Publications by authors named "Gereon Poschmann"

67 Publications

Lack of GABARAP-Type Proteins Is Accompanied by Altered Golgi Morphology and Surfaceome Composition.

Int J Mol Sci 2020 Dec 23;22(1). Epub 2020 Dec 23.

Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany.

GABARAP (γ-aminobutyric acid type A receptor-associated protein) and its paralogues GABARAPL1 and GABARAPL2 comprise a subfamily of autophagy-related Atg8 proteins. They are studied extensively regarding their roles during autophagy. Originally, however, especially GABARAPL2 was discovered to be involved in intra-Golgi transport and homotypic fusion of post-mitotic Golgi fragments. Recently, a broader function of mammalian Atg8s on membrane trafficking through interaction with various soluble -ethylmaleimide-sensitive factor-attachment protein receptors SNAREs was suggested. By immunostaining and microscopic analysis of the Golgi network, we demonstrate the importance of the presence of individual GABARAP-type proteins on Golgi morphology. Furthermore, triple knockout (TKO) cells lacking the whole GABARAP subfamily showed impaired Golgi-dependent vesicular trafficking as assessed by imaging of fluorescently labelled ceramide. With the Golgi apparatus being central within the secretory pathway, we sought to investigate the role of the GABARAP-type proteins for cell surface protein trafficking. By analysing the surfaceome compositionofTKOs, we identified a subset of cell surface proteins with altered plasma membrane localisation. Taken together, we provide novel insights into an underrated aspect of autophagy-independent functions of the GABARAP subfamily and recommend considering the potential impact of GABARAP subfamily proteins on a plethora of processes during experimental analysis of GABARAP-deficient cells not only in the autophagic context.
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http://dx.doi.org/10.3390/ijms22010085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7795684PMC
December 2020

The Puzzle of Metabolite Exchange and Identification of Putative Octotrico Peptide Repeat Expression Regulators in the Nascent Photosynthetic Organelles of .

Front Microbiol 2020 27;11:607182. Epub 2020 Nov 27.

Department of Biology, Institute of Microbial Cell Biology, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.

The endosymbiotic acquisition of mitochondria and plastids more than one billion years ago was central for the evolution of eukaryotic life. However, owing to their ancient origin, these organelles provide only limited insights into the initial stages of organellogenesis. The cercozoan amoeba contains photosynthetic organelles-termed chromatophores-that evolved from a cyanobacterium ∼100 million years ago, independently from plastids in plants and algae. Despite the more recent origin of the chromatophore, it shows tight integration into the host cell. It imports hundreds of nucleus-encoded proteins, and diverse metabolites are continuously exchanged across the two chromatophore envelope membranes. However, the limited set of chromatophore-encoded solute transporters appears insufficient for supporting metabolic connectivity or protein import. Furthermore, chromatophore-localized biosynthetic pathways as well as multiprotein complexes include proteins of dual genetic origin, suggesting that mechanisms evolved that coordinate gene expression levels between chromatophore and nucleus. These findings imply that similar to the situation in mitochondria and plastids, also in nuclear factors evolved that control metabolite exchange and gene expression in the chromatophore. Here we show by mass spectrometric analyses of enriched insoluble protein fractions that, unexpectedly, nucleus-encoded transporters are not inserted into the chromatophore inner envelope membrane. Thus, despite the apparent maintenance of its barrier function, canonical metabolite transporters are missing in this membrane. Instead we identified several expanded groups of short chromatophore-targeted orphan proteins. Members of one of these groups are characterized by a single transmembrane helix, and others contain amphipathic helices. We hypothesize that these proteins are involved in modulating membrane permeability. Thus, the mechanism generating metabolic connectivity of the chromatophore fundamentally differs from the one for mitochondria and plastids, but likely rather resembles the poorly understood mechanism in various bacterial endosymbionts in plants and insects. Furthermore, our mass spectrometric analysis revealed an expanded family of chromatophore-targeted helical repeat proteins. These proteins show similar domain architectures as known organelle-targeted expression regulators of the octotrico peptide repeat type in algae and plants. Apparently these chromatophore-targeted proteins evolved convergently to plastid-targeted expression regulators and are likely involved in gene expression control in the chromatophore.
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http://dx.doi.org/10.3389/fmicb.2020.607182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7729196PMC
November 2020

Insights in the Antimicrobial Potential of the Natural Nisin Variant Nisin H.

Front Microbiol 2020 20;11:573614. Epub 2020 Oct 20.

Institute of Biochemistry, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany.

Lantibiotics are a growing class of antimicrobial peptides, which possess antimicrobial activity against mainly Gram-positive bacteria including the highly resistant strains such as methicillin-resistant or vancomycin-resistant enterococci. In the last decades numerous lantibiotics were discovered in natural habitats or designed with bioengineering tools. In this study, we present an insight in the antimicrobial potential of the natural occurring lantibiotic nisin H from as well as the variant nisin H FI. We determined the yield of the heterologously expressed peptide and quantified the cleavage efficiency employing the nisin protease NisP. Furthermore, we analyzed the effect on the modification via mass spectrometry analysis. With standardized growth inhibition assays we benchmarked the activity of pure nisin H and the variant nisin H FI, and their influence on the activity of the nisin immunity proteins NisI and NisFEG from and the nisin resistance proteins NSR and NsrFP from COH1. We further checked the antibacterial activity against clinical isolates of , and via microdilution method. In summary, nisin H and the nisin H FI variant possessed better antimicrobial potency than the natural nisin A.
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http://dx.doi.org/10.3389/fmicb.2020.573614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7606277PMC
October 2020

Comparative Secretomics Gives Access to High Confident Secretome Data: Evaluation of Different Methods for the Determination of Bona Fide Secreted Proteins.

