Week 5 Flashcards
What is oxidative stress
Results form imbalance between the levels of reactive oxygen species and antioxidants
How can oxidative stress occur
If there is an excess level of ROS or a depleteion in AOX
What is an antioxidant
A molecule which protects a biological target against oxidative damage
What are the two groups of antioxidant
Enzymes (proteins) and low molecular weight non-protein organic molecules
What are the antioxidant enzymes
Catalases
Glutathione peroxidases
Peroxidases
Peroxiredoxins
SUperoxide dimutases
Thioredoxin
What are oxidants
Recative molecules that are produced both inside your body and the environment that can react with other cellular molecules such as protiens
What is the most common type of oxidant in the cells
Reactive Oxygen Species (ROS) it include Non-radicals and radicals
What are free radicals
ANy molecules containing one or more unpaired electrons
What are examples of non radicals
Hydrogen peroxide
Hypochlorous acid
Peroxynitrite
Singlet oxygen
ozone
What are some examples of radicals
Hydroxyl
Superoxide
Lipid peroxyl
Nitric oxide
What is oxygen in its ground state
Has two unpaired electrons with parallel spins (biradical)
What does oxygen being bi radical do
Unlikely to particpate in recations with stable organic molecules that are arranged in pairs with antiparallel spins unless oxygen is activated
How can oxygen be activated
If electrons are transferred to O2 one at a time producing mono-radical or non-radical molecules
What are the three types of ROS
Superoxide anion radical
Hydrogen peroxide
Hydroxyl radical
How are oxygen radicals formed
One electron reduction leads to the formation of superoxide anion radical then reductions of superoxide (addition of two electrons) produces hydrogen peroxide if it takes third electron you have hydroxyl radical
What happens once the hydroxyl radical is formed
can be reduced with an electron to from water (4 electron reduction of oxygen)
What are other ROS that can also be converted
Peroxynittrite (reaction of superoxide and nitric oxidie) and hypochlorous acid (hydrogen peroxide and chlorine)
Which radical is produced non-enzymatically
Hydroxyl radicals
How can hydroxyl radicals be produced
Radiation induced homolytic fission or photolytic cleavage
How cna hydroxyl radicals in vivo generate
Through a reacation of metal ions such as iron
How many grams of iron are in the body
4
What si the recation called that hydroxyl radicals are produced
Fenton reacation which is oxidation of a ferrous iron with hydrogen peroxide
What is ferric trivalent ions and ferrous ions
Ferrous iron is iron two and ferric iron is iron 3
What happens with the ferric trivalent ions formed in the fenton reacation
Need to be re-generated back to ferrous ions whihc happens in a recation with superoxide anion radical
What is a Harber-Weiss recation
Combing recations one and two
What does hydroxyl radicals require
Superoxide and hydrogen peroxide
What is the reduction potential
Measure in volts of the affinity of a substance for electrons as compared with hydrogen
What does high positive reduction potential mean
Thermodynamically they are able to oxidize molecules with lower or negative reduction potential by taking electrons from them
What does hydrogen peroxide have
lowest recativity and the highest stabality (measured by its half life) and highest intracellular concentration
What is the most reactive and dangerous ROS
Hydroxyl radical
What moleculer has intermediate activity
Superoxide
What is the dual function of ROS
Either be toxic and cause apoptosis or can also cause cell proliferation depending on the concentration
What type of lipid is susceptible to ROS
Unsaturtaed lipids
What is lipid preoxidation
Oxidative damage to lipids
What is initation for damage to lipid
Unstable lipid radical is produced when it recats with ROS to the allylic hydrogen and allylic carbon
What is an allyic carbon
Carbon atom bonded to a carbon atom of a double bond
What is the propagation stage
