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Structural basis for ubiquitin recognition by ubiquitin-binding zinc finger of FAAP20.
PUBLISHED: 03-24-2015
Several ubiquitin-binding zinc fingers (UBZs) have been reported to preferentially bind K63-linked ubiquitin chains. In particular, the UBZ domain of FAAP20 (FAAP20-UBZ), a member of the Fanconi anemia core complex, seems to recognize K63-linked ubiquitin chains, in order to recruit the complex to DNA interstrand crosslinks and mediate DNA repair. By contrast, it is reported that the attachment of a single ubiquitin to Rev1, a translesion DNA polymerase, increases binding of Rev1 to FAAP20. To clarify the specificity of FAAP20-UBZ, we determined the crystal structure of FAAP20-UBZ in complex with K63-linked diubiquitin at 1.9 Å resolution. In this structure, FAAP20-UBZ interacts only with one of the two ubiquitin moieties. Consistently, binding assays using surface plasmon resonance spectrometry showed that FAAP20-UBZ binds ubiquitin and M1-, K48- and K63-linked diubiquitin chains with similar affinities. Residues in the vicinity of Ala168 within the ?-helix and the C-terminal Trp180 interact with the canonical Ile44-centered hydrophobic patch of ubiquitin. Asp164 within the ?-helix and the C-terminal loop mediate a hydrogen bond network, which reinforces ubiquitin-binding of FAAP20-UBZ. Mutations of the ubiquitin-interacting residues disrupted binding to ubiquitin in vitro and abolished the accumulation of FAAP20 to DNA damage sites in vivo. Finally, structural comparison among FAAP20-UBZ, WRNIP1-UBZ and RAD18-UBZ revealed distinct modes of ubiquitin binding. UBZ family proteins could be divided into at least three classes, according to their ubiquitin-binding modes.
Authors: Elena García-Cano, Adi Zaltsman, Vitaly Citovsky.
Published: 03-26-2014
The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions. The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.
19 Related JoVE Articles!
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Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions
Authors: Jamie Snider, Saranya Kittanakom, Jasna Curak, Igor Stagljar.
Institutions: University of Toronto, University of Toronto, University of Toronto.
The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.
Cellular Biology, Issue 36, protein-protein interaction, membrane, split-ubiquitin, yeast, library screening, Y2H, yeast two-hybrid, MYTH
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siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells
Authors: John S. Bett, Adel F. M. Ibrahim, Amit K. Garg, Sonia Rocha, Ronald T. Hay.
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
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Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE
Authors: Michaela Weber, Friedemann Weber.
Institutions: Philipps-University Marburg.
Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, the PRRs RIG-I and PKR bind to specific viral RNA ligands. This first mediates conformational switching and oligomerization, and then enables activation of an antiviral interferon response. While methods to measure antiviral host gene expression are well established, methods to directly monitor the activation states of RIG-I and PKR are only partially and less well established. Here, we describe two methods to monitor RIG-I and PKR stimulation upon infection with an established interferon inducer, the Rift Valley fever virus mutant clone 13 (Cl 13). Limited trypsin digestion allows to analyze alterations in protease sensitivity, indicating conformational changes of the PRRs. Trypsin digestion of lysates from mock infected cells results in a rapid degradation of RIG-I and PKR, whereas Cl 13 infection leads to the emergence of a protease-resistant RIG-I fragment. Also PKR shows a virus-induced partial resistance to trypsin digestion, which coincides with its hallmark phosphorylation at Thr 446. The formation of RIG-I and PKR oligomers was validated by native polyacrylamide gel electrophoresis (PAGE). Upon infection, there is a strong accumulation of RIG-I and PKR oligomeric complexes, whereas these proteins remained as monomers in mock infected samples. Limited protease digestion and native PAGE, both coupled to western blot analysis, allow a sensitive and direct measurement of two diverse steps of RIG-I and PKR activation. These techniques are relatively easy and quick to perform and do not require expensive equipment.
Infectious Diseases, Issue 89, innate immune response, virus infection, pathogen recognition receptor, RIG-I, PKR, IRF-3, limited protease digestion, conformational switch, native PAGE, oligomerization
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Reconstitution Of β-catenin Degradation In Xenopus Egg Extract
Authors: Tony W. Chen, Matthew R. Broadus, Stacey S. Huppert, Ethan Lee.
