The secretome of primary human hepatocytes in an organotypic bioreactor culture for the identification of biomarkers of drug-induced hepatotoxicity

In the presented thesis, a workflow for analysis of the extracellular proteome, or “secretome”, of primary human hepatocytes by a shotgun proteomics approach was developed. The secretome analysis on hepatocytes is of special interest for the pharmaceutical industry as well as medical services because secreted proteins could serve as biomarkers for drug-induced hepatotoxicity. The avoidance of fetal calf serum, a common supplement used for in vitro cell culture was shown to be a prerequisite for efficient secretome analysis because supplemented proteins severely hamper the identification of secreted proteins using mass-spectrometry (MS). The removal of high abundant proteins from the sample by immunodepletion, a common strategy for proteomic analysis of human plasma, was shown to increase the number of identified proteins to almost two-fold the number of identifications obtained without prior immunodepletion, paving the way for in-depth analysis of the secretome. A prefractionation step at the level of proteins and the combination of different MS-types and ionization methods tremendously enhanced the identifications but at the cost of analysis time as well as the fraction of secreted proteins. The elaborated workflow without prior prefractionation was applied to a standard monolayer culture and a promising in vitro cultivation technique in a 3-dimensional bioreactor mimicking the microenvironment of a real liver. With 109 and 160 proteins identified in monolayer and bioreactor cultures, respectively, the number of proteins likely to be secreted by hepatocytes into the extracellular space was much higher than described in the current literature. Furthermore, proteome analysis on extracellular proteins in the applied culture conditions confirmed the tissue-like behavior of primary hepatocytes in the three-dimensional cultivation. Differentially expressed proteins in the bioreactor culture after application of the reference drug diclofenac were detected by label-free quantification and most of the detected differences could be traced back to the underlying mechanism of toxicity proposed for this drug. In summary, the presented work provides a basis for further in-depth analysis of the primary human hepatocytes secretome for in vitro drug-testing and demonstrates the valuable contributions of proteomics to toxicological research.In der vorliegenden Arbeit wurde eine experimentelle Strategie zur Analyse des extrazellulären Proteoms („Sekretom“) von primären humanen Hepatozyten mittels „shotgun“ proteomics entwickelt. Die Analyse des Sekretoms von Hepatozyten ist von besonderer Bedeutung für die pharmazeutische Industrie sowie das Gesundheitswesen, da sekretierte Proteine als Biomarker für arzneimittelinduzierte Lebertoxizität verwendet werden können. Es wurde gezeigt, dass die Vermeidung von foetalem Kälberserum, einem gewöhnlich in der in vitro Zellkultur verwendeten Zusatz, eine Grundvorraussetzung für die effiziente Analyse der sekretierten Proteine darstellt, da die zugesetzten Proteine erheblich die Identifizierung sekretierter Proteine mittels Massenspektrometrie erschweren. Desweiteren erhöhte die Entfernung hoch abundanter Proteine aus den Proben mit Hilfe einer Immunodepletion, wie sie oft in der Proteomanalyse von menschlichem Serum Verwendung findet, die Anzahl an identifizierten Proteinen fast um das Doppelte und ebnete damit den Weg für eine detailliertere Analyse des Sekretoms. Weiterhin konnte gezeigt werden, dass eine Vorfraktionierung der Proteine sowie die Kombination verschiedener Massenspektrometertypen bzw. Ionisationsmethoden die Anzahl an Identifizierungen deutlich steigert, allerdings auf Kosten der benötigten Analysezeit und des Anteils an sekretierten Proteinen. Die erarbeitete analytische Strategie ohne vorherige Fraktionierung wurde sowohl auf Standardkultivierungen in Zellkulturflaschen, als auch auf eine vielversprechende Bioreaktortechnik angewendet, wobei letztere die dreidimensionale Umgebung in einer echten Leber nachahmt. 109 bzw. 160 sekretierte Proteine konnten in Standard- bzw. Bioreaktokulturen identifiziert werden, wesentlich mehr als es derzeitig in der Literatur für primäre humane Hepatozyten beschrieben wird. Desweiteren bestätigte die Analyse des extrazellulären Proteoms in den verwendeten Kultivierungstechniken das gewebeähnliche Verhalten der primären Leberzellen in der dreidimensionalen Bioreaktorkultur. Differentiell exprimierte Proteine in mit der Referenzsubstanz Diclofenac behandelten Bioreaktorkulturen wurden mittels labelfreier Quantifizierung bestimmt und konnten auf die zugrundeliegenden Toxizitätsmechanismen zurückgeführt werden, die für dieses Medikament bekannt sind. Zusammenfassend legt die vorliegende Arbeit den Grundstein für weitere umfassende Analysen des Sekretoms von Leberzellen für in vitro Wirkstofftests und veranschaulicht den wertvollen Beitrag der Proteomik für die toxikologische Forschung

