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BIOC353 > Module 5- ROS And Cell Signalling > Flashcards

Module 5- ROS And Cell Signalling Flashcards

1
Q

What is a free radical

A

Any molecular species with one or more unpaired electrons- initiate chain reactions by moving an electron from another molecule to fill its own orbital

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2
Q

What is a reactive oxygen species (ROS)

A

Free radical plus non-radical oxygen species- chemical species that are able to react with targets to oxidise them

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3
Q

The two main places where ROS are generated

A

The ETC and NADPH oxidases (NOX)

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4
Q

The ETC and ROS

A

Biggest source of ROS- from electrons escaping transfer complexes and reacting with oxygen
Determined by flux through ETC, activity of cellular antioxidants and free Fe levels (fenton reaction)
Molecules that alter ETC flux regulate ROS production eg uncoupling protein activators, ETC complex inhibitors (metformin)
High ROS from mitochondria can cause oxidative damage to lipids and proteins

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5
Q

NOX and ROS

A

Transmembrane enzymes assembled from various protein subunits. Different isoforms
Facilitate electron transfer from NADPH to oxygen to produce superoxide
Many cellular locations= localisation of production
Superoxide is converted to hydrogen peroxide by SOD- used in cell signalling

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6
Q

What is NOX coupled to and what inhibits them

A

Coupled to angiotensin, growth and cytokine receptors
Inhibited by statins, angiotensin receptor blockers, growth receptor inhibitors, kinase inhibitors and NOX inhibitors

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7
Q

Measuring ROS

A

Direct measurement of individual oxidants eg EPR and fluorescent probes
Measuring oxidative products
Some assays like HORAC give overall measure of oxidative status
Hard to measure as they are so reactive

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8
Q

ROS products which are often measured

A

Commonly measured as they have a longer half life and can be measured by many different assays
Protein oxidation products- aa side chains sensitive to oxidation
Lipid oxidation
DNA oxidation- modification of bases and sugars, can induce breaks and DNA-protein cross links, if not repaired can lead to cancer and age-related disease

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9
Q

More on protein oxidation products and how they are measured

A

Generally more stable than lipid oxidation products
Can get direct oxidation, loss of thiols or formation of specific products
Measured through protein carbonyls, loss of protein thiols (both spectrophotometrically), ratio of GSH to GSSG or measure of glutathionated proteins

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10
Q

More on lipid oxidation products and how they are measured

A

Lipid hydroperoxides from oxidation of polyunsaturated fatty acids
Further breakdown into aldehydes eg MDA and HNE- can be measured with TBRAs assay and/or HPLC or GCMS
Isoprostanes from oxidation of arachidonic acid measured by GCMS or ELISA

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11
Q

TBAR spectrophotometric assay

A

Thiobarbituric acid reactive substances
Thiobarbituric acid reacts with MDA and other aldehydes to form a pink coloured product that absorbs at 532nm

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12
Q

What is an antioxidant

A

Any substance that delays, prevents or removes oxidative damage to a target molecule
Obtained from the diet and synthesised in vivo

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13
Q

Different ways antioxidants act

A

Scavenging of free radicals eg vitamin E or flavonoids
Metabolise the ROS eg antioxidant enzymes
Repair of oxidised lipids and proteins eg glutathione peroxidase and thioredoxins

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14
Q

Vitamin C as an antioxidant

A

Water soluble, mainly from citrus fruits
Strong reducing agent and scavenger of free radicals
Can regenerate vitamin E
Cofactor for a number of enzymes
2 oxidised forms, no good once it is oxidised
Body can regenerate reduced form from oxidised form using glutathione reductase

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15
Q

Vitamin E as an antioxidant

A

Alpha-tocopherol is the most potent form
High in vegetable oils, nuts and green veges
Lipophilid- found in membranes
Scavenger of lipid peroxide radical
Transported on lipoproteins eg LDL

