GST Fusion Protein Research Articles

Expression, Purification and Immunological Characterization of Recombinant Shiga Toxin A Subunit.

Shiga toxin family comprises toxins belonging to two major groups, Stx1 and Stx2, produced by the bacteria Shigella dysenteriae and some strains of Escherichia coli. Shiga toxins are the leading cause of diarrhea associated with life threatening hemolytic uremic syndrome (HUS). StxA is a ribosome inactivating protein (RIP) which inhibits the protein synthesis in most species of prokaryotes and eukaryotes. An in vitro expression system has not been reported to produce full-length biological active StxA subunit; hence substantial progress has been hampered. In the present study, a DNA fragment (955 bp Gene Bank Accn No HM017965) encoding for subunit A of Stx was amplified from Shigella dysenteriae type 1 and subsequently cloned in pGEX-5X-2 vector. We successfully produced recombinant StxA as GST fusion protein in Escherichia coli using pGEX-5X-2-STXA construct under IPTG induction. For the purpose of immunization the GST-tag was removed by factor Xa mediated endoproteolytic cleavage from GST-StxA. Antisera raised against rStxA in mice reacted with recombinant purified protein of rStxA and lysate of Shiga toxin. It was shown that antisera produced against rStxA significantly recognized Stx producing strains of S. dysenteriae and E. coli. The antiserum produced effectively neutralized the Shiga toxin's cytotoxicity towards Vero cells.

Abstract 1566: Association of a serum HPV16 IgG signature and risk of oropharyngeal cancer

Abstract Introduction: Human papillomavirus type 16 (HPV16) infection is associated with an increasing fraction of oropharyngeal cancer. The aim of our study was to determine the association of HPV16 antibodies (Abs) and oropharyngeal cancer (OPC) risk in sera obtained prior to clinical diagnosis. Methods: We identified 92 participants with incident oropharyngeal cancers and 460 age-, gender-, region- and year- matched controls from the Janus Serumbank in Norway. Serum samples were collected from cases, on average, 9.2 years before diagnosis (range, -0.6-14.9 years). 11 cases had serum samples from multiple time points. IgG Abs to the HPV16 antigens E1, E4, E5, E6, E7, L1, L2, and the N-terminal and C-terminal fragments of E2 (NE2, CE2) were quantified in blinded fashion using a custom RAPID ELISA assay. Full-length genes were expressed as GST fusion proteins with a mammalian protein expression system, incubated with sera, and bound IgG was measured. The ratio of relative luminescence units (RLU) values for each antigen to control GST protein was determined. Cutoff values were pre-established as 3 SD>median of 247 healthy controls. A logistic regression classifier of a 7-Ab biomarker panel (E1, NE2, CE2, E4, E5, E6, E7) for the predictive diagnosis of OPC was trained on an independent sample set, and applied to these samples. P-values were calculated by Fisher's exact test. Odds ratios (ORs) of cancer and 95% CIs were calculated. Results: HPV16 early antigen seropositivity was present in 13% of patients with OPC and 1% of controls (OR, 17.0; 95% CI, 5.0-74.0, p<0.0001). The probability of seropositivity was inversely associated with the time from serum collection and diagnosis (0-2 years, 84.9; >10 years, 5.4). Abs to CE2 were strongly associated with cases 0-2 years pre-diagnosis (OR, 112.0; 95% CI, 15.0-971.2, p<0.0001). Conclusion: HPV16 Abs to multiple early viral antigens are detectable years prior to diagnosis of oropharyngeal cancer, and the probability of seropositivity increases closer to diagnosis. A model for screening would help identify individuals at risk for whom interventions and surveillance may be evaluated. Citation Format: Karen S. Anderson, Hilde Langseth, Garrick Wallstrom, Marshall Posner, Julia N. Cheng, Rizwan Alam, Diego Chowell, Jon Mork. Association of a serum HPV16 IgG signature and risk of oropharyngeal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1566. doi:10.1158/1538-7445.AM2015-1566

Development of in vitro HSP90 foldase chaperone assay using a GST-fused Real-substrate, ZTL (ZEITLUPE)

