Human SERPINB9 blocking peptide

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.

Granzyme-mediated cell death is the major pathway for cytotoxic lymphocytes to kill virus-infected and tumor cells. In humans, five different granzymes (i.e. GrA, GrB, GrH, GrK, and GrM) are known that all induce cell death. Expression of intracellular serine protease inhibitors (serpins) is one of the mechanisms by which tumor cells evade cytotoxic lymphocyte-mediated killing. Intracellular expression of SERPINB9 by tumor cells renders them resistant to GrB-induced apoptosis. In contrast to GrB, however, no physiological intracellular inhibitors are known for the other four human granzymes. In the present study, we show that SERPINB4 formed a typical serpin-protease SDS-stable complex with both recombinant and native human GrM. Mutation of the P2-P1-P1' triplet in the SERPINB4 reactive center loop completely abolished complex formation with GrM and N-terminal sequencing revealed that GrM cleaves SERPINB4 after P1-Leu. SERPINB4 inhibited GrM activity with a stoichiometry of inhibition of 1.6 and an apparent second order rate constant of 1.3x10(4) M(-1) s(-1). SERPINB4 abolished cleavage of the macromolecular GrM substrates alpha-tubulin and nucleophosmin. Overexpression of SERPINB4 in tumor cells inhibited recombinant GrM-induced as well as NK cell-mediated cell death and this inhibition depended on the reactive center loop of the serpin. As SERPINB4 is highly expressed by squamous cell carcinomas, our results may represent a novel mechanism by which these tumor cells evade cytotoxic lymphocyte-induced GrM-mediated cell death.