The lipid radical recats with the molecular oxygen creating lipid peroxyl radical which recats with another lipid producing a different lipid radical lipid peroxide which initaties the next cycle of lipid oxidation
What is the termination stage
Lipid preoxidation is stopped by antioxidants
What happens if antioxidant mechanisms are imparied for lipids
The accumulated lipid peroxides exert toxic effects on cells membrane integrity fluditiy permeability change of enzyme activity and more
What is the most common DNA lesion
ROS induced oxidation of Guanine to 8-Oxoguanine
8-Oxoguanie can pair with Adenine which is modifying purines and pyrimidines mismatch pair
Can radiation also induce fragmentation
YES
What are the negative consequences of ROS induced damages to nucleic acids
Point mutations
Changes in gene expression
Single and double strand DNA breaks
What does oxidative damage do to protiens
Change in protien structure, function, turnover and loss or occasional gain of acitivity
change in enzyme activity change in ion transport proteolysis autoimmune respone
What does oxidative damage of proteins means
ROS-induced modifications individual amino acid and their functional groups
What can thiol groups on cysteine do
They react different types of ROS which lead to the formation of sulfoxidation products or intra protien disulfides they cna be dangerous to cells
What intensive oxidative stress do
Induce fragmentation of peptide chain and can aggregate due to cross linked recaations and alterd electrical charge and loss their activtiy
What is the glycocalyx coat
Surface of cells that are covered with glycans
What does oxidatvie damage to glycans mostly result in
Modfication of individual monsaccharides glycan celvaveg fragmentation degradation and accumation of AGE
What can these modifcations to glycagens do
Impairment of intracellular contacts (glycocalyx) induction of inflammation and modified functions of extracellular matrix
What are biomarkers of oxidative stress
products of oxidative damage
What are some biomarkers include
Lipids
DNA
carbohydrates
What are specalized biomarkers
Biomarkers relating to specific conditions exist
What are the organelles known to be sources of oxidative stress
Plasma membrane
Mitochondria
ER
Peroxisomes
Lysosomes
ROS direct or indirect products
BOTH and can function as parcirine signals
What is the major source of superoxide in the body
NADPH oxidase which is assembled in the plasma membrane
How many members of the NOX family oxidase
7
What is NOX2 a key component of
THe phagocyte NADPH oxidase
What are neutrophils
Cells of the inate immunity comprise white blood cells principle phaogcytic cells that migrate to site of injury or infenction recognize engluf and destory the invading pathogens
Can neutrophils generate ROS
YES which contribute to inactivation of pathogenic bacteria and fungi
How are neutrophils morpholgical different
oddly shaped nuclei with several loves and densenly stained granules
How many transmembrane domains do NOX family members share
6
What domains bind asymmetrical hemes
III and V each contain two histidine
What is heme
Iron-containing prosthetic group found in enzymes, ETC and oxygen binding pigments like haemoglobin
What do the heme groups do
confer functionality which can include oxygen carrying, oxygen reduction electron transfer and other process
What is the cytoplasmic C-terminal domain in NOX
FAD and NADPH binding domain
What kind of enzymes are NOX thought to be and what is the process
Single electron transporters passing electrons from NADPH to FAD first heme to the second heme and then finally to oxygen to reduce it into superoxide anion radical
Which enzymes contain Ca binding domains
NOX5 DUOX1 and DUOX2
Which domians contain peroxidases-like domain
DUOX1 and DUOX2 which gives them the name of dual oxidases
What NOX enzymes are similar in size and domains structure to NOX2
NOX1, NOX3, NOX4
What is NOX2 also known as
gp91phox
How do NOX1 NOX3 and NOX4 interact with superoxide
Electron transfer centers that you need to pass electrons from cytoplasmic NADPH to molecular oxygen which will form superoxide otuside the cell then it will recact to form hydrogen peroxide which is a substrate for peroxidases
