anti-FLAG Antibody Research Articles

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.

Efficient Screening of Combinatorial Peptide Libraries by Spatially Ordered Beads Immobilized on Conventional Glass Slides.

Screening of one-bead-one-compound (OBOC) libraries is a proven procedure for the identification of protein-binding ligands. The demand for binders with high affinity and specificity towards various targets has surged in the biomedical and pharmaceutical field in recent years. The traditional peptide screening involves tedious steps such as affinity selection, bead picking, sequencing, and characterization. Herein, we present a high-throughput “all-on-one chip” system to avoid slow and technically complex bead picking steps. On a traditional glass slide provided with an electrically conductive tape, beads of a combinatorial peptide library are aligned and immobilized by application of a precision sieve. Subsequently, the chip is incubated with a fluorophore-labeled target protein. In a fluorescence scan followed by matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry, high-affinity binders are directly and unambiguously sequenced with high accuracy without picking of the positive beads. The use of an optimized ladder sequencing approach improved the accuracy of the de-novo sequencing step to nearly 100%. The new technique was validated by employing a FLAG-based model system, identifying new peptide binders for the monoclonal M2 anti-FLAG antibody, and was finally utilized to search for IgG-binding peptides. In the present format, more than 30,000 beads can be screened on one slide.

Large-Scale, Quantitative Protein Assays on a High-Throughput DNA Sequencing Chip

High-throughput DNA sequencing techniques haveenabled diverse approaches for linking DNA sequence to biochemical function. In contrast, assays of protein function have substantial limitations in terms of throughput, automation, and widespread availability. We have adapted an Illumina high-throughput sequencing chip to display an immense diversity of ribosomally translated proteins and peptides and then carried out fluorescence-based functional assays directly on this flow cell, demonstrating that a single, widely available high-throughput platform can perform both sequencing-by-synthesis and protein assays. We quantified the binding of the M2 anti-FLAG antibody to a library of 1.3× 104 variant FLAG peptides, exploring non-additive effects of combinations of mutations and discovering a "superFLAG" epitope variant. We also measured the enzymatic activity of 1.56× 105 molecular variants of full-length human O6-alkylguanine-DNA alkyltransferase (SNAP-tag). This comprehensive corpus of catalytic rates revealed amino acid interactionnetworks and cooperativity, linked positive cooperativity to structural proximity, and revealed ubiquitous positively cooperative interactions with histidine residues.

Tag-Specific Affinity Purification of Recombinant Proteins by Using Molecularly Imprinted Polymers.

Epitope tagging is widely used to fuse a known epitope to proteins for which no affinity receptor is available by using recombinant DNA technology. One example is FLAG epitope (DYKDDDDK), which provides better purity and recoveries than the favorite poly histidine tag. However, purification requires using anti-FLAG antibody resins, the high cost and nonreusability of which restrict widespread use. One cost-effective solution is provided by the use of bioinspired anti-FLAG molecularly imprinted polymers (MIPs). This work describes the development of MIPs, based on the epitope approach, synthesized from the tetrapeptide DYKD as template that affords purification of FLAG-derived recombinant proteins. Polymer was optimized by using a combinatorial approach to select the functional monomer(s) and cross-linker(s), resulting in the best specific affinity toward FLAG and the peptide DYKD. The imprinted resin obtained was used to purify mCherry proteins tagged with either FLAG or DYKD epitopes from crude cell lysates. Both mCherry variants were highly efficiently purified ( R ≥ 95%, RSD ≤ 15%, n = 3) and impurities were removed. Unlike existing antibody-based resins, the proposed tag-imprinting strategy provides a general method for meeting the growing demand for efficient, inexpensive, and versatile materials for tagged proteins purification.

Tandem Immunoaffinity Purification Using Anti-FLAG and Anti-HA Antibodies.

