Knowledge based identification of essential signaling from genome-scale siRNA experiments

<p>Abstract</p> <p>Background</p> <p>A systems biology interpretation of genome-scale RNA interference (RNAi) experiments is complicated by scope, experimental variability and network signaling robustness. Over representation approaches (ORA), such as the Hypergeometric or z-score, are an established statistical framework used to associate RNA interference effectors to biologically annotated gene sets or pathways. These methods, however, do not directly take advantage of our growing understanding of the interactome. Furthermore, these methods can miss partial pathway activation and may be biased by protein complexes. Here we present a novel ORA, protein interaction permutation analysis (PIPA), that takes advantage of canonical pathways and established protein interactions to identify pathways enriched for protein interactions connecting RNAi hits.</p> <p>Results</p> <p>We use PIPA to analyze genome-scale siRNA cell growth screens performed in HeLa and TOV cell lines. First we show that interacting gene pair siRNA hits are more reproducible than single gene hits. Using protein interactions, PIPA identifies enriched pathways not found using the standard Hypergeometric analysis including the FAK <it>cytoskeletal remodeling pathway</it>. Different branches of the <it>FAK </it>pathway are distinctly essential in HeLa versus TOV cell lines while other portions are uneffected by siRNA perturbations. Enriched hits belong to protein interactions associated with cell cycle regulation, anti-apoptosis, and signal transduction.</p> <p>Conclusion</p> <p>PIPA provides an analytical framework to interpret siRNA screen data by merging biologically annotated gene sets with the human interactome. As a result we identify pathways and signaling hypotheses that are statistically enriched to effect cell growth in human cell lines. This method provides a complementary approach to standard gene set enrichment that utilizes the additional knowledge of specific interactions within biological gene sets. </p

HSV Usurps Eukaryotic Initiation Factor 3 Subunit M for Viral Protein Translation: Novel Prevention Target

Prevention of genital herpes is a global health priority. B5, a recently identified ubiquitous human protein, was proposed as a candidate HSV entry receptor. The current studies explored its role in HSV infection. Viral plaque formation was reduced by ∼90% in human cells transfected with small interfering RNA targeting B5 or nectin-1, an established entry receptor. However, the mechanisms were distinct. Silencing of nectin-1 prevented intracellular delivery of viral capsids, nuclear transport of a viral tegument protein, and release of calcium stores required for entry. In contrast, B5 silencing had no effect on these markers of entry, but inhibited viral protein translation. Specifically, viral immediate early genes, ICP0 and ICP4, were transcribed, polyadenylated and transported from the nucleus to the cytoplasm, but the viral transcripts did not associate with ribosomes or polysomes in B5-silenced cells. In contrast, immediate early gene viral transcripts were detected in polysome fractions isolated from control cells. These findings are consistent with sequencing studies demonstrating that B5 is eukaryotic initiation factor 3 subunit m (eIF3m). Although B5 silencing altered the polysome profile of cells, silencing had little effect on cellular RNA or protein expression and was not cytotoxic, suggesting that this subunit is not essential for host cellular protein synthesis. Together these results demonstrate that B5 plays a major role in the initiation of HSV protein translation and could provide a novel target for strategies to prevent primary and recurrent herpetic disease

Phosphorylation and Activation of the Plasma Membrane Na+/H+ Exchanger (NHE1) during Osmotic Cell Shrinkage

