Publications by authors named "Ruth Birner-Gruenberger"

102 Publications

Transcriptomic Profiling of Canine Atrial Fibrillation Models after One Week of Sustained Arrhythmia.

Circ Arrhythm Electrophysiol 2021 Jul 16. Epub 2021 Jul 16.

Faculty of Medicine, Université de Montréal & Montreal Heart Institute, Montreal, Quebec, Canada & Institute of Pharmacology, West German Heart and Vascular Center, Faculty of Medicine, University Duisburg-Essen, Essen, Germany.

- Atrial fibrillation (AF), the most common sustained arrhythmia, is associated with increased morbidity, mortality, and health-care costs. AF develops over many years and is often related to substantial atrial structural and electrophysiological remodeling. AF may lack symptoms at onset and atrial biopsy samples are generally obtained in subjects with advanced disease, so it is difficult to study earlier-stage pathophysiology in humans. - Here, we characterized comprehensively the transcriptomic (miRNAseq and mRNAseq) changes in the left atria of two robust canine AF-models after one week of electrically-maintained AF, without or with ventricular rate-control via atrioventricular node-ablation/ventricular pacing. - Our RNA-sequencing experiments identified thousands of genes that are differentially expressed, including a majority that have never before been implicated in AF. Gene-set enrichment analyses highlighted known (e.g. extracellular matrix structure organization) but also many novel pathways (e.g. muscle structure development, striated muscle cell differentiation) that may play a role in tissue remodeling and/or cellular trans-differentiation. Of interest, we found dysregulation of a cluster of non-coding RNAs, including many microRNAs but also the long non-coding RNA orthologue, located in the syntenic region of the imprinted human locus. Interestingly (in the light of other recent observations), our analysis identified gene-targets of differentially expressed microRNAs at the locus implicating glutamate signaling in AF pathophysiology. - Our results capture molecular events that occur at an early stage of disease development using well-characterized animal models, and may therefore inform future studies that aim to further dissect the causes of AF in humans.
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http://dx.doi.org/10.1161/CIRCEP.121.009887DOI Listing
July 2021

Targeted Chemotherapy of Glioblastoma Spheroids with an Iontronic Pump.

Adv Mater Technol 2021 May 12;6(5):2001302. Epub 2021 Apr 12.

Gottfried Schatz Research Center - Biophysics Medical University of Graz Graz 8010 Austria.

Successful treatment of glioblastoma multiforme (GBM), the most lethal tumor of the brain, is presently hampered by (i) the limits of safe surgical resection and (ii) "shielding" of residual tumor cells from promising chemotherapeutic drugs such as Gemcitabine (Gem) by the blood brain barrier (BBB). Here, the vastly greater GBM cell-killing potency of Gem compared to the gold standard temozolomide is confirmed, moreover, it shows neuronal cells to be at least 10-fold less sensitive to Gem than GBM cells. The study also demonstrates the potential of an electronically-driven organic ion pump ("GemIP") to achieve controlled, targeted Gem delivery to GBM cells. Thus, GemIP-mediated Gem delivery is confirmed to be temporally and electrically controllable with pmol min precision and electric addressing is linked to the efficient killing of GBM cell monolayers. Most strikingly, GemIP-mediated GEM delivery leads to the overt disintegration of targeted GBM tumor spheroids. Electrically-driven chemotherapy, here exemplified, has the potential to radically improve the efficacy of GBM adjuvant chemotherapy by enabling exquisitely-targeted and controllable delivery of drugs irrespective of whether these can cross the BBB.
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http://dx.doi.org/10.1002/admt.202001302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8218220PMC
May 2021

Blood Plasma Quality Control by Plasma Glutathione Status.

Antioxidants (Basel) 2021 May 27;10(6). Epub 2021 May 27.

Institute of Chemical Technologies and Analytics, Faculty of Technical Chemistry, Technische Universität Wien, 1060 Vienna, Austria.

Timely centrifugation of blood for plasma preparation is a key step to ensure high plasma quality for analytics. Delays during preparation can significantly influence readouts of key clinical parameters. However, in a routine clinical environment, a strictly controlled timeline is often not feasible. The next best approach is to control for sample preparation delays by a marker that provides a readout of the time-dependent degradation of the sample. In this study, we explored the usefulness of glutathione status as potential marker of plasma preparation delay. As the concentration of glutathione in erythrocytes is at least two orders of magnitude higher than in plasma, even the slightest leakage of glutathione from the cells can be readily observed. Over the 3 h observation period employed in this study, we observed a linear increase of plasma concentrations of both reduced (GSH) and oxidized glutathione (GSSG). Artificial oxidation of GSH is prevented by rapid alkylation with N-ethylmaleimide directly in the blood sampling vessel as recently published. The observed relative leakage of GSH was significantly higher than that of GSSG. A direct comparison with plasma lactate dehydrogenase activity, a widely employed hemolysis marker, clearly demonstrated the superiority of our approach for quality control. Moreover, we show that the addition of the thiol alkylating reagent NEM directly to the blood tubes does not influence downstream analysis of other clinical parameters. In conclusion, we report that GSH gives an excellent readout of the duration of plasma preparation and the associated pre-analytical errors.
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http://dx.doi.org/10.3390/antiox10060864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8226592PMC
May 2021

Adipose Triglyceride Lipase Loss Promotes a Metabolic Switch in A549 Non-Small Cell Lung Cancer Cell Spheroids.

Mol Cell Proteomics 2021 May 14;20:100095. Epub 2021 May 14.

