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  • 1. Alex, Amal
    et al.
    Piano, Valentina
    Polley, Soumitra
    Stuiver, Marchel
    Voss, Stephanie
    Ciossani, Giuseppe
    Overlack, Katharina
    Voss, Beate
    Wohlgemuth, Sabine
    Petrovic, Arsen
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Selenko, Philipp
    Musacchio, Andrea
    Maffini, Stefano
    Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology2019Ingår i: eLIFE, E-ISSN 2050-084X, Vol. 8, artikel-id e48287Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Delivery of native or chemically modified recombinant proteins into mammalian cells shows promise for functional investigations and various technological applications, but concerns that sub-cellular localization and functional integrity of delivered proteins may be affected remain high. Here, we surveyed batch electroporation as a delivery tool for single polypeptides and multi-subunit protein assemblies of the kinetochore, a spatially confined and well-studied subcellular structure. After electroporation into human cells, recombinant fluorescent Ndc80 and Mis12 multi-subunit complexes exhibited native localization, physically interacted with endogenous binding partners, and functionally complemented depleted endogenous counterparts to promote mitotic checkpoint signaling and chromosome segregation. Farnesylation is required for kinetochore localization of the Dynein adaptor Spindly. In cells with chronically inhibited farnesyl transferase activity, in vitro farnesylation and electroporation of recombinant Spindly faithfully resulted in robust kinetochore localization. Our data show that electroporation is well-suited to deliver synthetic and chemically modified versions of functional proteins, and, therefore, constitutes a promising tool for applications in chemical and synthetic biology.

  • 2. Carnero Corrales, Marjorie A.
    et al.
    Zinken, Sarah
    Konstantinidis, Georgios
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Chemical Genomics Center of the Max Planck Society, Dortmund, Germany.
    Rafehi, Muhammad
    Abdelrahman, Aliaa
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Chemical Genomics Center of the Max Planck Society, Dortmund, Germany.
    Janning, Petra
    Müller, Christa E.
    Laraia, Luca
    Waldmann, Herbert
    Thermal proteome profiling identifies the membrane-bound purinergic receptor P2X4 as a target of the autophagy inhibitor indophagolin2021Ingår i: Cell Chemical Biology, ISSN 2451-9456, E-ISSN 2451-9448, Vol. 28, nr 12, s. 1750-1757.e5Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Signaling pathways are frequently activated through signal-receiving membrane proteins, and the discovery ofsmall molecules targeting these receptors may yield insights into their biology. However, due to their intrinsicproperties,membrane protein targets often cannot be identified bymeans of established approaches, in particularaffinity-based proteomics, calling for the exploration of new methods. Here, we report the identification ofindophagolin as representative member of an indoline-based class of autophagy inhibitors through a targetagnosticphenotypic assay. Thermal proteome profiling and subsequent biochemical validation identified thepurinergic receptor P2X4 as a target of indophagolin, and subsequent investigations suggest that indophagolintargets further purinergic receptors. These results demonstrate that thermal proteome profiling may enable thede novo identification of membrane-bound receptors as cellular targets of bioactive small molecules.

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  • 3.
    Chen, Xi
    et al.
    Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Str. 15, Dortmund, Germany; Max Planck Institute for Molecular Physiology, Dortmund, Germany.
    Li, Fu
    Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Str. 15, Dortmund, Germany; Max Planck Institute for Molecular Physiology, Dortmund, Germany.
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Str. 15, Dortmund, Germany; Max Planck Institute for Molecular Physiology, Dortmund, Germany.
    Affinity conjugation for rapid and covalent labeling of proteins in live cells2019Ingår i: Proximity labeling: methods and protocols / [ed] Murat Sunbul; Andres Jäschke, New York: Humana Press, 2019, , s. 12s. 191-202Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    Protein labeling is enormously useful for characterization of protein function in live cells and study of the related cellular processes. Covalent labeling of protein using affinity conjugation confers stable and selective labeling of protein in cells. Affinity conjugation combines a specific ligand-protein interaction with a proximity-induced reaction to selectively label the protein of interest (POI) in the cell. Therefore, either a fluorogenic probe is directly introduced to the POI or a bioorthogonal group is incorporated to the POI, which is subsequently labeled with a fluorescent probe. Here, we describe a method for affinity conjugation of protein with a fluorogenic probe and a "tagging-then-labeling" approach by a combination of affinity conjugation with bioorthogonal reactions.

  • 4. Chen, Xi
    et al.
    Venkatachalapathy, Muthukumaran
    Dehmelt, Leif
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Multidirectional Activity Control of Cellular Processes by a Versatile Chemo-optogenetic Approach2018Ingår i: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 57, nr 37, s. 11993-11997Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The spatiotemporal dynamics of proteins or organelles plays a vital role in controlling diverse cellular processes. However, acute control of activity at distinct locations within a cell is challenging. A versatile multidirectional activity control (MAC) approach is presented, which employs a photoactivatable system that may be dimerized upon chemical inducement. The system comprises second-generation SLF*-TMP (S*T) and photocaged NvocTMP-Cl dimerizers; where, SLF*-TMP features a synthetic ligand of the FKBP(F36V) binding protein, Nvoc is a caging group, and TMP is the antibiotic trimethoprim. Two MAC strategies are demonstrated to spatiotemporally control cellular signaling and intracellular cargo transport. The novel platform enables tunable, reversible, and rapid control of activity at multiple compartments in living cells.

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  • 5. Chen, Xi
    et al.
    Venkatachalapathy, Muthukumaran
    Kamps, Dominic
    Weigel, Simone
    Kumar, Ravi
    Orlich, Michael
    Garrecht, Ruben
    Hirtz, Michael
    Niemeyer, Christof M.
    Wu, Yao-Wen
    Chemical Genomics Centre of the Max-Planck Society, Dortmund, Germany.
    Dehmelt, Leif
    “Molecular Activity Painting”: Switch-like, light-controlled perturbations inside living cells2017Ingår i: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 56, nr 21, s. 5916-5920Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Acute subcellular protein targeting is a powerful tool to study biological networks. However, signaling at the plasma membrane is highly dynamic, making it difficult to study in space and time. In particular, sustained local control of molecular function is challenging due to lateral diffusion of plasma membrane targeted molecules. Here we present “Molecular Activity Painting” (MAP), a novel technology which combines photoactivatable chemically induced dimerization (pCID) with immobilized artificial receptors. The immobilization of artificial receptors by surface-immobilized antibodies blocks lateral diffusion, enabling rapid and stable “painting” of signaling molecules and their activity at the plasma membrane with micrometer precision. Using this method, we show that painting of the RhoA-myosin activator GEF-H1 induces patterned acto-myosin contraction inside living cells.

