Additional file 3: Table S2. of The role of the two-component systems Cpx and Arc in protein alterations upon gentamicin treatment in Escherichia coli

Proteins regulated upon gentamicin treatment. (XLSX 210 kb

Additional file 4: Table S3. of The role of the two-component systems Cpx and Arc in protein alterations upon gentamicin treatment in Escherichia coli

Correlation studies between intensities of CpxA or ArcA and other detected proteins including WT condition, Cpx induction, and gentamicin treatment. (XLSX 496 kb

Cofilin phosphorylation regulates cofilin aggregate formation in endothelial cells.

<p>A) Endothelial cells were transfected with cofilin-DsRed2 (left panel) and activated with thrombin (1 U/ml) for 10 minutes (middle panel). Thrombin-treated cells were then incubated with Rho-kinase inhibitor (20 µM; Y27632) for 45 minutes. Aggregates of cofilin-DsRed2 were completely dissolved after thrombin treatment (middle panel) and reappeared again after incubation with Y27632 (right panel). B) Endothelial cells transfected with cofilin-EGFP were incubated with Y27632 (20 µM) for 45 minutes. Spike-like structures of cofilin-EGFP (left panel) disappeared after subsequent stimulation of cells with thrombin for 10 minutes (middle panel). Right panel, bar diagram,% of total cells with spike-like structures of cofilin-EGFP. Values are mean ± SEM of three independent experiments; *, <i>p</i><0.05.</p

Cofilin dephosphorylation stimulates cofilin oligomer formation in platelets.

<p>Effects of Y27632-treatment and thrombin stimulation. Human washed platelets were incubated with Y27632 (20 µM) or solvent (water) for 20 minutes and then stimulated by thrombin (0.5 U/ml). Platelets were lysed in Laemmli buffer for immunoblot analysis with anti-phospho-cofilin antibody or with lysis buffer containing BMOE (0.1 mM) for immunoblot analysis of cofilin oligomer with anti-cofilin antibody. A) Graphic representation of changes of cofilin phosphorylation in thrombin-stimulated platelets in the presence or absence of Y27632. Values are the mean ± SD for four independent experiments. Insert: representative anti-phospho cofilin immunoblot. B) Graphic representation of changes of cofilin oligomer (65 kDa) formation in thrombin-stimulated platelets in the presence or absence of Y27632. Values are the mean ± SD for four independent experiments. Insert: representative immunoblot of cofilin oligomer (65 kDa) formation.</p

The 65 kDa cofilin oligomer in endothelial cells and platelets does not contain actin.

<p>A) Lysates of cross-linked endothelial cells (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071769#pone-0071769-g001" target="_blank">Figure 1A</a>) were immunoblotted with an anti-cofilin antibody or an anti-actin antibody. A cofilin oligomer (∼65 kDa; indicated by asterisk) was observed in BMOE and BMH but not in DMSO-treated endothelial cells (right). An actin band of 43 kDa was observed in all cell lysates. No actin could be detected at the corresponding position of the cofilin oligomer (indicated by asterisk; left). B) Equimolar concentrations (20 µM) of cofilin and actin were incubated in PBS for one hour at room temperature, then cross-linked with BMOE and immunoblotted with anti-cofilin or anti-actin antibody. Both anti-cofilin antibody (left panel) and anti-actin antibody (right panel) were able to recognize a band of cross-linked actin-cofilin heterodimer (∼62 kDa; indicated by asterisk). C) Platelets and endothelial cells transfected with FLAG-tagged cofilin were incubated with BMOE, and cross-linked cofilin oligomers were immunoprecipitated with anti-cofilin antibody (platelets) or anti-FLAG antibody (endothelial cells). The immunoprecipitated cross-linked cofilin oligomer in platelets is indicated by an asterisk (left lane; left panel), which is absent in DMSO-treated platelets (middle lane). The cross-linked oligomer in BMOE-treated endothelial cell lysate (EC) is indicated by an asterisk (right lane). The immunoprecipitated cross-linked FLAG-tagged cofilin oligomer in transfected endothelial cells samples was observed in BMOE- but not in DMSO- treated cells (right panel; indicated by asterisk). D) The immunoprecipitated samples from platelets and endothelial cells were immunoblotted with an anti-actin antibody. No actin band could be detected at the corresponding position (∼65 kDa) of the cofilin oligomers (indicated by asterisk).</p

Low Dose Proteasome Inhibition Affects Alternative Splicing

Protein degradation by the ubiquitin proteasome system ensures controlled degradation of structural proteins, signaling mediators, and transcription factors. Inhibition of proteasome function by specific proteasome inhibitors results in dose-dependent cellular effects ranging from induction of apoptosis to protective stress responses. The present study seeks to identify nuclear regulators mediating the protective stress response to low dose proteasome inhibition. Primary human endothelial cells were treated with low doses of the proteasome inhibitor MG132 for 2 h, and proteomic analysis of nuclear extracts was performed. Using a 2-D differential in gel electrophoresis (DIGE) approach, we identified more than 24 splice factors to be differentially regulated by low dose proteasome inhibition. In particular, several isoforms of hnRNPA1 were shown to be increased, pointing toward altered posttranslational modification of hnRNPA1 upon proteasome inhibition. Elevated levels of splice factors were associated with a different alternative splicing pattern in response to proteasome inhibition as determined by Affymetrix exon array profiling. Of note, we observed alternative RNA processing for stress associated genes such as caspases and heat shock proteins. Our study provides first evidence that low dose proteasome inhibition affects posttranscriptional regulation of splice factors and early alternative splicing events

