Levels Of Ubiquitin Conjugates Research Articles

Hsp70 and Hsp110 Chaperones Promote Early Steps of Proteasome Assembly

Whereas assembly of the 20S proteasome core particle (CP) in prokaryotes apparently occurs spontaneously, the efficiency of this process in eukaryotes relies on the dedicated assembly chaperones Ump1, Pba1-Pba2, and Pba3-Pba4. For mammals, it was reported that CP assembly initiates with formation of a complete α-ring that functions as a template for β subunit incorporation. By contrast, we were not able to detect a ring composed only of a complete set of α subunits in S. cerevisiae. Instead, we found that the CP subunits α1, α2, and α4 each form independent small complexes. Purification of such complexes containing α4 revealed the presence of chaperones of the Hsp70/Ssa and Hsp110/Sse families. Consistently, certain small complexes containing α1, α2, and α4 were not formed in strains lacking these chaperones. Deletion of the SSE1 gene in combination with deletions of PRE9 (α3), PBA3, or UMP1 genes resulted in severe synthetic growth defects, high levels of ubiquitin-conjugates, and an accumulation of distinct small complexes with α subunits. Our study shows that Hsp70 and Hsp110 chaperones cooperate to promote the folding of individual α subunits and/or their assembly with other CP subunits, Ump1, and Pba1-Pba4 in subsequent steps.

26S proteasomes become stably activated upon heat shock when ubiquitination and protein degradation increase

Heat shock (HS) promotes protein unfolding, and cells respond by stimulating HS gene expression, ubiquitination of cell proteins, and proteolysis by the proteasome. Exposing HeLa and other cells to 43 °C for 2 h caused a twofold increase in the 26S proteasomes' peptidase activity assayed at 37 °C. This increase in activity occurred without any change in proteasome amount and did not require new protein synthesis. After affinity-purification from HS cells, 26S proteasomes still hydrolyzed peptides, adenosine 5'-triphosphate, and ubiquitinated substrates more rapidly without any evident change in subunit composition, postsynthetic modification, or association with reported proteasome-activating proteins. After returning HS cells to 37 °C, ubiquitin conjugates and proteolysis fell rapidly, but proteasome activity remained high for at least 16 h. Exposure to arsenite, which also causes proteotoxic stress in the cytosol, but not tunicamycin, which causes endoplasmic reticulum stress, also increased ubiquitin conjugate levels and 26S proteasome activity. Although the molecular basis for the enhanced proteasomal activity remains elusive, we studied possible signaling mechanisms. Proteasome activation upon proteotoxic stress required the accumulation of ubiquitinated proteins since blocking ubiquitination by E1 inhibition during HS or arsenite exposure prevented the stimulation of 26S activity. Furthermore, increasing cellular content of ubiquitin conjugates at 37 °C by inhibiting deubiquitinating enzymes with RA190 or b-AP15 also caused proteasome activation. Thus, cells respond to proteotoxic stresses, apparently in response to the accumulation of ubiquitinated proteins, by activating 26S proteasomes, which should help promote the clearance of damaged cell proteins.

Exposure of Mesenchymal Stem Cells to an Alzheimer's Disease Environment Enhances Therapeutic Effects.

Mesenchymal stem cells (MSCs) have emerged as a promising tool for the treatment of Alzheimer's disease (AD). Previous studies suggested that the coculture of human MSCs with AD in an in vitro model reduced the expression of amyloid-beta 42 (Aβ42) in the medium as well as the overexpression of amyloid-beta- (Aβ-) degrading enzymes such as neprilysin (NEP). We focused on the role of primed MSCs (human Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) exposed to an AD cell line via a coculture system) in reducing the levels of Aβ and inhibiting cell death. We demonstrated that mouse groups treated with naïve MSCs and primed MSCs showed significant reductions in cell death, ubiquitin conjugate levels, and Aβ levels, but the effects were greater in primed MSCs. Also, mRNA sequencing data analysis indicated that high levels of TGF-β induced primed-MSCs. Furthermore, treatment with TGF-β reduced Aβ expression in an AD transgenic mouse model. These results highlighted AD environmental preconditioning is a promising strategy to reduce cell death and ubiquitin conjugate levels and maintain the stemness of MSCs. Further, these data suggest that human WJ-MSCs exposed to an AD environment may represent a promising and novel therapy for AD.

Chronic over-expression of ubiquitin impairs learning, reduces synaptic plasticity, and enhances GRIA receptor turnover in mice.