The immunoaffinity purification of target proteins followed by the identification and characterization of associated proteins by mass spectrometry is a widely used technique. An immunoaffinity purification bears resemblance to a standard immunoprecipitation; however, the end product for mass spectrometric analysis in the femtomole (10-15) to attomole (10-18) range is required to be of exceptional purity. This high degree of sensitivity in detection renders it of extreme importance to eliminate most if not all of the nonspecific background proteins and can be achieved by performing a tandem affinity purification (TAP). In TAP, the cDNA of the target protein is engineered to contain at least two different epitope tags, and the target protein is extracted under nondenaturing conditions upon expression using an appropriate protein expression platform (CHO cells, HEK 293 cells, or yeast). The expressed protein is initially immunoprecipitated using an antibody against one epitope tag and is eluted in the presence of excess peptide by competition for antibody-binding sites, before being reimmunoprecipitated using an antibody that specifically recognizes the second epitope. These sequential immunoprecipitations significantly reduce the presence of associated nonspecific proteins. Numerous combinations of epitope tags have been applied for tandem affinity purification, and this protocol illustrates the use of tandem hemagglutinin (HA) and FLAG epitope tags. The first immunoprecipitation uses an anti-FLAG antibody followed by the elution in the presence of a competing FLAG peptide before the reimmunoprecipitation of the protein using an anti-HA antibody. Numerous high-quality antiepitope tag antibodies are commercially available from different antibody manufacturers.

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.

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.

Electrolysis Pumping on Centrifugal Microfluidic Platforms for Automation of Bioanalytical Assays

One of the advantages of Lab on a Disc (LoD), compared to Lab on a Chip (LoC), is the use of centrifugal forces to move liquids within the chambers and microchannels; eliminating the need of external mechanical pumps (e.g., syringe pump) and piping networks for the automatization of bioanalytical assays. However, the centrifugal force is unidirectional and therefore liquids can only be displaced from the center to the edge of the disc. This prevents the possibility of utilizing all the available space in the disc for the implementation of a complex microfluidic process. To overcome this limitation, we developed an electrolysis system to induce a pneumatic force to move liquids towards the center of a spinning disc or against the direction of centrifugal force. We developed a microfluidic disc that contains a series of electrolysis pumps and peptide microarrays to automate a multiplexed immunoassay for the detection of different antibodies e.g., Ha and anti-flag antibodies. The pumps on this centrifugal microfluidic platform are powered and controlled wirelessly using the electronics-LoD [1]. In general, the develop pumping system allows for the best use of the limited space of microfluidic disc and the automation of complex bioanalytical process for point of care applications. [1] S.M.T. Delgado, D.J. Kinahan, F.S. Sandoval, L.A.N. Julius, N.A. Kilcawley, J. Ducrée, D. Mager, Fully automated chemiluminescence detection using an electrified-Lab-on-a-Disc (eLoaD) platform, Lab Chip. 16 (2016) 4002–4011. doi:10.1039/C6LC00973E.

Planar cell polarity gene Fuz triggers apoptosis in neurodegenerative disease models.

Planar cell polarity (PCP) describes a cell-cell communication process through which individual cells coordinate and align within the plane of a tissue. In this study, we show that overexpression of Fuz, a PCP gene, triggers neuronal apoptosis via the dishevelled/Rac1 GTPase/MEKK1/JNK/caspase signalling axis. Consistent with this finding, endogenous Fuz expression is upregulated in models of polyglutamine (polyQ) diseases and in fibroblasts from spinocerebellar ataxia type 3 (SCA3) patients. The disruption of this upregulation mitigates polyQ-induced neurodegeneration in Drosophila We show that the transcriptional regulator Yin Yang 1 (YY1) associates with the Fuz promoter. Overexpression of YY1 promotes the hypermethylation of Fuz promoter, causing transcriptional repression of Fuz Remarkably, YY1 protein is recruited to ATXN3-Q84 aggregates, which reduces the level of functional, soluble YY1, resulting in Fuz transcriptional derepression and induction of neuronal apoptosis. Furthermore, Fuz transcript level is elevated in amyloid beta-peptide, Tau and α-synuclein models, implicating its potential involvement in other neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. Taken together, this study unveils a generic Fuz-mediated apoptotic cell death pathway in neurodegenerative disorders.

