Involvement of post-translational modification of neuronal plasticity-related proteins in hyperalgesia revealed by a proteomic analysis

To clarify roles of an endogenous pain modulatory system of the central nervous system (CNS) in hyperalgesia, we tried to identify qualitative and quantitative protein changes by a proteomic analysis using an animal model of hyperalgesia. Specifically, we first induced functional hyperalgesia on male Wistar rats by repeated cold stress (specific alternation of rhythm in temperature, SART). We then compared proteomes of multiple regions of CNS and the dorsal root ganglion between the hyperalgetic rats and non-treated ones by 2-D PAGE in the pI range of 4.0-7.0. We found that SART changed the proteomes prominently in the mesencephalon and cerebellum. We thus analyzed the two brain regions in more detail using gels with narrower pI ranges. As a result, 29 and 23 protein spots were significantly changed in the mesencephalon and the cerebellum, respectively. We successfully identified 12 protein spots by a MALDI-TOF/TOF MS and subsequent protein database searching. They included unc-18 protein homolog 67K, collapsin response mediator protein (CRMP)-2 and CRMP-4, which were reported to be involved in neurotransmitter release or axon elongation. Interestingly, mRNA expression levels of these three proteins were not changed significantly by the induction of hyperalgesia. Instead, we found that the detected changes in the protein spots are caused by the post-translational modification (PTM) of proteolysis or phosphorylation. Taken together, development of the hyperalgesia would be linked to PTM of these three CNS proteins. PTM regulation may be one of the useful ways to treat hyperalgesia.     

Pupylated proteins in Corynebacterium glutamicum revealed by MudPIT analysis

In a manner similar to ubiquitin, the prokaryotic ubiquitin‐like protein (Pup) has been shown to target proteins for degradation via the proteasome in mycobacteria. However, not all actinobacteria possessing the Pup protein also contain a proteasome. In this study, we set out to study pupylation in the proteasome‐lacking non‐pathogenic model organism Corynebacterium glutamicum. A defined pup deletion mutant of C. glutamicum ATCC 13032 grew aerobically as the parent strain in standard glucose minimal medium, indicating that pupylation is dispensable under these conditions. After expression of a Pup derivative carrying an aminoterminal polyhistidine tag in the Δpup mutant and Ni²⁺‐chelate affinity chromatography, pupylated proteins were isolated. Multidimensional protein identification technology (MudPIT) and MALDI‐TOF‐MS/MS of the elution fraction unraveled 55 proteins being pupylated in C. glutamicum and 66 pupylation sites. Similar to mycobacteria, the majority of pupylated proteins are involved in metabolism or translation. Our results define the first pupylome of an actinobacterial species lacking a proteasome, confirming that other fates besides proteasomal degradation are possible for pupylated proteins.     

Comprehensive quantitative proteome analysis of 20S proteasome subtypes from rat liver by isotope coded affinity tag and 2-D gel-based approaches

Quantitative protein profiling is an essential part of proteomics and requires technologies that accurately, reproducibly, and comprehensively identify and quantify proteins. Over the past years, many quantitative proteomic methods have been developed. Here, 20S proteasome subtypes isolated from rat were compared by four approaches based on the combination of isotope-coded affinity tag (ICAT), 2-DE, LC and ESI and MALDI MS: (i) 2-DE, (ii) ICAT/2-DE MALDI-MS, (iii) ICAT/LC-ESI-MS, (iv) ICAT/LC-MALDI-MS. A definite qualitative advantage of 2-DE gels was the separation of all known protein species, the identification of cysteine sulfoxide of alpha-4 (RC6-IS) and N-terminal acetylation of several subunits. Furthermore, quantitative differences between the standard subunits beta-2, and beta-5 and their immunosubunits were only detected by 2-DE image analysis revealing a higher replacement of standard- by immuno-beta-subunits in subtype IV. It was obvious that for relative quantification only protein spot and mass peaks with a certain level of intensity displayed acceptable values of SD. However, ICAT in conjunction with LC/MALDI-MS was the most accurate method for quantification. The experimental data of this investigation are accessible via http://www.mpiib-berlin.mpg.de/2D-PAGE/.     

