Defective glycosylation of coagulation factor XII underlies hereditary angioedema type III.

Hereditary angioedema type III (HAEIII) is a rare inherited swelling disorder that is associated with point mutations in the gene encoding the plasma protease factor XII (FXII). Here, we demonstrate that HAEIII-associated mutant FXII, derived either from HAEIII patients or recombinantly produced, is defective in mucin-type Thr309-linked glycosylation. Loss of glycosylation led to increased contact-mediated autoactivation of zymogen FXII, resulting in excessive activation of the bradykinin-forming kallikrein-kinin pathway. In contrast, both FXII-driven coagulation and the ability of C1-esterase inhibitor to bind and inhibit activated FXII were not affected by the mutation. Intravital laser-scanning microscopy revealed that, compared with control animals, both F12-/- mice reconstituted with recombinant mutant forms of FXII and humanized HAEIII mouse models with inducible liver-specific expression of Thr309Lys-mutated FXII exhibited increased contact-driven microvascular leakage. An FXII-neutralizing antibody abolished bradykinin generation in HAEIII patient plasma and blunted edema in HAEIII mice. Together, the results of this study characterize the mechanism of HAEIII and establish FXII inhibition as a potential therapeutic strategy to interfere with excessive vascular leakage in HAEIII and potentially alleviate edema due to other causes.

N-glycoproteomics: mass spectrometry-based glycosylation site annotation.

Glycosylations are ubiquitous and, in many cases, essential protein modifications. Yet comprehensive and detailed analysis of glycosylations on a proteome-wide scale is a daunting and still unsolved challenge. However, a common workflow has emerged over the last decade for large-scale N-glycosylation site annotation by application of proteomic methodology. Thereby, the qualitative and quantitative assessment of hundreds or thousands of modification sites is enabled. This review presents a short overview about common enrichment techniques and glycosylation site detection for N-glycopeptides, including benefits and challenges of analysis.

E. coli LoiP (YggG), a metalloprotease hydrolyzing Phe-Phe bonds.

YggG is a conserved lipoprotein localized to the outer membrane of Gram negative bacteria. Even though the expressed open reading frame has been identified previously, the Escherichia coli protein remained uncharacterized. We report that YggG of E. coli is a metalloprotease that cleaves its targets preferentially between Phe-Phe residues. Since the yggG promoter is upregulated when bacteria are subjected to media of low osmolarity, YggG was named LoiP (low osmolarity induced protease). LoiP has an intramolecular disulfide (S-S) bond that is formed even in the absence of the periplasmic oxido-reductase DsbA and proper membrane localization of LoiP can depend on another putative metalloprotease, YfgC.

STEAP1 is associated with the invasive and oxidative stress phenotype of Ewing tumors.

Ewing tumors comprise the second most common type of bone-associated cancer in children and are characterized by oncogenic EWS/FLI1 fusion proteins and early metastasis. Compelling evidence suggests that elevated levels of intracellular oxidative stress contribute to enhanced aggressiveness of numerous cancers, possibly including Ewing tumors. Using comprehensive microarray analyses and RNA interference, we identified the six-transmembrane epithelial antigen of the prostate 1 (STEAP1)-a membrane-bound mesenchymal stem cell marker of unknown function-as a highly expressed protein in Ewing tumors compared with benign tissues and show its regulation by EWS/FLI1. In addition, we show that STEAP1 knockdown reduces Ewing tumor proliferation, anchorage-independent colony formation as well as invasion in vitro and decreases growth and metastasis of Ewing tumor xenografts in vivo. Moreover, transcriptome and proteome analyses as well as functional studies revealed that STEAP1 expression correlates with oxidative stress responses and elevated levels of reactive oxygen species that in turn are able to regulate redox-sensitive and proinvasive genes. In synopsis, our data suggest that STEAP1 is associated with the invasive behavior and oxidative stress phenotype of Ewing tumors and point to a hitherto unanticipated oncogenic function of STEAP1.

Identification of Eps15 as antigen recognized by the monoclonal antibodies aa2 and ab52 of the Wuerzburg Hybridoma Library against Drosophila brain.

The Wuerzburg Hybridoma Library against the Drosophila brain represents a collection of around 200 monoclonal antibodies that bind to specific structures in the Drosophila brain. Here we describe the immunohistochemical staining patterns, the Western blot signals of one- and two-dimensional electrophoretic separation, and the mass spectrometric characterization of the target protein candidates recognized by the monoclonal antibodies aa2 and ab52 from the library. Analysis of a mutant of a candidate gene identified the Drosophila homolog of the Epidermal growth factor receptor Pathway Substrate clone 15 (Eps15) as the antigen for these two antibodies.

