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Liver – master and servant of serum proteome

  • Deniz Kuscuoglu
    Affiliations
    Medical Clinic III, Gastroenterology, Metabolic Diseases and Intensive Care, University Hospital RWTH Aachen, Aachen, Germany

    The Interdisciplinary Center for Clinical Research (IZKF), University Hospital Aachen, Aachen, Germany
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  • Sabina Janciauskiene
    Affiliations
    Department of Respiratory Medicine, Hannover Medical School, BREATH, German Center for Lung Research (DZL), Hannover, Germany
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  • Karim Hamesch
    Affiliations
    Medical Clinic III, Gastroenterology, Metabolic Diseases and Intensive Care, University Hospital RWTH Aachen, Aachen, Germany
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  • Johannes Haybaeck
    Affiliations
    Institute of Pathology, Medical University Graz, Graz, Austria

    Department of Pathology, Medical Faculty, Otto-von-Guericke University of Magdeburg, Magdeburg, Germany
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  • Christian Trautwein
    Affiliations
    Medical Clinic III, Gastroenterology, Metabolic Diseases and Intensive Care, University Hospital RWTH Aachen, Aachen, Germany
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  • Pavel Strnad
    Correspondence
    Corresponding author. Address: Department of Internal Medicine III and IZKF, University Hospital Aachen, Pauwelsstraße 30, D-52074 Aachen, Germany. Tel.: +49 (241) 80 35324; fax: +49 (241) 80 82455.
    Affiliations
    Medical Clinic III, Gastroenterology, Metabolic Diseases and Intensive Care, University Hospital RWTH Aachen, Aachen, Germany

    The Interdisciplinary Center for Clinical Research (IZKF), University Hospital Aachen, Aachen, Germany
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Published:April 27, 2018DOI:https://doi.org/10.1016/j.jhep.2018.04.018

      Summary

      Hepatocytes synthesise the majority of serum proteins. This production occurs in the endoplasmic reticulum (ER) and is adjusted by complex local and systemic regulatory mechanisms. Accordingly, serum levels of hepatocyte-made proteins constitute important biomarkers that reflect both systemic processes and the status of the liver. For example, C-reactive protein is an established marker of inflammatory reaction, whereas transferrin emerges as a liver stress marker and an attractive mortality predictor. The high protein flow through the ER poses a continuous challenge that is handled by a complex proteostatic network consisting of ER folding machinery, ER stress response, ER-associated degradation and autophagy. Various disorders disrupt this delicate balance and result in protein accumulation in the ER. These include chronic hepatitis B infection with overproduction of hepatitis B surface antigen or inherited alpha1-antitrypsin deficiency that give rise to ground glass hepatocytes and alpha1-antitrypsin aggregates, respectively. We review these ER storage disorders and their downstream consequences. The interaction between proteotoxic stress and other ER challenges such as lipotoxicity is also discussed. Collectively, this article aims to sharpen our view of liver hepatocytes as the central hubs of protein metabolism.

      Keywords

      Liver as protein secretory organ

      The liver represents the largest gland in the human body and is responsible for the production of the majority of plasma proteins including hormones, carrier proteins, apolipoproteins, as well as factors needed for blood coagulation and fibrinolysis (Table 1). Hepatocytes produce about 10–20 g of plasma proteins daily,
      • Quinlan G.J.
      • Martin G.S.
      • Evans T.W.
      Albumin: biochemical properties and therapeutic potential.
      under complex regulation that enables the repertoire of synthesised proteins to be adjusted to the needs of organism.
      • Jain S.
      • Gautam V.
      • Naseem S.
      Acute-phase proteins: as diagnostic tool.
      The levels of hepatocyte-made proteins can be monitored in the serum and constitute the basis of multiple clinically useful biomarkers. At the same time, the tremendous metabolic activity of hepatocytes requires both efficient folding/processing of the synthesised polypeptides and a quick disposal of the damaged species. This delicate system is susceptible to both inherited and acquired alterations that may lead to protein accumulation. In our article, we will review the proteostatic machinery of the liver and the alterations observed in various liver disorders and stress conditions.
      Table 1Overview of some of the liver-derived secretory proteins found in plasma.
      ProteinsFunctionHalf-life (Human)Serum levels
      Unless otherwise stated, data obtained from “Clinical chemistry laboratory: Reference range values in clinical chemistry, Professional services manual.” Baltimore, Department of Pathology, University of Maryland Medical System.
      Carrier proteins
       AlbuminRegulates osmotic pressure, binds fatty acids, hormones and drugs12–18 days
      • Quinlan G.J.
      • Martin G.S.
      • Evans T.W.
      Albumin: biochemical properties and therapeutic potential.
      35–50 g/L
       CeruloplasminCarries copper to cells6 days
      • Sternlieb I.
      • Morell A.G.
      • Tucker W.D.
      • Greene M.W.
      • Scheinberg I.H.
      The incorporation of copper into ceruloplasmin in vivo: studies with copper and copper.
      0.2–0.6 g/L
       HaptoglobinBinds free hemoglobin and transports it to the mononuclear phagocyte system4 days
      • Johnson A.M.
      • McMillan C.W.
      Protein turnover studies using in vivo labeling and immunoprecipitation.
      0.3–2.0 g/L
       HemopexinBinds and carries heme to hepatocytes7 days
      • Natelson S.
      • Natelson E.A.
      1–2 g/L
       Thyroxine-binding globulinTransports thyroid hormones5 days
      • Salvatore D.
      • Davies T.F.
      • Schlumberger M.-J.
      • Hay I.D.
      • Larsen P.R.
      Chapter 11 - Thyroid physiology and diagnostic evaluation of patients with thyroid disorders.
      12–30 mg/L
       TransthyretinTransports the thyroid hormone thyroxine and retinol-binding protein2–4 days
      • Shenkin A.
      Serum prealbumin: is it a marker of nutritional status or of risk of malnutrition?.
      100–400 mg/L
       TransferrinBinds and carries iron in the serum8 days
      • Chung M.C.M.
      Structure and function of transferrin.
      2.12–3.60 g/L
       ApolipoproteinsFacilitate formation and transport of lipid particles5–6 days
      • Rifai N.
      • Warnick G.R.
      0.94–2 g/L
      The concentration of apolipoprotein A1 (apo A-I) as a characteristic, most abundant lipoprotein is shown.
      Homeostatic proteins
       Coagulation cascade factors
        ProthrombinProteolytically cleaved form (thrombin) converts soluble fibrinogen to insoluble fibrin3 days
      • Chow B.K.
      • Ting V.
      • Tufaro F.
      • MacGillivray R.T.
      Characterization of a novel liver-specific enhancer in the human prothrombin gene.
      100 mg/L
        Fibrinogen
      Mutations in fibrinogen lead to the development of fibrinogen storage disease that is characterised by fibrinogen accumulation in the ER.
      After cleavage polymerizes to form insoluble fibrin clots4 days
      • Stein T.P.
      • Leskiw M.J.
      • Wallace H.W.
      Measurement of half-life human plasma fibrinogen.
      2–4 g/L
       Protease inhibitors
        α1-antitrypsin
      Mutations in α1-antitrypsin cause the inherited disorder α1-antitrypsin deficiency.
      Inhibits multiple serine proteases5–6 days
      • Makino S.
      • Reed C.E.
      Distribution and elimination of exogenous alpha1-antitrypsin.
      0.78–2.00 g/L
        α2-macroglobulinInhibits various proteases linked to fibrinolysis5–6 days
      • Blatrix C.
      • Amouch P.
      • Drouet J.
      • Steinbuch M.
      Study on the plasmatic elimination of the alpha2-macroglobulin-proteinase complexes.
      4–8 g/L
        Antitrombin IIIInactivates thrombin3 days
      • Collen D.
      • Schetz J.
      • de Cock F.
      • Holmer E.
      • Verstraete M.
      Metabolism of antithrombin III (heparin cofactor) in man: effects of venous thrombosis and of heparin administration.
      0.2 g/L
       Complement component factors (C1 to C9)Part of the innate immune system, enhances clearance of microbes and damaged cells2 days
      • Weinstein A.
      • Peters K.
      • Brown D.
      • Bluestone R.
      Metabolism of the third component of complement (C3) in patients with rheumatoid arthritis.
      ,
      The concentration of the most abundant classical family member (complement factor C3) is depicted.
      0.9–1.8 g/L
      The concentration of the most abundant classical family member (complement factor C3) is depicted.
      Hormones
       HepcidinRegulates the iron metabolismSeveral minutes
      • Xiao J.J.
      • Krzyzanski W.
      • Wang Y.M.
      • Li H.
      • Rose M.J.
      • Ma M.
      • et al.
      Pharmacokinetics of anti-hepcidin monoclonal antibody Ab 12B9m and hepcidin in cynomolgus monkeys.
      ,
      The data obtained from Cynomolgus Monkeys.16
      4–7 nM
      Reference ranges of serum hepcidin concentration are based on a large cohort study of healthy subjects.19
       ThrombopoietinRegulates the production of platelets45 min
      • Kuter D.J.
      • Rosenberg R.D.
      The reciprocal relationship of thrombopoietin (c-Mpl ligand) to changes in the platelet mass during busulfan-induced thrombocytopenia in the rabbit.
      ,
      The data were obtained from rabbits.17
      0.08–0.24 mg/L
      The average thrombopoietin concentration in healthy blood donors.20
       Insulin-like growth factor 1Regulates somatic growth and anabolic responses10 min
      • Morimoto L.M.
      • Newcomb P.A.
      • White E.
      • Bigler J.
      • Potter J.D.
      Variation in plasma insulin-like growth factor-1 and insulin-like growth factor binding protein-3: genetic factors.
      0.05–0.35 mg/L
      The reference range for serum IGF-1 (Insulin-like growth factor 1) is based on a study with a large number of healthy volunteers.21
      * Unless otherwise stated, data obtained from “Clinical chemistry laboratory: Reference range values in clinical chemistry, Professional services manual.” Baltimore, Department of Pathology, University of Maryland Medical System.
      ** The concentration of apolipoprotein A1 (apo A-I) as a characteristic, most abundant lipoprotein is shown.
      The concentration of the most abundant classical family member (complement factor C3) is depicted.
      †† The data obtained from Cynomolgus Monkeys.
      • Xiao J.J.
      • Krzyzanski W.
      • Wang Y.M.
      • Li H.
      • Rose M.J.
      • Ma M.
      • et al.
      Pharmacokinetics of anti-hepcidin monoclonal antibody Ab 12B9m and hepcidin in cynomolgus monkeys.
      Reference ranges of serum hepcidin concentration are based on a large cohort study of healthy subjects.
      • Galesloot T.E.
      • Vermeulen S.H.
      • Geurts-Moespot A.J.
      • Klaver S.M.
      • Kroot J.J.
      • van Tienoven D.
      • et al.
      Serum hepcidin: reference ranges and biochemical correlates in the general population.
      The data were obtained from rabbits.
      • Kuter D.J.
      • Rosenberg R.D.
      The reciprocal relationship of thrombopoietin (c-Mpl ligand) to changes in the platelet mass during busulfan-induced thrombocytopenia in the rabbit.
      # The average thrombopoietin concentration in healthy blood donors.
      • Wang J.C.
      • Chen C.
      • Lou L.H.
      • Mora M.
      Blood thrombopoietin, IL-6 and IL-11 levels in patients with agnogenic myeloid metaplasia.
      § The reference range for serum IGF-1 (Insulin-like growth factor 1) is based on a study with a large number of healthy volunteers.
      • Rosario P.W.
      Normal values of serum IGF-1 in adults: results from a Brazilian population.
      a Mutations in fibrinogen lead to the development of fibrinogen storage disease that is characterised by fibrinogen accumulation in the ER.
      b Mutations in α1-antitrypsin cause the inherited disorder α1-antitrypsin deficiency.

