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Non-canonical ATG8 conjugation in ESCRT-driven membrane remodeling processes
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.ORCID-id: 0000-0001-8504-9126
2024 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)Alternativ titel
Okonventionell ATG8-konjugering i ESCRT-drivna membranombyggnadsprocesser (Svenska)
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.

Ort, förlag, år, upplaga, sidor
Umeå: Umeå University, 2024. , s. 70
Nyckelord [en]
ATG8 conjugation, Endosomal Sorting Complex Required for Transport, membrane remodeling, chemical genetics
Nationell ämneskategori
Cellbiologi Biokemi och molekylärbiologi
Identifikatorer
URN: urn:nbn:se:umu:diva-222756ISBN: 978-91-8070-356-7 (tryckt)ISBN: 978-91-8070-357-4 (digital)OAI: oai:DiVA.org:umu-222756DiVA, id: diva2:1847289
Disputation
2024-04-26, Stora Hörsalen, KBC byggnad KBE303, 09:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2024-04-05 Skapad: 2024-03-27 Senast uppdaterad: 2024-03-27Bibliografiskt granskad
Delarbeten
1. Synthesis of 20-Membered Macrocyclic Pseudo-Natural Products Yields Inducers of LC3 Lipidation
Öppna denna publikation i ny flik eller fönster >>Synthesis of 20-Membered Macrocyclic Pseudo-Natural Products Yields Inducers of LC3 Lipidation
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2022 (Engelska)Ingår i: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 61, nr 11, artikel-id e202114328Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
Wiley-VCH Verlagsgesellschaft, 2022
Nationell ämneskategori
Organisk kemi
Identifikatorer
urn:nbn:se:umu:diva-192165 (URN)10.1002/anie.202114328 (DOI)000746469800001 ()34978373 (PubMedID)2-s2.0-85123464160 (Scopus ID)
Forskningsfinansiär
Vetenskapsrådet, 2018‐04585Knut och Alice Wallenbergs StiftelseGöran Gustafssons stiftelse för naturvetenskaplig och medicinsk forskning (KVA)
Tillgänglig från: 2022-02-04 Skapad: 2022-02-04 Senast uppdaterad: 2024-03-27Bibliografiskt granskad
2. Chemogenetic inhibition of IST1-CHMP1B interaction impairs endosomal recycling and promotes unconventional LC3 lipidation at stalled endosomes
Öppna denna publikation i ny flik eller fönster >>Chemogenetic inhibition of IST1-CHMP1B interaction impairs endosomal recycling and promotes unconventional LC3 lipidation at stalled endosomes
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(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nationell ämneskategori
Cellbiologi Biokemi och molekylärbiologi
Forskningsämne
cellforskning; biologisk kemi; biologi
Identifikatorer
urn:nbn:se:umu:diva-222750 (URN)10.1101/2023.08.28.555152 (DOI)
Tillgänglig från: 2024-03-27 Skapad: 2024-03-27 Senast uppdaterad: 2024-03-27
3. V. cholerae MakA is a cholesterol-binding pore-forming toxin that induces non-canonical autophagy
Öppna denna publikation i ny flik eller fönster >>V. cholerae MakA is a cholesterol-binding pore-forming toxin that induces non-canonical autophagy
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2022 (Engelska)Ingår i: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 221, nr 12, artikel-id e202206040Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
Rockefeller University Press, 2022
Nyckelord
cholesterol-binding, MakA, non-canonical autophagy, pore-forming toxin, Vibrio Cholerae
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:umu:diva-200014 (URN)10.1083/jcb.202206040 (DOI)000932911400001 ()36194176 (PubMedID)2-s2.0-85139366240 (Scopus ID)
Forskningsfinansiär
Knut och Alice Wallenbergs StiftelseEU, Europeiska forskningsrådetVetenskapsrådet, 2018-04585Göran Gustafssons stiftelse för naturvetenskaplig och medicinsk forskning (KVA)
Tillgänglig från: 2022-10-05 Skapad: 2022-10-05 Senast uppdaterad: 2024-03-27Bibliografiskt granskad
4. An ATG12-ATG5-TECPR1 E3-like complex regulates unconventional LC3 lipidation at damaged lysosomes
Öppna denna publikation i ny flik eller fönster >>An ATG12-ATG5-TECPR1 E3-like complex regulates unconventional LC3 lipidation at damaged lysosomes
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2023 (Engelska)Ingår i: EMBO Reports, ISSN 1469-221X, E-ISSN 1469-3178, Vol. 24, nr 9, artikel-id e56841Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
EMBO Press, 2023
Nyckelord
autophagy, lysophagy, lysosome, membrane repair, TECPR1
Nationell ämneskategori
Cell- och molekylärbiologi
Identifikatorer
urn:nbn:se:umu:diva-212078 (URN)10.15252/embr.202356841 (DOI)001018486400001 ()37381828 (PubMedID)2-s2.0-85163748819 (Scopus ID)
Forskningsfinansiär
EU, Europeiska forskningsrådetVetenskapsrådet, 2018-04585Vetenskapsrådet, 2022-02932Knut och Alice Wallenbergs StiftelseGöran Gustafssons stiftelse för naturvetenskaplig och medicinsk forskning (KVA)
Tillgänglig från: 2023-07-17 Skapad: 2023-07-17 Senast uppdaterad: 2024-03-27Bibliografiskt granskad

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Knyazeva, Anastasia

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