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12 Publications visible to you, out of a total of 12
Lysis buffer selection guidance for mass spectrometry-based global proteomics including studies on the intersection of signal transduction and metabolism (preprint)

Abstract (Expand)

Prerequisite for a successful proteomics experiment is a high-quality lysis of the sample of interest, resulting in a large number of identified proteins as well as a high coverage of protein sequences. … Therefore, the choice of suitable lysis conditions is crucial. Many buffers were previously employed in proteomics studies, yet a comprehensive comparison of lysate preparation conditions was so far missing. In this study, we compared the efficiency of four commonly used lysis buffers, containing the agents NP40, SDS, urea or GdnHCl, in four different types of biological samples (suspension and adherent cell lines, primary mouse cells and mouse liver tissue). After liquid chromatography-mass spectrometry (LC-MS) measurement and MaxQuant analysis, we compared chromatograms, intensities, number of identified proteins and the localization of the identified proteins. Overall, SDS emerged as the most reliable reagent, ensuring stable performance and reproducibility across diverse samples. Furthermore, our data advocated for a dual-sample lysis approach, including that the resulting pellet is lysed again after the initial lysis with a urea lysis buffer and subsequently both lysates are combined for a single LC-MS run to maximize the proteome coverage. However, none of the investigated lysis buffers proved to be superior in every category, indicating that the lysis buffer of choice depends on the proteins of interest and on the biological question. Further, we demonstrated with our systematic studies the establishment of conditions that allows to perform global proteomics and affinity purification-based interactome characterization from the same lysate. In sum our results provide guidance for the best-suited lysis buffer for mass spectrometry-based proteomics depending on the question of interest.

Authors: Barbara Helm, Pauline Hansen, Li Lai, Luisa Schwarzmüller, Simone M. Clas, Annika Richter, Max Ruwolt, Fan Liu, Dario Frey, Lorenza A. D’Alessandro, Wolf-Dieter Lehmann, Marcel Schilling, Dominic Helm, Dorothea Fiedler, Ursula Klingmüller

Date Published: 21st Feb 2024

Publication Type: Journal

Basal MET phosphorylation is an indicator of hepatocyte dysregulation in liver disease

Abstract (Expand)

Abstract Chronic liver diseases are worldwide on the rise. Due to the rapidly increasing incidence, in particular in Western countries, metabolic dysfunction-associated steatotic liver disease (MASLD) …otic liver disease (MASLD) is gaining importance as the disease can develop into hepatocellular carcinoma. Lipid accumulation in hepatocytes has been identified as the characteristic structural change in MASLD development, but molecular mechanisms responsible for disease progression remained unresolved. Here, we uncover in primary hepatocytes from a preclinical model fed with a Western diet (WD) an increased basal MET phosphorylation and a strong downregulation of the PI3K-AKT pathway. Dynamic pathway modeling of hepatocyte growth factor (HGF) signal transduction combined with global proteomics identifies that an elevated basal MET phosphorylation rate is the main driver of altered signaling leading to increased proliferation of WD-hepatocytes. Model-adaptation to patient-derived hepatocytes reveal patient-specific variability in basal MET phosphorylation, which correlates with patient outcome after liver surgery. Thus, dysregulated basal MET phosphorylation could be an indicator for the health status of the liver and thereby inform on the risk of a patient to suffer from liver failure after surgery.

Authors: Sebastian Burbano de Lara, Svenja Kemmer, Ina Biermayer, Svenja Feiler, Artyom Vlasov, Lorenza A D’Alessandro, Barbara Helm, Christina Mölders, Yannik Dieter, Ahmed Ghallab, Jan G Hengstler, Christiane Körner, Madlen Matz-Soja, Christina Götz, Georg Damm, Katrin Hoffmann, Daniel Seehofer, Thomas Berg, Marcel Schilling, Jens Timmer, Ursula Klingmüller

Date Published: 12th Jan 2024

Publication Type: Journal

Tumour cells can escape antiproliferative pressure by interferon-β through immunoediting of interferon receptor expression

Abstract (Expand)

