Analysis of the regulation of biological networks using quantitative proteomics

A protein network can be thought of as a graph with nodes and edges, where nodes represent proteins and edges represent protein-protein interactions. Neither proteins nor their interactions are stable constituents in the cell; they are constantly changing in response to external stimulation or internal programming. Changes in protein expression are regulated by transcription, translation and protein degradation, whereas protein interaction changes have been shown in focused studies to be regulated by post translational modification. To investigate the processes influencing the regulation of protein expression and interaction changes, mass spectrometry based proteomics was applied because it has two key advantages for the study of protein networks: 1) it directly detects peptides from the proteins, and so does not rely on antibodies or the generation of fusion proteins; 2) by combining mass spectrometry based proteomics with quantitative techniques, such as stable isotope labeling by amino acids in cell culture (SILAC), it is possible to quantify thousands of proteins in a single experiment. Here a systems biology approach was applied to investigate protein expression change, synthesis and degradation of proteins during cellular differentiation in two different cell lines. This allowed observing that protein expression during cellular differentiation is largely controlled by changes in the relative synthesis rate, whereas the relative degradation rate of the majority of proteins changed little. By comparing the data with previously published data of mRNA levels, there could be provide strong evidence that the generally poor correlation observed between transcript and protein levels can be explained once the protein synthesis and degradation rates are taken into account. To study how protein interactions change in response to perturbation, a novel approach was developed combining size exclusion chromatography (SEC) and protein correlation profiling (PCP)-SILAC. Stimulation with epidermal growth factor (EGF) caused 351 proteins to alter their interactions with other proteins and, interestingly, when compared to previously published phosphorylation data, these proteins tended to also have altered phosphorylation under similar experimental conditions. This approach allowed identification of protein interactions in numbers comparable to other high throughput techniques, but also enabled quantification of protein stoichiometry between proteins participating in multiple complexes.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat

Stent thrombosis is the primary cause of ST-segment elevation myocardial infarction following coronary stent implantation: a five year follow-up of the SORT OUT II study.

The widespread use of coronary stents has exposed a growing population to the risk of stent thrombosis, but the importance in terms of risk of ST-segment elevation myocardial infarctions (STEMIs) remains unclear.We studied five years follow-up data for 2,098 all-comer patients treated with coronary stents in the randomized SORT OUT II trial (mean age 63.6 yrs. 74.8% men). Patients who following stent implantation were readmitted with STEMI were included and each patient was categorized ranging from definite- to ruled-out stent thrombosis according to the Academic Research Consortium definitions. Multivariate logistic regression was performed on selected covariates to assess odds ratios (ORs) for definite stent thrombosis.85 patients (4.1%), mean age 62.7 years, 77.1% men, were admitted with a total of 96 STEMIs, of whom 60 (62.5%) had definite stent thrombosis. Notably, definite stent thrombosis was more frequent in female than male STEMI patients (81.8% vs. 56.8%, p =  .09), and in very late STEMIs (p = 0.06). Female sex (OR 3.53 [1.01-12.59]) and clopidogrel (OR 4.43 [1.03-19.01]) was associated with increased for definite stent thrombosis, whereas age, time since stent implantation, use of statins, initial PCI urgency (STEMI [primary PCI], NSTEMI/unstable angina [subacute PCI] or stable angina [elective PCI]), and glucose-lowering agents did not seem to influence risk of stent thrombosis.In a contemporary cohort of coronary stented patients, stent thrombosis was evident in more than 60% of subsequent STEMIs

Placental Sequestration of Plasmodium falciparum Malaria Parasites Is Mediated by the Interaction Between VAR2CSA and Chondroitin Sulfate A on Syndecan-1

During placental malaria, Plasmodium falciparum infected erythrocytes sequester in the placenta, causing health problems for both the mother and fetus. The specific adherence is mediated by the VAR2CSA protein, which binds to placental chondroitin sulfate (CS) on chondroitin sulfate proteoglycans (CSPGs) in the placental syncytium. However, the identity of the CSPG core protein and the cellular impact of the interaction have remain elusive. In this study we identified the specific CSPG core protein to which the CS is attached, and characterized its exact placental location. VAR2CSA pull-down experiments using placental extracts from whole placenta or syncytiotrophoblast microvillous cell membranes showed three distinct CSPGs available for VAR2CSA adherence. Further examination of these three CSPGs by immunofluorescence and proximity ligation assays showed that syndecan-1 is the main receptor for VAR2CSA mediated placental adherence. We further show that the commonly used placental choriocarcinoma cell line, BeWo, express a different set of proteoglycans than those present on placental syncytiotrophoblast and may not be the most biologically relevant model to study placental malaria. Syncytial fusion of the BeWo cells, triggered by forskolin treatment, caused an increased expression of placental CS-modified syndecan-1. In line with this, we show that rVAR2 binding to placental CS impairs syndecan-1-related Src signaling in forskolin treated BeWo cells, but not in untreated cells