L9 - Protein oxidation as a signalling mechanism I Flashcards
What are reactive oxygen species?
Oxygen molecules that are highly reactive because they have unpaired electrons
Because they are highly reactive, they are trying to steal electrons from substrates, to make them less reactive.
By doing this, they get reduced and the substrates they steal the electrons from become oxidised
What is the most unstable ROS?
Peroxide is the most unstable because it has two unpaired electrons and as such it is undetectable in the cell as it forms hydrogen peroxide
What is the most stable ROS?
Hydrogen peroxide is the most stable of these reactive oxygen species and this is the most important signalling molecule
What are the main ROS found in our cells?
Hydrogen peroxide (H2O2) Hydroxyl radical (•OH) Superoxide anion (O2-•)
OILRIG
Oxidation Is Loss Reduction is Gain (of electrons)
Cellular sources of ROS
There are two main sources of reactive oxygen species in the cell
1) Electron transport chain
2) NADPH oxidase complex
How does the electron transport chain create ROS?
During this oxidative phosphorylation event, there is the incomplete reduction of oxygen which can occur at complex I and complex III in the electron transport chain, and this forms superoxide.
All aerobically grown cells are constantly producing superoxide – up to 2% of all molecular oxygen forms superoxide
How the NADPH oxidase complex create ROS?
Deliberately produces reactive oxygen species
Within phagocytes, such as macrophages or neutrophils, if the white blood cells come across an infectious agent, then the NADPH oxidase complex is activated and uses electrons from NADPH to generate really high levels of superoxide within the phagosome.
These really high levels of superoxide are used as an anti-microbial killing mechanism.
This is also known as the respiratory burst
What happens to people if they have defects in their NADPH oxidase complex?
Can be really susceptible to lots of different infections
No anti-microbial killing mechanism
How does the superoxide formed by both the electron transport chain and NADPH oxidase complex form H2O2 or •OH?
Superoxide is also extremely reactive because of its unpaired electrons, so this will also actively form hydrogen peroxide.
In the presence of iron or copper, it can undergo the Fenton reaction and produce really toxic, highly reactive, hydroxyl radical species.
How do cells keep ROS at manageable levels?
Through the activity of antioxidant defences which are mainly the highly reactive antioxidant enzymes, such as superoxide dismutase and catalase
These keep the reactive oxygen species at a safe level
When is the cell in a state of oxidative stress?
When the ROS are exceeding the antioxidant capacity of the cell.
This could be due to the respiratory burst, for example, in the phagosome.
What does oxidative stress do to the cell?
The excess of ROS are then stealing electrons from all cellular substrates which causes the oxidation of proteins lipids and DNA.
This can be extremely damaging to the cell. ROS is one of the best understood triggers of apoptosis and cell death.
What is the free radial theory of ageing?
An accumulation of oxidative damage that occurs throughout the lifespan of an organism, contributes to the process of aging
This theory says that all the time, organisms are constantly exposed to endogenous sources of ROS and all exogenous sources of ROS
Endogenous sources of ROS
Mitochondria
Peroxisomes
NADPH oxidase
Exogenous sources of ROS
UV
Ionising radiation
Chemotherapy
Environmental toxins
Antioxidant defences in the free radial theory of ageing
Antioxidant defences try to prevent the damage but all the time there is an accumulation of oxidative damage and impaired physiological function and this is thought to contribute to the aging process
Are ROS always harmful?
NO
NADPH oxidase complex is now shown to be present in highly eukaryotic cell types
These NADPH oxidases (Nox enzymes) deliberately generate small levels of hydrogen peroxide to act as a signalling molecule that regulates cell growth, division, migration and differentiation
But it can also trigger the activation of transcription factors to cause the increase in expression of antioxidant genes to prevent the high damaging levels of hydrogen peroxide
Key player in protein oxidation in oxidative stress sensing & signalling
Key player = cysteine
H2O2 functions as a signalling molecule by targeting specific cysteine residues for oxidation
Will change the function of the protein so it then functions as a redox switch
Thiol (-SH) side chain is highly reactive
What can the oxidation of thiols regulate?
