Rupert L. Mayer

Purification and characterization of tyrosinase from walnut leaves (Juglans regia)

Graphical abstract Tyrosinase from walnut leaves (Juglans regia) corresponding to the known jrPPO1 sequence was purified and characterized. Two major tyrosinase forms differing only in their C-termini were identified. The first form (jrPPO1(Asp101 → Pro444)) is one amino acid shorter than the second form (jrPPO1(Asp101 → Arg445)).

Site-directed mutagenesis around the CuA site of a polyphenol oxidase from Coreopsis grandiflora (cgAUS1).

Aurone synthase from Coreopsis grandiflora (cgAUS1), catalyzing conversion of butein to sulfuretin in a type‐3 copper center, is a rare example of a polyphenol oxidase involved in anabolism. Site‐directed mutagenesis around the CuA site of AUS1 was performed, and recombinant enzymes were analyzed by mass spectrometry. Replacement of the coordinating CuA histidines with alanine resulted in the presence of a single copper and loss of diphenolase activity. The thioether bridge‐building cysteine and a phenylalanine over the CuA site, exchanged to alanine, have no influence on copper content but appear to play an important role in substrate binding.

Contribution of human fibroblasts and endothelial cells to the Hallmarks of Inflammation as determined by proteome profiling

In order to systematically analyze proteins fulfilling effector functionalities during inflammation, here we present a comprehensive proteome study of inflammatory activated primary human endothelial cells and fibroblasts. Cells were stimulated with interleukin 1-β and fractionated in order to obtain secreted, cytoplasmic and nuclear protein fractions. Proteins were submitted to a data-dependent bottom up analytical platform using a QExactive orbitrap and the MaxQuant software for protein identification and label-free quantification. Results were further combined with similarly generated data previously obtained from the analysis of inflammatory activated peripheral blood mononuclear cells. Applying a false discovery rate of less than 0.01 at both, peptide and protein level, a total of 8370 protein groups assembled from 117,599 peptides was identified; mass spectrometry data have been made fully accessible via ProteomeXchange with identifier PXD003406 to PXD003417.Comparative proteome analysis allowed us to determine common and cell type-specific inflammation signatures comprising novel candidate marker molecules and related expression patterns of transcription factors. Cardinal features of inflammation such as interleukin 1-β processing and the interferon response differed substantially between the investigated cells. Furthermore, cells also exerted similar inflammation-related tasks; however, by making use of different sets of proteins. Hallmarks of inflammation thus emerged, including angiogenesis, extracellular matrix reorganization, adaptive and innate immune responses, oxidative stress response, cell proliferation and differentiation, cell adhesion and migration in addition to monosaccharide metabolic processes, representing both, common and cell type-specific responsibilities of cells during inflammation.

Coffee consumption modulates inflammatory processes in an individual fashion

SCOPE Anti-inflammatory effects of coffee consumption have been reported to be caused by caffeine and adenosine receptor signaling. However, contradictory effects have been observed. Many kinds of chronic diseases are linked to inflammation; therefore a profound understanding of potential effects of coffee consumption is desirable. METHODS AND RESULTS We performed ex vivo experiments with eight individuals investigating peripheral blood mononuclear cells isolated from venous blood before and after coffee consumption, as well as in vitro experiments applying caffeine on isolated cells. After in vitro inflammatory stimulation of the cells, released cytokines, chemokines, and eicosanoids were determined and quantified using targeted mass spectrometric methods. Remarkably, the release of inflammation mediators IL6, IL8, GROA, CXCL2, CXCL5 as well as PGA2, PGD2, prostaglandin E2 (PGE2), LTC4, LTE4, and 15S-HETE was significantly affected after coffee consumption. While in several individuals coffee consumption or caffeine treatment caused significant downregulation of most inflammation mediators, in other healthy individuals exactly the opposite effects were observed. CONCLUSION Ruling out age, sex, coffee consumption habits, the metabolic kinetics of caffeine in blood and the individual amount of regulatory T cells or CD39 expression as predictive parameters, we demonstrated here that coffee consumption may have significant pro- or anti-inflammatory effects in an individual fashion.

