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Physiology 22: 373-379, 2007; doi:10.1152/physiol.00026.2007
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Physiology, Vol. 22, No. 6, 373-379, December 2007
© 2007 Int. Union Physiol. Sci./Am. Physiol. Soc.

REVIEW

Metabolic Control of Proteasome Function

Fengxue Zhang3, Andrew J. Paterson1,3, Ping Huang2, Kai Wang1 and Jeffrey E. Kudlow1,2,3

1 Departments of Physiology and Biophysics,
2 Pharmacology and Toxicology, and
3 Medicine, Division of Endocrinology, Diabetes and Metabolism, University of Alabama at Birmingham, Birmingham, Alabama kudlow{at}uab.edu


   Abstract
 
Proteasomes are major cellular proteases that are important for protein turnover and cell survival. Dysregulation of proteasome is related to many major human diseases. Regulation of the proteasome is beginning to be understood by the recent findings that proteasomes are modified and regulated by metabolic factors O-GlcNAcylation and PKA phosphorylation.


   Ubiquitin-Proteasome System and Its Regulation
Top
 Ubiquitin-Proteasome System and...
Metabolic Control of Proteasome
 Proteasome Might Play an...
 Proteasome Inhibition and Human...
 References
 
The proteome is in a dynamic state of synthesis and degradation. Controlling protein half life by destruction has emerged as a major cellular regulatory mechanism. The destruction process is carried out by two major proteolytic pathways involving either the lysosome or ubiquitin-proteasome system (UPS). The proteasome is an abundant major giant cellular organelle with protease activities that degrade intracellular proteins in an ATP-dependent manner. Not only does it remove abnormal proteins that may be misfolded, aged, or damaged by oxidation, it also regulates the half-life of the short-lived regulatory proteins such as cyclins that are involved in the control of cell cycle (27–29, 64, 74) and transcription regulators like β-catenin (1) and p53 (32, 49). Through its destruction function, the proteasome is thus involved in a variety of cellular processes such as protein quality control, cell cycle, antigen presentation, apoptosis, and cell signaling.

The intact 26S proteasome is composed of a cylindrical 670-kDa 20S core particle and two 19S regulatory particles at each end of the core cylinder. The core particle is composed of 28 subunits, {alpha} and β in type, which are arranged in four stacked heptameric rings ({alpha}1–7 β1–7 β1–7 {alpha}1–7). This core particle in the eukaryotic proteasome has three distinct catalytic activities: a chymotrypsin-like activity with preference for tyrosine or phenylalanine at the P1 position; a trypsin-like activity with preference for arginine or lysine at the P1 position; and a postglutamyl hydrolyzing activity with a preference for glutamate or aspartate at the P1 position. These catalytic activities each require an NH2-terminal threonine residue on their respective β-subunit to act as a nucleophile to coordinately cleave long proteins (23, 36, 44, 54, 81). The opening into the core is small enough that the protein substrates must be linearized before their entry into the catalytic cavity. The 19S regulatory particle (PA700) is 700 kDa in size and is composed of about 20 subunits. It binds to one or both ends of the 20S core particle. By recognizing and unraveling the ubiquitin-conjugated substrates (56) and perhaps by controlling the opening of the core particle (39), the 19S particle regulates the entry and degradation of the protein substrates in the proteolytic cavity of the core particle. The hexameric ring of the 19S particle that contacts with the outer {alpha} ring of the core particle is composed of six ATPases, which belongs to the AAA ATPase family. The six ATPases in the cap have been demonstrated to play important roles in the proteasome function, but they are not functionally redundant and have to work coordinately (67) to unfold the protein substrates and transport them into the proteolytic cavity of the 20S core (56), where proteolysis occurs.

