SH2-PLA: a sensitive in-solution approach for quantification of modular domain binding by proximity ligation and real-time PCR.

BACKGROUND: There is a great interest in studying phosphotyrosine dependent protein-protein interactions in tyrosine kinase pathways that play a critical role in many aspects of cellular function. We previously established SH2 profiling, a phosphoproteomic approach based on membrane binding assays that utilizes purified Src Homology 2 (SH2) domains as a molecular tool to profile the global tyrosine phosphorylation state of cells. However, in order to use this method to investigate SH2 binding sites on a specific target in cell lysate, additional procedures such as pull-down or immunoprecipitation which consume large amounts of sample are required. RESULTS: We have developed PLA-SH2, an alternative in-solution modular domain binding assay that takes advantage of Proximity Ligation Assay and real-time PCR. The SH2-PLA assay utilizes oligonucleotide-conjugated anti-GST and anti-EGFR antibodies recognizing a GST-SH2 probe and cellular EGFR, respectively. If the GST-SH2 and EGFR are in close proximity as a result of SH2-phosphotyrosine interactions, the two oligonucleotides are brought within a suitable distance for ligation to occur, allowing for efficient complex amplification via real-time PCR. The assay detected signal across at least 3 orders of magnitude of lysate input with a linear range spanning 1-2 orders and a low femtomole limit of detection for EGFR phosphotyrosine. SH2 binding kinetics determined by PLA-SH2 showed good agreement with established far-Western analyses for A431 and Cos1 cells stimulated with EGF at various times and doses. Further, we showed that PLA-SH2 can survey lung cancer tissues using 1 µl lysate without requiring phospho-enrichment. CONCLUSIONS: We showed for the first time that interactions between SH2 domain probes and EGFR in cell lysate can be determined in a microliter-scale assay using SH2-PLA. The obvious benefit of this method is that the low sample requirement allows detection of SH2 binding in samples which are difficult to analyze using traditional protein interaction assays. This feature along with short assay runtime makes this method a useful platform for the development of high throughput assays to determine modular domain-ligand interactions which could have wide-ranging applications in both basic and translational cancer research.

Fast rebinding increases dwell time of Src homology 2 (SH2)-containing proteins near the plasma membrane.

Receptor tyrosine kinases (RTKs) control a host of biological functions by phosphorylating tyrosine residues of intracellular proteins upon extracellular ligand binding. The phosphotyrosines (p-Tyr) then recruit a subset of ∼100 Src homology 2 (SH2) domain-containing proteins to the cell membrane. The in vivo kinetics of this process are not well understood. Here we use total internal reflection (TIR) microscopy and single-molecule imaging to monitor interactions between SH2 modules and p-Tyr sites near the cell membrane. We found that the dwell time of SH2 modules within the TIR illumination field is significantly longer than predictions based on chemical dissociation rate constants, suggesting that SH2 modules quickly rebind to nearby p-Tyr sites after dissociation. We also found that, consistent with the rebinding model, the effective diffusion constant is negatively correlated with the respective dwell time for different SH2 domains and the dwell time is positively correlated with the local density of RTK phosphorylation. These results suggest a mechanism whereby signal output can be regulated through the spatial organization of multiple binding sites, which will prompt reevaluation of many aspects of RTK signaling, such as signaling specificity, mechanisms of spatial control, and noise suppression.

Bisphenol A significantly modulates long-term depression in the hippocampus as observed by multi-electrode system.

OBJECTIVE: Low dose exposure to endocrine disrupters (environmental chemicals) may induce hormone-like effects on wildlife and humans. bisphenol A (BPA) might disturb the neuronal signaling regulated by endogenous estrogens. We investigated the rapid modulation effects of 10nM BPA, a typical endocrine disruptor, on long-term depression (LTD) of adult rat hippocampal slices. METHOD: LTD was induced by a transient perfusion of 30 µM NMDA for 3 min. And measured with multielectrode probes. RESULTS: A 30 min perfusion of 10 nM BPA rapidly enhanced LTD in CA1, however, BPA suppressed LTD in dentate gyrus (DG). An ERRγ antagonist, 4-OH-tamoxifen, suppressed LTD in CA1 and DG. Inhibitor of estrogen receptor ICI 182,780 did not disturb BPA effects. On the other hand, tributyltin (TBT), another endocrine disruptor, did not have any effect on LTD in CA1 and DG. CONCLUSION: ERRγ, but not estrogen receptors, is a high affinity BPA receptor in LTD processes, since the effect of BPA on LTD was suppressed by an ERRγ antagonist. A possible mechanisms of BPA-induced enhancement of LTD could be described with ERRγ, MAPK activation and phosphorylation of MMDA receptors.

