Spatial-proteomics reveals phospho-signaling dynamics at subcellular resolution.

Dynamic change in subcellular localization of signaling proteins is a general concept that eukaryotic cells evolved for eliciting a coordinated response to stimuli. Mass spectrometry-based proteomics in combination with subcellular fractionation can provide comprehensive maps of spatio-temporal regulation of protein networks in cells, but involves laborious workflows that does not cover the phospho-proteome level. Here we present a high-throughput workflow based on sequential cell fractionation to profile the global proteome and phospho-proteome dynamics across six distinct subcellular fractions.

High-resolution antibody array analysis of proteins from primary human keratinocytes and leukocytes.

Antibody array analysis of labeled proteomes has high throughput and is simple to perform, but validation remains challenging. Here, we used differential detergent fractionation and size exclusion chromatography in sequence for high-resolution separation of biotinylated proteins from human primary keratinocytes and leukocytes. Ninety-six sample fractions from each cell type were analyzed with microsphere-based antibody arrays and flow cytometry (microsphere affinity proteomics; MAP).

A high-throughput pipeline for validation of antibodies.

Western blotting (WB) is widely used to test antibody specificity, but the assay has low throughput and precision. Here we used preparative gel electrophoresis to develop a capture format for WB. Fractions with soluble, size-separated proteins facilitated parallel readout with antibody arrays, shotgun mass spectrometry (MS) and immunoprecipitation followed by MS (IP-MS).

MetaMass, a tool for meta-analysis of subcellular proteomics data.

We report a tool for the analysis of subcellular proteomics data, called MetaMass, based on the use of standardized lists of subcellular markers. We analyzed data from 11 studies using MetaMass, mapping the subcellular location of 5,970 proteins. Our analysis revealed large variations in the performance of subcellular fractionation protocols as well as systematic biases in protein annotation databases.

STAT1-dependent signal integration between IFNγ and TLR4 in vascular cells reflect pro-atherogenic responses in human atherosclerosis.

Signal integration between IFNγ and TLRs in immune cells has been associated with the host defense against pathogens and injury, with a predominant role of STAT1. We hypothesize that STAT1-dependent transcriptional changes in vascular cells involved in cross-talk between IFNγ and TLR4, reflect pro-atherogenic responses in human atherosclerosis. Genome-wide investigation identified a set of STAT1-dependent genes that were synergistically affected by interactions between IFNγ and TLR4 in VSMCs.

Data mining of atherosclerotic plaque transcriptomes predicts STAT1-dependent inflammatory signal integration in vascular disease.

Atherosclerotic plaque development involves multiple extra- and intra-cellular signals engaging cells from the immune system and from the vasculature. Pro-inflammatory pathways activated by interferon gamma (IFNγ) and toll-like receptor 4 (TLR4) ligands are profoundly involved in plaque formation and have been shown to involve cross-talk in all atheroma-interacting cell types leading to increased activation of signal transducer and activator of transcription-1 (STAT1) and elevated expression of pro-inflammatory mediators. Here we demonstrate that in Gene Expression Omnibus repository (GEO) deposited microarray datasets, obtained from human coronary and carotid atherosclerotic plaques, a significant increase in expression of pro-inflammatory and immunomodulatory genes can be detected.

In silico simulations of STAT1 and STAT3 inhibitors predict SH2 domain cross-binding specificity.

Signal transducers and activators of transcription (STATs) comprise a family of transcription factors that are structurally related and which participate in signaling pathways activated by cytokines, growth factors and pathogens. Activation of STAT proteins is mediated by the highly conserved Src homology 2 (SH2) domain, which interacts with phosphotyrosine motifs for specific contacts between STATs and receptors and for STAT dimerization. By generating new models for human (h)STAT1, hSTAT2 and hSTAT3 we applied comparative in silico docking to determine SH2-binding specificity of the STAT3 inhibitor stattic, and of fludarabine (STAT1 inhibitor).

STAT1 as a central mediator of IFNγ and TLR4 signal integration in vascular dysfunction.

Atherosclerosis is characterized by early endothelial dysfunction and altered vascular smooth muscle cells (VSMCs) contractility. The forming atheroma is a site of excessive production of cytokines and inflammatory ligands by various cell types that mediate inflammation and immune responses. Key factors contributing to early stages of plaque development are IFNγ and TLR4.

STAT1 as a novel therapeutical target in pro-atherogenic signal integration of IFNγ, TLR4 and IL-6 in vascular disease.

Inflammation participates importantly in host defenses against infectious agents and injury, but it also contributes to the pathophysiology of atherosclerosis. Recruitment of blood leukocytes to the injured vascular endothelium characterizes the initiation and progression of atherosclerosis and involves many inflammatory mediators, modulated by cells of both innate and adaptive immunity. The pro-inflammatory cytokine, interferon (IFN)-γ derived from T cells, is vital for both innate and adaptive immunity and is also expressed at high levels in atherosclerotic lesions.

STAT1-mediated signal integration between IFNγ and LPS leads to increased EC and SMC activation and monocyte adhesion.

Inflammation plays an important role in host defenses against infectious agents and injury, but it also contributes to the pathophysiology of atherosclerosis. Signal transducer and activated transcription 1 (STAT1) has been identified as a point of convergence for the cross talk between the pro-inflammatory cytokine interferon γ (IFNγ) and the Toll-like receptor-4 (TLR4) ligand LPS in immune cells. However, there is no information available on the role of STAT1 in TLR4-mediated progression of atherosclerosis and on potential synergism between lipopolysaccharides (LPS) and IFNγ signaling in cells from the vasculature.

Suppressor of cytokine signaling and accelerated atherosclerosis in kidney disease.

The prevalence of cardiovascular disease in patients with renal failure is extremely high and accounts for a large part of the morbidity and mortality. Inflammation participates importantly in host defense against infectious agents and injury, but also contributes to the pathophysiology of many diseases, including cardiovascular atherosclerosis, which is a main problem in patients with renal failure. Recruitment of blood leukocytes to the injured vascular endothelium characterizes the initiation and progression of atherosclerosis and involves many inflammatory mediators, modulated by cells of both innate and adaptive immunity.

Experimental model for observation of micromotion in cell culture.

It is known that the micromotion between implant and bone inhibits direct bone growth either on or into implant surfaces in vivo. Nevertheless, biocompatibility tests in vitro of biomaterials for bone/implant interfaces are mainly performed under static conditions. This work describes a dynamic, in vitro experimental simulation of the effect of mutual, small-scale implant surface-tissue displacement on adhered cells.