Glutationilacija – regulacijska uloga glutationa u fiziološkim procesima

Glutathione (γ-glutamyl-cysteinyl-glycine) is an intracellular thiol molecule and a potent antioxidant that participates in the toxic metabolism phase II biotransformation of xenobiotics. It can bind to a variety of proteins in a process known as glutathionylation. Protein glutathionylation is now recognised as one of important posttranslational regulatory mechanisms in cell and tissue physiology. Direct and indirect regulatory roles in physiological processes include glutathionylation of major transcriptional factors, eicosanoids, cytokines, and nitric oxide (NO). This review looks into these regulatory mechanisms through examples of glutathione regulation in apoptosis, vascularisation, metabolic processes, mitochondrial integrity, immune system, and neural physiology. The focus is on the physiological roles of glutathione beyond biotransformational metabolism.Glutation (γ-glutamil-cisteinil-glicin) stanični je tripeptid, tiolni spoj i jaki antioksidans koji sudjeluje u metabolizmu otrova i biotransformaciji ksenobiotika faze II. Može se vezati na različite proteine u procesu poznatom pod nazivom glutationilacija. Proteinska glutationilacija dokazano je jedan od važnih posttranslacijskih upravljačkih mehanizama u fiziologiji stanica i tkiva. Izravne i neizravne upravljačke uloge u fiziološkim procesima uključuju glutationilaciju glavnih transkripcijskih faktora, eikozanoida, citokina i dušikova oksida (NO). U ovom se preglednom radu razmatraju navedeni upravljački mehanizmi na primjerima regulacije glutationom u apoptozi, vaskularizaciji, metaboličkim procesima, mitohondrijskom integritetu, imunološkom sustavu i fiziologiji živčanog sustava. Težište je rada na novim opisanim fiziološkim ulogama glutationa, pored uobičajeno opisane uloge u biotransformacijskom metabolizmu

Purification of Reversibly Oxidized Proteins (PROP) Reveals a Redox Switch Controlling p38 MAP Kinase Activity

Oxidation of cysteine residues of proteins is emerging as an important means of regulation of signal transduction, particularly of protein kinase function. Tools to detect and quantify cysteine oxidation of proteins have been a limiting factor in understanding the role of cysteine oxidation in signal transduction. As an example, the p38 MAP kinase is activated by several stress-related stimuli that are often accompanied by in vitro generation of hydrogen peroxide. We noted that hydrogen peroxide inhibited p38 activity despite paradoxically increasing the activating phosphorylation of p38. To address the possibility that cysteine oxidation may provide a negative regulatory effect on p38 activity, we developed a biochemical assay to detect reversible cysteine oxidation in intact cells. This procedure, PROP, demonstrated in vivo oxidation of p38 in response to hydrogen peroxide and also to the natural inflammatory lipid prostaglandin J2. Mutagenesis of the potential target cysteines showed that oxidation occurred preferentially on residues near the surface of the p38 molecule. Cysteine oxidation thus controls a functional redox switch regulating the intensity or duration of p38 activity that would not be revealed by immunodetection of phosphoprotein commonly interpreted as reflective of p38 activity

Characterization of thioredoxin related protein of 14 kDa and its role in redox signaling

