Analysis of Dysferlin Direct Interactions with Putative Repair Proteins Links Apoptotic Signaling to Ca2+ Elevation via PDCD6 and FKBP8.

Quantitative surface plasmon resonance (SPR) was utilized to determine binding strength and calcium dependence of direct interactions between dysferlin and proteins likely to mediate skeletal muscle repair, interrupted in limb girdle muscular dystrophy type 2B/R2. Dysferlin canonical C2A (cC2A) and C2F/G domains directly interacted with annexin A1, calpain-3, caveolin-3, affixin, AHNAK1, syntaxin-4, and mitsugumin-53, with cC2A the primary target and C2F lesser involved, overall demonstrating positive calcium dependence. Dysferlin C2 pairings alone showed negative calcium dependence in almost all cases. Like otoferlin, dysferlin directly interacted via its carboxy terminus with FKBP8, an anti-apoptotic outer mitochondrial membrane protein, and via its C2DE domain with apoptosis-linked gene (ALG-2/PDCD6), linking anti-apoptosis with apoptosis. Confocal Z-stack immunofluorescence confirmed co-compartmentalization of PDCD6 and FKBP8 at the sarcolemmal membrane. Our evidence supports the hypothesis that prior to injury, dysferlin C2 domains self-interact and give rise to a folded, compact structure as indicated for otoferlin. With elevation of intracellular Ca2+ in injury, dysferlin would unfold and expose the cC2A domain for interaction with annexin A1, calpain-3, mitsugumin 53, affixin, and caveolin-3, and dysferlin would realign from its interactions with PDCD6 at basal calcium levels to interact strongly with FKBP8, an intramolecular rearrangement facilitating membrane repair.

Development of high-affinity nanobodies specific for NaV1.4 and NaV1.5 voltage-gated sodium channel isoforms.

Voltage-gated sodium channels, NaVs, are responsible for the rapid rise of action potentials in excitable tissues. NaV channel mutations have been implicated in several human genetic diseases, such as hypokalemic periodic paralysis, myotonia, and long-QT and Brugada syndromes. Here, we generated high-affinity anti-NaV nanobodies (Nbs), Nb17 and Nb82, that recognize the NaV1.4 (skeletal muscle) and NaV1.5 (cardiac muscle) channel isoforms. These Nbs were raised in llama (Lama glama) and selected from a phage display library for high affinity to the C-terminal (CT) region of NaV1.4. The Nbs were expressed in Escherichia coli, purified, and biophysically characterized. Development of high-affinity Nbs specifically targeting a given human NaV isoform has been challenging because they usually show undesired crossreactivity for different NaV isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNaV1.4 or CTNaV1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNaV1.4 and CTNaV1.5 with high affinity (KD ∼ 40-60 nM). In addition, as proof of concept, we show that Nb82 could detect NaV1.4 and NaV1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo NaV1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-NaV reagents to capture NaVs from cell lysates and as molecular visualization agents for NaVs.

Strategy for Tumor-Selective Disruption of Androgen Receptor Function in the Spectrum of Prostate Cancer.

PURPOSE: Testosterone suppression in prostate cancer is limited by serious side effects and resistance via restoration of androgen receptor (AR) functionality. ELK1 is required for AR-dependent growth in various hormone-dependent and castration-resistant prostate cancer models. The amino-terminal domain of AR docks at two sites on ELK1 to coactivate essential growth genes. This study explores the ability of small molecules to disrupt the ELK1-AR interaction in the spectrum of prostate cancer, inhibiting AR activity in a manner that would predict functional tumor selectivity. EXPERIMENTAL DESIGN: Small-molecule drug discovery and extensive biological characterization of a lead compound. RESULTS: We have discovered a lead molecule (KCI807) that selectively disrupts ELK1-dependent promoter activation by wild-type and variant ARs without interfering with ELK1 activation by ERK. KCI807 has an obligatory flavone scaffold and functional hydroxyl groups on C5 and C3'. KCI807 binds to AR, blocking ELK1 binding, and selectively blocks recruitment of AR to chromatin by ELK1. KCI807 primarily affects a subset of AR target growth genes selectively suppressing AR-dependent growth of prostate cancer cell lines with a better inhibitory profile than enzalutamide. KCI807 also inhibits in vivo growth of castration/enzalutamide-resistant cell line-derived and patient-derived tumor xenografts. In the rodent model, KCI807 has a plasma half-life of 6 hours, and maintenance of its antitumor effect is limited by self-induced metabolism at its 3'-hydroxyl. CONCLUSIONS: The results offer a mechanism-based therapeutic paradigm for disrupting the AR growth-promoting axis in the spectrum of prostate tumors while reducing global suppression of testosterone actions. KCI807 offers a good lead molecule for drug development.

