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Non-canonical ATG8 conjugation in ESCRT-driven membrane remodeling processes
Umeå University, Faculty of Science and Technology, Department of Chemistry.ORCID iD: 0000-0001-8504-9126
2024 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Okonventionell ATG8-konjugering i ESCRT-drivna membranombyggnadsprocesser (Swedish)
Abstract [en]

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.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2024. , p. 70
Keywords [en]
ATG8 conjugation, Endosomal Sorting Complex Required for Transport, membrane remodeling, chemical genetics
National Category
Cell Biology Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-222756ISBN: 978-91-8070-356-7 (print)ISBN: 978-91-8070-357-4 (electronic)OAI: oai:DiVA.org:umu-222756DiVA, id: diva2:1847289
Public defence
2024-04-26, Stora Hörsalen, KBC byggnad KBE303, 09:00 (English)
Opponent
Supervisors
Available from: 2024-04-05 Created: 2024-03-27 Last updated: 2024-03-27Bibliographically approved
List of papers
1. Synthesis of 20-Membered Macrocyclic Pseudo-Natural Products Yields Inducers of LC3 Lipidation
Open this publication in new window or tab >>Synthesis of 20-Membered Macrocyclic Pseudo-Natural Products Yields Inducers of LC3 Lipidation
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2022 (English)In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 61, no 11, article id e202114328Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2022
National Category
Organic Chemistry
Identifiers
urn:nbn:se:umu:diva-192165 (URN)10.1002/anie.202114328 (DOI)000746469800001 ()34978373 (PubMedID)2-s2.0-85123464160 (Scopus ID)
Funder
Swedish Research Council, 2018‐04585Knut and Alice Wallenberg FoundationGöran Gustafsson Foundation for Research in Natural Sciences and Medicine
Available from: 2022-02-04 Created: 2022-02-04 Last updated: 2024-03-27Bibliographically approved
2. Chemogenetic inhibition of IST1-CHMP1B interaction impairs endosomal recycling and promotes unconventional LC3 lipidation at stalled endosomes
Open this publication in new window or tab >>Chemogenetic inhibition of IST1-CHMP1B interaction impairs endosomal recycling and promotes unconventional LC3 lipidation at stalled endosomes
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(English)Manuscript (preprint) (Other academic)
National Category
Cell Biology Biochemistry and Molecular Biology
Research subject
cell research; biological chemistry; biology
Identifiers
urn:nbn:se:umu:diva-222750 (URN)10.1101/2023.08.28.555152 (DOI)
Available from: 2024-03-27 Created: 2024-03-27 Last updated: 2024-03-27
3. V. cholerae MakA is a cholesterol-binding pore-forming toxin that induces non-canonical autophagy
Open this publication in new window or tab >>V. cholerae MakA is a cholesterol-binding pore-forming toxin that induces non-canonical autophagy
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2022 (English)In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 221, no 12, article id e202206040Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Rockefeller University Press, 2022
Keywords
cholesterol-binding, MakA, non-canonical autophagy, pore-forming toxin, Vibrio Cholerae
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-200014 (URN)10.1083/jcb.202206040 (DOI)000932911400001 ()36194176 (PubMedID)2-s2.0-85139366240 (Scopus ID)
Funder
Knut and Alice Wallenberg FoundationEU, European Research CouncilSwedish Research Council, 2018-04585Göran Gustafsson Foundation for Research in Natural Sciences and Medicine
Available from: 2022-10-05 Created: 2022-10-05 Last updated: 2024-03-27Bibliographically approved
4. An ATG12-ATG5-TECPR1 E3-like complex regulates unconventional LC3 lipidation at damaged lysosomes
Open this publication in new window or tab >>An ATG12-ATG5-TECPR1 E3-like complex regulates unconventional LC3 lipidation at damaged lysosomes
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2023 (English)In: EMBO Reports, ISSN 1469-221X, E-ISSN 1469-3178, Vol. 24, no 9, article id e56841Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
EMBO Press, 2023
Keywords
autophagy, lysophagy, lysosome, membrane repair, TECPR1
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-212078 (URN)10.15252/embr.202356841 (DOI)001018486400001 ()37381828 (PubMedID)2-s2.0-85163748819 (Scopus ID)
Funder
EU, European Research CouncilSwedish Research Council, 2018-04585Swedish Research Council, 2022-02932Knut and Alice Wallenberg FoundationGöran Gustafsson Foundation for Research in Natural Sciences and Medicine
Available from: 2023-07-17 Created: 2023-07-17 Last updated: 2024-03-27Bibliographically approved

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Knyazeva, Anastasia

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