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991.
Alina C. Stiebler Johannes Freitag Kay O. Schink Thorsten Stehlik Britta A. M. Tillmann Julia Ast Michael B?lker 《PLoS genetics》2014,10(10)
Translation of mRNA into a polypeptide chain is a highly accurate process. Many prokaryotic and eukaryotic viruses, however, use leaky termination of translation to optimize their coding capacity. Although growing evidence indicates the occurrence of ribosomal readthrough also in higher organisms, a biological function for the resulting extended proteins has been elucidated only in very few cases. Here, we report that in human cells programmed stop codon readthrough is used to generate peroxisomal isoforms of cytosolic enzymes. We could show for NAD-dependent lactate dehydrogenase B (LDHB) and NAD-dependent malate dehydrogenase 1 (MDH1) that translational readthrough results in C-terminally extended protein variants containing a peroxisomal targeting signal 1 (PTS1). Efficient readthrough occurs at a short sequence motif consisting of a UGA termination codon followed by the dinucleotide CU. Leaky termination at this stop codon context was observed in fungi and mammals. Comparative genome analysis allowed us to identify further readthrough-derived peroxisomal isoforms of metabolic enzymes in diverse model organisms. Overall, our study highlights that a defined stop codon context can trigger efficient ribosomal readthrough to generate dually targeted protein isoforms. We speculate that beyond peroxisomal targeting stop codon readthrough may have also other important biological functions, which remain to be elucidated. 相似文献
992.
Syed Abid Ali Mehtab Alam Atiya Abbasi Hubert Kalbacher Thorsten J. Schaechinger Youtian Hu Cao Zhijian Wenxin Li Wolfgang Voelter 《International journal of peptide research and therapeutics》2014,20(1):19-32
Venomous fauna of the world is a unique source of protein and peptide toxins with a wide range of pharmacological and physiological activities targeting in particular the bio-signaling system. Thus the most widely known source of peptidyl neurotoxins used for callipering different ion channels (e.g. Na+, K+, Ca++ or Cl? etc.) as well as many medicinally important peptides which have been accepted as drugs or are in clinical trials originate from the venom of scorpions of Buthidae family. In the present study, structure–activity relationship of highly selective short-chain neurotoxin from scorpion Buthus sindicus (a common yellow scorpion of Sindh, Pakistan) has been established. The toxin named as Bs-KTx6 (4,115.4 Da) has been isolated, synthesized and found to be a potent as well as selective inhibitor of voltage gated potassium channel Kv1.3 (IC50 = 7.7 pM). The structural studies on channel selective modulators like Bs-KTx6 may serve as a possible template in establishing libraries of peptidyl toxins for treating diseases such as multiple sclerosis, type-1 diabetes mellitus, rheumatoid arthritis, allergic contact dermatitis, bone resorption due to periodontitis and delayed type hypersensitivity. It is also worth mentioning that the peptide based drugs have excellent safety profiles as well as selectivity compared to other non-protein molecules. In conclusion, identified peptide Bs-KTx6 is a specific as well as very selective blocker of Kv1.3 and provides a starting template for the synthesis of peptidyl drugs to treat autoimmune diseases. 相似文献
993.
