Phasing out the bad—How SQSTM1/p62 sequesters ubiquitinated proteins for degradation by autophagy

The degradation of misfolded, ubiquitinated proteins is essential for cellular homeostasis. These proteins are primarily degraded by the ubiquitin-proteasome system (UPS) and macroautophagy/autophagy serves as a backup mechanism when the UPS is overloaded. How autophagy and the UPS are coordinated is not fully understood. During the autophagy of misfolded, ubiquitinated proteins, referred to as aggrephagy, substrate proteins are clustered into larger structures in a SQSTM1/p62-dependent manner before they are sequestered by phagophores, the precursors to autophagosomes. We have recently shown that SQSTM1/p62 and ubiquitinated proteins spontaneously phase separate into micrometer-sized clusters in vitro. This enabled us to characterize the properties of the ubiquitin-positive substrates that are necessary for the SQSTM1/p62-mediated cluster formation. Our results suggest that aggrephagy is triggered by the accumulation of substrates with multiple ubiquitin chains and that the process can be inhibited by active proteasomes.     

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。     

Inhibition of Protein Deubiquitination by PR-619 Activates the Autophagic Pathway in OLN-t40 Oligodendroglial Cells

Protein aggregate formation may be the result of an impairment of the protein quality control system, e.g., the ubiquitin proteasome system (UPS) and the lysosomal autophagic pathway. For proteasomal degradation, proteins need to be covalently modified by ubiquitin and deubiquitinated before the substrates are proteolytically degraded. Deubiquitination is performed by a large family of proteases, the deubiquitinating enzymes (DUBs). DUBs display a variety of functions and their inhibition may have pathological consequences. Using the broad specificity DUB inhibitor PR-619 we previously have shown that DUB inhibition leads to an overload of ubiquitinated proteins, to protein aggregate formation and subsequent inhibition of the UPS. This study was undertaken to investigate whether PR-619 modulates autophagic functions to possibly compensate the failure of the proteasomal system. Using the oligodendroglial cell line OLN-t40 and a new oligodendroglial cell line stably expressing GFP-LC3, we show that DUB inhibition leads to the activation of autophagy and to the recruitment of LC3 and of the ubiquitin binding protein p62 to the forming aggresomes without impairing the autophagic flux. Furthermore, PR-619 induced the transport of lysosomes to the forming aggregates in a process requiring an intact microtubule network. Further stimulation of autophagy by rapamycin did not prevent PR-619 aggregate formation but rather exerted cytotoxic effects. Hence, inhibition of DUBs by PR-619 activated the autophagic pathway supporting the hypothesis that the UPS and the autophagy–lysosomal pathway are closely linked together.     

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.     

The proteasome subunit RPN10 functions as a specific receptor for degradation of the 26S proteasome by macroautophagy in Arabidopsis

The ubiquitin-proteasome system (UPS) and macroautophagy/autophagy are 2 main degradative routes, which are important for cellular homeostasis. In a study conducted by Marshall et al., the authors demonstrated that the UPS and autophagy converge in Arabidopsis (see the punctum in issue #11–10). In particular, they found that the 26S proteasome is degraded by autophagy, either nonselectively (induced by nitrogen starvation) or selectively (induced by proteasome inhibition). The selective phenotype is mediated through the proteasome subunit RPN10, which can bind both ubiquitin and ATG8. This newly identified autophagic degradation of the proteasome is termed “proteaphagy,” and the process reveals an interesting relationship between these degradative systems.     

Small molecule enhancers of autophagy for neurodegenerative diseases

Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, prion diseases and polyglutamine disorders, including Huntington's disease and various spinocerebellar ataxias, are associated with the formation of protein aggregates. These aggregates and/or their precursors are thought to be toxic disease-causing species. Autophagy is a major degradation pathway for intracytosolic aggregate-prone proteins, including those associated with neurodegeneration. It is a constitutive self-degradative process involved both in the basal turnover of cellular components and in response to nutrient starvation in eukaryotes. Enhancing autophagy may be a possible therapeutic strategy for neurodegenerative disorders where the mutant proteins are autophagy substrates. In cell and animal models, chemical induction of autophagy protects against the toxic insults of these mutant aggregate-prone proteins by enhancing their clearance. We will discuss various autophagy-inducing small molecules that have emerged in the past few years that may be leads towards the treatment of such devastating diseases.     

