Mysteries of α1-antitrypsin deficiency: emerging therapeutic strategies for a challenging disease

The classical form of α1-antitrypsin deficiency (ATD) is an autosomal co-dominant disorder that affects ~1 in 3000 live births and is an important genetic cause of lung and liver disease. The protein affected, α1-antitrypsin (AT), is predominantly derived from the liver and has the function of inhibiting neutrophil elastase and several other destructive neutrophil proteinases. The genetic defect is a point mutation that leads to misfolding of the mutant protein, which is referred to as α1-antitrypsin Z (ATZ). Because of its misfolding, ATZ is unable to efficiently traverse the secretory pathway. Accumulation of ATZ in the endoplasmic reticulum of liver cells has a gain-of-function proteotoxic effect on the liver, resulting in fibrosis, cirrhosis and/or hepatocellular carcinoma in some individuals. Moreover, because of reduced secretion, there is a lack of anti-proteinase activity in the lung, which allows neutrophil proteases to destroy the connective tissue matrix and cause chronic obstructive pulmonary disease (COPD) by loss of function. Wide variation in the incidence and severity of liver and lung disease among individuals with ATD has made this disease one of the most challenging of the rare genetic disorders to diagnose and treat. Other than cigarette smoking, which worsens COPD in ATD, genetic and environmental modifiers that determine this phenotypic variability are unknown. A limited number of therapeutic strategies are currently available, and liver transplantation is the only treatment for severe liver disease. Although replacement therapy with purified AT corrects the loss of anti-proteinase function, COPD progresses in a substantial number of individuals with ATD and some undergo lung transplantation. Nevertheless, advances in understanding the variability in clinical phenotype and in developing novel therapeutic concepts is beginning to address the major clinical challenges of this mysterious disorder.KEY WORDS: α1-antitrypsin deficiency, Autophagy, Liver disease     

Deficient and Null Variants of SERPINA1 Are Proteotoxic in a Caenorhabditis elegans Model of α1-Antitrypsin Deficiency

α1-antitrypsin deficiency (ATD) predisposes patients to both loss-of-function (emphysema) and gain-of-function (liver cirrhosis) phenotypes depending on the type of mutation. Although the Z mutation (ATZ) is the most prevalent cause of ATD, >120 mutant alleles have been identified. In general, these mutations are classified as deficient (<20% normal plasma levels) or null (<1% normal levels) alleles. The deficient alleles, like ATZ, misfold in the ER where they accumulate as toxic monomers, oligomers and aggregates. Thus, deficient alleles may predispose to both gain- and loss-of-function phenotypes. Null variants, if translated, typically yield truncated proteins that are efficiently degraded after being transiently retained in the ER. Clinically, null alleles are only associated with the loss-of-function phenotype. We recently developed a C. elegans model of ATD in order to further elucidate the mechanisms of proteotoxicity (gain-of-function phenotype) induced by the aggregation-prone deficient allele, ATZ. The goal of this study was to use this C. elegans model to determine whether different types of deficient and null alleles, which differentially affect polymerization and secretion rates, correlated to any extent with proteotoxicity. Animals expressing the deficient alleles, Mmalton, Siiyama and S (ATS), showed overall toxicity comparable to that observed in patients. Interestingly, Siiyama expressing animals had smaller intracellular inclusions than ATZ yet appeared to have a greater negative effect on animal fitness. Surprisingly, the null mutants, although efficiently degraded, showed a relatively mild gain-of-function proteotoxic phenotype. However, since null variant proteins are degraded differently and do not appear to accumulate, their mechanism of proteotoxicity is likely to be different to that of polymerizing, deficient mutants. Taken together, these studies showed that C. elegans is an inexpensive tool to assess the proteotoxicity of different AT variants using a transgenic approach.     

