RESUMO
Selenophosphate synthetases use selenium and ATP to synthesize selenophosphate. This is required for biological utilization of selenium, most notably for the synthesis of the non-canonical amino acid selenocysteine (Sec). Therefore, selenophosphate synthetases underlie all functions of selenoproteins, which include redox homeostasis, protein quality control, hormone regulation, metabolism, and many others. This protein family comprises two groups, SelD/SPS2 and SPS1. The SelD/SPS2 group represent true selenophosphate synthetases, enzymes central to selenium metabolism which are present in all Sec-utilizing organisms across the tree of life. Notably, many SelD/SPS2 proteins contain Sec as catalytic residue in their N-terminal flexible selenium-binding loop, while others replace it with cysteine (Cys). The SPS1 group comprises proteins originated through gene duplications of SelD/SPS2 in metazoa in which the Sec/Cys-dependent catalysis was disrupted. SPS1 proteins do not synthesize selenophosphate and are not required for Sec synthesis. They have essential regulatory functions related to redox homeostasis and pyridoxal phosphate, which affect signaling pathways for growth and differentiation. In this review, we summarize the knowledge about the selenophosphate synthetase family acquired through decades of research, encompassing their structure, mechanism, function, and evolution.
Assuntos
Selênio , Selenocisteína , Trifosfato de Adenosina/metabolismo , Cisteína , Hormônios , Ligases , Fosfatos , Fosfotransferases/genética , Fosfato de Piridoxal , Selênio/metabolismo , Compostos de Selênio , Selenocisteína/metabolismo , Selenoproteínas/metabolismoRESUMO
The trace element selenium is found in polypeptides as selenocysteine, the 21(st) amino acid that is co-translationally inserted into proteins at a UGA codon. In proteins, selenocysteine usually plays a role as an efficient redox catalyst. Trypanosomatids previously examined harbor a full set of genes encoding the machinery needed for selenocysteine biosynthesis and incorporation into three selenoproteins: SelK, SelT and, the parasite-specific, Seltryp. We investigated the selenoproteome of kinetoplastid species in recently sequenced genomes and assessed the in vivo relevance of selenoproteins for African trypanosomes. Database mining revealed that SelK, SelT and Seltryp genes are present in most kinetoplastids, including the free-living species Bodo saltans, and Seltryp was lost in the subgenus Viannia from the New World Leishmania. Homology and sinteny with bacterial sulfur dioxygenases and sulfur transferases suggest a putative role for Seltryp in sulfur metabolism. A Trypanosoma brucei selenocysteine synthase (SepSecS) null-mutant, in which selenoprotein synthesis is abolished, displayed similar sensitivity to oxidative stress induced by a short-term exposure to high concentrations of methylglyoxal or H2O2 to that of the parental wild-type cell line. Importantly, the infectivity of the SepSecS knockout cell line was not impaired when tested in a mouse infection model and compensatory effects via up-regulation of proteins involved in thiol-redox metabolism were not observed. Collectively, our data show that selenoproteins are not required for survival of African trypanosomes in a mammalian host and exclude a role for selenoproteins in parasite antioxidant defense and/or virulence. On this basis, selenoproteins can be disregarded as drug target candidates.
Assuntos
Kinetoplastida/metabolismo , Proteínas de Protozoários/genética , Selenocisteína/metabolismo , Selenoproteínas/genética , Transferases/genética , Trypanosoma brucei brucei/metabolismo , Animais , Mineração de Dados , Bases de Dados Genéticas , Deleção de Genes , Regulação da Expressão Gênica , Interações Hospedeiro-Parasita , Peróxido de Hidrogênio/farmacologia , Kinetoplastida/classificação , Kinetoplastida/efeitos dos fármacos , Kinetoplastida/crescimento & desenvolvimento , Camundongos , Filogenia , Proteoma/genética , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Aldeído Pirúvico/farmacologia , Selenoproteínas/deficiência , Transferases/deficiência , Trypanosoma brucei brucei/classificação , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/crescimento & desenvolvimento , Tripanossomíase Africana/parasitologia , Tripanossomíase Africana/patologiaRESUMO
Selenocysteine (Sec) is encoded by an UGA codon with the help of a SECIS element present in selenoprotein mRNAs. SECIS-binding protein (SBP2/SCBP-2) mediates Sec insertion, but the roles of its domains and the impact of its deficiency on Sec insertion are not fully understood. We used Caenorhabditis elegans to examine SBP2 function since it possesses a single selenoprotein, thioredoxin reductase-1 (TRXR-1). All SBP2 described so far have an RNA-binding domain (RBD) and a Sec-incorporation domain (SID). Surprisingly, C. elegans SBP2 lacks SID and consists only of an RBD. An sbp2 deletion mutant strain ablated Sec incorporation demonstrating SBP2 essentiality for Sec incorporation. Further in silico analyses of nematode genomes revealed conservation of SBP2 lacking SID and maintenance of Sec incorporation linked to TRXR-1. Remarkably, parasitic plant nematodes lost the ability to incorporate Sec, but retained SecP43, a gene associated with Sec incorporation. Interestingly, both selenophosphate synthetase (SPS) genes are absent in plant parasitic nematodes, while only Cys-containing SPS2 is present in Sec-incorporating nematodes. Our results indicate that C. elegans and the nematode lineage provide key insights into Sec incorporation and the evolution of Sec utilization trait, selenoproteomes, selenoproteins, and Sec residues. Finally, our study provides evidence of noncanonical translation initiation in C. elegans, not previously known for this well-established animal model.