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  • 6.
    Chen, Xi
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Chemical Genomics Centre of the Max Planck Society, Dortmund, Germany; Max Planck Institute of Molecular Physiology, Dortmund, Germany.
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Chemical Genomics Centre of the Max Planck Society, Dortmund, Germany; Max Planck Institute of Molecular Physiology, Dortmund, Germany.
    Tunable and Photoswitchable Chemically Induced Dimerization for Chemo-optogenetic Control of Protein and Organelle Positioning2018Ingår i: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 57, nr 23, s. 6796-6799Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The spatiotemporal dynamics of proteins and organelles play an important role in controlling diverse cellular processes. Optogenetic tools using photosensitive proteins and chemically induced dimerization (CID), which allow control of protein dimerization, have been used to elucidate the dynamics of biological systems and to dissect the complicated biological regulatory networks. However, the inherent limitations of current optogenetic and CID systems remain a significant challenge for the fine-tuning of cellular activity at precise times and locations. Herein, we present a novel chemo-optogenetic approach, photoswitchable chemically induced dimerization (psCID), for controlling cellular function by using blue light in a rapid and reversible manner. Moreover, psCID is tunable; that is, the dimerization and dedimerization degrees can be fine-tuned by applying different doses of illumination. Using this approach, we control the localization of proteins and positioning of organelles in live cells with high spatial (μm) and temporal (ms) precision.

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  • 7.
    Corkery, Dale
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Castro-Gonzalez, Sergio
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Knyazeva, Anastasia
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Herzog, Laura K.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    An ATG12-ATG5-TECPR1 E3-like complex regulates unconventional LC3 lipidation at damaged lysosomes2023Ingår i: EMBO Reports, ISSN 1469-221X, E-ISSN 1469-3178, Vol. 24, nr 9, artikel-id e56841Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Lysosomal membrane damage represents a threat to cell viability. As such, cells have evolved sophisticated mechanisms to maintain lysosomal integrity. Small membrane lesions are detected and repaired by the endosomal sorting complex required for transport (ESCRT) machinery while more extensively damaged lysosomes are cleared by a galectin-dependent selective macroautophagic pathway (lysophagy). In this study, we identify a novel role for the autophagosome-lysosome tethering factor, TECPR1, in lysosomal membrane repair. Lysosomal damage promotes TECPR1 recruitment to damaged membranes via its N-terminal dysferlin domain. This recruitment occurs upstream of galectin and precedes the induction of lysophagy. At the damaged membrane, TECPR1 forms an alternative E3-like conjugation complex with the ATG12-ATG5 conjugate to regulate ATG16L1-independent unconventional LC3 lipidation. Abolishment of LC3 lipidation via ATG16L1/TECPR1 double knockout impairs lysosomal recovery following damage.

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  • 8.
    Corkery, Dale
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Nadeem, Aftab
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Aung, Kyaw Min
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Hassan, Ahmed
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Liu, Tao
    Cervantes-Rivera, Ramón
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Lystad, Alf Håkon
    Wang, Hui
    Persson, Karina
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Puhar, Andrea
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Simonsen, Anne
    Uhlin, Bernt Eric
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wai, Sun Nyunt
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Vibrio cholerae cytotoxin MakA induces noncanonical autophagy resulting in the spatial inhibition of canonical autophagy2021Ingår i: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 134, nr 5, artikel-id jcs252015Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Autophagy plays an essential role in the defense against manymicrobial pathogens as a regulator of both innate and adaptive immunity. Some pathogens have evolved sophisticated mechanisms that promote their ability to evade or subvert host autophagy. Here, we describe a novel mechanism of autophagy modulation mediated by the recently discovered Vibrio cholerae cytotoxin, motility-associatedkilling factor A (MakA). pH-dependent endocytosis of MakA by host cells resulted in the formation of a cholesterol-rich endolysosomal membrane aggregate in the perinuclear region. Aggregate formation induced the noncanonical autophagy pathway driving unconventional LC3 (herein referring to MAP1LC3B) lipidation on endolysosomal membranes. Subsequent sequestration of the ATG12-ATG5-ATG16L1 E3-like enzyme complex, required for LC3 lipidation at the membranous aggregate, resulted in an inhibition of both canonical autophagy and autophagy-related processes, including the unconventional secretion of interleukin-1β (IL-1β). These findings identify a novel mechanismof host autophagy modulation and immune modulation employed by V. cholerae during bacterial infection.

  • 9.
    Corkery, Dale P.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    ATG12–ATG5-TECPR1: an alternative E3-like complex utilized during the cellular response to lysosomal membrane damage2024Ingår i: Autophagy, ISSN 1554-8627, E-ISSN 1554-8635, Vol. 20, nr 2, s. 443-444Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    ATG16L1 is an essential component of the Atg8-family protein conjugation machinery, providing membrane targeting for the ATG12–ATG5 conjugate. Recently, we identified an alternative E3-like complex that functions independently of ATG16L1. This complex utilizes the autophagosome-lysosome tethering factor TECPR1 for membrane targeting. TECPR1 is recruited to damaged lysosomal membranes via a direct interaction with sphingomyelin. At the damaged membrane, TECPR1 assembles into an E3-like complex with ATG12–ATG5 to regulate unconventional LC3 lipidation and promote efficient lysosomal repair.

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  • 10.
    Corkery, Dale P.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Eating while intoxicated: characterizing the molecular mechanism behind V. cholerae toxin MakA-regulated autophagy2023Ingår i: Autophagy, ISSN 1554-8627, E-ISSN 1554-8635, Vol. 19, nr 6, s. 1885-1886Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Extracellular pathogens utilize secreted virulence factors to regulate host cell function. Recently we characterized the molecular mechanism behind host macroautophagy/autophagy regulation by the Vibrio cholerae toxin MakA. Cholesterol binding at the plasma membrane induces MakA endocytosis and pH-dependent pore assembly. Membrane perforation of late endosomal membranes induces cellular membrane repair pathways and V-ATPase-dependent unconventional LC3 lipidation on damaged membranes.

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  • 11.
    Corkery, Dale
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Ursu, Andrei
    Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, Dortmund, Germany.
    Lucas, Belén
    Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, Dortmund, Germany.
    Grigalunas, Michael
    Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, Dortmund, Germany.
    Kriegler, Simon
    Physical Chemistry I – Biophysical Chemistry, Department of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Strasse 4a, 44227 Dortmund, Germany, Germany.
    Oliva, Rosario
    Physical Chemistry I – Biophysical Chemistry, Department of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Strasse 4a, 44227 Dortmund, Germany, Germany; Department of Chemical Sciences, University of Naples Federico II, Via Cintia 4, Naples, Italy.
    Dec, Robert
    Physical Chemistry I – Biophysical Chemistry, Department of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Strasse 4a, 44227 Dortmund, Germany, Germany.
    Koska, Sandra
    Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, Dortmund, Germany.
    Pahl, Axel
    Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, Dortmund, Germany.
    Sievers, Sonja
    Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, Dortmund, Germany.
    Ziegler, Slava
    Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, Dortmund, Germany.
    Winter, Roland
    Physical Chemistry I – Biophysical Chemistry, Department of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Strasse 4a, 44227 Dortmund, Germany, Germany.
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Waldmann, Herbert
    Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, Dortmund, Germany; Faculty of Chemistry and Chemical Biology, Technical University Dortmund, Otto-Hahn-Strasse 6, Dortmund, Germany.
    Inducin triggers LC3-lipidation and ESCRT-mediated lysosomal membrane repair2023Ingår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 24, nr 24, artikel-id e202300579Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Lipidation of the LC3 protein has frequently been employed as a marker of autophagy. However, LC3-lipidation is also triggered by stimuli not related to canonical autophagy. Therefore, characterization of the driving parameters for LC3 lipidation is crucial to understanding the biological roles of LC3. We identified a pseudo-natural product, termed Inducin, that increases LC3 lipidation independently of canonical autophagy, impairs lysosomal function and rapidly recruits Galectin 3 to lysosomes. Inducin treatment promotes Endosomal Sorting Complex Required for Transport (ESCRT)-dependent membrane repair and transcription factor EB (TFEB)-dependent lysosome biogenesis ultimately leading to cell death.

