Biomedical subjects

Cécile E Malnou

Publications and source records attributed to Cécile E Malnou.

5 recordsLinked to original sources

PubMed2005

Down-regulation of c-Fos/c-Jun AP-1 dimer activity by sumoylation.

The inducible transcriptional complex AP-1, composed of c-Fos and c-Jun proteins, is crucial for cell adaptation to many environmental changes. While its mechanisms of activation have been extensively studied, how its activity is restrained is poorly understood. We report here that lysine 265 of c-Fos is conjugated by the peptidic posttranslational modifiers SUMO-1, SUMO-2, and SUMO-3 and that c-Jun can be sumoylated on lysine 257 as well as on the previously described lysine 229. Sumoylation of c-Fos preferentially occurs in the context of c-Jun/c-Fos heterodimers. Using nonsumoylatable mutants of c-Fos and c-Jun as well as a chimeric protein mimicking sumoylated c-Fos, we show that sumoylation entails lower AP-1 transactivation activity. Interestingly, single sumoylation at any of the three acceptor sites of the c-Fos/c-Jun dimer is sufficient to substantially reduce transcription activation. The lower activity of sumoylated c-Fos is not due to inhibition of protein entry into the nucleus, accelerated turnover, and intrinsic inability to dimerize or to bind to DNA. Instead, cell fractionation experiments suggest that decreased transcriptional activity of sumoylated c-Fos is associated with specific intranuclear distribution. Interestingly, the phosphorylation of threonine 232 observed upon expression of oncogenically activated Ha-Ras is known to superactivate c-Fos transcriptional activity. We show here that it also inhibits c-Fos sumoylation, revealing a functional antagonism between two posttranslational modifications, each occurring within a different moiety of a bipartite transactivation domain of c-Fos. Finally we report that the sumoylation of c-Fos is a dynamic process that can be reversed via multiple mechanisms. This supports the idea that this modification does not constitute a final inactivation step that necessarily precedes protein degradation.

Binding Sites↗

PubMed2003

Effects of vaccine strain mutations in domain V of the internal ribosome entry segment compared in the wild type poliovirus type 1 context.

Initiation of poliovirus (PV) protein synthesis is governed by an internal ribosome entry segment structured into several domains including domain V, which is accepted to be important in PV neurovirulence because it harbors an attenuating mutation in each of the vaccine strains developed by A. Sabin. To better understand how these single point mutations exert their effects, we placed each of them into the same genomic context, that of PV type 1. Only the mutation equivalent to the Sabin type 3 strain mutation resulted in significantly reduced viral growth both in HeLa and neuroblastoma cells. This correlated with poor translation efficiency in vitro and could be explained by a structural perturbation of the domain V of the internal ribosome entry segment, as evidenced by RNA melting experiments. We demonstrated that reduced cell death observed during infection by this mutant is due to the absence of inhibition of host cell translation. We confirmed that this shut-off is correlated principally with cleavage of eIF4GII and not eIF4GI and that this cleavage is significantly impaired in the case of the defective mutant. These data support the previously reported conclusion that the 2A protease has markedly different affinities for the two eIF4G isoforms.

Base Sequence↗

PubMed2003

Comparison of the capacity of different viral internal ribosome entry segments to direct translation initiation in poly(A)-dependent reticulocyte lysates.

Polyadenylation stimulates translation of capped eukaryotic mRNAs and those carrying picornaviral internal ribosome entry segments (IRESes) in vivo. Rabbit reticulocyte lysates (RRL) reproduce poly(A)-mediated translation stimulation in vitro after partial depletion of ribosomes and ribosome-associated factors. Here, we have evaluated the effects of varying different parameters (extent of extract depletion, cleavage of eIF4G, concentrations of KCl, MgCl(2) and programming mRNA) on IRES-driven translation efficiency and poly(A)-dependency in ribosome-depleted RRL. For comparison, the study included a standard capped, polyadenylated mRNA. Dramatic differences were observed in the abilities of the different IRESes to direct translation in ribosome-depleted extracts. While the hepatitis A virus IRES was incapable of driving translation in physiological conditions in depleted RRL, mRNAs carrying the foot-and-mouth disease virus and hepatitis C virus IRESes were translated significantly better than a standard cellular mRNA in the same conditions. Indeed, the capacities of these IRESes to direct translation in ribosome-depleted RRL were similar to those reported previously in certain cell lines. Both the abilities of the IRESes to drive translation and their individual salt optima in ribosome-depleted extracts suggest that these elements have dramatically different affinities for some component(s) of the canonical translation machinery. Finally, using poliovirus as an example, we show that the ribosome-depleted system is well suited to the study of the translational capacity of naturally occurring IRES variants.