Automated high-content live animal drug screening using C. elegans expressing the aggregation prone serpin α1-antitrypsin Z

The development of preclinical models amenable to live animal bioactive compound screening is an attractive approach to discovering effective pharmacological therapies for disorders caused by misfolded and aggregation-prone proteins. In general, however, live animal drug screening is labor and resource intensive, and has been hampered by the lack of robust assay designs and high throughput work-flows. Based on their small size, tissue transparency and ease of cultivation, the use of C. elegans should obviate many of the technical impediments associated with live animal drug screening. Moreover, their genetic tractability and accomplished record for providing insights into the molecular and cellular basis of human disease, should make C. elegans an ideal model system for in vivo drug discovery campaigns. The goal of this study was to determine whether C. elegans could be adapted to high-throughput and high-content drug screening strategies analogous to those developed for cell-based systems. Using transgenic animals expressing fluorescently-tagged proteins, we first developed a high-quality, high-throughput work-flow utilizing an automated fluorescence microscopy platform with integrated image acquisition and data analysis modules to qualitatively assess different biological processes including, growth, tissue development, cell viability and autophagy. We next adapted this technology to conduct a small molecule screen and identified compounds that altered the intracellular accumulation of the human aggregation prone mutant that causes liver disease in α1-antitrypsin deficiency. This study provides powerful validation for advancement in preclinical drug discovery campaigns by screening live C. elegans modeling α1-antitrypsin deficiency and other complex disease phenotypes on high-content imaging platforms.     

Calreticulin enhances the secretory trafficking of a misfolded α-1-antitrypsin

α1-antitrypsin (AAT) regulates the activity of multiple proteases in the lungs and liver. A mutant of AAT (E342K) called ATZ forms polymers that are present at only low levels in the serum and induce intracellular protein inclusions, causing lung emphysema and liver cirrhosis. An understanding of factors that can reduce the intracellular accumulation of ATZ is of great interest. We now show that calreticulin (CRT), an endoplasmic reticulum (ER) glycoprotein chaperone, promotes the secretory trafficking of ATZ, enhancing the media:cell ratio. This effect is more pronounced for ATZ than with AAT and is only partially dependent on the glycan-binding site of CRT, which is generally relevant to substrate recruitment and folding by CRT. The CRT-related chaperone calnexin does not enhance ATZ secretory trafficking, despite the higher cellular abundance of calnexin-ATZ complexes. CRT deficiency alters the distributions of ATZ-ER chaperone complexes, increasing ATZ-BiP binding and inclusion body formation and reducing ATZ interactions with components required for ER-Golgi trafficking, coincident with reduced levels of the protein transport protein Sec31A in CRT-deficient cells. These findings indicate a novel role for CRT in promoting the secretory trafficking of a protein that forms polymers and large intracellular inclusions. Inefficient secretory trafficking of ATZ in the absence of CRT is coincident with enhanced accumulation of ER-derived ATZ inclusion bodies. Further understanding of the factors that control the secretory trafficking of ATZ and their regulation by CRT could lead to new therapies for lung and liver diseases linked to AAT deficiency.     

A novel endoplasmic reticulum adaptation is critical for the long-lived Caenorhabditis elegans rpn-10 proteasomal mutant

The loss of proteostasis due to reduced efficiency of protein degradation pathways plays a key role in multiple age-related diseases and is a hallmark of the aging process. Paradoxically, we have previously reported that the Caenorhabditis elegans rpn-10(ok1865) mutant, which lacks the RPN-10/RPN10/PSMD4 subunit of the 19S regulatory particle of the 26S proteasome, exhibits enhanced cytosolic proteostasis, elevated stress resistance and extended lifespan, despite possessing reduced proteasome function. However, the response of this mutant against threats to endoplasmic reticulum (ER) homeostasis and proteostasis was unknown. Here, we find that the rpn-10 mutant is highly ER stress resistant compared to the wildtype. Under unstressed conditions, the ER unfolded protein response (UPR) is activated in the rpn-10 mutant as signified by increased xbp-1 splicing. This primed response appears to alter ER homeostasis through the upregulated expression of genes involved in ER protein quality control (ERQC), including those in the ER-associated protein degradation (ERAD) pathway. Pertinently, we find that ERQC is critical for the rpn-10 mutant longevity. These changes also alter ER proteostasis, as studied using the C. elegans alpha-1 antitrypsin (AAT) deficiency model, which comprises an intestinal ER-localised transgenic reporter of an aggregation-prone form of AAT called ATZ. The rpn-10 mutant shows a significant reduction in the accumulation of the ATZ reporter, thus indicating that its ER proteostasis is augmented. Via a genetic screen for suppressors of decreased ATZ aggregation in the rpn-10 mutant, we then identified ecps-2/H04D03.3, a novel ortholog of the proteasome-associated adaptor and scaffold protein ECM29/ECPAS. We further show that ecps-2 is required for improved ER proteostasis as well as lifespan extension of the rpn-10 mutant. Thus, we propose that ECPS-2-proteasome functional interactions, alongside additional putative molecular processes, contribute to a novel ERQC adaptation which underlies the superior proteostasis and longevity of the rpn-10 mutant.     