Protein function
Activity
Cellular localisation
Example of when mammal cells are exposed to H2O2
ASK1 exists in mammals as a reduced inactive form in nonstress conditions
As soon as the cells are exposed to hydrogen peroxide, the kinase and transcription factor become oxidised, leading to key signalling outcomes
Once ASK1 is activated, it drives the activation of the P38 SAPK pathway
This allows the cells to survive the oxidative stress
What does SAPK mean?
Stress-activated protein kinase
Example of when yeast cells are exposed to H2O2
YAP1, in nonstress conditions is in a reduced inactive form
When exposed to H2O2, Yap1 becomes oxidised & activated - allowing it to accumulate in the nucleus and causes the expression of antioxidant genes, such as catalase
This allows the cells to survive the oxidative stress
What happens when the thiol group in cysteine is oxidised?
Sulphenic acid is formed - very reactive
First modification that occurs in response to low levels of H2O2
Sulphenic acid is very reactive - what does this mean?
This is so reactive and is very hard to capture inside the cell
Will form intramolecular disulphide bonds or intermolecular disulphide bonds - most common
However, can also form sulphenamide
How does sulphenic acid form INTRAmolecular disulphide bonds?
The sulphenic acid form can interact with another thiol group within the same protein to form an intramolecular disulphide bond
How does sulphenic acid form INTERmolecular disulphide bonds?
It can also react with a thiol on a separate protein to create an intermolecular disulphide bond
How does sulphenic acid form sulphenamide?
The sulphenic acid form can also interact with neighbouring amino groups to form the cyclical sulphenamide structure
What 3 oxidation events are reversible by the action of thioredoxin?
Thiol to sulphenic acid
Sulphenic acid to intermolecular bonds
Sulphenic acid to intramolecular bonds
What happens in response to high H2O2 levels?
Sulphenic acid can become further oxidised to sulphinic acid or under really high levels of oxidative stress can be oxidised to hyperoxidised sulphonic acid form
The sulphenamide can also be further oxidised to a sulphinamide form
What oxidation event can be reversed by the action of sulphiredoxin?
Sulphenic acid to sulphinic acid
What are the key features of signalling PTMs?
Specificity
Reversibility
What determines specificity in cysteine oxidation?
Cysteine is a relatively uncommon AA
Accessibility - looking for cysteine residues with free thiol groups on the surface of proteins and are accessible to ROS
Very few cysteine resides are redox sensitive
What is a pKa value?
Acid dissociation constant
The pH at which half the population is de-protonated
What does the pKa value of thiol groups within cysteine residues depend on?
Depends on whether it is a reactive cysteine residue (i.e. can become oxidised or not)
pKa values of thiol groups
Most protein thiols have pKa > 8
• Non reactive thiol groups
Redox sensitive thiols have pKa 3-5.5
• Few cysteine residues are in this environment
It is the local amino acid environment that determines reactivity of the cysteine residue
Why does cysteine oxidation need to be reversible?
Once the hydrogen peroxide signal is gone, we need to convert the oxidised active form of the protein to the inactive reduced form of the protein
Done by thioredoxin & sulphiredoxin
How does thioredoxin reverse cysteine oxidation?
Thioredoxin is oxidised when then reduced the protein back to the inactive reduced form
Thioredoxin becomes oxidised by reversing the oxidation state of the protein so then it itself needs to be reduced into the active form. This is undertaken by thioredoxin reductase
How does thioredoxin become active again?
Needs to be reduced by thioredoxin reductase
How does thioredoxin reductase work?
The main function of thioredoxin reductase is to keep thioredoxin in its reduced active state
Does this by using electrons from NADPH
This is a thioredoxin cycle
Thioredoxin reductase is a highly conserved protein found in all cells.
How does sulphiredoxin reverse cysteine oxidation?