The endolysosomal adaptor PLEKHM1 is a direct target for both mTOR and MAPK pathways

The lysosome is a cellular signalling hub at the point of convergence of endocytic and autophagic pathways, where the contents are degraded and recycled. Pleckstrin homology domain-containing family member 1 (PLEKHM1) acts as an adaptor to facilitate the fusion of endocytic and autophagic vesicles with the lysosome. However, it is unclear how PLEKHM1 function at the lysosome is controlled. Herein, we show that PLEKHM1 co-precipitates with, and is directly phosphorylated by, mTOR. Using a phospho-specific antibody against Ser432/S435 of PLEKHM1, we show that the same motif is a direct target for ERK2-mediated phosphorylation in a growth factor-dependent manner. This dual regulation of PLEKHM1 at a highly conserved region points to a convergence of both growth factor- and amino acid-sensing pathways, placing PLEKHM1 at a critical juncture of cellular metabolism

Lipidomics reveals seasonal shifts in a large-bodied hibernator, the brown bear

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Lipidomics Reveals Seasonal Shifts in a Large-Bodied Hibernator, the Brown Bear

Prior to winter, heterotherms retain polyunsaturated fatty acids (“PUFA”), resulting in enhanced energy savings during hibernation, through deeper and longer torpor bouts. Hibernating bears exhibit a less dramatic reduction (2–5°C) in body temperature, but lower their metabolism to a degree close to that of small hibernators. We determined the lipid composition, via lipidomics, in skeletal muscle and white adipose tissues (“WAT”), to assess lipid retention, and in blood plasma, to reflect lipid trafficking, of winter hibernating and summer active wild Scandinavian brown bears (Ursus arctos). We found that the proportion of monounsaturated fatty acids in muscle of bears was significantly higher during winter. During hibernation, omega-3 PUFAs were retained in WAT and short-length fatty acids were released into the plasma. The analysis of individual lipid moieties indicated significant changes of specific fatty acids, which are in line with the observed seasonal shift in the major lipid categories and can be involved in specific regulations of metabolisms. These results strongly suggest that the shift in lipid composition is well conserved among hibernators, independent of body mass and of the animals’ body temperature

Das Sekretom primärer humaner Hepatozyten in organsimulierender Bioreaktorkultur für die Identifizierung von Biomarkern arzneistoffinduzierter Lebertoxizität

