A novel serpin regulatory mechanism

SERPINB9 is reversibly inhibited by vicinal disulfide bond formation in the reactive center loop

Matthew S. J. Mangan, Catherina H. Bird, Dion Kaiserman, Anthony Y. Matthews, Corinne Hitchen, David L. Steer, Philip E. Thompson, Phillip I. Bird

Research output: Contribution to journalArticleResearchpeer-review

5 Citations (Scopus)

Abstract

The intracellular protease inhibitor, SERPINB9 (Sb9), is a regulator of the cytotoxic lymphocyte protease, granzyme B (GzmB). Although primarily involved in the destruction of compromised cells, recent evidence suggests that GzmB 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; 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.
Original languageEnglish
Pages (from-to)3626-3638
Number of pages13
JournalJournal of Biological Chemistry
Volume291
Issue number7
DOIs
Publication statusPublished - 12 Feb 2016

Keywords

  • immunotherapy
  • lysosome
  • natural killer cells (NK cells)
  • protease
  • reactive oxygen species (ROS)
  • serpin
  • T cell
  • SerpinB9
  • granzyme B
  • lysosomal membrane permeabilization

Cite this

Mangan, Matthew S. J. ; Bird, Catherina H. ; Kaiserman, Dion ; Matthews, Anthony Y. ; Hitchen, Corinne ; Steer, David L. ; Thompson, Philip E. ; Bird, Phillip I. / A novel serpin regulatory mechanism : SERPINB9 is reversibly inhibited by vicinal disulfide bond formation in the reactive center loop. In: Journal of Biological Chemistry. 2016 ; Vol. 291, No. 7. pp. 3626-3638.
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abstract = "The intracellular protease inhibitor, SERPINB9 (Sb9), is a regulator of the cytotoxic lymphocyte protease, granzyme B (GzmB). Although primarily involved in the destruction of compromised cells, recent evidence suggests that GzmB 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; 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.",
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A novel serpin regulatory mechanism : SERPINB9 is reversibly inhibited by vicinal disulfide bond formation in the reactive center loop. / Mangan, Matthew S. J.; Bird, Catherina H.; Kaiserman, Dion; Matthews, Anthony Y.; Hitchen, Corinne; Steer, David L.; Thompson, Philip E.; Bird, Phillip I.

In: Journal of Biological Chemistry, Vol. 291, No. 7, 12.02.2016, p. 3626-3638.

Research output: Contribution to journalArticleResearchpeer-review

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T2 - SERPINB9 is reversibly inhibited by vicinal disulfide bond formation in the reactive center loop

AU - Mangan, Matthew S. J.

AU - Bird, Catherina H.

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AU - Matthews, Anthony Y.

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N2 - The intracellular protease inhibitor, SERPINB9 (Sb9), is a regulator of the cytotoxic lymphocyte protease, granzyme B (GzmB). Although primarily involved in the destruction of compromised cells, recent evidence suggests that GzmB 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; 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.

AB - The intracellular protease inhibitor, SERPINB9 (Sb9), is a regulator of the cytotoxic lymphocyte protease, granzyme B (GzmB). Although primarily involved in the destruction of compromised cells, recent evidence suggests that GzmB 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; 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.

KW - immunotherapy

KW - lysosome

KW - natural killer cells (NK cells)

KW - protease

KW - reactive oxygen species (ROS)

KW - serpin

KW - T cell

KW - SerpinB9

KW - granzyme B

KW - lysosomal membrane permeabilization

UR - http://www.ncbi.nlm.nih.gov/pubmed/26670609

U2 - 10.1074/jbc.M115.699298

DO - 10.1074/jbc.M115.699298

M3 - Article

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SP - 3626

EP - 3638

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 1083-351X

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