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Hepatic Fibrosis and Carcinogenesis in a1-Antitrypsin Deficiency: A Prototype for Chronic Tissue Damage in Gain-of-Function Disorders

David H. Perlmutter and Gary A. Silverman

Departments of Pediatrics, Cell Biology, and Physiology, University of Pittsburgh School of Medicine, Children’s Hospital of Pittsburgh and Magee-Womens Hospital of UPMC, Pittsburgh, Pennsylvania 15224 Correspondence: [email protected]

In a1-antitrypsin (AT) deficiency, a point mutation renders a hepatic secretory glycoprotein prone to misfolding and polymerization. The mutant protein accumulates in the endoplas- mic reticulum of liver cells and causes hepatic fibrosis and hepatocellular carcinoma by a gain-of-function mechanism. Genetic and/or environmental modifiers determine whether an affected homozygote is susceptible to hepatic fibrosis/carcinoma. Two types of proteo- stasis mechanisms for such modifiers have been postulated: variation in the function of intracellular degradative mechanisms and/or variation in the signal transduction pathways that are activated to protect the cell from protein mislocalization and/or aggregation. In recent studies we found that carbamazepine, a drug that has been used safely as an anti- convulsant and mood stabilizer, reduces the hepatic load of mutant ATand hepatic fibrosis in a mouse model by enhancing autophagic disposal of this mutant protein. These results provide evidence that pharmacological manipulation of endogenous proteostasis mechan- isms is an appealing strategy for chemoprophylaxis in disorders involving gain-of-function mechanisms.

he classical form of a1-antitrypsin (AT) in 1963, we now know that this liver disease Tdeficiency is a relatively common genetic results from the accumulation of mutant AT disease that causes hepatic fibrosis and carcino- inside of liver cells. Ordinarily, wild-type AT is genesis by a gain-of-toxic function mechanism. secreted by liver cells into the blood so that it This disorder is the most common genetic liver can circulate to body fluids where it carries disease of childhood and a much more frequent out its primary function, inhibition of neutro- cause of liver disease in adults than previously phil elastase and perhaps several other serine recognized (Perlmutter 2002). From work that proteases released by neutrophils. AT is the ar- has been done since this disorder was discovered chetype of a family of proteins that have been

Editors: Richard Morimoto, Jeffrey Kelly, and Dennis Selkoe Additional Perspectives on Protein Homeostasis available at www.cshperspectives.org Copyright # 2011 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a005801 Cite this article as Cold Spring Harb Perspect Biol 2011;3:a005801

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D.H. Perlmutter and G.A. Silverman

called SERPINS because most of the members in many of the gain-of-function diseases there are serine protease inhibitors and share a rela- is evidence that mitochondrial dysfunction tively distinct mechanism for inactivating pro- plays a key role in the tissue damage. teases (Silverman et al. 2010; Whisstock et al. Lastly, we will discuss what is known about 2010). In the classical deficiency state, a point the other member of the SERPIN family with mutation leads to alteration in folding early in respect to mutations that lead to tissue damage the secretory pathway and renders the mutant by gain-of-function. Most of these occur less protein, ATZ, prone to polymerization and commonly and have not been as extensively aggregation. The mutant ATZ is relatively inef- investigated but have contributed to our under- fective in traversing the secretory pathway standing of gain-of-function mechanisms for with 10%–15% of newly synthesized mole- human disease. cules reaching the extracellular fluid and the remainder accumulating in the endoplasmic re- AT DEFICIENCY: ROLE OF PROTEIN ticulum (ER) of hepatocytes. Chronic hepatic AGGREGATION IN TISSUE DAMAGE fibrosis/cirrhosis and carcinogenesis is caused by the cellular sequellae from accumulation of The classical form of AT deficiency is character- the polymerized/aggregated mutant protein in ized by an autosomal codominant pattern of the ER by a gain-of-toxic function mechanism inheritance and affects 1 in 2000 to 3000 live (Perlmutter et al. 2007). births in most populations. AT is detected in One of the most striking aspects of this dis- the serum but it is reduced to 10%–15% of order is the variation in incidence and severity the normal serum levels. A point mutation leads of the liver disease among homozygotes. A to substitution of lysine for glutamate at resi- unique unbiased cohort study performed over due 342 and results in abnormal folding of 40 years after identifying homozygotes from the mutant protein, termed ATZ, in the earliest a newborn screening in Sweden has shown steps of the secretory pathway. The mutant that only 8%–10% develop severe liver disease protein also has an increased tendency to poly- (Piitulainen et al. 2005). This means that ge- merize, forming insoluble aggregates within netic and/or environmental modifiers deter- the rough endoplasmic reticulum and account- mine whether a homozygote is susceptible to, ing for the histological hallmark of the disease, or protected from, liver disease. We have long periodic acid-Schiff-positive, diastase-resistant postulated that these modifiers affect compo- globules in hepatocytes (reviewed in Perlmutter nents of the protein homeostasis machinery 2002). (Perlmutter 2002). Studies by Lomas et al. showed for the first In this review, we will discuss what is known time that the ATZproteinwas prone to polymer- about the gain-of-toxic function mechanism ization and aggregation (Lomas et al. 1992). A that is responsible for liver damage in AT defi- recent study by Yamasakiet al. which elucidated ciency and focus on at least four features that the crystal structure of a stable serpin dimer, has are prototypic of the gain-of-function disorders provided the basis for an interesting new model associated with other aggregation-prone pro- in which a domain swapping mechanism teins. First, damage occurs in a tissue, which is explains the formation of polymers and insolu- the site of synthesis of a misfolded, mislocalized, ble aggregates of ATZ (Yamasaki et al. 2008). and/oraggregation-prone protein. Second,pro- The model (Fig. 1) predicts that the final step tein homeostasis pathways, such as the protea- in the folding of the serpin molecule is the somal pathway, the autophagic and unfolded incorporation of strand 5a (s5a) into the central protein response, are activated in that tissue. b sheet A, while leaving the contiguous reactive Third, regulation and activityof these proteosta- center loop (RCL) and future s4a perched upon sismechanismsarelikelysitesofactionformodi- the top of the molecule where it serves as bait fiers that determine variation in the incidence for target peptidases (reviewed in Whisstock and severity of the clinical phenotype. Fourth, et al. 2000; Silverman et al. 2010). Because the

