Structure of Human Prostasin, a Target for the Regulation of Hypertension

Prostasin (also called channel activating protease-1 (CAP1)) is an extracellular serine protease implicated in the modulation of fluid and electrolyte regulation via proteolysis of the epithelial sodium channel. Several disease states, particularly hypertension, can be affected by modulation of epit...

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Bibliographic Details
Published in:The Journal of biological chemistry 2008-12, Vol.283 (50), p.34864-34872
Main Authors: Rickert, Keith W., Kelley, Paul, Byrne, Noel J., Diehl, Ronald E., Hall, Dawn L., Montalvo, Allison M., Reid, John C., Shipman, Jennifer M., Thomas, Bradley W., Munshi, Sanjeev K., Darke, Paul L., Su, Hua-Poo
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Apoproteins - chemistry
Crystallography, X-Ray - methods
Escherichia coli - metabolism
Guanidines - chemistry
Humans
Hypertension - metabolism
Molecular Conformation
Molecular Sequence Data
Protein Conformation
Protein Folding
Protein Renaturation
Protein Structure and Folding
Sequence Homology, Amino Acid
Serine Endopeptidases - chemistry
Serine Endopeptidases - genetics
Substrate Specificity
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Summary:Prostasin (also called channel activating protease-1 (CAP1)) is an extracellular serine protease implicated in the modulation of fluid and electrolyte regulation via proteolysis of the epithelial sodium channel. Several disease states, particularly hypertension, can be affected by modulation of epithelial sodium channel activity. Thus, understanding the biochemical function of prostasin and developing specific agents to inhibit its activity could have a significant impact on a widespread disease. We report the expression of the prostasin proenzyme in Escherichia coli as insoluble inclusion bodies, refolding and activating via proteolytic removal of the N-terminal propeptide. The refolded and activated enzyme was shown to be pure and monomeric, with kinetic characteristics very similar to prostasin expressed from eukaryotic systems. Active prostasin was crystallized, and the structure was determined to 1.45Å resolution. These apoprotein crystals were soaked with nafamostat, allowing the structure of the inhibited acyl-enzyme intermediate structure to be determined to 2.0Å resolution. Comparison of the inhibited and apoprotein forms of prostasin suggest a mechanism of regulation through stabilization of a loop which interferes with substrate recognition.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M805262200