Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a Förster resonance energy transfer (FRET) probe in which the ε subunit of the FoF1-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the ε subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.
22 Related JoVE Articles!
Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells
Institutions: University of Texas Health Science Center at Houston.
Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 Å. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.
Bioengineering, Issue 91, LRET, FRET, Luminescence Resonance Energy Transfer, Fluorescence Resonance Energy Transfer, glutamate receptors, acid sensing ion channel, protein conformation, protein dynamics, fluorescence, protein-protein interactions
Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1
Institutions: The Ohio State University, Tohoku University.
The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5
. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9
. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10
, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.
Cell Biology, Issue 48, BRCA1, homologous recombination, breast cancer, RNA interference, DNA repair
Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5
Institutions: Western University of Health Sciences, Western University of Health Sciences, Products.
An often-suggested mechanism of virus induced neuronal damage is oxidative stress. Astrocytes have an important role in controlling oxidative stress of the Central Nervous System (CNS). Astrocytes help maintain a homeostatic environment for neurons as well as protecting neurons from Reactive Oxygen Species (ROS). CM-H2DCFDA is a cell-permeable indicator for the presence of ROS. CM-H2
DCFDA enters the cell as a non-fluorescent compound, and becomes fluorescent after cellular esterases remove the acetate groups, and the compound is oxidized. The number of cells, measured by flow cytometry, that are found to be green fluorescing is an indication of the number of cells that are in an oxidative state. CM-H2
DCFDA is susceptible to oxidation by a large number of different ROS. This lack of specificity, regarding which ROS can oxidize CM-H2
DCFDA, makes this compound a valuable regent for use in the early stages of a pathogenesis investigation, as this assay can be used to screen for an oxidative cellular environment regardless of which oxygen radical or combination of ROS are responsible for the cellular conditions. Once it has been established that ROS are present by oxidation of CM-H2
DCFDA, then additional experiments can be performed to determine which ROS or combination of ROSs are involved in the particular pathogenesis process. The results of this study demonstrate that with the addition of hydrogen peroxide an increase in CM-H2
DCFDA fluoresce was detected relative to the saline controls, indicating that this assay is a valuable test for detecting an oxidative environment within G355-5 cells, a feline astrocyte cell line.
Neuroscience, Issue 53, Astrocytes, oxidative stress, flow cytometry, CM-H2DCFDA
Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells
Institutions: University of Rochester.
DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.
Cellular Biology, Issue 43, DNA repair, HR, NHEJ, mammalian cells
Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy
Institutions: State University of New York Upstate Medical University.
gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the Mgm101 protein is involved in the recombinational repair of mitochondrial DNA. Recent investigations have indicated that Mgm101 is related to the Rad52-type recombination protein family. These proteins form large oligomeric rings and promote the annealing of homologous single stranded DNA molecules. However, the characterization of Mgm101 has been hindered by the difficulty in producing the recombinant protein. Here, a reliable procedure for the preparation of recombinant Mgm101 is described. Maltose Binding Protein (MBP)-tagged Mgm101 is first expressed in Escherichia coli
. The fusion protein is initially purified by amylose affinity chromatography. After being released by proteolytic cleavage, Mgm101 is separated from MBP by cationic exchange chromatography. Monodispersed Mgm101 is then obtained by size exclusion chromatography. A yield of ~0.87 mg of Mgm101 per liter of bacterial culture can be routinely obtained. The recombinant Mgm101 has minimal contamination of DNA. The prepared samples are successfully used for biochemical, structural and single particle image analyses of Mgm101. This protocol may also be used for the preparation of other large oligomeric DNA-binding proteins that may be misfolded and toxic to bacterial cells.
Biochemistry, Issue 76, Genetics, Molecular Biology, Cellular Biology, Microbiology, Bacteria, Proteins, Mgm101, Rad52, mitochondria, recombination, mtDNA, maltose-binding protein, MBP, E. coli., yeast, Saccharomyces cerevisiae, chromatography, electron microscopy, cell culture
Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS
Institutions: Temple University , University of Washington.
Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O2•-
, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death1-6
. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-α (TNFα) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response7
. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O2•-
and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells8
. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others9,10
that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O2•-
that are important in the host defense mechanism11,12
. Although paracrine-derived O2•-
plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca2+
mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O2•-
to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O2•-
lead to calcium signaling in adjacent endothelial cells.