In the presented thesis, a workflow for analysis of the extracellular proteome, or “secretome”, of primary human hepatocytes by a shotgun proteomics approach was developed. The secretome analysis on hepatocytes is of special interest for the pharmaceutical industry as well as medical services because secreted proteins could serve as biomarkers for drug-induced hepatotoxicity. The avoidance of fetal calf serum, a common supplement used for in vitro cell culture was shown to be a prerequisite for efficient secretome analysis because supplemented proteins severely hamper the identification of secreted proteins using mass-spectrometry (MS). The removal of high abundant proteins from the sample by immunodepletion, a common strategy for proteomic analysis of human plasma, was shown to increase the number of identified proteins to almost two-fold the number of identifications obtained without prior immunodepletion, paving the way for in-depth analysis of the secretome. A prefractionation step at the level of proteins and the combination of different MS-types and ionization methods tremendously enhanced the identifications but at the cost of analysis time as well as the fraction of secreted proteins. The elaborated workflow without prior prefractionation was applied to a standard monolayer culture and a promising in vitro cultivation technique in a 3-dimensional bioreactor mimicking the microenvironment of a real liver. With 109 and 160 proteins identified in monolayer and bioreactor cultures, respectively, the number of proteins likely to be secreted by hepatocytes into the extracellular space was much higher than described in the current literature. Furthermore, proteome analysis on extracellular proteins in the applied culture conditions confirmed the tissue-like behavior of primary hepatocytes in the three-dimensional cultivation. Differentially expressed proteins in the bioreactor culture after application of the reference drug diclofenac were detected by label-free quantification and most of the detected differences could be traced back to the underlying mechanism of toxicity proposed for this drug. In summary, the presented work provides a basis for further in-depth analysis of the primary human hepatocytes secretome for in vitro drug-testing and demonstrates the valuable contributions of proteomics to toxicological research.In der vorliegenden Arbeit wurde eine experimentelle Strategie zur Analyse des extrazellulären Proteoms („Sekretom“) von primären humanen Hepatozyten mittels „shotgun“ proteomics entwickelt. Die Analyse des Sekretoms von Hepatozyten ist von besonderer Bedeutung für die pharmazeutische Industrie sowie das Gesundheitswesen, da sekretierte Proteine als Biomarker für arzneimittelinduzierte Lebertoxizität verwendet werden können. Es wurde gezeigt, dass die Vermeidung von foetalem Kälberserum, einem gewöhnlich in der in vitro Zellkultur verwendeten Zusatz, eine Grundvorraussetzung für die effiziente Analyse der sekretierten Proteine darstellt, da die zugesetzten Proteine erheblich die Identifizierung sekretierter Proteine mittels Massenspektrometrie erschweren. Desweiteren erhöhte die Entfernung hoch abundanter Proteine aus den Proben mit Hilfe einer Immunodepletion, wie sie oft in der Proteomanalyse von menschlichem Serum Verwendung findet, die Anzahl an identifizierten Proteinen fast um das Doppelte und ebnete damit den Weg für eine detailliertere Analyse des Sekretoms. Weiterhin konnte gezeigt werden, dass eine Vorfraktionierung der Proteine sowie die Kombination verschiedener Massenspektrometertypen bzw. Ionisationsmethoden die Anzahl an Identifizierungen deutlich steigert, allerdings auf Kosten der benötigten Analysezeit und des Anteils an sekretierten Proteinen. Die erarbeitete analytische Strategie ohne vorherige Fraktionierung wurde sowohl auf Standardkultivierungen in Zellkulturflaschen, als auch auf eine vielversprechende Bioreaktortechnik angewendet, wobei letztere die dreidimensionale Umgebung in einer echten Leber nachahmt. 109 bzw. 160 sekretierte Proteine konnten in Standard- bzw. Bioreaktokulturen identifiziert werden, wesentlich mehr als es derzeitig in der Literatur für primäre humane Hepatozyten beschrieben wird. Desweiteren bestätigte die Analyse des extrazellulären Proteoms in den verwendeten Kultivierungstechniken das gewebeähnliche Verhalten der primären Leberzellen in der dreidimensionalen Bioreaktorkultur. Differentiell exprimierte Proteine in mit der Referenzsubstanz Diclofenac behandelten Bioreaktorkulturen wurden mittels labelfreier Quantifizierung bestimmt und konnten auf die zugrundeliegenden Toxizitätsmechanismen zurückgeführt werden, die für dieses Medikament bekannt sind. Zusammenfassend legt die vorliegende Arbeit den Grundstein für weitere umfassende Analysen des Sekretoms von Leberzellen für in vitro Wirkstofftests und veranschaulicht den wertvollen Beitrag der Proteomik für die toxikologische Forschung

Functional translatome proteomics reveal converging and dose-dependent regulation by mtorc1 and eif2α

Regulation of translation is essential during stress. However, the precise sets of proteins regulated by the key translational stress responses—the integrated stress response (ISR) and mTORC1—remain elusive. We developed multiplexed enhanced protein dynamics (mePROD) proteomics, adding signal amplification to dynamic-SILAC and multiplexing, to enable measuring acute changes in protein synthesis. Treating cells with ISR/mTORC1-modulating stressors, we showed extensive translatome modulation with ∼20% of proteins synthesized at highly reduced rates. Comparing translation-deficient sub-proteomes revealed an extensive overlap demonstrating that target specificity is achieved on protein level and not by pathway activation. Titrating cap-dependent translation inhibition confirmed that synthesis of individual proteins is controlled by intrinsic properties responding to global translation attenuation. This study reports a highly sensitive method to measure relative translation at the nascent chain level and provides insight into how the ISR and mTORC1, two key cellular pathways, regulate the translatome to guide cellular survival upon stress

Real-time in situ viability assessment in a 3D bioreactor with liver cells using resazurin assay.

Three-dimensional cultivation of human cells is promising especially for long-term maintenance of specific functions and mimicking the in vivo tissue environment. However, direct viability assessment is very difficult in such systems. Commonly applied indirect methods such as glucose consumption, albumin or urea production are greatly affected by culture conditions, stress and time of cultivation and do not reflect the real time viability of the cells. In this study we established a real-time in situ viability assay namely; resazurin assay, in a 3D hollow-fiber bioreactor using human liver cells. Resazurin assay is based on the conversion of resazurin to a fluorescent dye by cytoplasmatic and mitochondrial enzymes. We show that the resazurin reagent in concentrations used in this study is non-toxic and could be rapidly removed out of the system. Moreover, we observed that dead cells do not affect the results of the assay. We optimized the assay on HepG2 cells and tested it with primary human hepatocytes. Moreover, we maintained primary human hepatocytes in the 3D bioreactor system in serum-free conditions and also assessed viability before and after the exposure to amiodarone using the resazurin assay. We show that this approach is applicable during long-term cultivation of cells in bioreactors under different conditions and can moreover be applied to pharmacological studies, e.g. investigation of chronic drug effects in such 3D bioreactors

