FLAG Tag Research Articles

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations.

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the cell by controlling protein stability, activity, interactions, and intracellular localization. They enable the cell to respond to signals and to adapt to changes in its environment. Alterations within these mechanisms can lead to severe pathological situations such as neurodegenerative diseases and cancers. The aim of the technique described here is to establish ub/ubls dependent PTMs profiles, rapidly and accurately, from cultured cell lines. The comparison of different profiles obtained from different conditions allows the identification of specific alterations, such as those induced by a treatment for example. Lentiviral mediated cell transduction is performed to create stable cell lines expressing a two-tags (6His and Flag) version of the modifier (ubiquitin or a ubl such as SUMO1 or Nedd8). These tags permit the purification of ubiquitin and therefore of ubiquitinated proteins from the cells. This is done through a two-step purification process: The first one is performed in denaturing conditions using the 6His tag, and the second one in native conditions using the Flag tag. This leads to a highly specific and pure isolation of modified proteins which are subsequently identified and semi-quantified by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) technology. Easy informatics analysis of MS data using Excel software enables the establishment of PTM profiles by eliminating background signals. These profiles are compared between each condition in order to identify specific alterations which will then be studied more specifically, starting with their validation by standard biochemistry techniques.

A novel porcine parvovirus DNA-launched infectious clone carrying stable double labels as an effective genetic platform

Porcine parvovirus (PPV) is one of the major pathogens causing reproductive failure of swine. However, its specific pathogenesis has not been fully elucidated. Infectious clone is a powerful tool for further studying the pathogenic mechanism of PPV. In the present study, a PPV infectious clone was constructed, and the clone carries His-tag and Flag-tag double-genetic marker at the end of the ns1 gene 3′ terminal and vp1 gene 5′ terminal, respectively. The PPV DNA fragment F1 (1-182) in 5′ end and the other PPV DNA fragment F2 (4788-5074) in 3′ end were synthesized and assembled to the lower copy plasmid to construct pKQLL(F1 + F2), while the PPV DNA genome as a template to amplify carrying tags sequence PPV middle DNA fragment F3 and F4 by introducing Flag and His tags sequence in primers. Subsequently, the fused fragment F3/F4 were cloned into the Stu I/Sna B I sites of pKQLL(F1 + F2) plasmid to assemble the complete full-length PPV DNA recombinant plasmids, named as pD-PPV. The pD-PPV was transfected into PK-15 cells to gain rescued PPV virus, designed as D-PPV. Moreover, D-PPV showed similar replicate capability and pathogenicity comparing to the wild-type parental PPV through in vitro and in vivo studies, and the double labels can effectively indicate the expression and localization of viral proteins. Finally, the rescued D-PPV was found to be a convenient tool for antiviral drug screening. These data indicated that the newly established reverse genetic system for PPV would be a useful tool for further studying the pathogenesis mechanisms of PPV, developing labeled vaccine and screening antiviral drug.

