Barbara Darnhofer

Proteomics reveals selective regulation of proteins in response to memory-related serotonin stimulation in Aplysia californica ganglia.

The marine mollusk Aplysia californica (Aplysia) is a powerful model for learning and memory due to its minimalistic nervous system. Key proteins, identified to be regulated by the neurotransmitter serotonin in Aplysia, have been successfully translated to mammalian models of learning and memory. Based upon a recently published large‐scale analysis of Aplysia proteomic data, the current study investigated the regulation of protein levels 24 and 48 h after treatment with serotonin in Aplysia ganglia using a 2‐D gel electrophoresis approach. Protein spots were quantified and protein‐level changes of selected proteins were verified by Western blotting. Among those were Rab GDP dissociation inhibitor alpha (RabGDIα), synaptotagmin‐1 and deleted in azoospermia‐associated protein (DAZAP‐1) in cerebral ganglia, calreticulin, RabGDIα, DAZAP‐1, heterogeneous nuclear ribonucleoprotein F (hnRNPF), RACK‐1 and actin‐depolymerizing factor (ADF) in pleural ganglia and DAZAP‐1, hnRNPF and ADF in pedal ganglia. Protein identity of the majority of spots was confirmed by a gel‐based mass spectrometrical method (FT‐MS). Taken together, protein‐level changes induced by the learning‐related neurotransmitter serotonin in Aplysia ganglia are described and a role for the abovementioned proteins in synaptic plasticity is proposed.

Enzyme discovery beyond homology: a unique hydroxynitrile lyase in the Bet v1 superfamily

Homology and similarity based approaches are most widely used for the identification of new enzymes for biocatalysis. However, they are not suitable to find truly novel scaffolds with a desired function and this averts options and diversity. Hydroxynitrile lyases (HNLs) are an example of non-homologous isofunctional enzymes for the synthesis of chiral cyanohydrins. Due to their convergent evolution, finding new representatives is challenging. Here we show the discovery of unique HNL enzymes from the fern Davallia tyermannii by coalescence of transcriptomics, proteomics and enzymatic screening. It is the first protein with a Bet v1-like protein fold exhibiting HNL activity, and has a new catalytic center, as shown by protein crystallography. Biochemical properties of D. tyermannii HNLs open perspectives for the development of a complementary class of biocatalysts for the stereoselective synthesis of cyanohydrins. This work shows that systematic integration of -omics data facilitates discovery of enzymes with unpredictable sequences and helps to extend our knowledge about enzyme diversity.

Recombinant production of a peroxidase-protein G fusion protein in Pichia pastoris

Streptococcal protein G (SpG) binds immunoglobulin G from a broad range of mammalian species with high affinity. Chemical conjugations of SpG to the reporter enzyme horseradish peroxidase (HRP) are commonly used in immunohistochemical applications. However, commercial HRP preparations are typically isolated from horseradish roots as varying mixtures of HRP isoenzymes with different biochemical properties, and chemical conjugation procedures lead to heterogeneous HRP-SpG preparations, partially including inactivated enzyme. A recombinant process allows the production of a well-defined HRP isoenzyme fused to SpG at constant 1:1 stoichiometry in a single step without the need for laborious chemical conjugation. By using state-of-the-art biotechnological tools, we produced a recombinant HRP-SpG fusion protein in Pichia pastoris in bioreactor cultivations. Purified HRP-SpG was tested successfully for functional binding of antibodies from different mammalian serums. Recombinant production of this novel well-defined fusion protein follows quality-by-design principles and facilitates the production of more reliable and cost-effective diagnostic kits.

Biotechnological advances towards an enhanced peroxidase production in Pichia pastoris

Horseradish peroxidase (HRP) is a high-demand enzyme for applications in diagnostics, bioremediation, biocatalysis and medicine. Current HRP preparations are isolated from horseradish roots as mixtures of biochemically diverse isoenzymes. Thus, there is a strong need for a recombinant production process enabling a steady supply with enzyme preparations of consistent high quality. However, most current recombinant production systems are limited at titers in the low mg/L range. In this study, we used the well-known yeast Pichia pastoris as host for recombinant HRP production. To enhance recombinant enzyme titers we systematically evaluated engineering approaches on the secretion process, coproduction of helper proteins, and compared expression from the strong methanol-inducible PAOX1 promoter, the strong constitutive PGAP promoter, and a novel bidirectional promoter PHTX1. Ultimately, coproduction of HRP and active Hac1 under PHTX1 control yielded a recombinant HRP titer of 132mg/L after 56h of cultivation in a methanol-independent and easy-to-do bioreactor cultivation process. With regard to the many versatile applications for HRP, the establishment of a microbial host system suitable for efficient recombinant HRP production was highly overdue. The novel HRP production platform in P. pastoris presented in this study sets a new benchmark for this medically relevant enzyme.

Apolipoproteins have a potential role in nasal mucus of allergic rhinitis patients: A proteomic study

Nasal mucus is a defense barrier against aeroallergens. We recently found apolipoproteins to be elevated in the nasal mucus of allergic rhinitis patients. Apolipoproteins are involved in lipid metabolism, have immunomodulatory properties, and may represent interesting novel biomarkers. This study aims to validate our findings and analyze whether the increased abundance of apolipoproteins in nasal mucus is a local or systemic phenomenon in allergic rhinitis.

