Binding Of Epigallocatechin-3-gallate Research Articles

Differences in binding behavior of (−)-epigallocatechin gallate to β-lactoglobulin heterodimers (AB) compared to homodimers (A) and (B)

The lipocalin β-lactoglobulin (β-LG) exists in different natural genetic variants--of which β-LG A and B are predominant in bovine milk. At physiological conditions the protein dimerizes--building homodimers of β-LG A and β-LG B and heterodimers of β-LG AB. Although β-LG is one of the most intensely characterized lipocalins, the interaction behavior of ligands with hetero- and homodimers of β-LG is largely unknown. The present findings revealed significant differences for hetero- and homodimers regarding ligand binding capacity as tested with a model ligand (i.e. surface binding (-)-epigallocatechin gallate (EGCG)). These findings were confirmed using FT-IR, where the addition of EGCG influenced the β-sheet backbone of homodimer A and B with significantly higher intensity compared to heterodimer AB. Further, shape analysis by SAXS revealed oligomerization of both types of dimers upon addition of EGCG; however, homodimer A and B produced significantly larger aggregates compared to the heterodimer AB. In summary, the present study revealed that EGCG showed significantly different interaction reactivity (binding sites, aggregation size and conformational changes) to the hetero and homodimers of β-LG in the order β-LG A > B > AB. The results suggest that conformational differences between homodimers and heterodimers strongly influence the EGCG binding ability. This may also occur with other polyphenols and ligands of β-LG and gives not only important information for β-LG binding studies, but may also apply for polymorphisms of other self-aggregating lipocalins.

Binding of epigallocatechin gallate to the laminin–β-integrin binding site decreases neural progenitor cell adhesion and migration: Adverse outcome pathway framework supporting neurodevelopmental toxicity research and risk assessment

Binding of epigallocatechin gallate to the laminin–β-integrin binding site decreases neural progenitor cell adhesion and migration: Adverse outcome pathway framework supporting neurodevelopmental toxicity research and risk assessment

Complexes of green tea polyphenol, epigalocatechin-3-gallate, and 2S albumins of peanut

2S albumins of peanuts are seed storage proteins, highly homologous in structure and described as major elicitors of anaphylactic reactions to peanut (allergens Ara h 2 and Ara h 6). Epigallocatechin-3-gallate (EGCG) is the most biologically potent polyphenol of green tea. Non-covalent interactions of EGCG with proteins contribute to its diverse biological activities. Here we used the methods of circular dichroism, fluorescence quenching titration, isothermal titration calorimetry and computational chemistry to elucidate interactions of EGCG and 2S albumins. Similarity in structure and overall fold of 2S albumins yielded similar putative binding sites and similar binding modes with EGCG. Binding affinity determined for Ara h 2 was in the range described for complexes of EGCG and other dietary proteins. Binding of EGCG to 2S albumins affects protein conformation, by causing an α-helix to β-structures transition in both proteins. 2S albumins of peanuts may be good carriers of physiologically active green tea catechin.

Au nanostructured surfaces for electrochemical and localized surface plasmon resonance-based monitoring of α-synuclein-small molecule interactions.

In this proof-of-concept study, the fabrication of novel Au nanostructured indium tin oxide (Au-ITO) surfaces is described for the development of a dual-detection platform with electrochemical and localized surface plasmon resonance (LSPR)-based biosensing capabilities. Nanosphere lithography (NSL) was applied to fabricate Au-ITO surfaces. Oligomers of α-synuclein (αS) were covalently immobilized to determine the electrochemical and LSPR characteristics of the protein. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were performed using the redox probe [Fe(CN)6](3-/4-) to detect the binding of Cu(II) ions and (-)-epigallocatechin-3-gallate (EGCG) to αS on the Au-ITO surface. Electrochemical and LSPR data were complemented by Thioflavin-T (ThT) fluorescence, surface plasmon resonance imaging (SPRi), and transmission electron microscopy (TEM) studies. EGCG was shown to induce the formation of amorphous aggregates that decreased the electrochemical signals. However, the binding of EGCG with αS increased the LSPR absorption band with a bathochromic shift of 10-15 nm. The binding of Cu(II) to αS enhanced the DPV peak current intensity. NSL fabricated Au-ITO surfaces provide a promising dual-detection platform to monitor the interaction of small molecules with proteins using electrochemistry and LSPR.

