Roles for the Ubiquitin-Proteasome Pathway in Protein Quality Control and Signaling in the Retina: Implications in the Pathogenesis of Age-Related Macular Degeneration

Abstract

The accumulation of damaged or postsynthetically modified proteins and dysregulation of inflammatory responses and angiogenesis in the retina/RPE are thought be etiologically related to formation of drusen and choroidal neovascularization (CNV), hallmarks of age-related macular degeneration (AMD). The ubiquitin proteasome pathway (UPP) plays crucial roles in protein quality control, cell cycle control and signal transduction. Selective degradation of aberrant proteins by the UPP is essential for timely removal of potentially cytotoxic damaged or otherwise abnormal proteins. Proper function of the UPP is thought to be required for cellular function. In contrast, age- or stress induced- impairment the UPP or insufficient UPP capacity may contribute to the accumulation of abnormal proteins, cytotoxicity in the retina, and AMD. Crucial roles for the UPP in eye development, regulation of signal transduction, and antioxidant responses are also established. Insufficient UPP capacity in retina and RPE can result in dysregulation of signal transduction, abnormal inflammatory responses and CNV. There are also interactions between the UPP and lysosomal proteolytic pathways (LPP). Means that modulate the proteolytic capacity are making their way into new generation of pharmacotherapies for delaying age-related diseases and may augment the benefits of adequate nutrition, with regard to diminishing the burden of AMD.

Introduction

The ubiquitin proteasome pathway (UPP) is a highly selective proteolytic system that plays crucial roles in protein quality control, cell cycle control, proliferation, development, signal transduction, transcriptional regulation, receptor down-regulation, and synaptic plasticity [1–12]. The observation that the retina, like many other tissues, accumulates damaged proteins upon aging, particularly in disease- related drusen [13–18], indicates that protein quality control is essential for retina heath. In this review we discuss the role of the UPP in retinal protein quality control and its implications in pathogenesis of age-related macular degeneration (AMD). Because of established crucial roles for the UPP in organogenesis and homeostasis, we also review the functions of the UPP in regulating signal transduction and responses to oxidative stresses in the retina/RPE as well as relationships to inflammatory response and choroidal nevasuclarization (CNV).

The simplest description of the UPP is as a two step process: identification of a substrate by ubiquitination and then degradation or processing of the ubiquitinated protein. The UPP begins with the attachment of a ubiquitin to a substrate protein [19, 20]. Ubiquitin is a highly conserved 76–amino acid polypeptide. Covalent attachment of ubiquitin to the protein substrate proceeds via a three-step cascade mechanism [8]. As illustrated in Figure. 1, ubiquitin is first activated by the ubiquitin-activating enzyme, E1, via formation of a high-energy thiol ester bond between C-terminal glycine residue of ubiquitin and an internal cysteine residue of E1. Next, one of dozens of ubiquitin-conjugating enzymes, E2s, transfers the activated ubiquitin, also via a ubiquitin thiol ester intermediate, to the substrate that is specifically bound to a member of the ubiquitin-protein ligase family, E3s. The E3 catalyzes the formation of an isopeptide bond between a carboxyl group at the C-terminus of ubiquitin and an ε-amine group of the substrate. In rare cases ubiquitin is transferred to the amino terminus of substrates, forming a peptide bond. There are two classes of E3s. One is called HECT, or homologous to the E6-associated protein carboxyl terminus of E6AP. Another is called RING E3, after really interesting gene. The latter often have multisubunit structures.

Figure 1. The Ubiquitin Proteasome Pathway.

Ubiquitin is activated in an ATP-requiring reaction that results in a Ub–“thiol ester”–of E1. The Ub is then transferred to E2 also as a thiol ester. In the presence of E3, substrate proteins have multiple ubiquitins attached. These conjugates are recognized and (usually) degraded in an ATP-dependent manner by the 26S proteasome. Isopeptidases and ubiquitin C-terminal hydrolases release ubiquitins. The ubiquitin is usually reused.

There are two genes in the human genome that encode different isoforms of E1 and each form has a distinct preference for E2s [21–24]. There are at least 37 genes in the human genome that encode distinctive E2s [25]. The number of the genes encoding E3s is over one thousand [26, 27]. The diversity of E2s and E3s and the combinatorial possibilities of various E2 and E3 in various cellular contexts provide the molecular basis for the exquisite substrate specificity of the UPP.

For efficient targeting of the substrate to the proteasome for degradation, multiple ubiquitins are often conjugated to the initial ubiquitin moiety to form polyubiquitin chains [12–14]. The first ubiquitin is conjugated to the ε-amino group of an internal lysine residue of the target protein. In the process of formation ubiquitin chains, additional ubiquitins are progressively conjugated to an internal lysine residue or the N-terminus of the previously conjugated ubiquitin molecule. Ubiquitin has 7 internal lysine residues. Together with the amine group at the N-terminus there are 8 possible positions for the second ubiquitin to attach. For the third ubiquitin, there are 15 possible positions of attachment. Thus, the structures of polyubiquitin chains in cells can be highly diverse. Different linkages of the polyubiquitin chains may have specific functions [28–32]. For example, K48-linked ubiquitin chains are often involved in targeting substrate for proteasomal degradation [33, 34] whereas K63-linked polyubiquitin chains are often involved in signal transduction and DNA repair [35, 36].

Whereas some ubiquitinated proteins are not degraded by the proteasome, there are also proteins that are degraded by the proteasome independent of ubiquitination. Both the 26S proteasome and the 20S proteasome can degrade some proteins in the absence of ubiquitination [37–41]. We consider the proteasome-dependent, but ubiquitin-independent protein degradation as a non-canonical UPP [42–48].

Role of the UPP in protein quality control

One of best understood functions of the UPP is for protein quality control: by selective degradation of aberrant proteins [49–52]. Both canonical and non-canonical UPP participate in the protein quality control process [9, 11, 42–48, 53–63]. Protein quality control is crucial because it prevents the accumulation of potentially toxic unfolded or misfolded proteins. Protein quality control is a post-translational process which involves folding of newly synthesized proteins and either refolding or degradation of proteins that fail to attain or maintain a native structure. Postsynthetically damaged or obsolete proteins must also be removed, most frequently by the UPP. A growing body of evidence indicates that accumulation of damaged or abnormal proteins is associated with various age-related diseases such as Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, cataract and AMD [16, 59, 64–71].

The accumulation of damaged or abnormal proteins may arise from their enhanced generation or deficiencies in repair or removal. The generation of abnormal proteins may be due to genetic mutation, failure to properly fold, or denaturation induced by various environmental stresses, including heat, oxidation, heavy metals, and light exposure. Unfolded or partially unfolded proteins are structurally unstable, and they tend to aggregate and precipitate. Accumulation of aggregation-prone proteins interferes with normal cellular functions including inhibiting or limiting access to the proteasome [16, 72–74]. To avoid disruption of cellular function by unfolded proteins, organisms have evolved multiple levels of protein quality control systems which recognize proteins with abnormal structures. These protein quality control mechanisms either refold the unfolded proteins to normal conformation or target them for degradation. Whereas molecular chaperones play central roles in the refolding of misfolded or unfolded proteins, intracellular proteolysis is responsible for the removal of unrepaired or obsolete proteins.

