Molecular Details of Retinal Guanylyl Cyclase 1/GCAP-2 Interaction

The rod outer segment guanylyl cyclase 1 (ROS-GC1) is an essential component of photo-transduction in the retina. In the light-induced signal cascade, membrane-bound ROS-GC1 restores cGMP levels in the dark in a calcium-dependent manner. With decreasing calcium concentration in the intracellular compartment, ROS-GC1 is activated via the intracellular site by guanylyl cyclase-activating proteins (GCAP-1/-2). Presently, the exact activation mechanism is elusive. To obtain structural insights into the ROS-GC1 regulation by GCAP-2, chemical cross-linking/mass spectrometry studies using GCAP-2 and three ROS-GC1 peptides were performed in the presence and absence of calcium. The majority of cross-links were identified with the C-terminal lobe of GCAP-2 and a peptide comprising parts of ROS-GC1's catalytic domain and C-terminal extension. Consistently with the cross-linking results, surface plasmon resonance and fluorescence measurements confirmed specific binding of this ROS-GC peptide to GCAP-2 with a dissociation constant in the low micromolar range. These results imply that a region of the catalytic domain of ROS-GC1 can participate in the interaction with GCAP-2. Additional binding surfaces upstream of the catalytic domain, in particular the juxtamembrane domain, can currently not be excluded

Widely Used Commercial ELISA Does Not Detect Precursor of Haptoglobin2, but Recognizes Properdin as a Potential Second Member of the Zonulin Family

Background: There is increasing evidence for the role of impaired intestinal permeability in obesity and associated metabolic diseases. Zonulin is an established serum marker for intestinal permeability and identical to pre-haptoglobin2. Here, we aimed to investigate the relationship between circulating zonulin and metabolic traits related to obesity. Methods: Serum zonulin was measured by using a widely used commercial ELISA kit in 376 subjects from the metabolically well-characterized cohort of Sorbs from Germany. In addition, haptoglobin genotype was determined in DNA samples from all study subjects. Results: As zonulin concentrations did not correlate to the haptoglobin genotypes, we investigated the specificity of the zonulin ELISA assay using antibody capture experiments, mass spectrometry, and Western blot analysis. Using serum samples that gave the highest or lowest ELISA signals, we detected several proteins that are likely to be captured by the antibody in the present kit. However, none of these proteins corresponds to pre-haptoglobin2. We used increasing concentrations of recombinant pre-haptoglobin2 and complement C3 as one of the representative captured proteins and the ELISA kit did not detect either. Western blot analysis using both the polyclonal antibodies used in this kit and monoclonal antibodies rose against zonulin showed a similar protein recognition pattern but with different intensity of detection. The protein(s) measured using the ELISA kit was (were) significantly increased in patients with diabetes and obesity and correlated strongly with markers of the lipid and glucose metabolism. Combining mass spectrometry and Western blot analysis using the polyclonal antibodies used in the ELISA kit, we identified properdin as another member of the zonulin family. Conclusion: Our study suggests that the zonulin ELISA does not recognize pre-haptoglobin2, rather structural (and possibly functional) analog proteins belonging to the mannose-associated serine protease family, with properdin being the most likely possible candidate

Acidosis-Induced Changes in Proteome Patterns of the Prostate Cancer-Derived Tumor Cell Line AT‑1

Under various pathological conditions, such as inflammation, ischemia and in solid tumors, physiological parameters (local oxygen tension or extracellular pH) show distinct tissue abnormalities (hypoxia and acidosis). For tumors, the prevailing microenvironment exerts a strong influence on the phenotype with respect to proliferation, invasion, and metastasis formation and therefore influences prognosis. In this study, we investigate the impact of extracellular metabolic acidosis (pH 7.4 versus 6.6) on the proteome patterns of a prostate cancer-derived tumor cell type (AT-1) using isobaric labeling and LC–MS/MS analysis. In total, 2710 proteins were identified and quantified across four biological replicates, of which seven were significantly affected with changes >50% and used for validation. Glucose transporter 1 and farnesyl pyrophosphatase were found to be down-regulated after 48 h of acidic treatment, and metallothionein 2A was reduced after 24 h and returned to control values after 48 h. After 24 and 48 h at pH 6.6, glutathione S transferase A3 and NAD­(P)H dehydrogenase 1, cellular retinoic acid-binding protein 2, and Na-bicarbonate transporter 3 levels were found to be increased. The changes in protein levels were confirmed by transcriptome and functional analyses. In addition to the experimental in-depth investigation of proteins with changes >50%, functional profiling (statistical enrichment analysis) including proteins with changes >20% revealed that acidosis upregulates GSH metabolic processes, citric acid cycle, and respiratory electron transport. Metabolism of lipids and cholesterol biosynthesis were downregulated. Our data comprise the first comprehensive report on acidosis-induced changes in proteome patterns of a tumor cell line

