Coordinate regulation of autophagy and the ubiquitin proteasome system by MTOR

Proteins in eukaryotic cells are continually being degraded to amino acids either by the ubiquitin proteasome system (UPS) or by the autophagic-lysosomal pathway. The breakdown of proteins by these 2 degradative pathways involves totally different enzymes that function in distinct subcellular compartments. While most studies of the UPS have focused on the selective ubiquitination and breakdown of specific cell proteins, macroautophagy/autophagy is a more global nonselective process. Consequently, the UPS and autophagy were traditionally assumed to serve distinct physiological functions and to be regulated in quite different manners. However, recent findings indicate that protein breakdown by these 2 systems is coordinately regulated by important physiological stimuli. The activation of MTORC1 by nutrients and hormones rapidly suppresses proteolysis by both proteasomes and autophagy, which helps promote protein accumulation, whereas in nutrient-poor conditions, MTORC1 inactivation causes the simultaneous activation of these 2 degradative pathways to supply the deprived cells with a source of amino acids. Also this selective breakdown of key anabolic proteins by the UPS upon MTORC1 inhibition can help limit growth-related processes (e.g., cholesterol biosynthesis). Thus, the collaboration of these 2 degradative systems, together with the simultaneous control of protein translation by MTORC1, provide clear advantages to the organism in both growth and starvation conditions.     

Induction of autophagy by proteasome inhibitor is associated with proliferative arrest in colon cancer cells

The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Blockade of UPS by proteasome inhibitors has been shown to activate autophagy. Recent evidence also suggests that proteasome inhibitors may inhibit cancer growth. In this study, the effect of a proteasome inhibitor MG-132 on the proliferation and autophagy of cultured colon cancer cells (HT-29) was elucidated. Results showed that MG-132 inhibited HT-29 cell proliferation and induced G₂/M cell cycle arrest which was associated with the formation of LC3⁺ autophagic vacuoles and the accumulation of acidic vesicular organelles. MG-132 also increased the protein expression of LC3-I and -II in a time-dependent manner. In this connection, 3-methyladenine, a Class III phosphoinositide 3-kinase inhibitor, significantly abolished the formation of LC3⁺ autophagic vacuoles and the expression of LC3-II but not LC3-I induced by MG-132. Taken together, this study demonstrates that inhibition of proteasome in colon cancer cells lowers cell proliferation and activates autophagy. This discovery may shed a new light on the novel function of proteasome in the regulation of autophagy and proliferation in colon cancer cells.     

SIRT2 interferes with autophagy-mediated degradation of protein aggregates in neuronal cells under proteasome inhibition

Abnormal protein aggregates have been suggested as a common pathogenesis of many neurodegenerative diseases. Two well-known protein degradation pathways are responsible for protein homeostasis by balancing protein biosynthesis and degradative processes: the ubiquitin–proteasome system (UPS) and autophagy-lysosomal system. UPS serves as the primary route for degradation of short-lived proteins, but large-size protein aggregates cannot be degraded by UPS. Autophagy is a unique cellular process that facilitates degradation of bulky protein aggregates by lysosome. Recent studies have demonstrated that autophagy plays a crucial role in the pathogenesis of neurodegenerative diseases characterized by abnormal protein accumulation, suggesting that regulation of autophagy may be a valuable therapeutic strategy for the treatment of various neurodegenerative diseases. Sirtuin-2 (SIRT2) is a class III histone deacetylase that is expressed abundantly in aging brain tissue. Here, we report that SIRT2 increases protein accumulation in murine cholinergic SN56 cells and human neuroblastoma SH-SY5Y cells under proteasome inhibition. Overexpression of SIRT2 inhibits lysosome-mediated autophagic turnover by interfering with aggresome formation and also makes cells more vulnerable to accumulated protein-mediated cytotoxicity by MG132 and amyloid beta. Moreover, MG132-induced accumulation of ubiquitinated proteins and p62 as well as cytotoxicity are attenuated in siRNA-mediated SIRT2-silencing cells. Taken together, these results suggest that regulation of SIRT2 could be a good therapeutic target for a range of neurodegenerative diseases by regulating autophagic flux.     