Molecular Biology, Issue 58, Reactive oxygen species, Calcium, paracrine superoxide, endothelial cells, confocal microscopy
EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1
Institutions: Delft University of Technology.
Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state.
The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor.
A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 µM. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution. A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor.
Biochemistry, Issue 93, Redox titration, electron paramagnetic resonance, Nar1, cofactor, iron-sulfur cluster, mononuclear iron, midpoint potential
Visualization of G3BP Stress Granules Dynamics in Live Primary Cells
Institutions: Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535.
SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly and fate is important to understand the cellular response to stress. The deficiency in key factors of SGs like G3BP (RasGAP SH3 domain Binding Protein) leads to developmental defects in mice and alterations of the Central Nervous System. To study the dynamics of SGs in cells from an organism, one can culture primary cells and follow the localization of a transfected tagged component of SGs. We describe time-lapse experiment to observe G3BP1-containing SGs in Mouse Embryonic Fibroblasts (MEFs). This technique can also be used to study G3BP-containing SGs in live neurons, which is crucial as it was recently shown that these SGs are formed at the onset of neurodegenerative diseases like Alzheimer's disease. This approach can be adapted to any other cellular body and granule protein component, and performed with transgenic animals, allowing the live study of granules dynamics for example in the absence of a specific factor of these granules.
Cellular Biology, Issue 87, Stress granule (SG), G3BP, primary cells, neurons
Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers
Institutions: University of Calgary, University of Calgary.
Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, oxidative stress, apoptosis, aging, mitochondrial encephalomyopathies and drug toxicity. Given this, technologies to measure cardiac mitochondrial function are in demand. One technique that employs an integrative approach to measure mitochondrial function is respirometric oxidative phosphorylation (OXPHOS) analysis.
The principle of respirometric OXPHOS assessment is centered around measuring oxygen concentration utilizing a Clark electrode. As the permeabilized fiber bundle consumes oxygen, oxygen concentration in the closed chamber declines. Using selected substrate-inhibitor-uncoupler titration protocols, electrons are provided to specific sites of the electron transport chain, allowing evaluation of mitochondrial function. Prior to respirometric analysis of mitochondrial function, mechanical and chemical preparatory techniques are utilized to permeabilize the sarcolemma of muscle fibers. Chemical permeabilization employs saponin to selectively perforate the cell membrane while maintaining cellular architecture.
This paper thoroughly describes the steps involved in preparing saponin-skinned cardiac fibers for oxygen consumption measurements to evaluate mitochondrial OXPHOS. Additionally, troubleshooting advice as well as specific substrates, inhibitors and uncouplers that may be used to determine mitochondria function at specific sites of the electron transport chain are provided. Importantly, the described protocol may be easily applied to cardiac and skeletal tissue of various animal models and human samples.
Physiology, Issue 48, cardiac fibers, mitochondria, oxygen consumption, mouse, methodology
Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism
Institutions: Ben-Gurion University of the Negev.
The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro
, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans
. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo
, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.
Biochemistry, Issue 82, aging, Caenorhabditis elegans, heat shock response, neurodegenerative diseases, protein folding homeostasis, proteostasis, stress, temperature-sensitive
Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins
Institutions: Virginia Commonwealth University, Virginia Commonwealth University, Virginia Commonwealth University, Virginia Commonwealth University.
Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1
. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2
). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4
, forming foci within 5-15 minutes5
. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6
. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9
. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6
. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11
. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12
. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.
Genetics, Issue 67, Molecular Biology, Cellular Biology, Biochemistry, DNA, Double-strand breaks, DNA damage response, proteins, live cell imaging, 3D cell imaging, confocal microscopy
Production and Detection of Reactive Oxygen Species (ROS) in Cancers
Institutions: Baylor College of Medicine.
Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2
(hydrogen peroxide), NO (nitric oxide), O2-
(oxide anion), peroxynitrite (ONOO-
), hydrochlorous acid (HOCl), and hydroxyl radical (OH-
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1
, autoimmune diseases 2
, etc…) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4
. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6
, migration 7
etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3
. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8
. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10
. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.