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  • 12.
    Corkery, Dale
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Dowaidar, Moataz
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)2021Ingår i: Autophagy, ISSN 1554-8627, E-ISSN 1554-8635, Vol. 17, nr 1, s. 1-382Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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 bona fide 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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  • 13. Foley, Daniel J.
    et al.
    Zinken, Sarah
    Corkery, Dale
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Laraia, Luca
    Pahl, Axel
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Waldmann, Herbert
    Phenotyping Reveals Targets of a Pseudo-Natural-Product Autophagy Inhibitor2020Ingår i: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 59, nr 30Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Pseudo-natural-product (NP) design combines natural product fragments to provide unprecedented NP-inspired compounds not accessible by biosynthesis, but endowed with biological relevance. Since the bioactivity of pseudo-NPs may be unprecedented or unexpected, they are best evaluated in target agnostic cell-based assays monitoring entire cellular programs or complex phenotypes. Here, the Cinchona alkaloid scaffold was merged with the indole ring system to synthesize indocinchona alkaloids by Pd-catalyzed annulation. Exploration of indocinchona alkaloid bioactivities in phenotypic assays revealed a novel class of azaindole-containing autophagy inhibitors, the azaquindoles. Subsequent characterization of the most potent compound, azaquindole-1, in the morphological cell painting assay, guided target identification efforts. In contrast to the parent Cinchona alkaloids, azaquindoles selectively inhibit starvation- and rapamycin-induced autophagy by targeting the lipid kinase VPS34.

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  • 14.
    Jia, Xiaotong
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Knyazeva, Anastasia
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Zhang, Yu
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Castro-Gonzalez, Sergio
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Nakamura, Shuhei
    Department of Genetics, Graduate School of Medicine, Osaka University, Osaka, Japan.
    Carlson, Lars-Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Wallenberg centrum för molekylär medicin vid Umeå universitet (WCMM). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Yoshimori, Tamotsu
    Department of Genetics, Graduate School of Medicine, Osaka University, Osaka, Japan.
    Corkery, Dale
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    V. cholerae MakA is a cholesterol-binding pore-forming toxin that induces non-canonical autophagy2022Ingår i: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 221, nr 12, artikel-id e202206040Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Pore-forming toxins (PFTs) are important virulence factors produced by many pathogenic bacteria. Here, we show that the Vibrio cholerae toxin MakA is a novel cholesterol-binding PFT that induces non-canonical autophagy in a pH-dependent manner. MakA specifically binds to cholesterol on the membrane at pH < 7. Cholesterol-binding leads to oligomerization of MakA on the membrane and pore formation at pH 5.5. Unlike other cholesterol-dependent cytolysins (CDCs) which bind cholesterol through a conserved cholesterol-binding motif (Thr-Leu pair), MakA contains an Ile-Ile pair that is essential for MakA-cholesterol interaction. Following internalization, endosomal acidification triggers MakA pore-assembly followed by ESCRT-mediated membrane repair and V-ATPase-dependent unconventional LC3 lipidation on the damaged endolysosomal membranes. These findings characterize a new cholesterol-binding toxin that forms pores in a pH-dependent manner and reveals the molecular mechanism of host autophagy manipulation.

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  • 15. Kaiser, Nadine
    et al.
    Corkery, Dale
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Laraia, Luca
    Waldmann, Herbert
    Modulation of autophagy by the novel mitochondrial complex I inhibitor Authipyrin2019Ingår i: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 27, nr 12, s. 2444-2448Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Autophagy ensures cellular homeostasis by the degradation of long-lived proteins, damaged organelles and pathogens. This catabolic process provides essential cellular building blocks upon nutrient deprivation. Cellular metabolism, especially mitochondrial respiration, has a significant influence on autophagic flux, and complex I function is required for maximal autophagy. In Parkinson’s disease mitochondrial function is frequently impaired and autophagic flux is altered. Thus, dysfunctional organelles and protein aggregates accumulate and cause cellular damage. In order to investigate the interdependency between mitochondrial function and autophagy, novel tool compounds are required. Herein, we report the discovery of a structurally novel autophagy inhibitor (Authipyrin) using a high content screening approach. Target identification and validation led to the discovery that Authipyrin targets mitochondrial complex I directly, leading to the potent inhibition of mitochondrial respiration as well as autophagy.

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  • 16. Kamps, Dominic
    et al.
    Koch, Johannes
    Juma, Victor O.
    Campillo-Funollet, Eduard
    Graessl, Melanie
    Banerjee, Soumya
    Mazel, Tomas
    Chen, Xi
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Chemical Genomics Centre of the Max-Planck Society, Dortmund, Germany.
    Portet, Stephanie
    Madzvamuse, Anotida
    Nalbant, Perihan
    Dehmelt, Leif
    Optogenetic Tuning Reveals Rho Amplification-Dependent Dynamics of a Cell Contraction Signal Network2020Ingår i: Cell Reports, E-ISSN 2211-1247, Vol. 33, nr 9, artikel-id 108467Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Local cell contraction pulses play important roles in tissue and cell morphogenesis. Here, we improve a chemo-optogenetic approach and apply it to investigate the signal network that generates these pulses. We use these measurements to derive and parameterize a system of ordinary differential equations describing temporal signal network dynamics. Bifurcation analysis and numerical simulations predict a strong dependence of oscillatory system dynamics on the concentration of GEF-H1, an Lbc-type RhoGEF, which mediates the positive feedback amplification of Rho activity. This prediction is confirmed experimentally via optogenetic tuning of the effective GEF-H1 concentration in individual living cells. Numerical simulations show that pulse amplitude is most sensitive to external inputs into the myosin component at low GEF-H1 concentrations and that the spatial pulse width is dependent on GEF-H1 diffusion. Our study offers a theoretical framework to explain the emergence of local cell contraction pulses and their modulation by biochemical and mechanical signals.

