Evidence That G-quadruplex DNA Accumulates in the Cytoplasm and Participates in Stress Granule Assembly in Response to Oxidative Stress.

Cells engage numerous signaling pathways in response to oxidative stress that together repair macromolecular damage or direct the cell toward apoptosis. As a result of DNA damage, mitochondrial DNA or nuclear DNA has been shown to enter the cytoplasm where it binds to "DNA sensors," which in turn initiate signaling cascades. Here we report data that support a novel signaling pathway in response to oxidative stress mediated by specific guanine-rich sequences that can fold into G-quadruplex DNA (G4DNA). In response to oxidative stress, we demonstrate that sequences capable of forming G4DNA appear at increasing levels in the cytoplasm and participate in assembly of stress granules. Identified proteins that bind to endogenous G4DNA in the cytoplasm are known to modulate mRNA translation and participate in stress granule formation. Consistent with these findings, stress granule formation is known to regulate mRNA translation during oxidative stress. We propose a signaling pathway whereby cells can rapidly respond to DNA damage caused by oxidative stress. Guanine-rich sequences that are excised from damaged genomic DNA are proposed to enter the cytoplasm where they can regulate translation through stress granule formation. This newly proposed role for G4DNA provides an additional molecular explanation for why such sequences are prevalent in the human genome.

Expression and purification of HER2 extracellular domain proteins in Schneider2 insect cells.

Overexpression of human epidermal growth factor receptor 2 (HER2/ErbB2/Neu) results in ligand independent activation of kinase signaling and is found in about 30% of human breast cancers, and is correlated with a more aggressive tumor phenotype. The HER2 extracellular domain (ECD) consists of four domains - I, II, III and IV. Although the role of each domain in the dimerization and activation of the receptor has been extensively studied, the role of domain IV (DIV) is not clearly understood yet. In our previous studies, we reported peptidomimetic molecules inhibit HER2:HER3 heterodimerization. In order to study the binding interactions of peptidomimetics with HER2 DIV in detail, properly folded recombinant HER2 protein in pure form is important. We have expressed and purified HER2 ECD and DIV proteins in the Drosophila melanogaster Schneider2 (S2) cell line. Using the commercial Drosophila expression system (DES), we transfected S2 cells with plasmids designed to direct the expression of secreted recombinant HER2 ECD and DIV proteins. The secreted proteins were purified from the conditioned medium by filtration, ultrafiltration, dialysis and nickel affinity chromatography techniques. The purified HER2 proteins were then analyzed using Western blot, mass spectrometry and circular dichroism (CD) spectroscopy.

Analysis of Protein-protein Interaction Interface between Yeast Mitochondrial Proteins Rim1 and Pif1 Using Chemical Cross-linking Mass Spectrometry.

Defining protein-protein contacts is a challenging problem and cross-linking is a promising solution. Here, we present a case of mitochondrial single strand binding protein Rim1 and helicase Pif1, an interaction first observed in immuno-affinity pull-down from yeast cells using Pif1 bait. We found that only the short succinimidyl-diazirine cross-linker or formaldehyde captured the interaction between recombinant Rim1 and Pif1. In addition, Pif1 needed to be stripped of its N-terminal and C-terminal domains, and Rim1's C-terminus needed to be modified for the cross-linked product to become visible. Our report is an example of a non-trivial analysis, where a previously identified stable interaction escapes initial capture with cross-linking agents and requires substantial modification to recombinant proteins and fine-tuning of the mass spectrometry-based methods for the cross-links to become detectable. We used high resolution mass spectrometry to detect the cross-linked peptides. A 1:1 mixture of 15N and 14N-labeled Rim1 was used to validate the cross-links by their mass shift in the LC-MS profiles. Two sites on Rim1 were confirmed: 1) the N-terminus, and 2) the K29 residue. Performing cross-linking with a K29A variant visibly reduced the cross-linked product. Further, K29A-Rim1 showed a five-fold lower affinity to single stranded DNA compared to wild-type Rim1. Both the K29A variant and wild type Rim1 showed similar degrees of stimulation of Pif1 helicase activity. We propose structural models of the Pif1-Rim1 interaction and discuss its functional significance. Our work represents a non-trivial protein-protein interface analysis and demonstrates utility of short and non-specific cross-linkers.

