Listeria monocytogenesis a foodborne intracellular bacterial pathogen leading to human listeriosis. Despite a high mortality rate and increasing antibiotic resistance no clinically approved vaccine againstListeriais available. AttenuatedListeriastrains offer protection and are tested as antitumor vaccine vectors, but would benefit from a better knowledge on immunodominant vector antigens. To identify novel antigens, we screen forListeriapeptides presented on the surface of infected human cell lines by mass spectrometry-based immunopeptidomics. In between more than 15,000 human self-peptides, we detect 68Listeriaimmunopeptides from 42 different bacterial proteins, including several known antigens. Peptides presented on different cell lines are often derived from the same bacterial surface proteins, classifying these antigens as potential vaccine candidates. Encoding these highly presented antigens in lipid nanoparticle mRNA vaccine formulations results in specific CD8⁺ T-cell responses and induces protection in vaccination challenge experiments in mice. Our results can serve as a starting point for the development of a clinical mRNA vaccine againstListeriaand aid to improve attenuatedListeriavaccines and vectors, demonstrating the power of immunopeptidomics for next-generation bacterial vaccine development.

Polyamines, such as putrescine, spermidine, and spermine, are physiologically important polycations, but the transporters responsible for their uptake in mammalian cells remain poorly characterized. Here, we reveal a new component of the mammalian polyamine transport system using CHO-MG cells, a widely used model to study alternative polyamine uptake routes and characterize polyamine transport inhibitors for therapy. CHO-MG cells present polyamine uptake deficiency and resistance to a toxic polyamine biosynthesis inhibitor methylglyoxal bis-(guanylhydrazone) (MGBG), but the molecular defects responsible for these cellular characteristics remain unknown. By genome sequencing of CHO-MG cells, we identified mutations in an unexplored gene, ATP13A3, and found disturbed mRNA and protein expression. ATP13A3 encodes for an orphan P5B-ATPase (ATP13A3), a P-type transport ATPase that represents a candidate polyamine transporter. Interestingly, ATP13A3 complemented the putrescine transport deficiency and MGBG resistance of CHO-MG cells, whereas its knockdown in WT cells induced a CHO-MG phenotype demonstrated as a decrease in putrescine uptake and MGBG sensitivity. Taken together, our findings identify ATP13A3, which has been previously genetically linked with pulmonary arterial hypertension, as a major component of the mammalian polyamine transport system that confers sensitivity to MGBG.

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.

Polyphenol oxidase (PPO) is a type-3 copper enzyme catalyzing the oxidation of phenolic compounds to their quinone derivates, which are further converted to melanin, a ubiquitous pigment in living organisms. In this study a plant originated tyrosinase was isolated from walnut leaves (Juglans regia) and biochemically characterized. It was possible to isolate and purify the enzyme by means of an aqueous two-phase extraction method followed by chromatographic purification and identification. Interestingly, the enzyme showed a rather high monophenolase activity considering that the main part of plant PPOs with some exceptions solely possess diphenolase activity. The average molecular mass of 39,047 Da (Asp(101)→Arg(445)) was determined very accurately by high resolution mass spectrometry. This proteolytically activated tyrosinase species was identified as a polyphenol oxidase corresponding to the known jrPPO1 sequence by peptide sequencing applying nanoUHPLC-ESI-MS/MS. The polypeptide backbone with sequence coverage of 96% was determined to start from Asp(101) and not to exceed Arg(445).

B cell chronic lymphocytic leukemia (B-CLL), the most common type of leukemia in adults, is still essentially incurable despite the development of novel therapeutic strategies. This reflects the incomplete understanding of the pathophysiology of this disease. A comprehensive proteome analysis of primary human B-CLL cells and B cells from younger as well as elderly healthy donors was performed. For comparison, the chronic B cell leukemia cell line JVM-13 was also included. A principal component analysis comprising 6,945 proteins separated these four groups, placing B cells of aged-matched controls between those of young donors and B-CLL patients, while identifying JVM-13 as poorly related cells. Mass spectrometric proteomics data have been made fully accessible via ProteomeXchange with identifier PXD006570-PXD006572, PXD006576, PXD006578, and PXD006589-PXD006591. Remarkably, B cells from aged controls displayed significant regulation of proteins related to stress management in mitochondria and ROS stress such as DLAT, FIS1, and NDUFAB1, and DNA repair, including RAD9A, MGMT, and XPA. ROS levels were indeed found significantly increased in B cells but not in T cells or monocytes from aged individuals. These alterations may be relevant for tumorigenesis and were observed similarly in B-CLL cells. In B-CLL cells, some remarkable unique features like the loss of tumor suppressor molecules PNN and JARID2, the stress-related serotonin transporter SLC6A4, and high expression of ZNF207, CCDC88A, PIGR and ID3, otherwise associated with stem cell phenotype, were determined. Alterations of metabolic enzymes were another outstanding feature in comparison to normal B cells, indicating increased beta-oxidation of fatty acids and increased consumption of glutamine. Targeted metabolomics assays corroborated these results. The present findings identify a potential proteome signature for immune senescence in addition to previously unrecognized features of B-CLL cells and suggest that aging may be accompanied by cellular reprogramming functionally relevant for predisposing B cells to transform to B-CLL cells.

Main conclusion Aurone synthase belongs to the novel group 2 polyphenol oxidases and the presented kinetic characterization suggests a differing aurone biosynthesis in Asteraceae species compared to snapdragon. Aurone synthases (AUS) are polyphenol oxidases (PPO) physiologically involved in the formation of yellow aurone pigments in petals of various Asteraceae species. They catalyze the oxidative conversion of chalcones into aurones. Latent (58.9 kDa) and active (41.6 kDa) aurone synthase from petals of C. grandiflora was purified by a quantitative removal of pigments using aqueous two-phase separation and several subsequent chromatographic steps. The purified enzymes were identified as cgAUS1 (A0A075DN54) and sequence analysis revealed that cgAUS1 is a member of a new group of plant PPOs. Mass determination experiments of intact cgAUS1 gave evidence that the C-terminal domain, usually shielding the active site of latent polyphenol oxidases, is linked to the main core by a disulfide bond. This is a novel and unique structural feature of plant PPOs. Proteolytic activation in vivo leads to active aurone synthase possessing a residual peptide of the C-terminal domain. Kinetic characterization of purified cgAUS1 strongly suggests a specific involvement in 4-deoxyaurone biosynthesis in Coreopsis grandiflora (Asteraceae) that differs in various aspects compared to the 4-hydroxyaurone formation in Antirrhinum majus (Plantaginaceae): cgAUS1 is predicted to be localized in the thylakoid lumen, it possesses exclusively diphenolase activity and the results suggest that aurone formation occurs at the level of chalcone aglycones. The latent enzyme exhibits allosteric activation which changes at a specific product concentration to a constant reaction rate. The presented novel structural and functional properties of aurone synthase provide further insights in the diversity and role of plant PPOs.