Institutions: Vanderbilt University Medical Center, Cincinnati Children's Hospital Medical Center, Vanderbilt University School of Medicine.
Xenopus laevis 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 ([35S]-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
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Reporter-based Growth Assay for Systematic Analysis of Protein Degradation
Authors: Itamar Cohen, Yifat Geffen, Guy Ravid, Tommer Ravid.
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
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Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae
Authors: Sheldon G. Watts, Justin J. Crowder, Samuel Z. Coffey, Eric M. Rubenstein.
Institutions: Ball State University, Cincinnati Children's Hospital.
Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.
Molecular Biology, Issue 96, Ubiquitin-proteasome system, Saccharomyces cerevisiae, budding yeast, growth assay, protein extracts, western blotting, yeast genetics, mutants, endoplasmic reticulum-associated degradation, protein degradation
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Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80
Authors: Kentaro Ohkuni, Yoshimitsu Takahashi, Munira A. Basrai.
Institutions: National Cancer Institute, National Institute of Health.
Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular function of many proteins. PTMs of kinetochore proteins that associate with centromeric DNA mediate faithful chromosome segregation to maintain genome stability. Biochemical approaches such as mass spectrometry and western blot analysis are most commonly used for identification of PTMs. Here, a protein purification method is described that allows the detection of both sumoylation and ubiquitination of the kinetochore proteins, Ndc10 and Ndc80, in Saccharomyces cerevisiae. A strain that expresses polyhistidine-Flag-tagged Smt3 (HF-Smt3) and Myc-tagged Ndc10 or Ndc80 was constructed and used for our studies. For detection of sumoylation, we devised a protocol to affinity purify His-tagged sumoylated proteins by using nickel beads and used western blot analysis with anti-Myc antibody to detect sumoylated Ndc10 and Ndc80. For detection of ubiquitination, we devised a protocol for immunoprecipitation of Myc-tagged proteins and used western blot analysis with anti-Ub antibody to show that Ndc10 and Ndc80 are ubiquitinated. Our results show that epitope tagged-protein of interest in the His-Flag tagged Smt3 strain facilitates the detection of multiple PTMs. Future studies should allow exploitation of this technique to identify and characterize protein interactions that are dependent on a specific PTM.
Microbiology, Issue 99, Saccharomyces cerevisiae, Kinetochore protein, Ndc10, Ndc80, Sumoylation, Ubiquitination, Post-translational modifications, Protein extracts
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Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration
Authors: Daniel Wehner, Christopher Jahn, Gilbert Weidinger.
Institutions: Ulm University.
The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta–catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.
Developmental Biology, Issue 100, Tetracycline-controlled transcriptional activation, TetON, zebrafish, Regeneration, fin, tissue-specific gene expression, doxycycline, cryosectioning, transgenic, Tol2, I-SceI, anesthesia
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Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin
Authors: Jyoti Srivastava, Diane Barber.
Institutions: University of California, San Francisco - UCSF.
The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape. Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling. A number of proteins can bind to the actin cytoskeleton. The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin. The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin. Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.
Biochemistry, Issue 13, F-actin, protein, in vitro binding, ultracentrifugation
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Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies
Authors: Luke A. Yates, Robert J. C. Gilbert.
Institutions: University of Oxford.
Kindlins are essential coactivators, with talin, of the cell surface receptors integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin β-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3's role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.
Virology, Issue 85, Heterologous protein expression, insect cells, Spodoptera frugiperda, baculovirus, protein purification, kindlin, cell adhesion
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Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays
Authors: F. Mark Dunning, Timothy M. Piazza, Füsûn N. Zeytin, Ward C. Tucker.
Institutions: BioSentinel Inc., Madison, WI.
Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample testing. Rapid BoNT testing of foodstuffs is needed during outbreak forensics, patient diagnosis, and food safety testing while accurate potency testing is required for BoNT-based drug product manufacturing and patient safety. The widely used mouse bioassay for BoNT testing is highly sensitive but lacks the precision and throughput needed for rapid and routine BoNT testing. Furthermore, the bioassay's use of animals has resulted in calls by drug product regulatory authorities and animal-rights proponents in the US and abroad to replace the mouse bioassay for BoNT testing. Several in vitro replacement assays have been developed that work well with purified BoNT in simple buffers, but most have not been shown to be applicable to testing in highly complex matrices. Here, a protocol for the detection of BoNT in complex matrices using the BoTest Matrix assays is presented. The assay consists of three parts: The first part involves preparation of the samples for testing, the second part is an immunoprecipitation step using anti-BoNT antibody-coated paramagnetic beads to purify BoNT from the matrix, and the third part quantifies the isolated BoNT's proteolytic activity using a fluorogenic reporter. The protocol is written for high throughput testing in 96-well plates using both liquid and solid matrices and requires about 2 hr of manual preparation with total assay times of 4-26 hr depending on the sample type, toxin load, and desired sensitivity. Data are presented for BoNT/A testing with phosphate-buffered saline, a drug product, culture supernatant, 2% milk, and fresh tomatoes and includes discussion of critical parameters for assay success.
Neuroscience, Issue 85, Botulinum, food testing, detection, quantification, complex matrices, BoTest Matrix, Clostridium, potency testing
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Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)
Authors: Xiaolin Tian, Mingwei Zhu, Long Li, Chunlai Wu.
Institutions: Louisiana State University Health Sciences Center.
Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.
Biochemistry, Issue 82, Drosophila, GAL4/UAS system, transgenic, Tandem Affinity Purification, protein-protein interaction, proteomics
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Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Authors: James Smadbeck, Meghan B. Peterson, George A. Khoury, Martin S. Taylor, Christodoulos A. Floudas.
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 (, 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
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Quantitative FRET (Förster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination
Authors: Yan Liu, Jiayu Liao.
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/KM. 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/KM 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/KM was obtained as (3.2 ± 0.55) x107 M-1s-1 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
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Identification of Protein Interacting Partners Using Tandem Affinity Purification
Authors: Dalan Bailey, Luis Urena, Lucy Thorne, Ian Goodfellow.
Institutions: Imperial College London .
A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8. As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter. Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.
Molecular Biology, Issue 60, TAP tagging, translation, eIF4E, proteomics, tandem affinity purification
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Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein
Authors: Ruth Greussing, Hermann Unterluggauer, Rafal Koziel, Andrea B. Maier, Pidder Jansen-Dürr.
Institutions: Institute for Biomedical Aging Research, Leiden University Medical Center.
Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. Maintenance of proteasome activity was implicated in many key cellular processes, like cell's stress response 3, cell cycle regulation and cellular differentiation 4 or in immune system response 5. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases 4, 6. Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging 7, 8, 9, 10. Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)–dgn fusion protein (GFP–dgn, unstable) and a variant carrying a frameshift mutation (GFP–dgnFS, stable 11) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP–dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry.
Cellular Biology, Issue 69, Molecular Biology, Medicine, Biomedical Engineering, Virology, proteasome activity, lentiviral particles, GFP-dgn, GFP-dgnFS, GFP, human diploid fibroblasts, flow cytometry, plasmid, vector
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The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis
Authors: Ewa Pol.
Institutions: GE Healthcare Bio-Sciences AB.
In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), a plant cysteine protease, by real-time label-free analysis using Biacore X100. Several cystatin B variants with point mutations in areas of interaction with papain, are produced. For each cystatin B variant we determine its specific binding concentration using calibration-free concentration analysis (CFCA) and compare the values obtained with total protein concentration as determined by A280. After that, the kinetics of each cystatin B variant binding to papain is measured using single-cycle kinetics (SCK). We show that one of the four cystatin B variants we examine is only partially active for binding. This partial activity, revealed by CFCA, translates to a significant difference in the association rate constant (ka) and affinity (KD), compared to the values calculated using total protein concentration. Using CFCA in combination with kinetic analysis in a structure-function study contributes to obtaining reliable results, and helps to make the right interpretation of the interaction mechanism.
Cellular Biology, Issue 37, Protein interaction, Surface Plasmon Resonance, Biacore X100, CFCA, Cystatin B, Papain
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Detection of Protein Ubiquitination
Authors: Yeun Su Choo, Zhuohua Zhang.
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
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Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains
Authors: Thomas Gaj, Jia Liu.
Institutions: The Scripps Research Institute, ShanghaiTech University.
Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.
Molecular Biology, Issue 97, protein delivery, cell-penetrating peptide, zinc-finger, protein transduction domain, chemical biology, molecular biology
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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