Sorting, recognition and activation of the misfolded protein ...

[Autophagy 4:2, 141-150; 16 February 2008]; ©2008 Landes Bioscience

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Sorting, recognition and activation of the misfolded protein degradation pathways through macroautophagy and the proteasome Wen-Xing Ding and Xiao-Ming Yin*

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Department of Pathology; University of Pittsburgh School of Medicine; Pittsburgh, Pennsylvania USA

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Key words: macroautophagy, proteasome, misfolded proteins, endoplasmic reticulum stress, unfolded protein response, ER-associated degra‑ dation, apoptosis, conformational disease, neurodegenerative disease, cancer

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recent studies in normal human fibroblasts under various growth conditions indicate that the proteasome can participate in the degra‑ dation of long‑lived proteins and the lysosome can also participate in the degradation of short‑lived proteins.2,3 Nevertheless, suppression of macroautophagy, which accounts for a major part of the lyso‑ somal degradation mechanisms, does not seem to perturb the bulk short‑lived protein degradation, even under serum starvation condi‑ tion,2 but does affect significantly long‑lived protein degradation under serum or amino‑acid starvation conditions,3 suggesting that macroautophagy is mainly for the degradation of long‑lived proteins. The reverse argument that long‑lived proteins are degraded only by macroautophagy is not true as indicated above and by a study using Atg5‑deficient cells.4 The nature of the short‑lived proteins vs. long‑lived proteins is often unclear.1,2 For example, short‑lived proteins could include those that are under tight regulation or those derived from the rapid breakdown of unwanted protein fragments. It is also possible that the dependence on the proteasome or the lysosome for degrading proteins of different half‑lives would also vary in different cells in vivo under different physiological conditions. Together with the above concerns, the differentiation of short‑lived vs. long‑lived proteins in protein degradation does not seem to confer significant functional and mechanistic insights. The proteasome mainly degrades ubiquitinated proteins, but also non-ubiquitinated proteins such as ornithine decarboxylase. Lysosome‑mediated protein degradation is dependent on multiple mechanisms, including autophagy (macroautophagy, microau‑ tophagy, and chaperone‑mediated autophagy) and heterophagy. It has been generally conceived that the proteasome‑mediated degradation possesses a high degree of specificity, whereas lysosomal degradation (particularly that mediated by macroautophagy) is non-selective in nature. However, emerging evidence suggests that there could be a level of selectivity in macroautophagic degradation of certain proteins. Thus the distinction of the two systems based on bulk versus selectivity may hamper our understanding of the degradation mechanisms. The advances in the understanding of the ubiquitin proteasome system (UPS) and the macroautophagy‑lysosome system (MALS) now allow an alternative way to categorize protein degrada‑ tion based on the function, rather than on the degradation kinetics and selectivity (Fig. 1). By clearly defining the different types of proteins that could be degraded by autophagy, we may be able to better understand the different mechanisms involved.

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Based on a functional categorization, proteins may be grouped into three types and sorted to either the proteasome or the macroautophagy pathway for degradation. The two pathways are mechanistically connected but their capacity seems different. Macroautophagy can degrade all forms of misfolded proteins whereas proteasomal degradation is likely limited to soluble ones. Unlike the bulk protein degradation that occurs during starvation, autophagic degradation of misfolded proteins can have a degree of specificity, determined by ubiquitin modification and the interactions of p62/SQSTM1 and HDAC6. Macroautophagy is initiated in response to endoplasmic reticulum (ER) stress caused by misfolded proteins, via the ER-activated autophagy (ERAA) pathway, which activates a partial unfolded protein response involving PERK and/or IRE1, and a calcium-mediated signaling cascade. ERAA serves the function of mitigating ER stress and suppressing cell death, which may be explored for controlling protein conformational diseases. Conversely, inhibition of ERAA may be explored for sensitizing resistant tumor cells to cytotoxic agents.

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Categorization of Proteins Based on Degradation Mechanisms and Functional Consequence

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There are two main protein degradation systems in eukaryotic cells, the proteasomes and the lysosomes. The half‑lives of intracel‑ lular proteins vary dramatically ranging from several minutes (e.g., the tumor suppressor p53) to several days (e.g., actin and myosin). Early studies defined two categories of proteins based on their degra‑ dation kinetics, the “short‑lived” (with a fast degradation rate) and the “long‑lived” (with a much slower degradation rate).1,2 Subsequent studies suggest that the short‑lived proteins are mainly degraded by proteasomes, whereas the long‑lived proteins are degraded by lyso‑ somes.1 However, this distinction is only relative. For example, two *Correspondence to: Xiao-Ming Yin; Department of Pathology; University of Pittsburgh School of Medicine; Scaife Hall, Room S739; 3550 Terrace Street; Pittsburgh, Pennsylvania 15231 USA; Tel.: 412.648.8436; Fax: 412.648.9564; Email: xmyin@ pitt.edu Submitted: 08/01/07; Revised: 10/18/07; Accepted: 10/19/07 Previously published online as an Autophagy E-publication: www.landesbioscience.com/journals/autophagy/article/5190 141 www.landesbioscience.com

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ER-coordinated protein degradation systems

