Roles of genistein in learning and memory during aging and neurological disorders.

Genistein (GEN) is a non-steroidal phytoestrogen that belongs to the isoflavone class. It is abundantly found in soy. Soy and its products are used as food components in many countries including India. The present review is focused to address roles of GEN in brain functions in the context of learning and memory as a function of aging and neurological disorders. Memory decline is one of the most disabling features observed during normal aging and age-associated neurodegenerative disorders namely Alzheimer's disease (AD) and Parkinson's disease (PD), etc. Anatomical, physiological, biochemical and molecular changes in the brain with advancement of age and pathological conditions lead to decline of cognitive functions. GEN is chemically comparable to estradiol and binds to estrogen receptors (ERs). GEN acts through ERs and mimics estrogen action. After binding to ERs, GEN regulates a plethora of brain functions including learning and memory; however detailed study still remains elusive. Due to the neuroprotective, anti-oxidative and anti-inflammatory properties, GEN is used to restore or improve memory functions in different animal models and humans. The present review may be helpful to understand roles of GEN in learning and memory during aging and neurological disorders, its direction of research and therapeutic perspectives.

Neuromodulating roles of estrogen and phytoestrogens in cognitive therapeutics through epigenetic modifications during aging.

Estrogen (E2) plays important role in regulating hippocampal learning and memory. The decline of E2 after menopause affects learning and memory and increases the risk of neurodegenerative diseases like Alzheimer's disease (AD). Additionally, from the estrogen receptor (ER) mediated gene regulation; E2 also regulates gene expression at the transcriptional and posttranscriptional levels through epigenetic modifications. E2 recruits a number of proteins called co-regulators at the promoter region of genes. These co-regulators act as chromatin modifiers, alter DNA and histone modifications and regulate gene expression. Several studies show that E2 regulates learning and memory by altering chromatin at the promoters of memory-linked genes. Due to structural similarities with E2 and low side effects, phytoestrogens are now used as neuroprotective agents to recover learning and memory in animal models as well as human subjects during aging and different neurological disorders. Growing evidence suggests that apart from anti-oxidative and anti-inflammatory properties, phytoestrogens also act as epigenetic modifiers and regulate gene expression through epigenetic modifications. The epigenetic modifying properties of phytoestrogens are mostly studied in cancer cells but very little is known regarding the regulation of synaptic plasticity genes, learning and memory, and neurological disorders. In this article, we discuss the epigenetic modifying properties of E2 and the roles of phytoestrogens as epigenetic modifiers in the brain to recover and maintain cognitive functions.

Estrogen Receptor α- and ß-Interacting Proteins Contain Consensus Secondary Structures: An Insilico Study.

BACKGROUND: Estrogen receptor (ER)α and ERß are ligand-activated transcription factors that regulate gene expression by binding to estrogen-responsive elements and interacting with several coregulators through protein-protein interactions. Usually, these coregulators bind to the various conserved and functional domains of the receptor through a consensus LXXLL sequence, although variations can be found. The interaction of receptor domains and the consensus motif can be a possible target for nuclear receptor (NR) pharmacology, since modifications in these are responsible for possible pathogenesis of various diseases. PURPOSE: The present study focuses on the secondary structure and conserved domains of the ERα and ERß interacting proteins, using bioinformatics tools and their relation to the function of the coregulators. METHODS: Bioinformatics-based prediction tools like STRING, PSIPRED, PROTPARAM and Conserved Domain Database (CDD) were used. The prediction tools utilized in this study basically determines the characteristics of a possible coregulator by using an already existing protein as a template and determines the presence of any conserved consensus sequence. Coregulators have been enlisted with the help of NCBI, STRING and iHOP. The secondary structures were analyzed using PSIPRED and conserved domains were determined using CDD. RESULTS: The analysis of the structure has shown the presence of conserved domains and homology between the various coregulators. Each interacting protein contains conserved domains like the nuclear coactivators' domain, the helix-loop-helix domain and the SRC domain. CONCLUSION: Such studies give the characteristic features of ERα and ERß interacting proteins and maybe useful to determine their family and uses in NR pharmacology in health and diseases.

Analysis of estrogen receptor ß interacting proteins using pull-down assay and MALDI-MS methods.

Estrogen mediates a plethora of functions through well-characterized estrogen receptor (ER)α and ERß after recruiting a number of interacting proteins. Various laboratories including ours have focused on the identification of ERß interacting proteins using different methods including matrix-assisted laser desorptive ionization (MALDI), which is a powerful technique in proteomics to identify new proteins present in low abundance. We have identified ERß interacting proteins resolved by one-dimensional preparatory sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and two-dimensional SDS-PAGE followed by MALDI-MS. In this chapter, a detailed method of pull-down assay, SDS-PAGE, MALDI-MS, and immunoblotting along with the use of software for identification of interacting proteins have been described. Such methods are useful to identify the interacting proteins, which may predict the function and molecular mechanism of action that is helpful for developing therapeutic strategies.

Estrogen receptor ß and its domains interact with casein kinase 2, phosphokinase C, and N-myristoylation sites of mitochondrial and nuclear proteins in mouse brain.