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.     

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.     

Reticulon 3 attenuates the clearance of cytosolic prion aggregates via inhibiting autophagy

Autophagy plays an important role in targeting cellular proteins, protein aggregates and organelles for degradation for cell survival. Autophagy dysfunction has been extensively described in neurodegenerative conditions linked to protein misfolding and aggregation. However, the role of autophagy in the prion disease process is unclear. Here, we show that when expressed in mouse neuroblastoma N2a cells, cytoplasmic PrP (cyPrP) aggregates lead to endoplasmic reticulum stress (ER stress), activation of reticulon 3 (RTN3), impairment of ubiquitin-proteasome system (UPS), induction of autophagy and apoptosis. RTN3 belongs to the reticulon family with the highest expression in the brain and RTN3 is often activated under ER stress. To assess the function of RTN3 in pathological conditions involving cyPrP protein misfolding, we knocked down the expression of RTN3 in cyPrP-transfected cells; unexpectedly, the inhibition of expression of RTN3 enhances the induction of autophagy resulted from cyPrP aggregates, and the process is mediated by the enhanced interaction between Bcl-2 and Beclin1 promoted by RTN3, which enhances Bcl-2-mediated inhibition of Beclin 1-dependent autophagy. Furthermore, down-regulation of RTN3 promoted the clearance of cyPrP aggregates, allowed the activity of the UPS to resume and alleviated ER stress; ultimately, apoptosis due to the cyPrP aggregates was inhibited. Together, these data suggest that RTN3 negatively regulates autophagy to block the clearance of cyPrP aggregates and provide a clue regarding the potential to induce autophagy for the treatment of prion disease and other neurodegenerative diseases such as Parkinson disease (PD), Alzheimer disease (AD) and Huntington disease (HD).     

A chimerical phagocytosis model reveals the recruitment by Sertoli cells of autophagy for the degradation of ingested illegitimate substrates

Phagocytosis and autophagy are typically dedicated to degradation of substrates of extrinsic and intrinsic origins respectively. Although overlaps between phagocytosis and autophagy were reported, the use of autophagy for ingested substrate degradation by nonprofessional phagocytes has not been described. Blood-separated tissues use their tissue-specific nonprofessional phagocytes for homeostatic phagocytosis. In the testis, Sertoli cells phagocytose spermatid residual bodies produced during germ cell differentiation. In the retina, pigmented epithelium phagocytoses shed photoreceptor tips produced during photoreceptor renewal. Spermatid residual bodies and shed photoreceptor tips are phosphatidylserine-exposing substrates. Activation of the tyrosine kinase receptor MERTK, which is implicated in phagocytosis of phosphatidylserine-exposing substrates, is a common feature of Sertoli and retinal pigmented epithelial cell phagocytosis. The major aim of our study was to investigate to what extent phagocytosis by Sertoli cells may be tissue specific. We analyzed in Sertoli cell cultures that were exposed to either spermatid residual bodies (legitimate substrates) or retina photoreceptor outer segments (illegitimate substrates) the course of the main phagocytosis stages. We show that whereas substrate binding and ingestion stages occur similarly for legitimate or illegitimate substrates, the degradation of illegitimate but not of legitimate substrates triggers autophagy as evidenced by the formation of double-membrane wrapping, MAP1LC3A-II/LC3-II clustering, SQSTM1/p62 degradation, and by marked changes in ATG5, ATG9 and BECN1/Beclin 1 protein expression profiles. The recruitment by nonprofessional phagocytes of autophagy for the degradation of ingested cell-derived substrates is a novel feature that may be of major importance for fundamentals of both apoptotic substrate clearance and tissue homeostasis.     

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.     