XLPM: efficient algorithm for the analysis of protein-protein contacts using chemical cross-linking mass spectrometry.

BACKGROUND: Chemical cross-linking is used for protein-protein contacts mapping and for structural analysis. One of the difficulties in cross-linking studies is the analysis of mass-spectrometry data and the assignment of the site of cross-link incorporation. The difficulties are due to higher charges of fragment ions, and to the overall low-abundance of cross-link species in the background of linear peptides. Cross-linkers non-specific at one end, such as photo-inducible diazirines, may complicate the analysis further. In this report, we design and validate a novel cross-linked peptide mapping algorithm (XLPM) and compare it to StavroX, which is currently one of the best algorithms in this class. RESULTS: We have designed a novel cross-link search algorithm -XLPM - and implemented it both as an online tool and as a downloadable archive of scripts. We designed a filter based on an observation that observation of a b-ion implies observation of a complimentary y-ion with high probability (b-y filter). We validated the b-y filter on the set of linear peptides from NIST library, and demonstrate that it is an effective way to find high-quality mass spectra. Next, we generated cross-linked data from an ssDNA binding protein, Rim1with a specific cross-linker disuccinimidyl suberate, and a semi-specific cross-linker NHS-Diazirine, followed by analysis of the cross-linked products by nanoLC-LTQ-Orbitrap mass spectrometry. The cross-linked data were searched by XLPM and StavroX and the performance of the two algorithms was compared. The cross-links were mapped to the X-ray structure of Rim1 tetramer. Analysis of the mixture of NHS-Diazirine cross-linked ¹5N and ¹4N-labeled Rim1 tetramers yielded ¹5N-labeled to ¹4N-labeled cross-linked peptide pairs, corresponding to C-terminus-to-N-terminus cross-linking, demonstrating interaction between different two Rim1 tetramers. Both XLPM and StavroX were successful in identification of this interaction, with XLPM leading to a better annotation of higher-charged fragments. We also put forward a new method of estimating specificity and sensitivity of identification of a cross-linked residue in the case of a non-specific cross-linker. CONCLUSIONS: The novel cross-link mapping algorithm, XLPM, considerably improves the speed and accuracy of the analysis compared to other methods. The quality selection filter based on b-to-y ions ratio proved to be an effective way to select high quality cross-linked spectra.

Large Scale Chemical Cross-linking Mass Spectrometry Perspectives.

The spectacular heterogeneity of a complex protein mixture from biological samples becomes even more difficult to tackle when one's attention is shifted towards different protein complex topologies, transient interactions, or localization of PPIs. Meticulous protein-by-protein affinity pull-downs and yeast-two-hybrid screens are the two approaches currently used to decipher proteome-wide interaction networks. Another method is to employ chemical cross-linking, which gives not only identities of interactors, but could also provide information on the sites of interactions and interaction interfaces. Despite significant advances in mass spectrometry instrumentation over the last decade, mapping Protein-Protein Interactions (PPIs) using chemical cross-linking remains time consuming and requires substantial expertise, even in the simplest of systems. While robust methodologies and software exist for the analysis of binary PPIs and also for the single protein structure refinement using cross-linking-derived constraints, undertaking a proteome-wide cross-linking study is highly complex. Difficulties include i) identifying cross-linkers of the right length and selectivity that could capture interactions of interest; ii) enrichment of the cross-linked species; iii) identification and validation of the cross-linked peptides and cross-linked sites. In this review we examine existing literature aimed at the large-scale protein cross-linking and discuss possible paths for improvement. We also discuss short-length cross-linkers of broad specificity such as formaldehyde and diazirine-based photo-cross-linkers. These cross-linkers could potentially capture many types of interactions, without strict requirement for a particular amino-acid to be present at a given protein-protein interface. How these shortlength, broad specificity cross-linkers be applied to proteome-wide studies? We will suggest specific advances in methodology, instrumentation and software that are needed to make such a leap.