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.

Targeting the Protein Quality Control (PQC) Machinery

“…Quercetin, in addition to Hsp27-targeted activities (see also section 2.2), causes Hsp70 inhibition, likely through inhibition of CK2 [50]. Biotinylated, active quercetin analogs prove that a direct Hsp70-quercetin binding is established in Hsp70-overexpressing cells [126]. Myricetin is identified as an Hsp70 ATPase inhibitor in an HTS campaign [89].…”

Chemical Proteomics Identifies Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1 as the Molecular Target of Quercetin in Its Anti-cancer Effects in PC-3 Cells

“…Many bioactive compounds have been found to be more promiscuous than originally anticipated, exerting desirable drug efficacy and undesirable toxic responses in parallel (8,32). The identification of protein-binding profiles of an active compound will provide invaluable information for deciphering both beneficial and cytotoxic interactions of drugs.…”

“…Binding of quercetin to MEK/Raf1 (in rat liver epithelial cells) or PI3K (in mouse JB6 P ϩ skin epidermal cells) suppresses the activity of the corresponding kinase induced by arsenite and tissue plasminogen activator, respectively (6,7). Quercetin has also been reported to bind casein kinase 2, one of the regulators that modulate global caspase signaling, and inhibit its activity (8,9). More recently, the heat shock proteins HSP70 and HSP90, RuvB-like 2 ATPases, and eIF-3 have also been identified as quercetin-binding proteins.…”

“…More recently, the heat shock proteins HSP70 and HSP90, RuvB-like 2 ATPases, and eIF-3 have also been identified as quercetin-binding proteins. All of these proteins have been considered potential targets in anti-cancer therapy; thus, there are numerous candidate mediators of the anti-cancer actions of quercetin (8,9).…”

Hunting Molecular Targets for Anticancer Reagents by Chemical Proteomics

“…O7-biotinylated AfBP probes of quercetin (BioQ) were prepared to purify possible binding proteins from cells. 53 Several possible quercetin protein targets, including HSP70, HSP90, ubiquitin-activating enzyme E1, splicesomal protein SPA-130, RuvBL2 ATPases, and eukaryotic translation initiation factors 3, have been identified from normal and heat-shocked Jurkat cells under stringent washing conditions. Most of the binding partners collected by PAL are potent cancer therapeutic targets accounting for the apoptotic and antitumor activities of quercetin.…”