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16
Q

Phenolic compounds as antioxidants

A

Present in apples, onions, tea, grapes, berries and red wine
Scavenge ROS and can chelate metal ions
Can act as Nrf2 activators
Eg curcu,in, resveratrol and anthocyanidins

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17
Q

What is Nrf2

A

Ubiquitously expressed TF involved in oxidative stress responses, highly regulated
Regulates over 200 genes containing ARE elements
Induces adaptive responses to stress
Post-translationally regulated by ubiquitin
Therapeutic target for preventing some major diseases eg cancer and diabetes

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18
Q

How is Nrf2 activity regulated in cells

A

Held in complex with Keap1 dimer in cytoplasm which has cys residues. This can bind to cullin E3/E2 ligase complex where Nrf2 is ubiquitinated and degraded
When under oxidative stress and cysteine’s in low pKa, they are oxidised which breaks complex with E3 and Keap-Nrf2, releasing Nrf2 to translocate to the nucleus

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19
Q

Structural side of Nrf2 and Keap binding

A

Nrf2 Neh region binds to KEAP DGR

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20
Q

Therapeutic regulation of Nrf2 activity

A

With electrophilic compounds which affect Keap1 oxidation state
PPI which interfere with Keap1-Nrf2
GSK-3 inhibitors which stop P of TrCP so it cant bind to Nrf2
Interfere with interactions so Nrf2 can be released and function as a TF

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21
Q

Naturally occurring Nrf2 activators

A

Phenolic compounds
Act as electrophilic modifiers of Keap1
Also activated by exercise

22
Q

Nrf2 target genes

A

Antioxidant and antioxidant cofactors eg NADPH
Also things involved in antioxidant synthesis such as glutathione synthesis

23
Q

H2O2 concentrations in cells- what happens at high and low ends

A

Level of this is important in cells, made in many pathways
Low level= physiological cell signalling molecule
High level= oxidative damage

24
Q

Some features on redox signalling

A

ROS modifications made to protein that regulate signalling pathways
ROS can directly activate/ deactivate or indirectly activate
Dependent on local conc of oxidant/antioxidant
Reduction can eliminate the regulation
Involves redox sensitive proteins with cystienes
Hyperoxidation can lead to irreversible oxidation

25
Q

Why is H2O2 implicated in redox signalling

A

Is the end product from super-oxide
Longer half life (stable)
Targets specific amino acids (cysteine)
Mobile and can pass through membranes
Production is regulated eg NOX coupled to receptors so regulation when receptors are switched on and compartmentalisation of production

26
Q

Cysteine in proteins

A

Cys in proteins at low pKa (acidic) and is sensitive to oxidation- can give H rapidly from self hydrol (SH) group
Highly reactive, can form cross-links with other cystienes in same or different proteins and can form cross-bridges

27
Q

H2O2 and cys oxidation

A

H2O2 generally oxidises cys to sulfenic acid which is reversible, this can be oxidised further with more H2O2 to sulfinic acid and sulfonic acid

28
Q

Peroxiredoxins

A

Ubiquitous and different isoforms (1-6) found in different locations
Reduce up to 90% of cellular peroxides
Many conformational forms
Have redox cys pair, not selenocysteines
Modulators of redox cell signalling responses

29
Q

Deficiency in different peroxiredoxins (Prx)

A

Prdx1 and 2 commonly leads to haemolytic anaemia and tumours (Prdx1)
Susceptibility to cardiac ischemia with Prdx 6
Prdx 3 deficiency is associated with insulin resistance, cardiac hypertrophy and kidney disease

30
Q

Peroxiredoxin catalytic mechanism and H2O2 (prdx3)

A

Thiol peroxides- reduces H2O2 to H2O
Peroxidate cys most sensitive so is oxidised to sulfenic form from H2O2
Resolving cys then oxidised and forms cross-link
Can be over-oxidised with more H2O2 into its sulfinic form

31
Q

Reduction of oxidised peroxiredoxin 3

A

Thioredoxin-2 is oxidised to reduce Prx3. Exchanges cross-links donating H from its cys which then form their own cross-links
Trx2 is then reduced by thioredoxin reductase 2 which uses NADPH cofactor to donate H

