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Database surveys in the vertebrate model organisms: chicken (Gallus gallus), western clawed frog (Xenopus tropicalis), anole lizard (Anolis carolinensis) and zebrafish (Danio rerio) indicate that in some of these species the number of actin paralogues differs from the well-established six paralogues in mouse (Mus musculus). To investigate differential functions of actins and for establishing disease models it is important to know how actins in the different model organisms relate to each other and whether the vertebrate actin family is truly limited to six groups. Primarily through synteny analyses we discovered that the vertebrate actin family consists of eight instead of six orthologous actin groups for which we propose improved gene nomenclature. We also established that α-skeletal muscle, γ-enteric smooth muscle and γ-cytoplasmic actin genes originated prior to tetrapods contradicting an earlier and widely accepted model of actin evolution. Our findings allow a more reliable predictive classification of actin paralogues in (non-mammalian) vertebrates and contribute to a better understanding of actin evolution as basis for biomedical research on actin-related diseases.

We here present The Online Protein Processing Resource (TOPPR; http://iomics.ugent.be/toppr/), an online database that contains thousands of published proteolytically processed sites in human and mouse proteins. These cleavage events were identified with COmbinded FRActional DIagonal Chromatography proteomics technologies, and the resulting database is provided with full data provenance. Indeed, TOPPR provides an interactive visual display of the actual fragmentation mass spectrum that led to each identification of a reported processed site, complete with fragment ion annotations and search engine scores. Apart from warehousing and disseminating these data in an intuitive manner, TOPPR also provides an online analysis platform, including methods to analyze protease specificity and substrate-centric analyses. Concretely, TOPPR supports three ways to retrieve data: (i) the retrieval of all substrates for one or more cellular stimuli or assays; (ii) a substrate search by UniProtKB/Swiss-Prot accession number, entry name or description; and (iii) a motif search that retrieves substrates matching a user-defined protease specificity profile. The analysis of the substrates is supported through the presence of a variety of annotations, including predicted secondary structure, known domains and experimentally obtained 3D structure where available. Across substrates, substrate orthologs and conserved sequence stretches can also be shown, with iceLogo visualization provided for the latter.

The T cell integrin receptor LFA-1 orchestrates adhesion between T cells and antigen-presenting cells (APCs), resulting in formation of a contact zone known as the immune synapse (IS) which is supported by the cytoskeleton. L-plastin is a leukocyte-specific actin bundling protein that rapidly redistributes to the immune synapse following T cell-APC engagement. We used single domain antibodies (nanobodies, derived from camelid heavy-chain only antibodies) directed against functional and structural modules of L-plastin to investigate its contribution to formation of an immune synapse between Raji cells and human peripheral blood mononuclear cells or Jurkat T cells. Nanobodies that interact either with the EF hands or the actin binding domains of L-plastin both trapped L-plastin in an inactive conformation, causing perturbation of IS formation, MTOC docking towards the plasma membrane, T cell proliferation and IL-2 secretion. Both nanobodies delayed Ser(5) phosphorylation of L-plastin which is required for enhanced bundling activity. Moreover, one nanobody delayed LFA-1 phosphorylation, reduced the association between LFA-1 and L-plastin and prevented LFA-1 enrichment at the IS. Our findings reveal subtle mechanistic details that are difficult to attain by conventional means and show that L-plastin contributes to immune synapse formation at distinct echelons.

Vertebrate nonmuscle cells express two actin isoforms: cytoplasmic β- and γ-actin. Because of the presence and localized translation of β-actin at the leading edge, this isoform is generally accepted to specifically generate protrusive forces for cell migration. Recent evidence also implicates β-actin in gene regulation. Cell migration without β-actin has remained unstudied until recently and it is unclear whether other actin isoforms can compensate for this cytoplasmic function and/or for its nuclear role. Primary mouse embryonic fibroblasts lacking β-actin display compensatory expression of other actin isoforms. Consistent with this preservation of polymerization capacity, β-actin knockout cells have unchanged lamellipodial protrusion rates despite a severe migration defect. To solve this paradox we applied quantitative proteomics revealing a broad genetic reprogramming of β-actin knockout cells. This also explains why reintroducing β-actin in knockout cells does not restore the affected cell migration. Pathway analysis suggested increased Rho-ROCK signaling, consistent with observed phenotypic changes. We therefore developed and tested a model explaining the phenotypes in β-actin knockout cells based on increased Rho-ROCK signaling and increased TGFβ production resulting in increased adhesion and contractility in the knockout cells. Inhibiting ROCK or myosin restores migration of β-actin knockout cells indicating that other actins compensate for β-actin in this process. Consequently, isoactins act redundantly in providing propulsive forces for cell migration, but β-actin has a unique nuclear function, regulating expression on transcriptional and post-translational levels, thereby preventing myogenic differentiation. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc., SCOPUS: ar.j, info:eu-repo/semantics/published

Quantitative proteomic comparisons require a sufficient number of samples to reach an acceptable level of significance. But 2D gel electrophoresis commonly results in incomplete data sets due to spots with missing values reducing thereby the number of parallel measurements for individual proteins. Here we investigated how many missing values per spot can be tolerated. The number of spots in common between all gels was found to decrease with the number of parallel gels in a non-linear fashion. Increasing numbers of missing values were associated with a moderate increase in the quantitative variation of spot volumes. Based on the missing value pattern in 20 gels we performed an analysis of the multiple testing power for the hypothetical scenario of a comparative 2DE study with six or twelve parallel gels. The calculation considered the statistical power of the individual spot as well as the number of spots included in the analysis. The power increased with inclusion of spots with higher number of missing values and showed an optimum at a specific minimum number of spot replicates. The results suggest that proteins with missing values can be included in a univariate analysis as long as a sufficient number of parallel gels are made.

