Biotin Ligase BirA Research Articles

A tissue-specific protein purification approach in Caenorhabditis elegans identifies novel interaction partners of DLG-1/Discs large.

BackgroundAffinity purification followed by mass spectrometry (AP/MS) is a widely used approach to identify protein interactions and complexes. In multicellular organisms, the accurate identification of protein complexes by AP/MS is complicated by the potential heterogeneity of complexes in different tissues. Here, we present an in vivo biotinylation-based approach for the tissue-specific purification of protein complexes from Caenorhabditis elegans. Tissue-specific biotinylation is achieved by the expression in select tissues of the bacterial biotin ligase BirA, which biotinylates proteins tagged with the Avi peptide.ResultsWe generated N- and C-terminal tags combining GFP with the Avi peptide sequence, as well as four BirA driver lines expressing BirA ubiquitously and specifically in the seam and hyp7 epidermal cells, intestine, or neurons. We validated the ability of our approach to identify bona fide protein interactions by identifying the known LGL-1 interaction partners PAR-6 and PKC-3. Purification of the Discs large protein DLG-1 identified several candidate interaction partners, including the AAA-type ATPase ATAD-3 and the uncharacterized protein MAPH-1.1. We have identified the domains that mediate the DLG-1/ATAD-3 interaction, and show that this interaction contributes to C. elegans development. MAPH-1.1 co-purified specifically with DLG-1 purified from neurons, and shared limited homology with the microtubule-associated protein MAP1A, a known neuronal interaction partner of mammalian DLG4/PSD95. A CRISPR/Cas9-engineered GFP::MAPH-1.1 fusion was broadly expressed and co-localized with microtubules.ConclusionsThe method we present here is able to purify protein complexes from specific tissues. We uncovered a series of DLG-1 interactors, and conclude that ATAD-3 is a biologically relevant interaction partner of DLG-1. Finally, we conclude that MAPH-1.1 is a microtubule-associated protein of the MAP1 family and a candidate neuron-specific interaction partner of DLG-1.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0286-x) contains supplementary material, which is available to authorized users.

In Vivo Biotinylation of the Toxoplasma Parasitophorous Vacuole Reveals Novel Dense Granule Proteins Important for Parasite Growth and Pathogenesis.

ABSTRACTToxoplasma gondii is an obligate intracellular parasite that invades host cells and replicates within a unique parasitophorous vacuole. To maintain this intracellular niche, the parasite secretes an array of dense granule proteins (GRAs) into the nascent parasitophorous vacuole. These GRAs are believed to play key roles in vacuolar remodeling, nutrient uptake, and immune evasion while the parasite is replicating within the host cell. Despite the central role of GRAs in the Toxoplasma life cycle, only a subset of these proteins have been identified, and many of their roles have not been fully elucidated. In this report, we utilize the promiscuous biotin ligase BirA* to biotinylate GRA proteins secreted into the vacuole and then identify those proteins by affinity purification and mass spectrometry. Using GRA-BirA* fusion proteins as bait, we have identified a large number of known and candidate GRAs and verified localization of 13 novel GRA proteins by endogenous gene tagging. We proceeded to functionally characterize three related GRAs from this group (GRA38, GRA39, and GRA40) by gene knockout. While Δgra38 and Δgra40 parasites showed no altered phenotype, disruption of GRA39 results in slow-growing parasites that contain striking lipid deposits in the parasitophorous vacuole, suggesting a role in lipid regulation that is important for parasite growth. In addition, parasites lacking GRA39 showed dramatically reduced virulence and a lower tissue cyst burden in vivo. Together, the findings from this work reveal a partial vacuolar proteome of T. gondii and identify a novel GRA that plays a key role in parasite replication and pathogenesis.

Site-specific, covalent immobilization of BirA by microbial transglutaminase: A reusable biocatalyst for in vitro biotinylation

A facile approach for the production of a reusable immobilized recombinant Escherichia coli biotin ligase (BirA) onto amine-modified magnetic microspheres (MMS) via covalent cross-linking catalyzed using microbial transglutaminase (MTG) was proposed in this study. The site-specifically immobilized BirA exhibited approximately 95% of enzymatic activity of the free BirA, and without a significant loss in intrinsic activity after 10 rounds of recycling (P > 0.05). In addition, the immobilized BirA can be easily recovered from the solution via a simple magnetic separation. Thus, the immobilized BirA may be of general use for in vitro biotinylation in an efficient and economical manner.