Diagnostic and Research Institute of Pathology, Medical University of Graz, Graz, Austria; Omics Center Graz, BioTechMed-Graz, Graz, Austria; Faculty of Technical Chemistry, Institute of Chemical Technologies and Analytics, Technische Universität Wien, Vienna, Austria. Electronic address:

Cancer cells undergo complex metabolic adaptations to survive and thrive in challenging environments. This is particularly prominent for solid tumors, where cells in the core of the tumor are under severe hypoxia and nutrient deprivation. However, such conditions are often not recapitulated in the typical 2D in vitro cancer models, where oxygen as well as nutrient exposure is quite uniform. The aim of this study was to investigate the role of a key neutral lipid hydrolase, namely adipose triglyceride lipase (ATGL), in cancer cells that are exposed to more tumor-like conditions. To that end, we cultured lung cancer cells lacking ATGL as multicellular spheroids in 3D and subjected them to comprehensive proteomics analysis and metabolic phenotyping. Proteomics data are available via ProteomeXchange with identifier PXD021105. As a result, we report that loss of ATGL enhanced growth of spheroids and facilitated their adaptation to hypoxia, by increasing the influx of glucose and endorsing a pro-Warburg effect. This was followed by changes in lipid metabolism and an increase in protein production. Interestingly, the observed phenotype was also recapitulated in an even more "in vivo like" setup, when cancer spheroids were grown on chick chorioallantoic membrane, but not when cells were cultured as a 2D monolayer. In addition, we demonstrate that according to the publicly available cancer databases, an inverse relation between ATGL expression and higher glucose dependence can be observed. In conclusion, we provide indications that ATGL is involved in regulation of glucose metabolism of cancer cells when grown in 3D (mimicking solid tumors) and as such could be an important factor of the treatment outcome for some cancer types. Finally, we also ratify the need for alternative cell culture models, as the majority of phenotypes observed in 3D and spheroids grown on chick chorioallantoic membrane were not observed in 2D cell culture.
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http://dx.doi.org/10.1016/j.mcpro.2021.100095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8214150PMC
May 2021

Mass Spectrometry-Based Redox and Protein Profiling of Failing Human Hearts.

Int J Mol Sci 2021 Feb 11;22(4). Epub 2021 Feb 11.

Faculty of Technical Chemistry, Institute of Chemical Technologies and Analytics, Vienna University of Technology-TU Wien, Getreidemarkt 9/164, 1060 Vienna, Austria.

Oxidative stress contributes to detrimental functional decline of the myocardium, leading to the impairment of the antioxidative defense, dysregulation of redox signaling, and protein damage. In order to precisely dissect the changes of the myocardial redox state correlated with oxidative stress and heart failure, we subjected left-ventricular tissue specimens collected from control or failing human hearts to comprehensive mass spectrometry-based redox and quantitative proteomics, as well as glutathione status analyses. As a result, we report that failing hearts have lower glutathione to glutathione disulfide ratios and increased oxidation of a number of different proteins, including constituents of the contractile machinery as well as glycolytic enzymes. Furthermore, quantitative proteomics of failing hearts revealed a higher abundance of proteins responsible for extracellular matrix remodeling and reduced abundance of several ion transporters, corroborating contractile impairment. Similar effects were recapitulated by an in vitro cell culture model under a controlled oxygen atmosphere. Together, this study provides to our knowledge the most comprehensive report integrating analyses of protein abundance and global and peptide-level redox state in end-stage failing human hearts as well as oxygen-dependent redox and global proteome profiles of cultured human cardiomyocytes.
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http://dx.doi.org/10.3390/ijms22041787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7916846PMC
February 2021

Comparative proteomics of common allergenic tree pollens of birch, alder, and hazel.

Allergy 2021 06 15;76(6):1743-1753. Epub 2021 Jan 15.

Diagnostic and Research Institute of Pathology, Medical University of Graz, Graz, Austria.

Background: In addition to known allergens, other proteins in pollen can aid the development of an immune response in allergic individuals. The contribution of the "unknown" protein allergens is apparent in phylogenetically related species where, despite of high homology of the lead allergens, the degree of allergenic potential can vary greatly. The aim of this study was to identify other potentially allergenic proteins in pollen of three common and highly related allergenic tree species: birch (Betula pendula), hazel (Corylus avellana) and alder (Alnus glutinosa).

Methods: For that purpose, we carried out a comprehensive, comparative proteomic screening of the pollen from the three species. In order to maximize protein recovery and coverage, different protein extraction and isolation strategies during sample preparation were employed.

Results: As a result, we report 2500-3000 identified proteins per each of the pollen species. Identified proteins were further used for a number of annotation steps, providing insight into differential distribution of peptidases, peptidase inhibitors and other potential allergenic proteins across the three species. Moreover, we carried out functional enrichment analyses that, interestingly, corroborated high species similarity in spite of their relatively distinct protein profiles.

Conclusion: We provide to our knowledge first insight into proteomes of two very important allergenic pollen types, hazel and alder, where not even transcriptomics data are available, and compared them to birch. Datasets from this study can be readily used as protein databases and as such serve as basis for further functional studies.
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http://dx.doi.org/10.1111/all.14694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8248232PMC
June 2021

Spatially Resolved Activity-based Proteomic Profiles of the Murine Small Intestinal Lipases.

Mol Cell Proteomics 2020 12 6;19(12):2104-2115. Epub 2020 Oct 6.

Institute of Chemical Technologies and Analytics, Vienna University of Technology, Vienna, Austria; Diagnostic and Research Institute of Pathology, Medical University Graz, Graz, Austria; BioTechMed-Graz, Graz, Austria. Electronic address:

Despite the crucial function of the small intestine in nutrient uptake our understanding of the molecular events underlying the digestive function is still rudimentary. Recent studies demonstrated that enterocytes do not direct the entire dietary triacylglycerol toward immediate chylomicron synthesis. Especially after high-fat challenges, parts of the resynthesized triacylglycerol are packaged into cytosolic lipid droplets for transient storage in the endothelial layer of the small intestine. The reason for this temporary storage of triacylglycerol is not completely understood. To utilize lipids from cytosolic lipid droplets for chylomicron synthesis in the endoplasmic reticulum, stored triacylglycerol has to be hydrolyzed either by cytosolic lipolysis or lipophagy. Interestingly, triacylglycerol storage and chylomicron secretion rates are unevenly distributed along the small intestine, with the proximal jejunum exhibiting the highest intermittent storage capacity. We hypothesize that correlating hydrolytic enzyme activities with the reported distribution of triacylglycerol storage and chylomicron secretion in different sections of the small intestine is a promising strategy to determine key enzymes in triacylglycerol remobilization. We employed a serine hydrolase specific activity-based labeling approach in combination with quantitative proteomics to identify and rank hydrolases based on their relative activity in 11 sections of the small intestine. Moreover, we identified several clusters of enzymes showing similar activity distribution along the small intestine. Merging our activity-based results with substrate specificity and subcellular localization known from previous studies, carboxylesterase 2e and arylacetamide deacetylase emerge as promising candidates for triacylglycerol mobilization from cytosolic lipid droplets in enterocytes.
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http://dx.doi.org/10.1074/mcp.RA120.002171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710144PMC
December 2020

Proteomic analysis of soluble protein extract of adult Toxocara cati.