32
Q

Job of thioredoxin-2

A

Repair oxidised proteins- does its reduction in the same way on many different oxidised proteins

33
Q

Example of ROS indirectly affecting a signalling pathway

A

Nrf2- oxidation of the keap adapter occurs, not the actual Nrf2 TF. So indirectly allows transcription of antioxidant genes

34
Q

What types of pathways does ROS directly affect and examples of these and why

A

Growth factor pathways
Eg EGFR signalling, insulin signalling and VEGF (vascular endothelial growth factor). RTK activation allows assembly of NOX in the membrane through PI3K (kinase making PIP3) and RAC1 (GTPase which stimulates NOX assembly)

35
Q

How does H2O2 affect EGFR signalling

A

EGFR is a RTK and when active, NOX is activated and generates super-oxide which is converted into H2O2 by SOD3 (extracellular SOD)
H2O2 interacts with phosphatases involved in the pathway: SHIP2 inhibited, which means PI3K isnt inhibited and can generate PIP3 and allow AKT phosphorylation. PTEN inhibited, allowing PIP3 to continue being generated and AKT phosphorylated
Inhibits PTP1B
All therefore allowing the growth signal to continue, without inhibitory regulation

36
Q

Experimental evidence of H2O2 affecting EGFR signalling

A

Staining shows that receptor is internalised (happens when activated) and membrane ruffling occurs when EGF added
ROS is produced from fluorescence when pathway is stimulated- added inhibitors of pathway and inhibitors of NOX and antioxidant that scavenges H2O2 and shows that ROS is definitely made from pathway stimulation
Also looked at P of downstream proteins from addition of EGF and H2O2 showing the same result
When adding catalase to get rid of H2O2 there is loss of downstream P so therefore the pathway does use H2O2

37
Q

ROS in insulin signalling

A

Get H2O2 from NOX activation from insulin
Oxidises PTEN which negatively regulates the pathway- so allows the AKT phosphorylation and downstream events
Oxidises PTB1B so stops inhibition of AKT phosphorylation
Also oxidises AKT PH domain which enhances its binding to PIP3 at the membrane
When too much ROS can also oxidise another domain of AKT and make it inactive= shows it is concentration dependent

38
Q

ROS and vit C in VEGF generation

A

HIF1 is a TF which in normal oxygen conditions is hydroxylated by proline hydroxylase and then targeted to a ub complex and degraded. Has precursors such as vit C which allow this inhibition and oxygen
Under hypoxia (low oxygen) or when ROS is around and inhibits the oxygen, no hydroxylation occurs so HIF-1 goes into nucleus and acts as TF for genes with hypoxia response elements allowing growth and proliferation eg produces VEGF and angiopoietin

39
Q

ROS in VEGF signalling

A

VEGF has GF receptor, when active allows NOX assembly
ROS can inhibit PTP activation, therefore PTP cannot inhibit the receptor and allows signalling, causes upregulation of TFs for growth and allows proliferatrion
When ROS levels are high it can inhibit and inactivate some TFs when in the nucleus

40
Q

Different models of H2O2 signalling involving antioxidant enzymes

A

Relay model: antioxidant is oxidised itself as is more prone to this than target and then transfers this oxidation to the target: need evidence scavenger does interact with target and that it is oxidised first (time)
Floodgate model: when there is too much H2O2 all of the antioxidant available gets oxidised and inactivated, allowing more H2O2 to be free to modify targets

41
Q

Prdx and HMGB1

A

See that it is in nucleus in control, then add LPS HMGB1 moves out of the nucleus into cytoplasm. Mutations to cys suspected to be involved in oxidation cause no secretion- fluorescence
Suspected Prdx involved in mediating HMGB1 oxidation- when no PrdxII there is less HMGB1 secretion, comes back when PrdxII added back= is important for HMGB1 secretion and probably involved in oxidation event- gel and serum levels

