Publications by authors named "Christian Münch"

42 Publications

A PROTAC targets splicing factor 3B1.

Cell Chem Biol 2021 May 11. Epub 2021 May 11.

Buchmann Institute for Molecular Life Sciences, Pharmaceutical Chemistry, Goethe University Frankfurt am Main, Max-von-Laue-Strasse 15. R. 3.652, 60438 Frankfurt am Main, Germany. Electronic address:

The proteolysis-targeting chimeras (PROTACs) are a new technology to degrade target proteins. However, their clinical application is limited currently by lack of chemical binders to target proteins. For instance, it is still unknown whether splicing factor 3B subunit 1 (SF3B1) is targetable by PROTACs. We recently identified a 2-aminothiazole derivative (herein O4I2) as a promoter in the generation of human pluripotent stem cells. In this work, proteomic analysis on the biotinylated O4I2 revealed that O4I2 targeted SF3B1 and positively regulated RNA splicing. Fusing thalidomide-the ligand of the cereblon ubiquitin ligase-to O4I2 led to a new PROTAC-O4I2, which selectively degraded SF3B1 and induced cellular apoptosis in a CRBN-dependent manner. In a Drosophila intestinal tumor model, PROTAC-O4I2 increased survival by interference with the maintenance and proliferation of stem cell. Thus, our finding demonstrates that SF3B1 is PROTACable by utilizing noninhibitory chemicals, which expands the list of PROTAC target proteins.
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http://dx.doi.org/10.1016/j.chembiol.2021.04.018DOI Listing
May 2021

SIK2 orchestrates actin-dependent host response upon infection.

Proc Natl Acad Sci U S A 2021 May;118(19)

Institute of Biochemistry II, Goethe University School of Medicine, 60590 Frankfurt am Main, Germany;

is an intracellular pathogen of a substantial global health concern. In order to identify key players involved in infection, we performed a global host phosphoproteome analysis subsequent to bacterial infection. Thereby, we identified the kinase SIK2 as a central component of the host defense machinery upon infection. SIK2 depletion favors the escape of bacteria from the -containing vacuole (SCV) and impairs Xenophagy, resulting in a hyperproliferative phenotype. Mechanistically, SIK2 associates with actin filaments under basal conditions; however, during bacterial infection, SIK2 is recruited to the SCV together with the elements of the actin polymerization machinery (Arp2/3 complex and Formins). Notably, SIK2 depletion results in a severe pathological cellular actin nucleation and polymerization defect upon infection. We propose that SIK2 controls the formation of a protective SCV actin shield shortly after invasion and orchestrates the actin cytoskeleton architecture in its entirety to control an acute infection after bacterial invasion.
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http://dx.doi.org/10.1073/pnas.2024144118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8126862PMC
May 2021

Virus systems biology: Proteomics profiling of dynamic protein networks during infection.

Adv Virus Res 2021 4;109:1-29. Epub 2021 Jan 4.

Institute of Biochemistry II, Faculty of Medicine, Goethe University, Frankfurt am Main, Germany; Frankfurt Cancer Institute, Frankfurt am Main, Germany; Cardio-Pulmonary Institute, Frankfurt am Main, Germany. Electronic address:

The host cell proteome undergoes a variety of dynamic changes during viral infection, elicited by the virus itself or host cell defense mechanisms. Studying these changes on a global scale by integrating functional and physical interactions within protein networks during infection is an important tool to understand pathology. Indeed, proteomics studies dissecting protein signaling cascades and interaction networks upon infection showed how global information can significantly improve understanding of disease mechanisms of diverse viral infections. Here, we summarize and give examples of different experimental designs, proteomics approaches and bioinformatics analyses that allow profiling proteome changes and host-pathogen interactions to gain a molecular systems view of viral infection.
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http://dx.doi.org/10.1016/bs.aivir.2020.12.001DOI Listing
January 2021

Characterization of Extracellular Vesicles from Preconditioned Human Adipose-Derived Stromal/Stem Cells.

Int J Mol Sci 2021 Mar 12;22(6). Epub 2021 Mar 12.

Division of Nephrology, Department of Internal Medicine III, University Hospital, Goethe-University, 60596 Frankfurt/M., Germany.

Cell-free therapy using extracellular vesicles (EVs) from adipose-derived mesenchymal stromal/stem cells (ASCs) seems to be a safe and effective therapeutic option to support tissue and organ regeneration. The application of EVs requires particles with a maximum regenerative capability and hypoxic culture conditions as an in vitro preconditioning regimen has been shown to alter the molecular composition of released EVs. Nevertheless, the EV cargo after hypoxic preconditioning has not yet been comprehensively examined. The aim of the present study was the characterization of EVs from hypoxic preconditioned ASCs. We investigated the EV proteome and their effects on renal tubular epithelial cells in vitro. While no effect of hypoxia was observed on the number of released EVs and their protein content, the cargo of the proteins was altered. Proteomic analysis showed 41 increased or decreased proteins, 11 in a statistically significant manner. Furthermore, the uptake of EVs in epithelial cells and a positive effect on oxidative stress in vitro were observed. In conclusion, culture of ASCs under hypoxic conditions was demonstrated to be a promising in vitro preconditioning regimen, which alters the protein cargo and increases the anti-oxidative potential of EVs. These properties may provide new potential therapeutic options for regenerative medicine.
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http://dx.doi.org/10.3390/ijms22062873DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7999156PMC
March 2021

SRSF3 and SRSF7 modulate 3'UTR length through suppression or activation of proximal polyadenylation sites and regulation of CFIm levels.

