Study of the molecular details of p53 redox-regulation using Fourier transform ion cyclotron resonance mass spectrometry
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Date
27/06/2011Author
Scotcher, Jenna
Metadata
Abstract
Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and superoxide
(O2
• −) have been shown to serve as messengers in biological signal transduction, and
many prokaryotic and eukaryotic proteins are now known to have their function
controlled via ROS-mediated oxidation reactions occurring on critical cysteine
residues.
The tumour-suppressor protein p53 is involved in the regulation of a diverse
range of cellular processes including apoptosis, differentiation, senescence, DNArepair,
cell-cycle arrest, autophagy, glycolysis and oxidative stress. However, little is
understood about the specific molecular mechanisms that allow p53 to discriminate
between these various different functions. p53 is a multiple cysteine-containing
protein and there is mounting evidence to suggest that redox-modification of p53 Cys
residues participate in control of its biological activity. Furthermore, p53 activity has
been linked to intracellular ROS levels.
Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS)
offers superior mass resolving power and mass measurement accuracy, which is
beneficial for the study of intact proteins and the characterisation of their posttranslational
modifications (PTMs). The primary goal of the work described in this
thesis was to employ FT-ICR mass spectrometry to investigate the molecular details
of p53 redox-regulation.
The relative reactivity of each of the ten cysteine residues in the DNA-binding
core domain of recombinant human p53 was characterised by treatment with the
Cys-alkylating reagent N-ethylmaleimide (NEM) under various conditions. A
combination of top-down and middle-down FT-ICR MS was used to unambiguously
identify Cys182 and Cys277 as sites of preferential alkylation. These results were
confirmed by site-directed mutagenesis. Interestingly, Cys182 and Cys277 have
previously been implicated in p53 redox-regulation. Alkylation beyond these two
residues was found to trigger rapid alkylation of the remaining Cys residues,
presumably accompanied by protein unfolding. These observations have implications
for the re-activation of mutant p53 with Cys-targeting compounds which result in the
death of cancer-cells.
Furthermore, the molecular interaction between p53 and the ROS hydrogen
peroxide was investigated. p53 was found to form two disulfide bonds upon
treatment with H2O2. An enrichment strategy was developed to purify oxidised p53
and top-down FT-ICR mass spectrometry revealed unambiguously that Cys176, 182,
238 and 242 were the oxidised residues. Interestingly, Cys176, 238 and 242 are Zn2+-
binding residues suggesting that p53 contains a zinc-redox switch. The mechanism of
H2O2 oxidation was investigated, and revealed that oxidation via an alternative
pathway results in indiscriminate over-oxidation of p53. Moreover, Cys176, 238 or
242 was shown to act as a nucleophile, and the intracellular antioxidant glutathione
(GSH) did not prevent oxidation of the Zn2+-binding Cys residues, providing further
evidence for a role in p53 redox-regulation.
This study has revealed hitherto unknown details regarding the chemistry of
cysteine residues within the important tumour-suppressor protein p53. Furthermore,
the analytical power of FT-ICR MS for the study of multiple Cys-containing proteins
has been very clearly demonstrated.