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

The Chromatin Remodelling Complex B-WICH Changes the Chromatin Structure and Recruits Histone Acetyl-Transferases to Active rRNA Genes

The chromatin remodelling complex B-WICH, which comprises the William syndrome transcription factor (WSTF), SNF2h, and nuclear myosin 1 (NM1), is involved in regulating rDNA transcription, and SiRNA silencing of WSTF leads to a reduced level of 45S pre-rRNA. The mechanism behind the action of B-WICH is unclear. Here, we show that the B-WICH complex affects the chromatin structure and that silencing of the WSTF protein results in a compaction of the chromatin structure over a 200 basepair region at the rRNA promoter. WSTF knock down does not show an effect on the binding of the rRNA-specific enhancer and chromatin protein UBF, which contributes to the chromatin structure at active genes. Instead, WSTF knock down results in a reduced level of acetylated H3-Ac, in particular H3K9-Ac, at the promoter and along the gene. The association of the histone acetyl-transferases PCAF, p300 and GCN5 with the promoter is reduced in WSTF knock down cells, whereas the association of the histone acetyl-transferase MOF is retained. A low level of H3-Ac was also found in growing cells, but here histone acetyl-transferases were present at the rDNA promoter. We propose that the B-WICH complex remodels the chromatin structure at actively transcribed rRNA genes, and this allows for the association of specific histone acetyl-transferases

Open-gate mutants of the mammalian proteasome show enhanced ubiquitin-conjugate degradation

When in the closed form, the substrate translocation channel of the proteasome core particle (CP) is blocked by the convergent N termini of α-subunits. To probe the role of channel gating in mammalian proteasomes, we deleted the N-terminal tail of α3; the resulting α3ΔN proteasomes are intact but hyperactive in the hydrolysis of fluorogenic peptide substrates and the degradation of polyubiquitinated proteins. Cells expressing the hyperactive proteasomes show markedly elevated degradation of many established proteasome substrates and resistance to oxidative stress. Multiplexed quantitative proteomics revealed ∼200 proteins with reduced levels in the mutant cells. Potentially toxic proteins such as tau exhibit reduced accumulation and aggregate formation. These data demonstrate that the CP gate is a key negative regulator of proteasome function in mammals, and that opening the CP gate may be an effective strategy to increase proteasome activity and reduce levels of toxic proteins in cells

Betacoronavirus Adaptation to Humans Involved Progressive Loss of Hemagglutinin-Esterase Lectin Activity

Human beta1-coronavirus (β1CoV) OC43 emerged relatively recently through a single zoonotic introduction. Like related animal β1CoVs, OC43 uses 9-O-acetylated sialic acid as receptor determinant. β1CoV receptor binding is typically controlled by attachment/fusion spike protein S and receptor-binding/receptor-destroying hemagglutinin-esterase protein HE. We show that following OC43's introduction into humans, HE-mediated receptor binding was selected against and ultimately lost through progressive accumulation of mutations in the HE lectin domain. Consequently, virion-associated receptor-destroying activity toward multivalent glycoconjugates was reduced and altered such that some clustered receptor populations are no longer cleaved. Loss of HE lectin function was also observed for another respiratory human coronavirus, HKU1. This thus appears to be an adaptation to the sialoglycome of the human respiratory tract and for replication in human airways. The findings suggest that the dynamics of virion-glycan interactions contribute to host tropism. Our observations are relevant also to other human respiratory viruses of zoonotic origin, particularly influenza A virus

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

Betacoronavirus Adaptation to Humans Involved Progressive Loss of Hemagglutinin-Esterase Lectin Activity

Human beta1-coronavirus (β1CoV) OC43 emerged relatively recently through a single zoonotic introduction. Like related animal β1CoVs, OC43 uses 9-O-acetylated sialic acid as receptor determinant. β1CoV receptor binding is typically controlled by attachment/fusion spike protein S and receptor-binding/receptor-destroying hemagglutinin-esterase protein HE. We show that following OC43's introduction into humans, HE-mediated receptor binding was selected against and ultimately lost through progressive accumulation of mutations in the HE lectin domain. Consequently, virion-associated receptor-destroying activity toward multivalent glycoconjugates was reduced and altered such that some clustered receptor populations are no longer cleaved. Loss of HE lectin function was also observed for another respiratory human coronavirus, HKU1. This thus appears to be an adaptation to the sialoglycome of the human respiratory tract and for replication in human airways. The findings suggest that the dynamics of virion-glycan interactions contribute to host tropism. Our observations are relevant also to other human respiratory viruses of zoonotic origin, particularly influenza A virus

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

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