Inhibition of the extracellular signal-regulated kinase signaling pathway is correlated with proteasome inhibitor suppression of coxsackievirus replication

The ubiquitin/proteasome system (UPS), a major intracellular protein degradation pathway, plays a critical role in coxsackieviral replication. To elucidate the mechanisms by which the UPS regulates viral replication, we studied the influence of proteasome inhibition on signaling through the extracellular signal-regulated kinase (ERK) pathway, a pathway which has been previously demonstrated to be necessary for coxsackieviral replication and contribute to virus-mediated pathogenesis. We found that proteasome inhibition reduced coxsackievirus-induced ERK phosphorylation in a dose-dependent manner, which is correlated with an induction of the mitogen-activated protein kinase phosphatase-1 (MKP-1). Blockade of MKP induction by short-interfering RNA attenuated the loss of ERK phosphorylation, and subsequently restored viral replication. Our results suggest that inhibition of the ERK signaling pathway contributes, as least in part, to proteasome inhibitor-mediated reduction of coxsackievirus replication, demonstrating a converging function of major intracellular signaling and protein degradation pathways in the regulation of viral replication.     

The role of autophagy in age-related neurodegeneration

Most age-related neurodegenerative diseases are characterized by accumulation of aberrant protein aggregates in affected brain regions. In many cases, these proteinaceous deposits are composed of ubiquitin conjugates, suggesting a failure in the clearance of proteins targeted for degradation. The 2 principal routes of intracellular protein catabolism are the ubiquitin proteasome system and the autophagy-lysosome system (autophagy). Both of these degradation pathways have been implicated as playing important roles in the pathogenesis of neurodegenerative disease. Here we describe autophagy and review the evidence suggesting that impairment of autophagy contributes to the initiation or progression of age-related neurodegeneration. We also review recent evidence indicating that autophagy may be exploited to remove toxic protein species, suggesting novel strategies for therapeutic intervention for a class of diseases for which no effective treatments presently exist.     

Receptors make the pathway choice for protein degradation

Damaged or aggregated proteins and organelles accumulate with age and contribute to various age-related pathologies including Alzheimer, Parkinson or Huntington diseases. In eukaryotic cells, there are 2 major pathways for degradation of the cytoplasm: The ubiquitin–proteasome system (UPS) and macroautophagy/autophagy. Both pathways can share the characteristic of initiating the process by ubiquitination of the substrate, but they utilize different ubiquitin receptors. In a paper described in a punctum in this issue, Lu et al. used the yeast Saccharomyces cerevisiae to demonstrate that the decision to use a particular pathway is made through a mechanism that depends on the receptors rather than the specific type of substrate ubiquitination.     

Autophagy Coupling Interplay: Can Improve Cellular Repair and Aging?

Regular protein synthesis is a needful and complex task for a healthy cell. Improper folding leads to the deposition of misfolded proteins in cells. Autophagy and ubiquitin–proteasome system (UPS) are the conserved intracellular degradation processes of eukaryotic cells. How exactly these two pathways cross talk to each other is unclear. We do not know how the impairment of autophagy or UPS leads to the disturbance in cellular homeostasis and contribute into cellular aging and neurodegeneration. Here in this review, we will focus on the functional interconnections of autophagy and UPS, and why their loss of function results in abnormal aggregation of misfolded proteotoxic species in cells. Finally, we enumerate and discuss the crucial inducers of autophagy pathways and elaborate their intersection steps, which have been considered to be advantageous in aging linked with the abnormal protein aggregation. The final goal of this review is to improve our current understanding about multifaceted properties and interactions of autophagy and UPS, which may provide new insights to identify novel therapeutic strategies for aging and neurodegenerative diseases.     

