Induction des gènes pro-inflammatoires suite à l'activation de PAR-2 à la membrane nucléaire

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal

Large-scale interaction profiling of PDZ domains through proteomic peptide-phage display using human and viral phage peptidomes

The human proteome contains a plethora of short linear motifs (SLiMs) that serve as binding interfaces for modular protein domains. Such interactions are crucial for signaling and other cellular processes, but are difficult to detect because of their low to moderate affinities. Here we developed a dedicated approach, proteomic peptide-phage display (ProP-PD), to identify domain-SLiM interactions. Specifically, we generated phage libraries containing all human and viral C-terminal peptides using custom oligonucleotide microarrays. With these libraries we screened the nine PSD-95/ Dlg/ZO-1 (PDZ) domains of human Densin-180, Erbin, Scribble, and Disks large homolog 1 for peptide ligands. We identified several known and putative interactions potentially relevant to cellular signaling pathways and confirmed interactions between fulllength Scribble and the target proteins β-PIX, plakophilin-4, and guanylate cyclase soluble subunit a-2 using colocalization and coimmunoprecipitation experiments. The affinities of recombinant Scribble PDZ domains and the synthetic peptides representing the C termini of these proteins were in the 1- to 40-μM range. Furthermore, we identified several well-established host-virus protein- protein interactions, and confirmed that PDZ domains of Scribble interact with the C terminus of Tax-1 of human T-cell leukemia virus with micromolar affinity. Previously unknown putative viral protein ligands for the PDZ domains of Scribble and Erbin were also identified. Thus, we demonstrate that our ProP-PD libraries are useful tools for probing PDZ domain interactions. The method can be extended to interrogate all potential eukaryotic, bacterial, and viral SLiMs and we suggest it will be a highly valuable approach for studying cellular and pathogen-host protein-protein interactions

Alterations of beta-adrenoceptor responsiveness in postischemic myocardium after 72 h of reperfusion.

To determine the effect of a completely developed reperfused myocardial infarction model on beta-adrenoceptor responsiveness, we induced a 90-min regional ischemia followed by 72 h of reperfusion in dog hearts. Regional myocardial blood flow was determined after 60 min of ischemia using radioactive microspheres. beta-adrenoceptor density was reduced in the ischemic endocardium (95+/-16 fmol/mg) and epicardium (160+/-13 fmol/mg) compared to the nonischemic region (304+/-21 fmol/mg). beta-adrenoceptor density in the ischemic endocardium varied with the degree of collateral blood flow measured (r2=0.79, P<0.05); this relation was the opposite of that in the ischemic epicardium (r2=0.77, P<0.05). Higher levels of tissue catecholamines and G protein-coupled receptor kinase 2 (GRK2) were observed in the ischemic epicardium as compared to nonischemic tissue. Forskolin-induced adenylyl cyclase activities were depressed in both ischemic regions as compared to nonischemic region, correlating with a reduction in regional myocardial blood flow. Using forskolin stimulation as covariate, no difference in isoproterenol-induced adenylyl cyclase activity was identified in the different regions. It is concluded that cAMP production induced by beta-adrenoceptor activation is dependent upon adenylyl cyclase enzyme activity rather than beta-adrenoceptor density in the ischemic myocardium. However, the density of the beta-adrenoceptor in the viable ischemic regions can be modified by the presence of GRK2 and tissue catecholamines, an index of regional sympathetic efferent postganglionic nerve terminal activity

Apoptosis detected in the amygdala following myocardial infarction in the rat.

BACKGROUND: Myocardial infarction (MI) contains a risk factor for developing episodes of Major Depressive Disorder (MDD). Apoptosis is commonly observed in the reperfused myocardial infarcted heart, and recent findings suggest the existence of apoptosis in MDD. Cytokines, which are released by ischemic myocardium and which may induce apoptosis, have been proposed as a possible cause for MDD. METHODS: Myocardial infarction was produced in anesthetized rats by a 40-minute occlusion of the left anterior descending coronary artery followed by 72 hours of reperfusion. Determination of apoptosis was done in the amygdala, hippocampus and vermis of MI and Sham rats treated or not with pentoxyfilline (PTX), a cytokine synthesis inhibitor (10 mg/kg/day intraperitoneal). RESULTS: Compared to Sham rats, the amygdala of MI rats showed significantly reduced P13K activity, increased Bax/Bcl-2 ratio, caspase-3 activity, and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling)-positive cells. The effect of MI on apoptosis was completely reversed in presence of PTX. No statistical difference was observed in the hippocampus and the vermis in the different groups for any of the biochemical measurements. CONCLUSIONS: These results indicated that MI induce apoptosis in amygdala by a cytokine-sensitive mechanism and may explain the MDD observed following myocardial infarction

Early effects of spinal cord transection on skeletal muscle properties.