The majority of intracellular proteins are either degraded by the UPP or by the lysosomal proteolytic pathway (LPP). Acting independently, these two proteolytic pathways have different substrate specificities [75]. In general, short lived regulatory proteins, including damaged proteins or otherwise partially unfolded proteins are degraded by the UPP. Proteins degraded by the UPP normally contain intrinsic degradation signals (also called degrons). These signals are highly diverse, ranging from unique peptide sequences to specific domains or single amino acid residues at the N-terminus of the proteins [76]. Certain modifications of internal residues such as methionine oxidation and protein carbonyl formation also increase the susceptibility of certain proteins to UPP-mediated degradation [53, 58, 61, 77]. Many membrane-bound or organelle-associated proteins are degraded by the LPP, either through endocytosis or autophagic mechanisms. Recent studies indicate that the UPP and LPP can also work in a coordinated manner, particularly when cells are stressed [16, 75, 78]. For example, oligo/monoubiquitination of receptor and other membrane proteins is required for efficient endocytosis [79, 80].

The selective degradation of abnormal proteins has been known for about 30 years [53, 56, 81, 82], and the role of the UPP in this process has been proposed since the discovery of this pathway [9, 11, 53–59, 61]. For example, various forms of abnormal proteins, such as mutant [83–87], truncated [62], deamidated [60], denatured [88, 89] and oxidized proteins are degraded in a proteasome-dependent manner [42, 53, 58, 61, 77, 90–95]. However, the mechanism by which the UPP distinguishes abnormal from native proteins remains an unsolved mystery. Although the 19S regulatory complex of the 26S proteasome interacts directly with denatured proteins [96–99], most proteins degraded by the proteasome are first tagged by a polyubiquitin chain. In most cases the E2s and E3s are jointly responsible for substrate recognition and ubiquitination [8, 20]. Thus, these enzymes are essential in selecting proteins for degradation. Interestingly, mutations in specific E2s lead to defects in degradation of proteins with different classes of hydrophobic degradation signals, suggesting that the UPP recognizes exposed hydrophobic regions in some proteins [100].

Recent studies indicate that molecular chaperones, in particular Hsp90/Hsp70, are utilized by the UPP to recognize abnormal proteins [88, 89, 101]. As indicated above and in Figure 2, various modifications may alter protein conformation, resulting in the exposure of hydrophobic regions. These are normally buried in properly folded proteins. Such hydrophobic regions may provide a signal for the discrimination of denatured or unfolded proteins from native proteins by Hsp90 or other chaperones [102]. The C-terminus of Hsc70-interacting protein (CHIP), a co-chaperone with ubiquitin ligase activity, can target chaperone-bound substrates for degradation. CHIP contains a C-terminal U-box domain and three N-terminal tetratricopeptide repeat (TPR) domains. The C-terminal U-box domain is responsible for the ubiquitin ligase activity and the N-terminal TPR domains are responsible for the interaction with Hsp70 and Hsp90. By interacting with molecular chaperones (Hsp90 or Hsp70) and ubiquitin conjugating enzymes (Ubc4/5), CHIP catalyzes the ubiquitination of chaperone-bound proteins. Thus, CHIP functions as a bridge or molecular switch between the refolding and degradation arms of the protein quality control system. In addition to CHIP, other chaperone-interacting ubiquitin ligases, such as parkin, also participate in ubiquitinating misfolded or unfolded proteins [103].

Figure 2. Recognition and degradation of oxidatively modified proteins by the UPP.

This model predicts that most, if not all, proteins have intrinsic signals for interaction with molecular chaperones or the ubiquitination system. These signals (red) are hidden in properly folded native proteins and they are not recognized by the protein quality control systems. Upon environmental stress, such as oxidation, proteins may be unfolded with exposure of the recognition signals, such as hydrophobic patches. Some of the oxidized (unfolded) proteins can be recognized and degraded by the 20S proteasome directly whereas others are recognized by molecular chaperones. With the help of other chaperones or co-factors, molecular chaperones are capable of refolding the denatured proteins in an ATP-dependent manner. If the denatured proteins cannot be refolded rapidly, the chaperone bound substrates are ubiquitinated (yellow triangles) by chaperone-interacting ubiquitin-ligases, such as CHIP. The ubiquitinated substrates are recognized and degraded by the 26S proteasome. If the ubiquitinated proteins are deubiquitinated by isopeptidases, the denatured proteins have a second chance to be refolded by molecular chaperones. The parallel/competitive functional relationship between the UPP and molecular chaperones assures the efficiency of the protein quality control systems to remove abnormal proteins.

A common goal of the UPP and molecular chaperones is to prevent the accumulation of abnormal proteins. Both pathways do so by recognizing proteins with abnormal structures. However, to date it is not possible to predict why some denatured proteins are refolded by chaperones whereas others are degraded by the UPP. It appears that cellular protein quality control systems have a triage mechanism. The first level of triage must be identification of the proteins that are damaged and require repair or removal. The quality control system must be able to distinguish between native (properly folded, and assembled) proteins and everything else that might be considered non-native or abnormal, including partially unfolded, misfolded, incorrectly modified, unassembled subunits of complexes, or proteins in incorrect cellular compartments. Once damaged proteins have been identified, a second decision must be made: Is the protein repairable? In principle, chaperones should have the first opportunity to repair of damaged proteins. The proteins that are damaged beyond repair could then be degraded by the UPP.

How is the decision made to refold or to degrade a protein? The ability of the UPP and chaperones to interact with the same damaged or misfolded protein allows these pathways to operate in a parallel or competitive manner to either refold or degrade a given target protein (Figure. 2). We and others propose that the fate of the damaged protein depends on the kinetics and affinity of interaction between the damaged proteins and molecular chaperones or the chaperone component of the UPP as well as upon the availability of the proteolytic machinery [16, 51, 52, 89]. If the chaperone-bound nonnative protein is efficiently refolded with the assistance of other cochaperones, such Hsp40 and p23, it can be removed from the triage system and returned to cellular function. However, if the chaperone-bound nonnative protein is not efficiently refolded by chaperones, the co-chaperone CHIP, which interacts with both chaperones and Ubc4/5 or other E2s, can bring the chaperone-bound proteins to the ubiquitination machinery. If the ubiquitinated proteins are deubiquitinated by isopeptidases, including those associated with the 26S proteasome, the denatured proteins would have a second chance to be refolded by chaperones. Thus, the relative activities of ubiquitination and deubiquitination also participate in determining the fate of denatured proteins [104].

The parallel or competitive mode of action between the UPP and chaperones in the protein quality control process appears to be energy inefficient, resulting in the undesirable destruction of some repairable proteins. This may explain why ~30% of nascent proteins are degraded before they are folded properly, sometimes with dire consequences [105]. For example, premature degradation of inefficiently folded mutant cystic fibrosis transmembrane conductance regulator (CFTR) (ΔF508) causes cystic fibrosis [83]. CHIP is one of the ubiquitin ligases that target the mutant CFTR for ubiquitination and degradation [106, 107]. Blockage of the ubiquitination and degradation of CFTR by inactivation of CHIP allows a pool of CFTRΔF508 to be correctly folded and partially restores cellular function [108]. Similarly, blocking the premature degradation of mutant glucocerebrosidase (N370S or L444P) by proteasome inhibitors enhances its correct folding and partially corrects the loss of function phenotypes of mutant glucocerebrosidase [68]. From a teleological perspective, the cost of destruction of partially functional and functional proteins can be justified by efficient elimination of gain of function toxic mutants or damaged proteins.

Another interesting aspect of regulation of the UPP-chaperone axis is that if one system fails, the other system can serve as the backup. For instance, it has been shown that inhibition of the UPP results in up-regulation of heat shock proteins [109–112]. Conversely, inhibition of chaperone activity also promotes the degradation of Hsp90 client proteins by the proteasome [113]. If both arms of the UPP-chaperone axis fail, the damaged proteins, including ubiquitinated form of damaged or modified proteins, will be shuttled to lysosome for degradation or they may accumulate in the insoluble fraction of the cell [16].