K. Marquardt M. Saha M. Mishina J. W. Young J. L. Brigman 《Genes, Brain & Behavior》2014,13(7):611-617
Glutamate neurotransmission via the N‐methyl‐d ‐aspartate receptor (NMDAR) is thought to mediate the synaptic plasticity underlying learning and memory formation. There is increasing evidence that deficits in NMDAR function are involved in the pathophysiology of cognitive dysfunction seen in neuropsychiatric disorders and addiction. NMDAR subunits confer different physiological properties to the receptor, interact with distinct intracellular postsynaptic scaffolding and signaling molecules, and are differentially expressed during development. Despite these known differences, the relative contribution of individual subunit composition to synaptic plasticity and learning is not fully elucidated. We have previously shown that constitutive deletion of GluN2A subunit in the mouse impairs discrimination and re‐learning phase of reversal when exemplars are complex picture stimuli, but spares acquisition and extinction of non‐discriminative visually cued instrumental response. To investigate the role of GluN2A containing NMDARs in executive control, we tested GluN2A knockout (GluN2AKO), heterozygous (GluN2AHET) and wild‐type (WT) littermates on an attentional set‐shifting task using species‐specific stimulus dimensions. To further explore the nature of deficits in this model, mice were tested on a visual discrimination reversal paradigm using simplified rotational stimuli. GluN2AKO were not impaired on discrimination or reversal problems when tactile or olfactory stimuli were used, or when visual stimuli were sufficiently easy to discriminate. GluN2AKO showed a specific and significant impairment in ventromedial prefrontal cortex‐mediated set‐shifting. Together these results support a role for GluN2A containing NMDAR in modulating executive control that can be masked by overlapping deficits in attentional processes during high task demands. 相似文献
994.
Katharina Homburg Patric Brandt Claudia Drees Thorsten Assmann 《Journal of Insect Conservation》2014,18(5):781-790
Species distribution models (SDMs), especially those basing on climatic parameters, have frequently been used to project future species ranges and to develop conservation strategies. As suggested by several authors, we considered both different dispersal abilities and different evolutionarily significant units (ESUs, as determined in an earlier genetic survey). For our study species, the flightless ground beetle Carabus irregularis, SDMs for two ESUs from the western and the Carpathian area of the distribution range showed immense, and deviating future range contractions reflecting divergent ecological requirements. As minimal dispersal SDMs resulted in a stronger decline of future ranges than the maximal dispersal models, low dispersal ability tended to strengthen the already high vulnerability of the cold-adapted mountain species to global warming. Areas shown in our maximal dispersal models as offering climatically suitable habitats for C. irregularis in the future should be considered as potential areas of action in future conservation planning (e.g. assisted migration or assisted colonisation). Thus, both dispersal scenarios and different (if applicable) ESUs should be considered when developing SDMs as useful tools for species conservation strategies adapted to species’ performance and differentiation patterns. 相似文献
995.
Novel fatty acid elongases and their use for the reconstitution of docosahexaenoic acid biosynthesis 总被引:1,自引:0,他引:1
Meyer A Kirsch H Domergue F Abbadi A Sperling P Bauer J Cirpus P Zank TK Moreau H Roscoe TJ Zähringer U Heinz E 《Journal of lipid research》2004,45(10):1899-1909
In algae, the biosynthesis of docosahexaenoic acid (22:6omega3; DHA) proceeds via the elongation of eicosapentaenoic acid (20:5omega3; EPA) to 22:5omega3, which is required as a substrate for the final Delta4 desaturation. To isolate the elongase specific for this step, we searched expressed sequence tag and genomic databases from the algae Ostreococcus tauri and Thalassiosira pseudonana, from the fish Oncorhynchus mykiss, from the frog Xenopus laevis, and from the sea squirt Ciona intestinalis using as a query the elongase sequence PpPSE1 from the moss Physcomitrella patens. The open reading frames of the identified elongase candidates were expressed in yeast for functional characterization. By this, we identified two types of elongases from O. tauri and T. pseudonana: one specific for the elongation of (Delta6-)C18-PUFAs and one specific for (Delta5-)C20-PUFAs, showing highest activity with EPA. The clones isolated from O. mykiss, X. laevis, and C. intestinalis accepted both C18- and C20-PUFAs. By coexpression of the Delta6- and Delta5-elongases from T. pseudonana and O. tauri, respectively, with the Delta5- and Delta4-desaturases from two other algae we successfully implemented DHA synthesis in stearidonic acid-fed yeast. This may be considered an encouraging first step in future efforts to implement this biosynthetic sequence into transgenic oilseed crops. 相似文献
996.