Genome Biol 2021 Mar 11;22(1):82. Epub 2021 Mar 11.

Institute for Molecular Bio Science, Goethe University Frankfurt, Max-von-Laue-Str. 13, 60438, Frankfurt, Germany.

Background: Alternative polyadenylation (APA) refers to the regulated selection of polyadenylation sites (PASs) in transcripts, which determines the length of their 3' untranslated regions (3'UTRs). We have recently shown that SRSF3 and SRSF7, two closely related SR proteins, connect APA with mRNA export. The mechanism underlying APA regulation by SRSF3 and SRSF7 remained unknown.

Results: Here we combine iCLIP and 3'-end sequencing and find that SRSF3 and SRSF7 bind upstream of proximal PASs (pPASs), but they exert opposite effects on 3'UTR length. SRSF7 enhances pPAS usage in a concentration-dependent but splicing-independent manner by recruiting the cleavage factor FIP1, generating short 3'UTRs. Protein domains unique to SRSF7, which are absent from SRSF3, contribute to FIP1 recruitment. In contrast, SRSF3 promotes distal PAS (dPAS) usage and hence long 3'UTRs directly by counteracting SRSF7, but also indirectly by maintaining high levels of cleavage factor Im (CFIm) via alternative splicing. Upon SRSF3 depletion, CFIm levels decrease and 3'UTRs are shortened. The indirect SRSF3 targets are particularly sensitive to low CFIm levels, because here CFIm serves a dual function; it enhances dPAS and inhibits pPAS usage by binding immediately downstream and assembling unproductive cleavage complexes, which together promotes long 3'UTRs.

Conclusions: We demonstrate that SRSF3 and SRSF7 are direct modulators of pPAS usage and show how small differences in the domain architecture of SR proteins can confer opposite effects on pPAS regulation.
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http://dx.doi.org/10.1186/s13059-021-02298-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948361PMC
March 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

Den molekularen Wirtszellveränderungen durch SARS-CoV-2 auf der Spur.

Biospektrum (Heidelb) 2021 12;27(1):40-45. Epub 2021 Feb 12.

Institut Biochemie II, Universitätsklinikum, Universität Frankfurt a. M., Theodor-Stern-Kai 7, D-60590 Frankfurt a. M., Deutschland.

Upon infection with SARS-CoV-2, a variety of changes happen inside the host cell. The virus hijacks host cell pathways for driving its own replication, while the host counteracts with response mechanisms. To gain a comprehensive understanding of COVID-19, caused by SARS-CoV-2 infection, and develop therapeutic strategies, it is crucial to observe these systematic changes in their entirety. In our recent studies, we followed the effects of SARS-CoV-2 infection on the human proteome, which led to the identification of several drugs that abolished viral proliferation in cells.
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http://dx.doi.org/10.1007/s12268-021-1535-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880639PMC
February 2021

Autophagy activation, lipotoxicity and lysosomal membrane permeabilization synergize to promote pimozide- and loperamide-induced glioma cell death.

Autophagy 2021 Jan 19:1-20. Epub 2021 Jan 19.

Experimental Neurosurgery, Goethe University Hospital Frankfurt/Main, Frankfurt, Germany.

Increasing evidence suggests that induction of lethal macroautophagy/autophagy carries potential significance for the treatment of glioblastoma (GBM). In continuation of previous work, we demonstrate that pimozide and loperamide trigger an ATG5- and ATG7 (autophagy related 5 and 7)-dependent type of cell death that is significantly reduced with cathepsin inhibitors and the lipid reactive oxygen species (ROS) scavenger α-tocopherol in MZ-54 GBM cells. Global proteomic analysis after treatment with both drugs also revealed an increase of proteins related to lipid and cholesterol metabolic processes. These changes were accompanied by a massive accumulation of cholesterol and other lipids in the lysosomal compartment, indicative of impaired lipid transport/degradation. In line with these observations, pimozide and loperamide treatment were associated with a pronounced increase of bioactive sphingolipids including ceramides, glucosylceramides and sphingoid bases measured by targeted lipidomic analysis. Furthermore, pimozide and loperamide inhibited the activity of SMPD1/ASM (sphingomyelin phosphodiesterase 1) and promoted induction of lysosomal membrane permeabilization (LMP), as well as release of CTSB (cathepsin B) into the cytosol in MZ-54 wild-type (WT) cells. Whereas LMP and cell death were significantly attenuated in and knockout (KO) cells, both events were enhanced by depletion of the lysophagy receptor VCP (valosin containing protein), supporting a pro-survival function of lysophagy under these conditions. Collectively, our data suggest that pimozide and loperamide-driven autophagy and lipotoxicity synergize to induce LMP and cell death. The results also support the notion that simultaneous overactivation of autophagy and induction of LMP represents a promising approach for the treatment of GBM.: ACD: autophagic cell death; AKT1: AKT serine/threonine kinase 1; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG14: autophagy related 14; CERS1: ceramide synthase 1; CTSB: cathepsin B; CYBB/NOX2: cytochrome b-245 beta chain; ER: endoplasmatic reticulum; FBS: fetal bovine serum; GBM: glioblastoma; GO: gene ontology; HTR7/5-HT7: 5-hydroxytryptamine receptor 7; KD: knockdown; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LAP: LC3-associated phagocytosis; LMP: lysosomal membrane permeabilization; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; RB1CC1: RB1 inducible coiled-coil 1; ROS: reactive oxygen species; RPS6: ribosomal protein S6; SMPD1/ASM: sphingomyelin phosphodiesterase 1; VCP/p97: valosin containing protein; WT: wild-type.
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http://dx.doi.org/10.1080/15548627.2021.1874208DOI Listing
January 2021

A Spatial and Functional Interaction of a Heterotetramer Survivin-DNA-PKcs Complex in DNA Damage Response.