Insertion site of FLAG on foot-and-mouth disease virus VP1 G-H loop affects immunogenicity of FLAG

The G-H loop of the foot-and-mouth disease virus (FMDV) virion contains certain dominant immunogenic epitopes, as well as an arginine-glycine-aspartic acid (RGD) motif that is recognized by cell surface integrin receptors. Previous experiments indicate that it is critical to maintain virus structural integrity when inserting an exogenous epitope into the surface of an FMDV structural protein. However, it remains to be determined how factors such as different insertion positions affect interactions among the virus, cells and host immune system. In this study, one infectious cDNA clone of the swine FMDV Cathay topotype strain O/CHA/90 was constructed. Then, a FLAG marker (DYKDDDDK) was inserted upstream (–4) or downstream (+10) of the RGD motif to generate tagged viruses vFLAG-O/CHA/90 or vO/CHA/90-FLAG, investigating the possibility of expressing foreign antigen and effect on its immunogenicity. Compared to the parental virus, both tagged viruses exhibited similar plaque phenotypes, suckling mouse pathogenicity and antigenicity. Additionally, the FLAG tag insertion position did not change the use of integrin-mediated cell entry by the tagged viruses. Interestingly, both tagged vaccines protected pigs against challenge with the parental virus O/CHA/90 and induced immune responses against FMDV in BALB/c mice and pigs, but only vaccination with vFLAG-O/CHA/90 generated anti-FLAG antibodies. Our findings demonstrated that two sites (RGD–4 and RGD+10) tolerated the insertion of an exogenous gene in the swine FMDV O/CHA/90 strain. However, only RGD–4 was a novel and appropriate inserting site which could tolerate exogenous FLAG. The resultant tagged virus is a promising candidate for FMD vaccine which can be differentiating infected from vaccinated animals (DIVA).

Identification of Novel Transcriptional Regulators of Uncoupling Protein 1 Using a Modified CRISPR-Cas9 Methodology

Obesity is an epidemiological dilemma due to its associated comorbidities such as diabetes, cancer, and heart disease. Because traditional weight loss strategies such as diet and exercise are thus far ineffective at curtailing the growing prevalence of obesity, new therapeutic approaches are being sought after. In this regard, brown adipose tissue (BAT) has garnered attention due to its ability to burn fat as fuel during thermogenesis, which is carried out by the mitochondrial uncoupling protein 1 (UCP1). Since UCP1 is mainly regulated at the level of transcription, our laboratory sought to identify novel transcriptional regulators of UCP1 using a modified CRISPR-Cas9 methodology termed engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) (Fujii and Fujita, 2015). enChIP takes advantage of dCas9, which lacks endonuclease activity while maintaining functional DNA binding activity, fused to a 3xFLAG tag that can be used for immunoprecipitation. For our study, we delivered five guide RNAs (gRNAs) targeting the Ucp1 promoter region in a mouse-derived mature brown adipocyte line that had been modified to endogenously express the dCas9-3XFLAG fusion protein. Following treatment with either vehicle or CL316,243, we immunoprecipitated the resulting Ucp1/dCas9/3XFLAG complex using an anti-FLAG antibody. We then utilized tandem liquid chromatography-mass spectrometry for an unbiased proteomic screen to evaluate proteins enriched by β3-adrenergic stimulation. Three biological replicates revealed 17 enriched proteins in mature adipocytes following β-adrenergic stimulation. These included histone variants and proteins involved in transcriptional regulation, such as H2A.Z and CRIP2, respectively. These identified proteins have the potential to be manipulated to regulate the expression of UCP1, thereby affecting the thermogenic function of BAT and systemic metabolism. Disclosure J. Darcy: None. C. Wang: None. M. Lundh: None. F. Shamsi: None. M. Lynes: None. B. Emanuelli: None. Y. Tseng: Other Relationship; Self; Chugai Pharmaceutical Co., Ltd.. Research Support; Self; MedImmune.

Repeat-Associated Non-ATG (RAN) Translation in Fuchs' Endothelial Corneal Dystrophy.