The Journey for the Total Chemical Synthesis of a 53 kDa Protein

Chemical protein synthesis has been proved as an efficient way to afford medium-sized proteins with high homogeneity in workable quantities for various biochemical, structural, and functional studies. In particular, chemical protein synthesis has enabled access to proteins that are difficult or impossible to prepare by molecular biology approaches, such as those with post-translational modifications and mirror-image proteins. One prominent example is related to ubiquitination, a well-known modification that mediates a variety of cellular processes (e.g., proteasomal degradation). Ubiquitination is considered as a modification that is difficult to introduce into proteins in a test tube to generate ubiquitin (Ub) conjugates with high homogeneity with respect to the chain length and the anchored Lys residue in workable quantities to perform the biochemical and biophysical studies. Chemical protein synthesis has emerged as a powerful approach to prepare Ub conjugates for studies aiming to understand ubiquitination in great detail and decipher its roles in cell processes. Nevertheless, in order to answer more challenging questions in this field, it has been clear that researchers must also prepare Ub conjugates with increased size and complexity. Employing solid-phase peptide synthesis and chemoselective ligation, chemical protein synthesis offers a powerful way to furnish polypeptides composed of 100-200 residues. However, to synthesize larger proteins such as Ub conjugates, longer and more segments are required. This on the other hand leads to difficulties related to solubility, purification, ligation, and late-stage modifications. These challenges have encouraged us to explore more practical synthetic tools to facilitate the synthesis of complex Ub conjugates. In this Account, we summarize the synthetic tools that we have developed to achieve these goals. These include (1) δ-mercaptolysine-mediated isopeptide chemical ligation, (2) chemical synthesis of Ub building blocks, (3) palladium-mediated deprotection of key side chains during proteinsynthesis, (4) one-pot ligation and desulfurization, and (5) improving the solubility of peptide segments. The developed chemical toolbox has been a key for our successes in the synthesis of diverse and complex Ub conjugates. In this Account, we describe our approaches for generating various Ub conjugates, including (1) the K48 tetra-Ub chain composed of 304 amino acids, (2) the ubiquitinated histones and their analogues made of >200 amino acids, (3) the di-Ub-SUMO-2 hybrid chain composed of 245 amino acids, and (4) the 53 kDa tetra-Ub-α-globin composed of 472 amino acids, which represents the largest protein composed of natural amino acids ever made using chemical protein synthesis. The last target, Flag-Ub-Ub-Ub-Myc-Ub-(HA-α-globin), was prepared in the labeled form where the proximal Ub and distal Ub in the chain were labeled with Myc and Flag tags, respectively, while the α-globin was labeled with the HA tag. Applying the tetra-Ub-α-globin in proteasomal degradation studies assisted us to shed light on the proteolytic signal and the fates of the Ub moieties in the chains. Although these developments have contributed to the synthesis of interesting and challenging targets related to Ub signaling, several other targets may enforce new synthetic challenges. Hence, there is still a need to optimize the current synthetic tools and explore novel synthetic approaches to facilitate this process.

A High-Resolution Map of SBP1 Interactomes in Plasmodium falciparum-infected Erythrocytes.

SummaryThe pathogenesis of malaria parasites depends on host erythrocyte modifications that are facilitated by parasite proteins exported to the host cytoplasm. These exported proteins form a trafficking complex in the host cytoplasm that transports virulence determinants to the erythrocyte surface; this complex is thus essential for malaria virulence. Here, we report a comprehensive interaction network map of this complex. We developed authentic, unbiased, highly sensitive proteomic approaches to determine the proteins that interact with a core component of the complex, SBP1 (skeleton-binding protein 1). SBP1 interactomes revealed numerous exported proteins and potential interactors associated with SBP1 intracellular trafficking. We identified several host-parasite protein interactions and linked the exported protein MAL8P1.4 to Plasmodium falciparum virulence in infected erythrocytes. Our study highlights the complicated interplay between parasite and host proteins in the host cytoplasm and provides an interaction dataset connecting dozens of exported proteins required for P. falciparum virulence.

Nipah and Hendra Virus Glycoproteins Induce Comparable Homologous but Distinct Heterologous Fusion Phenotypes.

Nipah and Hendra viruses (NiV and HeV) exhibit high lethality in humans and are biosafety level 4 (BSL-4) paramyxoviruses in the growing genus Henipavirus The attachment (G) and fusion (F) envelope glycoproteins are both required for viral entry into cells and for cell-cell fusion, which is pathognomonic of henipaviral infections. Here, we compared the fusogenic capacities between homologous and heterologous pairs of NiV and HeV glycoproteins. Importantly, to accurately measure their fusogenic capacities, as these depend on glycoprotein cell surface expression (CSE) levels, we inserted identical extracellular tags to both fusion (FLAG tags) or both attachment (hemagglutinin [HA] tags) glycoproteins. Importantly, these tags were placed in extracellular sites where they did not affect glycoprotein expression or function. NiV and HeV glycoproteins induced comparable levels of homologous HEK293T cell-cell fusion. Surprisingly, however, while the heterologous NiV F/HeV G (NF/HG) combination yielded a hypofusogenic phenotype, the heterologous HeV F/NiV G (HF/NG) combination yielded a hyperfusogenic phenotype. Pseudotyped viral entry levels primarily corroborated the fusogenic phenotypes of the glycoprotein pairs analyzed. Furthermore, we constructed G and F chimeras that allowed us to map the overall regions in G and F that contributed to these hyperfusogenic or hypofusogenic phenotypes. Importantly, the fusogenic phenotypes of the glycoprotein combinations negatively correlated with the avidities of F-G interactions, supporting the F/G dissociation model of henipavirus-induced membrane fusion, even in the context of heterologous glycoprotein pairs.IMPORTANCE The NiV and HeV henipaviruses are BSL-4 pathogens transmitted from bats. NiV and HeV often lead to human death and animal diseases. The formation of multinucleated cells (syncytia) is a hallmark of henipaviral infections and is caused by fusion of cells coordinated by interactions of the viral attachment (G) and fusion (F) glycoproteins. We found via various assays that viral entry and syncytium formation depend on the viral origin of the glycoproteins, with HeV F and NiV G promoting higher membrane fusion levels than their counterparts. This is important knowledge, since both viruses use the same bat vector species and potential coinfections of these or subsequent hosts may alter the outcome of disease.