Johann Schredelseker Anamika Dayal Thorsten Schwerte Clara Franzini-Armstrong Manfred Grabner 《The Journal of biological chemistry》2009,284(2):1242-1251
The paralyzed zebrafish strain relaxed carries a null mutation for
the skeletal muscle dihydropyridine receptor (DHPR) β1a
subunit. Lack of β1a results in (i) reduced membrane
expression of the pore forming DHPR α1S subunit, (ii)
elimination of α1S charge movement, and (iii) impediment of
arrangement of the DHPRs in groups of four (tetrads) opposing the ryanodine
receptor (RyR1), a structural prerequisite for skeletal muscle-type
excitation-contraction (EC) coupling. In this study we used relaxed
larvae and isolated myotubes as expression systems to discriminate specific
functions of β1a from rather general functions of β
isoforms. Zebrafish and mammalian β1a subunits quantitatively
restored α1S triad targeting and charge movement as well as
intracellular Ca2+ release, allowed arrangement of DHPRs in
tetrads, and most strikingly recovered a fully motile phenotype in
relaxed larvae. Interestingly, the cardiac/neuronal
β2a as the phylogenetically closest, and the ancestral
housefly βM as the most distant isoform to β1a
also completely recovered α1S triad expression and charge
movement. However, both revealed drastically impaired intracellular
Ca2+ transients and very limited tetrad formation compared with
β1a. Consequently, larval motility was either only partially
restored (β2a-injected larvae) or not restored at all
(βM). Thus, our results indicate that triad expression and
facilitation of 1,4-dihydropyridine receptor (DHPR) charge movement are common
features of all tested β subunits, whereas the efficient arrangement of
DHPRs in tetrads and thus intact DHPR-RyR1 coupling is only promoted by the
β1a isoform. Consequently, we postulate a model that presents
β1a as an allosteric modifier of α1S
conformation enabling skeletal muscle-type EC coupling.Excitation-contraction
(EC)3 coupling in
skeletal muscle is critically dependent on the close interaction of two
distinct Ca2+ channels. Membrane depolarizations of the myotube are
sensed by the voltage-dependent 1,4-dihydropyridine receptor (DHPR) in the
sarcolemma, leading to a rearrangement of charged amino acids (charge
movement) in the transmembrane segments S4 of the pore-forming DHPR
α1S subunit
(1,
2). This conformational change
induces via protein-protein interaction
(3,
4) the opening of the
sarcoplasmic type-1 ryanodine receptor (RyR1) without need of Ca2+
influx through the DHPR (5).
The release of Ca2+ from the sarcoplasmic reticulum via RyR1
consequently induces muscle contraction. The protein-protein interaction
mechanism between DHPR and RyR1 requires correct ultrastructural targeting of
both channels. In Ca2+ release units (triads and peripheral
couplings) of the skeletal muscle, groups of four DHPRs (tetrads) are coupled
to every other RyR1 and hence are geometrically arranged following the
RyR-specific orthogonal arrays
(6).The skeletal muscle DHPR is a heteromultimeric protein complex, composed of
the voltage-sensing and pore-forming α1S subunit and
auxiliary subunits β1a, α2δ-1, and
γ1 (7). While
gene knock-out of the DHPR γ1 subunit
(8,
9) and small interfering RNA
knockdown of the DHPR α2δ-1 subunit
(10-12)
have indicated that neither subunit is essential for coupling of the DHPR with
RyR1, the lack of the α1S or of the intracellular
β1a subunit is incompatible with EC coupling and accordingly
null model mice die perinatally due to asphyxia
(13,
14). β subunits of
voltage-gated Ca2+ channels were repeatedly shown to be responsible
for the facilitation of α1 membrane insertion and to be
potent modulators of α1 current kinetics and voltage
dependence (15,
16). Whether the loss of EC
coupling in β1-null mice was caused by decreased DHPR membrane
expression or by the lack of a putative specific contribution of the β
subunit to the skeletal muscle EC coupling apparatus
(17,
18) was not clearly resolved.