Ubiquitin is an essential signaling protein that controls many different cellular processes. While cellular ubiquitin levels normally cycle between pools of free and conjugated ubiquitin, the balance of these ubiquitin pools can be shifted by exposure to a variety of cellular stresses. Altered ubiquitin pools are also observed in several neurological disorders, suggesting that imbalances in ubiquitin homeostasis may contribute to neuronal dysfunction. To examine the effects of increased ubiquitin levels on the mammalian nervous system, we generated transgenic mice that express ubiquitin under the control of the Thy1.2 promoter. While we did not detect global changes in levels of ubiquitin conjugates in the hippocampus, we found that increasing ubiquitin levels reduced AMPA (GRIA1-4) receptor expression without affecting the levels of NMDA (GRIN) or GABAA receptors. Ubiquitin over-expression also negatively impacted hippocampus-dependent learning and memory as well as baseline excitability and synaptic plasticity at hippocampal CA3-CA1 synapses. These changes occurred in a dose-dependent manner in that mice with the highest levels of ubiquitin over-expression had the greatest deficits in synaptic function and were the most impaired in the learning and memory tasks. As chronic elevation of ubiquitin expression in neurons is sufficient to cause changes in synaptic function and cognition, altered ubiquitin homeostasis may be an important contributor to the stress-induced changes observed in neurological disorders.

Female-specific regulation of skeletal muscle mass by USP19 in young mice.

17β-Estradiol (E₂) is thought to be responsible for sex-specific differences in skeletal muscle mass. The biological function of E₂ is exerted through its binding to estrogen receptor α (ERα). The expression of ubiquitin-specific peptidase 19 (USP19) is upregulated during muscle atrophy and by E₂-activated ERα. Here, we investigated the involvement of USP19 in sex difference in muscle mass in young mice. Knockdown of USP19 in hindlimb muscles increased the mass and fiber size in soleus muscle in females but not males. Using Usp19 promoter reporter constructs, a functional half-estrogen response element (hERE) was identified in intron 1 of Usp19. ERα bound to hERE in an E₂-dependent manner in C2C12 myoblasts and in soleus muscle in ovariectomized (OVX) female mice. Furthermore, under normal physiological conditions, ERα bound to hERE in soleus muscle only in females. In contrast, administration of E₂ resulted in increased Usp19 mRNA expression, decreased muscle mass, and recruitment of ERα to hERE in soleus muscle in males. Knockdown of ERα in hindlimb muscles decreased Usp19 mRNA expression and increased the mass of soleus muscle only in females. Knockdown of USP19 resulted in increased levels of ubiquitin conjugates in soleus muscle in females. OVX increased the levels of ubiquitin conjugates and administration of E₂ decreased OVX-induced levels of ubiquitin conjugates. These results demonstrate that in soleus muscle in young female mice under physiological conditions, E₂ upregulates USP19 expression through ERα and consequently leads to decreases in ubiquitin conjugates and muscle mass.

β-Carotene Increases Muscle Mass and Hypertrophy in the Soleus Muscle in Mice.

Supplements and naturally occurring nutraceuticals effective for maintenance or enhancement of skeletal muscle mass are expected to contribute to prevention of decreased mobility and increased risk of developing metabolic diseases. However, information about available food components remains widely unavailable. In the present study, we investigated the effects of dietary β-carotene on the quantity and quality of skeletal muscle under physiological conditions. Male ddY mice (8 wk old) were orally administered β-carotene (0.5 mg once daily) for 14 d. Dietary β-carotene had no influence on body weight, but increased the soleus muscle/body weight ratio. The cross-sectional area (CSA) in muscle fibers of the soleus muscle was increased, indicating that administration of β-carotene induces muscle hypertrophy. In the soleus muscle of the β-carotene-administered mice, twitch force tended to be increased (p=0.06) and tetanic force was significantly increased, whereas specific force (force per CSA) remained unchanged. Dietary β-carotene increased the mRNA level of insulin-like growth factor 1 (Igf-1) as its splicing variant Igf-1ea, but had no influence on the liver Igf-1 mRNA level or serum IGF-1 level. β-Carotene promoted protein synthesis in the soleus muscle and reduced levels of ubiquitin conjugates, but had no influence on the mRNA levels of two atrogenes, Atrogin-1 and Murf1. On the other hand, β-carotene had no influence on the processing of the autophagy marker protein light chain 3. These results indicate that in mice, administration of β-carotene increases mass and induces functional hypertrophy in the soleus muscle, perhaps by promoting IGF-1-mediated protein synthesis and by reducing ubiquitin-mediated protein degradation.

