Publications by authors named "Anja Stefanski"

21 Publications

  • Page 1 of 1

The signal transducer CD24 suppresses the germ cell program and promotes an ectodermal rather than mesodermal cell fate in embryonal carcinomas.

Mol Oncol 2021 Jul 22. Epub 2021 Jul 22.

Department of Urology, Urological Research Laboratory, Translational UroOncology, Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, Germany.

Testicular germ cell tumors (GCTs) are stratified into seminomas and nonseminomas. Seminomas share many histological and molecular features with primordial germ cells, whereas the nonseminoma stem cell population-embryonal carcinoma (EC)-is pluripotent and thus able to differentiate into cells of all three germ layers (teratomas). Furthermore, ECs are capable of differentiating into extra-embryonic lineages (yolk sac tumors, choriocarcinomas). In this study, we deciphered the molecular and (epi)genetic mechanisms regulating expression of CD24, a highly glycosylated signaling molecule upregulated in many cancers. CD24 is overexpressed in ECs compared with other GCT entities and can be associated with an undifferentiated pluripotent cell fate. We demonstrate that CD24 can be transactivated by the pluripotency factor SOX2, which binds in proximity to the CD24 promoter. In GCTs, CD24 expression is controlled by epigenetic mechanisms, that is, histone acetylation, since CD24 can be induced by the application histone deacetylase inhibitors. Vice versa, CD24 expression is downregulated upon inhibition of histone methyltransferases, E3 ubiquitin ligases, or bromodomain (BRD) proteins. Additionally, three-dimensional (3D) co-cultivation of EC cells with microenvironmental cells, such as fibroblasts, and endothelial or immune cells, reduced CD24 expression, suggesting that crosstalk with the somatic microenvironment influences CD24 expression. In a CRISPR/Cas9 deficiency model, we demonstrate that CD24 fulfills a bivalent role in differentiation via regulation of homeobox, and phospho- and glycoproteins; that is, it is involved in suppressing the germ cell/spermatogenesis program and mesodermal/endodermal differentiation, while poising the cells for ectodermal differentiation. Finally, blocking CD24 by a monoclonal antibody enhanced sensitivity toward cisplatin in EC cells, including cisplatin-resistant subclones, highlighting CD24 as a putative target in combination with cisplatin.
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http://dx.doi.org/10.1002/1878-0261.13066DOI Listing
July 2021

FIP200 controls the TBK1 activation threshold at SQSTM1/p62-positive condensates.

Sci Rep 2021 Jul 5;11(1):13863. Epub 2021 Jul 5.

Institute of Molecular Medicine I, Medical Faculty, University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, Universitätsstr. 1, Building 22.03, 40225, Düsseldorf, Germany.

The protein kinase TBK1 is a central regulator of innate immune responses and autophagy, and ablation of either function has been linked to neuroinflammatory or degenerative diseases. Autophagy is an intracellular process that recycles old or damaged proteins and organelles. In recent years, the TBK1-dependent regulation of autophagy pathways has been characterized. However, the autophagy-dependent regulation of TBK1 activity awaits further clarification. Here, we observed that TBK1 is recruited to SQSTM1/p62-containing aggregates via the selective autophagy receptor TAX1BP1. In these aggregates, TBK1 phosphorylates SQSTM1/p62 at serine 403 and thus presumably regulates the efficient engulfment and clearance of these structures. We found that TBK1 activation is strongly increased if FIP200, a component of the autophagy-inducing ULK1 complex, is not present or cannot bind to TAX1BP1. Given our collective findings, we hypothesize that FIP200 ensures the inducible activation of TBK1 at SQSTM1/p62 condensates.
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http://dx.doi.org/10.1038/s41598-021-92408-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8257712PMC
July 2021

Phosphorylation of GAPVD1 Is Regulated by the PER Complex and Linked to GAPVD1 Degradation.

Int J Mol Sci 2021 Apr 6;22(7). Epub 2021 Apr 6.

Institute of Clinical Chemistry and Laboratory Diagnostics, Medical Faculty, University of Düsseldorf, 40225 Düsseldorf, Germany.