Insights into the relationship between the proteasome and autophagy in human and yeast cells

In eukaryotes, the ubiquitin-proteasome system (UPS) and autophagy are two major intracellular protein degradation pathways. Several lines of evidence support the emerging concept of a coordinated and complementary relationship between these two processes, and a particularly interesting finding is that the inhibition of the proteasome induces autophagy. Yet, there is limited knowledge of the regulation of the UPS by autophagy. In this study, we show that the disruption of ATG5 and ATG32 genes in yeast cells under both nutrient-deficient conditions as well as stress that causes mitochondrial dysfunction leads to an activation of proteasome. The same scenario occurs after pharmacological inhibition of basal autophagy in cultured human cells. Our findings underline the view that the two processes are interconnected and tend to compensate, to some extent, for each other's functions.     

BAG3-dependent noncanonical autophagy induced by proteasome inhibition in HepG2 cells

Emerging lines of evidence have shown that blockade of ubiquitin-proteasome system (UPS) activates autophagy. The molecular players that regulate the relationship between them remain to be elucidated. Bcl-2 associated athanogene 3 (BAG3) is a member of the BAG co-chaperone family that regulates the ATPase activity of heat shock protein 70 (HSP70) chaperone family. Studies on BAG3 have demonstrated that it plays multiple roles in physiological and pathological processes, including antiapoptotic activity, signal transduction, regulatory role in virus infection, cell adhesion and migration. Recent studies have attracted much attention on its role in initiation of autophagy. The current study, for the first time, demonstrates that proteasome inhibitors elicit noncanonical autophagy, which was not suppressed by inhibitors of class III phosphatidylinositol 3-kinase (PtdIns3K) or shRNA against Beclin 1 (BECN1). In addition, we demonstrate that BAG3 is ascribed to activation of autophagy elicited by proteasome inhibitors and MAPK8/9/10 (also known as JNK1/2/3 respectively) activation is also implicated via upregulation of BAG3. Moreover, we found that noncanonical autophagy mediated by BAG3 suppresses responsiveness of HepG2 cells to proteasome inhibitors.     

Unfolded Protein Response and Activated Degradative Pathways Regulation in GNE Myopathy

Although intracellular beta amyloid (Aβ) accumulation is known as an early upstream event in the degenerative course of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) myopathy, the process by which Aβdeposits initiate various degradative pathways, and their relationship have not been fully clarified. We studied the possible secondary responses after amyloid beta precursor protein (AβPP) deposition including unfolded protein response (UPR), ubiquitin proteasome system (UPS) activation and its correlation with autophagy system. Eight GNE myopathy patients and five individuals with normal muscle morphology were included in this study. We performed immunofluorescence and immunoblotting to investigate the expression of AβPP, phosphorylated tau (p-tau) and endoplasmic reticulum molecular chaperones. Proteasome activities were measured by cleavage of fluorogenic substrates. The expression of proteasome subunits and linkers between proteasomal and autophagy systems were also evaluated by immunoblotting and relative quantitative real-time RT-PCR. Four molecular chaperones, glucose-regulated protein 94 (GRP94), glucose-regulated protein 78 (GRP78), calreticulin and calnexin and valosin containing protein (VCP) were highly expressed in GNE myopathy. 20S proteasome subunits, three main proteasome proteolytic activities, and the factors linking UPS and autophagy system were also increased. Our study suggests that AβPP deposition results in endoplasmic reticulum stress (ERS) and highly expressed VCP deliver unfolded proteins from endoplasmic reticulum to proteosomal system which is activated in endoplasmic reticulum associated degradation (ERAD) in GNE myopathy. Excessive ubiquitinated unfolded proteins are exported by proteins that connect UPS and autophagy to autophagy system, which is activated as an alternative pathway for degradation.     