Imaging EGFR and HER3 through 89Zr-labeled MEHD7945A (Duligotuzumab).

Tumor resistance to treatment paved the way toward the development of single agent drugs that target multiple molecular signatures amplified within the malignancy. The discovered crosstalk between EGFR and HER3 as well as the role of HER3 in mediating EGFR resistance made these two receptor tyrosine kinases attractive targets. MEHD7945A or duligotuzumab is a single immunotherapy agent that dually targets both molecular signatures. In this study, a positron emission tomography (PET) companion diagnostic to MEHD7945A is reported and evaluated in pancreatic cancer. Tumor accretion and whole body pharmacokinetics of 89Zr-MEHD7945A were established. Specificity of the probe for EGFR and/or HER3 was further examined.

Analysis of Protein Interactions by Surface Plasmon Resonance.

Surface plasmon resonance is an optical technique that is utilized for detecting molecular interactions, such as interactions that occur between proteins or other classes of molecules. Binding of a mobile molecule (analyte) to a molecule immobilized on a thin metal film (ligand) changes the refractive index of the film. The angle of extinction of light that is completely reflected after polarized light impinges upon the film, is altered and monitored as a change in detector position for a dip in reflected intensity (the surface plasmon resonance phenomenon). Because the method strictly detects mass, there is no need to label the interacting components, thus eliminating possible changes of their molecular properties. In this chapter, we review essential SPR methodology and present applications to basic science and human disease.

Dopamine D1A directly interacts with otoferlin synaptic pathway proteins: Ca2+ and phosphorylation underlie an NSF-to-AP2mu1 molecular switch.

Dopamine receptors regulate exocytosis via protein-protein interactions (PPIs) as well as via adenylyl cyclase transduction pathways. Evidence has been obtained for PPIs in inner ear hair cells coupling D1A to soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE)-related proteins snapin, otoferlin, N-ethylmaleimide-sensitive factor (NSF), and adaptor-related protein complex 2, mu 1 (AP2mu1), dependent on [Ca2+] and phosphorylation. Specifically, the carboxy terminus of dopamine D1A was found to directly bind t-SNARE-associated protein snapin in teleost and mammalian hair cell models by yeast two-hybrid (Y2H) and pull-down assays, and snapin directly interacts with hair cell calcium-sensor otoferlin. Surface plasmon resonance (SPR) analysis, competitive pull-downs, and co-immunoprecipitation indicated that these interactions were promoted by Ca2+ and occur together. D1A was also found to separately interact with NSF, but with an inverse dependence on Ca2+ Evidence was obtained, for the first time, that otoferlin domains C2A, C2B, C2D, and C2F interact with NSF and AP2mu1, whereas C2C or C2E do not bind to either protein, representing binding characteristics consistent with respective inclusion or omission in individual C2 domains of the tyrosine motif YXXΦ. In competitive pull-down assays, as predicted by KD values from SPR (+Ca2+), C2F pulled down primarily NSF as opposed to AP2mu1. Phosphorylation of AP2mu1 gave rise to a reversal: an increase in binding by C2F to phosphorylated AP2mu1 was accompanied by a decrease in binding to NSF, consistent with a molecular switch for otoferlin from membrane fusion (NSF) to endocytosis (AP2mu1). An increase in phosphorylated AP2mu1 at the base of the cochlear inner hair cell was the observed response elicited by a dopamine D1A agonist, as predicted.

Antigen itself antibody: an alternative one-step immunoassay for measuring the anti-idiotypic antibody titer.

Immunoassay designs rely on the great specificity of antibodies and a suitable marker that facilitates generation of a quantitative signal. Currently, there is no reliable method for measuring the titers of an anti-idiotypic antibody. Our initial attempt to measure titers of mouse anti-idiotypic antibody after idiotypic vaccination with HM-1 killer toxin neutralizing monoclonal antibody (nmAb-KT) failed. Because the injected antigen, nmAb-KT, is a mouse IgG, using a commercial antibody to measure the antibody titer always gave a false positive signal against control mouse serum antibody in parallel with the antigen-treated immunized serum antibodies. To get a reliable and clearly differentiable signal by ELISA, idiotypic antigen was labeled with HRP and HRP-conjugated-nmAb-KT used to measure the antibody titers in the antigen-treated mice. Compared with control mice, signals were found in high anti-nmAb-KT IgG responses in test mice; however, untreated control mice had a significant amount of purified non-specific IgG. This method is amenable to long read lengths and will likely enable anti-idiotypic antibody titer measurement in a more specific and cost effective way without requiring commercial antibody.