Repressor protein period (PER) complexes play a central role in the molecular oscillator mechanism of the mammalian circadian clock. While the main role of nuclear PER complexes is transcriptional repression, much less is known about the functions of cytoplasmic PER complexes. We found with a biochemical screen for PER2-interacting proteins that the small GTPase regulator GTPase-activating protein and VPS9 domain-containing protein 1 (GAPVD1), which has been identified previously as a component of cytoplasmic PER complexes in mice, is also a bona fide component of human PER complexes. We show that in situ GAPVD1 is closely associated with casein kinase 1 delta (CSNK1D), a kinase that regulates PER2 levels through a phosphoswitch mechanism, and that CSNK1D regulates the phosphorylation of GAPVD1. Moreover, phosphorylation determines the kinetics of GAPVD1 degradation and is controlled by PER2 and a C-terminal autoinhibitory domain in CSNK1D, indicating that the regulation of GAPVD1 phosphorylation is a novel function of cytoplasmic PER complexes and might be part of the oscillator mechanism or an output function of the circadian clock.
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http://dx.doi.org/10.3390/ijms22073787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8038846PMC
April 2021

Subcellular Localization and Mitotic Interactome Analyses Identify SIRT4 as a Centrosomally Localized and Microtubule Associated Protein.

Cells 2020 08 24;9(9). Epub 2020 Aug 24.

Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany.

The stress-inducible and senescence-associated tumor suppressor SIRT4, a member of the family of mitochondrial sirtuins (SIRT3, SIRT4, and SIRT5), regulates bioenergetics and metabolism via NAD-dependent enzymatic activities. Next to the known mitochondrial location, we found that a fraction of endogenous or ectopically expressed SIRT4, but not SIRT3, is present in the cytosol and predominantly localizes to centrosomes. Confocal spinning disk microscopy revealed that SIRT4 is found during the cell cycle dynamically at centrosomes with an intensity peak in G and early mitosis. Moreover, SIRT4 precipitates with microtubules and interacts with structural (α,β-tubulin, γ-tubulin, TUBGCP2, TUBGCP3) and regulatory (HDAC6) microtubule components as detected by co-immunoprecipitation and mass spectrometric analyses of the mitotic SIRT4 interactome. Overexpression of SIRT4 resulted in a pronounced decrease of acetylated α-tubulin (K40) associated with altered microtubule dynamics in mitotic cells. SIRT4 or the N-terminally truncated variant SIRT4(ΔN28), which is unable to translocate into mitochondria, delayed mitotic progression and reduced cell proliferation. This study extends the functional roles of SIRT4 beyond mitochondrial metabolism and provides the first evidence that SIRT4 acts as a novel centrosomal/microtubule-associated protein in the regulation of cell cycle progression. Thus, stress-induced SIRT4 may exert its role as tumor suppressor through mitochondrial as well as extramitochondrial functions, the latter associated with its localization at the mitotic spindle apparatus.
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http://dx.doi.org/10.3390/cells9091950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564595PMC
August 2020

A Multiplex Assay for the Stratification of Patients with Primary Central Nervous System Lymphoma Using Targeted Mass Spectrometry.

Cancers (Basel) 2020 Jun 29;12(7). Epub 2020 Jun 29.

Institute of Molecular Medicine, Medical Faculty, Heinrich-Heine-University, 40225 Düsseldorf, Germany.

Primary central nervous system lymphomas (PCNSL) account for approximately 2% to 3% of all primary brain tumors. Until now, neuropathological tumor tissue analysis, most frequently gained by stereotactic biopsy, is still the diagnostic gold standard. Here, we rigorously analyzed two independent patient cohorts comprising the clinical entities PCNSL ( = 47), secondary central nervous system lymphomas (SCNSL; = 13), multiple sclerosis (MS, = 23), glioma ( = 10), other tumors ( = 17) and tumor-free controls ( = 21) by proteomic approaches. In total, we identified more than 1220 proteins in the cerebrospinal fluid (CSF) and validated eight candidate biomarkers by a peptide-centric approach in an independent patient cohort ( = 63). Thus, we obtained excellent diagnostic accuracy for the stratification between PCNSL, MS and glioma patients as well as tumor-free controls for three peptides originating from the three proteins VSIG4, GPNMB4 and APOC2. The combination of all three biomarker candidates resulted in diagnostic accuracy with an area under the curve (AUC) of 0.901 (PCNSL vs. MS), AUC of 0.953 (PCNSL vs. glioma) and AUC 0.850 (PCNSL vs. tumor-free control). In summary, the determination of VSIG4, GPNMB4 and APOC2 in CSF as novel biomarkers for supporting the diagnosis of PCNSL is suggested.
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http://dx.doi.org/10.3390/cancers12071732DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7407338PMC
June 2020

Novel FMRP interaction networks linked to cellular stress.