Processing treatments enhance the adsorption characteristics of epigallocatechin-3-gallate onto apple pomace

The adsorption capacity of epigallocatechin-3-gallate (EGCG) onto freeze dried and extruded apple pomace ground into powder was compared to that of wet apple pomace. The isothermal adsorption studies were carried out over a range of concentrations (100–1000mg/L) and temperatures (25°C, 40°C and 55°C). Freeze drying or extrusion of apple pomace enhanced its capacity to adsorb EGCG. Analyses of thermodynamic parameters showed that adsorption of EGCG to freeze dried pomace powder was an exothermic process whereas that to extruded pomace powder was endothermic. This is due to the altered character of the binding sites for EGCG as a result of process-induced changes, which contributed to different underlying mechanisms of binding of EGCG to the processed apple pomace. This suggests that the adsorption mechanism was dependent on the processing treatment used. Our study highlights that by choosing the appropriate processing treatments, the adsorption of EGCG to apple pomace could be improved.

Binding of (−)-epigallocatechin-3-gallate with thermally-induced bovine serum albumin/ι-carrageenan particles

Novel thermally-induced BSA/ι-carrageenan particles are used as a protective carrier for (−)-epigallocatechin-3-gallate (EGCG). The addition of EGCG to BSA/ι-carrageenan particles can highly quench the intrinsic fluorescence of BSA, which is explained in terms of the binding of EGCG to the hydrophobic pockets of BSA mainly through the hydrophobic force. According to the double logarithm equation, the binding constant is determined as 1.1×108M−1 for the binding of EGCG with BSA/ι-carrageenan particles. The high binding affinity is ascribed to both the molecular structure of EGCG and the partial unfolding state of BSA in BSA/ι-carrageenan particles. The circular dichroism spectra and calculated α-helix of BSA suggest that the bound EGCG leads to a more random secondary structure of BSA. Furthermore, BSA/ι-carrageenan particles are found to be superior to native BSA and pure BSA particles for improving the stability and radical scavenging activity of EGCG.

Bioefficacy of Tea Catechins Associated with Milk Caseins Tested Using Different In Vitro Digestion Models

Milk proteins, and in particular caseins, are avid binders of polyphenols. The objective of this work was to understand how the presence of a polyphenol–casein complex may affect the bioefficacy of tea catechins [epigallocatechin-3-gallate (EGCG)]. In vitro digestion was carried out using two different models: a batch model and a dynamic digestion model, using a simulator of the human intestinal microbial ecosystem (SHIME). SDS-PAGE electrophoresis of the digestates showed that the presence of EGCG did not affect hydrolysis of the caseins, regardless of the digestion model used. However, there was a slower digestion of the caseins in the dynamic model (SHIME) compared to the in vitro batch model. The cytotoxic effect of free and complexed EGCG was tested using adenocarcinoma cells (HT-29) and the HT29-MTX subclone. The HT29-MTX closely mimics the presence of the mucus barrier in the intestine. HT-29 cells showed a decrease in cell viability proportional to the concentration of ECGC, free or complexed and after digestion. The proliferation rate of HT29 assessed on digested EGCG-casein complexes indicated that the binding of EGCG and milk proteins does not affect its bioactivity. Antiproliferative activities were comparable to those for free EGCG, in spite of the lower recovery of EGCG after the various stages of digestion. Exposure of EGCG (free or complexed) to mucus-producing cells HT29-MTX did not show a decrease in viability, and the cell response was typical of normal cells. The presence of mucus also protected coculture monolayers of HT29-MTX and Caco-2. All the models demonstrated that the presence of EGCG–casein micelle complex did not affect the bioefficacy of the polyphenol.

