Preventing and reversing the cellular consequences of Z alpha-1 antitrypsin accumulation by targeting s4A

Research Article

Preventing and reversing the cellular consequences of Zalpha-1 antitrypsin accumulation by targeting s4A

Sam Alam1, Jicun Wang1, Sabina Janciauskiene2, Ravi Mahadeva1,⇑

1Department of Medicine, University of Cambridge, Level 5, Box 157, Addenbrookes Hospital, Hills Road, Cambridge CB2 0QQ, UK;2Department of Pulmonology, Hannover Medical School, Feodor-Lynen str 23, Hannover 30625, Germany

Background & Aims: The Z variant (Glu342Lys) of a1-antitryp-sin (AT) polymerizes and accumulates in the hepatocyte endo-plasmic reticulum (ER) predisposing to neonatal hepatitis andliver cirrhosis. The resultant secretory defect leaves the lungsvulnerable to elastolysis and early-onset emphysema. Our aimin this study was to evaluate the effect of targeting strand 4a(s4A) as a strategy to inhibit polymerization and restoreplasma secretion.Methods: HEK293 cells and HepG2 cells were transfected withZ-AT (Z-AT cells) or control M-AT (M-AT cells). The effect ofAc-TTAI-NH2 (4M), Ac-FLEAIG-NH2 (6M), and Ac-SEAAASTAVVIA-NH2 (12M) on preventing and reversing intracellular Z-AT poly-mers and secretion of AT was evaluated by pulse-chase/immu-noprecipitation, ELISA, and immunoblot with a polymer-specificantibody (ATZII). The ER overload response was assessed byRT-PCR for PERK, calnexin, and RGS16, and ELISA for NF-jB,IL-6, and IL-8.Results: All peptides prevented the intracellular accumulation ofZ-AT (4M > 6M > 12M) in comparison with control peptides, withdetection of the AT–Inhibitor complex in inclusion bodies. In sodoing, 4M also significantly increased the concentration ofsecreted Z-AT and the elastase inhibitory activity. Furthermore,the 4M peptide was able to reverse the intracellular aggregationof Z-AT. The ER accumulation of Z-AT was shown to induce PERK-dependent NF-jB, IL-6, IL-8, and RGS16 and calnexin; all of whichcould be abrogated effectively by 4M. 4M had no effect on apop-tosis or cell viability.

Journal of Hepatology 20

Keywords: Alpha-1 antitrypsin; Polymerization; Liver disease; Emphysema; Genetransfection.Received 18 November 2011; received in revised form 12 February 2012; accepted 27February 2012; available online 14 March 2012⇑ Corresponding author. Tel.: +44 1223 336842; fax: +44 1223 336846.E-mail address: [email protected] (R. Mahadeva).Abbreviations: AT, a1-antitrypsin; Z-AT (E342K;PiZ), AT deficiency; HEK-M- or Zcells, HEK293 cells expressing human either M- or Z-AT gene; Hep-M- or Z cells,HepG2 cells expressing human either M- or Z-AT gene; 4M, Ac-TTAI-NH2 4-merpeptide; 6M, Ac-FLEAIG-NH2 6-mer peptide; 12M, Ac-SEAAASTAVVIA-NH2 12-mer peptide; AT–I, AT–inhibitor; RCL, reactive centre loop; ER, endoplasmicreticulum; EOR, ER overload response; PERK, protein kinase RNA (PKR)-like ERkinase; RGS16, Regulator of G-protein Signalling (RGS) protein 16.

Conclusions: These findings are the first evidence that targetings4A can prevent the cellular accumulation and deleterious effectsof Z-AT and restore its plasma concentrations. As such, this is amajor step towards treatment of patients with Z-AT-relateddisease.� 2012 European Association for the Study of the Liver. Publishedby Elsevier B.V. All rights reserved.