FEBS J 2021 02 23;288(3):837-860. Epub 2020 Jun 23.

Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich Heine University, Düsseldorf, Germany.

Silencing of the fragile X mental retardation 1 (FMR1) gene and consequently lack of synthesis of FMR protein (FMRP) are associated with fragile X syndrome, which is one of the most prevalent inherited intellectual disabilities, with additional roles in increased viral infection, liver disease, and reduced cancer risk. FMRP plays critical roles in chromatin dynamics, RNA binding, mRNA transport, and mRNA translation. However, the underlying molecular mechanisms, including the (sub)cellular FMRP protein networks, remain elusive. Here, we employed affinity pull-down and quantitative LC-MS/MS analyses with FMRP. We identified known and novel candidate FMRP-binding proteins as well as protein complexes. FMRP interacted with 180 proteins, 28 of which interacted with its N terminus. Interaction with the C terminus of FMRP was observed for 102 proteins, and 48 proteins interacted with both termini. This FMRP interactome comprises known FMRP-binding proteins, including the ribosomal proteins FXR1P, NUFIP2, Caprin-1, and numerous novel FMRP candidate interacting proteins that localize to different subcellular compartments, including CARF, LARP1, LEO1, NOG2, G3BP1, NONO, NPM1, SKIP, SND1, SQSTM1, and TRIM28. Our data considerably expand the protein and RNA interaction networks of FMRP, which thereby suggest that, in addition to its known functions, FMRP participates in transcription, RNA metabolism, ribonucleoprotein stress granule formation, translation, DNA damage response, chromatin dynamics, cell cycle regulation, ribosome biogenesis, miRNA biogenesis, and mitochondrial organization. Thus, FMRP seems associated with multiple cellular processes both under normal and cell stress conditions in neuronal as well as non-neuronal cell types, as exemplified by its role in the formation of stress granules.
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http://dx.doi.org/10.1111/febs.15443DOI Listing
February 2021

The Autophagy-Initiating Kinase ULK1 Controls RIPK1-Mediated Cell Death.

Cell Rep 2020 04;31(3):107547

Institute of Molecular Medicine I, Medical Faculty, Heinrich Heine University, Düsseldorf, Germany. Electronic address:

Autophagy, apoptosis, and necroptosis are stress responses governing the ultimate fate of a cell. However, the crosstalk between these cellular stress responses is not entirely understood. Especially, it is not clear whether the autophagy-initiating kinase ULK1 and the cell-death-regulating kinase RIPK1 are involved in this potential crosstalk. Here, we identify RIPK1 as a substrate of ULK1. ULK1-dependent phosphorylation of RIPK1 reduces complex IIb/necrosome assembly and tumor necrosis factor (TNF)-induced cell death, whereas deprivation of ULK1 enhances TNF-induced cell death. We observe that ULK1 phosphorylates multiple sites of RIPK1, but it appears that especially phosphorylation of S357 within the intermediate domain of RIPK1 mediates this cell-death-inhibiting effect. We propose that ULK1 is a regulator of RIPK1-mediated cell death.
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http://dx.doi.org/10.1016/j.celrep.2020.107547DOI Listing
April 2020

Rapid Single-Step Affinity Purification of HA-Tagged Plant Mitochondria.

Plant Physiol 2020 02 9;182(2):692-706. Epub 2019 Dec 9.