Composition and topology of the endoplasmic reticulum-mitochondria encounter structure.

Eukaryotic cells contain multiple organelles, which are functionally and structurally interconnected. The endoplasmic reticulum-mitochondria encounter structure (ERMES) forms a junction between mitochondria and the endoplasmic reticulum (ER). Four ERMES proteins are known in yeast, the ER-anchored protein Mmm1 and three mitochondria-associated proteins, Mdm10, Mdm12 and Mdm34, with functions related to mitochondrial morphology and protein biogenesis. We mapped the glycosylation sites of ERMES and demonstrate that three asparagine residues in the N­terminal domain of Mmm1 are glycosylated. While the glycosylation is dispensable, the cytosolic C­terminal domain of Mmm1 that connects to the Mdm proteins is required for Mmm1 function. To analyze the composition of ERMES, we determined the subunits by quantitative mass spectrometry. We identified the calcium-binding GTPase Gem1 as a new ERMES subunit, revealing that ERMES is composed of five genuine subunits. Taken together, ERMES represents a platform that integrates components with functions in formation of ER-mitochondria junctions, maintenance of mitochondrial morphology, protein biogenesis and calcium binding.

Strong cation exchange chromatography for analysis of sialylated glycopeptides.

Glycosylations represent major and essential co- and post-translational modification forms of proteins and facilitate a multitude of functions such as cell-cell interactions as well as protein folding and stability. The analysis of protein glycosylation is still an enormous task due to the vast heterogeneity and multitude of different possible carbohydrate structures. The elucidation of glycosylation sites - the attachment points of carbohydrate structures to the polypeptide backbone - is often among the first necessary steps of analysis. Therefore, we here present a simple protocol for charge-based enrichment of sialylated glycopeptides by strong cation exchange chromatography and subsequent analysis of glycosylation sites by mass spectrometry.

Phosphoproteome analysis of the platelet plasma membrane.

Blood platelets are key players standing at the crossroads between physiologically occurring hemostasis and pathologic thrombus formation. As these cellular particles lack a nucleus, intra- and intercellular processes involved in platelet activity and function are almost exclusively regulated on the protein level. In particular, posttranslational protein modification by phosphorylation, which allows for a quick and highly dynamic transduction of cellular signals, is discussed in this context. In addition, since platelet activation and aggregation usually require surface contact with the surrounding tissue, special interest focuses on this contacting region, and hence on the subproteome of the platelet plasma membrane. In this chapter, we present a mass spectrometry-driven approach capable of dealing with the task of platelet plasma membrane proteomics and phosphoproteomics. The outlined protocols include strategies for the isolation and purification of plasma membrane proteins by aqueous two-phase partitioning and subsequent enrichment of phosphopeptides via titanium dioxide chromatography.

The good, the bad, the ugly: validating the mass spectrometric analysis of modified peptides.

Mass spectrometric characterization of protein modifications is usually based on single peptides. With the advent of large-scale PTM-focussed MS studies, vast amounts of data are generated continuously, providing biologists extremely valuable and virtually never-ending sources for targeted functional research. However, even more than for proteomics in general, appropriate strategies for quality control of the different steps of the analytical strategy are imperative to prevent functional researchers from doing Sisyphos work on false-positive and unconfident PTM assignments. Here, we describe strategies to address the important issue of quality control for PTM analysis on various levels of the analytical pipeline: sample preparation/processing, analysis/identification and finally data interpretation, for qualitative as well as quantitative studies.

Online dual gradient reversed-phase/porous graphitized carbon nanoHPLC for proteomic applications.

The analysis of proteolytic peptide mixtures is among the dominant tasks within proteomic workflows. In order to limit undersampling effects during mass spectrometric detection, online-coupled liquid chromatography is the method of choice, with reversed-phase chromatography being the most important separation mode. Since hydrophilic compounds such as short peptides and some glycosylated species as well as oligosaccharides from glycoproteomic workflows are commonly not accessible by this analytical setup, we hereby present a dual gradient system combining reversed-phase and porous graphitic carbon retention modes within a single nanoHPLC setup. Samples in the low femtomole range are analyzed consecutively first by reversed phase, and nonretained molecules are directly separated by porous graphitic carbon. Both gradient elution systems allow for online coupled mass spectrometric detection and are demonstrated to enable analysis of protein, peptide, and oligosaccharide mixtures within the same setup. Thereby, the accessible range for proteomic and glycoproteomic applications may be extended far beyond the limits of conventional reversed-phase nanoHPLC setups.

