? Numerous human diseases result from the failure of mutant soluble proteins to exit the endoplasmic reticulum (ER) including alpha-1-antitrypsin (a1AT) deficiency, a1AT, a serine proteinase inhibitor, functions to inhibit neutrophil elastase in the lung and prevent degradation of the extracellular matrix in the alveolar wall. Patients deficient in a1AT exhibit increased risk of developing lung and liver disease. The objective of this proposal is to elucidate the mechanism involved in export of a1AT from the ER. This proposal addresses an important gap in our knowledge by characterizing the function of a new class of membrane receptors that mobilize a1AT through the secretory pathway. We have identified two proteins, L6 and il-TMP, members of the tetraspan family. The experiments described in this proposal will test the hypotheses that (1) residues found in the C-terminus of a1AT are necessary for ER export, (2) that L6 functions to facilitate a1AT export from the ER in alveolar type II cells, and (3) that iI-TMP is a sorting receptor for a1AT in hepatocytes.
Specific Aim 1 will characterize residues in the a1AT C-terminus that direct ER export using site-directed mutagenesis. We will explore the hypothesis that an export signal on a1AT interacts with the luminal domain of a1AT receptors to mediate incorporation of a1AT into COPII vesicles.
Specific Aim 2 will characterize the role of L6 as a sorting receptor mediating the export of a1AT from the ER in alveolar type II cells using in vivo and in vitro approaches. We will generate a cell-specific knockout of L6 in mice to determine the potential importance of L6 in the maintenance of a1AT in the alveolar interstitium.
Specific Aim 3 will analyze the role of iI-TMP as a sorting receptor for a1AT export from the ER and transit through the secretory pathway in hepatocytes. A cell-specific iI-TMP knockout mouse will be generated to address the role of iI-TMP in maintenance of serum levels of a1AT. The results of these experiments will define for the first time the mechanism(s) involved in the export of a1AT from the ER in the lung and liver. It is anticipated that the data generated in this proposal will provide important insight into a1AT deficiency as well as a basic understanding of proteins in the secretory pathway essential for the maintenance of normal extracellular pools of a1AT. ? ?

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Lung Biology and Pathology Study Section (LBPA)
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Croxton, Thomas
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Scripps Research Institute
La Jolla
United States
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Bouchecareilh, Marion; Conkright, Juliana J; Balch, William E (2010) Proteostasis strategies for restoring alpha1-antitrypsin deficiency. Proc Am Thorac Soc 7:415-22
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Gurkan, Cemal; Balch, William E (2005) Recombinant production in baculovirus-infected insect cells and purification of the mammalian Sec13/Sec31 complex. Methods Enzymol 404:58-66