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1.  A New Mint1 Isoform, but Not the Conventional Mint1, Interacts with the Small GTPase Rab6 
PLoS ONE  2013;8(5):e64149.
Small GTPases of the Rab family are important regulators of a large variety of different cellular functions such as membrane organization and vesicle trafficking. They have been shown to play a role in several human diseases. One prominent member, Rab6, is thought to be involved in the development of Alzheimer’s Disease, the most prevalent mental disorder worldwide. Previous studies have shown that Rab6 impairs the processing of the amyloid precursor protein (APP), which is cleaved to β-amyloid in brains of patients suffering from Alzheimer’s Disease. Additionally, all three members of the Mint adaptor family are implied to participate in the amyloidogenic pathway. Here, we report the identification of a new Mint1 isoform in a yeast two-hybrid screening, Mint1 826, which lacks an eleven amino acid (aa) sequence in the conserved C-terminal region. Mint1 826, but not the conventional Mint1, interacts with Rab6 via the PTB domain. This interaction is nucleotide-dependent, Rab6-specific and influences the subcellular localization of Mint1 826. We were able to detect and sequence a corresponding proteolytic peptide derived from cellular Mint1 826 by mass spectrometry proving the absence of aa 495–505 and could show that the deletion does not influence the ability of this adaptor protein to interact with APP. Taking into account that APP interacts and co-localizes with Mint1 826 and is transported in Rab6 positive vesicles, our data suggest that Mint1 826 bridges APP to the small GTPase at distinct cellular sorting points, establishing Mint1 826 as an important player in regulation of APP trafficking and processing.
PMCID: PMC3667844  PMID: 23737971
2.  Dietary Effects on Cuticular Hydrocarbons and Sexual Attractiveness in Drosophila 
PLoS ONE  2012;7(12):e49799.
Dietary composition is known to have profound effects on many aspects of animal physiology, including lifespan, general health, and reproductive potential. We have previously shown that aging and insulin signaling significantly influence the composition and sexual attractiveness of Drosophila melanogaster female cuticular hydrocarbons (CHCs), some of which are known to be sex pheromones. Because diet is intimately linked to aging and to the activity of nutrient-sensing pathways, we asked how diet affects female CHCs and attractiveness. Here we report consistent and significant effects of diet composition on female CHC profiles across ages, with dietary yeast and sugar driving CHC changes in opposite directions. Surprisingly, however, we found no evidence that these changes affect female attractiveness. Multivariate comparisons among responses of CHC profiles to diet, aging, and insulin signaling suggest that diet may alter the levels of some CHCs in a way that results in profiles that are more attractive while simultaneously altering other CHCs in a way that makes them less attractive. For example, changes in short-chain CHCs induced by a high-yeast diet phenocopy changes caused by aging and by decreased insulin signaling, both of which result in less attractive females. On the other hand, changes in long-chain CHCs in response to the same diet result in levels that are comparable to those observed in attractive young females and females with increased insulin signaling. The effects of a high-sugar diet tend in the opposite direction, as levels of short-chain CHCs resemble those in attractive females with increased insulin signaling and changes in long-chain CHCs are similar to those caused by decreased insulin signaling. Together, these data suggest that diet-dependent changes in female CHCs may be sending conflicting messages to males.
PMCID: PMC3515564  PMID: 23227150
3.  Insulin Signaling Mediates Sexual Attractiveness in Drosophila 
PLoS Genetics  2012;8(4):e1002684.
Sexually attractive characteristics are often thought to reflect an individual's condition or reproductive potential, but the underlying molecular mechanisms through which they do so are generally unknown. Insulin/insulin-like growth factor signaling (IIS) is known to modulate aging, reproduction, and stress resistance in several species and to contribute to variability of these traits in natural populations. Here we show that IIS determines sexual attractiveness in Drosophila through transcriptional regulation of genes involved in the production of cuticular hydrocarbons (CHC), many of which function as pheromones. Using traditional gas chromatography/mass spectrometry (GC/MS) together with newly introduced laser desorption/ionization orthogonal time-of-flight mass spectrometry (LDI-MS) we establish that CHC profiles are significantly affected by genetic manipulations that target IIS. Manipulations that reduce IIS also reduce attractiveness, while females with increased IIS are significantly more attractive than wild-type animals. IIS effects on attractiveness are mediated by changes in CHC profiles. Insulin signaling influences CHC through pathways that are likely independent of dFOXO and that may involve the nutrient-sensing Target of Rapamycin (TOR) pathway. These results suggest that the activity of conserved molecular regulators of longevity and reproductive output may manifest in different species as external characteristics that are perceived as honest indicators of fitness potential.
