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 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.     

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     

Activation of endoplasmic reticulum-specific stress responses associated with the conformational disease Z alpha 1-antitrypsin deficiency

Conformational diseases are a class of disorders associated with aberrant protein accumulation in tissues and cellular compartments. Z alpha1-antitrypsin (A1AT) deficiency is a genetic disease associated with accumulation of misfolded A1AT in the endoplasmic reticulum (ER) of hepatocytes. We sought to identify intracellular events involved in the molecular pathogenesis of Z A1AT-induced liver disease using an in vitro model system of Z A1AT ER accumulation. We investigated ER stress signals induced by Z A1AT and demonstrated that both the ER overload response and the unfolded protein response were activated by mutant Z A1AT, but not wild-type M A1AT. Interestingly, activation of the unfolded protein response pathway required an additional insult, whereas NF-kappa B activation, a hallmark of the ER overload response, was constitutive. These findings have important implications for the design of future therapeutics for Z A1AT liver disease and may also impact on drug design for other conformational diseases.     

Mitochondrial autophagy and injury in the liver in alpha 1-antitrypsin deficiency

Homozygous, PIZZ alpha(1)-antitrypsin (alpha(1)-AT) deficiency is associated with chronic liver disease and hepatocellular carcinoma resulting from the toxic effects of mutant alpha(1)-anti-trypsin Z (alpha(1)-ATZ) protein retained in the endoplasmic reticulum (ER) of hepatocytes. However, the exact mechanism(s) by which retention of this aggregated mutant protein leads to cellular injury are still unknown. Previous studies have shown that retention of mutant alpha(1)-ATZ in the ER induces an intense autophagic response in hepatocytes. In this study, we present evidence that the autophagic response induced by ER retention of alpha(1)-ATZ also involves the mitochondria, with specific patterns of both mitochondrial autophagy and mitochondrial injury seen in cell culture models of alpha(1)-AT deficiency, in PiZ transgenic mouse liver, and in liver from alpha(1)-AT-deficient patients. Evidence for a unique pattern of caspase activation was also detected. Administration of cyclosporin A, an inhibitor of mitochondrial permeability transition, to PiZ mice was associated with a reduction in mitochondrial autophagy and injury and reduced mortality during experimental stress. These results provide evidence for the novel concept that mitochondrial damage and caspase activation play a role in the mechanism of liver cell injury in alpha(1)-AT deficiency and suggest the possibility of mechanism-based therapeutic interventions.     

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.     

Molecular basis of α1-antitrypsin deficiency revealed by the structure of a domain-swapped trimer

α(1)-Antitrypsin (α1AT) deficiency is a disease with multiple manifestations, including cirrhosis and emphysema, caused by the accumulation of stable polymers of mutant protein in the endoplasmic reticulum of hepatocytes. However, the molecular basis of misfolding and polymerization remain unknown. We produced and crystallized a trimeric form of α1AT that is recognized by an antibody specific for the pathological polymer. Unexpectedly, this structure reveals a polymeric linkage mediated by domain swapping the carboxy-terminal 34 residues. Disulphide-trapping and antibody-binding studies further demonstrate that runaway C-terminal domain swapping, rather than the s4A/s5A domain swap previously proposed, underlies polymerization of the common Z-mutant of α1AT in vivo.     

Transgenic expression of entire hepatitis B virus in mice induces hepatocarcinogenesis independent of chronic liver injury