A Simple Cross-Linking/Mass Spectrometry Workflow for Studying System-wide Protein Interactions

We present a cross-linking/mass spectrometry workflow for performing proteome-wide cross-linking analyses within 1 week. The workflow is based on the commercially available mass spectrometry-cleavable cross-linker disuccinimidyl dibutyric urea and can be employed by every lab having access to a mass spectrometer with tandem mass spectrometry capabilities. We provide an updated version 2.0 of the freeware software tool MeroX, available at www.StavroX.com, that allows us to conduct fully automated and reliable studies delivering insights into protein–protein interaction networks and protein conformations at the proteome level. We exemplify our optimized workflow for mapping protein–protein interaction networks in Drosophila melanogaster embryos on a system-wide level. From cross-linked Drosophila embryo extracts, we detected 29931 cross-link spectrum matches corresponding to 7436 unique cross-linked residues in biological triplicate experiments at a 1% false discovery rate. Among these, 1611 interprotein cross-linking sites were identified and yielded valuable information about protein–protein interactions. The 5825 remaining intraprotein cross-links yield information about the conformational landscape of proteins in their cellular environment.ISSN:1520-6882ISSN:0003-270

Dissociation Behavior of a TEMPO-Active Ester Cross-Linker for Peptide Structure Analysis by Free Radical Initiated Peptide Sequencing (FRIPS) in Negative ESI-MS

We have synthesized a homobifunctional amine-reactive cross-linking reagent, containing a TEMPO (2,2,6,6-tetramethylpiperidine-1-oxy) and a benzyl group (Bz), termed TEMPO-Bz-linker, to derive three-dimensional structural information of proteins. The aim for designing this novel cross-linker was to facilitate the mass spectrometric analysis of cross-linked products by free radical initiated peptide sequencing (FRIPS). In an initial study, we had investigated the fragmentation behavior of TEMPO-Bz-derivatized peptides upon collision activation in (+)-electrospray ionization collision-induced dissociation tandem mass spectrometry (ESI-CID-MS/MS) experiments. In addition to the homolytic NO-C bond cleavage FRIPS pathway delivering the desired odd-electron product ions, an alternative heterolytic NO-C bond cleavage, resulting in even-electron product ions mechanism was found to be relevant. The latter fragmentation route clearly depends on the protonation of the TEMPO-Bz-moiety itself, which motivated us to conduct (-)-ESI-MS, CID-MS/MS, and MS3 experiments of TEMPO-Bz-cross-linked peptides to further clarify the fragmentation behavior of TEMPO-Bz-peptide molecular ions. We show that the TEMPO-Bz-linker is highly beneficial for conducting FRIPS in negative ionization mode as the desired homolytic cleavage of the NO-C bond is the major fragmentation pathway. Based on characteristic fragments, the isomeric amino acids leucine and isoleucine could be discriminated. Interestingly, we observed pronounced amino acid side chain losses in cross-linked peptides if the cross-linked peptides contain a high number of acidic amino acids

Protein Interaction Network of Human Protein Kinase D2 Revealed by Chemical Cross-Linking/Mass Spectrometry