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Figure 1. Functional sorting of proteins for different degradation pathways. Protein degradation in the cell may serve different functions. Proteins may be grouped based on these functions from the degradation point of view and they are sorted into different degradation pathways accordingly. Hence, while Type I proteins are exclusively degraded by the ubiquitin proteasome system for the purpose of cellular regulation, and Type III proteins are exclusively degraded by macroautophagy for the purpose of nutrient recycling, Type II proteins are non-functional misfolded proteins that can be degraded by either system for the purpose of clearance and detoxification. Type II may be further subgrouped based on their solubility, order of protein organization and subcellular localization. It seems that only soluble misfolded proteins (Type IIa) can be degraded by the proteasome (pathway 1), in which ERAD is used for those proteins derived from the ER lumen. Macroautophagy could degrade all categories of misfolded proteins, although it may be more important for those in a higher order of organization (Type IIb and Type IIc). Most of the misfolded proteins would be also polyubiquitinated, which may not only provide the specificity for proteasomal degradation, but also for autophagic degradation as speculated (pathways 2 and 3). For the latter, the process is further facilitated by p62/SQSTM1 and HDAC6. The ubiquitination status of some Type IIa misfolded proteins in the cytosol, such as the IKK complex, and Type IIc misfolded proteins in the ER, such as the a1‑antitrypsin z mutant, is not clear, although it could be positive as well, in which case they would be degraded as in pathways 2 and 3. If not ubiquitinated, these proteins may be degraded via different recognition mechanisms (pathways 4 and 5).

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Accordingly, we would consider that the UPS is able to degrade apparently two types of proteins from the functional point of view (Fig. 1): Type I corresponds to fully functional proteins that are degraded as a regulatory mechanism, which would include those proteins involved in cell division, gene transcription, signal trans‑ duction and endocytosis;5 and Type II corresponds to nonfunctional misfolded proteins that are degraded as a clearance mechanism, which is well established for those misfolded proteins that are degraded through the ER‑associated degradation pathway (ERAD).6,7 Many of the Type 1 and Type 2 proteins would naturally have a fast degra‑ dation rate (short‑lived), due to the need for tight regulation or due to structural defects. Specificity of the protein to be degraded is in large part conferred by the ubiquitination process. Similarly, MALS could also degrade at least two types of proteins (Fig. 1): Type III proteins that would be functional and relatively long‑lived, but nevertheless would be non-specifically degraded in a bulk fashion during nutrient deprivation as a nutrient‑recycling mechanism,1,8 and Type II non-functional misfolded proteins. While the role of macroautophagy in the bulk degradation of Type III proteins has been long recognized, its participation in degrading non-functional misfolded proteins (Type II) is only recognized recently, first in the mammalian system and then confirmed in the yeast, Drosophila and C. elegans models of proteinopathy (protein conformational disease). 142

This newer character of MALS (referred to hereafter as autophagy for short) is the focus of this review, since it brings up several impor‑ tant issues. First, the two degradation systems (UPS and MALS) would be perhaps most intimately coupled with each other during the degradation of the misfolded protein (Type II), whereas they can be fully independent in their respective functions of degrading Type I and Type III proteins. Second, the endoplasmic reticulum (ER) becomes a key linker for the two systems as the ER is a large source of misfolded proteins and the ER initiates a broad cellular response to misfolded protein accumulation. Third, the removal of these proteins would require a certain level of specificity in order to detoxify the faulty proteins without causing bulk degradation of irrelevant yet functional proteins (Type III). Fourth, the misfolded proteins degraded by autophagy would be likely short‑lived in the monomeric form due to structural instability, although they could turn into long‑lived proteins if the molecules become oligomerized and aggregated. From this point, autophagy would be also involved in the degradation of short‑lived proteins. Fifth, because of the pathological significance of misfolded proteins in mammalian cells, the regulation of misfolded protein degradation by autophagy has a significant impact in the pathogenesis and treatment of conforma‑ tional diseases as well as other diseases, such as cancer.

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Table 1

Specific proteins known to be degraded by macroautophagy

Protein

Disease

Androgen receptor (polyglutamine expansion mutants)

Kennedy’s disease (x‑linked spinobulbar muscular atrophy)

Reference

a1‑antitrypsin (Z mutant, Glu342Lys)

a1‑antitrypsin deficiency

APP/b‑amyloid

Sporadic inclusion body myositis

Cu, Zn‑Superoxide dismutase (SOD1) (A4V, G85R, G93A mutants)

Familial amyotrophic lateral sclerosis

Dysferlin (L1341P mutant)

Limb Girdle muscular dystrophy type 2B, Miyoshi myopathy

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Fibrinogen g‑chain (R375W, Aguadilla mutant)

hypofibrinogenemia

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Huntingtin (polyglutamin expansion mutants)

Huntington’s disease

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19,38 102

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16,22–24,26

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IKKa, IKKb and IKKg NIK Familial Parkinson’s disease

Vasopressin (cys67stop mutant)

Autosomal dominant familial neurohypophyseal diabetes insipidus

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a‑synuclein (A30P, A53T mutants)

