of these proteins, are effectively cleared from cells treated with the autophagy inducer rapamycin, whereas treatment with the autophagy inhibitor 3methyladenine reduces their clearance [288,289]. Also the formation of polyQ inclusions can be reduced by autophagy induction in cell culture experiments [287]. In addition, pharmacological upregulation of macroautophagy has been shown to be effective at reducing neuronal aggregates and slowing down the progression of neurological symptoms in fly and mouse models of Huntington

disease [290,291], reinforcing the possible therapeutic implications of this cross-talk.

Conclusions Oxidized or otherwise modified proteins are often nonfunctional and need to be removed from the cellular protein pool. Increased protein modifications and/or impairment of proteolytic systems might lead to the accumulation of oxidized proteins and the formation of nondegradable aggregates, which in turn impair cellular functions, amplify further protein modifications, and contribute to pathological processes. On the other hand, under several conditions the toxicity of protein aggregates can be reduced by macroautophagical uptake into autophagosomes, which eventually lead to a (partial) degradation of the aggregate in lysosomes. However, aggregate formation is involved in aging and several pathologies, such as neurodegenerative diseases, but the physicochemical and cellular conditions that promote formation and growth of aggregates in vivo are still ambiguous. There is lack of knowledge on the cause of aggregate toxicity, on some aggregates/inclusions being more toxic than others, or on the mechanisms through which these structures cause damage to cells. Further research needs to focus on the underlying biochemical processes, development of early biomarkers, and research on therapy concepts to counteract these aggregation processes. References [1] Starkov, A. A. The role of mitochondria in reactive oxygen species metabolism and signaling. Ann. N. Y. Acad. Sci. 1147:37–52; 2008. [2] Gross, E.; Sevier, C. S.; Heldman, N.; Vitu, E.; Bentzur, M.; Kaiser, C. A.; et al. Generating disulfides enzymatically: reaction products and electron acceptors of the endoplasmic reticulum thiol oxidase Ero1p. Proc. Natl. Acad. Sci. USA 103:299–304; 2006. [3] Ockner, R. K.; Kaikaus, R. M.; Bass, N. M. Fatty-acid metabolism and the pathogenesis of hepatocellular carcinoma: review and hypothesis. Hepatology 18:669–676; 1993. [4] Schonfeld, P.; Dymkowska, D.; Wojtczak, L. Acyl-CoA-induced generation of reactive oxygen species in mitochondrial preparations is due to the presence of peroxisomes. Free Radic. Biol. Med. 47:503–509; 2009. [5] Lucero, H. A.; Kagan, H. M. Lysyl oxidase: an oxidative enzyme and effector of cell function. Cell Mol. Life Sci. 63:2304–2316; 2006. [6] Dickinson, A. C.; DeJordy, J. O.; Boutin, M. G.; Teres, D. Absence of generation of oxygen-containing free radicals with 40 -deoxydoxorubicin, a noncardiotoxic anthracycline drug. Biochem. Biophys. Res. Commun. 125: 584–591; 1984. [7] Esterbauer, H.; Schaur, R. J.; Zollner, H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic. Biol. Med. 11:81–128; 1991. [8] Sies, H. Oxidative stress: oxidants and antioxidants. Exp. Physiol. 82:291–295; 1997. [9] Jones, D. P. Redefining oxidative stress. Antioxid. Redox Signaling 8: 1865–1879; 2006. [10] Trachootham, D.; Alexandre, J.; Huang, P. Targeting cancer cells by ROSmediated mechanisms: a radical therapeutic approach? Nat. Rev. Drug Discovery 8:579–591; 2009. [11] Emerit, J.; Edeas, M.; Bricaire, F. Neurodegenerative diseases and oxidative stress. Biomed. Pharmacother. 58:39–46; 2004. [12] Paravicini, T. M.; Touyz, R. M. Redox signaling in hypertension. Cardiovasc. Res. 71:247–258; 2006. [13] Haigis, M. C.; Yankner, B. A. The aging stress response. Mol. Cell 40:333–344; 2010. [14] Peng, S. L.; Fatenejad, S.; Craft, J. Scleroderma: a disease related to damaged proteins? Nat. Med. 3:276–278; 1997. [15] Dean, R. T.; Gebicki, J.; Gieseg, S.; Grant, A. J.; Simpson, J. A. Hypothesis: a damaging role in aging for reactive protein oxidation products? Mutat. Res. 275:387–393; 1992. [16] Stadtman, E. R.; Levine, R. L. Protein oxidation. Ann. N. Y. Acad. Sci. 899: 191–208; 2000. [17] Fu, S. L.; Dean, R. T. Structural characterization of the products of hydroxyl-radical damage to leucine and their detection on proteins. Biochem. J. 324(Pt 1):41–48; 1997. [18] Dean, R. T.; Fu, S.; Stocker, R.; Davies, M. J. Biochemistry and pathology of radical-mediated protein oxidation. Biochem. J. 324(Pt 1):1–18; 1997. [19] Hawkins, C. L.; Davies, M. J. Generation and propagation of radical reactions on proteins. Biochim. Biophys. Acta 1504:196–219; 2001.

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