Post Translational Modification and Glutathionylation - Thesis Example

GLUTATHIONYLATION OF PROTEINS ABSTRACT Post-translational modification is an important step involved in the activation of proteins which are in turn essential for regulating cellular function and processes. Glutathionylation is a type of modification of proteins which involves the addition of glutathione molecule to the protein by a disulphide bond. The process of Glutathionylation not only stabilises the proteins but also makes it functionally active allowing it to play a major role in the biochemical reactions occurring within the cell such as detoxification and active defence against antioxidants. They are also known to be a part of cellular proliferation. Dysfunction in the synthesis of glutathione may lead to many complications in within a cell and may thereby lead to various pathological conditions in a human body such as cancer, pulmonary fibrosis, diabetes mellitus, etc., Thus to understand these clinical symptoms caused by glutathione a better understanding of glutathione and the process involved in the modification of proteins using glutathione is found to be essential. TABLE OF CONTENTS i. Abstract ……………………………………………………………………………….2 1. Introduction………………………………………………………………………………..5 2. Glutathione………………………………………………………………………………...7 2.1 Structure……………………………………………………………………………….7 2.2 Synthesis………………………………………………………………………………7 2.3 Glutathione metabolism……………………………………………………………….9 2.4 Function…...................................................................................................................10 3. Glutathionylation………………………………………………………………………...11 4. Protein Glutathionylation………………………………………………………………...12 5. Identification of glutathionylated proteins……………………………………………….15 5.1 Two-dimensional electrophoresis……………………………………………………15 5.2 Sypro ruby staining…………………………………………………………………..15 5.3 Oxyblot immunochemical detection…………………………………………………15 5.4 Image analysis………………………………………………………………………..16 5.5 Trypsin in-gel digestion……………………………………………………………...16 5.6 Mass spectroscopy…………………………………………………………………...16 5.7 Peptide sequence analysis……………………………………………………………16 6. Functional consequences of protein glutathionylation…………………………………..17 7. Structure change induced by protein glutathionylation………………………………….19 8. Diseases…………………………………………………………………………………..20 8.1 Diabetes………………………………………………………………………………20 8.2 Cardiovascular disorder……………………………………………………………...20 8.3 Lung disease………………………………………………………………………….21 8.4 Cancer………………………………………………………………………………..21 8.5 Neurodegenerative diseases………………………………………………………….21 9. Discussion………………………………………………………………………………..22 10. Conclusion……………………………………………………………………………….23 References……………………………………………………………………………………24 1. INTRODUCTION The current advances in modern science have greatly enhanced the understanding of modification of proteins and its impact on the cellular microenvironment along with its association with various diseases. Even though protein folding and re-folding are important steps in the synthesis of proteins the modification of these proteins are of prime importance since they provide the structural and functional diversity of the proteins. Post-translational modification of proteins influences a variety of cellular functions such as DNA repair, enzyme activity, protein-protein interactions, etc. ‘Oxidative stress’ is a condition in which the intracellular per oxidant – anti-oxidant ratio scale tips in the favour of per oxidants, which leads to the accumulation of oxygen radicals and other oxygen-derived species within the cells which in turn leads to conditions like ageing or a variety of human diseases such as cancer. ATP synthesis in a Eukaryotic cell generally produces superoxide anion free radicals and other active oxygen species such as OH ions or H2O2. Even though there are several mechanisms to prevent their accumulation at times the system may get overwhelmed leading to toxic cell damage. Glutathione is an important component in providing the cellular defences against oxidative stress. During oxidative stress the cell is known to produce increased glutathione. Its role against active oxygen species and reactive intermediates are quite unique. They not only act as reductants for hydrogen peroxides and organic hydrogen peroxides but also act as a nucleophile which conjugates electrophilic molecules. The reaction is catalysed by glutathione peroxidise. The excess glutathione disulphide produced due to the reaction is effectively removed by glutathione reductase [35]. After extensive research it has been proved that cell death due to conditions of oxidative stress may be caused due to depletion of glutathione in the cell. But the exact mechanism of cell death and glutathione depletion has not yet been clarified. To maintain the intracellular redox balance glutathione is attached to proteins by a thiol group. Protein – SSG + GSH → Protein – SH + GSSG The reaction is catalysed by thiol transferase. It has been noted that loss of protein thiols leads to glutathione depletion eventually leading to cell death. Thus glutathionylation of proteins is important to prevent cell death and damage. Deficiency of glutathione is reported as an autosomal recessive disorder which is found to be hereditary to certain group of people [30]. Glutathione is also known to protect the Eukaryotic cell from oxidative stress caused due to excessive exposure of metals [17]. 