      Folding and trafficking of secretory proteins

      The translation of secretory proteins starts on cytosolic ribosomes with the synthesis of a 16- to 30-residue long, mostly hydrophobic N-terminal signal sequence, which binds to a cytosolic protein-RNA complex termed the signal recognition particle (SRP) (Fig. 1). The SRP slows down the translation process and enables the entry of the nascent polypeptides directly into the endoplasmic reticulum (ER) lumen. This happens via interaction with a receptor, that is localised near to the ER translocation channel.
      • Reid D.W.
      • Nicchitta C.V.
      Diversity and selectivity in mRNA translation on the endoplasmic reticulum.
      In the ER lumen, imported polypeptides are then supplemented with preassembled glycans. Chaperones such as the chaperone binding immunoglobulin protein (BiP [HSPA5]), or protein disulfide isomerases (PDIs) provide early, co-translational folding assistance.
      • Xu C.
      • Ng D.T.
      Glycosylation-directed quality control of protein folding.
      At the same time, the N-terminal signal sequence is removed by the signal peptidase complex. The subsequent folding is facilitated by a series of glycosylation/deglycosylation events.
      • Leach M.R.
      • Williams D.B.
      Lectin-deficient calnexin is capable of binding class I histocompatibility molecules in vivo and preventing their degradation.
      First, the stepwise trimming of two glucose residues from N-linked glycans by ER-resident glucosidases I and II leads to the recognition of the peptides by the lectins calnexin and calreticulin. The interaction with both lectins retains the polypeptides in the ER and facilitates their folding, with the help of additional chaperones such as PDIs that enable the formation of disulphide bonds.
      • Feige M.J.
      • Hendershot L.M.
      Disulfide bonds in ER protein folding and homeostasis.
      Aberrant glycoproteins are re-glucosylated by glycoprotein glucosyltransferases, leading to their re-association with the calnexin-calreticulin folding machinery. Finally, the correctly folded proteins dissociate from the calnexin-calreticulin complex via removal of their terminal glucose residue. They are then directed to the ER-exit site. While almost all secreted plasma proteins are glycoproteins, some of them, such as albumin, are not. Like glycoproteins, they also contain an N-terminal recognition sequence that facilitates their entry into the ER.

      Otagiri MC, V.T.G. Albumin in Medicine: Pathological and Clinical Applications. 1st ed. 2016 Edition, Kindle Edition ed2016.

      However, their folding is distinct and typically proceeds without involvement of lectins via inter-domain interactions that are promoted by catalysts such as PDIs and peptidyl-prolyl isomerases.
      • Santra M.K.
      • Banerjee A.
      • Rahaman O.
      • Panda D.
      Unfolding pathways of human serum albumin: evidence for sequential unfolding and folding of its three domains.
      Figure thumbnail gr1
      Fig. 1Handling of secretory proteins by hepatocytes. (1) Secretory proteins contain a hydrophobic signal sequence that is recognised by the signal recognition particle (SRP). SRP binding leads to the translational arrest and a transfer of the mRNA-ribosome complex to the endoplasmic reticulum (ER) membrane. The synthesised polypeptide then enters the ER lumen through a translocon channel (composed either by Sec61 or Derlins). In the ER, the peptide is supplemented with preassembled glycans and the signal sequence is removed. (2) The folding of glycoproteins is mediated by a range of glycosylation/deglycosylation steps that are facilitated by the chaperones calnexin (CNX) and calreticulin (CRT). Non-glycosylated peptides are folded in CNX-/CRT-independent manner including chaperones BiP or ERdj3/6. (3) The correctly folded proteins dissociate from the folding machineries and are transported to the ER-exit site and subsequently either by COPII-dependent manner or by unconventional secretion pathway (UPS) to the Golgi apparatus. A failure in the folding process may lead to accumulation of misfolded proteins that are chelated by the chaperone binding immunoglobulin protein (BiP). (4) At the same time, BiP dissociates from the ER stress sensors, thus triggering unfolded protein response (UPR). The protein accumulation activates (both in UPR-dependent and –independent manner) a transcriptional programme that tries to restore the regulated protein flow through the ER. As another line of defence, the misfolded proteins are degraded either via (5) ER-associated degradation pathway (ERAD) or (6) autophagy. The former process is initiated by ER mannosidase I (ERManI) mediated cleavage of a mannose residue, that removes the proteins from the CNX-CRT folding cycle. After association with lectins and chaperones, that prevent protein aggregation, the polypeptides are translocated via the Sec61/Derlin-containing channel into the cytoplasm, where they become ubiquitinated and degraded via proteasome.
      Once the proteins are folded, they are brought to the ER-exit site, where they are packaged into vesicles that transport them to the Golgi apparatus. The conventional pathway utilises the spherical COPII-coated vesicles.
      • Ferro-Novick S.
      • Brose N.
      Nobel 2013 Physiology or medicine: traffic control system within cells.
      They have a diameter of ∼60–100 nm and mediate the bulk, anterograde export of a vast number of proteins
      • Gillon A.D.
      • Latham C.F.
      • Miller E.A.
      Vesicle-mediated ER export of proteins and lipids.
      . However, several cargo molecules, such as collagen, form large rigid structures that exceed the size of COPII-coated carriers. Specialized factors such as TANGO1 (MIA3) and cTAGE5 (MIA2) assist the export of those oversized cargoes in cooperation with COPII vesicles. In addition to the conventional pathway, secretory proteins can also be delivered via alternative routes known as unconventional protein secretion (UPS). UPS is mediated by distinct mechanisms such as pore-mediated secretion, autophagosomal-endosomal translocation or Golgi-bypass dependent secretion.
      • Rabouille C.
      Pathways of Unconventional Protein Secretion.
      Meanwhile, COPI vesicles mediate retrograde transport from the Golgi to the ER, retrieving escaped resident ER proteins as well as misfolded species.
      • Spang A.
      Retrograde traffic from the Golgi to the endoplasmic reticulum.