Institute of Plant Biochemistry, Cluster of Excellence on Plant Science (CEPLAS), Heinrich Heine University, Universitätsstrasse 1, 40225 Düsseldorf, Germany

Photosynthesis in plant cells would not be possible without the supportive role of mitochondria. However, isolating mitochondria from plant cells for physiological and biochemical analyses is a lengthy and tedious process. Established isolation protocols require multiple centrifugation steps and substantial amounts of starting material. To overcome these limitations, we tagged mitochondria in Arabidopsis () with a triple hemagglutinin tag for rapid purification via a single affinity-purification step. This protocol yields a substantial quantity of highly pure mitochondria from 1 g of Arabidopsis seedlings. The purified mitochondria were suitable for enzyme activity analyses and yielded sufficient amounts of proteins for deep proteomic profiling. We applied this method for the proteomic analysis of the Arabidopsis mutant deficient in the mitochondrial Glu transporter À BOUT DE SOUFFLE (BOU) and identified 27 differentially expressed mitochondrial proteins compared with tagged Col-0 controls. Our work sets the stage for the development of advanced mitochondria isolation protocols for distinct cell types.
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http://dx.doi.org/10.1104/pp.19.00732DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997695PMC
February 2020

Native aortic valve derived extracellular matrix hydrogel for three dimensional culture analyses with improved biomimetic properties.

Biomed Mater 2019 04 5;14(3):035014. Epub 2019 Apr 5.

Department of Cardiovascular Surgery, Medical Faculty, Heinrich Heine University, Moorenstr. 5, D-40225, Duesseldorf, Germany.

Introduction: Calcific aortic valve disease (CAVD) is the most common acquired heart valve disease with complex underlying pathomechanisms that are yet not fully understood. Three-dimensional (3D) cell culture models as opposed to conventional two-dimensional (2D) techniques may reveal new aspects of CAVD and serve as a transitional platform between conventional 2D cell culture and in vivo experiments.

Methods: Here we report on fabrication and characterization of a novel 3D hydrogel derived from cell-free native aortic valves. A detailed analysis containing protein composition, rheological behavior, cytotoxic and proliferative effects as well as results of 3D cell culture experiments are presented. Moreover, this aortic valve derived hydrogel (AVdH) is compared to commercially available biological extracellular matrix (ECM) components to evaluate and classify AVdH with respect to other currently used ECM solutions, i.e. Collagen type I and Matrigel.

Results: On the biochemical level, a complex composition of native proteins was detected. Using different techniques, including mass spectrometry with Gene Ontology network and enrichment analysis, different fundamental biological functions of AVdH were identified, including peptidase-, peptidase inhibitor-, growth- and binding activity. No cytotoxic effects were detected and AVdH showed positive effects on cell growth and proliferation in vitro when compared to Collagen type I and Matrigel.

Conclusion: These results suggest AVdH as an organotypic ECM supporting sophisticated 3D cell culture model studies, while mimicking the native environment of the aortic valve to a greater level for enhanced in vitro analyses.
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http://dx.doi.org/10.1088/1748-605X/ab0791DOI Listing
April 2019

Secretome analysis of human bone marrow derived mesenchymal stromal cells.

Biochim Biophys Acta Proteins Proteom 2019 04 1;1867(4):434-441. Epub 2019 Feb 1.

Molecular Proteomics Laboratory, Biomedical Research Center (BMFZ), Heinrich-Heine-University, Düsseldorf, Germany. Electronic address:

As an essential cellular component of the bone marrow (BM) microenvironment mesenchymal stromal cells (MSC) play a pivotal role for the physiological regulation of hematopoiesis, in particular through the secretion of cytokines and chemokines. Mass spectrometry (MS) facilitates the identification and quantification of a large amount of secreted proteins (secretome), but can be hampered by the false-positive identification of contaminating proteins released from dead cells or derived from cell medium. To reduce the likelihood of contaminations we applied an approach combining secretome and proteome analysis to characterize the physiological secretome of BM derived human MSC. Our analysis revealed a secretome consisting of 315 proteins. Pathway analyses of these proteins revealed a high abundance of proteins related to cell growth and/or maintenance, signal transduction and cell communication thereby representing key biological functions of BM derived MSC on protein level. Within the MSC secretome we identified several cytokines and growth factors such as VEGFC, TGF-β1, TGF-β2 and GDF6 which are known to be involved in the physiological regulation of hematopoiesis. By comparing the peptide patterns of secretomes and cell lysates 17 proteins were identified as candidates for proteolytic processing. Taken together, our combined MS work-flow reduced the likelihood of contaminations and enabled us to carve out a specific overview about the composition of the secretome from human BM derived MSC. This methodological approach and the specific secretome signature of BM derived MSC may serve as basis for future comparative analyses of the interplay of MSC and HSPC in patients with hematological malignancies.
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http://dx.doi.org/10.1016/j.bbapap.2019.01.013DOI Listing
April 2019

A proteomics approach for the identification of species-specific immunogenic proteins in the Mycobacterium abscessus complex.