2. GLUTATHIONE Glutathione is a tri-peptide with a simple sulphur compound. It is one of the most common non-protein thiol group found in most of the organisms. The most important function of glutathione is redox - haemostatic buffering. It also acts as a transducer for the cellular network for environmental signals. a. Structure Glutathione is a thiol tri-peptide with an amino acid sequence of ɣ-L-glutamyl, L-cysteinyl-glycine [27]. Glutathione in nature exists in two forms the thiol-reduced glutathione (GSH) and the disulphide oxidised form (GSSG). GSH is the most predominant form and is found in most of the mammalian tissue where as the GSSG form only occupies 1% of the total amount of glutathione. The cytosol, mitochondria and the endoplasmic reticulum are the 3 major reservoirs of glutathione in the eukaryotic cell. One of the unique features of glutathione is the peptide bond linking the glutamate and cysteine of the GSH by the ɣ-carboxyl group which can be subjected to hydrolysis only by the enzyme ɣ-glutamyltranspeptidase. The ɣ-glutamyltranspeptidase is present only on the outer surface of the cells which makes the GSH resistant to intracellular degradation making it possible to be metabolised only extracellularly by the organs with the particular enzyme. b. Synthesis Glutathione is synthesised in the cytosol of most of the Eukaryotic cell. They are also found in abundance in the mitochondria and the endoplasmic reticulum. There are two major steps involved in the synthesis of glutathione. Figure 1 – Glutathione synthesis pathway [3] The first step in the production of glutathione is the formation of ɣ-L-glutamylcysteine. This is also the rate limiting step and requires the breakdown of an ATP molecule. The reaction is catalysed by glutamate cysteine ligase in the presence of Mg2+ or Mn2+ ion. The glutamate cysteine ligase is a dimer which is composed of a catalytic component and a modifier component encoded by different genes. The modifier component is enzymatically inactive and plays an important role in the regulation of glutathione synthesis. It has been identified that the catalytic component of the enzyme is inactive under normal physiological conditions but is activated only when there is an increased demand of glutathione. The glutamate cysteine ligase is specific for the glutamyl moiety and is physiologically regulated by non-allosteric feedback competitive inhibition and the availability of the precursor which is L-cysteine. The second step in glutathione synthesis is catalysed by glutathione synthase. The enzyme is again a dimer with a molecular weight of 118,000 daltons. The first step with the glutamate cysteine ligase is considered the rate limiting step because the ɣ-L-glutamylcysteine is present in lesser quantity when compared to the glutathione synthase. Decreased levels of glutathione synthase may lead to decrease in glutathione levels which may result in serious damage and may lead to haemolytic anaemia, central nervous system damage and metabolic acidosis. In general the synthesis of glutathione depends on 2 major factors such as the availability of L-cysteine and the activity of glutamate cysteine ligase [19]. c. Glutathione metabolism Cytosol, mitochondria and endoplasmic reticulum are the various sites were glutathione is synthesised. Once synthesised its principle role is to provide protection against reactive oxygen and nitrogen species. It plays a vital role in the reduction of hydrogen peroxide and organic peroxides and is used as a co-substrate by glutathione peroxidise. Moreover, GSSG produced by the consumption of glutathione can be effectively reduced by the action of glutathione reductase or excreted from the cell [29]. GSSG + NADPH +H+ → 2GSH + NADP+ Glutathione exists in 3 major forms within the cell and they are GSH, GSSG and GSH-conjugate. These are then excreted to extracellular spaces and then hydrolysed and reabsorbed. The cysteine residues which are conjugated with xenobiotics get excreted through the faeces. Most of the glutathione S-conjugates are generally metabolised to their respective mercapturic acids and excreted in the urine and bile. Mostly the glutamate and glycine residues are recovered and recycled but cysteine residues are lost since they remain conjugated with the product and get excreted [20]. d. Functions The most important function of glutathione is to provide active protection against oxidative stress and against endogenous and exogenous toxins. But they also provide a variety of other functions such as Acts as signal transducers for cellular transcription, Maintains the thiol state of proteins, Acts as a source for cysteine reserve, Active participation in the metabolism of prostaglandins, leukotrienes and estrogens, Helps in the reduction of RNA’s to DNA’s. Helps to mature iron-sulphur clusters in proteins, and Mediates in copper and iron transfer [2]. 