Protein folding is one of the essential and fundamental processes involved in all living organisms wherein Heat-Shock Protein 90 (HSP90) served as a chaperone which plays important function in protein folding and further promotes refolding of denatured proteins. There are over six hundred identified substrates of HSP90 at present; however, there is a need to develop a specific folding assay method to test its in vitro foldase activity in case of difficulty in substrate activity measurement in vitro. In previous studies, it has been reported that HSP90 is necessary for the stabilization of ZEITLUPE (ZTL) which plays in the circadian clock and photomorphogenesis in Arabidopsis (Kim et al. 2011). Rhythmic oscillations of ZTL protein might be caused by denaturation and stabilization, thus, refolding activity of HSP90 is possibly involved in the oscillations of ZTL protein. However, the question whether HSP90 indeed promotes refolding of ZTL or not has not yet identified and therefore needs to be investigated. Recombinant glutathione-S transferase (GST) fused-ZTL in bacteria was produced and purified as the soluble GST-ZTL through treatments of both sarkosyl and Triton-X100 as ionic and nonionic detergents, respectively. Upon heat (45°C) treatment, the GST activity of GST-ZTL decreased significantly. After the 3 h heat-induced denaturation of GST-ZTL, the refolding of denatured GST-ZTL was enhanced upon addition of HSP90 in a dose-dependent manner. The results from this study showed that recombinant GST-fused protein can be possibly used for in vitro protein refolding assay method. Moreover, results revealed that Arabidopsis HSP90 can efficiently refolds the denatured GST-ZTL in vitro.

A Simple and Rapid Method for Expression and Purification of Functional TNF-α Using GST Fusion System.

Tumor necrosis factor alpha (TNF-α) is an inflammatory cytokine, involved in both physiological and pathological pathways. Although there have been various attempts to express and purify human TNF-α, the current work introduces a simple, rapid, and efficient method for its production without loss of biological activity. The protein was expressed based on GST-tagged fusion system in Escherichia coli under optimized condition. The expressed GST fusion protein was applied to glutathione affinity column and then, TNF-α was cleaved off the GST using thrombin protease. The purity of the product was more than 95% and further size exclusion chromatography slightly improved the purity. The purified human TNF-α was tested for its biological activity and structural analysis, using MTT assay (EC(50) of 4.1 ×10E-12 M in L929 cell death assay) and circular dichroism spectropolarimetry, respectively. The results showed that the method used in this study enables successful production of highly purified and fully functional TNF-α.

Preparation of human tau exon-2- and -10-specific monoclonal antibodies for the recognition of brain tau proteins in various mammals.

The aggregations of tau protein in brain tissue have been described in a large number of neurodegenerative diseases; however, due to the lack of tau isoform- or exon-specific antibodies, the exact situations under which various brain tau isoforms can be found and their exact contributions during disease progression remain unknown. Therefore, in this study, we prepared tau exon-specific monoclonal antibodies (mAbs) that recognize different mammalian tau isoforms. Briefly, 3 Balb/c mice were separately immunized (3 mice per antigen) with the recombinant GST-fusion proteins, GST-tE2 and GST-tE10. Two hybridoma cell lines, 4A8 and 3E12, secreting antibodies against human tau exon-2 and -10 were established using the hybridoma technique. The sensitivity and specificity of the prepared mAbs were evaluated using indirect ELISA and western blot analysis. The ability of the prepared mAbs, 4A8 and 3E12, to recognize endogenous tau protein in the brain tissues of various mammals was estimated by immunoprecipitation. Based on the results of various verification methods, we found that the prepared mAbs, 4A8 and 3E12, not only specifically reacted with the individual recombinant GST tau exon fusion proteins, but also correctly recognized the recombinant human tau isoforms containing respective exon sequences, as shown by western blot analysis. Furthermore, western blot analysis and immunoprecipitation assays verified that the mAbs, 4A8 and 3E12, recognized endogenous tau proteins in human brain tissue, as well as tau proteins in a series of mammalian tissues, including goat, bovine, rabbit, hamster and mouse. Thus, in the present study, using the hybridoma technique, we successfully prepared the mAbs, 4A8 against tau exon-2 and 3E12 against tau exon-10, which provide useful tools for determining potential alternations of tau isoforms in neurodegenerative diseases.