The degradation process of proteins by the UPS is divided into two steps: first, a specific recognition process, employing the ubiquitin conjugation cascade (30), and second, an indiscriminate destruction process, mediated by the proteolytic activities in the 26S proteasome. The proteins destined for degradation must be tagged covalently with a polyubiquitin chain. Ubiquitin is a protein of 76 amino acid residues with glycine (Gly) at the COOH terminus. Polyubiquitin is accomplished by the sequential action of three enzymes: ATP-dependent ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin-protein ligase E3. This cascade covalently links the COOH-terminal Gly-76 to the {varepsilon}-amino group of an internal lysine (Lys) residue of the protein. In the first step, the carboxyl group of Gly-76 of ubiquitin is activated by ubiquitin-activating enzyme E1. This step involves hydrolysis of ATP and the covalent binding of ubiquitin to cysteine (Cys) in the active site of E1. After activation, the ubiquitin is then transferred to the Cys in the active site of E2. And, finally, the ubiquitin is shuttled to a protein substrate in cooperation with or without ubiquitin ligase E3 to form an amido isopeptide bond between the carboxyl group of Gly-76 of ubiquitin and an {varepsilon}-amino group of an internal Lys residue in the protein substrate. The polyubiquitin chain is formed by adding another molecule of ubiquitin to the Lys48 of the ubiquitin molecule already linked to the protein. Although there is only one E1 ubiquitin-activating enzyme that is highly conserved, E2 and E3 exist as a multimember family. It is the combination of E2 and E3 that determines the specificity of the ubiquitinylation system (23, 36, 44, 54, 81).

Although the structure and function of the proteasome have been extensively investigated (80), the regulation of its activity remains elusive. The emphasis on regulation of proteolysis by the UPS has been on substrate recognition through polyubiquitination. However, the proteolytic activity of the proteasome itself might also be regulated by posttranslational modifications such as phosphorylation. The proteasomal activity in muscle cells has been found to be stimulated during starvation (34). Phosphorylation has been recognized in both core and regulatory subunits of the proteasome (5, 8, 9, 50, 65). One function of phosphorylation in the core particle (9) or some subunits in the 19S regulatory particle (70) has been related to the assembly of the intact 26S proteasome. Phosphorylation patterns in the proteasome are changeable, as observed by Bose et al. They found that gamma-interferon decreases the level of 26S proteasomes by upregulating three immuno beta-catalytic subunits of the 20S proteasome and the PA28 regulator (7). This treatment also decreases the level of phosphorylation of two proteasome {alpha} subunits, C8 ({alpha}7) and C9 ({alpha}3). Subsequently, they found that phosphorylation of C8 by protein kinase CK2 is essential in the formation and stability of 26S proteasomes (9). Satoh et al. found that phosphorylation of an approximately 45-kDa proteasome subunit by an unknown kinase is associated with proteasome activity (69). In another study, they reported that phosphorylation of this subunit is required for its association with core alpha-subunit C3 (70). Phosphorylation might also change the activity of proteasomes in the cell. Bardag-Gorce et al. (5) found that ethanol ingestion caused an inhibition of the chymotrypsin-like activity of the purified proteasome and resulted in hyperphosphorylation of proteasomal subunits, {alpha}3/C9 and {alpha}7/C8. They also found that chymotrypsin-like activity was inhibited by okadaic acid treatment, a serine/threonine phosphatase inhibitor, similar to ethanol feeding. The 26S proteasome fraction examined by isoelectric focusing gel electrophoresis revealed many hyperphosphorylated bands in the proteasomes from both the okadaic acid-treated and the ethanol-fed rat livers (5). As might be expected due to its complex structure, the regulation of proteasome must be very complicated.


   Metabolic Control of Proteasome
Top
 Ubiquitin-Proteasome System and...
 Metabolic Control of Proteasome
Proteasome Might Play an...
 Proteasome Inhibition and Human...
 References
 
Recent studies have provided new insights into the regulation of 26S proteasomes. Sumegi et al. reported in 2003 that the 26S proteasomes in Drosophila were extensively posttranslationally modified by O-GlcNAc (75). We showed that mammalian proteasomes were also modified by O-GlcNAc (85) and that the 19S proteasome complex was inhibited by O-GlcNAcylation. In another recent study (84), we showed that the proteasome is stimulated by PKA phosphorylation. These findings provide evidence that the proteasome is under metabolic control, linking together two important intracellular processes, glucose metabolism and protein turnover.

