Your SlideShare is downloading. ×
  • Like
Reactive Oxygen Species
Upcoming SlideShare
Loading in...5
×

Thanks for flagging this SlideShare!

Oops! An error has occurred.

×

Now you can save presentations on your phone or tablet

Available for both IPhone and Android

Text the download link to your phone

Standard text messaging rates apply

Reactive Oxygen Species

  • 421 views
Published

During favorable conditions, the level of reactive spices in the cell is limited to what is required for normal cellular activities. They act as important components of signaling pathways. Plants …

During favorable conditions, the level of reactive spices in the cell is limited to what is required for normal cellular activities. They act as important components of signaling pathways. Plants control some important processes such as defense, hormonal signaling and development by using them as signaling molecules. And An equilibrium is steblished between antioxidant system and ros formation. But when plant feels an external stress like, drought,cold, salt etc. the level of reactive specease increases above the basal level a situation that we call oxidative stress. These reactive molecules during oxidative stress, they react with biomolecules like as carbohydrates, unsaturated lipids, proteins, nucleic acids. Proteins are the most abundant cellular targets of the oxidative species, more than DNA and lipids, making up 68% of the oxidized molecules in the cell. Ros reacts with proteins which results in protein modification called redox PTMs.

Published in Science , Technology
  • Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
    Be the first to comment
No Downloads