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Conformational change is the key. Autophagy is able to degrade different misfolded proteins (see examples in Table 1). Interestingly, most of the known proteins that can be degraded by autophagy as a clearance mechanism are mutant proteins that are well known for their conformational instability and their propensity to form oligomers and aggregates. Thus it seems that misfolding and conformational change is a major factor leading to autophagic degradation. It has to be pointed out that proteins do not need to be mutated to become conformationally unstable for their subsequent degrada‑ tion by autophagy. Specific deletion of a key autophagy gene, atg5 or atg7, in neurons or hepatocytes of otherwise normal mice leads to an accumulation of polyubiquitinated proteins in these cells.9,10 These proteins are apparently non-mutated. However, they are likely misfolded since aggregates are subsequently developed in the autophagy‑deficient cells.9,10 The accumulation of these proteins suggests that their removal is not compensated by the proteasome (at least not efficiently). However, it is not clear what could cause the conformational instability of these proteins during development, which requires the constant activity of autophagy to remove these inclusions. In various in vitro systems, disrupting the ER environ‑ ment/function would cause the accumulation of misfolded proteins (see below). It is not unlikely that the developmental process could provide similar signals due to specific metabolic alterations that might stress the ER. Disruption of the interaction of HSP90 with its client proteins could lead to their conformational instability and the autophagic degradation of certain substrates. This is best illustrated by the degra‑ dation of the IkB kinase (IKK) complex (including IKKa, IKKb and IKKg)11 and NFkB inducing kinase (NIK)12 via autophagy, but not the proteasome, following treatment with Geldanamycin, a HSP90 inhibitor. This type of specific recognition of a target protein by the autophagy machinery has never been recognized before and the molecular mechanism is still at large. However, conformational instability of the IKK proteins following their dissociation from HSP90 could be certainly the first determining factor leading to their eventual targeting to autophagosomes.

Misfolded proteins may also be induced under stress, including oxidative stress, transfection and starvation. These proteins are also polyubiquitinated and can accumulate into cytoplasmic aggregates similar to the aggresome structure formed by conformationally mutated proteins. These structures are called aggresome‑like induced structure (ALIS).13 It is not clear exactly how these proteins become misfolded during stress. A similar structure, called dendritic cell ALIS (or DALIS), was first observed in dendritic cells during their matura‑ tion.14 DALIS is formed by defective ribosomal products (DRiPS).15 It is possible therefore that the proteins in ALIS in general may be modified or damaged under stress conditions, which leads to misfolding and polyubiquitination. Sorting misfolded proteins for different degradation pathways. Several misfolded proteins are known to be degraded by both the proteasome and autophagy, such as the polyglutamine expansion mutant of huntingtin, certain a‑synuclein mutants, a1‑antitrypsin z mutant, the Aguadilla mutant of fibrinogen and certain super‑ oxide dismutase 1 (SOD1) mutants.16‑21 Perhaps the polyglutamine expanded huntingtin mutants are the most studied. Autophagic removal of this mutant protein has been shown in mammalian cells,16,22‑24 and in mouse,25 Drosophila25 and C. elegans26 models of Huntington’s disease. Thus promoting autophagy, such as by rapa‑ mycin, enhances the clearance of these misfolded proteins in cells and improves the outcome of the disease in the animal models. How do the proteasome and autophagy coordinate in the degra‑ dation of these proteins? First it seems that most soluble misfolded proteins would be preferentially degraded by the proteasome, and by autophagy only if the proteasome capacity is exceeded20 (pathway 1, 2, Fig. 1). This notion is also supported by the observation that inhibition of the proteasome activates autophagy, which is critical for the removal of the overflowed polyubiquitinated misfolded proteins.27‑31 Second, the decision may be dependent on the ER stress level, and autophagic degradation will be activated if the ER stress level reaches a certain point (see next section). Third, the orga‑ nization of the misfolded protein can be important. Many misfolded proteins have the propensity to polymerize, forming aggregates and packing into aggresomes or inclusion bodies.32,33 Whereas the soluble monomeric form of the misfolded proteins (Type IIa, Fig. 1) can be degraded by the proteasome, the polymerized/aggregated form (Type IIb, Fig. 1) is often insoluble and cannot be accessed by the protea‑ some. In addition, aggregates are often toxic to the proteasome.27,34

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Coordination of the Ubiquitin Proteasome System and the Macroautophagy‑lysosome System in Misfolded Protein Degradation

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presentation of such an ER inclusion body together with an extended ER lumen in the case of a1‑antitrypsin‑deficiency is mani‑ fested as periodic acid‑Schiff+/diastase‑resistant globules in the liver cells.44 Nevertheless, the aggregated proteins could be still subjected to clearance by autophagy (pathway 5, Fig. 1),19,20,37,41,42 but how they end up in the autophagosome is a fascinating yet unresolved issue.45,46 The signaling mechanism for capture by autophagosomes may be different from that for cytoplasmic aggregated proteins (pathway 5 vs. 3, Fig. 1), although the presence of p62/SQSTM1 in both types of aggregates47 may instead suggest a conserved mechanism (see below). From this point of view, these ER trapped aggregated proteins may be categorized as Type IIc (Fig. 1). A recent study provides evidence indicating the co-localization of the autophagosome marker LC3 with the mutated a1‑antitrypsin protein and the ER.19 This may be reminiscent of what is described as ER‑phagy (reticulophagy), engulfment of expanded ER membrane by autophagy, during ER stress in yeast.48 Kruse et al has proposed that either autophagosomes form directly from the ER or the part of the ER containing the aggregated proteins buds off, which is then engulfed by the autophagosome.45 Indeed, an earlier study provides evidence that the autophagosome membrane could be derived from ER membranes.49 Overall, it seems that there could be intimate and dynamic interactions of ER membranes and autophagosomes during the clearance of this type of misfolded proteins aggregates (pathway 5, Fig. 1). Target recognition: Ubiquitination, p62 and HDAC6, one size fits all? There are two key issues in the removal of misfolded proteins by autophagy, the activation of autophagy (see next section) and the recognition of the target proteins by the phagophore. It has been generally considered that autophagic degradation is nonselective. However, it now seems that conformational change and subsequent polyubiquitination may confer some types of specificity to the misfolded proteins to be degraded by autophagy, although this is still debatable.50 Many of the soluble misfolded proteins (Type IIa) and the cytoplasmic insoluble aggregated proteins (Type IIb), including ALIS proteins, are often polyubiquitinated. Under the scenario that autophagy serves as a secondary, compensatory degradation pathway under stress conditions (pathway 2, 3, Fig. 1), the ubiquitination of the cargo proteins seems to occur prior to the autophagic engulf‑ ment, as it is meant for proteasomal degradation in the first place. The misfolded proteins accumulated in the Atg5‑ or Atg7‑deficient cells due to the disruption of constitutive autophagy are also polyu‑ biquitinated.9,10 But here whether the ubiquitination occurs before autophagic engulfment is less clear.50 Furthermore, whether IKK or NIK is ubiquitinated following HSP90 suppression is not known. The ubiquitination status for Type IIc misfolded proteins is also unclear. It is possible that if ubiquitination does not occur, or occurs only in the absence of autophagy, the recognition of these target proteins for autophagic degradation may follow a different pathway (pathway 4, 5, Fig. 1). However, if the ubiquitination does occur as in the stress condition, similar recognition pathways may be followed (pathway 2, 3, Fig. 1). Perhaps a stronger piece of evidence supporting the idea that ubiquitination could be a signaling event for the selective removal of misfolded proteins by autophagy is the finding that p62/SQSTM1 could serve as an important adaptor molecule, which recognizes both