PurposeThe strongest genetic association with Fuchs' endothelial corneal dystrophy (FECD) is the presence of an intronic (CTG·CAG)n trinucleotide repeat (TNR) expansion in the transcription factor 4 (TCF4) gene. Repeat-associated non-ATG (RAN) translation, an unconventional protein translation mechanism that does not require an initiating ATG, has been described in many TNR expansion diseases, including myotonic dystrophy type 1 (DM1). Given the similarities between DM1 and FECD, we wished to determine whether RAN translation occurs in FECD.MethodsAntibodies against peptides in the C-terminus of putative RAN translation products from TCF4 were raised and validated by Western blotting and immunofluorescence (IF). CTG·CAG repeats of various lengths in the context of the TCF4 gene were cloned in frame with a 3× FLAG tag and transfected in human cells. IF with antipeptide and anti-FLAG antibodies, as well as cytotoxicity and cell proliferation assays, were performed in these transfected cells. Corneal endothelium derived from patients with FECD was probed with validated antibodies by IF.ResultsCTG·CAG repeats in the context of the TCF4 gene are transcribed and translated via non-ATG initiation in transfected cells and confer toxicity to an immortalized corneal endothelial cell line. An antipeptide antibody raised against the C-terminus of the TCF4 poly-cysteine frame recognized RAN translation products by IF in cells transfected with CTG·CAG repeats and in FECD corneal endothelium.ConclusionsExpanded CTG·CAG repeats in the context of the third intron of TCF4 are transcribed and translated via non-ATG initiation, providing evidence for RAN translation in corneal endothelium of patients with FECD.

ATF6 ubiquitylation is required for its transcriptional activity and degradation

IntroductionATF6α is an endoplasmic reticulum (ER) transmembrane protein that senses the accumulation of misfolded proteins in the ER of cells during ER stress. Upon ER stress, ATF6α is proteolytically cleaved. The resulting N‐terminal fragment becomes an active transcription factor, i.e. N‐ATF6α, which induces genes that adaptively reprogram ER protein folding. N‐ATF6α exerts potent but transient transcriptional activation of its target genes; the transient nature of its activity is due in part to its rapid degradation upon engagement in transcription. The transcriptional activation domain (TAD) and degradation domain (degron) of N‐ATF6α map to the same N‐terminal region of N‐ATF6α, implying that transcriptional activity and degradation are linked. Moreover, mutations introduced into the TAD of N‐ATF6α that decrease its activity also decrease its degradation, indicating that N‐ATF6α degradation is coupled with its transcriptional activity. To elucidate how and why N‐ATF6α degradation and activation are coupled, we examined the mechanism of N‐ATF6α degradation, positing that ubiquitylation may target it for proteasome‐mediated degradation. Accordingly, this study focused primarily on delineating whether N‐ATF6α is ubiquitylated, and if so, what domain(s) of N‐ATF6α are ubiquitylated, as well as investigating the functional consequences of such ubiquitylation.MethodsHeLa cells were co‐transfected with DNA constructs encoding FLAG‐tagged N‐ATF6α and with constructs encoding HA‐ubiquitin. To examine ubiquitylation, FLAG‐N‐ATF6α was FLAG immunoprecipitated (IP'd), then subjected to SDS‐PAGE followed by immunoblotting (IB) with anti‐HA or anti‐FLAG antibody. Specific ubiquitylation, i.e. the level of ubiquitylation per quantity of ATF6, was defined as the ratio of HA/FLAG. A series of mutations was introduced into N‐ATF6α to map the ubiquitylation domain(s) on N‐ATF6α and to assess the functional effects of the mutations that exhibited altered ubiquitylation. Transfected N‐ATF6α function was determined by assessing its ability to induce canonical ATF6 target genes by IB.ResultsIn the absence of ER stress, wild type (WT) N‐ATF6α was ubiquitylated in a proteasome‐dependent manner in HeLa cells. A series of mutations in the N‐terminus of N‐ATF6α that progressively decreased its ability to induce ATF6 target genes coordinately decreased its specific ubiquitylation, as well as its degradation. Using this mutation approach, the ubiquitylation domain of N‐ATF6α was mapped to the N‐terminal 39 amino acids. To determine whether engagement in transcription affected ATF6 ubiquitylation, the effect of mutating the DNA binding domain (DBD) of WT N‐ATF6α was examined. Interestingly, the DBD mutation disengaged ATF6 from DNA, which led to a loss of its transcriptional activation, as expected, and to a dramatic increased its specific ubiquitylation.ConclusionN‐ATF6α is ubiquitylated in a domain within the first 39 amino acids of the N‐terminus. This ubiquitylation is required for full ATF6 activity as a transcription factor, as well as its degradation. Moreover, deubiquitylation, degradation and transcriptional engagement were shown to be coordinate processes.Support or Funding InformationNational Institutes of Health (NIH) grants R01 HL75573, R01 HL104535, R01 HL127439, and P01 HL085577This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A novel monitoring approach of antibody-peptide binding using “bending” capillary electrophoresis