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AT Deficiency as Prototype Gain-of-Function Disorder

A Serpin fold and function B Model of A-sheet polymer Final relaxed (cleaved) Initial encounter (Michaelis) Native stressed Original model of serpin-peptidase covalent complex with form polymer complex peptidase formation: loop- Reactive center A-sheet 6 stranded Peptidase loop (RCL) β-sheet A s5A

s4A/RCL

Normal folding

5 stranded β-sheet A

Mutation heat New model of polymer formation: A-sheet domain swapping stress

Short-lived polymerogenic Unfolded intermediate

C Model of domain-swapped polymer D Native serpin folding pathway

Figure 1. Serpin folding and misfolding. (A) Active serpins fold into a metastable state. Binding of target protease results in RCL cleavage. The serpin undergoes a radical conformational change (RCL/s4A incorporation into a-sheet A) that culminates in protease inhibition via distortion of the catalytic residues. (B) Loop-sheet model of serpin polymerization. The RCL of one molecule is inserted into the open a-sheet A of another. (C) Domain-swapped model of serpin polymerization. The model is based on the structure of a domain- swapped AT dimer, where s5A and s4A (RCL) of a donor molecule insert into the a-sheet A of a recipient. (D) Normal serpins may fold through a polymerogenic intermediate that is stabilized by certain mutations. Black dashed arrow indicates gap in a-sheet A that accommodates s5A to form the s4A (RCL) exposed native form in (A) or the s5A and s4A domain-swapped structure in (C). (Reproduced from Whisstock et al. 2010 and printed with permission from the American Society for Biochemistry and Molecular Biology # 2010.)

substitution of lysine for glutamate 342 that head-to-tail orientation predicts formation of a characterizes the ATZ molecule lies within s5a linear polymer in which the exposed s5a-RCL and because this substitution disrupts the usual loop from the second molecule inserts into the intramolecular interactions with lysine 290 on a-sheet A gap of a third ATZ molecule, and so s6a and threonine 203, folding is slowed and on. Finally, domain swapping requires that helix yields a more stable intermediate without inclu- I, which connects s6A to s5a, remains uncoiled. sion of s5a. The exposed s5a and contiguous Because this uncoiled domain is 50% hydro- RCL form a loop that inserts into the a-sheet phobic, this region may facilitate lateral associa- A gap present in a second partially unfolded tions among linear polymers, and enhance the ATZ molecule, thereby forming a more stable formation of the tangled aggregates seen in the six-stranded a-sheet structure. This insertion ER of liver cells in AT deficiency (Yamasaki is analogous to that observed in the cleaved et al. 2008). A delay in proper folding of the or latent conformations of AT in which the ATZ variant, rather than a complete failure in RCL loop has been inserted into sheet A. folding, provides an explanation for how a small Although the domain swapped structure is that number (10%–15%) of these molecules are of a self-terminating dimer, molecular modeling kinetically capable of reaching a conformation using the partially unfolded monomers in a that allows traversal of the secretory pathway,

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D.H. Perlmutter and G.A. Silverman

