New Therapeutic Approach for Targeting Hippo Signalling Pathway.

Nuclear localization signals are short amino acid sequences that target proteins for nuclear import. In this manuscript, we have generated a chimeric tri-functional peptide composed of a cell penetrating peptide (CPP), a nuclear localization sequence and an interfering peptide blocking the interaction between TEAD and YAP, two transcription factors involved in the Hippo signalling pathway, whose deregulation is related to several types of cancer. We have validated the cell penetration and nuclear localization by flow cytometry and fluorescence microscopy and shown that the new generated peptide displays an apoptotic effect in tumor cell lines thanks to the specific nuclear delivery of the cargo, which targets a protein/protein interaction in the nucleus.

Consensus designs and thermal stability determinants of a human glutamate transporter.

Human excitatory amino acid transporters (EAATs) take up the neurotransmitter glutamate in the brain and are essential to maintain excitatory neurotransmission. Our understanding of the EAATs' molecular mechanisms has been hampered by the lack of stability of purified protein samples for biophysical analyses. Here, we present approaches based on consensus mutagenesis to obtain thermostable EAAT1 variants that share up to ~95% amino acid identity with the wild type transporters, and remain natively folded and functional.

Structure and allosteric inhibition of excitatory amino acid transporter 1.

Human members of the solute carrier 1 (SLC1) family of transporters take up excitatory neurotransmitters in the brain and amino acids in peripheral organs. Dysregulation of the function of SLC1 transporters is associated with neurodegenerative disorders and cancer. Here we present crystal structures of a thermostabilized human SLC1 transporter, the excitatory amino acid transporter 1 (EAAT1), with and without allosteric and competitive inhibitors bound.

Preferential Extracellular Generation of the Active Parkinsonian Toxin MPP+ by Transporter-Independent Export of the Intermediate MPDP+.

Aims: 1-Methyl-4-phenyl-tetrahydropyridine (MPTP) is among the most widely used neurotoxins for inducing experimental parkinsonism. MPTP causes parkinsonian symptoms in mice, primates, and humans by killing a subpopulation of dopaminergic neurons. Extrapolations of data obtained using MPTP-based parkinsonism models to human disease are common; however, the precise mechanism by which MPTP is converted into its active neurotoxic metabolite, 1-methyl-4-phenyl-pyridinium (MPP(+)), has not been fully elucidated.

Surface charges of the membrane crucially affect regulation of Na,K-ATPase by phospholemman (FXYD1).

The human α1/His10-β1 isoform of Na,K-ATPase has been reconstituted as a complex with and without FXYD1 into proteoliposomes of various lipid compositions in order to study the effect of the regulatory subunit on the half-saturating Na⁺ concentration (K(½)) of Na⁺ ions for activation of the ion pump. It has been shown that the fraction of negatively charged lipid in the bilayer crucially affects the regulatory properties. At low concentrations of the negatively charged lipid DOPS (<10 %), FXYD1 increases K(½) of Na⁺ ions for activation of the ion pump.

Phospholemman (FXYD1) raises the affinity of the human α1β1 isoform of Na,K-ATPase for Na ions.

The human α(1)/His(10)-β(1) isoform of the Na,K-ATPase has been expressed in Pichia pastoris, solubilized in n-dodecyl-β-maltoside, and purified by metal chelate chromatography. The α(1)β(1) complex spontaneously associates in vitro with the detergent-solubilized purified human FXYD1 (phospholemman) expressed in Escherichia coli. It has been confirmed that FXYD1 spontaneously associates in vitro with the α(1)/His(10)-β(1) complex and stabilizes it in an active mode.

FXYD proteins stabilize Na,K-ATPase: amplification of specific phosphatidylserine-protein interactions.

FXYD proteins are a family of seven small regulatory proteins, expressed in a tissue-specific manner, that associate with Na,K-ATPase as subsidiary subunits and modulate kinetic properties. This study describes an additional property of FXYD proteins as stabilizers of Na,K-ATPase. FXYD1 (phospholemman), FXYD2 (γ subunit), and FXYD4 (CHIF) have been expressed in Escherichia coli and purified.

Investigation of electrogenic partial reactions in detergent-solubilized Na,K-ATPase.

A method for investigating electrogenic partial reactions in the pump cycle of membrane-bound P-type ATPases with electrochromic fluorescent dyes has been extended to detergent-solubilized native and purified recombinant Na,K-ATPase. As a first step, it has been shown here that the function and ion binding properties of the detergent-soluble and membrane-bound rabbit renal Na,K-ATPase are not significantly different. Thus, the new assay overcomes a previous limitation of the styryl dye method, in that the protein need not be embedded in a membrane at a high density.