172 Junctophilin-2 interacts with the cardiac L-Type voltage-gated calcium channel

Background Excitation contraction (E-C) coupling in cardiac muscle is regulated by the L-type voltage-gated calcium channels (LTCC) within the t-tubules, and surface membrane, and the ryanodine receptors (RyR2) localised to the junctional sarcoplasmic reticulum (jSR). The juxtaposition of the t-tubules and jSR forms a microdomain termed a dyad. It is now widely accepted that junctophilin-2 (JP2) acts as a protein bridge to maintain the dyad geometry. JP2 is anchored in the SR, via its C-terminal transmembrane domain, and is thought to span the cytosol and interact with the plasma membrane thereby constraining the dyad architecture to provide the optimal proximal distance between the LTCCs and RyR2s to facilitate effective calcium induced calcium release for cardiac contraction. More recently, data has emerged to suggest that JP2 has an additional role, influencing E-C coupling through an interaction with RyR2. Furthermore, there is evidence for an association between JP2 and the LTCC in skeletal muscle. However, the region of the LTCC involved in binding to JP2 was not established nor was whether there is an interaction between JP2 and the LTCC in the heart. Methods We employed yeast-2-hybrid (Y2H) (Hybrigenics) experiments to investigate JP2 binding partners using human heart cDNA library. Domains of the LTCC were expressed as GST-fusion proteins and JP2 full-length and domains were purified from E.coli . Pull-down experiments were employed to investigate the interaction between the JP2 and the LTCC protein domains. Results Data from Y2H experiments identified the C-terminal region within the ion channel pore of LTCCs as a JP2 binding partner. Therefore, we designed a series of constructs encompassing the C-terminal domain of the Cav1.2 subunit and expressed them as GST-fusion proteins. Employing purified JP2 we showed a direct interaction with the C-terminal tail of Ca v 1.2. Furthermore, we determined that the N-terminal portion of JP2 mediates this interaction. We also investigated whether this interaction is Ca 2+ dependent since we have previously reported that JP2 has a cation binding domain. Our experiments revealed that the JP2-Ca v 1.2 interaction is independent of [Ca 2+ ]. Conclusion This study has provided novel data revealing that not only is there a direct interaction between the cardiac LTCC and JP2 but that the binding domains involve the C-terminal region of Ca v 1.2 and the N-terminal domain of JP2. Significantly, the Ca v 1.2 C-terminal domain binds calmodulin (CaM), both apoCaM and Ca 2+ CaM, regulating both calcium dependent inactivation (CDI) and facilitation (CDF) of the channel. Therefore, our future studies are directed at understanding the significance of JP2 binding, for example whether JP2 influences channel function (CDI/CDF) and/or whether a JP2-Ca v 1.2 is important for LTCC localisation to the dyad.

Actinin-4 Governs Dendritic Spine Dynamics and Promotes Their Remodeling by Metabotropic Glutamate Receptors

Dendritic spines are dynamic, actin-rich protrusions in neurons that undergo remodeling during neuronal development and activity-dependent plasticity within the central nervous system. Although group 1 metabotropic glutamate receptors (mGluRs) are critical for spine remodeling under physiopathological conditions, the molecular components linking receptor activity to structural plasticity remain unknown. Here we identify a Ca(2+)-sensitive actin-binding protein, α-actinin-4, as a novel group 1 mGluR-interacting partner that orchestrates spine dynamics and morphogenesis in primary neurons. Functional silencing of α-actinin-4 abolished spine elongation and turnover stimulated by group 1 mGluRs despite intact surface receptor expression and downstream ERK1/2 signaling. This function of α-actinin-4 in spine dynamics was underscored by gain-of-function phenotypes in untreated neurons. Here α-actinin-4 induced spine head enlargement, a morphological change requiring the C-terminal domain of α-actinin-4 that binds to CaMKII, an interaction we showed to be regulated by group 1 mGluR activation. Our data provide mechanistic insights into spine remodeling by metabotropic signaling and identify α-actinin-4 as a critical effector of structural plasticity within neurons.

Desmocollin-2 alone forms functional desmosomal plaques, with the plaque formation requiring the juxtamembrane region and plakophilins.

The role of the juxtamembrane region of the desmocollin-2 cytoplasmic domain in desmosome formation was investigated by using gene knockout and reconstitution experiments. When a deletion construct of the desmocollin-2 juxtamembrane region was expressed in HaCaT cells, the mutant protein became localized linearly at the cell-cell boundary, suggesting the involvement of this region in desmosomal plaque formation. Then, desmocollin-2 and desmoglein-2 genes of epithelial DLD-1 cells were ablated by using the CRISPR/Cas9 system. The resultant knockout cells did not form desmosomes, but re-expression of desmocollin-2 in the cells formed desmosomal plaques in the absence of desmoglein-2 and the transfectants showed significant cell adhesion activity. Intriguingly, expression of desmocollin-2 lacking its juxtamembrane region did not form the plaques. The results of an immunoprecipitation and GST-fusion protein pull-down assay suggested the binding of plakophilin-2 and -3 to the region. Ablation of plakophilin-2 and -3 genes resulted in disruption of the plaque-like accumulation and linear localization of desmocollin-2 at intercellular contact sites. These results suggest that the juxtamembrane region of desmocollin-2 and plakophilins are involved in the desmosomal plaque formation, possibly through the interaction between this region and plakophilins.