Reduction of the sulphinic acid form to the sulphenic acid form and
This is undertaken by sulphiredoxin which is undertaken using an ATP mechanism to reduce the oxidised form
Problems in detecting protein oxidation
Oxidation is an unstable PTM
Breaking open cells triggers the artificial oxidation of many proteins
Solutions to the problems of detecting protein oxidation
Acid lysis
• By breaking cells open using acid – this lowers the pH so even the most reactive cysteine residues (pKa < 5) stay reduced (protonated)
The use of cysteine-binding chemical that only bind to reduced cysteine residues
What is the easiest oxidation event to detect?
Intermolecular disulphides
How does Tsa1 form intermolecular disulphides in response to H2O2?
Tsa1 detoxifies hydrogen peroxide and as a result becomes oxidised
Goes from a monomeric form to a dimeric form in response to H2O2
This can be visualised using SDS-PAGE and western blot
Intermolecular disulphide detection using SDS-PAGE and western blot
- Collect cells and lyse in acid to trap in the oxidation state
- Run extracts on a gel
- Transfer gel to a membrane
- Then use an antibody that will bind to Tsa1
- Before stress, without an oxidation species, Tsa1 is largely in the monomeric reduced state
- After introduction of hydrogen peroxide, the protein forms disulphide bond where there are two Tsa1 molecules (dimeric forms) and it runs slower on the gel
- Reducing agent DTT breaks disulphide bond
- Oxidation it detected by an increase in size and this is DTT sensitive
What is DTT?
Dithiolthreitol
Reducing agent that reduces disulphide bonds - breaks them
The use of cysteine binding chemicals that only bind to reduced cysteine residues
They range in molecular weight from the really small NEM molecules to AMS molecules to the much larger PEG-maleimidie molecules
NEM - MW = 0.1 kDa
AMS - MW = 0.5 kDa
PEG-Mal - MW = 2-5 kDa
Detection of protein oxidation using AMS
Method
1) Extract proteins from cells under acidic conditions
2) Resusend in buffer & AMS
3) Run extracts on PAGE
4) Western blotting to detect proteins of interest
Detection of protein oxidation using AMS
If the protein is reduced:
Both of the reduced thiol groups of cysteine will bind AMS
Leads to a higher MW due to AMS binding
Has slower mobility on SDS-PAGE
Detection of protein oxidation using AMS
If the protein is oxidised:
In response to hydrogen peroxide, the proteins oxidise so it will either produce a sulphenic acid form so only one AMS can bind or an intramolecular disulphide bond can form and this prevents AMS binding
Leads to a lower molecular weight as no AMS binding
Has faster mobility on SDS-PAGE
How can detection of protein oxidation on SDS-PAGE using AMS show that oxidation is reversible?
This oxidation event is reversible because when hydrogen peroxide is added, after one minute, it becomes oxidised but then by 10 minutes the oxidised form is lost.
This shows that it is not oxidised forever as there is thioredoxin there to reverse it
How can you detect intramolecular disulphide bonds?
This is using 2 different reagents that bind to reduced thiol residues
We block reduced cysteine residues with NEM (which has a really small molecular weight) so all the reduced thiols are blocked (protected).
We then reduce an oxidised thiol residue using DTT so the thiol groups are released and can now bind to a different thiol reagent called PEG-Mal (high molecular weight).
Identification of intramolecular di-sulphide bonds
If there is an intramolecular disulphide bond, then the protein will be much greater in size than if it is reduced - will move slower on a gel
What is OxiCAT?
A quantitative redox proteomic approach
Global analysis of what proteins are oxidised in the cell.
Can tell us which cysteines are oxidised
Process of OxiCAT?
Uses the same principle as described before to detect disulphides
1) Reduced cysteines residues are all blocked with light ICAT.
2) Any disulphides are reduced with DTT and this then releases the thiol groups.
3) Newly formed thiols are then labelled with the heavier ICAT form.
4) The oxidised forms labelled with heavier ICAT
Structure of ICAT
ICAT has a thiol binding group and a biotin tag
The different between the light and the heavy ICAT is the size of the linker region.
What is the biotin tag in ICAT?
Allows for purification of cysteine containing peptides