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  • 17. Klewer, Laura
    et al.
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Light-Induced Dimerization Approaches to Control Cellular Processes2019Ingår i: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 25, s. 12452-12463Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Light-inducible approaches provide a means to control biological systems with spatial and temporal resolution that is unmatched by traditional genetic perturbations. Recent developments of optogenetic and chemo-optogenetic systems for induced proximity in cells facilitate rapid and reversible manipulation of highly dynamic cellular processes and have become valuable tools in diverse biological applications. New expansions of the toolbox facilitate control of signal transduction, genome editing, "painting" patterns of active molecules onto cellular membranes, and light-induced cell cycle control. A combination of light- and chemically induced dimerization approaches have also seen interesting progress. Herein, an overview of optogenetic systems and emerging chemo-optogenetic systems is provided, and recent applications in tackling complex biological problems are discussed.

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  • 18.
    Knyazeva, Anastasia
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Corkery, Dale
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Shankar, Kasturika
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Wallenberg centrum för molekylär medicin vid Umeå universitet (WCMM). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Herzog, Laura K.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Zhang, Xuepei
    Chemical Proteomics Core Facility, Division of Chemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden; Chemical Proteomics Unit, SciLifeLab, Stockholm, Sweden; Chemical Proteomics, Swedish National Infrastructure for Biological Mass Spectrometry (BioMS), Stockholm, Sweden.
    Singh, Birendra
    Umeå universitet, Medicinska fakulteten, Institutionen för kirurgisk och perioperativ vetenskap, Anestesiologi och intensivvård.
    Niggemeyer, Georg
    Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
    Grill, David
    Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
    Gilthorpe, Jonathan D.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Gaetani, Massimiliano
    Chemical Proteomics Core Facility, Division of Chemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden; Chemical Proteomics Unit, SciLifeLab, Stockholm, Sweden; Chemical Proteomics, Swedish National Infrastructure for Biological Mass Spectrometry (BioMS), Stockholm, Sweden.
    Carlson, Lars-Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Wallenberg centrum för molekylär medicin vid Umeå universitet (WCMM). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Waldmann, Herbert
    Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany; Faculty of Chemistry and Chemical Biology, Technical University Dortmund, Dortmund, Germany.
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Chemogenetic inhibition of IST1-CHMP1B interaction impairs endosomal recycling and promotes unconventional LC3 lipidation at stalled endosomesManuskript (preprint) (Övrigt vetenskapligt)
  • 19.
    Konstantinidis, Georgios
    et al.
    Chemical Genomics Centre of the Max Planck Society, Dortmund, Germany; Max Planck Institute of Molecular Physiology, Dortmund, Germany.
    Sievers, Sonja
    Max Planck Institute of Molecular Physiology, Dortmund, Germany; Compound Management and Screening Center of the Max Planck Society, Dortmund, Germany.
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Chemical Genomics Centre of the Max Planck Society, Dortmund, Germany; Max Planck Institute of Molecular Physiology, Dortmund, Germany.
    Identification of novel autophagy inhibitors via cell-based high-content screening2019Ingår i: Autophagy in differentiation and tissue maintenance: methods and protocols / [ed] Kursad Turksen, Humana Press, 2019, , s. 9s. 187-195Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    Autophagy is a fundamental cellular catabolic pathway mediating the recycling of cellular components. Autophagy has been implicated in pathogenesis of diverse diseases such as neurodegeneration and cancer. Due to the therapeutic potential, the autophagy-modulating agents have profoundly enriched the spectrum of tools used to investigate autophagy. However, many of these compounds have additional off-target effects that may confound elucidation of autophagy in certain contexts. There remains high demand for highly specific and novel chemotypes that can be used to study the regulation mechanism of autophagy and contribute novel pharmacophores for therapeutic purposes. Here, we describe a cell-based quantitative high-content screening (HCS) for autophagy inhibitors using a human breast adenocarcinoma MCF7 cell line stably expressing EGFP-LC3, a bona fide marker of autophagy.

  • 20.
    Kowalczyk, Manuela
    et al.
    Department of Molecular Cell Biology, Center for Medical Biotechnology, University of Duisburg-Essen, Essen, Germany.
    Kamps, Dominic
    Department of Chemistry and Chemical Biology and Department of Systemic Cell Biology, TU Dortmund University and Max-Planck-Institute of Molecular Physiology, Dortmund, Germany.
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Dehmelt, Leif
    Department of Chemistry and Chemical Biology and Department of Systemic Cell Biology, TU Dortmund University and Max-Planck-Institute of Molecular Physiology, Dortmund, Germany.
    Nalbant, Perihan
    Department of Molecular Cell Biology, Center for Medical Biotechnology, University of Duisburg-Essen, Essen, Germany.
    Monitoring the Response of Multiple Signal Network Components to Acute Chemo-Optogenetic Perturbations in Living Cellsope2022Ingår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 23, nr 4, artikel-id e202100582Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cells process information via signal networks that typically involve multiple components which are interconnected by feedback loops. The combination of acute optogenetic perturbations and microscopy-based fluorescent response readouts enables the direct investigation of causal links in such networks. However, due to overlaps in spectra of photosensitive and fluorescent proteins, current approaches that combine these methods are limited. Here, we present an improved chemo-optogenetic approach that is based on switch-like perturbations induced by a single, local pulse of UV light. We show that this approach can be combined with parallel monitoring of multiple fluorescent readouts to directly uncover relations between signal network components. We present the application of this technique to directly investigate feedback-controlled regulation in the cell contraction signal network that includes GEF-H1, Rho and Myosin, and functional interactions of this network with tumor relevant RhoA G17 mutants.

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  • 21. Laraia, Luca
    et al.
    Friese, Alexandra
    Corkery, Dale
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Chemical Genomics Centre of the Max Planck Society, Dortmund, Germany.
    Konstantinidis, Georgios
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Chemical Genomics Centre of the Max Planck Society, Dortmund, Germany.
    Erwin, Nelli
    Hofer, Walter
    Karatas, Hacer
    Klewer, Laura
    Brockmeyer, Andreas
    Metz, Malte
    Schoelermann, Beate
    Dwivedi, Mridula
    Li, Lei
    Rios-Munoz, Pablo
    Koehn, Maja
    Winter, Roland
    Vetter, Ingrid R.
    Ziegler, Slava
    Janning, Petra
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Chemical Genomics Centre of the Max Planck Society, Dortmund, Germany.
    Waldmann, Herbert
    The cholesterol transfer protein GRAMD1A regulates autophagosome biogenesis2019Ingår i: Nature Chemical Biology, ISSN 1552-4450, E-ISSN 1552-4469, Vol. 15, nr 7, s. 710-720Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Autophagy mediates the degradation of damaged proteins, organelles and pathogens, and plays a key role in health and disease. Thus, the identification of new mechanisms involved in the regulation of autophagy is of major interest. In particular, little is known about the role of lipids and lipid-binding proteins in the early steps of autophagosome biogenesis. Using target-agnostic, high-content, image-based identification of indicative phenotypic changes induced by small molecules, we have identified autogramins as a new class of autophagy inhibitor. Autogramins selectively target the recently discovered cholesterol transfer protein GRAM domain-containing protein 1A (GRAMD1A, which had not previously been implicated in autophagy), and directly compete with cholesterol binding to the GRAMD1A StART domain. GRAMD1A accumulates at sites of autophagosome initiation, affects cholesterol distribution in response to starvation and is required for autophagosome biogenesis. These findings identify a new biological function of GRAMD1A and a new role for cholesterol in autophagy.

