Identification and Characterization of Flavonoid Target Proteins

Chapter 4

Identification and Characterization of Flavonoid Target Proteins

Downloaded by UNIV OF QUEENSLAND on October 8, 2015 | http://pubs.acs.org Publication Date: September 19, 2008 | doi: 10.1021/bk-2008-0993.ch004

Herwig O. Gutzeit and Markus Böhl Institut für Zoologie, TU Dresden, D-01062 Dresden, Germany

Flavonoids are known to interact with a host of cellular proteins in vitro and to affect their biological activity, but the relevance of the interaction in vivo has rarely been ascertained. To realistically assess the biological and pharmacological effects of flavonoids, a systematic approach to identify relevant cellular target proteins is required. We showed that the changing spectroscopic properties of target protein and ligand can be exploited to identify and characterize target proteins. In a proof-of-principle experiment actin was identified as a new and relevant flavonoid target protein.

The biological effects of flavonoids have been intensively studied due to their suspected beneficial effects on human health. The interaction of flavonoids with numerous cellular enzymes has been reported (1) and the picture emerged that each flavonoid is likely to affect a spectrum of cellular target proteins. However, the relevance of these studies for human health has often remained elusive. The complex interactions of quercetin with target proteins apparently result in profound physiological changes in the affected cells. This is reflected in an altered pattern of expressed proteins in human colon cancer cells in response to quercetin treatment as was shown recently in a proteomics approach (2). A causal analysis of flavonoid - induced effects on the cellular and tissue level has been hampered by lack of knowledge concerning the spectrum of relevant flavonoid-specific target proteins in a given cell or tissue. How can the flavonoid target proteins in a cell be identified systematically? The straightforward method is to immobilize the flavonoid of interest and to isolate and identify binding proteins by affinity chromatography (3). The limitation of this procedure is that the biochemical coupling of the flavonoid to, for example, © 2008 American Chemical Society In Functional Food and Health; Shibamoto, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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28 Sepharose beads alters the structure of the flavonoid and hence altered binding properties to the target proteins cannot be excluded. An alternative method that requires no modification of the flavonoid is based on the changing spectroscopic properties of both target protein and flavonoid ligand upon their specific interaction. The basic principle and a proof-of-principle experiment will be outlined in this contribution.


Materials and Methods Size Exclusion Chromatography Chromatography and fractionation was performed using a Biorad Biologic Workstation (MUnchen, Germany) equipped with a Pharmacia (Uppsala, Sweden) Superdex 200 gel filtration column. At an isocratic flow of 0.4 mL/min (200 mM KC1, 1.5 mM MgCl , 200 mM Tris-HCl, pH 7.5) the absorption at 280nm was continuously measured and fractions of 0.5 mL collected. A l l standard proteins, buffer constituents, flavonoids and solvents were supplied by Sigma (Steinheim, Germany). 2

Fluorescence Spectroscopy For each fraction obtained by chromatography the fluorescence intensity at Ex280nm/Em345nm was determined in 96 well testplates in the absence and in the presence of the tested flavonoids using a Tecan Infinite 200 microplate reader (Crailsheim, Germany). A more detailed description for this procedure is explained by Bohl et al. (7).

Results and Discussion The changing spectroscopic properties as a result of protein/ligand interaction can best be illustrated using 3 model proteins which were separated by column chromatography and the fractions analysed by fluorescence spectroscopy (Figure 1). Proteins typically show autofluorescence (e.g. BSA and lysozyme, Figure 1A) due to the presence of aromatic amino acids. In exceptional cases (e.g. ferritin, Figure 1A), the fluorescence may be suppressed by the presence of bound iron (Figure 1A). For the experimental analysis of protein autofluorescence the tryptophan (tip) residues are most relevant. When a flavonoid like genistein or quercetin binds to a trp-containing protein the autofluorescence is quenched (BSA, Figure IB) and the absence of quenching is

In Functional Food and Health; Shibamoto, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.


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Figure 1. Fractionation of 3 proteins: ferritin (440kDa), BSA (68kDa) und lysozyme (14kDa). A: The absorption at 280 nm was continuously measured and the intrinsic protein fluorescence determined in the collectedfractions (Ex280 nm/Em345 nm) B: Quenching of intrinsic protein fluorescence by quercetin and genistein (Ex280/Em345) C: Elicitation of flavonoidfluorescence by binding to BSA (Ex450/Em530 nm).



30 indicative for the absence of molecular interactions (lysozyme, Figure IB). The degree of quenching depends on the spectral properties of the ligand, the strength of protein/ligand interaction, the number of trp residues in the protein and their location and distance from the ligand binding site. The solvent of the flavonoid stock solutions (DMSO) contributes little to the quenching effect (Figure IB). There is another interesting spectroscopic change when theflavonoidligand interacts with a target protein: the binding of the flavonol quercetin and structurally related flavonoids may lead to the stabilisation of quercetin monoanion tautomers (4) and, as a result, to an elicitation of fluorescence in the visible spectrum (e.g. quercetin binding to BSA, Figure 1C). This property of quercetin allows to visualise target proteins in living cells (5) and, furthermore, to monitor quercetin metabolism in vital cells (6). Other flavonoids like the isoflavone genistein do not show this property (Figure 1C).

