p62 links the autophagy pathway and the ubiqutin–proteasome system upon ubiquitinated protein degradation

The ubiquitin–proteasome system (UPS) and autophagy are two distinct and interacting proteolytic systems. They play critical roles in cell survival under normal conditions and during stress. An increasing body of evidence indicates that ubiquitinated cargoes are important markers of degradation. p62, a classical receptor of autophagy, is a multifunctional protein located throughout the cell and involved in many signal transduction pathways, including the Keap1–Nrf2 pathway. It is involved in the proteasomal degradation of ubiquitinated proteins. When the cellular p62 level is manipulated, the quantity and location pattern of ubiquitinated proteins change with a considerable impact on cell survival. Altered p62 levels can even lead to some diseases. The proteotoxic stress imposed by proteasome inhibition can activate autophagy through p62 phosphorylation. A deficiency in autophagy may compromise the ubiquitin–proteasome system, since overabundant p62 delays delivery of the proteasomal substrate to the proteasome despite proteasomal catalytic activity being unchanged. In addition, p62 and the proteasome can modulate the activity of HDAC6 deacetylase, thus influencing the autophagic degradation.     

Synphilin-1 degradation by the ubiquitin-proteasome pathway and effects on cell survival

Parkinson's disease is characterized by loss of nigral dopaminergic neurons and the presence of cytoplasmic inclusions known as Lewy bodies. alpha-Synuclein and its interacting partner synphilin-1 are among constituent proteins in these aggregates. The presence of ubiquitin and proteasome subunits in these inclusions supports a role for this protein degradation pathway in the processing of proteins involved in this disease. To begin elucidating the kinetics of synphilin-1 in cells, we studied its degradation pathway in HEK293 cells that had been engineered to stably express FLAG-tagged synphilin-1. Pulse-chase experiments revealed that this protein is relatively stable with a half-life of about 16 h. Treatment with proteasome inhibitors resulted in attenuation of degradation and the accumulation of high molecular weight ubiquitinated synphilin-1 in immunoprecipitation/immunoblot experiments. Additionally, proteasome inhibitors stimulated the formation of peri-nuclear inclusions which were immunoreactive for synphilin-1, ubiquitin and alpha-synuclein. Cell viability studies revealed increased susceptibility of synphilin-1 over-expressing cells to proteasomal dysfunction. These observations indicate that synphilin-1 is ubiquitinated and degraded by the proteasome. Accumulation of ubiquitinated synphilin-1 due to impaired clearance results in its aggregation as peri-nuclear inclusions and in poor cell survival.     

Lower expression of catalytic and structural subunits of the proteasome contributes to decreased proteolysis in peripheral blood T lymphocytes during aging

Aging, in the immune system, is characterized by a decreased ability to respond to exogenous insults, resulting in increased susceptibility to infections and blunted response to vaccination. While significant age-associated deficits in immune function have been documented, the underlying molecular mechanisms are still being investigated. A consistent decline in the proteolytic activity of the proteasome has been demonstrated with advancing age, implicating an important role for the proteasome in immune senescence, by studies that largely employed proteasome-enriched preparations from cell lysates. With the availability of novel cell permeable active site probes designed specifically for assaying proteasomal activity in live cells, we now confirm our earlier data demonstrating lower catalytic activity of the proteasome in primary human T cells obtained from the elderly when compared to those from young donors. Loss in proteasomal catalytic activity translated into a loss in functional activity, as was observed in a degradation assay employing an ubiquitinated protein substrate, Ub-IkappaBalpha. Unlike fluorogenic peptide substrates, use of ubiquitinated protein substrates not only confer greater stringency in terms of proteasomal hydrolysis, but also involve the participation of the 19S regulatory component. This age-associated loss in proteasomal activity is accompanied by alteration in the levels of catalytic, structural and regulatory subunits, with no change in that of the 11S activator or the inhibitor PAAF1. Oxidative modification, such as carbonylation and lipid-peroxidation, of proteasomal subunits was also detected in T cells from the elderly. Thus, oxidative modification and lower levels of proteasomal subunits contribute to decreased proteolytic activity during immune-senescence.     

