Analysis of transglutaminase protein substrates by functional proteomics

Transglutaminases are calcium-dependent enzymes that catalyze a post-translational modification of proteins through the formation of epsilon -(gamma-glutamyl)lysine bonds. Although specific roles for transglutaminases have been described, recent findings have provided evidence that dysregulation of transglutaminases may contribute to many pathological processes including celiac disease and neurodegenerative diseases. A crucial step in the elucidation of biological and pathological roles of transglutaminases requires the identification of protein substrates. A strategy based on a functional proteomic analysis was set up using two well-characterized biotinylated transglutaminase substrates as affinity probes: 5-(biotinamido)pentylamine and the synthetic biotinylated peptide TVQQEL, the amino- and acyl-donor probes, respectively. A pool of known tissue type transglutaminase protein substrates was selected in order to test the procedure. Results obtained in this paper indicate that the whole strategy can be successfully applied in order to identify transglutaminases protein substrates as well as the amino acid site sensitive toward enzyme activity.     

PROTEOMER: A workflow‐optimized laboratory information management system for 2‐D electrophoresis‐centered proteomics

In recent years proteomics became increasingly important to functional genomics. Although a large amount of data is generated by high throughput large‐scale techniques, a connection of these mostly heterogeneous data from different analytical platforms and of different experiments is limited. Data mining procedures and algorithms are often insufficient to extract meaningful results from large datasets and therefore limit the exploitation of the generated biological information. In our proteomic core facility, which almost exclusively focuses on 2‐DE/MS‐based proteomics, we developed a proteomic database custom tailored to our needs aiming at connecting MS protein identification information to 2‐DE derived protein expression profiles. The tools developed should not only enable an automatic evaluation of single experiments, but also link multiple 2‐DE experiments with MS‐data on different levels and thereby helping to create a comprehensive network of our proteomics data. Therefore the key feature of our “PROTEOMER” database is its high cross‐referencing capacity, enabling integration of a wide range of experimental data. To illustrate the workflow and utility of the system, two practical examples are provided to demonstrate that proper data cross‐referencing can transform information into biological knowledge.     

A combined 15N tracing/proteomics study in Brassica napus reveals the chronology of proteomics events associated with N remobilisation during leaf senescence induced by nitrate limitation or starvation

Our goal was to identify the leaf proteomic changes which appeared during N remobilisation that were associated or not associated with senescence of oilseed rape in response to contrasting nitrate availability. Remobilisation of N and leaf senescence status were followed using ¹⁵N tracing, patterns of chlorophyll level, total protein content and a molecular indicator based on expression of senescence‐associated gene 12/Cab genes. Three phases associated with N remobilisation were distinguished. Proteomics revealed that 55 proteins involved in metabolism, energy, detoxification, stress response, proteolysis and protein folding, were significantly induced during N remobilisation. Four proteases were specifically identified. FtsH, a chloroplastic protease, was induced transiently during the early stages of N remobilisation. Considering the dynamics of N remobilisation, chlorophyll and protein content, the pattern of FtsH expression indicated that this protease could be involved in the degradation of chloroplastic proteins. Aspartic protease increased at the beginning of senescence and was maintained at a high level, implicating this protease in proteolysis during the course of leaf senescence. Two proteases, proteasome beta subunit A1 and senescence‐associated gene 12, were induced and continued to increase during the later phase of senescence, suggesting that these proteases are more specifically involved in the proteolysis processes occurring at the final stages of leaf senescence.     

Archaeal N-terminal protein maturation commonly involves N-terminal acetylation: a large-scale proteomics survey

We present the first large-scale survey of N-terminal protein maturation in archaea based on 873 proteomically identified N-terminal peptides from the two haloarchaea Halobacterium salinarum and Natronomonas pharaonis. The observed protein maturation pattern can be attributed to the combined action of methionine aminopeptidase and N-terminal acetyltransferase and applies to cytosolic proteins as well as to a large fraction of integral membrane proteins. Both N-terminal maturation processes primarily depend on the amino acid in penultimate position, in which serine and threonine residues are over represented. Removal of the initiator methionine occurs in two-thirds of the haloarchaeal proteins and requires a small penultimate residue, indicating that methionine aminopeptidase specificity is conserved across all domains of life. While N-terminal acetylation is rare in bacteria, our proteomic data show that acetylated N termini are common in archaea affecting about 15% of the proteins and revealing a distinct archaeal N-terminal acetylation pattern. Haloarchaeal N-terminal acetyltransferase reveals narrow substrate specificity, which is limited to cleaved N termini starting with serine or alanine residues. A comparative analysis of 140 ortholog pairs with identified N-terminal peptide showed that acetylatable N-terminal residues are predominantly conserved amongst the two haloarchaea. Only few exceptions from the general N-terminal acetylation pattern were observed, which probably represent protein-specific modifications as they were confirmed by ortholog comparison.     

