Peter C. Simons

Purification of glutathione S-transferases from human liver by glutathione-affinity chromatography.

Abstract Multiple forms of glutathione S -transferase (EC 2.5.1.18), a family of proteins involved both with bilirubin transport and with the detoxification of electrophiles, have been purified from human liver using a scheme which employs an affinity chromatography column prepared by coupling glutathione to epoxy-activated Sepharose. This procedure offers a convenient method for obtaining highly purified transferases in good yields.

[29] Purification of glutathione S-transferases by glutathione-affinity chromatography

Publisher Summary The glutathione S-transferases from a variety of sources have been purified by the use of conventional chromatographic procedures, as well as by affinity chromatography. When affinity chromatography is used, it is generally necessary to apply additional separation procedures because most of the glutathione S -transferases contain several similar forms of the enzyme. This chapter describes the preparation of a glutathione-affinity chromatography column that has been successfully used for the purification of the glutathiones-transferases from the human liver. This procedure can serve as the initial step in the isolation of the family of glutathione S -transferases from a crude mixture, or it may be used as a final purification step after the several forms of the glutathione S -transferases are separated from one another.

Estrogen receptors alpha (ESR1) and beta (ESR2) are expressed in circulating human lymphocytes.

Bone marrow thymocytes in part mediate the bone-preserving effects of estrogen by decreasing their production of osteoclast growth factors such as interleukin-1 and -6 and tumor necrosis factor alpha in the presence of physiological amounts of estradiol. Although several in vitro studies implicate the T-lymphocyte as a candidate mediator of estrogen signaling in the skeleton, whether these cells or any lymphocytes ordinarily express one or both nuclear estrogen receptors was previously unresolved. The purpose of our investigation was therefore to ascertain, by using real-time PCR, immmunoblotting, and cytometric techniques, if any of the nuclear estrogen receptors could be detected in normal peripheral blood mononuclear cells (PBMNC) collected from healthy volunteers. The results of immunoblotting experiments revealed that both estrogen receptor alpha (ESR1) and beta (ESR2) proteins are expressed in nuclei, but not in the cytoplasm of PBMNC harvested from all of the 15 healthy male and female volunteers (aged 23–50 years) we tested. PBMNCs contained mRNA coding for the two major full-length isoforms of ESR2 and the expression of ESR2 protein was localized within a lymphocyte subpopulation by cytometric analysis. Our data provide further evidence that lymphocytes and monocytes are responsive to estrogen and underscore its importance in modulating the immune response, as well as the vascular and skeletal health of men and women.

Flow Cytometry for Real-Time Measurement of Guanine Nucleotide Binding and Exchange by Ras-like GTPases

Ras-like small GTPases cycle between GTP-bound active and GDP-bound inactive conformational states to regulate diverse cellular processes. Despite their importance, detailed kinetic or comparative studies of family members are rarely undertaken due to the lack of real-time assays measuring nucleotide binding or exchange. Here we report a bead-based flow cytometric assay that quantitatively measures the nucleotide binding properties of glutathione-S-transferase (GST) chimeras for prototypical Ras family members Rab7 and Rho. Measurements are possible in the presence or absence of Mg(2+), with magnesium cations principally increasing affinity and slowing nucleotide dissociation rates 8- to 10-fold. GST-Rab7 exhibited a 3-fold higher affinity for guanosine diphosphate (GDP) relative to guanosine triphosphate (GTP) that is consistent with a 3-fold slower dissociation rate of GDP. Strikingly, GST-Rab7 had a marked preference for GTP with ribose ring-conjugated BODIPY FL. The more commonly used gamma-NH-conjugated BODIPY FL GTP analogue failed to bind to GST-Rab7. In contrast, both BODIPY analogues bound equally well to GST-RhoA and GST-RhoC. Comparisons of the GST-Rab7 and GST-RhoA GTP binding pockets provide a structural basis for the observed binding differences. In sum, the flow cytometric assay can be used to measure nucleotide binding properties of GTPases in real time and to quantitatively assess differences between GTPases.

Discovery of Regulators of Receptor Internalization with High-Throughput Flow Cytometry

We developed a platform combining fluorogen-activating protein (FAP) technology with high-throughput flow cytometry to detect real-time protein trafficking to and from the plasma membrane in living cells. The hybrid platform facilitates drug discovery for trafficking receptors such as G protein-coupled receptors and was validated with the β2-adrenergic receptor (β2AR) system. When a chemical library containing ∼1200 off-patent drugs was screened against cells expressing FAP-tagged β2ARs, all 33 known β2AR-active ligands in the library were successfully identified, together with a number of compounds that might regulate receptor internalization in a nontraditional manner. Results indicated that the platform identified ligands of target proteins regardless of the associated signaling pathway; therefore, this approach presents opportunities to search for biased receptor modulators and is suitable for screening of multiplexed targets for improved efficiency. The results revealed that ligands may be biased with respect to the rate or duration of receptor internalization and that receptor internalization may be independent of activation of the mitogen-activated protein kinase pathway.