Sp1 is a ubiquitous transcription factor that plays a vital role in the regulation of transcription from TATA-less promoters that generally encode housekeeping genes (58). Its activity is believed to be constitutive, but with fluctuations, since its stability in the cell is both proteasome and O-glycosylation related (24, 73). Sp1 has multiple O-glycosylation sites and becomes hyperglycosylated when cells are exposed to 5 mM glucosamine, whereas, under conditions of glucose starvation, stimulation with cAMP results in nearly complete deglycosylation of this protein (24). Correlating with this hypoglycosylated state, Sp1 is rapidly, proteolytically degraded by an enzyme(s) that can be inhibited by specific proteasome inhibitors, lactacystin and acetyl-leu-leu-norleucinal (LLnL), although it is stabilized in cells treated with glucose or glucosamine (24). This is also confirmed in a reconstituted in vitro system, which utilizes NRK cell nuclear extract (73). Sp1 was degraded in vitro by nuclear extracts derived from NRK cells under glucose starvation and forskolin treatment, and its degradation was blocked in nuclear extracts from NRK cells pretreated with glucosamine (73). In vitro degradation of Sp1 is ATP dependent and can be inhibited by proteasome-specific inhibitors lactacystin and LLnL. It has been demonstrated that the in vitro degradation of Sp1 in the NRK cell nuclear extract is a two-step process (73). Sp1 is first cleaved between residues leu56 and leu57 through endoproteolysis (44). The COOH-terminal fragment is then degraded by the proteasome, whereas the NH2-terminal fragment (SpX) remains intact. The characteristic degradation of Sp1 in NRK cell nuclear extracts is representative of proteasome function and can be used as an assay for the detection of proteasome activity in vitro. Proteasome activity can be detected by monitoring the change of the large Sp1 to the small SpX fragment.

In 2003, we reported that proteasome function is inhibited by O-GlcNAcylation (85). This modification of covalently adding a sugar moiety to the serine/threonine residues of proteins (16, 35) was the first report of an endogenous proteasome inhibitor. O-GlcNAc transferase (OGT) is the enzyme that adds the O-GlcNAc moiety to the protein (40, 41), and O-GlcNAcase is the enzyme that removes this modification (20, 22, 82). The substrate used by OGT, UDP-GlcNAc, is a product of the hexosamine biosynthesis pathway. As discussed above, we have shown before that the degradation of the transcription factor Sp1 in NRK cell nuclear extracts was found to be related to O-GlcNAcylation (24, 73). With extensive study, we found that the in vitro degradation of Sp1 was inhibited by pretreating NRK cell nuclear extract with OGT, regardless of the O-GlcNAcylation status of Sp1 itself. With the use of proteasome-specific fluorogenic peptide substrates, we found that the degradation of chymotrypsin-like peptide substrate suc-LLVY-AMC in the NRK cell nuclear extract was also inhibited by OGT pretreatment, evidence, as it turned out, that the resistance to Sp1 degradation was the result of proteasome loss of function due to O-GlcNAcylation. Interestingly, the degradation of another chymotrypsin-like peptide Z-GGL-AMC and trypsin-like peptide Boc-LSTR-AMC was not affected, which indicated that the proteolytic activity in the cavity of the proteasome core is intact. Using purified proteasomes, we were able to show that OGT acts on the 26S proteasome directly, although not on the 20S proteasome. Subsequently, it was shown that it was the ATPase activity of the 26S proteasome that was affected by OGT treatment, and more specifically, this inhibition is caused by the modification of Rpt2, one of the ATPases in the 19S cap. ATPase activity is necessary for the unfolding of protein substrates before their entry into the proteolytic channel in the core particle (39, 56), and that inhibition of ATPase activity will result in the blockade of the proteolytic function of the 26S proteasome. The inhibitory effect was confirmed in living cells by the use of a GFP fused to CL1 degron (GFP-degron) (6) as a reporter of proteasome-specific protein degradation, and GFP fluorescence accumulation was a measure of reduced proteasome function. Conversely, a knockdown of OGT by OGT-specific siRNA, lowered the O-GlcNAc modificaton of the proteasome and increased its activity, which resulted in the reduction of GFP-degron, whereas GFP alone was not affected. The inhibitory effect of OGT could be reversed byO -GlcNAcase.