Atorvastatin-mediated protection of the retina in a model of diabetes with hyperlipidemia.

Insulin resistance, a key feature of obesity, metabolic syndrome, and type 2 diabetes mellitus (T2DM), results in a variety of metabolic and vascular abnormalities. Metabolic disturbances associated with diabetes could contribute to disrupting the structural and (or) functional integrity of the retina. The effects of atorvastatin on retinal cells in hyperlipidemic T2DM rats have not yet been investigated. We used Goto-Kakizaki (GK) rats fed with an atherogenic diet (AD) for 4 months to investigate whether atorvastatin (administered for 1 month) would slow-down or reverse the progression of lesions in the diabetic retina. Fluorogenic substrates were used to measure the proteasome activities in retinal cells. The production of reactive oxygen species was determined by immunofluorescence in frozen retina sections, using dihydroethydium. Nitrotyrosine levels were assessed using immunohistochemistry. Protein levels of ubiquitin conjugates, free ubiquitin, and ubiquitin activating enzyme E1 were determined with Western blotting. Atorvastatin significantly reduced the levels of oxidative stress that were induced by the AD and restored the proteasome activities in the diabetic GK rats. Atorvastatin therapy significantly improved local oxidative stress levels in GK rats fed with AD. Atorvastatin can, at least in part, restore the ubiquitin proteasome system, and may represent a pharmacological approach to prevent some of the complications associated with diabetic retinopathy.

Patterns of ubiquitylation and SUMOylation associated with exposure to anoxia in embryos of the annual killifish Austrofundulus limnaeus.

Embryos of the annual killifish Austrofundulus limnaeus acquire extreme tolerance to anoxia during embryonic development. These embryos can survive environmental and cellular conditions that would likely result in death in the majority of vertebrate cells, despite experiencing a massive loss of ATP. It is highly likely that the initial response to anoxia must quickly alter cellular physiology to reprogram cell signaling and metabolic pathways to support anaerobiosis. Covalent protein modifications are a mechanism that can quickly act to effect large-scale changes in protein structure and function and have been suggested by others to play a key role in mammalian ischemia tolerance. Using Western blot analysis, we explored patterns of protein ubiquitylation and SUMOylation in embryos of A. limnaeus exposed to anoxia and anoxic preconditioning. Surprisingly, we report stage-specific protein ubiquitylation patterns that suggest different mechanisms for altering protein turnover in dormant and actively developing embryos that both survive long-term anoxia. Anoxic preconditioning does not appear to alter levels of ubiquitin conjugates in a unique manner. Global SUMOylation of proteins does not change in response to anoxia, but there are stage-specific changes in SUMOylation of specific protein bands. Contrary to other systems, global changes in protein SUMOylation may not be required to support long-term tolerance to anoxia in embryos of A. limnaeus. These data lead us to conclude that embryos of A. limnaeus respond to anoxia in a unique manner compared to other vertebrate models of anoxia tolerance and may provide novel mechanisms for engineering vertebrate tissues to survive long-term anoxia.

Interaction between misfolded PrP and the ubiquitin-proteasome system in prion-mediated neurodegeneration

Prion diseases are associated with the conformational conversion of cellular prion protein (PrP(C)) to pathological β-sheet isoforms (PrP(Sc)), which is the infectious agent beyond comprehension. Increasing evidence indicated that an unknown toxic gain of function of PrP(sc) underlies neuronal death. Conversely, strong evidence indicated that cellular prion protein might be directly cytotoxic by mediating neurotoxic signaling of β-sheet-rich conformers independent of prion replication. Furthermore, the common properties of β-sheet-rich isoform such as PrP(Sc) and β amyloid protein become the lynchpin that interprets the general pathological mechanism of protein misfolding diseases. Dysfunction of the ubiquitin-proteasome system (UPS) has been implicated in various protein misfolding diseases. However, the mechanisms of this impairment remain unknown in many cases. In prion disease, prion-infected mouse brains have increased levels of ubiquitin conjugates, which correlate with decreased proteasome function. Both PrP(C) and PrP(Sc) accumulate in cells after proteasome inhibition, which leads to increased cell death. A direct interaction between 20S core particle and PrP isoforms was demonstrated. Here we review the ability of misfolded PrP and UPS to affect each other, which might contribute to the pathological features of prion-mediated neurodegeneration.