Hepatocellular carcinoma (HCC), the third leading cause of cancer deaths worldwide, is most commonly caused by chronic hepatitis B virus (HBV) infection. However, whether HBV plays any direct role in carcinogenesis, other than indirectly causing chronic liver injury by inciting the host immune response, remains unclear. We have established two independent transgenic mouse lines expressing the complete genome of a mutant HBV ("preS2 mutant") that is found at much higher frequencies in people with HCC than those without. The transgenic mice show evidence of stress in the endoplasmic reticulum (ER) and overexpression of cyclin D1 in hepatocytes. These mice do not show any evidence of chronic liver injury, but by 2 years of age a majority of the male mice develop hepatocellular neoplasms, including HCC. Unexpectedly, we also found a significant increase in hepatocarcinogenesis independent of necroinflammation in a transgenic line expressing the entire wildtype HBV. As in the mutant HBV mice, HCC was found only in aged--2-year-old--mice of the wildtype HBV line. The karyotype in all the three transgenic lines appears normal and none of the integration sites of the HBV transgene in the mice is near an oncogene or tumor suppressor gene. The significant increase of HCC incidence in all the three transgenic lines--expressing either mutant or wildtype HBV--therefore argues strongly that in absence of chronic necroinflammation, HBV can contribute directly to the development of HCC.     

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.     

Histone Deacetylase Inhibitor (HDACi) Suberoylanilide Hydroxamic Acid (SAHA)-mediated Correction of α1-Antitrypsin Deficiency

α1-Antitrypsin (α1AT) deficiency (α1ATD) is a consequence of defective folding, trafficking, and secretion of α1AT in response to a defect in its interaction with the endoplasmic reticulum proteostasis machineries. The most common and severe form of α1ATD is caused by the Z-variant and is characterized by the accumulation of α1AT polymers in the endoplasmic reticulum of the liver leading to a severe reduction (>85%) of α1AT in the serum and its anti-protease activity in the lung. In this organ α1AT is critical for ensuring tissue integrity by inhibiting neutrophil elastase, a protease that degrades elastin. Given the limited therapeutic options in α1ATD, a more detailed understanding of the folding and trafficking biology governing α1AT biogenesis and its response to small molecule regulators is required. Herein we report the correction of Z-α1AT secretion in response to treatment with the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA), acting in part through HDAC7 silencing and involving a calnexin-sensitive mechanism. SAHA-mediated correction restores Z-α1AT secretion and serpin activity to a level 50% that observed for wild-type α1AT. These data suggest that HDAC activity can influence Z-α1AT protein traffic and that SAHA may represent a potential therapeutic approach for α1ATD and other protein misfolding diseases.     

Biosynthesis, processing, and secretion of M and Z variant human alpha 1-antitrypsin

The Z genetic variant of human alpha 1-antitrypsin (alpha 1AT) is associated with decreased serum alpha 1AT levels, hepatic inclusion bodies, and an increased risk of lung and liver disease. We studied the biosynthesis, processing, and secretion of normal and Z variant alpha 1AT in cell-free translation systems, reconstituted in vitro processing systems, and in the Xenopus oocyte secretory system. Human liver mRNA was prepared from normal subjects (PiMM) and from individuals homozygous for alpha 1AT deficiency (PiZZ). Cell-free translation resulted in the synthesis of 49,000-Da preproteins with a 23-amino acid signal sequence. The genetic variants were synthesized at comparable levels and could be distinguished on the basis of charge. The majority of the amino acids in the ZZ signal peptide were identified and found to be the same as those comprising the MM signal sequence. These proteins were co-translationally processed with similar efficiency by dog pancreas microsomes, producing 52,000-Da glycoproteins which were completely translocated across the endoplasmic reticulum membrane. When the human liver RNA preparations were injected into Xenopus oocytes, both of the alpha 1AT variants were synthesized intracellularly and alpha 1AT was detected in the medium of all oocytes injected with MM RNA. However, the Z variant accumulated within the microsomal vesicles of the cell and was undetectable or present at decreased levels in the medium. We conclude that the single amino acid substitution in the Z variant of alpha 1AT does not affect its synthesis or co-translational processing but that it strongly affects its transport from the rough endoplasmic reticulum through the secretory pathway.     

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.     