whereas the majority of mutant ATZ proteins et al. 2004). Mitochondrial depolarization fol- accumulate as polymers and insoluble aggregates lowed induction of ATZ expression in a cell within the ER. This domain swapping mecha- line model. Most importantly, cyclosporine A nism also provides an explanation for why poly- treatment of PiZ mice reduced mitochondrial merized ATZ in the ER does not activate the damage and mortality after the stress of starva- unfolded protein response, as the structure of tion (Teckman et al. 2004), providing further the polymer closely resembles relaxed and latent evidence for the concept that mitochondrial conformations that can be acquired by properly dysfunction is a sequelae of the primary cellular folded serpin monomers. defect in AT deficiency. In most cases, the liver disease associated BrdU labeling studies in the PiZ mouse with AT deficiency is slowly progressing and model have permitted some observations about characterized by fibrosis with low-grade inflam- hepatocellular proliferation in AT deficiency mation. The intrahepatic accumulation of ATZ (Rudnick et al. 2004). The results indicate also predisposes to dysplasia, adenomas, and that there is increased proliferation but almost carcinomas (Eriksson et al. 1986). The accumu- exclusively in globule-devoid hepatocytes. Con- lation of mutant ATZin the ER accounts for the sistent with this, most, if not all, of the adeno- periodic acid-Schiff-positive, diastase-resistant mas and carcinomas that occur in the human globules in hepatocytes. Compelling evidence disease arise in the globule-devoid cells (re- for a gain-of-toxic function mechanism of liver viewed in Rudnick and Perlmutter 2005; damage comes from the observation that mice Perlmutter 2006). It is not clear how these transgenic for the mutant human ATZ gene globule-devoid hepatocytes arise but they are develop liver disease (Dycaico et al. 1988; Carl- known to have less of the polymerized/aggre- son et al. 1989). The liver disease that develops in gated ATZ than globule-containing hepato- one of the transgenic models, the PiZ mouse, has cytes (An et al. 2005). We have speculated been more extensively characterized in recent that the globule-devoid hepatocytes are younger years and recapitulates many of the features of and have a selective proliferative advantage the disease that occurs in humans. In addition when in the presence of globule-containing to the intrahepatocytic globules, there is slowly hepatocytes. The globule-containing cells have progressing fibrosis, low-grade inflammation more aggregated ATZ and mitochondrial dys- and regeneration, mild steatosis, dysplasia, and function, which renders them relatively impaired carcinomatosis. Furthermore, the severity of in cell proliferation. Furthermore, the degree of this pathology varies with the background strain regenerative activity in the liver is directly pro- of the mouse (D.H. Perlmutter, unpubl.). These portional to the number of these globule- mice have normal levels of endogenous antipro- containing hepatocytes. Thus, we have theorized teases and so this liver disease must be attribut- that the globule-containing hepatocytes are “sick able to the gain-of-toxic function mechanism. but not dead” and are responsible for regenera- tive signaling but the globule-devoid hepatocytes are the predominant ones that can respond to AT DEFICIENCY: ROLE OF these signals. This cross talk presumably perpet- MITOCHONDRIAL DYSFUNCTION uates hepatocellular proliferation in the presence IN LIVER DISEASE of inflammation, creating a milieu that is prone The molecular basis for liver cell injury in AT to carcinogenesis. deficiency is still largely undefined. Detailed studies of liver pathology in the PiZ mouse AT DEFICIENCY: DETERMINANTS model showed significant increases in mito- OF TISSUE-SPECIFIC DAMAGE chondrial damage and mitochondrial injury together with caspase-3 activation in situ (Teck- ATdeficiency is also known as a cause of prema- man et al. 2004). Similar alterations were noted turely developing chronic obstructive pulmo- in the liver of AT-deficient patients (Teckman nary disease (COPD)/emphysema. In contrast

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AT Deficiency as Prototype Gain-of-Function Disorder

to the pathogenesis of liver disease, the mecha- pathways responsible for disposal of mutant nism of lung damage has long been thought ATZ and/or the protective signaling pathways to involve loss-of-function (Crystal 1990). The that are activated by accumulation of ATZ elastase-antielastase theory for the pathogene- in the ER (Perlmutter et al. 2007). This theory sis of COPD is based on the concept that in envisions “secondary” alterations that are sub- the absence of sufficient AT, neutrophil elastase, tle, in the sense that they are clinically silent and perhaps other neutrophil proteases, pro- when inherited or acquired by a host that has gressively destroy the connective tissue matrix not inherited the mutation of ATZ. The modi- of the lung. In the classical form of AT defi- fiers are likely to be heterogeneous among ciency this is because of the reduction in ATlev- patients and their families because this would els in the circulating blood as well as in body explain the rather remarkable diversity in natu- fluids and tissues such as lung. This is the basis ral history of liver disease that has been ob- for replacement therapy with purified AT that served. In many ways, this theory is based on is administered by intravenous infusion or the concept that subtle alterations in the protein by inhalation therapy to AT-deficient patients homeostasis network represent modifiers of with established COPD. cell and tissue damage that is caused by aggre- However, several observations have sug- gated proteins through gain-of-toxic function gested that gain-of-toxic function mechanisms (Fig. 2). One study has provided substantiation may contribute to lung injury. For example, for this theory by showing a delay in intracellu- ATZ polymers deposited in the lung from lar disposal of ATZ after gene transfer into cell the extracellular fluid may attract neutrophil lines from homozygotes with known liver dis- and neutrophilic inflammatory effectors that ease compared to cell lines from homozygotes probably increase the severity of lung damage that had no evidence of liver disease (Wu et al. (Mahadeva et al. 2005). Gain-of-toxic function 1994). could also theoretically result from the effects of mutant ATZ expression in bronchoalveolar macrophages and respiratory epithelial cells AT DEFICIENCY: ROLE OF PROTEASOMAL AND AUTOPHAGIC PATHWAYS AS in the same way it happens in hepatocytes (Hu MODIFIERS OF TISSUE DAMAGE and Perlmutter 2002). These effects could ex- plain the fact that replacement therapy has We hypothesized that pathways responsible for had relatively limited clinical efficacy to date. intracellular degradation of mutant ATZ that accumulate in the ER would be one category of protein homeostasis mechanisms that are AT DEFICIENCY: VARIATION IN CLINICAL altered by genetic and/or environmental modi- DISEASE AMONG HOMOZYGOTES IS fiers. To address this hypothesis, we and others BECAUSE OF GENETIC AND/OR sought to determine the mechanisms by which ENVIRONMENTAL MODIFIERS ATZ is degraded when it accumulates in the In the only unbiased clinical epidemiological ER. Early studies in both yeast and mammalian study of AT deficiency Sveger has shown that cell lines showed that the proteasome partici- only 8% of homozygotes for the classical form pates in degradation of mutant ATZ (Qu et al. of AT deficiency develop clinically significant 1996; Werner et al. 1996; Teckman et al. 2001). liver disease over the first three decades of life In fact, degradation of mutant ATZ by the pro- (Piitulainen et al. 2005). The results of this teasome was one of the original observations study have led to the concept that modifiers, that led to the recognition of the ER-associated either genetic or environmental, predispose degradation (ERAD) pathway. ERAD is a path- a subgroup of homozygotes to liver disease way by which proteins in the lumen or mem- and/or protect the remainder of the population brane of the ER are targeted for degradation by from liver disease. Furthermore, we have theo- retrograde translocation into cytoplasm where rized that these putative modifiers affect the ubiquitination and proteasomal degradation