Introduction

Alpha-1 antitrypsin (AT) is a member of the serine proteinaseinhibitor superfamily. It is primarily synthesized in hepatocytesand secreted into plasma from where it enters into the lungand protects the alveoli from unregulated neutrophil elastaseactivity [1,2]. The normal variant is termed M-AT accordingto its isoelectric point [1]. More than 95% of individuals withsevere AT deficiency are homozygous for Z-AT (E342K; PiZ),which is most common in Northern European populations [1–3]. The Z mutation, by disturbing the normal reactive centreloop (RCL)-b-sheet A tertiary relationship, promotes a highlyspecific intermolecular linkage whereby the RCL of one mole-cule inserts into b-sheet A of a second and so on, to form poly-mers [4]. It is now well established that Z-AT polymerizationand retention within the hepatocyte ER predispose to neonatalhepatitis and liver cirrhosis [4,5]. The resultant secretory defectpredisposes to early-onset emphysema [2]. Retrospective stud-ies have identified that up to 25% of those subjects withZ-AT may suffer from liver cirrhosis or liver cancer in lateadulthood [6]. In the US, approximately 45% of emphysemasubjects referred for lung transplantation are affected by Z-ATdeficiency [7]. Other than organ transplantation, there is noeffective treatment for Z-AT-related disease. Thus, preventingpolymerization and/or reducing aggregation of Z-AT have ther-apeutic potential, and a variety of strategies have been sug-gested [8–13].

One such approach is to utilize synthetic peptides to competewith the RCL for binding to b-sheet A (s4A) to prevent polymer-ization [14–16]. Although substantial progress has been madewith regard to optimizing these inhibitors in vitro, their effective-ness in vivo is unknown.

12 vol. 57 j 116–124

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JOURNAL OF HEPATOLOGY
Materials and methods (see Supplementary material)
Construction of pcDNA3.1-Z-AT and pcDNA3.1-M-AT plasmid vectors and genetransfection

Z-AT or M-AT cDNA was amplified by reverse transcription-polymerase chainreaction (RT-PCR) from pS64T-Z-AT or pS64T-M-AT, respectively (donated byDr. R. Foreman, University of Southampton), and cloned into pcDNA3.1 (BDBioscience, UK). HEK293 cells (0.5–1 � 106/well) and HepG2 were transfectedusing Lipofectamine™ 2000 (Invitrogen, UK), and labelled as HEK-Z or Hep-Z,respectively. The expression of fusion proteins Z-AT-GFP and M-AT-GFP in cellswas evaluated by ELISA and microscopy.

Electron microscopy

Pelleted cells were fixed with 2.5% glutaraldehyde, treated with 1% osmiumtetroxide and en bloc stained with 2% aqueous uranyl acetate and dehydratedfor electron microscopy analysis.

Binding of synthetic peptides to human M-AT and Z-AT in vitro

Synthetic blocking peptides [14,15] were purchased from Genosys Biotechnolo-gies, UK, unless stated; 12-mer Ac-SEAAASTAVVIA-NH2 (12M) (1287 Da), 6-merAc-FLEAIG-NH2 (6M) (739 Da), 4-mer Ac-TTAI-NH2 (4M) (459 Da) (Alta Biosci-ence, UK) and the corresponding unrelated peptides; Ac-HHLGGAKQAGDV-NH2

(12U) (1388 Da), Ac-GRGDTP-NH2 (6U) (691.65 Da), and Ac-WWWH-NH2 (4U)(768 Da) (Alta Bioscience, UK). Their binding to plasma purified M and Z-ATwas evaluated by Coomassie staining of 7.5% non-denaturing PAGE containing8 M urea (NUG) [14].

The effect of synthetic peptides on Z-AT cells

Prevention of polymers was assessed following treatment of transfected cells(HEK-Z or Hep-Z) with peptides. Cells were treated with peptides 24 h followingtransfection to assess reversal of polymerization (i.e. once polymers had accumu-lated). n = 3 for HEK cells and n = 5 for HepG2 cells.

Cell and inclusion body lysis

Cells were lysed with cell lysis buffer (see Supplementary material). The inclusionbodies were prepared by lysing the remaining cell pellet with inclusion lysis buf-fer [17].

Cell cytotoxicity assays

Mitochondrial viability and cell damage were assessed using an MTT and LDHassay, respectively.