Dynamics of the degradation of ubiquitinated proteins by proteasomes and autophagy: association with sequestosome 1/p62

Proteotoxicity resulting from accumulation of damaged/unwanted proteins contributes prominently to cellular aging and neurodegeneration. Proteasomal removal of these proteins upon covalent polyubiquitination is highly regulated. Recent reports proposed a role for autophagy in clearance of diffuse ubiquitinated proteins delivered by p62/SQSTM1. Here, we compared the turnover dynamics of endogenous ubiquitinated proteins by proteasomes and autophagy by assessing the effect of their inhibitors. Autophagy inhibitors bafilomycin A1, ammonium chloride, and 3-methyladenine failed to increase ubiquitinated protein levels. The proteasome inhibitor epoxomicin raised ubiquitinated protein levels at least 3-fold higher than the lysosomotropic agent chloroquine. These trends were observed in SK-N-SH cells under serum or serum-free conditions and in WT or Atg5(-/-) mouse embryonic fibroblasts (MEFs). Notably, chloroquine considerably inhibited proteasomes in SK-N-SH cells and MEFs. In these cells, elevation of p62/SQSTM1 was greater upon proteasome inhibition than with all autophagy inhibitors tested and was reduced in Atg5(-/-) MEFs. With epoxomicin, soluble p62/SQSTM1 associated with proteasomes and p62/SQSTM1 aggregates contained inactive proteasomes, ubiquitinated proteins, and autophagosomes. Prolonged autophagy inhibition (96 h) failed to elevate ubiquitinated proteins in rat cortical neurons, although epoxomicin did. Moreover, prolonged autophagy inhibition in cortical neurons markedly increased p62/SQSTM1, supporting its degradation mainly by autophagy and not by proteasomes. In conclusion, we clearly demonstrate that pharmacologic or genetic inhibition of autophagy fails to elevate ubiquitinated proteins unless the proteasome is affected. We also provide strong evidence that p62/SQSTM1 associates with proteasomes and that autophagy degrades p62/SQSTM1. Overall, the function of p62/SQSTM1 in the proteasomal pathway and autophagy requires further elucidation.     

Novel targets for Huntington's disease in an mTOR-independent autophagy pathway

Autophagy is a major clearance route for intracellular aggregate-prone proteins causing diseases such as Huntington's disease. Autophagy induction with the mTOR inhibitor rapamycin accelerates clearance of these toxic substrates. As rapamycin has nontrivial side effects, we screened FDA-approved drugs to identify new autophagy-inducing pathways. We found that L-type Ca2+ channel antagonists, the K+ATP channel opener minoxidil, and the G(i) signaling activator clonidine induce autophagy. These drugs revealed a cyclical mTOR-independent pathway regulating autophagy, in which cAMP regulates IP3 levels, influencing calpain activity, which completes the cycle by cleaving and activating G(s)alpha, which regulates cAMP levels. This pathway has numerous potential points where autophagy can be induced, and we provide proof of principle for therapeutic relevance in Huntington's disease using mammalian cell, fly and zebrafish models. Our data also suggest that insults that elevate intracytosolic Ca2+ (like excitotoxicity) inhibit autophagy, thus retarding clearance of aggregate-prone proteins.     

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.     

Regulation of p97 in the ubiquitin-proteasome system by the UBX protein-family

The AAA protein p97 is a central component in the ubiquitin-proteasome system, in which it is thought to act as a molecular chaperone, guiding protein substrates to the 26S proteasome for degradation. This function is dependent on association with cofactors that are specific to the different biological pathways p97 participates in. The UBX-protein family (ubiquitin regulatory X) constitutes the largest known group of p97 cofactors. We propose that the regulation of p97 by UBX-proteins utilizes conserved structural features of this family. Firstly, they act as scaffolding subunits in p97-containing multiprotein complexes, by providing additional interaction motifs. Secondly, they provide regulation of multiprotein complex assembly and we suggest two possible models for p97 substrate recruitment in the UPS pathway. Lastly, they impose constraints on p97 and its interaction with substrates and further cofactors. These features allow the regulation, within the UPS, of the competitive interactions on p97, a regulation that is crucial to allow the diverse functionality of p97.     

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.     

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.