Quantitative analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced proteome alterations in 5L rat hepatoma cells using isotope-coded protein labels

In an effort to contribute to a better understanding of the hepatic toxicity of the ubiquitous environmental pollutant and hepatocarcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a comprehensive quantitative proteome analysis was performed on 5L rat hepatoma cells exposed to 1 nM TCDD for 8 h. Changes in the abundances of individual protein species in TCDD-treated cells as compared to untreated cells were analysed using the nongel-based isotope-coded protein label (ICPL) method [Schmidt, A., Kellermann, J., Lottspeich, F., Proteomics 2005, 5, 4-15]. 89 proteins were identified as up- or down-regulated by TCDD. For the majority of the altered proteins, an impact of TCDD on their abundance had not been known before. Due to the physicochemical properties or the translational regulation of a large number of the affected proteins, their alteration would have escaped detection by gel-based methods for proteome analysis and by standard mRNA expression profiling, respectively. The identified proteins with TCDD-altered abundance include several proteins implicated in cell cycle regulation, growth factor signalling and the control of apoptosis. The results thus provide new starting-points for the investigation of specific aspects of the toxicity and carcinogenicity of dioxin in liver.     

Evaluation of the accuracy of protein quantification using isotope TMPP‐labeled peptides

N‐succinimidyloxycarbonylmethyl tris(2,4,6‐trimethoxyphenyl) phosphonium bromide (TMPP‐Ac‐OSu) reacts rapidly, mildly, and specifically with the N‐terminals of proteins and peptides. Thus, it can be developed as an ideal isotope‐coded tag to be used in quantitative proteomics. Here, we present a strategy for light and heavy TMPP‐based quantitative proteomic analysis, in which peptides in a mixture can be quantified using an on‐tip TMPP derivatization approach. To demonstrate the accuracy of this strategy, light and heavy TMPP‐labeled peptides were combined at different ratios and subsequently analyzed by LC‐MS/MS. The MS spectra and scatter plots show that peptide and protein ratios were both consistent with the mixed ratios. We observed a linear correlation between protein ratios and the predicted ratios. In comparison with SILAC method, the TMPP labeling method produced similarly accurate quantitative results with low CVs. In conclusion, our results suggest that this isotope‐coded TMPP method achieved accurate quantification and compatibility with IEF‐based separation. With the inherent advantages of TMPP derivatization, we believe that it holds great promise for future applications in quantitative proteomics analysis.     

Rapid proteomic remodeling of cardiac tissue caused by total body ionizing radiation

Accidental nuclear scenarios lead to environmental contamination of unknown level. Immediate radiation‐induced biological responses that trigger processes leading to adverse health effects decades later are not well understood. A comprehensive proteomic analysis provides a promising means to identify and quantify the initial damage after radiation exposure. Early changes in the cardiac tissue of C57BL/6 mice exposed to total body irradiation were studied, using a dose relevant to both intentional and accidental exposure (3 Gy gamma ray). Heart tissue protein lysates were analyzed 5 and 24 h after the exposure using isotope‐coded protein labeling (ICPL) and 2‐dimensional difference‐in‐gel‐electrophoresis (2‐D DIGE) proteomics approaches. The differentially expressed proteins were identified by LC‐ESI‐MS‐MS. Both techniques showed similar functional groups of proteins to be involved in the initial injury. Pathway analyses indicated that total body irradiation immediately induced biological responses such as inflammation, antioxidative defense, and reorganization of structural proteins. Mitochondrial proteins represented the protein class most sensitive to ionizing radiation. The proteins involved in the initial damage processes map to several functional categories involving cardiotoxicity. This prompts us to propose that these early changes are indicative of the processes that lead to an increased risk of cardiovascular disease after radiation exposure.     

