SwePub - sökning: WFRF:(Wu Yao Wen Professor)

Numrering	Referens	Omslagsbild	Hitta
1.	Jakobsson, J., et al. (författare) Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1 2021 Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 17:1, s. 1-382 Tidskriftsartikel (refereegranskat)
2.	Knyazeva, Anastasia, 1995- (författare) Non-canonical ATG8 conjugation in ESCRT-driven membrane remodeling processes 2024 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract ATG8 family proteins have the unique ability to conjugate to membrane lipids. Initially identified as a hallmark of autophagy, ATG8 lipidation is emerging as an important regulator of a growing list of non-degradative cellular functions. In this thesis we developed and applied novel chemical genetic approaches to perturb dynamic membrane remodeling processes and induce non-canonical ATG8 conjugation in cells. We investigated novel roles of ATG8 in membrane deformation processes carried out by the Endosomal Sorting Complex Requiredfor Transport (ESCRT) machinery.In Paper I, using a high-throughput phenotypic screening assay, we developed a collection of pseudo-natural product-based compounds which potently induce ATG8 lipidation in mammalian cells. The most potent compound, Tantalosin, induces ATG8 lipidation which is insensitive to simultaneous inhibition of autophagosome-lysosome fusion, suggesting a non-canonical function ofTantalosin-induced ATG8 conjugation.In Paper II we investigated the molecular target of Tantalosin. We found that Tantalosin targets the ESCRT-III protein IST1 and inhibits IST1-CHMP1B copolymer formation. This inhibition results in the impairment of transferrin receptor (TfR) recycling resulting in the rapid accumulation of the receptor inearly/sorting endosomes. At the same time, Tantalosin induces non-canonical ATG8 conjugation on stalled sorting endosomes containing TfR. This conjugation is dependent on the ATG16L1-ATG5-ATG12 complex which is recruited to stalled endosomes via ATG16L1-V-ATPase interaction.In Paper III and Paper IV we studied the induction of non-canonical ATG8 lipidation in response to endolysosomal membrane damage. We used two established membrane damaging agents: V. Cholerae cytotoxin MakA and the lysosomotropic compound, LLOMe. In Paper III we demonstrated that, at lowpH, MakA assembles into small pores in endosomal membranes which arerecognized by the ESCRT membrane repair machinery. Non-canonical ATG8 lipidation in response to MakA-induced pore formation is mediated by V-ATPase activity. In Paper IV we identified a novel player in the lysosomal damage response – TECRP1. TECPR1 is recruited to damaged membranes where it forms an alternative ATG16L1-independent E3 ligase complex with the ATG5-ATG12 conjugate and plays a role in the restoration of lysosomal integrity after damage.
3.	Alex, Amal, et al. (författare) Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology 2019 Ingår i: eLIFE. - : ELIFE SCIENCES PUBLICATIONS LTD. - 2050-084X. ; 8 Tidskriftsartikel (refereegranskat)abstract 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.
4.	Carnero Corrales, Marjorie A., et al. (författare) Thermal proteome profiling identifies the membrane-bound purinergic receptor P2X4 as a target of the autophagy inhibitor indophagolin 2021 Ingår i: Cell Chemical Biology. - : Elsevier. - 2451-9456 .- 2451-9448. ; 28:12, s. 1750-1757.e5 Tidskriftsartikel (refereegranskat)abstract 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.
5.	Chen, Xi, et al. (författare) Affinity conjugation for rapid and covalent labeling of proteins in live cells 2019 Ingår i: Proximity labeling. - New York : Humana Press. - 9781493995363 - 9781493995394 - 9781493995370 ; , s. 191-202 Bokkapitel (refereegranskat)abstract 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.
6.	Chen, Xi, et al. (författare) “Molecular Activity Painting” : Switch-like, light-controlled perturbations inside living cells 2017 Ingår i: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 56:21, s. 5916-5920 Tidskriftsartikel (refereegranskat)abstract 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.
7.	Corkery, Dale, et al. (författare) An ATG12-ATG5-TECPR1 E3-like complex regulates unconventional LC3 lipidation at damaged lysosomes 2023 Ingår i: EMBO Reports. - : EMBO Press. - 1469-221X .- 1469-3178. ; 24:9 Tidskriftsartikel (refereegranskat)abstract 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.