Proteomics 2021 Jan 17;21(2):e2000178. Epub 2020 Nov 17.

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

Secretome analysis is broadly applied to understand the interplay between cells and their microenvironment. In particular, the unbiased analysis by mass spectrometry-based proteomics of conditioned medium has been successfully applied. In this context, several approaches have been developed allowing to distinguish proteins actively secreted by cells from proteins derived from culture medium or proteins released from dying cells. Here, three different methods comparing conditioned medium and lysate by quantitative mass spectrometry-based proteomics to identify bona fide secreted proteins are evaluated. Evaluation in three different human cell lines reveals that all three methods give access to a similar set of bona fide secreted proteins covering a broad abundance range. In the analyzed primary cells, that is, mesenchymal stromal cells and normal human dermal fibroblasts, more than 70% of the identified proteins are linked to classical secretion pathways. Furthermore, 4-12% are predicted to be released by unconventional secretion pathways. Interestingly, evidence of release by ectodomain shedding in a large number of the remaining candidate proteins is found. In summary, it is convinced that comparative secretomics is currently the method of choice to obtain high-confident secretome data and to identify novel candidates for unconventional protein secretion which have been neglected so far.
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http://dx.doi.org/10.1002/pmic.202000178DOI Listing
January 2021

An Integrative Omics Approach Reveals Involvement of in Hepatic Metastatic Progression of Colorectal Cancer.

Cancers (Basel) 2020 Aug 22;12(9). Epub 2020 Aug 22.

Computational Biology and Systems Biomedicine Group, Biodonostia Health Research Institute, Calle Doctor Beguiristain s/n, 20014 San Sebastián, Spain.

(1) Background & Aims: The roles of different cells in the tumor microenvironment (TME) are critical to the metastatic process. The phenotypic transformation of the liver cells is one of the most important stages of the hepatic metastasis progression of colorectal cancer (CRC). Our aim was to identify the major molecules (i.e., genes, miRNAs and proteins) involved in this process. (2) Methods: We isolated and performed whole-genome analysis of gene, miRNA, and protein expression in three types of liver cells (Ito cells, Kupffer cells, and liver sinusoidal endothelial cells) from the TME of a murine model of CRC liver metastasis. We selected the statistically significant differentially expressed molecules using the Student's t-test with Benjamini-Hochberg correction and performed functional statistically-significant enrichment analysis of differentially expressed molecules with hypergeometric distribution using the curated collection of molecular signatures, MSigDB. To build a gene-miRNA-protein network centered in Brca1, we developed a software package (miRDiana) that collects miRNA targets from the union of the TargetScan, MicroCosm, mirTarBase, and miRWalk databases. This was used to search for miRNAs targeting . We validated the most relevant miRNAs with real-time quantitative PCR. To investigate BRCA1 protein expression, we built tissue microarrays (TMAs) from hepatic metastases of 34 CRC patients. (3) Results: Using integrated omics analyses, we observed that the gene is among the twenty transcripts simultaneously up-regulated in all three types of TME liver cells during metastasis. Further analysis revealed that is the last BRCA1-associated genome surveillance complex (BASC) gene activated in the TME. We confirmed this finding in human reanalyzing transcriptomics datasets from 184 patients from non-tumor colorectal tissue, primary colorectal tumor and colorectal liver metastasis of the GEO database. We found that the most probable sequence of cell activation during metastasis is Endothelial→Ito→Kupffer. Immunohistochemical analysis of human liver metastases showed the BRCA1 protein was co-localized in Ito, Kupffer, and endothelial cells in 81.8% of early or synchronous metastases. However, in the greater part of the metachronous liver metastases, this protein was not expressed in any of these TME cells. (4) Conclusions: These results suggest a possible role of the co-expression of BRCA1 in Ito, Kupffer, and sinusoidal endothelial cells in the early occurrence of CRC liver metastases, and point to BRCA1 as a potential TME biomarker.
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http://dx.doi.org/10.3390/cancers12092380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565528PMC
August 2020

Profiling of cis- and trans-acting factors supporting noncanonical splice site activation.

RNA Biol 2021 Jan 5;18(1):118-130. Epub 2020 Aug 5.

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

Recently, by combining transcriptomics with functional splicing reporter assays we were able to identify GT > GC > TT as the three highest ranked dinucleotides of human 5' splice sites (5'ss). Here, we have extended our investigations to the proteomic characterization of nuclear proteins that bind to canonical and noncanonical 5'ss. Surprisingly, we found that U1 snRNP binding to functional 5'ss sequences prevented components of the DNA damage response (DDR) from binding to the RNA, suggesting a close link between spliceosome arrangement and genome stability. We demonstrate that all tested noncanonical 5'ss sequences are bona-fide targets of the U2-type spliceosome and are bound by U1 snRNP, including U1-C, in the presence of splicing enhancers. The quantity of precipitated U1-C protein was similar for all noncanonical 5'ss dinucleotides, so that the highly different 5'ss usage was likely due to a later step after early U1 snRNP binding. In addition, we show that an internal GT at positions +5/+6 can be advantageous for splicing at position +1 of noncanonical splice sites. Likewise, and in agreement with previous observations, splicing inactive U1 snRNP binding sites could serve as splicing enhancers, which may also explain the higher abundance of U1 snRNPs compared to other U snRNPs. Finally, we observe that an arginine-serine (RS)-rich domain recruitment to stem loop I of the U1 snRNA is functionally sufficient to promote exon-definition and upstream 3'ss activation.
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http://dx.doi.org/10.1080/15476286.2020.1798111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7834088PMC
January 2021

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

Secretome Analysis of Mesenchymal Stem Cell Factors Fostering Oligodendroglial Differentiation of Neural Stem Cells In Vivo.