The physiologic requirement for a functional UPP for cellular protein quality control and responses to various stresses is indicated by observations that disruption of UPP in cells leads to hypersensitivity to various environmental insults, such as UV light, oxidation, heavy metals and amino acid analogs [55, 61, 114–119]. All these insults, like other protein precipitation or amyloid diseases, are associated with accumulation of damaged proteins and it is believed that the increased sensitivity to these insults is related to failure to remove the damaged proteins in a timely fashion. Various forms of damaged or modified proteins in the eyes are also selective degraded by the UPP and dysfunction of the UPP may contributes to age-related diseases, such as cataract and AMD [16, 56, 60, 120–122].

Interaction between the UPP and LPP in degradation of glycated proteins

Autophagy is a highly conserved housekeeping pathway that plays a critical role in the removal of damaged intracellular organelles or aberrant proteins that were not efficiently degraded by the UPP [16, 123, 124]. Other lysosomal proteolytic capacities may also be involved in recognition and removal of damaged proteins. In this article, these are included within the abbreviation LPP. Proteins that are cleared by autophagic processes are encapsulated into autophagosomes and make their way into lysosomes for degradation [125–130]. Efficient autophagy is dependent on a balance between the formation and elimination of autophagosomes. Disruption of any part of this pathway will cause autophagic dysfunction. There are few reports regarding autophagy in retina diseases, but it appears that a decline in autophagy and lysosomal activity are associated with retinal aging and AMD [131–133].

Both the UPP and LPP are involved in selective degradation of damaged proteins. Whereas the UPP is most efficient in the degradation of soluble cytoplasmic proteins, the LPP mainly degrades damaged organelles or protein aggregates. However, there is overlap for some substrates between the UPP and LPP. It appears that the UPP and LPP complement and back-up each other in removal of damaged proteins. Such a backup mechanism was indicated in a recent study regarding the degradation of glycated proteins in cultured RPE [16].

Glycation is an adverse posttranslational modification related of eye diseases. Non enzymatic attachment of sugars, or their reactive metabolites such as methyl glyoxal, alters the activity and stability target proteins [134, 135]. Glycated proteins and advanced glycation end products (AGE) accumulate at accelerating rates upon aging in the retina, specifically in drusen [13, 14, 18], as well as in the aging lens [16], and the process is accelerated under diabetic conditions [13, 136]. Consuming high glycemic index diets, one of major risk factor for development of AMD in humans [137–141], also accelerates the accumulation of AGE in eye tissues [15, 16]. Glycation induces irreversible changes in protein structures and leads to loss activity or aggregation and toxicity [136]. We determined the effects of glycation on protein degradation and found that glycated crystallins were resistant to degradation by the UPP and that glycative stress also diminished the activity of several components of the UPP [16]. While it remains to be elucidated how glycation prevents the degradation of UPP substrates, one of the possibilities is that glycation modifies lysine residues of the substrates and blocks their ubiquitination [142].

In cultured RPE, both the UPP and LPP are involved in removal of MGO-modified proteins. Inhibition of the proteasome or lysosome alone only partially slowed the degradation of MGO-modified protein [16]. When both the proteasome and lysosome were inhibited, there is greater accumulation of MGO-modified proteins [16]. Furthermore, when the proteasome was inhibited, ubiquitin-conjugates and MGO-modified proteins were enriched in the lysosome fraction, indicating that glycated and ubiquitinated proteins were delivered to the lysosome when the proteasome is not functional [16]. The interaction between UPP and lysomome was also observed in lens epithelial cells [143]. We found the HNE-modified proteins were ubiquitinated, but degraded by the lysosome. In RPE, inhibition of the proteasome results in accumulation of perinuclear aggregates positive for Hsp70, ubiquitin-protein conjugates and the lysosomal membrane protein LAMP-2 [144]. Our work also indicates that impairment of the UPP in RPE activates the autophagic pathway as indicated by the increase in levels of LC3II upon inhibition of the proteasome, or expression of K6W-ubiquitin [16]. The interaction between UPP and LPP also provides an opportunity to manipulate the activities of these degradation pathways for AMD prevention or treatment. For example, chemical induction of autophagy may help to prevent the accumulation of ubiquitin-containing aggregates. Enhancement of UPP function may also reduce the burden of the LPP and thus promotes the degradation of lipofuscin and dysfunctional organelles, such as damaged mitochondria, by the LPP [133].

The interaction between UPP and LPP and its physiologic significance is summarized in Figure 3. This scheme proposes that under normal homeostatic conditions, the UPP and LPP functions independently or in concert to maintain the proteome homeostasis. The proper functions of the UPP and LPP prevent the accumulation of abnormal proteins or adversely modified proteins, such as AGEs. However, upon chronic stress and aging these capacities are insufficient to maintain the proteome, or are themselves compromised. This results in accumulation of damaged proteins. Degrons, or amino acid residues that encode susceptibility for degradation on substrates may be blocked by adverse modifications. Thus, upon severe chronic stress there is a vicious cycle starting with glycative insult to substrates or proteolytic components, accumulation of cytotoxic damaged proteins, altered interactions between the UPP and LPP. Together these result in compromised cellular function, possibly including the development of AMD-like lesions.

Figure 3. Scheme of interaction between the ubiquitin-proteasome pathway (UPP) and lysosomal proteolytic pathway (LPP) in protein quality control and proposed ramifications of dysfunctions of these proteolytic pathways.

Environment stress, including consuming high glycemic index diet, causes protein denaturation or unfolding, resulting in exposure of degradation signals (deg) and triggering their ubiquitination and degradation. Majority of ubiquitinated proteins are degraded by the proteasome, but some ubiquitinated proteins are degraded by lysosome via autophagy, particularly when the function of the proteasome is impaired or overburdened. The rapid degradation of damaged proteins by concerned action of the UPP and LPP prevents the accumulation of cytotoxic damaged proteins and maintains the homeostasis of the proteome, such as those observed in young and healthy tissues. When the levels of damaged proteins overwhelmed the capacity of the UPP and LPP, damaged protein will accumulate. Some of the damaged protein may cross link forming the higher mass aggregates noted in the figures above. Accumulated oligomerized damaged proteins may impair the proteolytic machinery, further compromising the proteome and resulting in diseases related to accumulation of damaged proteins, such in the retina/RPE of AMD patients.

Role of the UPP in degradation of oxidized proteins

The retina has the highest metabolic rate and oxygen consumption in the body [71, 145]. The high metabolic rate and oxygen consumption is usually accompanied by generation of reactive oxygen species. Chronic exposure to light also increases the production of reactive oxygen species [146, 147]. Age-related accumulation of lipofuscin in RPE is another source of oxidative stress (Also see reviews by Sparrow, Mettu, Handa, Boulton in this issue). Lipofuscin is a mixture of non-degradable protein-lipid aggregates derived from the ingestion of photoreceptor outer segments [148]. A2E is a major fluorophore of lipofuscin and acts as a photosensitizer to generate reactive oxygen species inside the cells upon exposure to blue light [148–150]. An increasing body of literature indicates that oxidative stress is associated with the pathogenesis of AMD [151, 152]. Thus, relationship between oxidative stress and selective degradation of oxidized proteins is of keen interest to AMD researchers.