In recent years, procedures for selecting the N-terminal peptides of proteins with analysis by mass spectrometry have been established to characterize protease-mediated cleavage and protein α-N-acetylation on a proteomic level. As a pioneering technology, N-terminal combined fractional diagonal chromatography (COFRADIC) has been used in numerous studies in which these protein modifications were investigated. Derivatization of primary amines--which can include stable isotope labeling--occurs before trypsin digestion so that cleavage occurs after arginine residues. Strong cation exchange (SCX) chromatography results in the removal of most of the internal peptides. Diagonal, reversed-phase peptide chromatography, in which the two runs are separated by reaction with 2,4,6-trinitrobenzenesulfonic acid, results in the removal of the C-terminal peptides and remaining internal peptides and the fractionation of the sample. We describe here the fully matured N-terminal COFRADIC protocol as it is currently routinely used, including the most substantial improvements (including treatment with glutamine cyclotransferase and pyroglutamyl aminopeptidase to remove pyroglutamate before SCX, and a sample pooling scheme to reduce the overall number of liquid chromatography-tandem mass spectrometry analyses) that were made since its original publication. Completion of the N-terminal COFRADIC procedure takes ~5 d.

Initiation of protein translation is a well-studied fundamental process, albeit high-throughput and more comprehensive determination of the exact translation initiation sites (TIS) was only recently made possible following the introduction of positional proteomics techniques that target protein N-termini. Precise translation initiation is of crucial importance, as truncated or extended proteins might fold, function, and locate erroneously. Still, as already shown for some proteins, alternative translation initiation can also serve as a regulatory mechanism. By applying N-terminal COFRADIC (combined fractional diagonal chromatography), we here isolated N-terminal peptides of a Saccharomyces cerevisiae proteome and analyzed both annotated and alternative TIS. We analyzed this N-terminome of S. cerevisiae which resulted in the identification of 650 unique N-terminal peptides corresponding to database annotated TIS. Furthermore, 56 unique N(α)-acetylated peptides were identified that suggest alternative TIS (MS/MS-based), while MS-based evidence of N(α)-acetylation led to an additional 33 such peptides. To improve the overall sensitivity of the analysis, we also included the 5' UTR (untranslated region) in-frame translations together with the yeast protein sequences in UniProtKB/Swiss-Prot. To ensure the quality of the individual peptide identifications, peptide-to-spectrum matches were only accepted at a 99% probability threshold and were subsequently analyzed in detail by the Peptizer tool to automatically ascertain their compliance with several expert criteria. Furthermore, we have also identified 60 MS/MS-based and 117 MS-based N(α)-acetylated peptides that point to N(α)-acetylation as a post-translational modification since these peptides did not start nor were preceded (in their corresponding protein sequence) by a methionine residue. Next, we evaluated consensus sequence features of nucleic acids and amino acids across each of these groups of peptides and evaluated the results in the context of publicly available data. Taken together, we present a list of 706 annotated and alternative TIS for yeast proteins and found that under normal growth conditions alternative TIS might (co)occur in S. cerevisiae in roughly one tenth of all proteins. Furthermore, we found that the nucleic acid and amino acid features proximate to these alternative TIS favor either guanine or adenine nucleotides following the start codon or acidic amino acids following the initiator methionine. Finally, we also observed an unexpected high number of N(α)-acetylated peptides that could not be related to TIS and therefore suggest events of post-translational N(α)-acetylation.

Chlorosomes, the antenna complexes of green bacteria, are unique antenna systems in which pigments are organized in aggregates. Studies on isolated chlorosomes from Chlorobaculum tepidum based on SDS-PAGE, immunoblotting and molecular biology have revealed that they contain ten chlorosomal proteins, but no comprehensive information is available about the protein composition of the entire organelle. To extend these studies, chlorosomes were isolated from C. tepidum using three related and one independent isolation protocol and characterized by absorption spectroscopy, tricine SDS-PAGE, dynamic light scattering (DLS) and electron microscopy. Tricine SDS-PAGE showed the presence of more than 20 proteins with molecular weights ranging between 6 and 70 kDa. The chlorosomes varied in size. Their hydrodynamic radius (R(h) ) ranged from 51 to 75 nm and electron microscopy indicated that they were on average 140 nm wide and 170 nm long. Furthermore, the mass of 184 whole chlorosome organelles determined by scanning transmission electron microscopy ranged from 27 to 237 MDa being on average 88 (+/-28) MDa. In contrast their mass-per-area was independent of their size, indicating that there is a strict limit to chlorosome thickness. The average protein composition of the C. tepidum chlorosome organelles was obtained by MS/MS-driven proteomics and for the first time a detailed protein catalogue of the isolated chlorosomal proteome is presented. Based on the proteomics results for chlorosomes isolated by different protocols, four proteins that are involved in the electron or ion transport are proposed to be tightly associated with or incorporated into C. tepidum chlorosomes as well as the ten Csm proteins known to date.