How does NOX5 interact with superoxide
It has a calmodulin like domain for calcium which makes the enzyme activated by calcium to produces super oxidize
How does DUOX create Superoxide
Build off the NOX5 structure but adds an amino terminus an extra transmembrane a-helix followed by a peroxidases loacalized on the outside of the plasma membrane
Can NOX2 generate superoxide itself
NO
What is the multicomponent complex also called
Phagocyte NADPH oxidase
What are the five proteins that should be combined to activate gp91phox
p22phox p40phox p47phox p67phox and RAC
What is flavocytochrom b558
p22phox being a hetrodimer with NOX2 (sits in the lipid raft of the plasma membrane)
What forms a trimer to actiatre phagocyte
p40phox, p67phox, p47phox
What activates the pathway of NADPH oxidases
Induced by pathogens or inflammatory mediators or by some specefic compounds
What does fMLP stand for
N-formyl-L-methionyl-L-leucyl_phenylaaline bacteria wall oligopeptides
What are the 3 signlaing process nedded for this system
Protien phosphorylation lipid metabolsim and GTPase activation
What is the process of activation of phagocyte NADPH oxidase
Protien kinase requries for phosphorylation p47 which p47 will now pind too p22 lipid metabolsim is then requried to syntehzies PtdlnsP3 which makes change in the lipid compostion which stabalizes the enzymatic complex. You then need GTP to actiave RAC to stablize the enzymatic complex. p67 is also used to convert NADPH to NADP. It can now produce recative oxygen species
What is the job of p47
Organizer
What is the job of the p67
It is an activator
What is the job of p40
It is a second adaptor
What are the essential protein binding domains
AIR SH3 PRR PB1 tetratricopeptided repeat T and AD
What are the lipid binding domains
PX and lipid tail of RAC
How are p47 p67 and p40 associated with each other
Through PRR-SH3 and PB1-PB1 interactions
Why do p40/p47/p67 trimer stays in they cytsol
Because of auto-inhibitory region AIR p47phox is combined with its SH3 domain which prevents its binding to the PRR domain of p22phox
Why is RAC maintained in the cytsol
Its lipid tail is masked by the inhibitory protien RhoGDP-dissociation inhibitor which prevents the binding to the membrane
What also blocks the phox protiens from binding to the memrabne
The binding of SH3 in p47 and by PB1 in p40phox
What trigger en block
The phosphorylation of p47phox which serve as an organizer the phosphorylation induces a conformational change of p47 which unmasks the SH3 domains so now PRR of p22phox can bind to SH3 pX is now accesible to the membrane
How will p67 be activated
Should be oriented in a specefic postion which is controled by RAC and p40 through protien protien and protien lipid interacations
How do you activate RAC
GTP binds to RAC which can induce conformational changes which will promote dissociation of RhoGDI and membrane association through the tail which will now bind to p67 and induce a conformation change allow the activation domain to act on gp91
What is Chronic Granulomatous Disease
Mutation in the 5 structural genes of the NADPH oxidase expect RACT
A mutation in what accounts for the majority of cases in CGD
gp91phox then p47 lease being p40
What happens with patients who have CGD
Fail or are less able to generate superoxide and have a high sensitivity to bacterial and fungal infections
What are granulomas
Masses of immune cells that form at sites of infection or inflammation and can result in damage to those tissues
What causes a more server recation
gp91 phox-deficient
What are ROS-sensitive indicators
Nitroblue Tetrazolium and Dihydrohodamine
How can you tell if there is ROS with Nitroblue Tetrazolium
It is a yellow transparent color but when reduced it becomes insoluble and turns a deep purple and it shows that there is a production of oxygen radical without it they have CGD
What does the DHR test do
Measures ROS using flow cytometry uses a flow cytometry machine to detect signals from each cell.