Salubrinal对铜负荷诱导的PERK/eIF2α内质网应激信号通路介导肝细胞凋亡的干预作用

Wilson病是一种以肝损害为常见临床表现的常染色体隐性遗传铜代谢障碍性疾病,但铜蓄积导致的肝细胞损害的具体分子机制尚不明确。本研究拟采用硫酸铜模拟肝细胞铜负荷进行体外实验,选用Western印迹法测试铜负荷肝细胞内蛋白激酶R样内质网激酶(protein kinase R-like endoplasmic reticulum kinase, PERK)及p-eIF2α蛋白质的表达;并探究特异性eIF2α磷酸化抑制剂Salubrinal对铜负荷诱导的肝细胞凋亡、胱天蛋白酶-3活性、C/EBP同源蛋白（C/EBP homologous protein, CHOP）mRNA转录的影响。选用流式细胞仪测试肝细胞凋亡率;比色法测试肝细胞内的胱天蛋白酶-3活性;Real-time PCR法检测肝细胞内CHOP mRNA转录水平。本研究结果显示: (1) 铜负荷显示出时间依赖性地增加肝细胞内PERK及p-eIF2α蛋白质表达(P<0.05, P<0.01)。(2)相比较对照组,铜负荷培养肝细胞24 h明显增加了肝细胞的凋亡率(P<0.01),增强了肝细胞内的胱天蛋白酶-3活性,促进肝细胞内CHOP mRNA的转录,加入特异性eIF2α磷酸化抑制剂Salubrinal后可抑制上述过程。(3)相比较对照组,铜负荷促进肝细胞PERK及p-eIF2α蛋白质表达,加入特异性eIF2α磷酸化抑制剂Salubrinal后,对铜负荷诱导的肝细胞PERK蛋白质表达无影响(P>0.05),但可显著抑制铜负荷诱导的肝细胞p-eIF2α蛋白质表达(P<0.01)。本研究结果提示,铜负荷可以诱发肝细胞内质网应激,铜负荷引起的肝细胞凋亡机制与激活内质网应激PERK/eIF2α信号通路密切相关, Salubrinal具有干预该信号通路作用,能够抑制铜负荷后肝细胞的凋亡。     

Mutant Frizzled 4 associated with vitreoretinopathy traps wild-type Frizzled in the endoplasmic reticulum by oligomerization

nt signalling pathways regulate cell proliferation, cell fate and morphogenetic movements. Here, we demonstrate that the Frizzled (Fz) family of Wnt receptors, similarly to G-protein-coupled receptors (GPCRs), form specific homo- and hetero-oligomers. Two lines of evidence suggest that oligomerization occurs in the endoplasmic reticulum: first, a mutant allele of Fz4, encoding a truncated protein that is retained in the endoplasmic reticulum, is linked to the autosomal-dominant retinal degenerative disease, familial exudative vitreoretinopathy (FEVR). We show that this mutant form of Fz4 oligomerizes with wild-type Fz4, retains it in the endoplasmic reticulum and inhibits its signalling. Second, a derivative of Fz1 targeted to the endoplasmic reticulum traps wild-type Fz1 in the endoplasmic reticulum and blocks its signalling. These data support the hypothesis that oligomerization of mutant and wild-type Fz proteins occurs in the endoplasmic reticulum and may explain the genetic dominance of this FEVR allele.     

Accumulation of mutant alpha1-antitrypsin Z in the endoplasmic reticulum activates caspases-4 and -12, NFkappaB, and BAP31 but not the unfolded protein response

