title: A Novel Serpin Regulatory Mechanism: SerpinB9 IS REVERSIBLY INHIBITED BY VICINAL DISULFIDE BOND FORMATION IN THE REACTIVE CENTER LOOP.
abstract: The intracellular protease inhibitor Sb9 (SerpinB9) is a regulator of the cytotoxic lymphocyte protease GzmB (granzyme B). Although GzmB is primarily involved in the destruction of compromised cells, recent evidence suggests that it is also involved in lysosome-mediated death of the cytotoxic lymphocyte itself. Sb9 protects the cell from GzmB released from lysosomes into the cytosol. Here we show that reactive oxygen species (ROS) generated within cytotoxic lymphocytes by receptor stimulation are required for lyososomal permeabilization and release of GzmB into the cytosol. Importantly, ROS also inactivate Sb9 by oxidizing a highly conserved cysteine pair (P1-P1' in rodents and P1'-P2' in other mammals) in the reactive center loop to form a vicinal disulfide bond. Replacement of the P4-P3' reactive center loop residues of the prototype serpin, SERPINA1, with the P4-P5' residues of Sb9 containing the cysteine pair is sufficient to convert SERPINA1 into a ROS-sensitive GzmB inhibitor. Conversion of the cysteine pair to serines in either human or mouse Sb9 results in a functional serpin that inhibits GzmB and resists ROS inactivation. We conclude that ROS sensitivity of Sb9 allows the threshold for GzmB-mediated suicide to be lowered, as part of a conserved post-translational homeostatic mechanism regulating lymphocyte numbers or activity. It follows, for example, that antioxidants may improve NK cell viability in adoptive immunotherapy applications by stabilizing Sb9.
authors.affiliation: From the Department of Biochemistry & Molecular Biology, Biomedicine Discovery Institute, Monash University Clayton, Clayton, Victoria 3800 Australia and.
authors.forename: Matthew S J
authors.initials: MS
authors.lastname: Mangan
authors.affiliation: From the Department of Biochemistry & Molecular Biology, Biomedicine Discovery Institute, Monash University Clayton, Clayton, Victoria 3800 Australia and.
authors.forename: Catherina H
authors.initials: CH
authors.lastname: Bird
authors.affiliation: From the Department of Biochemistry & Molecular Biology, Biomedicine Discovery Institute, Monash University Clayton, Clayton, Victoria 3800 Australia and.
authors.forename: Dion
authors.initials: D
authors.lastname: Kaiserman
authors.affiliation: From the Department of Biochemistry & Molecular Biology, Biomedicine Discovery Institute, Monash University Clayton, Clayton, Victoria 3800 Australia and.
authors.forename: Anthony Y
authors.initials: AY
authors.lastname: Matthews
authors.affiliation: From the Department of Biochemistry & Molecular Biology, Biomedicine Discovery Institute, Monash University Clayton, Clayton, Victoria 3800 Australia and.
authors.forename: Corinne
authors.initials: C
authors.lastname: Hitchen
authors.affiliation: From the Department of Biochemistry & Molecular Biology, Biomedicine Discovery Institute, Monash University Clayton, Clayton, Victoria 3800 Australia and.
authors.forename: David L
authors.initials: DL
authors.lastname: Steer
authors.affiliation: the Department of Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University Parkville, Parkville, Victoria 3052, Australia.
authors.forename: Philip E
authors.initials: PE
authors.lastname: Thompson
authors.affiliation: From the Department of Biochemistry & Molecular Biology, Biomedicine Discovery Institute, Monash University Clayton, Clayton, Victoria 3800 Australia and phil.bird@monash.edu.
authors.forename: Phillip I
authors.identifier: http://orcid.org/0000-0002-6695-606X
authors.initials: PI
authors.lastname: Bird
mesh_terms: D000818:Animals; D017209:Apoptosis; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D003553:Cystine / Q000737:chemistry; D053804:Granzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D007694:Killer Cells, Natural / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D008247:Lysosomes / Q000201:enzymology / Q000378:metabolism; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D050505:Mutant Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015843:Serpins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D050505:Mutant Proteins; D010446:Peptide Fragments; D017382:Reactive Oxygen Species; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C102316:SERPINB9 protein, human; D015843:Serpins; D003553:Cystine; C509213:GZMB protein, human; D053804:Granzymes; C509214:Gzmb protein, mouse; C106610:Serpinb9 protein, mouse
keywords: SerpinB9; T cell; granzyme B; immunotherapy; lysosomal membrane permeabilization; lysosome; natural killer cells (NK cells); protease; reactive oxygen species (ROS); serpin