Author Summary
In nature, a myriad of specialized traits have evolved that are used for intraspecific communication and mate choice. We postulated that certain traits may have evolved to be attractive by virtue of their accurate representation of molecular pathways that are critical for determining evolutionary fitness. Insulin signaling (IIS) is one such pathway. It has been shown to modulate aging, reproduction, and stress resistance in several species and to contribute to variability of these traits in natural populations. We therefore asked whether IIS affected key sexual characteristics and overall attractiveness in the fruit fly Drosophila melanogaster. We found that IIS regulates cuticular hydrocarbons (the key pheromones in flies), that reduced IIS also reduced attractiveness, and that flies with increased IIS were significantly more attractive than wild-type animals. Further experiments revealed that these effects may also be influenced by a second conserved nutrient-sensitive pathway, the TOR pathway. We suggest that natural selection may have favored a plethora of species-specific sexual characteristics because they accurately represent a small number of influential pathways that determine longevity and reproductive output across taxa. In other words, it may be that, whether fly or human, beauty is more than skin-deep.
PMCID: PMC3343104  PMID: 22570625
5.  Male-Specific Transfer and Fine Scale Spatial Differences of Newly Identified Cuticular Hydrocarbons and Triacylglycerides in a Drosophila Species Pair 
PLoS ONE  2011;6(2):e16898.
We analyzed epicuticular hydrocarbon variation in geographically isolated populations of D. mojavensis cultured on different rearing substrates and a sibling species, D. arizonae, with ultraviolet laser desorption/ionization mass spectrometry (UV-LDI MS). Different body parts, i.e. legs, proboscis, and abdomens, of both species showed qualitatively similar hydrocarbon profiles consisting mainly of long-chain monoenes, dienes, trienes, and tetraenes. However, D. arizonae had higher amounts of most hydrocarbons than D. mojavensis and females of both species exhibited greater hydrocarbon amounts than males. Hydrocarbon profiles of D. mojavensis populations were significantly influenced by sex and rearing substrates, and differed between body parts. Lab food–reared flies had lower amounts of most hydrocarbons than flies reared on fermenting cactus substrates. We discovered 48 male- and species-specific hydrocarbons ranging in size from C22 to C50 in the male anogenital region of both species, most not described before. These included several oxygen-containing hydrocarbons in addition to high intensity signals corresponding to putative triacylglycerides, amounts of which were influenced by larval rearing substrates. Some of these compounds were transferred to female cuticles in high amounts during copulation. This is the first study showing that triacylglycerides may be a separate class of courtship-related signaling molecules in drosophilids. This study also extends the kind and number of epicuticular hydrocarbons in these species and emphasizes the role of larval ecology in influencing amounts of these compounds, many of which mediate courtship success within and between species.
PMCID: PMC3038915  PMID: 21369358
6.  Correction: Pheromonal and Behavioral Cues Trigger Male-to-Female Aggression in Drosophila 
PLoS Biology  2010;8(12):10.1371/annotation/1c19d040-9f9f-4b9f-b678-70f1fe387192.
PMCID: PMC2997046
7.  Pheromonal and Behavioral Cues Trigger Male-to-Female Aggression in Drosophila 
PLoS Biology  2010;8(11):e1000541.