Quantitative proteomics reveals protein kinases and phosphatases in the individual phases of contextual fear conditioning in the C57BL/6J mouse

A series of protein kinases and phosphatases (PKPs) have been linked to contextual fear conditioning (cFC) but information is mainly derived from immunochemical studies. It was therefore decided to use an explorative label-free quantitative proteomics approach to concomitantly determine PKPs in hippocampi of mice in the individual phases of cFC. C57BL/6J mice were divided into four groups: three training groups representing the acquisition, consolidation and retrieval phases of cFC and a foot shock control group. Using this approach we identified 32 protein kinases or phosphatases/phosphatase subunits with significantly changed protein levels in one or more training groups as compared to foot shock control. These include members of PKP signalling modules of mitogen-activated protein kinase (MAP3K10, RAF1, KSR2), Ca2+/calmodulin-dependent protein kinase (CaMKIIα, DAPK1), protein kinase C (PRKCD) and protein phosphatases 1, 2A, 2B(3) previously implicated in various learning paradigms. In addition, our analysis showed protein kinases WNK1, LYN, VRK1, ABL1, CDK4, CDKL3, SgK223 and ADCK1, and protein phosphatases PTPRF, ACP1, DNAJC6, SSH2 and UBASH3B that have not been directly linked to fear memory processes so far. Determination of PKPs in the individual cFC phases represents a valuable resource for interpretation of previous and design of future studies on PKPs in memory mechanisms.

Combined Proteome and Eicosanoid Profiling Approach for Revealing Implications of Human Fibroblasts in Chronic Inflammation

During inflammation, proteins and lipids act in a concerted fashion, calling for combined analyses. Fibroblasts are powerful mediators of chronic inflammation. However, little is known about eicosanoid formation by human fibroblasts. The aim of this study was to analyze the formation of the most relevant inflammation mediators including proteins and lipids in human fibroblasts upon inflammatory stimulation and subsequent treatment with dexamethasone, a powerful antiphlogistic drug. Label-free quantification was applied for proteome profiling, while an in-house established data-dependent analysis method based on high-resolution mass spectrometry was applied for eicosadomics. Furthermore, a set of 188 metabolites was determined by targeted analysis. The secretion of 40 proteins including cytokines, proteases, and other inflammation agonists as well as 14 proinflammatory and nine anti-inflammatory eicosanoids was found significantly induced, while several acylcarnithins and sphingomyelins were found significantly downregulated upon inflammatory stimulation. Treatment with dexamethasone downregulated most cytokines and proteases, abrogated the formation of pro- but also anti-inflammatory eicosanoids, and restored normal levels of acylcarnithins but not of sphingomyelins. In addition, the chemokines CXCL1, CXCL5, CXCL6, and complement C3, known to contribute to chronic inflammation, were not counter-regulated by dexamethasone. Similar findings were obtained with human mesenchymal stem cells, and results were confirmed by targeted analysis with multiple reaction monitoring. Comparative proteome profiling regarding other cells demonstrated cell-type-specific synthesis of, among others, eicosanoid-forming enzymes as well as relevant transcription factors, allowing us to better understand cell-type-specific regulation of inflammation mediators and shedding new light on the role of fibroblasts in chronic inflammation.

Proteome profiling of keratinocytes transforming to malignancy

To shed light on the multistep process of squamous cell carcinoma development and the underlying pathologic mechanisms, we performed comparative proteome analysis of keratinocytes, keratinocytes stimulated with Il‐1beta, and A431 epidermoid carcinoma cells. Fractionation of the cells into supernatant, nucleus, and cytoplasm was followed by protein separation, proteolytic digest, and nano‐LC separation, and fragmentation using an ion trap mass spectrometer. Specific bioinformatics tools were used to generate a list of keratinocyte‐specific proteins. Ninety percent of these proteins were found to be upregulated in keratinocytes versus the A431 cells. Classification of the identified proteins by biologic function and gene set enrichment analysis revealed that keratinocytes produced more proteins involved in cell differentiation, cell adhesion, cell junction, calcium ion, calmodulin binding, cytoskeleton organization, and cytokinesis, whereas A431 produced more proteins involved in cell cycle checkpoint, cell cycle process, RNA processing and transport, DNA damage and repair, RNA and DNA binding, and chromatin remodeling. The protein signatures of A431 and normal keratinocytes treated with IL‐1beta showed marked similarity, confirming that inflammation is an important step in malignant transformation in nonmelanoma skin cancer. Thus, proteome profiling and bioinformatic processing may support the understanding of the underlying mechanisms, with the potential to facilitate development of early biomarkers and patient‐tailored therapy.

Evaluation of inflammation-related signaling events covering phosphorylation and nuclear translocation of proteins based on mass spectrometry data

Peripheral blood mononuclear cells are important players in immune regulation relying on a complex network of signaling pathways. In this study, we evaluated the power of label-free quantitative shotgun proteomics regarding the comprehensive characterization of signaling pathways in such primary cells by studying regulation of protein abundance, post-translational modifications and nuclear translocation events. The effects of inflammatory stimulation and the treatment of stimulated cells with dexamethasone were investigated. Therefore, a previously published dataset accessible via ProteomeXchange consisting of 6901 identified protein groups was re-evaluated. These data enabled us to comprehensively map the c-JUN, ERK5 and NF-κB signaling cascade in a semi-quantitative fashion. Without the application of any enrichment, 3775 highly confident phosphopeptides derived from 1249 proteins including 66 kinases were identified. Efficient subcellular fractionation and subsequent comparative analysis identified previously unrecognized inflammation-associated nuclear translocation events of proteins such as histone-modifying proteins, zinc finger proteins as well as transcription factors. Profound effects of inflammatory stimulation and dexamethasone treatment on histone H3 and ZFP161 localization represent novel findings and were verified by immunofluorescence. In conclusion, we demonstrate that multiple regulatory events resulting from the activity of signaling pathways can be determined out of untargeted shotgun proteomics data. SIGNIFICANCE Relevant functional events such as phosphorylation and nuclear translocation of proteins were extracted from high-resolution mass spectrometry data and provided additional biological information contained in shotgun proteomics data.