Cyclic nucleotide-gated channel α-3 (CNGA3) interacts with stereocilia tip-link cadherin 23 + exon 68 or alternatively with myosin VIIa, two proteins required for hair cell mechanotransduction.

Previously, we obtained evidence for a photoreceptor/olfactory type of CNGA3 transcript in a purified teleost vestibular hair cell preparation with immunolocalization of CNGA3 protein to stereocilia of teleost vestibular and mammalian cochlear hair cells. The carboxyl terminus of highly Ca(2+)-permeable CNGA3 expressed in the mammalian organ of Corti and saccular hair cells was found to interact with an intracellular domain of microfibril interface-located protein 1 (EMILIN 1), a member of the elastin superfamily, also immunolocalizd to hair cell stereocilia (Selvakumar, D., Drescher, M. J., Dowdall, J. R., Khan, K. M., Hatfield, J. S., Ramakrishnan, N. A., and Drescher, D. G. (2012) Biochem. J. 443, 463-476). Here, we provide evidence for organ of Corti proteins, of Ca(2+)-dependent binding of the amino terminus of CNGA3 specifically to the carboxyl terminus of stereocilia tip-link protein CDH23 +68 (cadherin 23 with expressed exon 68) by yeast two-hybrid mating and co-transformation protocols, pulldown assays, and surface plasmon resonance analysis. Myosin VIIa, required for adaptation of hair cell mechanotransduction (MET) channel(s), competed with CDH23 +68, with direct Ca(2+)-dependent binding to the amino terminus of CNGA3. Based upon the premise that hair cell stereocilia tip-link proteins are closely coupled with MET, these results are consistent with the possibility that CNGA3 participates in hair-cell MET. Together with the demonstration of protein-protein interaction between HCN1 and tip-link protein protocadherin 15 CD3 (Ramakrishnan, N. A., Drescher, M. J., Barretto, R. L., Beisel, K. W., Hatfield, J. S., and Drescher, D. G. (2009) J. Biol. Chem. 284, 3227-3238; Ramakrishnan, N. A., Drescher, M. J., Khan, K. M., Hatfield, J. S., and Drescher, D. G. (2012) J. Biol. Chem. 287, 37628-37646), a protein-protein interaction for CNGA3 and a second tip-link protein, CDH23 +68, further suggests possible association of two different channels with a single stereocilia tip link.

CNGA3 is expressed in inner ear hair cells and binds to an intracellular C-terminus domain of EMILIN1.

The molecular characteristics of CNG (cyclic nucleotide-gated) channels in auditory/vestibular hair cells are largely unknown, unlike those of CNG mediating sensory transduction in vision and olfaction. In the present study we report the full-length sequence for three CNGA3 variants in a hair cell preparation from the trout saccule with high identity to CNGA3 in olfactory receptor neurons/cone photoreceptors. A custom antibody targeting the N-terminal sequence immunolocalized CNGA3 to the stereocilia and subcuticular plate region of saccular hair cells. The cytoplasmic C-terminus of CNGA3 was found by yeast two-hybrid analysis to bind the C-terminus of EMILIN1 (elastin microfibril interface-located protein 1) in both the vestibular hair cell model and rat organ of Corti. Specific binding between CNGA3 and EMILIN1 was confirmed with surface plasmon resonance analysis, predicting dependence on Ca2+ with Kd=1.6×10-6 M for trout hair cell proteins and Kd=2.7×10-7 M for organ of Corti proteins at 68 µM Ca2+. Pull-down assays indicated that the binding to organ of Corti CNGA3 was attributable to the EMILIN1 intracellular sequence that follows a predicted transmembrane domain in the C-terminus. Saccular hair cells also express the transcript for PDE6C (phosphodiesterase 6C), which in cone photoreceptors regulates the degradation of cGMP used to gate CNGA3 in phototransduction. Taken together, the evidence supports the existence in saccular hair cells of a molecular pathway linking CNGA3, its binding partner EMILIN1 (and ß1 integrin) and cGMP-specific PDE6C, which is potentially replicated in cochlear outer hair cells, given stereociliary immunolocalizations of CNGA3, EMILIN1 and PDE6C.

Peptide derived from anti-idiotypic single-chain antibody is a potent antifungal agent compared to its parent fungicide HM-1 killer toxin peptide.