Tyrosine nitration is the consequence of a complex machinery of formation and merging of oxygen and nitrogen radicals, and has been associated with both physiological pathways as well as with several human diseases. The latter turned this posttranslational protein modification into an interesting biomarker, being either a consequence of the disease or a factor contributing to the disease onset. However, the interpretation of MS and MS/MS data of peptides containing nitrotyrosine has proven to be very challenging and consequently, the risk of linking MS/MS spectra to incorrect peptide sequences exists and has been reported. Here, we discuss the causes of data misinterpretation and describe a general method to avoid mistakes of MS/MS spectrum misinterpretation. Central in our approach is the reduction of nitrotyrosine into aminotyrosine and the use of the Peptizer algorithm to inspect MS/MS quality-related assumptions.

The identification of peptides and proteins from fragmentation mass spectra is a very common approach in the field of proteomics. Contemporary high-throughput peptide identification pipelines can quickly produce large quantities of MS/MS data that contain valuable knowledge about the actual physicochemical processes involved in the peptide fragmentation process, which can be extracted through extensive data mining studies. As these studies attempt to exploit the intensity information contained in the MS/MS spectra, a critical step required for a meaningful comparison of this information between MS/MS spectra is peak intensity normalization. We here describe a procedure for quantifying the efficiency of different published normalization methods in terms of the quartile coefficient of dispersion (qcod) statistic. The quartile coefficient of dispersion is applied to measure the dispersion of the peak intensities between redundant MS/MS spectra, allowing the quantification of the differences in computed peak intensity reproducibility between the different normalization methods. We demonstrate that our results are independent of the data set used in the evaluation procedure, allowing us to provide generic guidance on the choice of normalization method to apply in a certain MS/MS pipeline application.

We describe a positional proteomics approach to simultaneously analyze N- and C-terminal peptides and used it to screen for human protein substrates of granzyme B and carboxypeptidase A4 in human cell lysates. This approach allowed comprehensive proteome studies, and we report the identification of 965 database-annotated protein C termini, 334 neo-C termini resulting from granzyme B processing and 16 neo-C termini resulting from carboxypeptidase A4 processing.

Proteolytic processing has recently received increased attention in the field of signal propagation and cellular differentiation. Because of its irreversible nature, protein cleavage has been associated with committed steps in cell function. One aspect of protease biology that boomed the past few years is the detailed characterization of protease substrates by both shotgun as well as targeted MS-driven proteomics techniques. The most promising techniques are discussed in this review and we further elaborate on the bioinformatics challenges that accompany mainly qualitative, MS-driven protease substrate degradome studies.

Caspase-3 and -7 are considered functionally redundant proteases with similar proteolytic specificities. We performed a proteome-wide screen on a mouse macrophage lysate using the N-terminal combined fractional diagonal chromatography technology and identified 46 shared, three caspase-3-specific, and six caspase-7-specific cleavage sites. Further analysis of these cleavage sites and substitution mutation experiments revealed that for certain cleavage sites a lysine at the P5 position contributes to the discrimination between caspase-7 and -3 specificity. One of the caspase-7-specific substrates, the 40 S ribosomal protein S18, was studied in detail. The RPS18-derived P6–P5′ undecapeptide retained complete specificity for caspase-7. The corresponding P6–P1 hexapeptide still displayed caspase-7 preference but lost strict specificity, suggesting that P′ residues are additionally required for caspase-7-specific cleavage. Analysis of truncated peptide mutants revealed that in the case of RPS18 the P4–P1 residues constitute the core cleavage site but that P6, P5, P2′, and P3′ residues critically contribute to caspase-7 specificity. Interestingly, specific cleavage by caspase-7 relies on excluding recognition by caspase-3 and not on increasing binding for caspase-7.

Recent developments in proteomics technology offer new opportunities for clinical applications in hospital or specialized laboratories including the identification of novel biomarkers, monitoring of disease, detecting adverse effects of drugs, and environmental hazards. Advanced spectrometry technologies and the development of new protein array formats have brought these analyses to a standard, which now has the potential to be used in clinical diagnostics. Besides standardization of methodologies and distribution of proteomic data into public databases, the nature of the human body fluid proteome with its high dynamic range in protein concentrations, its quantitation problems, and its extreme complexity present enormous challenges. Molecular cell biology (cytomics) with its link to proteomics is a new fast moving scientific field, which addresses functional cell analysis and bioinformatic approaches to search for novel cellular proteomic biomarkers or their release products into body fluids that provide better insight into the enormous biocomplexity of disease processes and are suitable for patient stratification, therapeutic monitoring, and prediction of prognosis. Experience from studies of in vitro diagnostics and especially in clinical chemistry showed that the majority of errors occurs in the preanalytical phase and the setup of the diagnostic strategy. This is also true for clinical proteomics where similar preanalytical variables such as inter- and intra-assay variability due to biological variations or proteolytical activities in the sample will most likely also influence the results of proteomics studies. However, before complex proteomic analysis can be introduced at a broader level into the clinic, standardization of the preanalytical phase including patient preparation, sample collection, sample preparation, sample storage, measurement, and data analysis is another issue which has to be improved. In this report, we discuss the recent advances and applications that fulfill the criteria for clinical proteomics with the focus on cellular proteomics (cytoproteomics) as related to preanalytical and analytical standardization and to quality control measures required for effective implementation of these technologies and analytes into routine laboratory testing to generate novel actionable health information. It will then be crucial to design and carry out clinical studies that can eventually identify novel clinical diagnostic strategies based on these techniques and validate their impact on clinical decision making.