Engineered Lactococcus lactis Secreting IL-23 Receptor-Targeted REX Protein Blockers for Modulation of IL-23/Th17-Mediated Inflammation.

Lactococcus lactis, a probiotic bacterium of food origin, has recently been demonstrated as a suitable strain for the production and in vivo delivery of therapeutically important proteins into the gut. We aimed to engineer recombinant L. lactis cells producing/secreting REX binding proteins that have been described as IL-23 receptor (IL-23R) blockers and IL-23R antagonists suppressing the secretion of cytokine IL-17A, a pivotal step in the T-helper Th17-mediated pro-inflammatory cascade, as well as in the development of autoimmune diseases, including inflammatory bowel disease (IBD). To reach this goal, we introduced cDNA sequences coding for REX009, REX115, and REX125 proteins into plasmid vectors carrying a Usp45 secretion signal, a FLAG tag sequence consensus, and a LysM-containing cA surface anchor (AcmA), thus allowing cell–surface peptidoglycan anchoring. These plasmids, or their non-FLAG/non-AcmA versions, were introduced into L. lactis host cells, thus generating unique recombinant L. lactis–REX strains. We demonstrate that all three REX proteins are expressed in L. lactis cells and are efficiently displayed on the bacterial surface, as tested by flow cytometry using an anti-FLAG antibody conjugate. Upon 10-fold concentration of the conditioned media, a REX125 secretory variant can be detected by Western blotting. To confirm that the FLAG/non-FLAG REX proteins displayed by L. lactis retain their binding specificity, cell-surface interactions of REX proteins with an IL-23R-IgG chimera were demonstrated by flow cytometry. In addition, statistically significant binding of secreted REX009 and REX115 proteins to bacterially produced, soluble human IL-23R was confirmed by ELISA. We conclude that REX-secreting L. lactis strains were engineered that might serve as IL-23/IL-23R blockers in an experimentally induced mouse model of colitis.

Virion-Associated Cholesterol Regulates the Infection of Human Parainfluenza Virus Type 3.

The matrix (M) proteins of paramyxoviruses bind to the nucleocapsids and cytoplasmic tails of glycoproteins, thus mediating the assembly and budding of virions. We first determined the budding characterization of the HPIV3 Fusion (F) protein to investigate the assembly mechanism of human parainfluenza virus type 3 (HPIV3). Our results show that expression of the HPIV3 F protein alone is sufficient to initiate the release of virus-like particles (VLPs), and the F protein can regulate the VLP-forming ability of the M protein. Furthermore, HPIV3F-Flag, which is a recombinant HPIV3 with a Flag tag at the C-terminus of the F protein, was constructed and recovered. We found that the M, F, and hemagglutinin-neuraminidase (HN) proteins and the viral genome can accumulate in lipid rafts in HPIV3F-Flag-infected cells, and the F protein mainly exists in the form of F1 in VLPs, lipid rafts, and purified virions. Furthermore, the function of cholesterol in the viral envelope and cell membrane was assessed via the elimination of cholesterol by methyl-β-cyclodextrin (MβCD). Our results suggest that the infectivity of HPIV3 was markedly reduced, due to defective internalization ability in the absence of cholesterol. These results reveal that HPIV3 might assemble in the lipid rafts to acquire cholesterol for the envelope of HPIV3, which suggests the that disruption of the cholesterol composition of HPIV3 virions might be a useful method for the design of anti-HPIV3 therapy.

Identification of druggable small molecule antagonists of the Plasmodium falciparum hexose transporter PfHT and assessment of ligand access to the glucose permeation pathway via FLAG-mediated protein engineering.

