The serpin class of protease inhibitors includes many of the protease inhibitors in blood, such as antithrombin and alpha-1 antitrypsin. Serpins are involved in the regulation of many physiological processes including fibrinolysis, complement activation, angiogenesis and inflammation. Central to serpin function is their unique inhibitory mechanism which involves a large conformational change from a metastable form to a stable form, and this mechanism imposes on serpins the unusual requirement that they fold to a metastable state. Folding, conformational change and function are inextricably linked in serpins, and understanding this linkage is essential for understanding normal serpin function as well as the pathologies associated with various serpin mutations. Misfolding is known to be the basis of many serpin-linked diseases including thrombosis and emphysema. The most common pathological serpin mutation in humans, the Z variant of alpha-1 antitrypsin, misfolds and polymerizes in hepatic cells where alpha-1 antitrypsin is primarily produced. Detailed knowledge of misfolded forms of alpha-1 antitrypsin and other serpins is essential for the design of effective therapeutic agents. The proposed work will employ the unique capabilities of hydrogen exchange and mass spectrometry to provide previously inaccessible information on the conformational dynamics of serpins in solution and the folding pathways of normal and pathological serpins.
Our specific aims are: 1) Measure the distribution of conformational flexibility in wild type alpha-1 antitrypsin and a number of mutants known to have compromised activity using hydrogen exchange/mass spectrometry. 2) Determine the folding pathway of alpha-1 antitrypsin by using hydrogen exchange/mass spectrometry to monitor the acquisition of native structure throughout the alpha-1 antitrypsin molecule during refolding. 3) Determine the folding pathway of the pathogenic Z variant of alpha-1 antitrypsin by hydrogen exchange/ mass spectrometry. Significance: Alpha-1 antitrypsin deficiency is a serious genetic disorder, with an estimated 100,000 individuals in the U.S. suffering from the severe form. This disorder can lead to both liver cirrhosis and emphysema. Polymerization of alpha-1 antitrypsin is the underlying cause of these symptoms. Determining the structure of the polymerization prone intermediate is an important step in developing therapeutic agents.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
7R01HL085469-06
Application #
8468255
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Croxton, Thomas
Project Start
2007-06-01
Project End
2013-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
6
Fiscal Year
2011
Total Cost
$300,000
Indirect Cost
Name
University of Maryland Baltimore
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
188435911
City
Baltimore
State
MD
Country
United States
Zip Code
21201
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