Characterization of the Activity and Biological Function of Human Protein Arginine Methyltransferase 9 (PRMT9)

Protein arginine methyltransferases (PRMTs) make up a family of enzymes that transfer methyl groups onto arginine residues and play important roles in DNA repair, splicing, transcriptional control and signaling. In mammals, a family of nine PRMTs are known. PRMT1,2,3,4,6, and 8 catalyze the formation of asymmetric dimethylarginine (ADMA) and w‐monomethylarginine (MMA), PRMT5 catalyzes symmetric dimethylarginine (SDMA) and MMA formation, and PRMT7 catalyzes only MMA formation. The last member of this family, PRMT9 (also designated PRMT10), has not previously been characterized. In humans, it is encoded on a gene on chromosome 4 in position 4q31. We have now expressed human PRMT9 both as a GST‐fusion protein in bacterial cells and as a GFP‐fusion protein in human HEK293 cells. For both expression systems, we have also prepared catalytic mutants to serve as controls for possible contamination of other types of PRMTs. Using an in vitro methylation assay, we show that both forms of PRMT9 catalyze the methylation of myelin basic protein and a GST‐fusion protein of the N‐terminal domain of human fibrillarin. Studies are in progress to characterize the type of activity and physiological substrates for PRMT9.

Lysophosphatidic acid (LPA) stimulates invasion and metastatic colonization of ovarian cancer cells through Rac activation

To investigate the mechanisms of lysophosphatidic acid (LPA) in stimulating invasion and metastatic colonization of ovarian cancer cells. The metastatic ability in vivo of ovarian cancer SK-OV3, HEY, OVCAR3, and IGROV1 cells was determined in tumor-bearing nude mouse models. Matrigel assay was used to detect the changes of response in vitro of ovarian cancer cells to LPA after Rac(-) or Rac(+) adenovirus treatment. LPA-induced Rho GTPase activation was detected by GST-fusion protein binding assay. The peritoneal metastatic colonization assay showed overt metastatic colonization in mice receiving SK-OV3 and HEY cell inoculation, indicating that they are invasive cells. Metastatic colonization was not detected in animals receiving OVCAR3 and IGROV1 cells, indicating that these cells are non-invasive cells. In the matrigel invasion assay, exposure to LPA led to a notably greater migratory response in metastatic SK-OV3 and HEY cells (Optical density: SK-OV3 cells: 0.594±0.023 vs. 1.697±0.049, P<0.01; HEY cells: 0.804±0.070 vs. 1.851±0.095, P<0.01). But LPA did little in the non-metastatic OVCAR3 and IGROV1 cells (Optical density A: OVCAR3 cells: 0.336±0.017 vs. 0.374±0.007, P>0.05; IGROV1 cells: 0.491±0.036 vs. 0.479±0.061, P>0.05). LPA migratory responses of ovarian cancer cells were closely related to their metastatic colonization capabilities (r = 0.983, P<0.05). Rac(-) blocked the LPA response of invasive SK-OV3 and HEY cells (LPA-induced fold increase of cell migration: SK-OV3 cells: 2.988±0.095 vs. 0.997±0.100,P=0.01; HEY cells: 2.404±0.059 vs. 0.901±0.072, P=0.01). But Rac(+) confered the non-invasive cells with LPA response and invasion capability (LPA-induced fold increase of cell migration: OVCAR3 cells: 1.072±0.080 vs. 1.898±0.078, P<0.01; IGROV1 cells: 1.002±0.044 vs. 2.141±0.057, P<0.05). Among Rho GTPases, only Rac activation was different between ovarian cancer cell lines with different metastatic capability after LPA stimulation: Cdc42 could not be activated in both the invasive and non-invasive cell lines. RhoA could be activated in both the invasive and non-invasive cell lines. Rac could be activated by LPA in the invasive ovarian cancer cell lines. However, Rac could not be activated in the non-invasive cell lines. Lysophosphatidic acid stimulates invasion and metastasis of ovarian cancer cells through Rac activation.

Determination of Rab5 activity in the cell by effector pull-down assay.

Rab5 targets to early endosomes and is a master regulator of early endosome fusion and endocytosis in all eukaryotic cells. Like other GTPases, Rab5 functions as a molecular switch by alternating between GTP-bound and GDP-bound forms, with the former being biologically active via interactions with multiple effector proteins. Thus the Rab5-GTP level in the cell reflects Rab5 activity in promoting endosome fusion and endocytosis and is indicative of cellular endocytic activity. In this chapter, we describe a Rab5 activity assay by using GST fusion proteins with the Rab5 effectors such as Rabaptin-5, Rabenosyn-5, and EEA1 that specifically bind to GTP-bound Rab5. We compare the efficiencies of the three GST fusion proteins in the pull-down of mammalian and fungal Rab5 proteins.

Glutathione-S-transferase (GST)-fusion based assays for studying protein-protein interactions.