Although the Plasmodium falciparum hexose transporter PfHT has emerged as a promising target for anti-malarial therapy, previously identified small-molecule inhibitors have lacked promising drug-like structural features necessary for development as clinical therapeutics. Taking advantage of emerging insight into structure/function relationships in homologous facilitative hexose transporters and our novel high throughput screening platform, we investigated the ability of compounds satisfying Lipinksi rules for drug likeness to directly interact and inhibit PfHT. The Maybridge HitFinder chemical library was interrogated by searching for compounds that reduce intracellular glucose by >40% at 10 μM. Testing of initial hits via measurement of 2-deoxyglucose (2-DG) uptake in PfHT over-expressing cell lines identified 6 structurally unique glucose transport inhibitors. WU-1 (3-(2,6-dichlorophenyl)-5-methyl-N-[2-(4-methylbenzenesulfonyl)ethyl]-1,2-oxazole-4-carboxamide) blocked 2-DG uptake (IC50 = 5.8 ± 0.6 μM) with minimal effect on the human orthologue class I (GLUTs 1–4), class II (GLUT8) and class III (GLUT5) facilitative glucose transporters. WU-1 showed comparable potency in blocking 2-DG uptake in freed parasites and inhibiting parasite growth, with an IC50 of 6.1 ± 0.8 μM and EC50 of 5.5 ± 0.6 μM, respectively. WU-1 also directly competed for N-[2-[2-[2-[(N-biotinylcaproylamino)ethoxy)ethoxyl]-4-[2-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl]-1,3-bis(mannopyranosyl-4-yloxy)-2-propylamine (ATB-BMPA) binding and inhibited the transport of D-glucose with an IC50 of 5.9 ± 0.8 μM in liposomes containing purified PfHT. Kinetic analysis revealed that WU-1 acts as a non-competitive inhibitor of zero-trans D-fructose uptake. Decreased potency for WU-1 and the known endofacial ligand cytochalasin B was observed when PfHT was engineered to contain an N-terminal FLAG tag. This modification resulted in a concomitant increase in affinity for 4,6-O-ethylidene-α-D-glucose, an exofacially directed transport antagonist, but did not alter the Km for 2-DG. Taken together, these data are consistent with a model in which WU-1 binds preferentially to the transporter in an inward open conformation and support the feasibility of developing potent and selective PfHT antagonists as a novel class of anti-malarial drugs.

401 Characterization of novel MAPK interactor with potential in therapeutic development

MAPK signaling pathways are integral to cell proliferation and apoptosis (e.g. via ERK), so identifying potential therapeutic targets that can regulate MAPK activation remains an area of active research. We first investigated MAPK interactors through BioID. BirA-tagged MAPK-1 was expressed in three melanoma cell lines (A375, SKMEL28, COLO829) with oncogenic BRAF mutations, under two conditions: with or without drug inhibition of MAPK activation. The biotin MAPK-1 proximal proteins are biotin labeled and then captured by streptavidin pull-down and detected by LC-MS. These proximal interactor proteins, detected by LC-MS, were all previously annotated in MAPK1 pathways with one notable exception—TICRR (treslin). The treslin protein is part of initiating DNA replication, which suggests that elucidating its relationship with MAPK activity could be biologically significant. To identify which treslin residues are possibly modified by MAPK, we used a two-pronged approach and, in live cells, used in vitro kinase and phosphoproteomic assays. For the first, we incubated recombinant treslin with active MAPK for increasing periods of time and detected phosphorylated residues on treslin through mass spectometry. To complement this approach, we also looked at MAPK pathway inhibition and its result on TICRR phosphorylation—A375 cells were exposed to pharmacologic inhibition of ERK, MERK, and RAFi, and the exogenously expressed treslin was captured by a FLAG tag and its phosphorylated residues determined through mass spectrometry. We found several candidate treslin residues that are site of MAPK phosphorylation. Our findings show that treslin’s relationship to MAPK is relatively uncharacterized yet that understanding presents possible biological and ultimately clinical benefits.

SAT-558 Tsh Modulation Of Bone Biology - Further Evidence From A Recombinant Tsh-β Variant.

