RESUMO
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.
Assuntos
Ubiquitina , Ubiquitinas , Anticódon , Proteínas de Transporte/metabolismo , Cisteína , Peroxirredoxinas , Enxofre/metabolismo , Ubiquitina/metabolismo , Ubiquitinas/metabolismoRESUMO
Protein S-persulfidation (P-SSH) is recognized as a common posttranslational modification. It occurs under basal conditions and is often observed to be elevated under stress conditions. However, the mechanism(s) by which proteins are persulfidated inside cells have remained unclear. Here we report that 3-mercaptopyruvate sulfur transferase (MPST) engages in direct protein-to-protein transpersulfidation reactions beyond its previously known protein substrates thioredoxin and MOCS3/Uba4, associated with H2S generation and transfer RNA thiolation, respectively. We observe that depletion of MPST in human cells lowers overall intracellular protein persulfidation levels and identify a subset of proteins whose persulfidation depends on MPST. The predicted involvement of these proteins in the adaptation to stress responses supports the notion that MPST-dependent protein persulfidation promotes cytoprotective functions. The observation of MPST-independent protein persulfidation suggests that other protein persulfidases remain to be identified.
Assuntos
Sulfurtransferases , Humanos , Cisteína , Sulfeto de Hidrogênio/metabolismo , Enxofre/metabolismoRESUMO
Fungal pathogens threaten ecosystems and human health. Understanding the molecular basis of their virulence is key to develop new treatment strategies. Here, we characterize NCS2*, a point mutation identified in a clinical baker's yeast isolate. Ncs2 is essential for 2-thiolation of tRNA and the NCS2* mutation leads to increased thiolation at body temperature. NCS2* yeast exhibits enhanced fitness when grown at elevated temperatures or when exposed to oxidative stress, inhibition of nutrient signalling, and cell-wall stress. Importantly, Ncs2* alters the interaction and stability of the thiolase complex likely mediated by nucleotide binding. The absence of 2-thiolation abrogates the in vivo virulence of pathogenic baker's yeast in infected mice. Finally, hypomodification triggers changes in colony morphology and hyphae formation in the common commensal pathogen Candida albicans resulting in decreased virulence in a human cell culture model. These findings demonstrate that 2-thiolation of tRNA acts as a key mediator of fungal virulence and reveal new mechanistic insights into the function of the highly conserved tRNA-thiolase complex.
Assuntos
RNA de Transferência , Saccharomyces cerevisiae , Animais , Humanos , Camundongos , Candida albicans/metabolismo , Ecossistema , Proteínas Fúngicas/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/patogenicidade , Enxofre/metabolismo , Virulência/genéticaRESUMO
The chemical modification of tRNA bases by sulfur is crucial to tune translation and to optimize protein synthesis. In eukaryotes, the ubiquitin-related modifier 1 (Urm1) pathway is responsible for the synthesis of 2-thiolated wobble uridine (U34 ). During the key step of the modification cascade, the E1-like activating enzyme ubiquitin-like protein activator 4 (Uba4) first adenylates and thiocarboxylates the C-terminus of its substrate Urm1. Subsequently, activated thiocarboxylated Urm1 (Urm1-COSH) can serve as a sulfur donor for specific tRNA thiolases or participate in ubiquitin-like conjugation reactions. Structural and mechanistic details of Uba4 and Urm1 have remained elusive but are key to understand the evolutionary branch point between ubiquitin-like proteins (UBL) and sulfur-relay systems. Here, we report the crystal structures of full-length Uba4 and its heterodimeric complex with its substrate Urm1. We show how the two domains of Uba4 orchestrate recognition, binding, and thiocarboxylation of the C-terminus of Urm1. Finally, we uncover how the catalytic domains of Uba4 communicate efficiently during the reaction cycle and identify a mechanism that enables Uba4 to protect itself against self-conjugation with its own product, namely activated Urm1-COSH.
Assuntos
Nucleotidiltransferases/química , RNA de Transferência/química , Enxofre/química , Sulfurtransferases/química , Ubiquitinas/química , Humanos , Nucleotidiltransferases/metabolismo , RNA de Transferência/metabolismo , Enxofre/metabolismo , Sulfurtransferases/metabolismo , Ubiquitinas/metabolismoRESUMO
Ribonucleotide modifications perform a wide variety of roles in synthesis, turnover and functionality of tRNA molecules. The presence of particular chemical moieties can refine the internal interaction network within a tRNA molecule, influence its thermodynamic stability, contribute novel chemical properties and affect its decoding behavior during mRNA translation. As the lack of specific modifications in the anticodon stem and loop causes disrupted proteome homeostasis, diminished response to stress conditions, and the onset of human diseases, the underlying modification cascades have recently gained particular scientific and clinical interest. Nowadays, a complicated but conclusive image of the interconnectivity between different enzymatic modification cascades and their resulting tRNA modifications emerges. Here we summarize the current knowledge in the field, focusing on the known instances of cross talk among the enzymatic tRNA modification pathways and the consequences on the dynamic regulation of the tRNA modificome by various factors. This article is part of a Special Issue entitled: SI: Regulation of tRNA synthesis and modification in physiological conditions and disease edited by Dr. Boguta Magdalena.
Assuntos
Processamento Pós-Transcricional do RNA , RNA de Transferência/metabolismo , Animais , Anticódon/genética , Endorribonucleases/metabolismo , Células Eucarióticas/metabolismo , Humanos , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/metabolismo , Conformação de Ácido Nucleico , Biossíntese de Proteínas , Estabilidade de RNA , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Neoplásico/metabolismo , RNA de Transferência/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Uridina/análogos & derivados , Uridina/genética , tRNA Metiltransferases/metabolismoRESUMO
Microtubules are hollow biopolymers of 25-nm diameter and are key constituents of the cytoskeleton. In neurons, microtubules are organized differently between axons and dendrites, but their precise organization in different compartments is not completely understood. Super-resolution microscopy techniques can detect specific structures at an increased resolution, but the narrow spacing between neuronal microtubules poses challenges because most existing labelling strategies increase the effective microtubule diameter by 20-40 nm and will thereby blend neighbouring microtubules into one structure. Here we develop single-chain antibody fragments (nanobodies) against tubulin to achieve super-resolution imaging of microtubules with a decreased apparent diameter. To test the resolving power of these novel probes, we generate microtubule bundles with a known spacing of 50-70 nm and successfully resolve individual microtubules. Individual bundled microtubules can also be resolved in different mammalian cells, including hippocampal neurons, allowing novel insights into fundamental mechanisms of microtubule organization in cell- and neurobiology.