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The ubiquitin-proteasome system is integrally involved in a number of cellular processes and undoubtedly serves many important functions. Not least, it is heavily involved in the modulation of the function and plasticity of synapses, leading, in some cases, to neurodegenerative disease when there are aberrations in the system. Although the structure of the ubiquitin proteasome system is now largely understood a number of questions remain about the neuronal proteasome and its distribution and composition within the brain. Understanding how the neuronal proteasome is regulated is therefore of vital importance as it is responsible for regulated proteolysis of the majority of intracellular proteins and yet heavily regulated itself and therefore able to be controlled.
The ubiquitin proteasome system is a major pathway for protein turnover in eukaryotic cells. When ubiquitin, a 76 amino acid protein, is covalently attached to substrate protein, it tags it for proteasomal degradation. This is a very complex and highly regulated process.
The dynamic E1-E2-E3 enzymatic cascade, as seen in Figure 1 can be summarised in the following steps. Firstly, ubiquitin is activated by an ubiquitin-activating enzyme, otherwise known as E1. The E1 enzyme generates a high-energy thioester intermediate, E1-S~ubiquitin, in an ATP-dependent reaction.
This, secondly, encourages the association of E1 with an ubiquitin carrier protein, known as E2, by causing a conformational change in the E1 enzyme. The activated ubiquitin is transferred to the E2 enzyme which then dissociates from the E1 enzyme (Huang et al., 2007).
Fig. 1. The ubiquitin-proteasome pathway. (A) Conjugation of ubiquitin to the target molecule. (B) Degradation of the tagged substrate by the 26S proteasome. (Ciechanover, 1998)
Thirdly, the activated ubiquitin is transferred to a substrate specific E3 enzyme. In mammals there are hundreds of different E3 enzymes which are largely responsible for ubiquitination specificity, due to the large variety of E2/E3 combinations. Each separate combination will target a specific group of proteins (Ciechanover, 1998). The E2/E3 combination will also determine the number and type of ubiquitin chain linkages that are possible (Kim et al., 2007).
Fourthly, an isopeptide linkage is created between the C-terminal carboxyl group of the ubiquitin and the Îµ-amino group on the lysine of the target protein which facilitates the transfer of the activated ubiquitin to the substrate protein. This is part of the one reaction between the E3 ligase and the substrate protein-E2 ubiquitin complex. Extra ubiquitin proteins may be added to form a substrate-anchored polyubiquitin chain. Additional ubiquitins are added via one of seven possible ubiquitin lysine linkages, although the most common is a lysine 48 linkage. The polyubiquitin chains that form may be linear or branched, although branched chains with a high number of linkages appear to prevent the protein being targeted for proteasomal degradation (Kim et al., 2007). Multi-ubiquitination may also occur if polyubiquitin chains are added at multiple lysine residues in the substrate protein (Haas and Broadie, 2008).
The type and attachment of the polyubiquitin chain is important as it determines whether or not the protein will be targeted for protein degradation. For a protein to be targeted to the proteasome for degradation a minimum of four lysine 48 ubiquitins are required (Thrower et al., 2000). Polyubiquitin chains linked to lysine 29 or 63 are the exception to the rule as they are also thought to target a substrate protein for proteasomal degradation (Johnson, 1995).
Fifthly, the substrate protein with attached polyubiquitin chain is bound to the ubiquitin receptor subunit in the 19S complex of the mammalian 26S proteasome. Once attached to the proteasome the protein can be degraded to shorter peptides by the 20S complex. The mammalian proteasome, 26S, is a complex made up of a central, catalytic component, 20S, and two regulatory complexes, 19S (Glickman and Ciechanover, 2002) although these can be substituted with other regulatory complexes. The proteasome is designed to almost exclusively recognise proteins that have been tagged with ubiquitin by the UPS, as described above. Ornithine decarboxylase, however, is degraded without prior ubiquitination after association with its inhibitor enzyme (Ciechanover, 1998).
The 20S proteasomal core is a 700 KDa complex, that consists of two Î± and two Î² subunits. Each Î± and Î² subunit itself has 7 subunits making a total of 28 subunits. The Î± and Î² subunits are heptameric rings that are stacked axially in the order: Î±1-7Î²1-7Î²1-7Î±1-7 (Haas and Broadie, 2008). Within the Î² subunits lie three important catalytic sites: trypsin-, chymotrypsin- and post-glutamyl peptidyl hydrolytic like. While they Î± rings are catalytically inactive they are still important, as they stabilise the two inner Î² rings. Additionally, they are important in the binding of the 20S complex to the 19S regulatory complexes (Ciechanover, 1998). The 20S proteasome exists in several forms as several of the Î² subunits can be replaced by inducible subunits, after the cell has been stimulated with interferon-Î³. Î²1, Î²2 and Î²5 can be replaced by LMP2, MECL-1 and LMP7 respectively (Eleuteri et al., 1997).