Characterization of alpha-1 antitrypsin

Proteins were quantified using BCA Protein Assay Reagent Kit (Pierce, UK).ELISA was used for quantification of all conformations AT, polymeric Z-ATand, elastase activity and Z and M-AT were purified from human plasma aspreviously described [18–22]. Western blot for AT was performed as previouslydescribed on a 7.5% non-denaturing gel, 8 M urea-7.5% non-denaturing gel or12% SDS–PAGE [18–22]. The presence of antitrypsin–inhibitor (AT–I) complexwas assessed on a novel Western blot of 8 M urea–12% SDS gel (Supplemen-tary Fig. 1A and B).

Intracellular signalling pathways

Semi-quantitative PCR was performed using specific PCR primers for proteinkinase RNA (PKR)-like ER kinase (PERK) (382 bp), calnexin (580 bp) and Regula-tor of G-protein Signalling (RGS) protein 16 (RGS16) (334 bp), Bcl-2 (464 bp),and Bax (427 bp). The house-keeping gene was GAPDH (330 bp) (see Supple-mentary material for oligonucleotide sequences and PCR conditions). Levelsof expression were analyzed using Image Processing and Analysis in Java (Ima-


geJ). ELISA for IL-6 and IL-8 was performed using the Human IL-6 DuoSet (R&DSystems, USA), and an in-house protocol, respectively [23]. Nuclear proteinextracts from Z-AT cells were assessed for NF-rB active site using NuclearExtract Kit (Active Motif, Belgium).

Metabolic labelling and immunoprecipitation

Distribution of AT was determined by pulse-chase experiments as detailedpreviously [11] with slight modification. Briefly, HEK-Z, HEK-Z + 4M, andHEK-Z + 4U-treated cells were incubated for 24 h and subsequently sub-jected to pulse-chase studies for 5 h. HEK-M cells were used as control.Separate monolayers were incubated in serum and methionine (2 mM)/cys-teine (2 mM)-free medium for 1 h at 37 �C followed by pulse labelling with100 lCi/ml of 35S-labelled Easy Tag Express protein labelling mix (PerkinEl-mer Life Sciences) for 1 h at 37 �C. Radioactive medium was removed bywashing with warm PBS twice and cells were chased in media containingmethionine (2 mM)/cysteine (2 mM) (complete media) for several differenttime periods; 0, 1, 2, 3, and 5 h. Supernatants were collected, cells wereharvested for cell lysates and inclusion bodies as detailed above, and sub-jected to immunoprecipitation using anti-a1-AT polyclonal antibody andprotein G-coupled Sepharose 4FF (Amersham Biosciences), followed by anal-ysis with 10% SDS–PAGE and autoradiography. Levels of expression wereanalyzed using ImageJ and expressed as relative to percentage control att = 0.

Statistical analysis

All statistical analyses were performed using SigmaStat and SPSS software(version 12.0.1, for Windows, SPSS Inc., Chicago, USA). The statistical signifi-cance was set at p value <0.05. Data are presented as mean (±SEM) unlessstated.

Results

Development of a cell model of Z and M-AT

HEK-M cells and Hep-M cells transfected with the M-AT generesulted in a high level of secretion of total AT into the superna-tant when assessed by ELISA. Z-AT transfected cells (HEK-Z andHep-Z cells) were characterized by a significantly low level ofAT secretion in comparison to M-AT cells due to retention ofZ-AT in inclusion bodies (Fig. 1A, top panel). Western blotanalysis confirmed the presence of Z-AT in inclusions of Z-ATcells alone (Fig. 1A, bottom panel).

Electron microscopy demonstrated significant distension ofthe rough ER in Z-AT cells in keeping with aggregation of Z-ATpolymers (Fig. 1B).

ELISA with the ATZ11 monoclonal antibody that detectspolymers and Western blot analysis revealed a significantamount of Z-AT polymers exclusively in the insoluble fractionfrom cell lysis of HEK-Z and Hep-Z cells (p 60.001) (Fig. 1C,top panel), and Western blot showed presence of oligomersand higher order polymers in the inclusion fractions (Fig. 1C,bottom panel).