Modulation of beta-adrenergic receptor (beta-AR) activity affects muscle mass and could have a role in the reduction of muscle mass observed following spinal cord transection (Tx). The aims of this study were to examine the early acute effects of Tx on muscle mass, total and myofibrillar protein concentrations, cytochrome c oxidase activity, and beta-AR density of skeletal muscle, to ascertain if any change in muscle properties could be related to beta-AR signalling events. Female Sprague-Dawley rats (n = 33; approximately 255 g) were randomly assigned to 4 experimental groups: control 4 d, control 8 d, Tx 4 d, and Tx 8 d. A complete Tx was performed surgically at the T10 cord level. Compared with controls, muscle mass and muscle - body mass ratios decreased significantly following Tx, with no significant change observed in total and myofibrillar protein concentrations. Spinal cord Tx also resulted in a significant decrease in plantaris cytochrome c oxidase activity by 24% at Tx 4 d and 28% at Tx 8 d (p < 0.05). Beta-AR density of the lateral gastrocnemius was unchanged; however, the beta-AR density of the forelimb triceps brachii m. was found to increase after Tx. Our results suggest that changes in muscle mass and cytochrome c oxidase activity rapidly occur after Tx and do not appear to be related to changes in beta-AR density

A systematic approach to identify novel cancer drug targets using machine learning, inhibitor design and high-throughput screening

Abstract We present an integrated approach that predicts and validates novel anti-cancer drug targets. We first built a classifier that integrates a variety of genomic and systematic datasets to prioritize drug targets specific for breast, pancreatic and ovarian cancer. We then devised strategies to inhibit these anti-cancer drug targets and selected a set of targets that are amenable to inhibition by small molecules, antibodies and synthetic peptides. We validated the predicted drug targets by showing strong anti-proliferative effects of both synthetic peptide and small molecule inhibitors against our predicted targets

Discovery of short linear motif-mediated interactions through phage display of intrinsically disordered regions of the human proteome

The intrinsically disordered regions of eukaryotic proteomes are enriched in short linear motifs (SLiMs), which are of crucial relevance for cellular signaling and protein regulation; many mediate interactions by providing binding sites for peptide-binding domains. The vast majority of SLiMs remain to be discovered highlighting the need for experimental methods for their large-scale identification. We present a novel proteomic peptide phage display (ProP-PD) library that displays peptides representing the disordered regions of the human proteome, allowing direct large-scale interrogation of most potential binding SLiMs in the proteome. The performance of the ProP-PD library was validated through selections against SLiM-binding bait domains with distinct folds and binding preferences. The vast majority of identified binding peptides contained sequences that matched the known SLiM-binding specificities of the bait proteins. For SHANK1 PDZ, we establish a novel consensus TxF motif for its non-C-terminal ligands. The binding peptides mostly represented novel target proteins, however, several previously validated protein-protein interactions (PPIs) were also discovered. We determined the affinities between the VHS domain of GGA1 and three identified ligands to 40-130 mu M through isothermal titration calorimetry, and confirmed interactions through coimmunoprecipitation using full-length proteins. Taken together, we outline a general pipeline for the design and construction of ProP-PD libraries and the analysis of ProP-PD-derived, SLiM-based PPIs. We demonstrated the methods potential to identify low affinity motif-mediated interactions for modular domains with distinct binding preferences. The approach is a highly useful complement to the current toolbox of methods for PPI discovery

Large-scale interaction profiling of PDZ domains through proteomic peptide-phage display using human and viral phage peptidomes

The human proteome contains a plethora of short linear motifs (SLiMs) that serve as binding interfaces for modular protein domains. Such interactions are crucial for signaling and other cellular processes, but are difficult to detect because of their low to moderate affinities. Here we developed a dedicated approach, proteomic peptide-phage display (ProP-PD), to identify domain–SLiM interactions. Specifically, we generated phage libraries containing all human and viral C-terminal peptides using custom oligonucleotide microarrays. With these libraries we screened the nine PSD-95/Dlg/ZO-1 (PDZ) domains of human Densin-180, Erbin, Scribble, and Disks large homolog 1 for peptide ligands. We identified several known and putative interactions potentially relevant to cellular signaling pathways and confirmed interactions between full-length Scribble and the target proteins β-PIX, plakophilin-4, and guanylate cyclase soluble subunit α-2 using colocalization and coimmunoprecipitation experiments. The affinities of recombinant Scribble PDZ domains and the synthetic peptides representing the C termini of these proteins were in the 1- to 40-μM range. Furthermore, we identified several well-established host–virus protein–protein interactions, and confirmed that PDZ domains of Scribble interact with the C terminus of Tax-1 of human T-cell leukemia virus with micromolar affinity. Previously unknown putative viral protein ligands for the PDZ domains of Scribble and Erbin were also identified. Thus, we demonstrate that our ProP-PD libraries are useful tools for probing PDZ domain interactions. The method can be extended to interrogate all potential eukaryotic, bacterial, and viral SLiMs and we suggest it will be a highly valuable approach for studying cellular and pathogen–host protein–protein interactions