自噬和泛素-蛋白酶体系统之间的交联

自噬和泛素-蛋白酶体系统作为细胞内最重要的两大降解途径,对细胞稳态及细胞正常生理功能的维持都具有十分重要的作用。目前,越来越多的证据显示,这两大降解途径之间存在多种交联方式。首先,自噬和泛素-蛋白酶体系统都能以泛素作为共同标签,从而将泛素化底物降解;其次,泛素化的蛋白酶体可以通过自噬被清除,自噬相关蛋白质也可以通过蛋白酶体系统被降解;再次,这两条途径在细胞内能协同降解同一种底物;最后,它们之间可以相互调节活性,任一条途径被干扰都将影响另一条途径的活性。自噬和泛素-蛋白酶体系统之间的交联对细胞稳态的维持至关重要。交联失调不仅导致细胞功能异常,还可引起多种疾病的发生。本文主要对自噬和泛素-蛋白酶体系统之间的交联方式及其分子机制进行阐述,有助于深入了解细胞的分解代谢过程,进一步理解细胞稳态的维持机制,继而加深对相关疾病病理机制的认识。     

Mature ribosomes are selectively degraded upon starvation by an autophagy pathway requiring the Ubp3p/Bre5p ubiquitin protease

Eukaryotic cells use autophagy and the ubiquitin-proteasome system (UPS) as their major protein degradation pathways. Whereas the UPS is required for the rapid degradation of proteins when fast adaptation is needed, autophagy pathways selectively remove protein aggregates and damaged or excess organelles. However, little is known about the targets and mechanisms that provide specificity to this process. Here we show that mature ribosomes are rapidly degraded by autophagy upon nutrient starvation in Saccharomyces cerevisiae. Surprisingly, this degradation not only occurs by a non-selective mechanism, but also involves a novel type of selective autophagy, which we term 'ribophagy'. A genetic screen revealed that selective degradation of ribosomes requires catalytic activity of the Ubp3p/Bre5p ubiquitin protease. Although ubp3Delta and bre5Delta cells strongly accumulate 60S ribosomal particles upon starvation, they are proficient in starvation sensing and in general trafficking and autophagy pathways. Moreover, ubiquitination of several ribosomal subunits and/or ribosome-associated proteins was specifically enriched in ubp3Delta cells, suggesting that the regulation of ribophagy by ubiquitination may be direct. Interestingly, ubp3Delta cells are sensitive to rapamycin and nutrient starvation, implying that selective degradation of ribosomes is functionally important in vivo. Taken together, our results suggest a link between ubiquitination and the regulated degradation of mature ribosomes by autophagy.     

Sustained Autophagy Contributes to Measles Virus Infectivity

The interplay between autophagy and intracellular pathogens is intricate as autophagy is an essential cellular response to fight against infections, whereas numerous microbes have developed strategies to escape this process or even exploit it to their own benefit. The fine tuned timing and/or selective molecular pathways involved in the induction of autophagy upon infections could be the cornerstone allowing cells to either control intracellular pathogens, or be invaded by them. We report here that measles virus infection induces successive autophagy signallings in permissive cells, via distinct and uncoupled molecular pathways. Immediately upon infection, attenuated measles virus induces a first transient wave of autophagy, via a pathway involving its cellular receptor CD46 and the scaffold protein GOPC. Soon after infection, a new autophagy signalling is initiated which requires viral replication and the expression of the non-structural measles virus protein C. Strikingly, this second autophagy signalling can be sustained overtime within infected cells, independently of the expression of C, but via a third autophagy input resulting from cell-cell fusion and the formation of syncytia. Whereas this sustained autophagy signalling leads to the autophagy degradation of cellular contents, viral proteins escape from degradation. Furthermore, this autophagy flux is ultimately exploited by measles virus to limit the death of infected cells and to improve viral particle formation. Whereas CD150 dependent virulent strains of measles virus are unable to induce the early CD46/GOPC dependent autophagy wave, they induce and exploit the late and sustained autophagy. Overall, our work describes distinct molecular pathways for an induction of self-beneficial sustained autophagy by measles virus.     