The second major finding on proteasome regulaton, we reported in 2007, was that 26S proteasome function is stimulated by another important metabolic regulator, PKA phosphorylation (84). PKA is activated by cAMP and is involved in many cellular processes. It stimulates gluconeogenesis in liver and is a critical kinase in the maintenance of blood glucose level. Using the Sp1 degradation system, we found that the 26S proteasome can be modified by cAMP-dependent protein kinase (PKA) and that this modification greatly stimulated the function of 26S proteasome. The degradation of Sp1 and both chymotrypsin-like and trypsin-like peptidase activities in NRK cell nuclear extracts were increased by PKA treatment in vitro or by forskolin treatment in vivo. The PKA-specific inhibitor H-89 attenuates the stimulatory effect of PKA and forskolin on the proteolytic activity of proteasome. By using purified 26S proteasomes, it was shown that PKA acted on the 26S proteasome directly and stimulated both chymotrypsin-like and trypsin-like peptidases. In vitro 32P labeling showed that Rpt6 (Sug1), another AAA ATPase in the 19S regulatory cap, was the subunit being modified by the kinase. Mutagenesis studies of Rpt6 showed that the residue being modified was Ser120. Using GFP-CL1 (degron) as reporter, it was shown that forskolin treatment greatly reduced the half-life of the fluorescent protein and that co-transfection with the Ser120 to Ala (Sug1S120A) mutated Rpt6, acting as a dominant negative, and blocked its degradation. The stimulatory effect of PKA and the phosphorylation of Rpt6 were reversed by protein phosphatase 1{gamma} (PP1{gamma}).

Which of the two metabolic processes dominate over the proteasome: the inhibitory effects of O-GlcNAc modification by OGT or the stimulatory effects of phosphorylation by PKA? We have shown that the inhibitory effect of OGT dominates, regardless of whether PKA was applied before or after OGT. This suggests that, for PKA to be effective, O-GlcNAc modification needs to be removed. However, forskolin treatment was found to dramatically reduce the O-GlcNAc levels on all proteins in these cells (13). An explanation for the ability of PKA to regulate O-GlcNAc status is its phosphorylation of GFAT1 and the inhibition of its enzymatic activity (13, 33). The inhibition of GFAT1 reduces the conversion of glucose into glucosamine via the hexosamine biosynthesis pathway, resulting in less UDP-GlcNAc substrate for O-GlcNAc transferase (OGT). Thus the activities of OGT and PKA are mutually linked and, working in concert, provide an example whereby the function of the 26S proteasome can be modulated in response to extracellular metabolic signals.

PKA might also act on the 20S core particle. In 1990, Pereira et al. reported that PKA co-purified with the multi-catalytic protease complex (MPC) (60). At least two subunits of 28 kDa and 27 kDa were phosphorylated by PKA. In 1996, Marambaud et al. reported that the alpha-secretase activity of proteasomes in HEK 293 cells was stimulated in a PKA-dependent manner (50). PKA enhanced the phosphorylation state and the chymotrypsin-like activity both in vitro and in vivo. Zong et al. reported that multiple subunits in the 20S core particle, including {alpha}1 and β2, were targets of PKA (86). The cellular functions of these modifications by PKA are still not clear, although they may affect the assembly of the 26S proteasome.


   Proteasome Might Play an Important Role in the Maintenance of Blood Glucose Level
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 Ubiquitin-Proteasome System and...
 Metabolic Control of Proteasome
 Proteasome Might Play an...
Proteasome Inhibition and Human...
 References
 