Views

Total Views
421
On SlideShare
0
From Embeds
0
Number of Embeds
0

Actions

Shares
Downloads
25
Comments
0
Likes
2

Embeds 0

No embeds

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
    No notes for slide
  • Topic of my presentation is Redox proteomics. Redox Proteomics is branch of science that deals with the study of Redox Proteome.
  • Before we see what is redox proteomics.. Let us recall some basic points. During favorable conditions, the level of reactive spices in the cell is limited to what is required for normal cellular activities. They act as important components of signaling pathways. Plants control some important processes such as defense, hormonal signaling and development by using them as signaling molecules. And An equilibrium is steblished between antioxidant system and ros formation. But when plant feels an external stress like, drought,cold, salt etc. the level of reactive specease increases above the basal level a situation that we call oxidative stress. These reactive molecules during oxidative stress, they react with biomolecules like as carbohydrates, unsaturated lipids, proteins, nucleic acids. Proteins are the most abundant cellular targets of the oxidative species, more than DNA and lipids, making up 68% of the oxidized molecules in the cell.Ros reacts with proteins which results in protein modification called redox PTMs. So the redox proteomics is an important area of plants proteomics that deals with the global identification of of Redox PTMs and charachterization of redox control systems. It involves mapping of….and exploroing.. Nitrosative stress, that is the icrease of RNS beyond the basal level is almost always accompanied with oxidative stress……. Sudy of redox proteome is significant to understand the plants response against various stress conditions and the mechanism of acclimation. Delicate equilibrium between .. When the level of ROS exceeds the defense mechanisms, a cell is said to be in a state of “oxidative stress.” The enhanced production of ROS during environmental stresses can pose a threat to cells by causing peroxidation of lipids, oxidation of proteins, damage to nucleic acids, enzyme inhibition, activation of programmed cell death (PCD) pathway and ultimately leading to death of the cells 
  • Now we will proceed with some questions like…this part that is how we can study redox proteome I will be covering in my pesentation of paperb.
  • Now we will proceed with some questions like…this part that is how we can study redox proteome I will be covering in my pesentation of paperb.
  • When they are overloaded…..aerobic organisms produce SOD…. This extremely reactive moleculecan initiate radical chain reactions and damage all types of biomolecules. Radical reactions will eventually stop by the formation of stable harmless radicals upon reaction with ascorbate, or with α-tocopherol in membranes.
  • Mitochondrial electron transport and reactive oxygen species formation.Reduced nicotinamide adenine dinucleotide (NADH) and reduced flavin adenine dinucleotide (FADH2) are reoxidized to nicotinamide adenine dinucleotide (NAD) and flavin adenine dinucleotide (FAD) at Complex I and II of the electron transport system in the inner mitochondrial membrane, which provides the electrons that reduce oxygen to H2O at Complex IV. Electron transport along the electron transport system from Complex I or II via ubiquinone (UQ), Complex III, cytochrome c (cyt c), and Complex IV provides the energy to actively pump protons from the mitochondrial matrix to the intermembrane space and establish a proton gradient and a membrane potential across an otherwise impermeable inner mitochondrial membrane. This membrane potential is the driving force for protons to reenter the mitochondrial matrix through Complex V and drive adenosine triphosphate (ATP) synthesis. Complexes I and III are potential sources of electron leakage and superoxide (O2· −) production. O2· −, possibly after reacting with nitric oxide (NO·) to form peroxynitrite (ONOO−), could further attenuate electron transport at Complex I and possibly lead to increased O2· − formation. ADP = adenosine diphosphate.
  • Normally, the electron flow from the excited photosystem centres is directed to NADP which is reduced to NADPH. It then enters the Calvin cycle and reduces the final electron acceptor, CO2 (Fig. 2B). The processes catalysed by rubisco (carboxylase-oxygenase) both produce and consume O2. If the ETC is overloaded, working under O2-rich conditions, the leakage of electrons leads to an inevitable yield of ROS. Moreover, chlorophyll molecules, being endowed with photosensitive properties, mediate ROS generation after the input of light energy. In particular, a part of the electron flow is diverted from ferredoxin to O2, reducing it to the superoxide free radical, Reduction of oxygen on the acceptor side of PSI, as a result of the photosynthetic transport of electrons, leads to the formation of superoxide anion, which can be further converted to H2O2 and HOd. Transfer of excitation energy from excited chlorophylls to oxygen in the light-harvesting complexes leads to the formation of 1O2. The production of ROS is enhanced by strong light and also by deceleration of the Calvin cycle. Arrows indicate the transport of electrons, whereas the dashed indicates the transfer of excitation energy. Fd, ferredoxin; FNR, ferredoxin-NADP+ reductase; OEC, oxygen evolving complex; PC, plastocyanin.
  • So here in this table I have tried to put all things at a place. Fe-S? aromatic amino acids? Dorect impacting the health of the cell. And the cell can either restablaize redox homeaostasis by the virtue of its control system or it can die…
  • Now we will proceed with some questions like…this part that is how we can study redox proteome I will be covering in my pesentation of paperb.
  • They work to maintain redox homeostasis in the cell. To understand the way they scavenge ROS let us look at this pathway that eliminates H2O2. although this antioxidant system is always active in the cell but as the ROS concentration increases their activity is also increased significantly.