Parkin deficiency increases the resistance of midbrain neurons and glia to mild proteasome inhibition: the role of autophagy and glutathione homeostasis

Parkin mutations in humans produce parkinsonism whose pathogenesis is related to impaired protein degradation, increased free radicals and abnormal neurotransmitter release. In this study, we have investigated whether partial proteasomal inhibition by epoxomicin, an ubiquitin proteasomal system (UPS) irreversible inhibitor, further aggravates the cellular effects of parkin suppression in midbrain neurons and glia. We observed that parkin null (PK‐KO) midbrain neuronal cultures are resistant to epoxomicin‐induced cell death. This resistance is due to increased GSH and DJ‐1 protein levels in PK‐KO mice. The treatment with epoxomicin increases, in wild type (WT) cultures, the pro‐apoptotic Bax/Bcl‐2 ratio, the phosphorylation of tau, and the levels of chaperones heat‐shock protein 70 and C‐terminal Hsc‐interacting protein, but none of these effects took place in epoxomicin‐treated PK‐KO cultures. Poly‐ubiquitinated proteins increased more in WT than in PK‐KO‐treated neuronal cultures. Parkin accumulated in WT neuronal cultures treated with epoxomicin. Markers of autophagy, such as LC3II/I, were increased in naïve PK‐KO cultures, and further increased after treatment with epoxomicin, implying that the blockade of the proteasome in PK‐KO neurons triggers the enhancement of autophagy. The treatment with l ‐buthionine‐S,R‐sulfoximine and the inhibition of autophagy, however, reverted the increase resistance to epoxomicin of the PK‐KO cultures. We also found that PK‐KO glial cells, stressed by growth in defined medium and depleted of GSH, were more susceptible to epoxomicin induced cell death than WT glia treated similarly. This susceptibility was linked to reduced GSH levels and less heat‐shock protein 70 response, and to activation of p‐serine/threonine kinase protein signaling pathway as well as to increased poly‐ubiquitinated proteins. These data suggest that mild UPS inhibition is compensated by other mechanisms in PK‐KO midbrain neurons. However the depletion of GSH, as happens in stressed glia, suppresses the protection against UPS inhibition‐induced cell death. Furthermore, GSH inhibition regulated differentially UPS activity and in old PK‐KO mice, which have depletion of GSH, UPS activity is decreased in comparison with that of old‐WT.     

Anti- and pro-microbial roles of autophagy in plant-bacteria interactions

Macroautophagy/autophagy and the ubiquitin-proteasome system (UPS) are major proteolytic pathways that are increasingly recognized as battlegrounds during host-microbe interactions in eukaryotes. In plants, the UPS has emerged as central component of innate immunity and is manipulated by bacterial pathogens to enhance virulence. Autophagy has been ascribed a similar importance for anti-bacterial immunity in animals, but the contribution of autophagy to host-bacteria interactions remained elusive in plants. Here, we present and discuss our recent findings that revealed anti- and pro-bacterial roles of autophagy pathways during bacterial infection in the model plant Arabidopsis thaliana. We discovered that selective autophagy mediated by the autophagy cargo receptor AT4G24690/NBR1 limits growth of Pseudomonas syringae pv. tomato DC3000 (Pst) by suppressing the establishment of an aqueous extracellular space (‘water-soaking’). In turn, Pseudomonas employs the effector protein HopM1 to activate autophagy and proteasome degradation (‘proteaphagy’), thereby enhancing its pathogenicity. Thus, our study demonstrates that distinct selective autophagy pathways contribute to host immunity and bacterial pathogenesis during Pst infection and provide evidence for an intimate crosstalk between the proteasome and autophagy system in plant-bacterial interactions.     

Time-Point Dependent Activation of Autophagy and the UPS in SOD1G93A Mice Skeletal Muscle

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by a selective loss of motor neurons together with a progressive muscle weakness. Albeit the pathophysiological mechanisms of the disease remain unknown, growing evidence suggests that skeletal muscle can be a target of ALS toxicity. In particular, the two main intracellular degradation mechanisms, autophagy and the ubiquitin-proteasome degradative system (UPS) have been poorly studied in this tissue. In this study we investigated the activation of autophagy and the UPS as well as apoptosis in the skeletal muscle from SOD1G93A mice along disease progression. Our results showed a significant upregulation of proteasome activity at early symptomatic stage, while the autophagy activation was found at presymptomatic and terminal stages. The mRNA upregulated levels of LC3, p62, Beclin1, Atg5 and E2f1 were only observed at symptomatic and terminal stages, which reinforced the time-point activation of autophagy. Furthermore, no apoptosis activation was observed along disease progression. The combined data provided clear evidence for the first time that there is a time-point dependent activation of autophagy and UPS in the skeletal muscle from SOD1G93A mice.     