The dynamic actin cytoskeleton, consisting of six actin isoforms in mammals and a variety of actin binding proteins is essential for all developmental processes and for the viability of the adult organism. Actin isoform specific functions have been proposed for muscle contraction, cell migration, endo- and exocytosis and maintaining cell shape. However, these specific functions for each of the actin isoforms during development are not well understood. Based on transgenic mouse models, we will discuss the expression patterns of the six conventional actin isoforms in mammals during development and adult life. Ablation of actin genes usually leads to lethality and affects expression of other actin isoforms at the cell or tissue level. A good knowledge of their expression and functions will contribute to fully understand severe phenotypes or diseases caused by mutations in actin isoforms.

Zonula occludens proteins (ZO) are postsynaptic density protein-95 discs large-zonula occludens (PDZ) domain-containing proteins that play a fundamental role in the assembly of tight junctions and establishment of cell polarity. Here, we show that the second PDZ domain of ZO-1 and ZO-2 binds phosphoinositides (PtdInsP) and we identified critical residues involved in the interaction. Furthermore, peptide and PtdInsP binding of ZO PDZ2 domains are mutually exclusive. Although lipid binding does not seem to be required for plasma membrane localisation of ZO-1, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2) binding to the PDZ2 domain of ZO-2 regulates ZO-2 recruitment to nuclear speckles. Knockdown of ZO-2 expression disrupts speckle morphology, indicating that ZO-2 might play an active role in formation and stabilisation of these subnuclear structures. This study shows for the first time that ZO isoforms bind PtdInsPs and offers an alternative regulatory mechanism for the formation and stabilisation of protein complexes in the nucleus.

In proteomics, rapid developments in instrumentation led to the acquisition of increasingly large data sets. Correspondingly, ProDaC was founded in 2006 as a Coordination Action project within the 6th European Union Framework Programme to support data sharing and community-wide data collection. The objectives of ProDaC were the development of documentation and storage standards, setup of a standardized data submission pipeline and collection of data. Ending in March 2009, ProDaC has delivered a comprehensive toolbox of standards and computer programs to achieve these goals.

N α -terminal acetylation is one of the most common protein modifications in eukaryotes. The COmbined FRActional DIagonal Chromatography (COFRADIC) proteomics technology that can be specifically used to isolate N-terminal peptides was used to determine the N-terminal acetylation status of 742 human and 379 yeast protein N termini, representing the largest eukaryotic dataset of N-terminal acetylation. The major N-terminal acetyltransferase (NAT), NatA, acts on subclasses of proteins with Ser-, Ala-, Thr-, Gly-, Cys- and Val- N termini. NatA is composed of subunits encoded by y ARD1 and y NAT1 in yeast and h ARD1 and h NAT1 in humans. A yeast ard1 -Δ nat1 -Δ strain was phenotypically complemented by h ARD1 h NAT1 , suggesting that yNatA and hNatA are similar. However, heterologous combinations, h ARD1 y NAT1 and y ARD1 h NAT1 , were not functional in yeast, suggesting significant structural subunit differences between the species. Proteomics of a yeast ard1 -Δ nat1 -Δ strain expressing hNatA demonstrated that hNatA acts on nearly the same set of yeast proteins as yNatA, further revealing that NatA from humans and yeast have identical or nearly identical specificities. Nevertheless, all NatA substrates in yeast were only partially N-acetylated, whereas the corresponding NatA substrates in HeLa cells were mainly completely N-acetylated. Overall, we observed a higher proportion of N-terminally acetylated proteins in humans (84%) as compared with yeast (57%). N-acetylation occurred on approximately one-half of the human proteins with Met-Lys- termini, but did not occur on yeast proteins with such termini. Thus, although we revealed different N-acetylation patterns in yeast and humans, the major NAT, NatA, acetylates the same substrates in both species.

We showed earlier that human beta-thymosin 15 (Tb15) is up-regulated in prostate cancer, confirming studies from others that propagated Tb15 as a prostate cancer biomarker. In this first report on mouse Tb15, we show that, unlike in humans, four Tb15-like isoforms are present in mouse. We used phylogenetic analysis of deuterostome beta-thymosins to show that these four new isoforms cluster within the vertebrate Tb15-clade. Intriguingly, one of these mouse beta-thymosins, Tb15r, consists of two beta-thymosin domains. The existence of such a repeat beta-thymosin is so far unique in vertebrates, though common in lower eukaryotes. Biochemical data indicate that Tb15r potently sequesters actin. In a cellular context, Tb15r behaves as a bona fide beta-thymosin, lowering central stress fibre content. We reveal that a complex genomic organization underlies Tb15r expression: Tb15r results from read-through transcription and alternative splicing of two tandem duplicated mouse Tb15 genes. Transcript profiling of all mouse beta-thymosin isoforms (Tb15s, Tb4 and Tb10) reveals that two isoform switches occur between embryonic and adult tissues, and indicates Tb15r as the major mouse Tb15 isoform in adult cells. Tb15r is present also in mouse prostate cancer cell lines. This insight into the mouse Tb15 family is fundamental for future studies on Tb15 in mouse (prostate) cancer models.