Binding of synthetic peptides to human M-AT and Z-AT

Analysis by Coomassie staining on 7.5% non-denaturing PAGEcontaining 8 M urea (NUG) showed that 12M binds to bothM-AT and Z-AT, 6M selectively binds to plasma Z-AT, but notM-AT, and 4M preferentially binds to Z-AT as previouslydescribed [14,15]. All selected unrelated peptides failed to bindto M-AT or Z-AT (Fig. 1D).

2 vol. 57 j 116–124 117

553672

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6U- 4U4M - 4U4M -

Fig. 1. Analysis of AT in M-and Z-AT cells. (A) (Top panel) ELISA shows that M-AT protein is mainly secreted, whereas Z-AT protein is retained in inclusion bodies of Z-ATcells, (bottom panel) Western blot showing AT inclusions in HEK-Z cells. ⁄M or Z vs. vector controls, ⁄⁄for inclusions M vs. Z. (B) Representative EM image demonstrateddistended ER in HEK-Z cells (arrow). (C) (Top panel) ELISA detected polymeric Z-AT in Z-AT inclusions, shown to be oligomers and higher order polymers by Western blot(bottom panel). (D) Control 12M and 4M form AT–Inhibitor (AT–I) complex with human M-AT and Z-AT in vitro. Control 6M selectively forms AT–I complex with Z-AT.Unrelated peptides (U) had no effect.

Research Article

The effect of peptides on the formation of AT polymers in inclusionbodies

4M inhibited the amount of Z-AT polymers in the insolublefraction of Z-AT cells in a dose-dependent fashion and theunrelated peptide 4U had no such effect (Fig. 2A and C, leftpanel). Twenty micrograms of 4M completely prevented poly-mer formation in inclusions of both HEK-Z and Hep-Z cells.More detailed examination revealed that 4M was able to pre-vent the formation of polymers from a very early stage (Sup-plementary Fig. 2A). 6M and 12M also reduced the amountof polymers in the insoluble fraction in a dose-dependent man-ner (Supplementary Fig. 2B and C) but were less effective than4M; HEK-Z, 94.1% and 65.0% reduction, respectively and Hep-Z,89.54% and 70.54%, respectively (Fig. 2B). Using a novel NUG–SDS–PAGE, we were able to confirm that the prevention of

118 Journal of Hepatology 201

polymer formation by 4M was due to binding to b-sheet Awith the formation of an AT–I complex (Fig. 2C, right panel).

Assessment of cell cytotoxicity

Cytotoxicity assays for mitochondrial function (MTT assay)and cell membrane damage (LDH assay) show that 4M(20 lg) had no effect on Z-AT cell integrity or viability asdemonstrated by cell viability over 99% (Fig. 2D and E). Thiswas further supported by low expression of an anti-apoptotic(Bcl-2) (Supplementary Fig. 2D) and a proapoptotic (Bax)(Supplementary Fig. 2E) message and the stable meanratio between Bcl-2:Bax mRNA (Fig. 2F). Taken together, ourdata conclusively show that 4M had no cytotoxic or prolifer-ative effect on both HEK-Z and Hep-Z cell model systems at24 h.

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A B

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8 M urea gel section

12% SDS gel section Monomeric

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Fig. 2. 4M prevents intracellular accumulation of Z-AT. (A) 4M demonstrated significant dose-related reduction in inclusion body polymers in HEK-Z and Hep-Z cellscompared to respective control Z-AT cells; HEK-Z, p <0.007 for all doses and Hep-Z, p = 0.038. (B) 4M, 6M and 12M significantly reduced inclusion body polymers comparedto Z-AT, p 60.001, 0.008 and 0.021, respectively. (C) Western blot demonstrating (left panel) prevention of inclusion body Z-AT polymers by 4M, (right panel) formation ofAT–I complex in inclusions from 4M-treated Z-AT cells. (D) MTT assay showing the percentage of viable cells as measured by integrity of mitochondrial function; 4M is notcytotoxic, (n = 3). (E) LDH release assay shows no evidence of cell membrane injury. (F) 4M has no effect on mRNA expression of Bcl-2 and Bax in HEK-Z cells (ratio is meanof n = 3). Unrelated peptides (U) had no significant effect. ⁄Z vs. Z + peptide dose (lg). (A–C, n = 3) for HEK-M and Z, (A–C, n = 5) for Hep-M and Z.