Wild type and mutant amyloid precursor proteins influence downstream effects of proteasome and autophagy inhibition

Cells rely on complementary proteolytic pathways including the ubiquitin–proteasome system and autophagy to maintain proper protein degradation. There is known to be considerable interplay between them, whereby the loss of one clearance system results in compensatory changes in other proteolytic pathways of the cell. Disturbances in proteolysis are known to occur in Alzheimer's disease, and potentially contribute to neurophysiological and neurodegenerative processes. Currently, few data are available on how the presence of wild type and mutant amyloid precursor protein (APPwt and APPmut) potentially alters the reciprocal interplay between the different intracellular proteolytic pathways. This study used human SH-SY5Y neuronal cell lines, and SH-SY5Y transfected with either APPwt or APPmut (valine-to-glycine substitution at position 717), in order to explore if the presence of APPwt or APPmut altered the downstream effects of pharmacological proteasome or autophagy inhibition. The occurrence of APPwt or APPmut was observed to disturb proteasome or autophagy activities upon treatment with proteasome inhibitors or authophagy inhibitors. Interestingly, APPwt and APPmut expression was observed to significantly and robustly enhance the induction in cathepsin B following the administration of an established proteasome inhibitor. The presence of APPwt and APPmut also significantly reduced the elevation in ubiquitinated proteins following proteasome inhibitor treatments. Our data strongly suggest that APP is able to affect the downstream effects of protease inhibition in neural cells including enhancement of cathepsin B activity, with these changes in cathepsin B significantly and inversely related to the levels of ubiquitinated protein.     

Adaptive autophagy in Alexander disease-affected astrocytes

The ubiquitin-proteasome and autophagy-lysosomal pathways are the two main routes of protein and organelle clearance in eukaryotic cells. The proteasome system is responsible for unfolded, short-lived proteins, which precludes the clearance of oligomeric and aggregated proteins, whereas macroautophagy, a process generally referred to as autophagy, mediates mainly the bulk degradation of long-lived cytoplasmic proteins, large protein complexes or organelles.(1) Recently, the autophagy-lysosomal pathway has been implicated in neurodegenerative disorders as an important pathway for the clearance of abnormally accumulated intracellular proteins, such as huntingtin, tau and mutant and modified alpha-synuclein.(1-6) Our recent study illustrated the induction of adaptive autophagy in response to mutant glial fibrillary acidic protein (GFAP) accumulation in astrocytes, in the brains of patients with Alexander disease (AxD), and in mutant GFAP knock-in mouse brains.(7) This autophagic response is negatively regulated by mammalian target of rapamycin (mTOR). The activation of p38 MAPK by GFAP accumulation is responsible for mTOR inactivation and the induction of autophagy. We also found that the accumulation of GFAP impairs proteasome activity.(8) In this commentary we discuss the potential compensatory relationship between an impaired proteasome and activated autophagy, and propose that the MLK-MAPK (mixed lineage kinase-mitogen-activated protein kinase) cascade is a regulator of this crosstalk.     

Felix Hoppe-Seyler Lecture 2000. The ubiquitin system and the N-end rule pathway

Eukaryotes contain a highly conserved multienzyme system which covalently links a small protein, ubiquitin, to a variety of intracellular proteins that bear degradation signals recognized by this system. The resulting ubiquitin-protein conjugates are degraded by the 26S proteasome, an ATP-dependent protease. Pathways that involve ubiquitin play major roles in a huge variety of processes, including cell differentiation, cell cycle, and responses to stress. In this article we briefly review the design of the ubiquitin system, and describe two recent advances, the finding that ubiquitin ligases interact with specific components of the 26S proteasome, and the demonstration that peptides accelerate their uptake into cells by activating the N-end rule pathway, one of several proteolytic pathways of the ubiquitin system.     

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.     

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.     