Recently, other β-functions were identified in skeletal muscle using the
β1-null mutant zebrafish relaxed
(19,
20). Like the
β1-knock-out mouse
(14) zebrafish
relaxed is characterized by complete paralysis of skeletal muscle
(21,
22). While
β1-knock-out mouse pups die immediately after birth due to
respiratory paralysis (14),
larvae of relaxed are able to survive for several days because of
oxygen and metabolite diffusion via the skin
(23). Using highly
differentiated myotubes that are easy to isolate from these larvae, the lack
of EC coupling could be described by quantitative immunocytochemistry as a
moderate ∼50% reduction of α1S membrane expression
although α1S charge movement was nearly absent, and, most
strikingly, as the complete lack of the arrangement of DHPRs in tetrads
(19). Thus, in skeletal muscle
the β subunit enables EC coupling by (i) enhancing α1S
membrane targeting, (ii) facilitating α1S charge movement,
and (iii) enabling the ultrastructural arrangement of DHPRs in tetrads.The question arises, which of these functions are specific for the skeletal
muscle β1a and which ones are rather general properties of
Ca2+ channel β subunits. Previous reconstitution studies made
in the β1-null mouse system
(24,
25) using different β
subunit constructs (26) did
not allow differentiation between β-induced enhancement of non-functional
α1S membrane expression and the facilitation of
α1S charge movement, due to the lack of information on
α1S triad expression levels. Furthermore, the β-induced
arrangement of DHPRs in tetrads was not detected as no ultrastructural
information was obtained.In the present study, we established zebrafish mutant relaxed as
an expression system to test different β subunits for their ability to
restore skeletal muscle EC coupling. Using isolated myotubes for in
vitro experiments (19,
27) and complete larvae for
in vivo expression studies
(28-31)
and freeze-fracture electron microscopy, a clear differentiation between the
major functional roles of β subunits was feasible in the zebrafish
system. The cloned zebrafish β1a and a mammalian (rabbit)
β1a were shown to completely restore all parameters of EC
coupling when expressed in relaxed myotubes and larvae. However, the
phylogenetically closest β subunit to β1a, the
cardiac/neuronal isoform β2a from rat, as well as the
ancestral βM isoform from the housefly (Musca
domestica), could recover functional α1S membrane
insertion, but led to very restricted tetrad formation when compared with
β1a, and thus to impaired DHPR-RyR1 coupling. This impairment
caused drastic changes in skeletal muscle function.The present study shows that the enhancement of functional
α1S membrane expression is a common function of all the
tested β subunits, from β1a to even the most distant
βM, whereas the effective formation of tetrads and thus proper
skeletal muscle EC coupling is an exclusive function of the skeletal muscle
β1a subunit. In context with previous studies, our results
suggest a model according to which β1a acts as an allosteric
modifier of α1S conformation. Only in the presence of
β1a, the α1S subunit is properly folded to
allow RyR1 anchoring and thus skeletal muscle-type EC coupling. 相似文献
997.
Viral inhibitor of apoptosis vFLIP/K13 protects endothelial cells against superoxide-induced cell death
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Thurau M Marquardt G Gonin-Laurent N Weinländer K Naschberger E Jochmann R Alkharsah KR Schulz TF Thome M Neipel F Stürzl M 《Journal of virology》2009,83(2):598-611
Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposi's sarcoma (KS). HHV-8 encodes an antiapoptotic viral Fas-associated death domain-like interleukin-1β-converting enzyme-inhibitory protein (vFLIP/K13). The antiapoptotic activity of vFLIP/K13 has been attributed to an inhibition of caspase 8 activation and more recently to its capability to induce the expression of antiapoptotic proteins via activation of NF-κB. Our study provides the first proteome-wide analysis of the effect of vFLIP/K13 on cellular-protein expression. Using comparative proteome analysis, we identified manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant and an important antiapoptotic enzyme, as the protein most strongly upregulated by vFLIP/K13 in endothelial cells. MnSOD expression was also upregulated in endothelial cells upon infection with HHV-8. Microarray analysis confirmed that MnSOD is also upregulated at the RNA level, though the differential expression at the RNA level was much lower (5.6-fold) than at the protein level (25.1-fold). The induction of MnSOD expression was dependent on vFLIP/K13-mediated activation of NF-κB, occurred in a cell-intrinsic manner, and was correlated with decreased intracellular superoxide accumulation and increased resistance of endothelial cells to superoxide-induced death. The upregulation of MnSOD expression by vFLIP/K13 may support the survival of HHV-8-infected cells in the inflammatory microenvironment in KS. 相似文献
998.