Abstract Type I interferons (IFNs) play a central role not only in innate immunity against viral infection, but also in the antitumour response, e.g. through a direct impact on cell proliferation. …act on cell proliferation. Particularly for cancer arising in the context of chronic inflammation, constant exposure to IFNs may constitute a strong selective pressure during tumour evolution. Expansion of neoplastic subclones resistant to the antiproliferative effects of IFNs may contribute to immunoediting of tumours, leading to more aggressive disease. Experimental evidence for this development of IFN-insensitivity has been scarce and its molecular mechanism is unclear. In this study we demonstrate that six weeks exposure of cells to IFN-β in vitro reduces their sensitivity to its antiproliferative effects, and that this phenotype was stable for up to four weeks. Furthermore, we observed substantial differences in cellular sensitivity to growth inhibition by IFN-β in a panel of ten different liver cancer cell lines, most prominently in a pair of highly dedifferentiated cell lines, and least in cells from well-differentiated tumours. In both, long-term IFN selection and in dedifferentiated tumour cell lines, we found IFNAR2 expression to be substantially reduced, suggesting the receptor complex to be a sensitive target amenable to immunoediting. Beyond new insights into possible molecular processes in tumour evolution, these findings might prove valuable for the development of biomarkers allowing to stratify tumours for their sensitivity to IFN treatment in the context of patient tailored therapies.

Authors: Felix Hiebinger, Aiste Kudulyte, Huanting Chi, Sebastian Burbano De Lara, Doroteja Ilic, Barbara Helm, Hendrik Welsch, Viet Loan Dao Thi, Ursula Klingmüller, Marco Binder

Date Published: 1st Dec 2023

Publication Type: Journal

Tumour cells can escape antiproliferative pressure by interferon-β through immunoediting of interferon receptor expression (preprint)

Abstract (Expand)

Type I interferons (IFNs) play a central role not only in innate immunity against viral infection, but also in the antitumour response. Apart from indirect immune-modulatory and anti-angiogenic effects, … they have direct impact on cell proliferation. Particularly for cancer arising in the context of chronic inflammation, constant exposure to IFNs may constitute a strong selective pressure during tumour evolution. Expansion of neoplastic subclones or -populations that developed resistance to the antiproliferative effects of IFNs might constitute an important contribution to immunoediting of the cancer cells leading to more aggressive and metastasising disease. Experimental evidence for this development of IFN-insensitivity has been scarce and its molecular mechanism is unclear. In this study we demonstrate that prolonged (six weeks) exposure of cells to IFN-β in vitro reduces their sensitivity to its antiproliferative effects, and that this phenotype was stable for up to four weeks. Furthermore, we observed substantial differences in cellular sensitivity to growth inhibition by IFN-β in a panel of ten different liver cancer cell lines of varying malignity. IFN-resistance was most prominent in a pair of highly dedifferentiated cell lines, and least in cells from well-differentiated tumours, fostering the hypothesis of IFN-driven immunoediting in advanced cancers. In both settings, long-term IFN selection in vitro as well as in dedifferentiated tumour cell lines, we found IFNAR expression to be substantially reduced, suggesting the receptor complex, in particular IFNAR2, to be a sensitive target amenable to immunoediting. Beyond new insights into possible molecular processes in tumour evolution, these findings might prove valuable for the development of biomarkers allowing to stratify tumours for their sensitivity to IFN treatment in the context of patient tailored therapies.

Authors: Felix Hiebinger, Aiste Kudulyte, Huanting Chi, Sebastian Burbano De Lara, Barbara Helm, Hendrik Welsch, Viet Loan Dao Thi, Ursula Klingmüller, Marco Binder

Date Published: 24th Aug 2023

Publication Type: Journal

Basal MET Phosphorylation is an Indicator of Hepatocyte Dysregulation in Liver Disease (preprint)

Abstract (Expand)

