L9 - Protein oxidation as a signalling mechanism I

Question 1

What are reactive oxygen species?

Answer 1

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

Question 2

What is the most unstable ROS?

Answer 2

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

Question 3

What is the most stable ROS?

Answer 3

Hydrogen peroxide is the most stable of these reactive oxygen species and this is the most important signalling molecule

Question 4

What are the main ROS found in our cells?

Answer 4

Hydrogen peroxide (H2O2)
Hydroxyl radical (•OH)
Superoxide anion (O2-•)

Question 5

OILRIG

Answer 5

Oxidation Is Loss Reduction is Gain (of electrons)

Question 6

Cellular sources of ROS

Answer 6

There are two main sources of reactive oxygen species in the cell

1) Electron transport chain
2) NADPH oxidase complex

Question 7

How does the electron transport chain create ROS?

Answer 7

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

Question 8

How the NADPH oxidase complex create ROS?

Answer 8

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

Question 9

What happens to people if they have defects in their NADPH oxidase complex?

Answer 9

Can be really susceptible to lots of different infections

No anti-microbial killing mechanism

Question 10

How does the superoxide formed by both the electron transport chain and NADPH oxidase complex form H2O2 or •OH?

Answer 10

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.

Question 11

How do cells keep ROS at manageable levels?

Answer 11

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

Question 12

When is the cell in a state of oxidative stress?

Answer 12

When the ROS are exceeding the antioxidant capacity of the cell.

This could be due to the respiratory burst, for example, in the phagosome.

Question 13

What does oxidative stress do to the cell?

Answer 13

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.

Question 14

What is the free radial theory of ageing?

Answer 14

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

Question 15

Endogenous sources of ROS

Answer 15

Mitochondria
Peroxisomes
NADPH oxidase

Question 16

Exogenous sources of ROS

Answer 16

UV
Ionising radiation
Chemotherapy
Environmental toxins

Question 17

Antioxidant defences in the free radial theory of ageing

Answer 17

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

Question 18

Are ROS always harmful?

Answer 18

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

Question 19

Key player in protein oxidation in oxidative stress sensing & signalling

Answer 19

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

Question 20

What can the oxidation of thiols regulate?

Answer 20

Protein function
Activity
Cellular localisation

Question 21

Example of when mammal cells are exposed to H2O2

Answer 21

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

Question 22

What does SAPK mean?

Answer 22

Stress-activated protein kinase

Question 23

Example of when yeast cells are exposed to H2O2

Answer 23

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

Question 24

What happens when the thiol group in cysteine is oxidised?

Answer 24

Sulphenic acid is formed - very reactive

First modification that occurs in response to low levels of H2O2

Question 25

Sulphenic acid is very reactive - what does this mean?

Answer 25

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

Question 26

How does sulphenic acid form INTRAmolecular disulphide bonds?

Answer 26

The sulphenic acid form can interact with another thiol group within the same protein to form an intramolecular disulphide bond

Question 27

How does sulphenic acid form INTERmolecular disulphide bonds?

Answer 27

It can also react with a thiol on a separate protein to create an intermolecular disulphide bond

Question 28

How does sulphenic acid form sulphenamide?

Answer 28

The sulphenic acid form can also interact with neighbouring amino groups to form the cyclical sulphenamide structure

Question 29

What 3 oxidation events are reversible by the action of thioredoxin?

Answer 29

Thiol to sulphenic acid

Sulphenic acid to intermolecular bonds

Sulphenic acid to intramolecular bonds

Question 30

What happens in response to high H2O2 levels?

Answer 30

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

Question 31

What oxidation event can be reversed by the action of sulphiredoxin?

Answer 31

Sulphenic acid to sulphinic acid

Question 32

What are the key features of signalling PTMs?

Answer 32

Specificity

Reversibility

Question 33

What determines specificity in cysteine oxidation?

Answer 33

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

Question 34

What is a pKa value?

Answer 34

Acid dissociation constant

The pH at which half the population is de-protonated

Question 35

What does the pKa value of thiol groups within cysteine residues depend on?

Answer 35

Depends on whether it is a reactive cysteine residue (i.e. can become oxidised or not)

Question 36

pKa values of thiol groups

Answer 36

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

Question 37

Why does cysteine oxidation need to be reversible?

Answer 37

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

Question 38

How does thioredoxin reverse cysteine oxidation?

Answer 38

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

Question 39

How does thioredoxin become active again?

Answer 39

Needs to be reduced by thioredoxin reductase

Question 40

How does thioredoxin reductase work?

Answer 40

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.

Question 41

How does sulphiredoxin reverse cysteine oxidation?

Answer 41

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

Question 42

Problems in detecting protein oxidation

Answer 42

Oxidation is an unstable PTM

Breaking open cells triggers the artificial oxidation of many proteins

Question 43

Solutions to the problems of detecting protein oxidation

Answer 43

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

Question 44

What is the easiest oxidation event to detect?

Answer 44

Intermolecular disulphides

Question 45

How does Tsa1 form intermolecular disulphides in response to H2O2?

Answer 45

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

Question 46

Intermolecular disulphide detection using SDS-PAGE and western blot

Answer 46

• 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

Question 47

What is DTT?

Answer 47

Dithiolthreitol

Reducing agent that reduces disulphide bonds - breaks them

Question 48

The use of cysteine binding chemicals that only bind to reduced cysteine residues

Answer 48

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

Question 49

Detection of protein oxidation using AMS

Method

Answer 49

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

Question 50

Detection of protein oxidation using AMS

If the protein is reduced:

Answer 50

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

Question 51

Detection of protein oxidation using AMS

If the protein is oxidised:

Answer 51

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

Question 52

How can detection of protein oxidation on SDS-PAGE using AMS show that oxidation is reversible?

Answer 52

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

Question 53

How can you detect intramolecular disulphide bonds?

Answer 53

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).

Question 54

Identification of intramolecular di-sulphide bonds

Answer 54

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

Question 55

What is OxiCAT?

Answer 55

A quantitative redox proteomic approach

Global analysis of what proteins are oxidised in the cell.

Can tell us which cysteines are oxidised

Question 56

Process of OxiCAT?

Answer 56

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

Question 57

Structure of ICAT

Answer 57

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.

Question 58

What is the biotin tag in ICAT?

Answer 58

Allows for purification of cysteine containing peptides