Glutathione-S-transferase (GST)-fusion proteins have become an effective reagent to use in the study of protein-protein interactions. GST-fusion proteins can be produced in bacterial and mammalian cells in large quantities and purified rapidly. GST can be coupled to a glutathione matrix, which permits its use as an effective affinity column to study interactions in vitro or to purify protein complexes in cells expressing the GST-fusion protein. Here, we provide a technical description of the utilization of GST-fusion proteins as both a tool to study protein-protein interactions and also as a means to purify interacting proteins.

Study on Binding Affinity of a Glutathione &amp;lt;i&amp;gt;S&amp;lt;/i&amp;gt;-Transferase (GST) Fusion Protein to DNA Probe

The aims of this work were to: i) purify GST-fusion protein from bacterial cell extracts of Escherichia coli; ii) quantify the protein by SDS PAGE and Bradford assay; iii) determine protein-DNA interaction of the purified protein by Electrophoretic Mobility Shift Assay. Bacterial culture prepared by inoculation of a single E. coli colony that had a GST fusion protein (gst: six-X10 hd) constructed by ligation of the six-7-hd (X10) sequence into the BamHI and EcoRI sites of the vector pGEX-2T, grown overnight, was sonicated using Cole-Palmer Ultrasonic Homogenizer. Fusion protein was eluted from the beads with Tris-glutathione buffer (50 mM Tris [pH 8.1], 20 mM glutathione), which contained reduced Glutathione. SDS-PAGE was used to calculate the extracted bound protein. Total protein quantification was then estimated by the Bradford assay. Bovine Serum Albumin (BSA) absorbance values were used to plot the standard curve used to calculate the concentrations of the sample proteins. Nylon membrane was used for the electrophoretic transfer; membrane was cross linked and detected by Pierce’s Chemiluminescent Nucleic Acid Detection module. Results showed that X10 gave a strong band shift observed in Lanes 6 and 7 for both 200 ng and 400 ng elute 1 samples; however, there was no shift in the bands for the wild-type, positive control. The concentration of the elute 1 was obtained by the Bradford assay as 242.52 ng/μl and that of elute 2 was 106.30 ng/μl. Similarly, the result obtained by gel analysis was 300 ng/μl (0.3 μg/μl) and 150 ng/μl (0.15 μg/μl) for elutes 1 and 2 respectively.

RIAM Interacts with the Hematopoietic-Specific Adaptor Protein Gads and Forms a LAT-Independent Node of Signal Integration That Regulates Activation of PLC-γ1

TCR stimulation triggers the activation of protein tyrosine kinases resulting in phosphorylation of the adaptor protein LAT. SLP-76, interacts constitutively with PLC-γ1 and with the SH3 domain of Gads, which via its SH2 domain mediates inducible recruitment of SLP-76 and PLC-γ1 to LAT, upon T cell activation. PLC-γ1 hydrolyzes phosphatidylinositol-4, 5 bisphosphate [PI(4,5)P2], generating inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), second messengers responsible for mediating intracellular calcium release and activation of downstream signals. The adaptor protein RIAM constitutively interacts with PLC-γ1 and is required for PLC-γ1 activation. RIAM is a multidomain protein with a small N-terminus proline-rich region, two coiled-coiled regions, sequential Ras association (RA) and pleckstrin homology (PH) domains, and a large C-terminus proline-rich region, which interacts with PLC-γ1. The RA domain of RIAM has specificity for Rap1-GTP whereas the PH domain binds to the PLC-γ1 substrate PI(4,5)P2. The RA-PH domain region of RIAM functions as a single structural unit and mediates translocation of RIAM to the plasma membrane upon T cell activation. Previously, we determined that RIAM deficiency results in impaired activation of PLC-γ1 in spite of the formation of the PLC-γ1-SLP-76-LAT complex, suggesting perhaps somewhat paradoxically, that PLC-γ1-SLP-76-LAT signalosome is not sufficient to mediate distal signaling in the absence of RIAM. This observation indicated that RIAM mediates its effects at a level distal to SLP-76-LAT or through a signaling pathway parallel but distinct from SLP-76-Gads-LAT. Here we investigated whether RIAM forms a signalosome parallel to PLC-γ1-SLP-76-Gads and whether such pathway might be involved in the activation of PLC-γ1. Using primary T lymphocytes and Jurkat T cells stimulated via TCR/CD3 and CD28 we determined that RIAM constitutively interacted with Gads as determined by immunoprecipitation with RIAM-specific antibody followed by Gads immunoblot. To determine whether the interaction between RIAM and Gads was direct, we employed an in vitro protein association assay. Glutathione S-transferase (GST) and GST-fusion protein of Gads were coupled to glutathione-sepharose and incubated with [35S]methionine-labeled RIAM or luciferase, as negative control. Gads bound to [35S]methionine-labeled RIAM indicating that RIAM interacts directly with Gads. We further examined domain-specific interaction of RIAM with endogenous Gads using GST fusion proteins of RIAM. We determined a constitutive interaction between Gads and GST fusion proteins of full-length RIAM or C-terminus region of RIAM. Although a number of tyrosine phosphorylated proteins were associated with the RIAM-Gads complex upon T cell activation, LAT was not detected among the components of this complex as determined by immunoblot with anti-phosphotyrosine-specific or LAT-specific antibodies. Using a GST fusion protein of the RA-PH domain of RIAM we determined that, surprisingly, Gads displayed activation-dependent interaction with the RA-PH domain, which mediates the recruitment of RIAM to the plasma membrane upon T cell activation. Furthermore, in addition to Gads, SLP-76 and PLC-γ1 were recruited to the RA-PH domain of RIAM in activated T cells. To determine whether RIAM and Gads had a synergistic effect on IL-2 transcription, we performed luciferase-based reporter assays using a reporter construct driven by the entire IL-2 promoter or by NFAT binding sequences. We found that RIAM and Gads had a synergistic effect on IL-2 and on NFAT-mediated transcriptional activation, which depends on PLC-γ1. Thus, via its C-terminus region, RIAM directly and constitutively interacts with Gads. In addition, via its RA-PH domain, RIAM mediates an activation-dependent interaction with Gads and serves as a docking site recruiting the PLC-γ1-SLP-76-Gads complex to the plasma membrane in a LAT-independent manner. These findings indicate a crosstalk between RIAM and SLP-76 in the activation of PLC-γ1 and reveal a previously unidentified, alternative signaling pathway leading to Gads-SLP-76 recruitment to the plasma membrane of activated T cells in a LAT-independent manner. DisclosuresNo relevant conflicts of interest to declare.