Development of a biotinylated broad-specificity single-chain variable fragment antibody and a sensitive immunoassay for detection of organophosphorus pesticides.

Organophosphorus pesticides (OPs) are the most widely used pesticides in agriculture, and OP residues have been broadly reported in food and environmental samples. The aim of this study is to develop a recombinant antibody-based broad-specificity immunoassay for OPs. A phage display library was prepared from a mouse pre-immunized with a generic immunogen of OPs, and a single-chain variable fragment (scFv) antibody was selected. The selected scFv antibody was fused with biotin acceptor domain (BAD) and overexpressed as an inclusion body in Escherichia coli BL21 (DE3). Then, the protein was refolded by stepwise urea gradient dialysis and biotinylated in vitro by E. coli biotin ligase (BirA). Subsequently, the scFv-BAD protein was purified from the biotinylated system with high yield (66.7mgL(-1)) and confirmed by SDS-PAGE and Western blot. Based on the biotinylated scFv-BAD, a sensitive and broad-specificity competitive indirect enzyme-linked immunosorbent assay (ciELISA) for detection of OPs was developed. The cross-reactivity (CR) studies demonstrated that the ciELISA described here exhibited the broadest detection spectrum for OPs up to now, and 30 OPs could be determined with 50% inhibition value (IC50) values ranging from 19.4 to 515.2ngmL(-1). Moreover, the developed ciELISA was used for the recovery study of the spiked samples and showed satisfactory recoveries. Graphical Abstract Schematic diagram of the development of biotinylated broad-specificity single-chain variable fragment antibody-based immunoassay for organophosphorus pesticides.

A simple and robust approach to immobilization of antibody fragments

Antibody fragments, such as the single-chain variable fragment (scFv), have much potential in research and diagnostics because of their antigen-binding ability similar to a full-sized antibody and their ease of production in microorganisms. Some applications of antibody fragments require immobilization on a surface, and we have established a simple immobilization method that is based on the biotin-streptavidin interaction and does not require a separate purification step. We genetically fused two biotinylation tags—the biotin carboxyl carrier protein (BCCP) or the AviTag minimal sequence—to six different scFvs (scFv13R4, scFvD10, scFv26-10, scFv3, scFv5, and scFv12) for site-specific biotinylation in vivo by endogenous biotin ligases produced by Escherichia coli. The biotinylated scFvs were immobilized onto streptavidin-coated plates directly from cell lysates, and immobilization was detected through enzyme-linked immunosorbent assays. All scFvs fusions were successfully immobilized, and scFvs biotinylated via the BCCP tag tended to immobilize better than those biotinylated via the AviTag, even when biotinylation efficiency was improved with the biotin ligase BirA. The ability of immobilized scFvs to bind antigens was confirmed using scFv13R4 and scFvD10 with their respective targets β-galactosidase and bacteriophage lambda head protein D (gpD). The immobilized scFv13R4 bound to β-galactosidase at the same level for both biotinylation tags when the surface was saturated with the scFv, and immobilized scFvs retained their functionality for at least 100days after immobilization. The simplicity and robustness of our method make it a promising approach for future applications that require antibody fragment immobilization.

Identification of Interactions in the NMD Complex Using Proximity-Dependent Biotinylation (BioID).

Proximity-dependent trans-biotinylation by the Escherichia coli biotin ligase BirA mutant R118G (BirA*) allows stringent streptavidin affinity purification of proximal proteins. This so-called BioID method provides an alternative to the widely used co-immunoprecipitation (co-IP) to identify protein-protein interactions. Here, we used BioID, on its own and combined with co-IP, to identify proteins involved in nonsense-mediated mRNA decay (NMD), a post-transcriptional mRNA turnover pathway that targets mRNAs that fail to terminate translation properly. In particular, we expressed BirA* fused to the well characterized NMD factors UPF1, UPF2 and SMG5 and detected by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) the streptavidin-purified biotinylated proteins. While the identified already known interactors confirmed the usefulness of BioID, we also found new potentially important interactors that have escaped previous detection by co-IP, presumably because they associate only weakly and/or very transiently with the NMD machinery. Our results suggest that SMG5 only transiently contacts the UPF1-UPF2-UPF3 complex and that it provides a physical link to the decapping complex. In addition, BioID revealed among others CRKL and EIF4A2 as putative novel transient interactors with NMD factors, but whether or not they have a function in NMD remains to be elucidated.