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It has long been recognized that proteasome functions are often impaired in pathological conditions where aggregates prevail.27,34,35 Notably, autophagic compartment were frequently found in these cases.27,36,37 In fact, it now becomes clear that aggregated proteins have to be removed via the autophagy pathway (pathway 3, 5, Fig. 1). Studies on misfolded proteins that could be in both soluble and insoluble forms, such as the Aguadilla mutant of fibrinogen g‑chain and the a1‑antitrypsin z mutant,19,21,38 clearly show that whereas the soluble form of the mutant (Type IIa) can be degraded via the proteasome and autophagy, the insoluble form of the mole‑ cules (Type IIb) is only degraded by autophagy (Fig. 1). Finally, some Type IIa misfolded proteins may be preferentially degraded by autophagy without ever being targeted to the protea‑ some even though they may be accessible to the latter (pathway 4, Fig. 1). For example, the IKK complex11 and NIK12 are almost exclusively targeted to the autophagosome following the disruption of their interaction with HSP90, a chaperone important for their functional conformation. In addition, deletion of a key autophagy gene, atg5 or atg7, in the mouse neuronal cells or hepatocytes leads to the accumulation of polyubiquitinated proteins.9,10 Unless one assumes that the proteasome capacity in these cells has already been exceeded by these proteins in the normal neurons or hepatocytes, one would have to consider that these proteins are actually preferentially degraded by autophagy, which cannot be compensated effectively by the proteasome when the autophagy is suppressed. These proteins are clearly Type IIa misfolded proteins to start, as they are soluble, and diffusely distributed in the cells at the early stage of autophagy inhi‑ bition, but become aggregated and insoluble (Type IIb) later on.9,10 Thus the nature of the proteins to be degraded and how they are conformationally different may have an impact on which degra‑ dation pathway is chosen. Additional evidence emerges from the study on the degradation of ALIS/DALIS. DALIS can be cleared by the proteasome.13 Puromycin‑induced ALIS can be cleared by both the proteasome and autophagy,13 but oxidative stress‑induced ALIS in pancreatic cells of diabetic rats can only be cleared up by autophagy.39 Ubiquitination is observed in both cases and thus other factors would have to account for the differential clearance. Location, location, location. There is another interesting issue coming from the examination of the proteins known to be degraded by autophagy, that is, the cellular location where the misfolded proteins are located and degraded. Misfolded proteins, such as the poly‑glutamine expanded mutants mainly reside in the cytosol and they could be directly sequestered by autophagosomes in the cyto‑ plasm. Misfolded proteins derived from the ER, if soluble, can be degraded by the proteasome via the ERAD pathway in which the misfolded proteins are retro‑transported to the cytosol and ubiq‑ uitinated along the way6,7 (pathway 1, Fig. 1). If they are degraded by autophagy, e.g., in response to ER stress or proteasome inhibi‑ tion,30,40 these proteins would likely be retro‑transported to the cytosol as in the ERAD, but they are now taken up by the autopha‑ gosome. However, some of the misfolded proteins, such as the a1‑an‑ titrypsin z mutant,19,37 the Aguadilla mutant of fibrinogen g‑chain,20 the Cys67stop mutant of vasopressin,41 and the L1341P mutant of dysferlin42 can polymerize within the ER and may not be retro‑transported out of the ER. Thus they form inclusion bodies in the ER, such as Russell’s body.43 The histological 144

