A model of full-length RAGE in complex with S100B.

The receptor for advanced glycation end products (RAGE) is an immunoglobulin-type multiligand transmembrane protein expressed in numerous cell types, including the central nervous system cells. RAGE interaction with S100B, released during brain tissue damage, leads to RAGE upregulation and initialization of a spiral proinflammatory associated with different neural disorders. Here, we present the structural characterization of the hetero-oligomeric complex of the full-length RAGE with S100B, obtained by a combination of mass spectrometry-based methods and molecular modeling. We predict that RAGE functions as a tightly packed tetramer exposing a positively charged surface formed by V domains for S100B binding. Based on HDX results we demonstrate an allosteric coupling of the distal extracellular V domains and the transmembrane region, indicating a possible mechanism of signal transmission by RAGE across the membrane. Our model provides an insight into RAGE-ligand interactions, providing a basis for the rational design of the therapeutic modifiers of its activity.

Evaluation of Aptamers as Affinity Reagents for an Enhancement of SRM-Based Detection of Low-Abundance Proteins in Blood Plasma.

Selected reaction monitoring (SRM) is a mass spectrometric technique characterized by the exceptionally high selectivity and sensitivity of protein detection. However, even with this technique, the quantitative detection of low- and ultralow-abundance proteins in blood plasma, which is of great importance for the search and verification of novel protein disease markers, is a challenging task due to the immense dynamic range of protein abundance levels. One approach used to overcome this problem is the immunoaffinity enrichment of target proteins for SRM analysis, employing monoclonal antibodies. Aptamers appear as a promising alternative to antibodies for affinity enrichment. Here, using recombinant protein SMAD4 as a model target added at known concentrations to human blood plasma and SRM as a detection method, we investigated a relationship between the initial amount of the target protein and its amount in the fraction enriched with SMAD4 by an anti-SMAD4 DNA-aptamer immobilized on magnetic beads. It was found that the aptamer-based enrichment provided a 30-fold increase in the sensitivity of SRM detection of SMAD4. These results indicate that the aptamer-based affinity enrichment of target proteins can be successfully employed to improve quantitative detection of low-abundance proteins by SRM in undepleted human blood plasma.

Formation of highly stable multinuclear AgnSn clusters in zinc fingers disrupts their structure and function.

Silver (Ag(i)) binding to consensus zinc fingers (ZFs) causes Zn(ii) release inducing a gradual disruption of the hydrophobic core, followed by an overall conformational change and formation of highly stable AgnSn clusters. A compact eight-membered Ag4S4 structure formed by a CCCC ZF is the first cluster example reported for a single biological molecule. Ag(i)-induced conformational changes of ZFs can, as a consequence, affect transcriptional regulation and other cellular processes.

Enhanced oligomerization of full-length RAGE by synergy of the interaction of its domains.

The pattern recognition receptor RAGE (receptor for advanced glycation end-products) transmits proinflammatory signals in several inflammation-related pathological states, including vascular diseases, cancer, neurodegeneration and diabetes. Its oligomerization is believed to be important in signal transduction, but RAGE oligomeric structures and stoichiometries remain unclear. Different oligomerization modes have been proposed in studies involving different truncated versions of the extracellular parts of RAGE. Here, we provide basic characterization of the oligomerization patterns of full-length RAGE (including the transmembrane (TM) and cytosolic regions) and compare the results with oligomerization modes of its four truncated fragments. For this purpose, we used native mass spectrometry, analytical ultracentrifugation, and size-exclusion chromatography coupled with multi-angle light scattering. Our results confirm known oligomerization tendencies of separate domains and highlight the enhanced oligomerization properties of full-length RAGE. Mutational analyses within the GxxxG motif of the TM region show sensitivity of oligomeric distributions to the TM sequence. Using hydrogen-deuterium exchange, we mapped regions involved in TM-dependent RAGE oligomerization. Our data provide experimental evidence for the major role of the C2 and TM domains in oligomerization, underscoring synergy among different oligomerization contact regions along the RAGE sequence. These results also explain the variability of obtained oligomerization modes in RAGE fragments.

Brochosomins and other novel proteins from brochosomes of leafhoppers (Insecta, Hemiptera, Cicadellidae).

Brochosomes (BS) are secretory granules resembling buckyballs, produced intracellularly in specialized glandular segments of the Malpighian tubules and forming superhydrophobic coatings on the integuments of leafhoppers (Hemiptera, Cicadellidae). Their composition is poorly known. Using a combination of SDS-PAGE, LC-MS/MS, next-generation sequencing (RNAseq) and bioinformatics we demonstrate that the major structural component of BS of the leafhopper Graphocephala fennahi Young is a novel family of 21-40-kDa secretory proteins, referred to herein as brochosomins (BSM), apparently cross-linked by disulfide bonds. At least 28 paralogous BSM were identified in a transcriptome assembly of this species, most of which were detected in BS. Multiple additional BS-associated proteins (BSAP), possibly loosely attached to the outer and inner surfaces of BS, were also identified; some of these were glycine-, tyrosine- and proline-rich. BSM and BSAP together accounted for half of the 100 most expressed transcripts in the Malpighian tubules of G. fennahi. Except for several minor BSAP possibly related to cyclases, BSM and BSAP had no homologs among known proteins, thus representing taxonomically restricted gene families (orphans). Searching in 50 whole-body transcriptome assemblies of Hemiptera found homologs of BSM in representatives of all five families of the superfamily Membracoidea (Cicadellidae, Myerslopiidae, Aetalionidae, Membracidae, and Melizoderidae), but not in other lineages. Among the identified proteins only BSM were shared in common between all 17 surveyed leafhoppers known to produce BS. Combined CHN elemental and aminoacid analyses estimated the total protein content of BS from the integument of G. fennahi to be 60-70%.