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  • 22. Laraia, Luca
    et al.
    Garivet, Guillaume
    Foley, Daniel J.
    Kaiser, Nadine
    Mueller, Sebastian
    Zinken, Sarah
    Pinkert, Thomas
    Wilke, Julian
    Corkery, Dale
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Pahl, Axel
    Sievers, Sonja
    Janning, Petra
    Arenz, Christoph
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Rodriguez, Raphael
    Waldmann, Herbert
    Image-Based Morphological Profiling Identifies a Lysosomotropic, Iron-Sequestering Autophagy Inhibitor2020Ingår i: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 59, s. 5721-5729Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Chemical proteomics is widely applied in small-molecule target identification. However, in general it does not identify non-protein small-molecule targets, and thus, alternative methods for target identification are in high demand. We report the discovery of the autophagy inhibitor autoquin and the identification of its molecular mode of action using image-based morphological profiling in the cell painting assay. A compound-induced fingerprint representing changes in 579 cellular parameters revealed that autoquin accumulates in lysosomes and inhibits their fusion with autophagosomes. In addition, autoquin sequesters Fe2+ in lysosomes, resulting in an increase of lysosomal reactive oxygen species and ultimately cell death. Such a mechanism of action would have been challenging to unravel by current methods. This work demonstrates the potential of the cell painting assay to deconvolute modes of action of small molecules, warranting wider application in chemical biology.

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  • 23. Laraia, Luca
    et al.
    Ohsawaa, Kosuke
    Konstantinidis, Georgios
    Robke, Lucas
    Wu, Yao-Wen
    Chemical Genomics Center of the Max Planck Society Otto-Hahn-Str.15, 44227 Dortmund.
    Kumar, Kamal
    Waldmann, Herbert
    Discovery of Novel Cinchona‐Alkaloid‐Inspired Oxazatwistane Autophagy Inhibitors2017Ingår i: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 56, nr 8, s. 2145-2150Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The cinchona alkaloids are a privileged class of natural products and are endowed with diverse bioactivities. However, for compounds with the closely‐related oxazatricyclo[4.4.0.0]decane (“oxazatwistane”) scaffold, which are accessible from cinchonidine and quinidine by means of ring distortion and modification, biological activity has not been identified. We report the synthesis of an oxazatwistane compound collection through employing state‐of‐the‐art C−H functionalization, and metal‐catalyzed cross‐coupling reactions as key late diversity‐generating steps. Exploration of oxazatwistane bioactivity in phenotypic assays monitoring different cellular processes revealed a novel class of autophagy inhibitors termed oxautins, which, in contrast to the guiding natural products, selectively inhibit autophagy by inhibiting both autophagosome biogenesis and autophagosome maturation.

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  • 24. Li, Fang-Yi
    et al.
    Zhang, Zhen-Feng
    Voss, Stephanie
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Chemical Genomics Centre of the Max Planck Society, Dortmund, Germany; Max-Planck-Institute of Molecular Physiology, Dortmund, Germany.
    Zhao, Yu-Fen
    Li, Yan-Mei
    Chen, Yong-Xiang
    Inhibition of K-Ras4B-plasma membrane association with a membrane microdomain-targeting peptide2020Ingår i: Chemical Science, ISSN 2041-6520, E-ISSN 2041-6539, Vol. 11, nr 3, s. 826-832Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The association of K-Ras4B protein with plasma membrane (PM) is required for its signaling activity. Thus, direct inhibition of K-Ras4B-PM interaction could be a potential anti-Ras therapeutic strategy. However, it remains challenging to modulate such protein-PM interaction. Based on Ras isoform-specific PM microdomain localization patterns, we have developed a potent and isoform-selective peptide inhibitor, Memrasin, for detachment of K-Ras4B from the PM. Memrasin is one of the first direct inhibitors of K-Ras4B-PM interaction, and consists of a membrane l(d) region-binding sequence derived from the C-terminal region of K-Ras4B and an endosome-escape enhancing motif that can aggregate on membrane. It forms peptide-enriched domains in the l(d) region, abrogates the tethering of K-Ras4B to the PM and accordingly impairs Ras signaling activity, thereby efficiently decreasing the viability of several human lung cancer cells in a dose-responsive and K-Ras dependent manner. Memrasin provides a useful tool for exploring the biological function of K-Ras4B on or off the PM and a potential starting point for further development into anti-Ras therapeutics.

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  • 25.
    Li, Fu
    et al.
    Max-Planck-Institute of Molecular Physiology, Dortmund, Germany.
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Imaging of spatial cycling of Rab GTPase in the cell2021Ingår i: Rab GTPases: methods and protocols / [ed] Guangpu Li, Nava Segev, Humana Press, 2021, 2, , s. 11s. 105-115Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    Rab GTPases (>60 members in human) function as master regulators of intracellular membrane trafficking. To fulfill their functions, Rab proteins need to localize on specific membranes in cells. It remains elusive how the distinct spatial distribution of Rab GTPases in the cell is regulated. To make a global assessment on the subcellular localization of Rab1, we determined kinetic parameters of the spatial cycling of Rab1 in live cells using photoactivatable fluorescent proteins and live cell imaging. We found that the switching between GTP- and GDP-binding states, which is governed by guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), GDP dissociation inhibitor (GDI) and GDI displacement factor (GDF), is a major determinant for Rab1’s ability to effectively cycle between cellular compartments and eventually for its subcellular distribution. Herein, we describe the method for monitoring Rab1 dynamics in live cells. This approach can be used to study spatial cycling of other Rab GTPases.

