Jordan B. Fishman

A trans Golgi-derived exocytic coated vesicle can contain both newly synthesized cholinesterase and internalized transferrin

We used a cholinesterase-mediated density shift protocol to investigate the movement of internalized transferrin (Tf) through endo- and exocytic coated vesicles (CVs) in the perfused rat liver. Upon internalization, exogenous 125I-Tf was found in endocytic CVs but not in cholinesterase-containing (i.e., exocytic) CVs (0-40 min). Between 1 and 2 hr, 125I-Tf began to appear in exocytic CVs. The origin of the exocytic CV was further investigated. After perfusion of the liver with asialotransferrin, the exocytic CVs were shown to contain resialylated Tf, indicating that the trans Golgi was the origin of this class of CVs. The resialylated Tf accumulated in the extracellular medium with kinetics very similar to the time course for appearance of Tf in cholinesterase-containing, exocytic CVs, suggesting that these CVs are directly involved in the transfer of material from the trans Golgi to the cell surface.

Pyroglutamate-Aβ 3 and 11 colocalize in amyloid plaques in Alzheimer's disease cerebral cortex with pyroglutamate-Aβ 11 forming the central core.

N-terminal truncated amyloid beta (Aβ) derivatives, especially the forms having pyroglutamate at the 3 position (AβpE3) or at the 11 position (AβpE11) have become the topic of considerable study. AβpE3 is known to make up a substantial portion of the Aβ species in senile plaques while AβpE11 has received less attention. We have generated very specific polyclonal antibodies against both species. Each antibody recognizes only the antigen against which it was generated on Western blots and neither recognizes full length Aβ. Both anti-AβpE3 and anti-AβpE11 stain senile plaques specifically in Alzheimers disease cerebral cortex and colocalize with Aβ, as shown by confocal microscopy. In a majority of plaques examined, AβpE11 was observed to be the dominant form in the innermost core. These data suggest that AβpE11 may serve as a generating site for senile plaque formation.

The effect of bovine serum albumin on the synthesis of prostaglandin and incorporation of [3H]acetate into platelet-activating factor

The binding of fatty acids by bovine serum albumin (BSA) is well documented. However, the interaction between the synthesis of prostaglandins (PGs) and the trapping of arachidonate released from cellular lipid stores is not as well understood. In this communication, we relate the trapping of fatty acids to the synthesis of PGs and the incorporation of [3H]acetate into platelet-activating factor (PAF). Our results show that, as determined by radioimmunoassay, BSA inhibits bradykinin (BK) (5 ng/ml) and ionophore A23187 (10 microM)-stimulated synthesis of PGs in human embryo lung fibroblasts (IMR-90) in a concentration-dependent manner. Experiments using prelabel with [3H]arachidonate followed by extraction and thin-layer chromatography show that, in the presence of 2 mg/ml BSA, IMR-90 release essentially only fatty acid following stimulation with bradykinin. Little if any prostaglandin and no endoperoxide are detected. In the same experiment, in absence of BSA, about 70% of the released label is detected as prostaglandin. alpha-Cyclodextrin, another trapper of fatty acid, inhibits PG synthesis in much the same way. BSA and alpha-cyclodextrin also inhibit prostacyclin synthesis in endothelial cells derived from the calf pulmonary artery. However, the inhibition of PG synthesis in these cells is not as complete as that in the IMR-90. In contrast to the effect of the trappers on PG synthesis, BSA and alpha-cyclodextrin are observed to potentiate BK- and ionophore-stimulated incorporation of [3H]acetate into PAF in the endothelial cells. The labeled PAF is not released from the cells in either the presence or absence of the trappers, leading us to conclude that BSA causes an increase in acetate-labeled cellular PAF by trapping released fatty acid.