Literature regarding the degradation of oxidized proteins in retina and RPE is scanty. Most data regarding the degradation of oxidized proteins were collected in lens or cultured lens epithelial cells. H₂O₂ is a natural oxidant in the eye [153–155] and is often used to model oxidative stress in the eye. Exposure of proteins or cells to H₂O₂ results in generation of protein carbonyls, disulfide and non-disulfide-linked aggregates [58, 61, 90, 156–158]. We found that oxidized, as compared with native, crystallins are preferentially degraded [90, 156, 157, 159]. Preferential degradation of oxidized proteins was also observed in non-ocular cells. While there is consensus that the proteasome plays a key role in selective degradation of oxidized proteins [47, 48, 61, 63, 77, 92, 93, 160–166], the extent to which ubiquitination is involved in the degradation of oxidized proteins remains controversial. Many publications suggest that suggest that oxidized proteins are degraded by the 20S proteasome independent of ubiquitin [42, 45, 46, 91–95, 165, 167–170]. However, there is also solid evidence that supports the involvement of ubiquitination in degradation of oxidized proteins [53, 58, 61, 77, 90, 104, 171].

Evidence that support the involvement of the UPP in degradation of oxidized proteins includes the preferential ubiquitination of certain oxidized proteins [53, 58, 61, 77, 171] and accumulation of ubiquitinated species of oxidized proteins upon proteasome inhibition [58, 61]. The stabilization of ubiquitinated species of oxidized proteins by proteasome inhibitors indicates that these ubiquitin conjugates are en route to proteasomal degradation. A transient increase in levels of ubiquitinated proteins that correlated with increased intracellular proteolysis during recovery from mild oxidative stress [11, 57] also implies that the UPP is involved in restoring protein homeostasis during recovery from oxidative stress. A recent study provided direct evidence for the involvement of ubiquitination in proteasomal degradation of oxidized proteins [104]. In that study, the authors demonstrated that USP14, a proteasome-associated deubiquitinating enzyme, is a negative regulator of UPP by its trimming of the ubiquitin chain of ubiquitinated substrates. They further demonstrated that inhibition of USP14 not only promoted the degradation of classic substrates of the UPP, but also promoted the clearance oxidized proteins and enhanced the resistance to oxidative stress [104], indicating that the UPP is involved in targeting oxidized proteins for degradation.

Glutathiolation is another type of oxidative damage. Glutathiolation results in formation of mixed disulfides in response to oxidative stress [172]. Glutathiolation is reversible when cellular reducing potential is restored. Glutathiolated protein was degraded more rapidly than unmodified protein by the UPP [63]. Consistent with the involvement of ubiquitination in the degradation of glutathiolated proteins, we found that addition Ubc4, a ubiquitin conjugating enzyme, to cell lysates promotes the degradation of glutathiolated γC-crystallin, whereas addition of a dominant negative ubiquitin to cell lysates inhibits the degradation of glutathiolated proteins [63].

While data regarding degradation of oxidized proteins in retina/RPE by the UPP is scanty at present, the conserved nature of functions of the UPP in most tissues examined suggests that comparable phenomena will be obtained with retina samples. Given the prominent etiologic relationship between oxidation, increase in levels of oxidized proteins in the neural retina and RPE, aggregate accumulation, and retinopathy, this should be a high priority [17, 173].

Role of the UPP in retinal development

A growing body of data shows that the UPP plays critical roles in mammalian and fly eye differentiation and development. A role for ubiquitination in neurite outgrowth was presaged by experiments in PC-12 cells [174] and developing Xenopus retinal ganglion cells [175]. The roles for the UPP in axon guidance or steering are also established in mammalian systems [7, 176].

Controlled degradation of transcription factors or signaling molecules by the UPP is essential for proper retinal development. For example, UPP-mediated degradation of microphthalmia-associated transcription factor (Mitf) is required for retina/RPE differentiation. Mitf plays essential roles in proliferation, survival, and differentiation of neural crest-derived melanocytes during retinal development [177, 178]. The degradation of Mitf depends on a specific degron called a PEST sequence [178]

Formation of the retina has been most extensively studied in Drosophila. The fly eye is composed of ~750 unit eyes called ommatidia, arranged in a hexagonal pattern. Each ommatidium has eight photoreceptor neurons and six support cells. During larval life, the eye disc changes from an undifferentiated epithelial sheet of cells to a highly patterned neuroepithelium through a series of ordered differentiation steps. The first cells to be specified are the clusters of photoreceptors, which will each give rise to an ommatidial unit. During late larval and early pupal stages, the photoreceptors retreat below the apical surface of the eye as they recruit the next cell type, the four cone cells, each of which in turn recruit two primary pigment cells. The remaining cells will form the interommatidial lattice of secondary pigment cells, tertiary pigment cells and mechanosensory bristles that separate neighboring ommatidia and set the precise hexagonal pattern of the adult eye. In order to allow for proper alignment following primary pigment cell differentiation, approximately 30% of the secondary and tertiary pigment cells must be removed by apoptosis. If these excess cells are not eliminated, the adult eye appears roughened because ommatidial units are separated by a varying number of cells.

The apoptotic editing of retinal cells is a highly regulated process. Many mutants which cause abnormal apoptosis and abnormal eyes are found to be related to aberrations in UPP function, indicating that the UPP plays an important role in regulating these apoptotic pathways. Some members of the inhibitors of apoptosis (IAP) are members of the E3 family and provide a critical barrier to apoptosis by binding to and inactivating caspases. For example, the drosophila IAP, DIAP, is an E3 that blocks apoptosis by promoting the ubiquitination and degradation of Dronc, an upstream caspase [179]. Caspase-mediated cleavage of DIAP1, at position 20 converts the stable, longer DIAP1 into the highly unstable, N-terminal Asn-bearing, DIAP1 [180]. An SCF is involved in degradation of DIAP and the F-box for this degradative process appears to be Drosophila Morgue [181, 182]. Reaper (Rpr) and Grim, but not Hid, also promote the degradation of DIAP1 in vivo [182]. Thus, the degradation of DIAP1 is mediated through sequential caspase activity and subsequently N-end rule-dependent UPP activity. This coordinated mutual destruction of DIAP and associated active caspases is essential for suppression of apoptosis.

Another family of E3s involved in retinal development includes the widely conserved SINA (seven in absentia) protein and its mammalian orthologs (Siah, seven in absentia homolog). These E3s facilitate degradation of a wide range of cellular proteins, including β-catenin, a protein with dual roles in development. β-catenin is a cell junction protein thought to link the actin cytoskeleton to cell–cell adhesion, and also a nuclear protein that modulates transcription in response to the Wnt signaling pathway [183, 184]. Over expression of the Xenopus homolog Xsiah-2 during development results in a small eye characterized by a reduced size of the lens, the retina, and the pigmented epithelium.

As indicated above, ubiquitination can also be a signal for endocytosis. The Notch receptor is part of an intercellular signaling pathway which is required for proper eye development [185]. The only known ligand for Notch expressed in the pupal eye is Delta. Neuralized acts as an E3, triggering endocytosis of Delta, and inhibition of Drosophila neurogenesis [186, 187]. Delta is expressed at high levels in cone cells and at lower levels in primary pigment cells and in some lattice cells [188]. Whereas polyubiquitination usually results in degradation of substrates, Delta appears to be monoubiquitinated at the cell surface [186, 187]. Such monoubiquitination is consistent with Delta being endocytosed and delivered to lysosomes [79]. Planar cell polarity (PCP) is a common feature in many epithelia, reflected in cellular organization within the plane of an epithelium. In the Drosophila eye, Frizzled (Fz)/PCP signaling induces cell-fate specification of the R3/R4 photoreceptors through regulation of Notch activation in R4. Neuralized is asymmetrically expressed within the R3/R4 pair and the asymmetric expression is controlled in a Fz/PCP-dependent manner. The Fz/PCP-dependent neuralized expression in R3 ensures the proper R3/R4 specification [189].

