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Sequestration of mutated alpha1-antitrypsin into inclusion bodies is a cell-protective mechanism to maintain endoplasmic reticulum function.

ABSTRACT: A variant alpha1-antitrypsin with E342K mutation has a high tendency to form intracellular polymers, and it is associated with liver disease. In the hepatocytes of individuals carrying the mutation, alpha1-antitrypsin localizes both to the endoplasmic reticulum (ER) and to membrane-surrounded inclusion bodies (IBs). It is unclear whether the IBs contribute to cell toxicity or whether they are protective to the cell. We found that in hepatoma cells, mutated alpha1-antitrypsin exited the ER and accumulated in IBs that were negative for autophagosomal and lysosomal markers, and contained several ER components, but not calnexin. Mutated alpha1-antitrypsin induced IBs also in neuroendocrine cells, showing that formation of these organelles is not cell type specific. In the presence of IBs, ER function was largely maintained. Increased levels of calnexin, but not of protein disulfide isomerase, inhibited formation of IBs and lead to retention of mutated alpha1-antitrypsin in the ER. In hepatoma cells, shift of mutated alpha1-antitrypsin localization to the ER by calnexin overexpression lead to cell shrinkage, ER stress, and impairment of the secretory pathway at the ER level. We conclude that segregation of mutated alpha1-antitrypsin from the ER to the IBs is a protective cell response to maintain a functional secretory pathway.

SUBMITTER: Granell S

PROVIDER: S-EPMC2230602 | biostudies-literature | 2008 Feb

REPOSITORIES: biostudies-literature

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Sequestration of mutated alpha1-antitrypsin into inclusion bodies is a cell-protective mechanism to maintain endoplasmic reticulum function.

Granell Susana S Baldini Giovanna G Mohammad Sameer S Nicolin Vanessa V Narducci Paola P Storrie Brian B Baldini Giulia G

Molecular biology of the cell 20071128 2

A variant alpha1-antitrypsin with E342K mutation has a high tendency to form intracellular polymers, and it is associated with liver disease. In the hepatocytes of individuals carrying the mutation, alpha1-antitrypsin localizes both to the endoplasmic reticulum (ER) and to membrane-surrounded inclusion bodies (IBs). It is unclear whether the IBs contribute to cell toxicity or whether they are protective to the cell. We found that in hepatoma cells, mutated alpha1-antitrypsin exited the ER and ac ...[more]

PMID: 18045994

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Project description:BACKGROUND:Alpha-1-antitrypsin (AAT) deficiency (AATD) of Z, Mmalton, Siiyama type is associated with liver storage of the mutant proteins and liver disease. The Z variant can be diagnosed on isoelectric focusing (IEF) while Mmalton and Siiyama may be missed or misdiagnosed with this technique. Therefore, molecular analysis is mandatory for their characterization. In particular, that holds true for the Mmalton variant as on IEF profile it resembles the wild M2 subtype. METHODS:This is a retrospective analysis involving review of medical records and of liver biopsy specimens from a series of Mmalton, Z and Siiyama Alpha-1-antitrypsin deficiency patients. The review has been implemented by additional histological stains, electron microscopic observations and 3-D modeling studies of the sites of the mutations. RESULTS:Z, Mmalton and Siiyama liver specimen contained characteristic intrahepatocytic PAS-D globules. The globules differed in the three variants as only Mmalton cases showed dark basophilic precipitates within the AAT inclusions. The precipitates were visualized in haematoxylin-eosin (H.E.) stained preparations and corresponded to calcium precipitates as demonstrated by von Kossa staining. On immunohistochemistry, ZAAT inclusions were stained by polyclonal as well as monoclonal noncommercial anti-AAT antibody (AZT11), whilst Mmalton and Siiyama inclusion bodies remained negative with the monoclonal anti-Z antibody. 3-D protein analysis allowed to predict more severe misfolding of the Mmalton molecule as compared to Z and Siiyama that could trigger anomalous interaction with endoplasmic reticulum chaperon proteins, namely calcium binding proteins. CONCLUSIONS:Mmalton AAT inclusion bodies contain calcium precipitates inside them that allow the differential diagnosis with Siiyama and ZAAT inclusions in routine histological sections. The study has confirmed the specificity of the monoclonal AZT11 for the Z mutant. Thus, the combination of these two features is crucial for the distinction between the three variants and for predicting the genotype, whose confirmation would definitely require molecular analysis. Our study provides new data on the pathomorphogenesis of Mmalton inclusion bodies whose mineralization could play a central role in disease pathogenesis of Mmalton that is distinct from the Z and Siiyama variants. Calcium is known to be a major effector of cell death either via the increased intracellular concentration or the alteration of homeostasis.

Project description:BackgroundExcessive inflammation, hemolysis, and accumulation of labile heme play an essential role in the pathophysiology of multi-organ dysfunction syndrome (MODS) in sepsis. Alpha1-antitrypsin (AAT), an acute phase protein with heme binding capacity, is one of the essential modulators of host responses to inflammation. In this study, we evaluate the putative protective effect of AAT against MODS and mortality in a mouse model of polymicrobial abdominal sepsis.MethodsPolymicrobial abdominal sepsis was induced in C57BL/6N mice by cecal ligation and puncture (CLP). Immediately after CLP surgery, mice were treated intraperitoneally with three different forms of human AAT-plasma-derived native (nAAT), oxidized nAAT (oxAAT), or recombinant AAT (recAAT)-or were injected with vehicle. Sham-operated mice served as controls. Mouse survival, bacterial load, kidney and liver function, immune cell profiles, cytokines/chemokines, and free (labile) heme levels were assessed. In parallel, in vitro experiments were carried out with resident peritoneal macrophages (MPMΦ) and mouse peritoneal mesothelial cells (MPMC).ResultsAll AAT preparations used reduced mortality in septic mice. Treatment with AAT significantly reduced plasma lactate dehydrogenase and s-creatinine levels, vascular leakage, and systemic inflammation. Specifically, AAT reduced intraperitoneal accumulation of free heme, production of cytokines/chemokines, and neutrophil infiltration into the peritoneal cavity compared to septic mice not treated with AAT. In vitro experiments performed using MPMC and primary MPMΦ confirmed that AAT not only significantly decreases lipopolysaccharide (LPS)-induced pro-inflammatory cell activation but also prevents the enhancement of cellular responses to LPS by free heme. In addition, AAT inhibits cell death caused by free heme in vitro.ConclusionData from the septic CLP mouse model suggest that intraperitoneal AAT treatment alone is sufficient to improve sepsis-associated organ dysfunctions, preserve endothelial barrier function, and reduce mortality, likely by preventing hyper-inflammatory responses and by neutralizing free heme.

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Sequestration of mutated alpha1-antitrypsin into inclusion bodies is a cell-protective mechanism to maintain endoplasmic reticulum function.

Publications

Sequestration of mutated alpha1-antitrypsin into inclusion bodies is a cell-protective mechanism to maintain endoplasmic reticulum function.

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