Ubiquitin-dependent and -independent proteasomal degradation of apoB associated with endoplasmic reticulum and Golgi apparatus,respectively, in HepG2 cells

Studies in hepatocyte cultures indicate that apolipoprotein (apo) B-100 production is regulated largely by intracellular degradation and the proteasome pathway is a major mechanism for the degradation. In the present study, we have examined the detailed itinerary of apoB degradation through its secretory pathway in HepG2 cells. We found that ubiquitin-dependent proteasomal degradation of apoB largely occurred on the cytosolic surface of rough and smooth endoplasmic reticulum (ER) and that a small proportion of apoB was dislodged from the secretory organelles into the cytosolic compartment where it underwent ubiquitination for proteasomal degradation. The transmembrane conformation of apoB persisted as the protein was transported through the Golgi apparatus. We further demonstrated that proteasomal degradation of apoB was associated the Golgi apparatus but Golgi-associated apoB was not ubiquitinated, indicating an ubiquitin-independent proteasomal degradation of apoB is associated with this organelle. We conclude that apoB undergoes proteasomal degradation while going through different compartments of the secretory pathway; further, ER-associated proteasomal degradation of apoB in the ER is ubiquitin-dependent whereas that occurring in the Golgi is ubiquitin-independent.     

Autoregulation of the 26S proteasome by in situ ubiquitination

The 26S proteasome degrades ubiquitinated proteins, and proteasomal degradation controls various cellular events. Here we report that the human 26S proteasome is ubiquitinated, by which the ubiquitin receptors Adrm1 and S5a, the ATPase subunit Rpt5, and the deubiquitinating enzyme Uch37 are ubiquitinated in situ by proteasome-associating ubiquitination enzymes. Ubiquitination of these subunits significantly impairs the 26S proteasome''s ability to bind, deubiquitinate, and degrade ubiquitinated proteins. Moreover, ubiquitination of the 26S proteasome can be antagonized by proteasome-residing deubiquitinating enzymes, by the binding of polyubiquitin chains, and by certain cellular stress, indicating that proteasome ubiquitination is dynamic and regulated in cells. We propose that in situ ubiquitination of the 26S proteasome regulates its activity, which could function to adjust proteasomal activity in response to the alteration of cellular ubiquitination levels.     

BAG3 induces the sequestration of proteasomal clients into cytoplasmic puncta: Implications for a proteasome-to-autophagy switch

Eukaryotic cells use autophagy and the ubiquitin–proteasome system as their major protein degradation pathways. Upon proteasomal impairment, cells switch to autophagy to ensure proper clearance of clients (the proteasome-to-autophagy switch). The HSPA8 and HSPA1A cochaperone BAG3 has been suggested to be involved in this switch. However, at present it is still unknown whether and to what extent BAG3 can indeed reroute proteasomal clients to the autophagosomal pathway. Here, we show that BAG3 induces the sequestration of ubiquitinated clients into cytoplasmic puncta colabeled with canonical autophagy linkers and markers. Following proteasome inhibition, BAG3 upregulation significantly contributes to the compensatory activation of autophagy and to the degradation of the (poly)ubiquitinated proteins. BAG3 binding to the ubiquitinated clients occurs through the BAG domain, in competition with BAG1, another BAG family member, that normally directs ubiquitinated clients to the proteasome. Therefore, we propose that following proteasome impairment, increasing the BAG3/BAG1 ratio ensures the “BAG-instructed proteasomal to autophagosomal switch and sorting” (BIPASS).     

Stress-induced polyubiquitination of proteasomal ubiquitin receptors targets the proteolytic complex for autophagic degradation

Ubiquitin (Ub) is a small protein (8 kDa) found in all eukaryotic cells, which is conjugated covalently to numerous proteins, tagging them for recognition by a downstream effector. One of the best characterized functions of Ub is targeting proteins for either selective degradation by the proteasome, or for bulk degradation by the autophagy-lysosome system. The executing arm of the UPS is the 26S proteasome, a large multicatalytic complex. While much is known about the synthesis and assembly of the proteasome's subunits, the mechanism(s) underlying its removal has remained obscure, similar to that of many other components of the ubiquitin-proteasome system. Our recent study identified autophagy as the degrading mechanism for the mammalian proteasome, mostly under stress conditions. Amino acid starvation induces specific ubiquitination of certain 19S proteasomal subunits that is essential for its binding to SQSTM1/p62, the protein that shuttles the ubiquitinated proteasome to the autophagic machinery. SQSTM1 delivers ubiquitinated substrates for proteasomal degradation via interaction of its PB1 domain with the 19S proteasomal subunit PSMD4/Rpn10, in situations where the proteasome serves as a “predator." In contrast, we found that the UBA domain of SQSTM1 is essential for its interaction with the ubiquitinated proteasome and its delivery to the autophagosome, rendering the proteasome a “prey.”     