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the mutant androgen receptor in a Drosophila model of Kennedy’s disease.31 Furthermore, autophagy that is activated following protea‑ some inhibition is also dependent on HDAC6 in Drosophila.31 The latter observation suggests that HDAC6 may be important for the autophagic degradation of both the Type IIb aggregated misfolded proteins (pathway 3, Fig. 1), and the Type IIa soluble misfolded proteins (pathway 2, Fig. 1), as both will be generated following proteasome inhibition. Whether HDAC6 could participate in the autophagic degradation of Type IIc aggregated proteins is not known, although the detection of p62 in the a1‑antitrypsin aggregates47 would imply that HDAC6 could be also involved. How HDAC6 participates in the autophagic degradation of misfolded proteins is not clear. There is some evidence suggesting that this effect is also mediated by its activity on microtubules, in which HDAC6 is responsible for the retrograde transport of some autophagy component, such as the lysosome, to the microtubule organizing center where the aggresomes locate, thereby facilitating the autophagic degradation of the aggresome.29 In addition, the two functions of HDAC6 in promoting aggresomes and autophagy could be coupled and could represent one mechanism. In this sense, autophagic degradation may require the formation of aggresomes promoted by HDAC6. This notion would be consistent with the function of p62, in which p62 and HDAC6 may work in synergy to pack the misfolded proteins together and facilitate their interactions with the phagophore, thus providing the specificity required for the degradation. Another implication of this scenario is that aggresomes may actually be more sensitive to autophagic degradation than the other forms of misfolded proteins. Certainly one cannot rule out that HDAC6 promotes autophagy in a way unrelated to the aggresome formation if one considers the possibility that HDAC6‑dependent autophagy can also degrade misfolded proteins that do not form aggresomes or do so before they become aggregated. However, even in this scenario, HDAC6 could still be part of the target recognition mechanism based on its ability to interact with the ubiquitinated proteins. Theses issues would need to be determined in future studies.

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the ubiquitin moiety of the misfolded proteins and the LC3/Atg8 on the autophagosome.51,52 The C‑terminal part of p62 contains an ubiquitin‑associated (UBA) domain, which binds to ubiquitin noncovalently.53 Overexpression of p62 in cells could form large p62 positive cytoplasmic bodies, which are also positive for ubiquitin and are dependent on the UBA domain.51 This seems to suggest that p62 could attract ubiquitinated proteins to form large complexes, which can be further enlarged through the ability of p62 to polym‑ erize. Interestingly, p62 had been found in inclusion bodies in many protein aggregate diseases, including Lewy bodies (a‑synuclein, Parkinson’s disease), neurofibrillary tangles (tau, Alzheimer’s diseases), huntingtin aggregates (Huntington’s disease), and aggregates seen in a1‑antitrypsin deficiency.47,54 These cytoplasmic inclusion bodies/ aggregates are caused by the aggregated misfolded mutant proteins and are in most cases ubiquitin‑positive. They are similar in many ways to, and could be the same as, the aggresome defined in cultured cells, which consists of the packed polyubiquitinated misfolded protein aggregates formed around the microtubule organizing center (MTOC) in a dynein‑dependent manner.32 These mutant protein aggregates (Type IIb and IIc) are the target of autophagic degradation and thus the role of p62 in mediating this degradation becomes crucial. Indeed, p62 can also directly bind to LC3/Atg8 via a sequence (aa.321‑342) located near the UBA domain.52 LC3 and p62 can co-localize with mutant huntingtin, forming a shell around the misfolded proteins51 and seem to be co-degraded in autolysosomes.52 An interesting observation is that even very large p62‑positive structures (2 mm) could be degraded by autophagy in cultured cells,52 which might imply that not only the aggregates could be degraded by autophagy, but also the large‑sized inclusion bodies or aggresomes could be removed by this mechanism. This possibility is further supported by a study on HDAC629 (see below). In addition, p62 is also required for the autophagic removal of ALIS.52 Finally, it has been widely observed that the level of p62 is significantly increased following the inhibition of autophagy,50,51,55 suggesting that it is constantly degraded by the autophagy machinery even at the basal level. This observation would also suggest that the polyubiquitination of the misfolded proteins accumulated in the Atg5‑ and Atg7‑deficient neuron and hepatocytes9,10 would be meaningful (for the subsequent recognition by p62). In addition to p62, HDAC6 is another protein that has been found to bind to the polyubiquitinated misfolded protein via its BUZ domain.56 It could be detected in the aggresomes formed by the misfolded DF508 mutant of cystic fibrosis transmembrane conducting regulator (CFTR)56 or by the polyglutamine expanded mutant of huntingtin,29 and in the aggresomes formed following proteasome inhibition.29,56 It also localizes with a‑synuclein and ubiquitin in the Lewy bodies of Parkinson’s disease and Dementia with Lewy Bodies.56 HDAC6 seems to be important for the aggresome formation as inhibition of HDAC6 disrupts aggresomes.56,57 HDAC6 is a micro‑ tubule‑associated deacetylase and can bind to the dynein motors,56 which are known to be important for the retrograde transportation of aggregated proteins to the microtubule organizing center to form the aggresome.32 Thus HDAC6 may contribute to aggresome formation through its effects on the dynein motors. However, perhaps more striking is that HDAC6 is also required for autophagic degrada‑ tion of the aggregated mutant huntingtin in cultured cells29 and www.landesbioscience.com

The Endoplasmic Reticulum Serves as the Central Linker Connecting the Proteasome and Autophagy During Misfolded Protein Degradation Misfolded proteins activates autophagy via ER stress. As discussed above, misfolded proteins could be degraded by the protea‑ some or via the autophagy pathway and there are several factors determining whether autophagy is called upon for their degradation. However, the ultimate issue is whether autophagy could be promptly activated in response to the build up of misfolded protein. It seems that the ER plays an essential role in this activation and the reason for this involvement seems to be self‑explanatory, i.e., to release ER stress caused by the misfolded proteins. There are intricate relationships among the ER, the proteasome and autophagy (Fig. 2). The endoplasmic reticulum performs impor‑ tant functions in protein post‑translational modifications, protein folding and oligomerization. Proteins may fail to be properly modi‑ fied or folded due to mutations or ER dysfunction. The abnormal proteins (Type IIa) would be exported to the cytosol to be degraded mainly by the proteasomes in the so‑called ER‑associated degradation (ERAD) pathway6,7 (pathway 1, Fig. 1). In the case of proteasome