Site-directed immobilization of a genetically engineered anti-methotrexate antibody via an enzymatically introduced biotin label significantly increases the binding capacity of immunoaffinity columns.

In this study, the effect of random vs. site-directed immobilization techniques on the performance of antibody-based HPLC columns was investigated using a single-domain camelid antibody (VHH) directed against methotrexate (MTX) as a model system. First, the high flow-through support material POROS-OH was activated with disuccinimidyl carbonate (DSC), and the VHH was bound in a random manner via amines located on the protein's surface. The resulting column was characterized by Frontal Affinity Chromatography (FAC). Then, two site-directed techniques were explored to increase column efficiency by immobilizing the antibody via its C-terminus, i.e., away from the antigen-binding site. In one approach, a tetra-lysine tail was added, and the antibody was immobilized onto DSC-activated POROS. In the second site-directed approach, the VHH was modified with the AviTag peptide, and a biotin-residue was enzymatically incorporated at the C-terminus using the biotin ligase BirA. The biotinylated antibody was subsequently immobilized onto NeutrAvidin-derivatized POROS. A comparison of the FAC analyses, which for all three columns showed excellent linearity (R(2)>0.999), revealed that both site-directed approaches yield better results than the random immobilization; the by far highest efficiency, however, was determined for the immunoaffinity column based on AviTag-biotinylated antibody. As proof of concept, all three columns were evaluated for quantification of MTX dissolved in phosphate buffered saline (PBS). Validation using UV-detection showed excellent linearity in the range of 0.04-12μM (R(2)>0.993). The lower limit of detection (LOD) and lower limit of quantification (LLOQ) were found to be independent of the immobilization strategy and were 40nM and 132nM, respectively. The intra- and inter-day precision was below 11.6%, and accuracy was between 90.7% and 112%. To the best of our knowledge, this is the first report of the AviTag-system in chromatography, and the first application of immunoaffinity chromatography for the analysis of MTX.

Differential Signaling through p190 and p210 Forms of BCR-ABL Fusion Proteins Revealed By Proteomic Analysis

Chromosomal translocations involving chromosome 9q34 and 22q11 generate the BCR-ABL1 fusion gene. The location of the translocation within the BCR gene dictates which exons are excluded or included in the resulting fusion with the ABL1 gene. The most common translocations produce three main protein products with molecular weights of 190, 210, and 230 kD. Interestingly, the p190 and p210 BCR-ABL1 forms are associated with different clinical characteristics. Specifically, BCR-ABL1+ acute lymphoblastic leukemia (ALL) cases typically harbor the p190 form, whereas chronic myelogenous leukemia (CML) cases typically harbor the p210 form. Despite a great deal of study over many decades devoted to the BCR-ABL1 molecule, neither phosphorylation of putative target proteins at site-specific resolution nor protein-protein interaction differences between the p190 and the p210 forms is well understood, preventing thorough understanding of the signaling programs employed by these variants.Therefore, to examine signaling differences between p190 and p210 we chose a homogeneous system to investigate global phosphorylation of tyrosine residues. Ba/F3 cell lines, which are normally dependent on IL-3 for growth, were transduced with either the p190 or p210 form of BCR-ABL1. Subsequently, these cell lines were selected for successful growth in cytokine free media, indicating that the BCR-ABL1 fusions were signaling and inducing aberrant growth. These two BCR-ABL1 cell lines, as well as the parental Ba/F3 line, were grown in SILAC (stable isotope labeling by amino acids in cell culture) media and lysates were enriched for phosphotyrosine (pTyr) peptides and submitted for mass spectrometric analysis. The resulting 3-state SILAC dataset includes quantitation on 355 pTyr sites in three pairwise comparisons; p190 vs. p210 as well as each BCR-ABL1 cell line vs. its parental control. Many of the known BCR-ABL1 associated phosphorylation sites are recapitulated in our dataset. To our knowledge we report the first data on pTyr site-specific differences between p190 and p210 forms within in the same cell type. Surprisingly, 78 pTyr sites are differentially regulated between p190 and p210, with notable examples including pTyr sites on TEC, SHC1, PEAK1, PTPN6 and LYN. Additionally, sites in the kinase domain of JAK2 and the DNA binding domain of STAT6 showed a reciprocal relationship in p190 vs. p210, suggesting the differential involvement of JAK-STAT pathway. Notably, 28 pTyr sites were differentially regulated exclusively in either p190 or p210 BCR-ABL1 Ba/F3 cells, suggesting that there may be signaling pathways unique to p190 and p210, respectively.To attempt to identify direct substrates of BCR-ABL1 and detect differential binding of p190 and p210, we used the BioID system (Roux et al., JBC 2012). In this system, a specialized biotin ligase (BirA) is used to label adjacent or interacting proteins with a biotin moiety on lysine residues. Specifically, Ba/F3 cells transduced with BirA-tagged p190 or untagged p190 were grown in SILAC media and whole cell lysates were subjected streptavidin immunoprecipitation followed by mass spectrometry. Forty-four proteins were enriched over the control and known interactors such as SHC1, DOK2, and GAB1 were detected, along with several putative novel interactors. Combining both the phosphorylation and interactome datasets revealed all these proteins are hyper-phosphorylated compared to parental cell lines, suggesting these are direct substrates or in proximal complexes with p190 BCR-ABL1. A direct comparison of p190 and p210 interactomes using BioID is pending, and will further elucidate potential differences between these important fusion protein isoforms. In sum, our data suggests that p190 and p210 have at least partially independent signaling cascades that are mediated by differential protein-protein interactions, which may help explain the observed association of p190 with BCR-ABL1+ ALL and p210 with CML. DisclosuresNo relevant conflicts of interest to declare.