Comp Immunol Microbiol Infect Dis 2020 Dec 13;73:101528. Epub 2020 Aug 13.

Department of Pathobiology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Iran. Electronic address:

Toxocara cati is a cat roundworm and the causative agent of toxocariasis as a cosmopolitan zoonotic disease. As no information has been reported so far, identification of T. cati proteins can be useful for the development of new diagnostic strategies. This study was conducted to identify the major proteins in the adult T. cati tegument using bi-dimensional electrophoresis (2-DE) and shotgun proteomics. A total proteins were identified, among them the metabolic enzymes were the largest group, including: Enolase, triose phosphate isomerase, fructose-bisphosphate aldolase, aldehyde dehydrogenase. The other important protein groups recognized in T. cati, belong to the HSP-family, the structure and motor proteins, such as actin. The role of these proteins have been implicated in parasite-host interactions and modulating cellular immune response, immune regulation in evasion mechanisms of the host immune response. Characterizing T. cati adult proteins play a key role not only in host-parasite interactions, but also in the discovery of drug targets, subunit vaccines against toxocariasis, immunodiagnostic kits for toxocariasis and the identification of novel immuno-modulators that can form the next generation of therapeutic possibilities for inflammatory diseases.
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http://dx.doi.org/10.1016/j.cimid.2020.101528DOI Listing
December 2020

Plasma proteins facilitates placental transfer of polystyrene particles.

J Nanobiotechnology 2020 Sep 9;18(1):128. Epub 2020 Sep 9.

Department of Obstetrics and Gynecology, Medical University of Graz, Auenbruggerplatz 14, 8036, Graz, Austria.

Background: Nanoparticles, which are exposed to biological fluids are rapidly interacting with proteins and other biomolecules forming a corona. In addition to dimension, charge and material the distinct protein corona influences the interplay of nanoparticles with tissue barriers. In this study we were focused on the impact of in situ formed human plasma protein corona on the transfer of 80 nm polystyrene nanoparticles (PS-particles) across the human placenta. To study materno-to fetal PS transfer we used the human ex vivo placental perfusion approach, which represents an intact and physiological tissue barrier. To analyze the protein corona of PS particles we performed shotgun proteomics of isolated nanoparticles before and after tissue exposure.

Results: Human plasma incubated with PS-particles of 80 nm and subsequent formed protein corona enhanced the transfer across the human placenta compared to PS-corona formed by bovine serum albumin and dextran which served as a control. Quantitative and qualitative changes of plasma proteins determined the changes in PS transfer across the barrier. Based on the analysis of the PS-proteome two candidate proteins, namely human albumin and immunoglobulin G were tested if these proteins may account for the enhanced PS-transfer across the placenta. Interestingly, the protein corona formed by human albumin significantly induced the transfer of PS-particles across the tissue compared to the formed IgG-corona.

Conclusion: In total we demonstrate the PS corona dynamically and significantly evolves upon crossing the human placenta. Thus, the initial composition of PS particles in the maternal circulation is not predictive for their transfer characteristics and performance once beyond the barrier of the placenta. The precise mechanism of these effects remains to be elucidated but highlights the importance of using well designed biological models when testing nanoparticles for biomedical applications.
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http://dx.doi.org/10.1186/s12951-020-00676-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7487953PMC
September 2020

The Positive Association between Plasma Myristic Acid and ApoCIII Concentrations in Cardiovascular Disease Patients Is Supported by the Effects of Myristic Acid in HepG2 Cells.

J Nutr 2020 10;150(10):2707-2715

Department of Medicine, University of Verona, Verona, Italy.

Background: In the settings of primary and secondary prevention for coronary artery disease (CAD), a crucial role is played by some key molecules involved in triglyceride (TG) metabolism, such as ApoCIII. Fatty acid (FA) intake is well recognized as a main determinant of plasma lipids, including plasma TG concentration.

Objectives: The aim was to investigate the possible relations between the intakes of different FAs, estimated by their plasma concentrations, and circulating amounts of ApoCIII.

Methods: Plasma samples were obtained from 1370 subjects with or without angiographically demonstrated CAD (mean ± SD age: 60.6 ± 11.0 y; males: 75.8%; BMI: 25.9 ± 4.6 kg/m2; CAD: 73.3%). Plasma lipid, ApoCIII, and FA concentrations were measured. Data were analyzed by regression models adjusted for FAs and other potential confounders, such as sex, age, BMI, diabetes, smoking, and lipid-lowering therapies. The in vitro effects of FAs were tested by incubating HepG2 hepatoma cells with increasing concentrations of selected FAs, and the mRNA and protein contents in the cells were quantified by real-time RT-PCR and LC-MS/MS analyses.

Results: Among all the analyzed FAs, myristic acid (14:0) showed the most robust correlations with both TGs (R = 0.441, P = 2.6 × 10-66) and ApoCIII (R = 0.327, P = 1.1 × 10-31). By multiple regression analysis, myristic acid was the best predictor of both plasma TG and ApoCIII variability. Plasma TG and ApoCIII concentrations increased progressively at increasing concentrations of myristic acid, independently of CAD diagnosis and gender. Consistent with these data, in the in vitro experiments, an ∼2-fold increase in the expression levels of the ApoCIII mRNA and protein was observed after incubation with 250 μM myristic acid. A weaker effect (∼30% increase) was observed for palmitic acid, whereas incubation with oleic acid did not affect ApoCIII protein or gene expression.

Conclusions: Plasma myristic acid is associated with increased ApoCIII concentrations in cardiovascular patients. In vitro experiments indicated that myristic acid stimulates ApoCIII expression in HepG2 cells.
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http://dx.doi.org/10.1093/jn/nxaa202DOI Listing
October 2020

Folding Assessment of Incorporation of Noncanonical Amino Acids Facilitates Expansion of Functional-Group Diversity for Enzyme Engineering.

Chemistry 2020 Sep 4;26(54):12338-12342. Epub 2020 Sep 4.

Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010, Graz, Austria.