Int J Mol Sci 2020 Jun 18;21(12). Epub 2020 Jun 18.

Department of Neurology, Medical Faculty, Heinrich-Heine-University, D-40225 Düsseldorf, Germany.

Mesenchymal stem cell (MSC)-secreted factors have been shown to significantly promote oligodendrogenesis from cultured primary adult neural stem cells (aNSCs) and oligodendroglial precursor cells (OPCs). Revealing underlying mechanisms of how aNSCs can be fostered to differentiate into a specific cell lineage could provide important insights for the establishment of novel neuroregenerative treatment approaches aiming at myelin repair. However, the nature of MSC-derived differentiation and maturation factors acting on the oligodendroglial lineage has not been identified thus far. In addition to missing information on active ingredients, the degree to which MSC-dependent lineage instruction is functional in vivo also remains to be established. We here demonstrate that MSC-derived factors can indeed stimulate oligodendrogenesis and myelin sheath generation of aNSCs transplanted into different rodent central nervous system (CNS) regions, and furthermore, we provide insights into the underlying mechanism on the basis of a comparative mass spectrometry secretome analysis. We identified a number of secreted proteins known to act on oligodendroglia lineage differentiation. Among them, the tissue inhibitor of metalloproteinase type 1 (TIMP-1) was revealed to be an active component of the MSC-conditioned medium, thus validating our chosen secretome approach.
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http://dx.doi.org/10.3390/ijms21124350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352621PMC
June 2020

The GABARAP Co-Secretome Identified by APEX2-GABARAP Proximity Labelling of Extracellular Vesicles.

Cells 2020 06 16;9(6). Epub 2020 Jun 16.

Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany.

The autophagy-related ATG8 protein GABARAP has not only been shown to be involved in the cellular self-degradation process called autophagy but also fulfils functions in intracellular trafficking processes such as receptor transport to the plasma membrane. Notably, available mass spectrometry data suggest that GABARAP is also secreted into extracellular vesicles (EVs). Here, we confirm this finding by the immunoblotting of EVs isolated from cell culture supernatants and human blood serum using specific anti-GABARAP antibodies. To investigate the mechanism by which GABARAP is secreted, we applied proximity labelling, a method for studying the direct environment of a protein of interest in a confined cellular compartment. By expressing an engineered peroxidase (APEX2)-tagged variant of GABARAP-which, like endogenous GABARAP, was present in EVs prepared from HEK293 cells-we demonstrate the applicability of APEX2-based proximity labelling to EVs. The biotinylated protein pool which contains the APEX2-GABARAP co-secretome contained not only known GABARAP interaction partners but also proteins that were found in APEX2-GABARAP's proximity inside of autophagosomes in an independent study. All in all, we not only introduce a versatile tool for co-secretome analysis in general but also uncover the first details about autophagy-based pathways as possible biogenesis mechanisms of GABARAP-containing EVs.
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http://dx.doi.org/10.3390/cells9061468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7349886PMC
June 2020

Regulates Transcription and the Epigenetic Landscape via POLE and DMAP1 while Deficiency or Pharmacological Inhibition Sensitizes Germ Cell Tumor Cells to ATR Inhibition.

Cancers (Basel) 2020 Apr 7;12(4). Epub 2020 Apr 7.

Department of Urology, Urological Research Lab, Translational UroOncology, University Hospital Düsseldorf, 40225 Düsseldorf, Germany.

Germ cell tumors (GCTs) are the most common solid malignancies found in young men. Although they generally have high cure rates, metastases, resistance to cisplatin-based therapy, and late toxicities still represent a lethal threat, arguing for the need of new therapeutic options. In a previous study, we identified downregulation of the chromatin-remodeling SWI/SNF complex member ARID1A as a key event in the mode of action of the histone deacetylase inhibitor romidepsin. Additionally, the loss-of-function mutations re-sensitize different tumor types to various drugs, like EZH2-, PARP-, HDAC-, HSP90- or ATR-inhibitors. Thus, ARID1A presents as a promising target for synthetic lethality and combination therapy. In this study, we deciphered the molecular function of ARID1A and screened for the potential of two pharmacological ARID1A inhibitors as a new therapeutic strategy to treat GCTs. By CRISPR/Cas9, we generated -deficient GCT cells and demonstrate by mass spectrometry that is putatively involved in regulating transcription, DNA repair and the epigenetic landscape via DNA Polymerase POLE and the DNA methyltransferase 1-associated protein DMAP1. Additionally, deficiency or pharmacological inhibition increased the efficacy of romidepsin and considerably sensitized GCT cells, including cisplatin-resistant subclones, towards ATR inhibition. Thus, targeting ARID1A in combination with romidepsin and ATR inhibitors presents as a new putative option to treat GCTs.
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http://dx.doi.org/10.3390/cancers12040905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226530PMC
April 2020

The Proteomic Landscape of Cysteine Oxidation That Underpins Retinoic Acid-Induced Neuronal Differentiation.

J Proteome Res 2020 May 3;19(5):1923-1940. Epub 2020 Apr 3.

Institute for Molecular Medicine, Medical Faculty, Heinrich Heine University Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany.

The initial phases of neuronal differentiation are key to neuronal function. A particularly informative model to study these initial phases are retinoic acid-stimulated SH-SY5Y cells. Although these progressions are associated with redox-sensitive processes, it is largely undefined how the cellular proteome underpins redox dynamics and the management of reactive oxygen species. Here, we map the global cysteine-based redox landscape of SH-SY5Y cells using quantitative redox proteomics. We find evidence that redox alterations occurred early in differentiation and affect the expression of neuronal marker proteins and the extension of neurites. The spatiotemporal analysis of reactive oxygen species suggests a NOX2-dependent peak in cytoplasmic superoxide anions/hydrogen peroxide generation 2 h after retinoic acid stimulation. At the same time point, 241 out of 275 proteins with an altered cysteine redox state are reversibly oxidized in response to retinoic acid. Our analyses pinpoint redox alterations of proteins involved in the retinoic acid homeostasis and cytoskeletal dynamics.
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http://dx.doi.org/10.1021/acs.jproteome.9b00752DOI Listing
May 2020

Extracellular Redox Regulation of α7β Integrin-Mediated Cell Migration Is Signaled via a Dominant Thiol-Switch.

