RESUMO
Ubiquitin related modifier 1 (Urm1) is a unique eukaryotic member of the ubiquitin-fold (UbF) protein family and conserved from yeast to humans. Urm1 is dual-functional, acting both as a sulfur carrier for thiolation of tRNA anticodons and as a protein modifier in a lysine-directed Ub-like conjugation also known as urmylation. Although Urm1 conjugation coincides with oxidative stress and targets proteins like 2-Cys peroxiredoxins from yeast (Ahp1) and fly (Prx5), it was unclear how urmylation proceeds molecularly and whether it is affected by the activity of these antioxidant enzymes. An in-depth study of Ahp1 urmylation in yeast from our laboratory (Brachmann et al., 2020) uncovered that promiscuous lysine target sites and specific redox requirements determine the Urm1 acceptor activity of the peroxiredoxin. The results clearly show that the dimer interface and the 2-Cys based redox-active centers of Ahp1 are affecting the Urm1 conjugation reaction. Together with in vivo assays demonstrating that high organic peroxide concentrations can prevent Ahp1 from being urmylated, Brachmann et al. provide insights into a potential link between Urm1 utilization and oxidant defense of cells. Here, we highlight these major findings and discuss wider implications with regards to an emerging link between Urm1 conjugation and redox biology. Moreover, from these studies we propose to redefine our perspective on Urm1 and the molecular nature of urmylation, a post-translational conjugation that may not be that ubiquitin-like after all.
RESUMO
The yeast peroxiredoxin Ahp1, like related anti-oxidant enzymes in other species, undergoes urmylation, a lysine-directed conjugation to ubiquitin-like modifier Urm1. Ahp1 assembles into a homodimer that detoxifies peroxides via forming intersubunit disulfides between peroxidatic and resolving cysteines that are subsequently reduced by the thioredoxin system. Although urmylation coincides with oxidative stress, it is unclear how this modification happens on a molecular level and whether it affects peroxiredoxin activity. Here, we report that thioredoxin mutants decrease Ahp1 urmylation in yeast and each subunit of the oxidized Ahp1 dimer is modified by Urm1 suggesting coupling of urmylation to dimerization. Consistently, Ahp1 mutants unable to form dimers, fail to be urmylated as do mutants that lack the peroxidatic cysteine. Moreover, Ahp1 urmylation involves at least two lysine residues close to the catalytic cysteines and can be prevented in yeast cells exposed to high organic peroxide concentrations. Our results elucidate redox requirements and molecular determinants critical for Ahp1 urmylation, thus providing insights into a potential link between oxidant defense and Urm1 utilization in cells.
Assuntos
Mutação , Peroxirredoxinas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Domínio Catalítico , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Modelos Moleculares , Oxirredução , Peróxidos/metabolismo , Peroxirredoxinas/química , Peroxirredoxinas/genética , Conformação Proteica , Multimerização Proteica , Saccharomyces cerevisiae/genéticaRESUMO
Transfer RNA (tRNA) is subject to a multitude of posttranscriptional modifications which can profoundly impact its functionality as the essential adaptor molecule in messenger RNA (mRNA) translation. Therefore, dynamic regulation of tRNA modification in response to environmental changes can tune the efficiency of gene expression in concert with the emerging epitranscriptomic mRNA regulators. Several of the tRNA modifications are required to prevent human diseases and are particularly important for proper development and generation of neurons. In addition to the positive role of different tRNA modifications in prevention of neurodegeneration, certain cancer types upregulate tRNA modification genes to sustain cancer cell gene expression and metastasis. Multiple associations of defects in genes encoding subunits of the tRNA modifier complex Elongator with human disease highlight the importance of proper anticodon wobble uridine modifications (xm5U34) for health. Elongator functionality requires communication with accessory proteins and dynamic phosphorylation, providing regulatory control of its function. Here, we summarized recent insights into molecular functions of the complex and the role of Elongator dependent tRNA modification in human disease.