Tamo gdje završava drama mentaliteta započinje drama političkog trenutka. Ivan Vidić, Octopussy, HNK, Zagreb & Veliki bijeli zec, ZeKaeM

<p><b>Copyright information:</b></p><p>Taken from "Host-pathogen systems biology: logical modelling of hepatocyte growth factor and induced c-Met signal transduction"</p><p>http://www.biomedcentral.com/1752-0509/2/4</p><p>BMC Systems Biology 2008;2():4-4.</p><p>Published online 14 Jan 2008</p><p>PMCID:PMC2254585.</p><p></p

Structural Dynamics of Lys11-Selective Deubiquitinylase Cezanne‑1 during the Catalytic Cycle

Deubiquitinylating enzymes (DUBs) regulate the deubiquitinylation process of post-translationally modified proteins and thus control protein signaling in various cellular processes. The DUB Cezanne-1 catalyzes the cleavage of the iso-peptide bond of Lys11-linked polyubiquitin chains with high selectivity. Crystal structures of Cezanne-1 in different states provide important insight regarding the complex formation and global changes during the catalytic cycle but are lacking details of dynamics and control of activation. Activity-based probes are used to isolate intermediate states upon forming covalent bonds with the DUB active site. Those, however, may lead to structures that are non-native. Conformational changes of Cezanne-1, during its process of activation and proteolytic activity, are investigated using all-atom molecular dynamics (MD) simulations of the ubiquitin-free, diubiquitin-bound, and monoubiquitin-bound Cezanne-1 DUB for a total of ∼18 μs. Our results show that ubiquitin-free Cezanne-1 dynamically shuttles between catalytically competent and incompetent states which suggests that its activation is independent of substrate binding. The catalytically competent substrate-free Cezanne-1 promotes distal ubiquitin substrate access to the catalytic center. The subsequent binding of the proximal ubiquitin shifts the equilibrium toward the catalytically competent state of the dyad, thereby promoting proteolysis of the iso-peptide bond. After cleavage of the scissile bond, sequential dissociation of first the proximal ubiquitin induces the inactivation of Cezanne-1. The subsequent release of the distal ubiquitin fully reconstitutes the inactive substrate-free state of Cezanne-1. The process of activation and catalytic turnover of DUB Cezanne-1 is a multistage cycle with several critical dynamic transitions that cannot be characterized based on protein structures alone. Activity-based probes of cysteine proteases lead to non-native protein–protein contacts, which need to be resolved in order to be able to issue statements about physiological states and substrate binding

Cyld^−/− mice are protected from lethal listeriosis and severe liver pathology.

<p>(A) C57BL/6 Cyld^−/− (n = 10) and WT (n = 10) mice were infected i.v. with 5×10⁵ Lm and survival rates were monitored until day 20 p.i. (p<0.005 for WT vs. CYLD^−/− mice). One of two representative experiments is shown. (B) A macroscopic examination of livers from WT and Cyld^−/− mice showed severe haemorrhage in WT but not in Cyld^−/− mice at day 5 p.i. (C–F) Histopathology of WT (C, D) and Cyld^−/− mice (E, F) at day 5 p.i. (C, E) Immunohistochemistry with α-Lm antiserum in a WT (C) and Cyld^−/− mouse (E) (slight counterstaining with hemalum, bar 5 µm). In (C), * marks necrosis surrounded by clusters of Lm. (D, F) PAS staining of a WT (D) and Cyld^−/− (F) mouse (bar 10 µm). In (D), * indicates a large area of necrosis. In (F), * indicates a well-defined inflammatory focus. In (B–F), three mice per group were analysed and representative data are shown. The experiment was performed twice. (G) CFUs were determined in the liver of Lm-infected WT and Cyld^−/− mice at the indicated time points p.i. (* p<0.05, ** p<0.01). Data show the combined results of two independent experiments with a total of 8–9 mice per experimental group and time point. The mean of each experimental group is shown by a bar and each symbol represents one mouse. (H) The liver enzymes aspartate transaminase (AST) and alanine transaminase (ALT) were determined in serum at day 5 p.i. (* p<0.05 and ** p<0.01 for WT vs. Cyld^−/− mice). Data show the mean ± SD of 5 mice per experimental group from one of two representative experiments.</p

Inhibition of fibrin production abolished protection and increased the hepatic bacterial load of Cyld^−/− mice.