  • Catalase presence?
  • Location? Study asadahelliwell pathway. Here, Ascorbic acid needs to regenerated to eliminate rapidly formed H2o2.Ascorbic acid provides e-… glutathione helping for continutious regeneration of ascorbic acid by accepting and realinsing proton.
  • So this is how the redox homeostasis in a plant cell is maintained for the normal functioning of the cell. Molecularwieght? It plays very important role in asadahelliwell pathway that eliminates excees h2o2 from cell.
  • Acceptor-side photoinhibition[edit]Strong light causes the reduction of the plastoquinone pool, which leads to protonation and double reduction (and double protonation) of the QA electron acceptor of Photosystem II. The protonated and double-reduced forms of QA do not function in electron transport.Inhibition of the activity of photosystem II (PSII) under strong light is referred to as photoinhibition. This phenomenon is due to an imbalance between the rate of photodamage to PSII and the rate of the repair of damaged PSII.
  • Strong light causes the reduction of the plastoquinone pool, which leads to protonation and double reduction (and double protonation) of the QA electron acceptor of Photosystem II. The protonated and double-reduced forms of QA do not function in electron transport. This hypothesis was supported by observation where people observed that r OEC has been chemically removed or destroyed in an active PSII unit
  • If the oxygen-evolving complex is chemically inactivated, then the remaining electron transfer activity of PSII becomes very sensitive to light.A photon absorbed by the manganese ions of the oxygen-evolving complex triggers inactivation of the oxygen-evolving complex. Further inhibition of the remaining electron transport reactions occurs like in the donor-side mechanism. P680 starts oxidising other components of PSII unspecifically thus leading to donr side inactivation.
  • Uncoupling proteins, members of the mitochondrial carrier family, are present in mitochondrialinner membrane and mediate free fatty acid-activated, purine-nucleotide-inhibited H+re-uptake. The fifth enzyme is a terminal oxidase called ‘alternative oxidase’ (AOX). It catalyses direct electron transfer from ubiquinol to molecular oxygen (Møller, 2001). All these alternative oxidoreductases do not couple electron transport to protontranslocation across the inner mitochondrial membrane and therefore, seem to catalyse energy-wasting reactions. However, it is believed that these reactions are important, possibly because they are the basis for overflow-protection mechanisms of the respiratory chain in plant cells under certain physiological conditions (Møller, 2001). According to the chemiosmotic energy-transduction concept, in respiring mitochondria electrontransport through the respiratory chain is coupled to pumping of protons from the mitochondrial matrix, generating a proton electrochemicalgradient (H + ) across the inner membrane [1] (Fig. 1). This protonmotive force drives the protons back into the matrix through theF1F0-ATP synthase resulting in phosphorylation of ADP and supports energy requiring processes,
  • Among amino acids, the most oxidation-susceptible residues are the sulphur-containing ones, cysteine and methionine. Except the sulphonic acid all other three modifications are reversible hence are significant as they can participate in signaling. An oxidised protein can act as oxidant when the change is reversible.S
  • The most reactive oxygen species in biology is the Hod radical, which has a constant oxidation rate for proteins which is comparable to the rate of diffusion. It leads to non-specific protein oxidation, whereas all other ROS, having lower oxidation rates, react more specifically (Davies, 2005). it is thought that the formation of protein disulphides can protect the protein from further, potentially more damaging, oxidation. Duringcatalysis, these ubiquitous enzymes are occasionally inactivated by the substrate-dependent oxidation of the catalytic cysteine to the sulphinic acid (-SO2H) form, and are reactivated by reduction by sulphiredoxin (Srx). In In plants, 2-Cys-Prxs constitute the most abundant peroxiredoxins and are located in chloroplasts. Here we can see proteins like peroxiredoxins after being oxidised by ROS can be re reduced and thus participate in signaling that helps plant withstand oxidative stress.
  • methionine sulphoxide reductases. In many proteins, oxidation of a few surface methionines has little effect on protein structure and function, but accumulation of methionine damage can cause changes in protein hydrophobicity and conformation, with concomitant effects on protein function. However, the ready enzymatic regeneration of methionine sulphoxide has led to the suggestion that methionine acts as an antioxidant to protect other amino acid residues from more deleterious damage
  • There are other reports also that suggest that reversible methionine sulphoxidation will turn out to be an important regulatory mechanism..
  • Protein carbonylation is the second most abundant protein modification after modification that involve sulphurcontanining amino acids. Side chains can be oxodatively converted into aldehydes an d ketons .. inactivation, crosslinking or breakdown of proteins
  • Total contribution of protein modifications to oxidative status in plant cells. When the presence of ROS and RNS species is limited,protein modifications mean intracellular communication and eventually the start of a real oxidative stress response. A ROS and RNS excess, noteffectively countered by scavenger species, causes protein carbonylations, the formation of reactive groups, backbone cleavage, proteinunfolding, and loss of functionality. A severe imbalance in ROS concentration inside the cellular environment leads to an alteration ofcellular function and even to cellular death.