Green Tea and One of Its Constituents, Epigallocatechine-3-gallate, Are Potent Inhibitors of Human 11β-hydroxysteroid Dehydrogenase Type 1

The microsomal enzyme 11β-hydroxysteroid deydrogenase type 1 (11β-HSD1) catalyzes the interconversion of glucocorticoid receptor-inert cortisone to receptor- active cortisol, thereby acting as an intracellular switch for regulating the access of glucocorticoid hormones to the glucocorticoid receptor. There is strong evidence for an important aetiological role of 11β-HSD1 in various metabolic disorders including insulin resistance, diabetes type 2, hypertension, dyslipidemia and obesity. Hence, modulation of 11β-HSD1 activity with selective inhibitors is being pursued as a new therapeutic approach for the treatment of the metabolic syndrome. Since tea has been associated with health benefits for thousands of years, we sought to elucidate the active principle in tea with regard to diabetes type 2 prevention. Several teas and tea specific polyphenolic compounds were tested for their possible inhibition of cortisone reduction with human liver microsomes and purified human 11β-HSD1. Indeed we found that tea extracts inhibited 11β-HSD1 mediated cortisone reduction, where green tea exhibited the highest inhibitory potency with an IC50 value of 3.749 mg dried tea leaves per ml. Consequently, major polyphenolic compounds from green tea, in particular catechins were tested with the same systems. (−)-Epigallocatechin gallate (EGCG) revealed the highest inhibition of 11β-HSD1 activity (reduction: IC50 = 57.99 µM; oxidation: IC50 = 131.2 µM). Detailed kinetic studies indicate a direct competition mode of EGCG, with substrate and/or cofactor binding. Inhibition constants of EGCG on cortisone reduction were Ki = 22.68 µM for microsomes and Ki = 18.74 µM for purified 11β-HSD1. In silicio docking studies support the view that EGCG binds directly to the active site of 11β-HSD1 by forming a hydrogen bond with Lys187 of the catalytic triade. Our study is the first to provide evidence that the health benefits of green tea and its polyphenolic compounds may be attributed to an inhibition of the cortisol producing enzyme 11β-HSD1.

Molecular and thermodynamic basis for EGCG‐Keratin interaction‐part II: Experimental investigation

In Part I, we reported all‐atom, fully solvated molecular dynamics (MD) simulations of epigallocatechin‐3‐gallate (EGCG) binding to keratin. Herein, we report the second part of experimental investigation on EGCG binding to keratin using ultrafiltration and isothermal titration calorimetry (ITC). The thermodynamic equilibrium of EGCG binding to keratin has been quantitatively determined using ultrafiltration and high‐performance liquid chromatography−UV/vis. The relationship confirms multilayer binding of EGCG to keratin which was observed in MD simulations. By combining the ultrafiltration and ITC data, the thermodynamic parameters of EGCG binding to keratin have been quantified. The obtained free energy of the first layer binding (ΔG = −6.37 kcal mol−1) is shown in excellent agreement with that obtained from computer simulations (ΔG = −6.20 kcal mol−1) presented in Part I. © 2013 American Institute of Chemical Engineers AIChE J, 59: 4824–4827, 2013

Interactions between tea catechins and casein micelles and their impact on renneting functionality

Many studies have shown that tea catechins bind to milk proteins. This research focused on the association of tea polyphenols with casein micelles, and the consequences of the interactions on the renneting behaviour of skim milk. It was hypothesized that epigallocatechin-gallate (EGCG), the main catechin present in green tea, forms complexes with the casein micelles and that the association modifies the processing functionality of casein micelles. The binding of EGCG to casein micelles was quantified using HPLC. The formation of catechin–casein micelles complexes affected the rennet induced gelation of milk, and the effect was concentration dependent. Both the primary as well as the secondary stage of gelation were affected. These experiments clearly identify the need for a better understanding of the effect of tea polyphenols on the processing functionality of casein micelles, before milk products can be used as an appropriate platform for delivery of bioactive compounds.