The plasma glucose concentration has to be maintained. This is one of the functions of liver, so that tissues that are obligated for glucose utilization can receive this nutrient. The finding that proteasome function is under metabolic control might explain the origination of glycogenic amino acids in the starvation state (FIGURE 1Go). During starvation, PKA levels are elevated in both liver and muscle. Promoted by the elevated PKA, liver cells first engage in glycogenolysis to release free glucose, from glycogen, into blood vessels. However, when the stores of glycogen are depleted, the liver engages in gluconeogenesis to generate de novo glucose. One of the important sources for gluconeogenesis is glycogenic amino acids. Without the consumption of food, glycogenic amino acids can only be obtained from the degradation of proteins in peripheral tissues, and the principle source of proteins is muscle. To survive, the organism will sacrifice part of its muscle protein until enough food is found. Thus muscle protein can be regarded as another source for calorie storage (57). Mechanisms on how the muscle is signaled to release these glycogenic amino acids to the liver is beginning to be understood by our finding that proteasome function is affected by O-GlcNAcylation and PKA phosphorylation. During starvation, insulin levels are suppressed and counter-regulatory hormones are elevated. In muscle, the suppression of insulin results in less glucose transport, whereas the counterregulatory hormones elevate cAMP levels. With less glucose, there is less substrate for glutamine:fructose-6-phosphate (GFAT) to convert to glucosamine-6-phosphate. Furthermore, the elevated cAMP levels increase the activity of PKA. PKA in turn inactivates GFAT1 (13) by phosphorylation, causing even less entry of the glucose into the hexosamine pathway. Limited by the availability of UDP-GlcNAc substrate, OGT is relatively less active than the O-GlcNAcase, which causes the removal of the O-GlcNAc modification from proteins, and this results in proteins (13, 24, 73), including those in the proteasome (85), becoming less O-GlcNAcylated. By removing O-GlcNAc modification in Rpt2, PKA is able to stimulate the proteasome activity through the phosphorylation of Rpt6 at Ser120. The increased proteasome activity allows it to degrade ubiquitinated protein substrates in muscle (17, 18, 34, 51, 78, 83), providing the glycogenic amino acids to be used by the liver for gluconeogenesis. The converse applies in the fed state.


Figure 1
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  		FIGURE 1. Proteasome is under metabolic control and might be involved in the maintenance of blood glucose level

 

   Proteasome Inhibition and Human Diseases
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 Ubiquitin-Proteasome System and...
 Metabolic Control of Proteasome
 Proteasome Might Play an...
 Proteasome Inhibition and Human...
References
 
Inhibition of the proteasome can induce apoptosis (FIGURE 2Go). Apoptosis has been induced with the application of proteasome inhibitors in various mammalian cells, such as neurons (12, 59, 62), epithelial cells (48), aortic endothelial cells (21), vascular smooth muscle cells (37), and tumor cells (3, 31, 55, 72). The mechanism by which proteasome inhibitors induce apoptosis is unclear and may involve multiple factors. Apoptosis results from the activation of a set of cysteine proteases, the caspases. Two major apoptosis pathways have been found in mammalian cells (26). One is the death receptor pathway, which is triggered by the binding of CD95 ligand to CD95, followed by the activation of caspase-9 and caspase-3. The other pathway is via the mitochondria, which is triggered by the accumulation of p53 and the release of cytochrome c from mitochondria, leading to the activation of caspase-8 and caspase-3. Tumor suppressor protein p53 is normally ubiquitinylated and degraded by the proteasome (71). Mdm2 functions as the ubiquitin ligase and plays a major role in p53 ubiquitinylation and subsequent degradation (25, 32, 42). Accumulation of p53 will lead to cell cycle arrest and apoptosis. Pro-apoptotic factors such as Bax (14) and an active form of bid (11) are also substrates of the proteasome. Inhibition of proteasomes will cause the accumulation of these factors and the subsequent induction of apoptosis. Proteasome inhibition can also lead to the inhibition of NF-{kappa}B activation, which is involved in the transcription regulation of some anti-apoptotic factors such as IAP-1/2 and Bcl family members (76). Proteasome can be regarded as an anti-apoptotic factor in this way. It degrades pro-apoptotic factors but promotes the expression of anti-apoptotic factors. The accumulation of p53 (15, 47) and other pro-apoptotic factors such as Bax, followed by release of cytochrome c into the cytoplasm, and activation of caspase-mediated apoptosis (43) have all been documented in the proteasome inhibitor-induced apoptotic process. These findings strongly suggest that proteasome inhibition induces apoptosis through the mitochondria pathway. Another study has shown that MG132 and other proteasome inhibitors led to a steady increase in activity of c-Jun NH2-terminal kinase JNK1, which is known to initiate the apoptotic program in response to certain stresses (52).