Virginia Edgcomb William Orsi Chesley Leslin Slava S. Epstein John Bunge Sunok Jeon Michail M. Yakimov Anke Behnke Thorsten Stoeck 《Extremophiles : life under extreme conditions》2009,13(1):151-167
Environmental factors restrict the distribution of microbial eukaryotes but the exact boundaries for eukaryotic life are not
known. Here, we examine protistan communities at the extremes of salinity and osmotic pressure, and report rich assemblages
inhabiting Bannock and Discovery, two deep-sea superhaline anoxic basins in the Mediterranean. Using a rRNA-based approach,
we detected 1,538 protistan rRNA gene sequences from water samples with total salinity ranging from 39 to 280 g/Kg, and obtained evidence that this DNA was endogenous to the extreme habitat sampled. Statistical analyses indicate that the
discovered phylotypes represent only a fraction of species actually inhabiting both the brine and the brine-seawater interface,
with as much as 82% of the actual richness missed by our survey. Jaccard indices (e.g., for a comparison of community membership)
suggest that the brine/interface protistan communities are unique to Bannock and Discovery basins, and share little (0.8–2.8%)
in species composition with overlying waters with typical marine salinity and oxygen tension. The protistan communities from
the basins’ brine and brine/seawater interface appear to be particularly enriched with dinoflagellates, ciliates and other
alveolates, as well as fungi, and are conspicuously poor in stramenopiles. The uniqueness and diversity of brine and brine-interface
protistan communities make them promising targets for protistan discovery. 相似文献
999.
Distribution of Roseobacter RCA and SAR11 lineages and distinct bacterial communities from the subtropics to the Southern Ocean 总被引:1,自引:0,他引:1
Helge-Ansgar Giebel Thorsten Brinkhoff Walter Zwisler Natascha Selje Meinhard Simon 《Environmental microbiology》2009,11(8):2164-2178
We assessed the composition of the bacterioplankton in the Atlantic sector of the Southern Ocean in austral fall and winter and in New Zealand coastal waters in summer. The various water masses between the subtropics/Agulhas–Benguela boundary region and the Antarctic coastal current exhibited distinct bacterioplankton communities with the highest richness in the polar frontal region, as shown by denaturing gradient gel electrophoresis of 16S rRNA gene fragments. The SAR11 clade and the Roseobacter clade‐affiliated (RCA) cluster were quantified by real‐time quantitative PCR. SAR11 was detected in all samples analysed from subtropical waters to the coastal current and to depths of > 1000 m. In fall and winter, this clade constituted < 3% to 48% and 4–28% of total bacterial 16S rRNA genes respectively, with highest fractions in subtropical to polar frontal regions. The RCA cluster was only present in New Zealand coastal surface waters not exceeding 17°C, in the Agulhas–Benguela boundary region (visited only during the winter cruise), in subantarctic waters and in the Southern Ocean. In fall, this cluster constituted up to 36% of total bacterial 16S rRNA genes with highest fractions in the Antarctic coastal current and outnumbered the SAR11 clade at most stations in the polar frontal region and further south. In winter, the RCA cluster constituted lower proportions than the SAR11 clade and did not exceed 8% of total bacterial 16S rRNA genes. In fall, the RCA cluster exhibited significant positive correlations with latitude and ammonium concentrations and negative correlations with concentrations of nitrate, phosphate, and for near‐surface samples also with chlorophyll a, biomass production of heterotrophic prokaryotes and glucose turnover rates. The findings show that the various water masses between the subtropics and the Antarctic coastal current harbour distinct bacterioplankton communities. They further indicate that the RCA cluster, despite the narrow sequence similarity of > 98% of its 16S rRNA gene, is an abundant component of the heterotrophic bacterioplankton in the Southern Ocean, in particular in its coldest regions. 相似文献
1000.