Real-time monitoring of NKCC2 endocytosis by total internal reflection fluorescence (TIRF) microscopy.

The apical Na-K-2Cl cotransporter (NKCC2) mediates NaCl reabsorption by the thick ascending limb (TAL). The amount of NKCC2 at the apical membrane of TAL cells is determined by exocytic delivery, recycling, and endocytosis. Surface biotinylation allows measurement of NKCC2 endocytosis, but it has low time resolution and does not allow imaging of the dynamic process of endocytosis. We hypothesized that total internal reflection fluorescence (TIRF) microscopy imaging of labeled NKCC2 would allow monitoring of NKCC2 endocytosis in polarized Madin-Darby canine kidney (MDCK) and TAL cells. Thus we generated a NKCC2 construct containing a biotin acceptor domain (BAD) sequence between the transmembrane domains 5 and 6. Once expressed in polarized MDCK or TAL cells, surface NKCC2 was specifically biotinylated by exogenous biotin ligase (BirA). We also demonstrate that expression of a secretory form of BirA in TAL cells induces metabolic biotinylation of NKCC2. Labeling biotinylated surface NKCC2 with fluorescent streptavidin showed that most apical NKCC2 was located within small discrete domains or clusters referred to as "puncta" on the TIRF field. NKCC2 puncta were observed to disappear from the TIRF field, indicating an endocytic event which led to a decrease in the number of surface puncta at a rate of 1.18 ± 0.16%/min in MDCK cells, and a rate 1.09 ± 0.08%/min in TAL cells (n = 5). Treating cells with a cholesterol-chelating agent (methyl-β-cyclodextrin) completely blocked NKCC2 endocytosis. We conclude that TIRF microscopy of labeled NKCC2 allows the dynamic imaging of individual endocytic events at the apical membrane of TAL cells.