Based on anti-idiotypic network theory in light of the need for new antifungal drugs, we attempted to identify biologically active fragments from HM-1 yeast killer toxin and its anti-idiotypic antibody and to compare their potency as an antifungal agent. Thirteen overlapping peptides from HM-1 killer toxin and six peptides from its anti-idiotypic single-chain variable fragment (scFv) antibodies representing the complementarity determining regions were synthesized. The binding affinities of these peptides were investigated and measured by Dot blot and surface plasmon resonance analysis and finally their antifungal activities were investigated by inhibition of growth, colony forming unit assay. Peptide P6, containing the potential active site of HM-1 was highly capable of inhibiting the growth of Saccharomyces cerevisiae but was less effective on pathogenic fungi. However, peptide fragments derived from scFv antibody exerted remarkable inhibitory effect on the growth of pathogenic strains of Candida and Cryptococcus species in vitro. One scFv-derived decapeptide (SP6) was selected as the strongest killer peptide for its high binding affinity and antifungal abilities on both Candida and Cryptococcus species with IC(50) values from 2.33 × 10(-7) M to 36.0 × 10(-7) M. SP6 peptide activity was neutralized by laminarin, a ß-1,3-glucan molecule, indicating this peptide derived from scFv anti-idiotypic antibody retains antifungal activity through interaction with cell wall ß-glucan of their target fungal cells. Experimental evidence strongly suggested the possibility of development of anti-idiotypic scFv peptide-based antifungal agents which may lead to improve therapeutics for the management of varieties of fungal infections.

Recombinant single-chain anti-idiotypic antibody: an effective fungal beta-1,3-glucan synthase inhibitor.

Recombinant single-chain fragment variable anti-idiotypic antibodies were produced to represent the internal image of HM-1 killer toxin and were used as novel and effective antifungal agents to inhibit in vitro beta-1,3-glucan synthase and cell growth. The mechanism of cytocidal activity of anti-idiotypic antibodies was investigated and was compared with the actions of aculeacin A and papulacandin B, the most common antibiotics acting as beta-1,3-glucan synthase inhibitors. The degree of inhibition of beta-1,3-glucan synthase by both antibodies and antibiotics were examined for yeasts Saccharomyces cerevisiae A451, Cryptococcus albidus NBRC 0612 and Candida albicans IFM 40215. Although the mechanism of actions of the anti-idiotypic antibodies and antibiotics seems identical, the IC(50) values for the various yeasts used in this study confirmed that anti-idiotypic antibodies could be used as more effective fungal beta-1,3-glucan synthase inhibitors than those of antibiotics.

The role of the histidine-35 residue in the cytocidal action of HM-1 killer toxin.

Diethylpyrocarbonate modification and site-directed mutagenesis studies of histidine-35 in HM-1 killer toxin (HM-1) have shown that a specific feature, the imidazole side chain of histidine-35, is essential for the expression of the killing activity. In subcellular localization experiments, wild-type HM-1 was in the membrane fraction of Saccharomyces cerevisiae BJ1824, but not the HM-1 analogue in which histidine-35 was replaced by alanine (H35A HM-1). Neither wild-type nor H35A HM-1 was detected in cellular fractions of HM-1-resistant yeast S. cerevisiae BJ1824 rhk1Delta : : URA3 and HM-1-insensitive yeast Candida albicans even after 1 h incubation. H35A HM-1 inhibited the activity of partially purified 1,3-beta-glucan synthase from S. cerevisiae A451, and its extent was almost the same as wild-type HM-1. Co-immunoprecipitation experiments showed that wild-type and H35A HM-1 directly interact with the 1,3-beta-glucan synthase complex. These results strongly suggest that histidine-35 has an important role in the cytocidal action of HM-1 that participates in the binding process to the HM-1 receptor protein on the cell membrane, but it is not essential for the interaction with, and inhibition of, 1,3-beta-glucan synthase.

Inhibition of fungal beta-1,3-glucan synthase and cell growth by HM-1 killer toxin single-chain anti-idiotypic antibodies.