Assessment of secreted AT

The secretory defect secondary to intracellular retention of Z-ATresults in a reduced supernatant AT concentration and elastaseinhibitory capacity in both HEK-Z and Hep-Z cells compared withM-AT cells. Following treatment with 4M, the Z-AT concentrationin the supernatant significantly increased compared to Z-ATtransfection alone, HEK-Z vs. HEK-Z + 4M, 42 ± 63 vs.2306 ± 176 ng/ml, p 60.001 (Fig. 3A, top panel) and Hep-Z vs.Hep-Z + 4M, 145.6 ± 56.6 vs. 2476.6 ± 261.4 ng/ml, p 60.001(Fig. 3A, bottom panel). To further evaluate the effect of the pep-


tide on secretion of AT, we performed pulse-chase labelling inHEK-Z cells following treatment with 4M. There was a time-dependent (t = 0, 1, 2, 3, and 5 h) decrease in the amount of ATfrom cell lysates, which was paralleled by a time-dependentincrease in the supernatant (Fig. 3B). As expected, there was atime-dependent increase in the aggregation of AT in the inclusionbodies of HEK-Z cells, but not HEK-Z + 4M cells, suggesting that4M had prevented formation of aggregates in the inclusionbodies. Statistical analysis of the relative densitometric values(expressed as percentage control at t = 0) show a significantlyincreased rate of AT disappearance from HEK-Z + 4M lysates

2 vol. 57 j 116–124 119

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Research Article

120 Journal of Hepatology 2012 vol. 57 j 116–124

compared to HEK-Z at all time points; t = 1, 2, 3, and 5 h,p = 0.008, 0.006, 0.008, and 0.002, respectively, with a half-lifeof 2.66 h and 4.33 h for HEK-Z + 4M and HEK-Z, respectively.The disappearance in the amount of AT from lysates could beattributed to the secretion of AT to the supernatant (Fig. 3C).
Assessment of the activity of secreted AT

The AT in the supernatant of HEK-Z + 4M cells had an increasedability to inhibit neutrophil elastase compared with Z-AT cellswithout 4M (Fig. 3D). This secreted AT was also able to form anSDS-stable complex with neutrophil elastase (Fig. 3F), and theAT–I complex was not detectable in the supernatant (Supplemen-tary Fig. 3). The AT secreted from HEK-Z + 4M was as active asHEK-M-AT (Fig. 3E).

Reversal of AT polymers by polymerization inhibitors

The addition of inhibitors 24 h after the formation of polymersshowed that they were all able to significantly reverse Z-AT poly-mers in inclusions. 4M was the most potent followed by 6M and12M; HEK-Z cells, 93.1%, 74.3%, and 63.4% reversal, respectively;p = 0.003 and 0.013 for 4M vs. 6M and 12M, respectively andHep-Z cells, 94.3%, 71.7%, and 63.6% reversal, respectively;p = 0.017 and 0.034 for 4M vs. 6M and 12M, respectively (Fig. 3F).

Accumulation of Z-AT and cellular activation

The relationship between the intracellular aggregation of mis-folded proteins and endoplasmic reticulum (ER) stress has beenintensively studied [24]. ER stress response includes activationof PERK and we found that ER retention of Z-AT resulted in a2.3- and 3.04-fold upregulation of PERK mRNA in HEK-Z andHep-Z cells, respectively (p = 0.005 and <0.001, respectively)(Fig. 4A). ER retention of Z-AT cells was further supported byupregulation of both calnexin (Fig. 4B) and RGS16 mRNA(Fig. 4C) in Z-AT cells compared to vehicle, a 1.53-fold(p = 0.003) and 1.3-fold (p = 0.015) increase, respectively.

