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. In addition, the peptide has an anti-tumoral effect in vivo in xenograft models of breast cancer. The chimeric peptide designed in the current study shows encouraging prospects for developing nuclear anti- neoplastic drugs.Facultad de Ciencias Médica

SHMT2-mediated mitochondrial serine metabolism drives 5-FU resistance by fueling nucleotide biosynthesis

5-Fluorouracil (5-FU) is a key component of chemotherapy for colorectal cancer (CRC). 5-FU efficacy is established by intracellular levels of folate cofactors and DNA damage repair strategies. However, drug resistance still represents a major challenge. Here, we report that alterations in serine metabolism affect 5-FU sensitivity in in vitro and in vivo CRC models. In particular, 5-FU-resistant CRC cells display a strong serine dependency achieved either by upregulating endogenous serine synthesis or increasing exogenous serine uptake. Importantly, regardless of the serine feeder strategy, serine hydroxymethyltransferase-2 (SHMT2)-driven compartmentalization of one-carbon metabolism inside the mitochondria represents a specific adaptation of resistant cells to support purine biosynthesis and potentiate DNA damage response. Interfering with serine availability or affecting its mitochondrial metabolism revert 5-FU resistance. These data disclose a relevant mechanism of mitochondrial serine use supporting 5-FU resistance in CRC and provide perspectives for therapeutic approaches

Regulation der Na,K-ATPase durch FXYD1

The Na,K-ATPase is an integral membrane protein present in virtually all animal cells, where it actively transports Na+ and K+ ions across the plasma membrane using ATP as energy source. For every ATP molecule hydrolyzed, the enzyme pumps three Na+ ions out of and two K+ ions into the cell. Because of its fundamental role in many physiological processes, the Na,K-ATPase is the target of specific regulatory mechanisms. Among them, the enzyme is modulated by the interaction with the so-called FXYD proteins, a group of short transmembrane polypeptides named after the invariant extra¬cellular motif FXYD. All mammalian members of the FXYD family are known to associate with the Na,K-ATPase and modulate its properties in a tissue- and isozyme-specific way. FXYD1, also known as phospholemman, has been first identified as the major substrate for protein kinases A and C in the heart. Subsequently, it has been discovered to associate with specific isozymes of the Na,K-ATPase and modulate the enzyme activity in heart and skeletal muscle as well as kidneys and brain.So far, the effects of FXYD1 on the Na,K-ATPase have been investigated mainly in intact cells, both heterologous systems and native cells. These systems allow a better characterization of the physiological effects of FXYD1, but are of limited use for the investigation of the functional and structural interactions between FXYD1 and the enzyme. A purification procedure of the human α1/His10-β1 and α2/His10-β1 isozymes of the Na,K-ATPase expressed in yeast P. pastoris has been recently developed by the group of Steven Karlish at the Weizmann Institute of Science. The purified, detergent-solubilized α1/His10-β1 can be in vitro reconstituted with purified, detergent-solubilized human FXYD1 expressed in E. coli to obtain the α1/His10-β1/FXYD1 complex. The purified recombinant preparations provide a system that enables us to work under well defined conditions and without interference by other cellular components. Unlike in native cells, the effects of FXYD1 on the different isozymes of the Na,K-ATPase can be investigated separately. Moreover, since the phosphorylation state of FXYD1 in the purified preparations is easily controllable, the functional role of the protein kinases-mediated phosphorylation of FXYD1 can be investigated. Therefore, these systems allow the performance of a detailed functional analysis of the effects of FXYD1 on the Na,K-ATPase.The biophysical techniques based on the fluorescence of external dyes available in our lab allow a thorough characterization of the transport cycle of the Na,K-ATPase. Among them, the electrochromic styryl dye RH421 enables us to monitor the ion movements inside the membrane domain of the enzyme, allowing the detection of ion binding and ion release during the transport cycle. Moreover, the time course of the signals provides information about the kinetics of the processes involved. In contrast, the voltage-sensitive dye Oxonol VI can be successfully applied to detect the ion transport of the Na,K-ATPase reconstituted in lipid vesicles.In a first step of the current study, the dye RH421 has been applied to the purified α1/His10-β1 and α2/His10-β1 preparations to ensure that it is suitable to investigate the ion-binding kinetics of detergent-solubilized ion pumps and that the functional properties of the purified recombinant enzymes do not differ significantly from those of the membrane-bound native Na,K-ATPase. Afterwards, the dye RH421 has been applied in steady-state and time-resolved kinetic measurements to characterize the effects of FXYD1 on the different partial reactions of the transport cycle of the α1/β1 isozyme of the Na,K-ATPase. These experiments have shown a single kinetic property affected by the presence of FXYD1: in both the enzyme conformations, E1 and P-E2, the Na+-binding affinity is increased of ~ 20-30%. In the final part of the study, the influence of the membrane and its lipid composition on the effect of FXYD1 on the Na+-binding affinity of the enzyme has been investigated with the voltage-sensitive dye Oxonol VI in proteoliposomes containing either α1/His10-β1 or α1/His10-β1/FXYD1. These experiments have revealed an unexpected role of the lipid environment surrounding the complex in the interaction of FXYD1 with the enzyme, probably related to the cytoplasmic segment of the regulatory protein

