HDAC6 at the intersection of autophagy, the ubiquitin-proteasome system and neurodegeneration

The two major intracellular catabolic pathways, the ubiquitin-proteasome system (UPS) and macroautophagy (autophagy), have each been implicated as playing roles in neurodegenerative proteinopathies. We have investigated the relationship between the UPS and autophagy using Drosophila models of neurodegenerative diseases. We identified histone deacetylase 6 (HDAC6) as a genetic modifier of polyglutamine-induced neurodegeneration and determined that its mechanism of action is autophagy-dependent. The ability of HDAC6 to suppress degeneration has been extended to additional neurodegenerative disease models, including a fly model expressing pathological Abeta fragments, presented here, but is not a universal modifier of degenerative phenotypes. Importantly, HDAC6 was also found to suppress degeneration associated with proteasome mutations in an autophagy-dependent manner, revealing a compensatory relationship between these two degradation pathways. Our findings indicate that HDAC6 facilitates degradation of potentially noxious protein substrates, contributing vitally to the neuroprotective role of autophagy.     

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.     

HDAC6 at the Intersection of Autophagy,the Ubiquitin-proteasome System,and Neurodegeneration

The two major intracellular catabolic pathways, the ubiquitin-proteasome system (UPS) and macroautophagy (autophagy), have each been implicated as playing roles in neurodegenerative proteinopathies1, 2. We have investigated the relationship between the UPS and autophagy using Drosophila models of neurodegenerative diseases. We identified histone deacetylase 6 (HDAC6) as a genetic modifier of polyglutamine-induced neurodegeneration and determined that its mechanism of action is autophagy-dependent3. The ability of HDAC6 to suppress degeneration has been extended to additional neurodegenerative disease models, including a fly model expressing pathologic Aβ fragments presented here, but is not a universal modifier of degenerative phenotypes. Importantly, HDAC6 was also found to suppress degeneration associated with proteasome mutations in an autophagy-dependent manner, revealing a compensatory relationship between these two degradation pathways. Our findings indicate that HDAC6 facilitates degradation of potentially noxious protein substrates, contributing vitally to the neuroprotective role of autophagy.     

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。     

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.     

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.     

Alpha-synuclein and Protein Degradation Systems: a Reciprocal Relationship

An increasing wealth of data indicates a close relationship between the presynaptic protein alpha-synuclein and Parkinson’s disease (PD) pathogenesis. Alpha-synuclein protein levels are considered as a major determinant of its neurotoxic potential, whereas secreted extracellular alpha-synuclein has emerged as an additional important factor in this regard. However, the manner of alpha-synuclein degradation in neurons remains contentious. Both the ubiquitin–proteasome system (UPS) and the autophagy–lysosome pathway (ALP)—mainly macroautophagy and chaperone-mediated autophagy—have been suggested to contribute to alpha-synuclein turnover. Additionally, other proteases such as calpains, neurosin, and metalloproteinases have been also proposed to have a role in intracellular and extracellular alpha-synuclein processing. Both UPS and ALP activity decline with aging and such decline may play a pivotal role in many neurodegenerative conditions. Alterations in these major proteolytic pathways may result in alpha-synuclein accumulation due to impaired clearance. Conversely, increased alpha-synuclein protein burden promotes the generation of aberrant species that may impair further UPS or ALP function, generating thus a bidirectional positive feedback loop leading to neuronal death. In the current review, we summarize the recent findings related to alpha-synuclein degradation, as well as to alpha-synuclein-mediated aberrant effects on protein degradation systems. Identifying the factors that regulate alpha-synuclein association to cellular proteolytic pathways may represent potential targets for therapeutic interventions in PD and related synucleinopathies.     

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.     

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.     

A novel link between autophagy and the ubiquitin-proteasome system

Autophagy and the ubiquitin-proteasome system (UPS) are the major routes for intracellular protein degradation. These two pathways were previously thought to be largely distinct. Here we summarize our recent work that demonstrates that long-term autophagy inhibition slows the clearance of short-lived UPS-specific substrates, like p53. This is caused by the accumulation of p62 after autophagy inhibition. These data suggest that the ramifications of a block in autophagy may be much wider than what was previously thought. Rather than simply decreasing clearance of autophagic substrates, while UPS flux is undisturbed, the cell will have to contend with a decrease in clearance by both major routes.     

Autophagy and the ubiquitin-proteasome system: collaborators in neuroprotection

Protein degradation is an essential cellular function that, when dysregulated or impaired, can lead to a wide variety of disease states. The two major intracellular protein degradation systems are the ubiquitin-proteasome system (UPS) and autophagy, a catabolic process that involves delivery of cellular components to the lysosome for degradation. While the UPS has garnered much attention as it relates to neurodegenerative disease, important links between autophagy and neurodegeneration have also become evident. Furthermore, recent studies have revealed interaction between the UPS and autophagy, suggesting a coordinated and complementary relationship between these degradation systems that becomes critical in times of cellular stress. Here we describe autophagy and review evidence implicating this system as an important player in the pathogenesis of neurodegenerative disease. We discuss the role of autophagy in neurodegeneration and review its neuroprotective functions as revealed by experimental manipulation in disease models. Finally, we explore potential parallels and connections between autophagy and the UPS, highlighting their collaborative roles in protecting against neurodegenerative disease.     

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.     

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.     

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.     

The dependency of autophagy and ubiquitin proteasome system during skeletal muscle atrophy

Among the four proteolytic systems in the cell, autophagy and the ubiquitin-proteasome system (UPS) are the main proteolytic events that allow for the removal of cell debris and proteins to maintain cellular homeostasis. Previous studies have revealed that these systems perform their functions independently of each other. However, recent studies indicate the existence of regulatory interactions between these proteolytic systems via ubiquitinated tags and a reciprocal regulation mechanism with several crosstalk points. UPS plays an important role in the elimination of short-lived/soluble misfolded proteins, whereas autophagy eliminates defective organelles and persistent insoluble protein aggregates. Both of these systems seem to act independently; however, disruption of one pathway affects the activity of the other pathway and contributes to different pathological conditions. This review summarizes the recent findings on direct and indirect dependencies of autophagy and UPS and their execution at the molecular level along with the important drug targets in skeletal muscle atrophy.     

The ubiquitin-proteasome system in cardiac dysfunction

Since proteins play crucial roles in all biological processes, the finely tuned equilibrium between their synthesis and degradation regulates cellular homeostasis. Controlling the quality of proteome informational content is essential for cell survival and function. After initial synthesis, membrane and secretory proteins are modified, folded, and assembled in the endoplasmic reticulum, whereas other proteins are synthesized and processed in the cytosol. Cells have different protein quality control systems, the molecular chaperones, which help protein folding and stabilization, and the ubiquitin-proteasome system (UPS) and lysosomes, which degrade proteins. It has generally been assumed that UPS and lysosomes are regulated independently and serve distinct functions. The UPS degrades both cytosolic, nuclear proteins, and myofibrillar proteins, whereas the lysosomes degrade most membrane and extracellular proteins by endocytosis as well as cytosolic proteins and organelles via autophagy. Over the last two decades, the UPS has been increasingly recognized as a major system in several biological processes including cell proliferation, adaptation to stress and cell death. More recently, activation or impairment of the UPS has been reported in cardiac disease and recent evidence indicate that autophagy is a key mechanism to maintain cardiac structure and function. This review mainly focuses on the UPS and its various components in healthy and diseased heart, but also summarizes recent data suggesting parallel activation of the UPS and autophagy in cardiac disease.     

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.