42
Q

Model of PrdxII mediated HMGB1 oxidation and secretion

A

Inflammatory stimuli causes H2O2 into cell and formation of NOX= more H2O2
H2O2 into nucleus where it oxidises PrdxII. PrdxII then oxidises HMGB1 and is reduced itself
HMGB1 oxidised forms disulfide and exported by CRM1 into cytoplasm and then into extracellular region

43
Q

Prdx and ASK1- showing redox relay

A

Saw in gel that 100uM H2O2 (amount in cells under stress) causes ASK1 oxidation due to dimer formation
Over time with same amount of H2O2 saw increases p38 phosphorylation (downstream of ASK1)
When PrdxI removed see loss of p38 phosphorylation
Pulldowns sow PrdxI and ASK1 interact with each other
Over time see Prdx1 oxidation occurs then ASK1 oxidation then phospho p38. Phospho p38 decreases as Prdx is hyperoxidated

44
Q

STAT in signal transduction

A

TF involved in growth- promotes tumour growth too
Activated by cytokines, some GPCR and TLR4 pathway
JAK tyr kinase interacts with STAT= STAT is phosphorylated and localised to nucleus where it acts as TF for cell survival, proliferation and immune responses

45
Q

STAT3 and oxidation and PrdxII

A

When looking at PrdxII, see it is oxidised due to monomer forming. When oxidised can also interact with other things, do see that it interacts with other molecules on the gel. When did 2D gel, saw it interacts with TrxI and STAT3
PrdxII oxidised STAT3 through relay method- can be reduced by Trx which it interacts with or Trx can reduce STAT3
Also have evidence that STAT3 can be oxidised in the floodgate model also when PrdxII is all oxidised

46
Q

Examples of antioxidant therapies

A

Whole foods (fruit and veg eg Mediterranean diet)
Single oxidant supplements, vitamin E/C, coenzyme Q, polyphenols
GSH enhancers
Nrf2 activators
Antioxidant enzyme mimetics
Nox inhibitors
Uncouplers

47
Q

Why was it expected vitamin E would prevent/ slow development of heart disease

A

Oxidised LDL promotes inflammation in artery wall, initiating atherosclerosis
Through LPA causing cytokines (promoting macrophages) and ox fatty acids binding to TLR4
Vit E carried on LDL particle as is lipid-soluble, thought would oxidise the lipids in particle
Didn’t work

48
Q

Why was it expected vitamin c would prevent/ slow development of cancer

A

Vitamin C is a cofactor of proline hydroxylase, causing hydroxylation of HIF TF meaning it is ub and degraded
When not degraded, HIF acts as TF for genes promoting tumour survival and growth. Increased vit C thought to increase its hydroxylation
Didn’t work

49
Q

Antioxidant vitamins and cardiovascular disease

A

Did not work- p-values were not significant and made no difference
No evidence to support the use of vitamin and antioxidant supplements for prevention of cardiovascular disease

50
Q

Why havent antioxidants worked

A

Need to thin, of the right choice based on pathways. Also single oxidants have been used in trials, need a range and need relays for the system to work as need multiple things working together

51
Q

Things to consider when designing antioxidant therapies

A

What is the right choice
Will any one antioxidant work
Is oxidative stress a cause or consequence of the disease
Bioavailability: can we get high enough concentrations in the right place?
What the disease too advanced for antioxidants to have an effect
Prior antioxidant status of individuals
Do antioxidants interfere with redox signalling

52
Q

Research on antioxidants and health-promoting effects of physical exercise in humans with diabetes

A

Know oxidative stress is involved in diabetes and know exercise enhances insulin signalling responses in diabetics
Looked in those that had been doing exercise regime and those that hadn’t
Found that antioxidants prevent exercise-dependent induction of insulin sensitivity by preventing mediators of insulin sensitivity and the anti-oxidant response
Antioxidants also likely interfere with potentiation of insulin signalling and nrf2 as remove the ROS which allow these

BIOC353 (5 decks)

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