      Degradation of damaged secretory proteins

      When proteins fail to achieve their three-dimensional conformations, they are off-loaded from the folding pathway and become degraded either via ER-associated degradation (ERAD) or autophagy (Fig. 1). The removal of misfolded proteins by ERAD requires their recognition and translocation into the cytoplasm, where they become polyubiquitinated, extracted from the membrane and finally degraded by the proteasome.
      • Brodsky J.L.
      Cleaning up: ER-associated degradation to the rescue.
      Depending on the localisation of the misfolded substrate, ERAD can be subdivided into ERAD-L (luminal), -C (cytosolic), -M (membrane) and –T (translocon-associated). While the transition from folding to degradation is far from being understood, it is assumed that it is activated by the prolonged association of the substrate with BiP or its cofactors (e.g., ERdj3 [DNAJB11]/ERdj6 [DNAJC3]). In fact, it has been shown that prolonged folding promotes the recruitment of the ERAD players, such as ERdj4 (DNAJB9), ERdj5 (DNAJC10) and VCP (also known as p97), to the side of targeted substrate.
      • Otero J.H.
      • Lizak B.
      • Hendershot L.M.
      Life and death of a BiP substrate.
      As the first step towards ERAD, ER mannosidase I (ERManI [MAN1B1]) cleaves a mannose residue, thereby removing the proteins from the calnexin-calreticulin refolding cycle. It also promotes the interaction with the protein EDEM1, which facilitates the binding to ERAD lectins OS9 and XTP3B (ERLEC1).
      • Christianson J.C.
      • Shaler T.A.
      • Tyler R.E.
      • Kopito R.R.
      OS-9 and GRP94 deliver mutant alpha1-antitrypsin to the Hrd1-SEL1L ubiquitin ligase complex for ERAD.
      • Oda Y.
      • Hosokawa N.
      • Wada I.
      • Nagata K.
      EDEM as an acceptor of terminally misfolded glycoproteins released from calnexin.
      During these steps, BiP prevents the substrates from aggregation and keeps them in a soluble state.
      • Zhang J.X.
      • Braakman I.
      • Matlack K.E.
      • Helenius A.
      Quality control in the secretory pathway: the role of calreticulin, calnexin and BiP in the retention of glycoproteins with C-terminal truncations.
      In comparison to glycoproteins, the recognition and degradation of non-glycosylated proteins is much less well understood, but seems to involve BiP as well.
      • Otero J.H.
      • Lizak B.
      • Hendershot L.M.
      Life and death of a BiP substrate.
      Moreover, glycosylated proteins may use glycosylation-independent degradation pathways during stress conditions or if they are severely misfolded.
      Hepatocytes harbor a complex proteostatic network that facilitates the processing and secretion of correctly folded proteins and a proper disposal of the damaged ones. The latter is carried out by ERAD and autophagy.
      The translocation of proteins into the cytoplasm is a complex, multi-step process with Sec61 proteins and Derlins being the best-known translocation channels. At the same time as translocation, polyubiquitination is carried out directly at the ER membrane by E3 ligase complexes such as Hrd1-SEL1L. Ubiquitinated proteins are recognised by the AAA+ ATPase VCP/p97 machinery that mediates their removal from the ER membrane. After being segregated from the membrane, substrates are transported to the proteasomes via cytosolic molecular chaperones, such as heat shock 70 kDa proteins (Hsp70s)
      • Park S.H.
      • Bolender N.
      • Eisele F.
      • Kostova Z.
      • Takeuchi J.
      • Coffino P.
      • et al.
      The cytoplasmic Hsp70 chaperone machinery subjects misfolded and endoplasmic reticulum import-incompetent proteins to degradation via the ubiquitin-proteasome system.
      or BAG6 complexes.
      • Akahane T.
      • Sahara K.
      • Yashiroda H.
      • Tanaka K.
      • Murata S.
      Involvement of Bag6 and the TRC pathway in proteasome assembly.
      Although proteasome-dependent degradation constitutes a crucial type of disposal for secretory cells, the pore-size limitations of the retro-translocation channel and of the 26S proteasome restrict the degradation of large protein aggregates.
      Unlike the proteasome, macroautophagy (herein simply referred to as autophagy) can degrade even large protein aggregates, as well as the organelles. It begins with the formation of a double membrane vesicle known as the autophagosome that encloses cytosolic material and later fuses with the lysosome, which contains the enzymes needed for degradation.
      • Ward C.
      • Martinez-Lopez N.
      • Otten E.G.
      • Carroll B.
      • Maetzel D.
      • Singh R.
      • et al.
      Autophagy, lipophagy and lysosomal lipid storage disorders.
      Autophagosome formation is facilitated by a ubiquitin-like conjugation system involving the autophagy-related genes (ATGs). At the end of this process, conjugation of LC3 (MAP1LC3A)/GABARAP family proteins with phosphatidylethanolamine mediates the closure of the autophagosome. While bulk autophagy (non-selective autophagy) sequesters the cytosolic material randomly, selective autophagy encloses the cargo selectively with the help of adapter molecules such as p62 (SQSTM1), NBR1 or optineurin. For example, p62 binds to ubiquitinated proteins and recruits them to autophagosomes by interacting with LC3. Depending on the target structure, several types of autophagy such as mitophagy, lipophagy etc. have been described.
      • Madrigal-Matute J.
      • Cuervo A.M.
      Regulation of liver metabolism by autophagy.
      As such, ER-phagy refers to a subtype that recycles dysfunctional or unnecessary ER components. Compared to other pathways, the ER-phagy mediated cargo segregation is less well understood.
      • Song S.
      • Tan J.
      • Miao Y.
      • Zhang Q.
      Crosstalk of ER stress-mediated autophagy and ER-phagy: involvement of UPR and the core autophagy machinery.
      However, a recent study identified the ER-resident receptor FAM134B (RETREG1) as a critical mediator of ER-phagy in mammals.
      • Khaminets A.
      • Heinrich T.
      • Mari M.
      • Grumati P.
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      • et al.
      Regulation of endoplasmic reticulum turnover by selective autophagy.
      Autophagy is induced in response to a wide range of stimuli such as starvation, proteotoxic, metabolic or pathogen-associated stress (reviewed in detail in Hurley and Young, 2017
      • Hurley J.H.
      • Young L.N.
      Mechanisms of autophagy initiation.
      ). The classic regulators include the protein kinases AMPK (PRKAA1) and ULK1 and mammalian target of rapamycin complex 1 (mTORC1). For example, during starvation or energy depletion, the declining ATP/AMP ratios lead to AMPK activation, inhibition of mTORC1 and subsequent activation of autophagy.
      Although proteasome- and autophagy-mediated degradation represent two distinct pathways, several lines of evidence suggest that they closely cooperate.
      • Arndt V.
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      • Dreiseidler M.
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      • Hesse M.
      • et al.
      Chaperone-assisted selective autophagy is essential for muscle maintenance.
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      • Behl C.
      Protein quality control during aging involves recruitment of the macroautophagy pathway by BAG3.
      For instance, the Hsp70 co-chaperones BAG1 and BAG3 are known to regulate both pathways. During aging, a switch from BAG1 to BAG3 occurs and results in an increase in autophagic activity.
      • Gamerdinger M.
      • Hajieva P.
      • Kaya A.M.
      • Wolfrum U.
      • Hartl F.U.
      • Behl C.
      Protein quality control during aging involves recruitment of the macroautophagy pathway by BAG3.
      Similarly, CHIP (STUB1), an ubiquitin ligase, acts as a dual player targeting misfolded proteins for both autophagy- and proteasome-dependent degradation.
      • Arndt V.
      • Dick N.
      • Tawo R.
      • Dreiseidler M.
      • Wenzel D.
      • Hesse M.
      • et al.
      Chaperone-assisted selective autophagy is essential for muscle maintenance.
      Last but not least, some misfolded proteins escape from ERAD, for example when they fail to interact with BiP and are then targeted for autophagosomal degradation.
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      Degradation of mutated bovine pancreatic trypsin inhibitor in the yeast vacuole suggests post-endoplasmic reticulum protein quality control.
      Despite these interesting findings, further studies are needed to elucidate the precise interplay between both degradational pathways.

      Response to accumulation of misfolded proteins

      A dysregulation in, or overload of, the folding-degradational pathway leads to the accumulation of misfolded proteins in the ER lumen and triggers an adaptive cellular response named the unfolded protein response (UPR) (Fig. 1, Fig. 2). It is induced by three ER stress sensors activating transcription factor 6 (ATF6), inositol-requiring enzyme 1α (IRE1α [ERN1]) and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK [EIF2AK3]) (reviewed in Wang and Kaufman, 2016).
      • Wang M.
      • Kaufman R.J.
      Protein misfolding in the endoplasmic reticulum as a conduit to human disease.
      Under steady state conditions, they are complexed with BiP, which keeps them in an inactive state. When misfolded proteins accumulate, they recruit BiP and thereby disrupt its association with the ER stress sensors. The dissociation triggers oligomerisation and autophosphorylation of IRE1α and PERK, and leads to the translocation of ATF6 to the Golgi apparatus (Fig. 2). While BiP-dependent UPR activation constitutes the characteristic pathway, alternative routes have also been described that include a direct interaction of IRE1α or PERK with misfolded proteins or the activation of IRE1α via Hsp47 (SERPINH1), the major collagen chaperone.
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      An unfolded protein-induced conformational switch activates mammalian IRE1.
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      • et al.
      Interactome screening identifies the ER luminal chaperone Hsp47 as a regulator of the unfolded protein response transducer IRE1alpha.
      • Wang P.
      • Li J.
      • Tao J.
      • Sha B.
      The luminal domain of the ER stress sensor protein PERK binds misfolded proteins and thereby triggers PERK oligomerization.
      Figure thumbnail gr2
      Fig. 2Endoplasmic reticulum stress response. Endoplasmic reticulum (ER) stress response is regulated by the availability of the central chaperone BiP. Under basal conditions, BiP binds to the ER stress sensors ATF6α, IRE1α and PERK. Accumulation of misfolded proteins leads to its dissociation from these sensors and their activation. The activation of IRE1α unleashes both its kinase and endoribonuclease functions. The latter causes decay of mRNAs (RIDD; regulated IRE1-dependent decay of mRNA) and a cleavage of XBP1 mRNA into the transcriptionally active form. Spliced XBP1 (sXBP1) transits into the nucleus and enhances the expression of genes related to ER trafficking / ER-associated degradation (ERAD). In the case of ATF6α, the dissociation from BiP leads to translocation to Golgi complex, where it becomes proteolytically cleaved. Its cytosolic fragment then induces the expression of various chaperones. PERK phosphorylates eIF2alpha, thereby inhibiting translation of proteins. Although phosphorylation of eIF2α reduces the overall mRNA translation, it also enhances the translation of specific mRNAs bearing short upstream open reading frames (uORFs) that boost protein transport or antioxidant stress response. While all these steps promote the survival of the cells, the prolonged stress induces apoptotic cell death mainly through the PERK–eIF2α–ATF4-CHOP pathway. CHOP induces the synthesis of pro-apoptotic genes. IRE1α also promotes apoptosis by inducing ASK1 and JNK. Other strategies of the ER to cope with stress include the activation of the transcription factor NF-κB and the liver specific transcription factor CREBH. While the former is induced by the efflux of Ca2+ and reactive oxygen intermediates from the ER, CREBH is proteolytically cleaved and its N-terminal fragment stimulates the production of acute-phase response (APR) genes.
      UPR leads to attenuation of protein synthesis as well as an increase in folding, transport and disposal capacity of the ER.
      • Wang M.
      • Kaufman R.J.
      Protein misfolding in the endoplasmic reticulum as a conduit to human disease.
      Under conditions of ER stress, IRE1α activates both its kinase and endoribonuclease activity. The nuclease activity of IRE1α mediates degradation of a specific subset of ER-localised mRNAs through a mechanism known as regulated IRE1-dependent decay, thus limiting new protein translation. At the same time, it causes a non-canonical splicing of XBP1 mRNA. The transcriptionally active, spliced form of XBP1 (sXBP1) transits into the nucleus and enhances the expression of genes related to ER trafficking. Similarly, the proteolytic cleavage of ATF6, that occurs after its translocation from the ER to the Golgi apparatus, yields a cytosolic fragment that induces the expression of various chaperones such as BiP and GRP94 [HSP90B1]). Additionally, PERK phosphorylates eIF2α (EIF2A), thereby inhibiting translation of proteins and resulting in cell cycle arrest. Although phosphorylation of eIF2α reduces the overall mRNA translation, it enhances the transcription of specific genes carrying small open reading frames that collectively boost protein transport or the antioxidant stress response, in order to enhance the ER folding capacity. Finally, proteolytic cleavage of CREBH (CREB3L3) leads to translocation of its N-terminal fragment to the nucleus, where it stimulates the production of acute-phase response (APR) genes.
      • Luebke-Wheeler J.
      • Zhang K.
      • Battle M.
      • Si-Tayeb K.
      • Garrison W.
      • Chhinder S.
      • et al.
      Hepatocyte nuclear factor 4alpha is implicated in endoplasmic reticulum stress-induced acute phase response by regulating expression of cyclic adenosine monophosphate responsive element binding protein H.
      • Zhang K.
      • Shen X.
      • Wu J.
      • Sakaki K.
      • Saunders T.
      • Rutkowski D.T.
      • et al.
      Endoplasmic reticulum stress activates cleavage of CREBH to induce a systemic inflammatory response.
      If these steps fail to restore the homeostasis in the ER, the prolonged stress induces apoptotic cell death, mainly through the PERK–eIF2α–ATF4-CHOP pathway. Under conditions of persistent ER stress, CHOP (DDIT3) expression eventually promotes the pro-apoptotic genes, e.g. BIM (BCL2L11), DR5 (TNFRSF10B), and represses the expression of BCL2, which altogether sensitise the cell and trigger apoptosis. Independent of its endoribonuclease activity, IRE1α also promotes apoptosis by inducing the activation of procaspase-12, and the kinases, ASK1 [MAP3K5] and c-Jun N-terminal kinase (JNK [MAPK8]).
      • Urano F.
      • Wang X.
      • Bertolotti A.
      • Zhang Y.
      • Chung P.
      • Harding H.P.
      • et al.
      Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1.
      • Nishitoh H.
      • Matsuzawa A.
      • Tobiume K.
      • Saegusa K.
      • Takeda K.
      • Inoue K.
      • et al.
      ASK1 is essential for endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats.
      Despite promoting apoptosis, it has been shown recently that IRE1α activation also favours the development of obesity-associated hepatocellular carcinoma (HCC) via activation of STAT3 and NF-kB signalling.
      • Wu Y.
      • Shan B.
      • Dai J.
      • Xia Z.
      • Cai J.
      • Chen T.
      • et al.
      Dual role for inositol-requiring enzyme 1alpha in promoting the development of hepatocellular carcinoma during diet-induced obesity.
      Another strategy the ER utilises to cope with stress is the activation of the transcription factor NF-κB
      • Hidvegi T.
      • Schmidt B.Z.
      • Hale P.
      • Perlmutter D.H.
      Accumulation of mutant alpha1-antitrypsin Z in the endoplasmic reticulum activates caspases-4 and -12, NFkappaB, and BAP31 but not the unfolded protein response.
      • Pahl H.L.
      • Baeuerle P.A.
      The ER-overload response: activation of NF-kappa B.
      that is induced by the efflux of Ca2+ and reactive oxygen intermediates from the ER.
      • Yoon S.
      • Woo S.U.
      • Kang J.H.
      • Kim K.
      • Shin H.J.
      • Gwak H.S.
      • et al.
      NF-kappaB and STAT3 cooperatively induce IL6 in starved cancer cells.
      The integrated stress response (ISR) leads to the activation of eIF2α kinases that in turn trigger the phosphorylation of eIF2α.
      • Pakos-Zebrucka K.
      • Koryga I.
      • Mnich K.
      • Ljujic M.
      • Samali A.
      • Gorman A.M.
      The integrated stress response.
      To date, four distinct eIF2α kinases, i.e. HRI ((EIF2AK1), PKR (EIF2AK2), PERK (EIF2AK3) and GCN2 (EIF2AK4) have been identified. Like the UPR, the ISR leads to a decrease in global protein synthesis and the induction of certain genes, such as the transcription factor ATF4 that promotes cellular recovery. Therefore, these strategies cooperate to enable survival from proteotoxic stress.