α1-Antitrypsin deficiency and the hepatocytes – An elegans solution to drug discovery

Hepatocytes are metabolically active cells of the liver that play an important role in the biosynthesis of proteins including α1-antitrypsin. Mutations in the α1-antitrypsin gene can lead to protein misfolding, polymerization/aggregation and retention of protein within the endoplasmic reticulum of hepatocytes. The intracellular accumulation of α1-antitrypsin aggregates can lead to liver disease and increased likelihood of developing hepatocellular carcinomas. Of note, only ∼10% of individuals with α1-antitrypsin-deficiency develop severe liver disease suggesting that there are other genetic and/or environmental factors that determine disease outcome. The nematode, Caenorhabditis elegans, is a powerful genetic model organism to study molecular aspects of human disease. In this review, we discuss the functional similarities between the intestinal cells of C. elegans and human hepatocytes and how a C. elegans model of α1-antitrypsin-deficiency can be used as a tool for identifying genetic modifiers and small molecule drugs.     

Brivanib Attenuates Hepatic Fibrosis In Vivo and Stellate Cell Activation In Vitro by Inhibition of FGF,VEGF and PDGF Signaling

MethodsIn vivo, we induced liver fibrosis by bile duct ligation (BDL), chronic carbon tetrachloride (CCl₄), and chronic thioacetamide (TAA) administration. Liver fibrosis was examined by immunohistochemistry and Western immunoblotting. In vitro, we used LX-2 human hepatic stellate cells (HSCs) to assess the effect of brivanib on stellate cell proliferation and activation.ResultsAfter in vivo induction with BDL, CCl₄, and TAA, mice treated with brivanib showed reduced liver fibrosis and decreased expression of collagen Iα1 and α-smooth muscle actin in the liver. In vitro, brivanib decreased proliferation of HSCs induced by platelet-derived growth factor (PDGF), VEGF, and FGF. Brivanib also decreased stellate cell viability and inhibited PDGFBB-induced phosphorylation of its cognate receptor.ConclusionBrivanib reduces liver fibrosis in three different animal models and decreases human hepatic stellate cell activation. Brivanib may represent a novel therapeutic approach to treatment of liver fibrosis and prevention of liver cancer.     

The Role of Autophagy in Alpha-1-Antitrypsin Deficiency: A Specific Cellular Response in Genetic Diseases Associated with Aggregation-Prone Proteins

In the classical form of alpha-1-antitrypsin (AT) deficiency a point mutation renders aggregation-prone properties on a hepatic secretory protein. The mutant ATZ protein in retained in the endoplasmic reticulum (ER) of liver cells rather than secreted into the blood and body fluids where it ordinarily functions as an inhibitor of neutrophil proteases. A loss-of-function mechanism allows the neutrophil proteases to slowly destroy the connective tissue matrix of the lung, resulting in premature development of pulmonary emphysema as early as the third decade of life. A gain-of-toxic function mechanism is responsible for liver inflammation and carcinogenesis. Indeed this deficiency is the most common genetic cause of liver disease in children in the US. It also causes chronic liver inflammation and carcinoma that manifests itself later in life. However, the majority of affected homozygotes apparently escape liver disease. This last observation has led to the concept that genetic and/or environmental modifiers affect the disposal of mutant ATZ within the ER or affect the protective cellular responses activated by accumulation of ATZ in the ER and, in turn, these modifiers determine which homozygotes develop liver inflammation and carcinoma. In this article I review a series of studies published over the last 6 years showing that autophagy is specifically activated by ER accumulation of ATZ and that autophagy plays a critical role in the disposal of this mutant protein. Further, I review data suggesting that the autophagy is specifically designed for the cellular response to aggregated ATZ and aggregated proteins in general.     