Chronic liver diseases are worldwide on the rise. Due to the rapidly increasing incidence, in particular in Western countries, Non-alcoholic fatty liver disease (NAFLD) is gaining importance as the … disease can develop into hepatocellular carcinoma. Lipid accumulation in hepatocytes has been identified as the characteristic structural change in NAFLD development, but molecular mechanisms responsible for disease progression remained unresolved. Here, we uncover in primary hepatocytes from a preclinical model fed with a Western diet (WD) a strong downregulation of the PI3K-AKT pathway and an upregulation of the MAPK pathway. Dynamic pathway modeling of hepatocyte growth factor (HGF) signal transduction combined with global proteomics identifies that an elevated basal MET phosphorylation rate is the main driver of altered signaling leading to increased proliferation of WD-hepatocytes. Model-adaptation to patient-derived hepatocytes reveal patient-specific variability in basal MET phosphorylation, which correlates with patient outcome after liver surgery. Thus, dysregulated basal MET phosphorylation could be an indicator for the health status of the liver and thereby inform on the risk of a patient to suffer from liver failure after surgery.

Authors: Sebastian Burbano De Lara, Svenja Kemmer, Ina Biermayer, Svenja Feiler, Artyom Vlasov, Lorenza D'Alessandro, Barbara Helm, Yannik Dieter, Ahmed Ghallab, Jan Hengstler, Professor Dr. med. Katrin Hoffmann, Marcel Schilling, Jens Timmer, Ursula Klingmüller

Date Published: 4th Jul 2023

Publication Type: Journal

Tolerance of repeated toxic injuries of murine livers is associated with steatosis and inflammation

Abstract (Expand)

Abstract The human liver has a remarkable capacity to regenerate and thus compensate over decades for fibrosis caused by toxic chemicals, drugs, alcohol, or malnutrition. To date, no protective mechanisms …rition. To date, no protective mechanisms have been identified that help the liver tolerate these repeated injuries. In this study, we revealed dysregulation of lipid metabolism and mild inflammation as protective mechanisms by studying longitudinal multi-omic measurements of liver fibrosis induced by repeated CCl 4 injections in mice ( n  = 45). Based on comprehensive proteomics, transcriptomics, blood- and tissue-level profiling, we uncovered three phases of early disease development—initiation, progression, and tolerance. Using novel multi-omic network analysis, we identified multi-level mechanisms that are significantly dysregulated in the injury-tolerant response. Public data analysis shows that these profiles are altered in human liver diseases, including fibrosis and early cirrhosis stages. Our findings mark the beginning of the tolerance phase as the critical switching point in liver response to repetitive toxic doses. After fostering extracellular matrix accumulation as an acute response, we observe a deposition of tiny lipid droplets in hepatocytes only in the Tolerant phase. Our comprehensive study shows that lipid metabolism and mild inflammation may serve as biomarkers and are putative functional requirements to resist further disease progression.

Authors: Seddik Hammad, Christoph Ogris, Amnah Othman, Pia Erdoesi, Wolfgang Schmidt-Heck, Ina Biermayer, Barbara Helm, Yan Gao, Weronika Piorońska, Christian H. Holland, Lorenza A. D’Alessandro, Carolina de la Torre, Carsten Sticht, Sherin Al Aoua, Fabian J. Theis, Heike Bantel, Matthias P. Ebert, Ursula Klingmüller, Jan G. Hengstler, Steven Dooley, Nikola S. Mueller

Date Published: 1st Jul 2023

Publication Type: Journal

Hepatocytes reprogram liver macrophages involving control of TGF-β activation, influencing liver regeneration and injury

Abstract (Expand)

Background: Macrophages play an important role in maintaining liver homeostasis and regeneration. However, it is not clear to what extent the different macrophage populations of the liver differ in … terms of their activation state and which other liver cell populations may play a role in regulating the same. Methods: Reverse transcription PCR, flow cytometry, transcriptome, proteome, secretome, single cell analysis, and immunohistochemical methods were used to study changes in gene expression as well as the activation state of macrophages in vitro and in vivo under homeostatic conditions and after partial hepatectomy. Results: We show that F4/80+/CD11bhi/CD14hi macrophages of the liver are recruited in a C-C motif chemokine receptor (CCR2)–dependent manner and exhibit an activation state that differs substantially from that of the other liver macrophage populations, which can be distinguished on the basis of CD11b and CD14 expressions. Thereby, primary hepatocytes are capable of creating an environment in vitro that elicits the same specific activation state in bone marrow–derived macrophages as observed in F4/80+/CD11bhi/CD14hi liver macrophages in vivo. Subsequent analyses, including studies in mice with a myeloid cell–specific deletion of the TGF-β type II receptor, suggest that the availability of activated TGF-β and its downregulation by a hepatocyte-conditioned milieu are critical. Reduction of TGF-βRII-mediated signal transduction in myeloid cells leads to upregulation of IL-6, IL-10, and SIGLEC1 expression, a hallmark of the activation state of F4/80+/CD11bhi/CD14hi macrophages, and enhances liver regeneration. Conclusions: The availability of activated TGF-β determines the activation state of specific macrophage populations in the liver, and the observed rapid transient activation of TGF-β may represent an important regulatory mechanism in the early phase of liver regeneration in this context.