Several additional E3s appear to be involved in signaling pathways which control eye development. Hedgehog and Decapentaplegic pathways direct the progressive differentiation of the eye imaginal disc. Levels of Hedgehog and Decapentaplegic are controlled by the transcription factor Cubitus interruptus (Ci). A HECT domain E3 regulates levels of Hedgehog and Decapentaplegic by regulating levels of Ci [190]. Levels of Ci, along with Arm, and CycE are also regulated by the SCF E3, with a Cul1 backbone [191]. In cells anterior to the morphogenetic furrow, Ci proteolytic processing requires the activity of the Nedd8-modified, Cul1-based SCF^Slimb E3 complex. Nedd8 is a ubiquitin-like protein and is involved in regulating SCF E3 function by modifying SCF components or Ubc3. In cells posterior to morphogenetic furrow, Ci degradation is controlled by a mechanism that requires the activity of Cul3, another member of the Cullin family. This posterior Ci degradation mechanism is activated by Hedgehog signaling. Cul3 suppresses Hedgehog signaling through downregulating the level of Ci. High-level Hedgehog signaling promotes Cul3-dependent Ci degradation, leading to the downregulation of Hedgehog signaling. This process is manifested in controlling cell proliferation during Drosophila retinal development. Consistent with a role for Cul3 in controlling levels of Ci and eye development, Cul3 mutation resulted in an accumulation of Ci and an increase in the population of interommatidial cells. This is corroborated by the finding that the phenotype of Cul3 mutation can be suppressed by depleting endogenous Ci [192].

Deubiquitinating enzymes or isopeptidases also play critical roles in retinal development. The first deubiquitinating enzyme found in retina is a product of the Uch-L1 gene, also known as PGP 9.5 [193–195]. Uch-L1 also exhibits an ubiquitin ligase activity [196]. The two opposing enzymatic activities of ubiquitination and deubiquitination control the degradation of α-synuclein and affect Parkinson’s disease susceptibility [197]. However, the physiological role for Uch-L1 in retina remains to be defined.

Another deubiquitinating enzyme, Uch-L3, displays 52% amino acid identity to Uch-L1 [198]. A murine Uch-L3 deletion mutant displays retinal degeneration, muscular degeneration, and mild growth retardation. In the normal retina, Uch-L3 was enriched in the photoreceptor inner segment. Curiously, the retina of Uch-L3-deficient mice showed no significant morphological abnormalities during retinal development. However, prominent retinal degeneration became manifested after 3 weeks of age with massive apoptosis of photoreceptors. Retina degeneration in Uch-L3 deficient mice was associated with an increase in immunoreactivities for manganese superoxide dismutase, cytochrome c oxidase I, and apoptosis-inducing factor in the inner segment, indicating Uch-L3 deficient may trigger mitochondrial oxidative stress [199].

Deubiquitinating enzymes are also involved in establishing the number of photoreceptors as well as in guiding the photoreceptor neuron projection to different synaptic layers in the brain. During eye development in Drosophila, axons from photoreceptor neurons 1–6 terminate in the lamina, while axons from photoreceptor neurons 7 and 8 pass through the lamina and stop in the medulla. As the photoreceptor neuron axons enter the lamina, they encounter both glial cells and neurons. Mutation of a ubiquitin hydrolase, nonstop, results in altered developmental cues from glial cells and disrupts targeting of photoreceptor neurons 1–6. Particularly, this suggests that ubiquitin-hydrolase activities in glial cells, provide the initial stop signal promoting growth cone termination in the lamina [200].

Fat facets is another essential deubiquitinating enzyme that limits to 8 photoreceptor cells in each facet of the Drosophila compound eye, by preventing the proteasomal degradation of specific substrates such as Liquid facets [201–204] and beta-catenin. Liquid facets (Lqf) or epsin, the eukaryotic homolog, appears to bridge 3 components: the cell membrane, a transmembrane protein to be internalized, and the core endocytic complex during endocytosis [205, 206]. Retention of liquid facets facilitates endocytosis in order to generate an inhibitory signal that prevents ectopic photoreceptor determination. It remains to be determined if neuralized and liquid facets activities are related with respect to functions in endocytosis. The enhanced endocytosis due to deubiquitination by the Fat facets protein is in contrast to enhanced endocytosis noted upon ubiquitination of many plasma membrane proteins [207]. Interestingly, Fam [208] the mouse homolog of Fat facets, can substitute for Fat facets in all of its essential functions in Drosophila [201]. Biochemical experiments implicate Af-6 and beta-catenin as substrates for Fam [209]. Af-6 is a tight junction protein thought to mediate the effects of Ras signaling on cell-cell junctions.

Role of the UPP in visual signaling

The UPP also plays important roles in visual signaling. Light signals are transduced via GTP-binding proteins such as transducin. Transducin and other GTP-binding proteins are essential heterotrimeric (Tα, β, γ) signaling molecules, which dissociate into Tα-GTP and Tβγ upon light activation by heptahelical membrane receptors such as rhodopsin. Ubiquitination of Tγ occurs when it is dissociated from Tα. Although only Tγ is ubiquitinated, the entire Tβγ subunit complex is degraded [210, 211].

Ubiquitination of Tγ is selectively catalyzed by UbcH5 and UbcH7. Interestingly, Tβγ association with the photoreceptor-specific protein phosducin following light-induced Tα, β, γ dissociation blocks Tβγ ubiquitination and its subsequent degradation. Conversely, inhibition of phosducin-Tβγ complex formation by phosphorylation of phosducin (by Ca2+/calmodulin-dependent protein kinase II) restores Tβγ ubiquitination and degradation. Thus, it appears that Tβγ is a substrate of the UPP and that phosducin serves to protect Tβγ following the light-dependent dissociation of Tα, β, γ [211].

These observations suggest a UPP-dependent mechanism in which the cycle of phosducin phosphorylation/dephosphorylation regulates the light sensitivity of the photoreceptor by controlling the amount of transducin available for activation. Since formation of the phosducin-T complex reduces the availability of free Tβγ for re-association with Tα GDP, (a requirement for subsequent transducin activation), such a mechanism might be important for photoreceptor light adaptation, during which potentially saturating levels of light input to photoreceptor cells are countered by desensitization [212]. A potential problem with this hypothesis is that most phosducin is localized not in the outer segments of photoreceptors, where rhodopsin is present and where phototransduction occurs, but in the inner segments, which are primarily responsible for the metabolic functions of the cell. This hypothesis and these data may be reconciled by the observation that upon continuous illumination, up to 90% of both Tα and Tβγ translocate from the photoreceptor outer segment to the inner segment [213].

Role of UPP in regulating signal transduction

Efficient and proper cell signalling are essential for retina health. One of the most important functions of the UPP is to regulate intracellular signaling process by conditional degradation of regulators of signaling pathways. [122, 214–217]. The signal pathways that are controlled by the UPP include, but are not limited to, Notch pathway [218, 219], Wnt/PCP pathway [220, 221], NF-κB pathway [216, 222], STAT pathway [223, 224], MAP kinase signal pathways [225–227] and hypoxia-induced signaling pathway [228, 229]. Proper regulation of these signal pathways is essential for development and visual functions. Dysregulation of these signal pathways may leads to retinal degeneration.

Role of the UPP in regulating NF-κB signaling pathway

Inflammation appears to be involved in the pathogenesis of AMD. NF-κB is a master regulator of genes that control immune and inflammatory responses [230] (see reviews by Gorin, Handa, Sparrow). Relationships between the UPP and the NF-κB pathway have been extensively investigated. The UPP is required for both activation and termination of this signal pathway [122].

