In previous studies, we have characterized a new hormonal pathway involving a mitochondrial T3 receptor (p43) acting as a mitochondrial transcription factor and consequently stimulating mitochondrial activity and mitochondrial biogenesis. We have established the involvement of this T3 pathway in the regulation of in vitro myoblast differentiation.We have generated mice overexpressing p43 under control of the human α-skeletal actin promoter. In agreement with the previous characterization of this promoter, northern-blot and western-blot experiments confirmed that after birth p43 was specifically overexpressed in skeletal muscle. As expected from in vitro studies, in 2-month old mice, p43 overexpression increased mitochondrial genes expression and mitochondrial biogenesis as attested by the increase of mitochondrial mass and mt-DNA copy number. In addition, transgenic mice had a body temperature 0.8°C higher than control ones and displayed lower plasma triiodothyronine levels. Skeletal muscles of transgenic mice were redder than wild-type animals suggesting an increased oxidative metabolism. In line with this observation, in gastrocnemius, we recorded a strong increase in cytochrome oxidase activity and in mitochondrial respiration. Moreover, we observed that p43 drives the formation of oxidative fibers: in soleus muscle, where MyHC IIa fibers were partly replaced by type I fibers; in gastrocnemius muscle, we found an increase in MyHC IIa and IIx expression associated with a reduction in the number of glycolytic fibers type IIb. In addition, we found that PGC-1α and PPARδ, two major regulators of muscle phenotype were up regulated in p43 transgenic mice suggesting that these proteins could be downstream targets of mitochondrial activity. These data indicate that the direct mitochondrial T3 pathway is deeply involved in the acquisition of contractile and metabolic features of muscle fibers in particular by regulating PGC-1α and PPARδ.
In previous studies, we characterized a new hormonal pathway involving a mitochondrial T3 receptor (p43) acting as a mitochondrial transcription factor. In in vitro and in vivo studies, we have shown that p43 increases mitochondrial transcription and mitochondrial biogenesis. In addition, p43 overexpression in skeletal muscle stimulates mitochondrial respiration and induces a shift in metabolic and contractile features of muscle fibers which became more oxidative. Here we have studied the influence of p43 overexpression in skeletal muscle of mice during aging. We report that p43 overexpression initially increased mitochondrial mass. However, after the early rise in mitochondrial DNA occurring at 2 months of age in transgenic mice, we observed a progressive decrease of mitochondrial DNA content which became 2-fold lower at 23 months of age relatively to control animals. Moreover, p43 overexpression induced an oxidative stress characterized by a strong increase of lipid peroxidation and protein oxidation in quadriceps muscle, although antioxidant enzyme activities (catalase and superoxide dismutase) were stimulated. In addition, muscle atrophy became detectable at 6 months of age, probably through a stimulation of the ubiquitin proteasome pathway via two muscle-specific ubiquitin ligases E3, Atrogin-1/MAFbx and MuRF1. Taken together, these results demonstrate that a prolonged stimulation of mitochondrial activity induces muscle atrophy. In addition, these data underline the importance of a tight control of p43 expression and suggest that a deregulation of the direct T3 mitochondrial pathway could be one of the parameters involved in the occurrence of sarcopenia.
ABSTRACT Guanine-quadruplexes (G4s) are non-canonical DNA structures that play important protective and regulatory roles within cells, influencing, for instance, gene expression. Although the secondary structure of many human G4s is well characterized, in several gene-promoter regions multiple G4s are located in close proximity and may form three-dimensional structures which could ultimately influence their biological roles. In this contribution, we analyze the interplay between the three neighboring G4s present in the c- KIT proto-oncogene promoter, namely WK1, WSP and WK2. In particular, we highlight how these three G4s are structurally linked and how their crosstalk favors the formation of a parallel structure for WSP, differently from what observed for this isolated G4 in solution. Relying on all-atom molecular dynamic simulations exceeding the μs time-scale and using enhanced sampling methods, we provide the first computationally-resolved structure of a well-organized G4 cluster in the promoter of a crucial gene involved in cancer development. Our results indicate that neighboring G4s influence their mutual three-dimensional arrangement and provide a powerful tool to predict and interpret complex DNA structures that ultimately can be used as starting point for drug discovery purposes.