Modulation of the Protein Kinase Cδ Interaction with the “d” Subunit of F1F0-ATP Synthase in Neonatal Cardiac Myocytes DEVELOPMENT OF CELL-PERMEABLE, MITOCHONDRIALLY TARGETED INHIBITOR AND FACILITATOR PEPTIDES

The F1F0-ATP synthase provides ∼90% of cardiac ATP, yet little is known regarding its regulation under normal or pathological conditions. Previously, we demonstrated that protein kinase Cδ (PKCδ) inhibits F1F0 activity via an interaction with the “d” subunit of F1F0-ATP synthase (dF1F0) in neonatal cardiac myocytes (NCMs) (Nguyen, T., Ogbi, M., and Johnson, J. A. (2008) J. Biol. Chem. 283, 29831–29840). We have now identified a dF1F0-derived peptide (NH2-2AGRKLALKTIDWVSF16-COOH) that inhibits PKCδ binding to dF1F0 in overlay assays. We have also identified a second dF1F0-derived peptide (NH2-111RVREYEKQLEKIKNMI126-COOH) that facilitates PKCδ binding to dF1F0. Incubation of NCMs with versions of these peptides containing HIV-Tat protein transduction and mammalian mitochondrial targeting sequences resulted in their delivery into mitochondria. Preincubation of NCMs, with 10 nm extracellular concentrations of the mitochondrially targeted PKCδ-dF1F0 interaction inhibitor, decreased 100 nm 4β-phorbol 12-myristate 13-acetate (4β-PMA)-induced co-immunoprecipitation of PKCδ with dF1F0 by 50 ± 15% and abolished the 30 nm 4β-PMA-induced inhibition of F1F0-ATPase activity. A scrambled sequence (inactive) peptide, which contained HIV-Tat and mitochondrial targeting sequences, was without effect. In contrast, the cell-permeable, mitochondrially targeted PKCδ-dF1F0 facilitator peptide by itself induced the PKCδ-dF1F0 co-immunoprecipitation and inhibited F1F0-ATPase activity. In in vitro PKC add-back experiments, the PKCδ-F1F0 inhibitor blocked PKCδ-mediated inhibition of F1F0-ATPase activity, whereas the facilitator induced inhibition. We have developed the first cell-permeable, mitochondrially targeted modulators of the PKCδ-dF1F0 interaction in NCMs. These novel peptides will improve our understanding of cardiac F1F0 regulation and may have potential as therapeutics to attenuate cardiac injury.

Limiting angiotensin II signaling with a cell‐penetrating peptide mimicking the second intracellular loop of the angiotensin II type‐I receptor

A cell‐penetrating peptide consisting of the second intracellular loop (IC2) of the angiotensin II (AngII) type‐I receptor (AT1) linked to the HIV‐transactivating regulatory protein (TAT) domain was used to identify the role of this motif In intracellular signal transduction. HEK‐293 cells stably transfected with AT1R cDNA and primary cultures of human pulmonary artery smooth muscle cells expressing endogenous AT1 receptor were exposed to the cell‐penetrating peptide construct, and the effect on angiotensin II signaling was determined. The AT1 IC2 peptide effectively inhibited AngII‐stimulated phosphatidylinositol turnover and calcium influx. It also limited the activation of Akt/PKB as determined by an inhibition of phosphorylation of Akt at Ser473, and completely abolished the AngII‐dependent activation of the transcriptional factor NFκB. In contrast, the AT1 IC2 peptide had no effect on AngII/AT1 receptor activation of ERK. These results illustrate the potential of using cell‐penetrating peptides to both delineate receptor‐mediated signal transduction and to selectively regulate G protein‐coupled receptor signaling.

A Systematic Approach to Identify Biased Agonists of the Apelin Receptor through High-Throughput Screening:

Biased agonists are defined by their ability to selectively activate distinct signaling pathways of a receptor, and they hold enormous promise for the development of novel drugs that specifically elicit only the desired therapeutic response and avoid potential adverse effects. Unfortunately, most high-throughput screening (HTS) assays are designed to detect signaling of G protein–coupled receptors (GPCRs) downstream of either G protein or β-arrestin–mediated signaling but not both. A comprehensive drug discovery program seeking biased agonists must employ assays that report on the activity of each compound at multiple discrete pathways, particularly for HTS campaigns. Here, we report a systematic approach to the identification of biased agonists of human apelin receptor (APJ). We synthesized 448 modified versions of apelin and screened them against a cascade of cell-based assays, including intracellular cAMP and β-arrestin recruitment to APJ, simultaneously. The screen yielded potent and highly selective APJ agonists. Representative hits displaying preferential signaling via either G-protein or β-arrestin were subjected to a battery of confirmation assays. These biased agonists will be useful as tools to probe the function and pharmacology of APJ and provide proof of concept of our systematic approach to the discovery of biased ligands. This approach is likely universally applicable to the search for biased agonists of GPCRs.