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D.H. Perlmutter and G.A. Silverman

Protected Susceptible

Synthesis Secretion Synthesis Secretion α α 1AT Z Protein in globules 1AT Z Protein in globules

Golgi Golgi Transcription Translation Transcription Translation

RER RER DNA RNA DNA RNA Nucleus Nucleus

ER Degradation Cellular protective ER Degradation responses Cellular protective • UPR responses • Autophagy • UPR • NFκB • Autophagy • Regeneration • NFκB • Fibrosis • Regeneration • Caspases • Fibrosis • ? Others • Caspases • ? Others

Figure 2. Cellular factors that determine whether an AT-deficient individual is protected or susceptible to liver disease. In the susceptible host, there is greater accumulation of insoluble ATZ in the ER because of subtle alter- ations in putative proteostasis network regulatory mechanisms. Here these proteostasis regulatory mechanisms are envisioned as either ER degradation pathways or cellular protective responses.

take place (reviewed in Brodsky and Wojcikie- Autophagy is a catabolic process by which wicz 2009). We know now that there are differ- the cell digests its internal constituents to gener- ent types of ERAD for luminal and membrane ate amino acids in response to nutrient starva- proteins and there may even be further subspe- tion and other stress states. It also appears to cialization for different types of luminal pro- play a role in homeostasis, cell growth, and teins but the exact chaperones and proteins differentiation. It begins with the formation that recognize ATZ and mediate its transport of a membranous platform around a targeted into the cytoplasm are not yet elucidated. Stud- region of the cell. This platform becomes a ies using a cell-free microsomal transloca- double-membrane vesicle as it envelopes cyto- tion system suggest that ATZ degradation by plasm together with parts of or entire subcellu- the proteasome occurs by both ubiquitin- lar organelles. Eventually this autophagosome dependent and ubiquitin-independent mecha- fuses with the lysosome for degradation of its nisms (Teckman et al. 2000). contents. Nevertheless, there was evidence from these Thus, the presence of abundant autophago- studies of the proteasomal pathway in mamma- somes in the liver in AT deficiency and its mod- lian cell lines, cell-free microsomal transloca- els raised the possibility that autophagy could tion systems, and yeast that this pathway could be involved in degrading the mutant ATZ that not completely account for disposal of ATZ. is retained in the ER. Indeed the initial study Autophagy was first implicated in AT deficiency went on to show that disposal of ATZ was par- when a marked increased in autophagosomes tially abrogated by chemical inhibitors of auto- were observed in fibroblast cell lines engineered phagy, including 3-methyladenine, wortmannin for expression of ATZ(Teckmanand Perlmutter and LY-294002 (Teckman and Perlmutter 2000). 2000). Increased autophagosomes were ob- However, because these drugs have other cellu- served in the liver of PiZ mice and in liver biopsy lar effects and therein cannot be considered specimens from patients (Teckman and Perl- specific for autophagy we sought to provide mutter 2000). genetic evidence that autophagy participated

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AT Deficiency as Prototype Gain-of-Function Disorder