Abstract 2235: Epigallocatechin-gallate suppresses tumorigenesis by directly targeting Pin1.

Abstract The most active anticancer component in green tea is epigallocatechin-3-gallate (EGCG). The human peptidyl prolyl cis/trans isomerase (Pin1) plays a critical role in oncogenic signaling. Herein, we report the X-ray crystal structure of the Pin1/EGCG complex resolved at 1.9 Å resolution. Notably, the structure revealed the presence of EGCG in both the WW and PP1ase domains of Pin1. The direct binding of EGCG with Pin1 was confirmed and the interaction inhibited Pin1 PP1ase activity. In addition, proliferation of cells expressing Pin1 was inhibited and tumor growth in a xenograft mouse model was suppressed. The binding of EGCG with Arg17 in the WW domain prevented the binding of c-Jun, a well-known Pin1 substrate. EGCG treatment corresponded with a decreased abundance of cyclin D1 and diminution of TPA-induced AP-1 or NFκB promoter activity in cells expressing Pin1. Overall, these results showed that EGCG directly suppresses the tumor promoting effect of Pin1. Citation Format: Darya V. Urusova, Jung-Hyun Shim, Dong Joon Kim, Sung Keun Jung, Tatyana Zykova, Andria Carper, Ann M. Bode, Zigang Dong. Epigallocatechin-gallate suppresses tumorigenesis by directly targeting Pin1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2235. doi:10.1158/1538-7445.AM2013-2235

The potential role of caveolin in intracellular signaling for cardiac protection of (−)‐epigallocatechin‐3‐gallate

In this study, we showed the putative mechanism for the cardio‐protection of (−)‐epigallocatechin‐3‐gallate (EGCg). H9c2 cardiac myoblasts exposed to H2O2 decreased cell viability by increasing reactive oxygen species and cytosolic Ca2+, and imposed cell cycle arrest at G1‐S phase via the signaling pathway for glycogen synthase kinase‐3β/β‐catenin/cyclin D1. All these H2O2‐induced cardiac cell injuries could be counteracted by EGCg pretreatment. To determine the binding of EGCg to H9c2 cells, EGFP (enhanced green fluorescence protein) was ectopically expressed in the cells. In overexpressed cells EGFP formed the protein complexes with 67 kD laminin receptor, caveolin‐1 and ‐3, γ‐actin, myosin IX, and vimentin. EGFP fluorescence measurements showed that EGCg bound to intact, Triton X‐100 soluble fraction, and Triton X‐100 insoluble fraction of H9c2 cells with a dissociation constant (Kd) of ~ 86, ~126, and ~42 μM, respectively. H9c2 cells pretreated with H2O2 increased the binding affinity of EGCg (Kd = 47μM). Using a rat model of myocardial infarction involving left anterior descending coronary artery ligation we showed involvement of caveolin‐3 in the cardio‐protection of green tea polyphenols against myocardial ischemia. Taken together, caveolins might serve as rafts for intracellular signaling to mediate cardiac protection of EGCg.

Electroanalysis of the interaction between (−)-epigallocatechin-3-gallate (EGCG) and amyloid-β in the presence of copper

The misfolding of amyloid-beta (Aβ) peptide is one of the pathological hallmarks of Alzheimer's disease (AD). Polyphenols are strong antioxidants and metal chelators, with characteristics that are of beneficial therapeutic values for their development as candidates targeting neurodegenerative and metal-induced diseases. We have demonstrated here the electrochemical properties of a green tea component, (-)-epigallocatechin-3-gallate (EGCG), and its potent activity on Aβ peptides. Characterization of early interactions (≤48 h) between EGCG and Aβ was conducted using square wave voltammetry (SWV). The interaction of Cu(ii) ions with the Tyr-10 residue of Aβ was shown to be affected by surrounding His residues. Morphological changes due to the binding of EGCG and Cu(II) were also elucidated using transmission electron microscopy (TEM). Electroanalytical techniques are promising for facilitating the investigation of metals and flavonoids in drug screening studies.