The binding of the 19S regulatory complexes, also known an as PA700, is an ATP dependent reaction. The complex contains 20 subunits, six ATPase subunits - Rpt1-Rpt6 - and fourteen non-ATPase subunits - Rpn1-Rpn14. A non-ATPase subunit, Rpn10 binds to polyubiquitin via an ubiquitin interaction motif (Pickart and Cohen, 2004) and is responsible for substrate recognition in the proteasome, although it is thought that a supplementary, presently unknown subunit may also be involved (Ciechanover, 1998). 19S complexes are additionally involved in the regulation of the entry of the substrate into the proteasome (DeMartino and Gillette, 2007). It is thought that entry of the substrate into the proteasome is dependent on ATP-dependent structural rearrangement of the 20S proteasome complex subsequent to the association with the 19S complex, as there are no obvious entry pores in the 20S proteasome complex unlike in the Thermoplasma proteasome which contains an entry pore at each end of the cylinder (Ciechanover, 1998).
There are also several 11S (PA28) regulatory complexes that can replace the 19S regulatory complexes and associate with the 20S proteasome: PA28Î±, PA28Î² and PA28Î³ (Hill et al., 2002). Unlike association with the 19S complex, the formation is an ATP-independent reaction. However, the 11S-20S-11S complex will only digest peptides as they do not bind to polyubiquitin chains and so cannot digest the ubiquitin-conjugated intact proteins. It acts downstream to the 26S proteasome and breaks down large peptides created by the 26S complex. A hybrid, 19S-20S-11S proteasome also exists and has higher levels of proteolytic activity as it is able to perform proteolysis on poly-ubiquitinated proteins and then further break them down, as described above (Hendil et al., 1998; Cascio et al., 2002).
Sixthly, there is a recycling of ubiquitin. This final but important step releases ubiquitin and restores the cellular ubiquitin pool. Ubiquitin is released from the Lys residues of proteolytic products by de-ubiquitinating enzymes which can be divided into two main classes: ubiquitin C-terminal hydrolases (UCH) and ubiquitin-specific proteases (UBP), isopeptidases. UCHs are generally involved in the release of ubiquitin from small molecules: for example, amines. UBPs are much larger enzymes, ~100 kDa (as opposed to ~25 kDa) that catalyse the release of ubiquitin from larger cellular proteins or free polyubiquitin chains. It is thought that some even recognise distinct tagged substrates (Wilkinson, 1997). The action of these enzymes is also important for the proofreading of proteins that may have been ubiquitinated mistakenly. This inhibits proteolysis, but the majority of deubiquitinating enzymes stimulate proteolysis, by releasing ubiquitin into the cell, or by editing polyubiquitin chains and correcting them, so that they can then be recognised by the 26S proteasome and broken down.
The ubiquitin-proteasome system serves a number of important functions and is integrally involved in a variety of cellular processes. Perhaps most importantly, the UPS serves to modulate the function and plasticity of synapses (Yi and Ehlers, 2007; Tai and Schuman, 2008). Inhibition of proteasomal activity has been shown to impair synaptic plasticity and learning in animals (Fonseca et al., 2006; Lopez-Salon et al., 2001). The UPS is involved in the regulation of the synapse, both pre- and postsynaptically. Willeumier et al. (2006) describe the involvement of the UPS in the regulation of vesicle release presynaptically while Ehlers (2003) demonstrated the effect of the UPS on restructuring of the postsynaptic density (PSD).
The proteasome is also important in the removal of altered forms of protein, clearing damaged or misfolded proteins (Davies, 2001; Goldberg, 2003). Impairment of the activity of the UPS has implications for neurodegenerative diseases such as Parkinson's disease where intraneuronal aggregates of misfolded proteins, in this case Î±-synuclein build up (Bedford et al., 2008). These aggregates include ubiquitinated proteins, tagged for degradation. A similar example is Alzheimer's disease where aggregates or hyperphosphorylated tau are found (Rubinsztein, 2006).