Recently, the in-capillary electrophoresis assay has been applied to variety kinds of analyses owing to its multiple functional integrating features, including mixing of samples, reaction process of the mixtures, and the separation and detection in one capillary system. However, the micro-reactor still has its limitations to the currently available applications, especially the mixing step of the samples inside the capillary could not be well controlled automatically or manually. Herein, we have developed a novel capillary electrophoresis assay for the detection of antibody-peptide binding inside a bending capillary. Its efficacy was monitored using an anti-FLAG M2 antibody and its ligand conjugated with FAM dye (FAM-DYKD). The antibody and the peptide were mixed inside the bending capillary with sequential injections. It was found that the numbers of semi-circle on the capillary interfered by the antibody and peptide binding dynamic. Additionally, an online competition assay was performed, which further validated the efficacy of the bending capillary device on monitoring the dynamic binding between the antigen and antibody. In summary, our data suggests that the novel assay is a practical approach in monitoring the antibody-antigen complex formation at a nano-scale. It could be applied to detect any biomolecule-biomolecule interaction as a general strategy.

Advancing the immunoaffinity platform AFFIRM to targeted measurements of proteins in serum in the pg/ml range

There is a great need for targeted protein assays with the capacity of sensitive measurements in complex samples such as plasma or serum, not the least for clinical purposes. Proteomics keeps generating hundreds of biomarker candidates that need to be transferred towards true clinical application through targeted verification studies and towards clinically applicable analysis formats. The immunoaffinity assay AFFIRM (AFFInity sRM) combines the sensitivity of recombinant single chain antibodies (scFv) for targeted protein enrichment with a specific mass spectrometry readout through selected reaction monitoring (SRM) in an automated workflow. Here we demonstrate a 100 times improved detection capacity of the assay down to pg/ml range through the use of oriented antibody immobilization to magnetic beads. This was achieved using biotin-tagged scFv coupled to streptavidin coated magnetic beads, or utilizing the FLAG tag for coupling to anti-FLAG antibody coated magnetic beads. An improved multiplexing capacity with an 11-plex setup was also demonstrated compared to a previous 3-plex setup, which is of great importance for the analysis of panels of biomarker targets.

O-GlcNAc on PKCζ Inhibits the FGF4-PKCζ-MEK-ERK1/2 Pathway via Inhibition of PKCζ Phosphorylation in Mouse Embryonic Stem Cells.

SummaryMouse embryonic stem cells (ESCs) differentiate into multiple cell types during organismal development. Fibroblast growth factor 4 (FGF4) signaling induces differentiation from ESCs via the phosphorylation of downstream molecules such as mitogen-activated protein kinase/extracellular signal-related kinase (MEK) and extracellular signal-related kinase 1/2 (ERK1/2). The FGF4-MEK-ERK1/2 pathway is inhibited to maintain ESCs in the undifferentiated state. However, the inhibitory mechanism of the FGF4-MEK-ERK1/2 pathway in ESCs is uncharacterized. O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is a post-translational modification characterized by the attachment of a single N-acetylglucosamine (GlcNAc) to the serine and threonine residues of nuclear or cytoplasmic proteins. Here, we showed that the O-GlcNAc on the phosphorylation site of PKCζ inhibits PKCζ phosphorylation (activation) and, consequently, the FGF4-PKCζ-MEK-ERK1/2 pathway in ESCs. Our results demonstrate the mechanism for the maintenance of the undifferentiated state of ESCs via the inhibition of the FGF4-PKCζ-MEK-ERK1/2 pathway by O-GlcNAcylation on PKCζ.

Nisin-induced expression of recombinant T cell epitopes of major Japanese cedar pollen allergens in Lactococcus lactis.