Assuntos
Adaptação Biológica/genética , Caenorhabditis elegans/metabolismo , Evolução Molecular , Inativação Gênica , Redes e Vias Metabólicas/genética , Selenocisteína/metabolismo , Sequência de Aminoácidos , Animais , Pareamento de Bases , Sequência de Bases , Caenorhabditis elegans/genética , Códon de Terminação , Dados de Sequência Molecular , Filogenia , RNA de Transferência/genética , RNA de Transferência/metabolismo , Selenocisteína/genética , Selenoproteínas/genéticaRESUMO
In bacteria selenocysteyl-tRNA(sec) (SelC) is synthesized by selenocysteine synthase (SelA). Here we show by fluorescence anisotropy binding assays and electron microscopical symmetry analysis that the SelA-tRNA(sec) binding stoichiometry is of one tRNA(sec) molecule per SelA monomer (1:1) rather than the 1:2 value proposed previously. Negative stain transmission electron microscopy revealed a D5 pointgroup symmetry for the SelA-tRNA(sec) assembly both with and without tRNA(sec) bound. Furthermore, SelA can associate forming a supramolecular complex of stacked decamer rings, which does not occur in the presence of tRNA(sec). We discuss the structure-function relationships of these assemblies and their regulatory role in bacterial selenocysteyl-tRNA(sec) synthesis.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Aminoacil-RNA de Transferência/genética , Transferases/genética , Sequência de Bases , Ligação Competitiva , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Polarização de Fluorescência , Cinética , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Substâncias Macromoleculares/ultraestrutura , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA de Protozoário/química , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/metabolismo , Selenocisteína/genética , Selenocisteína/metabolismo , Transcrição Gênica , Transferases/química , Transferases/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMO
The thioredoxin (Trx) system, involving redox active Trxs and thioredoxin reductases (TrxRs), sustain a number of important Trx-dependent pathways. These redox active proteins support several processes crucial for cell function, cell proliferation, antioxidant defense, and redox-regulated signaling cascades. Methylmercury (MeHg) is an important environmental toxicant that has a high affinity for thiol groups and can cause oxidative stress. The Trx system is the major system responsible for maintaining the redox state of cells and this function involves thiol reduction mediated by selenol groups in TrxRs. MeHg has a great affinity to thiols and selenols, thus the potential toxic effects of MeHg on TrxR inhibition were determined in the current study. A single administration of MeHg (1, 5, and 10 mg/Kg) caused a marked inhibition of kidney TrxR activity, while significant inhibition was observed in the liver after exposure to 5 and 10 mg/Kg of MeHg. TrxR activity was determined 24 h after MeHg. In the brain, MeHg did not inhibit TrxR activity. In vitro exposure to MeHg indicated that MeHg inhibits cerebral (IC(50), 0.158 µM), hepatic (IC(50), 0.071 µM), and renal TrxR activity (IC(50), 0.078 µM). The results presented herein demonstrated for the first time that renal and hepatic TrxRs can serve as an in vivo target for MeHg. This study suggests that MeHg can bind to selenocysteine residues present in the catalytic site of TrxR, in turn causing enzyme inhibition that can compromise the redox state of cells.
Assuntos
Inibidores Enzimáticos/farmacologia , Compostos de Metilmercúrio/farmacologia , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Inibidores Enzimáticos/metabolismo , Técnicas In Vitro , Concentração Inibidora 50 , Rim/efeitos dos fármacos , Rim/enzimologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Compostos de Metilmercúrio/metabolismo , Camundongos , Selenocisteína/metabolismoRESUMO
Selenophosphate synthetase (EC 2.7.9.3), the product of the selD gene, produces the biologically active selenium donor compound, monoselenophosphate, from ATP and selenide, for the synthesis of selenocysteine. The kinetoplastid Leishmania major and Trypanosoma brucei selD genes were cloned and the SELD protein overexpressed and purified to apparent homogeneity. The selD gene in L. major and T. brucei are respectively 1197 and 1179 bp long encoding proteins of 399 and 393 amino acids with molecular masses of 42.7 and 43 kDa. The molecular mass of 100 kDa for both (L. major and T. brucei) SELDs is consistent with dimeric proteins. The kinetoplastid selD complement Escherichia coli (WL400) selD deletion confirming it is a functional enzyme and the specific activity of these enzymes was determined. A conserved Cys residue was identified both by multiple sequence alignment as well as by functional complementation and activity assay of the mutant (Cys to Ala) forms of the SELD identifying this residue as essential for the catalytic function.
Assuntos
Leishmania major/enzimologia , Fosfotransferases/química , Proteínas de Protozoários/química , Selenocisteína/metabolismo , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cisteína/metabolismo , Teste de Complementação Genética , Leishmania major/metabolismo , Dados de Sequência Molecular , Fosfotransferases/isolamento & purificação , Fosfotransferases/metabolismo , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência , Trypanosoma brucei brucei/metabolismoRESUMO
The thioredoxin and glutathione systems play a central role in thiol-disulfide redox homeostasis in many organisms by providing electrons to essential enzymes, and defence against oxidative stress. These systems have recently been characterized in platyhelminth parasites, and the emerging biochemical scenario is the existence of linked processes with the enzyme thioredoxin glutathione reductase supplying reducing equivalents to both pathways. In contrast to their hosts, conventional thioredoxin reductase and glutathione reductase enzymes appear to be absent. Analysis of published data and expressed-sequence tag databases indicates the presence of linked thioredoxin-glutathione systems in the cytosolic and mitochondrial compartments of these parasites.