Identification of a Protein Kinase C-dependent phosphorylation site involved in sensitization of TRPV4 channel.

Transient Receptor Potential (TRP) proteins are non-selective cation channels performing diverse cellular functions. TRPV1 and TRPV4, two calcium-permeable channels of the vanilloid subfamily of TRP proteins, are activated by various physical and chemical stimuli, including noxious heat and mechanical stress, respectively. These channels are also required for exaggerated sensation of painful stimuli, condition referred to as hyperalgesia, which is frequently associated with inflammation. Phosphorylation of TRPV1, involving Protein Kinase C (PKC) and Protein Kinase A (PKA), appears to be the predominant mechanism for channel sensitization and development of heat hyperalgesia. PKC and PKA pathways have also been implicated in the sensitization of TRPV4, but the respective phosphorylation sites remain unknown. Using mass spectrometry, we report now that TRPV4 is phosphorylated on serine 824 by the PKC-activating phorbol 12-myristate 13-acetate. This phosphorylation is prevented by a PKC inhibitor, confirming the involvement of PKC. Ser824, located in the carboxy-terminal cytosolic tail of TRPV4, is also phosphorylated after activation of the PKA pathway by forskolin, albeit less potently. Substitution of Ser824 with aspartic acid, mimicking phosphorylation at this site, increased TRPV4-mediated calcium influx in resting and in stimulated cells, underlining the importance of this residue in TRPV4 regulation. Thus PKC, and possibly PKA, phosphorylate TRPV4 at Ser824 leading to the enhancement of TRPV4 channel function. Our findings suggest an important role of this phosphorylation in TRPV4 sensitization and the development of hyperalgesia.

Characterization of a novel interaction between vasodilator-stimulated phosphoprotein and Abelson interactor 1 in human platelets: a concerted computational and experimental approach.

OBJECTIVE: The goal of this study was systematic profiling of vasodilator-stimulated phosphoprotein (VASP)-Ena/VASP homology 1 (EVH1) interactors in human platelets using a combined in silico and in vitro approach. METHODS AND RESULTS: Exploiting the information of the comprehensive proteome catalogue in the PlateletWeb database (http://plateletweb.bioapps.biozentrum.uni-wuerzburg.de/PlateletWeb.php), we performed a motif search of all sequences and identified potential target sites of class I EVH1 domains in human platelet proteins. Performing affinity purification with VASP-EVH1 domain and the lysates of platelets, we examined complex partners by mass spectrometry. Combining the results of both analyses, we identified Abelson interactor 1 (Abi-1) as a novel EVH1 domain-specific interaction partner of VASP in human platelets and investigated this interaction by yeast 2-hybrid mutational studies and immunoprecipitation. Immunofluorescence microscopy indicated colocalization of both proteins at the lamellipodia of spread human platelets, suggesting a role in reorganizing the cytoskeleton during spreading. CONCLUSIONS: The combination of experimental and computational interactome research has emerged as a valuable tool for the analysis of protein-protein interaction networks and facilitates the discovery and characterization of novel interactions as detailed here for Abi-1 and VASP in human platelets. System biological approaches can be expected to play an important role in basic and clinical platelet research, as they offer the potential to analyze signal transduction beyond the scope of established pathways.

Integrin-dependent translocation of LASP-1 to the cytoskeleton of activated platelets correlates with LASP-1 phosphorylation at tyrosine 171 by Src-kinase.

During platelet adhesion, the complex cytoskeletal structure is rearranged resulting in the formation of F-actin-based filopodia and lamellipodia. Stimulatory platelet signalling pathways include binding of integrin alpha(IIb)beta(3) to fibrinogen followed by activation of protein tyrosine kinases (PTK) and phosphorylation of downstream signalling proteins. In this study, we demonstrate that the scaffolding and F-actin binding protein LASP-1 undergoes tyrosine phosphorylation in thrombin-stimulated human platelets. By means of specific inhibitors we identified Src-kinase as the primary enzyme phosphorylating LASP-1 in intact cells. These data were confirmed in platelet model cells (A5-CHO cells), constitutively expressing integrin alpha(IIb)beta(3). Fibrinogen-mediated cell stimulation resulted in a similar tyrosine phosphorylation of transiently transfected LASP-1. Site directed mutagenesis identified tyrosine 171 as the Src-kinase phosphorylation site. Immunofluorescence microscopic analysis of these cells revealed a relocation of LASP-1 to focal contacts and the leading edge of the membrane upon fibrinogen activation and tyrosine 171 phosphorylation. This translocation was also seen in adherent platelets. Concomitant with adhesion, LASP-1 translocated from the cytosol along the arms of the pseudopodia into the leading lamellae of the spreading platelets, indicating a crucial role of the protein in platelet cytoskeleton rearrangement.

