Intracellular Serine Protease Inhibitor SERPINB4 Inhibits Granzyme M-Induced Cell Death

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 1.3×104 M−1s−1. SERPINB4 abolished cleavage of the macromolecular GrM substrates α-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

Freedom of Expression: Positive or Negative Freedom? Helping Understanding the Boundaries of a Cherished Right

Freedom of expression is a key value of a liberal democracy. In the Netherlands, like other democracies, freedom of expression has become a hot topic. Events such as the assassinations of Pim Fortuyn (2002) and Theo van Gogh (2004), the Danish cartoons affaire (2005) and the Paris Charlie Hebdo shooting (2015) have ignited debates concerning the scope and limits of the right to freedom of expression. There are roughly speaking two prevalent opinions in the current heated debate about freedom of expression: either free speech is under attack or there is simply too much of it. This brings me to the following main question of the article: what are the justifications to curtail freedom of expression in a liberal democracy? I will show in this article that Isaiah Berlin´s ideas of freedom are extremely useful in exploring the boundaries of free speech. After presenting a case dealing with the boundaries of freedom of expression, I will explain the two concepts of liberty as designed by Isaiah Berlin. I will demonstrate that, generally speaking, we value freedom, when it becomes practically meaningful, when it is a condition in which people have the opportunity to become or do something else. I will analyze Berlin’s preference for negative freedom and discuss his criticism of positive freedom. Next, I will analyze whether freedom of expression is a positive or negative freedom. This outcome helps to better understand appropriate boundaries on freedom of expression

Mutation of the SERPINB4 RCL at the P2-P1-P1′ positions completely abolishes complex formation with human GrM.

<p>(<b>A</b>) A RCL-mutant of SERPINB4 was employed in which the amino acids at the putative P2(Glu³⁵³)-P1(Leu³⁵⁴)-P1′(Ser³⁵⁵) positions were mutated into P2(Gln³⁵³)-P1(Gly³⁵⁴)-P1′(Ala³⁵⁵). Purified recombinant GrM (0.9 µM), GrM-SA (0.9 µM), SERPINB4 (0.9 µM), and SERPINB4 RCL-mutant (0.9 µM) were incubated for 1 h at 37°C. All samples were treated with PNGase F, separated by SDS-PAGE, and immunoblotted for GrM. Bound antibodies were visualized using DAB. (<b>B</b>) Cell lysates of 293T cells transfected with C-terminal GFP-conjugated SERPINB4 or an empty vector (mock) were incubated with recombinant GrM (0.5 µM) for 1 h at 37°C. Subsequently, samples were immunoblotted for GFP. SERPINB4-GFP represents the full length protein, whereas SERPINB4*-GFP depicts the C-terminal cleavage product. (<b>C</b>) Schematic representation of C-terminal GFP-conjugated SERPINB4, including the GrM cleavage site after Leu³⁵⁴ at the P1-position in the RCL.</p

SERPINB4 forms a typical serpin-protease SDS-stable complex with recombinant and native human GrM.

<p>(<b>A</b>) Purified recombinant GrM (1.8 µM) and SERPINB4 (1.8 µM) were incubated for 1 h at 37°C. All samples were treated with PNGase F, separated by SDS-PAGE, and analyzed by SimplyBlue staining. (<b>B</b>) Cell lysate of the NK cell line KHYG-1 (33 µg) was incubated with recombinant SERPINB4 (0, 50, 150, and 450 ng) for 2 h at 37°C. Subsequently, samples were immunoblotted for native GrM. GrM, SERPINB4, GrM-SERPINB4 complexes, and cleaved SERPINB4 (SERPINB4*) are indicated.</p

Overexpression of SERPINB4 in HeLa cells inhibits NK cell-mediated cell death.

<p>HeLa cells stably transfected with SERPINB4 or SERPINB4 RCL-mutant were loaded with the fluorescent cell staining dye CFDA-SE and co-cultured with KHYG-1 NK cells in varying E∶T ratio's for 16 h at 37°C. Cells were stained with PI and analyzed by flow cytometry. HeLa cells were separated from KHYG-1 cells by gating for CFDA-SE positive cells. Depicted is the percentage of specific cytotoxicity (mean ± SD, <i>n</i> = 3, * <i>p</i><0.05, ** <i>p</i><0.005).</p

Overexpression of SERPINB4 in HeLa cells inhibits GrM-induced cell death.

<p>(<b>A</b>) RT-PCR analysis of stably transfected HeLa cells for SERPINB4 and GAPDH mRNA expression (upper and lower panel, respectively). (<b>B</b>) Immunoblot analysis of SERPINB4 protein expression by HeLa cells stably transfected with pcDNA3 SERPINB4, pcDNA3 SERPINB4 RCL-mutant or pcDNA3 empty vector. (<b>C</b>) HeLa cells stably transfected with SERPINB4 or SERPINB4 RCL-mutant were treated with the indicated combinations of a sublytic dose of SLO (500 ng/ml), recombinant GrM (0.5 µM), and/or recombinant GrM-SA (0.5 µM) for 16 h at 37°C. Viable cells were quantified using the methylene blue assay. Data represent the percentages of viable cells as compared to HeLa cells that overexpressed SERPINB4 RCL-mutant and were treated with buffer only, which was set as 100%. Figure represents the mean ± SD of 4 independent experiments; * <i>p</i><0.05. (<b>D</b>) HeLa cells stably transfected with SERPINB4 or SERPINB4 RCL-mutant were treated with the indicated combinations of a sublytic dose of SLO (500 ng/ml), recombinant GrM (1 µM), and/or recombinant GrM-SA (1 µM) for 20 h at 37°C. Cell viability was determined using flow cytometry, with AnnexinV and PI negative cells considered viable (mean ± S.D., <i>n</i> = 3, ** <i>p</i><0.005). (<b>E</b>) HeLa cells stably transfected with SERPINB4 or SERPINB4 RCL-mutant were treated with the indicated combinations of a sublytic dose of SLO (500 ng/ml), recombinant GrM (1 µM), and/or recombinant GrM-SA (1 µM) for 4 h at 37°C. Total cell lysates were immunoblotted using antibodies against nucleophosmin and nm23H-1 (which served as a loading control).</p