Interaction of (–)-Epigallocatechin Gallate with Lysozyme-Conjugated Silver Nanoparticles

The interaction of lysozyme (Lyz)-conjugated silver (Ag) nanoparticles with (-)-epigallocatechin gallate (EGCG), one of the major components of green tea, has been investigated. Interaction of a protein with ligand/drug molecules perturbs the conformation of secondary and tertiary structures of the protein. We have demonstrated the conformational changes in the tertiary structures of the Lyz molecules on EGCG binding using surface-enhanced Raman scattering (SERS) and circular dichroism (CD) spectroscopic measurements. From the analysis of the amide I band of Lyz in SERS and CD spectra, the site of interaction of EGCG with protein molecules in Lyz-conjugated Ag particles has been identified. Spectroscopic evidence for the conformational response of Trp62 and Trp63, in the β-domain of the protein, to the binding of EGCG has been discussed.

Thermodynamic Analysis of the Molecular Interactions between Amyloid β-Protein Fragments and (−)-Epigallocatechin-3-gallate

(-)-Epigallocatechin-3-gallate (EGCG) has been proven effective in preventing the aggregation of amyloid β-protein 42 (Aβ42), and the thermodynamic interactions between Aβ42 and EGCG have been studied in our previous work ( J. Phys. Chem. B 2010, 114, 11576). Herein, to further probe the interactions between different regions of Aβ42 and EGCG, three Aβ42 fragments (i.e., Aβ1-16, Aβ1-30, and Aβ31-42) were synthesized, and the thermodynamic interactions between each of the fragments and EGCG at different EGCG and salt concentrations were investigated by isothermal titration calorimetry. The results indicate that, although hydrogen bonding and hydrophobic interaction are both involved in the interactions between Aβ42 and EGCG, hydrogen bonding mainly happens in Aβ1-16 while hydrophobic interaction mainly happens in Aβ17-42. It is found that when Aβ42 and its fragments are saturated by EGCG, their thermodynamic parameters have linear relationships. The saturated binding stoichiometry (N(s)) for Aβ42 is the sum of the N(s) values for Aβ1-30 and Aβ31-42, while ΔH(s), ΔS(s), and ΔG(s) for Aβ42 are half the sum of the values for Aβ1-30 and Aβ31-42. The result suggests that there are no specific interactions and binding sites in the Aβ42 and EGCG binding. The orders of ΔH(s) and TΔS(s) values for the Aβ fragments are determined as Aβ17-42 > Aβ31-42 > Aβ1-30 > Aβ1-16. Moreover, there is significant enthalpy-entropy compensation in the binding of EGCG to Aβ42 and its fragments, resulting in insignificant change of ΔG with the change of the solution environment. The research has shed new light on the molecular mechanisms of the interactions between EGCG and Aβ42.

Experimental and Theoretical Studies on the Binding of Epigallocatechin Gallate to Purified Porcine Gastric Mucin