Antioxidants (Basel) 2020 Mar 10;9(3). Epub 2020 Mar 10.

Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, 48149 Münster, Germany.

While adhering to extracellular matrix (ECM) proteins, such as laminin-111, cells temporarily produce hydrogen peroxide at adhesion sites. To study the redox regulation of α7β1 integrin-mediated cell adhesion to laminin-111, a conserved cysteine pair within the α-subunit hinge region was replaced for alanines. The molecular and cellular effects were analyzed by electron and atomic force microscopy, impedance-based migration assays, flow cytometry and live cell imaging. This cysteine pair constitutes a thiol-switch, which redox-dependently governs the equilibrium between an extended and a bent integrin conformation with high and low ligand binding activity, respectively. Hydrogen peroxide oxidizes the cysteines to a disulfide bond, increases ligand binding and promotes cell migration toward laminin-111. Inversely, extracellular thioredoxin-1 reduces the disulfide, thereby decreasing laminin binding. Mutation of this cysteine pair into the non-oxidizable hinge-mutant shows molecular and cellular effects similar to the reduced wild-type integrin, but lacks redox regulation. This proves the existence of a dominant thiol-switch within the α subunit hinge of α7β1 integrin, which is sufficient to implement activity regulation by extracellular redox agents in a redox-regulatory circuit. Our data reveal a novel and physiologically relevant thiol-based regulatory mechanism of integrin-mediated cell-ECM interactions, which employs short-lived hydrogen peroxide and extracellular thioredoxin-1 as signaling mediators.
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http://dx.doi.org/10.3390/antiox9030227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139957PMC
March 2020

Propionic Acid Shapes the Multiple Sclerosis Disease Course by an Immunomodulatory Mechanism.

Cell 2020 03 10;180(6):1067-1080.e16. Epub 2020 Mar 10.

Department of Neurology, Ruhr University Bochum, St. Josef Hospital Bochum, Bochum 44791, Germany. Electronic address:

Short-chain fatty acids are processed from indigestible dietary fibers by gut bacteria and have immunomodulatory properties. Here, we investigate propionic acid (PA) in multiple sclerosis (MS), an autoimmune and neurodegenerative disease. Serum and feces of subjects with MS exhibited significantly reduced PA amounts compared with controls, particularly after the first relapse. In a proof-of-concept study, we supplemented PA to therapy-naive MS patients and as an add-on to MS immunotherapy. After 2 weeks of PA intake, we observed a significant and sustained increase of functionally competent regulatory T (Treg) cells, whereas Th1 and Th17 cells decreased significantly. Post-hoc analyses revealed a reduced annual relapse rate, disability stabilization, and reduced brain atrophy after 3 years of PA intake. Functional microbiome analysis revealed increased expression of Treg-cell-inducing genes in the intestine after PA intake. Furthermore, PA normalized Treg cell mitochondrial function and morphology in MS. Our findings suggest that PA can serve as a potent immunomodulatory supplement to MS drugs.
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http://dx.doi.org/10.1016/j.cell.2020.02.035DOI Listing
March 2020

Impaired integrin α /β -mediated hepatocyte growth factor release by stellate cells of the aged liver.

Aging Cell 2020 04 11;19(4):e13131. Epub 2020 Mar 11.

Clinic of Gastroenterology, Hepatology and Infectious Diseases, Heinrich Heine University, Düsseldorf, Germany.

Hepatic blood flow and sinusoidal endothelial fenestration decrease during aging. Consequently, fluid mechanical forces are reduced in the space of Disse where hepatic stellate cells (HSC) have their niche. We provide evidence that integrin α /β is an important mechanosensor in HSC involved in shear stress-induced release of hepatocyte growth factor (HGF), an essential inductor of liver regeneration which is impaired during aging. The expression of the integrin subunits α and β decreases in liver and HSC from aged rats. CRISPR/Cas9-mediated integrin α and β knockouts in isolated HSC lead to lowered HGF release and impaired cellular adhesion. Fluid mechanical forces increase integrin α and laminin gene expression whereas integrin β remains unaffected. In the aged liver, laminin β2 and γ1 protein chains as components of laminin-521 are lowered. The integrin α knockout in HSC reduces laminin expression via mechanosensory mechanisms. Culture of HSC on nanostructured surfaces functionalized with laminin-521 enhances Hgf expression in HSC, demonstrating that these ECM proteins are critically involved in HSC function. During aging, HSC acquire a senescence-associated secretory phenotype and lower their growth factor expression essential for tissue repair. Our findings suggest that impaired mechanosensing via integrin α /β in HSC contributes to age-related reduction of ECM and HGF release that could affect liver regeneration.
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http://dx.doi.org/10.1111/acel.13131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7189994PMC
April 2020

Protein complexes including PGRMC1 and actin-associated proteins are disrupted by AG-205.

Biochem Biophys Res Commun 2020 03 21;524(1):64-69. Epub 2020 Jan 21.