Abstract B04: In vivo BioID identifies novel Myc interacting partners

Abstract Myc oncoprotein is a major driver of cancer initiation and progression, and thus targeting its activity would mark a key therapeutic advance. In a genetic preclinical mouse model, systemic Myc inhibition using the dominant-negative Myc mutant, termed Omomyc, showed that Ras-driven lung cancer could be eradicated without any harmful long-term effects to the animal. However, developing an anti-cancer agent that directly binds and inhibits Myc has not been possible, to date. Therefore, new strategies are required to inhibit Myc in cancer. Understanding the Myc interactome may unravel novel approaches to target Myc in cancer. The BioID proximity-based biotin labeling technique was recently developed for the characterization of protein-protein interaction networks. In BioID, the protein of interest is expressed as a fusion partner biotin ligase (BirA*), which activates biotin. The active biotin reacts with lysine residues on nearby polypeptides. Following a stringent cell lysis and streptavidin-sepharose pulldown, biotinylated proteins can be identified using MS. To date, this method has been applied to a number of different polypeptides expressed in cultured cells. Here we report the adaptation of BioID to the identification of protein-protein interactions surrounding the Myc oncoprotein in human cells grown both under standard culture conditions and in mice as tumor xenografts. Notably, in vivo BioID yielded >100 high confidence Myc interacting proteins, including >30 known binding partners such as MAX (Myc-associated factor X), TRRAP (transformation/transcription domain-associated protein), the enhancer of polycomb homologs 1 and 2 (EPC1, EPC2), lysine acetyltransferase 5 (KAT5). Putative novel Myc interactors include components of the STAGA/KAT5 and SWI/SNF chromatin remodelling complexes (see Penn lab abstract Tu et al), DNA repair and replication factors, general transcription and elongation factors, and transcriptional co-regulators such as the DNA helicase chromodomain 8 (CHD8). Providing additional confidence in these findings, ENCODE ChIP-seq datasets highlight significant coincident binding throughout the genome for the Myc interactors identified here, and we validate the previously unreported CHD8 (an ATP-dependent helicase)-Myc interaction using both a yeast two hybrid analysis and the proximity-based ligation assay (PLA). Additionally, we also validate Myc-BRD4 and Myc-TRIM24 interaction by PLA. In sum, here we identify bona fide interacting partners of Myc in vivo by use of BioID. Our study shows for the first time Myc interactome in vivo, understanding these interactors will shed more light on Myc oncogenesis, which can be used to therapeutically target Myc in cancer. Citation Format: Dharmendra Dingar, Manpreet Kalkat, Pak-Kei Chan, Swneke D. Bailey, Tharan Srikumar, William B. Tu, Etienne Coyaud, Romina Ponzielli, Max Kolyar, Igor Jurisica, Annie Huang, Mathieu Lupien, Brian Raught, Linda Z. Penn. In vivo BioID identifies novel Myc interacting partners. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B04.

Probing the Biology of Giardia intestinalis Mitosomes Using In Vivo Enzymatic Tagging.

Giardia intestinalis parasites contain mitosomes, one of the simplest mitochondrion-related organelles. Strategies to identify the functions of mitosomes have been limited mainly to homology detection, which is not suitable for identifying species-specific proteins and their functions. An in vivo enzymatic tagging technique based on the Escherichia coli biotin ligase (BirA) has been introduced to G. intestinalis; this method allows for the compartment-specific biotinylation of a protein of interest. Known proteins involved in the mitosomal protein import were in vivo tagged, cross-linked, and used to copurify complexes from the outer and inner mitosomal membranes in a single step. New proteins were then identified by mass spectrometry. This approach enabled the identification of highly diverged mitosomal Tim44 (GiTim44), the first known component of the mitosomal inner membrane translocase (TIM). In addition, our subsequent bioinformatics searches returned novel diverged Tim44 paralogs, which mediate the translation and mitosomal insertion of mitochondrially encoded proteins in other eukaryotes. However, most of the identified proteins are specific to G. intestinalis and even absent from the related diplomonad parasite Spironucleus salmonicida, thus reflecting the unique character of the mitosomal metabolism. The in vivo enzymatic tagging also showed that proteins enter the mitosome posttranslationally in an unfolded state and without vesicular transport.

Proximity Utilizing Biotinylation of Nuclear Proteins in vivo.