Protein design is limited by the diversity of functional groups provided by the canonical protein "building blocks". Incorporating noncanonical amino acids (ncAAs) into enzymes enables a dramatic expansion of their catalytic features. For this, quick identification of fully translated and correctly folded variants is decisive. Herein, we report the engineering of the enantioselectivity of an esterase utilizing several ncAAs. Key for the identification of active and soluble protein variants was the use of the split-GFP method, which is crucial as it allows simple determination of the expression levels of enzyme variants with ncAA incorporations by fluorescence. Several identified variants led to improved enantioselectivity or even inverted enantiopreference in the kinetic resolution of ethyl 3-phenylbutyrate.
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http://dx.doi.org/10.1002/chem.202002077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7590180PMC
September 2020

Transgene integration causes RARB downregulation in homozygous Tg4-42 mice.

Sci Rep 2020 04 14;10(1):6377. Epub 2020 Apr 14.

QPS Austria GmbH, Parkring 12, 8074, Grambach, Austria.

Alzheimer's disease can be modelled by different transgenic mouse strains. To gain deeper insight into disease model mechanisms, the previously described Tg4-42 mouse was analysed for transgene integration. On RNA/DNA level the transgene integration resulted in more than 20 copy numbers and further caused a deletion of exon 2 of the retinoic acid receptor beta. These findings were also confirmed on protein level with highly decreased retinoic acid receptor beta protein levels in homozygous Tg4-42 mice and may have an impact on the previously described phenotype of homozygous Tg4-42 mice to be solely dependent on amyloid-ß 4-42 expression. Since hemizygous mice show no changes in RARB protein levels it can be concluded that the previously described phenotype of these mice should not be affected by the retinoic acid receptor beta gene knockout. In order to fully understand the results of transgenesis, it is extremely advisable to determine the genome integration site and the basic structure of the inserted transgenes. This can be carried out for instance by next-generation sequencing techniques. Our results thus suggest that a detailed characterization of new disease models using the latest genomics technologies prior to functional studies could be a valuable tool to avoid an unexpected genetic influence on the animals' phenotype that is not only based on the inserted transgene. This would also significantly improve the selection of mouse models that are best suited for therapeutic development and basic research.
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http://dx.doi.org/10.1038/s41598-020-63512-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7156671PMC
April 2020

Nasal mucus proteome and its involvement in allergic rhinitis.

Expert Rev Proteomics 2020 03 8;17(3):191-199. Epub 2020 Apr 8.

Diagnostic and Research Institute of Pathology, Diagnostic and Research Center of Molecular Medicine, Medical University of Graz, Graz, Austria.

: Nasal mucus is the first line defense barrier against various pathogens including allergens. Proteins in nasal mucus maybe used as biomarkers for diagnosis or future therapeutic strategies. Proteomics opens the possibility to investigate whole human proteomes.: We aimed to analyze the existing literature on nasal mucus and nasal secretions proteomic approaches especially in allergic rhinitis. A PubMed/Medline search was conducted entering the following keywords and combinations: "nasal mucus", "nasal lavage fluid," nasal secretions," "nasal swabs," "allergic rhinitis," "proteins," and "proteomics.": The majority of studies focus on single proteins or protein groups mainly using ELISA techniques. Four studies met the criteria using mass spectrometry in the analysis of nasal mucus proteomes in rhinologic diseases. In these studies, 7, 35, 267, and 430 proteins were identified, respectively. These four studies are discussed in this review and put in relation to seven other proteomic studies that focus on nasal lavage fluid and nasal secretions obtained by swabs or filter paper. To put it in a nutshell, proteomics facilitates the investigation of the nasal secretome and its role in healthy and diseased state and as potential biomarkers for new diagnostic or therapeutic approaches.
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http://dx.doi.org/10.1080/14789450.2020.1748502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261402PMC
March 2020

Addressing Glutathione Redox Status in Clinical Samples by Two-Step Alkylation with N-ethylmaleimide Isotopologues.

Metabolites 2020 Feb 16;10(2). Epub 2020 Feb 16.

Institute of Chemical Technologies and Analytics, Faculty of Technical Chemistry, Vienna University of Technology-TU Wien, Getreidemarkt 9/164, 1060 Vienna, Austria.

Determination of the ratio of reduced to oxidized glutathione is of profound clinical interest in assessing the oxidative status of tissues and body fluids. However, this ratio is not yet a routine clinical parameter due to the analytically challenging interconversion of reduced (free) glutathione to oxidized (bound) glutathione. We aimed to facilitate this ratio determination in order to aid its incorporation as a routine clinical parameter. To this end, we developed a simple derivatization route that yields different isotopologues of N-ethylmaleimide alkylated glutathione from reduced and oxidized glutathione (after its chemical reduction) for mass spectrometric analysis. A third isotopologue can be used as isotopic standard for simultaneous absolute quantification. As all isotopologues have similar chromatographic properties, matrix effects arising from different sample origins can only impact method sensitivity but not quantification accuracy. Robustness, simplified data analysis, cost effectiveness by one common standard, and highly improved mass spectrometric sensitivity by conversion of oxidized glutathione to an alkylated glutathione isotopologue are the main advantages of our approach. We present a method fully optimized for blood, plasma, serum, cell, and tissue samples. In addition, we propose production of N-ethylmaleimide customized blood collection tubes to even further facilitate the analysis in a clinical setting.
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http://dx.doi.org/10.3390/metabo10020071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7074022PMC
February 2020

Alteration of protein profile in cerebral cortex of rats exposed to bisphenol a: a proteomics study.

Neurotoxicology 2020 05 1;78:1-10. Epub 2020 Feb 1.

Pharmaceutical Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran; Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. Electronic address:

Bisphenol A (BPA) is one of the most widely used chemicals in plastic industry, which enters the human body through occupational and food contact. We studied the protein changes in rat cerebral cortex to evaluate the neurotoxicity of BPA. Twenty-four adult male rats were randomly selected and divided into four groups and each group respectively received 0, 0.5, 5 and 50 mg/kg of BPA for 4 weeks orally. To determine the oxidative status, reduced glutathione content and the level of malondialdehyde were measured in brain cortical tissue. The proteins of each sample extracted and separated on a two-dimensional acrylamide gel electrophoresis. From the obtained protein map, the 10 most altered protein spots were used for mass spectroscopy analysis. The lipid peroxidation in both doses of 0.5 and 5 mg/kg was significantly higher than the control group, but the glutathione content had no significant difference between the groups. Based on the results of the MS data analysis by the MASCOT database search engine, 10 proteins with altered intensity were identified as pyruvate kinase, alpha-enolase, aconitate hydratase, creatine kinase B-type, phosphatidylethanolamine-binding protein 1, 14-3-3 protein eta, guanine nucleotide-binding protein subunit beta-1, dihydropyrimidinase-related protein 2, glutamine synthetase and the neurofilament light polypeptide. There are several reports suggesting that the increase or decrease in the level and activity of these 10 proteins, similar to those observed in this study, is related to some neurological and psychosocial disorders including neurodegenerative diseases, schizophrenia, depression, epilepsy and some brain tumors.
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http://dx.doi.org/10.1016/j.neuro.2020.01.013DOI Listing
May 2020

HDAC inhibition improves cardiopulmonary function in a feline model of diastolic dysfunction.