We have previously shown that TSH has modulatory influences on osteoblasts and osteoclasts and can act as a bone protection ligand. We have also shown that a novel TSH-β variant protein is produced locally by murine and human bone marrow macrophages (Endocrinology 154:4919-26, 2013 and Endocrinology 157:3658-67, 2016). Furthermore, we have also shown by molecular modelling that both TSH-β and TSH-βv can bind to the native TSH receptor. In order to characterize the biological activity of this TSH-β variant we have inserted the gene sequence together with a FLAG tag into the P-secTag2 vector which was then cloned into mammalian HEK-293 cells. Western blot analysis identified 17KD and a 12KD proteins corresponding to the wild type TSH-β and the TSH-βv nucleotide sequence in cell culture lysates and, less so in supernatants, by using a FLAG monoclonal antibody and a TSH-β polyclonal antibody. Protein bands of ~34 KD and 24 KD were observed only in the absence of DTT suggesting dimeric forms of TSH-β and TSH-βv respectively. Detection by Coomassie staining followed by sequence analysis using mass spectrometry confirmed the identity of these proteins after column purification. Furthermore, cyclic AMP responses were generated by both supernatant preparations with an 8-fold increase in luciferase activity with TSH-β and a 4-fold increase with TSH-βv. These data demonstrate the inherent bioactivity of single TSH-β chains which was mirrored by TSH-βv. Local paracrine action by macrophage derived TSH-βv on osteoblasts and osteoclasts is, therefore, most likely and raises the possibility of this variant form having significant osteoprotective activity.

An N-terminal Flag-tag impairs TPP1 regulation of telomerase function

The shelterin protein complex protects natural chromosome ends from being recognized as DNA damage sites and also regulates the synthesis of telomeric repeats by telomerase. TPP1, a shelterin subunit that is essential for telomerase extension of telomeres, has been studied intensively in recent years. Many such studies utilize epitope tagged TPP1, but it is unclear how the tags may affect the multiple cellular functions of TPP1. Here we analyzed the effect of adding a 3x Flag epitope tag to the N- or C-terminus of TPP1. While the position of the tag did not affect TPP1's interaction within the shelterin complex or its localization to telomeres, the N-terminal Flag tag on TPP1 impaired telomerase function, resulting in reduced telomerase processivity in vitro and a failure to stimulate telomere elongation in vivo. The C-terminally Flag-tagged TPP1, in contrast, behaved similarly to untagged TPP1 in all functional aspects examined. These findings suggest that caution is required when utilizing epitope tagged TPP1 to study its regulation of telomerase function.

Conditional Synaptic Vesicle Markers for Drosophila.

The release of neurotransmitters from synaptic vesicles (SVs) at pre-synaptic release sites is the principle means by which information transfer between neurons occurs. Knowledge of the location of SVs within a neuron can thus provide valuable clues about the location of neurotransmitter release within a neuron and the downstream neurons to which a given neuron is connected, important information for understanding how neural circuits generate behavior. Here the development and characterization of four conditional tagged SV markers for Drosophila melanogaster is presented. This characterization includes evaluation of conditionality, specificity for SV localization, and sensitivity of detection in diverse neuron subtypes. These four SV markers are genome-edited variants of the synaptic vesicle-specific protein Rab3. They depend on either the B2 or FLP recombinases for conditionality, and incorporate GFP or mCherry fluorescent proteins, or FLAG or HA epitope tags, for detection.

Establishing Cell Lines Overexpressing DR3 to Assess the Apoptotic Response to Anti-mitotic Therapeutics.

Studying the function of a gene of interest can be achieved by manipulating its level of expression, such as decreasing its expression with knockdown cell lines or increasing its expression with overexpression cell lines. Transient and stable transfection are two methods that are often used for exogenous gene expression. Transient transfection is only useful for short-term expression, whereas stable transfection allows exogenous genes to be integrated into the host cell genome where it will be continuously expressed. As a result, stable transfection is usually employed for research into long-term genetic regulation. Here we describe a simple protocol to generate a stable cell line overexpressing tagged death receptor 3 (DR3) to explore DR3 function. We picked single clones after a retroviral infection in order to maintain the homogeneity and purity of the stable cell lines. The stable cell lines generated using this protocol render DR3-deficient HT29 cells sensitive to antimitotic drugs, thus reconstituting the apoptotic response in HT29 cells. Moreover, the FLAG tag on DR3 compensates for the unavailability of good DR3 antibody and facilitates the biochemical study of the molecular mechanism by which antimitotic agents induce apoptosis.

CRISPR/Cas9-Mediated Genetic Engineering of Hybridomas for Creation of Antibodies that Allow for Site-Specific Conjugation.

Covalent conjugation of chemical moieties to antibodies has numerous applications, including antibody-drug conjugates, antibody conjugation for diagnostics, and more. Most nonspecific chemical conjugation methods ligate onto any of a number of sites on the antibody, leading to multiple conjugated species, many of which perturb antibody function. To solve these problems, we used CRISPR/Cas9-edited hybridomas to introduce a Sortase tag (LPXTG) and a Flag tag at the 3' end of the CH3 heavy chain region of a mouse monoclonal antibody. The Flag tag allows easy purification of the antibody, while the LPXTG is then acted on by the bacterial transpeptidase Sortase to site-specifically add on any of a number of chemical moieties that possess a triglycine repeat. This technique thus allows rapid production of an antibody onto which a wide array of chemical moieties can be site-specifically conjugated.