Animals↗

PubMed2002

Quasispecies heterogeneity and constraints on the evolution of the 5' noncoding region of hepatitis C virus (HCV): relationship with HCV resistance to interferon-alpha therapy.

Hepatitis C virus (HCV) polyprotein translation depends on direct internal entry of the 40S ribosomal subunit mediated by an internal ribosome entry segment (IRES) located in the 5' noncoding (5'NC) region of the viral genome. HCV is genetically heterogeneous and is characterized by the existence of a quasispecies distribution of the virus population within a single infected individual. Cloning and sequencing strategies were used to characterize 5'NC quasispecies genetically. Similar to coding regions, the HCV 5'NC region was distributed as a quasispecies, but it appeared to be subjected to stronger conservatory constraints than other regions of the HCV genome, probably due to the need for structural (and functional) conservation of the IRES. Indeed, most of the quasispecies substitutions were in unpaired regions of the IRES or clustered such that base-pairing was maintained, whereas only 21% were expected to result in a loss of base-pairing. Quasispecies-related structural changes could be predicted in the core cruciform of IRES domain III composed of the RNA helices which extend from the four-way junction JIIIabc, mostly in minor variants, but sometimes in major ones. The results presented here suggest the simultaneous presence in infected patients of a mixture of genetically distinct but closely related IRES sequences that may have different structures. No significant genetic changes of 5'NC quasispecies were observed after interferon-alpha treatment, except in patients with mixed genotype infection who cleared one of the infecting strains during therapy, suggesting that the quasispecies distribution of IRES sequences does not play a role in HCV resistance to interferon-alpha therapy. In contrast, the overall quasispecies distribution of HCV genomes (including IRES sequences) might participate in regulation of hepatic and extrahepatic HCV replication.

5' Untranslated Regions↗

PubMed2002

Poliovirus internal ribosome entry segment structure alterations that specifically affect function in neuronal cells: molecular genetic analysis.

Translation of poliovirus RNA is driven by an internal ribosome entry segment (IRES) present in the 5' noncoding region of the genomic RNA. This IRES is structured into several domains, including domain V, which contains a large lateral bulge-loop whose predicted secondary structure is unclear. The primary sequence of this bulge-loop is strongly conserved within enteroviruses and rhinoviruses: it encompasses two GNAA motifs which could participate in intrabulge base pairing or (in one case) could be presented as a GNRA tetraloop. We have begun to address the question of the significance of the sequence conservation observed among enterovirus reference strains and field isolates by using a comprehensive site-directed mutagenesis program targeted to these two GNAA motifs. Mutants were analyzed functionally in terms of (i) viability and growth kinetics in both HeLa and neuronal cell lines, (ii) structural analyses by biochemical probing of the RNA, and (iii) translation initiation efficiencies in vitro in rabbit reticulocyte lysates supplemented with HeLa or neuronal cell extracts. Phenotypic analyses showed that only viruses with both GNAA motifs destroyed were significantly affected in their growth capacities, which correlated with in vitro translation defects. The phenotypic defects were strongly exacerbated in neuronal cells, where a temperature-sensitive phenotype could be revealed at between 37 and 39.5 degrees C. Biochemical probing of mutated domain V, compared to the wild type, demonstrated that such mutations lead to significant structural perturbations. Interestingly, revertant viruses possessed compensatory mutations which were distant from the primary mutations in terms of sequence and secondary structure, suggesting that intradomain tertiary interactions could exist within domain V of the IRES.

Amino Acid Motifs↗