8.	Corkery, Dale, et al. (författare) Inducin triggers LC3-lipidation and ESCRT-mediated lysosomal membrane repair 2023 Ingår i: ChemBioChem. - : Wiley-VCH Verlagsgesellschaft. - 1439-4227 .- 1439-7633. ; 24:24 Tidskriftsartikel (refereegranskat)abstract 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.
9.	Corkery, Dale P., et al. (författare) ATG12–ATG5-TECPR1 : an alternative E3-like complex utilized during the cellular response to lysosomal membrane damage 2024 Ingår i: Autophagy. - : Taylor & Francis. - 1554-8627 .- 1554-8635. ; 20:2, s. 443-444 Tidskriftsartikel (refereegranskat)abstract 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.
10.	Corkery, Dale P., et al. (författare) Eating while intoxicated : characterizing the molecular mechanism behind V. cholerae toxin MakA-regulated autophagy 2023 Ingår i: Autophagy. - : Taylor & Francis. - 1554-8627 .- 1554-8635. ; 19:6, s. 1885-1886 Tidskriftsartikel (refereegranskat)abstract 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.
11.	Corkery, Dale, et al. (författare) Vibrio cholerae cytotoxin MakA induces noncanonical autophagy resulting in the spatial inhibition of canonical autophagy 2021 Ingår i: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 134:5 Tidskriftsartikel (refereegranskat)abstract 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.
12.	Foley, Daniel J., et al. (författare) Phenotyping Reveals Targets of a Pseudo-Natural-Product Autophagy Inhibitor 2020 Ingår i: Angewandte Chemie International Edition. - : Wiley-VCH Verlagsgesellschaft. - 1433-7851 .- 1521-3773. ; 59:30 Tidskriftsartikel (refereegranskat)abstract 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.
13.	Jia, Xiaotong, et al. (författare) V. cholerae MakA is a cholesterol-binding pore-forming toxin that induces non-canonical autophagy 2022 Ingår i: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 221:12 Tidskriftsartikel (refereegranskat)abstract 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.
14.	Kaiser, Nadine, et al. (författare) Modulation of autophagy by the novel mitochondrial complex I inhibitor Authipyrin 2019 Ingår i: Bioorganic & Medicinal Chemistry. - : Elsevier BV. - 0968-0896 .- 1464-3391. ; 27:12, s. 2444-2448 Tidskriftsartikel (refereegranskat)abstract 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.
15.	Kamps, Dominic, et al. (författare) Optogenetic Tuning Reveals Rho Amplification-Dependent Dynamics of a Cell Contraction Signal Network 2020 Ingår i: Cell Reports. - : Cell Press. - 2211-1247. ; 33:9 Tidskriftsartikel (refereegranskat)abstract 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.
16.	Klewer, Laura, et al. (författare) Light-Induced Dimerization Approaches to Control Cellular Processes 2019 Ingår i: Chemistry - A European Journal. - : Wiley. - 0947-6539 .- 1521-3765. ; 25, s. 12452-12463 Tidskriftsartikel (refereegranskat)abstract 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.
17.	Knyazeva, Anastasia, 1995-, et al. (författare) A chemical inhibitor of IST1-CHMP1B interaction impairs endosomal recycling and induces noncanonical LC3 lipidation 2024 Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 121:17 Tidskriftsartikel (refereegranskat)abstract The endosomal sorting complex required for transport (ESCRT) machinery constitutes multisubunit protein complexes that play an essential role in membrane remodeling and trafficking. ESCRTs regulate a wide array of cellular processes, including cytokinetic abscission, cargo sorting into multivesicular bodies (MVBs), membrane repair, and autophagy. Given the versatile functionality of ESCRTs, and the intricate organizational structure of the ESCRT machinery, the targeted modulation of distinct ESCRT complexes is considerably challenging. This study presents a pseudonatural product targeting IST1-CHMP1B within the ESCRT-III complexes. The compound specifically disrupts the interaction between IST1 and CHMP1B, thereby inhibiting the formation of IST1-CHMP1B copolymers essential for normal-topology membrane scission events. While the compound has no impact on cytokinesis, MVB sorting, or biogenesis of extracellular vesicles, it rapidly inhibits transferrin receptor recycling in cells, resulting in the accumulation of transferrin in stalled sorting endosomes. Stalled endosomes become decorated by lipidated LC3, suggesting a link between noncanonical LC3 lipidation and inhibition of the IST1-CHMP1B complex.