Differential proteomic analysis during the vegetative phase change and the floral transition in Malus domestica

In order to identify the proteomic changes of apple (Malus domestica Borkh.) during the vegetative phase change and the floral transition, leaf protein of juvenile, adult vegetative and reproductive phase in a seedling ('Jonathan' × 'Golden Delicious') was extracted and analyzed by 2-D electrophoresis and Matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Seventy two gel spots with significant expression differences between ontogenetic phases were obtained. Five protein spots were only detected in leaves of juvenile phase and 11 were not; 17 spots were found exclusively in adult vegetative leaves; and only one spot solely appeared in reproductive leaves while 12 did not. Twenty six of the differentially expressed proteins identified were involved in photosynthesis. Seven enzymes were related to respiration and carbohydrate metabolism. Fifteen other proteins also presented qualitative or quantitative differences among developmental phases. The spatial distribution of one differentially expressed protein, serine hydroxymethyltransferase, was confirmed by enzyme linked immunosorbent assay and immunohistochemistry. These results strongly support the idea that the vegetative phase change and the floral transition are regulated independently during developmental process.     

A blue dive: from ‘blue fingers’ to ‘blue silver’. A comparative overview of staining methods for in-gel proteomics

Gel-based proteomics are the most useful method for protein separation, even when compared with gel-free proteomics. Proteomic analysis by 2D gel electrophoresis (2-DE) with immobilized pH gradients is in turn the best approach to large-scale protein-expression screening. Spots visualization is pivotal for protein identification by mass spectrometry. Commonly used staining methods with excellent mass spectrometry compatibility are coomassie brilliant blue (CBB) or fluorescent dyes. In this study, an implementation of ‘blue silver’ colloidal CBB staining, characterized by high sensitivity and immediate low background, is discussed. The sensitivity of classical, colloidal and ‘blue silver’ CBB staining methods was compared on monodimensional and 2-DE gels. The implementation of the ‘blue silver’ method performs better, provided the physical state of the micelles is respected. An example of a 2-DE of human urine treated with combinatorial peptide ligand libraries demonstrates that implemented ‘blue silver’ can evidence the complexity of the sample.     

Glioma proteomics: Status and perspectives

High grade gliomas are the most common brain tumors in adults and their malignant nature makes them the fourth biggest cause of cancer death. Major efforts in neuro-oncology research are needed to reach similar progress in treatment efficacy as that achieved for other cancers in recent years. In addition to the urgent need to identify novel effective drug targets against malignant gliomas, the search for glioma biomarkers and grade specific protein signatures will provide a much needed contribution to diagnosis, prognosis, treatment decision and assessment of treatment response. Over the past years glioma proteomics has been attempted at different levels, including proteome analysis of patient biopsies and bodily fluids, of glioma cell lines and animal models. Here we provide an extensive review of the outcome of these studies in terms of protein identifications (protein numbers and regulated proteins), with an emphasis on the methods used and the limitations of the studies with regard to biomarker discovery. This is followed by a perspective on novel technologies and on the potential future contribution of proteomics in a broad sense to understanding glioma biology.     

Primary style protein expression in the self-incompatible/compatible apricot by the 2D-DIGE technique

In order to explore the molecular mechanism underlying self-incompatibility (SI) in the apricot (Prunus armeniaca L.) at the proteome level, we examined the style proteomes at different stages of flower development: small bud, big bud, 24h after self-pollination and 24h after cross-pollination with cultivar Badanshui in the SI apricot cultivar Xinshiji and the self-compatible (SC) apricot cultivar Katy by 2D fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). About 1500 style protein spots were detected; 66 were expressed differently in the four stages in Xinshiji. About 1600 style protein spots were detected; 143 were expressed differently in the four stages of flower development in Katy. In Xinshiji, one protein was expressed specifically, four proteins showed up-regulated expression and twenty-nine proteins showed down-regulated expression in the cross-pollinated style compared to the self-pollinated style. Thirteen proteins were identified unambiguously. In Katy, three proteins were expressed specifically, five proteins showed up-regulated expression and thirteen proteins showed down-regulated expression in the cross-pollinated style compared to self-pollinated style. Seven proteins were identified unambiguously. The different reactions of the style at the proteomic level were triggered in Xinshiji and Katy by self pollen and non-self pollen.     