School of Biomedical Sciences, Charles Sturt University, Wagga, Wagga, NSW, 2650, Australia. Electronic address:

PGRMC1 is a protein from the MAPR family with a range of cellular functions. PGRMC1 has been described to play a role in fertility, neuroprotection, steroidogenesis, membrane trafficking and in cancer cell biology. PGRMC1 represents a likely key regulator of cell metabolism and proliferation, as well as a potential target for anti-cancer therapies. To further understand the functions of PGRMC1 and the mechanism of the small molecule inhibitor of PGRMC1, AG-205, proteins differentially bound to PGRMC1 were identified following AG-205 treatment of MIA PaCa-2 cells. Our results suggest that AG-205 influences PGRMC1 interactions with the actin cytoskeleton. The binding of two PGRMC1-associated proteins that support this, RACK1 and alpha-Actinin-1, was reduced following AG-205 treatment. The biology associated with PGRMC1 binding partners identified here merits further investigation.
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http://dx.doi.org/10.1016/j.bbrc.2019.12.108DOI Listing
March 2020

OutCyte: a novel tool for predicting unconventional protein secretion.

Sci Rep 2019 12 19;9(1):19448. Epub 2019 Dec 19.

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

The prediction of protein localization, such as in the extracellular space, from high-throughput data is essential for functional downstream inference. It is well accepted that some secreted proteins go through the classic endoplasmic reticulum-Golgi pathway with the guidance of a signal peptide. However, a large number of proteins have been found to reach the extracellular space by following unconventional secretory pathways. There remains a demand for reliable prediction of unconventional protein secretion (UPS). Here, we present OutCyte, a fast and accurate tool for the prediction of UPS, which for the first time has been built upon experimentally determined UPS proteins. OutCyte mediates the prediction of protein secretion in two steps: first, proteins with N-terminal signals are accurately filtered out; second, proteins without N-terminal signals are classified as UPS or intracellular proteins based on physicochemical features directly generated from their amino acid sequences. We are convinced that OutCyte will play a relevant role in the annotation of experimental data and will therefore contribute to further characterization of the extracellular nature of proteins by considering the commonly neglected UPS proteins.OutCyte has been implemented as a web server at www.outcyte.com.
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http://dx.doi.org/10.1038/s41598-019-55351-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6923414PMC
December 2019

Unconventional protein secretion: The hidden pathways.

Biochim Biophys Acta Proteins Proteom 2019 12 6;1867(12):140272. Epub 2019 Sep 6.

Molecular Proteomics Laboratory (MPL), Institute for Molecular Medicine, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf D-40225, Germany. Electronic address:

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http://dx.doi.org/10.1016/j.bbapap.2019.140272DOI Listing
December 2019

Shaping the lipid composition of bacterial membranes for membrane protein production.

Microb Cell Fact 2019 Aug 10;18(1):131. Epub 2019 Aug 10.

Institute of Biochemistry, Heinrich-Heine-University Duesseldorf, Universitaetsstr. 1, 40225, Duesseldorf, Germany.

Background: The overexpression and purification of membrane proteins is a bottleneck in biotechnology and structural biology. E. coli remains the host of choice for membrane protein production. To date, most of the efforts have focused on genetically tuning of expression systems and shaping membrane composition to improve membrane protein production remained largely unexplored.

Results: In E. coli C41(DE3) strain, we deleted two transporters involved in fatty acid metabolism (OmpF and AcrB), which are also recalcitrant contaminants crystallizing even at low concentration. Engineered expression hosts presented an enhanced fitness and improved folding of target membrane proteins, which correlated with an altered membrane fluidity. We demonstrated the scope of this approach by overproducing several membrane proteins (4 different ABC transporters, YidC and SecYEG).

Conclusions: In summary, E. coli membrane engineering unprecedentedly increases the quality and yield of membrane protein preparations. This strategy opens a new field for membrane protein production, complementary to gene expression tuning.
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http://dx.doi.org/10.1186/s12934-019-1182-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6689329PMC
August 2019

Pitfalls and opportunities in the characterization of unconventionally secreted proteins by secretome analysis.

Biochim Biophys Acta Proteins Proteom 2019 12 12;1867(12):140237. Epub 2019 Jun 12.

Molecular Proteomics Laboratory (MPL), Biomedical Research Center (BMFZ), Heinrich-Heine-University, Düsseldorf, Germany; Institute for Molecular Medicine, University Hospital Düsseldorf, Germany. Electronic address:

Proteins are released from cells by different secretory pathways. The secretory pathway via the ER-Golgi route - realized by a signal sequence - is referred to as "classical secretion". In contrast, alternative secretory pathways were summarized as "unconventional protein secretion". Until now, unconventional protein secretion was lacking attention due to the absence of detailed mechanistic insight and limited experimental access. However, there is a growing number of experimental data showing that a large proportion of secreted proteins is released by these alternative routes. Secretomics - the analysis of all secreted proteins of a cell population - offers the opportunity to gain more functional insight into unconventional protein secretion. Several pitfalls in secretome analysis starting with the analyzed cell model and sample preparation to data analysis have to be considered for detailed characterization of the secretome. Here, we highlight the investigation of secretomes by quantitative LC-MS/MS analysis and discuss pitfalls and opportunities in the characterization of unconventionally secreted proteins by secretome analysis.
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http://dx.doi.org/10.1016/j.bbapap.2019.06.004DOI Listing
December 2019

Adipokinome Signatures in Obese Mouse Models Reflect Adipose Tissue Health and Are Associated with Serum Lipid Composition.

Int J Mol Sci 2019 May 24;20(10). Epub 2019 May 24.

Institute of Clinical Biochemistry and Pathobiochemistry, German Diabetes Center at the Heinrich-Heine-University Duesseldorf, Leibniz Center for Diabetes Research; 40225 Duesseldorf, Germany.