in disposal of ATZ. For this we engineered an that chronic liver disease can be caused by accu- embryonic fibroblast cell line (MEF) from an mulation of an aggregation-prone protein in the ATG5-null mouse for expression of ATZ(Kami- ER and that autophagy is specialized for the dis- moto et al. 2006). The results showed a marked posal of aggregation-prone proteins that accu- delay in degradation of ATZ in the ATG5-null mulate in the ER. compared to the wild type MEFs. Furthermore, The presence of increased hepatic autopha- in the ATG5-null cell line it became possible gosomes in AT deficiency could potentially in- to observe massive accumulation of ATZ with dicate that there is some defect in clearance of very large inclusions throughout the cytoplasm. autophagosomes rather than an increase in for- Therefore, in addition to providing definitive mation of autophagosomes. We have recently evidence that it contributes to the disposal of investigated this issue by monitoring LC3 iso- ATZ, these studies suggest that autophagy plays form conversion in a cell line model with indu- a “homeostatic” role in the AT-deficient state by cible expression of ATZ. The results indicate preventing toxic cytoplasmic accumulation of that ATZ expression is sufficient to elicit LC3 ATZ through piecemeal digestion of insoluble conversion and that conversion is further accen- aggregates. tuated by the presence of lysosomal inhibitors A study by Kruse et al using a completely (Hidvegi et al. 2010). Thus, at least within the different strategy also showed the importance context of a cell line model, accumulation of of autophagy in disposal of ATZ (Kruse et al. ATZ in the ER results in increased formation 2006a). This group expressed human ATZ in a of autophagosomes and increased autophagic library of yeast mutants and screened for flux without a defect in clearance. mutants that were impaired for degradation of Several lines of evidence suggest that there ATZ. One of the mutant corresponded to the are one or more other pathways that contribute yeast homolog of mammalian autophagy to disposal of ATZ. A pathway that depends on protein ATG6. In the absence of this ATG6 a tyrosine phosphatase activity has been de- homolog or the homolog of ATG16 there was scribed (Cabral et al. 2002). The studies of a marked delay in disposal of human ATZ. Kruse et al indicated that ATZ could be trans- These studies were particularly revealing ported to the trans-Golgi and then targeted to because delay in degradation of ATZ was most the lysosome of yeast but a comparable pathway apparent in the autophagy-deficient yeast has not been described in mammalian cells strains when ATZ was expressed at high levels. (Kruse et al. 2006a). At lower levels of expression, ATZwas degraded Modifiers that affect the function of any of at a rate not significantly different from that in these pathways could increase susceptibility to wild type yeast. These results are consistent liver disease according to our theory. Indeed, a with the notion that at lower levels of expression single nucleotide polymorphism (SNP) in the ATZin the ER is predominantly soluble and can downstream flanking region of ER mannosi- be degraded by the proteasome whereas at dase I has been implicated in early-onset liver higher levels of expression it is more likely to disease among AT-deficient individuals (Pan transition to insoluble polymers/aggregates et al. 2009). Because ER mannosidase I plays a that require autophagy for disposal. role in ERAD a polymorphism that affects its Kruse et al. also discovered that a mutant function would be a prime candidate for a subunit of fibrinogen that forms insoluble modifier of liver disease in AT deficiency. How- aggregates in the ER of liver cells in an inherited ever, further epidemiological studies are needed form of fibrinogen deficiency depends on auto- to determine if this SNP is truly affecting liver phagy for disposal (Kruse et al. 2006b). This disease susceptibility and further cellular stud- type of fibrinogen deficiency has been associ- ies are needed to identify how it alters ATZ ac- ated with a chronic liver disease characterized cumulation. A SNP in the upstream flanking by distinct fibrillar aggregates in the ER of liver region of the AT gene itself has also been impli- cells. These results substantiate the concept cated in liver disease susceptibility (Chappell

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et al. 2008). There is no reason to believe that the AT Saar variant that accumulates in the ER this SNP would affect the disposal of ATZ. but does not polymerize. Thus, autophagy is The logical explanation for the effect of this activated when ATZ accumulates in the ER polymorphism would be to increase expression and the autophagic pathway then plays a critical of ATZ but the published results did not sub- role in disposal of ATZ and preventing massive stantiate that notion. Furthermore, the study intracellular aggregates. could have led to an entirely different conclu- Activation of NFkB is another hallmark sion with a legitimate alternate way of classify- of the cellular response to ATZ accumulation ing one of the patient groups. (Hidvegi et al. 2005). One of the most interest- ing aspects of the NFkB signaling pathway under these circumstances is that it is associ- AT DEFICIENCY: ROLE OF CELLULAR ated with a rather limited set of downstream RESPONSE PATHWAYS ACTIVATED BY transcriptional targets (Hidvegi et al. 2007). ACCUMULATION OF ATZ IN THE ER Indeed the most significant change in expres- According to our theory for pathogenesis of sion that could be attributable to NFkBis liver disease in AT deficiency, modifiers would down-regulation of Egr-1, a transcription factor also be predicted to alter the functioning of that is essential for hepatocyte proliferation and cellular signaling pathways that might be ac- the hepatic regenerative response (Liao et al. tivated to protect the cell from aggregated 2004). Our most recent studies have indicated proteins. To address this we began a series of that the down-regulation of Egr-1 in the liver studies designed to determine what signaling of the Z mouse when ATZexpression is induced pathways were activated when ATZaccumulated is directly attributable to the action of NFkB in the ER. We reasoned that this would require (A. Mukherjee, D.H. Perlmutter, unpubl.). Fur- cell line and mouse model systems with induci- thermore, the complex of proteins that as- ble rather than constitutive expression of ATZ sembles to form NFkB when ATZ accumulates because the latter would potentially permit in the ER has a profile that is entirely distinct adaptations that could obscure the primary from that which forms when cells are treated signaling effects. A series of studies using these with TNF or tunicamycin (A. Mukherjee and kinds of systems have shown that the autophagic D.H. Perlmutter, unpubl.). Finally, mating of response and the NFkB signaling pathway but the PiZ mouse to a mouse model with condi- not the unfolded protein response are activated tional hepatocyte-specific deficiency of NFkB when ATZ accumulates in the ER (Hidvegi activity shows more severe inflammation, fibro- et al. 2005; Kamimoto et al. 2006). Activation sis, steatosis, dysplasia and more hepatocytes of the autophagic response was shown by inves- with globules (A. Mukherjee, T. Hidvegi, D.H. tigating the liver of a novel mouse model with Perlmutter, unpubl.), indicating that NFkB hepatocyte-specific inducible expression of signaling is intended to protect the liver from ATZ, the Z mouse, bred onto the GFP-LC3 the effects of ATZ accumulation. Together, the mouse background. LC3 is an autophagosomal data on NFkB suggest that it plays a particu- membrane-specific protein and so the GFP-LC3 larly important role in the effects of ATZ on mouse makes green fluorescent autophago- cell proliferation, survival and ultimately the somes. Green fluorescent autophagosomes predisposition to hepatic carcinogenesis in AT appear in the liver of the GFP-LC3 mouse only deficiency. after 24 hours of starvation. In the Z x GFP-LC3 The absence of the unfolded protein re- mouse green fluorescent autophagosomes are sponse (UPR) is also noteworthy (Hidvegi seen merely by allowing hepatocyte expression et al. 2005; Hidvegi et al. 2007). For one, it of the ATZ gene to be induced (Kamimoto et al. means that activation of autophagy and NFkB 2006). GFPþ autophagosomes are not seen in in cells which accumulate mutant ATZ is inde- the liver of the Saar x GFP-LC3 mouse, which pendent of the UPR, another distinct character- has hepatocyte-specific inducible expression of istic of the cellular response in the ATdeficiency