Microbes Infect 2019 Apr - May;21(3-4):154-162. Epub 2018 Nov 13.

Institute of Pharmaceutical Biology and Biotechnology, Heinrich Heine University, Universitaetsstr. 1, 40225, Duesseldorf, Germany.

The Mycobacterium abscessus complex can cause fatal pulmonary disease, especially in cystic fibrosis patients. Diagnosing M. abscessus complex pulmonary disease is challenging. Immunologic assays specific for M. abscessus are not available. In this study seven clinical M. abscessus complex strains and the M. abscessus reference strain ATCC19977 were used to find species-specific proteins for their use in immune assays. Six strains showed rough and smooth colony morphotypes simultaneously, two strains only showed rough mophotypes, resulting in 14 separate isolates. Clinical isolates were submitted to whole genome sequencing. Proteomic analysis was performed on bacterial lysates and culture supernatant of all 14 isolates. Species-specificity for M. abscessus complex was determined by a BLAST search for proteins present in all supernatants. Species-specific proteins underwent in silico B- and T-cell epitope prediction. All clinical strains were found to be M. abscessus ssp. abscessus. Mutations in MAB_4099c as a likely genetic basis of the rough morphotype were found in six out of seven clinical isolates. 79 proteins were present in every supernatant, of which 12 are exclusively encoded by all members of M. abscessus complex plus Mycobacterium immunogenum. In silico analyses predicted B- and T-cell epitopes in all of these 12 species-specific proteins.
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http://dx.doi.org/10.1016/j.micinf.2018.10.006DOI Listing
February 2020

SIRT4 interacts with OPA1 and regulates mitochondrial quality control and mitophagy.

Aging (Albany NY) 2017 10;9(10):2163-2189

Institut für Biochemie und Molekularbiologie II, Medizinische Fakultät der Heinrich-Heine-Universität, Düsseldorf, Germany.

The stress-responsive mitochondrial sirtuin SIRT4 controls cellular energy metabolism in a NAD-dependent manner and is implicated in cellular senescence and aging. Here we reveal a novel function of SIRT4 in mitochondrial morphology/quality control and regulation of mitophagy. We report that moderate overexpression of SIRT4, but not its enzymatically inactive mutant H161Y, sensitized cells to mitochondrial stress. CCCP-triggered dissipation of the mitochondrial membrane potential resulted in increased mitochondrial ROS levels and autophagic flux, but surprisingly led to increased mitochondrial mass and decreased Parkin-regulated mitophagy. The anti-respiratory effect of elevated SIRT4 was accompanied by increased levels of the inner-membrane bound long form of the GTPase OPA1 (L-OPA1) that promotes mitochondrial fusion and thereby counteracts fission and mitophagy. Consistent with this, upregulation of endogenous SIRT4 expression in fibroblast models of senescence either by transfection with miR-15b inhibitors or by ionizing radiation increased L-OPA1 levels and mitochondrial fusion in a SIRT4-dependent manner. We further demonstrate that SIRT4 interacts physically with OPA1 in co-immunoprecipitation experiments. Overall, we propose that the SIRT4-OPA1 axis is causally linked to mitochondrial dysfunction and altered mitochondrial dynamics that translates into aging-associated decreased mitophagy based on an unbalanced mitochondrial fusion/fission cycle.
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http://dx.doi.org/10.18632/aging.101307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5680561PMC
October 2017

Surveying the Oligomeric State of Arabidopsis thaliana Chloroplasts.

Mol Plant 2017 01 26;10(1):197-211. Epub 2016 Oct 26.

Institute for Plant Biochemistry, Cluster of Excellence on Plant Sciences (CEPLAS), Heinrich-Heine-University, Universitätsstraße 1, 40225 Düsseldorf, Germany.