CRISPR/Cas9-Mediated Gene Tagging: A Step-by-Step Protocol.

CRISPR/Cas9 provides a simple and powerful tool for modifying almost any DNA of interest. One promising application of the CRISPR/Cas9 system is for tagging genes with a fluorescence marker or tag peptides. For such a purpose, FLAG, HIS, and HA tags or fluorescence proteins (EGFP, BFP, RFP, etc.) have been broadly used to tag endogenous genes of interest. The advantages of generating fluorescence tagging proteins are to provide easy tracing of the subcellular locations, real-time monitoring the expression and dynamics of the protein in different conditions, which cannot be achieved using traditional immunostaining or biochemistry assays. However, the generation of such a gene-tagged cell line could be technically challenging. In this chapter, we demonstrate the generation of tagging the porcine GAPDH (pGAPDH) gene GFP by CRISPR/Cas9-based homology-directed repair.

HnRNP U and DDX47 Are Novel FANCD2 Interactors That May Help to Resolve R-Loops during Mild Replication Stress

Background: Fanconi anemia proteins, encoded by at least 22genes (FANCA-W), constitute the Interstrand Cross Link (ICL) repair pathway. While FANCD2 is a master regulator of ICL repair, it accumulates at common fragile sites (CFS) during mild replication stress stimulated by low-dose Aphidicolin (APH) treatment. A recent study indicated that FANCD2 is required for efficient genome replication across the CFS regions. FANCD2 is also implicated in the regulation of R-loops levels. R-loops, which consist of DNA: RNA hybrids and displaced single-stranded DNA, are physiologically relevant in the genome and associate with immunoglobulin class switching, replication of mitochondrial DNA as well as transcriptional promoters or terminators. However, in any case, untimely formation of R-loops is a major threat to genome instability. Furthermore, it has been reported that R-loops which are induced by common slicing factor mutations in cases with myelodysplastic syndrome are linked to compromised proliferation of hematopoietic progenitors. It is also interesting to note that a recent study shows an interaction of FANCD2 with splicing factor 3B1 (SF3B1) and proposes their role in organizing chromatin domains to ensure coordination of replication and co-transcriptional processes.Methods: To examine the genome-wide distribution of FANCD2 protein, we set out to create a derivative of human osteosarcoma cell line, U2OS, which incorporated a 3×FLAG tag into the FANCD2 termination codon by genome editing. We performed chromatin-immunoprecipitation and sequencing (ChIP-Seq) analysis, and provide a genome-wide landscape of replication stress response involving FANCD2 in this cell line. Moreover, we purified the FANCD2 complex and analyzed by liquid chromatography-tandem mass spectrometry, and confirmed this interaction by co-immunoprecipitation (Co-IP) and proximal ligation assay (PLA) with FANCD2-3xFLAG. R-loops levels were assayed as the number of S9.6 (anti DNA:RNA hybrid antibody) stained foci per nucleus.Results: FANCD2 accumulation mostly occurs in the central portion of large transcribed genes, including CFS, and its accumulation appeared to be dependent on R-loop formation induced by transcription-replication collisions during mild replication stress. Moreover, our mass spectrometry analysis identified that FANCD2 interacts with several RNA processing factors including heterogeneous nucleoprotein U (hnRNP U), or DEAD box protein 47 (DDX47). We confirmed the interaction of these factors with FANCD2 by Co-IP as well as PLA. It was previously reported that defects in RNA-processing factors result in R-loop accumulation associated genome instability. Indeed, we found that treatment with siRNA against hnRNP U or DDX47 resulted in the increased number of the S9.6 foci. Furthermore, FANCD2 and hnRNP U or DDX47 appeared to function in an epistatic manner in suppressing APH-induced transcription-replication collisions as detected by PLA between PCNA and RNA polymerase II.Conclusion: We suggest that FANCD2 protects genome stability by recruiting RNA processing enzymes, including hnRNP U or DDX47, to resolve or prevent accumulation of R-loops induced by transcription-replication collisions during mild replication stress. Thus, our study may provide a novel insight to understand the mechanism of bone marrow failure and leukemogenesis in Fanconi anemia patients. DisclosuresTakaori-Kondo:Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria; Janssen Pharmaceuticals: Honoraria.