Sci Transl Med 2020 01;12(525)

Cardiovascular Research Center, Lewis Katz School of Medicine, Temple University, Philadelphia, PA 19140, USA.

Heart failure with preserved ejection fraction (HFpEF) is a major health problem without effective therapies. This study assessed the effects of histone deacetylase (HDAC) inhibition on cardiopulmonary structure, function, and metabolism in a large mammalian model of pressure overload recapitulating features of diastolic dysfunction common to human HFpEF. Male domestic short-hair felines ( = 31, aged 2 months) underwent a sham procedure ( = 10) or loose aortic banding ( = 21), resulting in slow-progressive pressure overload. Two months after banding, animals were treated daily with suberoylanilide hydroxamic acid (b + SAHA, 10 mg/kg, = 8), a Food and Drug Administration-approved pan-HDAC inhibitor, or vehicle (b + veh, = 8) for 2 months. Echocardiography at 4 months after banding revealed that b + SAHA animals had significantly reduced left ventricular hypertrophy (LVH) ( < 0.0001) and left atrium size ( < 0.0001) versus b + veh animals. Left ventricular (LV) end-diastolic pressure and mean pulmonary arterial pressure were significantly reduced in b + SAHA ( < 0.01) versus b + veh. SAHA increased myofibril relaxation ex vivo, which correlated with in vivo improvements of LV relaxation. Furthermore, SAHA treatment preserved lung structure, compliance, blood oxygenation, and reduced perivascular fluid cuffs around extra-alveolar vessels, suggesting attenuated alveolar capillary stress failure. Acetylation proteomics revealed that SAHA altered lysine acetylation of mitochondrial metabolic enzymes. These results suggest that acetylation defects in hypertrophic stress can be reversed by HDAC inhibitors, with implications for improving cardiac structure and function in patients.
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http://dx.doi.org/10.1126/scitranslmed.aay7205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065257PMC
January 2020

A Broad Spectrum Protein Glycosylation System Influences Type II Protein Secretion and Associated Phenotypes in .

Front Microbiol 2019 3;10:2780. Epub 2019 Dec 3.

Institute of Molecular Biosciences, University of Graz, Graz, Austria.

Protein secretion plays a crucial role for bacterial pathogens, exemplified by facultative human-pathogen , which secretes various proteinaceous effectors at different stages of its lifecycle. Accordingly, the identification of factors impacting on protein secretion is important to understand the bacterial pathophysiology. PglL, a predicted oligosaccharyltransferase of , has been recently shown to exhibit -glycosylation activity with relaxed glycan specificity in an engineered system. By engineering strains to express a defined, undecaprenyl diphosphate-linked glycoform precursor, we confirmed functional -linked protein glycosylation activity of PglL in . We demonstrate that PglL is required for the glycosylation of multiple proteins, including periplasmic chaperones such as DegP, that are required for efficient type II-dependent secretion. Moreover, defined deletion mutants and complementation strains provided first insights into the physiological role of -linked protein glycosylation in . RbmD, a protein with structural similarities to PglL and other established oligosaccharyltransferases (OTases), was also included in this phenotypical characterization. Remarkably, presence or absence of PglL and RbmD impacts the secretion of proteins via the type II secretion system (T2SS). This is highlighted by altered cholera toxin (CT) secretion, chitin utilization and biofilm formation observed in Δ and Δ single or double mutants. This work thus establishes a unique connection between broad spectrum -linked protein glycosylation and the efficacy of type II-dependent protein secretion critical to the pathogen's lifecycle.
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http://dx.doi.org/10.3389/fmicb.2019.02780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6901666PMC
December 2019

Outgrowth, proliferation, viability, angiogenesis and phenotype of primary human endothelial cells in different purchasable endothelial culture media: feed wisely.

Histochem Cell Biol 2019 Nov 21;152(5):377-390. Epub 2019 Sep 21.

Department of Obstetrics and Gynaecology, Medical University of Graz, Graz, Austria.

Function and dysfunction of endothelial cells are regulated by a multitude of factors. Endothelial cell research often requires in vitro cell culture experiments. Hence, various culture media specifically designed to promote endothelial cell growth are available. These strikingly differ in their composition: complex media contain endothelial cell growth supplement (ECGS), an extract produced of bovine brain with undefined amounts of biologically active compounds, whilst defined media contain selected growth factors in defined concentrations. We here compared the effect of seven purchasable endothelial cell culture media on colony outgrowth, proliferation, viability, in vitro angiogenesis and phenotype of mature primary human endothelial cells using feto-placental endothelial cells isolated from chorionic arteries (fpEC). The effect of media on colony outgrowth was additionally tested on umbilical cord blood-derived endothelial progenitor cells (ECFCs). Outgrowth, purity, proliferation and viability differed between media. Outgrowth of fpEC and ECFCs was best in a defined medium containing EGF, FGF2 and VEGF. By contrast, established fpEC isolations proliferated best in complex media containing ECGS, heparin and ascorbic acid. Also viability of cells was higher in complex media. In vitro angiogenesis was most intense in a defined medium containing the highest number of individual growth factors. FACS analysis of surface markers for endothelial cell subtypes revealed that endothelial phenotype of fpEC was unaffected by media composition. Our data demonstrate the fundamental effect of endothelial cell culture media on primary cell isolation success and behaviour. Whether the composition of supplements is suitable also for individual experiments needs to be tested specifically.
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http://dx.doi.org/10.1007/s00418-019-01815-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6842357PMC
November 2019

Cysteine oxidation triggers amyloid fibril formation of the tumor suppressor p16.