IntroductionThe human genome consists of roughly 30,000 genes coding for over 500,000 different proteins, of which more than 10,000 proteins can be produced by the cell at any given time (the cellular “proteome”). It has been estimated that over 80% of proteins do not operate alone, but in complexes. These protein-protein interactions (PPI) are regulated by several mechanisms. For example, post-translational modifications (methylation, acetylation, phosphorylation, or ubiquitination) or metal-binding can lead to conformational changes that alter the affinity and kinetic parameters of the interaction. Many PPIs are part of larger cellular networks of interactions or interactomes. Indeed, these interactions are at the core of the entire interactomics system of any living cell, and so, aberrant PPIs are the basis of multiple diseases, such as neurodegenerative diseases and cancer. The objective of this study was to develop a method of monitoring protein-protein interactions and proximity dependence in vivo.MethodsThe biotin ligase BirA was fused to the protein of interest, and the Biotin Acceptor Peptide (BAP) was fused to an interacting partner to make the detection of its biotinylation possible by western blot or mass spectrometry.ResultsUsing several experimental systems (BirA.A + BAP.B), we showed that the biotinylation is interaction/proximity dependent. Here, A and B are the next nuclear proteins used in the experiments – 3 paralogues of heterochromatin protein HP1α (CBX5), HP1β (CBX1), HP1γ (CBX3), wild type and transcription mutant factor Kap1, translesion DNA polymerase PolH and E3, ubiquitin ligase RAD18, Proliferative Cell Nuclear Antigen (PCNA), ubiquitin Ub, SUMO-2/3, different types and isoforms of histones H2A, H2Az, H3.1, H3.3, CenpA, H2A.BBD, and macroH2A. The variant of this approach is termed PUB-NChIP (Proximity Utilizing Biotinylation with Native Chromatin Immuno-precipitation) and is designed to purify and study the protein composition of chromatin in proximity to the nuclear protein of interest. Using the RAD18 protein as a model, we demonstrated that the RAD18-proximal chromatin is enriched in some H4 acetylated species. Moreover, the RAD18-proximal chromatin containing a replacement histone H2Az has a different pattern of H4 acetylation.ConclusionProgress in the last decade in cancer drug therapy has led us to the conclusion that the nucleus of eukaryotic cells is an active site for many cellular processes important to the development of cancer. These processes include changes in genetic and epigenetic landscape (e. g. methylation of DNA, modification of histones) and the expression levels of transcription factors, which regulates gene products (e.g. hypoxia-inducible factor 1α (HIF-1α) in chronic anemia, etc.) where protein-protein interactions play important role. Understanding the nature of protein-protein interactions may improve design strategies for small-molecule PPI modulators. PPI assay technologies that closely reflect physiological conditions hold the key to developing specific anti-cancer drugs.

Directional Trans-Synaptic Labeling of Specific Neuronal Connections in Live Animals.

Understanding animal behavior and development requires visualization and analysis of their synaptic connectivity, but existing methods are laborious or may not depend on trans-synaptic interactions. Here we describe a transgenic approach for in vivo labeling of specific connections in Caenorhabditis elegans, which we term iBLINC. The method is based on BLINC (Biotin Labeling of INtercellular Contacts) and involves trans-synaptic enzymatic transfer of biotin by the Escherichia coli biotin ligase BirA onto an acceptor peptide. A BirA fusion with the presynaptic cell adhesion molecule NRX-1/neurexin is expressed presynaptically, whereas a fusion between the acceptor peptide and the postsynaptic protein NLG-1/neuroligin is expressed postsynaptically. The biotinylated acceptor peptide::NLG-1/neuroligin fusion is detected by a monomeric streptavidin::fluorescent protein fusion transgenically secreted into the extracellular space. Physical contact between neurons is insufficient to create a fluorescent signal, suggesting that synapse formation is required. The labeling approach appears to capture the directionality of synaptic connections, and quantitative analyses of synapse patterns display excellent concordance with electron micrograph reconstructions. Experiments using photoconvertible fluorescent proteins suggest that the method can be utilized for studies of protein dynamics at the synapse. Applying this technique, we find connectivity patterns of defined connections to vary across a population of wild-type animals. In aging animals, specific segments of synaptic connections are more susceptible to decline than others, consistent with dedicated mechanisms of synaptic maintenance. Collectively, we have developed an enzyme-based, trans-synaptic labeling method that allows high-resolution analyses of synaptic connectivity as well as protein dynamics at specific synapses of live animals.

Force dependent biotinylation of myosin IIA by α-catenin tagged with a promiscuous biotin ligase.

Tissues and organs undergo constant physical perturbations and individual cells must respond to mechanical forces to maintain tissue integrity. However, molecular interactions underlying mechano-transduction are not fully defined at cell-cell junctions. This is in part due to weak and transient interactions that are likely prevalent in force-induced protein complexes. Using in situ proximal biotinylation by the promiscuous biotin ligase BirA tagged to α-catenin and a substrate stretch cell chamber, we sought to identify force-dependent molecular interactions surrounding α-catenin, an actin regulator at the sites of cadherin mediated cell-cell adhesion. While E-cadherin, β-catenin, vinculin and actin localize with α-catenin at cell-cell contacts in immuno-fluorescent staining, only β-catenin and plakoglobin were biotinylated, suggesting that this proximal biotinylation is limited to the molecules that are in the immediate vicinity of α-catenin. In mechanically stretched samples, increased biotinylation of non-muscle myosin IIA, but not myosin IIB, suggests close spatial proximity between α-catenin and myosin IIA during substrate stretching. This force-induced biotinylation diminished as myosin II activity was inhibited by blebbistatin. Taken together, this promising technique enables us to identify force sensitive complexes that may be essential for mechano-responses in force bearing cell adhesion.