ER stress in HEK-Z and Hep-Z cells also led to significantupregulation of NF-jB activity by 8 h (p = 0.001) (Fig. 4D). InHEK-Z and Hep-Z cells there was significant secretion of thepotent pro-inflammatory cytokine IL-6, p 60.001, for both(Fig. 4E, top panel) and the neutrophil chemoattractant IL-8, p60.001, for both (Fig. 4E, bottom panel) in Z-AT cells. 4M was ableto significantly inhibit Z-AT-induced PERK, calnexin and RGS16mRNA expression, as well as Z-AT-related NF-jB activity andIL-6 and IL-8 secretion at 24 h and 48 h, p <0.001 for both(Fig. 4A–E). Z-AT-induced PERK mRNA was also significantlyreduced by 6M, but not by 12M (Supplementary Fig. 4A and B).6M, but not 12M, also significantly inhibited Z-AT polymer-induced NF-jB activity at 16 and 24 h (Supplementary Fig. 4C

Fig. 3. 4M facilitates secretion of Z-AT and reverses established intracellular polyminclusion body AT at 24 h; p 60.001. ⁄M-AT control vs. Z-AT control; ⁄⁄Z-AT + 4M vs. Z-Aprevented accumulation of Z-AT into inclusions as seen in HEK-Z. (C) Levels of AT frhistograms. Results are expressed as relative to percentage control, t = 0 (100%), showingAT from HEK-Z + 4M cells into the supernatant. (D) Elastase inhibitory activity in the suactivity in the supernatant from Z-AT cells was reduced compared to M-AT, but was reto HEK-M. (F) 4M improved functional secreted Z-AT as shown by the antitrypsin–dissociated established polymer Z-AT from inclusions than 6M or 12M in HEK-Z, p = 0.0

3


and D), and reduced IL-6 and IL-8 levels, but less efficiently ascompared to 4M (Fig. 4E and Supplementary Fig. 4E and F). Takentogether, these results suggest that 4M was significantly potentcompared to 12M at all time points studied; p <0.04 for all mea-surements, as well as a more effective inhibitor of PERK and NF-jB activation than 6M.

Discussion

We have found for the first time that a 4M peptide targeting s4awas highly effective at preventing and reversing the intracellularaggregation of polymers, and inhibiting the associated cellularactivation in two transfected cell lines.

In line with its small size and high affinity for Z-AT, 4M was amore effective inhibitor of intracellular Z-AT polymerizationcompared to 6M or 12M. The mechanism that underlies inhibi-tion of polymer formation by 4M is likely to be due to bindingto b-sheet A as evidenced by the formation of an AT–I complexin the inclusion bodies of Z-AT cells. Furthermore, 4M not onlyabrogated the pathologic polymerization of Z-AT, but was alsoable to dissociate over 90% of the existing polymers. Interestingly,12M was also able to significantly prevent polymerizationdespite its requirements in vitro for a high molar excess overAT, prolonged incubation periods and its non-specific bindingto other serpins. 6M and 12M were also able to dissociate existingpolymers albeit to a lesser extent. These findings demonstrate theadded value of screening for compounds in a cell system com-pared to biochemical assays.

Z-AT accumulates in the ER of Z-AT cells and as such, stress tothe ER can activate the ER overload response (EOR) [25]. A keystep in the regulation of the EOR involves transmembrane trans-ducers for sensing ER stress such as PERK, calnexin and RGS16messages. In particular, activation of PERK leads to phosphoryla-tion of eukaryotic translation initiation factor 2a (eIF2a), whichcauses general inhibition of protein synthesis and induction ofthe transcription factor ATF4 that binds to the amino acidresponse element [26]. Previous studies had also suggested thatER stress can activate NF-jB and thereby contribute to the devel-opment of inflammation [27]. Our data shows that the expressionof the Z-AT gene results in misfolded Z-AT proteins, which have atendency to form polymers, aggregate in the ER, and resulting indeficient secretion of AT. ER accumulation of Z-AT polymers isassociated with activation of the ER resident transmembrane pro-tein PERK, which when activated induces NF-jB activity, in keep-ing with activation of the EOR [25,28]. This in turn is linked toexcess inflammatory activity of the Z-AT cell, as demonstratedby significant secretion of IL-6 and IL-8. Whilst there may beother influences in vivo, in our model, increased IL-6 activitycould perpetuate the liver injury by increasing translation ofZ-AT via its well characterized IL-6 promoter [29] that would sub-