There are several NF-κB proteins and all share an approximately 300-amino-acid Rel homology domain (RHD) that regulates DNA binding, nuclear localization and dimerization [231]. NF-κB family members form homo- and heterodimers, including p65/RelA, c-Rel, RelB, p105/p50 and p100/p52. NF-κB proteins are sequestered in the cytoplasm as latent complexes by inhibitory proteins, or I-κBs, that prevent NF-κB nuclear translocation and DNA binding [232]. Members of the I-κB family, consisting of I-κBα, I-κBβ, I-κBε, NF-κB1, NF-κB2, I-κBζ (also known as MAIL) and Bcl-3, all contain a series of ankyrin repeat domains that mediate binding with NF-κB subunits [233]. The NF-κB1 and NF-κB2 proteins, also known as p105 and p100, respectively, are precursor forms of the p50 and p52 NF-κB subunits [225].

There are two unique NF-κB signaling pathways, termed canonical and noncanonical. These have distinct biological roles [234]. The canonical NF-κB pathway plays an important role in innate and adaptive immunity and cell survival. In this pathway proinflammatory cytokines such as TNF-α or IL-1β, or pathogen-derived components such as bacterial lipopolysaccharide (LPS), trigger the rapid nuclear translocation of NF-κB subunits [230, 234]. A wide variety of stimuli activate canonical NF-κB signaling, all of which converge at the IκB kinase (IKK), consisting of catalytic subunits IKKα and IKKβ and the regulatory subunit IKKγ (also known as NEMO) [235, 236]. IKK phosphorylates I-κB proteins, triggering their ubiquitination and degradation by the proteasome, thus allowing NF-κB to enter the nucleus and activate stimulus-specific gene programs [237].

Ubiquitination is also involved in activation of IKK. For example, during TNFα-induced NF-κB activation, TNFα binds to TNF receptor 1 (TNFR1) and results in ubiquitination of receptor-interacting protein 1 (RIP1) [238, 239]. TNF-receptor-associated factors (TRAF) are a class of ring finger E3s that play an important role in IKK activation. Activated TRAFs function as ubiquitin ligases (E3) for RIP1 ubiquitination through K63-linked polyubiquitin chain [240, 241]. A20 acts the deubiquitinating enzyme to disassemble K63-linked polyubiquitin chain. The polyubiquitin chains of ubiquitinated RIP1 recruit IKKγ/Nemo in the IKK complex [236, 241, 242]. This ubiquitination requires the dimeric E2 enzyme complex Ubc13/Uev1a as well as the transforming growth factor receptor-β-activated kinase 1 (TAK1) and its regulatory subunits TAB1 and TAB2 [35].

TRAF6 also undergoes oligomerization and K63-linked polyubiquitination. This leads to the recruitment of the TAB1-TAB2-TAK1 complex and subsequent activation of TAK1, which in turn activates IKKβ by phosphorylation [243, 244]. The activated IKK complex phosphorylates I-κBα at two sites (32 and 36) [245], triggering its ubiquitination and degradation [246–248].

UPP activity is also required for the regulation of the noncanonical NF-κB pathway. The MAP 3 kinase MAP3K14, also known as NF-κB inducing kinase (NIK) is a central player in the noncanonical pathway [249]. A multi-subunit E3 ubiquitin ligase complex consisting of TRAF2, TRAF3, cIAP1 and cIAP2 promotes the degradation of NIK, thus, maintaining it at extremely low levels in most cell types [250]. If NIK is not degraded in time, it phosphorylates IKKα, and triggers the proteasome dependent processing of NF-κB2 [251]. NF-κB2 is processed by the proteasome to generate p52, which together with RelB regulates a distinct subset of target genes [234].

In addition to regulating immune and inflammation responses, the NF-κB pathway is essential for survival. Many pro-survival genes, such as survivin, Bcl-2, and inhibitors of apoptosis, are under the control of NF-κB [252–254]. The activation of NF-κB in cultured ocular cells also requires UPP activity [121, 122, 255, 256]. We found that sustained oxidative stress impairs UPP function and inhibits NF-κB activation [256]. Inhibition of NF-κB activation during recovery from transient oxidative stress reduces the cell viability [256]. Inhibition of the UPP in RPE also suppresses the expression of NF-κB-regulated genes, such as MCP-1 and IL-6 [121, 257].

The role of the UPP in regulating expression of angiogenic factors

While establishing and maintaining blood flow in the inner and outer retina is essential for retinal health, unscheduled angiogenesis is a major problem, particularly in the aging or diseased retina. The hypoxia inducible factor (HIF) signaling cascade controls vasculature adaptations to hypoxia, including the formation of new blood vessels. HIF is an α-heterodimer that was first recognized as a DNA-binding factor that mediates hypoxia-inducible activity of the erythropoietin 3′ enhancer [258, 259]. In addition, the HIF signaling pathway is a key regulator of many other processes, including transcription of an array of pro-angiogenic factors, such as VEGF, TGF-β3, and various components of glucose transport and glycolysis. These are generally thought to be exploited to overcome vascular insufficiency [260–262]. Both the HIF-α and HIF-α subunits exist as a series of isoforms encoded by distinct genetic loci. HIF-α subunits are constitutive nuclear proteins, whereas HIF-α subunits are inducible by hypoxia. While regulation of HIF-α can occur at the mRNA level [258], it is primarily regulated through post-translational modification and UPP-mediated degradation of HIFα. Oxygen-dependent hydroxylation at two prolyl residues (Pro402 and Pro564 in human HIF-1α) mediates its interactions with E3, von Hippel–Lindau (VHL), which targets HIF-1α for rapid proteasomal degradation [263–265]. In addition to VHL, a recent study showed that CHIP, another E3, is also involved in degradation of HIF-1α in RPE. But CHIP-mediated ubiquitination and degradation of HIF-1α is independent of prolyl hydroxylation [266].

Deubiquitinating enzymes are also involved in regulating the stability of HIF-1α by removing ubiquitin from the substrate. For example, VHL-interacting deubiquitinating enzyme 2 (VDU2) interacts with HIF-1α and specifically deubiquitinates and stabilizes HIF-1α [267]. Thus, overexpression of VDU2 increases expression of HIF-1α targeted genes, such as VEGF. The balance between the VHL-mediated ubiquitination and VDU2-mediated deubiquitination of HIF-1α provides another level of control for HIF-1α stabilization.

Failure of degradation of HIF-1α increases the expression and secretion of VEGF and other pro-angiogenesis factors and abnormal angiogenesis, such as those observed in AMD and diabetic retinopathy [121, 268, 269]. The discoveries regarding HIF-1α signaling, together with new drugs that can control ubiquitination, deubiquitination and degradation provide new avenues for AMD therapy [270].

Redox regulation of UPP activity in eye

The capacity of the UPP to degrade proteins in cells or tissues is altered in response to environmental insults, including oxidative stress. It has been demonstrated in several cell types that exposure to various forms of oxidative stress results in a transient increase in the intracellular degradation of both short-lived and long-lived cellular proteins. An increase in substrate availability is one reason for the increased intracellular protein degradation in response to oxidative stress. Enhancement of ubiquitination and degradation capacities in response to oxidative stress is another reason for the increased intracellular protein degradation [11, 57, 91, 119, 167, 271, 272]. We demonstrated that both E1 and E2 activities of the ubiquitin conjugation system increase in response to mild oxidative stress [11, 58, 173] and Pickering et al demonstrated that the levels and activity of the proteasome, including immunoproteasome, increased upon adaption to mild oxidative stress [272]. The AMD-related increase in immunoporteasome in retina might be also related to oxidative stress [273].

