Alpha-1-antitrypsin mutant Z protein content in individual hepatocytes correlates with cell death in a mouse model

Abstract

Alpha-1-antitrypsin (a1AT) deficiency is caused by homozygosity for the a1AT mutant Z gene and occurs in 1 in 2000 births. The Z mutation confers an abnormal conformation on the protein, resulting in an accumulation within the endoplasmic reticulum of hepatocytes rather than appropriate secretion. The accumulation of the mutant protein is strikingly heterogeneous within the liver. Homozygous ZZ children and adults have an increased risk of chronic liver disease, which is thought to result from this variable intracellular accumulation of the a1AT mutant Z protein. Previous reports have suggested that autophagy, mitochondrial injury, apoptosis, and other pathways may be involved in the mechanism of hepatocyte injury, although the interplay of these mechanisms in vivo is unclear. In this study, we examine a well-characterized in vivo model of a1AT mutant Z liver injury, the PiZ mouse, to better understand the pathways involved in this disease. The results show an increase in the stimulation of the apoptotic cascade in hepatocytes, the magnitude of which strongly correlates to the absolute amount of the a1AT mutant Z protein accumulated within the individual cell. Increases in apoptotic regulatory proteins are also detected. Conclusion: These data, combined with previous work, permit for the first time the construction of a hypothetical hepatocellular injury cascade for this disease involving mitochondrial injury, caspase activation, and apoptosis, which takes into account the heterogeneous nature of the mutant Z protein accumulation within the liver. Further development of this hypothetical cascade will focus future research on this and other metabolic liver diseases. T he genetic disease alpha-1-antitrypsin (a1AT) deficiency is caused by homozygosity for the a1AT mutant Z gene and occurs in 1 in 2000 births. 1 The Z mutation confers an abnormal conformation on the nascent polypeptide, resulting in an accumulation of the mutant protein within the endoplasmic reticulum (ER) of hepatocytes rather than the appropriate, highly efficient secretion of the wild-type (WT) protein. Homozygous, ZZ individuals have an increased risk of chronic liver disease and hepatocellular carcinoma resulting from this intracellular accumulation of the a1AT mutant Z protein.Studies of the a1AT mutant Z protein molecule have shown that the nascent polypeptide has a tendency to form unique protein homopolymers. 1,2 Although this loop-sheet insertion mechanism of a1AT mutant Z protein polymerization, in which the reactive site loop of one molecule inserts into a surface groove in a neighboring molecule, does not involve the formation of covalent bonds, physical-chemical studies of these polymers suggest that this conformation is highly favored. It is proposed and supported by some published data that the liver injury in humans with a1AT deficiency is directly related to the hepatic accumulation of the polymerized a1AT mutant Z protein. [3][4][5] Our laboratory and others have reported a series of studies that have begun to examine the mechani...

Spontaneous hepatic repopulation in transgenic mice expressing mutant human α1-antitrypsin by wild-type donor hepatocytes

“…Quantitative RT-PCR revealed that both groups of hepatocytes expressed similar AAT-Z mRNA levels (data not shown). In contrast, Western blot analysis showed a much higher AAT-Z content in the globulecontaining hepatocytes, which was consistent with the results of Lindblad et al, who also showed that the globule-containing cells had a higher rate of apoptosis than globule-devoid hepatocytes (15).…”

Autophagic disposal of the aggregation-prone protein that causes liver inflammation and carcinogenesis in α-1-antitrypsin deficiency

“…42 Markers of apoptosis were found to be more pronounced in hepatocytes with greater levels of insoluble ATZ. Furthermore, stimulation of the extrinsic apoptotic pathway with antibody to fas resulted in increased apoptosis almost exclusively within the globulecontaining cells.…”

“…Indeed, the data in this recent study shows an increase in one antiapoptotic protein, cFLIP, in cells with higher levels of insoluble ATZ. 42 This would mean that the globule-devoid hepatocytes were even more resistant than normal hepatocytes to apoptosis. This is probably an important distinction because relative resistance to apoptosis in either of these cellular compartments could potentially contribute to the mechanism of carcinogenesis.…”

γ375W fibrinogen-synthesizing CHO cells indicate the accumulation of variant fibrinogen within endoplasmic reticulum

“…In brief, the report suggested the possibility that HERSD associated with variant fibrinogenemia might be caused by decreasing function of the autophagic pathway. Moreover, molecular mechanisms of A1AT homozygous Z mutation causing hepatocyte injury have been studied extensively and showed increased ER retention as a conformational disease [24], activation of ER-specific stress responses [25], up-regulated mitochondrial autophagy [26], or mitochondrial injury and apoptosis [27]. Our γ375W-CHO cells indicated cytoplasmic aberrant inclusion bodies with large granular or fibrous forms and this suggested that variant fibrinogen was retained and aggregated within the dilated ER.…”