Binding of epigallocatechin gallate (EGCG) to highly purified short side-chain porcine gastric mucin similar to human MUC6 type has been studied by ultraviolet-visible absorption spectroscopy (UV-vis), ultrafiltration isothermal titration microcalorimetry (ITC) and transmission electron microscopy (TEM). The thermodynamic equilibrium of EGCG binding to mucin has been quantitatively determined using ultrafiltration and high-performance liquid chromatography (HPLC)-UV/vis. The relationship suggests multilayer binding rather than simple Langmuir monolayer binding of EGCG. By combining the ultrafiltration and ITC data, the thermodynamic parameters of EGCG binding to mucin have been obtained. The binding constant for the first layer is about an order of magnitude higher than that of the consecutive multilayers. Negative entropy indicates multilayer of EGCG formed. Hydrogen bonding may be responsible for the multilayer formation. Increasing temperature resulted in a decrease in the binding affinity, further suggesting that hydrogen bonds dominated the interaction energy. A TEM micrograph of the EGCG-mucin complex revealed a monodispersion of blobs similar to pure mucin solution but with relatively bigger size (about twice). It is proposed that the EGCG-mucin binding process occurs by single and/or cluster of EGCG molecules driven to the surface of the two hydrophobic globules of mucin by hydrophobic interaction followed by hydrogen bond interaction between EGCG and mucin. Further adsorption of EGCG molecules onto bound EGCG molecules to form multilayers can also occur. This fits well with the observations that EGCG-mucin interaction followed a multilayer adsorption isotherm, the energy released is dominated by hydrogen bonds, and no large aggregates were formed.

Comparison of Three Amyloid Assembly Inhibitors: The Sugar scyllo-Inositol, the Polyphenol Epigallocatechin Gallate, and the Molecular Tweezer CLR01

Many compounds have been tested as inhibitors or modulators of amyloid β-protein (Aβ) assembly in hope that they would lead to effective, disease-modifying therapy for Alzheimer's disease (AD). These compounds typically were either designed to break apart β-sheets or selected empirically. Two such compounds, the natural inositol derivative scyllo-inositol and the green-tea-derived flavonoid epigallocatechin gallate (EGCG), currently are in clinical trials. Similar to most of the compounds tested thus far, the mechanism of action of scyllo-inositol and EGCG is not understood. Recently, we discovered a novel family of assembly modulators, Lys-specific molecular tweezers, which act by binding specifically to Lys residues and modulate the self-assembly of amyloid proteins, including Aβ, into formation of nontoxic oligomers by a process-specific mechanism (Sinha, S., Lopes, D. H., Du, Z., Pang, E. S., Shanmugam, A., Lomakin, A., Talbiersky, P., Tennstaedt, A., McDaniel, K., Bakshi, R., Kuo, P. Y., Ehrmann, M., Benedek, G. B., Loo, J. A., Klarner, F. G., Schrader, T., Wang, C., and Bitan, G. (2011) Lysine-specific molecular tweezers are broad-spectrum inhibitors of assembly and toxicity of amyloid proteins. J. Am. Chem. Soc.133, 16958-16969). Here, we compared side-by-side the capability of scyllo-inositol, EGCG, and the molecular tweezer CLR01 to inhibit Aβ aggregation and toxicity. We found that EGCG and CLR01 had comparable activity whereas scyllo-inositol was a weaker inhibitor. Exploration of the binding of EGCG and CLR01 to Aβ using heteronuclear solution-state NMR showed that whereas CLR01 bound to the two Lys and single Arg residues in Aβ monomers, only weak, nonspecific binding was detected for EGCG, leaving the binding mode of the latter unresolved.

Site Specific Interaction of the Polyphenol EGCG with the SEVI Amyloid Precursor Peptide PAP(248–286)