      Regulation of hepatocellular protein synthesis under stress conditions

      To adjust the hepatocellular protein production to the needs of the organism,
      • Strnad P.
      • Tacke F.
      • Koch A.
      • Trautwein C.
      Liver – guardian, modifier and target of sepsis.
      the repertoire of synthesised species is modified by various cytokines. This stress-related alteration is termed the APR and leads to enhanced production of proteins that are called acute-phase proteins (APPs) or positive acute-phase reactants.
      • Kushner I.
      The phenomenon of the acute phase response.

      Kushner I. Acute phase reactants. In: Furst DE, ed. UpToDate: UpToDate; 2017.

      At the same time, several proteins tend to decrease under these circumstances and are therefore termed negative acute-phase reactants or anti-APPs.
      • Jain S.
      • Gautam V.
      • Naseem S.
      Acute-phase proteins: as diagnostic tool.
      The classic negative APPs are albumin, transferrin, or transthyretin, while the positive APPs include C-reactive protein (CRP), serum amyloid A, fibrinogen, hepcidin and ferritin.

      Kushner I. Acute phase reactants. In: Furst DE, ed. UpToDate: UpToDate; 2017.

      Based on the predominant inducer, APPs can be further subdivided into type I and type II acute-phase proteins.
      • Moshage H.
      Cytokines and the hepatic acute phase response.
      Type I APPs (e.g. complement C3) are regulated by IL-1 like cytokines, such as IL-1α (IL1A), IL-1β (IL1B) or TNFα (TNF). Type II APPs comprise fibrinogen, α1-antitrypsin, and hepcidin. Their production is stimulated via the IL-6 family cytokines, such as IL-6, IL-11, oncostatin M or leukemia inhibitory factor. While the cytokines described are typically produced by inflammatory cells and modify the production of hepatocellular proteins through systemic inflammation, several autocrine mechanisms have also been described. For example, UPR components such as XBP1 were shown to regulate APP production.
      • Argemi J.
      • Kress T.R.
      • Chang H.C.Y.
      • Ferrero R.
      • Bertolo C.
      • Moreno H.
      • et al.
      X-box binding protein 1 regulates unfolded protein, acute-phase, and DNA damage responses during regeneration of mouse liver.
      • Reimold A.M.
      • Etkin A.
      • Clauss I.
      • Perkins A.
      • Friend D.S.
      • Zhang J.
      • et al.
      An essential role in liver development for transcription factor XBP-1.
      Liver hepatocytes synthesize the majority of serum protein and the production is adjusted to the physiological needs of the human body by inducing the acute-phase proteins (APPs) and down-regulating the anti-APPs.
      With regard to downstream signalling, several important hepatocyte-specific transcription factors are involved in the UPR. These include, the ER-bound CREBH protein that is predominantly expressed in the liver and is activated by pro-inflammatory cytokines, such as TNFα under ER stress conditions.
      • Zhang K.
      • Shen X.
      • Wu J.
      • Sakaki K.
      • Saunders T.
      • Rutkowski D.T.
      • et al.
      Endoplasmic reticulum stress activates cleavage of CREBH to induce a systemic inflammatory response.
      Hepatocyte nuclear factors constitute a family of liver specific transcription factors that can be both induced and suppressed by cytokine signalling and mediate both induction of APPs and a reduction in anti-APPs.
      • Wang B.
      • Cai S.R.
      • Gao C.
      • Sladek F.M.
      • Ponder K.P.
      Lipopolysaccharide results in a marked decrease in hepatocyte nuclear factor 4 alpha in rat liver.
      Although the function of APPs is only partially understood, they are believed to protect the body from stresses and are often involved in microbial defence. As such, induction of APPs via a protein-rich diet improved the survival of seriously burned patients mainly via the APP-mediated readjustment of the immunologic response.
      • Alexander J.W.
      • MacMillan B.G.
      • Stinnett J.D.
      • Ogle C.K.
      • Bozian R.C.
      • Fischer J.E.
      • et al.
      Beneficial effects of aggressive protein feeding in severely burned children.
      Conversely, inhibition of APP production by ablation of the IL-6-downstream effector gp130 (IL6STP1) resulted in significantly increased mortality in a mouse model of polymicrobial infection because of a dysregulated inflammatory reaction. Notably, this phenotype was reversed by the addition of the classic APP, serum amyloid A.
      • Sander L.E.
      • Sackett S.D.
      • Dierssen U.
      • Beraza N.
      • Linke R.P.
      • Muller M.
      • et al.
      Hepatic acute-phase proteins control innate immune responses during infection by promoting myeloid-derived suppressor cell function.
      As another example, the IL-6-inducible hormone hepcidin causes the retention of iron in macrophages, thereby shielding it from the invading bacteria and preventing the spread of infection.
      • Nicolas G.
      • Chauvet C.
      • Viatte L.
      • Danan J.L.
      • Bigard X.
      • Devaux I.
      • et al.
      The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation.
      Finally, CRP promotes early defence against infection by activating the complement system.
      • Mantovani A.
      • Garlanda C.
      • Doni A.
      • Bottazzi B.
      Pentraxins in innate immunity: from C-reactive protein to the long pentraxin PTX3.
      While the induction of APPs under stress conditions likely has an important stress-protective function, it represents a significant ER challenge and an important mechanism of disease-related hepatocellular proteotoxic stress. However, the relationship between the APR and the UPR is more complex. For example, some UPR components such as XBP1 are required for robust APP production under certain stress conditions.
      • Argemi J.
      • Kress T.R.
      • Chang H.C.Y.
      • Ferrero R.
      • Bertolo C.
      • Moreno H.
      • et al.
      X-box binding protein 1 regulates unfolded protein, acute-phase, and DNA damage responses during regeneration of mouse liver.
      • Reimold A.M.
      • Etkin A.
      • Clauss I.
      • Perkins A.
      • Friend D.S.
      • Zhang J.
      • et al.
      An essential role in liver development for transcription factor XBP-1.
      Given the tight relationship between the UPR and the APR, future studies should analyse whether any of the known APPs may also serve as non-invasive sensors of hepatic UPR.