Redox Biol 2020 01 3;28:101316. Epub 2019 Sep 3.

Center for Molecular Medicine, Molecular Cancer Research, University Medical Center Utrecht, Universiteitsweg 100, 3584CG, Utrecht, The Netherlands. Electronic address:

The tumor suppressor p16 induces cell cycle arrest and senescence in response to oncogenic transformation and is therefore frequently lost in cancer. p16 is also known to accumulate under conditions of oxidative stress. Thus, we hypothesized it could potentially be regulated by reversible oxidation of cysteines (redox signaling). Here we report that oxidation of the single cysteine in p16 in human cells occurs under relatively mild oxidizing conditions and leads to disulfide-dependent dimerization. p16 is an all α-helical protein, but we find that upon cysteine-dependent dimerization, p16 undergoes a dramatic structural rearrangement and forms aggregates that have the typical features of amyloid fibrils, including binding of diagnostic dyes, presence of cross-β sheet structure, and typical dimensions found in electron microscopy. p16 amyloid formation abolishes its function as a Cyclin Dependent Kinase 4/6 inhibitor. Collectively, these observations mechanistically link the cellular redox state to the inactivation of p16 through the formation of amyloid fibrils.
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http://dx.doi.org/10.1016/j.redox.2019.101316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6812003PMC
January 2020

Irreversible oxidative post-translational modifications in heart disease.

Expert Rev Proteomics 2019 08 30;16(8):681-693. Epub 2019 Jul 30.

Institute of Pathology, Diagnostic and Research Center for Molecular Biomedicine, Medical University of Graz , Graz , Austria.

: Development of specific biomarkers aiding early diagnosis of heart failure is an ongoing challenge. Biomarkers commonly used in clinical routine usually act as readouts of an already existing acute condition rather than disease initiation. Functional decline of cardiac muscle is greatly aggravated by increased oxidative stress and damage of proteins. Oxidative post-translational modifications occur already at early stages of tissue damage and are thus regarded as potential up-coming disease markers. : Clinical practice regarding commonly used biomarkers for heart disease is briefly summarized. The types of oxidative post-translational modification in cardiac pathologies are discussed with a special focus on available quantitative techniques and characteristics of individual modifications with regard to their stability and analytical accessibility. As irreversible oxidative modifications trigger protein degradation pathways or cause protein aggregation, both influencing biomarker abundance, a chapter is dedicated to their regulation in the heart.
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http://dx.doi.org/10.1080/14789450.2019.1645602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6816499PMC
August 2019

Endothelial lipase increases antioxidative capacity of high-density lipoprotein.

Biochim Biophys Acta Mol Cell Biol Lipids 2019 10 17;1864(10):1363-1374. Epub 2019 Jun 17.

Otto Loewi Research Center, Division of Physiological Chemistry, Medical University of Graz, Neue Stiftingtalstraße 6/3, 8010 Graz, Austria.

Endothelial lipase (EL) is a strong determinant of structural and functional properties of high-density lipoprotein (HDL). We examined whether the antioxidative capacity of HDL is affected by EL. EL-modified HDL (EL-HDL) and control EV-HDL were generated by incubation of HDL with EL- overexpressing or control HepG2 cells. As determined by native gradient gel electrophoresis, electron microscopy, and small-angle X-ray scattering EL-HDL is smaller than EV-HDL. Mass spectrometry revealed an enrichment of EL-HDL with lipolytic products and depletion of phospholipids and triacylglycerol. Kinetics of conjugated diene formation and HPLC-based malondialdehyde quantification revealed that EL-HDL exhibited a significantly higher resistance to copper ion-induced oxidation and a significantly higher capacity to protect low-density lipoprotein (LDL) from copper ion-induced oxidation when compared to EV-HDL. Depletion of the lipolytic products from EL-HDL abolished the capacity of EL-HDL to protect LDL from copper ion-induced oxidation, which could be partially restored by lysophosphatidylcholine enrichment. Proteomics of HDL incubated with oxidized LDL revealed significantly higher levels of methionine 136 sulfoxide in EL-HDL compared to EV-HDL. Chloramine T (oxidizes methionines and modifies free thiols), diminished the difference between EL-HDL and EV-HDL regarding the capacity to protect LDL from oxidation. In absence of LDL small EV-HDL and EL-HDL exhibited higher resistance to copper ion-induced oxidation when compared to respective large particles. In conclusion, the augmented antioxidative capacity of EL-HDL is primarily determined by the enrichment of HDL with EL-generated lipolytic products and to a lesser extent by the decreased HDL particle size and the increased activity of chloramine T-sensitive mechanisms.
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http://dx.doi.org/10.1016/j.bbalip.2019.06.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6699986PMC
October 2019

Low cardiac lipolysis reduces mitochondrial fission and prevents lipotoxic heart dysfunction in Perilipin 5 mutant mice.

Cardiovasc Res 2020 02;116(2):339-352

Institute of Molecular Biosciences, University of Graz, Heinrichstrasse 31, 8010 Graz, Austria.

Aims: Lipotoxic cardiomyopathy in diabetic and obese patients typically encompasses increased cardiac fatty acid (FA) uptake eventually surpassing the mitochondrial oxidative capacity. Lowering FA utilization via inhibition of lipolysis represents a strategy to counteract the development of lipotoxic heart dysfunction. However, defective cardiac triacylglycerol (TAG) catabolism and FA oxidation in humans (and mice) carrying mutated ATGL alleles provokes lipotoxic heart dysfunction questioning a therapeutic approach to decrease cardiac lipolysis. Interestingly, decreased lipolysis via cardiac overexpression of Perilipin 5 (Plin5), a binding partner of ATGL, is compatible with normal heart function and lifespan despite massive cardiac lipid accumulation. Herein, we decipher mechanisms that protect Plin5 transgenic mice from the development of heart dysfunction.

Methods And Results: We generated mice with cardiac-specific overexpression of Plin5 encoding a serine-155 to alanine exchange (Plin5-S155A) of the protein kinase A phosphorylation site, which has been suggested as a prerequisite to stimulate lipolysis and may play a crucial role in the preservation of heart function. Plin5-S155A mice showed a substantial increase in cardiac TAG and ceramide levels, which was comparable to mice overexpressing non-mutated Plin5. Lipid accumulation was compatible with normal heart function even under mild stress. Plin5-S155A mice showed reduced cardiac FA oxidation but normal ATP production and changes in the Plin5-S155A phosphoproteome compared to Plin5 transgenic mice. Interestingly, mitochondrial recruitment of dynamin-related protein 1 (Drp1) was markedly reduced in cardiac muscle of Plin5-S155A and Plin5 transgenic mice accompanied by decreased phosphorylation of mitochondrial fission factor, a mitochondrial receptor of Drp1.