Cancer Res 2021 May 6;81(9):2304-2317. Epub 2021 Jan 6.

Department of Radiotherapy and Oncology, University Hospital, Goethe University Frankfurt, Germany.

Substantial evidence has shown that overexpression of the inhibitor of apoptosis protein (IAP) survivin in human tumors correlates significantly with treatment resistance and poor patient prognosis. Survivin serves as a radiation resistance factor that impacts the DNA damage response by interacting with DNA-dependent protein kinase (DNA-PKcs). However, the complexity, molecular determinants, and functional consequences of this interrelationship remain largely unknown. By applying coimmunoprecipitation and flow cytometry-based Förster resonance energy transfer assays, we demonstrated a direct involvement of the survivin baculovirus IAP repeat domain in the regulation of radiation survival and DNA repair. This survivin-mediated activity required an interaction of residues S20 and W67 with the phosphoinositide 3-kinase (PI3K) domain of DNA-PKcs. molecular docking and dynamics simulation analyses, kinase assays, and large-scale mass spectrometry suggested a heterotetrameric survivin-DNA-PKcs complex that results in a conformational change within the DNA-PKcs PI3K domain. Overexpression of survivin resulted in enhanced PI3K enzymatic activity and detection of differentially abundant phosphopeptides and proteins implicated in the DNA damage response. The survivin-DNA-PKcs interaction altered the S/T-hydrophobic motif substrate specificity of DNA-PKcs with a predominant usage of S/T-P phosphorylation sites and an increase of DNA-PKcs substrates including Foxo3. These data demonstrate that survivin differentially regulates DNA-PKcs-dependent radiation survival and DNA double-strand break repair via formation of a survivin-DNA-PKcs heterotetrameric complex. SIGNIFICANCE: These findings provide insight into survivin-mediated regulation of DNA-PKcs kinase and broaden our knowledge of the impact of survivin in modulating the cellular radiation response. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/9/2304/F1.large.jpg.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-2931DOI Listing
May 2021

BAG3 Proteomic Signature under Proteostasis Stress.

Cells 2020 11 4;9(11). Epub 2020 Nov 4.

Institute of Pathobiochemistry, The Autophagy Lab, University Medical Center of the Johannes Gutenberg University, Duesbergweg 6, 55128 Mainz, Germany.

The multifunctional HSP70 co-chaperone BAG3 (BCL-2-associated athanogene 3) represents a key player in the quality control of the cellular proteostasis network. In response to stress BAG3 specifically targets aggregation-prone proteins to the perinuclear aggresome and promotes their degradation via BAG3-mediated selective macroautophagy. To adapt cellular homeostasis to stress, BAG3 modulates and functions in various cellular processes and signaling pathways. Noteworthy, dysfunction and deregulation of BAG3 and its pathway are pathophysiologically linked to myopathies, cancer, and neurodegenerative disorders. Here, we report a BAG3 proteomic signature under proteostasis stress. To elucidate the dynamic and multifunctional action of BAG3 in response to stress, we established BAG3 interactomes under basal and proteostasis stress conditions by employing affinity purification combined with quantitative mass spectrometry. In addition to the identification of novel potential BAG3 interactors, we defined proteins whose interaction with BAG3 was altered upon stress. By functional annotation and protein-protein interaction enrichment analysis of the identified potential BAG3 interactors, we confirmed the multifunctionality of BAG3 and highlighted its crucial role in diverse cellular signaling pathways and processes, ensuring cellular proteostasis and cell viability. These include protein folding and degradation, gene expression, cytoskeleton dynamics (including cell cycle and transport), as well as granulostasis, in particular.
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http://dx.doi.org/10.3390/cells9112416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7694386PMC
November 2020

Aprotinin Inhibits SARS-CoV-2 Replication.

Cells 2020 10 30;9(11). Epub 2020 Oct 30.

Institute for Medical Virology, University Hospital, Goethe University, 60596 Frankfurt am Main, Germany.

Severe acute respiratory syndrome virus 2 (SARS-CoV-2) is the cause of the current coronavirus disease 19 (COVID-19) pandemic. Protease inhibitors are under consideration as virus entry inhibitors that prevent the cleavage of the coronavirus spike (S) protein by cellular proteases. Herein, we showed that the protease inhibitor aprotinin (but not the protease inhibitor SERPINA1/alpha-1 antitrypsin) inhibited SARS-CoV-2 replication in therapeutically achievable concentrations. An analysis of proteomics and translatome data indicated that SARS-CoV-2 replication is associated with a downregulation of host cell protease inhibitors. Hence, aprotinin may compensate for downregulated host cell proteases during later virus replication cycles. Aprotinin displayed anti-SARS-CoV-2 activity in different cell types (Caco2, Calu-3, and primary bronchial epithelial cell air-liquid interface cultures) and against four virus isolates. In conclusion, therapeutic aprotinin concentrations exert anti-SARS-CoV-2 activity. An approved aprotinin aerosol may have potential for the early local control of SARS-CoV-2 replication and the prevention of COVID-19 progression to a severe, systemic disease.
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http://dx.doi.org/10.3390/cells9112377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692688PMC
October 2020

Unbiased translation proteomics upon cell stress.