Targeted Quantitative Screening of Chromosome 18 Encoded Proteome in Plasma Samples of Astronaut Candidates.

This work was aimed at estimating the concentrations of proteins encoded by human chromosome 18 (Chr 18) in plasma samples of 54 healthy male volunteers (aged 20-47). These young persons have been certified by the medical evaluation board as healthy subjects ready for space flight training. Over 260 stable isotope-labeled peptide standards (SIS) were synthesized to perform the measurements of proteins encoded by Chr 18. Selected reaction monitoring (SRM) with SIS allowed an estimate of the levels of 84 of 276 proteins encoded by Chr 18. These proteins were quantified in whole and depleted plasma samples. Concentration of the proteins detected varied from 10-6 M (transthyretin, P02766) to 10-11 M (P4-ATPase, O43861). A minor part of the proteins (mostly representing intracellular proteins) was characterized by extremely high inter individual variations. The results provide a background for studies of a potential biomarker in plasma among proteins encoded by Chr 18. The SRM raw data are available in ProteomeXchange repository (PXD004374).

Threonine versus isothreonine in synthetic peptides analyzed by high-resolution liquid chromatography/tandem mass spectrometry.

RATIONALE: One of the problems in proteogenomic research aimed at identification of variant peptides is the presence of peptides with amino acid isomers of different origin in the analyzed samples. Among the most challenging examples are peptides with threonine and isothreonine (homoserine) in their sequences. Indeed, the latter residue may appear in vitro as a methionine substitution during sample preparation for shotgun proteome analysis. Yet, this substitution of Met to isoThr is not encoded genetically and should be unambiguously distinguished from, e.g., point mutations in proteins that result in Met conversion to Thr. METHODS: In this work we compared tandem mass (MS/MS) spectra produced by an Orbitrap mass spectrometer of Thr- and isoThr-containing tryptic peptides and found a distinctive feature in their collisionally activated fragmentation patterns. RESULTS: Up to 84% of MS/MS spectra for peptides containing isoThr residues have been positively specified. We also studied the differences in retention times for peptides containing Thr isoforms that can be further used for their distinction. CONCLUSIONS: Threonine can be distinguished from isothreonine by its retention time and HCD fragmentation pattern, specifically relative intensity of the bn - product ion, which can be further used in proteomic research. Copyright © 2016 John Wiley & Sons, Ltd.

Spaceflight Effects on Cytochrome P450 Content in Mouse Liver.

Hard conditions of long-term manned spaceflight can affect functions of many biological systems including a system of drug metabolism. The cytochrome P450 (CYP) superfamily plays a key role in the drug metabolism. In this study we examined the hepatic content of some P450 isoforms in mice exposed to 30 days of space flight and microgravity. The CYP content was established by the mass-spectrometric method of selected reaction monitoring (SRM). Significant changes in the CYP2C29, CYP2E1 and CYP1A2 contents were detected in mice of the flight group compared to the ground control group. Within seven days after landing and corresponding recovery period changes in the content of CYP2C29 and CYP1A2 returned to the control level, while the CYP2E1 level remained elevated. The induction of enzyme observed in the mice in the conditions of the spaceflight could lead to an accelerated biotransformation and change in efficiency of pharmacological agents, metabolizing by corresponding CYP isoforms. Such possibility of an individual pharmacological response to medication during long-term spaceflights and early period of postflight adaptation should be taken into account in space medicine.

Methionine to isothreonine conversion as a source of false discovery identifications of genetically encoded variants in proteogenomics.

Searching deep proteome data for 9 NCI-60 cancer cell lines obtained earlier by Moghaddas Gholami et al. (Cell Reports, 2013) against a database from cancer genomes returned a variant tryptic peptide fragment 57-72 of molecular chaperone HSC70, in which methionine residue at 61 position is replaced by threonine, or isothreonine (homoserine), residue. However, no traces of the corresponding genetic alteration were found in the cell line genomes reported by Abaan et al. (Cancer Research, 2013). Studying on the background of this modification led us to conclude that a conversion of methionine into isothreonine resulted from iodoacetamide treatment of the probe during a sample preparation step. We found that up to 10% of methionine containing peptides experienced the above conversion for the datasets under study. The artifact was confirmed by model experiment with bovine albumin, where three of four methionine residues were partly converted to isothreonine by conventional iodoacetamide treatment. This experimental side reaction has to be taken into account when searching for genetically encoded peptide variants in the proteogenomics studies. BIOLOGICAL SIGNIFICANCE: A lot of effort is currently put into proteogenomics of cancer. Studies detect non-synonymous cancer mutations at protein level by search of high-throughput LC-MS/MS data against customized genomic databases. In such studies, much attention is paid to potential false positive identifications. Here we describe one possible cause of such false identifications, an artifact of sample preparation which mimics methionine to threonine nucleic acid-encoded variant. The methionine to isothreonine conversion should be taken into consideration for correct interpretation of proteogenomic data.