Modulation of synaptic plasticity by brain estrogen in the hippocampus.

The hippocampus is a center for learning and memory as well as a target of Alzheimer's disease in aged humans. Synaptic modulation by estrogen is essential to understand the molecular mechanisms of estrogen replacement therapy. Because the local synthesis of estrogen occurs in the hippocampus of both sexes, in addition to the estrogen supply from the gonads, its functions are attracting much attention. Hippocampal estrogen modulates memory-related synaptic plasticity not only slowly but also rapidly. Slow actions of 17ß-estradiol (17ß-E2) occur via classical nuclear receptors (ERα or ERß), while rapid E2 actions occur via synapse-localized ERα or ERß. Elevation or decrease of the E2 concentration changes rapidly the density and morphology of spines in CA1-CA3 neurons. ERα, but not ERß, drives this enhancement/suppression of spinogenesis. Kinase networks are involved downstream of ERα. The long-term depression but not the long-term potentiation is modulated rapidly by changes of E2 level. Determination of the E2 concentration in the hippocampus is enabled by mass-spectrometry in combination with derivatization methods. The E2 level in the hippocampus is as high as approx. 8 nM for the male and 0.5-2 nM for the female, which is much higher than that in circulation. Therefore, hippocampus-derived E2 plays a major role in modulation of synaptic plasticity. Many hippocampal slice experiments measure the restorative effects of E2 by supplementation of E2 to E2-depleted slices. Accordingly, isolated slice experiments can be used as in vitro models of in vivo estrogen replacement therapy for ovariectomized female animals with depleted circulating estrogen.

Perinatal Exposure of Bisphenol A Differently Affects Dendritic Spines of Male and Female Grown-Up Adult Hippocampal Neurons.

Perinatal exposure to Bisphenol A (BPA) at a very low dose may modulate the development of synapses of the hippocampus during growth to adulthood. Here, we demonstrate that perinatal exposure to 30 µg BPA/kg per mother's body weight/day significantly altered the dendritic spines of the grownup rat hippocampus. The density of the spine was analyzed by imaging of Lucifer Yellow-injected CA1 glutamatergic neurons in adult hippocampal slices. In offspring 3-month male hippocampus, the total spine density was significantly decreased by BPA exposure from 2.26 spines/µm (control, no BPA exposure) to 1.96 spines/µm (BPA exposure). BPA exposure considerably changed the normal 4-day estrous cycle of offspring 3-month females, resulting in a 4∼5 day estrous cycle with 2-day estrus stages in most of the subjects. In the offspring 3-month female hippocampus, the total spine density was significantly increased by BPA exposure at estrus stage from 2.04 spines/µm (control) to 2.25 spines/µm (BPA exposure). On the other hand, the total spine density at the proestrus stage was moderately decreased from 2.33 spines/µm (control) to 2.19 spines/µm (BPA exposure). Thus, after the perinatal exposure to BPA, the total spine density in males became lower than that in females. Concerning the BPA effect on the morphology of spines, the large-head spine was significantly changed with its significant decrease in males and moderate change in females.

Rapid modulation of synaptic plasticity by estrogens as well as endocrine disrupters in hippocampal neurons.

Estrogen modulates memory-related synaptic plasticity not only slowly but also rapidly in the hippocampus. However, molecular mechanisms of the rapid action are yet largely unknown. We here describe rapid modulation of representative synaptic plasticity, i.e., long-term depression (LTD), long-term potentiation (LTP) and spinogenesis, by 17beta-estradiol, selective estrogen agonists as well as endocrine disrupters. The authors demonstrated that 1-10 nM estradiol induced rapid enhancement of LTD within 1 h in not only CA1 but also CA3 and dentate gyrus (DG). On the other hand, the modulation of LTP by estradiol was not statistically significant. The total density of spines was increased in CA1 pyramidal neurons, within 2 h after application of estradiol. The total density of thorns (postsynaptic spine-like structure) was, however, decreased by estradiol in CA3 pyramidal neurons. Both the increase of spines in CA1 and the decrease of thorns in CA3 were completely suppressed by Erk MAP kinase inhibitor. Only ERalpha agonist PPT induced the same enhancement/suppression effect as estradiol on both LTD and spinogenesis in CA1 and CA3. ERbeta agonist DPN induced completely different results. ERalpha localized in spines and presynapses of principal glutamatergic neurons in CA1, CA3 and DG. The same ERalpha was also located in nuclei and cytoplasm. Identification of ERalpha was successfully performed using purified RC-19 antibody. Non-purified ERalpha antisera, however, reacted significantly with unknown proteins, resulting in wrong immunostaining different from real ERalpha distribution. An issue of 'endocrine disrupters' (1-100 nM low dose of environmental chemicals), which are artificial xenoestrogenic or anti-xenoestrogenic substances, has emerged as a social and environmental problem. Endocrine disrupters were found to significantly modulate LTD and spinogenesis. Bisphenol A (BPA) and diethylstilbestrol (DES) enhanced LTD in CA1 and CA3. The total spine density was significantly increased by BPA and DES in CA1. Most probable receptors for BPA and DES may be Ralpha; however, other receptors might also be involved.