3. GLUTATHIONYLATION IN CELLS Glutathione is one of the most important low molecular mass thiol tri-peptide group found in abundance in most of the cell types. Glutathionylation is a type of post translational modification in which the cysteine residues of the proteins are added to the glutathione tri-peptide [4]. The trigger to glutathionylation is nitrosative or oxidative stress within a particular cell. Glutathionylation not only reduces stress but also plays an important role in cellular processes such as protein modification and re-cycling of protein thiols. They are also known to have a significant role to play in cell signalling pathways involved in tumours and in viral infections [6]. Glutathionylation is also known to induce redox signalling mechanism in plants. Glutathione acts as a thiol buffer to regulate plant stress response. Glutathionylation is a reversible post translational modification which forms mixed disulphide bonds between the protein and GSH. ROS and oxidised glutathione acts as a trigger for the modification to begin. Glutathionylation not only protects the proteins from irreversible oxidation and modulates its activity [22]. Glutathionylation is not only an important process in plants but it is of vital importance in human and animal cells as well. Since all types of proteins are susceptible to glutathionylation its role is quite diverse in the human body. During oxidative stress it acts as a switch for energy metabolism. Glutathionylation plays an important role in the expression of cancer by the prevention of the dimerisation of the tumour suppressor p53. It prevents apoptosis by protecting caspase. Some of the diseases caused due to oxidative stress are cardiovascular disorders, cancer, neurodegenerative diseases and lung diseases are caused due to the extensive occurrence of glutathionylation in human cells [18]. 4. PROTEIN GLUTATHIONYLATION Even though various mechanisms of protein glutathionylation have been proposed in vitro, the exact mechanism involved in vivo is still under debate. In vitro the glutathionylation is studied under conditions of stress but due to recent advances in proteomic studies has helped in the study of a number of targets for glutathionylation in a cell without the induction of external stress. Thus by this method a better understanding of the mechanism of protein glutathionylation under conditions of physiological and oxidative stress can be better understood and analysed. During protein glutathionylation the sensitivity of the given cysteine residue may depend upon specific features such as the accessibility of the solvent, reactivity or the microenvironment surrounding the target cysteine residue. Glutathione has several targets in a cell in which most of it functions as a protector or as a signal transducer [13]. In vitro, glutathionylation is a spontaneous reaction which involves a number of glutathione products. One of the basic glutathione reaction is one in which there occurs n exchange of a thiol group leading to the formation of a disulphide bond between the cysteine group of the protein and the glutathione molecule. This reaction is given as: Protein – SH + GSSG ↔ Protein – SSG + GSH The equilibrium constant of this reaction determines that 50% of the target protein in this reaction occurs in the glutathionylated form [14]. The equilibrium constant of this reaction is given as KOH. It is determined that most of the cysteine protein residues susceptible to glutathionylation have a KOH value around or below 1 which implies the equimolar levels of GSH and GSSG. This state does not occur during normal physiological conditions but only during increased stress levels. In plant tissues the GSH/GSSG ratio ranges between 10 to 20 but these values may differ with different cell types since each type of cell have different redox states. Hence the redox states in the chloroplast, mitochondria and the cytosol are higher than the redox states in the endoplasmic reticulum. The GSH/GSSG ratio can be established by using fluorescent probes which are usually redox sensitive [36]. It is a well established fact that the redox state of glutathione is influenced by the accumulation of GSSG in the cell which is caused as a result of oxidation under varying conditions of stress such as ozone, drought, pathogen attack, etc. Thus it is established that the glutathione pool is considered to be the most sensitive indicator of hydrogen peroxide in the cell in nature. Thus this condition leads to a decrease in the GSH/GSSG ratio leading to the accumulation of GSSG within the cell. The accumulation of GSSG leads to the spontaneous process of glutathionylation [10]. In vivo the most common mechanism involved in protein glutathionylation generally involved activation of a thiol derivative which is mostly sulfenic acid. Peroxynitrite or hydrogen peroxide easily converts the two electron oxidation of the protein thiols to sulfenic acid. Protein cysteine which undergoes glutathionylation is generally acidic in nature and after they form sulfenic acid and sulfenates they form sulfinates and sulfonates. Mixed disulphides are produced due to the spontaneous reaction of the GSH with the thiol group of proteins. Sulfentes are unstable products and hence they are responsible for the reversible oxidation of protein cysteine residues [15]. In plants, glutathionylation may also occur as a result of the