Adipocyte and hepatic lipid metabolism govern whole-body metabolic homeostasis, whereas a disbalance of de novo lipogenesis (DNL) in fat and liver might lead to obesity, with severe co-morbidities. Nevertheless, some obese people are metabolically healthy, but the "protective" mechanisms are not yet known in detail. Especially, the adipocyte-derived molecular mediators that indicate adipose functionality are poorly understood. We studied transgenic mice (alb-SREBP-1c) with a "healthy" obese phenotype, and obob mice with hyperphagia-induced "sick" obesity to analyze the impact of the tissue-specific DNL on the secreted proteins, i.e., the adipokinome, of the primary adipose cells by label-free proteomics. Compared to the control mice, adipose DNL is reduced in both obese mouse models. In contrast, the hepatic DNL is reduced in obob but elevated in alb-SREBP-1c mice. To investigate the relationship between lipid metabolism and adipokinomes, we formulated the "liver-to-adipose-tissue DNL" ratio. Knowledge-based analyses of these results revealed adipocyte functionality with proteins, which was involved in tissue remodeling or metabolism in the alb-SREBP-1c mice and in the control mice, but mainly in fibrosis in the obob mice. The adipokinome in "healthy" obesity is similar to that in a normal condition, but it differs from that in "sick" obesity, whereas the serum lipid patterns reflect the "liver-to-adipose-tissue DNL" ratio and are associated with the adipokinome signature.
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http://dx.doi.org/10.3390/ijms20102559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6567124PMC
May 2019

HDAC5 Expression in Urothelial Carcinoma Cell Lines Inhibits Long-Term Proliferation but Can Promote Epithelial-to-Mesenchymal Transition.

Int J Mol Sci 2019 Apr 30;20(9). Epub 2019 Apr 30.

Department of Urology, Medical Faculty, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany.

Class I histone deacetylases (HDACs) generally promote cell proliferation and tumorigenesis, whereas class IIA HDACs like HDAC4 and HDAC5 may promote or impede cancer development in a tissue-dependent manner. In urothelial carcinoma (UC), HDAC5 is often downregulated. Accordingly, HDAC5 was weakly expressed in UC cell lines suggesting a possible tumor-suppressive function. We therefore characterized the effects of stable HDAC5 expression in four UC cell lines (RT112, VM-Cub-1, SW1710 and UM-UC-3) with different phenotypes reflecting the heterogeneity of UC, by assessing proliferation, clonogenicity and migration ability. Further, we detailed changes in the proteome and transcriptome by immunoblotting, mass spectrometry and RNA sequencing analysis. We observed that HDAC5 overexpression in general decreased cell proliferation, but in one cell line (VM-Cub-1) induced a dramatic change from an epitheloid to a mesenchymal phenotype, i.e., epithelial-mesenchymal transition (EMT). These phenotypical changes were confirmed by comprehensive proteomics and transcriptomics analyses. In contrast to HDAC5, overexpression of HDAC4 exerted only weak effects on cell proliferation and phenotypes. We conclude that overexpression of HDAC5 may generally decrease proliferation in UC, but, intriguingly, may induce EMT on its own in certain circumstances.
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http://dx.doi.org/10.3390/ijms20092135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6539474PMC
April 2019

Control of Drosophila Growth and Survival by the Lipid Droplet-Associated Protein CG9186/Sturkopf.

Cell Rep 2019 03;26(13):3726-3740.e7

Institute for Mathematical Modeling of Biological Systems, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany; Systems Biology of Lipid Metabolism, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany. Electronic address:

Lipid droplets (LDs) are the universal cellular storage organelles for esterified neutral lipids. The increasing number of characterized LD-associated proteins attained LDs with hitherto unexpected functions on top of their classical role as energy depot. Here, we characterize the LD-associated protein CG9186 of Drosophila by a CRISPR/Cas9-derived mutant fly line. While the mutant flies only showed a mild triacylglycerol storage phenotype, they were highly protected from desiccation stress, likely linked to a reduced locomotor activity and altered cuticular hydrocarbons. Both parameters depend on juvenile hormone (JH) signaling. Together with an observed interaction between CG9186 and JH-degrading enzymes, our results suggest that CG9186 participates in endocrine physiology regulation. In support of this hypothesis, CG9186 mutant flies show an altered expression of JH target genes and fail to adjust their developmental rate to dietary yeast-to-sugar ratio changes. Our results thus link LDs to organismic physiology regulation.
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http://dx.doi.org/10.1016/j.celrep.2019.02.110DOI Listing
March 2019

Quantitative insights into the cyanobacterial cell economy.

Elife 2019 02 4;8. Epub 2019 Feb 4.

Laboratory of Adaptive Biotechnologies, Global Change Research Institute CAS, Brno, Czech Republic.

Phototrophic microorganisms are promising resources for green biotechnology. Compared to heterotrophic microorganisms, however, the cellular economy of phototrophic growth is still insufficiently understood. We provide a quantitative analysis of light-limited, light-saturated, and light-inhibited growth of the cyanobacterium sp. PCC 6803 using a reproducible cultivation setup. We report key physiological parameters, including growth rate, cell size, and photosynthetic activity over a wide range of light intensities. Intracellular proteins were quantified to monitor proteome allocation as a function of growth rate. Among other physiological acclimations, we identify an upregulation of the translational machinery and downregulation of light harvesting components with increasing light intensity and growth rate. The resulting growth laws are discussed in the context of a coarse-grained model of phototrophic growth and available data obtained by a comprehensive literature search. Our insights into quantitative aspects of cyanobacterial acclimations to different growth rates have implications to understand and optimize photosynthetic productivity.
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http://dx.doi.org/10.7554/eLife.42508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391073PMC
February 2019

A Proteome Approach Reveals Differences between Fertile Women and Patients with Repeated Implantation Failure on Endometrial Level⁻Does hCG Render the Endometrium of RIF Patients?

Int J Mol Sci 2019 Jan 19;20(2). Epub 2019 Jan 19.