BAR-Bodies: B-Cell Receptor Antigens As the Targeting Moiety of Antibodies in Substitution for the Variable Region of Heavy and Light Chains

BackgroundChronic antigenic stimulation of the B-cell receptor (BCR) seems to play a critical role in the pathogenesis of B-cell lymphomas. We recently identified ARS2 and LRPAP1 as the autoantigenic targets of the B-cell receptors of approximately 25% of diffuse large B cell lymphomas (DLBCLs) of the ABC type and 45% of mantle cell lymphomas (MCLs), respectively. These BCR antigens can be used to target lymphoma cells in an approach we designated as BAR (B-cell receptor antigens for reverse targeting). The optimal therapeutic format BARs can be integrated in has yet to be found. Since the most established approach to deliver therapeutic payloads to specific targets are antibodies which have well-defined pharmacokinetics, we constructed and tested an antibody like construct (BAR-body) incorporating the DLBCL-BAR ARS2 in substitution for the variable domains of the heavy and light chains.Material and methodsTo create the ARS2 BAR-body, we exchanged the heavy and light chain variable region sequences of an IgG1 antibody with a sequence of similar length (approximately 120 amino acids) of the ARS2 protein (aa 343 - 466) containing the DLBCL reactive epitope (aa 343 - 375). The construct was assembled in a pCR2.1 vector, then transferred to a pSfi FLAG Tag vector for fusion with the FLAG tag and transfected into HEK293 cells for production. Purification of the BAR-body was performed via anti-FLAG antibody affinity chromatography. The BAR-body was detected by western blot analysis and binding capacity to the ARS2-reactive lymphoma cell lines U2932 and OCI-Ly3 and the not ARS2-reactive control DLBCL cell line TMD8 was assessed by flow cytometry. ARS2 BAR-body induced cytotoxicity of lymphoma cells with an ARS2 reactive BCR was measured by LDH release assays with human PBMCs as effector cells at an E:T ratio of 10:1.ResultsWe cloned, expressed and characterized an ARS2 containing BAR-body incorporating 4 molecules of the lymphoma-reactive epitope of ARS2 resulting in an antibody like construct using a BAR (ARS2) as binding moiety instead of normal variable regions. The ARS2 BAR-body could successfully be cloned and expressed as confirmed by western blot analysis, which showed the construct at approximately 150 kD as was to be expected. The BAR-body bound specifically to the ARS2-reactive lymphoma cell lines U2932 and OCI-Ly3 and did not bind to the DLBCL cell line TMD8, which has a B-cell receptor of different specificity or to lymphoma cell lines of different entities. In LDH release assays with 5 x 104 PBMCs and 5 x 103 lymphoma cells (E:T ratio of 10:1) the ARS2 BAR-body induced PBMC mediated specific lysis of the ARS2 reactive lymphoma cell lines U2932 and OCI-Ly3 but not the control DLBCL cell line TMD8 starting at a concentration of 0,1µg/ml. Cytotoxic effects were dose dependent, reached a maximum of 50% specific lysis at a concentration of 1µg/ml and did not increase at concentrations of 10µg/ml.ConclusionHere, we show that BARs can substitute for the variable domains as binding moiety in antibody like constructs to target the BCR of B-cell lymphomas. Because approaches using their specific cognate antigen for targeting the malignant B cells have an exclusive specificity for the BCR of the malignant clone, they can be expected to be less toxic than the currently available antibody derived therapies targeting B-cells, because they leave normal B-lymphocytes unaffected. By incorporating BARs into the well-known format of an antibody we hope to capitalize on years of experience with this therapeutic format from conducting and interpreting in vivo experiments to the translation of the BAR approach into the clinic. DisclosuresStilgenbauer:Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

A novel in-capillary assay for dynamically monitoring fast binding between antibody and peptides using CE.

Nowadays, despite numerous techniques available for the detection of antibody-peptide binding, sensitivity and detection limit inflow still remains a major challenge. Herein, we report a strategy for the detection of binding inflow of monoclonal anti-FLAG M2 antibody and 5,6-carboxyfluorescein-labeled FLAG tag in capillary. Antibody and peptides were sequentially injected into the capillary where they mixed and bound together. Capillary electrophoresis with fluorescence detection was employed to monitor the binding process, and the results showed that the efficacy of the antibody-peptide binding in capillary (in-capillary assay) is affected by the stoichiometry. Compared to the out-capillary assay, this novel assay showed different KD values and required a very short detection time, thus showing great potential for rapid detection as well as other applications. Additionally, our novel assay can be used to investigate the stability of the antibody-peptide complex. The addition of excess DYKDDDDK or TAMRA-DYKDDDDK, with similar binding priorities with M2, can leads to dissociation of the complex with a two-step mechanism including dissociation and association. We believed that our developed method can be extended to monitor wide range of other biomolecule interactions.