Effect of glycosylation on the protein pattern in 2-D-gel electrophoresis

Single proteins, when analyzed with 2-D-PAGE, often show multiple spots due to PTMs. In gels of human body fluids, the spot patterns facilitate the assignment and identification of the proteins. We analyzed serums from patients with congenital disorders of glycosylation (CDG) in which glycoproteins are strongly impacted and exhibit highly distinguishable spot patterns compared to healthy controls. We detected a typical protein pattern for alpha1-acid glycoprotein (AGP) and transferrin (Trf) that are markers for CDG. AGP contains five glycosylation sites which results in a complex microheterogeneity of the glycoprotein. On the other hand, in Trf, a glycoprotein with only two glycosylation sites, mainly biantennary complex-type-N-linked glycans are bound. We used 2-D-PAGE, MALDI-TOF-MS, and ESI-MS for the analysis of these glycoproteins and their corresponding glycans. In AGP, the heterogenic glycosylation of the different glycosylation sites is responsible for the complex spot pattern. In contrast to AGP, the protein spots of Trf cannot be explained by glycosylation. We found strong evidence that oxidation of cysteine is responsible for the spot pattern. This study contradicts the commonly accepted assumption that the multiple protein spots of Trf observed in 2-D-PAGE are due, as in AGP, to the glycosylation of the protein.     

A “fluorescence switch” technique increases the sensitivity of proteomic detection and identification of S‐nitrosylated proteins

Protein S‐nitrosylation is a reversible post‐translational modification of protein cysteines that is increasingly being considered as a signal transduction mechanism. The “biotin switch” technique marked the beginning of the study of the S‐nitrosoproteome, based on the specific replacement of the labile S‐nitrosylation by a more stable biotinylation that allowed further detection and purification. However, its application for proteomic studies is limited by its relatively low sensitivity. Thus, typical proteomic experiments require high quantities of protein extracts, which precludes the use of this method in a number of biological settings. We have developed a “fluorescence switch” technique that, when coupled to 2‐DE proteomic methodologies, allows the detection and identification of S‐nitrosylated proteins by using limited amounts of starting material, thus significantly improving the sensitivity. We have applied this methodology to detect proteins that become S‐nitrosylated in endothelial cells when exposed to S‐nitroso‐L ‐cysteine, a physiological S‐nitrosothiol, identifying already known S‐nitrosylation targets, as well as proteins that are novel targets. This “fluorescence switch” approach also allowed us to identify several proteins that are denitrosylated by thioredoxin in cytokine‐activated RAW264.7 (murine macrophage) cells. We believe that this method represents an improvement in order to approach the identification of S‐nitrosylated proteins in physiological conditions.     

A new approach for detecting C‐terminal amidation of proteins and peptides by mass spectrometry in conjunction with chemical derivatization

We describe a mass spectrometric method for distinguishing between free and modified forms of the C‐terminal carboxyl group of peptides and proteins, in combination with chemical approaches for the isolation of C‐terminal peptides and site‐specific derivatization of the C‐terminal carboxyl group. This method could most advantageously be exploited to discriminate between peptides having C‐terminal carboxyl groups in the free (COOH) and amide (CONH₂) forms by increasing their mass difference from 1 to 14 Da by selectively converting the free carboxyl group into methylamide (CONHCH₃). This method has been proven to be applicable to peptides containing aspartic and glutamic acids, because all the carboxyl groups except the C‐terminal one are inert to derivatization, according to oxazolone chemistry. The efficiency of the method is illustrated by a comparison of the peaks of processed peptides obtained from a mixture of adrenomedullin, calcitonin, and BSA. Among these components of the mixture, only the C‐terminal peptide of BSA exhibited the mass shift of 13 Da upon treatment, eventually unambiguously validating the C‐terminal amide structures of adrenomedullin and calcitonin. The possibility of extending this method for the analysis of C‐terminal PTMs is also discussed.     