Looking for a needle in a haystack: Cellular proteins that may interact with the tyrosine-based sorting signal of the TGEV S protein

The spike protein S of transmissible gastroenteritis virus, an Alphacoronavirus, contains a tyrosine-based sorting signal that is responsible for ERGIC retention and may be important for a correct viral assembly process. To find out whether the S protein interacts with cellular proteins via this sorting signal, a pulldown assay with GST fusion proteins was performed. Filamin A has been identified as a putative interaction candidate. Immunofluorescence assays confirmed a co-localization between the TGEV S protein and filamin A. Further experiments have to be performed to prove a significant impact of filamin A on TGEV infection. Different approaches of several researchers for the identification of cellular interaction candidates relevant for coronavirus replication are summarized. These results may help in the future to identify the role of cellular proteins during coronavirus assembly at the ER-Golgi intermediate compartment.

A Cytosolic Relay of Heat Shock Proteins HSP70-1A and HSP90β Monitors the Folding Trajectory of the Serotonin Transporter

Mutations in the C terminus of the serotonin transporter (SERT) disrupt folding and export from the endoplasmic reticulum. Here we examined the hypothesis that a cytosolic heat shock protein relay was recruited to the C terminus to assist folding of SERT. This conjecture was verified by the following observations. (i) The proximal portion of the SERT C terminus conforms to a canonical binding site for DnaK/heat shock protein of 70 kDa (HSP70). A peptide covering this segment stimulated ATPase activity of purified HSP70-1A. (ii) A GST fusion protein comprising the C terminus of SERT pulled down HSP70-1A. The interaction between HSP70-1A and SERT was visualized in live cells by Förster resonance energy transfer: it was restricted to endoplasmic reticulum-resident transporters and enhanced by an inhibitor that traps HSP70-1A in its closed state. (iv) Co-immunoprecipitation confirmed complex formation of SERT with HSP70-1A and HSP90β. Consistent with an HSP relay, co-chaperones (e.g. HSC70-HSP90-organizing protein) were co-immunoprecipitated with the stalled mutants SERT-R607A/I608A and SERT-P601A/G602A. (v) Depletion of HSP90β by siRNA or its inhibition increased the cell surface expression of wild type SERT and SERT-F604Q. In contrast, SERT-R607A/I608A and SERT-P601A/G602A were only rendered susceptible to inhibition of HSP70 and HSP90 by concomitant pharmacochaperoning with noribogaine. (vi) In JAR cells, inhibition of HSP90 also increased the levels of SERT, indicating that endogenously expressed transporter was also susceptible to control by HSP90β. These findings support the concept that the folding trajectory of SERT is sampled by a cytoplasmic chaperone relay.