We investigated the interaction network of human PKD2 in the cytosol and in Golgi-enriched subcellular protein fractions by an affinity enrichment strategy combined with chemical cross-linking/mass spectrometry (MS). Analysis of the subproteomes revealed the presence of distinct proteins in the cytosolic and Golgi fractions. The covalent fixation of transient or weak interactors by chemical cross-linking allowed capturing interaction partners that might otherwise disappear during conventional pull-down experiments. In total, 31 interaction partners were identified for PKD2, including glycogen synthase kinase-3 beta (GSK3B), 14–3–3 protein gamma (YWHAG), and the alpha isoform of 55 kDa regulatory subunit B of protein phosphatase 2A (PPP2R2A). Remarkably, the entire seven-subunit Arp2/3 complex (ARPC1B, ARPC2, ARPC3, ARPC4, ARPC5, ACTR3, ACTR2) as well as ARPC1A and ARPC5L, which are putative substitutes of ARPC1B and ARPC5, were identified. We provide evidence of a direct protein–protein interaction between PKD2 and Arp2/3. Our findings will pave the way for further structural and functional studies of PKD2 complexes, especially the PKD2/Arp2/3 interaction, to elucidate the role of PKD2 for transport processes at the <i>trans</i>-Golgi network. Data are available via ProteomeXchange with identifiers PXD003909 (enrichment from cytosolic fractions), PXD003913 (enrichment from Golgi fractions), and PXD003917 (subcellular fractionation)

Automated Assignment of MS/MS Cleavable Cross-Links in Protein 3D-Structure Analysis

CID-MS/MS cleavable cross-linkers hold an enormous potential for an automated analysis of cross-linked products, which is essential for conducting structural proteomics studies. The created characteristic fragment ion patterns can easily be used for an automated assignment and discrimination of cross-linked products. To date, there are only a few software solutions available that make use of these properties, but none allows for an automated analysis of cleavable cross-linked products. The MeroX software fills this gap and presents a powerful tool for protein 3D-structure analysis in combination with MS/MS cleavable cross-linkers. We show that MeroX allows an automatic screening of characteristic fragment ions, considering static and variable peptide modifications, and effectively scores different types of cross-links. No manual input is required for a correct assignment of cross-links and false discovery rates are calculated. The self-explanatory graphical user interface of MeroX provides easy access for an automated cross-link search platform that is compatible with commonly used data file formats, enabling analysis of data originating from different instruments. The combination of an MS/MS cleavable cross-linker with a dedicated software tool for data analysis provides an automated workflow for 3D-structure analysis of proteins. MeroX is available at www.StavroX.com

Monitoring Solution Structures of Peroxisome Proliferator-Activated Receptor β/δ upon Ligand Binding

<div><p>Peroxisome proliferator-activated receptors (PPARs) have been intensively studied as drug targets to treat type 2 diabetes, lipid disorders, and metabolic syndrome. This study is part of our ongoing efforts to map conformational changes in PPARs in solution by a combination of chemical cross-linking and mass spectrometry (MS). To our best knowledge, we performed the first studies addressing solution structures of full-length PPAR-β/δ. We monitored the conformations of the ligand-binding domain (LBD) as well as full-length PPAR-β/δ upon binding of two agonists. (Photo-) cross-linking relied on (i) a variety of externally introduced amine- and carboxyl-reactive linkers and (ii) the incorporation of the photo-reactive amino acid <i>p</i>-benzoylphenylalanine (Bpa) into PPAR-β/δ by genetic engineering. The distances derived from cross-linking experiments allowed us to monitor conformational changes in PPAR-β/δ upon ligand binding. The cross-linking/MS approach proved highly advantageous to study nuclear receptors, such as PPARs, and revealed the interplay between DBD (DNA-binding domain) and LDB in PPAR-β/δ. Our results indicate the stabilization of a specific conformation through ligand binding in PPAR-β/δ LBD as well as full-length PPAR-β/δ. Moreover, our results suggest a close distance between the <i>N</i>- and <i>C</i>-terminal regions of full-length PPAR-β/δ in the presence of GW1516. Chemical cross-linking/MS allowed us gaining detailed insights into conformational changes that are induced in PPARs when activating ligands are present. Thus, cross-linking/MS should be added to the arsenal of structural methods available for studying nuclear receptors.</p></div

High-resolution 3D-structures of PPAR-β/δ.

<p>(A) X-ray structure of PPAR-β/δ LBD (pdb 3TKM), bound to agonist GW0742; The activation function helix 2 (AF2, helix 12) is shown in red, the flexible Ω-loop is shown in orange; the amino acids replaced by Bpa are shown as green sticks; the ligand GW0742 is shown in stick representation in magenta. Helices containing amino acids that are involved in cross-linking are colored (helix 1: light blue; helix 2: light pink; helix 4: pale cyan; helix 8: pale green; helix 10: wheat; helix 11: pale yellow). (B) NMR structure of PPAR-β/δ DBD (pdb 2ENV).</p