<p>(A) WB analysis of hepatic fibrin production in uninfected and infected WT and Cyld^−/− mice. GAPDH was used as loading control. (B) Quantification of fibrin (± SD) was performed from WB data of uninfected and Lm-infected WT and Cyld^−/−, respectively, which were treated with warfarin as indicated. The results present 3 mice per experimental group. (C) The survival rate of uninfected and infected mice, which were treated with warfarin as indicated, was monitored until day 10 of infection (n = 10 per experimental group). Survival of infected Cyld^−/−, uninfected Cyld^−/− mice treated with warfarin, and WT mice treated with warfarin, respectively, was significantly increased as compared to infected WT mice without warfarin treatment (p<0.001 for all groups). (D) CFUs were determined in the liver of Lm-infected WT and Cyld^−/− mice, which were treated with warfarin as indicated, at day 5 p.i. (* p<0.05, n = 5 per experimental group). Data show the mean ± SD. In (C) and (D) one of two representative experiments is shown.</p

IL-6 neutralization abolishes increased STAT3 activation, fibrin production, survival and pathogen control in Lm-infected Cyld^−/− mice.

<p>(A) The survival rates of rat IgG and α-IL-6-treated Lm-infected WT and Cyld^−/− mice (n = 7 per experimental group) are shown. The survival rate of IgG-treated Cyld^−/− mice (p<0.05) but not of the other groups was significantly increased as compared to rat IgG-treated WT mice. (B) CFUs were determined in the liver of Lm-infected rat IgG and IL-6-treated WT and Cyld^−/− mice at day 5 p.i. (n = 5 per experimental group; * p<0.05). Data show the mean ± SD from one of two representative experiments. (C) Proteins were isolated from livers of infected rat IgG and IL-6-treated WT and Cyld^−/− mice (n = 3 per experimental group) at day 5 p.i. WB analysis for CYLD, pSTAT3, fibrin and GAPDH was performed and representative data are shown. (D, E). Quantification of hepatic pSTAT3 (D) and fibrin (E) (± SD) was performed from WB data of rat IgG and α-IL-6-treated WT and Cyld^−/− mice, respectively. The results present 3 mice of each experimental group.</p

CYLD impairs IL-6 and IFN-γ production and leukocyte recruitment in listeriosis.

<p>(A) The serum concentrations of IL-10, IL-17, TNF, IFN-γ, IL-6, IL-4, and IL-2 were determined in Lm-infected WT and Cyld^−/− mice by a cytometric bead assay at day 5 p.i. (* p<0.05, ** p<0.01). Symbols represent individual mice from two representative experiments. (B–G) Quantitative RT-PCR analysis of hepatic (B–D) and splenic (E–G) IL-6, IFN-γ, and NOX2 mRNA expression. Data show the increase of the respective mRNA expression of Lm-infected over uninfected mice of the same mouse strain. Data represent the mean ± SD of 5 mice. Data from of two representative experiments are shown. (H) The number of different leukocyte populations was determined in cells isolated from livers of uninfected and Lm-infected WT and Cyld^−/− mice. Data show the mean ± SD of CD45⁺ cell populations from 5 mice per experimental group. Data from one of two representative experiments are shown (* p<0.05 and ** p<0.001 for WT vs. Cyld^−/− mice).</p

Host-pathogen systems biology: logical modelling of hepatocyte growth factor and induced c-Met signal transduction-7

<p><b>Copyright information:</b></p><p>Taken from "Host-pathogen systems biology: logical modelling of hepatocyte growth factor and induced c-Met signal transduction"</p><p>http://www.biomedcentral.com/1752-0509/2/4</p><p>BMC Systems Biology 2008;2():4-4.</p><p>Published online 14 Jan 2008</p><p>PMCID:PMC2254585.</p><p></p

Reduced activation of p65, JAK2, STAT3, p38 MAPK and fibrin production in livers of <i>Listeria</i>-infected WT mice.