extract proteins from isolated nuclei of human HL-60

I fractionate proteins by column chromatography

identify fractions with proteins that show fluorescence quenching with quercetin

I separate proteins by SDS gel-electrophoresis

I identification of proteins by MALDI-MS

I verify flavonoid/protein interaction with purified protein

Figure 2. Outline of the experimental strategy to identify quercetin target proteins by means of the changing spectroscopic properties when proteins interact specifically with a flavonoid. The quenching of autofluorescence by flavonoid ligands is a sensitive and a generally applicable method to analyze target/ligand interactions. Can target proteins be isolated and identified out of a complex protein mixture by virtue of fluorescence quenching? In a pilot experiment that is summarized in Figure 2,



31 we showed that this is indeed possible (7). As starting material we chose nuclear proteins of human leukemia cells (HL-60 cell line) since in previous experiments with HL-60 cells it had become clear that a high concentration of quercetin target proteins is present in cell nuclei. The extracted nuclear proteins were fractionated by column chromatography and each fraction analyzed by fluorescence spectroscopy. Fractions of nuclear proteins which showed strong fluorescence quenching after the addition of quercetin were subjected to SDS gel electrophoresis and, finally, single stained protein bands were cut out of the gel and the protein(s) identified by mass spectrometry (in collaboration with B. Hoflack, BIOTEC Dresden). Amongst the identified proteins were different DNA binding proteins like high mobility group proteins and also actin. Because this protein can easily be obtained in pure form, the molecular interaction with quercetin was analyzed further. Actin is well-known for its cytoskeletal functions but the protein also plays an essential role in transcription (8,9). In a collaborative effort (70) we verified by fluorescence spectroscopy that quercetin binds specifically to actin. With respect to the actin activity we could show that actin polymerisation is inhibited by quercetin (25 \iM) in vitro and this also applies to the nuclear function since transcription is inhibited using an in vitro transcription test system. If this inhibitory activity also leads to functional deficiencies in living cells remains to be shown. How reliable is the method and what are the limitations? The identification of the flavonoid target proteins hinges, in part, on the sophistication of the applied fractionation procedure. Ideally, each fraction should contain only one predominant protein. As discussed above, not all proteins are amenable to an analysis by fluorescence spectroscopy and, for that reason, affinity chromatography of target proteins by immobilized flavonoids is a useful technique that may provide valuable additional information. The latter approach, however, suffers from the unavoidable modification of the flavonoid structure and there are many examples that small structural changes may substantially alter the biological activity in a given test system. The flavonoid/protein interaction is specific and a discrete binding site could be defined in all cases that were analysed in sufficient detail. For example, the binding of some estrogenic flavonoids like genistein to the mammalian estradiol receptors (a and/or P) is well understood (11). When known target proteins of flavonoids are compared, it is apparent that flavonoids do not necessarily target proteins with similar functions and ligand structures, but also interact with proteins which are functionally unrelated and possess different ligand binding sites (Table I). For example, quercetin inhibits the activity of quite different proteins, like actin, phospholipase A2 and myosin II ATPase. In actin and myosin, quercetin appears to bind at the ATP binding site while in phospholipase A2 the binding site is in the phospholipid substrate pocket (13). The synthetic naringenin derivative 6-(l,l-dimethylallyl)-naringenin is not only highly estrogenic and


32 Table I. Examples for Different Biological Activities of Structurally Related Flavonoids at 25 uM Concentrations

y y ^ o H


OH

OH

OH

0

quercetin

epigallocatechin

naringenin

6-(1,1-dimethylaHyl)narlngenln

phospholipase A2

inhibition

no effect

inhibition

inhibition

myosin II ATPase

inhibition

inhibition

no effect

inhibition

actin polymerisation

inhibition

stimulation

n.d.

n.d.

inhibition

stimulation

n.d.

n.d.

transcription (nuclear function of actin)

A

0

single flavonoid may affect

very different proteins and cellular functions

while

structurally similar flavonoids may not possess this activity or even produce opposite effects, n. d.: not determined.

binds to the steroid ligand pocket of the estradiol receptor (14) but also inhibits phospholipase A2 and myosin II ATPase. Other structurally related flavonoids do not show these effects or even have opposite effects and show stimulatory instead of inhibitory activity (compare quercetin with epigallocatechin concerning actin-dependent functions; Table I). Such molecular promiscuity is pharmacologically considered undesirable. However, the numerous reports on beneficial effects of flavonoids should remind us that some flavonoids are apparently able to influence the cellular physiology in a positive way under certain pathological circumstances (e.g. anti inflammatory, (anti)estrogenic, anti-oxidative etc.). While the exact number of relevant target proteins remains unknown it is clear that the number of affected proteins is not small. However, only a few of these target protein may be sufficient to produce the beneficial effects on human health. Pharmacological research traditionally focuses on the specific inhibition of a single target protein, yet there is no a priori reason why a multi-hit approach as exemplified by flavonoids is inherently inferior to an attempted (but mostly not achieved) single-hit-approach. The systematic identification of flavonoid target proteins will give valuable information concerning the molecular mechanisms that give rise to the observed biological effects and it may also change our way of thinking about the pharmacological use of flavonoids.


References 1. 2. 3. 4.


5. 6. 7. 8.

9.

10. 11. 12.

13. 14.

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Identification and Characterization of Flavonoid Target Proteins

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