Redox signalling and reactive oxygen and nitrogen species

Question 1

What are free radicals

Answer 1

they have an unpaired valence shell electron

Free radicals want to pair this electron

This is done through them ripping electrons from biological molecules

Free radicals oxidise biological molecules and become reduced – this is redox

Question 2

What are ROS and RNS a part of

Answer 2

consist of a large family of charged and uncharged compounds which all have the ability to oxidise other molecules

Superoxide anions are catalysed by an enzyme in hydrogen peroxide (superoxide dismutase)

A side reaction is the formation of a hydroxyl radical – this can cause damage to membranes and DNA (which can cause mutations)

Question 3

What is key features of free radicals

Answer 3

highly reactive and unstable

Question 4

How are free radicals controlled

Answer 4

antioxidants neutralise ROS / RNS (act as free radical
sponges) – can be endogenous (produced by cells) or exogenous (diet)

Question 5

What are examples of endogenous antioxidants

Answer 5

Endogenous enzymes and small molecular weight
thiols – glutathione, superoxide dismutase (SOD),
catalase, thioredoxin, peroxiredoxin

Question 6

What are examples of exogneous antioxidants

Answer 6

Vitamin C - green & red chilli peppers, oranges, kiwi
& broccoli
Vitamin E - sunflower seeds, paprika, red chilli
powder, nuts, dried fruits & olives
Vitamin A - liver, sweet potatoes, carrots & spinach
Others - flavonoids (green tea), zinc

Question 7

What is oxidative stress

Answer 7

When generation of free radicals is greater than the antioxidant capacity of the cell – this causes oxidative stress

Question 8

What are the benefits of oxidative stress

Answer 8

ROS and RNS are central second messengers in many transduction cascades

Question 9

What enzymes do ROS/RNS regulate

Answer 9

Kinases

Phosphatases

G-proteins

Membrane proteins (receptors)

Transcription factors

Metabolic enzymes

Question 10

How can ROS/RNS be generated on demand

Answer 10

Specific enzymes generate ROS/RNS

Specific enzymes can also remove the effect of ROS/RNS to reset the cell

Question 11

How is nitric oxide damaging to the body

Answer 11

Can target a haem group so is damaging to red blood cells – can prevent oxygen from binding to oxygen causing ischaemia

Question 12

What is another name for nitric oxide

Answer 12

Endothelium-derived relaxing factor (EDRF) caused relaxation of blood vessels – this was found to be nitric oxide

Question 13

How can nitric oxide be used in treatment

Answer 13

Advanced medicines (ischemic heart disease) drugs which liberate nitric oxide (nitroglycerin) - this can treat angina (chest pain caused by the heart muscle not getting enough oxygen)

Question 14

How is nitric oxide generated in cells

Answer 14

Nitric oxide in cells is generated from the enzymatic oxidation of the amino acid arginine

This is catalysed by nitric oxide synthase (NOS)

Question 15

What are the different tissue specific NOS enzymes

Answer 15

1 – nNOS = found in neural/brain, endometrium, skeletal muscle

2 – iNOS – inducible, found in macrophages, liver, smooth muscle, regulated by LPS, cytokines and glucocorticoids

3 – eNOS – found in endothelium, brain, heart and is regulated by phosphorylation

Question 16

What does nitric oxide do to smooth muscle

Answer 16

vasodilation

Question 17

What does the phosphorlyation and dephosphorlyation of mysoin light chains cause

Answer 17

Phosphorylation of myosin light chain by myosin light chain kinase (MLCK) increases ATPase activity of myosin = contraction

Dephosphorylation of myosin light chain by myosin light chain phosphatase (MLCP) decreases ATPase activity of myosin = relaxation

Question 18

What does nitric oxide do to smooth muscle

Answer 18

Nitric oxide inhibits the contraction of smooth muscle

This is due to the activation of PKG which reduces the level of calcium in the cells

PKG activates calcium regulating proteins

Question 19

How can nitric oxide regulate protein activity if proteins lack a haem group

Answer 19

If on the surface of a protein, cysteine and tyrosine can be modified by RNS and ROS

If this modification induces a conformational change in the active site or prevents protein being regulated by altering the chemical composition of a phosphorylation site –> protein activity can be modulated