The localization of estrogen receptor (ER)ß in mitochondria suggests ERß-dependent regulation of genes, which is poorly understood. Here, we analyzed the ERß interacting mitochondrial as well as nuclear proteins in mouse brain using pull-down assay and matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS). In the case of mitochondria, ERß interacted with six proteins of 35-152 kDa, its transactivation domain (TAD) interacted with four proteins of 37-172 kDa, and ligand binding domain (LBD) interacted with six proteins of 37-161 kDa. On the other hand, in nuclei, ERß interacted with seven proteins of 30-203 kDa, TAD with ten proteins of 31-160 kDa, and LBD with fourteen proteins of 42-179 kDa. For further identification, these proteins were cleaved by trypsin into peptides and analyzed by MALDI-MS using mascot search engine, immunoprecipitation, immunoblotting, and far-Western blotting. To find the consensus binding motifs in interacting proteins, their unique tryptic peptides were analyzed by the motif scan software. All the interacting proteins were found to contain casein kinase (CK) 2, phosphokinase (PK)C phosphorylation, and N-myristoylation sites. These were further confirmed by peptide pull-down assays using specific mutations in the interacting sites. Thus, the present findings provide evidence for the interaction of ERß with specific mitochondrial and nuclear proteins through consensus CK2, PKC phosphorylation, and N-myristoylation sites, and may represent an essential step toward designing selective ER modulators for regulating estrogen-mediated signaling.

AIB1 shows variation in interaction with ERßTAD and expression as a function of age in mouse brain.

Estrogen mediates its multiple functions in the brain through the recruitment of a number of interacting proteins. In this paper, we report the identification of 160 kD interacting nuclear protein of estrogen receptor (ER)ß-transactivation domain (TAD) as amplified in breast cancer 1(AIB1) by pull down assay, immunoblotting, far-western analysis and immunoprecipitation. Further we show the age dependent interaction and expression of AIB1 in the brain of young (6 weeks), adult (25 weeks) and old (70 weeks) AKR strain mice of both sexes. The immunoprecipitation data revealed higher interaction of AIB1 in young than adult and old male mice. In contrast, the interaction was low in young, increased in adult but decreased in old female. However, immunoblotting showed age related increase in the expression of AIB1 in both male and female mice. Further, the level of interaction of AIB1 with ERßTAD in young and old male was significantly higher than female of same age, whereas the expression of AIB1 in adult and old female was significantly higher than male of same age. These data suggest that such age dependent variation in the interaction of AIB1 with ERßTAD and its expression may be helpful to regulate estrogen-mediated gene functions during aging and neurodegenerative diseases.

Overexpression of mouse estrogen receptor-ß decreases but its transactivation and ligand binding domains increase the growth characteristics of E. coli.

Escherichia coli is one of the most common and widely used prokaryotic hosts for the expression of recombinant proteins. The overexpression of recombinant proteins occasionally increases bacterial growth but sometimes reduces it and becomes lethal to the host cells. Here, we report the overexpression of mouse ER-ß and its domains in the prokaryotic expression system and its opposite effect on the growth characteristics of E. coli. ER-ß protein was immunologically detected as a 53 kDa his-tag protein in the pellet of the bacterial lysate. Its overexpression, as reflected by the total protein content and expression pattern, resulted in the decrease of bacterial growth. However, the overexpression of ER-ß transactivation domain (TAD) using pIVEX and ligand binding domain (LBD) using pRSETA in E. coli BL21 (DE3) show opposite pattern. TAD was immunologically detected as 20 kDa and LBD as 22 kDa protein in the supernatant of the bacterial lysate and their overexpression increased the bacterial growth.

NMR analysis reveals 17ß-estradiol induced conformational change in ERß ligand binding domain expressed in E. coli.

Nuclear magnetic resonance (NMR) spectroscopy is a useful biophysical technique to study the ligand-protein interaction. In this report, we have used bacterially produced ERß and its domains for studying the functional analysis of ligand-protein interaction. Briefly, ERß and its transactivation domain (TAD) and ligand binding domain (LBD) were subcloned and overexpressed using a prokaryotic expression system. The recombinant proteins were purified using Ni(+2)-IDA affinity chromatography and analyzed by NMR. Purified ERß and TAD show similar conformation in the absence or presence of 17ß-estradiol. However, LBD shows altered conformation in the presence of 17ß-estradiol. These findings suggest that ERß produced in bacteria exhibits a conformation such that its LBD remains masked and consequently it binds less to 17ß-estradiol. Such study may help to develop the therapeutic approaches for controlling the estradiol-mediated gene expression in hormone dependent diseases.

Interaction of Estrogen Receptor Associated Protein (ERAP) 140 with ER beta decreases but its expression increases in aging mouse cerebral cortex.

Following binding to cognate ligand, estrogen receptor (ER) beta interacts with specific responsive elements of the target genes and recruits a host of nuclear proteins for hormone dependent gene regulation. However, it is poorly known which proteins interact with ER beta in mouse brain and whether their interaction and expression change with age. In this report, we have used his-tag mouse ER beta for interaction with nuclear proteins of cerebral cortex of young (6 +/- 1 weeks), adult (25 +/- 2 weeks), and old (70 +/- 5 weeks) female mice. We have identified estrogen receptor-associated protein (ERAP) 140 as one of the interacting proteins and studied its interaction by pull down immunoblotting, far-Western blotting and immunoprecipitation, and expression by western blotting. The data show that ERAP 140 interacts with ER beta and its interaction decreases but its expression increases with age in mouse cerebral cortex, suggesting its role in estrogen-mediated brain functions during aging.