Topokaryotyping – a proposal for a novel approach to study nuclear organization

Gene positioning in the nucleus plays an important role in gene activity and genome stability, both in norm and pathology. We propose a new principle for the analysis of the large scale chromatin organization at the single-cell level, termed Topokaryotyping. It is based on coexpression in cells of (i) biotin ligase BirA targeted to a particular intranuclear domain (via its fusion with a protein-marker of this domain) together with (ii) BAP-histone fusion. The application of biotin pulse chase followed by (i) generation of mitotic spreads, (ii) detection of the biotin label and (iii) karyotyping technique to identify the chromosomes should provide information on the association of particular chromosome regions with the nuclear domain under study. We discuss potential advantages of the proposed approach as compared to other methods to study genome topology in the nucleus.

Structural and Functional Study of d-Glucuronyl C5-epimerase

Heparan sulfate (HS) is a glycosaminoglycan present on the cell surface and in the extracellular matrix, which interacts with diverse signal molecules and is essential for many physiological processes including embryonic development, cell growth, inflammation, and blood coagulation. D-glucuronyl C5-epimerase (Glce) is a crucial enzyme in HS synthesis, converting D-glucuronic acid to L-iduronic acid to increase HS flexibility. This modification of HS is important for protein ligand recognition. We have determined the crystal structures of Glce in apo-form (unliganded) and in complex with heparin hexasaccharide (product of Glce following O-sulfation), both in a stable dimer conformation. A Glce dimer contains two catalytic sites, each at a positively charged cleft in C-terminal α-helical domains binding one negatively charged hexasaccharide. Based on the structural and mutagenesis studies, three tyrosine residues, Tyr(468), Tyr(528), and Tyr(546), in the active site were found to be crucial for the enzymatic activity. The complex structure also reveals the mechanism of product inhibition (i.e. 2-O- and 6-O-sulfation of HS keeps the C5 carbon of L-iduronic acid away from the active-site tyrosine residues). Our structural and functional data advance understanding of the key modification in HS biosynthesis.

Analysis of HIV-1 Gag protein interactions via biotin ligase tagging.

We have examined the interactions of wild-type (WT) and matrix protein-deleted (ΔMA) HIV-1 precursor Gag (PrGag) proteins in virus-producing cells using a biotin ligase-tagging approach. To do so, WT and ΔMA PrGag proteins were tagged with the Escherichia coli promiscuous biotin ligase (BirA*), expressed in cells, and examined. Localization patterns of PrGag proteins and biotinylated proteins overlapped, consistent with observations that BirA*-tagged proteins biotinylate neighbor proteins that are in close proximity. Results indicate that BirA*-tagged PrGag proteins biotinylated themselves as well as WT PrGag proteins in trans. Previous data have shown that the HIV-1 Envelope (Env) protein requires an interaction with MA for assembly into virions. Unexpectedly, ΔMA proteins biotinylated Env, whereas WT BirA*-tagged proteins did not, suggesting that the presence of MA made Env inaccessible to biotinylation. We also identified over 50 cellular proteins that were biotinylated by BirA*-tagged PrGag proteins. These included membrane proteins, cytoskeleton-associated proteins, nuclear transport factors, lipid metabolism regulators, translation factors, and RNA-processing proteins. The identification of these biotinylated proteins offers new insights into HIV-1 Gag protein trafficking and activities and provides new potential targets for antiviral interference. We have employed a novel strategy to analyze the interactions of the HIV-1 structural Gag proteins, which involved tagging wild-type and mutant Gag proteins with a biotin ligase. Expression of the tagged proteins in cells allowed us to analyze proteins that came in close proximity to the Gag proteins as they were synthesized, transported, assembled, and released from cells. The tagged proteins biotinylated proteins encoded by the HIV-1 pol gene and neighbor Gag proteins, but, surprisingly, only the mutant Gag protein biotinylated the HIV-1 Envelope protein. We also identified over 50 cellular proteins that were biotinylated, including membrane and cytoskeletal proteins and proteins involved in lipid metabolism, nuclear import, translation, and RNA processing. Our results offer new insights into HIV-1 Gag protein trafficking and activities and provide new potential targets for antiviral interference.

