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

Abstract

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 reconstitute the covalent attachment of thiocarboxylated Urm1 to various cellular target proteins in vitro, revealing that, unlike other known UBLs, this process is E2/E3-independent and requires oxidative stress. Furthermore, we present the crystal structures of the peroxiredoxin Ahp1 before and after the covalent attachment of Urm1. Surprisingly, we show that urmylation is accompanied by the transfer of sulfur to cysteine residues in the target proteins, also known as cysteine persulfidation. Our results illustrate the role of the Uba4-Urm1 system as a key evolutionary link between prokaryotic SCPs and the UBL modifications observed in modern eukaryotes.

The logic of protein post‐translational modifications (PTMs): Chemistry, mechanisms and evolution of protein regulation through covalent attachments

“…This hypothesis is supported by the similarity between components of the eukaryotic ubiquitin and prokaryotic sulphur carrier systems [212][213][214][215] and, even more strikingly, by the case of the eukaryotic protein Urm1, a ubiquitin-like ʻmolecular fossil' that can act both as a sulphur carrier and a protein modification. [216][217][218][219][220] A different example of repurposing an existing resource (this timean enzymatic activity) for a PTM reaction is provided by the Pupylation pathway of Mycobacterium tuberculosis. [42] Here, the writer enzyme PafA, which ligates a small intrinsically disordered protein called Pup to substrates, is related to metabolic enzymes including glutamine synthetase, which catalyse the attachment of a glutamyl moiety to an amino group in a biosynthetic pathway.…”

“…This hypothesis is supported by the similarity between components of the eukaryotic ubiquitin and prokaryotic sulphur carrier systems [ 212–215 ] and, even more strikingly, by the case of the eukaryotic protein Urm1, a ubiquitin‐like ʻmolecular fossil’ that can act both as a sulphur carrier and a protein modification. [ 216–220 ]…”

In the moonlight: non-catalytic functions of ubiquitin and ubiquitin-like proteases

“…Notwithstanding, ATG8, ATG12 and UFM1 only have a single C-terminal glycine ( Aichem and Groettrup, 2020 ). In addition, URM1 has been shown to form covalent bonds with various target proteins through a process that does not rely on E2/E3 enzymes ( Ravichandran et al, 2022 ). Furthermore, numerous UBLs such as SUMO, ISG15, FAT10 and ATG12 have been demonstrated to perform indispensable functions by engaging with substrates in a non-covalent manner ( Perng and Lenschow, 2018 ; Pang et al, 2019 ; Aichem and Groettrup, 2020 ; González-Prieto et al, 2021 ).…”

Discovery and characterization of noncanonical E2-conjugating enzymes

“…E2s that functionally interact with RING E3 ligases have intrinsic reactivity toward lysine, the canonical ubiquitylation target. However, other hydroxyl-containing amino acid and biomolecules, such as serine, threonine, sugars, and the bacterial liposaccharide, have also been found to be targeted by ubiquitin (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23) and the ubiquitin-like protein URM1 (24). The isopeptide bond formed between the ubiquitin C terminus and the amine present in the lysine side chain is very stable over a range of temperatures and pH.…”