Conserved and nonconserved features of the folding pathway of hisactophilin, a beta-trefoil protein

Based on previous studies of interleukin-1beta (IL-1beta) and both acidic and basic fibroblast growth factors (FGFs), it has been suggested that the folding of beta-trefoil proteins is intrinsically slow and may occur via the formation of essential intermediates. Using optical and NMR-detected quenched-flow hydrogen/deuterium exchange methods, we have measured the folding kinetics of hisactophilin, another beta-trefoil protein that has < 10% sequence identity and unrelated function to IL-1beta and FGFs. We find that hisactophilin can fold rapidly and with apparently two-state kinetics, except under the most stabilizing conditions investigated where there is evidence for formation of a folding intermediate. The hisactophilin intermediate has significant structural similarities to the IL-1beta intermediate that has been observed experimentally and predicted theoretically using a simple, topology-based folding model; however, it appears to be different from the folding intermediate observed experimentally for acidic FGF. For hisactophilin and acidic FGF, intermediates are much less prominent during folding than for IL-1beta. Considering the structures of the different beta-trefoil proteins, it appears that differences in nonconserved loops and hydrophobic interactions may play an important role in differential stabilization of the intermediates for these proteins.     

Identification and expression of an autosomal paralogue of ribosomal protein S4, X-linked,in mice: Potential involvement of testis-specific ribosomal proteins in translation and spermatogenesis

Recent studies have revealed heterogeneity in the structure of eukaryotic cytoplasmic ribosomes, including a difference in protein composition. It has been proposed that this heterogeneity, or the specialized ribosome, contributes to tissue development and homeostasis through selective mRNA translation, although this remains largely unclear. Our previous proteomic survey of rodent ribosomes found the testis-specific ribosomal proteins L10-like and L39-like, which are paralogues of the X-linked ribosomal proteins L10 and L39, respectively. We have hypothesized that the rodent testis provides a good model for examining the possible functional importance of ribosome heterogeneity. In the present study, a new paralogue of X-linked ribosomal protein S4 has been identified in the mouse testis. The gene encoding this paralogue was autosomal, intronless and expressed predominantly in the testis. It appeared that this paralogue was included in polysomes as a component of the ribosome. Although these properties were similar to those of the ribosomal proteins L10-like and L39-like, this S4 paralogue and L10-like showed partially different expression patterns in spermatogenic cells. These findings are discussed in relation to the unique evolution of genes encoding a paralogue of ribosomal protein S4 in mammals and to the significance of testis-specific paralogues of ribosomal proteins in active ribosomes during spermatogenesis.     

Fructose-bisphosphate aldolase and enolase from Echinococcus granulosus: genes, expression patterns and protein interactions of two potential moonlighting proteins

Glycolytic enzymes, such as fructose-bisphosphate aldolase (FBA) and enolase, have been described as complex multifunctional proteins that may perform non-glycolytic moonlighting functions, but little is known about such functions, especially in parasites. We have carried out in silico genomic searches in order to identify FBA and enolase coding sequences in Echinococcus granulosus, the causative agent of cystic hydatid disease. Four FBA genes and 3 enolase genes were found, and their sequences and exon-intron structures were characterized and compared to those of their orthologs in Echinococcus multilocularis, the causative agent of alveolar hydatid disease. To gather evidence of possible non-glycolytic functions, the expression profile of FBA and enolase isoforms detected in the E. granulosus pathogenic larval form (hydatid cyst) (EgFBA1 and EgEno1) was assessed. Using specific antibodies, EgFBA1 and EgEno1 were detected in protoscolex and germinal layer cells, as expected, but they were also found in the hydatid fluid, which contains parasite's excretory-secretory (ES) products. Besides, both proteins were found in protoscolex tegument and in vitro ES products, further suggesting possible non-glycolytic functions in the host-parasite interface. EgFBA1 modeled 3D structure predicted a F-actin binding site, and the ability of EgFBA1 to bind actin was confirmed experimentally, which was taken as an additional evidence of FBA multifunctionality in E. granulosus. Overall, our results represent the first experimental evidences of alternative functions performed by glycolytic enzymes in E. granulosus and provide relevant information for the understanding of their roles in host-parasite interplay.     

