Urm1 couples sulfur transfer to ubiquitin-like protein function in oxidative stress.

T he posttranslational modification of proteins with ubiquitin and ubiquitin-like proteins (collectively referred to as UBLs) has emerged as a major regulatory mechanism in eukaryotes. UBLs are characterized by a core β-grasp fold and an essential carboxy terminal glycine within a di-glycine motif (1). These features are also found in several prokaryotic sulfur carriers, suggestive of an evolutionary relationship to UBLs (2–5). A case in point is the UBL Urm1 (ubiquitin-related modifier 1). Urm1 is known to be conjugated to the peroxiredoxin Ahp1 (6), but its sequence and structure more closely resembles bacterial sulfur carrier proteins (7). In PNAS, work by Van der Veen et al. provides important insight into the mechanism of Urm1 conjugation that highlights its position as an ancient UBL in eukaryotes and solidifies its roles as a posttranslational protein modifier involved in oxidative stress response mechanisms (8). All UBLs require ATP-dependent activation through their cognate E1 enzyme before conjugation onto target proteins (Fig. 1). After the initial formation of a UBL-adenylate in the nucleotide binding pocket of the E1 through its carboxy terminal glycine, the UBL is transferred onto an acceptor sulfhydryl—the E1 catalytic cysteine—to form a high-energy thiolester intermediate (9, 10). The ultimate covalent transfer of the UBL onto its target proteins involves additional specialized enzymes—E2s (conjugating enzymes) and sometimes E3s (ligases)—which receive the UBL through a trans-thiolation reaction using other active site cysteines, thereby providing additional layers of specificity and regulation. UBLs are usually transferred, via their carboxyl terminus, onto the ε-amine of lysine side chains of their substrates in a site-specific manner to form a covalent iso-peptide bond. The discovery of Urm1 in 2000 relied on the observation that prokaryotic sulfur carrier proteins, such as MoaD and ThiS, contain the characteristic UBL di-glycine motif and require ATP-dependent activation through a mechanism similar to E1 enzymes (7) (Fig. 1). These proteins function as sulfur donors in biosynthetic pathways, with MoaD involved in molybdenum cofactor (Moco) synthesis and ThiS in thiamine biosynthesis (2, 5, 11, 12). In contrast to the mechanism described above for UBLs, ATP-dependent activation promotes the formation of an acyl disulfide with MoeB and ThiS, respectively (Fig. 1). This leads to the formation of a thiocarboxylate that then provides sulfur necessary for subsequent reactions. How is Urm1 activated? Whereas the Urm1 E1 (Uba4 in budding yeast, MOCS3 in humans) was originally proposed to form a thiolester linkage to the carboxyl terminus of Urm1 (7), more recent in vitro studies determined that Urm1 forms an acyl disulfide through its E1, leading to the formation of thiocarboxylated Urm1 (ref. 13; Fig. 1). This mechanism requires two cysteine residues: one that has sulfurtransferase activity and another with adenylyltransferase activity. This is not entirely surprising, given that MOCS3 is also involved in the biosynthesis of Moco in humans (13–15). Thus, these observations suggest that Urm1 activation is more similar to that of a prokaryotic sulfur carrier protein than that of a UBL. This function has been linked to the downstream thiolation of certain tRNAs during oxidative stress, where their modification alters their decoding specificity (16–19). Urm1 also functions as a UBL to covalently modify proteins. Unlike other UBL systems, however, enzymes other than an E1 have not been identified; thus, it remained unclear how activated Urm1 is transferred onto proteins, the nature of these conjugates, and how residues on these proteins are selected for modification. Although previous studies detected covalent Urm1 conjugates in budding yeast, only a single substrate, the peroxiredoxin Ahp1, has been identified (6, 7). Genetic studies implicated Urm1 and Ahp1 in oxidative stress response mechanisms (6, 20). Van der Veen et al. significantly extend these observations by demonstrating that oxidative stress specifically induces the formation of Urm1–protein conjugates in both yeast and human cells (8). How do Fig. 1. Juxtaposition of Urm1 as a prokaryotic sulfur carrier and a eukaryotic UBL. The shaded areas indicate overlapping aspects of the pathways, illustrating that Urm1 seems to use features common to both ubiquitin and MoaD for its activation and conjugation. The Urm1–E2 intermediate indicated in brackets is still hypothetical but may account for the hydroxylamine sensitivity of Urm1–protein conjugates formed in response to oxidative stress.