reaction of nitro-glutathione with a protein thiol. It is relatively stable and acts as a reservoir for nitrogen. Thus it may either trigger nitrosylation or glutathionylation depending on the target but the exact mechanism involved in the switch between nitrosylation and glutathionylation is still unclear and is yet to be established. Glycine decarboxylase and GrxS12 are examples of plant proteins which can undergo glutathionylation due to nitro-glutathione [32]. Theoretically it has been established that the protein thiols may first be nitrosylated before undergoing glutathionylation by glutathione. Thus in this case nitrosylation acts as an activator of the protein before glutathionylation. Even though the theory is quite possible the experimental evidence is still under speculation [8]. Except the GSSG – dependent mechanism of protein glutathionylation all other mechanism of glutathionylation in vivo deliver similar results as the production of the glutathionylated product of the target protein in the form of Protein – SSG. The stability of the product is mainly established by the redox state of the intermediate product and the glutathionylated protein and GSH [24]. Glutathionylation of proteins are considered to be spontaneous reactions but in nature they are extremely slow processes and are often undetectable. Once enzymatically accelerated the target proteins which undergoes glutathionylation does not equilibrate with the glutathione pool in vitro but this may not be the case in vivo since both occurs under different conditions and stress levels. Protein glutathionylation in vivo can be brought about by glutaredoxins. Glutaredoxins are also considered as effective deglutathionylation in vitro as well [34]. 5. IDENTIFICATION OF GLUTATHIONYLATED PROTEINS The identification of glutathionylated proteins is important in the analysis of various conditions of stress which may in turn lead to diseased conditions within the human body. There are specific methods to identify proteins that are sensitive to glutathionylation. The sensitivity is based mainly due to the presence of cysteine residues which undergoes oxidation when the particular cell is exposed to varying conditions of stress [9]. There are various steps for the identification of glutathionylated proteins and they are: a. Two – dimensional electrophoresis Samples of the glutathionylated proteins are prepared as per protocol and sonicated. Using the ready strips the electrophoresis is then conducted at 200C at 5v for 16 hrs. After electrophoresis iso-electric focusing was performed with varying degrees of gradients. The strips were again subjected to the second dimensional separation. Then the strips were equilibrated using the specific buffer. b. Sypro ruby staining The gel was allowed to stain in methanol and acetic acid for 20 mins and allowed to stain overnight using sypro ruby gel stain. c. Oxyblot immunochemical detection For immunoblot analysis the gel obtained previously was transferred to a nitrocellulose membrane and blot transfer was carried out. The membranes were then blocked using specific solutions and incubated using specific monoclonal anti-bodies which is rabbit monoclonal anti-GSH. The membrane was then washed and incubated with the secondary anti-body anti-rabbit alkaline phosphatise. The blots were then dried and scanned using adobe Photoshop. d. Image analysis The nitrocellulose membrane was scanned and visualised under UV trans-illuminator. The images obtained were used for the comparison of protein expression by 2D gels and immune-reactivity with 2D blots. The results were read using PDQuest software. e. Trypsin In-Gel digestion The images obtained from the image analysis of the gel showed a significant increase in the presence of glutathionylated proteins. The gel was then washed with the specific buffers and treated with trypsin overnight at 370C. f. Mass spectroscopy The trypsin digested protein spots obtained from mass peptide fingerprints was determined using a mass spectrometer operated in the reflectron mode. g. Peptide sequence analysis Using the Swiss prot database along with the MASCOT search engine the mass peptide fragment obtained from the trypsin fragments was identified. Thus by using this method the size of the proteins was determined along with the pI range which is based on the position of the gel [26]. Profilin is an example of a cytoskeleton protein which has an increased rate of glutathionylation due to the presence of oxidants but proved to be quite elusive under normal experimental conditions for identification. But today with the help of two dimensional electrophoresis and labelled protein detection using monoclonal anti-bodies which are in turn identified using phospho-imaging and later identified using mass spectroscopy. Thus in this way great number of such cytoskeleton proteins have been identified and analysed [11]. 