Gaining insights into the Bcr-Abl activity-independent mechanisms of resistance to imatinib mesylate in KCL22 cells: A comparative proteomic approach

Imatinib mesylate is a potent inhibitor of Bcr-Abl tyrosine kinase, an oncoprotein that plays a key role in the development of chronic myeloid leukemia. Consequently, imatinib is used as front-line therapy for this disease. A major concern in imatinib treatment is the emergence of resistance to the drug. Here we used the imatinib-resistant KCL22R and imatinib-sensitive KCL22S cells in which none of the known resistance mechanisms has been detected and hence novel Bcr-Abl activity-independent mechanisms could be envisaged. We characterized proteins that were differentially expressed between the KCL22R and KCL22S cells. Using two-dimensional differential gel electrophoresis coupled with mass spectrometry and Western blot analysis we identified 51 differentially expressed proteins: 27 were over-expressed and 24 were under-expressed in KCL22R versus KCL22S cells. Several of these proteins are likely to be involved in such survival mechanisms as modulation of redox balance and activation of anti-apoptotic pathways mediated by NF-κB and Ras-MAPK signaling. The data reported may be useful for further studies on mechanisms of imatinib resistance and for the screening of biomarkers to develop new combinatorial therapeutic approaches.     

Proteomic analysis of global protein expression changes in the endothelin-1 rat model for cerebral ischemia: Rescue effect of mild hypothermia

Mild hypothermia is a promising neuroprotective therapy in stroke management. However, little is known about its effects on the global protein expression patterns in brain regions affected by ischemic stroke. We investigated protein expression changes associated with the neuroprotective effects of hypothermia via a functional proteomics approach through the analysis of the core (striatum) and the penumbra (cortex) after an ischemic insult in rats induced by endothelin-1 (Et-1). Functional outcome, infarct volume and related global protein expression changes were assessed 24 h after the insult using two-dimensional difference gel electrophoresis. Mild hypothermia, induced 20 min after endothelin-1 infusion, improved the neurological outcome, reflected by a 36% reduction in infarct volume and a significantly better neurological deficit score. Hypothermia was typically associated with opposite protein expression changes inthe cortex to those induced by stroke under normothermic conditions, but not in the striatum. The main cellular processes rescued by hypothermia and potentially involved in the protection of the cortex are cellular assembly and organization, followed by cell signaling, thereby confirming that hypothermia is neuroprotective through multiple molecular and cellular pathways.     

Actin protein up-regulated upon infection and development of the filarial parasite, Wuchereria bancrofti (Spirurida: Onchocercidae), in the vector mosquito, Culex quinquefasciatus (Diptera: Culicidae)

Detection and identification of humoral proteins, which are up-regulated in Culex quinquefasciatus upon infection by Wuchereria bancrofti, is important in tracing out the biochemical consequences of the filarial parasite development in the vector mosquito. Analysis of the haemolymph of infected mosquitoes through SDS-PAGE and RP-HPLC showed up-regulation of five proteins of molecular weights 40, 66, 22, 14, and 7-kDa. Among these, only the 40-kDa was unknown and the others were comparable with those already reported as transferrin, attacin, lysozyme, and defensin, respectively. In the present study, the 40-kDa protein up-regulated upon infection was identified as actin through nano-LC-MS/MS analysis. Actin is known to be one of the cytoskeletal proteins up-regulated in the haemolymph, as part of the innate immune system, of Escherichia coli challenged Drosophila melanogaster larvae. For the first time, we have observed an increased level of actin in the haemolymph of W. bancrofti-infected Cx. quinquefasciatus. However, the exact mechanism of actin involvement in the immune system of this mosquito is yet to be studied.     

