Publications by authors named "Katarzyna Nieścierowicz"

5 Publications

  • Page 1 of 1

The ligand-bound state of a G protein-coupled receptor stabilizes the interaction of functional cholesterol molecules.

J Lipid Res 2021 Feb 26;62:100059. Epub 2021 Feb 26.

Univ. Grenoble Alpes, CNRS, CEA, IBS, Grenoble, France. Electronic address:

Cholesterol is a major component of mammalian plasma membranes that not only affects the physical properties of the lipid bilayer but also is the function of many membrane proteins including G protein-coupled receptors. The oxytocin receptor (OXTR) is involved in parturition and lactation of mammals and in their emotional and social behaviors. Cholesterol acts on OXTR as an allosteric modulator inducing a high-affinity state for orthosteric ligands through a molecular mechanism that has yet to be determined. Using the ion channel-coupled receptor technology, we developed a functional assay of cholesterol modulation of G protein-coupled receptors that is independent of intracellular signaling pathways and operational in living cells. Using this assay, we discovered a stable binding of cholesterol molecules to the receptor when it adopts an orthosteric ligand-bound state. This stable interaction preserves the cholesterol-dependent activity of the receptor in cholesterol-depleted membranes. This mechanism was confirmed using time-resolved FRET experiments on WT OXTR expressed in CHO cells. Consequently, a positive cross-regulation sequentially occurs in OXTR between cholesterol and orthosteric ligands.
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http://dx.doi.org/10.1016/j.jlr.2021.100059DOI Listing
February 2021

Decoding the Heart through Next Generation Sequencing Approaches.

Genes (Basel) 2018 Jun 7;9(6). Epub 2018 Jun 7.

International Institute of Molecular and Cell Biology in Warsaw, 02-109 Warsaw, Poland.

Vertebrate organs develop through a complex process which involves interaction between multiple signaling pathways at the molecular, cell, and tissue levels. Heart development is an example of such complex process which, when disrupted, results in congenital heart disease (CHD). This complexity necessitates a holistic approach which allows the visualization of genome-wide interaction networks, as opposed to assessment of limited subsets of factors. Genomics offers a powerful solution to address the problem of biological complexity by enabling the observation of molecular processes at a genome-wide scale. The emergence of next generation sequencing (NGS) technology has facilitated the expansion of genomics, increasing its output capacity and applicability in various biological disciplines. The application of NGS in various aspects of heart biology has resulted in new discoveries, generating novel insights into this field of study. Here we review the contributions of NGS technology into the understanding of heart development and its disruption reflected in CHD and discuss how emerging NGS based methodologies can contribute to the further understanding of heart repair.
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http://dx.doi.org/10.3390/genes9060289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6027153PMC
June 2018

Tuning the allosteric regulation of artificial muscarinic and dopaminergic ligand-gated potassium channels by protein engineering of G protein-coupled receptors.

Sci Rep 2017 02 1;7:41154. Epub 2017 Feb 1.

Institut de Biologie Structurale (IBS), 38044 Grenoble, France.

Ligand-gated ion channels enable intercellular transmission of action potential through synapses by transducing biochemical messengers into electrical signal. We designed artificial ligand-gated ion channels by coupling G protein-coupled receptors to the Kir6.2 potassium channel. These artificial channels called ion channel-coupled receptors offer complementary properties to natural channels by extending the repertoire of ligands to those recognized by the fused receptors, by generating more sustained signals and by conferring potassium selectivity. The first artificial channels based on the muscarinic M2 and the dopaminergic D2 receptors were opened and closed by acetylcholine and dopamine, respectively. We find here that this opposite regulation of the gating is linked to the length of the receptor C-termini, and that C-terminus engineering can precisely control the extent and direction of ligand gating. These findings establish the design rules to produce customized ligand-gated channels for synthetic biology applications.
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http://dx.doi.org/10.1038/srep41154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5286527PMC
February 2017

Ion channel reporter for monitoring the activity of engineered GPCRs.

Methods Enzymol 2015 21;556:425-54. Epub 2015 Mar 21.

Institut de Biologie Structurale (IBS), University of Grenoble Alpes, Grenoble, France; CNRS, IBS, LabEx ICST, Grenoble, France; CEA, IBS, Grenoble, France. Electronic address:

Ion channel-coupled receptor (ICCR) is a recent technology based on the fusion of G protein-coupled receptors (GPCRs) to an ion channel. Binding of ligands on the GPCR triggers conformational changes of the receptor that are mechanically transmitted to the ion channel gates, generating an electrical signal easily detectable with conventional electrophysiological techniques. ICCRs are heterologously expressed in Xenopus oocytes and offers several advantages such as: (i) real-time recordings on single cells, (ii) standard laboratory environment and inexpensive media for Xenopus oocytes maintenance, (iii) absence of protein purification steps, (iv) sensitivity to agonists and antagonists in concentration-dependent manner, (v) compatibility with a Gi/o protein activation assay based on Kir3.x channels, and (vi) ability to detect receptor activation independently of intracellular effectors. This last characteristic of ICCRs led to the development of a functional assay for G protein-"uncoupled" receptors such as GPCRs optimized for crystallization by alteration of their third intracellular (i3) loop. One of the most widely used approaches consists in replacing the i3 loop with the T4 phage lysozyme (T4L) domain that obstructs the access of G proteins to their binding site. We recently demonstrated that the ICCR technology can functionally characterize GPCRs(T4L). Two-electrode voltage-clamp (TEVC) recordings revealed that apparent affinities and sensitivities to ligands are not affected by T4L insertion, while ICCRs(T4L) displayed a partial agonist phenotype upon binding of full agonists, suggesting that ICCRs could detect intermediate-active states. This chapter aims to provide exhaustive details from molecular biology steps to electrophysiological recordings for the design and the characterization of ICCRs and ICCRs(T4L).
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http://dx.doi.org/10.1016/bs.mie.2014.12.017DOI Listing
January 2016

Functional assay for T4 lysozyme-engineered G protein-coupled receptors with an ion channel reporter.

Structure 2014 Jan 21;22(1):149-55. Epub 2013 Nov 21.

University Grenoble Alpes, Institut de Biologie Structurale (IBS), F-38027 Grenoble, France; Le Centre National de la Recherche Scientifique (CNRS), IBS, LabEx ICST, F-38027 Grenoble, France; Direction des Sciences du Vivant du Comissariat à l'Energie Atomique (CEA), F-38027 Grenoble, France. Electronic address:

Structural studies of G protein-coupled receptors (GPCRs) extensively use the insertion of globular soluble protein domains to facilitate their crystallization. However, when inserted in the third intracellular loop (i3 loop), the soluble protein domain disrupts their coupling to G proteins and impedes the GPCRs functional characterization by standard G protein-based assays. Therefore, activity tests of crystallization-optimized GPCRs are essentially limited to their ligand binding properties using radioligand binding assays. Functional characterization of additional thermostabilizing mutations requires the insertion of similar mutations in the wild-type receptor to allow G protein-activation tests. We demonstrate that ion channel-coupled receptor technology is a complementary approach for a comprehensive functional characterization of crystallization-optimized GPCRs and potentially of any engineered GPCR. Ligand-induced conformational changes of the GPCRs are translated into electrical signal and detected by simple current recordings, even though binding of G proteins is sterically blocked by the added soluble protein domain.
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http://dx.doi.org/10.1016/j.str.2013.10.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3947161PMC
January 2014