Japanese cedar pollinosis is a seasonal allergic disease caused by two major pollen allergens: Cry j 1 and Cry j 2 antigens. To develop an oral vaccine to treat pollinosis, we constructed recombinant Lactococcus lactis harboring the gene encoding fused T cell epitopes from the Cry j 1 and Cry j 2 antigens. The recombinant T cell epitope peptide was designed to contain the fused cholera toxin B subunit as an adjuvant and a FLAG tag at the C-terminus. An expression plasmid was constructed by inserting the T cell epitope peptide gene into the multiple cloning sites of plasmid pNZ8148, an Escherichia coli-L. lactis shuttle vector. The constructed plasmid was transformed into L. lactis NZ9000 for expression induced by nisin, an antibacterial peptide from L. lactis. The expression of the epitope peptide was induced with 10-40ng/mL nisin, and the expressed T cell epitope peptide was detected by western blot analysis using an anti-FLAG antibody and an antibody against the T cell epitopes. The concentration of the epitope peptide was estimated to be ~22mg/L of culture in the presence of 40ng/mL nisin, although it varied depending on the nisin concentration, the culture time, and the bacterial concentration when nisin was added. The expression of the recombinant epitope peptide in L. lactis, an organism generally recognized as safe, as demonstrated in this study, may contribute to the development of an oral vaccine for the treatment of pollinosis.

The Effect of SIN1 and Microtubules on Insulin Induced PKC ζ Activation.

BackgroundProtein kinase C zeta (PKC ζ) plays an important role in insulin induced glycometabolism and insulin receptor (IR) associated signaling pathways. The full activation of PKC ζ depends on its translocation from cytosol to membrane and phosphorylation at Thr410. However, the mechanism of PKC ζ activation remains elusive. In this study, the effect of SIN1 and microtubules on insulin-induced PKC ζ activation was investigated.Material/MethodsHepG2 cells were stimulated with insulin for co-immunoprecipitation (co-IP) assay. The immunocomplex was captured by using anti-PKC ζ, anti-SIN1 or anti-FLAG antibodies and was subjected to western blotting analysis for detecting PKC ζ, SIN1, and β-tubulin protein expression level. The cells were intervened by small interfering RNA (siRNA) that targeted exon regions of SIN1. Then the glucose uptake ratio after cells were stimulated by insulin was measured. The PKC ζ insulin receptor levels in the membranes were analyzed. Cells stained with anti-PKC ζ, anti-SIN1 antibodies and probed with molecular probes were observed by immunofluorescence confocal microscopy.ResultsSIN1 interacted and co-located with PKC ζ by pleckstrin homology (PH) domain. Downregulation of SIN1 severely impaired PKC ζ translocation and phosphorylation induced by insulin. PKC ζ co-immunoprecipitated with β-tubulin at different intervals upon insulin stimulus, and the activation of PKC ζ was affected by paclitaxel and nocodazole.ConclusionsPKC ζ translocated from cytosol to membrane depending on SIN1, which suggested that PKC ζ may be activated directly by PI3K and the reaction probably carried out on microtubules in HepG2 cells.

Modified whole-mount in situ hybridisation and immunohistochemistry protocols without removal of the vitelline membrane in the appendicularian Oikopleura dioica.

The appendicularian Oikopleura dioica is a planktonic chordate that retains a tadpole shape throughout its life. Its simple and transparent body, invariant cell lineages, fast development and available genome and transcriptome resources make it a promising model organism for research in developmental biology. However, large-scale analysis of gene expression in O. dioica is limited owing to the laborious and time-consuming process of manual removal of the vitelline membrane, because devitellinisation of pre-hatching embryos causes failure of normal development. Therefore, in this study, modified procedures were developed for whole-mount in situ hybridisation (WISH) and immunohistochemistry (WIHC). This protocol enables rapid mRNA or protein detection without a manual devitellination step for each specimen. The critical procedure is brief treatment of the vitelline membrane of living embryos with 0.05% actinase E before fixation. Two minutes of treatment was optimal for the penetration of antisense RNA probes and antibodies through the vitelline membrane. This WISH protocol was applicable for chromogenic and fluorescent tyramide signal amplification reactions. Using the new protocol, we found eight genes with tissue-specific expression in the tail muscle, trunk epidermis, heart, pharynx, oesophagus, stomach or gill openings of developing larvae. This procedure also allowed for the detection of exogenous FLAG-tagged histone-enhanced green fluorescent protein by WIHC using anti-FLAG antibody. This study provides a useful and convenient tool for studying spatial and temporal gene expression patterns in this simple chordate model and should facilitate handling large amounts of genetic data from transcriptome-based approaches and other techniques such as treatments with chemical inhibitors.