RESUMO
A foot-and-mouth disease virus (FMDV) DNA-launched reporter replicon containing a luciferase gene was used to assess the impact of non-structural (NS) protein 3A on viral replication. Independent deletions within the N-terminal region (amino acid [aa] residues 6 to 24) and the central hydrophobic region (HR, aa 59 to 76) of FMDV NS protein 3A were engineered, and luciferase activity in lysates of control and mutated replicon-transfected cells was measured. Triple alanine replacements of the N-terminal triplet Arg 18- His 19 -Glu 20 and a single alanine substitution of the highly charged Glu 20 residue both resulted in a 70-80% reduction in luciferase activity when compared with wild-type controls. Alanine substitution of the 17 aa present in the central HR, on the other hand, resulted in complete inhibition of luciferase activity and in the accumulation of the mutated 3A within the cell nucleus according to immunofluorescence analysis. Our results suggest that both the aa sequence around the putatively exposed hydrophilic E20 residue at the N-terminus of the protein and the hydrophobic tract located between aa 59 and 76 are of major relevance for maintaining the functionality of the 3A protein and preventing its mislocalization into the cell nucleus.
Assuntos
Vírus da Febre Aftosa/genética , Replicon , Proteínas não Estruturais Virais/química , Replicação Viral/genética , Sequência de Aminoácidos , Aminoácidos/química , Aminoácidos/metabolismo , Animais , Linhagem Celular , Núcleo Celular/virologia , Cricetinae , Replicação do DNA , Febre Aftosa/virologia , Vírus da Febre Aftosa/química , Vírus da Febre Aftosa/fisiologia , Expressão Gênica , Interações Hidrofóbicas e Hidrofílicas , Luciferases , Mutação , Domínios Proteicos , Deleção de Sequência , Proteínas não Estruturais Virais/genéticaRESUMO
The Mediator complex is a greater than 1-megadalton complex, composed of about 30 subunits and found in most eukaryotes, whose main role is to transmit signals from DNA-bound transcription factors to RNA Polymerase II. The proteasome is emerging as an important regulator of transcription during both initiation and elongation. It is increasing the number of cases where the proteolysis of transcriptional activators by the proteasome activates their function. This counterintuitive phenomenon was called "activation by destruction." Here, we show that, in Arabidopsis (Arabidopsis thaliana), PHYTOCHROME AND FLOWERING TIME1 (PFT1), the MEDIATOR25 (MED25) subunit of the plant Mediator complex, is degraded by the proteasome and that proteasome-mediated PFT1 turnover is coupled to its role in stimulating the transcription of FLOWERING LOCUS T, the plant florigen, which is involved in the process of flowering induction. We further identify two novel RING-H2 proteins that target PFT1 for degradation. We show that MED25-BINDING RING-H2 PROTEIN1 (MBR1) and MBR2 bind to PFT1 in yeast (Saccharomyces cerevisiae) and in vitro, and they promote PFT1 degradation in vivo, in a RING-H2-dependent way, typical of E3 ubiquitin ligases. We further show that both MBR1 and MBR2 also promote flowering by PFT1-dependent and -independent mechanisms. Our findings extend the phenomenon of activation by destruction to a Mediator subunit, adding a new mechanism by which Mediator subunits may regulate downstream genes in specific pathways. Furthermore, we show that two novel RING-H2 proteins are involved in the destruction of PFT1, adding new players to this process in plants.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Ligação a DNA , Flores/genética , Flores/fisiologia , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Dados de Sequência Molecular , Mutação/genética , Ligação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteólise , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Leukaemia inhibitory factor (LIF) or Oncostatin M (OSM), both mitogens for Swiss mouse 3T3 cells, triggers initiation of DNA synthesis without the requirement for mevalonic acid. Thus, Lovastatin (LOV), an inhibitor of the hydroxy methylglutaryl CoA (HMGCoA) reductase, does not block LIF or OSM induced DNA replication and cell multiplication. In contrast, increasing concentrations of LOV from 1 to 60 microM block the mitogenic action of PGF(2alpha) by decreasing the number of cells capable of entering S-phase and dividing. This inhibition by LOV can be reversed by addition of mevanolactone (MEV), an analogue of mevalonic acid. Thus, LIF or OSM triggers initiation of DNA replication independently of mevalonic acid synthesis and therefore without the involvement of isoprenylation of various signalling proteins.