Mol Cell Oncol 2020 18;7(4):1763150. Epub 2020 May 18.

Institute of Biochemistry II, Faculty of Medicine, Goethe University, Frankfurt Am Main, Germany.

The mammalian target of rapamycin and the integrated stress response are central cellular hubs regulating translation upon stress. The precise proteins and pathway specificity of translation targets of these pathways remained largely unclear. We recently described a new method for quantitative translation proteomics and found that both pathways control translation of the same sets of proteins.
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http://dx.doi.org/10.1080/23723556.2020.1763150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7469668PMC
May 2020

Growth Factor Receptor Signaling Inhibition Prevents SARS-CoV-2 Replication.

Mol Cell 2020 10 11;80(1):164-174.e4. Epub 2020 Aug 11.

Institute of Medical Virology, University Hospital Frankfurt, Frankfurt am Main, Germany. Electronic address:

SARS-CoV-2 infections are rapidly spreading around the globe. The rapid development of therapies is of major importance. However, our lack of understanding of the molecular processes and host cell signaling events underlying SARS-CoV-2 infection hinders therapy development. We use a SARS-CoV-2 infection system in permissible human cells to study signaling changes by phosphoproteomics. We identify viral protein phosphorylation and define phosphorylation-driven host cell signaling changes upon infection. Growth factor receptor (GFR) signaling and downstream pathways are activated. Drug-protein network analyses revealed GFR signaling as key pathways targetable by approved drugs. The inhibition of GFR downstream signaling by five compounds prevents SARS-CoV-2 replication in cells, assessed by cytopathic effect, viral dsRNA production, and viral RNA release into the supernatant. This study describes host cell signaling events upon SARS-CoV-2 infection and reveals GFR signaling as a central pathway essential for SARS-CoV-2 replication. It provides novel strategies for COVID-19 treatment.
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http://dx.doi.org/10.1016/j.molcel.2020.08.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418786PMC
October 2020

COVID-19-Related Coagulopathy-Is Transferrin a Missing Link?

Diagnostics (Basel) 2020 Jul 30;10(8). Epub 2020 Jul 30.

Institute for Medical Virology, University Hospital, Goethe University, 60596 Frankfurt am Main, Germany.

SARS-CoV-2 is the causative agent of COVID-19. Severe COVID-19 disease has been associated with disseminated intravascular coagulation and thrombosis, but the mechanisms underlying COVID-19-related coagulopathy remain unknown. The risk of severe COVID-19 disease is higher in males than in females and increases with age. To identify gene products that may contribute to COVID-19-related coagulopathy, we analyzed the expression of genes associated with the Gene Ontology (GO) term "blood coagulation" in the Genotype-Tissue Expression (GTEx) database and identified four procoagulants, whose expression is higher in males and increases with age (ADAMTS13, F11, HGFAC, KLKB1), and two anticoagulants, whose expression is higher in females and decreases with age (C1QTNF1, SERPINA5). However, the expression of none of these genes was regulated in a proteomics dataset of SARS-CoV-2-infected cells and none of the proteins have been identified as a binding partner of SARS-CoV-2 proteins. Hence, they may rather generally predispose individuals to thrombosis without directly contributing to COVID-19-related coagulopathy. In contrast, the expression of the procoagulant transferrin (not associated to the GO term "blood coagulation") was higher in males, increased with age, and was upregulated upon SARS-CoV-2 infection. Hence, transferrin warrants further examination in ongoing clinic-pathological investigations.
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http://dx.doi.org/10.3390/diagnostics10080539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7459734PMC
July 2020

The Lipid Receptor G2A (GPR132) Mediates Macrophage Migration in Nerve Injury-Induced Neuropathic Pain.

Cells 2020 07 21;9(7). Epub 2020 Jul 21.

Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Branch for Translational Medicine and Pharmacology TMP, Theodor-Stern-Kai 7, 60596 Frankfurt am Main, Germany.

Nerve injury-induced neuropathic pain is difficult to treat and mechanistically characterized by strong neuroimmune interactions, involving signaling lipids that act via specific G-protein coupled receptors. Here, we investigated the role of the signaling lipid receptor G2A (GPR132) in nerve injury-induced neuropathic pain using the robust spared nerve injury (SNI) mouse model. We found that the concentrations of the G2A agonist 9-HODE (9-Hydroxyoctadecadienoic acid) are strongly increased at the site of nerve injury during neuropathic pain. Moreover, G2A-deficient mice show a strong reduction of mechanical hypersensitivity after nerve injury. This phenotype is accompanied by a massive reduction of invading macrophages and neutrophils in G2A-deficient mice and a strongly reduced release of the proalgesic mediators TNFα, IL-6 and VEGF at the site of injury. Using a global proteome analysis to identify the underlying signaling pathways, we found that G2A activation in macrophages initiates MyD88-PI3K-AKT signaling and transient MMP9 release to trigger cytoskeleton remodeling and migration. We conclude that G2A-deficiency reduces inflammatory responses by decreasing the number of immune cells and the release of proinflammatory cytokines and growth factors at the site of nerve injury. Inhibiting the G2A receptor after nerve injury may reduce immune cell-mediated peripheral sensitization and may thus ameliorate neuropathic pain.
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http://dx.doi.org/10.3390/cells9071740DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7409160PMC
July 2020

Instrument Logic Increases Identifications during Mutliplexed Translatome Measurements.

Anal Chem 2020 06 29;92(12):8041-8045. Epub 2020 May 29.

Institute of Biochemistry II, Faculty of Medicine, Goethe University, 60590 Frankfurt am Main, Germany.