The Endosomal Protein-Sorting Receptor Sortilin Has a Role in Trafficking α-1 Antitrypsin

Up to 1 in 3000 individuals in the United States have α-1 antitrypsin deficiency, and the most common cause of this disease is homozygosity for the antitrypsin-Z variant (ATZ). ATZ is inefficiently secreted, resulting in protein deficiency in the lungs and toxic polymer accumulation in the liver. However, only a subset of patients suffer from liver disease, suggesting that genetic factors predispose individuals to liver disease. To identify candidate factors, we developed a yeast ATZ expression system that recapitulates key features of the disease-causing protein. We then adapted this system to screen the yeast deletion mutant collection to identify conserved genes that affect ATZ secretion and thus may modify the risk for developing liver disease. The results of the screen and associated assays indicate that ATZ is degraded in the vacuole after being routed from the Golgi. In fact, one of the strongest hits from our screen was Vps10, which can serve as a receptor for the delivery of aberrant proteins to the vacuole. Because genome-wide association studies implicate the human Vps10 homolog, sortilin, in cardiovascular disease, and because hepatic cell lines that stably express wild-type or mutant sortilin were recently established, we examined whether ATZ levels and secretion are affected by sortilin. As hypothesized, sortilin function impacts the levels of secreted ATZ in mammalian cells. This study represents the first genome-wide screen for factors that modulate ATZ secretion and has led to the identification of a gene that may modify disease severity or presentation in individuals with ATZ-associated liver disease.     

Retention of mutant alpha(1)-antitrypsin Z in endoplasmic reticulum is associated with an autophagic response

Although there is evidence for specific subcellular morphological alterations in response to accumulation of misfolded proteins in the endoplasmic reticulum (ER), it is not clear whether these morphological changes are stereotypical or if they depend on the specific misfolded protein retained. This issue may be particularly important for mutant secretory protein alpha(1)-antitrypsin (alpha(1)AT) Z because retention of this mutant protein in the ER can cause severe target organ injury, the chronic hepatitis/hepatocellular carcinoma associated with alpha(1)AT deficiency. Here we examined the morphological changes that occur in human fibroblasts engineered for expression and ER retention of mutant alpha(1)ATZ and in human liver from three alpha(1)AT-deficient patients. In addition to marked expansion and dilatation of ER, there was an intense autophagic response. Mutant alpha(1)ATZ molecules were detected in autophagosomes by immune electron microscopy, and intracellular degradation of alpha(1)ATZ was partially reduced by chemical inhibitors of autophagy. In contrast to mutant CFTRDeltaF508, expression of mutant alpha(1)ATZ in heterologous cells did not result in the formation of aggresomes. These results show that ER retention of mutant alpha(1)ATZ is associated with a marked autophagic response and raise the possibility that autophagy represents a mechanism by which liver of alpha(1)AT-deficient patients attempts to protect itself from injury and carcinogenesis.     

The role of autophagy in alpha-1-antitrypsin deficiency: a specific cellular response in genetic diseases associated with aggregation-prone proteins