Preparation of Antibody Using Caprylic Acid

Caprylic acid has been used to enrich IgG from serum, ascites, and cell culture supernatants by precipitating the non-IgG serum proteins. By precipitating all of the unwanted serum proteins rather than the antibodies, the tendency of antibodies to aggregate when precipitated is avoided. This method should not be used with antibody sources that contain low concentrations of antibody, such as many cell culture supernatants, owing to the potential loss of high-affinity antibodies, which may be bound by the caprylic acid. After centrifugation, IgG remains in the supernatant, which can be used as the starting material for the next step in a multistep antibody purification protocol.

Isolation of IgY from Chicken Eggs

Because IgY does not interact with either Protein A or Protein G, more traditional methods must be used for its isolation. To isolate chicken IgY from the yolk of chicken eggs, the yolks and the whites must be separated much in the way a cook does. Minimizing the egg white proteins is an important part of this process. Although eggs can be accumulated for short periods of time, IgY must be isolated from the yolk within 2-3 wk to avoid fouling of the material. In the first section of this protocol, sodium sulfate is used to precipitate protein fractions differentially from the chicken egg yolk. The second section describes an alternative differential precipitation technique that uses Polyethylene Glycol 6000 instead of sodium sulfate.

Abstract LB-024: Optimized Sox-based sensors for continuous, homogeneous, and quantitative monitoring of protein kinase activity

Introduction: Protein kinases are a diverse group of over 500 enzymes whose dysregulation lies at the center of many human diseases, spanning all therapeutic areas. Oncology is the most active area, where 30% of all drug development efforts are focused on protein kinases. Although 30 drugs have been approved by the FDA, and another 120 are in clinical trials, these are predominately ATP-competitive inhibitors. More recently, there has been a surge in the generation of kinase inhibitors with different modes of action, where new tools are needed to effectively and efficiently characterize inhibitor mechanism of action, predict drug potency and to drive decisions earlier in the drug development process. We developed a simple yet powerful method for the generation of sensors that can be used for the continuous, quantitative and homogenous detection of kinase activity to enable target discovery and drug development. Experimental Procedures: We have harnessed chelation-enhanced fluorescence by integrating the sulfonamido-oxine (Sox) chromophore into high-throughput peptide synthesis methods to create degenerate peptide libraries containing the consensus sequence for Ser, Thr or Tyr kinases that are implicated in cancer. Kinase reactions included 10 μM substrate, 10 mM MgCl2 and 1 mM ATP and fluorescence intensity was monitored in kinetic mode using a Biotek Synergy Neo2 microplate reader (excitation 360 nm, emission wavelength of 485-505 nm). Results: By exploiting the continuous, quantitative and homogeneous nature of Sox-based detection, we demonstrate the ability to rapidly identify novel optimum substrates simply from observing and analyzing the resulting progress curve (fluorescence intensity over time). Performance measures included higher reaction rates, lower Km9s, lower and flatter backgrounds, improved signal/background, increased sensitivity (down to low pM levels), and increased specificity. We identified highly generic substrates (for robust detection of 80 Tyrosine kinases) and highly-selective substrates (for quantitative detection of targeted kinases in crude cell or tissue lysates for profiling, potency assessments and SAR). We highlight recently developed novel substrates to monitor activity of high-profile tyrosine kinases, including the EGFR and multiple clinically-relevant mutants and Tec-family kinases (BTK, ITK, TEC, TXK, BMX), and serine/threonine kinases, including MAP4K9s, MAPKs, CDKs and DRAK1/2. Conclusions: The generation of robust activity-based assays, especially where previously only binding assay formats were available, opens up new areas of the kinome for effective drug discovery. The Sox-based assay technology is ideal for elucidating drug mechanism of action, potency, kinase activation and profiling, and therefore can be applied across the entire target discovery and drug development workflow. These developments provide a quantum improvement in performance and productivity that is needed to address the challenges and opportunities of next generation protein kinase inhibitors. These assays run on commonly available microplate instruments, providing access across the cancer research and drug development community. Citation Format: Erik M. Schaefer, Susan Cornell-Kennon, Eric Lamsa, Erik McCauley, Eric Berg, Jordan Fishman, Barbara Imperiali. Optimized Sox-based sensors for continuous, homogeneous, and quantitative monitoring of protein kinase activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-024. doi:10.1158/1538-7445.AM2017-LB-024