Epigenome profiling of specific plant cell types using a streamlined INTACT protocol and ChIP-seq.

Plants consist of many functionally specialized cell types, each with its own unique epigenome, transcriptome, and proteome. Characterization of these cell type-specific properties is essential to understanding cell fate specification and the responses of individual cell types to the environment. In this chapter we describe an approach to map chromatin features in specific cell types of Arabidopsis thaliana using nuclei purification from individual cell types with the INTACT method (isolation of nuclei tagged in specific cell types) followed by chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq). The INTACT system employs two transgenes to generate affinity-labeled nuclei in the cell type of interest, and these tagged nuclei can then be selectively purified from tissue homogenates. The primary transgene encodes the nuclear tagging fusion protein (NTF), which consists of a nuclear envelope-targeting domain, the green fluorescent protein, and a biotin ligase recognition peptide, while the second transgene encodes the E. coli biotin ligase (BirA), which selectively biotinylates NTF. Expression of NTF and BirA in a specific cell type thus yields nuclei that are coated with biotin and can be purified by virtue of their affinity for streptavidin-coated magnetic beads. Compared with the original INTACT nuclei purification protocol, the procedure presented here is greatly simplified and shortened. After nuclei purification, we provide detailed instructions for chromatin isolation, shearing, and immunoprecipitation. Finally, we present a low input ChIP-seq library preparation protocol based on the nano-ChIP-seq method of Adli and Bernstein, and we describe multiplex Illumina sequencing of these libraries to produce high quality, cell type-specific epigenome profiles at a relatively low cost. The procedures given here are optimized for Arabidopsis but should be easily adaptable to other plant species.

Probing mammalian centrosome structure using BioID proximity-dependent biotinylation.

Understanding the structure and function of the centrosome will require identification of its constituent components and a detailed characterization of the interactions among these components. Here, we describe the application of proximity-dependent biotin identification (BioID) to identify spatial and temporal relationships among centrosome proteins. The BioID method relies on protein fusions to a promiscuous mutant of the Escherichia coli biotin ligase BirA, which biotinylates proteins that are in a ∼10nm labeling radius of the enzyme. The biotinylated proteins are captured by affinity and are identified by mass spectrometry. Proteins identified in this way are referred to as "proximity interactors." Application of BioID to a set of centrosome proteins demonstrated the utility of this approach in overcoming inherent limitations in probing centrosome structure. These studies also demonstrated the potential of BioID for building large-scale proximity interaction maps among centrosome proteins. In this chapter, we describe the work flow for identification of proximity interactions of centrosome proteins, including materials and methods for the generation and characterization of a BirA*-fusion protein expression plasmid, expression of BirA*-fusion proteins in cells, and purification and identification of proximity partners by mass spectrometry.

Isolation, cloning, and expression of E. coli BirA gene for biotinylation applications.

Background:The key enzyme in biotin-(strept) avidin systems, Escherichia coli BirA biotin ligase, is currently obtained by overexpression of the long protein-tagged versions of the gene to prevent its toxic effect in E. coli. Herein we describe a rather simple and efficient system for expression of E. coli BirA without the application of long-tag proteins.Materials and Methods:The coding sequence of BirA gene was isolated by polymerase chain reaction using DNA extract of E. coli-DH5α as template. BirA amplicon harboring a GS-linker at its C-terminal was cloned into NdeI-XhoI sites of pET24a(+) vector under control of T7 promoter and upstream of the vector-derived 6xHis-tag. pET24-BirA transformed BL21-cells were induced for protein expression by IPTG and analyzed by SDS-PAGE and Western blotting. Protein expression yields were assessed by image analysis of the SDS-PAGE scans using ImageJ software.Result:Agarose gel electrophoresis indicated proper size of the BirA gene amplicon (963 bp) and accuracy of the recombinant pET24-BirA construct. Sequence alignment analysis indicated identical sequence (100%) of our isolate with that of the standard E. coli-K12 BirA gene sequence (accession number: NC_000913.3). SDS-PAGE and Western blot results indicated specific expression of the 36.6 kDa protein corresponding to the BirA protein. Image analysis estimated a yield of 12% of total protein for the BirA expression.Conclusions:By application of pET24a(+) we achieved relatively high expression of BirA in E. coli without application of any long protein-tags. Introduction of the present expression system may provide more readily available source of BirA enzyme for (strept) avidin–biotin applications and studies.