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1.
Nat Chem ; 7(12): 968-79, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26587712

ABSTRACT

Copper is a transition metal that plays critical roles in many life processes. Controlling the cellular concentration and trafficking of copper offers a route to disrupt these processes. Here we report small molecules that inhibit the human copper-trafficking proteins Atox1 and CCS, and so provide a selective approach to disrupt cellular copper transport. The knockdown of Atox1 and CCS or their inhibition leads to a significantly reduced proliferation of cancer cells, but not of normal cells, as well as to attenuated tumour growth in mouse models. We show that blocking copper trafficking induces cellular oxidative stress and reduces levels of cellular ATP. The reduced level of ATP results in activation of the AMP-activated protein kinase that leads to reduced lipogenesis. Both effects contribute to the inhibition of cancer cell proliferation. Our results establish copper chaperones as new targets for future developments in anticancer therapies.


Subject(s)
Cell Proliferation/drug effects , Copper/metabolism , Metallochaperones/antagonists & inhibitors , Molecular Chaperones/antagonists & inhibitors , Neoplasms/metabolism , Amino Acid Sequence , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Copper Transport Proteins , Drug Discovery , Gene Knockdown Techniques , Humans , Metallochaperones/chemistry , Metallochaperones/genetics , Metallochaperones/metabolism , Mice , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Molecular Sequence Data , Oxidative Stress/drug effects , Sequence Alignment , Xenograft Model Antitumor Assays
2.
New Phytol ; 202(1): 198-208, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24372442

ABSTRACT

Zinc plays a central role in all living cells as a cofactor for enzymes and as a structural element enabling the adequate folding of proteins. In eukaryotic cells, metals are highly compartmentalized and chelated. Although essential to characterize the mechanisms of Zn(2+) homeostasis, the measurement of free metal concentrations in living cells has proved challenging and the dynamics are difficult to determine. Our work combines the use of genetically encoded Förster resonance energy transfer (FRET) sensors and a novel microfluidic technology, the RootChip, to monitor the dynamics of cytosolic Zn(2+) concentrations in Arabidopsis root cells. Our experiments provide estimates of cytosolic free Zn(2+) concentrations in Arabidopsis root cells grown under sufficient (0.4 nM) and excess (2 nM) Zn(2+) supply. In addition, monitoring the dynamics of cytosolic [Zn(2+) ] in response to external supply suggests the involvement of high- and low-affinity uptake systems as well as release from internal stores. In this study, we demonstrate that the combination of genetically encoded FRET sensors and microfluidics provides an attractive tool to monitor the dynamics of cellular metal ion concentrations over a wide concentration range in root cells.


Subject(s)
Arabidopsis/metabolism , Cytosol/metabolism , Fluorescence Resonance Energy Transfer/methods , Imaging, Three-Dimensional/methods , Plant Roots/metabolism , Zinc/metabolism , Extracellular Space/metabolism , Intracellular Space/metabolism , Perfusion , Plant Roots/cytology , Plant Roots/growth & development
3.
PLoS One ; 8(12): e82009, 2013.
Article in English | MEDLINE | ID: mdl-24312622

ABSTRACT

Magnesium has important structural, catalytic and signaling roles in cells, yet few tools exist to image this metal ion in real time and at subcellular resolution. Here we report the first genetically encoded sensor for Mg(2+), MagFRET-1. This sensor is based on the high-affinity Mg(2+) binding domain of human centrin 3 (HsCen3), which undergoes a transition from a molten-globular apo form to a compactly-folded Mg(2+)-bound state. Fusion of Cerulean and Citrine fluorescent domains to the ends of HsCen3, yielded MagFRET-1, which combines a physiologically relevant Mg(2+) affinity (K d = 148 µM) with a 50% increase in emission ratio upon Mg(2+) binding due to a change in FRET efficiency between Cerulean and Citrine. Mutations in the metal binding sites yielded MagFRET variants whose Mg(2+) affinities were attenuated 2- to 100-fold relative to MagFRET-1, thus covering a broad range of Mg(2+) concentrations. In situ experiments in HEK293 cells showed that MagFRET-1 can be targeted to the cytosol and the nucleus. Clear responses to changes in extracellular Mg(2+) concentration were observed for MagFRET-1-expressing HEK293 cells when they were permeabilized with digitonin, whereas similar changes were not observed for intact cells. Although MagFRET-1 is also sensitive to Ca(2+), this affinity is sufficiently attenuated (K d of 10 µM) to make the sensor insensitive to known Ca(2+) stimuli in HEK293 cells. While the potential and limitations of the MagFRET sensors for intracellular Mg(2+) imaging need to be further established, we expect that these genetically encoded and ratiometric fluorescent Mg(2+) sensors could prove very useful in understanding intracellular Mg(2+) homeostasis and signaling.