Regulation of protein turnover by heat shock proteins

Protein turnover reflects the balance between synthesis and degradation of proteins, and it is a crucial process for the maintenance of the cellular protein pool. The folding of proteins, refolding of misfolded proteins, and also degradation of misfolded and damaged proteins are involved in the protein quality control (PQC) system. Correct protein folding and degradation are controlled by many different factors, one of the most important of which is the heat shock protein family. Heat shock proteins (HSPs) are in the class of molecular chaperones, which may prevent the inappropriate interaction of proteins and induce correct folding. On the other hand, these proteins play significant roles in the degradation pathways, including endoplasmic reticulum-associated degradation (ERAD), the ubiquitin–proteasome system, and autophagy. This review focuses on the emerging role of HSPs in the regulation of protein turnover; the effects of HSPs on the degradation machineries ERAD, autophagy, and proteasome; as well as the role of posttranslational modifications in the PQC system.     

p62 filaments capture and present ubiquitinated cargos for autophagy

The removal of misfolded, ubiquitinated proteins is an essential part of the protein quality control. The ubiquitin‐proteasome system (UPS) and autophagy are two interconnected pathways that mediate the degradation of such proteins. During autophagy, ubiquitinated proteins are clustered in a p62‐dependent manner and are subsequently engulfed by autophagosomes. However, the nature of the protein substrates targeted for autophagy is unclear. Here, we developed a reconstituted system using purified components and show that p62 and ubiquitinated proteins spontaneously coalesce into larger clusters. Efficient cluster formation requires substrates modified with at least two ubiquitin chains longer than three moieties and is based on p62 filaments cross‐linked by the substrates. The reaction is inhibited by free ubiquitin, K48‐, and K63‐linked ubiquitin chains, as well as by the autophagosomal marker LC3B, suggesting a tight cross talk with general proteostasis and autophagosome formation. Our study provides mechanistic insights on how substrates are channeled into autophagy.     

TNFα modulates protein degradation pathways in rheumatoid arthritis synovial fibroblasts

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory and destructive disease of the joint. The synovial lining consists of two main types of cells: synovial fibroblasts and macrophages. The macrophage-derived cytokine TNFα stimulates RA synovial fibroblasts to proliferate and produce growth factors, chemokines, proteinases and adhesion molecules, making them key players in the RA disease process. If proteins are not correctly folded, cellular stress occurs that can be relieved in part by increased degradation of the aberrant proteins by the proteasome or autophagy. We hypothesized that the activity of the protein degradation pathways would be increased in response to TNFα stimulation in RA synovial fibroblasts compared with control fibroblasts.

Methods

Endoplasmic reticulum (ER) stress markers were examined in synovial fibroblasts by immunoblotting and PCR. Use of the autophagy and proteasome protein degradation pathways in response to TNFα stimulation was determined using a combination of experiments involving chemical inhibition of the autophagy or proteasome pathways followed by immunoblotting for the autophagy marker LC3, measurement of proteasome activity and long-lived protein degradation, and determination of cellular viability.

Results

RA synovial fibroblasts are under acute ER stress, and the stress is increased in the presence of TNFα. Autophagy is the main pathway used to relieve the ER stress in unstimulated fibroblasts, and both autophagy and the proteasome are more active in RA synovial fibroblasts compared with control fibroblasts. In response to TNFα, the autophagy pathway but not the proteasome is consistently stimulated, yet there is an increased dependence on the proteasome for cell viability. If autophagy is blocked in the presence of TNFα, an increase in proteasome activity occurs in RA synovial fibroblasts but not in control cells.

Conclusions

TNFα stimulation of synovial fibroblasts results in increased expression of ER stress markers. Survival of synovial fibroblasts is dependent on continuous removal of proteins by both the lysosome/autophagy and ubiquitin/proteasome protein degradation pathways. Both pathways are more active in RA synovial fibroblasts compared with control fibroblasts. These results may provide a better understanding of the mechanism of TNFα on prolonging the survival of synovial fibroblasts in RA tissue.     