<p>(A) Proteins were isolated from livers of uninfected (d0) and Lm-infected WT and Cyld^−/− mice (6 h p.i.). Protein lysates were immunoprecipitated with STAT3 and WB was performed for STAT3 and K63-linked ubiquitin. (B, C) Proteins were isolated from livers of uninfected (d0) and Lm-infected WT and Cyld^−/− mice at days 1, 3, and 5 p.i. WB were incubated with α-CLYD, α-p-p65, α-p65, α-pSTAT3, α-STAT3, α-p-p38MAPK, α-p38MAPK, and α-PAI-1 (B) and fibrin (C). GAPDH was used as loading control. Three to four mice were analysed per group and representative data from one of two independent experiments are shown. (D) Quantification of fibrin (± SD) was performed from WB data of uninfected and Lm-infected WT and Cyld^−/−, respectively. The results present 3 mice per group and time point.</p

Therapeutic Cyld siRNA treatment protects WT mice from lethal listeriosis.

<p>(A) WT and Cyld^−/− mice were i.v. infected with 5×10⁵ Lm. Infected mice were treated as indicated 24 h p.i. At day 5 p.i., proteins were isolated from the liver (n = 3 per experimental group) and CYLD, pSTAT3 fibrin, and GAPDH production was analysed by WB. (B, C, D) Quantification of CYLD (B), pSTAT3 (C) and fibrin (D) (± SD) was performed from WB data of the indicated groups. The results present 3 mice per experimental group. (E) The survival rates of Lm-infected untreated Cyld^−/− and Cyld siRNA-treated WT mice were significantly were increased as compared to untreated WT mice (p<0.01 for Cyld^−/− vs. WT mice, p<0.05 for Cyld siRNA treated WT vs. WT mice). Ten mice per group were analysed until day 10 p.i. (F) CFUs were determined in the liver at day 5 p.i. Five mice were analysed per group and data show the mean ± SD (* p<0.05, ** p<0.01). In (A–G) data from one of two representative experiments are shown. (G) Macroscopic analysis showed haemorrhage of untreated and control siRNA-treated infected WT mice. Haemorrhage was reduced in WT mice treated with Cyld siRNA and was absent from untreated Cyld^−/− mice.</p

CYLD diminishes NF-κB-dependent IL-6 production, ROS production and pathogen control of Lm-infected macrophages.

<p>(A–C) BMDM were isolate from WT and Cyld^−/− mice and stimulated with IFN-γ (100 U/ml). Indicated groups were infected with Lm (MOI of 5∶1) and treated with IKK inhibitor (10 µM for 4 h followed by 1 µM for 20 h), respectively. (A) After 24 h, the amount of intracellular Lm was determined in 1×10⁶ BMDM. (B) ROS production was analysed by flow cytometry in Lm-infected macrophages 24 h after infection. (C) The supernatant was harvested from uninfected and infected macrophages after 24 h and analysed for IL-6 by CBA. In (A–C), data show the mean ± SD of triplicate wells; * p<0.05, ** p<0.01, *** p<0.005, **** p<0.001. (D) Proteins were isolated from uninfected and Lm-infected BMDM at the indicated time points. Cells were stimulated with IFN-γ and IKK inhibitor VII as indicated. WBs were incubated with α-p-p65, α-p65, and α-GAPDH as loading control. Representative WBs from a total of three independent experiments are shown. (E) Quantification of p-p65 intensity (± SD) was performed from WB data of uninfected and Lm-infected BMDM, which were stimulated as described in (D). The results present pooled data from 3 independent experiments.</p