Abstract 1311: Notch1 inhibits retinoblastoma (RB) via direct interaction with RB pocket domain

Abstract Notch signaling is involved in many biological processes, ranging from cellular proliferation to differentiation and apoptosis. Mammals have four Notch genes (Notch1-4) that encode heterodimeric membrane receptors. The transmembrane subunit is cleaved to generate an intracellular active subunit (NotchIC). Gain-of-function Notch1 mutations are found in a majority of T-ALL, some adult chronic lymphocytic leukemia and some breast cancers. The Retinoblastoma (RB) protein is a tumor suppressor and negative regulator of the cell cycle. Cellular and viral proteins that interact with RB possess a LxCxE and/or ExxxDLF motifs or closely related motifs. Notch1IC affects the phosphorylation and total amount of RB in T-ALL and mouse erythroleukemia cells. Oncoproteins such as E7 and SV40 large T-antigen bind and inactivate RB but also upregulate Notch1 in some systems. Notch1 contains an AxCxD motif, a conserved negatively charged cluster similar to viral oncoproteins in ankyrin repeat 1 and an ExxLEDL motif. We tested whether Notch1 interacts with RB and determinated the binding motif responsible of this interaction. We quantified Notch transcriptional activity using CBF-1 luciferase. We performed co-immunoprecipitation experiments in T-ALL and other cell lines with elevated Notch1IC expression. To map the Notch1-RB interaction, several GST fusion proteins were constructed and used in interaction assays. Total cellular lysate of breast cancer cells transiently expressing Notch1IC with different point mutations in AxCxD and/or ExxLEDL motifs, were used for co-immunoprecipitation. Luciferase assay showed that exogenous expression of RB significantly inhibits ICN-induced stimulation of CBF1 as well as basal CBF1 activity. We found that Notch1IC containing putative LxCxE interacts with the pocket region of RB-GST and does not appear to bind or interact with other domains of RB. Co-immunoprecipitation experiments demonstrated binding of unphosphorylated RB and Notch1IC in T-ALL and other cell lines. Mutations in Notch1IC in the two putative motifs do not completely abolish interaction with RB. These mutants appear to bind preferentially the N-terminal region of RB, suggesting the presence of two binding sites or a larger complex including other nuclear proteins. We showed direct interaction of RB and Notch1IC by GST-fusion proteins and co-immunoprecipitation in T-ALL and cells. We also showed interaction of AxCxD and ExxLEDL Notch1 motifs with RB. We conclude that inactivation of RB may contribute to the oncogenic activity of Notch1IC. Citation Format: Filomena T. Papa, Ingrid Espinoza, Nicole Darack-Gutyon, Antonio Giordano, Antonio De Luca, Christian R. Gomez, Lucio Miele. Notch1 inhibits retinoblastoma (RB) via direct interaction with RB pocket domain. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1311. doi:10.1158/1538-7445.AM2014-1311

Abstract 4929: Radiolabeled cCPE for molecular imaging of tight junction changes during breast oncogenesis

Abstract Expression of Claudin-4, a member of the Claudin protein family that is integral in adherens and tight junction complexes, is widely dysregulated in epithelial malignancies and is overexpressed in a number of premalignant precursor lesions, including those of cancers of the breast, pancreas and prostate. Increased Claudin-4 expression in breast cancer is also associated with poor prognosis. Optical imaging of Claudin-4 has been shown previously to be an effective way to detect precancerous lesions of the pancreas, using a fluorescently labelled form of a non-cytotoxic c-terminal fragment of Clostridium perfringens enterotoxin (cCPE, aa184-319), the natural ligand for Claudin-4. Here, we demonstrate whole-body quantitative SPECT imaging of Claudin-4 in preneoplastic breast cancer tissue using 111In-labelled cCPE. Our aim is to improve overall outcome by detection of lesions at an earlier stage. cCPE was produced as a GST-fusion protein from a pGEX plasmid. cCPE.GST or GST was conjugated to the metal ion chelator, benzylDTPA, to allow radiolabelling with 111In. Radiolabeling yield was &amp;gt;95%. Affinity of radiolabelled cCPE-GST for Claudin-4 was confirmed by binding to Claudin-4 expressing MDA-MB-468 and SQ20b cells, compared to Claudin-4 negative HT1080 cells. Radioactivity associated with MDA-MB-468 cells was 24.1±0.9 times higher for 111In-cCPE.GST vs. 111In-GST; P&amp;lt;0.001. Association of 111In-cCPE.GST was 4.7±0.2 times higher with MDA-MB-468 vs. HT1080 cells; P&amp;lt;0.001. 111In-cCPE.GST, but not 111In-GST, was internalised in MDA-MB-468 and SQ20b cells, but not in HT1080 cells. Athymic balb/c mice carrying subcutaneous xenograft tumors of MDA-MB-468 or HT1080 cells were injected intravenously with 5 µg 111In-labelled cCPE.GST or GST (5 MBq). After 24 h, SPECT/CT images were acquired, and the amount of radioactivity in selected organs was determined after dissection. Uptake of 111In-cCPE.GST in Claudin-4 positive MDA-MD-468 tumors was significantly higher compared to 111In-GST or HT1080 tumors (26.14±12.31 vs. 4.84±1.29 vs. 2.36±1.25 percent of the injected dose per gram of tumor tissue (%ID/g); p=0.021). No other significant differences were observed in any of the examined organs. Balb/neuT mice, expressing rat neuT under mmtv promotor control, spontaneously obtain tumorous lesions within their mammary fat pads over the course of 130 days. Balb/neuT mice were imaged monthly using 111In-cCPE.GST or 111In-GST. Mammary fat pads in mice aged around 90 days, bore aplastic lesions which were positive for Claudin-4, as determined by immunohistochemistry, and attracted 111In-cCPE.GST (3-5 %ID/g), but not 111In-GST (&amp;lt;1 %ID/g). In summary, 111In-cCPE.GST targets Claudin-4 expression in frank tumors and preneoplastic tissue, and cCPE imaging may be used as an early detection tool for breast, prostate, pancreas. Citation Format: Michael Mosley, James Knight, Albrecht Neesse, Patrick Michl, Veerle Kersemans, Bart Cornelissen. Radiolabeled cCPE for molecular imaging of tight junction changes during breast oncogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4929. doi:10.1158/1538-7445.AM2014-4929