ers. (A) 4M increased the concentration of Z-AT in the supernatant and reducedT control. (B) 4M increased the kinetics of AT secretion from Z-AT cells. 4M also

om pulse-chase for lysates, supernatants and inclusion bodies are presented ina time-dependent aggregation into inclusion bodies of HEK-Z cells and secretion ofpernatant from M-AT cells was greater than vector control (⁄). Elastase inhibitorystored by 4M⁄⁄. (E) Elastase activity in the supernatant HEK-Z + 4M was similarelastase (AT–E) complex (lane 9) on Western blot (Top). 4M more potently

03 and 0.013, respectively and Hep-Z, p = 0.009 and 0.021, respectively (Bottom).

2 vol. 57 j 116–124 121

A B

C

E F

D

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

Neg

ativ

e

Neg

ativ

e

Posi

tive

Vect

or Z Z

+ 4M Z

+ 4U

Posi

tive

Vect

or Z Z

+ 4M Z

+ 4U


Neg

ativ

e

Neg

ativ

e

Posi

tive

Vect

or Z Z

+ 4M Z

+ 4U

Posi

tive

Vect

or Z Z

+ 4M Z

+ 4U


Neg

ativ

e

Neg

ativ

e

Posi

tive

Vect

or Z Z

+ 4M Z

+ 4U

Posi

tive

Vect

or Z Z

+ 4M Z

+ 4U


PER

K m

RN

A re

lativ

e to

GAP

DH

**

*

**

*

GAPDH (330 bp)

PERK (382 bp) MK W P V Z 4M 4U MK W P V Z 4M 4U

HEK Hep

MK W P V Z 4M 4U MK W P V Z 4M 4U

HEK Hep

MK W P V Z 4M 4U MK W P V Z 4M 4U

HEK Hep

GAPDH (330 bp)

Calnexin (580 bp)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Cal

nexi

n m

RN

A re

lativ

e to

GAP

DH

**

*

**

*

GAPDH (330 bp)

RGS16 (334 bp)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

RG

S16

mR

NA

rela

tive

to G

APD

H

*

**

*

**

**

* *

* ** ** **

* *

* ** ** **

**

Media-HEK Vector-HEK-Z HEK-Z + 4M HEK-Z + 4U

Media-Hep Vector-HEK-Z HEK-Z + 4M HEK-Z + 4U

0.0 0.2 0.4 0.6 0.8 1.0 1.2

0.0 0.2 0.4 0.6 0.8 1.0 1.2

Rel

ativ

e N

F-κB

act

ivity

(O

.D. a

t 450

nm

)R

elat

ive

NF-

κB a

ctiv

ity

(O.D

. at 4

50 n

m)

Time (h) 1 4 8 16 24 48

0 100 200 300 400 500 600

Vehicle-HEK

HEK-M 4M 4U HEK-Z 4M 4U Vehicle-Hep

Hep-Z 4M 4U

Vehicle-HEK

HEK-M 4M 4U HEK-Z 4M 4U Vehicle-Hep

Hep-Z 4M 4U

Controls Controls HEK-M + 20 µg peptide

HEK-Z + 20 µg peptide

Controls Hep-Z + 20 µg peptide

IL-6

(pg/

ml) 1d

2d

** ** *

*

** ** *

*

0 500

1000 1500 2000 2500 3000 3500 4000 4500

IL-8

(pg/

ml)