  • 26.
    Nanda, Suchet
    et al.
    Fakultät für Chemie und Chemische Biologie, TU Dortmund University, Dortmund, Germany; Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
    Calderon, Abram
    Fakultät für Chemie und Chemische Biologie, TU Dortmund University, Dortmund, Germany; Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
    Sachan, Arya
    Fakultät für Chemie und Chemische Biologie, TU Dortmund University, Dortmund, Germany.
    Duong, Thanh-Thuy
    Fakultät für Chemie und Chemische Biologie, TU Dortmund University, Dortmund, Germany; Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
    Koch, Johannes
    Department of Molecular Cell Biology, Center of Medical Biotechnology, University of Duisburg-Essen, Essen, Germany.
    Xin, Xiaoyi
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Solouk-Stahlberg, Djamschid
    Fakultät für Chemie und Chemische Biologie, TU Dortmund University, Dortmund, Germany; Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Nalbant, Perihan
    Department of Molecular Cell Biology, Center of Medical Biotechnology, University of Duisburg-Essen, Essen, Germany.
    Dehmelt, Leif
    Fakultät für Chemie und Chemische Biologie, TU Dortmund University, Dortmund, Germany.
    Rho GTPase activity crosstalk mediated by Arhgef11 and Arhgef12 coordinates cell protrusion-retraction cycles2023Ingår i: Nature Communications, E-ISSN 2041-1723, Vol. 14, nr 1, artikel-id 8356Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Rho GTPases play a key role in the spatio-temporal coordination of cytoskeletal dynamics during cell migration. Here, we directly investigate crosstalk between the major Rho GTPases Rho, Rac and Cdc42 by combining rapid activity perturbation with activity measurements in mammalian cells. These studies reveal that Rac stimulates Rho activity. Direct measurement of spatio-temporal activity patterns show that Rac activity is tightly and precisely coupled to local cell protrusions, followed by Rho activation during retraction. Furthermore, we find that the Rho-activating Lbc-type GEFs Arhgef11 and Arhgef12 are enriched at transient cell protrusions and retractions and recruited to the plasma membrane by active Rac. In addition, their depletion reduces activity crosstalk, cell protrusion-retraction dynamics and migration distance and increases migration directionality. Thus, our study shows that Arhgef11 and Arhgef12 facilitate exploratory cell migration by coordinating cell protrusion and retraction by coupling the activity of the associated regulators Rac and Rho.

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  • 27.
    Niggemeyer, Georg
    et al.
    Max Planck Institute of Molecular Physiology, Department of Chemical Biology, Otto-Hahn-Strasse 11, Dortmund, Germany; Technical University Dortmund, Faculty of Chemistry, Chemical Biology, Otto-Hahn-Strasse 6, Dortmund, Germany.
    Knyazeva, Anastasia
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Gasper, Raphael
    Max Planck Institute of Molecular Physiology, Crystallography and Biophysics Unit, Otto-Hahn-Strasse 11, Dortmund, Germany.
    Corkery, Dale
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Bodenbinder, Pia
    Max Planck Institute of Molecular Physiology, Department of Chemical Biology, Otto-Hahn-Strasse 11, Dortmund, Germany; Technical University Dortmund, Faculty of Chemistry, Chemical Biology, Otto-Hahn-Strasse 6, Dortmund, Germany.
    Holstein, Julian J.
    Technical University Dortmund, Faculty of Chemistry, Chemical Biology, Otto-Hahn-Strasse 6, Dortmund, Germany; Technical University Dortmund, Faculty of Chemistry, Inorganic Chemistry, Otto-Hahn-Strasse 6, Dortmund, Germany.
    Sievers, Sonja
    Compound Management and Screening Center (COMAS), Otto-Hahn-Strasse 11, Dortmund, Germany.
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Waldmann, Herbert
    Max Planck Institute of Molecular Physiology, Department of Chemical Biology, Otto-Hahn-Strasse 11, Dortmund, Germany; Technical University Dortmund, Faculty of Chemistry, Chemical Biology, Otto-Hahn-Strasse 6, Dortmund, Germany.
    Synthesis of 20-Membered Macrocyclic Pseudo-Natural Products Yields Inducers of LC3 Lipidation2022Ingår i: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 61, nr 11, artikel-id e202114328Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Design and synthesis of pseudo-natural products (PNPs) through recombination of natural product (NP) fragments in unprecedented arrangements enables the discovery of novel biologically relevant chemical matter. With a view to wider coverage of NP-inspired chemical and biological space, we describe the combination of this principle with macrocycle formation. PNP-macrocycles were synthesized efficiently in a stereoselective one-pot procedure including the 1,3-dipolar cycloadditions of different dipolarophiles with dimeric cinchona alkaloid-derived azomethine ylides formed in situ. The 20-membered bis-cycloadducts embody 18 stereocenters and an additional fragment-sized NP-structure. After further functionalization, a collection of 163 macrocyclic PNPs was obtained. Biological investigation revealed potent inducers of the lipidation of the microtubule associated protein 1 light chain 3 (LC3) protein, which plays a prominent role in various autophagy-related processes.

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  • 28.
    Pantoom, Supansa
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Translational Neurodegeneration Section "Albrecht-kossel", Department of Neurology, University Medical Center Rostock, Rostock, Germany.
    Konstantinidis, Georgios
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Crete, Greece.
    Voss, Stephanie
    Han, Hongmei
    Hofnagel, Oliver
    Li, Zhiyu
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    RAB33B recruits the ATG16L1 complex to the phagophore via a noncanonical RAB binding protein2021Ingår i: Autophagy, ISSN 1554-8627, E-ISSN 1554-8635, Vol. 17, nr 9, s. 2290-2304Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Autophagosome formation is a fundamental process in macroautophagy/autophagy, a conserved self-eating mechanism in all eukaryotes, which requires the conjugating ATG (autophagy related) protein complex, ATG12-ATG5-ATG16L1 and lipidated MAP1LC3/LC3 (microtubule associated protein 1 light chain 3). How the ATG12-ATG5-ATG16L1 complex is recruited to membranes is not fully understood. Here, we demonstrated that RAB33B plays a key role in recruiting the ATG16L1 complex to phagophores during starvation-induced autophagy. Crystal structures of RAB33B bound to the coiled-coil domain (CCD) of ATG16L1 revealed the recognition mechanism between RAB33B and ATG16L1. ATG16L1 is a novel RAB-binding protein (RBP) that can induce RAB proteins to adopt active conformation without nucleotide exchange. RAB33B and ATG16L1 mutually determined the localization of each other on phagophores. RAB33B-ATG16L1 interaction was required for LC3 lipidation and autophagosome formation. Upon starvation, a fraction of RAB33B translocated from the Golgi to phagophores and recruited the ATG16L1 complex. In this work, we reported a new mechanism for the recruitment of the ATG12-ATG5-ATG16L1 complex to phagophores by RAB33B, which is required for autophagosome formation.

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  • 29. Pantoom, Supansa
    et al.
    Yang, Aimin
    Wu, Yao-Wen
    Chemical Genomics Centre of the Max Planck Society, Dortmund, Germany; Max-Planck-Institute of Molecular Physiology, Dortmund, Germany; Institute of Chemical Biology and Precision Therapy, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China.
    Lift and cut: Anti-host autophagy mechanism of Legionella pneumophila2017Ingår i: Autophagy, ISSN 1554-8627, E-ISSN 1554-8635, Vol. 13, nr 8, s. 1467-1469Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    RavZ, an effector protein of pathogenic Legionella pneumophila, inhibits host macroautophagy/autophagy by deconjugation of lipidated LC3 proteins from phosphatidylethanolamine (PE) on the autophagosome membrane. The mechanism for how RavZ specifically recognizes and deconjugates the lipidated LC3s is not clear. To understand the structure-function relationship of LC3-deconjugation by RavZ, we prepared semisynthetic LC3 proteins modified with different fragments of PE or 1-hexadecanol (C16). We find that RavZ activity is strictly dependent on the conjugated PE structure and RavZ extracts LC3-PE from the membrane before deconjugation. Structural and biophysical analysis of RavZ-LC3 interactions suggest that RavZ initially recognizes LC3-PE on the membrane via its N-terminal LC3-interacting region (LIR) motif. RavZ specifically targets to autophagosome membranes by interaction with phosphatidylinositol 3-phosphate (PtdIns3P) via its C-terminal domain and association with membranes via the hydrophobic α3 helix. The α3 helix is involved in extraction of the PE moiety and docking of the fatty acid chains into the lipid-binding site of RavZ, which is related in structure to that of the phospholipid transfer protein Sec14. The LIR interaction and lipid binding facilitate subsequent proteolytic cleavage of LC3-PE. The findings reveal a novel mode of host-pathogen interaction.