Low Dose Proteasome Inhibition Affects Alternative Splicing

Protein degradation by the ubiquitin proteasome system ensures controlled degradation of structural proteins, signaling mediators, and transcription factors. Inhibition of proteasome function by specific proteasome inhibitors results in dose-dependent cellular effects ranging from induction of apoptosis to protective stress responses. The present study seeks to identify nuclear regulators mediating the protective stress response to low dose proteasome inhibition. Primary human endothelial cells were treated with low doses of the proteasome inhibitor MG132 for 2 h, and proteomic analysis of nuclear extracts was performed. Using a 2-D differential in gel electrophoresis (DIGE) approach, we identified more than 24 splice factors to be differentially regulated by low dose proteasome inhibition. In particular, several isoforms of hnRNPA1 were shown to be increased, pointing toward altered posttranslational modification of hnRNPA1 upon proteasome inhibition. Elevated levels of splice factors were associated with a different alternative splicing pattern in response to proteasome inhibition as determined by Affymetrix exon array profiling. Of note, we observed alternative RNA processing for stress associated genes such as caspases and heat shock proteins. Our study provides first evidence that low dose proteasome inhibition affects posttranscriptional regulation of splice factors and early alternative splicing events

Endogenous and recombinant human His-cofilin has an intracellular disulphide bond.

<p>A) Recombinant human His-cofilin, platelet, and endothelial cell lysates were subjected to SDS-PAGE under reducing (with ß-mercapto-ethanol; +ßM) and non-reducing conditions (without ß-mercapto-ethanol; -ßM) and then immunoblotted with anti-cofilin antibody. Monomeric cofilin showed higher electrophoretic mobility under non-reducing conditions than under reducing conditions. Moreover, oligomers of different molecular masses of recombinant His-cofilin but not of endogenous cofilin were observed under non-reducing condition, suggesting the involvement of intermolecular disulphide bonding in <i>in vitro</i> oligomerization. B) Recombinant human cofilin (with and without His-tag) were subjected to SDS-PAGE under reducing and non-reducing conditions and analyzed by Coomassie blue staining. Both types of recombinant cofilin demonstrate a higher electrophoretic mobility under non-reducing conditions as compared to reducing conditions.</p

DataSheet_1_Immunoproteasomes control activation of innate immune signaling and microglial function.pdf

Microglia are the resident immune cells of the central nervous system (CNS) and play a major role in the regulation of brain homeostasis. To maintain their cellular protein homeostasis, microglia express standard proteasomes and immunoproteasomes (IP), a proteasome isoform that preserves protein homeostasis also in non-immune cells under challenging conditions. The impact of IP on microglia function in innate immunity of the CNS is however not well described. Here, we establish that IP impairment leads to proteotoxic stress and triggers the unfolded and integrated stress responses in mouse and human microglia models. Using proteomic analysis, we demonstrate that IP deficiency in microglia results in profound alterations of the ubiquitin-modified proteome among which proteins involved in the regulation of stress and immune responses. In line with this, molecular analysis revealed chronic activation of NF-κB signaling in IP-deficient microglia without further stimulus. In addition, we show that IP impairment alters microglial function based on markers for phagocytosis and motility. At the molecular level IP impairment activates interferon signaling promoted by the activation of the cytosolic stress response protein kinase R. The presented data highlight the importance of IP function for the proteostatic potential as well as for precision proteolysis to control stress and immune signaling in microglia function.</p

Dynamic adaptation of myocardial proteome during heart failure development

<div><p>Heart failure (HF) development is characterized by huge structural changes that are crucial for disease progression. Analysis of time dependent global proteomic adaptations during HF progression offers the potential to gain deeper insights in the disease development and identify new biomarker candidates. Therefore, hearts of TAC (transverse aortic constriction) and sham mice were examined by cardiac MRI on either day 4, 14, 21, 28, 42, and 56 after surgery (n = 6 per group/time point). At each time point, proteomes of the left (LV) and right ventricles (RV) of TAC and sham mice were analyzed by mass spectrometry (MS). In TAC mice, systolic LV heart function worsened from day 4 to day 14, remained on a stable level from day 14 to day 42, and showed a further pronounced decline at day 56. MS analysis identified in the LV 330 and in RV 246 proteins with altered abundance over time (TAC vs. sham, fc≥±2). Functional categorization of proteins disclosed the time-dependent alteration of different pathways. Heat shock protein beta-7 (HSPB7) displayed differences in abundance in tissue and serum at an early stage of HF. This study not only provides an overview of the time dependent molecular alterations during transition to HF, but also identified HSPB7 as a novel blood biomarker candidate for the onset of cardiac remodeling.</p></div