1d 2d

** **

*

*

** **

* *

Expression

RER

Abnormal Z-AT folding

Polymerization +aggregation in ER

Deficient secretion

Inhibitor of polymerization

NF-κB

PERK

IL-6

IL-6 promoter

Liver disease Emphysema

Additional factor

RGS16

Calnexin

Z-AT gene

Research Article

122 Journal of Hepatology 2012 vol. 57 j 116–124

sequently lead to further aggregation and accumulation of Z-AT,cellular overload and disease (Fig. 4F). Upregulation of RGS16 inZ-AT cells provided further evidence of activation of EOR asRGS16 is a specific marker for EOR associated with accumulationof Z-AT [30]. Calnexin is a ubiquitously expressed transmem-brane chaperone in all cells containing ER that transiently bindsnewly synthesized mono-glucosylated and misfolded glycopro-teins. AT is a well characterized cargo protein of calnexin [31].Here, we demonstrate that significant upregulation of PERK, caln-exin and RGS16 by Z-AT is efficiently inhibited by 4M. Takentogether, these results suggest that 4M is highly effective inblocking the pathogenic cellular activation associated with Z-ATaggregation and ER stress.
The accumulation of Z-AT in hepatocytes results in plasmadeficiency, which predisposes to uncontrolled proteolysis withinthe lungs, and early-onset emphysema [1]. We found that treat-ment with 4M resulted in an increase in secreted concentrationsof Z-AT, and an improvement in elastase inhibitory capacitywithout the detection of AT–I in the supernatant. This suggeststhat following binding of TTAI to Z-AT, there is resumption ofnormal ER processing of the AT–I complex, and gradual releaseof native AT (Fig. 4F).

Other strategies have been assessed for treatment of Z-AT-related liver disease [8–13]; most recently, targeting a lateralhydrophobic cavity to prevent polymerization, and enhancingclearance of Z-AT aggregates by drugs promoting autophagy[10,11]. These approaches have shown a reduction in the intracel-lular aggregation of Z-AT, but did not assess the reversal of poly-mers. In common with TTAI, rapamycin, and carbamazepine werealso able to reduce NF-jB [11,13]. However, unlike TTAI none ofthese other strategies resulted in any change in plasma concen-trations in Z-AT. Improvement in elastase inhibitory capacityand secreted AT concentrations and reduction in intracellularaggregation of Z-AT with our approach, unlike others, areuniquely attractive for preventing and treating both the liverand lung sequelae of Z-AT. In addition, 4M is not cytotoxic oraffects cell proliferation or apoptosis; necessary prerequisitesfor further clinical assessment.

In summary, our data have shown for the first time that Ac-TTAI-NH2 has the potent ability to prevent and reverse intracellularZ-AT aggregation, and to significantly restore secreted functionalAT in vivo. Moreover, by inhibiting polymerization, we were ableto prevent the cellular consequences of Z-AT accumulation, andthus shed new light on the pathways of cellular activation relatedto Z-AT. These exciting data demonstrate the potential for targetings4A as a therapy, as a real prospect to ameliorate both liver and pul-monary dysfunction in PiZZ homozygotes.

Conflict of interest

The authors who have taken part in this study declared that theydo not have anything to disclose regarding funding or conflict ofinterest with respect to this manuscript.

Fig. 4. Z-AT cells show activation of EOR signalling pathways, which are inhibited by 4E) Upregulation of PERK-mediated NF-jB activity and subsequent production of inflamminhibits PERK expression, NF-jB activity and IL-6 and IL-8, (n = 3). (A–D) ⁄Z vs. vector,peptides had no effect. (n = 3) for HEK-M and Z, (n = 5) for Hep-M and Z. (F) Modelhomozygotes.

3


Acknowledgements

Cambridge NIHR Biomedical Research Centre, and Research grantfrom German Research Foundation (SFB587, A18).We thank Dr. Anne L’Hernault, Department of Medicine, Univer-sity of Cambridge, for her helpful advice with the pulse-chase andimmunoprecipitation protocols.

Supplementary data

Supplementary data associated with this article can be found, in theonline version, at http://dx.doi.org/10.1016/j.jhep. 2012.02.025.

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Documents

Preventing and reversing the cellular consequences of Z alpha-1 antitrypsin accumulation by targeting s4A