Blue native-PAGE (BN-PAGE) resolves protein complexes in their native state. When combined with immunoblotting, it can be used to identify the presence of high molecular weight complexes at high resolution for any protein, given a suitable antibody. To identify proteins in high molecular weight complexes on a large scale and to bypass the requirement for specific antibodies, we applied a tandem mass spectrometry (MS/MS) approach to BN-PAGE-resolved chloroplasts. Fractionation of the gel into six bands allowed identification and label-free quantification of 1000 chloroplast proteins with native molecular weight separation. Significantly, this approach achieves a depth of identification comparable with traditional shotgun proteomic analyses of chloroplasts, indicating much of the known chloroplast proteome is amenable to MS/MS identification under our fractionation scheme. By limiting the number of fractionation bands to six, we facilitate scaled-up comparative analyses, as we demonstrate with the reticulata chloroplast mutant displaying a reticulated leaf phenotype. Our comparative proteomics approach identified a candidate interacting protein of RETICULATA as well as effects on lipid remodeling proteins, amino acid metabolic enzymes, and plastid division machinery. We additionally highlight selected proteins from each sub-compartment of the chloroplast that provide novel insight on known or hypothesized protein complexes to further illustrate the utility of this approach. Our results demonstrate the high sensitivity and reproducibility of this technique, which is anticipated to be widely adaptable to other sub-cellular compartments.
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http://dx.doi.org/10.1016/j.molp.2016.10.011DOI Listing
January 2017

MicroRNA-138 promotes acquired alkylator resistance in glioblastoma by targeting the Bcl-2-interacting mediator BIM.

Oncotarget 2016 Mar;7(11):12937-50

Laboratory of Molecular Neuro-Oncology, Department of Neurology, University Hospital and University of Zurich, Zurich, Switzerland.

Glioblastoma is the most aggressive brain tumor in adults with a median survival below 12 months in population-based studies. The main reason for tumor recurrence and progression is constitutive or acquired resistance to the standard of care of surgical resection followed by radiotherapy with concomitant and adjuvant temozolomide (TMZ/RT→TMZ). Here, we investigated the role of microRNA (miRNA) alterations as mediators of alkylator resistance in glioblastoma cells. Using microarray-based miRNA expression profiling of parental and TMZ-resistant cultures of three human glioma cell lines, we identified a set of differentially expressed miRNA candidates. From these, we selected miR-138 for further functional analyses as this miRNA was not only upregulated in TMZ-resistant versus parental cells, but also showed increased expression in vivo in recurrent glioblastoma tissue samples after TMZ/RT→TMZ treatment. Transient transfection of miR-138 mimics in glioma cells with low basal miR-138 expression increased glioma cell proliferation. Moreover, miR-138 overexpression increased TMZ resistance in long-term glioblastoma cell lines and glioma initiating cell cultures. The apoptosis regulator BIM was identified as a direct target of miR-138, and its silencing mediated the induced TMZ resistance phenotype. Altered sensitivity to apoptosis played only a minor role in this resistance mechanism. Instead, we identified the induction of autophagy to be regulated downstream of the miR-138/BIM axis and to promote cell survival following TMZ exposure. Our data thus define miR-138 as a glioblastoma cell survival-promoting miRNA associated with resistance to TMZ therapy in vitro and with tumor progression in vivo.
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http://dx.doi.org/10.18632/oncotarget.7346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4914333PMC
March 2016

Redox proteomics reveal stress responsive proteins linking peroxiredoxin-1 status in glioma to chemosensitivity and oxidative stress.

Biochim Biophys Acta 2015 Jun 4;1854(6):624-31. Epub 2014 Dec 4.

Molecular Proteomics Laboratory, BMFZ, Universität Düsseldorf, Germany.

The combined deletion of chromosomal arms 1p and 19q has been described as a prognostic marker for oligodendroglial tumors. These tumors show a better response to chemotherapy and radiotherapy. Recently, we found a lower abundance of peroxiredoxin 1 (PRDX1) in oligodendroglial tumors with 1p/19q deletion, suggesting a potential role of this enzyme in the clearance of therapy induced reactive oxygen species (ROS). Here, we confirmed the importance of PRDX1 in tumor cell survival by PRDX1 knockdown and overexpression in A-172 cells treated with the alkylating agent bis-chloroethyl nitrosourea (BCNU). Overexpression of PRDX1 resulted in a higher resistance of cells to BCNU treatment. In addition, BCNU challenged cells showed higher levels of ROS in PRDX1 knockdown cells. We applied a modified version of the redox two dimensional difference gel electrophoresis approach to analyze ROS mediated effects on protein thiols after BCNU treatment by labeling protein thiols with fluorescent dyes. Altogether eleven proteins were identified showing PRDX1 dependent altered labeling, many of them have been previously linked to stress response processes. Furthermore, 30 additional potentially redox active proteins were identified. The majority of them is involved in therapy associated processes like cellular stress response, DNA damage and regulation of cell death and therewith suggests that tumor cells maintain a network of redox sensitive proteins to escape chemotherapy. This article is part of a Special Issue entitled: Medical Proteomics.
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http://dx.doi.org/10.1016/j.bbapap.2014.11.011DOI Listing
June 2015