Single-chain variable-fragment (scFv) anti-idiotypic antibodies of an HM-1 killer toxin (HM-1) from the yeast Williopsis saturnus var. mrakii IFO 0895 have been produced by recombinant DNA technology from the splenic lymphocytes of mice immunized by idiotypic vaccination with a neutralizing monoclonal antibody (nMAb-KT). The fungicidal activity of scFv anti-idiotypic antibodies against the isolates of four Candida species was assessed by MIC analysis. scFv antibodies were fungicidal at concentrations of 1.56 to 12.5 microg/ml in vitro against four Candida species. The scFv antibodies exerted a strong candidacidal activity in vitro, with 50% inhibitory concentration (IC(50)) values ranging from 7.3 x 10(-8) to 16.0 x 10(-8) M, and were neutralized by adsorption with nMAb-KT. Furthermore, all scFv antibodies effectively inhibited fungal beta-1,3-glucan synthase activity in vitro, with IC(50) values ranging from 2.0 x 10(-8) to 22.7 x 10(-8) M, values which almost coincide with the values that are inhibitory to the growth of fungal cells. Binding assays showed that the scFv antibodies specifically bind to nMAb-KT, and this binding pattern was confirmed by surface plasmon resonance analysis. The binding ability was further demonstrated by the competition observed between scFv antibodies and HM-1 to bind nMAb-KT. To the best of our knowledge, this is the first study to show that an antifungal anti-idiotypic antibody, in the form of recombinant scFv, potentially inhibits beta-1,3-glucan synthase activity.

Inhibition of beta-1,3-glucan synthase and cell growth of Cryptococcus species by recombinant single-chain anti-idiotypic antibodies.

Recombinant single-chain fragment variable (scFv) anti-idiotypic antibodies were produced to represent the internal image of a HM-1 killer toxin, which is characterized by a wide spectrum of anti-fungal activity through inhibiting beta-1,3-glucan synthase (GS). We examined if scFv antibodies are active against Cryptococcus species, a human pathogen of increasing medical importance. The anti-cryptococcal activity of scFv antibodies and HM-1 were assessed by MIC analysis for C. neoformans IFM 40215 and C. albidus NBRC 0612 cells. The scFv antibodies had strong anti-cryptococcal activity in vitro with IC50 at 1.07 x 10(-7) to 2.60 x 10(-7) M for C. neoformans and C. albidus. Furthermore, the scFv antibodies potentially inhibited GS of C. neoformans with IC50 at 1.27 x 10(-7) to 2.27 x 10(-7) M. Both the anti-fungal and anti-GS activities of the scFv antibodies were markedly neutralized by the monoclonal antibody that neutralizes HM-1 killer toxin.

Identification and characterization of a neutralizing monoclonal antibody for the epitope on HM-1 killer toxin.

Killer toxin-neutralizing monoclonal antibody (nmAb-KT) against HM-1 killer toxin (HM-1) produced by yeast Williopsis saturnus var. mrakii IFO 0895 reduces both the killing and glucan synthase inhibitory activity of HM-1. nmAb-KT is classified as IgG1kappa and has been shown to be ineffective against HYI killer toxin produced by the related yeast W. saturnus var. saturnus IFO 0117. To determine the epitope for nmAb-KT, overlapping peptides were synthesized from the primary structure of HM-1. nmAb-KT reacted with peptides P5 (33NVHWMVTGGST43), P6 (39TGGSTDGKQG48) and P7 (44DGKQGCATIWEGS56), which represent the middle region of the HM-1 sequence. P6 reacted most strongly with nmAb-KT. Combined analysis by immunoblotting, surface plasmon resonance (SPR) analysis and yeast growth inhibition assay showed that nmAb-KT recognizes a specific epitope within peptide P6. The K(d) value of nmAb-KT against HM-1 and P6 were determined to be 5.48 x 10(-9) M and 1.47 x 10(-6) M by SPR analysis, respectively. These results strongly indicate that nmAb-KT binds to HM-1 at the sequence 41GSTDGK46, and not to HYI at the same position. The potential active site of HM-1 involved in the killing activity against sensitive yeast is discussed.

Monoclonal antibodies and sandwich ELISA for quantitation of HM-1 killer toxin.

To establish a method for quantitative analysis of HM-1 killer toxin (HM-1), two purified mouse monoclonal antibodies, 1F1 and 4A2, and rabbit polyclonal antiserum against HM-1 were prepared. Both monoclonal antibodies were classified as IgG1(kappa) subtype, and did not neutralize the killing activity of HM-1. By SPOTs analysis, the epitope of 1F1 was found in the sequence of CDPNTG with a corresponding sequence of 11-16 from N-terminal amino acid residues of HM-1, but the epitope of 4A2 was not determined. Using 4A2 and polyclonal antiserum, the sandwich enzyme-linked immunosorbent assay (ELISA) was applied to establish the quantitative determination of HM-1. The concentration of HM-1 was determined successfully at the range of 2.5-100 ng/ml. But in the case of 1F1, the method was not established. Genes were constructed to apply the system to the measurement of the secreted concentrations of mutant HM-1, and it was evident that the production of mutant toxins varied among HM-1 mutant genes. The findings of this study are unique in determinimg the epitope of monoclonal antibody against HM-1, and in quantifying the HM-1 using the spot analysis and sandwich ELISA methods.