By genetically manipulating both pheromonal profiles and behavioral patterns, we find that Drosophila males showed a complete reversal in their patterns of aggression towards other males and females
Appropriate displays of aggression rely on the ability to recognize potential competitors. As in most species, Drosophila males fight with other males and do not attack females. In insects, sex recognition is strongly dependent on chemosensory communication, mediated by cuticular hydrocarbons acting as pheromones. While the roles of chemical and other sensory cues in stimulating male to female courtship have been well characterized in Drosophila, the signals that elicit aggression remain unclear. Here we show that when female pheromones or behavior are masculinized, males recognize females as competitors and switch from courtship to aggression. To masculinize female pheromones, a transgene carrying dsRNA for the sex determination factor transformer (traIR) was targeted to the pheromone producing cells, the oenocytes. Shortly after copulation males attacked these females, indicating that pheromonal cues can override other sensory cues. Surprisingly, masculinization of female behavior by targeting traIR to the nervous system in an otherwise normal female also was sufficient to trigger male aggression. Simultaneous masculinization of both pheromones and behavior induced a complete switch in the normal male response to a female. Control males now fought rather than copulated with these females. In a reciprocal experiment, feminization of the oenocytes and nervous system in males by expression of transformer (traF) elicited high levels of courtship and little or no aggression from control males. Finally, when confronted with flies devoid of pheromones, control males attacked male but not female opponents, suggesting that aggression is not a default behavior in the absence of pheromonal cues. Thus, our results show that masculinization of either pheromones or behavior in females is sufficient to trigger male-to-female aggression. Moreover, by manipulating both the pheromonal profile and the fighting patterns displayed by the opponent, male behavioral responses towards males and females can be completely reversed. Therefore, both pheromonal and behavioral cues are used by Drosophila males in recognizing a conspecific as a competitor.
Author Summary
As in other species, the fruit fly Drosophila melanogaster uses chemical signals in the form of pheromones to recognize the species and sex of another individual. Males typically fight with other males and do not attack females. While the roles of pheromonal and other sensory cues in stimulating courtship towards females have been extensively studied, the signals that elicit aggression towards other males remain unclear. In this work, we use genetic tools to show that masculinization of female pheromones is sufficient to trigger aggression from wild type males towards females. Surprisingly, males also attacked females that displayed male patterns of aggression, even if they show normal female pheromonal profiles, indicating that pheromones are not the only cues important for identifying another animal as an opponent. By simultaneously manipulating pheromones and behavioral patterns of opponents, we can completely switch the behavioral response of males towards females and males. These results demonstrate that not only pheromonal but also behavioral cues can serve as triggers of aggression, underlining the importance of behavioral feedback in the manifestation of social behaviors.
PMCID: PMC2990703  PMID: 21124886
8.  A new male sex-pheromone and novel cuticular cues for chemical communication in Drosophila 
Current biology : CB  2009;19(15):1245-1254.
In many insect species, cuticular hydrocarbons serve as pheromones that can mediate complex social behaviors. In Drosophila melanogaster, several hydrocarbons including the male sex pheromone 11-cis-vaccenyl acetate (cVA) and female-specific 7,11-dienes influence courtship behavior and can function as cues for short-term memory associated with the mating experience. Behavioral and physiological studies suggest that other unidentified chemical communication cues are likely to exist. To more fully characterize the hydrocarbon profile of the D. melanogaster cuticle, we applied direct ultraviolet laser desorption/ionization orthogonal time-of-flight mass spectrometry (UV-LDI-o-TOF MS) and analyzed the surface of intact fruit flies at a spatial resolution of approximately 200 μm.
We report the chemical and spatial characterization of 28 species of cuticular hydrocarbons, including a new major class of oxygen-containing compounds. Using UV-LDI MS, pheromones previously shown to be expressed exclusively by one sex, e.g. cVA, 7,11-heptacosadiene, and 7,11-nonacosadiene, appear to be found on both male and female flies. In males, cVA co-localizes at the tip of the ejaculatory bulb with a second acetylated hydrocarbon named CH503. We describe the chemical structure of CH503 as 3-O-acetyl-1,3-dihydroxy-octacosa-11,19-diene and show one behavioral role for this compound as a long-lived inhibitor of male courtship. Like cVA, CH503 is transferred from males to females during mating. Unlike cVA, CH503 remains on the surface of females for at least 10 days.
