Quantitative isolation of ?1AT mutant Z protein polymers from human and mouse livers and the effect of heat

Abstract

Alpha-1-antitrypsin (␣1AT) deficiency in its most common form is caused by homozygosity for the ␣1AT mutant Z gene. This gene encodes a mutant Z secretory protein, primarily synthesized in the liver, that assumes an abnormal conformation and accumulates within hepatocytes causing liver cell injury. Studies have shown that mutant ␣1ATZ protein molecules form unique protein polymers. These Z protein polymers have been hypothesized to play a critical role in the pathophysiology of liver injury in this disease, although a lack of quantitative methods to isolate the polymers from whole liver has hampered further analysis. In this study, we demonstrate a quantitative ␣1ATZ polymer isolation technique from whole liver and show that the hepatocellular periodic acid-Schiff-positive globular inclusions that are the histopathological hallmark of this disease are composed almost entirely of the polymerized ␣1ATZ protein. Furthermore, we examine the previously proposed but untested hypothesis that induction of ␣1ATZ polymerization by the heat of physiological fever is part of the mechanism of hepatic ␣1ATZ protein accumulation. The results, however, show that fever-range temperature elevations have no detectable effect on steady-state levels of intrahepatic Z protein polymer in a model in vivo system. In conclusion, methods to separate insoluble protein aggregates from liver can be used for quantitative isolation of ␣1ATZ T he most common form of alpha-1-antitrypsin (␣1AT) deficiency is caused by homozygosity for the ␣1AT mutant Z gene. [1][2][3][4][5][6][7][8][9][10][11] The Z mutation confers an abnormal conformation on the nascent polypeptide, which is synthesized in the liver, resulting in accumulation within the endoplasmic reticulum (ER) of hepatocytes. ZZ individuals have an increased risk of chronic liver disease and hepatocellular carcinoma resulting from this intracellular accumulation of mutant ␣1ATZ protein.Studies of the mutant ␣1ATZ protein molecule have shown that the nascent polypeptide has the tendency to form unique protein homopolymers. 2,[12][13][14][15][16] Although the "loop sheet" insertion mechanism of ␣1ATZ polymerization-in which the reactive site loop of one molecule inserts into a surface groove in a neighboring moleculedoes not involve the formation of covalent bonds, physical/chemical studies of these polymers suggest that this conformation is highly favored. It has been widely hypothesized that liver injury in humans with ␣1AT deficiency is directly related to the hepatic accumulation of the polymerized ␣1ATZ protein, rather than to the monomeric form. Furthermore, in vitro studies of ␣1ATZ molecules show that the equilibrium favoring formation of polymers increases as temperature increases. 13 These data have been used to propose that progressive liver disease in ZZ humans is directly related to increased ␣1ATZ polymers formed during episodes of physiological fever. 1,8 Our laboratory has reported a series of studies that have begun to examine the mechanism of liver cell injury in ␣1AT deficie...

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

“…With previously published biochemical techniques, the intracellular content of the a1AT mutant Z intracellular monomer and polymer protein was determined for the cells in each side-scatter quartile. 11 The result is shown in Fig. 1D as a quantitative immunoblot of the a1AT mutant Z protein.…”

“…A monomer-polymer assay was performed as previously described. 11 Histologic and immunohistochemistry analysis was performed with standard techniques, as described. [3][4][5]11 Immunoblots were performed as previously described in triplicate with antibodies previously noted to be useful in these systems.…”

“…11 Histologic and immunohistochemistry analysis was performed with standard techniques, as described. [3][4][5]11 Immunoblots were performed as previously described in triplicate with antibodies previously noted to be useful in these systems. 4,5,8 For the Jo2 antibody treatment, mice were given a single intraperitoneal injection of 6 m of anti-Jo2 antibody (DB Bioscience).…”

A novel monoclonal antibody to characterize pathogenic polymers in liver disease associated with α1-antitrypsin deficiency

“…Z a 1 -antitrypsin was purified from hepatic inclusions as described 20 and confirmed by nondenaturing PAGE.…”

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

“…40 These results have led us to hypothesize (Figure 4) that the pathogenesis of hepatic carcinoma involves the interplay of globule-containing cells that are 'sick but not yet dead' and chronically stimulating the globule-devoid cells 'in trans' in the presence of inflammation. 40 We know that the globule-devoid cells have accumulated aggregated mutant ATZ 41 and activated a number of cellular 'alarm' pathways, including ER-and mitochondrial caspases, NF-kB and autophagy. 27 Furthermore, we know that the globule-containing cells are relatively impaired in cell proliferation.…”