How stemlike are sphere cultures from long-term cancer cell lines? Lessons from mouse glioma models.

J Neuropathol Exp Neurol 2014 Nov;73(11):1062-77

From the Laboratory of Molecular Neuro-Oncology, Departments of Neurology (MA, PR, MW) and Neurosurgery (MA, KF); University Hospital Zurich, and Neuroscience Center (MA, KF, PR, MW), University of Zurich, Zurich, Switzerland; Interdisciplinary Institute for Bioinformatics, University of Leipzig, Leipzig, Germany (EW, HB); Molecular Proteomics Laboratory, Center for Biological and Medical Research (BMFZ) (AS, KS); Department of Neuropathology and German Cancer Consortium (DKTK), partner site Essen/Düsseldorf (KK, GR); and Institute for Molecular Medicine (KS), Heinrich Heine University, Düsseldorf, Germany.

Cancer stem cells may mediate therapy resistance and recurrence in various types of cancer, including glioblastoma. Cancer stemlike cells can be isolated from long-term cancer cell lines, including glioma lines. Using sphere formation as a model for cancer cell stemness in vitro, we derived sphere cultures from SMA-497, SMA-540, SMA-560, and GL-261 glioma cells. Gene expression and proteomics profiling demonstrated that sphere cultures uniformly showed an elevated expression of stemness-associated genes, notably including CD44. Differences in neural lineage marker expression between nonsphere and sphere cultures were heterogeneous except for a uniform reduction of β-III-tubulin in sphere cultures. All sphere cultures showed slower growth. Self-renewal capacity was influenced by medium conditions but not nonsphere versus sphere culture phenotype. Sphere cultures were more resistant to irradiation, whereas both nonsphere and sphere cultures were highly resistant to temozolomide. Nonsphere cells formed more aggressive tumors in syngeneic mice than sphere cells in all models except SMA-560. There were no major differences in vascularization or infiltration by T cells or microglia/macrophages between nonsphere and sphere cell-derived tumors implanted in syngeneic hosts. Together, these data indicate that mouse glioma cell lines may be induced in vitro to form spheres that acquire features of stemness, but they do not exhibit a uniform biologic phenotype, thereby challenging the view that they represent a superior model system.
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http://dx.doi.org/10.1097/NEN.0000000000000130DOI Listing
November 2014

The fate of b-ions in the two worlds of collision-induced dissociation.

Biochim Biophys Acta 2013 Dec 28;1834(12):2843-8. Epub 2013 Aug 28.

Molecular Proteomics Laboratory, Biologisch-Medizinisches Forschungszentrum (BMFZ), Heinrich-Heine-Universität, Düsseldorf, Germany. Electronic address:

Fragment analysis of proteins and peptides by mass spectrometry using collision-induced dissociation (CID) revealed that the pairwise generated N-terminal b- and C-terminal y-ions have different stabilities resulting in underrepresentation of b-ions. Detailed analyses of large-scale spectra databases and synthetic peptides underlined these observations and additionally showed that the fragmentation pattern depends on utilized CID regime. To investigate this underrepresentation further we systematically compared resonant excitation energy and beam-type CID facilitated on different mass spectrometer platforms: (i) quadrupole time-of-flight, (ii) linear ion trap and (iii) three-dimensional ion trap. Detailed analysis of MS/MS data from a standard tryptic protein digest revealed that b-ions are significantly underrepresented on all investigated mass spectrometers. By N-terminal acetylation of tryptic peptides we show for the first time that b-ion cyclization reaction significantly contributes to b-ion underrepresentation even on ion trap instruments and accounts for at most 16% of b-ion loss.
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http://dx.doi.org/10.1016/j.bbapap.2013.08.007DOI Listing
December 2013

Tra2-mediated recognition of HIV-1 5' splice site D3 as a key factor in the processing of vpr mRNA.