Abstract 17079: MYBPC3 Truncation Mutations Cause Contractile Dysregulation in iPSC-Derived Cardiomyocytes

The mechanism of MYBPC3 (encoding cardiac myosin binding protein C, MyBP-C) truncation mutations, the most common genetic cause of hypertrophic cardiomyopathy, has been incompletely resolved. We hypothesized that truncating MYBPC3 mutations cause myofibrillar protein assembly defects and/or contractile dysfunction. Methods and Results: Control iPSCs were CRISPR/Cas9-edited to create cell lines with homozygous (homCT) and heterozygous (hetCT) C-terminal MYBPC3 truncation mutations and heterozygous MYBPC3 promoter knock-out (hetPROM). HetCT was further edited to add an N-terminal flag tag (FhetCT). Purified cardiomyocytes were assayed at day ~25. RNAseq showed a 50% reduction in MYBPC3 mRNA in hetCT (p<0.01). Full ablation of MyBP-C was demonstrated in homCT but MyBP-C content was not reduced in hetCT or hetPROM by quantitative mass spectrometry. Immunofluorescence and co-IP of FhetCT showed no truncated MyBP-C. Sarcomere assembly, quantified by aligned myofilament number after replating onto 7:1 rectangular micropatterns, did not differ between lines; homCT (23±3), hetCT (23±3), hetPROM (22±3 myofilaments), control (22±3), p=NS. Quantitative mass spectrometry demonstrated that the stoichiometry of other major thick and thin filament proteins was not altered (all p=NS). Maximum force was reduced in homCT (866±366 uN, p<0.001) and hetCT (1213±545 uN, p=0.03) vs controls (1509±441 uN), and not significantly different for hetPROM (1238±360 uN, p=0.07), when measured in single micropatterned iPSC-CMs on 8.7 kPa hydrogels by traction force microscopy. Time to peak contraction was shorter in homCT (0.27 s, p=0.02) vs control (0.42 sec), but not in hetCT (0.46 s) and hetPROM (0.49 s). Contractile deceleration time was reduced only in homCT (0.12 s vs 0.25 s, p=0.002). Conclusions: Heterozygous MYBPC3 truncation mutations result in haploinsufficent mRNA with steady state compensation of MYBP3 protein and no MYBPC3 truncated peptide. Even complete ablation of MyBP-C loss does not alter the overall stoichiometry of other sarcomeric thick and thin filament proteins. MyBP-C ablation and heterozygous truncating mutations impair maximal contractile force, strongly implicating contractile dysregulation as the primary mechanism of MYBPC3-HCM.

A method using angiotensin converting enzyme immobilized on magnetic beads for inhibitor screening

Angiotensin converting enzyme (ACE), fusing with FLAG tag, was overexpressed in human embryonic kidney 293T cells. This recombinant FLAG-tagged ACE was immobilized on anti-FLAG antibody coated magnetic beads by affinity method in crude cell lysate for the first time. The enzyme-immobilized magnetic beads (ACE-MB), without further cleavage procedure, were used directly to establish a cost-effective and reliable method for screening ACE inhibitors by coupling with fluorescence detection. The enzymatic activity of the ACE-MB was validated based on its Michaelian kinetic behavior towards hippuryl-histidyl-leucine by UHPLC-MS/MS method firstly. Then, several conditions were optimized including amount of magnetic beads, incubation temperature and time in the procedure of ACE immobilization and amount of ACE-MB in the microplate operation. Moreover, this screening assay was validated with Z′ factors between 0.71 and 0.81 using four known ACE inhibitors (captopril, lisinopril, fosinopril and fosinoprilat). The developed method was applied for the screening of ACE inhibitors from a small compound library of 45 natural products. As a result, epiberberine and fangchinoline with certain ACE inhibitory activities were screened out in the assay and validated. The results demonstrate the usefulness of this screening method using ACE immobilized on magnetic beads and the advantage of great efficiency with respect to both time and reagents for screening ACE inhibitors.