Speaking more generally, a variety of cellular components are also regulated by the proteasome including steroid receptors, heat shock proteins, cytoskeletal proteins and apolipoprotein (Ciechanover, 1998). The UPS is also involved in the immune and stress responses of the cell (Zeng et al., 2005), signal transduction (May and Ghosh, 1998) and even the prevention of apoptosis (Breitschopf et al., 2000). The proteasome and the UPS as a whole are evidently critical for the correct functioning of the cell.Â
Little is known about the proteasome content of the brain. Tai et al. (2010) purified 26S proteasomes from rat cortex and identified the standard 26S subunits but also 28 proteasome-interacting proteins, thought to be regulators or cofactors. These differed to those found in other tissues, such as rat muscle, highlighting the importance of the need to investigate the neuronal proteasome further. Proteasome degradation serves a significantly different function in the muscle than the neuron and protein composition is also very different in the two tissues which would be why different proteasome-interacting proteins between the two tissues. Doubly-capped 26S, singly-capped 26S and 20S proteasomes were found in the rat cortex, and the level of doubly-capped 26S proteasomes was found to be significantly higher in the cortex than other tissue such as the kidney and liver.
However, there are differences in proteasome composition even within the brain. The ratio of doubly-capped 26S proteasomes to singly-capped (or hybrid) 26S proteasomes was significantly higher in cytosolic tissue than in synaptic tissue (Tai et al., 2010. Both doubly- and singly-capped 26S proteasomes serve to break down ubiquitinated protein (Kriegenburgh et al., 2008) but other functional differences are currently unclear. Although, Rechstainer and Hill (2005) demonstrate that a singly-capped 26S proteasome has a difference in structure, which exposes an Î± subunit on the 20S complex, which in turn allows other regulators (for example, PA28 or PA200) to interact with the proteasome. Again, the effect that regulatory factors have on the neuronal proteasome is not well understood but it is believed they may stimulate the breakdown of specific substrates. For example, PA28Î³ is thought to stimulate the ubiquitin-independent breakdown of the SRC-3, a transcriptional coactivator (Li et al., 2006). Perhaps, the singly-capped 26S proteasome when associated to various regulatory complexes actually has a more specialised role within different subcellular locations - for example, the synapse (Tai et al., 2010).
A study by Zeng et al. (2005) studied catalytic activity of the proteasome within the brain and found differential activity between different brain regions. Alterations in proteasome activity have been found in ageing animals, all three types (chymotrypsin- trypsin- and PGPH- like) of proteasome activity have been shown to decrease with age (Keller et al., 2000). Proteasomal activity was most significantly decreased in the substantia nigra which may be linked to a higher level of basal oxidative stress (Zeng et al., 2005). Although catalytic inactivation is age-related event some tissues are evidently more vulnerable to alterations in proteasomal activity than others.
There are several proteasome interacting proteins which are unique to the synaptic 26S proteasome. These may contribute to the regulation of proteolysis in a synapse-specific manner and include, amongst others, TAX1BP1, a ubiquitin-binding protein (Iha et al., 2008), and SNAP-25, a synaptic vesicle protein (DeBello et al., 1995). Tai et al. (2010) suggest that TAX1BP1 mediates substrate-proteasome association at the synapse and that SNAP-25 may be the means by which the proteasome controls synaptic vesicle release. Another proteasome interacting protein, ECM29 is present only in the cytosolic 26S proteasome, not the synaptic 26S proteasome. ECM29 is believed to stabilise the attachment of the 19S complex to the 20S complex (Kleijnen et al., 2007). It may also be involved in stimulating the creation of the doubly-capped 26S proteasome (Tai et al., 2010).
The proteasome is responsive to the needs of any one cell, as can be seen by concentrations of proteasome localised to concentrations of substrate. Furthermore, protein degradation and the UPS have been shown to regulate changes in synaptic strength and it is this that underlies synaptic plasticity. It is possible, therefore, that the UPS itself is regulated by synaptic activity.
Djakovic et al. (2009) demonstrate that the neuronal proteasome can be rapidly and dynamically regulated by synaptic activity. They used drugs such as tetrodotoxi (TTX) to put a blockade on action potentials in hippocampal neurons which resulted in an inhibition of proteasomal activity. Bicuculline, on the other hand, was used to up-regulate action potentials and resulted in a significantly increased proteasomal activity. The increased proteasome activity was a result, at least in part, of entry of calcium through NMDA receptors and L-type voltage-gated calcium channels (VGCCs). This is in turn, was dependent on the activity of calcium/calmodulin-dependent protein kinase II (CaMKII). CaMKIIÎ± acts to phosphorylate Rpt6, a subunit of the 19S regulatory complex of the 26S proteasome. Proteasome function can be significantly increased, therefore, by neuronal activity and it is apparent that CaMKII is a key regulator in this process.