Reversible reduction/oxidation (redox) reactions play key roles in cellular signaling pathways. Particularly cysteine residues in proteins can be modified by reactive oxygen-, nitrogen- or sulfur species (ROS, RNS, RSS), thereby altering the functions of the respective proteins. These modifications can be reversed by two major reductive systems in mammalian cells – the thioredoxin (Trx) and glutathione (GSH) systems. Both contain various representatives of the Trx fold family of proteins, among them the name-giving Trxs being the most prominent. In the cytosolic Trx system, electrons are transferred from NADPH to Trx reductase 1 (TrxR1) and subsequently to Trx1, which reduces a multitude of cellular substrates. Thioredoxin-related protein of 14 kDa (TRP14, TXNDC17) is a sparsely characterized, but evolutionarily well-conserved member of the Trx system. The studies comprising this thesis examined TRP14 in several aspects of redox signaling. In Paper I we investigated the inhibition of TrxR1 by noble metal compounds and their effect on cancer cell survival. Inhibition of the Trx system as anti-cancer strategy is thought to attenuate the antioxidant capacity of cancer cells, thereby leading to cell death. We found that gold (Au), platinum (Pt), and palladium (Pd) compounds all inhibited TrxR1 in vitro, but in a cellular context, the inhibition and cytotoxicity were mainly dependent on the ligand substituents and cellular uptake. Furthermore, we found a covalent crosslink between TrxR1 and TRP14 upon treatment of cells with the antitumor agent cisplatin. We concluded that noble metals are potent TrxR1 inhibitors but Pt compounds, especially cisplatin, trigger highly specific cellular effects, including the covalent complex formation. In Paper II we studied the role of the Trx system in reactivation of oxidized protein tyrosine phoshatases (PTPs) in platelet derived growth factor (PDGF) signaling. Using fibroblasts that lacked TrxR1 (Txnrd1 -/-), we found both an increased oxidation of PTP1B and phosphorylation of the PDGF β receptor (PDGF βR). Consequently, we showed that both Trx1 and TRP14, coupled to TrxR1, are able to reduce oxidized PTP1B in vitro. This study demonstrated that the Trx system, including both Trx1 and TRP14, impacts the oxidation of specific PTPs and can thereby modulate PDGF signaling. In Paper III we established TRP14 as an efficient TrxR1-dependent reductase and denitrosylase. Using several low molecular weight disulfide compounds, we found that, dependent on the substrate, TRP14 can be at least as efficient as Trx1. We also suggested TRP14 instead of Trx1 to be a major intracellular cystine reductase, because Trx1 does not reduce cystine once a preferred substrate such as insulin is present. Acting in parallel with Trx1, we also provide evidence of TRP14 being an efficient cellular reductase for nitrosylated proteins and concluded that TRP14 should be considered as an integral part of the Trx system. In Paper IV we developed a novel method for the detection of protein persulfides named Protein Persulfide Detection Protocol, ProPerDP. The formation of persulfide (-SSH) moieties at regulatory cysteine residues is emerging as a major pathway of hydrogen sulfide (H2S) mediated redox signaling. Using ProPerDP we discovered that both the Trx and the GSH system are potent reduction pathways for poly- and persulfides in cells. These studies reinforce the notion that TrxR1-dependent pathways are not only mediated via its wellknown substrate Trx1. We show that TRP14 is yet another cytosolic oxidoreductase with various intracellular functions, including reduction of PTPs, disulfides, nitrosothiols and persulfides. TRP14 is thereby potentially involved in a variety of different redox signaling pathways

Use of Proteomics to Probe Dynamic Changes in Cyanobacteria

Cyanobacteria are unicellular photosynthetic microorganisms that capture and convert light energy to chemical energy, which is the precursor for feed, fuel, and food. These oxygenic phototrophs appear blue-green in color due to the blue bilin pigments in their phycobilisomes and green chlorophyll pigments in their photosystems. They also have diverse morphologies, and thrive in terrestrial, marine water, fresh water, as well as extreme environments. Cyanobacteria have developed a number of protective mechanisms and adaptive responses that allow the photosynthetic process to operate optimally under diverse and extreme conditions. Prolonged deprivation of essential nutrients, such as nitrogen and sulfur, commonly found in the natural environments cyanobacteria grow in, can disrupt crucial metabolic activities and promote the production of lethal reactive oxygen species. The dynamic remodeling of protein complexes and structures facilitates adaptation to environmental stresses, however, specific protein modifications are poorly understood. Synthetic and systems biology approaches have been used to study how photosynthetic microorganisms optimize their cellular metabolism in response to adverse environmental conditions. To gain insights on how cyanobacteria cope with environmental changes, we created a global proteomics map of redox-sensitive amino acid residues and examined the degradation of light harvesting apparatus in cyanobacteria. These studies offered significant insights into the broad redox regulation and protein degradation, advancing knowledge of how photosynthetic microbial cells dynamically rely on protective mechanisms to survive changing environmental conditions

Disulfides as redox switches : from molecular mechanisms to functional significance

The molecular mechanisms underlying thiol-based redox control are poorly defined. Disulfide bonds between Cys residues are commonly thought to confer extra rigidity and stability to their resident protein, forming a type of proteinaceous spot weld. Redox biologists have been redefining the role of disulfides over the last 30&ndash;40 years. Disulfides are now known to form in the cytosol under conditions of oxidative stress. Isomerization of extracellular disulfides is also emerging as an important regulator of protein function. The current paradigm is that the disulfide proteome consists of two subproteomes: a structural group and a redox-sensitive group. The redoxsensitive group is less stable and often associated with regions of stress in protein structures. Some characterized redox-active disulfides are the helical CXXC motif, often associated with thioredoxin-fold proteins; and forbidden disulfides, a group of metastable disulfides that disobey elucidated rules of protein stereochemistry. Here we discuss the role of redox-active disulfides as switches in proteins.<br /

Papel de Peroxirredoxina 6 (PRDX6) humana en proliferación, migración e invasión en líneas celulares de hepatocarcinoma