Medical Center University of Düsseldorf, Department of OB/GYN and REI (UniKiD), Moorenstrasse 5, 40225 Düsseldorf, Germany.

Background: The molecular signature of endometrial receptivity still remains barely understood, especially when focused on the possible benefit of therapeutical interventions and implantation-related pathologies. Therefore, the protein composition of tissue and isolated primary cells (endometrial stromal cells, ESCs) from endometrial scratchings of ART (Assisted Reproductive Techniques) patients with repeated implantation failure (RIF) was compared to volunteers with proven fertility during the time of embryo implantation (LH + 7). Furthermore, an analysis of the endometrial tissue of fertile women infused with human chorionic gonadotropin (hCG) was conducted.

Methods: Endometrial samples ( = 6 RIF, = 10 fertile controls) were split into 3 pieces: 1/3 each was frozen in liquid nitrogen, 1/3 fixed in PFA and 1/3 cultured. Protein lysates prepared from fresh frozen tissue were processed for mass spectrometric analysis.

Results: Three proteins (EPPK1, BCLAF1 and PTMA) showed a statistically altered abundance in the endometrial tissue of RIF patients. Furthermore, pathways like metabolism, immune system, ferroptosis and the endoplasmic reticulum were altered in RIF patients. Remarkably, endometrial tissues of RIF patients showed a significantly higher (-value = 9 × 10) protein intensity correlation (Pearson's correlation coefficient = 0.95) compared to fertile women (Pearson's correlation coefficient = 0.88). The in vivo infusion of hCG stimulated proteins of endocytosis, HIF1 signalling and chemokine production. Notably, patients suffering from RIF had a clinical pregnancy rate of 19% after the intrauterine infusion of hCG before embryo transfer (ET) compared to their failed previous cycles.

Conclusion: Our study showed for the first time that the endometrial proteome composition of RIF patients differs from fertile controls during the window of implantation. The intrauterine infusion of hCG prior to an embryo transfer might improve the chemokine triggered embryo-endometrial dialogue and intensify the angiogenesis and immune response. From a clinical point of view, the hCG infusion prior to an embryo transfer might increase the pregnancy rate of RIF patients.
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http://dx.doi.org/10.3390/ijms20020425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6358950PMC
January 2019

Secretome analysis of nerve repair mediating Schwann cells reveals Smad-dependent trophism.

FASEB J 2019 04 28;33(4):4703-4715. Epub 2018 Dec 28.

Department of Neurology, Medical Faculty, Biomedical Research Center, Heinrich-Heine-University, Düsseldorf, Germany.

Schwann cells promote nerve regeneration by adaptation of a regenerative phenotype referred to as repair mediating Schwann cell. Down-regulation of myelin proteins, myelin clearance, formation of Bungner's bands, and secretion of trophic factors characterize this cell type. We have previously shown that the sphingosine-1-phosphate receptor agonist Fingolimod/FTY720P promotes the generation of this particular Schwann cell phenotype by activation of dedifferentiation markers and concomitant release of trophic factors resulting in enhanced neurite growth of dorsal root ganglion neurons. Despite its biomedical relevance, a detailed characterization of the corresponding Schwann cell secretome is lacking, and the impact of FTY720P on enhancing neurite growth is not defined. Here, we applied a label-free quantitative mass spectrometry approach to characterize the secretomes derived from primary neonatal and adult rat Schwann cells in response to FTY720P. We identified a large proportion of secreted proteins with a high overlap between the neonatal and adult Schwann cells, which can be associated with biologic processes such as development, axon growth, and regeneration. Moreover, FTY720P-treated Schwann cells release proteins downstream of Smad signaling known to support neurite growth. Our results therefore uncover a network of trophic factors involved in glial-mediated repair of the peripheral nervous system.-Schira, J., Heinen, A., Poschmann, G., Ziegler, B., Hartung, H.-P., Stühler, K., Küry, P. Secretome analysis of nerve repair mediating Schwann cells reveals Smad-dependent trophism.
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http://dx.doi.org/10.1096/fj.201801799RDOI Listing
April 2019

Cholestasis induced liver pathology results in dysfunctional immune responses after arenavirus infection.

Sci Rep 2018 08 15;8(1):12179. Epub 2018 Aug 15.

Department of Molecular Medicine II, Medical Faculty, Heinrich Heine University, Universitätsstrasse. 1, 40225, Düsseldorf, Germany.

Immune responses are critical for defense against pathogens. However, prolonged viral infection can result in defective T cell immunity, leading to chronic viral infection. We studied immune activation in response to arenavirus infection during cholestasis using bile duct ligation (BDL). We monitored T cell responses, virus load and liver pathology markers after infection with lymphocytic choriomeningitis virus (LCMV). BDL mice failed to induce protective anti-viral immunity against LCMV and consequently exhibited chronic viral infection. BDL mice exhibited reduced anti-viral T cell immunity as well as reduced type 1 interferon production early after LCMV infection. Consistently, the presence of serum from BDL mice reduced the responsiveness of dendritic cell (DC) and T cell cultures when compared to Sham controls. Following fractionation and mass spectrometry analyses of sera, we identified several serum factors to be upregulated following BDL including bilirubin, bile acids, 78 kDa Glucose regulated protein (GRP78) and liver enzymes. Bilirubin and GRP78 were capable of inhibiting DC and T cell activation. In this work, we demonstrate that liver damage mediated by cholestasis results in defective immune induction following arenavirus infection.
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http://dx.doi.org/10.1038/s41598-018-30627-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6093869PMC
August 2018

Laminin-521 promotes quiescence in isolated stellate cells from rat liver.

Biomaterials 2018 10 7;180:36-51. Epub 2018 Jul 7.

