Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.
17 Related JoVE Articles!
Reporter-based Growth Assay for Systematic Analysis of Protein Degradation
Institutions: The Hebrew University of Jerusalem.
Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.
Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3
-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3
gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.
This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3
-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways.
Cellular Biology, Issue 93, Protein Degradation, Ubiquitin, Proteasome, Baker's Yeast, Growth kinetics, Doubling time
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
Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog
Institutions: Lehigh University.
Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein substrates. The histone tails represent one of the most heavily modified stretches within all human proteins. Peptidyl-arginine deiminase 4 (PAD4) has been shown to convert arginine residues into the non-genetically encoded citrulline residue. Few assays described to date have been operationally facile with satisfactory sensitivity. Thus, the lack of adequate assays has likely contributed to the absence of potent non-covalent PAD4 inhibitors. Herein a novel fluorescence-based assay that allows for the monitoring of PAD4 activity is described. A pro-fluorescent substrate analog was designed to link PAD4 enzymatic activity to fluorescence liberation upon the addition of the protease trypsin. It was shown that the assay is compatible with high-throughput screening conditions and has a strong signal-to-noise ratio. Furthermore, the assay can also be performed with crude cell lysates containing over-expressed PAD4.
Chemistry, Issue 93, PAD4, PADI4, citrullination, arginine, post-translational modification, HTS, assay, fluorescence, citrulline
Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach
Institutions: University of Pittsburgh, University of Pittsburgh.
Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro
in an undifferentiated state and they are pluripotent, thus having the potential to differentiate into multiple lineages. Such properties make ESCs extremely attractive for cell based therapy and regenerative treatment applications 1
. However for its full potential to be realized the cells have to be differentiated into mature and functional phenotypes, which is a daunting task. A promising approach in inducing cellular differentiation is to closely mimic the path of organogenesis in the in vitro
setting. Pancreatic development is known to occur in specific stages 2
, starting with endoderm, which can develop into several organs, including liver and pancreas. Endoderm induction can be achieved by modulation of the nodal pathway through addition of Activin A 3
in combination with several growth factors 4-7
. Definitive endoderm cells then undergo pancreatic commitment by inhibition of sonic hedgehog inhibition, which can be achieved in vitro
by addition of cyclopamine 8
. Pancreatic maturation is mediated by several parallel events including inhibition of notch signaling; aggregation of pancreatic progenitors into 3-dimentional clusters; induction of vascularization; to name a few. By far the most successful in vitro
maturation of ESC derived pancreatic progenitor cells have been achieved through inhibition of notch signaling by DAPT supplementation 9
. Although successful, this results in low yield of the mature phenotype with reduced functionality. A less studied area is the effect of endothelial cell signaling in pancreatic maturation, which is increasingly being appreciated as an important contributing factor in in-vivo pancreatic islet maturation 10,11
The current study explores such effect of endothelial cell signaling in maturation of human ESC derived pancreatic progenitor cells into insulin producing islet-like cells. We report a multi-stage directed differentiation protocol where the human ESCs are first induced towards endoderm by Activin A along with inhibition of PI3K pathway. Pancreatic specification of endoderm cells is achieved by inhibition of sonic hedgehog signaling by Cyclopamine along with retinoid induction by addition of Retinoic Acid. The final stage of maturation is induced by endothelial cell signaling achieved by a co-culture configuration. While several endothelial cells have been tested in the co-culture, herein we present our data with rat heart microvascular endothelial Cells (RHMVEC), primarily for the ease of analysis.
Stem Cell Biology, Issue 61, Human embryonic stem cells, Endothelial cells, Pancreatic differentiation, Co-culture
Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples
Institutions: Institute for Hepatitis and Virus Research, Thomas Jefferson University , Drexel University College of Medicine, Van Andel Research Institute, Serome Biosciences Inc..
In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6
. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12
. In fact, most current cancer biomarkers, such as the L3 fraction of α-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15
, and CA199 for pancreatic cancer 16, 17
are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al.
has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18
. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis
-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4
in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20
. This method has been used successfully in multiple different labs 1, 7, 13, 19-31
. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.
Molecular Biology, Issue 63, Glycoproteins, glycan-binding protein, specific protein glycosylation, multiplexed high-throughput glycan blocked antibody microarray
Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification
Institutions: Seattle University, Seattle University.
In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1
. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4
). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2
. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3
. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5
. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.
Biochemistry, Issue 55, hydrophobic interaction chromatography, liquid chromatography, green fluorescent protein, GFP, scouting, protein purification, Bio-Rad DuoFlow, FPLC
Reconstitution Of β-catenin Degradation In Xenopus Egg Extract
Institutions: Vanderbilt University Medical Center, Cincinnati Children's Hospital Medical Center, Vanderbilt University School of Medicine.
egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus
egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3
. Herein, a method is described for isolating Xenopus
egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, β-catenin. Two different methods are described to assess β-catenin protein degradation in Xenopus
egg extract. One method is visually informative ([35
S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess β-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus
egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of β-catenin degradation.
Molecular Biology, Issue 88, Xenopus laevis, Xenopus egg extracts, protein degradation, radiolabel, luciferase, autoradiography, high-throughput screening
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro
model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2
on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3
cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro
BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
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
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
siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells
Institutions: University of Dundee, University of Dundee.
Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription. To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA “ubiquitome” library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question. Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter. An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested. Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.
Biochemistry, Issue 87, siRNA screening, ubiquitin, UBL, ubiquitome, hypoxia, HIF1A, High-throughput, mammalian cells, luciferase reporter
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
Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells
Institutions: Monash University.
