Dataset Information

E2/E3-independent ubiquitin-like protein conjugation by Urm1 is directly coupled to cysteine persulfidation

ABSTRACT: Posttranslational modifications by ubiquitin-like proteins (UBL) are essential for nearly all cellular processes. Ubiquitin related modifier 1 (Urm1) is a non-canonical UBL which plays a key role in tRNA anticodon thiolation as a sulfur carrier protein (SCP). While Urm1 has also been observed to conjugate directly to target proteins like other UBLs, the mechanism of attachment as well as how the SCP properties of Urm1 may impact its conjugation are unknown. Here, we reconstitute the covalent attachment of Urm1 to various cellular target proteins in vitro, revealing that, unlike other known UBLs, this process is E2/E3-independent and requires oxidative stress conditions. We determined the crystal structures of the peroxiredoxin Ahp1 before and after covalent attachment of Urm1. Strikingly, we show that this conjugation process results in persulfidation of a cysteine residue in the target protein. Demonstrating that Urm1 actively transfers sulfur atoms to proteins as part of its conjugation reaction. Our results redefine Urm1 as a key evolutionary link between prokaryotic SCPs and the plethora of UBL modifications observed in modern eukaryotes, and demonstrate a critical role for Urm1 in protecting proteins during oxidative stress.

INSTRUMENT(S): micrOTOF-Q II, Q Exactive

ORGANISM(S): Chaetomium Thermophilum (ncbitaxon:209285) Saccharomyces Cerevisiae (ncbitaxon:4932) Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Sebastian Glatt

PROVIDER: MSV000088390 | MassIVE | Tue Nov 16 03:44:00 GMT 2021

REPOSITORIES: MassIVE

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E2/E3-independent ubiquitin-like protein conjugation by Urm1 is directly coupled to cysteine persulfidation.

Ravichandran Keerthiraju E KE Kaduhr Lars L Skupien-Rabian Bozena B Shvetsova Ekaterina E Sokołowski Mikołaj M Krutyhołowa Rościsław R Kwasna Dominika D Brachmann Cindy C Lin Sean S Guzman Perez Sebastian S Wilk Piotr P Kösters Manuel M Grudnik Przemysław P Jankowska Urszula U Leidel Sebastian A SA Schaffrath Raffael R Glatt Sebastian S

The EMBO journal 20220914 20

Post-translational modifications by ubiquitin-like proteins (UBLs) are essential for nearly all cellular processes. Ubiquitin-related modifier 1 (Urm1) is a unique UBL, which plays a key role in tRNA anticodon thiolation as a sulfur carrier protein (SCP) and is linked to the noncanonical E1 enzyme Uba4 (ubiquitin-like protein activator 4). While Urm1 has also been observed to conjugate to target proteins like other UBLs, the molecular mechanism of its attachment remains unknown. Here, we reconst ...[more]

PMID: 36102610

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Project description:Eukaryotes may experience oxygen deprivation under several physiological and pathological conditions. Because oxygen shortage leads to a reduction in cellular energy production, all the eukaryotes studied so far evolved ways to conserve energy via suppression of metabolism. However, the molecular physiology of animals that naturally and repeatedly experience anoxia is poorly studied. One such animal is the symbiotic marine nematode Laxus oneistus. This thrives, invariably coated by its sulfur-oxidizing bacterium Candidatus Thiosymbion oneisti, in anoxic sulfidic or hypoxic sand. Here, by applying transcriptomics and proteomics, we discovered that, irrespective of the oxygen concentration it is subjected to, this nematode is mostly and constitutively engaging in ubiquitination-mediated proteolysis, energy generation, oxidative stress response and immune response. Furthermore, by incubating L. oneistus under sulfidic anoxic or hypoxic conditions, we found that, under the first condition, the worm upregulated genes involved in ribosome biogenesis, proteolytic systems (autophagy and the ubiquitin-proteasome system), chaperones, detoxification and lectins. Degradation pathways were likely induced to recycle damaged cellular components (mitochondria) and misfolded proteins into nutrients, whereas lectins were likely expressed to promote the attachment of its anaerobic, thiotrophic symbiont. Besides, L. oneistus appeared to survive oxygen deprivation by using alternative electron carriers (rhodoquinone) and acceptors (fumarate) to rewire the electron transfer chain. On the other hand, under hypoxia, genes involved in costly processes (e.g., development, amino acid biosynthesis, feeding behaviour, mating) were upregulated. Intriguingly, the worm’s Toll-like innate immunity pathway and a number of immune effectors (e.g., Bacterial Permeability Increasing proteins, fungicides) were upregulated in the presence of oxygen. In conclusion, we hypothesize that L. oneistus survives in anoxic sulfidic sand by overexpressing degradation processes, rewiring oxidative phosphorylation and by reinforcing its coat of bacterial sulfur-oxidizers. In the presence of oxygen, instead, it appears to exploit glycolysis to eat, mate and grow. Moreover, oxygen appears to affect L. oneistus immune system in such way, as to favor specific attachment of its anaerobic partner in anoxia and to produce broad-range antimicrobials when oxygen is present.