18.	Knyazeva, Anastasia, et al. (författare) Chemogenetic inhibition of IST1-CHMP1B interaction impairs endosomal recycling and promotes unconventional LC3 lipidation at stalled endosomes Annan publikation (övrigt vetenskapligt/konstnärligt)
19.	Konstantinidis, Georgios, et al. (författare) Identification of novel autophagy inhibitors via cell-based high-content screening 2019 Ingår i: Autophagy in differentiation and tissue maintenance. - New York, NY : Humana Press. - 9781493987474 - 9781493993826 - 9781493987481 ; , s. 187-195 Bokkapitel (refereegranskat)abstract 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. (författare) Monitoring the Response of Multiple Signal Network Components to Acute Chemo-Optogenetic Perturbations in Living Cellsope 2022 Ingår i: ChemBioChem. - : John Wiley & Sons. - 1439-4227 .- 1439-7633. ; 23:4 Tidskriftsartikel (refereegranskat)abstract 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.
21.	Laraia, Luca, et al. (författare) Discovery of Novel Cinchona‐Alkaloid‐Inspired Oxazatwistane Autophagy Inhibitors 2017 Ingår i: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 56:8, s. 2145-2150 Tidskriftsartikel (refereegranskat)abstract 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.
22.	Laraia, Luca, et al. (författare) Image-Based Morphological Profiling Identifies a Lysosomotropic, Iron-Sequestering Autophagy Inhibitor 2020 Ingår i: Angewandte Chemie International Edition. - : Wiley-VCH Verlagsgesellschaft. - 1433-7851 .- 1521-3773. ; 59, s. 5721-5729 Tidskriftsartikel (refereegranskat)abstract 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.
23.	Laraia, Luca, et al. (författare) The cholesterol transfer protein GRAMD1A regulates autophagosome biogenesis 2019 Ingår i: Nature Chemical Biology. - : Nature Publishing Group. - 1552-4450 .- 1552-4469. ; 15:7, s. 710-720 Tidskriftsartikel (refereegranskat)abstract 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.
24.	Li, Fu, et al. (författare) Imaging of spatial cycling of Rab GTPase in the cell 2021. - 2 Ingår i: Rab GTPases. - New York, NY : Humana Press. - 9781071613450 - 9781071613467 ; , s. 105-115 Bokkapitel (refereegranskat)abstract 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.
25.	Li, Fang-Yi, et al. (författare) Inhibition of K-Ras4B-plasma membrane association with a membrane microdomain-targeting peptide 2020 Ingår i: Chemical Science. - : Royal Society of Chemistry. - 2041-6520 .- 2041-6539. ; 11:3, s. 826-832 Tidskriftsartikel (refereegranskat)abstract 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.
26.	Nanda, Suchet, et al. (författare) Rho GTPase activity crosstalk mediated by Arhgef11 and Arhgef12 coordinates cell protrusion-retraction cycles 2023 Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1 Tidskriftsartikel (refereegranskat)abstract 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.
27.	Niggemeyer, Georg, et al. (författare) Synthesis of 20-Membered Macrocyclic Pseudo-Natural Products Yields Inducers of LC3 Lipidation 2022 Ingår i: Angewandte Chemie International Edition. - : Wiley-VCH Verlagsgesellschaft. - 1433-7851 .- 1521-3773. ; 61:11 Tidskriftsartikel (refereegranskat)abstract 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.
28.	Pantoom, Supansa, et al. (författare) Lift and cut: Anti-host autophagy mechanism of Legionella pneumophila 2017 Ingår i: Autophagy. - : Taylor & Francis Group. - 1554-8627 .- 1554-8635. ; 13:8, s. 1467-1469 Tidskriftsartikel (refereegranskat)abstract 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.
29.	Pantoom, Supansa, et al. (författare) RAB33B recruits the ATG16L1 complex to the phagophore via a noncanonical RAB binding protein 2021 Ingår i: Autophagy. - : Taylor & Francis. - 1554-8627 .- 1554-8635. ; 17:9, s. 2290-2304 Tidskriftsartikel (refereegranskat)abstract 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.
30.	Robke, Lucas, et al. (författare) Discovery of the novel autophagy inhibitor aumitin that targets mitochondrial complex I 2018 Ingår i: Chemical Science. - : Royal Society of Chemistry. - 2041-6520 .- 2041-6539. ; 9:11, s. 3014-3022 Tidskriftsartikel (refereegranskat)abstract 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.
31.	Robke, Lucas, et al. (författare) Phenotypic Identification of a Novel Autophagy Inhibitor Chemotype Targeting Lipid Kinase VPS34 2017 Ingår i: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 56:28, s. 8153-8157 Tidskriftsartikel (refereegranskat)abstract 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.