Here, we report that Cdk5 activation is stimulated by insulin and plays a key role in the regulation of GLUT4-mediated glucose uptake in 3T3-L1 adipocytes. Insulin activation of Cdk5 requires PI3K signaling. Insulin-activated Cdk5 phosphorylates E-Syt1, a 5 C2-domain protein-related to the synaptotagmins that is induced during adipocyte differentiation. Phosphorylated E-Syt1 associates with GLUT4, an event inhibited by the Cdks inhibitor roscovitine. Cdk5 silencing inhibits glucose uptake by 3T3-L1 adipocytes. These studies elucidate a previously unknown activity of Cdk5 and demonstrate the involvement of this kinase in the regulation of insulin-dependent glucose uptake in adipocytes.

Caspases are key players in various cellular processes, such as apoptosis, proliferation and differentiation, and in pathological conditions including cancer and inflammation. Although caspases preferentially cleave C-terminal of aspartic acid residues, their action is restricted generally to one or a few sites per protein substrate. Caspase-specific substrate recognition appears to be determined by the substrate sequences adjacent to the scissile bond. Knowledge of these substrates and the generated fragments is crucial for a thorough understanding of the functional implications of caspase-mediated proteolysis. In addition, insight into the cleavage specificity might assist in designing inhibitors that target disease-related caspase activities. Here, we critically review recently published procedures used to generate a proteome-wide view of caspase substrates.

Gelsolin is an actin-binding protein (82 kDa) consisting of six repeated segments (S1-S6), each approximately 120 residues long. It interacts with phospholipids and we previously showed that phosphatidylinositol 4,5-bisphosphate promotes phosphorylation of gelsolin by the tyrosine kinase c-Src. We used a combination of different methods, such as thin-layer chromatography and anti-phosphotyrosine-agarose immunoprecipitation of phosphopeptides combined with matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS) and post source decay (PSD) analysis, to identify the phosphorylation sites in gelsolin. The major phosphorylation site (Tyr438) was located in subdomain 4 (S4). Phosphorylation of gelsolin in the gelsolin-actin2 complex was inhibited by 90%. Gelsolin phosphorylation by c-Src in the presence of lysophosphatidic acid also revealed Tyr438 as the most prominent site. Additional minor sites were found using the anti-phosphotyrosine bead immunoprecipitation method followed by MALDI-MS and PSD analysis. These sites, representing approximately 5% of the total phosphate incorporation, were identified as Tyr59, Tyr382, Tyr576, and Tyr624. Based on these results we generated antibodies which specifically recognize Tyr438 phosphorylated gelsolin.

Syntenin-1 is a tandem PDZ protein that binds a diverse array of signaling molecules that are often associated with cell adhesion and intracellular trafficking. With the use of a MS-based functional proteomics approach, we identified several members of the aminoacyl-tRNA synthetase macromolecular (ARS) complex in a syntenin-1 pull down assay. Interaction of these proteins with syntenin-1 was confirmed by co-immunoprecipitation from cultured cells. We demonstrate a direct interaction of syntenin-1 with lysyl-tRNA synthetase (KRS), which contains a PDZ binding motif at its C-terminus. This motif is important for the interaction of the entire complex with syntenin-1. A point mutation in the PDZ2 domain of syntenin-1 abrogates interaction with KRS. As a result, other components of the ARS complex no longer co-immunoprecipitate with syntenin-1. We further show that syntenin-1 regulates KRS activity. These findings suggest that syntenin-1 is an adaptor modulating the activity of KRS.

Proteases are important pharmaceutical targets for new drugs because of their involvement in numerous disease processes. This study evaluates whether photophysically encoded microparticles carrying fluorescently labeled protease substrates (peptides) at their surface show potential for detecting proteases in a sample. Layer-by-layer (LbL) polyelectrolyte coatings, containing a red-labeled peptidic trypsin substrate, are carefully designed and applied at the surface of the encoded microparticles. The peptide-loaded LbL coatings lose their red fluorescence upon incubation in a trypsin solution, indicating that LbL-coated microparticles show potential to screen for the presence of active proteases in biological samples.

We analysed the involvement of proteases during taxol-mediated cell death of human A549 non-small-cell lung carcinoma cells using a proteomics approach that specifically targets protein N termini and further detects newly formed N termini that are the result of protein processing. Our analysis revealed 27 protease-mediated cleavages, which we divided in sites C-terminal to aspartic acid (Asp) and sites C-terminal to non-Asp residues, as the result of caspase and non-caspase protease activities, respectively. Remarkably, some of the former were insensitive to potent pancaspase inhibitors, and we therefore suggest that previous inhibitor-based studies that report on the caspase-independent nature of taxol-induced cell death should be judged with care. Furthermore, many of the sites C-terminal to non-Asp residues were also uniquely observed in a model of cytotoxic granule-mediated cell death and/or found by in vitro cataloging human mu-calpain substrates using a similar proteomics technique. This thus raises the hypothesis that killing tumor cells by chemotherapy or by immune cells holds similar non-Asp-specific proteolytic components with strong indications to calpain activity.