Comparative proteomics of paired vocal fold and oral mucosa fibroblasts

Injuries of the vocal folds frequently heal with scar formation, which can have lifelong detrimental impact on voice quality. Current treatments to prevent or resolve scars of the vocal fold mucosa are highly unsatisfactory. In contrast, the adjacent oral mucosa is mostly resistant to scarring. These differences in healing tendency might relate to distinct properties of the fibroblasts populating oral and vocal fold mucosae. We thus established the in vitro cultivation of paired, near-primary vocal fold fibroblasts (VFF) and oral mucosa fibroblasts (OMF) to perform a basic cellular characterization and comparative cellular proteomics. VFF were significantly larger than OMF, proliferated more slowly, and exhibited a sustained TGF-β1-induced elevation of pro-fibrotic interleukin 6. Cluster analysis of the proteomic data revealed distinct protein repertoires specific for VFF and OMF. Further, VFF displayed a broader protein spectrum, particularly a more sophisticated array of factors constituting and modifying the extracellular matrix. Conversely, subsets of OMF-enriched proteins were linked to cellular proliferation, nuclear events, and protection against oxidative stress. Altogether, this study supports the notion that fibroblasts sensitively adapt to the functional peculiarities of their respective anatomical location and presents several molecular targets for further investigation in the context of vocal fold wound healing. Biological significance Mammalian vocal folds are a unique but delicate tissue. A considerable fraction of people is affected by voice problems, yet many of the underlying vocal fold pathologies are sparsely understood at the molecular level. One such pathology is vocal fold scarring - the tendency of vocal fold injuries to heal with scar formation -, which represents a clinical problem with highly suboptimal treatment modalities. This study employed proteomics to obtain comprehensive insight into the protein repertoire of vocal fold fibroblasts, which are the cells that predominantly synthesize the extracellular matrix in both physiological and pathophysiological conditions. Protein profiles were compared to paired fibroblasts from the oral mucosa, a neighboring tissue that is remarkably resistant to scarring. Bioinformatic analyses of the data revealed a number of pathways as well as single proteins (e.g. ECM-remodeling factors, transcription factors, enzymes) that were significantly different between the two fibroblast types. Thereby, this study has revealed novel interesting molecular targets which can be analyzed in the future for their impact on vocal fold wound healing.

Proteomic characterization of the abdominal ganglion of Aplysia californica—A protein resource for neuroscience

Aplysia californica (AC) is a widely used model for testing learning and memory. Although ESTs have been generated, proteomics studies on AC proteins are limited. Studies at the protein level, however, are mandatory, not only due to the fact that studies at the nucleic acid level are not allowing conclusions about PTMs. A gel‐based proteomics method was therefore applied to carry out protein profiling in abdominal ganglia from AC. Abdominal ganglia were extirpated, proteins extracted and run on 2DE with subsequent in‐gel digestion with trypsin, chymotrypsin, and partially by subtilisin. Peptides were identified using a nano‐LC‐ESI‐LTQ‐FT‐mass spectrometer. MS/MS data were analyzed by searching the NCBI nonredundant public AC EST database and the NCBI nonredundant public AC protein database. A total of 477 different proteins represented by 363 protein spots were detected and were assigned to different protein pathways as for instance signaling (receptors, protein kinases, and phosphatases), metabolism, protein synthesis, handling and degradation, cytoskeleton and structural, oxido‐redox, heat shock and chaperone, hypothetical, predicted and unnamed proteins. The generation of a protein map of soluble proteins shows the existence of so far hypothetical and predicted proteins and is allowing and challenging further work at the protein level, in particular in the field of neuroscience.

Characterization of a lipid droplet protein from Yarrowia lipolytica that is required for its oleaginous phenotype

Oleaginous microorganisms are characterized by their ability to store high amounts of triacylglycerol (TAG) in intracellular lipid droplets (LDs). In this work, we characterized a protein of the oleaginous yeast Yarrowia lipolytica that is associated with LD and plays a role in the regulation of TAG storage. This protein is required for the oleaginous phenotype of Y. lipolytica because deletion of the coding gene results in a strongly reduced TAG content of the mutant. Therefore, we named it Oleaginicity Inducing LD protein, Oil1. Furthermore, a mutant overexpressing OIL1 accumulates more TAG than the wild type and is delayed in TAG lipolysis when this process is stimulated. We found that Oil1p plays a role in protecting the TAG content of the LD from degradation through lipases under conditions where the cell aims at building up its TAG reserves. Heterologous expression studies showed that Oil1p rescued the phenotype of a Saccharomyces cerevisiae mutant deleted for the perilipin-like protein Pln1p and that its expression in COS-7 cells resulted in increased TAG accumulation, similar to the phenotype of a perilipin 1 expressing control strain. Despite this phenotypical parallels to mammalian perilipins, Oil1p is not a member of this protein family and its activity does not depend on phosphorylation. Rather, our results suggest that ubiquitination might contribute to the function of Oil1p in Y. lipolytica and that a different mechanism evolved in this species to regulate TAG homeostasis.

Effect of non-canonical amino acids on protein-carbohydrate interactions: Structure, dynamics and carbohydrate affinity of a lectin engineered with fluorinated tryptophan analogs.

Protein–carbohydrate interactions play crucial roles in biology. Understanding and modifying these interactions is of major interest for fighting many diseases. We took a synthetic biology approach and incorporated noncanonical amino acids into a bacterial lectin to modulate its interactions with carbohydrates. We focused on tryptophan, which is prevalent in carbohydrate binding sites. The exchange of the tryptophan residues with analogs fluorinated at different positions resulted in three distinctly fluorinated variants of the lectin from Ralstonia solanacearum. We observed differences in stability and affinity toward fucosylated glycans and rationalized them by X-ray and modeling studies. While fluorination decreased the aromaticity of the indole ring and, therefore, the strength of carbohydrate–aromatic interactions, additional weak hydrogen bonds were formed between fluorine and the ligand hydroxyl groups. Our approach opens new possibilities to engineer carbohydrate receptors.