The Na+/H+ Exchanger isoform 1 (NHE1) is a highly versatile, broadly distributed and precisely controlled transport protein that mediates volume and pH regulation in most cell types. NHE1 phosphorylation contributes to Na+/H+ exchange activity in response to phorbol esters, growth factors or protein phosphatase inhibitors, but has not been observed during activation by osmotic cell shrinkage (OCS). We examined the role of NHE1 phosphorylation during activation by OCS, using an ideal model system, the Amphiuma tridactylum red blood cell (atRBC). Na+/H+ exchange in atRBCs is mediated by an NHE1 homolog (atNHE1) that is 79% identical to human NHE1 at the amino acid level. NHE1 activity in atRBCs is exceptionally robust in that transport activity can increase more than 2 orders of magnitude from rest to full activation. Michaelis-Menten transport kinetics indicates that either OCS or treatment with the phosphatase inhibitor calyculin-A (CLA) increase Na+ transport capacity without affecting transport affinity (Km = 44 mM) in atRBCs. CLA and OCS act non-additively to activate atNHE1, indicating convergent, phosphorylation-dependent signaling in atNHE1 activation. In situ 32P labeling and immunoprecipitation demonstrates that the net phosphorylation of atNHE1 is increased 4-fold during OCS coinciding with a more than 2-order increase in Na+ transport activity. This is the first reported evidence of increased NHE1 phosphorylation during OCS in any vertebrate cell type. Finally, liquid chromatography and mass spectrometry (LC-MS/MS) analysis of atNHE1 immunoprecipitated from atRBC membranes reveals 9 phosphorylated serine/threonine residues, suggesting that activation of atNHE1 involves multiple phosphorylation and/or dephosphorylation events

High-sensitivity Orbitrap mass analysis of intact macromolecular assemblies

The analysis of intact protein assemblies in native-like states by mass spectrometry offers a wealth of information on their biochemical and biophysical properties. Here we show that the Orbitrap mass analyzer can be used to measure protein assemblies of molecular weights approaching one megadalton with sensitivity down to the detection of single ions. Minor instrumental modifications enabled the measurement of various protein assemblies with outstanding mass-spectral resolution

Structural characterization of Ni–Al (111) interface by surface x-ray absorption spectroscopy

The evolution of the Ni/Al(111) interface has been studied in situ by x-ray absorption spectroscopy at the Ni–K edge. Ni films were deposited on bulk Al(111) with thickness ranging from 2 monolayers (ML) up to 30 ML. The aim was to determine the diffusion length of Ni and the phases that have formed. Ni diffused spontaneously at room temperature to a depth that we estimated to be of the order of 11 ML. The structure of Ni/Al(111) mixed interface has been characterized by x-ray absorption spectroscopy. With respect to previous studies on Al(110) the first phase formed on Al(111) is Al3Ni2-like instead of AlNi-like. Accordingly to previous observations, an AlNi3 phase forms on top of Al3Ni2 after the deposition of the first few monolayers. We propose that the pure Ni growth observed after deposition of 11 ML is due to the presence of the AlNi3 aluminide that acts as a diffusion barrier preventing deeper Ni penetration into Al at room temperature

High-sensitivity Orbitrap mass analysis of intact macromolecular assemblies

The analysis of intact protein assemblies in native-like states by mass spectrometry offers a wealth of information on their biochemical and biophysical properties. Here we show that the Orbitrap mass analyzer can be used to measure protein assemblies of molecular weights approaching one megadalton with sensitivity down to the detection of single ions. Minor instrumental modifications enabled the measurement of various protein assemblies with outstanding mass-spectral resolution

High resolution proteome analysis of ctyoglobulins using Fourier transforme-ion cyclotron resonance mass spectrometry.

Cryoglobulins are cold-precipitable serum immunoglobulins associated with a number of infectious, autoimmune and neoplastic disorders such as hepatitis C, Waldenstrom's macroglobulinemia, multiple myeloma, chronic lymphocytic leukemia, and rheumatoid arthritis. The mechanism(s) of cryoprecipitation has remained obscure hitherto, which has prompted recent intensive efforts on the identification of cryoglobulin components. In the present study, two-dimensional gel electrophoresis (2-DE) combined with high resolution Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometry has been applied as a powerful approach for the analysis of cryoglobulins. While FT-ICR mass spectrometry has been shown to enable the high resolution identification and structure analysis of biopolymers using both electrospray (ESI) and matrix-assisted laser desorption ionization (MALDI), the recently developed MALDI-FT-ICR source is shown here to provide high (sub-ppm) mass determination accuracy and isotopic fine structure as particular advantages in the identification of proteins. The main protein components in a serum cryoprecipitate from a patient with hepatitis C virus (HCV) infection and presenting type II cryogobulinemia are immunoglobulin (Ig)M and IgG which were identified by MALDI-FT-ICR MS analysis after separation by 2-DE as mu- and gamma-heavy chains, kappa- and lambda-light chains, and J-chains. Furthermore, complementarity determining regions CDR1 and CDR2 from monoclonal IgM-RF variable region (V)L were directly identified using accurate mass determinations by FT-ICR-MS. The presence of Spalpha was ascertained as an IgM-associated protein in the serum cryoprecipitate from a patient with HCV infection

