Biotinylated quercetin as an intrinsic photoaffinity proteomics probe for the identification of quercetin target proteins

Abstract

Quercetin is a flavonoid natural product, that is, found in many foods and has been found to have a wide range of medicinal effects. Though a number of quercetin binding proteins have been identified, there has been no systematic approach to identifying all potential targets of quercetin. We describe an O7-biotinylated derivative of quercetin (BioQ) that can act as a photoaffinity proteomics reagent for capturing quercetin binding proteins, which can then be identified by LC-MS/MS. BioQ was shown to inhibit heat induction of HSP70 with almost the same efficiency as quercetin, and to both inhibit and photocrosslink to CK2 kinase, a known target of quercetin involved in activation of the heat shock transcription factor. BioQ was also able to pull down a number of proteins from unheated and heated Jurkat cells following UV irradiation that could be detected by both silver staining and Western blot analysis with an anti-biotin antibody. Analysis of the protein bands by trypsinization and LC-MS/MS led to the identification of heat shock proteins HSP70 and HSP90 as possible quercetin target proteins, along with ubiquitin-activating enzyme, a spliceosomal protein, RuvB-like 2 ATPases, and eukaryotic translation initiation factor 3. In addition, a mitochondrial ATPase was identified that has been previously shown to be a target of quercetin. Most of the proteins identified have also been previously suggested to be potential anticancer targets, suggesting that quercetin's antitumor activity may be due to its ability to inhibit multiple target proteins.

Figure 1

Strategy for pull down and identification of quercetin target proteins. A) Structure of quercetin and derivatives. BioQ was designed for pulling down protein targets of quercetin. B) Steps involved in pulling down and identifying quercetin target proteins with BioQ.

Figure 2

Two synthetic routes to BioQ.

Figure 3

Structural characterization of BioQ by COSY, HMQC, and HMBC. Through bond correlations A, B, C, D were observed in ¹H-¹H COSY, one bond ¹H-¹³C correlations were made by HMQC, and multiple through bond correlations F, H, J, and K were by HMBC. See Fig. S1-S3 for the 2D spectra.

Figure 4

Ability of BioQ to inhibit heat shock induction of HSP70 in Jurkat cells. Jurkat cells were treated with 145 μM quercetin (Q), DMSO vehicle (V) or 145 μM BioQ (BQ) for 2 h prior to a 30 min 43°C heat shock (HS) and then allowed to recover at 37°C for 8 h before Western blot analysis of HSP70 and actin levels. Unheated Jurkat cells (C) and heated Jurkat cells in the absence of any additive (-) were used as controls. The results were reproducible.

Figure 5

Casein Kinase II pull down by BioQ. Increasing concentrations of CK2 (lanes 3-6: 17.5, 35, 52.5, 70 nM) in 400 μL, 10 mM PBS buffer (pH 7.2), were incubated with 150 μM BioQ and then irradiated at 365 nM for 30 min in the (a) absence or (b) presence of 10 mM MgCl₂ and 2 μM ATP. The biotinylated proteins were then pulled down by streptavidin beads and subj ected to a denaturing wash prior to electrophoresis. L is a Fermentas prestained protein ladder. Lane 1 in panel (a) and lane 2 in panel (b) were controls that contained 0.05 μg CK2, while lane 2 in panel (a) and lane 1 in panel (b) contained 0.1 μg of CK2. α and β refer to the positions of the α and β subunits of CK2, and k refers to the position of contaminating keratin proteins.

Figure 6

SDS PAGE of proteins pulled down from Jurkat cells incubated with BioQ that were UV irradiated following lysis. Left: Silver stained 12% SDS polyacrylamide electrophoresis gel of proteins. Lanes 1 and 2: proteins pulled down from lysates of normal or heat shocked Jurkat cells by the streptavidin beads in the absence of BioQ and UV irradiation. Lanes 3 and 4: proteins pulled down from lysates of normal or heat shocked cells that had been incubated with 150 μM BioQ in the absence of UV irradiation. Lanes 5 and 6: proteins pulled down from lysates of normal or heat shocked cells that had been incubated with BioQ and UV irradiated for 30 min following lysis, and then subjected to a denaturing wash. Lane 7: proteins pulled down under the same conditions as for lane 6, except that the lysate was not UV irradiated. Right: Western blot of corresponding lanes with anti-biotin antibody. Legend: heat, heat shock applied after incubation with BioQ but before lysis; wash, washing beads with cell lysis buffer; denaturing wash, washing beads with 2% SDS buffer.

Figure 7

SDS PAGE of proteins pulled down from Jurkat cells incubated with BioQ that were irradiated prior to lysis. Left: Proteins pulled down with streptavidin beads from Jurkat cells under similar sets of conditions as in the gel in Figure 6. Right: Western blot with anti-biotin antibody. Legend the same as in Figure 6.