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Figure 2. The ER commands the degradation of misfolded proteins by the proteasome and autophagy. Misfolded proteins are degraded by the ubiquitin proteasome system (UPS) and the macroautophagy‑lysosome system (MALS). Both degradation systems are activated in response to ER stress caused by misfolded proteins. Compensatory activation of MALS during UPS inhibition is also mediated by the misfolded protein‑induced ER stress. The compensatory effect of the proteasome during autophagy inhibition has not been reported, suggesting that proteasome degradation is a more limited mechanism for clearing misfolded proteins (see Fig. 1). ER stress activates proteasome degradation by the ER‑associated degradation (ERAD) pathway, which is mediated by the unfolded protein response (UPR) involving all three branches of ATF6, PERK and IRE1. On the other hand, ER stress activates MALS by the ER‑activated autophagy (ERAA) pathway, which is mediated by a limited UPR involving PERK and/or IRE1, and UPR‑independent mechanisms. ER calcium leakage is a major UPR‑independent mechanism, which may activate multiple downstream mechanisms including CaMMKb/AMPK, calpain, and DAPk. In addition, molecules regulating G protein signaling, such as RGS16, have also been implicated in the activation of ERAA.

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UPR promotes protein folding via the up‑regulated ER protein chaperones and the degradation of misfolded proteins via the upreg‑ ulation of ERAD components.58,59 Except for the ATF6 pathway,64 both the PERK and IRE1 path‑ ways have been reported to be involved in ERAA. PERK and its target molecule, eIF2a, was found to be critical for LC3‑I conversion to LC3‑II in the process of autophagy induced by the huntingtin poly‑ glutamine expansion mutant23 or the dysferlin L1341P mutant.42 The huntingtin mutant could transcriptionally upregulate CHOP and Atg12 in an eIF2a phosphorylation‑dependent manner.23 Atg12 is an ubiquitin‑like protein and modifies Atg5 through a covalent binding, which in turn is critical to autophagosome formation.66 On the other hand, autophagy induced by agents that directly disturb the ER environment and functions, such as A23187, tunica‑ mycin or thapsigargin, in mammalian cells seems to be dependent on IRE1 signaling,30,64 although PERK‑eIF2a signaling has also been noted.23 IRE1 also plays a critical role in autophagy induced by proteasome inhibitors.30 Notably, IRE1‑mediated UPR is conserved in yeast,58,59 in which ER stress can induce autophagy.48,65 Although it has not been formally shown that in yeast IRE1 is responsible for ER‑stress induced autophagy, since the deletion of yeast IRE1 leads to low viability, it is likely that this pathway is involved since this is the only UPR pathway in yeast.48,65 In the yeast system, Atg1 activity is increased by ER stress and can be responsible for the autophagy activation.65 In the mammalian system, the kinase activity of IRE1 is required for autophagy induction.64 IRE1 can activate a transcription factor, XBP‑1, via its endoribonuclease activity67,68 and activation of a MAP3K, ASK‑1,69 and a stress kinase, JNK, via its association with TRAF2.70 We have found that markers of autophagy were activated to the wild‑type levels in XBP‑1‑deficient MEFs exposed to proteasome inhibitors, A23187, thapsigargin or tunicamycin.30 On the other hand, we find that JNK can participate in proteasome inhibitor‑induced autophagy. JNK has also been found important in autophagy induced by the ER stress inducers, A23187 and thapsi‑ gargin.30,64 The role of JNK in autophagy has also been implicated in other studies.71,72 How JNK may contribute to autophagy is not known at the present time, although it is tempting to speculate that it may suppress the mTOR pathway via its kinase activity. The ER and the UPR pathways could be an integration center for various signals that activate autophagy. In fact, an early study shows that autophagy induced by starvation or herpes simplex virus infection can be also regulated by the eIF2a pathway in mamma‑ lian cells, which is mediated by the non-ER‑resident eIF2a kinase GCN2 and PKR, respectively.73 Interestingly, although proteasome inhibitors could also activate GCN2 and consequently modulate eIF2a activity,74 this pathway does not seem to participate in the autophagy induction by these chemicals (Ding and Yin, unpublished observations). It is not clear how different UPR pathways could be differentially activated by autophagic stimuli and subsequently inte‑ grated to initiate the downstream autophagy response. It is important to address these questions in future studies. Signaling pathway of ERAA, beyond UPR. The unfolded protein response is not the only activating mechanism for ERAA. In fact, the a1‑antitrypsin z mutant does not elicit the UPR at all, despite the fact that it could induce a potent autophagic response.19,44,62 Since this mutant polymerizes and is trapped in the ER (Type IIc, Fig. 1), it still induces severe ER stress as evidenced by significant