The preventive effect of β-carotene on denervation-induced soleus muscle atrophy in mice

Muscle atrophy increases the production of reactive oxygen species and the expression of atrophy-related genes, which are involved in the ubiquitin-proteasome system. In the present study, we investigated the effects of β-carotene on oxidative stress (100 μM-H2O2)-induced muscle atrophy in murine C2C12 myotubes. β-Carotene (10 μM) restored the H2O2-induced decreased levels of myosin heavy chain and tropomyosin (P< 0·05, n 3) and decreased the H2O2-induced increased levels of ubiquitin conjugates. β-Carotene reduced the H2O2-induced increased expression levels of E3 ubiquitin ligases (Atrogin-1 and MuRF1) and deubiquitinating enzymes (USP14 and USP19) (P< 0·05, n 3) and attenuated the H2O2-induced nuclear localisation of FOXO3a. Furthermore, we determined the effects of β-carotene on denervation-induced muscle atrophy. Male ddY mice (8 weeks old, n 30) were divided into two groups and orally pre-administered micelle with or without β-carotene (0·5 mg once daily) for 2 weeks, followed by denervation in the right hindlimb. β-Carotene was further administered once daily until the end of the experiment. At day 3 after denervation, the ratio of soleus muscle mass in the denervated leg to that in the sham leg was significantly higher in β-carotene-administered mice than in control vehicle-administered ones (P< 0·05, n 5). In the denervated soleus muscle, β-carotene administration significantly decreased the expression levels of Atrogin-1, MuRF1, USP14 and USP19 (P< 0·05, n 5) and the levels of ubiquitin conjugates. These results indicate that β-carotene attenuates soleus muscle loss, perhaps by repressing the expressions of Atrogin-1, MuRF1, USP14 and USP19, at the early stage of soleus muscle atrophy.

Regulation of the Proteasome by Neuronal Activity and Calcium/Calmodulin-dependent Protein Kinase II

Protein degradation via the ubiquitin proteasome system has been shown to regulate changes in synaptic strength that underlie multiple forms of synaptic plasticity. It is plausible, therefore, that the ubiquitin proteasome system is itself regulated by synaptic activity. By utilizing live-cell imaging strategies we report the rapid and dynamic regulation of the proteasome in hippocampal neurons by synaptic activity. We find that the blockade of action potentials (APs) with tetrodotoxin inhibited the activity of the proteasome, whereas the up-regulation of APs with bicuculline dramatically increased the activity of the proteasome. In addition, the regulation of the proteasome is dependent upon external calcium entry in part through N-methyl-D-aspartate receptors and L-type voltage-gated calcium channels and requires the activity of calcium/calmodulin-dependent protein kinase II (CaMKII). Using in vitro and in vivo assays we find that CaMKII stimulates proteasome activity and directly phosphorylates Rpt6, a subunit of the 19 S (PA700) subcomplex of the 26 S proteasome. Our data provide a novel mechanism whereby CaMKII may regulate the proteasome in neurons to facilitate remodeling of synaptic connections through protein degradation.

E1 Ubiquitin-Activating Enzyme UBA-1 Plays Multiple Roles throughout C. elegans Development

Poly-ubiquitination of target proteins typically marks them for destruction via the proteasome and provides an essential mechanism for the dynamic control of protein levels. The E1 ubiquitin-activating enzyme lies at the apex of the ubiquitination cascade, and its activity is necessary for all subsequent steps in the reaction. We have isolated a temperature-sensitive mutation in the Caenorhabditis elegans uba-1 gene, which encodes the sole E1 enzyme in this organism. Manipulation of UBA-1 activity at different developmental stages reveals a variety of functions for ubiquitination, including novel roles in sperm fertility, control of body size, and sex-specific development. Levels of ubiquitin conjugates are substantially reduced in the mutant, consistent with reduced E1 activity. The uba-1 mutation causes delays in meiotic progression in the early embryo, a process that is known to be regulated by ubiquitin-mediated proteolysis. The uba-1 mutation also demonstrates synthetic lethal interactions with alleles of the anaphase-promoting complex, an E3 ubiquitin ligase. The uba-1 mutation provides a sensitized genetic background for identifying new in vivo functions for downstream components of the ubiquitin enzyme cascade, and it is one of the first conditional mutations reported for the essential E1 enzyme in a metazoan animal model.