Estrogen synthesis in the brain--role in synaptic plasticity and memory.

Estrogen and androgen are synthesized from cholesterol locally in hippocampal neurons of adult animals. These neurosteroids are synthesized by cytochrome P450s and hydroxysteroid dehydrogenases (HSDs) and 5alpha-reductase. The expression levels of enzymes are as low as 1/200-1/50,000 of those in endocrine organs, however these numbers are high enough for local synthesis. Localization of P450(17alpha), P450arom, 17beta-HSD and 5alpha-reductase is observed in principal glutamatergic neurons in CA1, CA3 and the dendate gyrus. Several nanomolar levels of estrogen and androgen are observed in the hippocampus. Estrogen modulates memory-related synaptic plasticity not only slowly but also rapidly in the hippocampus. Rapid action of 17beta-estradiol via membrane receptors is demonstrated for spinogenesis and long-term depression (LTD). The enhancement of LTD by 1-10nM estradiol occurs within 1 h. The density of spine is increased in CA1 pyramidal neurons within 2h after application of estradiol. The density of spine-like structure is, however, decreased by estradiol in CA3 pyramidal neurons. ERalpha, but not ERbeta, induces the same enhancement/suppression effects on both spinogenesis and LTD.

Local production of sex hormones and their modulation of hippocampal synaptic plasticity.

It is believed that sex hormones are synthesized in the gonads and reach the brain via the blood circulation. In contrast with this view, the authors have demonstrated that sex hormones are also synthesized locally in the hippocampus and that these steroids act rapidly to modulate neuronal synaptic plasticity. The authors demonstrated that estrogens are locally synthesized from cholesterol through dehydroepiandrosterone and testosterone in adult hippocampal neurons. Significant expression of mRNA for P450(17alpha), P450arom, and other steroidogenic enzymes was demonstrated. Localization of P450(17alpha) and P450arom was observed in synapses of principal neurons. In contrast to the slow action of gonadal estradiol, hippocampal neuron-derived estradiol may act locally and rapidly within the neurons. For example, 1 to 10 nM estradiol rapidly enhances long-term depression (LTD). The density of thin spines is selectively increased within two hours upon application of estradiol in pyramidal neurons. Estrogen receptor ERalpha agonist has the same enhancing effect as estradiol on both LTD and spinogenesis. Localization of ERalpha in spines in addition to nuclei of principal neurons implies that synaptic ERalpha is responsible for rapid modulation of synaptic plasticity by endogenous estradiol. Activin A, a peptide sex hormone, may also play a role as a local endogenous modulator of synaptic plasticity.

Expression and Purification of SH2 Domains Using Baculovirus Expression System.

Recombinant proteins expressed in bacteria are sometimes insoluble, aggregated, and incorrectly folded. For those Src homology 2 (SH2) domains that are insoluble in bacteria, baculovirus-insect cell expression systems can be an alternative to produce soluble and functionally active proteins. We describe a protocol for cloning and purification of GST-tagged SH2 domains using the Bac-to-Bac baculovirus expression system.

Functionally Altered SH2 Domains for Biochemical Studies: Loss-of-Function Mutant and Domain Concatenation.

Recombinant modular protein domains have been a convenient proteomics tool for deciphering protein-protein interactions and elucidating the role of protein modifications in cell signaling. To obtain reliable experimental data, these protein domain probes require sufficient specificity and sensitivity. Since naturally evolved protein domains do not always have optimal biochemical characteristics for in vitro assays, functional alterations such as improved affinity are sometimes needed. In this chapter, we describe preparation of loss-of-function and concatenated (tandem) SH2 domains that should be widely applicable to both high- and low-throughput phosphoproteomics studies.