Novel eye-specific calmodulin methylation characterized by protein mapping in Drosophila melanogaster

Post-translational methylation of the epsilon-amino group of lysine residues regulates a number of protein functions. Calmodulin, a key modulator of intracellular calcium signaling, is methylated on lysine 115 in many species. Although the amino acid sequence of calmodulin is highly conserved in eukaryotes, it has been shown that lysine 115 is not methylated in Drosophila calmodulin and no other methylation site has been reported. In this study, we characterized in vivo modification states of Drosophila calmodulin using proteomic methodology involving the protein mapping of microdissected Drosophila tissues on 2-D gels. We found that Drosophila calmodulin was highly expressed in methylated forms in the compound eye, whereas its methylation was hardly detected in other tissues. We identified that lysine 94 located in an EF-hand III is the methylation site in Drosophila calmodulin. The predominance of methylated calmodulin in the compound eye may imply the involvement of calmodulin in photoreceptor-specific functions through methylation.     

In vivo post-translational modifications of recombinant mussel adhesive protein in insect cells

Mussel adhesive proteins (MAPs) have been suggested as promising bioadhesives for diverse application fields, including medical uses. Previously, we successfully constructed and produced a new type of functional recombinant MAP, fp-151, in a prokaryotic Escherichia coli expression system. Even though the E. coli-derived MAP showed several excellent features, such as high production yield and efficient purification, in vitro enzymatic modification is required to convert tyrosine residues to l-3,4-dihydroxyphenyl alanine (dopa) molecules for its adhesive ability, due to the intrinsic inability of E. coli to undergo post-translational modification. In this work, we produced a soluble recombinant MAP in insect Sf9 cells, which are widely used as an effective and convenient eukaryotic expression system for eukaryotic foreign proteins. Importantly, we found that insect-derived MAP contained converted dopa residues by in vivo post-translational modification. In addition, insect-derived MAP also had other post-translational modifications including phosphorylation of serine and hydroxylation of proline that originally occurred in some natural MAPs. To our knowledge, this is the first report on in vivo post-translational modifications of MAP containing dopa and other modified amino acid residues.     

Back to the future—The value of single protein species investigations

In proteomics, in the past years, there was a focus on high throughput and reaching of large numbers of identified proteins with the basic discourse of protein expression. To avoid the impression of producing pure lists attempts are usually made to correlate proteins changed in amount between two biological situations to different pathways or protein interactions. This discourse is based on two simplifications, which limit the applicability of proteomics drastically: (i) it is sufficient to quantify a protein from several enzymatic digestion products; (ii) a biological situation is sufficiently described, if a peptide with its PTM is identified, resulting in long lists of modified peptides with data amounts, which are not anymore made accessible for the reader of a publication. The elucidation of N‐terminal methylation of proteasome subunit Rpt1 in yeast by Kimura et al. (Proteomics 2013, 13, 3167–3174) , which represents the focus on one protein, shows the value of solid chemical analysis with a complete data documentation and paves the way to proteomics based on the protein speciation discourse.     

Proteomic analysis of the proliferative and secretory phases of the human endometrium: protein identification and differential protein expression

Proteomic analyses of the proliferative and secretory phases of the human endometrium were carried out to identify proteins and discover differentially expressed proteins using isotope-coded affinity tags, three stages of chromatographic separation and online tandem mass spectrometry (MS/MS). From an initial list of 346 proteins identified by ProICAT, manual inspection of MS/MS spectra and confirmatory searches pared the list down to 119 positively identified proteins. Only five of the proteins showed consistent differential expression. The utility of some of these proteins as indicators of true differential expression in the endometrium is open to discussion. The two proteins with unquestionable differential expressions in the secretory endometrium are: glutamate NMDA receptor subunit zeta 1 precursor and FRAT1. Some of the proteins that show no differential expression have previously been examined in gene-expression studies with similar conclusions.     

Regulation of the nucleocytoplasmic trafficking of viral and cellular proteins by ubiquitin and small ubiquitin-related modifiers

Nucleocytoplasmic trafficking of many cellular proteins is regulated by nuclear import/export signals as well as post-translational modifications such as covalent conjugation of ubiquitin and small ubiquitin-related modifiers (SUMOs). Ubiquitination and SUMOylation are rapid and reversible ways to modulate the intracellular localisation and function of substrate proteins. These pathways have been co-opted by some viruses, which depend on the host cell machinery to transport their proteins in and out of the nucleus. In this review, we will summarise our current knowledge on the ubiquitin/SUMO-regulated nuclear/subnuclear trafficking of cellular proteins and describe examples of viral exploitation of these pathways.     

