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- Knyazeva, Anastasia, 1995-
(author)
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Non-canonical ATG8 conjugation in ESCRT-driven membrane remodeling processes
- 2024
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Doctoral thesis (other academic/artistic)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.
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| 3. |
- Alex, Amal, et al.
(author)
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Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology
- 2019
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In: eLIFE. - : ELIFE SCIENCES PUBLICATIONS LTD. - 2050-084X. ; 8
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Journal article (peer-reviewed)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.
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| 4. |
- Carnero Corrales, Marjorie A., et al.
(author)
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Thermal proteome profiling identifies the membrane-bound purinergic receptor P2X4 as a target of the autophagy inhibitor indophagolin
- 2021
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In: Cell Chemical Biology. - : Elsevier. - 2451-9456 .- 2451-9448. ; 28:12, s. 1750-1757.e5
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Journal article (peer-reviewed)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.
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| 5. |
- Chen, Xi, et al.
(author)
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Affinity conjugation for rapid and covalent labeling of proteins in live cells
- 2019
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In: Proximity labeling. - New York : Humana Press. - 9781493995363 - 9781493995394 - 9781493995370 ; , s. 191-202
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Book chapter (peer-reviewed)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.
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| 6. |
- Chen, Xi, et al.
(author)
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“Molecular Activity Painting” : Switch-like, light-controlled perturbations inside living cells
- 2017
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In: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 56:21, s. 5916-5920
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Journal article (peer-reviewed)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.
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| 7. |
- Corkery, Dale, et al.
(author)
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An ATG12-ATG5-TECPR1 E3-like complex regulates unconventional LC3 lipidation at damaged lysosomes
- 2023
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In: EMBO Reports. - : EMBO Press. - 1469-221X .- 1469-3178. ; 24:9
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Journal article (peer-reviewed)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.
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| 8. |
- Corkery, Dale, et al.
(author)
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Inducin triggers LC3-lipidation and ESCRT-mediated lysosomal membrane repair
- 2023
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In: ChemBioChem. - : Wiley-VCH Verlagsgesellschaft. - 1439-4227 .- 1439-7633. ; 24:24
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Journal article (peer-reviewed)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.
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| 9. |
- Corkery, Dale P., et al.
(author)
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ATG12–ATG5-TECPR1 : an alternative E3-like complex utilized during the cellular response to lysosomal membrane damage
- 2024
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In: Autophagy. - : Taylor & Francis. - 1554-8627 .- 1554-8635. ; 20:2, s. 443-444
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Journal article (peer-reviewed)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.
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| 10. |
- Corkery, Dale P., et al.
(author)
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Eating while intoxicated : characterizing the molecular mechanism behind V. cholerae toxin MakA-regulated autophagy
- 2023
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In: Autophagy. - : Taylor & Francis. - 1554-8627 .- 1554-8635. ; 19:6, s. 1885-1886
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Journal article (peer-reviewed)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.
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