      Hepatocyte-produced serum proteins as disease markers

      CRP is an APP used as a marker of systemic inflammation (e.g., infection, rheumatologic disease). As part of the innate immune response, CRP is rapidly expressed within hours after injury or infection,
      • Black S.
      • Kushner I.
      • Samols D.
      C-reactive protein.
      in response to bacterial/intrinsic compounds known as pathogen/damage-associated molecular patterns. Both IL-1β and IL-6, and the transcription factors C/EBPβ (CEBP) and C/EBPδ (CEBPD) are critical for CRP induction.
      • Black S.
      • Kushner I.
      • Samols D.
      C-reactive protein.
      CRP levels between 3 and 10 mg/L indicate low-grade inflammation which may originate from various stresses such as obesity.
      • Kushner I.
      • Rzewnicki D.
      • Samols D.
      What does minor elevation of C-reactive protein signify?.
      In contrast, CRP is most efficiently induced by bacterial infection and consequently might be used to distinguish it from viral infection. Since CRP synthesis may become impaired in advanced liver disease, alternative inflammatory markers should be used in this situation.
      • Strnad P.
      • Tacke F.
      • Koch A.
      • Trautwein C.
      Liver – guardian, modifier and target of sepsis.
      Stress-modified serum proteins such as CRP or transferrin constitute clinical useful disease markers.
      Albumin is quantitatively the most important plasma protein, accounting for nearly 50% of total plasma protein.
      • Quinlan G.J.
      • Martin G.S.
      • Evans T.W.
      Albumin: biochemical properties and therapeutic potential.
      The serum albumin concentration mainly reflects the rate of its synthesis and in the absence of inflammation and protein-losing conditions (e.g. nephrotic syndrome), it can be used as a surrogate of hepatic synthetic activity. Accordingly, albumin is part of the commonly used Child-Turcotte-Pugh score that assesses the liver function in cirrhotics and constitutes a useful mortality predictor.
      • Albers I.
      • Hartmann H.
      • Bircher J.
      • Creutzfeldt W.
      Superiority of the Child-Pugh classification to quantitative liver function tests for assessing prognosis of liver cirrhosis.
      Additionally, albumin is a negative APP
      • Strnad P.
      • Tacke F.
      • Koch A.
      • Trautwein C.
      Liver – guardian, modifier and target of sepsis.
      and its synthesis is suppressed by TNFα and IL-1.
      • Strnad P.
      • Tacke F.
      • Koch A.
      • Trautwein C.
      Liver – guardian, modifier and target of sepsis.
      Consequently, serum albumin levels are particularly low among patients with inflammation.
      • Gabay C.
      • Kushner I.
      Acute-phase proteins and other systemic responses to inflammation.
      In line with this, a meta-analysis showed that albumin was an independent and dose-dependent predictor of poor outcome in acutely ill patients with various underlying diseases.
      • Vincent J.L.
      • Dubois M.J.
      • Navickis R.J.
      • Wilkes M.M.
      Hypoalbuminemia in acute illness: is there a rationale for intervention? A meta-analysis of cohort studies and controlled trials.
      Transferrin is the major iron-binding protein in the plasma.
      • Ganz T.
      • Nemeth E.
      Iron homeostasis in host defence and inflammation.
      This function is of key importance since unbound iron is highly reactive and causes oxidative stress. However, transferrin is downregulated in systemic inflammation, which may lead to an increased availability of iron for the invading microorganisms.
      • Strnad P.
      • Tacke F.
      • Koch A.
      • Trautwein C.
      Liver – guardian, modifier and target of sepsis.
      • Ritchie R.F.
      • Palomaki G.E.
      • Neveux L.M.
      • Navolotskaia O.
      • Ledue T.B.
      • Craig W.Y.
      Reference distributions for the negative acute-phase serum proteins, albumin, transferrin and transthyretin: a practical, simple and clinically relevant approach in a large cohort.
      Of note, transferrin was suggested to constitute a promising prognostic marker in various clinical conditions. In decompensated cirrhosis, low serum transferrin levels were associated with increased mortality
      • Bruns T.
      • Nuraldeen R.
      • Mai M.
      • Stengel S.
      • Zimmermann H.W.
      • Yagmur E.
      • et al.
      Low serum transferrin correlates with acute-on-chronic organ failure and indicates short-term mortality in decompensated cirrhosis.
      and similar findings were made in patients with acute-on-chronic liver failure.
      • Bruns T.
      • Nuraldeen R.
      • Mai M.
      • Stengel S.
      • Zimmermann H.W.
      • Yagmur E.
      • et al.
      Low serum transferrin correlates with acute-on-chronic organ failure and indicates short-term mortality in decompensated cirrhosis.
      • Maras J.S.
      • Maiwall R.
      • Harsha H.C.
      • Das S.
      • Hussain M.S.
      • Kumar C.
      • et al.
      Dysregulated iron homeostasis is strongly associated with multiorgan failure and early mortality in acute-on-chronic liver failure.
      In acute liver failure, low serum transferrin was associated with worse outcomes,
      • Anastasiou O.E.
      • Kalsch J.
      • Hakmouni M.
      • Kucukoglu O.
      • Heider D.
      • Korth J.
      • et al.
      Low transferrin and high ferritin concentrations are associated with worse outcome in acute liver failure.
      as it was in critically ill patients.
      • Tacke F.
      • Nuraldeen R.
      • Koch A.
      • Strathmann K.
      • Hutschenreuter G.
      • Trautwein C.
      • et al.
      Iron parameters determine the prognosis of critically Ill patients.
      These findings warrant further investigation into the function of transferrin in human disease.

      Diseases associated with ER stress and ER protein accumulation

      ER stress is an integral part of multiple liver diseases including (but not limited to) viral hepatitis, alcoholic liver disease and non-alcoholic fatty liver disease.
      • Baiceanu A.
      • Mesdom P.
      • Lagouge M.
      • Foufelle F.
      Endoplasmic reticulum proteostasis in hepatic steatosis.
      • Fu S.
      • Watkins S.M.
      • Hotamisligil G.S.
      The role of endoplasmic reticulum in hepatic lipid homeostasis and stress signaling.
      • Malhi H.
      • Kaufman R.J.
      Endoplasmic reticulum stress in liver disease.
      Given the epidemics of metabolic liver diseases, as well as the important function of the liver as the central metabolic regulator, there is currently a significant focus on lipid-related ER stress.
      • Baiceanu A.
      • Mesdom P.
      • Lagouge M.
      • Foufelle F.
      Endoplasmic reticulum proteostasis in hepatic steatosis.
      • Fu S.
      • Watkins S.M.
      • Hotamisligil G.S.
      The role of endoplasmic reticulum in hepatic lipid homeostasis and stress signaling.
      In contrast, the aim of the current review is to raise awareness of the liver as the central hub of protein metabolism and accordingly, we will focus on liver disorders that associate with protein accumulation in the ER and the associated ER stress.
      Several human diseases challenge the ER proteostatic network and result in protein accumulation within the ER. Most prominent examples are the inherited alpha1-antitrypsin deficiency and chronic hepatitis B infection, that lead to retention of mutated AAT and hepatitis B surface protein, respectively.

      Chronic hepatitis B

      Chronic hepatitis B virus (HBV) infection often leads to accumulation of hepatitis B surface (HBs) protein in the ER of hepatocytes.
      • Strnad P.
      • Nuraldeen R.
      • Guldiken N.
      • Hartmann D.
      • Mahajan V.
      • Denk H.
      • et al.
      Broad spectrum of hepatocyte inclusions in humans, animals, and experimental models.
      In the haematoxylin and eosin (H&E) staining, this retention gives rise to granular, glassy appearing inclusions that are known as ground glass hepatocytes (GGH) (Fig. 3A–B). GGHs arise because of a disordered production of HBs protein isoforms.
      • Strnad P.
      • Nuraldeen R.
      • Guldiken N.
      • Hartmann D.
      • Mahajan V.
      • Denk H.
      • et al.
      Broad spectrum of hepatocyte inclusions in humans, animals, and experimental models.
      Under normal circumstances, HBV synthesises three HBs protein isoforms termed as large, middle and small that result from translation at three different start codons. Mutations in the HBs gene or its promoter region result in overproduction of the large HBs isoform that becomes trapped in the ER where it forms filamentous particles.
      • Chisari F.V.
      Hepatitis B virus transgenic mice: insights into the virus and the disease.
      Of note, this proteotoxic challenge remains unaffected by current treatments for HBV, namely nucleoside/nucleotide analogues, since these drugs do not inhibit HBs production.
      • Ringelhan M.
      • O'Connor T.
      • Protzer U.
      • Heikenwalder M.
      The direct and indirect roles of HBV in liver cancer: prospective markers for HCC screening and potential therapeutic targets.
      Figure thumbnail gr3
      Fig. 3Characteristic hepatocellular endoplasmic reticulum-storage diseases. Ground glass hepatocytes (GGHs, depicted by arrows) are visualised via (A) haematoxylin and eosin (H&E) staining and (B) immunohistochemistry with an antibody against hepatitis B surface protein in a patient with chronic hepatitis B infection. (C) H&E and (D) periodic acid-Schiff after diastase digestion (PAS-D) stained liver sections of a patient with severe alpha1-antitrypsin deficiency (PiZZ genotype). The arrows indicate the alpha1-antitrypsin (AAT) aggregates. (E) H&E staining reveals hepatocytes with amorphous, fibrillar pale bodies (arrows) in a patient with hepatocellular carcinoma (HCC). (F) These inclusions are not labelled in an immunohistochemistry staining with an antibody against glutamine synthetase.
      GGHs can be subdivided into type I and II inclusions. Type I GGHs are sporadically distributed throughout the liver, whereas type II GGHs are clustered. The latter are considered preneoplastic lesions that may progress to HCC. In contrast, the biological significance of type I GGHs, that often emerge at an earlier stage of HBV infection, is less clear.
      • Su I.J.
      • Wang L.H.
      • Hsieh W.C.
      • Wu H.C.
      • Teng C.F.
      • Tsai H.W.
      • et al.
      The emerging role of hepatitis B virus pre-S2 deletion mutant proteins in HBV tumorigenesis.
      To obtain a cellular model of GGHs, multiple studies transfected hepatoma cell lines with genetically-altered HBs constructs, resulting in HBs accumulation in the ER. In a likely attempt to degrade the HBs via ERAD, the engineered cell lines displayed an induction in EDEM family members.
      • Lazar C.
      • Macovei A.
      • Petrescu S.
      • Branza-Nichita N.
      Activation of ERAD pathway by human hepatitis B virus modulates viral and subviral particle production.
      In addition, the transfection caused ER stress with PERK activation and induction of JNK.
      • Wang H.C.
      • Wu H.C.
      • Chen C.F.
      • Fausto N.
      • Lei H.Y.
      • Su I.J.
      Different types of ground glass hepatocytes in chronic hepatitis B virus infection contain specific pre-S mutants that may induce endoplasmic reticulum stress.
      It also provided the cells a growth advantage because of activation of Akt/mTOR signalling.
      • Yang J.C.
      • Teng C.F.
      • Wu H.C.
      • Tsai H.W.
      • Chuang H.C.
      • Tsai T.F.
      • et al.
      Enhanced expression of vascular endothelial growth factor-A in ground glass hepatocytes and its implication in hepatitis B virus hepatocarcinogenesis.
      Moreover, an impaired induction of Nrf2/antioxidative stress response was observed, leading to elevated levels of reactive oxygen species and activation of JNK signalling.
      • Peiffer K.H.
      • Akhras S.
      • Himmelsbach K.
      • Hassemer M.
      • Finkernagel M.
      • Carra G.
      • et al.
      Intracellular accumulation of subviral HBsAg particles and diminished Nrf2 activation in HBV genotype G expressing cells lead to an increased ROI level.
      In a hepatic progenitor cell line, an HBs transfection resulted in altered ER homeostasis, elevated intracellular Ca2+ levels and led to increased tumour growth in a xenograft model.
      • Yen T.T.
      • Yang A.
      • Chiu W.T.
      • Li T.N.
      • Wang L.H.
      • Wu Y.H.
      • et al.
      Hepatitis B virus PreS2-mutant large surface antigen activates store-operated calcium entry and promotes chromosome instability.
      To study the pathogenic consequences of HBs protein accumulation in vivo, a transgenic mouse model overexpressing the large HBs isoform (often referred to as lineage 50–4 or Bri 44) has been generated. These Bri 44 mice display retention of HBs in the ER and inclusions that are morphologically indistinguishable from human GGHs.
      • Chisari F.V.
      • Filippi P.
      • McLachlan A.
      • Milich D.R.
      • Riggs M.
      • Lee S.
      • et al.
      Expression of hepatitis B virus large envelope polypeptide inhibits hepatitis B surface antigen secretion in transgenic mice.
      The sustained overproduction of HBs isoforms causes hepatocellular injury coupled with enhanced oxidative DNA damage, the development of nodular hyperplasia and HCC.
      • Chisari F.V.
      • Klopchin K.
      • Moriyama T.
      • Pasquinelli C.
      • Dunsford H.A.
      • Sell S.
      • et al.
      Molecular pathogenesis of hepatocellular carcinoma in hepatitis B virus transgenic mice.
      Of note, the severity of the proteotoxicity correlated with the intrahepatic concentration of HBs.
      • Chisari F.V.
      Hepatitis B virus transgenic mice: insights into the virus and the disease.
      Mechanistically, persistent ER stress with a sustained PERK activation and attenuated IRE1α/ATF6 signalling have been observed.
      • Churin Y.
      • Roderfeld M.
      • Stiefel J.
      • Wurger T.
      • Schroder D.
      • Matono T.
      • et al.
      Pathological impact of hepatitis B virus surface proteins on the liver is associated with the host genetic background.
      The degradation of HBs by autophagy seemed to be protective and its impairment in mice with a heterozygous disruption of the Becn1 gene accelerated the development of liver tumours.
      • Qu X.
      • Yu J.
      • Bhagat G.
      • Furuya N.
      • Hibshoosh H.
      • Troxel A.
      • et al.
      Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene.
      These findings might be of human relevance since signs of impaired autophagy, including high levels of p62 and/or an activated mTOR pathway, were observed in patients with chronic HBV infection as well as HBV-related HCC.
      • Teng C.F.
      • Wu H.C.
      • Tsai H.W.
      • Shiah H.S.
      • Huang W.
      • Su I.J.
      Novel feedback inhibition of surface antigen synthesis by mammalian target of rapamycin (mTOR) signal and its implication for hepatitis B virus tumorigenesis and therapy.
      • Xiang X.
      • Qin H.G.
      • You X.M.
      • Wang Y.Y.
      • Qi L.N.
      • Ma L.
      • et al.
      Expression of P62 in hepatocellular carcinoma involving hepatitis B virus infection and aflatoxin B1 exposure.
      Given that tumours do not spontaneously form in mice carrying the entire HBV genome under basal conditions, HBs accumulation rather than balanced expression of all viral proteins seems to underlie the observed liver phenotype.
      • Zheng Y.
      • Chen W.L.
      • Louie S.G.
      • Yen T.S.
      • Ou J.H.
      Hepatitis B virus promotes hepatocarcinogenesis in transgenic mice.
      While these data clearly point towards the role of proteotoxic stress in the pathogenesis of HBV-related liver injury, its relative contribution compared to other HBV-related pathologies remains to be clarified.