6. FUNCTIONAL CONSEQUENCES OF PROTEIN GLUTATHIONYLATION Protein glutathionylation is a spontaneous response to stress which occurs in most of the Eukaryotic cell. The glutathionylation of proteins not only protects the protein from oxidation but performs a host of functions within the cell. The most fascinating thing about the process of glutathionylation is that it is known to have different functional consequences on different target proteins. Some of the most important and notable functional consequences on the target proteins due to glutathionylation are a. The target proteins modified at the cysteine residue by the thiol group of the glutathione thereby modifying the protein in such a way to participate in the regulatory functions within the cell. For example, some of the glutathionylated proteins are known to participate in cell signalling pathways [7]. b. It is a well known fact that the process of glutathionylation is known to be in its element under conditions of oxidative stress. During oxidative stress the cell is found to contain increased number of hydrogen peroxide and nitro oxidative species which may induce the damage of proteins within the cell. Glutathionylation is one such process which prevents the damage of the target proteins by protecting the sensitive protein thiols from some irreversible forms of oxidation [25]. c. Glutathione being an important agent for stress control has to be found in enough quantity at any time required for spontaneous reactions. Glutathionylation not only reserves glutathione by binding with the proteins but also makes the availability of glutathione during dire situations especially under conditions of stress [16]. Other than the above mentioned functional uses glutathionylation is known to have other consequences as well and they are: Since the glutathionylation occurs during conditions of stress it prevents the activity of certain types of proteins during such conditions. This is one of the most important consequences of glutathionylation since it effectively reduces the production of unwanted proteins and converts the energy used in these processes to the removal of the oxidative species produced due to stress. This is because most of the enzymes which are susceptible to glutathionylation are inhibited by the post-translational modifications. For example, the DNA binding of p53 is effectively inhibited by glutathionylation. It is also known to inhibit the transcription factors of NF – κB and AP-1 which is brought about by the modification of p50 and c-Jun subunits [33]. Glutathionylation also decreases the rate of polymerisation of actin and also decreases the dimerisation of HIV Proteases [28]. On contrary to the above statement, glutathione is also known to increase the functional activities of proteins. This can be proved by a great number of examples. The enzymes activated by the process are mainly due to the thiol/disulphide exchange with GSSG. For example, in case of the HIV protease glutathionylation of cys67 increases the activity of the protein whereas glutathionylation of cys95 decreases the activity of the protein. Glutathionylation is also known to increase the activity of S100A1 thereby enhancing its calcium binding activity and also the calcium channel activity of ryanodine receptors [1]. 7. STRUCTURE CHANGE INDUCED BY PROTIEN GLUTATHIONYLATION Glutathionylation of proteins not only induces functional changes in a given protein but is also known to induce structural changes as well. These structural changes may also lead to changes in the functions and molecular mechanism of the given protein. Glutathionylation may lead to the inhibition of certain enzymes by the addition of thiol groups to certain cysteine groups [12]. The addition of glutathione to a protein not only blocks the free cysteine residue in the protein but also causes difference in the steric hindrance and the iso-electric point of the protein. It is established that glutathionylation generally makes the proteins acidic which is due to the addition of glutamic acid residue to the protein from the glutathione. Thus glutathionylation is completely different from cystenylation and is found to affect the proteins differently. For example, different forms of S-thionylation of protein kinase C may produce different results on the protein. The evidence of structural changes induced by glutathionylation of proteins is proved by circular dichroism and is established in human cyclophilin A and E.coli cobalamine – independent methionine synthase [21]. 8. GLUTATHIONYLATION AND THE DISORDERS CAUSED DUE TO ITS IMPAIRMENT IN HUMANS Oxidative stress in the human body is characterised by many diseases which may result due to the interruption or the mere diversion from the normal redox signalling haemostasis. S – Glutathionylation is responsible for the regulation of a large number of proteins some of which are responsible for causing diabetes, cancer and various other diseases. a. Diabetes Diabetes is characterised by the chronic increase in the blood glucose level. This is a type of disorder which may arise due to complications in multiple mechanisms and may be caused by different types of oxidative stress. The glucose level in the body is generally regulated by four major pathways which can be disrupted by an increase in the production of superoxide. The overproduction of the mitochondrial super-oxides may act as inhibitors to these pathways. ROS along with NADPH oxidase acts as a primary source of signalling in diabetes. The glutathionylated proteins are known to play a major role in diabetes