In the classical form of alpha-1-antitrypsin (AT) deficiency a point mutation renders aggregation-prone properties on a hepatic secretory protein. The mutant ATZ protein in retained in the endoplasmic reticulum (ER) of liver cells rather than secreted into the blood and body fluids where it ordinarily functions as an inhibitor of neutrophil proteases. A loss-of-function mechanism allows the neutrophil proteases to slowly destroy the connective tissue matrix of the lung, resulting in premature development of pulmonary emphysema as early as the third decade of life. A gain-of-toxic function mechanism is responsible for liver inflammation and carcinogenesis. Indeed this deficiency is the most common genetic cause of liver disease in children in the US. It also causes chronic liver inflammation and carcinoma that manifests itself later in life. However, the majority of affected homozygotes apparently escape liver disease. This last observation has led to the concept that genetic and/or environmental modifiers affect the disposal of mutant ATZ within the ER or affect the protective cellular responses activated by accumulation of ATZ in the ER and, in turn, these modifiers determine which homozygotes develop liver inflammation and carcinoma. In this article I review a series of studies published over the last six years showing that autophagy is specifically activated by ER accumulation of ATZ and that it plays a critical role in the disposal of this mutant protein. Indeed, the most recent studies suggest that there is specialization of the autophagic pathway in that it is specifically activated by, and designed for disposal of, the aggregated forms of ATZ while the proteasome is specialized for disposal of soluble forms of ATZ. Together, these studies provide further evidence for the importance of autophagy in the cellular adaptive response to aggregated proteins in general.     

Caenorhabditis elegans Is a Model Host for Listeria monocytogenes

Here we report that Caenorhabditis elegans nematodes fed Listeria monocytogenes die over the course of several days, as a consequence of an accumulation of bacteria in the worm intestine. Mutant strains previously shown to be important for virulence in mammalian models were also found to be attenuated in their virulence in C. elegans. However, ActA, which is required for actin-based intracellular motility, appears to be dispensable during infection of C. elegans, indicating that L. monocytogenes remains extracellular in C. elegans.     

Antifungal Efficacy during Candida krusei Infection in Non-Conventional Models Correlates with the Yeast In Vitro Susceptibility Profile

The incidence of opportunistic fungal infections has increased in recent decades due to the growing proportion of immunocompromised patients in our society. Candida krusei has been described as a causative agent of disseminated fungal infections in susceptible patients. Although its prevalence remains low among yeast infections (2–5%), its intrinsic resistance to fluconazole makes this yeast important from epidemiologic aspects. Non mammalian organisms are feasible models to study fungal virulence and drug efficacy. In this work we have used the lepidopteran Galleria mellonella and the nematode Caenorhabditis elegans as models to assess antifungal efficacy during infection by C. krusei. This yeast killed G. mellonella at 25, 30 and 37°C and reduced haemocytic density. Infected larvae melanized in a dose-dependent manner. Fluconazole did not protect against C. krusei infection, in contrast to amphotericin B, voriconazole or caspofungin. However, the doses of these antifungals required to obtain larvae protection were always higher during C. krusei infection than during C. albicans infection. Similar results were found in the model host C. elegans. Our work demonstrates that non mammalian models are useful tools to investigate in vivo antifungal efficacy and virulence of C. krusei.     

A Liposomal Drug Platform Overrides Peptide Ligand Targeting to a Cancer Biomarker,Irrespective of Ligand Affinity or Density

One method for improving cancer treatment is the use of nanoparticle drugs functionalized with targeting ligands that recognize receptors expressed selectively by tumor cells. In theory such targeting ligands should specifically deliver the nanoparticle drug to the tumor, increasing drug concentration in the tumor and delivering the drug to its site of action within the tumor tissue. However, the leaky vasculature of tumors combined with a poor lymphatic system allows the passive accumulation, and subsequent retention, of nanosized materials in tumors. Furthermore, a large nanoparticle size may impede tumor penetration. As such, the role of active targeting in nanoparticle delivery is controversial, and it is difficult to predict how a targeted nanoparticle drug will behave in vivo. Here we report in vivo studies for α_vβ₆-specific H2009.1 peptide targeted liposomal doxorubicin, which increased liposomal delivery and toxicity to lung cancer cells in vitro. We systematically varied ligand affinity, ligand density, ligand stability, liposome dosage, and tumor models to assess the role of active targeting of liposomes to α_vβ₆. In direct contrast to the in vitro results, we demonstrate no difference in in vivo targeting or efficacy for H2009.1 tetrameric peptide liposomal doxorubicin, compared to control peptide and no peptide liposomes. Examining liposome accumulation and distribution within the tumor demonstrates that the liposome, and not the H2009.1 peptide, drives tumor accumulation, and that both targeted H2009.1 and untargeted liposomes remain in perivascular regions, with little tumor penetration. Thus H2009.1 targeted liposomes fail to improve drug efficacy because the liposome drug platform prevents the H2009.1 peptide from both actively targeting the tumor and binding to tumor cells throughout the tumor tissue. Therefore, using a high affinity and high specificity ligand targeting an over-expressed tumor biomarker does not guarantee enhanced efficacy of a liposomal drug. These results highlight the complexity of in vivo targeting.     