Transcript

  • 1. Redox Proteomics Satya Prakash M.Phil Student
  • 2. Redox proteomics ? global identification of post-translational modifications related to protein oxidoreduction (redox PTMs) and characterization of redox control systems Mapping of protein damage caused by oxidative and nitrosative stress Exploring catalytic mechanisms and signaling pathways involving redox PTMs Examples of: ROS= H2O2, HO● , O2 ●- , 1O2 etc. RNS= NO ● ,ONOO- etc.
  • 3. How & where How? formation Control & scavengingWhat? Modifications How? Techniques Research findings
  • 4. How & where formation Molecular oxygen ( O2 ) Single electron donor O2 e- O2 ●- 1) Electron flow from ETC 2) Photo-excitation of chlorophyll molecules 3) Peroxidases
  • 5. O2 e- O2 ●- Electron flow from ETC of in chloroplast & mitochondria O2 ●- ͢ H2O2 ͢ OH● ͢ radical of chain reactions SOD Fe2+ ,Cu2+ Ascorbate α-tocopherol } Fenton reaction
  • 6. ROS production in mitochondrion J. Cell Biol., 2011, 194 :1 7–15
  • 7. During overloading of ETC
  • 8. Respiratory burst oxidase homolog, O2 ●- producing NADPH oxidase located at the plasmalemma, and the cell wall/apoplast peroxidases, amine oxidases, and oxalate oxidases are important components of the ROS-generating system.
  • 9. S. No . ROS Note Substrate/reacts with Concentration in plant tissue Production site PROCESS 1. H2O2 Relatively stable enzymes, proteins Micromolar to low millimolar Peroxisomes Mitochondria & chloroplast Photorespiration, Fatty-acid β- oxidation, Glyoxylate cycle & Respiration. 2. HO● Short half life, Most reactive All types of cellular components Very low Mitochondria, Chloroplast, cytosol, peroxisome), Respiration 3. O2 ●- Short half life Protein Fe-S centers Very low Peroxisome, Chloroplast & Mitochondria Photorespiration, Fatty-acid β- oxidation, glyoxylate cycle, Respiration & Photosynthesis. 4. 1O2 Short half life Conjugated double bonds( as found in PUFAs) & aromatic amino acids. Very low Chloroplast Photosynthesis.
  • 10. How? Control & scavenging Accumulation of ROS molecules in the cell is controlled by: 1. Several enzymatic and non-enzymatic antioxidants 2. Limiting electron flow through ETC
  • 11. ROS Scavenging antioxidants Enzymatic Antioxidants  SOD  CAT  APX  MDHAR  DHAR  GR Non-enzymatic Antioxidants GSH AA Carotenoids Tocopherols  Resveratrol  NAD & NADP  Phenolics  Flavonoids  Proline
  • 12. The enzyme SOD belongs to the group of metalloenzymes. O2 •− Three isozymes of SOD: • Copper/zinc SOD (Cu/Zn-SOD; cytosol, chloroplast, mitochondria and peroxisome), • Manganese SOD (Mn-SOD; mitochondria) • Iron SOD (Fe-SOD; chloroplasts). O2 + H2O2. SOD Superoxide Dismutase (SOD)
  • 13. Catalase (CAT) • It is a ubiquitous tetrameric heme-containing enzyme. 2H2O2 • CATs have a very fast turnover rate, but a much lower affinity for H2O2 than APX. • The peroxisomes are major sites of Hydrogen peroxide production. • Three isoforms of CAT found in maize (present on different chromosomes and independently regulated). 2H2O + O2 CATALASE
  • 14. Asada-Halliwell pathway (hydrogen peroxide sacvenging and ascorbic acid regeneration).
  • 15. ROS Enymatic antioxidant Non-enzymatic antioxidant 1O2 β-carotene , Tocoferol, Proline H2O2 Catalase, , MDHAR DHAR,GR NAD & NAD, GSH, Ascorbic acid O2 ●- SOD HO● Proline, ascorbic acid, tocoferol
  • 16.  photoinhibition of photosystem II in the chloroplast and  activation of the alternative oxidase and uncoupling proteins in mitochondria Limiting electron flow through ETC