Matrix protein 2 (M2) of influenza A is a tetrameric type III membrane protein that functions as a proton-selective channel. The extracellular domain (M2e) has remained nearly invariable since the first human influenza strain was isolated in 1933. By linking a modified form of the leucine zipper of the yeast transcription factor GCN4 to M2e, we obtained a recombinant tetrameric protein, M2e-tGCN4. This protein mimics the quaternary structure of the ectodomain of the natural M2 protein. M2e-tGCN4 was purified, biochemically characterized, and used to immunize BALB/c mice. High M2e-specific serum IgG antibody titers were obtained following either intraperitoneal or intranasal administration. Immunized mice were protected fully against a potentially lethal influenza A virus challenge. Antibodies raised by M2e-tGCN4 immunization specifically bound to the surface of influenza-infected cells and to an M2-expressing cell line. Using a M2e peptide competition enzyme-linked immunosorbent assay with M2-expressing cells as target, we obtained evidence that M2e-tGCN4 induces antibodies that are specific for the native tetrameric M2 ectodomain. Therefore, fusion of an oligomerization domain to the extracellular part of a transmembrane protein allows it to mimic the natural quaternary structure and can promote the induction of oligomer-specific antibodies.

Binding of GH to its receptor induces rapid phosphorylation of conserved tyrosine motifs that function as recruitment sites for downstream signaling molecules. Using mammalian protein-protein interaction trap (MAPPIT), a mammalian two-hybrid method, we mapped the binding sites in the GH receptor for signal transducer and activator of transcription 5 (STAT5) a and b and for the negative regulators of cytokine signaling cytokine-inducible Src-homology 2 (SH2)-containing protein (CIS) and suppressor of cytokine signaling 2 (SOCS2). Y534, Y566, and Y627 are the major recruitment sites for STAT5. A non-overlapping recruitment pattern is observed for SOCS2 and CIS with positions Y487 and Y595 as major binding sites, ruling out SOCS-mediated inhibition of STAT5 activation by competition for shared binding sites. More detailed analysis revealed that CIS binding to the Y595, but not to the Y487 motif, depends on both its SH2 domain and the C-terminal part of its SOCS box, with a critical role for the CIS Y253 residue. This functional divergence of the two CIS/SOCS2 recruitment sites is also observed upon substitution of the Y+1 residue by leucine, turning the Y487, but not the Y595 motif into a functional STAT5 recruitment site.

Methylglyoxal is a cytotoxic metabolite that is produced in vivo mainly from glycolysis. Increased production of methylglyoxal can be induced by tumor necrosis factor and occurs in a number of pathological conditions, including diabetes and neurodegenerative disorders. Methylglyoxal is highly reactive and can modify proteins, which results in the formation of advanced glycation end products. Yet, we, and others, have recently proposed a role for methylglyoxal as a signaling molecule. In this study, we show that methylglyoxal inhibits TNF-induced NF-κB activation and NF-κB-dependent reporter gene expression by inhibiting the DNA binding capacity of NF-κB p65. Methylglyoxal slightly delayed, but did not inhibit, TNF-induced degradation of IκBα and strongly inhibited TNF-induced NF-κB-dependent re-synthesis of IκBα. The TNF-induced nuclear translocation of NF-κB p65 was also delayed, but not inhibited, in the presence of methylglyoxal. TNF-induced phosphorylation of p65 was not affected by methylglyoxal. We show that the conserved Cys 38 residue, which is located in the DNA binding loop of NF-κB p65 and responsible for the redox regulation of the transcription factor, is involved in the methylglyoxal-mediated inhibition of p65 DNA-binding. Furthermore, overexpression of p65 inhibited TNF-induced cell death; however, in the presence of exogenously added methylglyoxal, overexpression of p65 caused far greater TNF-induced cell death. These findings suggest that methylglyoxal provides another control mechanism for modulating the expression of NF-κB-responsive genes and that methylglyoxal may be responsible for tipping the balance towards TNF-induced cell death in cells with constitutive NF-κB activation.

Caspase-14 belongs to a conserved family of aspartate-specific proteinases. Its expression is restricted almost exclusively to the suprabasal layers of the epidermis and the hair follicles. Moreover, the proteolytic activation of caspase-14 is associated with stratum corneum formation, implicating caspase-14 in terminal keratinocyte differentiation and cornification. Here, we show that the skin of caspase-14-deficient mice was shiny and lichenified, indicating an altered stratum-corneum composition. Caspase-14-deficient epidermis contained significantly more alveolar keratohyalin F-granules, the profilaggrin stores. Accordingly, caspase-14-deficient epidermis is characterized by an altered profilaggrin processing pattern and we show that recombinant caspase-14 can directly cleave profilaggrin in vitro. Caspase-14-deficient epidermis is characterized by reduced skin-hydration levels and increased water loss. In view of the important role of filaggrin in the structure and moisturization of the skin, the knockout phenotype could be explained by an aberrant processing of filaggrin. Importantly, the skin of caspase-14-deficient mice was highly sensitive to the formation of cyclobutane pyrimidine dimers after UVB irradiation, leading to increased levels of UVB-induced apoptosis. Removal of the stratum corneum indicate that caspase-14 controls the UVB scavenging capacity of the stratum corneum.