      Alpha1-antitrypsin deficiency

      Inherited alpha1-antitrypsin deficiency (AATD) is a genetic disorder caused by mutations within the SERPINA1 gene, which encodes the AAT protein. The most clinically recognised form of severe AATD is a homozygous Z (Glu342Lys) mutation termed PiZZ, which affects 1:1,200–1:3,000 newborns in Europe. It predisposes individuals to the development of emphysema and a liver disease that manifests in two different ways. The neonatal form is characterised by jaundice and increased serum aminotransferase levels and progresses to life-threatening liver disease in about 5% of children.
      • Sveger T.
      Liver disease in alpha1-antitrypsin deficiency detected by screening of 200,000 infants.
      While the PiZZ carriers are mostly asymptomatic during early adulthood, some of them develop liver fibrosis and cirrhosis later.
      • Eriksson S.
      • Carlson J.
      • Velez R.
      Risk of cirrhosis and primary liver cancer in alpha 1-antitrypsin deficiency.
      According to autopsy studies in adults, the lifetime risk of cirrhosis may be as high as 50%,

      Eriksson S. Alpha1-antitrypsin deficiency: natural course and therapeutic strategies. Falk Symposium 115; 2000; Dordrecht, The Netherlands.

      while others have suggested a lower risk.
      • Clark V.C.
      • Dhanasekaran R.
      • Brantly M.
      • Rouhani F.
      • Schreck P.
      • Nelson D.R.
      Liver test results do not identify liver disease in adults with alpha(1)-antitrypsin deficiency.
      Histopathologic examination of liver specimens from patients with inherited AATD typically shows the intracellular globules that stain positive with periodic acid-Schiff-diastase. Although AAT aggregates can also be seen in H&E staining, they are quite indistinct and can easily be missed.
      • Strnad P.
      • Nuraldeen R.
      • Guldiken N.
      • Hartmann D.
      • Mahajan V.
      • Denk H.
      • et al.
      Broad spectrum of hepatocyte inclusions in humans, animals, and experimental models.
      As the underlying mechanism, the Z variant of AAT leads to AAT protein polymerisation and accumulation within the ER (Fig. 3C–D), resulting in ER dysfunction.
      • Perlmutter D.H.
      Alpha1-antitrypsin deficiency: a misfolded secretory protein variant with unique effects on the endoplasmic reticulum.
      In this regard, ERGIC-53 (LMAN1), which is an export receptor for wild-type AAT, does not recognise the Z variant.
      • Nyfeler B.
      • Reiterer V.
      • Wendeler M.W.
      • Stefan E.
      • Zhang B.
      • Michnick S.W.
      • et al.
      Identification of ERGIC-53 as an intracellular transport receptor of alpha1-antitrypsin.
      One of the main consequences of AAT polymerisation is the release of Ca2+ from the ER and the activation of NF-κB, a central mediator of inflammation.
      • Pahl H.L.
      • Baeuerle P.A.
      The ER-overload response: activation of NF-kappa B.
      In a seeming contradiction to the above considerations, several studies reported that cells containing AAT polymers do not show a constitutive activation of the UPR,
      • Graham K.S.
      • Le A.
      • Sifers R.N.
      Accumulation of the insoluble PiZ variant of human alpha 1-antitrypsin within the hepatic endoplasmic reticulum does not elevate the steady-state level of grp78/BiP.
      • Ordonez A.
      • Snapp E.L.
      • Tan L.
      • Miranda E.
      • Marciniak S.J.
      • Lomas D.A.
      Endoplasmic reticulum polymers impair luminal protein mobility and sensitize to cellular stress in alpha1-antitrypsin deficiency.
      but these cells are sensitised to UPR activation by a second hit.
      • Ordonez A.
      • Snapp E.L.
      • Tan L.
      • Miranda E.
      • Marciniak S.J.
      • Lomas D.A.
      Endoplasmic reticulum polymers impair luminal protein mobility and sensitize to cellular stress in alpha1-antitrypsin deficiency.
      A likely explanation is that the rapid polymerisation of the mutant AAT does not lead to significant exposure of hydrophobic amino acids and therefore does not deplete the cellular BiP levels.
      • Hidvegi T.
      • Schmidt B.Z.
      • Hale P.
      • Perlmutter D.H.
      Accumulation of mutant alpha1-antitrypsin Z in the endoplasmic reticulum activates caspases-4 and -12, NFkappaB, and BAP31 but not the unfolded protein response.
      However, this may change in the presence of a second hit, thereby accounting for the “UPR susceptibility” of cells with AAT polymers. To further highlight the importance of a functioning ER proteostatic network in AATD, a recent study by Joly et al.
      • Joly P.
      • Vignaud H.
      • Di Martino J.
      • Ruiz M.
      • Garin R.
      • Restier L.
      • et al.
      ERAD defects and the HFE-H63D variant are associated with increased risk of liver damages in Alpha 1-antitrypsin deficiency.
      identified two variants of HRD1 (SYVN1) (an ERAD- associated protein) to be associated with liver damage in PiZZ AATD individuals.
      The main evidence that liver disease in AATD is caused by gain-of-function protein toxicity comes from the studies using PiZ mice transgenic for the human Z AAT.
      • Carlson J.A.
      • Rogers B.B.
      • Sifers R.N.
      • Finegold M.J.
      • Clift S.M.
      • DeMayo F.J.
      • et al.
      Accumulation of PiZ alpha 1-antitrypsin causes liver damage in transgenic mice.
      Their liver pathology resembles the human disease, including intrahepatic Z AAT globules, fibrosis, mild steatosis, mild inflammation, and increased incidence of HCC.
      • Hidvegi T.
      • Ewing M.
      • Hale P.
      • Dippold C.
      • Beckett C.
      • Kemp C.
      • et al.
      An autophagy-enhancing drug promotes degradation of mutant alpha1-antitrypsin Z and reduces hepatic fibrosis.
      The PiZ mice also display an increased amount of autophagosomes, structural alterations in mitochondria, and activation of NF-κB signalling.
      • Hidvegi T.
      • Schmidt B.Z.
      • Hale P.
      • Perlmutter D.H.
      Accumulation of mutant alpha1-antitrypsin Z in the endoplasmic reticulum activates caspases-4 and -12, NFkappaB, and BAP31 but not the unfolded protein response.
      Moreover, a recent study indicated that PiZ production leads to reduced expression of hepatocyte nuclear factor-4α, a major driver of liver zonation.
      • Piccolo P.
      • Annunziata P.
      • Soria L.R.
      • Attanasio S.
      • Barbato A.
      • Castello R.
      • et al.
      Down-regulation of hepatocyte nuclear factor-4alpha and defective zonation in livers expressing mutant Z alpha1-antitrypsin.
      Consequently, PiZ mice and PiZZ individuals had a perturbed liver zonation and a defect in urea generation.
      • Piccolo P.
      • Annunziata P.
      • Soria L.R.
      • Attanasio S.
      • Barbato A.
      • Castello R.
      • et al.
      Down-regulation of hepatocyte nuclear factor-4alpha and defective zonation in livers expressing mutant Z alpha1-antitrypsin.
      Studies investigating the mechanisms of Z AAT degradation suggest that both proteasomal and autophagic pathways play important roles. Experiments with mammalian cell lines revealed that cells could dispose of Z AAT polymers by ERAD
      • Werner E.D.
      • Brodsky J.L.
      • McCracken A.A.
      Proteasome-dependent endoplasmic reticulum-associated protein degradation: an unconventional route to a familiar fate.
      and Z AAT protein was one of the first identified substrates of ERAD.
      • Brodsky J.L.
      • Wojcikiewicz R.J.
      Substrate-specific mediators of ER associated degradation (ERAD).
      The ERAD-mediated degradation is facilitated by ERManI and the overexpression of this enzyme accelerated the degradation of the Z variant of AAT.
      • Hosokawa N.
      • Tremblay L.O.
      • You Z.
      • Herscovics A.
      • Wada I.
      • Nagata K.
      Enhancement of endoplasmic reticulum (ER) degradation of misfolded Null Hong Kong alpha1-antitrypsin by human ER mannosidase I.
      In contrast, chemical inhibitors of the ERManI (e.g., kifunensine) stabilised misfolded AAT.
      • Wu Y.
      • Swulius M.T.
      • Moremen K.W.
      • Sifers R.N.
      Elucidation of the molecular logic by which misfolded alpha 1-antitrypsin is preferentially selected for degradation.
      The enzymatically inactive homologue of ERManI called EDEM1 also enhanced the degradation of misfolded AAT.
      • Hosokawa N.
      • Wada I.
      • Natsuka Y.
      • Nagata K.
      EDEM accelerates ERAD by preventing aberrant dimer formation of misfolded alpha1-antitrypsin.
      Autophagy is the second major pathway degrading Z AAT polymers.
      • Teckman J.H.
      • Perlmutter D.H.
      Retention of mutant alpha(1)-antitrypsin Z in endoplasmic reticulum is associated with an autophagic response.
      Additional evidence for the role of autophagy in Z AAT removal comes from autophagy-deficient [Atg5-null] murine embryonic fibroblast cell lines
      • Kamimoto T.
      • Shoji S.
      • Hidvegi T.
      • Mizushima N.
      • Umebayashi K.
      • Perlmutter D.H.
      • et al.
      Intracellular inclusions containing mutant alpha1-antitrypsin Z are propagated in the absence of autophagic activity.
      and Atg6-null yeast strains,
      • Kruse K.B.
      • Brodsky J.L.
      • McCracken A.A.
      Characterization of an ERAD gene as VPS30/ATG6 reveals two alternative and functionally distinct protein quality control pathways: one for soluble Z variant of human alpha-1 proteinase inhibitor (A1PiZ) and another for aggregates of A1PiZ.
      that both displayed an impaired clearance of Z AAT protein. In PiZ mice, autophagy-enhancing drugs promoted Z AAT elimination and attenuated hepatic fibrosis.
      • Hidvegi T.
      • Ewing M.
      • Hale P.
      • Dippold C.
      • Beckett C.
      • Kemp C.
      • et al.
      An autophagy-enhancing drug promotes degradation of mutant alpha1-antitrypsin Z and reduces hepatic fibrosis.
      In summary, the current evidence indicates that the proteasomal pathway is mainly responsible for the degradation of soluble Z AAT whereas autophagy is essential for clearance of polymers.