  • 17. (Source: Wikipedia)
  • 18. Päivi Sarvikas UNIVERSITY OF TURKU PS II 1. Acceptor-side photoinhibition 2. Donor-side photoinhibition 3. Manganese mechanism 4. Singlet oxygen mechanisms 5. Low-light mechanism OEC-less mutant of Scenedesmus have shown high sensitivity to light (Keren et al. 1995).
  • 19. 1. Acceptor-side photoinhibition 2. Donor-side photoinhibition 3. Manganese mechanism 4. Singlet oxygen mechanisms 5. Low-light mechanism Singlet oxygen is a highly reactive oxygen species with potential to damage the D1 protein in vivo (Telfer et al. 1999). (P680+/ pheo-) ͢ *(3P680)
  • 20. Energy dissipation in mitochondrion (Zhu et al, 2011) They are the basis for overflow-protection mechanisms of the respiratory chain in plant cells under certain physiological conditions (Møller, 2001).
  • 21. RNS NO● is formed non-enzymatically in plants during normal cell metabolism both in the presence of ascorbate and as a by-product in nitrate reductase (NR) activities under hypoxic conditions; enzymatically, it is formed from nitrite by the activity of nitrite: nitric oxide reductase (Ni- NOR), and from L-arginine and oxygen by putative nitric oxide synthase (NOS). At low concentrations, NO● acts as a chemical messenger of the defenses against stress and pathogens, integrating and differentiating time- dependent responses
  • 22. At high concentrations and in the presence of ROS,NO● reacts to give rise to reactive nitrogen species (RNS) and in so doing creates oxidative and nitrosative stress. In particular, NO● reacts with superoxide forming peroxynitrite and peroxynitrous acid. Peroxynitrite (ONOO- ) decomposes rapidly generating hydroxyl (HO●) and nitrogen dioxide (NO●2). The latter initiates lipid peroxidation, oxidizes sulphhydryls, and nitrates the aromatic residues of proteins NO●2 is considered an inhibitor of catalase and ascorbate peroxidase, enzymes reputed to be involved in the control of ROS .
  • 23. What? Modifications Sulphur-containing residues: cysteines and methionines The thiol of cysteine can be oxidized to: • disulphide (PSSP), • sulphenic acid (PSOH), • Sulphinic acid (PSO2H) or • sulphonic acid (PSO3H)
  • 24. 2-cysteine peroxiredoxins (Prx) are known to be antioxidants that reduce peroxides through a thiol-based mechanism ((Jeong et al., 2006) reduction by sulphiredoxin (Srx). 2-Cys-Prx redox status and sulphiredoxin are parts of a signalling mechanism participating in plant responses to oxidative stress (Rey et al., 2007). (Rinalducci et al,2008)
  • 25. Like cysteine, methionine is also one of the most readily oxidized amino acids, owing to the presence of sulphur, and is susceptible to attack by most reactive oxygen or nitrogen species (Vogt, 1995). methionine sulphoxide reductases (Rinalducci et al,2008)
  • 26. • In A. thaliana, a null mutation in a gene encoding a cytosolic isoform of the enzyme showed increased ROS content, lipid peroxidation, and protein oxidation at the end of a long night, which was clearly stressful to the plant (Bechtold et al., 2004). • Small heat shock protein of chloroplasts. This protein is inactivated by methionine sulphoxidation, and reactivated by reduction catalysed by the enzyme peptide methionine sulphoxide reductase using thioredoxin (Trx) as the reductant (Gustavsson et al., 2002).
  • 27. (Rinalducci et al,2008)
  • 28. Protein carbonylation 1. Lysine, 2. arginine, 3. proline, and 4. threonine aldehydes or ketone group Impact: inactivation, crosslinking or breakdown of proteins