32.	Wen, Bo, et al. (författare) Comparison for the effects of different components of temperature variability on mortality : A multi-country time-series study 2024 Ingår i: Environment International. - : Elsevier. - 0160-4120 .- 1873-6750. ; 187 Tidskriftsartikel (refereegranskat)abstract Background: Temperature variability (TV) is associated with increased mortality risk. However, it is still unknown whether intra-day or inter-day TV has different effects. Objectives: We aimed to assess the association of intra-day TV and inter-day TV with all-cause, cardiovascular, and respiratory mortality.Methods: We collected data on total, cardiovascular, and respiratory mortality and meteorology from 758 locations in 47 countries or regions from 1972 to 2020. We defined inter-day TV as the standard deviation (SD) of daily mean temperatures across the lag interval, and intra-day TV as the average SD of minimum and maximum temperatures on each day. In the first stage, inter-day and intra-day TVs were modelled simultaneously in the quasi-Poisson time-series model for each location. In the second stage, a multi-level analysis was used to pool the location-specific estimates.Results: Overall, the mortality risk due to each interquartile range [IQR] increase was higher for intra-day TV than for inter-day TV. The risk increased by 0.59% (95% confidence interval [CI]: 0.53, 0.65) for all-cause mortality, 0.64% (95% CI: 0.56, 0.73) for cardiovascular mortality, and 0.65% (95% CI: 0.49, 0.80) for respiratory mortality per IQR increase in intra-day TV0–7 (0.9 °C). An IQR increase in inter-day TV0–7 (1.6 °C) was associated with 0.22% (95% CI: 0.18, 0.26) increase in all-cause mortality, 0.44% (95% CI: 0.37, 0.50) increase in cardiovascular mortality, and 0.31% (95% CI: 0.21, 0.41) increase in respiratory mortality. The proportion of all-cause deaths attributable to intra-day TV0–7 and inter-day TV0–7 was 1.45% and 0.35%, respectively. The mortality risks varied by lag interval, climate area, season, and climate type.Conclusions: Our results indicated that intra-day TV may explain the main part of the mortality risk related to TV and suggested that comprehensive evaluations should be proposed in more countries to help protect human health.
33.	Wu, Yao, et al. (författare) Fluctuating temperature modifies heat-mortality association around the globe 2022 Ingår i: The Innovation. - : Cell Press. - 2666-6758. ; 3:2 Tidskriftsartikel (refereegranskat)abstract Studies have investigated the effects of heat and temperature variability (TV) on mortality. However, few assessed whether TV modifies the heat-mortality association. Data on daily temperature and mortality in the warm season were collected from 717 locations across 36 countries. TV was calculated as the standard deviation of the average of the same and previous days’ minimum and maximum temperatures. We used location-specific quasi-Poisson regression models with an interaction term between the cross-basis term for mean temperature and quartiles of TV to obtain heat-mortality associations under each quartile of TV, and then pooled estimates at the country, regional, and global levels. Results show the increased risk in heat-related mortality with increments in TV, accounting for 0.70% (95% confidence interval [CI]: −0.33 to 1.69), 1.34% (95% CI: −0.14 to 2.73), 1.99% (95% CI: 0.29–3.57), and 2.73% (95% CI: 0.76–4.50) of total deaths for Q1–Q4 (first quartile–fourth quartile) of TV. The modification effects of TV varied geographically. Central Europe had the highest attributable fractions (AFs), corresponding to 7.68% (95% CI: 5.25–9.89) of total deaths for Q4 of TV, while the lowest AFs were observed in North America, with the values for Q4 of 1.74% (95% CI: −0.09 to 3.39). TV had a significant modification effect on the heat-mortality association, causing a higher heat-related mortality burden with increments of TV. Implementing targeted strategies against heat exposure and fluctuant temperatures simultaneously would benefit public health.