One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell populations. These cultures contain the cells of interest, but are also contaminated with undifferentiated ESCs, non-neural derivatives and other neuronal subtypes. This limits their use in in vitro
and in vivo
applications, such as in vitro
modeling for drug discovery or cell replacement therapy. To help overcome this, reporter cell lines, which offer a means to visualize, track and isolate cells of interest, can be engineered. However, to achieve this in human embryonic stem cells via conventional homologous recombination is extremely inefficient. This protocol describes targeting of the Pituitary homeobox 3
) locus in human embryonic stem cells using custom designed zinc-finger nucleases, which introduce site-specific double-strand DNA breaks, together with a PITX3
-specific DNA donor vector. Following the generation of the PITX3
reporter cell line, it can then be differentiated using published protocols for use in studies such as in vitro
Parkinson’s disease modeling or cell replacement therapy.
Molecular Biology, Issue 90, Electroporation, human embryonic stem cell, genome editing, reporter cell line, midbrain dopaminergic neurons
Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells
Institutions: Stanford University School of Medicine, Stanford University School of Medicine.
Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson’s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro
for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro
disease modeling and drug screening and in vivo
cell transplantation therapy for PD.
Neuroscience, Issue 91, dopaminergic neuron, substantia nigra pars compacta, midbrain, Parkinson’s disease, directed differentiation, human pluripotent stem cells, floor plate
Quantitative FRET (Förster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination
Institutions: University of California, Riverside .
Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein activity and have diverse roles in many biological processes. For example, SUMO covalently modifies a large number or proteins with important roles in many cellular processes, including cell-cycle regulation, cell survival and death, DNA damage response, and stress response 1-5. SENP, as SUMO-specific protease, functions as an endopeptidase in the maturation of SUMO precursors or as an isopeptidase to remove SUMO from its target proteins and refresh the SUMOylation cycle 1,3,6,7
The catalytic efficiency or specificity of an enzyme is best characterized by the ratio of the kinetic constants, kcat
. In several studies, the kinetic parameters of SUMO-SENP pairs have been determined by various methods, including polyacrylamide gel-based western-blot, radioactive-labeled substrate, fluorescent compound or protein labeled substrate 8-13
. However, the polyacrylamide-gel-based techniques, which used the "native" proteins but are laborious and technically demanding, that do not readily lend themselves to detailed quantitative analysis. The obtained kcat
from studies using tetrapeptides or proteins with an ACC (7-amino-4-carbamoylmetylcoumarin) or AMC (7-amino-4-methylcoumarin) fluorophore were either up to two orders of magnitude lower than the natural substrates or cannot clearly differentiate the iso- and endopeptidase activities of SENPs.
Recently, FRET-based protease assays were used to study the deubiquitinating enzymes (DUBs) or SENPs with the FRET pair of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) 9,10,14,15
. The ratio of acceptor emission to donor emission was used as the quantitative parameter for FRET signal monitor for protease activity determination. However, this method ignored signal cross-contaminations at the acceptor and donor emission wavelengths by acceptor and donor self-fluorescence and thus was not accurate.
We developed a novel highly sensitive and quantitative FRET-based protease assay for determining the kinetic parameters of pre-SUMO1 maturation by SENP1. An engineered FRET pair CyPet and YPet with significantly improved FRET efficiency and fluorescence quantum yield, were used to generate the CyPet-(pre-SUMO1)-YPet substrate 16
. We differentiated and quantified absolute fluorescence signals contributed by the donor and acceptor and FRET at the acceptor and emission wavelengths, respectively. The value of kcat
was obtained as (3.2 ± 0.55) x107
of SENP1 toward pre-SUMO1, which is in agreement with general enzymatic kinetic parameters. Therefore, this methodology is valid and can be used as a general approach to characterize other proteases as well.
Bioengineering, Issue 72, Biochemistry, Molecular Biology, Proteins, Quantitative FRET analysis, QFRET, enzyme kinetics analysis, SENP, SUMO, plasmid, protein expression, protein purification, protease assay, quantitative analysis
Adjustable Stiffness, External Fixator for the Rat Femur Osteotomy and Segmental Bone Defect Models
Institutions: Queensland University of Technology, RISystem AG.
The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there has been great clinical interest in improving bone healing by altering the mechanical environment through the fixation stability around the lesion. One constraint of preclinical animal research in this area is the lack of experimental control over the local mechanical environment within a large segmental defect as well as osteotomies as they heal. In this paper we report on the design and use of an external fixator to study the healing of large segmental bone defects or osteotomies. This device not only allows for controlled axial stiffness on the bone lesion as it heals, but it also enables the change of stiffness during the healing process in vivo.
The conducted experiments have shown that the fixators were able to maintain a 5 mm femoral defect gap in rats in vivo
during unrestricted cage activity for at least 8 weeks. Likewise, we observed no distortion or infections, including pin infections during the entire healing period. These results demonstrate that our newly developed external fixator was able to achieve reproducible and standardized stabilization, and the alteration of the mechanical environment of in vivo
rat large bone defects and various size osteotomies. This confirms that the external fixation device is well suited for preclinical research investigations using a rat model in the field of bone regeneration and repair.
Medicine, Issue 92, external fixator, bone healing, small animal model, large bone defect and osteotomy model, rat model, mechanical environment, mechanobiology.
Detection of Protein Ubiquitination
Institutions: The Sanford Burnham Institute for Medical Research.
Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo
and test tubes in vitro
. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.
Cell Biology, Biochemistry, Issue 30, ubiquitination, cultured cell, in vitro system, immunoprecipitation, immunoblotting, ubiquitin, posttranslational modification