Oxygenated hydrocarbons comprise one major previously undescribed class of compounds on the Drosophila cuticular surface. In addition to cVA, a newly-discovered long chain acetate, CH503, serves as a mediator of courtship-related chemical communication.
PMCID: PMC2726907  PMID: 19615904
9.  Rapid metabolic profiling of Nicotiana tabacum defence responses against Phytophthora nicotianae using direct infrared laser desorption ionization mass spectrometry and principal component analysis 
Plant Methods  2010;6:14.
Successful defence of tobacco plants against attack from the oomycete Phytophthora nicotianae includes a type of local programmed cell death called the hypersensitive response. Complex and not completely understood signaling processes are required to mediate the development of this defence in the infected tissue. Here, we demonstrate that different families of metabolites can be monitored in small pieces of infected, mechanically-stressed, and healthy tobacco leaves using direct infrared laser desorption ionization orthogonal time-of-flight mass spectrometry. The defence response was monitored for 1 - 9 hours post infection.
Infrared laser desorption ionization orthogonal time-of-flight mass spectrometry allows rapid and simultaneous detection in both negative and positive ion mode of a wide range of naturally occurring primary and secondary metabolites. An unsupervised principal component analysis was employed to identify correlations between changes in metabolite expression (obtained at different times and sample treatment conditions) and the overall defence response.
A one-dimensional projection of the principal components 1 and 2 obtained from positive ion mode spectra was used to generate a Biological Response Index (BRI). The BRI obtained for each sample treatment was compared with the number of dead cells found in the respective tissue. The high correlation between these two values suggested that the BRI provides a rapid assessment of the plant response against the pathogen infection. Evaluation of the loading plots of the principal components (1 and 2) reveals a correlation among three metabolic cascades and the defence response generated in infected leaves. Analysis of selected phytohormones by liquid chromatography electrospray ionization mass spectrometry verified our findings.
The described methodology allows for rapid assessment of infection-specific changes in the plant metabolism, in particular of phenolics, alkaloids, oxylipins, and carbohydrates. Moreover, potential novel biomarkers can be detected and used to predict the quality of plant infections.
PMCID: PMC2904756  PMID: 20534155
10.  Shiga Toxin Receptor Gb3Cer/CD77: Tumor-Association and Promising Therapeutic Target in Pancreas and Colon Cancer 
PLoS ONE  2009;4(8):e6813.
Despite progress in adjuvant chemotherapy in the recent decades, pancreatic and colon cancers remain common causes of death worldwide. Bacterial toxins, which specifically bind to cell surface-exposed glycosphingolipids, are a potential novel therapy. We determined the expression of globotriaosylceramide (Gb3Cer/CD77), the Shiga toxin receptor, in human pancreatic and colon adenocarcinomas.
Methodology/Principal Findings
Tissue lipid extracts of matched pairs of cancerous and adjacent normal tissue from 21 pancreatic and 16 colon cancer patients were investigated with thin-layer chromatography overlay assay combined with a novel mass spectrometry approach. Gb3Cer/CD77 was localized by immunofluorescence microscopy of cryosections from malignant and corresponding healthy tissue samples. 62% of pancreatic and 81% of colon adenocarcinomas showed increased Gb3Cer/CD77 expression, whereas 38% and 19% of malignant pancreas and colon tissue, respectively, did not, indicating an association of this marker with neoplastic transformation. Also, Gb3Cer/CD77 was associated with poor differentiation (G>2) in pancreatic cancer (P = 0.039). Mass spectrometric analysis evidenced enhanced expression of Gb3Cer/CD77 with long (C24) and short chain fatty acids (C16) in malignant tissues and pointed to the presence of hydroxylated fatty acid lipoforms, which are proposed to be important for receptor targeting. They could be detected in 86% of pancreatic and about 19% of colon adenocarcinomas. Immunohistology of tissue cryosections indicated tumor-association of these receptors.
Enhanced expression of Gb3Cer/CD77 in most pancreatic and colon adenocarcinomas prompts consideration of Shiga toxin, its B-subunit or B-subunit-derivatives as novel therapeutic strategies for the treatment of these challenging malignancies.