Whereas mild oxidative stress up-regulates the activity of the UPP, all components of the UPP (E1, E2s, some E3s, proteasome and deubiquitinating enzymes) can be targets of extensive oxidative insults because they all have sulfhydryl groups as critical features of their active sites [10, 173, 274–280]. Exposure to oxidative stress rapidly depletes reduced glutathione (GSH) and elevates the levels of oxidized glutathione (GSSG). GSSG reacts with the cysteine residues in the active site of E1 and E2, forming mixed disulfide bonds, and blocking their binding to ubiquitin [10]. In addition, other types of modifications, such as S-nitrosylation, can inactivate these enzymes [275]. When oxidants are removed from the medium and cells are allowed to recover, the GSH level is restored, and the levels of ubiquitin conjugates, together with the ability to form the conjugates are also restored [10, 57, 274]. Moreover, after several hours of recovery from oxidation, there is hyperactivation of E1 and increased ubiquitination and proteolysis relative to levels found in untreated cells [11, 57, 281]. Thus, intracellular proteolysis can show a biphasic response to oxidative stress. Mild to moderate oxidative stress increases susceptibility of proteins to degradation and enhances the proteolytic capacity, therefore, promoting intracellular protein degradation. In contrast, extensive but non lethal oxidative stress impairs the functions of the UPP reduces intracellular protein degradation [10, 11, 57, 91, 167, 256, 274] and induces intracellular accumulation and aggregation of damaged or abnormal, potentially cytotoxic, proteins.

The regulation of ubiquitin conjugation activity by redox status was corroborated by treatment of RPE cells with diamide and these experiments also led to a new definition for an oxidative stress message. Diamide specifically oxidizes sulfhydryl groups and results in an elevation of GSSG/GSH ratios. It also caused dose-dependent inhibition of ubiquitination and ubiquitin-dependent protein degradation [274]. Thus, the GSSG/GSH ratio is the molecular redox message that regulates the cellular ubiquitin conjugating activity. The down-regulation of ubiquitin conjugating activity upon oxidative stress is due to reversible S-thiolation of E1 and E2 enzymes by increased levels of GSSG. The transient inhibition of the ubiquitin conjugation system by reversible thiolation may also have a protective role: by preventing ubiquitination and degradation of essential, reversibly modified, proteins upon oxidative stress [63]. It may also be perceived that glutathiolation of ubiquitin conjugating enzymes is a protective mechanism, which blocks the cysteine residue from irreversible oxidation and allowing for recovery of activity when redox status is restored.

The proteasome is also a target of oxidative stress. Reactive oxygen species and reactive lipid peroxidation products, such as 4-hydroxynonenal (HNE), impair the proteasome [95, 276–279, 282]. It appears that that the 26S proteasome is more susceptible than the 20S proteasome to oxidative inactivation [277]. The relative susceptibility of the ubiquitination system vs the proteasome to oxidative stress was also compared in RPE [167]. The proteasome is more susceptible to oxidative inactivation than the ubiquitination enzymes in RPE [173]. Sustained exposure to as low as 50 μM H₂O₂ resulted in 30–50% inactivation of the proteasome, whereas these levels of oxidative stress had no detectable effect on ubiquitin conjugation enzymes and ubiquitination activity. Given that the 26S proteasome is more susceptible to oxidative stress than the 20S proteasome [283], the loss of the peptidase activity of the proteasome upon oxidative stress is most likely due to inactivation of the 26S proteasome. Oxidative inactivation of the 26S proteasome prior to inactivation of the ubiquitination system may explain why levels of endogenous ubiquitin conjugates increase in response to oxidative stress in various types of cells [11, 57, 58, 61, 281, 284–289]. Age-related inactivation or inhibition of the proteasome [170, 290] may also contribute to the increased levels of ubiquitin conjugates in old tissues [284, 285, 291]. Constant light exposure also resulted in increase in levels of ubiquitin conjugates in retina [292], probably due to impairment of the proteasome. Furthermore, extensive oxidative insults may result in non-degradable cross-linked protein aggregates and these may interfere with the functions of the proteasome indirectly [74, 282, 293–297]. This would have the result of insufficient proteasome capacity even if the proteasome per se was not inactivated by the oxidative stress.

Interaction between the UPP and cellular antioxidant systems

Cellular antioxidant systems provide the primary defence mechanisms for cells to cope with oxidative stress via directly quenching reactive oxygen species. Just as the UPP is affected by cellular redox status, the UPP also regulates cellular redox status. Regulation of intracellular redox status by the UPP is mediated by degradation of nuclear factor-E2-related factor 2 (Nrf2), an important transcription factor involved in the transcriptional activation of antioxidant enzymes [298–302](see reviews by Pennesi and Handa in this issue). Nrf2 binds to the antioxidant-response element (ARE) and regulates ARE-mediated expression of antioxidant enzymes, such as catalase, superoxide dismutase, NAD(P)H:quinone oxidoreductase, certain forms of glutathione-S-transferases, and γ-glutamate cysteine ligase regulatory subunit [303–305].

As with many other transcription factors, the abundance of Nrf2 is regulated by UPP-mediated degradation [299]. The Kelch-like ECH-associating protein (Keap1) is a cytosolic inhibitor of Nrf2 [306, 307]. The main function of Keap1 is to serve as an adapter for the cullin 3-dependent E3 [308, 309]. Keap1 binds to Cul3 via its N-terminal BTB/POZ domain and binds to Nrf2 via its C-terminal Kelch domain [310], leading to the ubiquitination and degradation of Nrf2 through the 26S proteasome [298, 307, 311, 312].

Under normal cellular conditions, Nrf2 is constantly ubiquitinated by the cytosolic Keap1/Cul3/Rbx1 complex and degraded by the proteasome. When a cell is exposed to oxidative stress, Nrf2 dissociates from the Keap1/Cul3 complex and translocates into the nucleus, leading to activation of ARE-mediated gene expression [313].

A recent study indicates that a specific ubiquitin conjugating enzyme, UbcM2, acts as a redox sensor and a regulator of Nrf2 activation [314]. Oxidation of the cys-136 residue of UbcM2 increases its interaction with Nrf2 and results in Nrf2 stabilization. When the function of the UPP was impaired or inhibited, Nrf2 accumulated and the ARE is activated [311, 315–317]. The stabilization of Nrf2 with up-regulation of antioxidant enzymes explains why partial inhibition of the UPP results in cytoprotection under certain conditions [311, 315–320]. Nrf2 activation also enhances the expression of proteasome subunits and increases proteasome activity [321]. The activation of Nrf2 may explain why pre-treatment of cultured neocortical neurons with low doses of proteasome inhibitors led to increased, rather than decreased, proteasome activity [322].

In corroboration of essential roles for this antioxidant pathway in the eye, RPE are more susceptible to environmental stress in Nrf2 knockout mice [323, 324] and development of AMD-like lesions at later stage of life [325]. Additional elucidation of the interrelationship between the UPP and antioxidant system will bring new drug targets for AMD and other oxidation-induced diseases.

Impairment or dysregulation of the UPP: implications in AMD pathogenesis

Given the essential roles of the UPP in protein quality control and signaling transduction, it is anticipated that impairment or dysregulation of the UPP in retina/RPE would have several consequences. Impairment of the UPP may contribute to accumulation and aggregation of various forms of damaged proteins, such as those observed in subretinal deposits. However, dysregulated induction of UPP activity may promote the degradation of functional proteins and impair vision functions. For example, inflammation-related degradation of rhodopsin causes deficiency of this critical phototransduction protein and abnormal retinal function [326].