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AT Deficiency as Prototype Gain-of-Function Disorder

state. With what is known about the mechanism stabilizer. We found that this drug enhanced by which the UPR is initiated it has always been the autophagic disposal of ATZ in cell line and relatively easy to understand how polymerized mouse models of AT deficiency (Fig. 3) (Hid- and aggregated would not elicit the UPR. vegi et al. 2010). Furthermore, CBZ reduced Results of the recent structural studies by Hun- the hepatic load of ATZ and hepatic fibrosis in tington and colleagues provide an explanation vivo in the mouse model (Hidvegi et al. 2010). for why soluble monomeric ATZ does not get These results indicate that CBZ can be tested recognized by the UPR apparatus. Those results immediately in clinical trials for treatment of suggest that the monomeric ATZ intermediate AT deficiency-associated liver disease and pro- adopts a conformation that resembles the wild vide a proof-in-principle for the therapeutic type molecule and therein would not be recog- use of autophagy enhancers. This concept has nized as unfolded (Yamasaki et al. 2008). been further substantiated by recent results Genomic analysis of liver from the Z mouse from high-content screening of drug libraries has identified other changes in gene expression using a novel C. elegans model of AT deficiency. that are attributable to the cellular response to In a pilot screen, 4 of 6 hit compounds are ATZ(Hidvegi et al. 2007). One of these changes, known to be autophagy enhancers (Gosai et al. up-regulation of regulator of G signaling 16 2010). These results are also particularly excit- (RGS 16), may represent a mechanism by which ing because they provide evidence for the con- autophagy is activated in the liver in AT defi- cept that the endogenous protein homeostasis ciency. We have found that the RGS16 response machinery can be used to prevent tissue damage is characteristic and specific for ATZ. Because from mutant proteins. RGS16 antagonizes Gai3 and Gai3 plays a Ultimately, it would be ideal to investigate role in inhibiting hepatic autophagy (Gohla these drugs together with small molecules that et al. 2007), we have hypothesized that increased alter the conformation of the mutant ATZ in RGS16 when ATZ accumulates in the ER leads such a way that insoluble ATZ is destroyed in to reversal of the inhibition of autophagy that the liver and soluble ATZ is secreted, thereby would otherwise pertain in resting hepatocytes. preventing both liver and lung disease in AT There is still relatively limited information about deficiency. The recently described domain- how RGS16 is up-regulated and where in the cell swapping mechanism for polymerization of it acts to antagonize Gai3. ATZ (Yamasaki et al. 2008) provides a basis for believing that only relatively minor changes in the kinetics of folding and degradation would AT DEFICIENCY: NOVEL THERAPEUTIC be needed to facilitate secretion of the soluble STRATEGIES BASED ON GAIN-OF-TOXIC forms of ATZ. FUNCTION MECHANISMS The autophagic response would appear to be an ideal target for potential therapies because OTHER SERPINOPATHIES THAT CAUSE HUMAN DISEASE BY GAIN-OF-FUNCTION it plays a role in disposal of mutant ATZ, is par- MECHANISMS ticularly specialized for disposal of the insoluble aggregated forms of ATZ that are integral to the Although mutations in AT have been the most pathogenesis of hepatic fibrosis and carcinoma- widely studied, missense mutations in other tosis, and because the autophagic response is serpin family members are increasingly recog- specifically activated by accumulation of ATZ nized as causes of human disease. Most of these in the ER. For this reason, we recently investi- mutations cluster in three key regions of the ser- gated one of a list of FDA-approved drugs that pin scaffold; the shutter region, the proximal are known to enhance autophagy. Carbamaze- hinge region and the distal hinge region (Stein pine (CBZ) was selected because it has the and Carrell 1995). The proximal and distal most extensive safety profile, having been used hinge regions help maintain the RCL in its for years as an anticonvulsant and mood active conformation above the body of the

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Synthesis Secretion

Golgi Transcription

Translation Proteasome

Lysosome

DNA RNA Rough endoplasmic reticulum

Autophagy Nucleus CBZ

Figure 3. Cellular action of carbamazepine (CBZ). CBZ enhances autophagic disposal of insoluble ATZ (red globules). Soluble ATZ may be secreted (top) or transported by ERAD for degradation by the proteasome.