Determination of Phosphorus-, Copper-, and Zinc-Containing Human Brain Proteins by LA-ICPMS and MALDI-FTICR-MS

Human brain proteins containing phosphorus, copper, and zinc were detected directly in protein spots in gels of a human brain sample after separation by two-dimensional gel electrophoresis using laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS). A powerful laser ablation system with cooled laser ablation chamber was coupled to a double-focusing sector field ICPMS. The separated protein spots in 2D gels were fast screened using the optimized microanalytical LA-ICPMS technique measured at medium mass resolution with a focused laser beam (wavelength, 213 nm; diameter of laser crater, 50 mum; and laser power density, 3 x 10(9) W cm(-2)) with respect to selected three essential elements. Of 176 protein spots in 2D gel from a human brain sample, phosphorus, copper, and zinc were detected in 31, 43, and 49 protein spots, respectively. For the first time, uranium as a naturally occurring radioactive element was found in 20 selected protein spots. The detection limits for P, S, Cu, Zn and U were determined in singular protein spots with 0.0013, 1.29, 0.029, 0.063, and 0.000 01 mg g(-1), respectively. A combination of LA-ICPMS with matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS) was applied for the identification of selected protein spots from human brain protein separated by 2D gel electrophoresis. Combining MALDI-FTICR-MS for the structure analysis of metal- and phosphorus-containing human brain proteins with LA-ICPMS, the direct analysis of heteroelements on separated proteins in 2D gels can be performed. For quantification of analytical LA-ICPMS data, the number of sulfur atoms per protein (and following the sulfur concentration) determined by MALDI-FTICR-MS was used for internal standardization. From the known sulfur concentration in protein, the concentration of other heteroelements was calculated. In addition, the number of phosphorylation and the phosphorylation sites of phosphorylated proteins in the human brain sample detected by LA-ICPMS were determined by MALDI-FTICR-MS. This technique allows the study of posttranslational modifications in human brain proteins

High resolution proteome analysis of cryoglobulins using Fourier transform-ion cyclotron resonance mass spectrometry

Cryoglobulins are cold-precipitable serum immunoglobulins associated with a number of infectious, autoimmune and neoplastic disorders such as hepatitis C, Waldenstrom's macroglobulinemia, multiple myeloma, chronic lymphocytic leukemia, and rheumatoid arthritis. The mechanism(s) of cryoprecipitation has remained obscure hitherto, which has prompted recent intensive efforts on the identification of cryoglobulin components. In the present study, two-dimensional gel electrophoresis (2-DE) combined with high resolution Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometry has been applied as a powerful approach for the analysis of cryoglobulins. While FT-ICR mass spectrometry has been shown to enable the high resolution identification and structure analysis of biopolymers using both electrospray (ESI) and matrix-assisted laser desorption ionization (MALDI), the recently developed MALDI-FT-ICR source is shown here to provide high (sub-ppm) mass determination accuracy and isotopic fine structure as particular advantages in the identification of proteins. The main protein components in a serum cryoprecipitate from a patient with hepatitis C virus (HCV) infection and presenting type II cryogobulinemia are immunoglobulin (Ig)M and IgG which were identified by MALDI-FT-ICR MS analysis after separation by 2-DE as mu- and gamma-heavy chains, kappa- and lambda-light chains, and J-chains. Furthermore, complementarity determining regions CDR1 and CDR2 from monoclonal IgM-RF variable region (V)L were directly identified using accurate mass determinations by FT-ICR-MS. The presence of Spalpha was ascertained as an IgM-associated protein in the serum cryoprecipitate from a patient with HCV infection