Expression and Production of SH2 Domain Proteins.

The Src Homology 2 (SH2) domain lies at the heart of phosphotyrosine signaling, coordinating signaling events downstream of receptor tyrosine kinases (RTKs), adaptors, and scaffolds. Over a hundred SH2 domains are present in mammals, each having a unique specificity which determines its interactions with multiple binding partners. One of the essential tools necessary for studying and determining the role of SH2 domains in phosphotyrosine signaling is a set of soluble recombinant SH2 proteins. Here we describe methods, based on a broad experience with purification of all SH2 domains, for the production of SH2 domain proteins needed for proteomic and biochemical-based studies such as peptide arrays, mass-spectrometry, protein microarrays, reverse-phase microarrays, and high-throughput fluorescence polarization (HTP-FP). We describe stepwise protocols for expression and purification of SH2 domains using GST or poly His-tags, two widely adopted affinity tags. In addition, we address alternative approaches, challenges, and validation studies for assessing protein quality and provide general characteristics of purified human SH2 domains.

Estimation of membrane permeability and intracellular diffusion coefficients.

We propose a method for estimating membrane permeability and the intracellular diffusion coefficient using pulsed-gradient spin-echo measurement in combination with numerical simulation. The diffusion signal attenuation of leukocytes was measured with 4pi(2)q(2)(Delta-delta/3) values up to 6000 s/mm(2). For numerical simulations, the cell was modeled as a 15 x 15 microm(2) square with various membrane permeabilities and intracellular diffusion coefficients. Minimization of the difference in signal attenuations between the measurement and the simulation enabled estimations of these unknown parameters for the leukocytes. A cell sample of 2.17 x 10(8) cells/mL had a membrane permeability and an intracellular diffusion coefficient of 23 mum/s and 8.9 x 10(-4) mm(2)/s, respectively.

Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases.

While the affinities and specificities of SH2 domain-phosphotyrosine interactions have been well characterized, spatio-temporal changes in phosphosite availability in response to signals, and their impact on recruitment of SH2-containing proteins in vivo, are not well understood. To address this issue, we used three complementary experimental approaches to monitor phosphorylation and SH2 binding in human A431 cells stimulated with epidermal growth factor (EGF): 1) phospho-specific mass spectrometry; 2) far-Western blotting; and 3) live cell single-molecule imaging of SH2 membrane recruitment. Far-Western and MS analyses identified both well-established and previously undocumented EGF-dependent tyrosine phosphorylation and binding events, as well as dynamic changes in binding patterns over time. In comparing SH2 binding site phosphorylation with SH2 domain membrane recruitment in living cells, we found in vivo binding to be much slower. Delayed SH2 domain recruitment correlated with clustering of SH2 domain binding sites on the membrane, consistent with membrane retention via SH2 rebinding.

Acquisition of ischemic tolerance by repetitive transcranial magnetic stimulation in the rat hippocampus.

We investigated the acquisition of ischemic tolerance in the rat hippocampus using repetitive transcranial magnetic stimulation (rTMS). Rats received 1000 pulses/day for 7 days, and the field excitatory postsynaptic potentials were measured in the hippocampal CA1. After slices were exposed to ischemic conditions, long-term potentiation (LTP) was induced. The LTP of the stimulated group was enhanced compared with the LTP of the sham control group in each ischemic condition, suggesting that rTMS has the potential to protect hippocampal function from ischemia.

Acute repetitive transcranial magnetic stimulation reactivates dopaminergic system in lesion rats.

Repetitive transcranial magnetic stimulation (rTMS) offers potential benefit as a therapeutic treatment for neurological and psychiatric disorders. However, the mechanism underlying the therapeutic effects of rTMS is still unknown. In this study, we investigated the rescue effects of rTMS in the lesioned rats by administering the neurotoxin MPTP (l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine). The rats received rTMS (10 trains of 25 pulses/s for 8 s) 48 h after MPTP injection, and tyrosine hydroxylase (TH) and NeuN expressions were investigated in the substantia nigra. The functional observational battery-hunched posture score for the MPTP-rTMS group was significantly lower and the number of rearing events was higher compared with the MPTP-sham group, these behavioral parameters revert to control levels. These results suggest that rTMS treatment reactivates the dopaminergic system in lesion rats.

Development of energy and time parameters in the walking of healthy human infants.