Growth factor receptor signaling inhibition prevents SARS-CoV-2 replication

SARS-CoV-2 infections are rapidly spreading around the globe. The rapid development of therapies is of major importance. However, our lack of understanding of the molecular processes and host cell signaling events underlying SARS-CoV-2 infection hinder therapy development. We employed a SARS-CoV-2 infection system in permissible human cells to study signaling changes by phospho-proteomics. We identified viral protein phosphorylation and defined phosphorylation-driven host cell signaling changes upon infection. Growth factor receptor (GFR) signaling and downstream pathways were activated. Drug-protein network analyses revealed GFR signaling as key pathway targetable by approved drugs. Inhibition of GFR downstream signaling by five compounds prevented SARS-CoV-2 replication in cells, assessed by cytopathic effect, viral dsRNA production, and viral RNA release into the supernatant. This study describes host cell signaling events upon SARS-CoV-2 infection and reveals GFR signaling as central pathway essential for SARS-CoV-2 replication. It provides with novel strategies for COVID-19 treatment

Profiling the murine SUMO proteome in response to cardiac ischemia and reperfusion injury

SUMOylation is a reversible posttranslational modification pathway catalyzing the conjugation of small ubiquitin-related modifier (SUMO) proteins to lysine residues of distinct target proteins. SUMOylation modifies a wide variety of cellular regulators thereby affecting a multitude of key processes in a highly dynamic manner. The SUMOylation pathway displays a hallmark in cellular stress-adaption, such as heat or redox stress. It has been proposed that enhanced cellular SUMOylation protects the brain during ischemia, however, little is known about the specific regulation of the SUMO system and the potential target proteins during cardiac ischemia and reperfusion injury (I/R). By applying left anterior descending (LAD) coronary artery ligation and reperfusion in mice, we detect dynamic changes in the overall cellular SUMOylation pattern correlating with decreased SUMO deconjugase activity during I/R injury. Further, unbiased system-wide quantitative SUMO-proteomics identified a sub-group of SUMO targets exhibiting significant alterations in response to cardiac I/R. Notably, transcription factors that control hypoxia- and angiogenesis-related gene expression programs, exhibit altered SUMOylation during ischemic stress adaptation. Moreover, several components of the ubiquitin proteasome system undergo dynamic changes in SUMO conjugation during cardiac I/R suggesting an involvement of SUMO signaling in protein quality control and proteostasis in the ischemic heart. Altogether, our study reveals regulated candidate SUMO target proteins in the mouse heart, which might be important in coping with hypoxic/proteotoxic stress during cardiac I/R injury

Biotransformation of diclofenac and effects on the metabolome of primary human hepatocytes upon repeated dose exposure.

In vitro repeated dose testing for the assessment of chronic drug-induced effects is a huge challenge in preclinical pharmaceutical drug development. Chronic toxicity results in discontinuation of therapy or post-marketing withdrawal of drugs despite in vivo preclinical screening. In case of hepatotoxicity, due to limited long term viability and functionality of primary hepatocytes, chronic hepatic effects are difficult to detect. In this study, we maintained primary human hepatocytes in a serum-free cultivation medium for more than 3 weeks and analyzed physiology, viability and drug metabolizing capacities of the hepatocytes. Moreover, we assessed acute (24 h) diclofenac toxicity in a range of (10-1000 muM) concentrations. The chronic (9 repeated doses) toxicity at one clinically relevant and another higher concentration (6.4 and 100 muM) was also tested. We investigated phase I and II metabolism of diclofenac upon repeated dose exposure and analyzed effects on the cellular exometabolome. Acute 24 h assessment revealed toxicity only for the highest tested concentration (1 mM). Upon repeated dose exposure, toxic effects were observed even at a low, clinically relevant concentration (6.4 muM). Biotransformation pathways were active for 3 weeks and diclofenac-acylglucuronide was detected as the predominant metabolite. Dose dependent diclofenac-induced effects on exometabolome, such as on the production of lactate and 3-hydroxybutyric acid as well as glucose and galactose metabolism, were observed upon nine repeated doses. Summarizing, we show that repeated dose testing on long-term functional cultures of primary human hepatocytes may be included for the assessment of long term toxic effects in preclinical screening and can potentially help replace/reduce in vivo animal testing