Bingol and Schuman (2006) show in turn that neuronal activity affects the activity of the UPS in dendrites, not just synapses. As a result of synaptic stimulation, there was a redistribution of proteasomes: from dendritic shafts to synaptic spines. This is thought to be dependent on the activity of NMDA receptors. Therefore, synaptic activity regulates proteasomal activity locally, within the dendrites - as it stimulates both the recruitment and also the sequestration of proteasomes which results in a redistribution of the protein composition of the synapse.
Furthermore, Tai et al. (2010) show that the UPS activity of a neuron as a whole can be affected by neuronal activity. After treating neurons with NMDA, whole-cell lysates showed a decrease in the level of ubiquitin conjugates. This is possibly a result of either a decreased level of ubiquitination or an increased level of substrate degradation. Their results showed decreased levels of 26S proteasomes, which is thought to be a result of degradation of 19S complexes. When 19S particles dissociate from the 20S catalytic core, they can then be degraded by the proteasome, when induced by NMDA. This is a possible mechanism for suppression of proteolysis. Tai et al. (2010) also demonstrated a selective degradation of the 19S complex but the detailed mechanism is not full understood. Selective degradation of the 19S complex results in a shift to 20S proteasome complexes from 26S complexes. However, it is thought that 20S proteasome complexes cannot degrade ubiquitinated proteins in vivo alone as entry of protein substrates into its proteolytic chamber is blocked by the N-terminal residues of the Î±-subunits which make a "gate" (Smith et al., 2007). There is important evidence to suggest that the degradation of unfolded/oxidant-damaged proteins is dependent on a ubiquitin requiring mechanism that involves the 26S proteasome and VCP/p97 complexes (Medicherla and Goldberg, 2008). It is therefore unclear whether an increased number of individual 20S proteasome complexes improves the proteolytic function of a neuron.
There is also an increasing amount of evidence that the composition, and presumably function, of the neuronal proteasome may be regulated by levels of closely associated proteasome-interacting proteins. Studies have shown that posttranslational modifications: for example, glycosylation or phosphorylation have an effect on proteasomes in muscle cells, amongst others, but it is unknown whether the same effect is seen in the neuronal proteasome (Glickman and Raveh, 2005; Schmidt et al., 2005).
Perhaps most important are three E3s (KCMF1, HUWE1 and UBE3A) and five DUBs (USP5, USP7, USP13, USP14 and UCH37) (Tai et al., 2010) that are thought to improve the efficiency of proteasome function by determining substrate specificity or insuring that there is a rapid removal of released ubiquitin chains - they inhibit proteasomal activity (Finley, 2009).
There are hundreds of E3s and yet only a very few of them are associated with the neuronal proteasome and so can be considered to be important in the regulation of the proteasome. Knockout mice with UBE3A mutations show aberrations in the morphology of the synapse, glutamate receptor endocytosis and long-term potentiation (Greer et al., 2010). UBE3A mutations are also linked to Angelman syndrome, a genetic disorder associated with mental retardation. HUWE1 mutations are also linked to synaptic dysfunction and X-linked mental retardation. Synaptic defects in both UBE3A and HUWE1 mutations are possibly caused by altered function of the neuronal proteasome (Tai et al., 2010). When the 26S proteasome is exposed to NMDA, both UBE3A and HUWE1 dissociate and are degraded - a possible mechanism for regulation of the ubiquitin proteasome system.
In mammals both USP14 and UCH37, deubiquitinating enzymes (DUBs) have been shown to influence protein degradation as a result of their interaction with the 26S proteasome, catalysing the degradation of the ubiquitin chain and also the recycling of free ubiquitin (Koulich et al., 2008). USP14 also has an important regulatory role: it regulates "gate" opening in the 20S proteasome with the assistance of the ATPases (Peth et al., 2009). Animal models have shown that mutations of USP14 lead to defects in synaptic ubiquitination - these mice are called ataxia (ax1 mice) (Wilson et al., 2002). Chen et al. (2009) reported ax1 mice with reduced levels of free ubiquitin and ubiquitin conjugates and changes in short-term synaptic plasticity - this, therefore, may be due to the altered functioning of the proteasome that degraded the protein but do not recycle the ubiquitin.
The proteasome is an ATP-dependent protease that degrades polyubiquitinated substrates. Although it is evidently very important physiologically, its regulation and detailed structural organisation are still relatively poorly understood. However, the neuronal proteasome is clearly the subject of intense regulation and the increasing numbers of proteasome interacting proteins that have been identified suggest that regulation of the neuronal proteasome is even more complex and diverse than previously thought.