Recent advances in yeast organelle and membrane proteomics.

Yeast proteome research comprises two different aspects: with respect to systemic fungal infections (fungemias), invasive candidiasis, for instance by Candida albicans, is among the most common causes of morbidity and mortality particularly in the expanding population of immunocompromised patients, which rises a high medical and pharmaceutical interest in this facultative pathogenic organism. Apart from its clinical relevance, yeast research moreover provides an indispensable source of knowledge regarding fundamental biochemical processes of eukaryotic cells. In this context, the budding yeast Saccharomyces cerevisiae is, in addition to its multiple industrial applications, one of the most extensively used microorganisms and serves as the best understood eukaryotic model system so far. Consequently, numerous studies have focused on gaining insight into the yeast proteome, with protein MS providing a very efficient technology to cope with this task since it enables both protein identification and differential quantification of cellular material. In this review we present an overview of recent advances in yeast organelle and membrane proteomics focusing on the cell wall, plasma membrane, mitochondria and vacuole.

Analysis of tyrosine-phosphorylated proteins in rat brain mitochondria.

Mitochondrial protein phosphorylation is emerging as a central event in mitochondrial signaling. In particular, tyrosine phosphorylation is proving to be an unappreciated mechanism involved in regulation of mitochondrial functions. Tyrosine kinases and phosphatases have been identified in mitochondrial compartments and there is a steadily increasing number of new identified tyrosine-phosphorylated proteins implicated in a wide spectrum of mitochondrial functions. The deciphering of the tyrosine phosphorylation signaling in mitochondria is strictly linked to the definition of the entire mitochondrial tyrosine phosphoproteome. This chapter describes methods to analyze tyrosine phosphorylation in brain mitochondria: identification of new substrates by biochemical and mass spectrometry approaches and bioinformatic tools to analyze the potential effect of tyrosine phosphorylation on the structure/activity of a protein.

Platelet membrane proteomics: a novel repository for functional research.

Being central players in thrombosis and hemostasis, platelets react in manifold and complex ways to extracellular stimuli. Cell-matrix and cell-cell interactions are mandatory for initial adhesion as well as for final development of stable plugs. Primary interfaces for interactions are plasma membrane proteins, of which many have been identified over the past decades in individual studies. However, due to their enucleate structure, platelets are not accessible to large-scale genomic screens and thus a comprehensive inventory of membrane proteins is still missing. For this reason, we here present an advanced proteomic setup for the detailed analysis of enriched platelet plasma membranes and the so far most complete collection of platelet membrane proteins. In summary, 1282 proteins were identified, of which more than half are termed to be of membrane origin. This study provides a brief overview of gene ontology subcellular and functional classification, as well as interaction network analysis. In addition, the mass spectrometric data were used to assemble a first tentative relative quantification of large-scale data on the protein level. We therefore estimate the presented data to be of major interest to the platelet research field and to support rational design of functional studies.

N-glycosylation site analysis of human platelet proteins by hydrazide affinity capturing and LC-MS/MS.

A starting point for many glycosylation analysis pathways is marked by the determination of the respectivecarbohydrate attachment sites to the polypeptide backbone of proteins. Several methods have been reported for this purpose in the past, commonly divided into a three-step approach (1) affinity purification of glycoproteins/-peptides, (2) processing/trimming of the glycopeptides and (3) elucidation of the glycan attachment site by mass spectrometry. For N-glycosylation site analysis the last two steps are usually similar, while methods differ in the affinity purification step. Here, we describe the oxidative derivatisation of carbohydrate moieties and covalent trapping of glycopeptides on hydrazide functionalized beads. This method is suitable for large scale analysis of glycoproteins as well as isolated glycoproteins and can be applied readily to a number of different samples. In the described protocol, the elucidation of N-glycosylation sites of human platelet proteins is demonstrated as an example.

The Wuerzburg hybridoma library against Drosophila brain.