Authors: Stephanie D. Wolf, Christian Ehlting, Sophia Müller-Dott, Gereon Poschmann, Patrick Petzsch, Tobias Lautwein, Sai Wang, Barbara Helm, Marcel Schilling, Julio Saez-Rodriguez, Mihael Vucur, Kai Stühler, Karl Köhrer, Frank Tacke, Steven Dooley, Ursula Klingmüller, Tom Luedde, Johannes G. Bode

Date Published: 2023

Publication Type: Journal

Erythropoietin-driven dynamic proteome adaptations during erythropoiesis prevent iron overload in the developing embryo.

Abstract (Expand)

Erythropoietin (Epo) ensures survival and proliferation of colony-forming unit erythroid (CFU-E) progenitor cells and their differentiation to hemoglobin-containing mature erythrocytes. A lack of … Epo-induced responses causes embryonic lethality, but mechanisms regulating the dynamic communication of cellular alterations to the organismal level remain unresolved. By time-resolved transcriptomics and proteomics, we show that Epo induces in CFU-E cells a gradual transition from proliferation signature proteins to proteins indicative for differentiation, including heme-synthesis enzymes. In the absence of the Epo receptor (EpoR) in embryos, we observe a lack of hemoglobin in CFU-E cells and massive iron overload of the fetal liver pointing to a miscommunication between liver and placenta. A reduction of iron-sulfur cluster-containing proteins involved in oxidative phosphorylation in these embryos leads to a metabolic shift toward glycolysis. This link connecting erythropoiesis with the regulation of iron homeostasis and metabolic reprogramming suggests that balancing these interactions is crucial for protection from iron intoxication and for survival.

Authors: S. Chakraborty, G. Andrieux, P. Kastl, L. Adlung, S. Altamura, M. E. Boehm, L. E. Schwarzmuller, Y. Abdullah, M. C. Wagner, B. Helm, H. J. Grone, W. D. Lehmann, M. Boerries, H. Busch, M. U. Muckenthaler, M. Schilling, U. Klingmuller

Date Published: 20th Sep 2022

Publication Type: Journal

Deciphering signal transduction networks in the liver by mechanistic mathematical modelling

Abstract (Expand)

In health and disease, liver cells are continuously exposed to cytokines and growth factors. While individual signal transduction pathways induced by these factors were studied in great detail, the … cellular responses induced by repeated or combined stimulations are complex and less understood. Growth factor receptors on the cell surface of hepatocytes were shown to be regulated by receptor interactions, receptor trafficking and feedback regulation. Here, we exemplify how mechanistic mathematical modelling based on quantitative data can be employed to disentangle these interactions at the molecular level. Crucial is the analysis at a mechanistic level based on quantitative longitudinal data within a mathematical framework. In such multi-layered information, step-wise mathematical modelling using submodules is of advantage, which is fostered by sharing of standardized experimental data and mathematical models. Integration of signal transduction with metabolic regulation in the liver and mechanistic links to translational approaches promise to provide predictive tools for biology and personalized medicine.