Down-regulation of proteolytic complexes following EBV activation in BL cells

In Burkitt’s lymphoma cells, Epstein Barr virus (EBV) latency products interact with the ubiquitin–proteasome system to promote episomal maintenance and immunological evasion while the tripeptidylpeptidase II (TPPII) functions as an alternative protease. In the present study, we have examined the activities and levels of the proteasome and TPPII complex in Raji and in Akata cells after induction of EBV lytic cycle. The results show that the chymotrypsin-like and caspase-like activities of the proteasome were substantially reduced in Raji and Akata cells. Similarly, TPPII activity was diminished in both cell lines but was recovered in Akata cells at longer time after induction. Protein levels of the α/β subunits of the 20S proteasome and TPPII concentration decreased to different extents after EBV activation, whereas the ubiquitin binding S6’ subunit of the 19S regulatory complex increased three to fourfold along with the levels of ubiquitin-conjugates. Collectively, these observations demonstrate impairment of two major cellular proteolytic systems at the onset of EBV lytic infection.

HRpn13/ADRM1/GP110 is a novel proteasome subunit that binds the deubiquitinating enzyme, UCH37

The 26S proteasome catalyzes the degradation of most proteins in mammalian cells. To better define its composition and associated regulatory proteins, we developed affinity methods to rapidly purify 26S proteasomes from mammalian cells. By this approach, we discovered a novel 46-kDa (407 residues) subunit of its 19S regulatory complex (previously termed ADRM1 or GP110). As its N-terminal half can be incorporated into the 26S proteasome and is homologous to Rpn13, a 156-residue subunit of the 19S complex in budding yeast, we renamed it human Rpn13 (hRpn13). The C-terminal half of hRpn13 binds directly to the proteasome-associated deubiquitinating enzyme, UCH37, and enhances its isopeptidase activity. Knockdown of hRpn13 in 293T cells increases the cellular levels of ubiquitin conjugates and decreases the degradation of short-lived proteins. Surprisingly, an overproduction of hRpn13 also reduced their degradation. Furthermore, transfection of the C-terminal half of hRpn13 slows proteolysis and induces cell death, probably by acting as a dominant-negative form. Thus in human 26S proteasomes, hRpn13 appears to be important for the binding of UCH37 to the 19S complex and for efficient proteolysis.

Protein Quality Control by the Ubiquitin Proteolytic Pathway: Roles in Resistance to Oxidative Stress and Disease

AbstractThere is now consensus that the accumulation of oxidatively modified proteins is cytotoxic and causally related to several age‐related diseases including the amyloid diseases and age‐related cataracts. There is also general agreement that the ubiquitin proteolytic pathway (UPP) provides a quality control mechanism to limit accumulation of modified proteins. We asked if and how oxidative stress is related to the function of the ubiquitin proteolytic pathway, and vice versa, with the objective of obtaining information that can lead to the development of strategies to delay age‐related “amyloid” or “protein precipitation” diseases such as cataracts and age‐related macular degeneration. Elevated levels of ubiquitin conjugates were observed when human, rabbit, bovine, and rat lens, retina, liver cells or tissues were exposed to mild oxidative stress, which was created by exposure to paraquat, diamide, peroxide, light together with lipofuscin, and radiomimetic drugs. The increase in ubiquitin conjugates derived from an increase in substrates as well as by hyperactivation of E1, rather than inactivation of the proteasome. Using a novel glutathiolated substrate, γC‐crystallin, we demonstrated that the UPP shows a previously unrecognized selectivity for such specifically oxidatively modified proteins. Selectivity of the pathway for other oxidatively modified proteins, specifically for protein carbonyls, was indicated in assays that employed the ubiquitin conjugation‐competent, but degradation‐resistant ubiquitin variant K6W‐ubiquitin. These experiments showed that failure to execute ubiquitin‐dependent proteolysis renders cells more susceptible to oxidative‐stress‐related cytotoxicity. Activity of the pathway is regulated in part by cellular redox status, specifically as affected by GSSG. Ubiquitination is enhanced when GSSG/GSH ratios are 0.02–0.15. Since there is potentiation of ubiquitination even when GSSG/GSH ratios are indistinguishable from basal levels, it appears that ubiquitination provides one of the most sensitive indicators of oxidative stress. Ubiquitination is attenuated when GSSG/GSH rises &gt;0.2 and does not occur when GSSG/GSH ⩾ 2.9. The data indicate that inhibition of the pathway, which occurs upon aging, is associated with accumulation rather than the timely degradation of ubiquitin conjugates. They further suggest that if the system fails to keep up with production of substrates, high mass ubiquitin conjugates may accumulate and precipitate in cytotoxic aggregates such as are seen in many age‐related syndromes, including lens cataracts or in lipofuscin and drusen in the aging retina.