Recently, a 39 amino acid peptide fragment from prostatic acid phosphatase has been isolated from seminal fluid that can enhance infectivity of the HIV virus by up to 4-5 orders of magnitude. PAP(248-286) is effective in enhancing HIV infectivity only when it is aggregated into amyloid fibers termed SEVI. The polyphenol EGCG (epigallocatechin-3-gallate) has been shown to disrupt both SEVI formation and HIV promotion by SEVI, but the mechanism by which it accomplishes this task is unknown. Here, we show that EGCG interacts specifically with the side chains of monomeric PAP(248-286) in two regions (K251-R257 and N269-I277) of primarily charged residues, particularly lysine. The specificity of interaction to these two sites is contrary to previous studies on the interaction of EGCG with other amyloidogenic proteins, which showed the nonspecific interaction of EGCG with exposed backbone sites of unfolded amyloidogenic proteins. This interaction is specific to EGCG as the related gallocatechin (GC) molecule, which shows greatly decreased antiamyloid activity, exhibits minimal interaction with monomeric PAP(248-286). The EGCG binding was shown to occur in two steps, with the initial formation of a weakly bound complex followed by a pH dependent formation of a tightly bound complex. Experiments in which the lysine residues of PAP(248-286) have been chemically modified suggest the tightly bound complex is created by Schiff-base formation with lysine residues. The results of this study could aid in the development of small molecule inhibitors of SEVI and other amyloid proteins.

Green tea polyphenol EGCG suppresses lung cancer cell growth through upregulating miR-210 expression caused by stabilizing HIF-1

(-)-Epigallocatechin-3-gallate (EGCG) has been reported to affect many cellular regulatory pathways. This study aims to determine whether EGCG could target microRNA (miRNA), one of the mechanisms for cells to achieve subtle change in multiple targets. We found that, in both human and mouse lung cancer cells in culture, EGCG specifically upregulated the expression of miR-210, a major miRNA regulated by HIF-1α. Furthermore, we found that overexpression of miR-210 led to reduced cell proliferation rate and anchorage-independent growth as well as reduced sensitivity to EGCG. On the mechanisms of miR-210 regulation by EGCG, we demonstrated that the regulation was mediated through the hypoxia-response element in miR-210 promoter. Consistently, the upregulation of miR-210 was found to be correlated with the stabilized HIF-1α in lung cancer cell lines after EGCG treatment. This EGCG-induced stabilization of HIF-1α was further shown by the stabilization of HA-tagged HIF-1α but not the P402A/P564A-mutated HIF-1α by EGCG, suggesting that EGCG targets the oxygen-dependent degradation (ODD) domain. Direct evidence was obtained by affinity binding assay showing that EGCG specifically binds HIF-1α with a K(d) = 3.47 μM. This result suggests that EGCG binding interferes with the hydroxylation of key Pro residues in the ODD domain, preventing HIF-1α from the Pro hydroxylation-dependent ubiquitination and subsequent proteosome-mediated degradation. In summary, our results demonstrated, for the first time, the elevation of miR-210 by EGCG in lung cancer cell lines and this is mediated by the stabilization of HIF-1α. This event contributes to the anticancer activity of EGCG.

Epigallocatechin gallate (EGCG) inhibits type II phosphatidylinositol 4-kinases: A key component in pathways of phosphoinositide turnover

Type II phosphatidylinositol (PtdIns) 4-kinases produce PtdIns 4-phosphate, an early key signaling molecule in phosphatidylinositol cycle, which is indispensable for T cell activation. Type II PtdIns 4-kinase alpha and beta have similar biochemical properties. To distinguish these isoforms Epigallocatechin gallate (EGCG) has been evaluated as a specific inhibitor. EGCG is the major active catechin in green tea having anti-inflammatory, antiatherogenic and cancer chemopreventive properties. The precise mechanism of actions and molecular targets of EGCG in early signaling cascades are not well understood. In the present study, we have shown that EGCG inhibits type II PtdIns 4-kinases (α and β isoforms) and PtdIns 3-kinase activity in vitro. EGCG directly bind to both alpha and beta isoforms of type II PtdIns 4-kinases with a Kd of 2.62 μM and 1.02 μM, respectively. Type II PtdIns 4-kinase-EGCG complex have different binding pattern at its excited state. Both isoforms showed significant change in helicity upon binding with EGCG. EGCG modulates its effect by interacting with ATP binding pocket; the residues likely to be involved in EGCG binding were predicted by Autodock. Our findings suggest that EGCG inhibits two isoforms and could be a key to regulate T cell activation.