Clinic of Gastroenterology, Hepatology and Infectious Diseases, Heinrich Heine University Düsseldorf, Moorenstraße 5, 40225 Düsseldorf, Germany. Electronic address:

The laminin α5 protein chain is an element of basement membranes and important to maintain stem cells. Hepatic stellate cells (HSC) are liver-resident mesenchymal stem cells, which reside in a quiescent state on a basement membrane-like structure in the space of Dissé. In the present study, laminin α5 chain was detected in the space of Dissé of normal rat liver. Since HSC are critical for liver regeneration and can contribute to fibrosis in chronic liver diseases, the effect of laminins on HSC maintenance was investigated. Therefore, isolated rat HSC were seeded on uncoated polystyrene (PS) or PS coated with either laminin-521 (PS/LN-521) or laminin-211 (PS/LN-211). PS/LN-521 improved HSC adhesion and better preserved their retinoid stores as well as quiescence- and stem cell-associated phenotype, whereas HSC on PS/LN-211 or PS developed into myofibroblasts-like cells. To improve the homogeneity as well as the presentation of laminin molecules on the culture surface to HSC, laminin-functionalized, gold-nanostructured glass surfaces were generated. This approach further enhanced the expression of quiescence-associated genes in HSC. In conclusion, the results indicate that LN-521 supports the quiescent state of HSC and laminin α5 can be regarded as an important element of their niche in the space of Dissé.
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http://dx.doi.org/10.1016/j.biomaterials.2018.07.008DOI Listing
October 2018

Corrigendum to 'Impaired Ca cycling of nonischemic myocytes contributes to sarcomere dysfunction early after myocardial infarction' [J. Mol. Cell. Cardiol. 119 (2018) 28-39].

J Mol Cell Cardiol 2018 08 12;121:304. Epub 2018 Jun 12.

Institute of Pharmacology and Clinical Pharmacology, University Hospital Düsseldorf, Germany; Cardiovascular Research Institute Düsseldorf (CARID), Heinrich-Heine-University, Düsseldorf, Germany. Electronic address:

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http://dx.doi.org/10.1016/j.yjmcc.2018.06.004DOI Listing
August 2018

Impaired Ca cycling of nonischemic myocytes contributes to sarcomere dysfunction early after myocardial infarction.

J Mol Cell Cardiol 2018 06 16;119:28-39. Epub 2018 Apr 16.

Institute of Pharmacology and Clinical Pharmacology, University Hospital Düsseldorf, Germany; Cardiovascular Research Institute Düsseldorf (CARID), Heinrich-Heine-University, Düsseldorf, Germany. Electronic address:

Changes in the nonischemic remote myocardium of the heart contribute to left ventricular dysfunction after ischemia and reperfusion (I/R). Understanding the underlying mechanisms early after I/R is crucial to improve the adaptation of the viable myocardium to increased mechanical demands. Here, we investigated the role of myocyte Ca handling in the remote myocardium 24 h after 60 min LAD occlusion. Cardiomyocytes isolated from the basal noninfarct-related parts of wild type mouse hearts demonstrated depressed beat-to-beat Ca handling. The amplitude of the Ca transients as well as the kinetics of Ca transport were reduced by up to 25%. These changes were associated with impaired sarcomere contraction. While expression levels of Ca regulatory proteins were unchanged in remote myocardium compared to the corresponding regions of sham-operated hearts, mobility shift analyses of phosphorylated protein showed 2.9 ± 0.4-fold more unphosphorylated phospholamban (PLN) monomers, the PLN species that inhibits the Ca ATPase SERCA2a (P ≤ 0.001). Phospho-specific antibodies revealed normal phosphorylation of PLN at T17 in remote myocardium, but markedly reduced phosphorylation at its PKA-dependent phosphorylation site, S16 (P ≤ 0.01). The underlying cause involved enhanced activity of protein phosphatases, particularly PP2A (P ≤ 0.01). In contrast, overall PKA activity was normal. The PLN interactome, as determined by co-immunoprecipitation and mass spectrometry, and the phosphorylation state of PKA targets other than PLN were also unchanged. Isoproterenol enhanced cellular Ca cycling much stronger in remote myocytes than in healthy controls and improved sarcomere function. We conclude that the reduced phosphorylation state of PLN at S16 impairs myocyte Ca cycling in the remote myocardium 24 h after I/R and contributes to contractile dysfunction.
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http://dx.doi.org/10.1016/j.yjmcc.2018.04.004DOI Listing
June 2018

Targeting liver sinusoidal endothelial cells with miR-20a-loaded nanoparticles reduces murine colon cancer metastasis to the liver.

Int J Cancer 2018 08 15;143(3):709-719. Epub 2018 Mar 15.

Department of Cell Biology and Histology, Faculty of Medicine and Nursery, University of Basque Country, UPV/EHU, Leioa, Spain.

Phenotypic transformation of liver sinusoidal endothelial cells is one of the most important stages of liver metastasis progression. The miRNA effects on liver sinusoidal endothelial cells during liver metastasis have not yet been studied. Herein, whole genome analysis of miRNA expression in these cells during colorectal liver metastasis revealed repressed expression of microRNA-20a. Importantly, downregulation of miR-20a occurs in parallel with upregulation of its known protein targets. To restore normal miR-20a levels in liver sinusoidal endothelial cells, we developed chondroitin sulfate-sorbitan ester nanoparticles conjugated with miR-20a in a delivery system that specifically targets liver sinusoidal endothelial cells. The restoration of normal mir-20a levels in these cells induced downregulation of the expression of its protein targets, and this also resulted in a reduction of in vitro LSEC migration and a reduction of in vivo activation and tumor-infiltrating capacity and ability of the tumor decreased by ∼80% in a murine liver metastasis model.
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http://dx.doi.org/10.1002/ijc.31343DOI Listing
August 2018