Characterization of 5-HT1A receptors and their complexes with G-proteins in budded baculovirus particles using fluorescence anisotropy of Bodipy-FL-NAN-190.

Bodipy-FL-NAN-190 was found to be well suited for characterization of ligand binding to 5-HT1A receptors expressed in budded baculovirus particles, as binding is accompanied by large increases in fluorescence intensity and anisotropy. This ligand appears to bind rapidly (t1/2,ass<1 min), reversibly (t1/2,diss∼6 min) and has high affinity (Kd=0.30 ± 0.13 nM). This fluorescence anisotropy assay based on Bodipy-FL-NAN-190 binding to baculovirus particles was also a suitable assay system for the pharmacological characterization of non-labelled serotonergic ligands, as well as being sensitive to the presence of G-proteins and guanine nucleotides. Coexpression of αi subunits of human G-proteins in baculovirus particles resulted in the appearance of significantly greater proportion of nucleotide sensitive high affinity agonist binding sites. There were no significant differences between αi1 and αi3 subtypes, while ligand binding in the presence of αi2 had higher sensitivity to GDP and Mn(2+).

Biarsenical ligands bind to endogenous G-protein α-subunits and enable allosteric sensing of nucleotide binding.

BACKGROUND: Heterotrimeric G-proteins relay extracellular signals to intracellular effector proteins. Multiple methods have been developed to monitor their activity; including labeled nucleotides and biosensors based on genetically engineered G-proteins. Here we describe a method for monitoring unlabeled nucleotide binding to endogenous G-proteins α-subunits in a homogeneous assay based on the interaction of 4',5'-bis(1,2,3-dithioarsolan-2-yl)-2',7'-difluorofluorescein (F2FlAsH) with G-protein α-subunits. RESULTS: The biarsenic fluorescent ligand F2FlAsH binds to various wild-type G-protein α-subunits (αi1, αi2, αi3, αslong, αsshort, αolf, αq, α13) via high affinity As-cysteine interactions. This allosteric label enables real time monitoring of the nucleotide bound states of α-subunits via changes in fluorescence anisotropy and intensity of their F2FlAsH-complexes. We have found that different α-subunits displayed different signal amplitudes when interacting with F2FlAsH, being more sensitive to nucleotide binding to αi, αs, αolf and αq than to α13. Addition of nucleotides to F2FlAsH-labeled α-subunits caused concentration-dependent effects on their fluorescence anisotropy. pEC50 values of studied nucleotides depended on the subtype of the α-subunit and were from 5.7 to 8.2 for GTPγS, from 5.4 to 8.1 for GppNHp and from 4.8 to 8.2 for GDP and lastly up to 5.9 for GMP. While GDP and GMP increased the fluorescence anisotropy of F2FlAsH complexes with αi-subunits, they had the opposite effect on the other αßγM complexes studied. CONCLUSIONS: Biarsenical ligands interact allosterically with endogenous G-protein α-subunits in a nucleotide-sensitive manner, so the presence or absence of guanine nucleotides has an effect on the fluorescence anisotropy, intensity and lifetime of F2FlAsH-G-protein complexes.

Characterization of heterotrimeric nucleotide-depleted Gα(i)-proteins by Bodipy-FL-GTPγS fluorescence anisotropy.

Recombinant heterotrimeric G-protein α(i1), α(i2) and α(i3) subunits were purified in GDP-depleting conditions by affinity chromatography using StrepII-tagged ß1γ2 subunits. Real-time monitoring of fluorescence anisotropy of Bodipy-FL-GTPγS was used for characterization of nucleotide binding properties and inactivation of the purified proteins. All GDP-depleted α(i) were unstable at room temperature and therefore nucleotide binding could be characterized only in a nonequilibrium state. In comparison to Mg²âº, Mn²âº inhibited nucleotide binding to all α(i)-heterotrimers studied and accelerated nucleotide release. Mn²âº had stabilizing effect on the nucleotide free state of the α(i1) subunit, whereas both Mn²âº as well as G-protein activation by mastoparan destabilized the α(i2) subunit.

Millimolar Mn2+ influences agonist binding to 5-HT1A receptors by inhibiting guanosine nucleotide binding to receptor-coupled G-proteins.

Manganese is an essential trace element but its overexposure causes poisoning (called manganism) that shares several symptoms with Parkinson's disease, but with a mechanism that is still not well understood: in addition to involvement of the dopaminergic system, both serotonergic and peptiergic systems have been implicated. In the present report we have studied the influence of Mn(2+) on 5-HT(1A) receptor signaling complexes in rat brain and found that Mn(2+) in millimolar concentration caused an increase of high-affinity agonist binding to rat hippocampal membranes in comparison with experiments in the presence of Mg(2+), but not in rat cortical membranes and in Sf9 cell membranes expressing 5-HT(1A) receptors and G(i1) heterotrimers. Activation of G proteins with 30µM GTPγS turned all 5-HT(1A) receptors in these preparations into a low-affinity state for agonist binding in the presence of 1mM Mg(2+), but not in the presence of 1mM Mn(2+) in rat hippocampal membranes. However, if 1µM GTPγS was used for G protein activation, a substantial amount of high affinity agonist binding was detected in the presence of Mn(2+) also in cortical membranes and Sf9 cells, but not with Mg(2+) or EDTA. Comparison of the abilities of GDP and GTPγS to modulate high affinity agonist binding to 5-HT(1A) receptors indicated that both nucleotides were almost 10-fold less potent in the presence of MnCl(2) compared to MgCl(2). This means that by inhibiting guanosine nucleotide binding to G proteins in complex with 5-HT(1A) receptors, Mn(2+) acts as an enhancer for agonist binding and signal transduction. As the influence of Mn(2+) resembles the hypersensitivity of dopaminergic system in Parkinsonial models, it can be proposed that at least some symptoms of manganism are connected with a change of signal transduction complex caused by manganese-nucleotide complexes.

Medical bioremediation: a concept moving toward reality.

Abstract A major driver of aging is catabolic insufficiency, the inability of our bodies to break down certain substances that accumulate slowly throughout the life span. Even though substance buildup is harmless while we are young, by old age the accumulations can reach a toxic threshold and cause disease. This includes some of the most prevalent diseases in old age-atherosclerosis and macular degeneration. Atherosclerosis is associated with the buildup of cholesterol and its oxidized derivatives (particularly 7-ketocholesterol) in the artery wall. Age-related macular degeneration is associated with carotenoid lipofuscin, primarily the pyridinium bisretinoid A2E. Medical bioremediation is the concept of reversing the substance accumulations by using enzymes from foreign species to break down the substances into forms that relieve the disease-related effect. We report on an enzyme discovery project to survey the availability of microorganisms and enzymes with these abilities. We found that such microorganisms and enzymes exist. We identified numerous bacteria having the ability to transform cholesterol and 7-ketocholesterol. Most of these species initiate the breakdown by same reaction mechanism as cholesterol oxidase, and we have used this enzyme directly to reduce the toxicity of 7-ketocholesterol, the major toxic oxysterol, to cultured human cells. We also discovered that soil fungi, plants, and some bacteria possess peroxidase and carotenoid cleavage oxygenase enzymes that effectively destroy with varied degrees of efficiency and selectivity the carotenoid lipofuscin found in macular degeneration.