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inhibition or overloading, or the failure of ERAD pathway, accu‑ mulated misfolded proteins in the ER lumen induce ER stress.58‑61 Misfolded proteins that cannot be exported out of the ER (Type IIc), such as the a1‑antitrypsin z mutant, would directly stress the ER as well.62 Notably, misfolded protein aggregates in the cytosol (Type IIb), such as the mutant huntingtin, are endogenous inhibitors of the proteasome and ERAD.23,34 These aggregates can thus also induce ER stress,23,58,59,63 which in turn leads to a further accumulation of misfolded proteins in the ER. Clinically, ER stress is frequently observed in pathological conditions where protein misfolding is caused by genetic mutations either in the molecule to be processed or in the machinery processing the molecule’s folding.60,61 Directly stressing the ER using chemicals that disturb the ER envi‑ ronment, such as tunicamycin, thapsigargin or A23187, can induce autophagy in mammalian cells23,40,64 and in yeast.48,65 Autophagy induced by the proteasome inhibition is also via ER stress as the result of the misfolded protein that is built‑up.30 Like ERAD, this ER activated autophagy (dubbed as ERAA here as a process parallel to that mediated by the UPS, also called ERAD‑II42) is important for the removal of misfolded proteins, and suppressing ERAA under these conditions increases the accumulation of misfolded proteins and enhances ER stress. Signaling pathway of ERAA, a novel function of the unfolded protein response. ERAA is activated in response to ER stress. Current studies indicate that it can be mediated by multiple signals. Not surprisingly, the unfold protein response (UPR), which is the major signaling pathway for ERAD, is critically involved in ERAA as well. The UPR is the major protective and compensatory mecha‑ nism during ER stress. It is mediated by the ATF6, PERK and IRE1 pathways,58,59 which lead to translational attenuation and selective upregulation of a number of bZip transcription factors.58,59 The 146

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to increased cell death.56,57 Sequestering the misfolded proteins at different stages by autophagy would perhaps cause an even greater protection. Indeed, suppression of autophagy significantly increases cell death caused by proteasome inhibitors30 or ER function inhibitors.40,64 In addition, in cells overexpressing specific misfolded proteins, such as the mutant huntingtin,22,23,25 a1‑anti‑trypsin z mutant,19 mutant vasopressin,41 or mutant androgen receptor,31 promotion of autophagy reduces cell death whereas inhibition leads to increased cell death. The benefits of autophagy in improving the pathological conditions caused by the misfolded proteins could be clearly demonstrated in animal models of Huntington’s disease25 or Kennedy’s diseases.31 The degenerative phenotype seen in Drosophila in which the general proteasome system is disrupted could be also rescued by autophagy.31 How do the toxic misfolded proteins cause cell death? As discussed above, one of the most significant effects of misfolded proteins is the induction of ER stress.63,90 Other than the classical ER function inhibitors, such as A23187 or tunicamycin,59,91 and the misfolded proteins,23,63 proteasome inhibitors are also known to induce ER stress,30,92,93 obviously due to their effects on the accumulation of misfolded proteins. The induction of the UPR in many of the cases indicates the activation of compensatory mechanisms. However this will be at the expense of normal physiological functions. In addition, proteasome function is often inhibited by the misfolded proteins so that the protective effects of ERAD could be limited. Furthermore, in some cases, such as for the a1‑antitrypsin z mutant, there is no induction of the protective UPR,44,62 despite the fact that there is activation of ER‑originated caspase 4 or caspase 12.62 Thus, if the stress is excessive, persistent or fails to be restricted. ER de‑compen‑ sation can lead to cell death.88,89 An important function of ERAA in removing misfolded proteins is to protect the ER and mitigate ER stress. In the cases of both proteasome inhibitors and ER function inhibitors, suppression of autophagy promotes ER stress as indicated by ER dilation/expansion, and cellular vacuolization.30,40 The benefit of ERAA in this aspect clearly has an implication in ameliorating conformational diseases, such as Huntington’s disease and a1‑antitrypsin deficiency. Suppression of autophagy enhances the death stimulation. By ameliorating ER stress, autophagy suppresses cell death upstream of the death signaling pathway. Autophagy is thus able to reduce the strength of the death signals emitting from the ER. This could be important as cells may be differentially sensitive to the same death stimulation, e.g., due to the stronger expression of a protective molecule or the reduced expression of a death‑promoting molecule at the downstream level. ER stress‑induced apoptosis is in large part mediated by the mitochondria pathway,88,89 which is ultimately dependent on the activity of the two multi‑domain pro‑death Bcl‑2 family proteins, Bax and Bak.94 Recent studies, however, suggest that Bax and Bak are not necessarily equivalent in their responses to death simulation. Thus proteasome inhibitors can easily induce apoptosis in a Bax‑positive colon cancer cell line (HCT116), but not in the syngeneic Bax‑deficient cells,95,96 which express Bak.97,98 Suppressing autophagy enhances apoptotic death not only in the Bax‑positive cells, but also in Bax‑deficient cells.30,40 This observa‑ tion would be consistent with the explanation that autophagy helps to reduce death stimulation to the level that it will only activate the more sensitive Bax, but not the less sensitive Bak, which could