Protein-polymer conjugation via ligand affinity and photoactivation of glutathione S-transferase.

A photoactivated, site-selective conjugation of poly(ethylene glycol) (PEG) to the glutathione (GSH) binding pocket of glutathione S-transferase (GST) is described. To achieve this, a GSH analogue (GSH-BP) was designed and chemically synthesized with three functionalities: (1) the binding affinity of GSH to GST, (2) a free thiol for polymer functionalization, and (3) a photoreactive benzophenone (BP) component. Different molecular weights (2 kDa, 5 kDa, and 20 kDa) of GSH-BP modified PEGs (GSBP-PEGs) were synthesized and showed conjugation efficiencies between 52% and 76% to GST. Diazirine (DA) PEG were also prepared but gave conjugation yields lower than for GSBP-PEGs. PEGs with different end-groups were also synthesized to validate the importance of each component in the end-group design. End-groups included glutathione (GS-PEG) and benzophenone (BP-PEG). Results showed that both GSH and BP were crucial for successful conjugation to GST. In addition, conjugations of 5 kDa GSBP-PEG to different proteins were investigated, including bovine serum albumin (BSA), lysozyme (Lyz), ubiquitin (Ubq), and GST-fused ubiquitin (GST-Ubq) to ensure specific binding to GST. By combining noncovalent and covalent interactions, we have developed a new phototriggered protein–polymer conjugation method that is generally applicable to GST-fusion proteins.

Isolation of dihydroflavonol 4-reductase cDNA clones from Angelonia x angustifolia and heterologous expression as GST fusion protein in Escherichia coli.

Blue Angelonia × angustifolia flowers can show spontaneous mutations resulting in white/blue and white flower colourations. In such a white line, a loss of dihydroflavonol 4-reductase (DFR) activity was observed whereas chalcone synthase and flavanone 3-hydroxylase activity remained unchanged. Thus, cloning and characterization of a DFR of Angelonia flowers was carried out for the first time. Two full length DFR cDNA clones, Ang.DFR1 and Ang.DFR2, were obtained from a diploid chimeral white/blue Angelonia × angustifolia which demonstrated a 99% identity in their translated amino acid sequence. In comparison to Ang.DFR2, Ang.DFR1 was shown to contain an extra proline in a proline-rich region at the N-terminus along with two exchanges at the amino acids 12 and 26 in the translated amino acid sequence. The recombinant Ang.DFR2 obtained by heterologous expression in yeast was functionally active catalyzing the NADPH dependent reduction of dihydroquercetin (DHQ) and dihydromyricetin (DHM) to leucocyanidin and leucomyricetin, respectively. Dihydrokaempferol (DHK) in contrast was not accepted as a substrate despite the presence of asparagine in a position assumed to determine DHK acceptance. We show that substrate acceptance testing of DFRs provides biased results for DHM conversion if products are extracted with ethyl acetate. Recombinant Ang.DFR1 was inactive and functional activity could only be restored via exchanges of the amino acids in position 12 and 26 as well as the deletion of the extra proline. E. coli transformation of the pGEX-6P-1 vector harbouring the Ang.DFR2 and heterologous expression in E. coli resulted in functionally active enzymes before and after GST tag removal. Both the GST fusion protein and purified DFR minus the GST tag could be stored at −80°C for several months without loss of enzyme activity and demonstrated identical substrate specificity as the recombinant enzyme obtained from heterologous expression in yeast.