Fetal articular cartilage regeneration versus adult fibrocartilaginous repair: secretome proteomics unravels molecular mechanisms in an ovine model

ABSTRACT Osteoarthritis (OA), a degenerative joint disease characterized by progressive cartilage degeneration, is one of the leading causes of disability worldwide owing to the limited regenerative capacity of adult articular cartilage. Currently, there are no disease-modifying pharmacological or surgical therapies for OA. Fetal mammals, in contrast to adults, are capable of regenerating injured cartilage in the first two trimesters of gestation. A deeper understanding of the properties intrinsic to the response of fetal tissue to injury would allow us to modulate the way in which adult tissue responds to injury. In this study, we employed secretome proteomics to compare fetal and adult protein regulation in response to cartilage injury using an ovine cartilage defect model. The most relevant events comprised proteins associated with the immune response and inflammation, proteins specific for cartilage tissue and cartilage development, and proteins involved in cell growth and proliferation. Alarmins S100A8, S100A9 and S100A12 and coiled-coil domain containing 88A (CCDC88A), which are associated with inflammatory processes, were found to be significantly upregulated following injury in adult, but not in fetal animals. By contrast, cartilage-specific proteins like proteoglycan 4 were upregulated in response to injury only in fetal sheep postinjury. Our results demonstrate the power and relevance of the ovine fetal cartilage regeneration model presented here for the first time. The identification of previously unrecognized modulatory proteins that plausibly affect the healing process holds great promise for potential therapeutic interventions. Summary: Secretome proteomics identifies differential regulation of inflammation modulators during fetal and adult articular cartilage defect healing, offering novel strategies for therapy.

Transplantation of human amnion prevents recurring adhesions and ameliorates fibrosis in a rat model of sciatic nerve scarring

Peripheral nerve fibrosis and painful adhesions are common, recurring pathological sequelae following injury. In this study, vital human amnion (hAM), an increasingly interesting biomaterial for regenerative medicine, was investigated as a novel therapy. hAM was first analyzed in vitro regarding its anti-adhesive characteristics. Then, the reflected region of hAM which was identified as more suitable, was transplanted into female Sprague Dawley rats with recurring sciatic nerve scarring (n = 24) and compared with untreated controls (n = 30) at one, four and twelve weeks. Immune response and fibrosis were investigated by (immuno)histochemical analysis. Nerve structure was examined and function determined using electrophysiology and gait analysis. Here we identified strongly reduced adhesions in the hAM-treated rats, displaying a significant difference at four weeks post transplantation compared to untreated controls (p = .0052). This correlated with the in vitro cell attachment test on hAM explants, which demonstrated a distinctly limited ability of fibroblasts to adhere to amniotic epithelial cells. Upon hAM transplantation, significantly less intraneural fibrosis was identified at the later time points. Moreover, hAM-treated rats exhibited a significantly higher sciatic functional index (SFI) after four weeks compared to controls (p < .05), which indicated a potentially pro-regenerative effect of hAM. As a possible explanation, an impact of hAM on the endogenous immune response, including T cell and macrophage subsets, was indicated. We conclude that hAM is strongly effective against recurring nerve scarring and induces an anti-fibrotic and pro-regenerative effect, making it highly promising for treating adhesion-related disorders. STATEMENT OF SIGNIFICANCE Abnormal fibrotic bonding of tissues, frequently involving peripheral nerves, affects millions of people worldwide. These so-called adhesions usually cause severe pain and drastically reduce quality of life. To date, no adequate treatment exists and none is routinely used in the clinical practice. In this study, vital human amnion, the innermost of the fetal membranes, was transplanted in a rat model of peripheral nerve scarring and recurring adhesions as novel therapeutic approach. Amniotic cells have already demonstrated to feature stem-cell like properties and produce pro-regenerative factors, which makes the amnion an increasingly promising biomaterial for regenerative medicine. We identified that its transplantation was very effective against peripheral nerve scarring and distinctly reduced recurring adhesions. Moreover, we identified a pro-regenerative effect. This study showed that the amnion is a highly promising novel therapeutic approach for adhesion-related disorders.