Proteomics of proteasome complexes and ubiquitinated proteins

Ubiquitin-proteasome-mediated protein degradation is central to the regulation of many important biological processes, including cell cycle progression, apoptosis and DNA repair. Recognition and degradation of ubiquitinated substrates by the 26S proteasome is tightly regulated to maintain normal cell growth. Disruption of the proteasomal degradation pathway has been implicated in a wide range of human diseases. Although the ubiquitin-proteasome system has been intensively investigated, many key questions remain unanswered in regard to its components and regulation of its activities. A key step towards a full understanding of the pathway is to investigate the proteasome complex subunit composition, heterogeneity, post-translational modifications, assembly, proteasome interaction networks and degradation substrates. Mass spectrometry-based proteomic approaches have been successfully applied for unraveling the details of the proteasome complexes and their substrates in an unprecedented fashion. An overview of the current knowledge of the proteasomal degradation pathway based on mass spectrometry approaches is presented.     

The ubiquitin-proteasome system regulates membrane fusion of yeast vacuoles

Ubiquitination is known to regulate early stages of intracellular vesicular transport, without proteasomal involvement. We now show that, in yeast, ubiquitination regulates a late-stage, membrane fusion, with proteasomal involvement. A known proteasome mutant had a vacuolar fragmentation phenotype in vivo often associated with vacuolar membrane fusion defects, suggesting a proteasomal role in fusion. Inhibiting vacuolar proteasomes interfered with membrane fusion in vitro, showing that fusion cannot occur without proteasomal degradation. If so, one would expect to find ubiquitinated proteins on vacuolar membranes. We found a small number of these, identified the most prevalent one as Ypt7 and mapped its two major ubiquitination sites. Ubiquitinated Ypt7 was linked to the degradation event that is necessary for fusion: vacuolar Ypt7 and vacuolar proteasomes were interdependent, ubiquitinated Ypt7 became a proteasomal substrate during fusion, and proteasome inhibitors reduced fusion to greater degree when we decreased Ypt7 ubiquitination. The strongest model holds that fusion cannot proceed without proteasomal degradation of ubiquitinated Ypt7. As Ypt7 is one of many Rab GTPases, ubiquitin-proteasome regulation may be involved in membrane fusion elsewhere.     

pVHL-independent ubiquitination of HIF1alpha and its stabilization by cobalt ion

Recent studies have shown that hypoxia-inducible factor1alpha (HIF1alpha) is ubiquitinated by an E3-ligase complex containing von Hippel-Lindau gene product (pVHL) after which it is targeted for proteasomal degradation. In this study, we showed that HIF1alpha was stabilized in the pVHL-deficient cell line 786-0 treated with a proteasome inhibitor or Co(2+). This suggests that HIF1alpha is also ubiquitinated by a pVHL-independent pathway and that its stability is regulated by Co(2+). Indeed, using the COS cell expression system, we confirmed that HIF1alpha is ubiquitinated at the N-terminal region by a pVHL-independent pathway and that its degradation is inhibited by Co(2+). We also demonstrated that Co(2+) binds to both PAS domains in the N-terminal region of HIF1alpha. These observations imply that the stability of HIF1alpha is regulated by an additional pathway through the cobalt binding of PAS domains.     

Proteasomal turnover of p21Cip1 does not require p21Cip1 ubiquitination

The Cdk inhibitor p21Cip1 is an unstable protein. Pharmacologic inhibition of the proteasome increases the half-life of p21 from less than 30 min to more than 2 hr and results in the accumulation of p21-ubiquitin conjugates. To determine whether ubiquitination was required for proteasomal degradation of p21, we constructed mutant versions of p21 that were not ubiquitinated in vivo. Remarkably, these mutants remained unstable and increased in abundance upon proteasome inhibition, indicating that direct ubiquitination of p21 is not necessary for its turnover by the proteasome. The frequently observed correlation between protein ubiquitination and proteasomal degradation is insufficient to conclude that ubiquitination is a prerequisite for degradation.     