Regulation of brain-type creatine kinase by AMP-activated protein kinase: Interaction,phosphorylation and ER localization

AMP-activated protein kinase (AMPK) and cytosolic brain-type creatine kinase (BCK) cooperate under energy stress to compensate for loss of adenosine triphosphate (ATP) by either stimulating ATP-generating and inhibiting ATP-consuming pathways, or by direct ATP regeneration from phosphocreatine, respectively. Here we report on AMPK-dependent phosphorylation of BCK from different species identified by in vitro screening for AMPK substrates in mouse brain. Mass spectrometry, protein sequencing, and site-directed mutagenesis identified Ser6 as a relevant residue with one site phosphorylated per BCK dimer. Yeast two-hybrid analysis revealed interaction of active AMPK specifically with non-phosphorylated BCK. Pharmacological activation of AMPK mimicking energy stress led to BCK phosphorylation in astrocytes and fibroblasts, as evidenced with a highly specific phospho-Ser6 antibody. BCK phosphorylation at Ser6 did not affect its enzymatic activity, but led to the appearance of the phosphorylated enzyme at the endoplasmic reticulum (ER), close to the ER calcium pump, a location known for muscle-type cytosolic creatine kinase (CK) to support Ca²⁺-pumping.     

Alterations in protein and gene expression within the barrel cortices of ZnT3 knockout mice: experience-independent and dependent changes

Much evidence exists for the involvement of vesicular zinc in neurotransmission and cortical plasticity. Recent studies have reported that mice deficient in zinc transporter-3 protein (ZnT3) and thus, vesicular zinc, have significant behavioural and biochemical deficits. Here, we examined whether phenotypic differences existed in the barrel cortices of ZnT3 KO mice using functional proteomics and quantitative PCR. Additionally, by manipulating whisker input, we also investigated experience-dependent changes in protein and gene expression, thereby assaying how cortical plasticity is different in the absence of vesicular zinc. The GABA metabolizing protein ABAT was observed in lower abundances consistently in KO mice. Several presynaptic proteins were identified that were abundant in differing amounts between the WT and KO groups in an experience-dependent manner. At baseline, we observed a decrease in the relative expression of Dlg4, Grin2a, Mt3, and Ntrkb genes in KO mice. The reduced expression of Nrtkb persisted with whisker plucking. These data demonstrate that fundamental changes in the expression of proteins and genes important in neurotransmission occur in the absence of vesicular zinc. Furthermore, the complement of experience-dependent changes were different between WT and KO mice, indicating that the lack of vesicular zinc affects the process of cortical plasticity.     

Quality control of Photosystem II: Cleavage and aggregation of heat-damaged D1 protein in spinach thylakoids

Moderate heat stress (40 °C, 30 min) on spinach thylakoids induced cleavage of the D1 protein, producing an N-terminal 23-kDa fragment, a C-terminal 9-kDa fragment, and aggregation of the D1 protein. A homologue of Arabidopsis FtsH2 protease, which is responsible for degradation of the damaged D1 protein, was abundant in the stroma thylakoids. Two processes occurred in the thylakoids in response to heat stress: dephosphorylation of the D1 protein in the stroma thylakoids, and aggregation of the phosphorylated D1 protein in the grana. Heat stress also induced the release of the extrinsic PsbO, P and Q proteins from Photosystem II, which affected D1 degradation and aggregation significantly. The cleavage and aggregation of the D1 protein appear to be two alternative processes influenced by protein phosphorylation/dephosphorylation, distribution of FtsH, and intactness of the thylakoids.     

Dynamics of phosphodiesterase-induced cAMP dissociation from protein kinase A: Capturing transient ternary complexes by HDXMS

cAMP signaling is a fundamental cellular process necessary for mediating responses to hormonal stimuli. In contrast to cAMP-dependent activation of protein kinase A (PKA), an important cellular target, far less is known on termination in cAMP signaling, specifically how phosphodiesterases (PDEs) facilitate dissociation and hydrolysis of bound cAMP. In this study, we have probed the dynamics of a ternary complex of PKA and a PDE–RegA with an excess of a PDE-nonhydrolyzable cAMP analog, Sp-cAMPS by amide hydrogen/deuterium exchange mass spectrometry (HDXMS). Our results highlight how HDXMS can be used to monitor reactions together with mapping conformational dynamics of transient signaling complexes. Our results confirm a two-state model for active RegA-mediated dissociation of bound cAMP. Further, our results reveal that Sp-cAMPS and RegA mediate mutually exclusive interactions with the same region of PKA and at specific concentrations of Sp-cAMPS, RegA is capable of blocking Sp-cAMPS reassociation to PKA. This provides a molecular basis for how PDEs modulate levels of intracellular cAMP so that PKA is better suited to responding to fluxes rather than constant levels of cAMP. This study underscores how HDXMS can be a powerful tool for monitoring reactions together with mapping conformational dynamics in signaling proteins. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.