Accumulation of ubiquitin or ubiquitin conjugates is prominent in age-related sub-RPE deposits, i.e. drusen, basal laminar deposits (BLD) and inclusion bodies in retinas in patients with Ataxia type II [292, 327, 328], as well as in ganglion cells in rats with chloroquine retinopathy [329, 330]. The presence of ubiquitin in drusen—without active forms of ubiquitin-processing enzymes—raises the possibility that certain proteins become ubiquitinated within RPE but that further degradation of the ubiquitin-protein complexes does not occur [18, 330–332]. Several “amyloid diseases” are associated with accumulation rather than the timely degradation of ubiquitin conjugates derived from a myriad of, but as yet mostly uncharacterized, protein substrates. Lewy bodies are ubiquitin-containing inclusions in many premature neurological aging syndromes. Studies show that α-synuclein, a neuron specific molecule of unknown functions is a substrate for the UPP and is found within neurofibrilary deposits in brains of patients with Parkinson-dementia and abnormal eye function [333]. This corroborates the concept that limited UPP activity is causally related to the “amyloid diseases”. How these deposits are related to physiologic dysfunction is not known, but it would appear that drusen-related AMD can be included among these age-related “amyloid diseases”.

As in many other tissues, proteasome activity in retina decreases with aging [17, 334]. The age-related decline in proteasome activity may be a consequence of oxidative stress, since the decline in proteasome activity was accompanied by increased levels of oxidatively modified proteins, including nitrotyrosine and 4-hydroxy-2-nonenal (HNE) modified proteins [17]. In a cell culture system we demonstrated that the proteasome in RPE is more susceptible to oxidative stress than the ubiquitin conjugating system. Oxidative stress, including photo-oxidation, is often associated with accumulation of ubiquitinated proteins, both in neural retina and RPE [10, 122, 173, 217, 292]. Impairment of the UPP in RPE not only resulted in accumulation of oxidatively modified proteins [173], but also increased the expression and secretion of pro-angiogenesis and pro-inflammation factors [121, 122, 217]. Impairment of the UPP in RPE resulted in accumulation of HIF-1α and subsequently increased the expression of VEGF and other HIF-regulated genes [121]. The increased VEGF expression and secretion may trigger retinal and choroidal angiogenesis, such as those observed in wet AMD and proliferative retinopathy.

Prolonged inhibition of the UPP in RPE also triggers the expression of IL-8, a potent pro-inflammation and pro-agiogenesis factor [122, 217]. Disruption or up-regulation of NF-κB, p38 MAP kinase or PI3K signaling pathways also appears to be involved in the increased expression of IL-8 upon inhibition of the UPP [122, 217]. The inhibition of NF-κB signaling by proteasome inhibition is also related to the down-regulation of MCP-1, an important monocyte chemotactic protein [121, 335]. MCP-1 deficiency in mice causes AMD-like lesions, such as accumulation of lipofuscin in RPE, photoreceptor atrophy and CNV [336]. Thus, down-regulation of MCP-1 expression upon proteasome inhibition may be a factor for AMD pathogenesis. The relationship between impairment of the UPP and enhanced inflammation is not limited in the eyes. A recent study demonstrated that a mutation in the human proteasome subunit beta type 8 gene (PSMB8) that encodes the immunoproteasome subunit β5i was detected in patients with Nakajo-Nishimura syndrome, a disease with characteristics of chronic inflammation [337]. The β5i mutant is not efficiently incorporated during immunoproteasome biogenesis, resulting in reduced proteasome activity and accumulation of ubiquitinated and oxidized proteins within cells expressing immunoproteasomes [337]. As a result, the level of IL-6 and IFN-γ inducible protein-10 in patient sera is markedly increased. Levels of phospho-p38 and the secretion of IL-6 are increased in cells that harbor this mutant.

Despite much suggestive evidence, direct evidence for UPP impairment in AMD pathogenesis is still lacking. Ethen et al compared proteasome activity in retina from donor eyes with different grades of AMD and found that the chymotrypsin-like activity of the proteasome increased in neurosensory retina with disease progression [273]. Increased proteasome activity was correlated with a dramatic increase in the inducible subunits of the immunoproteasome, suggest the proteasome was transformed in retina of AMD patients, probably due to local inflammation or oxidative stress. Compared to the standard proteasome, the immunoproteasome has higher chymotrypsin activity [338], but the trypsin-like and caspase-like activity of the proteasome are comparable [338]. The transformation of the proteasome may be a mechanism for the increase chymotrypsin-like activity in the retina of AMD patients. Induction of immonoproteasome was also observed in retina and brain upon cytotoxic T lymphocytes induced damage [339]. The up-regulation of immunoproteasome in response to retinal and brain injury implies that the immonoproteasome may have a role in neuronal protection or and/or repair of damage. Consistent with the notion, immunoproteasome deficient RRE are more susceptible to oxidative stress [338].

Taken together, these data imply that age-or stress-related impairment of the UPP may contribute to pathogenesis of AMD and other age-related eye diseases. As illustrated in Figure 4, age-or stress-related impairment of the UPP may be etiologically linked to AMD pathogenesis via multiple UPP-requiring mechanisms.

Figure 4. The hypothesized molecular mechanisms for the potential link between impairment of the UPP in RPE and the pathogenesis of AMD.

Oxidative stress may reduce UPP activity in RPE. Impairment of the UPP in RPE decreases NF-κB and STAT transcription activity by stabilization of I-κB and SOCS, respectively. Impairment of the UPP enhances HIF activity by stabilization of HIFα. Impairment of the UPP also activates p38 and JNK MAP kinases via indirect mechanisms. Changes in activities of these signaling pathways will alter the expression of many genes, such as MCP-1, complement factor H, IL-8 and VEGF. The altered expression of these genes may trigger the development of AMD-related phenotypes, such as local inflammation, activation of the complement system, accumulation of drusen and choroidal neovascularization.

Protecting the UPP from oxidative stress may be a valid strategy for prolonging retina function

Age-or stress-related impairment of the UPP appears to be a contributing factor for the age-related eye diseases, such as AMD (Fig 4). Any means that can preserve or control the functions of the UPP should be beneficial. Since oxidative stress is major cause of AMD and dysfunction of the UPP, adequate dietary intake nutritional antioxidants may protect the UPP from oxidative inactivation (see review by Weikel in this issue). Indeed, we recently found that supplementation of lutein, zeaxanthin or vitamin E to cultured RPE can protect the proteasome from photooxidative inactivation in cultured RPE (Bian et al, manuscript submitted). Lutein and zeaxanthin supplementation also attenuated photooxidation-induced accumulation of ubiquitin conjugates and alteration in inflammatory response, such as expression and secretion of MCP-1, complement factor H and IL-8 (Bian et al, manuscript submitted). The protection of the UPP from oxidative inactivation may be one of mechanisms for the protective effects of lutein, zeaxanthin and other nutritional anti-oxidants in reducing the risk of AMD. Consuming low glycemic index diets would also help to preserve the function of the UPP via decreasing glycative/oxidative stress and reducing burdens of the UPP. In addition to antioxidant nutrients, pharmaceutical activators of the UPP or LPP may also be used to promote the degradation of abnormal proteins and reduce the risk for AMD.

Conclusions and future perspectives

The UPP is involved in regulating many cellular functions, from protein quality control to signal transduction and stress responses. A fully functional UPP is essential for eye development and maintaining the normal functions of the eye. Aging or stress-related impairment of the UPP in the retina/RPE appears to result in accumulation of abnormal or damaged proteins, which contribute to drusen formation and accumulation. Impairment of the UPP also disrupts signal transduction pathways, which in turn, alters inflammation responses and angiogenesis, common features of AMD-related lesions. Any means that can protect the UPP from impairment or reduce the burden of the UPP would be a beneficial for the eye health. Sufficient intake of nutritional antioxidants, such as lutein, zeaxanthin, vitamins C, E and zinc may protect the proteasome from oxidative inactivation and preserve UPP function in the eye, and reduce the risk for AMD. Consuming low glycemic index diets would also help to preserve the function of UPP via decreasing glycative/oxidative stress and reducing burdens of abnormal proteins on the UPP. The interaction between nutrition and UPP, as well as between UPP and other protective mechanisms warrant comprehensive investigation.