including metabolism, homeostatic and redox regulation, transport, protein folding and even cell death. However, complications in glutathionylation of these proteins may lead to exocytosis of insulin causing diabetes. b. Cardiovascular Diseases During conditions of oxidative stress in the cardiovascular system, the glutathionylated proteins are produced as a result of critical cell signals. These glutathionylated proteins are known to regulate numerous physiological processes which are found to be crucial in the maintenance of cardiovascular homeostasis. Any impairment in the production of these glutathionylated proteins may lead to three major cardiovascular disorders such as the myocardial infarction, cardiac hypertrophy and atherosclerosis. The condition of oxidative stress leading to cardiovascular disorders is termed as ischemia or reperfusion injury. c. Lung Diseases Oxidative stress may cause chronic damage to lung tissues thereby leading to a number of physiological diseases such as asthma, cystic fibrosis, chronic obstructive pulmonary diseases, fibrotic diseases and cancer of the lungs. Even though it is well known that these diseases are caused as a result of increase in the production of glutathionylated proteins the exact mechanism or the pathway is not well understood. In most of the cases the actual involvement of glutathionylated proteins and their specific roles remain quite uncertain [23]. d. Cancer Alterations in the cellular redox state in humans may lead to numerous degenerative diseases including cancer. Under normal conditions the cell is maintains a redox balance with high concentrations of anti-oxidants along with its enzymes to reduce the effect of oxidative stress caused due to the presence of ROS. It has been well established that the cancer cell are known to have increased oxidative stress levels leading to increased metabolic activity which in turn leads to mitochondrial dysfunction and oncogenic stimulation [31]. e. Neurodegenerative Diseases Protein glutathionylation caused due to oxidative stress is one of the major reasons for neurodegenerative diseases by disrupting the normal signalling pathway thereby increasing the progress of the disease. The disorder is caused by gradual progressive degeneration of neurons. Aging is also one such process caused by mitochondrial dysfunction and activation of cell death pathway. Alzheimer’s and Parkinson diseases are a few examples of the same [26]. 9. DISCUSSION Glutathionylation of proteins has been extensively studied over the years but still the exact understanding of the reversible S-glutathionylation and its regulatory mechanism is still not yet well understood. A number of pathways in which glutathione is used has been well identified but some are still under speculation. Immunohistology of various cells in the highly stressed conditions were known to show high concentrations of glutathionylated proteins. Thus, it has been proved that oxidative stress enhances glutathionylation of NOS and ROS which is in turn responsible for the cellular nitroso-redox imbalance which continuously stimulates the susceptible thiols to undergo oxidation or production of glutathionylated modifications [8]. The scope for investigation in the aspect of glutathionylation is vast since the better understanding of the subject may help in the development of effective anti-oxidant therapies. Biomarkers have been used for identifying the anti-oxidants and for analysing their role in the removal of the oxidative species within the particular cell [11]. Therefore, investigations involving cell culture and unicellular organisms can be carried out to identify the mechanism involved in the various pathways of glutathionylation. Moreover, the development in molecular biology along with the production of cell lines with deleted genes or chimeric lines may provide more information on the subject. Finally, the experience, skill, interest and the facilities involved may prove to be crucial in the understanding of the process of glutathionylation so that major breakthrough can be made in the field of medical science [5]. 10. CONCLUSION Glutathionylation of proteins in the Eukaryotic cell is a well studied subject but a clear understanding of all the pathways involved is yet to be obtained. Research is on for further and better understanding of the various pathways and functions involved in the process of glutathionylation. As discussed above glutathionylation has a wide array of functions within the human body and is associated with a number of physiological disorders such as cancer, cardiovascular disorder, neurodegenerative disorders and lung diseases. Thus a better understanding of the subject may help us to solve many medical issues. The proper understanding of the pathways involved in the process of glutathionylation not only helps us to understand the reasons and causes of the disease but will also prove to be a trigger to find the cure for the diseases thereby decreasing the sufferings of many patients who are in a dire need for such treatments. Glutathionylation in plants is also well studied to understand the various pathways that are activated during conditions of stress. 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