Mechanisms of cross-talk between the ubiquitin-proteasome and autophagy-lysosome systems

The ubiquitin proteasome system (UPS) and macroautophagy (hereafter called autophagy) were, for a long time, regarded as independent degradative pathways with few or no points of interaction. This view started to change recently, in the light of findings that have suggested that ubiquitylation can target substrates for degradation via both pathways. Moreover, perturbations in the flux through either pathway have been reported to affect the activity of the other system, and a number of mechanisms have been proposed to rationalise the link between the UPS and autophagy. Here we critically review these findings and outline some outstanding issues that still await clarification.     

Combined autophagy and proteasome inhibition

The efficacy of proteasome inhibition for myeloma is limited by therapeutic resistance, which may be mediated by activation of the autophagy pathway as an alternative mechanism of protein degradation. Preclinical studies demonstrate that autophagy inhibition with hydroxychloroquine augments the antimyeloma efficacy of the proteasome inhibitor bortezomib. We conducted a phase I trial combining bortezomib and hydroxychloroquine for relapsed or refractory myeloma. We enrolled 25 patients, including 11 (44%) refractory to prior bortezomib. No protocol-defined dose-limiting toxicities occurred, and we identified a recommended phase 2 dose of hydroxychloroquine 600 mg twice daily with standard doses of bortezomib, at which we observed dose-related gastrointestinal toxicity and cytopenias. Of 22 patients evaluable for response, 3 (14%) had very good partial responses, 3 (14%) had minor responses, and 10 (45%) had a period of stable disease. Electron micrographs of bone marrow plasma cells collected at baseline, after a hydroxychloroquine run-in, and after combined therapy showed therapy-associated increases in autophagic vacuoles, consistent with the combined effects of increased trafficking of misfolded proteins to autophagic vacuoles and inhibition of their degradative capacity. Combined targeting of proteasomal and autophagic protein degradation using bortezomib and hydroxychloroquine is therefore feasible and a potentially useful strategy for improving outcomes in myeloma therapy.     

Amelioration of neuronal cell death in a spontaneous obese rat model by dietary restriction through modulation of ubiquitin proteasome system

Dietary restriction (DR) has been shown to increase longevity, delay onset of aging, reduce DNA damage and oxidative stress and prevent age-related decline of neuronal activity. We previously reported the role of altered ubiquitin proteasome system (UPS) in the neuronal cell death in a spontaneous obese rat model (WNIN/Ob rat). In this study, we investigated the effect of DR on obesity-induced neuronal cell death in a rat model. Two groups of 40-day-old WNIN/Ob rats were either fed ad libitum (Ob) or pair-fed with lean. The lean phenotype of WNIN/Ob rats served as ad libitum control. These animals were maintained for 6.5 months on their respective diet regime. At the end of the study, cerebral cortex was collected and markers of UPS, endoplasmic reticulum (ER) stress and autophagy were analyzed by quantitative real-time polymerase chain reaction, immunoblotting and immunohistochemistry. Chymotrypsin-like activity of proteasome was assayed by the fluorimetric method. Apoptotic cells were analyzed by TUNEL assay. DR improved metabolic abnormalities in obese rats. Alterations in UPS (up-regulation of UCHL1, down-regulation of UCHL5, declined proteasomal activity), increased ER stress, declined autophagy and increased expression of α-synuclein, p53 and BAX were observed in obese rats and DR alleviated these changes in obese rats. Further, DR decreased TUNEL-positive cells. In conclusion, DR in obese rats could not only restore the metabolic abnormalities but also preserved neuronal health in the cerebral cortex by preventing alterations in the UPS.     

