L10 - Protein oxidation as a signalling mechanism II: Transcription factors regulated by oxidation Flashcards
Yap1 transcription factor
Yap1 is needed for the cells to survive.
Before stress the yap1 transcription factor is diffusely present around the cell but upon exposing the cells to hydrogen peroxide, there is rapid accumulation of yap1 in the nucleus.
Therefore, we knew that in response to hydrogen peroxide, this transcription factor changed its cellular localisation
Structure of the Yap1 transcription factor
Each of the N-CRD and the C-CRD contain 3 cysteine residues
CRD = cysteine rich domain
Within the C-CRD there is also a nuclear export sequence (NES) which interacts with the nuclear export factor to drive the transcription factor into the nucleus
In the DNA binding leucine zipper at the N-terminus there is a nuclear localisation sequence (NLS)
What happens when you get mutations in the N-CRD & the C-CRD?
Two different mutants were made – either all three cysteine residues in the N-CRD were mutated or all cysteine residues in the C-CRD were mutated.
In both cases, the oxidation of Yap1 is lost
How do we know Yap1 is oxidised in response to hydrogen peroxide?
When hydrogen peroxide was added to the cells, the mobility of Yap1 changed and became oxidised, showing that this protein is oxidised in response to hydrogen peroxide.
Reduced form binds AMS so is heavier and runs slower on SDS-PAGE
Potential structural consequences of oxidation
Oxidation of Yap1 prevents its interaction with the Crm1 nuclear export factor
1) When hydrogen peroxide is added, there is activation of the transcription factor by oxidation of the cysteine residues.
2) This drives a structural conformational change in the protein that is masking the nuclear export sequence bound within the C-CRD and preventing interaction with the Crm1 nuclear export factor.
3) Yap1 is thus retained in the nucleus and it allows the expression of catalase and superoxide dismutase.
Yap1 is not directly oxidised itself by hydrogen peroxide
Instead, the protein Gpx3 is initially oxidised forming a sulphenic acid group on Gpx3.
Once Gpx3 is oxidised, it can form an intermolecular disulphide bond with the cysteine 598 found within the C-terminal domain of Yap1 forming a Yap1-Gpx3 mixed disulphide.
Genetic studies showed that a third protein, ybp1 (yap binding protein 1) was needed to bind to the C terminal domain for the oxidation event to occur.
The mechanism of this is not completely understood, but the hypothesis is that Ybp1 keeps the structure of the C-terminal domain that allows for the oxidation event to occur
Which 2 cysteine residues are critical for Yap1 oxidation?
C303 & C598
As when they are mutated they lose the oxidation & the protein runs as if it hasn’t been treated with hydrogen peroxide
Stepwise oxidation of Yap1
SUMMARY
- Upon exposure to H2O2, the catalytic site cysteine Cys36 of Gpx3 is oxidised to a reactive sulphenic acid (Cys36-SOH)
- Cys36-SOH subsequently reacts with Cys598 of Yap1 c-CRD to form a mixed Yap1-Gpx3 disulphide intermediate. This also requires Ybp1 binding to Yap1.
- Rearrangement of this disulphide by intramolecular thiol-disulphide exchange with Cys303 of Yap1p then leads to the formation of the first intramolecular disulphide bond between Cys303-Cys598 in Yap1 (and the regeneration of reduced Gpx3).
- Triggered by this intramolecular disulphide bond formation, Yap1p undergoes further oxidation events resulting in conformational changes that mask the NES and disrupt the Yap1p–Crm1 interaction.
- This allows for Yap1 nuclear accumulation and anti-oxidant gene expression.
Upon return to non-stress conditions, disulphide bonds are reduced by the thioredoxin- system, leading to the re-exposure of the NES and Yap1 nuclear export
The analogous transcription factor Cap1, in the pathogenic fungus Candida albicans, is regulated in the same way as Yap1
In the same way as yap1 by the Gpx3 protein and the ybp1 protein.
• Gpx3 becomes oxidised
• Gpx3 forms mixed disulphide with Cap1
• This initiates the intracellular disulphides in Cap1
Oxidation has been show to drive the nuclear localisation of cap1 and this is not seen in either the ybp1 or the gpx3 null cells
Because of this, there is no expression of the key antioxidant enzymes which are catalase and superoxide dismutase
The Cap1 transcription factor in the pathogenic fungus Candida albicans is important for this yeast to survive ROS-mediated killing by macrophages
When a candida cell encounters a macrophage, the candida cells get taken up by the macrophages really quickly but then the candida change shape to form projections (long filaments) that eventually pierce the membrane of the macrophage, killing it.
It seems that the cap1 transcription factor is important for this pathogen to survive phagocytosis by macrophages.
Within a macrophage, there is an NADPH oxidase complex which produces high levels of superoxide anions and the cap1 transcription factor becomes activated in the candida cell following exposure to the ROS, reducing the antioxidant enzymes, allowing for the survival and filamentation within the macrophage
OxyR transcription factor in bacteria is regulated by oxidation
The OxyR transcriptional regulator senses low amounts of intracellular H2O2 and maintains H2O2 levels within safe limits
In the presence of iron H2O2 can undergo the Fenton reaction to produce hydroxyl radicals and these are extremely dangerous
Cysteine 199 has a very low pKa value so is extremely reactive
Upon exposure to hydrogen peroxide, cysteine 199 forms an intramolecular disulphide with a different cysteine residue in the protein (C208) and this changes the overall structure of protein, which now allows it to bind RNA polymerase.
When RNA polymerase is brought to the promoters, there is the expression of key antioxidant genes, for example, katG (catalase)
AMS experiment showing oxidation of OxyR in bacteria
Gel contains extracts that have been resuspended in buffers containing thiol binding agent AMS and then the extracts have been analysed by SDS-PAGE and western blotting
Oxidised OxyR has a faster mobility on SDS-PAGE because oxidised form has a lower molecular weight because it doesn’t bind AMS
E. coli OxyR mechanism
OxyR is constantly bound to the promoters of the genes that it regulates but it needs to recruit RNA polymerase to stimulate the production of mRNA
1) Cys199 is oxidised at low H2O2 to sulphenic acid
2) Oxidation results in the flipping out of Cys199 from its hydrophobic pocket
3) Triggers intramolecular disulphide bond formation with Cys208
4) This drives significant conformational changes which allows OxyR to now bind RNA polymerase
Mammalian Nrf2 transcription factor is also regulated by oxidation
Under non-stress conditions Nrf2 forms a tight complex with the Cys-rich protein Keap1
Keap1 negatively regulates Nrf2 by retaining in the cytoplasm and targeting it for ubiquitin-mediated proteasomal degradation
Keap1 has 27 Cys residues in the human protein – only 3 are redox active
Exposure to H2O2 induces both intramolecular and intermolecular disulpfides.
The intermolecular disulphide promotes conformational changes within Keap1 which allows Nrf2 release and stabilisation
What does the Nrf2 transcription factor do?
Major transcription factor regulating ROS protective genes in multicellular eukaryotes
Nrf2 forms functional dimers with other transcription factors, and these dimers regulate numerous genes, many of which have antioxidant functions.