Three groups of evolutionarily conserved proteins have been implicated in the establishment of epithelial cell polarity: the apically-localized proteins of the Par (Par3-Par6-aPKC-Cdc42) and Crumbs groups (Crb3-PALS1-PATJ) and the basolaterally localized proteins of the Dlg group (Dlg1-Scribble-Lgl). During epithelial morphogenesis, these proteins participate in a complex network of interdependent interactions that define the position and functional organization of adherens junctions and tight junctions. However, the biochemical pathways through which they control polarity are poorly understood. In this study, we identify an interaction between endogenous hDlg1 and MPP7, a previously uncharacterized MAGUK-p55 subfamily member. We find that MPP7 targets to the lateral surface of epithelial cells via its L27N domain, through an interaction with hDlg1. Loss of either hDlg1 or MPP7 from epithelial Caco-2 cells results in a significant defect in the assembly and maintenance of functional tight junctions. We conclude that the formation of a complex between hDlg1 and MPP7 promotes epithelial cell polarity and tight junction formation.

Characterization of protein N-terminal peptides supports the quality assessment of data derived from genomic sequences (e.g., the correct assignment of start codons) and hints to in vivo N-terminal modifications such as N-terminal acetylation and removal of the initiator methionine. The current work represents the first large-scale identification of N-terminal peptides from prokaryotes, of the two halophilic euryarchaeota Halobacterium salinarum and Natronomonas pharaonis. Two methods were used that specifically allow the characterization of protein N-terminal peptides: combined fractional diagonal chromatography (COFRADIC) and strong cation exchange chromatography (SCX), both known to enrich for N-terminally blocked peptides. In addition to these specific methods, N-terminal peptide identifications were extracted from our previous genome-wide proteomic data. Combining all data, 606 N-terminal peptides from Hbt. salinarum and 328 from Nmn. pharaonis were reliably identified. These results constitute the largest available dataset holding identified and characterized protein N-termini for prokaryotes (archaea and bacteria). They allowed the validation/improvement of start codon assignments as automatic gene finders tend to misassign start codons for GC-rich genomes. In addition, the dataset allowed unravelling N-terminal protein maturation in archaea, showing that 60% of the proteins undergo methionine cleavage and that-in contrast to current knowledge-Nalpha-acetylation is common in the archaeal domain of life with 13-18% of the proteins being Nalpha-acetylated. The protein sets described in this paper are available by FTP and might be used as reference sets to test the performance of new gene finders.

The previously reported COmbined FRActional DIagonal Chromatography (COFRA-DIC) methodology, in which a subset of peptides representative for their parent proteins are sorted, is particularly powerful for whole proteome analysis. This peptide-centric technology is built around diagonal chromatography, where peptide separations are crucial. This paper presents high efficiency peptide separations, in which four 250 x 2.1 mm, 5 microm Zorbax 300SB-C18 columns (total length 1 m) were coupled at operating temperatures of 60'C using a dedicated LC oven and conventional LC equipment. The high efficiency separations were combined with the COFRADIC procedure. This extremely powerful combination resulted, for the analysis of serum, in an increase in the uniquely identified peptide sequences by a factor of 2.6, compared to the COFRADIC procedure on a 25 cm column. This is a reflection of the increased peak capacity obtained on the 1 m column, which was calculated to be a factor 2.7 higher than on the 25 cm column. Besides more efficient sorting, less ion suppression was noticed.

To identify apoptotic targets of HtrA2/Omi, we purified recombinant HtrA2/Omi and its catalytically inactive S306A mutant. Lysates of human Jurkat T lymphocytes incubated with either wild-type recombinant HtrA2/Omi or the S306A mutant were screened using the gel-free COFRADIC approach that isolates peptides covering the N-terminal parts of proteins. Analysis of the 1162 proteins identified by mass spectrometry yielded 15 HtrA2/Omi substrates of potential physiological relevance together holding a total of 50 cleavage sites. Several processing events were validated by incubating purified recombinant HtrA2/Omi with in vitro translated substrates or with Jurkat cell lysates. In addition, the generated set of cleavage sites was used to assess the protein substrate specificity of HtrA2/Omi. Our results suggest that HtrA2/Omi has a rather narrow cleavage site preference and that cytoskeletal proteins are prime targets of this protease. Keywords: COFRADIC • HtrA2 • Omi • apoptosis • substrate

The three dimensional structures of profilins from invertebrates and vertebrates are remarkably similar despite low sequence similarity. Their evolutionary relationship remains thus enigmatic. A phylogenetic analysis of profilins from Deuterostoma indicates that profilin III and IV isoforms each form distinct groups. Profilin IV is most related to invertebrate profilins and originated prior to vertebrate evolution whereas separation of profilin I, II and III isoforms occurred early in vertebrate evolution. Viral profilins are most similar to profilin III. In silico analysis of representative profilin gene structures corroborates the phylogenetic result and we discuss this in terms of biochemical differences.

Diagonal peptide chromatography consists of two consecutive, identical peptide separations with in between an enzymatic or chemical alteration of the side-chain structure of selected peptides. Such selected and altered peptides acquire different chromatographic properties thereby segregating from non-altered peptides in a series of secondary peptide separations. Originally described by Brown and Hartley in 1966, we have modified the technique such that it can be used for higher throughput gel-free proteomics. Our technique is termed COmbined FRActional DIagonal Chromatography (COFRADIC) and exploits evoked differences of the hydrophobicity of peptides in reverse-phase liquid chromatography. One important advantage of COFRADIC is its versatility: by changing the alteration reaction, different classes of peptides are sorted and finally analyzed. We previously published protocols and applications for separating methionyl, cysteinyl, amino terminal and phosphorylated peptides. In this review, we assess the potential of COFRADIC for the analysis of several posttranslational modifications emphasizing on in vivo protein processing events. Additional modifications that can be analyzed include phosphorylation and N-glycosylation. The potential of COFRADIC for isolating peptides holding such modified amino acids are discussed here.