serpin where it is available for peptidase bind- the RCL of a serpin binds to the position in ing. These regions are also important for carry- a-sheet C vacated by the distal hinge movement ing out the serpin inhibitory mechanism by required to form the latent molecule (loop-C- facilitating the movement and insertion of the sheet dimer). proximal portion of the RCL into the strand 4 At least five different serpins causing human position of a-sheet A (Huntington et al. 2000). diseases have been found with mutations in one This RCL movement translocates the bound of the three critical regions, but most cluster peptidase and traps it as a stable covalent inter- in the shutter region (Stein and Carrell 1995; mediate (Huntington et al. 2000). The shutter Gooptu and Lomas 2009). Heterozygous muta- region is comprised of several structural ele- tions (L55Por P229A) in a1-antichymotrypsin, ments, which regulate the sliding of a-sheet A an inhibitor of cathepsin G and mast cell chy- strands to allow for insertion of the RCL after mase, can result in misfolded protein accu- peptidase cleavage. Mutations in these regions mulation in the liver and chronic obstructive result in serpin dimerization and higher order pulmonary disease analogous to that observed polymerization by domain swapping as de- with AT deficiency (Faber et al. 1993; Poller scribed above for ATZ or possibly by a loop- et al. 1993). A homozygous mutation of heparin sheet insertion (Fig. 1) (Lomas et al. 1992; cofactor II (E428K), an inhibitor of thrombin, Yamasaki et al. 2008). In the loop-sheet inser- was associated with a pro-thrombotic state, tion model, the serpin folds normally, but desta- especially in the presence of a heterozygous bilizing mutations allow for premature opening antithrombin deficiency (Villa et al. 1999; of the A-sheet and insertion of the intact RCL Corral et al. 2004a). This mutation, which is from the same (latent conformation) or from identical to that of ATZ, impaired mutant pro- a different serpin molecule (loop-A-sheet A tein secretion to circulating levels that were dimer). Latent serpins can also polymerize as 5% of normal. An autosomal dominant

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AT Deficiency as Prototype Gain-of-Function Disorder

mutation of C1 inhibitor, which regulates the 1999a; Davis et al. 1999b). In cases with PME, classical complement and bradykinin cascades, neurons in the cerebellum and the cerebellar is the cause of hereditary angioedema. Several dentate nucleus are also involved (Davis et al. of the mutations (A436T, V451M, F455S, and 2002). These inclusions (Collins bodies) are P476S) lead to impaired secretion, intracellular comprised almost exclusively of misfolded degradation, and polymerization (Aulak et al. and/or polymerized neuroserpin and are lo- 1993; Eldering et al. 1995; Verpy et al. 1995). cated within dilated cisternae of the rough Autosomal dominant mutations in antithrom- endoplasmic reticulum (Davis et al. 1999a; bin lead to thrombosis. Although many of these Davis et al. 1999b). Hence, this disorder has mutations lead to misfolding and impaired been described as familial encephalopathy secretion, others lead to the formation of latent with neuroserpin inclusion bodies (FENIB) species in the circulation (Bruce et al. 1994; (Davis et al. 1999a; Davis et al. 1999b). At least Zhou et al. 1999; Corral et al. 2004b). The latent five different missense neuroserpin mutations conformation is caused by instability of native have been identified, and they all involve the structure, which allows for insertion of the shutter region. Based on the nature of the amino intact RCL into b-sheet A and displacement of acid substitutions and their location within the s1C. Not only are latent molecules unable to serpin scaffold, these mutations are predicted inhibit thrombin, they form inactive dimers to confer different degrees of instability relative with wild-type proteins via a-stand linkages as to the native fold (Davis et al. 2002). Indeed, loop-C-sheet dimers as described above (Bruce the more destabilizing mutations (G392R. et al. 1994; Zhou et al. 1999; Corral et al. 2004b). G392E.H338R .S52R.S49P) are associated Thus, circulating mutant proteins in the latent with a greater number and more pervasive dis- conformation predispose to thrombosis by tribution of Collins bodies and an earlier onset further decreasing the concentration of active and a more severe progression of dementia and inhibitor. PME (Davis et al. 2002; Gooptu and Lomas In contrast to AT deficiency associated with 2009). This correlation was supported by transi- shutter region mutations, those in the hepati- ent transfection of the mutant genes into cell cally synthesized proteins heparin cofactor II, lines, and showing that the more destabilizing C1 inhibitor, and antithrombin do not appear mutations resulted in the formation of longer to cause liver disease and intracellular inclu- polymers and a greater degree of ER retention sions despite causing protein misfolding, poly- (Miranda et al. 2004; Miranda et al. 2008). merization, and impaired secretion. In part, this Because of their metastable active confor- difference may be caused by a concentration mation, all wild-type serpins are capable of effect, as the normal hepatic synthesis of these polymerizing in vitro in a concentration and latter proteins is only approximately 10% that temperature dependent process (Dafforn et al. of AT (Gooptu and Lomas 2009). 1999). Under certain conditions, this process Mutations in the neuroserpin gene are the appears to occur in vivo. Megsin (SERPINB7) cause of an autosomal dominant neurode- is an intracellular serpin that is expressed in generative disorder characterized by progressive the mesangial cells within the glomerulus of deterioration of cognition, memory, and visu- the kidney (Miyata et al. 1998). In IgA and dia- ospatial skills (Davis et al. 1999a; Davis et al. betic nephropathy, this serpin is up-regulated 1999b). Some patients also develop progressive and appears to drive mesangial cell proliferation myoclonus epilepsy (PME) (Takao et al. 2000). and mesangial matrix deposition leading to glo- Histopathological examination of affected indi- merulosclerosis (Suzuki et al. 1999). Transgenic viduals show the accumulation of PAS positive, mice over expressing human megsin also de- diastase resistant intracellular inclusion bodies velop glomerular sclerosis (Miyata et al. 2002). within neurons located in the deeper layers of However, transgenic rats expressing the same the cerebral cortex and subcortical nuclei, transgene develop intracellular PAS inclusion including the substantia nigra (Davis et al. bodies within glomerular epithelial, tubular