Authors: Lorenza A. D’Alessandro, Ursula Klingmüller, Marcel Schilling

Date Published: 30th Jun 2022

Publication Type: Journal

MSPypeline: a python package for streamlined data analysis of mass spectrometry-based proteomics

Abstract (Expand)

Abstract Summary Mass spectrometry-based proteomics is increasingly employed in biology and medicine. To generate reliable information from large datasets and ensure comparability of results, it is …rge datasets and ensure comparability of results, it is crucial to implement and standardize the quality control of the raw data, the data processing steps and the statistical analyses. MSPypeline provides a platform for importing MaxQuant output tables, generating quality control reports, data preprocessing including normalization and performing exploratory analyses by statistical inference plots. These standardized steps assess data quality, provide customizable figures and enable the identification of differentially expressed proteins to reach biologically relevant conclusions. Availability and implementation The source code is available under the MIT license at https://github.com/siheming/mspypeline with documentation at https://mspypeline.readthedocs.io. Benchmark mass spectrometry data are available on ProteomeXchange (PXD025792). Supplementary information Supplementary data are available at Bioinformatics Advances online.

Authors: Simon Heming, Pauline Hansen, Artyom Vlasov, Florian Schwörer, Stephen Schaumann, Paulina Frolovaitė, Wolf-Dieter Lehmann, Jens Timmer, Marcel Schilling, Barbara Helm, Ursula Klingmüller

Date Published: 2022

Publication Type: Journal

Cell-to-cell variability in JAK2/STAT5 pathway components and cytoplasmic volumes defines survival threshold in erythroid progenitor cells.

Abstract (Expand)

Survival or apoptosis is a binary decision in individual cells. However, at the cell-population level, a graded increase in survival of colony-forming unit-erythroid (CFU-E) cells is observed upon … stimulation with erythropoietin (Epo). To identify components of Janus kinase 2/signal transducer and activator of transcription 5 (JAK2/STAT5) signal transduction that contribute to the graded population response, we extended a cell-population-level model calibrated with experimental data to study the behavior in single cells. The single-cell model shows that the high cell-to-cell variability in nuclear phosphorylated STAT5 is caused by variability in the amount of Epo receptor (EpoR):JAK2 complexes and of SHP1, as well as the extent of nuclear import because of the large variance in the cytoplasmic volume of CFU-E cells. 24-118 pSTAT5 molecules in the nucleus for 120 min are sufficient to ensure cell survival. Thus, variability in membrane-associated processes is sufficient to convert a switch-like behavior at the single-cell level to a graded population-level response.

Authors: L. Adlung, P. Stapor, C. Tonsing, L. Schmiester, L. E. Schwarzmuller, L. Postawa, D. Wang, J. Timmer, U. Klingmuller, J. Hasenauer, M. Schilling

Date Published: 10th Aug 2021

Publication Type: Journal

Disentangling molecular mechanisms regulating sensitization of interferon alpha signal transduction.

Abstract (Expand)

Tightly interlinked feedback regulators control the dynamics of intracellular responses elicited by the activation of signal transduction pathways. Interferon alpha (IFNalpha) orchestrates antiviral … responses in hepatocytes, yet mechanisms that define pathway sensitization in response to prestimulation with different IFNalpha doses remained unresolved. We establish, based on quantitative measurements obtained for the hepatoma cell line Huh7.5, an ordinary differential equation model for IFNalpha signal transduction that comprises the feedback regulators STAT1, STAT2, IRF9, USP18, SOCS1, SOCS3, and IRF2. The model-based analysis shows that, mediated by the signaling proteins STAT2 and IRF9, prestimulation with a low IFNalpha dose hypersensitizes the pathway. In contrast, prestimulation with a high dose of IFNalpha leads to a dose-dependent desensitization, mediated by the negative regulators USP18 and SOCS1 that act at the receptor. The analysis of basal protein abundance in primary human hepatocytes reveals high heterogeneity in patient-specific amounts of STAT1, STAT2, IRF9, and USP18. The mathematical modeling approach shows that the basal amount of USP18 determines patient-specific pathway desensitization, while the abundance of STAT2 predicts the patient-specific IFNalpha signal response.

Authors: F. Kok, M. Rosenblatt, M. Teusel, T. Nizharadze, V. Goncalves Magalhaes, C. Dachert, T. Maiwald, A. Vlasov, M. Wasch, S. Tyufekchieva, K. Hoffmann, G. Damm, D. Seehofer, T. Boettler, M. Binder, J. Timmer, M. Schilling, U. Klingmuller

Date Published: 23rd Jul 2020

Publication Type: Journal

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