Adenine nucleotides are small, abundant molecules that bind numerous proteins involved in pivotal cellular processes. These nucleotides are co-factors or substrates for enzymes, regulators of protein function, or structural binding motifs. The identification of nucleotide-binding sites on a proteome-wide scale is tempting in view of the high number of nucleotide-binding proteins, their large in vivo concentration differences, and the various functions they exert. Here, we report on a functional, chemical, gel-free proteomics technology that allows the identification of protein adenine nucleotide-binding site(s) in cell lysates. Our technology uses a synthetic ATP analogue, 5'-p-fluorosulfonylbenzoyladenosine (FSBA), as an affinity/activity-based probe for nucleotide-binding sites. When applied on a cellular level, 185 different FSBA-labeled sites in a human Jurkat cell lysate were identified. Functional and structural aspects of the use of FSBA on a proteome-wide scale are discussed.

Hypothalamic leptin receptor signalling plays a central role in weight regulation by controlling fat storage and energy expenditure. In addition, leptin also has direct effects on peripheral cell types involved in regulation of diverse body functions including immune response, bone formation and reproduction. Previous studies have demonstrated the important role of SOCS3 (suppressor of cytokine signalling 3) in leptin physiology. Here, we show that CIS (cytokine-inducible SH2 protein) and SOCS2 can also interact with the leptin receptor. Using MAPPIT (mammalian protein-protein interaction trap), a cytokine receptor-based two-hybrid method operating in intact cells, we show specific binding of CIS with the conserved Y985 and Y1077 motifs in the cytosolic domain of the leptin receptor. SOCS2 only interacts with the Y1077 motif, but with higher binding affinity and can interfere with CIS and STAT5a prey recruitment at this site. Furthermore, although SOCS2 does not associate with Y985 of the leptin receptor, we find that SOCS2 can block interaction of CIS with this position. This unexpected interference can be explained by the direct binding of SOCS2 on the CIS SOCS box, whereby elongin B/C recruitment is crucial to suppress CIS activity.

The HeadPiece (HP) domain, present in several F-actin-binding multi-domain proteins, features a well-conserved, solvent-exposed PXWK motif in its C-terminal subdomain. The latter is an autonomously folding subunit comprised of three alpha-helices organised around a hydrophobic core, with the sequence motif preceding the last helix. We report the contributions of each conserved residue in the PXWK motif to human villin HP function and structure, as well as the structural implications of the naturally occurring Pro to Ala mutation in dematin HP. NMR shift perturbation mapping reveals that substitution of each residue by Ala induces only minor, local perturbations in the full villin HP structure. CD spectroscopic thermal analysis, however, shows that the Pro and Trp residues in the PXWK motif afford stabilising interactions. This indicates that, in addition to the residues in the hydrophobic core, the Trp-Pro stacking within the motif contributes to HP stability. This is reinforced by our data on isolated C-terminal HP subdomains where the Pro is also essential for structure formation, since the villin, but not the dematin, C-terminal subdomain is structured. Proper folding can be induced in the dematin C-terminal subdomain by exchanging the Ala for Pro. Conversely, the reverse substitution in the villin C-terminal subdomain leads to loss of structure. Thus, we demonstrate a crucial role for this proline residue in structural stability and folding potential of HP (sub)domains consistent with Pro-Trp stacking as a more general determinant of protein stability.

Differentiating neurons extend membrane protrusions that develop into growing neurites. The driving force for neurite outgrowth is the dynamic actin cytoskeleton, which is regulated by actin-binding proteins. In this study, we describe for the first time, the role of profilin I and its ligand interactions in neuritogenesis of PC12 cells. High-level overexpression of wild-type profilin I had an inhibitory effect on neurite outgrowth. Low levels of profilin I did not disturb this process, but these cells developed many more filopodia along the neurite shafts. Low-level overexpression of mutant forms of profilin I changed one or more aspects of PC12 differentiation. Expression of a profilin I mutant that is defective in actin binding (profilin IR74E) decreased neurite length and strongly inhibited filopodia formation. Cells expressing mutants defective in binding proline-rich ligands (profilin IW3A and profilin IR136D) differentiated faster, developed more and longer neurites and more branches. The profilin IR136D mutant, which is also defective in phosphatidylinositol 4,5-bisphosphate binding, enhanced neurite outgrowth even in the absence of NGF. Parental PC12 cells treated with the ROCK inhibitor Y27632, differentiate faster and display longer neurites and more branches. Similar effects were seen in cells expressing profilin IWT, profilin IW3A and profilin IR74E. By contrast, the profilin IR136D-expressing cells were insensitive to the ROCK inhibitor, suggesting that regulation of profilin I by phosphatidylinositol 4,5-bisphosphate metabolism is crucial for proper neurite outgrowth. Taken together, our data show the importance of the interaction of profilin I with actin, proline-rich proteins and phosphatidylinositol 4,5-bisphosphate in neuronal differentiation of PC12 cells.