Proteasome activity declines in aged macrophages

The ubiquitin-proteasome pathway is involved in regulation of a variety of biologically important processes including antigen presentation by macrophages (Mφ). Age-related decrease in proteasome activity has been reported in other tissues. However, the effect of aging on the ubiquitin-proteasome pathway in Mφ has not been systematically studied. Thioglycollate-elicited Mφ from young (4–6 mo) and Old (24–26 mo) male C57BL/6NIA mice were collected by lavage. Mφ from old mice exhibited significantly lower activity of all three proteasome peptidases [chymotrypsin-like activity (young vs old, Mean ± SE): 100 ± 13 vs 62 ± 4, p<0.05; trypsin-like activity: 100 ± 4 vs 56 ± 3, p<0.01; acidic activity: 100 ± 7 vs 58 ± 6, p<0.01]. The age-related decrease in proteasome activity was not due to decreased expression of proteasome subunits, since levels of C2 and S1 subunits were higher in old Mφ than young (C2: 100 ± 20 vs 167 ± 13, p<0.05; S1: 100 ± 15 vs 183 ± 20, p<0.01) while there was no significant age-related difference in levels of α5, LMP2 and LMP7. The decrease in proteasome activity was associated with an increase in the levels of ubiquitin conjugates. Furthermore, the levels of 2 ubiquitination-related enzymes, E1 and E2 (Ubc7), were significantly higher in old Mφ than young. The age-related decline in proteasome activity and the paralleled increase in levels of ubiquitin conjugates indicate that the function of the ubiquitin-proteasome pathway is impaired in the aged Mφ. The decrease in proteasome activity in Mφ might contribute to the age-related decline in the ability of Mφ to present antigens to T cells, a proteasome dependent process. Supported by USDA #58-1950-9-001 and NIA #R01 AG009140-10A1.

Vertebrate freezing survival: Regulation of the multicatalytic proteinase complex and controls on protein degradation

The wood frog , Rana sylvatica, survives weeks of whole body freezing during winter hibernation, expressing numerous metabolic adaptations that deal not only with freezing but with its consequences including organ ischemia and cellular dehydration. The present study analyzes the 20s multicatalytic proteinase (MCP) complex from skeletal muscle to determine how protein degradation is managed in the ischemic frozen state . MCP was partially purified and assayed fluorometrically using three AMC-labeled substrates to compare multiple states: control (5 °C acclimated), 24 h frozen at −2.5 °C, 4 or 8 h thawed at 5 °C, 8 h anoxia, and 40% dehydration. MCP from frozen frogs showed significantly different K m and V max values compared with controls; e.g., K m Z-LLE-AMC increased by 45% during freezing and 52% under anoxia whereas V max decreased by 40%. After thawing, K m was restored and V max rose by 2.2-fold. Incubations promoting protein kinase or phosphatase action on MCP showed that phosphatase treatment strongly increased V max implicating reversible phosphorylation in MCP regulation during freeze–thaw. Western blotting showed a 36% decrease in MCP protein in muscle from frozen frogs. The 20s MCP preferentially degrades oxidatively-damaged proteins and evidence of impaired function during freezing came from a 1.4-fold increase in protein carbonyl content in muscle and liver during freezing. Ubiquitin and ubiquitin conjugate levels were unchanged in muscle but changed markedly in liver during freeze–thaw.

Use of RNA interference and complementation to study the function of the Drosophila and human 26S proteasome subunit S13.