  • 30. Robke, Lucas
    et al.
    Futamura, Yushi
    Konstantinidis, Georgios
    Wilke, Julian
    Aono, Harumi
    Mahmoud, Zhwan
    Watanabe, Nobumoto
    Wu, Yao-Wen
    Chemical Genomics Centre of the Max-Planck-Society, Otto-Hahn-Str. 15, 44227 Dortmund, Germany.
    Osada, Hiroyuki
    Laraia, Luca
    Waldmann, Herbert
    Discovery of the novel autophagy inhibitor aumitin that targets mitochondrial complex I2018Ingår i: Chemical Science, ISSN 2041-6520, E-ISSN 2041-6539, Vol. 9, nr 11, s. 3014-3022Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Macroautophagy is a conserved eukaryotic process for degradation of cellular components in response to lack of nutrients. It is involved in the development of diseases, notably cancer and neurological disorders including Parkinson's disease. Small molecule autophagy modulators have proven to be valuable tools to dissect and interrogate this crucial metabolic pathway and are in high demand. Phenotypic screening for autophagy inhibitors led to the discovery of the novel autophagy inhibitor aumitin. Target identification and confirmation revealed that aumitin inhibits mitochondrial respiration by targeting complex I. We show that inhibition of autophagy by impairment of mitochondrial respiration is general for several mitochondrial inhibitors that target different mitochondrial complexes. Our findings highlight the importance of mitochondrial respiration for autophagy regulation.

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  • 31. Robke, Lucas
    et al.
    Laraia, Luca
    Corrales, Marjorie A. Carnero
    Konstantinidis, Georgios
    Muroi, Makoto
    Richters, André
    Winzker, Michael
    Engbring, Tobias
    Tomassi, Stefano
    Watanabe, Nobumoto
    Osada, Hiroyuki
    Rauh, Daniel
    Waldmann, Herbert
    Wu, Yao-Wen
    Chemical Genomics Centreof the Max-Planck-Society Otto-Hahn-Strasse15, 44227 Dortmund.
    Engel, Julian
    Phenotypic Identification of a Novel Autophagy Inhibitor Chemotype Targeting Lipid Kinase VPS342017Ingår i: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 56, nr 28, s. 8153-8157Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Autophagy is a critical regulator of cellular homeostasis and metabolism. Interference with this process is considered a new approach for the treatment of disease, in particular cancer and neurological disorders. Therefore, novel small‐molecule autophagy modulators are in high demand. We describe the discovery of autophinib, a potent autophagy inhibitor with a novel chemotype. Autophinib was identified by means of a phenotypic assay monitoring the formation of autophagy‐induced puncta, indicating accumulation of the lipidated cytosolic protein LC3 on the autophagosomal membrane. Target identification and validation revealed that autophinib inhibits autophagy induced by starvation or rapamycin by targeting the lipid kinase VPS34.

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  • 32. Voss, Stephanie
    et al.
    Li, Fu
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Strasse 15, 44227 Dortmund, Germany; Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
    Raetz, Andreas
    Roeger, Matthias
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Strasse 15, 44227 Dortmund, Germany; Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
    Spatial Cycling of Rab GTPase, Driven by the GTPase Cycle, Controls Rab's Subcellular Distribution2019Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 58, nr 4, s. 276-285Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Rab GTPases (>60 members in humans) function as master regulators of intracellular membrane trafficking. Correct and specific localization of Rab proteins is required for their function. How the distinct spatial distribution of Rab GTPases in the cell is regulated remains elusive. To globally assess the subcellular localization of Rab1, we determined kinetic parameters of two pathways that control the spatial cycles of Rabl, i.e., vesicular transport and GDP dissociation inhibitor (GDI)-mediated recycling. We demonstrate that the switching between GTP and GDP binding states, which is governed by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), GDI, and GDI displacement factor (GDF), is a major determinant of Rab1's ability to effectively cycle between cellular compartments and eventually its subcellular distribution. In silico perturbations of vesicular transport, GEFs, GAPs, GDI, and GDF using a mathematical model with simplified cellular geometries showed that these regulators play an important role in the subcellular distribution and activity of Rab1.

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  • 33.
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Controlling cellular activities with light2023Ingår i: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 20, s. 357-358Artikel i tidskrift (Refereegranskat)
  • 34.
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Spatiotemporal Imaging of Small GTPase Activity Using Conformational Sensors for GTPase Activity (COSGA)2021Ingår i: Ras Activity and Signaling: Methods and Protocols / [ed] Rubio I., Prior I., Humana Press, 2021, Vol. 2262, s. 259-267Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    Small GTPases cycle between active GTP bound and inactive GDP bound forms in live cells. They act as molecular switches and regulate diverse cellular processes at different times and locations in the cell. Spatiotemporal visualization of their activity provides important insights into dynamics of cellular signaling. Conformational sensors for GTPase activity (COSGAs) are based on the conserved GTPase fold and have been used as a versatile approach for imaging small GTPase activity in the cell. Conformational changes upon GDP/GTP binding can be visualized directly in solution, on beads, or in live cells using COSGA by fluorescence lifetime imaging microscopy (FLIM) technique. Herein, we describe the construction of COSGA for imaging K-Ras GTPase activity in live cells.

  • 35.
    Wu, Yao-Wen
    et al.
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Chemical Genomics Centre of the Max Planck Society, Dortmund, Germany; Max Planck Institute of Molecular Physiology, Dortmund, Germany.
    Li, Fu
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Chemical Genomics Centre of the Max Planck Society, Dortmund, Germany; Max Planck Institute of Molecular Physiology, Dortmund, Germany.
    Bacterial interaction with host autophagy2019Ingår i: Virulence, ISSN 2150-5594, E-ISSN 2150-5608, Vol. 10, nr 1, s. 352-362Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Autophagy is a conserved and fundamental cellular process mainly to recycle or eliminate dysfunctional cellular organelles or proteins. As a response to cellular stress, autophagy is used as a defense mechanism to combat the infection with pathogenic bacteria. However, many intracellular bacteria have developed diverse mechanisms to evade recognition, to manipulate the autophagic pathway, and to hijack the autophagosomal compartment for replication. In this review, we discuss recent understandings on how bacteria interact with host autophagy.