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D.H. Perlmutter and G.A. Silverman

(proximal and distal), and collecting duct cells Chappell S, Hadzic N, Stockley R, Guetta-Baranes T, Mor- (Inagi et al. 2005). The PAS inclusions were gan K, Kalsheker N. 2008. A polymorphism of the alpha 1-antitrypsin gene represents a risk factor for liver dis- positive for megsin by immunohistochemistry. ease. Hepatology 47: 127–132. Similar inclusions were found in pancreatic Corral J, Aznar J, Gonzalez-Conejero R, Villa P, Minano A, acinar cells and islet a cells. These animals devel- Vaya A, Carrell RW, Huntington JA, Vicente V. 2004a. Homozygous deficiency of heparin cofactor II: relevance oped progressive renal failure and diabetes and of P17 glutamate residue in serpins, relationship with die within about 10 weeks. Because many serpins conformational diseases, and role in thrombosis. Circula- are acute phase reactants, these data suggest that tion 110: 1303–1307. inflammatory reactions that drive serpin over Corral J, Huntington JA, Gonzalez-Conejero R, Mushunje A, Navarro M, Marco P, Vicente V, Carrell RW. 2004b. expression may be complicated by the deleteri- Mutations in the shutter region of antithrombin result ous consequences of serpin polymerization. in formation of disulfide-linked dimers and severe There is still relatively limited information venous thrombosis. J Thromb Haemost 2: 931–939. about the role of protein homeostasis path- Crystal RG. 1990. aAlpha 1-Antitrypsin deficiency, emphy- sema, and liver disease. Genetic basis and strategies for ways in tissue injury that occurs in these other therapy. J Clin Invest 85: 1343–1352. serpinopathies and whether variations in the Dafforn TR, Mahadeva R, Elliott PR, Sivasothy P, Lomas function of endogenous proteostasis pathways DA. 1999. A kinetic mechanism for the polymerization determine the incidence and severity of tissue of alpha 1-antitrypsin. J Biol Chem 274: 9548–9555. Davis RL, Holohan PD, Shrimpton AE, Tatum AH, Daucher injury. We suspect that the principles affecting J, Collins GH, Todd R, Bradshaw C, Kent P, Feiglin D, the classical form of AT deficiency will also et al. 1999a. Familial encephalopathy with neuroserpin apply to these disorders. Variation in the func- inclusion bodies. Am J Pathol 155: 1901–1913. tion of proteostasis pathways would be a likely Davis RL, Shrimpton AE, Carrell RW,Lomas DA, Gerhard L, Baumann B, Lawrence DA, Yepes M, Kim TS, Ghetti B, mechanism for genetic and environmental mod- et al. 2002. Association between conformational muta- ifiers of these disorders. 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Hepatic Fibrosis and Carcinogenesis in α1-Antitrypsin Deficiency: A Prototype for Chronic Tissue Damage in Gain-of-Function Disorders

David H. Perlmutter and Gary A. Silverman

Cold Spring Harb Perspect Biol 2011; doi: 10.1101/cshperspect.a005801 originally published online January 12, 2011

Subject Collection Protein Homeostasis

Proteome-Scale Mapping of Perturbed The Amyloid Phenomenon and Its Significance in Proteostasis in Living Cells Biology and Medicine Isabel Lam, Erinc Hallacli and Vikram Khurana Christopher M. Dobson, Tuomas P.J. Knowles and Michele Vendruscolo Pharmacologic Approaches for Adapting A Chemical Biology Approach to the Chaperome Proteostasis in the Secretory Pathway to in Cancer−−HSP90 and Beyond Ameliorate Protein Conformational Diseases Tony Taldone, Tai Wang, Anna Rodina, et al. Jeffery W. Kelly Cell-Nonautonomous Regulation of Proteostasis Proteostasis in Viral Infection: Unfolding the in Aging and Disease Complex Virus−Chaperone Interplay Richard I. Morimoto Ranen Aviner and Judith Frydman The Autophagy Lysosomal Pathway and The Proteasome and Its Network: Engineering for Neurodegeneration Adaptability Steven Finkbeiner Daniel Finley and Miguel A. Prado Functional Modules of the Proteostasis Network Functional Amyloids Gopal G. Jayaraj, Mark S. Hipp and F. Ulrich Hartl Daniel Otzen and Roland Riek Protein Solubility Predictions Using the CamSol Chaperone Interactions at the Ribosome Method in the Study of Protein Homeostasis Elke Deuerling, Martin Gamerdinger and Stefan G. Pietro Sormanni and Michele Vendruscolo Kreft Recognition and Degradation of Mislocalized Mechanisms of Small Heat Shock Proteins Proteins in Health and Disease Maria K. Janowska, Hannah E.R. Baughman, Ramanujan S. Hegde and Eszter Zavodszky Christopher N. Woods, et al. The Nuclear and DNA-Associated Molecular Structure, Function, and Regulation of the Hsp90 Chaperone Network Machinery Zlata Gvozdenov, Janhavi Kolhe and Brian C. Maximilian M. Biebl and Johannes Buchner Freeman

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