Substrate recognition in selective autophagy and the ubiquitin–proteasome system

Dynamic protein turnover through regulated protein synthesis and degradation ensures cellular growth, proliferation, differentiation and adaptation. Eukaryotic cells utilize two mechanistically distinct but largely complementary systems — the 26S proteasome and the lysosome (or vacuole in yeast and plants) — to effectively target a wide range of proteins for degradation. The concerted action of the ubiquitination machinery and the 26S proteasome ensures the targeted and tightly regulated degradation of a subset of commonly short-lived cellular proteins. Autophagy is a distinct degradation pathway, which transports a highly heterogeneous set of cargos in dedicated vesicles, called autophagosomes, to the lysosome. There the cargo becomes degraded and its molecular building blocks are recycled. While general autophagy randomly engulfs portions of the cytosol, selective autophagy employs dedicated cargo adaptors to specifically enrich the forming autophagosomes for a certain type of cargo as a response to various intra- or extracellular signals. Selective autophagy targets a wide range of cargos including long-lived proteins and protein complexes, organelles, protein aggregates and even intracellular microbes. In this review we summarize available data on cargo recognition mechanisms operating in selective autophagy and the ubiquitin–proteasome system (UPS), and emphasize their differences and common themes. Moreover, we derive general regulatory principles underlying cargo recognition in selective autophagy, and describe the system-wide crosstalk between these two cellular protein degradation systems. This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.     

A novel crosstalk between two major protein degradation systems

Eukaryotes have two major intracellular protein degradation pathways, namely the ubiquitin-proteasome system (UPS) and autophagy. Inhibition of proteasomal activities has been previously shown to induce autophagy, indicating a coordinated and complementary relationship between these two systems. However, little is known about the regulation of the UPS by autophagy. In this study, we showed for the first time that proteasomes were activated in response to pharmacological inhibition of autophagy as well as disruption of autophagy-related genes by RNA interference under nutrient-deficient conditions in cultured human colon cancer cells. The induction was evidenced by the increased proteasomal activities and the upregulation of proteasomal subunits, including the proteasome β5 subunit, PSMB5. Co-inhibition of the proteasome and autophagy also synergistically increased the accumulation of polyubiquitinated proteins. Collectively, our findings suggest that proteasomes are activated in a compensatory manner for protein degradation upon autophagy inhibition. Our studies unveiled a novel regulatory mechanism between the two protein degradation pathways.     

MURF2B,a Novel LC3-Binding Protein,Participates with MURF2A in the Switch between Autophagy and Ubiquitin Proteasome System during Differentiation of C2C12 Muscle Cells

The ubiquitin proteasome system and macroautophagy are proteolytic pathways essential in the maintenance of cellular homeostasis during differentiation and remodelling of skeletal muscle. In both pathways, proteins to be degraded are tagged with polyubiquitin. In skeletal muscles, the MURF2 proteins display E3 ubiquitin ligase structure suggesting that they may covalently attach ubiquitin polypeptides to still unknown target proteins. So far only MURF2A isoforms were studied and shown to interact with p62/SQSTM1, a protein implicated in macroautophagic and ubiquitin proteasome system degradations. Here, we analyzed the MURF2B and MURF2A proteins and show that the ratio of the isoforms changes during differentiation of muscle C2C12 cells and that the shift of the isoforms expression follows the sequential activation of autophagic or proteasomal degradation. We also show that MURF2B has a functional domain needed for its interaction with LC3, a protein needed for autophagic vesicles formation. Using specific MURF2 RNAi cells we observed that MURF2A and MURF2B are both needed for the formation of autophagosomes and that in the absence of MURF2B, the cells expressing MURF2A display an activated ubiquitin proteasome system implicated in the degradation of p62/SQSTM1 by UPS. Altogether, our results indicate that MURF2A and MURF2B proteins could participate in the molecular switch between the two ubiquitin degradative pathways.