Pulsed Stable Isotope Labeling in Cell culture (SILAC) approaches allow measurement of protein dynamics, including protein translation and degradation. However, its use for quantifying acute changes has been limited due to low labeled peptide stoichiometry. Here, we describe the use of instrument logic to select peaks of interest via targeted mass differences (TMD) for overcoming this limitation. Comparing peptides artificially mixed at low heavy-to-light stoichiometry measured using standard data dependent acquisition with or without TMD revealed 2-3-fold increases in identification without significant loss in quantification precision for both MS and MS methods. Our benchmarked method approach increased throughput by reducing the necessary machine time. We anticipate that all pulsed SILAC measurements, combined with tandem mass tagging (TMT) or not, would greatly benefit from instrument logic based approaches.
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http://dx.doi.org/10.1021/acs.analchem.0c01749DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7537974PMC
June 2020

Proteomics of SARS-CoV-2-infected host cells reveals therapy targets.

Nature 2020 07 14;583(7816):469-472. Epub 2020 May 14.

Institute of Biochemistry II, Faculty of Medicine, Goethe University, Frankfurt am Main, Germany.

A new coronavirus was recently discovered and named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Infection with SARS-CoV-2 in humans causes coronavirus disease 2019 (COVID-19) and has been rapidly spreading around the globe. SARS-CoV-2 shows some similarities to other coronaviruses; however, treatment options and an understanding of how SARS-CoV-2 infects cells are lacking. Here we identify the host cell pathways that are modulated by SARS-CoV-2 and show that inhibition of these pathways prevents viral replication in human cells. We established a human cell-culture model for infection with a clinical isolate of SARS-CoV-2. Using this cell-culture system, we determined the infection profile of SARS-CoV-2 by translatome and proteome proteomics at different times after infection. These analyses revealed that SARS-CoV-2 reshapes central cellular pathways such as translation, splicing, carbon metabolism, protein homeostasis (proteostasis) and nucleic acid metabolism. Small-molecule inhibitors that target these pathways prevented viral replication in cells. Our results reveal the cellular infection profile of SARS-CoV-2 and have enabled the identification of drugs that inhibit viral replication. We anticipate that our results will guide efforts to understand the molecular mechanisms that underlie the modulation of host cells after infection with SARS-CoV-2. Furthermore, our findings provide insights for the development of therapies for the treatment of COVID-19.
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http://dx.doi.org/10.1038/s41586-020-2332-7DOI Listing
July 2020

Loss of mitochondrial ClpP, Lonp1, and Tfam triggers transcriptional induction of Rnf213, a susceptibility factor for moyamoya disease.

Neurogenetics 2020 07 28;21(3):187-203. Epub 2020 Apr 28.

Experimental Neurology, Goethe University Medical School, 60590, Frankfurt am Main, Germany.

Human RNF213, which encodes the protein mysterin, is a known susceptibility gene for moyamoya disease (MMD), a cerebrovascular condition with occlusive lesions and compensatory angiogenesis. Mysterin mutations, together with exposure to environmental trigger factors, lead to an elevated stroke risk since childhood. Mysterin is induced during cell stress, to function as cytosolic AAA+ ATPase and ubiquitylation enzyme. Little knowledge exists, in which context mysterin is needed. Here, we found that genetic ablation of several mitochondrial matrix factors, such as the peptidase ClpP, the transcription factor Tfam, as well as the peptidase and AAA+ ATPase Lonp1, potently induces Rnf213 transcript expression in various organs, in parallel with other components of the innate immune system. Mostly in mouse fibroblasts and human endothelial cells, the Rnf213 levels showed prominent upregulation upon Poly(I:C)-triggered TLR3-mediated responses to dsRNA toxicity, as well as upon interferon gamma treatment. Only partial suppression of Rnf213 induction was achieved by C16 as an antagonist of PKR (dsRNA-dependent protein kinase). Since dysfunctional mitochondria were recently reported to release immune-stimulatory dsRNA into the cytosol, our results suggest that mysterin becomes relevant when mitochondrial dysfunction or infections have triggered RNA-dependent inflammation. Thus, MMD has similarities with vasculopathies that involve altered nucleotide processing, such as Aicardi-Goutières syndrome or systemic lupus erythematosus. Furthermore, in MMD, the low penetrance of RNF213 mutations might be modified by dysfunctions in mitochondria or the TLR3 pathway.
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http://dx.doi.org/10.1007/s10048-020-00609-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7283203PMC
July 2020

Serine-dependent redox homeostasis regulates glioblastoma cell survival.

Br J Cancer 2020 04 17;122(9):1391-1398. Epub 2020 Mar 17.

Dr. Senckenberg Institute of Neurooncology, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany.

Background: The amino acid serine is an important substrate for biosynthesis and redox homeostasis. We investigated whether glioblastoma (GBM) cells are dependent on serine for survival under conditions of the tumour microenvironment.

Methods: Serine availability in GBM cells was modulated pharmacologically, genetically and by adjusting serine and glycine concentrations in the culture medium. Cells were investigated for regulation of serine metabolism, proliferation, sensitivity to hypoxia-induced cell death and redox homeostasis.

