Endoplasmic reticulum polymers impair luminal protein mobility and sensitize to cellular stress in alpha1-antitrypsin deficiency

Abstract

Point mutants of α1-antitrypsin form ordered polymers that are retained as inclusions within the endoplasmic reticulum (ER) of hepatocytes in association with neonatal hepatitis, cirrhosis and hepatocellular carcinoma. These inclusions cause cell damage and predispose to ER stress in the absence of the classical unfolded protein response (UPR). The pathophysiology underlying this ER stress was explored by generating cell models that conditionally express wildtype α1-antitrypsin, two mutants that cause polymer-mediated inclusions and liver disease (E342K [the Z allele] and H334D) and a truncated mutant (Null Hong Kong, NHK) that induces classical ER stress and is removed by ER associated degradation. Expression of the polymeric mutants resulted in gross changes in the ER luminal environment that recapitulated the changes seen in liver sections from individuals with PI*ZZ α1-antitrypsin deficiency. In contrast expression of NHK α1-antitrypsin caused electron lucent dilatation and expansion of the ER throughout the cell. Photobleaching microscopy in live cells demonstrated a decrease in the mobility of soluble luminal proteins in cells that express E342K and H334D α1-antitrypsin when compared to those that express wildtype and NHK α1-antitrypsin (0.34±0.05, 0.22±0.03, 2.83±0.30 and 2.84±0.55 μm2/s respectively). There was no effect on protein mobility within ER membranes indicating that cisternal connectivity was not disrupted. Polymer expression alone was insufficient to induce the UPR but the resulting protein overload rendered cells hypersensitive to ER stress induced by either tunicamycin or glucose depletion. Conclusion Changes in protein diffusion provide an explanation for the cellular consequences of ER protein overload in mutants that cause inclusion body formation and α1-antitrypsin deficiency.

Update on alpha-1 antitrypsin deficiency: New therapies

“…Misfolded proteins within the ER lumen usually trigger adaptive measures termed the unfolded protein response (UPR). Indeed, terminally misfolded truncated variants of α 1 -antitrypsin (e.g., α 1 -antitrypsin NullHongKong and Saar) constitutively activate the UPR [32,33]. Surprisingly, the accumulation of Z α 1 -antitrypsin polymers within the ER is not associated with UPR activation in cell lines that recapitulate hepatocyte phenotypes or in transgenic mice, possibly because polymers are structurally ordered (Fig.…”

“…Instead Z α 1 -antitrypsin expression activates NFκB by a calcium-mediated pathway that is independent of the UPR [32][33][34][35]. We have termed this the 'ordered protein response' [35] and others have shown that it results in the release of IL-6 and IL-8 [34].…”

“…These cytokines are well-recognised mediators of both acute and chronic inflammatory responses and so may play pathogenic roles in both the liver and lung disease associated with α 1 -antitrypsin deficiency. Moreover, cells expressing Z α 1 -antitrypsin display a more marked UPR when stressed with a 'second hit' [33,34]. We have proposed that this results from α 1 -antitrypsin polymers increasing the viscosity within the ER thereby reducing the mobility of chaperones and hence their ability to neutralise the effect of a second insult [33].…”

Alpha-1 Antitrypsin Augmentation Therapy Corrects Accelerated Neutrophil Apoptosis in Deficient Individuals

“…Although many investigators agree that mutations of the AAT protein can lead to the formation of polymers that are retained within the ER of hepatocytes in ZZ-AATD (3,53), much debate exists regarding the presence of the classical unfolded protein response (UPR), a cellular stress response that leads to attenuation of protein translation and enhancement of ER protein folding in AATD (54,55). Rather, opinion is now moving toward the concept that retention of ordered polymers of AAT within the ER of hepatocytes may predispose cells to ER stress and fully activate the UPR as misfolded protein levels increase (2). Of interest, whereas these findings are well recognized in primary hepatic and various transfected cell line models, the characteristics of ER stress in immune cells from ZZ-AATD patients are less well studied.…”

“…AAT deficiency (AATD) is a lethal hereditary disorder characterized by low plasma levels of AAT, with the most common form associated with the Z allele, or homozygous ZZ phenotype (1). Accumulation and disrupted luminal mobility of polymerized aggregates of mutant Z-AAT protein within the endoplasmic reticulum (ER) are implicated in liver cirrhosis and chronic hepatitis (2,3). The most common manifestations of the disease, however, affect the lung, and were initially thought to be due to the loss of the natural antiprotease screen, resulting in early onset emphysema in the ZZ phenotype.…”