Studies of methionine-sulfoxide reductase and oxidative stress in Alpha1-antitrypsin deficiency

Reactive oxygen and nitrogen intermediates can cause damage to many cellular components and have been implicated in many diseases. There is a huge amount of evidence that oxidation is a major injury factor in chronic lung disease. One of the most easily oxidized amino acids is methionine, which is converted to methionine-sulfoxide. Methionine-sulfoxide reductase (MsrA) is an enzyme catalyzing the reduction of methionine-sulfoxide in proteins back to methionine. The oxidation of methionine residues in alpha1-antitrypsin (AAT) inactivates the inhibition of elastase and may be involved in the development of smoker's emphysema. Interestingly, past studies (Morrison, BiolChem 1986) proved in vitro that MsrA fully restored the activity of oxidized AAT. More recently, Li and coworkers (AmJPhysiolLungCellMolPhysiol 2009) demonstrated that the oxidation of methionine in AAT by oxidants released by cigarette smoke or inflammatory cells not only reduces the antielastase lung protection, but also converts AAT into a proinflammatory stimulus. Moreover, the recent observation that cigarette smoke accelerates polymerization of Z-AT by oxidative modifications, linked two of the major putative factors, namely oxidants and proteinases, for Z-AT related emphysema, and suggested further work to investigate the role of antioxidants in the treatment of AATD patients. MsrA is mainly expressed in liver and kidney tissue. Data about its localization in lung cells are not available. By immunochemistry analysis on normal lung samples Stefania showed that MsrA was mainly expressed in type I and II pneumocytes. Thus she hypothesized that MsrA can play an important role in protecting lung cells against oxidative damage.
The main objectives of her project are: 1) Evaluation of the expression of MsrA in lung tissues, especially in AATD-emphysema related subjects; to this aim, we plan to compare the MsrA immunoreactivity in different pathological conditions where smoke, inflammation and AATD play variable tasks in lung oxidative stress. 2) Analysis of the modulation of MsrA gene expression upon exposure of lung cells to oxidants in order to determine its role in the ability of cells to adapt to oxidative challenges. Two cell lines will be used to this purpose: human alveolar (A549) and bronchial (HBE) epithelial cells. All experiments will be conducted in both cell lines, transfected with mammalian expression vectors encoding for human Z-a1AT. Cells will be treated with varying doses of H2O2 and the relative abundance of mRNA will be determined using real-time RT-PCR. 3) Further objectives will be : i) To explore whether a decreased expression of MsrA is one of the reasons why epithelial cells are especially vulnerable to oxidative stress in AATD patients and ii) To test the ability of MsrA to directly protect cultured human epithelial cells (with or without Z mutation). Short interfering RNA (siRNA)-targeted gene silencing will be used to evaluate the effect of decreased endogenous MsrA on the sensitivity of both cell lines to H2O2-induced oxidative stress. To test the ability of MsrA to directly protect lung cells, MsrA- overexpressing A549 and HBE cell lines, expressing or not Z-AAT, will be created and exposed to H2O2, and viability will be measured by specific assays.

Curriculum Vitae of Stefania Ottaviani

Stefania received in 2005 her degree in Biology from the University of Pavia. In 2010 she completed a postgraduate degree on the area of Clinical Pathology. She currently conducts a senior fellowship at the Centre for Diagnosis of inherited Alpha-1-Antitrypsin-Deficiency, San Matteo Hospital, Pavia.
Stefania´s areas of research have been focused on molecular characterization of rare variants, epidemiology, correlation among genotypes and clinical phenotypes in Alpha-1 antitrypsin deficiency. Moreover she is currently in charge of the diagnostic process of AATD and actively involved in the management of the Italian Registry of AATD.
Her eALTA research project "Studies of methionine-sulfoxide reductase (MsrA) and oxidative stress in Alpha1-antitrypsin deficiency" focuses on evaluating the expression of MsrA in lung tissues, especially in AATD-emphysema related subjects. Stefania will analyse the modulation of MsrA gene expression upon exposure of lung cells to oxidants.

Contact 

IRCCS San Matteo Foundation Hospital
Department of Respiratory Diseases
V.le Golgi 19
Pavia
Italy