Module 5- ROS And Cell Signalling Flashcards

Question

Why is H2O2 implicated in redox signalling

Answer 1

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

Answer 2

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

Answer 3

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

Answer 4

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

Answer 5

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

Answer 6

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

Answer 7

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

Answer 8

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

Answer 9

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

Answer 10

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)

Answer 11

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

Answer 12

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

Answer 13

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

Answer 14

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

Answer 15

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

Answer 16

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

Answer 17

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

Answer 18

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

Answer 19

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

Answer 20

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

Answer 21

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

Answer 22

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

Answer 23

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

Answer 24

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

Answer 25

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

Answer 26

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

Answer 27

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

Answer 28

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