Results: Hypoxia-induced expression of phosphoglycerate dehydrogenase (PHGDH) and the mitochondrial serine hydroxymethyltransferase (SHMT2) was observed in three of five tested glioma cell lines. Nuclear factor erythroid 2-related factor (Nrf) 2 activation also induced PHGDH and SHMT2 expression in GBM cells. Low levels of endogenous PHGDH as well as PHGDH gene suppression resulted in serine dependency for cell growth. Pharmacological inhibition of PHGDH with CBR-5884 reduced proliferation and sensitised cells profoundly to hypoxia-induced cell death. This effect was accompanied by an increase in reactive oxygen species and a decrease in the NADPH/NADP ratio. Similarly, hypoxia-induced cell death was enhanced by PHGDH gene suppression and reduced by PHGDH overexpression.

Conclusions: Serine facilitates adaptation of GBM cells to conditions of the tumour microenvironment and its metabolism could be a plausible therapeutic target.
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http://dx.doi.org/10.1038/s41416-020-0794-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7188854PMC
April 2020

Maintaining protein stability of ∆Np63 via USP28 is required by squamous cancer cells.

EMBO Mol Med 2020 04 4;12(4):e11101. Epub 2020 Mar 4.

Department of Biochemistry and Molecular Biology, Protein Stability and Cancer Group, University of Würzburg, Würzburg, Germany.

The transcription factor ∆Np63 is a master regulator of epithelial cell identity and essential for the survival of squamous cell carcinoma (SCC) of lung, head and neck, oesophagus, cervix and skin. Here, we report that the deubiquitylase USP28 stabilizes ∆Np63 and maintains elevated ∆NP63 levels in SCC by counteracting its proteasome-mediated degradation. Impaired USP28 activity, either genetically or pharmacologically, abrogates the transcriptional identity and suppresses growth and survival of human SCC cells. CRISPR/Cas9-engineered in vivo mouse models establish that endogenous USP28 is strictly required for both induction and maintenance of lung SCC. Our data strongly suggest that targeting ∆Np63 abundance via inhibition of USP28 is a promising strategy for the treatment of SCC tumours.
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http://dx.doi.org/10.15252/emmm.201911101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7136964PMC
April 2020

Functional Translatome Proteomics Reveal Converging and Dose-Dependent Regulation by mTORC1 and eIF2α.

Mol Cell 2020 02 4;77(4):913-925.e4. Epub 2019 Dec 4.

Institute of Biochemistry II, Faculty of Medicine, Goethe University, Frankfurt am Main, Germany; Frankfurt Cancer Institute, Frankfurt am Main, Germany; Cardio-Pulmonary Institute, Frankfurt am Main, Germany. Electronic address:

Regulation of translation is essential during stress. However, the precise sets of proteins regulated by the key translational stress responses-the integrated stress response (ISR) and mTORC1-remain elusive. We developed multiplexed enhanced protein dynamics (mePROD) proteomics, adding signal amplification to dynamic-SILAC and multiplexing, to enable measuring acute changes in protein synthesis. Treating cells with ISR/mTORC1-modulating stressors, we showed extensive translatome modulation with ∼20% of proteins synthesized at highly reduced rates. Comparing translation-deficient sub-proteomes revealed an extensive overlap demonstrating that target specificity is achieved on protein level and not by pathway activation. Titrating cap-dependent translation inhibition confirmed that synthesis of individual proteins is controlled by intrinsic properties responding to global translation attenuation. This study reports a highly sensitive method to measure relative translation at the nascent chain level and provides insight into how the ISR and mTORC1, two key cellular pathways, regulate the translatome to guide cellular survival upon stress.
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http://dx.doi.org/10.1016/j.molcel.2019.11.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033560PMC
February 2020

TA*p63 and GTAp63 achieve tighter transcriptional regulation in quality control by converting an inhibitory element into an additional transactivation domain.

Cell Death Dis 2019 09 17;10(10):686. Epub 2019 Sep 17.

Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance and Cluster of Excellence Macromolecular Complexes (CEF), Goethe University, Frankfurt/Main, Germany.

The p53 homolog p63 plays important roles in development of epithelial tissues and quality control in germ cells. These two functions are executed by two distinct isoforms of p63. They are created by different promotors resulting in isoforms having either an N-terminal transactivation domain (TAp63) or a truncated form (ΔNp63). In addition to these two N-terminal isoforms a third one with an even longer N-terminus, named TA*p63, has been found. A fourth N-terminal isoform, GTAp63, that closely resembles TA*p63 was discovered in male germ cells where it is involved in genetic quality control. Here, we characterize TA*p63α and GTAp63α and show that their N-terminal extensions stabilize the closed and only dimeric conformation adopted by the shorter TAp63α protein. Both proteins can be activated by the two kinases Chk2 and CK1 resulting in the open tetrameric state. In this conformation, the N-terminal extension acts as an additional transactivation domain enhancing transcriptional activity. Through this mechanism, the difference in transcriptional activity between the repressed and the active state of the protein gets enhanced relative to TAp63α. Finally, we show by mass spectrometry that TA*p63α is expressed in the breast cancer cell line Sum159 at the protein level together with mutant p53. Upon doxorubicin treatment, TA*p63α gets activated, providing a potential new tool to fight cancer.
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http://dx.doi.org/10.1038/s41419-019-1936-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6746776PMC
September 2019

Novel Method for NTC Thermistor Production by Aerosol Co-Deposition and Combined Sintering.

Sensors (Basel) 2019 Apr 5;19(7). Epub 2019 Apr 5.

Department of Functional Materials, Universität Bayreuth, 95440 Bayreuth, Germany.