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  • 36.
    Wu, Yao-Wen
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Waldmann, Herbert
    Toward the role of cholesterol and cholesterol transfer protein in autophagosome biogenesis2019Ingår i: Autophagy, ISSN 1554-8627, E-ISSN 1554-8635, Vol. 15, nr 12, s. 2167-2168Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A forward chemical genetic approach led to identification of autogramins as novel autophagy inhibitors. Autogramins selectively target the cholesterol transfer protein GRAMD1A (GRAM domain containing 1A). Autogramins compete with cholesterol binding to the StART domain of GRAMD1A, thereby inhibiting its cholesterol transfer activity. GRAMD1A associates with phosphatidylinositol monophosphate via its GRAM domain. GRAMD1A accumulates at autophagosome initiation sites upon starvation. This protein is involved in cholesterol distribution in response to starvation and is required for autophagosome biogenesis. Therefore, we identify a novel function of GRAMD1A and a new role of cholesterol in macroautophagy/autophagy.

  • 37.
    Xin, Xiaoyi
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Zhang, Yu
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Gaetani, Massimiliano
    Division of Physiological Chemistry I, Chemical Proteomics Core Facility, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden; Chemical Proteomics, Science for Life Laboratory (SciLifeLab), Stockholm, Sweden.
    Lundström, Susanna L.
    Division of Physiological Chemistry I, Chemical Proteomics Core Facility, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm 17177, Sweden;Chemical Proteomics, Science for Life Laboratory (SciLifeLab), Stockholm, Sweden.
    Zubarev, Roman A.
    Division of Physiological Chemistry I, Chemical Proteomics Core Facility, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm 17177, Sweden;Chemical Proteomics, Science for Life Laboratory (SciLifeLab), Stockholm, Sweden.
    Zhou, Yuan
    School of Medical Technology, Xuzhou Medical University, Xuzhou, China.
    Corkery, Dale P.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Ultrafast and selective labeling of endogenous proteins using affinity-based benzotriazole chemistry2022Ingår i: Chemical Science, ISSN 2041-6520, E-ISSN 2041-6539, Vol. 13, nr 24, s. 7240-7246Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Chemical modification of proteins is enormously useful for characterizing protein function in complex biological systems and for drug development. Selective labeling of native or endogenous proteins is challenging owing to the existence of distinct functional groups in proteins and in living systems. Chemistry for rapid and selective labeling of proteins remains in high demand. Here we have developed novel affinity labeling probes using benzotriazole (BTA) chemistry. We showed that affinity-based BTA probes selectively and covalently label a lysine residue in the vicinity of the ligand binding site of a target protein with a reaction half-time of 28 s. The reaction rate constant is comparable to the fastest biorthogonal chemistry. This approach was used to selectively label different cytosolic and membrane proteins in vitro and in live cells. BTA chemistry could be widely useful for labeling of native/endogenous proteins, target identification and development of covalent inhibitors.

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  • 38. Yang, Aimin
    et al.
    Hacheney, Inken
    Wu, Yao-Wen
    Chemical Genomics Centre of the Max Planck Society, Dortmund, Germany; Max-Planck-Institute of Molecular Physiology, Dortmund, Germany.
    Semisynthesis of autophagy protein LC3 conjugates2017Ingår i: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 25, nr 18, s. 4971-4976Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Autophagy is a conserved catabolic process involved in the elimination of proteins, organelles and pathogens. Autophagosome formation is the key process in autophagy. Lipidated Atg8/LC3 proteins that are conjugated to phosphatidylethanolamine (PE) play a key role in autophagosome biogenesis. To understand the function of Atg8/LC3-PE in autophagosome formation and host-pathogen interaction requires preparation and structural manipulation of lipidated Atg8/LC3 proteins. Herein, we report the semisynthesis of LC3 proteins and mutants with modifications of different PE fragments or lipids using native chemical ligation and aminolysis approaches.

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  • 39. Yang, Aimin
    et al.
    Pantoom, Supansa
    Wu, Yao-Wen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Chemical Genomics Centre of the Max Planck Society, Dortmund, Germany; Max Planck Institute of Molecular Physiology, Dortmund, Germany.
    Distinct Mechanisms for Processing Autophagy Protein LC3-PE by RavZ and ATG4B2020Ingår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 21, nr 23, s. 3377-3382Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Autophagy is a conserved catabolic process involved in the elimination of proteins, organelles and pathogens in eukaryotic cells. Lipidated LC3 proteins that are conjugated to phosphatidylethanolamine (PE) play a key role in autophagosome biogenesis. Endogenous ATG4-mediated deconjugation of LC3-PE is required for LC3 recycling. However, theLegionellaeffector RavZ irreversibly deconjugates LC3-PE to inhibit autophagy. It is not clear how ATG4 and RavZ process LC3-PE with distinct modes. Herein, a series of semisynthetic LC3-PE proteins containing C-terminal mutations or insertions were used to investigate the relationship of the C-terminal structure of LC3-PE with ATG4/RavZ-mediated deconjugation. Using a combination of molecular docking and biochemical assays, we found that Gln116, Phe119 and Gly120 of LC3-PE are required for cleavage by both RavZ and ATG4B, whereas Glu117(LC3) is specific to cleavage by RavZ. The molecular ruler mechanism exists in the active site of ATG4B, but not in RavZ. Met63 and Gln64 at the active site of RavZ are involved in accommodating LC3 C-terminal motif. Our findings show that the distinct binding modes of the LC3 C-terminal motif (116-120) with ATG4 and RavZ might determine the specificity of cleavage site.

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  • 40. Yang, Aimin
    et al.
    Pantoom, Supansa
    Wu, Yao-Wen
    Institute of Chemical Biology and Precision Therapy, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China; Chemical Genomics Centre of the Max Planck Society, Dortmund, Germany; Max-Planck-Institute of Molecular Physiology, Dortmund, Germany.
    Elucidation of the anti-autophagy mechanism of the Legionella effector RavZ using semisynthetic LC3 proteins2017Ingår i: eLIFE, E-ISSN 2050-084X, Vol. 6, artikel-id e23905Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Autophagy is a conserved cellular process involved in the elimination of proteins and organelles. It is also used to combat infection with pathogenic microbes. The intracellular pathogen Legionella pneumophila manipulates autophagy by delivering the effector protein RavZ to deconjugate Atg8/LC3 proteins coupled to phosphatidylethanolamine (PE) on autophagosomal membranes. To understand how RavZ recognizes and deconjugates LC3-PE, we prepared semisynthetic LC3 proteins and elucidated the structures of the RavZ:LC3 interaction. Semisynthetic LC3 proteins allowed the analysis of structure-function relationships. RavZ extracts LC3-PE from the membrane before deconjugation. RavZ initially recognizes the LC3 molecule on membranes via its N-terminal LC3-interacting region (LIR) motif. The RavZ α3 helix is involved in extraction of the PE moiety and docking of the acyl chains into the lipid-binding site of RavZ that is related in structure to that of the phospholipid transfer protein Sec14. Thus, Legionella has evolved a novel mechanism to specifically evade host autophagy.

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