Tobias Reinl Manfred Nimtz Claudia Hundertmark Thorsten Johl Gy?rgy Kéri Jürgen Wehland Henrik Daub Lothar J?nsch 《Molecular & cellular proteomics : MCP》2009,8(12):2778-2795
Stimulated by its physiological ligand, hepatocyte growth factor, the transmembrane receptor tyrosine kinase Met activates a signaling machinery that leads to mitogenic, motogenic, and morphogenic responses. Remarkably, the food-borne human pathogen Listeria monocytogenes also promotes autophosphorylation of Met through its virulence factor internalin B (InlB) and subsequently exploits Met signaling to induce phagocytosis into a broad range of host cells. Although the interaction between InlB and Met has been studied in detail, the signaling specificity of components involved in InlB-triggered cellular responses remains poorly characterized. The analysis of regulated phosphorylation events on protein kinases is therefore of particular relevance, although this could not as yet be characterized systematically by proteomics. Here, we implemented a new pyridopyrimidine-based strategy that enabled the efficient capture of a considerable subset of the human kinome in a robust one-step affinity chromatographic procedure. Additionally, and to gain functional insights into the InlB/Met-induced bacterial invasion process, a quantitative survey of the phosphorylation pattern of these protein kinases was accomplished. In total, the experimental design of this study comprises affinity chromatographic procedures for the systematic enrichment of kinases, as well as phosphopeptides; the quantification of all peptides based on the iTRAQTM reporter system; and a rational statistical strategy to evaluate the quality of phosphosite regulations. With this improved chemical proteomics strategy, we determined and relatively quantified 143 phosphorylation sites detected on 94 human protein kinases. Interestingly, InlB-mediated signaling shows striking similarities compared with the natural ligand hepatocyte growth factor that was intensively studied in the past. In addition, this systematic approach suggests a new subset of protein kinases including Nek9, which are differentially phosphorylated after short time (4-min) treatment of cells with the Met-activating InlB321. Thus, this quantitative phosphokinome study suggests a general, hypothesis-free concept for the detection of dynamically regulated protein kinases as novel signaling components involved in host-pathogen interactions.The human food-borne pathogen Listeria monocytogenes has evolved mechanisms to cross the intestinal, placental, and blood-brain barriers with severe consequences for pregnant women, newborns, and immunocompromised individuals. As a facultative intracellular pathogen, L. monocytogenes invades host cells within minutes, thus escaping the humoral arm of adaptive immunity. In this protective host niche, the organism replicates and spreads from cell to cell through the formation of so-called membrane protrusions. L. monocytogenes utilizes two different molecular routes to invade non-professional phagocytotic cells. (i) Internalin A binds to the cell adhesion molecule E-cadherin, resulting in the initial penetration of intestinal tissue (1, 2). (ii) In contrast, internalin B (InlB)1 contributes to the systemic infection of the host, promoting the invasion of a broader range of cell types including hepatocytes (3) and endothelial cells (4). A basic GW motif at the C terminus mediates the attachment of InlB to the bacterial cell wall, but the non-covalent nature of this interaction also allows the partial release of InlB into the environment (5, 6). GW domains of soluble InlB interact with glycosaminoglycans (7) and the complement receptor qC1q-R (8) on the host cell surface, although these interactions seem to be dispensable for the process of listerial invasion. In contrast, the N-terminal region of InlB comprising the cap, leucine-rich repeat, and inter-repeat domains (termed InlB321) constitutes structural features that stimulate the bacterial ingestion into the host cell cytosol. The horseshoe-like shape of InlB321 allows binding to and activation of the transmembrane tyrosine kinase Met, which is also the receptor for the host growth factor, hepatocyte growth factor (HGF). Although InlB binds to a different region of Met compared with HGF, it exploits the Met signaling capabilities, ultimately leading to actin cytoskeleton rearrangements, membrane engulfment, and uptake of the pathogen. InlB induces a rapid autophosphorylation in the kinase domain of Met (9) followed by recruitment of specific adapter molecules initiating signal transduction via prominent downstream components such as PI3K and the Raf-Erk pathway (10). Moreover, immobilized InlB321 is sufficient to induce the efficient uptake of latex