What happens if they only see one peak in DHR
This means that the patient has CGD because it is only showing the no flourscent cells that are active
What happens if you see two peaks in DHR
The patient is healthy because the once you have activation NADPH oxidase normal neturophills will then produce superoxide radical and that will oxidize DHR to highly fluorescen molecules
Where else can large amount of ROS come from
Generated in the mitochondrial as a leakage from the ETC especially from complexes I and III that create superoxide
Where is superoxide formed in complex I and complex III
Complex I is only within the matrix whereas complex II is released both into the matrix and the mitochondrial space
How does the mitochondria protect against ROS
Uses antioxidant enzymes
What are antioxidant enzymes the mitochondria uses
Manganese superoxide dismutatse which converts superoxide to hydrogen peroxide CAT an GPx and Prx3 to inacative or catbolize hydrogen peroxide
What can heme serve as
ROS in cells
What is haemoglobin
Produces a constant flux of superoxide and carries oxygen through your body
When does Haemoglobin release superoxide
Due to electron delocalization between heme divalent iron Fe2 and O2 which can then lead to the formation of Fe3
How do the blood cells prevent possible toxic effects
They contain high levels of antioxidant enzymes such as peroxiredoxins and superoxide dismutase
What happens when human erythrocytes are exposed to hydrogen peroxised
produce echinocytes they are small and throny
What is hydrogen peroxide
Spontatneous or enzymatic
How is spontaneous hydrogen peroxide formed
Superoxide anion radical due to the dismutation recation can happen slow but still work in cells it is catalyzed by enzymes called superocide dismutases
Why is SOD important in antioxidant defense
Converts a highly reacative superoxide into less active non radical hydrogen peroxide
What are the three types of SOD and where are they located
SOD1 (in the cytoplasm) SOD2(in the mitochondria) SOD3 depends on copper zinc or maganese as cofactors
How else can hydrogen peroxide be generated
Enzymes called oxidases which catalyze an oxidation-reduction reacation molecular oxygen is the receptor
What are peroxisomes
Generation and scavenging of hydrogen peroxide
How can you see peroxisomes
With GFP using fluorescence microscopy as mutiple dots in the cytoplasm
What do peroxisomes contain
Hydrogen peroxide-degrading enzymes and SOD
What does Catalsae do
Directly incatvaties hydrogen peroxide converting it to water and oxygen
What does peroxidase do
Utlize hydrogen peroxide as a a substrate for the oxidation of certain organic compounds
What does peroxiredoxin 5 do
Major scavenger of highly eacative peroxyntirie
What happens to peroxisomes when under oxidatve stress
Tubular peroxisms they have a higher surface and can accomodate more antioxidant enzymes to prevent oxidative damage
What does NAC do
Peroxismes wont become tubular because it is preventing oxidative stress
What does P450 do
Produce ROS as by products it is also involved in metabolic conversion of lipophilic substrates by oxdizing them to become more polar and soluble to become secreted from cells
How is ROS produced in cytochrome P450
Heme ferrous/ferric states there are two where there are leakeges of a large fraction of the activated oxygen from the enzyme without substrate oxidation
What system is P450 apart of
monoxygenase system oxidizes a substrate in a complex process involving the electron transfer from NADPH through NADPH-cytochrome reducated to cytochrome P450
What does the autoxidation shunt produce
Fe Iron (II) superoxide autocidation of the oxy ferrous complex
What does the peroxide shunt produce
Hydrogen peroxide by the protonation of the peroxyctochrome ferric complexi (III)
What is myeloperoxidase
Specefic enzyme of neutrophils localized in azurophil granulaes and helps destroy invading pathogens
What does MPO catalzye
Production of highly recative HOCl in a recation of hydrogen peroxide and cholrine ions
Is HOCL powerful
Yes more powerful than hydrogen peroxide and shows how moderate ROS can be converted to more aggresive molecules
What is peroxynitrite
Nitric oxide and superoxide
What is NO
A free radical generated under phsyiological conditions in all mammal cells called NOS
What are the three types of NOS
Enothelila neuronal and inducible but function in the same way and produce NO and citrulline from arginine you need oxygen and NADPH as an electron donor
What can NO do
Vasodilationa and neutrotransmission and antimicrobial and antitumor activties
What can NO do
Depletion of SH groups
Oxidation
Nitration