      Pale bodies in HCC

      In 1980, Stromeyer et al. described HCC cells displaying ground glass cytoplasm in H&E staining reminiscent of GGHs
      • Stromeyer F.W.
      • Ishak K.G.
      • Gerber M.A.
      • Mathew T.
      Ground-glass cells in hepatocellular carcinoma.
      and these structures were later named pale bodies (PBs).
      • Craig J.R.
      • Peters R.L.
      • Edmondson H.A.
      • Omata M.
      Fibrolamellar carcinoma of the liver: a tumor of adolescents and young adults with distinctive clinico-pathologic features.
      PBs fill the cytoplasmic area and consequently often displace the cell nuclei to the cell periphery (Fig. 3E-F). Immunohistochemically, PBs strongly react with antibodies directed against fibrinogen.
      • Nakashima O.
      • Sugihara S.
      • Eguchi A.
      • Taguchi J.
      • Watanabe J.
      • Kojiro M.
      Pathomorphologic study of pale bodies in hepatocellular carcinoma.
      Ultrastructurally, they consist of nonmembrane-bound amorphous, granular, or fibrillar inclusions within the rough ER.
      • Stromeyer F.W.
      • Ishak K.G.
      • Gerber M.A.
      • Mathew T.
      Ground-glass cells in hepatocellular carcinoma.
      • Nakashima O.
      • Sugihara S.
      • Eguchi A.
      • Taguchi J.
      • Watanabe J.
      • Kojiro M.
      Pathomorphologic study of pale bodies in hepatocellular carcinoma.
      In HCC, PBs are found in 5–6% cases,
      • Nakashima O.
      • Sugihara S.
      • Eguchi A.
      • Taguchi J.
      • Watanabe J.
      • Kojiro M.
      Pathomorphologic study of pale bodies in hepatocellular carcinoma.
      • Moon W.S.
      • Yu H.C.
      • Chung M.J.
      • Kang M.J.
      • Lee D.G.
      Pale bodies in hepatocellular carcinoma.
      however they were observed in up to 50% of fibrolamellar carcinomas, which constitute a specific HCC subtype seen in younger, non-cirrhotic individuals.
      • Craig J.R.
      • Peters R.L.
      • Edmondson H.A.
      • Omata M.
      Fibrolamellar carcinoma of the liver: a tumor of adolescents and young adults with distinctive clinico-pathologic features.
      PBs are thought to arise because of acquired structural abnormalities in the accumulating proteins, either caused by a somatic mutation or a dysfunctional or overwhelmed secretory apparatus.
      • Nakashima O.
      • Sugihara S.
      • Eguchi A.
      • Taguchi J.
      • Watanabe J.
      • Kojiro M.
      Pathomorphologic study of pale bodies in hepatocellular carcinoma.

      Rare disorders

      Fibrinogen storage disease (FSD) and alpha1-antichymotrypsin (AACT [SERPINA3]) inclusions are less common examples of ER storage disorders.
      • Strnad P.
      • Nuraldeen R.
      • Guldiken N.
      • Hartmann D.
      • Mahajan V.
      • Denk H.
      • et al.
      Broad spectrum of hepatocyte inclusions in humans, animals, and experimental models.
      Fibrinogen is a secreted protein produced by hepatocytes. Mutations affecting the C-terminal region of one of the three fibrinogen genes cause its retention in the ER.
      • Brennan S.O.
      • Maghzal G.
      • Shneider B.L.
      • Gordon R.
      • Magid M.S.
      • George P.M.
      Novel fibrinogen gamma375 Arg–>Trp mutation (fibrinogen aguadilla) causes hepatic endoplasmic reticulum storage and hypofibrinogenemia.
      Fibrinogen-containing aggregates can be detected in H&E stained sections as irregularly, round shaped structures with eosinophilic nature. Besides the inherited form of FSD, acquired deposits were also reported.
      • Marucci G.
      • Morandi L.
      • Macchia S.
      • Betts C.M.
      • Tardio M.L.
      • Dal Monte P.R.
      • et al.
      Fibrinogen storage disease without hypofibrinogenaemia associated with acute infection.
      • Simsek Z.
      • Ekinci O.
      • Cindoruk M.
      • Karakan T.
      • Degertekin B.
      • Akyol G.
      • et al.
      Fibrinogen storage disease without hypofibrinogenemia associated with estrogen therapy.
      FSDs are thought to lead to a gain-of-function proteotoxicity,
      • Kruse K.B.
      • Dear A.
      • Kaltenbrun E.R.
      • Crum B.E.
      • George P.M.
      • Brennan S.O.
      • et al.
      Mutant fibrinogen cleared from the endoplasmic reticulum via endoplasmic reticulum-associated protein degradation and autophagy: an explanation for liver disease.
      • Puls F.
      • Goldschmidt I.
      • Bantel H.
      • Agne C.
      • Brocker V.
      • Dammrich M.
      • et al.
      Autophagy-enhancing drug carbamazepine diminishes hepatocellular death in fibrinogen storage disease.
      therefore stimulation of their degradation constitutes a potential treatment strategy. In this respect, a recent human investigation demonstrated that a pharmacological stimulation of autophagy by carbamazepine resulted in reduction of these aggregates and liver injury.
      • Puls F.
      • Goldschmidt I.
      • Bantel H.
      • Agne C.
      • Brocker V.
      • Dammrich M.
      • et al.
      Autophagy-enhancing drug carbamazepine diminishes hepatocellular death in fibrinogen storage disease.
      AACT is an abundant serine protease inhibitor primarily produced in the liver.
      • Gooptu B.
      • Lomas D.A.
      Conformational pathology of the serpins: themes, variations, and therapeutic strategies.
      Like AAT, AACT mutations may lead to retention of AACT in the ER
      • Lindmark B.
      • Millward-Sadler H.
      • Callea F.
      • Eriksson S.
      Hepatocyte inclusions of alpha 1-antichymotrypsin in a patient with partial deficiency of alpha 1-antichymotrypsin and chronic liver disease.
      and to low AACT serum levels. Compared to AAT aggregates, AACT inclusions are smaller in size (1–6 µm) and cannot be visualised by H&E staining. While AACT inclusions have been found in patients with cryptogenic liver cirrhosis,
      • Lindmark B.
      • Eriksson S.
      Partial deficiency of alpha 1-antichymotrypsin is associated with chronic cryptogenic liver disease.
      the exact pathomechanisms of their proteotoxicity still remain to be elucidated.