Proteasomal inhibition leads to formation of ubiquitin/alpha-synuclein-immunoreactive inclusions in PC12 cells

Proteasomal dysfunction has been recently implicated in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease and diffuse Lewy body disease. We have developed an in vitro model of proteasomal dysfunction by applying pharmacological inhibitors of the proteasome, lactacystin or ZIE[O-tBu]-A-leucinal (PSI), to dopaminergic PC12 cells. Proteasomal inhibition caused a dose-dependent increase in death of both naive and neuronally differentiated PC12 cells, which could be prevented by caspase inhibition or CPT-cAMP. A percentage of the surviving cells contained discrete cytoplasmic ubiquitinated inclusions, some of which also contained synuclein-1, the rat homologue of human alpha-synuclein. However the total level of synuclein-1 was not altered by proteasomal inhibition. The ubiquitinated inclusions were present only within surviving cells, and their number was increased if cell death was prevented. We have thus replicated, in this model system, the two cardinal pathological features of Lewy body diseases, neuronal death and the formation of cytoplasmic ubiquitinated inclusions. Our findings suggest that inclusion body formation and cell death may be dissociated from one another.     

Cytoplasmic lipid droplets are sites of convergence of proteasomal and autophagic degradation of apolipoprotein B

Lipid esters stored in cytoplasmic lipid droplets (CLDs) of hepatocytes are used to synthesize very low-density lipoproteins (VLDLs), into which apolipoprotein B (ApoB) is integrated cotranslationally. In the present study, by using Huh7 cells, derived from human hepatoma and competent for VLDL secretion, we found that ApoB is highly concentrated around CLDs to make "ApoB-crescents." ApoB-crescents were seen in <10% of Huh7 cells under normal conditions, but the ratio increased to nearly 50% after 12 h of proteasomal inhibition by N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal. Electron microscopy showed ApoB to be localized to a cluster of electron-lucent particles 50-100 nm in diameter adhering to CLDs. ApoB, proteasome subunits, and ubiquitinated proteins were detected in the CLD fraction, and this ApoB was ubiquitinated. Interestingly, proteasome inhibition also caused increases in autophagic vacuoles and ApoB in lysosomes. ApoB-crescents began to decrease after 12-24 h of proteasomal inhibition, but the decrease was blocked by an autophagy inhibitor, 3-methyladenine. Inhibition of autophagy alone caused an increase in ApoB-crescents. These observations indicate that both proteasomal and autophagy/lysosomal degradation of ApoB occur around CLDs and that the CLD surface functions as a unique platform for convergence of the two pathways.     

Ubiquitination of RhoA by Smurf1 promotes neurite outgrowth

The Rho-family of small GTPases consists of essential regulators of neurite outgrowth, axonal pathfinding, and dendritic arborization. Previous work has demonstrated in non-neuronal cell types that Smurf1, an E3 ubiquitin ligase, regulates cell polarity and protrusive activity via PKCzeta-dependent recruitment to cellular protrusion sites, and subsequent ubiquitination and proteasomal degradation of RhoA. In this study, we show that Smurf1 enhances neurite outgrowth in Neuro2a neuroblastoma cells. We demonstrate that RhoA is ubiquitinated, and that Smurf1 and RhoA physically interact in vivo. Interestingly, Smurf1 overexpression in Neuro2a cells dramatically reduces RhoA protein levels during dibutyric cyclic AMP, but not retinoic acid induced neurite outgrowth. This Smurf1-dependent reduction in RhoA protein levels was abrogated using the general proteasome inhibitor MG132, suggesting that RhoA is targeted for ubiquitination and degradation via Smurf1. Together, our data suggest that localized regulation of different subsets of Rho GTPases by specific guidance signals results in an intracellular asymmetry of RhoA activity, which could regulate neurite outgrowth and guidance.     

Redox regulation of human Rac1 stability by the proteasome in human aortic endothelial cells

Rac1 has been shown to activate a NADPH oxidase complex producing superoxide anions in a variety of mammalian cell types. We evaluated the impact of Rac1-induced reactive oxygen species production on the turnover of Rac1 itself in human aortic endothelial cells. The concentration of a constitutively active mutant of Rac1 (Rac1(V12)) was increased by treatment of the cells with diphenylene iodinium (DPI), an inhibitor of the NADPH oxidase. Such an effect was not observed for the dominant negative form of Rac1 (Rac1(N17)). We showed a decrease in proteolytic degradation of Rac1(V12) in the presence of DPI, and showed that short term treatment with H(2)O(2) reverses the effect of DPI. We found that proteasome inhibitors (lactacystin and MG132) increased Rac1(V12) protein level. In support of this finding, we have identified in the primary sequence of Rac1 a potential destruction box domain, which is known to be a signal for protein degradation mediated by the ubiquitin/proteasome system. We show that Rac1(V12) is ubiquitinated before degradation. By contrast Rac1(N17) induces an accumulation of the ubiquitinated form of Rac1. These results suggest that Rac1 activation of NADPH oxidase is necessary for the proteolytic degradation of Rac1 itself.     