J Virol 2013 Mar 19;87(5):2721-34. Epub 2012 Dec 19.

Institute for Virology, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.

Small noncoding HIV-1 leader exon 3 is defined by its splice sites A2 and D3. While 3' splice site (3'ss) A2 needs to be activated for vpr mRNA formation, the location of the vpr start codon within downstream intron 3 requires silencing of splicing at 5'ss D3. Here we show that the inclusion of both HIV-1 exon 3 and vpr mRNA processing is promoted by an exonic splicing enhancer (ESE(vpr)) localized between exonic splicing silencer ESSV and 5'ss D3. The ESE(vpr) sequence was found to be bound by members of the Transformer 2 (Tra2) protein family. Coexpression of these proteins in provirus-transfected cells led to an increase in the levels of exon 3 inclusion, confirming that they act through ESE(vpr). Further analyses revealed that ESE(vpr) supports the binding of U1 snRNA at 5'ss D3, allowing bridging interactions across the upstream exon with 3'ss A2. In line with this, an increase or decrease in the complementarity of 5'ss D3 to the 5' end of U1 snRNA was accompanied by a higher or lower vpr expression level. Activation of 3'ss A2 through the proposed bridging interactions, however, was not dependent on the splicing competence of 5'ss D3 because rendering it splicing defective but still competent for efficient U1 snRNA binding maintained the enhancing function of D3. Therefore, we propose that splicing at 3'ss A2 occurs temporally between the binding of U1 snRNA and splicing at D3.
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http://dx.doi.org/10.1128/JVI.02756-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3571381PMC
March 2013

Proteome-wide identification of mycobacterial pupylation targets.

Mol Syst Biol 2010 Jul;6:386

EMBL-Hamburg Unit, European Molecular Biology Laboratory, Hamburg, Germany.

Mycobacteria use a unique system for covalently modifying proteins based on the conjugation of a small protein, referred to as prokaryotic ubiquitin-like protein (PUP). In this study, we report a proteome-wide analysis of endogenous pupylation targets in the model organism Mycobacterium smegmatis. On affinity capture, a total of 243 candidate pupylation targets were identified by two complementary proteomics approaches. For 41 of these protein targets, direct evidence for a total of 48 lysine-mediated pupylation acceptor sites was obtained by collision-induced dissociation spectra. For the majority of these pupylation targets (38 of 41), orthologous genes are found in the M. tuberculosis genome. Interestingly, approximately half of these proteins are involved in intermediary metabolism and respiration pathways. A considerable fraction of the remaining targets are involved in lipid metabolism, information pathways, and virulence, detoxification and adaptation. Approximately one-third of the genes encoding these targets are located in seven gene clusters, indicating functional linkages of mycobacterial pupylation targets. A comparison of the pupylome under different cell culture conditions indicates that substrate targeting for pupylation is rather dynamic.
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http://dx.doi.org/10.1038/msb.2010.39DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2925521PMC
July 2010

Simultaneous extraction of nucleic acids and proteins from tissue specimens by ultracentrifugation: A protocol using the high-salt protein fraction for quantitative proteome analysis.

Proteomics 2009 Nov;9(21):4985-90

Medizinisches Proteom-Center, Ruhr-Universität Bochum, Bochum, Germany.

Comprehensive molecular profiling of human tumor tissue specimens at the DNA, mRNA and protein level is often obstructed by a limited amount of available material. Homogenization of frozen tissue samples in guanidine isothiocyanate followed by ultracentrifugation over cesium chloride allows the simultaneous extraction of high-molecular weight DNA and RNA. Here, we present a protocol for quantitative proteome analysis using the high-salt protein fraction obtained as supernatant after ultracentrifugation for nucleic acid extraction. We applied this method to extracts from primary human brain tumors and demonstrate its successful application for protein expression profiling in these tumors using 2-D DIGE, MS and Western blotting.
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http://dx.doi.org/10.1002/pmic.200800902DOI Listing
November 2009
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