The Proteasome Participates in Degradation of Mutant α1-Antitrypsin Z in the Endoplasmic Reticulum of Hepatoma-derived Hepatocytes

Abstract

Because retention of mutant ␣ 1 -antitrypsin (␣ 1 -AT) Z in the endoplasmic reticulum (ER) is associated with liver disease in ␣ 1 -AT-deficient individuals, the mechanism by which this aggregated glycoprotein is degraded has received considerable attention. In previous studies using stable transfected human fibroblast cell lines and a cell-free microsomal translocation system, we found evidence for involvement of the proteasome in degradation of Chem. 275, 25015-25022) found that degradation of ␣ 1 -ATZ in a stable transfected murine hepatoma cell line was inhibited by tyrosine phosphatase inhibitors, but not by the proteasomal inhibitor lactacystin and concluded that the proteasome was only involved in ER degradation of ␣ 1 -ATZ in nonhepatocytic cell types or in cell types with levels of ␣ 1 -AT expression that are substantial lower than that which occurs in hepatocytes. To examine this important issue in further detail, in this study we established rat and murine hepatoma cell lines with constitutive and inducible expression of ␣ 1 -ATZ. In each of these cell lines degradation of ␣ 1 -ATZ was inhibited by lactacystin, MG132, epoxomicin, and clasto-lactacystin ␤-lactone. Using the inducible expression system to regulate the relative level of ␣ 1 -ATZ expression, we found that lactacystin had a similar inhibitory effect on degradation of ␣ 1 -ATZ at high and low levels of ␣ 1 -AT expression. Although there is substantial evidence that other mechanisms contribute to ER degradation of ␣ 1 -ATZ, the data reported here indicate that the proteasome plays an important role in many cell types including hepatocytes.The classical and most common form of ␣ 1 -antitrypsin (␣ 1 -AT) 1 deficiency is a relatively unique genetic disease in that it is associated with injury to one tissue, pulmonary emphysema, by a loss-of-function mechanism and injury to another tissue, chronic hepatitis/hepatocellular carcinoma, by a gain-of-function mechanism. Many studies have provided evidence that emphysema results from lack of the anti-elastase activity of ␣ 1 -AT in the lung (reviewed in Refs. 1 and 2). Liver disease is due to toxic effects of aggregated ␣ 1 -ATZ retained in the ER of liver parenchymal cells. The gain-of-function mechanism is most clearly demonstrated by experiments in mice transgenic for human ␣ 1 -ATZ. These mice develop liver injury and hepatocellular carcinoma despite the fact that they have their own endogenous anti-elastases (3-5).The mutant Z allele of ␣ 1 -AT is characterized by a single nucleotide substitution, which results in the replacement of glutamate 342 by a bulky lysine residue (1, 2). The studies of Carrell and Lomas (6, 7) have shown that this substitution renders the ␣ 1 -AT molecule more susceptible to polymerization and that highly ordered aggregates accumulate in the ER of liver cells.One interesting observation, arising from unbiased nationwide screening studies of ␣ 1 -AT deficiency in Sweden, indicates that only 10 -15% of deficient individuals develop clinically significant liver disease (8,9...

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

“…[16][17][18] It is still not clear how the ATZ is transported from the lumen of the ER to the proteasome in the cytoplasm. Although retrograde translocation through the microsomal import channel has been proposed for some ER proteins, there is growing evidence for a mechanism in which the proteasome, as part of a multiprotein complex that forms a platform on the cytosolic side of the ER membrane, directly mediates extraction of substrates from the ER membrane.…”

“…Although retrograde translocation through the microsomal import channel has been proposed for some ER proteins, there is growing evidence for a mechanism in which the proteasome, as part of a multiprotein complex that forms a platform on the cytosolic side of the ER membrane, directly mediates extraction of substrates from the ER membrane. 19,20 Nevertheless, these early studies in yeast, mammalian cell lines and cell-free systems all indicated that the proteasomal pathway could not fully account for disposal of ATZ [16][17][18][21][22][23] -that is in the absence of proteasomal activity there was clear-cut residual and time-dependent degradation of ATZ.…”

Alpha-1-antitrypsin deficiency: A new paradigm for hepatocellular carcinoma in genetic liver disease

“…Studies in many systems have indicated that the proteosomal system is involved. [17][18][19][20][21] This involvement appears to include both the classical ubiquitin-dependent proteosomal mechanism as well as a ubiquitin-independent proteosomal mechanism. 19 However, exactly how ␣1ATZ on the luminal side of the ER membrane is accessed by the proteosome from the cytoplasm is not clear.…”

Characterization of anERADGene asVPS30/ATG6Reveals Two Alternative and Functionally Distinct Protein Quality Control Pathways: One for Soluble Z Variant of Human α-1 Proteinase Inhibitor (A1PiZ) and Another for Aggregates of A1PiZ

“…The classical form of A1Pi deficiency results from the Z variant (A1PiZ) that contains a K342E substitution (Bathurst et al, 1984;Crystal, 1990). A1PiZ homozygous individuals can develop emphysema via a loss-of-function mechanism because the altered conformation of A1PiZ results in its recognition and degradation by ERAD (Wu et al, 1994;Werner et al, 1996;Teckman et al, 2001). A subset of homozygous individuals (12-15%) also experience liver disease (Sveger, 1988) via a gain-offunction mechanism because A1PiZ accumulates and aggregates within the ER of hepatocytes (Lomas et al, 1992, Lomas andMahadeva, 2002; reviewed in Perlmutter, 2003).…”