      ER stress in other diseases

      While we have focussed on conditions leading to proteotoxicity-related ER stress, hepatic ER stress also occurs in various metabolic disorders, such as obesity, diabetes or non-alcoholic fatty liver disease.
      • Malhi H.
      • Kaufman R.J.
      Endoplasmic reticulum stress in liver disease.
      Several lines of evidence demonstrate a crosstalk between the metabolic and proteotoxic ER stresses.
      • Wang M.
      • Kaufman R.J.
      Protein misfolding in the endoplasmic reticulum as a conduit to human disease.
      • Stordeur C.
      • Puth K.
      • Saenz J.P.
      • Ernst R.
      Crosstalk of lipid and protein homeostasis to maintain membrane function.
      The latter can be induced pharmacologically via blocking the exit of proteins from the ER either by inhibiting protein glycosylation (tunicamycin, Brefeldin A) or by depleting Ca2+ stores (thapsigargin).
      • Pahl H.L.
      • Baeuerle P.A.
      The ER-overload response: activation of NF-kappa B.
      • Wojtowicz K.
      • Szaflarski W.
      • Januchowski R.
      • Zawierucha P.
      • Nowicki M.
      • Zabel M.
      Inhibitors of N-glycosylation as a potential tool for analysis of the mechanism of action and cellular localisation of glycoprotein P.
      Most studies have been carried out in yeast, in which pharmacological or genetic disruption of ER protein metabolism resulted in lipid accumulation.
      • Stordeur C.
      • Puth K.
      • Saenz J.P.
      • Ernst R.
      Crosstalk of lipid and protein homeostasis to maintain membrane function.
      • Fei W.
      • Wang H.
      • Fu X.
      • Bielby C.
      • Yang H.
      Conditions of endoplasmic reticulum stress stimulate lipid droplet formation in Saccharomyces cerevisiae.
      Subsequent work demonstrated that this relationship is likely conserved from yeast to higher eukaryotes.
      • Yamamoto K.
      • Takahara K.
      • Oyadomari S.
      • Okada T.
      • Sato T.
      • Harada A.
      • et al.
      Induction of liver steatosis and lipid droplet formation in ATF6alpha-knockout mice burdened with pharmacological endoplasmic reticulum stress.
      • Zhang K.
      • Wang S.
      • Malhotra J.
      • Hassler J.R.
      • Back S.H.
      • Wang G.
      • et al.
      The unfolded protein response transducer IRE1alpha prevents ER stress-induced hepatic steatosis.
      For example, the treatment of mice with the aforementioned drugs induced hepatic steatosis.
      • Yamamoto K.
      • Takahara K.
      • Oyadomari S.
      • Okada T.
      • Sato T.
      • Harada A.
      • et al.
      Induction of liver steatosis and lipid droplet formation in ATF6alpha-knockout mice burdened with pharmacological endoplasmic reticulum stress.
      • Zhang K.
      • Wang S.
      • Malhotra J.
      • Hassler J.R.
      • Back S.H.
      • Wang G.
      • et al.
      The unfolded protein response transducer IRE1alpha prevents ER stress-induced hepatic steatosis.
      Similar findings were obtained for patients with hyperhomocysteinemia.
      • Gaull G.
      • Sturman J.A.
      • Schaffner F.
      Homocystinuria due to cystathionine synthase deficiency: enzymatic and ultrastructural studies.
      Although the exact molecular mechanism remains unknown, it has been suggested that excess homocysteine leads to protein modification and misfolding
      • Outinen P.A.
      • Sood S.K.
      • Pfeifer S.I.
      • Pamidi S.
      • Podor T.J.
      • Li J.
      • et al.
      Homocysteine-induced endoplasmic reticulum stress and growth arrest leads to specific changes in gene expression in human vascular endothelial cells.
      • Werstuck G.H.
      • Lentz S.R.
      • Dayal S.
      • Hossain G.S.
      • Sood S.K.
      • Shi Y.Y.
      • et al.
      Homocysteine-induced endoplasmic reticulum stress causes dysregulation of the cholesterol and triglyceride biosynthetic pathways.
      thereby inducing ER stress.
      • Werstuck G.H.
      • Lentz S.R.
      • Dayal S.
      • Hossain G.S.
      • Sood S.K.
      • Shi Y.Y.
      • et al.
      Homocysteine-induced endoplasmic reticulum stress causes dysregulation of the cholesterol and triglyceride biosynthetic pathways.
      The stress-activated genes stimulate lipogenesis that results in increased hepatic levels of cholesterol and triglycerides.
      • Werstuck G.H.
      • Lentz S.R.
      • Dayal S.
      • Hossain G.S.
      • Sood S.K.
      • Shi Y.Y.
      • et al.
      Homocysteine-induced endoplasmic reticulum stress causes dysregulation of the cholesterol and triglyceride biosynthetic pathways.
      In addition to proteotoxic stress, other forms of ER stress including lipotoxic stress, are an integral component of multiple liver disorders. Emerging evidences suggest that a simultaneous presence of different stress forms likely accelerates the development of the associated disorders.
      The interaction between protein and lipid metabolism seems to be reciprocal, in that multiple reports revealed the adverse impact of metabolic stresses on proteotoxic disorders.
      • Bowlus C.L.
      • Willner I.
      • Zern M.A.
      • Reuben A.
      • Chen P.
      • Holladay B.
      • et al.
      Factors associated with advanced liver disease in adults with alpha1-antitrypsin deficiency.
      • Eigenbrodt M.L.
      • McCashland T.M.
      • Dy R.M.
      • Clark J.
      • Galati J.
      Heterozygous alpha 1-antitrypsin phenotypes in patients with end stage liver disease.
      • Regev A.
      • Guaqueta C.
      • Molina E.G.
      • Conrad A.
      • Mishra V.
      • Brantly M.L.
      • et al.
      Does the heterozygous state of alpha-1 antitrypsin deficiency have a role in chronic liver diseases? Interim results of a large case-control study.
      For example, obesity promoted the development of a liver phenotype in patients with severe AATD (homozygous PiZZ genotype).
      • Bowlus C.L.
      • Willner I.
      • Zern M.A.
      • Reuben A.
      • Chen P.
      • Holladay B.
      • et al.
      Factors associated with advanced liver disease in adults with alpha1-antitrypsin deficiency.
      Similarly, in patients with non-alcoholic fatty liver disease, the simultaneous presence of a heterozygous AAT mutation (PiMZ genotype) is associated with liver cirrhosis.
      • Eigenbrodt M.L.
      • McCashland T.M.
      • Dy R.M.
      • Clark J.
      • Galati J.
      Heterozygous alpha 1-antitrypsin phenotypes in patients with end stage liver disease.
      • Regev A.
      • Guaqueta C.
      • Molina E.G.
      • Conrad A.
      • Mishra V.
      • Brantly M.L.
      • et al.
      Does the heterozygous state of alpha-1 antitrypsin deficiency have a role in chronic liver diseases? Interim results of a large case-control study.
      Moreover, feeding of the Bri 44 mice with methionine-choline-deficient diet caused more pronounced liver steatosis.
      • Fu M.M.
      • Sun R.
      • Tian Z.G.
      • Wei H.M.
      Increased susceptibility to experimental steatohepatitis induced by methionine-choline deficiency in HBs-Tg mice.
      As a potential underlying mechanism, a recent study illustrated that IRE1 can integrate the sensing of both protein folding stress and lipid bilayer stress.
      • Halbleib K.
      • Pesek K.
      • Covino R.
      • Hofbauer H.F.
      • Wunnicke D.
      • Hanelt I.
      • et al.
      Activation of the unfolded protein response by lipid bilayer stress.
      Given these promising data, further studies should analyse the exact effect of specific proteotoxic disorders on the handling of lipids by hepatocytes.
      Cholestatic liver disease and haemochromatosis also induce ER stress and thereby may aggravate proteotoxic disorders.
      • Lawless M.W.
      • Mankan A.K.
      • White M.
      • O'Dwyer M.J.
      • Norris S.
      Expression of hereditary hemochromatosis C282Y HFE protein in HEK293 cells activates specific endoplasmic reticulum stress responses.
      • Liu Y.
      • Lee S.Y.
      • Neely E.
      • Nandar W.
      • Moyo M.
      • Simmons Z.
      • et al.
      Mutant HFE H63D protein is associated with prolonged endoplasmic reticulum stress and increased neuronal vulnerability.
      • Mencin A.
      • Seki E.
      • Osawa Y.
      • Kodama Y.
      • De Minicis S.
      • Knowles M.
      • et al.
      Alpha-1 antitrypsin Z protein (PiZ) increases hepatic fibrosis in a murine model of cholestasis.
      For instance, bile duct ligation, a commonly used model of obstructive cholestasis, led to a stronger liver injury in mice overexpressing mutant AAT, likely because of the increased levels of apoptosis.
      • Mencin A.
      • Seki E.
      • Osawa Y.
      • Kodama Y.
      • De Minicis S.
      • Knowles M.
      • et al.
      Alpha-1 antitrypsin Z protein (PiZ) increases hepatic fibrosis in a murine model of cholestasis.
      Additionally, liver explant series and case reports showed an accelerated disease course in individuals with AATD who simultaneously harboured mutations in the hemochromatosis (HFE) gene.
      • Anand S.
      • Schade R.R.
      • Bendetti C.
      • Kelly R.
      • Rabin B.S.
      • Krause J.
      • et al.
      Idiopathic hemochromotosis and alpha-1-antitrypsin deficiency: coexistence in a family with progressive liver disease in the proband.
      • Elzouki A.N.
      • Hultcrantz R.
      • Stal P.
      • Befrits R.
      • Eriksson S.
      Increased PiZ gene frequency for alpha 1 antitrypsin in patients with genetic haemochromatosis.
      Of note, overexpression of mutant HFE protein in cell lines resulted in chronic ER stress.
      • Lawless M.W.
      • Mankan A.K.
      • White M.
      • O'Dwyer M.J.
      • Norris S.
      Expression of hereditary hemochromatosis C282Y HFE protein in HEK293 cells activates specific endoplasmic reticulum stress responses.
      • Liu Y.
      • Lee S.Y.
      • Neely E.
      • Nandar W.
      • Moyo M.
      • Simmons Z.
      • et al.
      Mutant HFE H63D protein is associated with prolonged endoplasmic reticulum stress and increased neuronal vulnerability.

      Outlook

      As highlighted in this review, liver hepatocytes constitute an important hub of protein metabolism and a source of various clinical biomarkers. However, this continuous challenge makes hepatocytes susceptible to proteotoxic stress, which plays an important part in the development of multiple liver disorders. Further research is needed to delineate the consequences of proteotoxic ER stress in the liver, as well as the exact interplay between different ER challenges. In particular, the impact of proteotoxic stress on hepatic lipid metabolism needs to be further addressed.

      Financial support

      This work was supported by the German Research Foundation grant STR 1095/4–1 , IZKF research group funding, Else Kröner Exzellenzstipendium (to P.S.) and SFB/TRR57 (to P.S. and C.T.)

      Conflict of interest

      The authors declare no conflicts of interest that pertain to this work.

      Authors contributions

      Drafting of the manuscript: PS, DK. Critical revision of the manuscript for important intellectual content: all authors. Obtained funding and study supervision: PS. Technical or material support: all authors.

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