Introduction Advanced breast cancers do not respond well to therapies and represent a relevant focus for studying molecular mechanisms involved in the tumour progression and drug resistance. The transcription factor NF-κB is often activated constitutively in aggressive breast cancer cells and plays a significant role by inducing many target genes involved in tumour progression and drug resistance. Mechanisms controlling constitutive NF-kB activation are not all clearly understood. Among them, repression of the gene encoding the NF-κB inhibitor, IκBα is not well known. This protein controls NF-κB activation by sequestering it in the cytoplasmic compartment. The present study reports the identification of the hnRNP K/J protein, which is initially known for its role in mRNA splicing and translation, as a repressor of the IκBα gene expression. Material and methods Identification of hnRNP K/J protein on the IκBα promoter was carried out by DNA pull-down coupled with a mass spectrometry analysis, and chromatin immunoprecipitation (ChIP) using a specific polyclonal antibodies. The hnRNP K/J protein was overexpressed in breast cancer cell lines by transient transfection, and consequence on the IκBα expression and the proximal IκBα promoter activity was evaluated by RT-qPCR and gene reporter assay, respectively. The hnRNP K/J protein localization was visualised in cells by Western blotting using nuclear and cytoplasmic extracts. Results and discussions The IκBα gene is expressed higher in nonaggressive compared to aggressive breast cancer cells. We used previous data showing the importance of the proximal promoter at position −495 from the transcription site for DNA pull-down with nuclear protein extract from MCF-7 cells. Mass spectrometry analysis led to identify hnRNP K/J protein, whose the binding to this region of the proximal IκBα promoter was confirmed by ChIP. The IκBα expression at mRNA level and proximal IκBα promoter activity was strongly decreased in hnRNP K/J-overexpressing breast cancer cells, in contrast to the respective parental cells, suggesting the role of hnRNP K/J protein as a gene repressor. Conclusion The identification of hnRNP K/J as a repressor of IκBα gene expression depicts a new molecular mechanism, which may contribute to the high constitutive NF-kB activation in aggressive breast cancer cells and suggests to take it into account in the development of new therapies targeting NF-kB pathway in advanced breast cancers.
Abstract We report a molecular modeling study, coupled with spectroscopy experiments, on the behavior of two well known organic dyes, nile blue and nile red, when interacting with B-DNA. In particular, we evidence the presence of two competitive binding modes, for both drugs. However their subsequent photophysical behavior is different and only nile blue is able to induce DNA photosensitization via an electron transfer mechanism. Most notably, even in the case of nile blue, its sensitization capabilities strongly depend on the environment resulting in a single active binding mode: the minor groove. Fluorescence spectroscopy confirms the presence of competitive interaction modes for both sensitizers, while the sensitization via electron transfer, is possible only in the case of nile blue.
Abstract Human telomeric DNA, in G‐quadruplex (G4) conformation, is characterized by a remarkable structural stability that confers it the capacity to resist to oxidative stress producing one or even clustered 8‐oxoguanine (8oxoG) lesions. We present a combined experimental/computational investigation, by using circular dichroism in aqueous solutions, cellular immunofluorescence assays and molecular dynamics simulations, that identifies the crucial role of the stability of G4s to oxidative lesions, related also to their biological role as inhibitors of telomerase, an enzyme overexpressed in most cancers associated to oxidative stress.
The maintaining of the precise regulation of iron homeostasis is fundamental to assure cells viability. In this context the interplay between the messenger RNA Iron Response element (IRE) and the iron response protein (IRP) is crucial to regulate the expression of ferritin and hence the level of labile free iron pool. We have shown, using a combination of molecular modeling and experimental techniques, that tris bipyridine iron complexes (AIM3) interact specifically with the IRE RNA stem-loop in solution, without inducing noticeable structural deformations. Furthermore, we have also shown that, at a cellular level, this interaction may be traced back to the downregulation of ferritin translation, probably due to the stabilization of the IRE stem-loops thus favoring its binding to IRP or by inhibiting the downstream recruitment of ribosomal subunits.
The behavior of the structural parameters of DNA considering different levels of methylation in CpG islands is studied by means of full-atom molecular dynamics simulations and electronic circular dichroism, both in an artificial model system and in a gene promoter sequence. It is demonstrated that methylation although intrinsically brings quite local perturbations may, if its level is high enough, induce cooperative effects that strongly modify the DNA backbone torsional parameters altering the helicity as compared to the nonmethylated case. Because methylation of the CpG island is correlated with the regulation of gene expression, understanding the structural modifications induced in DNA is crucial to characterize all the fine equilibria into play in epigenetics phenomena.
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