alpha1-Antitrypsin Portland, a bioengineered serpin highly selective for furin: application as an antipathogenic agent.
The important role of furin in the proteolytic activation of many pathogenic molecules has made this endoprotease a target for the development of potent and selective antiproteolytic agents. Here, we demonstrate the utility of the protein-based inhibitor alpha1-antitrypsin Portland (alpha1-PDX) as an antipathogenic agent that can be used prophylactically to block furin-dependent cell killing by Pseudomonas exotoxin A. Biochemical analysis of the specificity of a bacterially expressed His- and FLAG-tagged alpha1-PDX (alpha1-PDX/hf) revealed the selectivity of the alpha1-PDX/hf reactive site loop for furin (Ki, 600 pM) but not for other proprotein convertase family members or other unrelated endoproteases. Kinetic studies show that alpha1-PDX/hf inhibits furin by a slow tight-binding mechanism characteristic of serpin molecules and functions as a suicide substrate inhibitor. Once bound to furin's active site, alpha1-PDX/hf partitions with equal probability to undergo proteolysis by furin at the C-terminal side of the reactive center -Arg355-Ile-Pro-Arg358- downward arrow or to form a kinetically trapped SDS-stable complex with the enzyme. This partitioning between the complex-forming and proteolytic pathways contributes to the ability of alpha1-PDX/hf to differentially inhibit members of the proprotein convertase family. Finally, we propose a structural model of the alpha1-PDX-reactive site loop that explains the high degree of enzyme selectivity of this serpin and which can be used to generate small molecule furin inhibitors.
Jean
F
F
Vollum Institute, Oregon Health Sciences University, Portland, OR 97201, USA.
Stella
K
K
Thomas
L
L
Liu
G
G
Xiang
Y
Y
Reason
A J
AJ
Thomas
G
G
D036002:ADP Ribose Transferases; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors; D005098:Exotoxins / Q000037:antagonists & inhibitors; D045683:Furin; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D011550:Pseudomonas aeruginosa; D011994:Recombinant Proteins / Q000494:pharmacology; D013379:Substrate Specificity; D013381:Subtilisins / Q000037:antagonists & inhibitors*; D037521:Virulence Factors; D000515:alpha 1-Antitrypsin / Q000235:genetics / Q000494:pharmacology*; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000900:Anti-Bacterial Agents; D001427:Bacterial Toxins; D005098:Exotoxins; D045683:Furin; D011994:Recombinant Proteins; D013381:Subtilisins; D037521:Virulence Factors; D000515:alpha 1-Antitrypsin; D000097668:Pseudomonas aeruginosa Exotoxin A; C113421:alpha 1-antitrypsin Portland