Glutathione-S-transferase-green fluorescent protein fusion protein reveals slow dissociation from high site density beads and measures free GSH

Glutathione, a ubiquitous tripeptide, is an important cellular constituent, and measurement of reduced and oxidized glutathione is a measure of the redox state of cells. Glutathione‐S‐transferase (GST) fusion proteins bind naturally to beads derivatized with glutathione, and elution of such bead‐bound fusion proteins with buffer containing millimolar glutathione is a commonly used method of protein purification. Many protein–protein interactions have been established by using GST fusion proteins and measuring binding of fusion protein binding partners by GST pulldown assays, usually monitored by Western blot methodology.

Simultaneous in vitro Molecular Screening of Protein - Peptide Interactions by Flow Cytometry Using Six Bcl-2 Family Proteins as Examples

The B-cell lymphoma-2 (Bcl-2) family contains six antiapoptotic members, each with a hydrophobic pocket in which Bcl-2 homology region 3 (BH3) helices bind. This binding quenches apoptotic signals from activated BH3 family members. Many tumor cells either have increased expression of one of these six proteins or become overexpressed under treatment. Six fusion proteins made up of glutathione-S-transferase and each of the Bcl-2 members are bound individually to six glutathione bead sets, each set being easily distinguished by its different intensity of red fluorescence. The coated bead sets are washed, combined and incubated with green fluorescent Bim-BH3 peptide and a small molecule in 10-μl wells for 1 h. The green fluorescence signal for each bead set is resolved, and selective inhibitors are expected to reduce the signal for individual bead sets. Each 384-well plate is analyzed in 12 min, measuring 200 of 2,000 beads (∼10%) of each type per well.

Duplexed, bead-based competitive assay for inhibitors of protein kinases.

Many cellular signal transduction cascades have protein kinases as critical components. Small molecule protein kinase inhibitors can be effective as laboratory probes and drugs. Methods that allow two or more kinases to be evaluated simultaneously for inhibition by a small molecule would allow unequivocal tests of specificity and selectivity of action of the small molecule.

Some Mechanistic Insights into GPCR Activation from Detergent‐Solubilized Ternary Complexes on Beads

The binding of full and partial agonist ligands (L) to G protein-coupled receptors (GPCRs) initiates the formation of ternary complexes with G proteins [ligand-receptor-G protein (LRG) complexes]. Cyclic ternary complex models are required to account for the thermodynamically plausible complexes. It has recently become possible to assemble solubilized formyl peptide receptor (FPR) and beta(2)-adrenergic receptor (beta(2)AR) ternary complexes for flow cytometric bead-based assays. In these systems, soluble ternary complex formation of the receptors with G proteins allows direct quantitative measurements which can be analyzed in terms of three-dimensional concentrations (molarity). In contrast to the difficulty of analyzing comparable measurements in two-dimensional membrane systems, the output of these flow cytometric experiments can be analyzed via ternary complex simulations in which all of the parameters can be estimated. An outcome from such analysis yielded lower affinity for soluble ternary complex assembly by partial agonists compared with full agonists for the beta(2)AR. In the four-sided ternary complex model, this behavior is consistent with distinct ligand-induced conformational states for full and partial agonists. Rapid mix flow cytometry is used to analyze the subsecond dynamics of guanine nucleotide-mediated ternary complex disassembly. The modular breakup of ternary complex components is highlighted by the finding that the fastest step involves the departure of the ligand-activated GPCR from the intact G protein heterotrimer. The data also show that, under these experimental conditions, G protein subunit dissociation does not occur within the time frame relevant to signaling. The data and concepts are discussed in the context of a review of current literature on signaling mechanism based on structural and spectroscopic (FRET) studies of ternary complex components.

A Pan-GTPase Inhibitor as a Molecular Probe.

Overactive GTPases have often been linked to human diseases. The available inhibitors are limited and have not progressed far in clinical trials. We report here a first-in-class small molecule pan-GTPase inhibitor discovered from a high throughput screening campaign. The compound CID1067700 inhibits multiple GTPases in biochemical, cellular protein and protein interaction, as well as cellular functional assays. In the biochemical and protein interaction assays, representative GTPases from Rho, Ras, and Rab, the three most generic subfamilies of the GTPases, were probed, while in the functional assays, physiological processes regulated by each of the three subfamilies of the GTPases were examined. The chemical functionalities essential for the activity of the compound were identified through structural derivatization. The compound is validated as a useful molecular probe upon which GTPase-targeting inhibitors with drug potentials might be developed.