Preparation of amino-acid-type selective isotope labeling of protein expressed in Pichia pastoris

We report the culture conditions for successful amino-acid-type selective (AATS) isotope labeling of protein expressed in Pichia pastoris (P. pastoris). Rhodostomin (Rho), a six disulfide-bonded protein expressed in P. pastoris with the correct fold, was used to optimize the culture conditions. The concentrations of [alpha-15N] selective amino acid, nonlabeled amino acids, and ammonium chloride, as well as induction time, were optimized to avoid scrambling and to increase the incorporation rate and protein yield. The optimized protocol was successfully applied to produce AATS isotope-labeled Rho. The labeling of [alpha-15N]Cys has a 50% incorporation rate, and all 12 cysteine resonances were observed in HSQC spectrum. The labeling of [alpha-15N]Leu, -Lys, and -Met amino acids has an incorporation rate greater than 65%, and the expected number of resonances in the HSQC spectra were observed. In contrast, the labeling of [alpha-15N]Asp and -Gly amino acids has a low incorporation rate and the scrambling problem. In addition, the culture condition was successfully applied to label dendroaspin (Den), a four disulfide-bonded protein expressed in P. pastoris. Therefore, the described condition should be generally applicable to other proteins produced in the P. pastoris expression system. This is the first report to present a protocol for AATS isotope labeling of protein expressed in P. pastoris for NMR study.     

Structural transformation-mediated dimerization of caspase recruitment domain revealed by the crystal structure of CARD-only protein in frog virus 3

Caspase recruitment domain (CARD)-only proteins (COPs), regulate apoptosis, inflammation, and innate immunity. They inhibit the assembly of NOD-like receptor complexes such as the inflammasome and NODosome, which are molecular complexes critical for caspase-1 activation. COPs are known to interact with either caspase-1 CARD or RIP2 CARD via a CARD-CARD interaction, and inhibit caspase-1 activation or further downstream signaling. In addition to the human COPs, Pseudo-ICE, INCA, and ICEBERG, several viruses also contain viral COPs that help them escape the host immune system. To elucidate the molecular mechanism of host immunity inhibition by viral COPs, we solved the structure of a viral COP for the first time. Our structure showed that viral COP forms a structural transformation-mediated dimer, which is unique and has not been reported in any structural study of a CARD domain. Based on the current structure, and the previously solved structures of other death domain superfamily members, we propose that structural transformation-mediated dimerization might be a new strategy for dimer assembly in the death domain superfamily.     

Interaction of transmembrane helices in ATP synthase subunit a in solution as revealed by spin label difference NMR

Subunit a in the membrane traversing F₀ sector of Escherichia coli ATP synthase is known to fold with five transmembrane helices (TMHs) with residue 218 in TMH IV packing close to residue 248 in TMH V. In this study, we have introduced a spin label probe at Cys residues substituted at positions 222 or 223 and measured the effects on the Trp ?NH indole NMR signals of the seven Trp residues in the protein. The protein was purified and NMR experiments were carried out in a chloroform-methanol-H₂O (4:4:1) solvent mixture. The spin label at positions 222 or 223 proved to broaden the signals of W231, W232, W235 and W241 located at the periplasmic ends of TMH IV and TMH V and the connecting loop between these helices. The broadening of W241 would require that the loop residues fold back on themselves in a hairpin-like structure much like it is predicted to fold in the native membrane. Placement of the spin label probe at several other positions also proved to have broadening effects on some of these Trp residues and provided additional constraints on folding of TMH IV and TMH V. The effects of the 223 probes on backbone amide resonances of subunit a were also measured by an HNCO experiment and the results are consistent with the two helices folding back on themselves in this solvent mixture. When Cys and Trp were substituted at residues 206 and 254 at the cytoplasmic ends of TMHs IV and V respectively, the W254 resonance was not broadened by the spin label at position 206. We conclude that the helices fold back on themselves in this solvent system and then pack at an angle such that the cytoplasmic ends of the polypeptide backbone are significantly displaced from each other.