Sixteen infants were analyzed longitudinally from the onset of independent walking to 3 years of age using time parameters, speed and energy recovery. Considerable variation and irregularities were observed in many parameters of infant walking, especially until 13 months of age when infants had difficulty in walking steadily step by step. Infant walking until 3 years of age was characterized by a small braking duration, caused mainly by the forward inclination of the trunk, a large relative stance phase duration, which maintained static balance, short stride length, due to the small range of the lower limb joint angle, and a small recovery of external energy. These characteristics were also predominantly evident until 13 months of age. The small recovery characteristic of infants was caused by flexed lower limb joints, pronounced irregularities in energy output, and in younger infants, slow speed. The maximum recovery up until 2 years of age, though smaller than in adults, appeared at about 0.45 dimensionless speed, which is about the same speed that adults in particular naturally and at which their maximum recovery appeared. The forward inclination of the trunk and the lower limb joint angle, influenced the development of many characteristics of bipedal walking.

Estrogen receptor KO mice study on rapid modulation of spines and long-term depression in the hippocampus.

Rapid modulation of hippocampal synaptic plasticity through synaptic estrogen receptors is an essential topic. We analyzed estradiol-induced modulation of CA1 dendritic spines using adult male ERαKO and ERßKO mice. A 2h treatment of estradiol particularly increased the density of middle-head spines (diameter 0.3-0.4 µm) in wild type mouse hippocampal slices. The enhancement of spinogenesis was completely suppressed by MAP kinase inhibitor. Estradiol-induced increase in middle-head spines was observed in ERßKO mice (which express ERα), but not in ERαKO, indicating that ERα is necessary for the spinogenesis. Direct observation of the dynamic estradiol-induced enhancing effect on rapid spinogenesis was performed using time-lapse imaging of spines in hippocampal live slices from yellow fluorescent protein expressed mice. Both appearance and disappearance of spines occurred, and the number of newly appeared spines was significantly greater than that of disappeared spines, resulting in the net increase of the spine density within 2h. As another type of synaptic modulation, we observed that estradiol rapidly enhanced N-methyl-D-aspartate (NMDA)-induced long-term depression (LTD) in CA1 of the wild type mouse hippocampus. In contrast, estradiol did not enhance NMDA-LTD in ERαKO mice, indicating the involvement of ERα in the estrogen signaling. This article is part of a Special Issue entitled SI: Brain and Memory.

The effect of repetitive transcranial magnetic stimulation on long-term potentiation in rat hippocampus depends on stimulus intensity.

We investigated the effect of repetitive transcranial magnetic stimulation (rTMS) on long-term potentiation (LTP) in the rat hippocampus. Rats were magnetically stimulated at a rate of 1000 pulses/day for 7 days by a round coil, in which the peak magnetic fields at the center of the coil were 0.75 and 1.00 T. LTP enhancement was observed only in the 0.75-T rTMS group, while no change was observed in the 1.00-T rTMS group. These results suggest that the effect of rTMS on LTP depends on the stimulus intensity.

Control of orientation of rat Schwann cells using an 8-T static magnetic field.

Schwann cells aid in neuronal regeneration in the peripheral nervous system via guiding the regrowth of axons. In this study, we investigated the magnetic orientation of Schwann cells, and of a mixture of Schwann cells and collagen, after an 8-tesla magnetic field exposure. We obtained cultured Schwann cells from dissected sciatic nerves of neonatal rats. After 60 h of magnetic field exposure, Schwann cells oriented parallel to the magnetic fields. In contrast, the mixture of Schwann cells and collagen, Schwann cells oriented in the direction perpendicular to the magnetic field after 2 h of magnetic field exposure. In this case, Schwann cells aligned along the collagen fiber oriented by magnetic fields. The magnetic control of Schwann cell alignment is useful in medical engineering applications such as nerve regeneration.

A new method to destruct targeted cells using magnetizable beads and pulsed magnetic force.

We investigated a new method to destruct targeted cells using magnetizable beads and pulsed magnetic force. The cells were combined with the beads by an antigen-antibody reaction (cell/bead/antibody complex), aggregated by a magnet, and stimulated by a magnetic stimulator. The viability of the aggregated and stimulated cell/bead/antibody complexes was significantly decreased, and the cells were destructed by the penetration of the beads into the cells or rupturing of the cells by the beads. These results suggest that magnetic aggregation and pulsed magnetic stimulation can effectively damage only the cells targeted by an antigen-antibody reaction.