34.	Wu, Yao, et al. (författare) Global, regional, and national burden of mortality associated with short-term temperature variability from 2000–19 : a three-stage modelling study 2022 Ingår i: The Lancet Planetary Health. - : Elsevier. - 2542-5196. ; 6:5, s. e410-e421 Tidskriftsartikel (refereegranskat)abstract Background: Increased mortality risk is associated with short-term temperature variability. However, to our knowledge, there has been no comprehensive assessment of the temperature variability-related mortality burden worldwide. In this study, using data from the MCC Collaborative Research Network, we first explored the association between temperature variability and mortality across 43 countries or regions. Then, to provide a more comprehensive picture of the global burden of mortality associated with temperature variability, global gridded temperature data with a resolution of 0·5° × 0·5° were used to assess the temperature variability-related mortality burden at the global, regional, and national levels. Furthermore, temporal trends in temperature variability-related mortality burden were also explored from 2000–19.Methods: In this modelling study, we applied a three-stage meta-analytical approach to assess the global temperature variability-related mortality burden at a spatial resolution of 0·5° × 0·5° from 2000–19. Temperature variability was calculated as the SD of the average of the same and previous days’ minimum and maximum temperatures. We first obtained location-specific temperature variability related-mortality associations based on a daily time series of 750 locations from the Multi-country Multi-city Collaborative Research Network. We subsequently constructed a multivariable meta-regression model with five predictors to estimate grid-specific temperature variability related-mortality associations across the globe. Finally, percentage excess in mortality and excess mortality rate were calculated to quantify the temperature variability-related mortality burden and to further explore its temporal trend over two decades.Findings: An increasing trend in temperature variability was identified at the global level from 2000 to 2019. Globally, 1 753 392 deaths (95% CI 1 159 901–2 357 718) were associated with temperature variability per year, accounting for 3·4% (2·2–4·6) of all deaths. Most of Asia, Australia, and New Zealand were observed to have a higher percentage excess in mortality than the global mean. Globally, the percentage excess in mortality increased by about 4·6% (3·7–5·3) per decade. The largest increase occurred in Australia and New Zealand (7·3%, 95% CI 4·3–10·4), followed by Europe (4·4%, 2·2–5·6) and Africa (3·3, 1·9–4·6).Interpretation: Globally, a substantial mortality burden was associated with temperature variability, showing geographical heterogeneity and a slightly increasing temporal trend. Our findings could assist in raising public awareness and improving the understanding of the health impacts of temperature variability. Funding: Australian Research Council, Australian National Health & Medical Research Council.
35.	Wu, Yao-Wen, Professor, et al. (författare) Bacterial interaction with host autophagy 2019 Ingår i: Virulence. - : Informa UK Limited. - 2150-5594 .- 2150-5608. ; 10:1, s. 352-362 Forskningsöversikt (refereegranskat)abstract 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.
36.	Wu, Yao-Wen, Professor (författare) Controlling cellular activities with light 2023 Ingår i: Nature Methods. - : Nature Publishing Group. - 1548-7091 .- 1548-7105. ; 20, s. 357-358 Tidskriftsartikel (refereegranskat)
37.	Wu, Yao-Wen, Professor (författare) Spatiotemporal Imaging of Small GTPase Activity Using Conformational Sensors for GTPase Activity (COSGA) 2021 Ingår i: Ras Activity and Signaling. - New York, NY : Humana Press. - 9781071611890 - 9781071611920 - 9781071611906 ; , s. 259-267 Bokkapitel (refereegranskat)abstract 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.
38.	Wu, Yao-Wen, Professor, et al. (författare) Toward the role of cholesterol and cholesterol transfer protein in autophagosome biogenesis 2019 Ingår i: Autophagy. - : Taylor & Francis Group. - 1554-8627 .- 1554-8635. ; 15:12, s. 2167-2168 Tidskriftsartikel (refereegranskat)abstract 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.
39.	Xin, Xiaoyi, et al. (författare) Ultrafast and selective labeling of endogenous proteins using affinity-based benzotriazole chemistry 2022 Ingår i: Chemical Science. - : Royal Society of Chemistry. - 2041-6520 .- 2041-6539. ; 13:24, s. 7240-7246 Tidskriftsartikel (refereegranskat)abstract 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.
40.	Yang, Aimin, et al. (författare) Distinct Mechanisms for Processing Autophagy Protein LC3-PE by RavZ and ATG4B 2020 Ingår i: ChemBioChem. - : Wiley-VCH Verlagsgesellschaft. - 1439-4227 .- 1439-7633. ; 21:23, s. 3377-3382 Tidskriftsartikel (refereegranskat)abstract 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.
41.	Yang, Aimin, et al. (författare) Elucidation of the anti-autophagy mechanism of the Legionella effector RavZ using semisynthetic LC3 proteins 2017 Ingår i: eLIFE. - 2050-084X. ; 6 Tidskriftsartikel (refereegranskat)abstract 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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