Question 20

How can proteins be controlled using Cys residues

Answer 20

If a protein has a solvent accessible Cys, it has potential to be redox modified

Question 21

What happens to oncogenic growth factors during oxidative stress

Answer 21

Redox active oncogenic growth factor signalling pathways, apoptotic or epigenetic proteins may be more (or less) active during oxidative stress

Question 22

What bonds are susceptible to redox modification

Answer 22

S-S = disulphide
S-SG = S-glutathionylation
S-On = sulphination
S-NO = S-ntrosylation

Question 23

What can redox modification result in

Answer 23

These modifications may
potentially alter protein
activity i.e. activate,
deactivate or alter ligand
binding

Question 24

What is S-nitrosylation

Answer 24

When RNS oxidise cysteine

Question 25

What does S-nitrosylation cause

Answer 25

Numerous proteins have been shown to be regulated in this way Includes signalling proteins (like Ras, a G-protein) and metabolic proteins (like BCAT)

Question 26

What can RNS also nitrate along with cysteine

Answer 26

RNS can also nitrate tyrosine which can alter protein function in the same way as SNO

Question 27

What continuously generates ROS

Answer 27

mitochondria Electron leakage from the transport chain creates superoxide from molecular oxygen

Question 28

How is superoxide generated

Answer 28

generation by NADPH oxidase ROS generation may be induced NADPH oxidase (NOX) can generate superoxide on command

Question 29

Where do electrons come from to generate superoxide

Answer 29

NADPH

Question 30

What does superoxide dismutase do

Answer 30

generation of hydrogen peroxide

Question 31

Why can superoxide not move around the cell freely

Answer 31

Superoxide is negatively charged so cannot diffuse across the membrane Therefore it is restricted to the compartment it was generated in

Question 32

How does superoxide dismutase enable superoxide to move around the cell

Answer 32

Superoxide dismutase converts superoxide into hydrogen peroxide (which is uncharged) - this can diffuse freely through membranes by aquaporins

Question 33

What are key features of hydrogen peroxide

Answer 33

Hydrogen peroxide is stable and has the capacity to oxidise proteins and alter their activity

Question 34

What are targets of ROS

Answer 34

cysteine thiols [-SH]

Question 35

What can oxidation of thiol groups by ROS lead to

Answer 35

Oxidation of the thiol group by ROS can yield SOH (sulphenic acid), SO2H (sluphinic acid), SO3H (sulphonic acid), sulphinyl amide and disulphide bonds

Question 36

Where can disulphide bonds be found

Answer 36

Disulphides can be intramolecular (within the protein) or intermolecular (between proteins) – for intramolecular we look for proteins with CXXC motifs

Question 37

What is a key feature of thiol groups

Answer 37

Thiol groups are reductive and can donate electrons easily - is an antioxidant Cys thiol exists in reduced (SH) and oxidised forms (SOnH, SNO and disulphide S-S)

Question 38

How does glutathione (GSH) effect thiol groups

Answer 38

Under ROS and RNS, GSH can form ‘mixed disulphides’ with protein cysteine's This is termed S-glutathionylation and can result in altered protein activity

Question 39

What is the reduced form of glutathione (GSH)

Answer 39

GSSG glutathione disulphide

Question 40

How is the CXXC motif affected by ROS

Answer 40

Hydrogen peroxide can oxidise this motif and form intramolecular disulphuide Antioxidant enzymes reduce the disulphide restoring BCAT activity (which has the CXXC motif)

Question 41

What else can reduce the CXXC motif other than hydrogen peroxide

Answer 41

Thioredoxins (Trx) can reduce the CXXC motif disulphide bonds This will switch protein activity back and can therefore be used as a signal

Question 42

What motif does thioredoxin (Trx)

Answer 42

Trx also has a CXXC motif – this is oxidised when it reduces a disulphide Regeneration of Trx requires a reductase and electrons from NADPH

Question 43

What is an important ROS signalling cascade

Answer 43

Binding of growth factor to RTK causes activation of intracellular signalling cascade This causes proliferation and survival (oncogenic pathway) PTEN contains a redox active cysteine in the active site and when oxidised by ROS inactivates PTEN activity The loss of PTEN activity means that cell survival is enhanced since : PI3K --> Akt --> mTOR – this pathway becomes more active