PMCID: PMC2730034  PMID: 19714252
11.  New Insights into the Glycosylation of the Surface Layer Protein SgsE from Geobacillus stearothermophilus NRS 2004/3a▿  
Journal of Bacteriology  2006;188(22):7914-7921.
The surface of Geobacillus stearothermophilus NRS 2004/3a cells is covered by an oblique surface layer (S-layer) composed of glycoprotein subunits. To this S-layer glycoprotein, elongated glycan chains are attached that are composed of [→2)-α-l-Rhap-(1→3)-β-l-Rhap-(1→2)-α-L-Rhap-(1→] repeating units, with a 2-O-methyl modification of the terminal trisaccharide at the nonreducing end of the glycan chain and a core saccharide as linker to the S-layer protein. On sodium dodecyl sulfate-polyacrylamide gels, four bands appear, of which three represent glycosylated S-layer proteins. In the present study, nanoelectrospray ionization time-of-flight mass spectrometry (MS) and infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry were adapted for analysis of this high-molecular-mass and water-insoluble S-layer glycoprotein to refine insights into its glycosylation pattern. This is a prerequisite for artificial fine-tuning of S-layer glycans for nanobiotechnological applications. Optimized MS techniques allowed (i) determination of the average masses of three glycoprotein species to be 101.66 kDa, 108.68 kDa, and 115.73 kDa, (ii) assignment of nanoheterogeneity to the S-layer glycans, with the most prevalent variation between 12 and 18 trisaccharide repeating units, and the possibility of extension of the already-known →3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→ core by one additional rhamnose residue, and (iii) identification of a third glycosylation site on the S-layer protein, at position threonine-590, in addition to the known sites threonine-620 and serine-794. The current interpretation of the S-layer glycoprotein banding pattern is that in the 101.66-kDa glycoprotein species only one glycosylation site is occupied, in the 108.68-kDa glycoprotein species two glycosylation sites are occupied, and in the 115.73-kDa glycoprotein species three glycosylation sites are occupied, while the 94.46-kDa band represents nonglycosylated S-layer protein.
PMCID: PMC1636307  PMID: 16963578
12.  Enterohaemorrhagic Escherichia coli haemolysin is cleaved and inactivated by serine protease EspPα 
Environmental Microbiology  2011;13(5):1327-1341.
The haemolysin from enterohaemorrhagic Escherichia coli (EHEC-Hly) and the serine protease EspPα are putative virulence factors of EHEC. We investigated the interplay between these secreted factors and demonstrate that EspPα cleaves the 107 kDa large EHEC-Hly. Degradation was observed when purified EspPα was added to a growing culture of an EHEC-Hly-expressing strain, with isolated proteins and with coexpressing strains, and was independent of the EHEC serotype. EHEC-Hly breakdown occurred as a multistage process with the formation of characteristic fragments with relative molecular masses of ∼82 kDa and/or ∼84 kDa and ∼34 kDa. The initial cleavage occurred in the N-terminal hydrophobic domain of EHEC-Hly between Leu235 and Ser236 and abolished its haemolytic activity. In a cellular infection system, the cytolytic potential of EHEC-Hly-secreting recombinant strains was abolished when EspPα was coexpressed. EHEC in contact with human intestinal epithelial cells simultaneously upregulated their EHEC-Hly and EspP indicating that both molecules might interact under physiological conditions. We propose the concept of bacterial effector molecule interference (BEMI), reflecting the concerted interplay of virulence factors. Interference between effector molecules might be an additional way to regulate virulence functions and increases the complexity of monomolecular phenotypes.
PMCID: PMC3472028  PMID: 21352460
13.  Liquid AP-UV-MALDI Enables Stable Ion Yields of Multiply Charged Peptide and Protein Ions for Sensitive Analysis by Mass Spectrometry** 
PMCID: PMC3592991  PMID: 23341077
AP-MALDI; liquid matrices; mass spectrometry; multiply charged peptides

Results 1-13 (13)