Nuclear importation of Mariner transposases among eukaryotes: motif requirements and homo-protein interactions

Mariner-like elements (MLEs) are widespread transposable elements in animal genomes. They have been divided into at least five sub-families with differing host ranges. We investigated whether the ability of transposases encoded by Mos1, Himar1 and Mcmar1 to be actively imported into nuclei varies between host belonging to different eukaryotic taxa. Our findings demonstrate that nuclear importation could restrict the host range of some MLEs in certain eukaryotic lineages, depending on their expression level. We then focused on the nuclear localization signal (NLS) in these proteins, and showed that the first 175 N-terminal residues in the three transposases were required for nuclear importation. We found that two components are involved in the nuclear importation of the Mos1 transposase: an SV40 NLS-like motif (position: aa 168 to 174), and a dimerization sub-domain located within the first 80 residues. Sequence analyses revealed that the dimerization moiety is conserved among MLE transposases, but the Himar1 and Mcmar1 transposases do not contain any conserved NLS motif. This suggests that other NLS-like motifs must intervene in these proteins. Finally, we showed that the over-expression of the Mos1 transposase prevents its nuclear importation in HeLa cells, due to the assembly of transposase aggregates in the cytoplasm.     

Effects of Decreasing Mitochondrial Volume on the Regulation of the Permeability Transition Pore

The permeability transition pore (PTP) is a Ca²⁺-sensitive mitochondrial inner membrane channel involved in several models of cell death. Because the matrix concentration of PTP regulatory factors depends on matrix volume, we have investigated the role of the mitochondrial volume in PTP regulation. By incubating rat liver mitochondria in media of different osmolarity, we found that the Ca²⁺ threshold required for PTP opening dramatically increased when mitochondrial volume decreased relative to the standard condition. This shrinkage-induced PTP inhibition was not related to the observed changes in protonmotive force, or pyridine nucleotide redox state and persisted when mitochondria were depleted of adenine nucleotides. On the other hand, mitochondrial volume did not affect PTP regulation when mitochondria were depleted of Mg²⁺. By studying the effects of Mg²⁺, cyclosporin A (CsA) and ubiquinone 0 (Ub₀) on PTP regulation, we found that mitochondrial shrinkage increased the efficacy of Mg²⁺ and Ub₀ at PTP inhibition, whereas it decreased that of CsA. The ability of mitochondrial volume to alter the activity of several PTP regulators represents a hitherto unrecognized characteristic of the pore that might lead to a new approach for its pharmacological modulation.     

Inducible dimerization of RET reveals a specific AKT deregulation in oncogenic signaling

Dominant-activating mutations in the RET (rearranged during transfection) proto-oncogene, a receptor tyrosine kinase, are causally associated with the development of multiple endocrine neoplasia type 2A (MEN2A) syndrome. Such oncogenic RET mutations induce its ligand-independent constitutive activation, but whether it spreads identical signaling to ligand-induced signaling is uncertain. To address this question, we designed a cellular model in which RET can be activated either by its natural ligand, or alternatively, by controlled dimerization of the protein that mimics MEN2A dimerization. We have shown that controlled dimerization leaves proximal RET signaling intact but impacts substantially on the tuning of the distal AKT kinase activation (delayed and sustained). In marked contrast, distal activation of ERK remained unaffected. We further demonstrated that specific temporal adjustment of ligand-induced AKT activation is dependent upon a lipid-based cholesterol-sensitive environment, and this control step is bypassed by MEN2A RET mutants. Therefore, these studies revealed that MEN2A mutations propagate previously unappreciated subtle differences in signaling pathways and unravel a role for lipid rafts in the temporal regulation of AKT activation.     

Mitochondrial NADH redox potential impacts the reactive oxygen species production of reverse Electron transfer through complex I

There is substantial evidence that Reactive Oxygen Species (ROS) play a major part in cell functioning. Although their harmfulness through oxidative stress is well documented, their role in signaling and sensing as an oxidative signal still needs to be investigated. In most cells, the mitochondrial Electron Transport Chain (ETC) is the primary source of ROS. The production of ROS by reverse electron transfer through complex I has been demonstrated both in an experimental context but also in many pathophysiological situations. Therefore, understanding the mechanisms that regulate this ROS production is of great interest to control its harmful effects. We used nigericin, Pi and valinomycin as tools to modulate the pH gradient (?pH) and the membrane potential (?Ψ) of the protonmotive force (?p) in liver and muscle mitochondria to accurately determine how these parameters control the ROS production. We show that a high ?Ψ is the “sine qua none” condition for ROS production from the reverse electron transfer (RET) through the complex I. However, a high ?Ψ is not the only condition governing ROS production. Indeed, using tools that modulate the mitochondrial NADH level, we also demonstrate that ROS production is directly related to the mitochondrial redox potential when the membrane potential is almost stable.