In alpha(1)-antitrypsin (alpha1AT) deficiency, a polymerogenic mutant form of the secretory glycoprotein alpha1AT, alpha1ATZ, is retained in the endoplasmic reticulum (ER) of liver cells. It is not yet known how this results in liver injury in a subgroup of deficient individuals and how the remainder of deficient individuals escapes liver disease. One possible explanation is that the "susceptible" subgroup is unable to mount the appropriate protective cellular responses. Here we examined the effect of mutant alpha1ATZ on several potential protective signaling pathways by using cell lines with inducible expression of mutant alpha1AT as well as liver from transgenic mice with liver-specific inducible expression of mutant alpha1AT. The results show that ER retention of polymerogenic mutant alpha1ATZ does not result in an unfolded protein response (UPR). The UPR can be induced in the presence of alpha1ATZ by tunicamycin excluding the possibility that the pathway has been disabled. In striking contrast, ER retention of nonpolymerogenic alpha1AT mutants does induce the UPR. These results indicate that the machinery responsible for activation of the UPR can distinguish the physical characteristics of proteins that accumulate in the ER in such a way that it can respond to misfolded but not relatively ordered polymeric structures. Accumulation of mutant alpha1ATZ does activate specific signaling pathways, including caspase-12 in mouse, caspase-4 in human, NFkappaB, and BAP31, a profile that was distinct from that activated by nonpolymerogenic alpha1AT mutants.     

Hepatitis B virus pre-S2 mutant surface antigen induces degradation of cyclin-dependent kinase inhibitor p27Kip1 through c-Jun activation domain-binding protein 1

The hepatitis B virus (HBV) large surface antigen (LHBS) mutant with deletion at the pre-S(2) region accumulates in endoplasmic reticulum (ER) and is associated with HBV-induced hepatocellular carcinogenesis. In this study, we found that the pre-S(2) LHBS mutant directly interacts with the Jun activation domain-binding protein 1 (JAB1). Association of pre-S(2) LHBS with JAB1 dissociated JAB1 from the JAB1/IRE1 complex in ER. The free (active) JAB1 then translocated into cell nuclei and rendered the Cdk inhibitor p27(Kip1) to cytosolic proteasome for degradation. The pre-S(2) LHBS mutant induced hyperphosphorylation of tumor suppressor retinoblastoma (RB) via cyclin-dependent kinase 2 (Cdk2), a downstream molecule regulated by p27(Kip1). This effect is independent of the ER stress signaling pathway. The transgenic mice carrying the pre-S(2) mutant LHBS gene also exhibited Cdk2 activation, p27(Kip1) degradation, as well as RB hyperphosphorylation. The mouse hepatocytes exhibited morphologic abnormalities such as chromatin condensation, multinucleation, and dysplasia of hepatocytes. In summary, the pre-S(2) LHBS mutant causes p27(Kip1) degradation through direct interaction with JAB1. The pre-S(2) mutant LHBS is suggested to be a potential oncoprotein for HBV-related hepatocellular carcinoma.     

The role of the endoplasmic reticulum in the accumulation of beta-amyloid peptide in Alzheimer's disease

Increased cerebral levels of Abeta(42) peptide, either as soluble or aggregated forms, are suggested to play a key role in the pathogenesis of Alzheimer's disease (AD). The identification of genetic defects in presenilins and beta-amyloid precursor protein (beta-APP) has led to the development of cellular and animal models that have helped in understanding aspects of the pathophysiology of the inherited early onset forms of AD. However, the majority of AD cases are sporadic with no clear or defined genetic basis. While genetic mutations are responsible for the accumulation of Abeta in early onset AD, the causative factors for accumulation of Abeta in the late onset AD forms are not known. This raises the possibility that Abeta accumulation in the absence of genetic mutations might result from abnormalities that indirectly affect Abeta production or its clearance. Currently, there is no consensus as to what are the mechanisms by which Abeta accumulates or as to which mechanisms underlie Abeta-induced neuronal death in AD. In this review, I will first describe the physiological role of endoplasmic reticulum in the cell and review some of the data supporting dysfunction of the endoplasmic reticulum as an early event leading to Abeta accumulation in familial AD. I will also discuss the possible role of oxidative stress and other factors as contributors in Abeta accumulation by reducing the clearance of Abeta from the endoplasmic reticulum. Finally, I will summarize data that show the endoplasmic reticulum stress as a mechanism underlying exogenous Abeta neurotoxicity.