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

A method to investigate 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 that the protein need not be embedded in a membrane at a high density. As an example of an application of this method, transport properties of recombinant Na,K-ATPase purified from yeast cells have been studied. We have investigated and compared Na+ and K+-binding properties of purified detergent-soluble human α1/his-β1 and α2/his-β1 isoforms of the sodium pump. The only significant difference found with respect to ion binding between both isoforms is an almost three-fold lower affinity for K+ binding in the E2P state of the α2/his-β1 isoform. This technique should be readily applicable to various other P-type ATPases, or transport proteins such as carriers or ion channels that can be purified in a detergent-soluble active form

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 %), little or no effect of FXYD1 on the K(½) of Na⁺ ions is observed. Depending on ionic strength and lipid composition of the proteoliposomes, FXYD1 can alter the K(½) of Na⁺ ions by up to twofold. We propose possible molecular mechanisms to explain the regulatory effects of FXYD1 and the influence of charged lipid and protein phosphorylation. In particular, the positively charged C-terminal helix of FXYD1 appears to be highly mobile and may interact with the cytoplasmic N domain of the α-subunit, the interaction being strongly affected by phosphorylation at Ser68 and the surface charge of the membrane

Consensus designs and thermal stability determinants of a human glutamate transporter

International audienceHuman 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. Structural analyses of EAAT1 and the consensus designs using hydrogen-deuterium exchange linked to mass spectrometry show that small and highly cooperative unfolding events at the inter-subunit interface rate-limit their thermal denaturation, while the transport domain unfolds at a later stage in the unfolding pathway. Our findings provide structural insights into the kinetic stability of human glutamate transporters, and introduce general approaches to extend the lifetime of human membrane proteins for biophysical analyses

Structure and allosteric inhibition of excitatory amino acid transporter 1

International audienceHuman 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 their functions is associated to neurodegenerative disorders and cancer. Here we present the first crystal structures of a thermostabilized human SLC1 transporter, the excitatory amino acid transporter 1 (EAAT1), with and without allosteric and competitive inhibitors bound. The structures show novel architectural features of the human transporters, including intra-and extracellular domains with potential roles in transport function, as well as regulation by lipids and post-translational modifications. The coordination of the inhibitor in the structures and the change in the transporter dynamics measured by hydrogen-deuterium exchange mass spectrometry, reveal an allosteric mechanism of inhibition, whereby the transporter is locked in the outward-facing states of the transport cycle. Our results provide unprecedented insights into the molecular mechanisms of function and pharmacology of human SLC1 transporters. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use