A novel three-stage process to produce NTCR sensors is presented. In this process, an uncalcined powder mixture of NiO and Mn₂O₃ was deposited onto an alumina substrate via aerosol co-deposition (AcD). Then, an electrode structure was screen-printed onto the surface and the composite film was sintered in a multifunctional temperature treatment. Thereby, the sintering of the electrode, the formation of the NiMn₂O₄ spinel and the removal of film strains took place simultaneously. This enabled a significant reduction in energy demand and workload. The manufactured sensors, both as first prototypes, as well as miniaturized chip components, were characterized by a single-phase cubic NiMn₂O₄ spinel structure, mechanical stability and electrical properties that were similar to those of classical NiMn₂O₄ bulk ceramics or tempered aerosol deposited (AD) NiMn₂O₄ films. Particularly noteworthy was the high reproducibility and low variation of the NTCR parameters, such as the specific resistivity at 25 °C , the electrical resistance at 25 °C and the thermistor constant . The NTCR parameters were as aging-stable as for NiMn₂O₄ bulk ceramics or tempered NiMn₂O₄ AD-films and could even be further improved by thermal post-treatment.
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http://dx.doi.org/10.3390/s19071632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6480331PMC
April 2019

Biglycan evokes autophagy in macrophages via a novel CD44/Toll-like receptor 4 signaling axis in ischemia/reperfusion injury.

Kidney Int 2019 03 31;95(3):540-562. Epub 2019 Jan 31.

Pharmazentrum Frankfurt/ZAFES, Institut für Allgemeine Pharmakologie und Toxikologie, Klinikum der JW Goethe-Universität Frankfurt am Main, Frankfurt, Germany. Electronic address:

Biglycan, a small leucine-rich proteoglycan, acts as a danger signal and is classically thought to promote macrophage recruitment via Toll-like receptors (TLR) 2 and 4. We have recently shown that biglycan signaling through TLR 2/4 and the CD14 co-receptor regulates inflammation, suggesting that TLR co-receptors may determine whether biglycan-TLR signaling is pro- or anti-inflammatory. Here, we sought to identify other co-receptors and characterize their impact on biglycan-TLR signaling. We found a marked increase in the number of autophagic macrophages in mice stably overexpressing soluble biglycan. In vitro, stimulation of murine macrophages with biglycan triggered autophagosome formation and enhanced the flux of autophagy markers. Soluble biglycan also promoted autophagy in human peripheral blood macrophages. Using macrophages from mice lacking TLR2 and/or TLR4, CD14, or CD44, we demonstrated that the pro-autophagy signal required TLR4 interaction with CD44, a receptor involved in adhesion, migration, lymphocyte activation, and angiogenesis. In vivo, transient overexpression of circulating biglycan at the onset of renal ischemia/reperfusion injury (IRI) enhanced M1 macrophage recruitment into the kidneys of Cd44 and Cd44 mice but not Cd14 mice. The biglycan-CD44 interaction increased M1 autophagy and the number of renal M2 macrophages and reduced tubular damage following IRI. Thus, CD44 is a novel signaling co-receptor for biglycan, an interaction that is required for TLR4-CD44-dependent pro-autophagic activity in macrophages. Interfering with the interaction between biglycan and specific TLR co-receptors could represent a promising therapeutic intervention to curtail kidney inflammation and damage.
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http://dx.doi.org/10.1016/j.kint.2018.10.037DOI Listing
March 2019

Loss of the selective autophagy receptor p62 impairs murine myeloid leukemia progression and mitophagy.

Blood 2019 01 29;133(2):168-179. Epub 2018 Nov 29.

Department of Medicine, Hematology/Oncology, Goethe University, Frankfurt, Germany.

Autophagy maintains hematopoietic stem cell integrity and prevents malignant transformation. In addition to bulk degradation, selective autophagy serves as an intracellular quality control mechanism and requires autophagy receptors, such as p62 (SQSTM1), to specifically bridge the ubiquitinated cargos into autophagosomes. Here, we investigated the function of p62 in acute myeloid leukemia (AML) in vitro and in murine in vivo models of AML. Loss of p62 impaired expansion and colony-forming ability of leukemia cells and prolonged latency of leukemia development in mice. High p62 expression was associated with poor prognosis in human AML. Using quantitative mass spectrometry, we identified enrichment of mitochondrial proteins upon immunoprecipitation of p62. Loss of p62 significantly delayed removal of dysfunctional mitochondria, increased mitochondrial superoxide levels, and impaired mitochondrial respiration. Moreover, we demonstrated that the autophagy-dependent function of p62 is essential for cell growth and effective mitochondrial degradation by mitophagy. Our results highlight the prominent role of selective autophagy in leukemia progression, and specifically, the importance of mitophagy to maintain mitochondrial integrity.
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http://dx.doi.org/10.1182/blood-2018-02-833475DOI Listing
January 2019

Thermal Treatment of Aerosol Deposited NiMn₂O₄ NTC Thermistors for Improved Aging Stability.

Sensors (Basel) 2018 Nov 15;18(11). Epub 2018 Nov 15.

Department of Functional Materials, Universität Bayreuth, 95440 Bayreuth, Germany.

This paper examines the influence of a short-term thermal treatment of aerosol deposited negative temperature coefficient (NTC) thermistor films on the NTCR characteristics and their long-term stability with different electrode materials. An aerosol deposition of a spinel-based NiMn₂O₄ powder on alumina substrates with screen-printed AgPd and Au interdigital electrode structures was performed. The manufactured components of the typical size of 1206 were tempered in a moderate temperature range of 200 °C to 800 °C and aged for 1000 h at 125 °C in air. Based on - measurements in a high-precision silicone oil thermostat bath and high temperature XRD analyses, the influence of the thermal treatment was analyzed and discussed. A 60-min tempering at 400 °C proved to be optimal, as both the NTCR parameters and their ageing stability could be significantly improved. The findings are explained.
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http://dx.doi.org/10.3390/s18113982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6263728PMC
November 2018

The different axes of the mammalian mitochondrial unfolded protein response.

