Mass Spectrometry Analysis of Nrf2-Keap1 Chemoprevention Signaling Pathway
thesisposted on 10.12.2012, 00:00 by Chenqi Hu
Chemoprevention is an active cancer preventive strategy to inhibit, delay or reverse human carcinogenesis using naturally occurring or synthetic chemical agents. Dietary natural products and diet-derived agents are potential chemopreventive agents since they are relatively safe and exhibit no or low toxicity. Activation of the transcription factor NF-E2-related factor-2 (Nrf2), leading to up-regulation of the antioxidant response element (ARE) for cellular defense against reactive oxygen species and xenobiotic electrophiles, is an important strategy for cancer chemoprevention. Numerous potential ARE activators have been identified, many of which are abundant in natural plants. However, how these chemopreventive agents activate ARE through Nrf2-Keap1 signaling pathway is still unclear. In this dissertation, mass spectrometry was employed to 1) screen natural product extracts for possible chemopreventive agents that can activate ARE-mediated gene expression; and 2) facilitate clarification of the Nrf2-Keap1 signaling pathway mechanism in terms of cysteine alkylation, reversibility of the cysteine-electrophile adducts and protein 3D structure. Firstly, in the new screening method, electrophiles that reversibly modify Keap1 protein can be trapped by β-mercaptoethanol (BME) and identified by mass spectrometry as adducts. Use of high resolution accurate mass measurement on a HPLC-ion trap-TOF mass spectrometer (IT-TOF MS) and automated peak detection software, permitted approximately 20-fold more sensitive detection than our previously developed MALDI-TOF MS-based assay and improved other aspects of that assay. Next, in order to reconcile the conflicting data in vitro and in vivo data regarding which Keap1 cysteine residues become modified for some ARE activators such as sulforaphane, a revised sample preparation protocol was developed. Our studies indicate that iodoacetamide treatment usually used during sample preparation promotes dissociation of sulforaphane-cysteine adducts, especially adducts with C151 of Keap1. By eliminating the iodoacetamide treatment step and reducing sample preparation time, we showed that C151 is detected as one of the four most reactive cysteine residues in Keap1 towards sulforaphane. In addition, the effect of iodoacetamide treatment on the other five activators was evaluated and the streamlined protocol was used to investigate the Keap1 cysteine alkylation pattern of PGA2 and 15d-PGJ2. Two Keap1 C151-independent ARE activators. Subsequently, to support the Keap1-Cul3 interaction alteration mechanistic study, peptide mapping studies was carried out to probe the Keap1 conformation change that alters cysteine modification patterns of wt Keap1 and Keap1 C151W mutant before and after Cul3 binding. The differentially modification of C636 of Cul3 implies a change in the Keap1-Cul3 interaction induced by the modification of Keap1 C151, apart from a simple disruption of the Keap1-Cul3 complex, changing the current model in the field. Finally, considering the unavailability of the three dimensional structure of either Keap1 or Cul3 protein, we developed a cross-linking coupled with mass spectrometry method to probe directly the Keap1 and Cul3 structure, as well as their interaction interface. We found that the cross-linker bis(sulfosuccinimidyl) suberate (BS3) is more suitable for intermolecular cross-linking studies than is glutaraldehyde due to its longer spacer and reaction specificity. With a much faster scanning speed, more efficient fragmentation and high mass accuracy of both precursor and product ions, the LTQ Orbitrap Velos is a more suitable instrument for cross-linked peptides identification than the LTQ FT-ICR mass spectrometer.