The S13 subunit (also called Pad1, Rpn11, and MPR1) is a component of the 19S complex, a regulatory complex essential for the ubiquitin-dependent proteolytic activity of the 26S proteasome. To address the functional role of S13, we combined double-stranded RNA interference (RNAi) against the Drosophila proteasome subunit DmS13 with expression of wild-type and mutant forms of the homologous human gene, HS13. These studies show that DmS13 is essential for 26S function. Loss of the S13 subunit in metazoan cells leads to increased levels of ubiquitin conjugates, cell cycle defects, DNA overreplication, and apoptosis. In vivo assays using short-lived proteasome substrates confirmed that the 26S ubiquitin-dependent degradation pathway is compromised in S13-depleted cells. In complementation experiments using Drosophila cell lines expressing HS13, wild-type HS13 was found to fully rescue the knockdown phenotype after DmS13 RNAi treatment, while an HS13 containing mutations (H113A-H115A) in the proposed isopeptidase active site was unable to rescue. A mutation within the conserved MPN/JAMM domain (C120A) abolished the ability of HS13 to rescue the Drosophila cells from apoptosis or DNA overreplication. However, the C120A mutant was found to partially restore normal levels of ubiquitin conjugates. The S13 subunit may possess multiple functions, including a deubiquitinylating activity and distinct activities essential for cell cycle progression that require the conserved C120 residue.

Removal of Oxidatively Damaged Proteins from Lens Cells by the Ubiquitin-Proteasome Pathway

Understanding how oxidized proteins are removed is important since accumulation of such damaged proteins is causally related to cellular and organismic dysfunction, disease and aging. Previous work showed that activity of the ubiquitin-proteasome pathway (UPP) in lens cells increased during recovery from oxidative stress (44: J. Biol. Chem. 272, 23086–93). In this study we sought to determine if the up-regulation of the UPP during recovery from oxidative stress has a role in selective removal of oxidized proteins from the cells. In cells which were not exposed to peroxide, inhibition of the proteasome with MG132 or clasto-lactacystin β-lactone had little effect on protein carbonyl levels. However, inhibition of the proteasome in the 20μM peroxide-treated cells caused an approximate 60% increase in levels of protein carbonyl and an approximate 100% increase in levels of ubiquitin conjugates. The carbonyl-containing proteins that accumulated in the presence of the proteasome inhibitor co-localized with high molecular mass ubiquitin-protein conjugates. Furthermore, isolated carbonyl-containing proteins from H2O2-treated cells were ubiquitinated, and ubiquitin-conjugates were enriched with carbonyl-containing proteins. The diminished effect of proteasome inhibitors on protein carbonyl levels, together with the robust increase in ubiquitin-protein conjugates and accompanied increases in oxidized proteins, upon exposure to 60μM H2O2indicate that the proteasomal step of the UPP is more susceptible to oxidative inactivation than the ubiquitination step. In fact, oxidative stress is associated with a hyperactivation of the ubiquitin-activating enzyme. These data indicate that the UPP plays a role in removal of oxidatively damaged proteins from cells and that attenuation of the UPP activity may result in cytotoxic accumulation of damaged proteins, possibly including the ubiquitinated forms.

Calorie restriction, stress and the ubiquitin-dependent pathway in mouse livers

Calorie restriction (R) is the only known method to delay the aging process and extend mean and maximal lifespan in rodents. R has been shown to delay the age-related accumulation of damaged proteins and to protect organisms from various stresses which can produce damaged proteins. Such stresses include irradiation, heat shock, and oxidative stress. The ubiquitin- and ATP-dependent proteolytic pathway (UPP) has been associated with the degradation of abnormal and/or damaged proteins. We examined the effect of diet and oxidative stress on activities of the UPP in supernatants from livers taken from 23-month-old Emory mice which had been exposed to an in-vivo injection of paraquat. Paraquat induces oxidative stress by generating superoxide radicals. In livers from non-stressed animals, steady-state levels of endogenous ubiquitin conjugates, de novo conjugate formation, and E1 and E2 activities were significantly lower in R animals than in control (C) animals. However, after exposure to paraquat, levels of endogenous ubiquitin conjugates were significantly higher in R versus C animals, and de novo conjugate formation and E1 and E2 activities in R animals rose to levels which were indistinguishable from levels of these activities noted in C animals. R was associated with an increased ability to degrade β-lactoglobulin by the UPP after an oxidative stress was imposed. Ability to degrade β-lactoglobulin by the C or R livers in non-stressed animals was not significantly different. Taken together, these data indicate that oxidative stress in R animals is associated with enhanced levels of ubiquitin conjugates and that this enhancement may be due to an increase in UPP activity. These data also indicate that the ability to form ubiquitin conjugates and the UPP system does not change with oxidative stress in C animals. The latter is consistent with prior reports that suggests that older C animals may already be in a state of enhanced oxidative stress and that activities of the UPP provide a sensitive indicator of levels of cellular redox status.