Medicine, Issue 57, reactive oxygen species (ROS), stress, ischemia, cancer, chemotherapy, immune response
Analysis of Oxidative Stress in Zebrafish Embryos
Institutions: University of Torino, Vesalius Research Center, VIB.
High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo
system to perform such studies and present a protocol to measure in vivo
oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo
: i) a “whole embryo ROS-detection method” for qualitative measurement of oxidative stress and ii) a “single-cell ROS detection method” for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.
Developmental Biology, Issue 89, Danio rerio, zebrafish embryos, endothelial cells, redox state analysis, oxidative stress detection, in vivo ROS measurements, FACS (fluorescence activated cell sorter), molecular probes
Methods to Assess Subcellular Compartments of Muscle in C. elegans
Institutions: University of Nottingham.
Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans
is an established model for understanding the genomic regulation of biological processes of interest. This worm’s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans
is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo
. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo
. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans
provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo
in any other organism.
Developmental Biology, Issue 93, Physiology, C. elegans, muscle, mitochondria, sarcomeres, ageing
Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry
Institutions: University of Florida , University of Florida , University of Florida , University of Florida .
strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum
but also on Brassica
species, as well as on Arabidopsis thaliana
, a genetically tractable host plant1,2
. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3
. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3
. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4
. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4
. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5
. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7
. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9
. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10
. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst
DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.
Genetics, Issue 61, Pseudomonas syringae pv. tomato (Pst), redox proteome, cysteine-reactive tandem mass tag (cysTMT), LTQ-Orbitrap mass spectrometry
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Institutions: Princeton University.
The aim of de novo
protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo
protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (http://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer
Institutions: Max Planck Institute for Psycholinguistics, Donders Institute for Brain, Cognition and Behaviour.
Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis
luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.
Cellular Biology, Issue 87, Protein-protein interactions, Bioluminescence Resonance Energy Transfer, Live cell, Transfection, Luciferase, Yellow Fluorescent Protein, Mutations
A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments
Institutions: Delft University of Technology, Delft University of Technology.
This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli
and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9
addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli
to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native β-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2
) of the alkane hydroxylase system from Gordonia
were transformed into E. coli
. For the conversion of long-chain alkanes (C15-C36), theladA
gene from Geobacillus thermodenitrificans
was implemented. For the required further steps of the degradation process, ADH
and ALDH (
originating from G. thermodenitrificans
) were introduced10,11
. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli
K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFDα and β, both from Pyrococcus horikoshii
OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFDα and β improve the protein folding process e.g.
under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n
-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli
to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.
Bioengineering, Issue 68, Microbiology, Biochemistry, Chemistry, Chemical Engineering, Oil remediation, alkane metabolism, alkane hydroxylase system, resting cell assay, prefoldin, Escherichia coli, synthetic biology, homologous interaction mapping, mathematical model, BioBrick, iGEM
High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli
Institutions: Aix-Marseille Université, Commissariat à l'énergie atomique et aux énergies alternatives (CEA) Saclay, France.
Escherichia coli (E. coli)
is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4 ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (www.venomics.eu), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli
cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.
Bioengineering, Issue 89, E. coli, expression, recombinant, high throughput (HTP), purification, auto-induction, immobilized metal affinity chromatography (IMAC), tobacco etch virus protease (TEV) cleavage, disulfide bond isomerase C (DsbC) fusion, disulfide bonds, animal venom proteins/peptides
Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice
Institutions: University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, Emory University School of Medicine, University of North Carolina School of Medicine.
Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro
using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro
. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo
. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo
tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro
and in vivo
and may be useful in preclinical drug development for these devastating diseases.
Neuroscience, Issue 90, astrocytoma, cortical astrocytes, genetically engineered mice, glioblastoma, neural stem cells, orthotopic allograft
The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin
Institutions: European Institute of Oncology.
Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics
), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP
in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.
Biochemistry, Issue 86, chromatin, histone post-translational modifications (hPTMs), epigenetics, mass spectrometry, proteomics, SILAC, chromatin immunoprecipitation , histone variants, chromatome, hPTMs cross-talks
Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets
Institutions: University of Alabama at Birmingham.
Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging. Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis. This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry. An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.
Immunology, Issue 85, bioenergetics, translational, mitochondria, oxidative stress, reserve capacity, leukocytes