Subject(s)
Biosensing Techniques/methods , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Fluorescence Resonance Energy Transfer , Magnesium/metabolism , Calcium-Binding Proteins/chemistry , HEK293 Cells , Humans , Magnesium/pharmacology , Models, Molecular , Mutagenesis , Protein Folding/drug effects , Protein Structure, Tertiary , Spectrometry, Fluorescence
4.
Biochem Soc Trans ; 41(5): 1201-5, 2013 Oct.
Article in English | MEDLINE | ID: mdl-24059509

ABSTRACT

Proteins that switch between distinct conformational states are ideal to monitor and control molecular processes within the complexity of biological systems. Inspired by the modular architecture of natural signalling proteins, our group explores generic design strategies for the construction of FRET-based sensor proteins and other protein switches. In the present article, I show that designing FRET sensors based on mutually exclusive domain interactions provides a robust method to engineer sensors with predictable properties and an inherently large change in emission ratio. The modularity of this approach should make it easily transferable to other applications of protein switches in fields ranging from synthetic biology, optogenetics and molecular diagnostics.


Subject(s)
Biosensing Techniques , Protein Engineering , Protein Interaction Domains and Motifs/genetics , Proteins/genetics , Fluorescence Resonance Energy Transfer , Proteins/chemistry , Synthetic Biology
5.
Angew Chem Int Ed Engl ; 51(36): 9176-80, 2012 Sep 03.
Article in English | MEDLINE | ID: mdl-22865679

ABSTRACT

A most able label: Labeled aptamers can be cross-linked to their target structures in a light-dependent and highly specific manner as a result of a new strategy termed aptamer-based affinity labeling (ABAL) of proteins. The aptamer-protein complexes can be enriched in vitro, from a cellular lysate and from the surface of living cells, opening new ways to study aptamer interactions in biological contexts.


Subject(s)
Affinity Labels/chemistry , Aptamers, Nucleotide/chemistry , Proteins/chemistry , Cell Line, Tumor , Humans , Proteins/metabolism , Proto-Oncogene Proteins c-met/chemistry , Proto-Oncogene Proteins c-met/metabolism , SELEX Aptamer Technique , Ultraviolet Rays
6.
Chem Commun (Camb) ; 47(43): 11879-81, 2011 Nov 21.
Article in English | MEDLINE | ID: mdl-21986860

ABSTRACT

Introduction of a (Cys)(4) metal binding site at the dimerization interface of two fluorescent protein domains yields a chelating FRET sensor protein that shows a 2500-fold selectivity for Cd(2+) over Zn(2+) by taking advantage of their different ionic radii.


Subject(s)
Cadmium/analysis , Fluorescence Resonance Energy Transfer , Recombinant Proteins/chemistry , Zinc/chemistry , Binding Sites , Cell Line , Dimerization , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Hydrogen-Ion Concentration , Protein Structure, Tertiary , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
7.
Nat Chem Biol ; 7(8): 519-27, 2011 Jul 18.
Article in English | MEDLINE | ID: mdl-21769099

ABSTRACT

Aptamers are useful for allosteric regulation because they are nucleic acid-based structures in which ligand binding induces conformational changes that may alter the function of a connected oligonucleotide at a distant site. Through this approach, a specific input is efficiently converted into an altered output. This property makes these biomolecules ideally suited to function as sensors or switches in biochemical assays or inside living cells. The ability to select oligonucleotide-based recognition elements in vitro in combination with the availability of nucleic acids with enzymatic activity has led to the development of a wide range of engineered allosteric aptasensors and aptazymes. Here, we discuss recent progress in the screening, design and diversity of these conformational switching oligonucleotides. We cover their application in vitro and for regulating gene expression in both prokaryotes and eukaryotes.


Subject(s)
Aptamers, Nucleotide/metabolism , Computers, Molecular , Gene Expression Regulation/physiology , Protein Engineering , Allosteric Regulation/physiology
9.
Curr Opin Chem Biol ; 14(2): 231-7, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20036601

ABSTRACT

The ability to image the concentration of transition metals in living cells in real time is important for understanding transition metal (TM) homeostasis and its involvement in diseases. Genetically encoded fluorescent sensor proteins are attractive because they do not require cell-invasive procedures, can be targeted to different locations in the cell, and allow ratiometric detection. Important progress in the development of Zn(2+) sensors has allowed sensitive detection of the very low free concentrations of Zn(2+) in single cells, both in the cytosol and various organelles. Together with other recent advances in chemical biology, these tools seem particularly useful to interrogate the dynamics and compartmentation of TM homeostasis.


Subject(s)
Biosensing Techniques/methods , Transition Elements/analysis , Zinc/analysis , Animals , Fluorescence Resonance Energy Transfer/methods , Humans , Transition Elements/metabolism , Zinc/metabolism
10.
Nat Methods ; 6(10): 737-40, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19718032

ABSTRACT

We developed genetically encoded fluorescence resonance energy transfer (FRET)-based sensors that display a large ratiometric change upon Zn(2+) binding, have affinities that span the pico- to nanomolar range and can readily be targeted to subcellular organelles. Using this sensor toolbox we found that cytosolic Zn(2+) was buffered at 0.4 nM in pancreatic beta cells, and we found substantially higher Zn(2+) concentrations in insulin-containing secretory vesicles.


Subject(s)
Biological Assay/methods , Fluorescence Resonance Energy Transfer/methods , Homeostasis/physiology , Molecular Probe Techniques , Protein Engineering/methods , Recombinant Proteins/metabolism , Zinc/metabolism , Animals , Cell Line , Insulin-Secreting Cells/metabolism , Rats , Recombinant Proteins/analysis , Zinc/analysis
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