Abstract
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 α1-antitrypsin Portland (α1-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 Hisand FLAG-tagged α1-PDX (α1- PDX/hf) revealed the selectivity of the α1-PDX/hf reactive site loop for furin (K(i), 600 pM) but not for other proprotein convertase family members or other unrelated endoproteases. Kinetic studies show that α1-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, α1-PDX/hf partitions with equal probability to undergo proteolysis by furin at the C-terminal side of the reactive center - Arg355-Ile-Pro-Arg358-↓ 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 α1-PDX/hf to differentially inhibit members of the proprotein convertase family. Finally, we propose a structural model of the α1-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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 7293-7298 |
| Number of pages | 6 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Volume | 95 |
| Issue number | 13 |
| DOIs | |
| State | Published - Jun 23 1998 |
| Externally published | Yes |
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ASJC Scopus subject areas
- General
- Genetics
Cite this
α1-Antitrypsin Portland, a bioengineered serpin highly selective for furin : Application as an antipathogenic agent. / Jean, François; Stella, Kori; Thomas, Laurel; Liu, Gseping; Xiang, Yang Kevin; Reason, Andrew J.; Thomas, Gary.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 95, No. 13, 23.06.1998, p. 7293-7298.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - α1-Antitrypsin Portland, a bioengineered serpin highly selective for furin
T2 - Application as an antipathogenic agent
AU - Jean, François
AU - Stella, Kori
AU - Thomas, Laurel
AU - Liu, Gseping
AU - Xiang, Yang Kevin
AU - Reason, Andrew J.
AU - Thomas, Gary
PY - 1998/6/23
Y1 - 1998/6/23
N2 - 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 α1-antitrypsin Portland (α1-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 Hisand FLAG-tagged α1-PDX (α1- PDX/hf) revealed the selectivity of the α1-PDX/hf reactive site loop for furin (K(i), 600 pM) but not for other proprotein convertase family members or other unrelated endoproteases. Kinetic studies show that α1-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, α1-PDX/hf partitions with equal probability to undergo proteolysis by furin at the C-terminal side of the reactive center - Arg355-Ile-Pro-Arg358-↓ 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 α1-PDX/hf to differentially inhibit members of the proprotein convertase family. Finally, we propose a structural model of the α1-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.
AB - 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 α1-antitrypsin Portland (α1-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 Hisand FLAG-tagged α1-PDX (α1- PDX/hf) revealed the selectivity of the α1-PDX/hf reactive site loop for furin (K(i), 600 pM) but not for other proprotein convertase family members or other unrelated endoproteases. Kinetic studies show that α1-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, α1-PDX/hf partitions with equal probability to undergo proteolysis by furin at the C-terminal side of the reactive center - Arg355-Ile-Pro-Arg358-↓ 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 α1-PDX/hf to differentially inhibit members of the proprotein convertase family. Finally, we propose a structural model of the α1-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.
UR - http://www.scopus.com/inward/record.url?scp=0032560449&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0032560449&partnerID=8YFLogxK
U2 - 10.1073/pnas.95.13.7293
DO - 10.1073/pnas.95.13.7293
M3 - Article
C2 - 9636142
AN - SCOPUS:0032560449
VL - 95
SP - 7293
EP - 7298
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 13
ER -