Liver-Specific Expressions of HBx and src in the p53 Mutant Trigger Hepatocarcinogenesis in Zebrafish

Hepatocarcinogenesis is a multistep process that starts from fatty liver and transitions to fibrosis and, finally, into cancer. Many etiological factors, including hepatitis B virus X antigen (HBx) and p53 mutations, have been implicated in hepatocarcinogenesis. However, potential synergistic effects between these two factors and the underlying mechanisms by which they promote hepatocarcinogenesis are still unclear. In this report, we show that the synergistic action of HBx and p53 mutation triggers progressive hepatocellular carcinoma (HCC) formation via src activation in zebrafish. Liver-specific expression of HBx in wild-type zebrafish caused steatosis, fibrosis and glycogen accumulation. However, the induction of tumorigenesis by HBx was only observed in p53 mutant fish and occurred in association with the up-regulation and activation of the src tyrosine kinase pathway. Furthermore, the overexpression of src in p53 mutant zebrafish also caused hyperplasia, HCC, and sarcomatoid HCC, which were accompanied by increased levels of the signaling proteins p-erk, p-akt, myc, jnk1 and vegf. Increased expression levels of lipogenic factors and the genes involved in lipid metabolism and glycogen storage were detected during the early stages of hepatocarcinogenesis in the HBx and src transgenic zebrafish. The up-regulation of genes involved in cell cycle regulation, tumor progression and other molecular hallmarks of human liver cancer were found at later stages in both HBx and src transgenic, p53 mutant zebrafish. Together, our study demonstrates that HBx and src overexpression induced hepatocarcinogenesis in p53 mutant zebrafish. This phenomenon mimics human HCC formation and provides potential in vivo platforms for drug screening for therapies for human liver cancer.     

Ubiquitin ligase gp78 increases solubility and facilitates degradation of the Z variant of alpha-1-antitrypsin

Deficiency of circulating alpha-1-antitrypsin (AAT) is the most widely recognized abnormality of a proteinase inhibitor that causes lung disease. AAT-deficiency is caused by mutations of the AAT gene that lead to AAT protein retention in the endoplasmic reticulum (ER). Moreover, the mutant AAT accumulated in the ER predisposes the homozygote to severe liver injuries, such as neonatal hepatitis, juvenile cirrhosis, and hepatocellular carcinoma. Despite the fact that mutant AAT protein is subject to ER-associated degradation (ERAD), yeast genetic studies have determined that the ubiquitination machinery, Hrd1/Der3p-cue1p-Ubc7/6p, which plays a prominent role in ERAD, is not involved in degradation of mutant AAT. Here we report that gp78, a ubiquitin ligase (E3) pairing with mammalian Ubc7 for ERAD, ubiquitinates and facilitates degradation of ATZ, the classic deficiency variant of AAT having a Z mutation (Glu 342 Lys). Unexpectedly, gp78 over-expression also significantly increases ATZ solubility. p97/VCP, an AAA ATPase essential for retrotranslocation of misfolded proteins from the ER during ERAD, is involved in gp78-mediated degradation of ATZ. Surprisingly, unlike other ERAD substrates that cause ER stress leading to apoptosis when accumulated in the ER, ATZ, in fact, increases cell proliferation when over-expressed in cells. This effect can be partially inhibited by gp78 over-expression. These data indicate that gp78 assumes multiple unique quality control roles over ATZ, including the facilitation of degradation and inhibition of aggregation of ATZ.