Evidence for proteasomal degradation of Kv1.5 channel protein

BACKGROUND: The voltage-gated potassium channel Kv1.5 plays a critical role in the maintenance of the membrane potential. While protein degradation is one of the major mechanisms for the regulation of channel functions, little is known on the degradation mechanism of Kv1.5. METHODS AND RESULTS: Kv1.5 was expressed in COS cells and its degradation, intracellular localization, and channel activities were assessed by pulse-chase analysis, immunofluorescence, and patch clamp techniques, respectively. Expressed Kv1.5 had a half-life time of approximately 6.7 h, which was prolonged by the proteasome inhibitors of MG132, ALLN, proteasomal inhibitor 1, or lactacystine, but not by a lysosomal inhibitor chloroquine. MG132 increased the protein level of Kv1.5, as well as the level of its ubiquitinated form in a dose-dependent manner. Similar effects of MG132 on endogenous Kv1.5 were seen in cultured rat atrial cells. Within a cell, Kv1.5 was mainly localized in both the endoplasmic reticulum and Golgi apparatus. MG132 increased the immunoreactivity of Kv1.5 in these compartments and also increased Ik(ur) currents through the cell-surface Kv1.5. Pretreatment with either brefeldin A or colchicine abolished MG132-induced increase in Ik(ur) currents. CONCLUSION: Kv1.5 is degraded by the proteasome. The inhibition of the proteasome increased Ik(ur) currents secondary to stabilization of the channel protein in the endoplasmic reticulum/Golgi apparatus.     

Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry

Ubiquitination regulates a host of cellular processes by labeling proteins for degradation, but also by functioning as a regulatory, nonproteolytic posttranslational modification. Proteome-wide strategies to monitor changes in ubiquitination profiles are important to obtain insight into the various cellular functions of ubiquitination. Here we describe generation of stable cell lines expressing a tandem hexahistidine-biotin tag (HB-tag) fused to ubiquitin for two-step purification of the ubiquitinated proteome under fully denaturing conditions. Using this approach we identified 669 ubiquitinated proteins from HeLa cells, including 44 precise ubiquitin attachment sites on substrates and all seven possible ubiquitin chain-linkage types. To probe the dynamics of ubiquitination in response to perturbation of the ubiquitin/proteasome pathway, we combined ubiquitin profiling with quantitative mass spectrometry using the stable isotope labeling with amino acids in cell culture (SILAC) strategy. We compared untreated cells and cells treated with the proteasome inhibitor MG132 to identify ubiquitinated proteins that are targeted to the proteasome for degradation. A number of proteasome substrates were identified. In addition, the quantitative approach allowed us to compare proteasome targeting by different ubiquitin chain topologies in vivo. The tools and strategies described here can be applied to detect changes in ubiquitination dynamics in response to various changes in growth conditions and cellular stress and will contribute to our understanding of the ubiquitin/proteasome system.     

Phosphorylated IkappaBalpha is a component of Lewy body of Parkinson's disease

Ubiquitin is one of the major components of Lewy bodies (LB), the pathological hallmark of Parkinson's disease (PD). Here, we identified that a phosphorylated form of IkappaBalpha (pIkappaBalpha), an inhibitor of NF-kappaB, and SCF(beta-TrCP), the ubiquitin ligase of pIkappaBalpha, are components of LB in brains of PD patients. In vitro studies identified those proteins in the ubiquitin- and alpha-synuclein (known as the major component of LB)-positive LB-like inclusions generated in dopaminergic SH-SY5Y cells treated with MG132, a proteasome inhibitor. Intriguingly, IkappaBalpha migration into such ubiquitinated inclusions in cells treated with MG132 was inhibited by a cell-permeable peptide known to block phosphorylation of IkappaBalpha, although this peptide did not influence cell viability under proteasomal inhibition. Our results indicate that phosphorylation of IkappaBalpha plays a role in the formation of IkappaBalpha-containing inclusions caused by proteasomal dysfunction, and that the generation of such inclusion is independent of cell death caused by impairment of proteasome.