beads into the host cell (11, 12). Recently, the structure of the InlB321-Met complex was solved at the atomic level, unambiguously demonstrating that InlB321 is mandatory but also sufficient to activate Met signaling (13).Numerous molecular studies of signaling components have been reported, and a complex protein network downstream of Met has been compiled (14). However, the molecular interactions defined so far are still insufficient to derive the InlB-induced signal transduction pathway resulting in uptake of Listeria. As a basic signaling principle, protein kinase-catalyzed phosphorylation regulates virtually every function of substrate proteins, i.e. protein-protein interactions, localization, activity, and stability. With more than 500 members, the superfamily of protein kinases is among the largest protein families encoded by the human genome (15). The functional mechanisms regulated by kinase-mediated phosphorylations on substrate proteins are also involved in the activity control of the kinases themselves. Studying these modifications directly at the kinase level enables classification of their activated states, and their systematic investigation by proteomics has already been used to detect and correlate kinases with potential functions in cell cycle control and cancer biology (16, 17). A detailed knowledge of InlB/Met-affected phosphorylation sites of proteins from the kinase superfamily would contribute to a better understanding of the listerial invasion strategy in addition to complementing our knowledge of the Met signaling pathway.Phosphorylation sites can be detected during the process of automatic peptide sequencing in well established bottom-up proteome approaches. However, the substoichiometric nature and poor ionization properties of phosphopeptides usually require purification strategies such as IMAC to optimize analysis by mass spectrometry (18). Furthermore, the complexity of the total phosphoproteome requires the pre-enrichment of protein kinases as a prerequisite for characterization of the low abundance family members. We and others have demonstrated that the highly conserved ATP-binding region of protein kinases offers possibilities for their systematic purification based on immobilized ATP-competitive small molecule inhibitors with broad kinase selectivity. In combination with phosphopeptide enrichment, this strategy has proven to be highly appropriate for a comprehensive LC-MS/MS-based phosphorylation site analysis of these key signaling components (17, 19, 20). To characterize the role of protein kinases as key regulatory elements in signaling pathways, the acquisition of quantitative peptide data of both the phosphorylated and unmodified proteins is required. Powerful isotopic labeling approaches such as SILAC (21) and iTRAQTM (22) have been devised and successfully applied to dissect cell and signaling states mainly at the substrate protein level (23, 24), but they are also beginning to support the in-depth characterization of the human kinome (17, 20, 25). Because the detection of individually regulated phosphopeptides has to cope with the so-called “one-hit wonder” problem in proteomics, the interpretation of single peptide regulation requires that particular attention must be paid to the process of statistical raw data evaluation. We have recently established a validated statistical strategy for the quality control of quantitative MS methods used in this study (26). In total, this bioinformatics work flow normalizes unequal sample amounts, corrects isotopic impurities of iTRAQ labeling reagents, and importantly can calculate the reliability of regulatory data based on the actual signal-to-noise properties of the mass spectrometer used.The synthesis of an optimized affinity resin as a base for a robust single step capture of protein kinases was the starting point in this study and allowed the systematic analyses of this enzyme class in human epithelial cells. In the following, we explain the biochemical strategy established for the quantitative characterization of phosphorylation events at these kinases in the context of infection. The dissection of one representative data set shows the potential of the selected strategy but also underscores the necessity of our statistical approach for evaluating the regulatory information based on iTRAQ reporter ions. Finally, we apply the total approach to analyze protein kinases systematically in the Met receptor kinase pathway exploited by the invasin InlB from L. monocytogenes. The majority of unambiguously regulated phosphorylation events are in accordance with our existing knowledge about the HGF/Met pathway. Furthermore, this study suggests novel candidates such as Nek9 involved in signal networks exploited in the process of listerial invasion. 相似文献