This review describes the present state of a project to identify and characterize novel nervous system proteins by using monoclonal antibodies (mAbs) against the Drosophila brain. Some 1,000 hybridoma clones were generated by injection of homogenized Drosophila brains or heads into mice and fusion of their spleen cells with myeloma cells. Testing the mAbs secreted by these clones identified a library of about 200 mAbs, which selectively stain specific structures of the Drosophila brain. Using the approach "from antibody to gene", several genes coding for novel proteins of the presynaptic terminal were cloned and characterized. These include the "cysteine string protein" gene (Csp, mAb ab49), the "synapse-associated protein of 47 kDa" gene (Sap47, mAbs nc46 and nb200), and the "Bruchpilot" gene (brp, mAb nc82). By a "candidate" approach, mAb nb33 was shown to recognize the pigment dispersing factor precursor protein. mAbs 3C11 and pok13 were raised against bacterially expressed Drosophila synapsin and calbindin-32, respectively, after the corresponding cDNAs had been isolated from an expression library by using antisera against mammalian proteins. Recently, it was shown that mAb aa2 binds the Drosophila homolog of "epidermal growth factor receptor pathway substrate clone 15" (Eps15). Identification of the targets of mAbs na21, ab52, and nb181 is presently attempted. Here, we review the available information on the function of these proteins and present staining patterns in the Drosophila brain for classes of mAbs that either bind differentially in the eye, in neuropil, in the cell-body layer, or in small subsets of neurons. The prospects of identifying the corresponding antigens by various approaches, including protein purification and mass spectrometry, are discussed.

Tyrosine phosphorylation modulates the activity of TRPV4 in response to defined stimuli.

Src family tyrosine kinases (SFKs) regulate the function of several transient receptor potential (TRP) family members, yet their role in the regulation of the vanilloid subfamily member 4 protein (TRPV4) remains controversial. TRPV4 is a calcium-permeable channel activated by numerous physical and chemical stimuli. Here we show that SFKs mediate tyrosine phosphorylation of TRPV4 in different cell lines. Using mass spectrometric analysis, we identified two novel phosphorylation sites in the cytosolic N- and C-terminal tails of TRPV4. Substitution of either tyrosine with phenylalanine led to a substantial reduction in the overall tyrosine phosphorylation level of TRPV4, suggesting that these two tyrosines constitute major phosphorylation sites. Both mutants efficiently localized to the plasma membrane, indicating that neither tyrosine is required for trafficking of TRPV4 in the secretory pathway. Analysis of the channel function demonstrated a crucial role of the N-terminal tyrosine residue in the activation of TRPV4 by heat, mechanical (shear) stress, hypotonic cell swelling, and phorbol 12-myristate 13-acetate, but not in the activation by synthetic ligand 4alpha-phorbol 12,13-didecanoate. Furthermore, the response of TRPV4 to phorbol 12-myristate 13-acetate was SFK-dependent. Because the SFK-mediated phosphorylation of the N-terminal tyrosine occurred before TRPV4 activation, tyrosine phosphorylation appears to sensitize rather than activate this channel. Reactive oxygen species, known to mediate inflammatory pain, strongly up-regulated TRPV4 phosphorylation in the presence of SFKs. Our findings indicate that tyrosine phosphorylation of TRPV4 represents an important modulatory mechanism, which may underlie the recently described function of TRPV4 in inflammatory hyperalgesia.

The catalytic activity of protein-disulfide isomerase requires a conformationally flexible molecule.

Protein-disulfide isomerase (PDI) catalyzes the formation of the correct pattern of disulfide bonds in secretory proteins. A low resolution crystal structure of yeast PDI described here reveals large scale conformational changes compared with the initially reported structure, indicating that PDI is a highly flexible molecule with its catalytic domains, a and a', representing two mobile arms connected to a more rigid core composed of the b and b' domains. Limited proteolysis revealed that the linker between the a domain and the core is more susceptible to degradation than that connecting the a' domain to the core. By restricting the two arms with inter-domain disulfide bonds, the molecular flexibility of PDI, especially that of its a domain, was demonstrated to be essential for the enzymatic activity in vitro and in vivo. The crystal structure also featured a PDI dimer, and a propensity to dimerize in solution and in the ER was confirmed by cross-linking experiments and the split green fluorescent protein system. Although sedimentation studies suggested that the self-association of PDI is weak, we hypothesize that PDI exists as an interconvertible mixture of monomers and dimers in the endoplasmic reticulum due to its high abundance in this compartment.