Authors:
Christian Münch

BMC Biol 2018 07 26;16(1):81. Epub 2018 Jul 26.

Institute of Biochemistry II, Goethe University - Medical Faculty, University Hospital, Frankfurt am Main, Germany.

Mitochondria are sensitive to numerous environmental stresses, which can lead to activation of mitochondrial stress responses (MSRs). Of particular recent interest has been the mitochondrial unfolded protein response (UPR), activated to restore protein homeostasis (proteostasis) upon mitochondrial protein misfolding. Several axes of the UPR have been described, creating some confusion as to the nature of the different responses. While distinct molecularly, these different axes are likely mutually beneficial and activated in parallel. This review aims at describing and distinguishing the different mammalian MSR/UPR axes to define key processes and members and to examine the involvement of protein misfolding.
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http://dx.doi.org/10.1186/s12915-018-0548-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6060479PMC
July 2018

AT 101 induces early mitochondrial dysfunction and HMOX1 (heme oxygenase 1) to trigger mitophagic cell death in glioma cells.

Autophagy 2018 21;14(10):1693-1709. Epub 2018 Jul 21.

a Experimental Neurosurgery , Goethe University Hospital Frankfurt/Main , Germany.

In most cases, macroautophagy/autophagy serves to alleviate cellular stress and acts in a pro-survival manner. However, the effects of autophagy are highly contextual, and autophagic cell death (ACD) is emerging as an alternative paradigm of (stress- and drug-induced) cell demise. AT 101 ([-]-gossypol), a natural compound from cotton seeds, induces ACD in glioma cells as confirmed here by CRISPR/Cas9 knockout of ATG5 that partially, but significantly rescued cell survival following AT 101 treatment. Global proteomic analysis of AT 101-treated U87MG and U343 glioma cells revealed a robust decrease in mitochondrial protein clusters, whereas HMOX1 (heme oxygenase 1) was strongly upregulated. AT 101 rapidly triggered mitochondrial membrane depolarization, engulfment of mitochondria within autophagosomes and a significant reduction of mitochondrial mass and proteins that did not depend on the presence of BAX and BAK1. Conversely, AT 101-induced reduction of mitochondrial mass could be reversed by inhibiting autophagy with wortmannin, bafilomycin A and chloroquine. Silencing of HMOX1 and the mitophagy receptors BNIP3 (BCL2 interacting protein 3) and BNIP3L (BCL2 interacting protein 3 like) significantly attenuated AT 101-dependent mitophagy and cell death. Collectively, these data suggest that early mitochondrial dysfunction and HMOX1 overactivation synergize to trigger lethal mitophagy, which contributes to the cell killing effects of AT 101 in glioma cells.

Abbreviations: ACD, autophagic cell death; ACN, acetonitrile; AT 101, (-)-gossypol; BAF, bafilomycin A; BAK1, BCL2-antagonist/killer 1; BAX, BCL2-associated X protein; BH3, BCL2 homology region 3; BNIP3, BCL2 interacting protein 3; BNIP3L, BCL2 interacting protein 3 like; BP, Biological Process; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; CC, Cellular Component; Con, control; CQ, chloroquine; CRISPR, clustered regularly interspaced short palindromic repeats; DMEM, Dulbecco's Modified Eagle Medium; DTT, 1,4-dithiothreitol; EM, electron microscopy; ER, endoplasmatic reticulum; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; GO, Gene Ontology; HAcO, acetic acid; HMOX1, heme oxygenase 1; DKO, double knockout; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry; LPL, lipoprotein lipase, MEFs, mouse embryonic fibroblasts; mPTP, mitochondrial permeability transition pore; MTG, MitoTracker Green FM; mt-mKeima, mito-mKeima; MT-ND1, mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1; PBS, phosphate-buffered saline; PE, phosphatidylethanolamine; PI, propidium iodide; PRKN, parkin RBR E3 ubiquitin protein ligase; SDS, sodium dodecyl sulfate; SQSTM1/p62, sequestome 1; STS, staurosporine; sgRNA, single guide RNA; SILAC, stable isotope labeling with amino acids in cell culture; TFA, trifluoroacetic acid, TMRM, tetramethylrhodamine methyl ester perchlorate; WM, wortmannin; WT, wild-type.
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http://dx.doi.org/10.1080/15548627.2018.1476812DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6135628PMC
October 2019

Publisher Correction: Hitchhiking on selective autophagy.

Nat Cell Biol 2018 08;20(8):990

Institute of Biochemistry II, Goethe University - Medical Faculty, University Hospital, Frankfurt am Main, Germany.

In the version of this News & Views originally published, owing to a technical error some incorrect characters were introduced into the second sentence of the main text, starting "Autophagy can be divided into non-selective...", where "[email protected]" should have read 'can'. This has now been corrected in all versions of this News & Views.
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http://dx.doi.org/10.1038/s41556-018-0051-1DOI Listing
August 2018

Hitchhiking on selective autophagy.

Nat Cell Biol 2018 02;20(2):122-124

Institute of Biochemistry II, Goethe University - Medical Faculty, University Hospital, Frankfurt am Main, Germany.

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http://dx.doi.org/10.1038/s41556-018-0036-0DOI Listing
February 2018