The proteasome controls presynaptic differentiation through modulation of an on-site pool of polyubiquitinated conjugates

Differentiation of the presynaptic terminal is a complex and rapid event that normally occurs in spatially specific axonal regions distant from the soma; thus, it is believed to be dependent on intra-axonal mechanisms. However, the full nature of the local events governing presynaptic assembly remains unknown. Herein, we investigated the involvement of the ubiquitin–proteasome system (UPS), the major degradative pathway, in the local modulation of presynaptic differentiation. We found that proteasome inhibition has a synaptogenic effect on isolated axons. In addition, formation of a stable cluster of synaptic vesicles onto a postsynaptic partner occurs in parallel to an on-site decrease in proteasome degradation. Accumulation of ubiquitinated proteins at nascent sites is a local trigger for presynaptic clustering. Finally, proteasome-related ubiquitin chains (K11 and K48) function as signals for the assembly of presynaptic terminals. Collectively, we propose a new axon-intrinsic mechanism for presynaptic assembly through local UPS inhibition. Subsequent on-site accumulation of proteins in their polyubiquitinated state triggers formation of presynapses.     

Regulation of proteasome activity in health and disease

The ubiquitin–proteasome system (UPS) is the primary selective degradation system in the nuclei and cytoplasm of eukaryotic cells, required for the turnover of myriad soluble proteins. The hundreds of factors that comprise the UPS include an enzymatic cascade that tags proteins for degradation via the covalent attachment of a poly-ubiquitin chain, and a large multimeric enzyme that degrades ubiquitinated proteins, the proteasome. Protein degradation by the UPS regulates many pathways and is a crucial component of the cellular proteostasis network. Dysfunction of the ubiquitination machinery or the proteolytic activity of the proteasome is associated with numerous human diseases. In this review we discuss the contributions of the proteasome to human pathology, describe mechanisms that regulate the proteolytic capacity of the proteasome, and discuss strategies to modulate proteasome function as a therapeutic approach to ameliorate diseases associated with altered UPS function. This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.     

Illuminating the ubiquitin/proteasome system

The ubiquitin/proteasome system (UPS) is responsible for the regulated processive degradation of proteins residing in the cytosol, nucleus, and endoplasmic reticulum. The two central players are ubiquitin, a small protein that is conjugated to substrates, and the proteasome, a large multi-subunit proteolytic complex that executes degradation of ubiquitylated proteins. Ubiquitylation and proteasomal degradation are highly dynamic processes. During the last decade, many researchers have started taking advantage of fluorescent proteins, which allow studying the dynamic nature of this system in the context of its natural environment: the living cell. In this review, we will summarize studies that have implemented this approach to examine the UPS and discuss novel insights in the dynamic organization of the UPS.     

In quiescence of fission yeast,autophagy and the proteasome collaborate for mitochondrial maintenance and longevity

Regulation of proliferation and quiescence in response to intra- or extracellular environmental signals are important for medicine and basic biology. Quiescence is relevant to tumorigenesis and tissue regeneration, and the maintenance of post-mitotic cells is important with regard to a number of senescence-related diseases such as neurodegeneration. We employ fission yeast, Schizosaccharomyces pombe, as a model to study quiescence and longevity as this lower eukaryote has a long chronological life span (over months) in quiescence that is induced by nitrogen starvation. We recently reported that autophagy and the proteasome cooperate in proper mitochondrial maintenance in the quiescent phase. Such cooperativity is not found in proliferating cells. In quiescence, the proteasome is required for normal mitochondrial functions; inactivation of the proteasome results in a large accumulation of reactive oxygen species (ROS), diminished mitochondrial function, and the elevation of proteins and compounds having anti-oxidant activities. Autophagy contributes to preventing the lethal accumulation of ROS by degrading mitochondria, the primary source of ROS. Our results indicate that the degradation of mitochondria by autophagy during proteasome dysfunction is a defense mechanism of quiescenct cells against the accumulation of ROS.