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ER distention75 and ER‑initiated caspase activation.62 Thus, the induction of autophagy would still be a cellular response to the ER stress. A very recent study has found that there is a marked increase in the expression of RGS16 in cells and patient samples harboring this mutant.44 RGS16 is a member of the Regulators of G protein Signaling family and can bind to Gai3,76 which is known to be able to regulate autophagy.77,78 This study suggests that RGS16 may be able to promote the autophagic effect of Gai3.44 It is not clear why the polymerized a1‑antitrypsin z mutant would activate a different pathway other than the UPR. This may not be simply due to their location within the ER as a Type IIc mutant protein (Fig. 1), since another Type IIc mutant, the dysferlin L1341P mutant, could acti‑ vate the PERK‑eIF2a pathway.42 It would be worthwhile to examine and compare the nature of the two mutant proteins and their effect on ER stress in more details. The a1‑antitrypsin z mutant may not be the only ER stress stim‑ ulant that does not elicit the UPR. Vitamin D compounds that can induce ERAA do not seem to have the capacity to induce the UPR, or at least not a full scale UPR, based on the expression level of CHOP and Bip.79 However, they can induce ER calcium leakage, like the other ER stress inducers, thapsigargin and ionomycin.80 ER‑derived calcium can be another signal for autophagy activation. Indeed, the calcium/calmodulin‑dependent kinase kinase‑b (CaMKKb) pathway is activated by calcium and in turn activates AMPK, which leads to the suppression of mTOR.80 As mTOR is a major suppressor of autophagy in both yeast and mammalian cells,8,81,82 its suppression during ER stress could be a potent mechanism of autophagy activa‑ tion. It is interesting to note that the calcium‑CaMKKb pathway alone may be sufficient to activate ERAA without the involvement of the UPR, as indicated by the effect of vitamin D compounds.79 Several other ER‑related signaling molecules, such as the inosi‑ toal‑1,4,5‑triphosphate (IP3) receptor that activates ER calcium release,83 and the calcium activated calpain84 and death associated protein kinase (DAPkinase)85 also regulate autophagy in various conditions including ER stress. Finally, Beclin 1/Atg6 can be local‑ ized at the ER and an important Beclin 1 regulator, Bcl‑2, needs to be localized at the ER to be functional as an anti‑autophagy agent.86 Bcl‑2 can regulate ER calcium storage as well.80,87 Thus there is a complex network regulating and executing ER calcium‑related effects on autophagy. A recent review by Høyer‑Hensen and Jäättelä79 has discussed these factors in greater details. Thus as ERAD is activated for misfolded protein degradation by the proteasome, ERAA is activated for misfolded protein degradation by autophagy. While both ER‑mediated mechanisms involve the UPR pathway, ERAA apparently can employ additional signaling molecules independent of UPR (Fig. 2).

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ERAA Regulates Cell Survival and has Broad Clinical Significance ERAA Mitigates ER Stress. What is the significance of ER‑activated autophagy in the removal of misfolded proteins? Obviously, seques‑ tering and clearing the misfolded proteins will reduce the toxicity of these proteins. It is well known that the accumulation of the misfolded proteins will eventually cause cell death.88,89 The forma‑ tion of the aggresome is considered to be a protective mechanism perhaps by restricting the intracellular distribution of the misfolded proteins. Disrupting aggresomes, e.g, by inhibiting HDAC6, leads www.landesbioscience.com

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Acknowledgements

The author’s own work was in part supported by the NIH (CA83817, CA111456 and NS45252). References

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nevertheless be activated with stronger death stimulations.98 Thus, suppression of autophagy could lead to a stronger death signal and therefore cell death in otherwise resistant cells. This last notion could be important in applying autophagy in treating diseases other than those traditionally affected by misfolded proteins, i.e., protein conformational diseases. One potential applica‑ tion would be in cancer therapy. While it is still controversial as to whether autophagy plays a protective role or a detrimental role in cancer development or cancer response to therapeutic agents,99 there are ample cases where autophagy has been clearly shown to protect cancer cells against killing by p53,100 metabolic stress101 or chemicals that induce ER stress, such as proteasome inhibitors or ER function inhibitors.30,40,64 The study on the Bax‑positive and Bax‑negative cells, discussed above, illustrates that inhibiting autophagy at the same time as inhibiting the proteasome would overcome the resis‑ tance of certain tumor cells to the proteasome inhibitor, enhance cell death further in the sensitive cells and improve the overall thera‑ peutic efficacy. The development of resistance to cell death is very common in tumor cells. Various approaches are being developed to sensitize the cells to cytotoxic agents. Blocking autophagy for the purpose of sensitization has the advantage of acting upstream of the death signaling pathway as discussed above. Thus we have also observed that in the case of ER stress‑induced cell death, suppressing autophagy not only enhances apoptosis, but also non-apoptotic cell death.40 There is also an additional advantage of suppressing autophagy during proteasome inhibition, in that cell death may be enhanced only in the tumor cells, but not in the non-transformed cells.40 It is possible that this may be related to the different degrees of misfolded proteins accumulated in these cells with more in the cancer cells, as indicated by the different levels of ER stress.40 Autophagy may actually have detrimental effects in normal cells where the misfolded protein level is low. The exact mechanisms affecting such a different outcome by ERAA are yet to be determined. Nevertheless, these observations suggest that the differential impact of autophagy in cancer cells vs. non-transformed cells can be further explored for tumor‑specific therapy. In conclusion, the functional coupling of autophagy to the UPS provides a complementary mechanism for degrading misfolded proteins, mitigating ER stress and reducing cell death, in which the ER serves as a critical central regulator linking both degradation machineries together. The signaling events regarding autophagy induction are just being revealed. Clearly a lot more work needs to be done to understand the activation of autophagy by various types of misfolded proteins and the recognition of these proteins by the autophagy machinery. However, this multi‑lateral relationship of the three systems is clearly crucial to the homeostasis of protein metabo‑ lism and to the pathogenesis and treatment of a number of human diseases.

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A recent study (Olzmann JA, et al. J Cell Biol 2007; 178:102538 and Olzmann JA, Chin LS. Autophagy 2007; In press) indicates that Parkin, an E3 ligase involved in autosomal recessive Parkinson’s disease, together with heterodimeric E2 enzyme UbcH13/Uevla, can mediate K63-linked polyubiquitination of misfolded proteins. This leads to the interaction of the misfolded protein with HDAC6, which promotes the sequestration of the misfolded protein into the aggresome and subsequent clearance by autophagy 148

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