Conclusions: This study suggests that low cardiac lipolysis is associated with reduced mitochondrial fission and may represent a strategy to combat the development of lipotoxic heart dysfunction.
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http://dx.doi.org/10.1093/cvr/cvz119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7338219PMC
February 2020

Proteomic Analysis of Vocal Fold Fibroblasts Exposed to Cigarette Smoke Extract: Exploring the Pathophysiology of Reinke's Edema.

Mol Cell Proteomics 2019 08 22;18(8):1511-1525. Epub 2019 May 22.

‡Division of Phoniatrics, Medical University of Graz, Graz, Austria; §§Takeda, Vienna, Austria.

Reinke's edema is a smoking-associated, benign, mostly bilateral lesion of the vocal folds leading to difficulties in breathing and voice problems. Pronounced histological changes such as damaged microvessels or immune cell infiltration have been described in the vocal fold connective tissue, the Thus, vocal fold fibroblasts, the main cell type of the , have been postulated to play a critical role in disease mediation. Yet information about the pathophysiology is still scarce and treatment is only surgical, symptomatic. To explore the pathophysiology of Reinke's edema, we exposed near-primary human vocal fold fibroblasts to medium conditioned with cigarette smoke extract for 24 h as well as 4 days followed by quantitative mass spectrometry.Proteomic analyses after 24 h revealed that cigarette smoke increased proteins previously described to be involved in oxidative stress responses in other contexts. Correspondingly, gene sets linked to metabolism of xenobiotics and reactive oxygen species were significantly enriched among cigarette smoke-induced proteins. Among the proteins most downregulated by cigarette smoke, we identified fibrillar collagens COL1A1 and COL1A2; this reduction was validated by complementary methods. Further, we found a significant increase of UDP-glucose 6-dehydrogenase, generating a building block for biosynthesis of hyaluronan, another crucial component of the vocal fold In line with this result, hyaluronan levels were significantly increased because of cigarette smoke exposure. Long term treatment of 4 days did not lead to significant changes.The current findings corroborate previous studies but also reveal new insights in possible disease mechanisms of Reinke's edema. We postulate that changes in the composition of the vocal folds' extracellular matrix -reduction of collagen fibrils, increase of hyaluronan- may lead to the clinical findings. This might ease the identification of better, disease-specific treatment options.
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http://dx.doi.org/10.1074/mcp.RA119.001272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6683006PMC
August 2019

Tsr4 and Nap1, two novel members of the ribosomal protein chaperOME.

Nucleic Acids Res 2019 07;47(13):6984-7002

Institute of Molecular Biosciences, University of Graz, Humboldtstrasse 50, 8010 Graz, Austria.

Dedicated chaperones protect newly synthesized ribosomal proteins (r-proteins) from aggregation and accompany them on their way to assembly into nascent ribosomes. Currently, only nine of the ∼80 eukaryotic r-proteins are known to be guarded by such chaperones. In search of new dedicated r-protein chaperones, we performed a tandem-affinity purification based screen and looked for factors co-enriched with individual small subunit r-proteins. We report the identification of Nap1 and Tsr4 as direct binding partners of Rps6 and Rps2, respectively. Both factors promote the solubility of their r-protein clients in vitro. While Tsr4 is specific for Rps2, Nap1 has several interaction partners including Rps6 and two other r-proteins. Tsr4 binds co-translationally to the essential, eukaryote-specific N-terminal extension of Rps2, whereas Nap1 interacts with a large, mostly eukaryote-specific binding surface of Rps6. Mutation of the essential Tsr4 and deletion of the non-essential Nap1 both enhance the 40S synthesis defects of the corresponding r-protein mutants. Our findings highlight that the acquisition of eukaryote-specific domains in r-proteins was accompanied by the co-evolution of proteins specialized to protect these domains and emphasize the critical role of r-protein chaperones for the synthesis of eukaryotic ribosomes.
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http://dx.doi.org/10.1093/nar/gkz317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6648895PMC
July 2019

Olfactory cleft proteome does not reflect olfactory performance in patients with idiopathic and postinfectious olfactory disorder: A pilot study.

Sci Rep 2018 12 3;8(1):17554. Epub 2018 Dec 3.

Department of Otorhinolaryngology, Medical University of Graz, Auenbruggerplatz 26, 8036, Graz, Austria.

Technical advances including liquid chromatography-tandem mass spectrometry and its data analysis enable detailed proteomic analysis of the nasal mucus. Alterations of the nasal mucus proteome may provoke substantial changes of the nasal physiology and have already been associated with rhinologic diseases such as allergic rhinitis. This study was conducted as a pilot study to map the olfactory cleft proteome using current techniques for proteomic analysis. Furthermore, we aimed to investigate proteomic changes as potential biomarkers in patients suffering from idiopathic and postinfectious olfactory disorders compared to healthy controls. Seven patients with idiopathic hyposmia and anosmia, seven patients with postinfectious hyposmia and anosmia and seven healthy controls were included in this study. In total, 1117 different proteins were detected in at least five patients in at least one group. Results of this study did not reveal significant differences regarding the proteomic composition of the olfactory cleft mucus between patients versus healthy controls. Among proteins involved in olfactory perception the G protein family was detected but also found unchanged between groups. Investigation of protein composition by liquid chromatography-tandem mass spectrometry enabled us to perform an in-depth analysis of the olfactory cleft mucus proteome regarding the diversity of different proteins in individual patients. However untargeted proteomics of the olfactory cleft mucus may not be an applicable approach to develop biomarkers for olfactory disorders. Targeted analyses of distinct proteins known to be involved in olfactory perception but not detected by our approach, e.g. odorant binding proteins, may provide more information regarding pathophysiology of olfactory diseases.
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http://dx.doi.org/10.1038/s41598-018-35776-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277379PMC
December 2018

High density lipoprotein cholesterol and proteome in SR-B1 KO mice: lost in precipitation.

J Transl Med 2018 11 12;16(1):309. Epub 2018 Nov 12.