Figure 2
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  		FIGURE 2. Inhibition of proteasome induces apoptosis
Green represents the survival pathway, and red represents cell death pathways. Proteasomes have an anti-apoptotic characteristic, activating the NF-{kappa}B pathway by degrading phosphorylated I-{kappa}B and by degrading pro-apoptotic factors such as p53, Bax, activated bid, and caspase-3, as indicated by the asterisk.

 
Since proteasome inhibitors can induce cell death, they have been proposed as a class of novel anticancer agents (2, 3, 38). The newly developed anticancer agent Bortezomib (originally PS341 and marketed as VELCADE by Millennium Pharmaceuticals) is a synthetic proteasome inhibitor. It is the first of the proteasome inhibitors to be approved by FDA for clinical use in the treatment of multiple myeloma (63, 68). Bortezomib is a modified boronic dipeptide with a molecular weight of 384.24 and formula C19H25BN4O4. It is substantially cytotoxic to a broad range of human tumor cells (3) and functions as an inhibitor of the 26S proteasome by selectively and reversibly inhibiting its chymotrypsin-like activity. This function allows the drug to inhibit the degradation of proteins critically involved in the regulation of cell proliferation and survival. These mechanisms have been most thoroughly investigated in myeloma cells in which disruption of these pathways also deregulates signaling molecules critical to the interactions between the myeloma cells and the bone marrow microenvironment, ultimately leading to growth inhibition and apoptosis (63).

However, although proteasome inhibitors have been used successfully to treat human cancer, severe side effects are expected due to the lack of selectivity among cell types. To have better control of the apoptotic process, it is necessary to inhibit proteasomes selectively in cancer cells. This might be achieved by extending the studies on proteasome regulation. The proteasome is a complicated complex consisting of about 50 subunits. It is imaginable that its regulation must be equally complicated. The finding that proteasomes are regulated by metabolic factors such as O-GlcNAc and PKA phosphorylation may be only the beginning for unravelling the complexity of its regulation. We expect that more factors involved in proteasome regulation will be characterized in the future and that these alternative factors might be able to inhibit proteasome function more selectively in a variety of cancer cells.

Although proteasome inhibition can be used to treat human cancer, it might also contribute to the development of human aging-related diseases. Proteasomes play important roles in the maintenance of proteomic homeostasis by the destruction of the oxidized or aged proteins. Inhibition of proteasome will cause the accumulation of the damaged proteins so as to affect the cell function and survival. Dysregulation of the proteasome has been documented in a variety of major human diseases such as neurodegenerative disorders (10, 19), cataracts (4), muscle atrophy (53, 61, 77), etc. Proteasome posttranslational modification might be an important contributor of this dysregulation. Although the modifications are universal, the role of these modifications of the proteasome may be different in diseases in different tissues. Although proteasome activity was found to be elevated in starvation-induced muscle atrophy, its activity might be depressed in the other diseases such as Alzheimer’s and cataracts. Without endogenous proteasome inhibitors, however, the underlying mechanisms for the dysregulation are difficult to unravel. Posttranslational modifications such as O-GlcNAc and PKA might provide a useful tool to study the consequence of proteasome inhibition in vivo. Increased O-GlcNAc, the endogenous inhibitor of proteasome (85), has been shown to induce apoptosis. Glucosamine and/or STZ treatment, an O-GlcNAcase inhibitor (66, 79) that causes the accumulation of O-GlcNAc, caused the accumulation of p53 and apoptosis in β-cells (45) and hippocampal pyramidal neurons as well (46), suggesting a role of O-GlcNAc inhibition of proteasome in β-cell failure and Alzheimer’s. Mutant Rpt6S120A has been shown to act as a dominant negative and inhibits proteasome function in the living cell (84). It might be used as a transgene to be expressed in specific tissues such as brain, β-cell, muscle, lens, etc. By inhibiting proteasomes, the role of PKA phosphorylation of proteasome subunit Rpt6 in human diseases will be observed. Protein aggregates, which are often seen in neurodegeneration, inhibit the function of the proteasome (6, 85). Perhaps advantage can be taken of an activated cAMP/PKA mechanism to overcome this inhibition and to develop as a series of new therapeutic agents.


   References
Top
 Ubiquitin-Proteasome System and...
 Metabolic Control of Proteasome
 Proteasome Might Play an...
 Proteasome Inhibition and Human...
 References
 

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