Department of Nutrition, Diabetes and Metabolism, Pontificia Universidad Católica de Chile, Santiago, Chile.

Scavenger receptor class B type 1 (SR-B1) plays an essential role in high density lipoprotein (HDL) metabolism. SR-B1 deficient (SR-B1 KO) mice are prone to atherosclerosis and exhibit abnormally large, cholesterol-rich, dysfunctional HDL. In a recent issue of J Transl Med, Cao et al. described results of proteomics analyses of HDL isolated from wild-type (WT) and SR-B1 KO mice using precipitation of large lipoproteins with polyethylene glycol (PEG). They report abnormalities in SR-B1 KO HDL protein components that correlate with HDL function. In this commentary, we describe and discuss the differences in the results published by Cao et al. and those obtained in a recent study from our laboratory using shotgun proteomics of HDL of SR-B1 KO mice isolated by ultracentrifugation. We propose that different HDL purification procedures used may account for the discrepancies observed. We show that SR-B1 KO HDL purification using either PEG or dextran sulfate precipitation results in enrichment of small HDL subclasses, and may therefore underestimate alterations in lipoprotein composition or function. Compared to HDL obtained by ultracentrifugation, HDL isolated by PEG precipitation show a lower ApoE/ApoA-I proportion and reduced cholesterol content. HDL protein components described by Cao et al. or our laboratory are mostly inconsistent: only 33 HDL proteins were detected in both datasets, whereas a significant number of proteins were only identified by Cao et al. (n = 43) or Contreras-Duarte et al. (n = 26) datasets. The relative abundance of HDL-associated peptide and protein levels in WT vs SR-B1 HDL were also highly different in both datasets. This study indicates that caution must be taken when interpreting results from HDL isolated by chemical precipitation.
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http://dx.doi.org/10.1186/s12967-018-1683-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6233513PMC
November 2018

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

Biochim Biophys Acta Mol Cell Biol Lipids 2018 10 25;1863(10):1193-1205. Epub 2018 Jul 25.

Institute of Molecular Biosciences, NAWI Graz, University of Graz, Humboldtstrasse 50/II, 8010 Graz, Austria. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbalip.2018.07.010DOI Listing
October 2018

Prazosin induced lysosomal tubulation interferes with cytokinesis and the endocytic sorting of the tumour antigen CD98hc.

Biochim Biophys Acta Mol Cell Res 2018 09 15;1865(9):1211-1229. Epub 2018 Jun 15.

Research Unit Functional Proteomics and Metabolic Pathways, Institute of Pathology, Medical University of Graz, Auenbruggerplatz 25, 8036 Graz, Austria; Omics Center Graz, BioTechMed-Graz, Stiftingtalstrasse 24, 8010 Graz, Austria.

The quinazoline based drug prazosin (PRZ) is a potent inducer of apoptosis in human cancer cells. We recently reported that PRZ enters cells via endocytosis and induces tubulation of the endolysosomal system. In a proteomics approach aimed at identifying potential membrane proteins with binding affinity to quinazolines, we detected the oncoprotein CD98hc. We confirmed shuttling of CD98hc towards lysosomes and upregulation of CD98hc expression in PRZ treated cells. Gene knockout (KO) experiments revealed that endocytosis of PRZ still occurs in the absence of CD98hc - suggesting that PRZ does not enter the cell via CD98hc but misroutes the protein towards tubular lysosomes. Lysosomal tubulation interfered with completion of cytokinesis and provoked endoreplication. CD98hc KO cells showed reduced endoreplication capacity and lower sensitivity towards PRZ induced apoptosis than wild type cells. Thus, loss of CD98hc does not affect endocytosis of PRZ and lysosomal tubulation, but the ability for endoreplication and survival of cells. Furthermore, we found that glutamine, lysomototropic agents - namely chloroquine and NHCl - as well as inhibition of v-ATPase, interfere with the intracellular transport of CD98hc. In summary, our study further emphasizes lysosomes as target organelles to inhibit proliferation and to induce cell death in cancer. Most importantly, we demonstrate for the first time that the intracellular trafficking of CD98hc can be modulated by small molecules. Since CD98hc is considered as a potential drug target in several types of human malignancies, our study possesses translational significance suggesting, that old drugs are able to act on a novel target.
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http://dx.doi.org/10.1016/j.bbamcr.2018.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6070144PMC
September 2018

A Semi-Rationally Engineered Bacterial Pyrrolysyl-tRNA Synthetase Genetically Encodes Phenyl Azide Chemistry.

Biotechnol J 2019 Mar 11;14(3):e1800125. Epub 2018 Jun 11.

Acib - Austrian Centre of Industrial Biotechnology, Petersgasse 14, A-8010 Graz, Austria.

The site-specific incorporation of non-canonical amino acids (ncAAs) at amber codons requires an aminoacyl-tRNA synthetase and a cognate amber suppressor tRNA (tRNA ). The archaeal tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii and the pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina mazei have been extensively engineered to accept a versatile set of ncAAs. The PylRS/tRNA pair from the bacterium Desulfitobacterium hafniense is functional in Escherichia coli, however, variants of this PylRS have not been reported yet. In this study, the authors describe a bacterial PylRS from Desulfitobacterium hafniense, which the authors engineered for the reactive ncAA para-azido-l-phenylalanine (DhAzFRS) using a semi-rational approach. DhAzFRS preferred para-azido-l-phenylalanine to the canonical l-phenylalanine as the substrate. In addition, the authors demonstrate the functionality in E. coli of a hybrid DhAzFRS carrying the first 190 N-terminal amino acids of the Methanosarcina mazei PylRS. These results suggest that bacterial and archaeal PylRSs can be "mixed and matched" to tune their substrate specificity.
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http://dx.doi.org/10.1002/biot.201800125DOI Listing
March 2019

Effect of Noncanonical Amino Acids on Protein-Carbohydrate Interactions: Structure, Dynamics, and Carbohydrate Affinity of a Lectin Engineered with Fluorinated Tryptophan Analogs.

ACS Chem Biol 2018 08 12;13(8):2211-2219. Epub 2018 Jun 12.

Austrian Centre of Industrial Biotechnology , Petersgasse 14 , 8010 Graz , Austria.

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.
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http://dx.doi.org/10.1021/acschembio.8b00377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102642PMC
August 2018
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