Reactive oxygen species (ROS) have been considered toxic waste of cellular metabolism and enzymatic activities. However, nowadays it is well-known their tight regulation and the wide range of specific targets that they have in biological systems. Mainly, ROS are responsible for reversible oxidative modifications in proteins leading to appropriate cell signaling in aerobic cells. ROS balance, between production and removal by antioxidant cellular defense, is maintained under normal conditions, for the correct functionality of cellular processes. However, uncontrolled production of ROS generates oxidative stress, one of the main factors involved in cancer disease. Tumorigenesis is described as the gaining of malignant properties which dysregulate cell signaling maintaining a sustained proliferation, evading cell death, and doting cells with new characteristics. Tumoral cells can reactivate the developmental epithelium-mesenchymal transition (EMT) process, and it is here when tumoral cells carry out the “cadherin switch”, gain motility capacity and develop extracellular matrix degrader properties through metalloproteinases (MMP) resulting in metastasis. It is widely assumed that malignant cells exhibit higher levels of ROS than normal cells, contributing to all these protumoral properties. Surprisingly, these uncontrolled levels of ROS that are harmful to normal cells do not have cytotoxic effects on tumor cells but instead lead to exacerbated cell signaling. This is only possible due to the increase of antioxidants in tumoral cells that not only controls the exacerbated induction of ROS but allow to achieve protumorigenic signaling and at the same time avoid cell death. In fact, the antioxidant master regulator NRF2 is a potential contributor to all described cancer hallmarks. Consequently, downregulation of antioxidant defenses has been postulated as a very promising antitumor strategy in cancer, alone or in combination with conventional treatments. Peroxiredoxins are important enzymes of cellular antioxidant defense whose function consists of reducing H2O2, peroxynitrite, and alkyl hydroperoxides. Within them, PRDX6, through its peroxidase activity, is the only one that also reduces phospholipids hydroperoxides derived from lipid peroxidation. Moreover, PRDX6 through its calcium independent phospholipase A2 (aiPLA2) and lisophosphatidylcholine acyl transferase (LPCAT) activities is involved in repairing cell membranes. In addition, PRDX6 is overexpressed in cancer cells. Although its role in tumors remains unclear, PRDX6 seems to protect against cytotoxic effects of ROS as well as contribute to the synthesis of some tumoral-related lipokines. Moreover, PRDX6 is described to support prooxidant activity of NOX1 whose effects seem to be related to the malignant phenotype of certain tumoral cells. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, sorafenib and other drugs represent the only treatment at advanced stages, but they have reduced effects since hepatoma cells rapidly acquire resistance to these drugs leading to the urgent necessity of finding new therapeutic options. The aim of this thesis is to decipher protumoral functions of PRDX6 in HCC using epithelioid HepG2 and mesenchymal SNU475 cell lines. For that, cells were knocked out for PRDX6 through CRISPR/Cas9 technology. Moreover, PRDX6 was overexpressed in conjunction with NOX1 system in SNU475 cells to explore the malignant effects of this partnership in HCC. After PRDX6 removal, both cell lines showed a redox status disruption, increased ROS, lack of NRF2 induction and lower Grx1 levels. Redox proteomic analysis detected 218 peptides more oxidized and 36 more reduced in HepG2 cells after PRDX6 deprivation. Among them, the two extra cysteines of Grx1, Cys79 and Cys83 were more oxidized in knockout cells which can have drastic consequences for Grx1 activity. In both cell lines, Seahorse analysis showed impaired mitochondrial and glycolytic functionality when PRDX6 was absent, leading to metabolic reprogramming. Furthermore, a decreased proliferative rate without apoptotic effects was exhibited in those cell lines. Interestingly, cell cycle studies identified a G2/M arrest as the cause of slow proliferation in the absence of PRDX6, with involvement of PCNA and GSK3b. The proliferation protein PCNA was repressed in HepG2PRDX6-/- or SNU475PRDX6-/- cell lines by oxidative changes or by lower expression, respectively. Importantly, SNU475PRDX6-/- cells presented a GSK3b inactivation in Ser9 produced by phosphorylation of AKT. Successfully, these PCNA levels and GSK3b activation were recovered once PRDX6 was again present in SNU475PRDX6-/- cell line. The malignant phenotype was also affected since both knockout cells presented a reduced expression of EMT markers through the “cadherin switch”. Additionally, SNU475 cells exhibited repressed intracellular and extracellular MMP2 enzymatic activities with lower migratory and invasive capacities after PRDX6 deficiency. The malignant effects of prooxidant capacity of PRDX6 through NOX1 were also evaluated in SNU475 cell line. Surprisingly, PRDX6 supported NOX1 activity through a possible stabilization of NOXA1 component. Thus, PRDX6 main activities, phospholipase and peroxidase, were essential to generate higher NOX1-ROS derived production which actively contributed to an increased expression of EMT markers, migratory and invasive capacities in SNU475 cells. All these changes supported the role of PRDX6 in tumor development that was demonstrated in xenograft models since its absence reduced the tumor formation capacity of these HCC cell lines. Consequently, these results point to PRDX6 as a good candidate for antitumoral strategies. In fact, preliminary data from the use of specific miRNA against PRDX6 contained in nanoliposomes showed lower tumoral formation capacity of HepG2 cells in xenograft models. Thus, this strategy of targeting PRDX6 in vivo could be a very promising antitumoral treatment alone or in presence of drugs as sorafenib in HCC patients.Las especies reactivas de oxígeno (EROS) han sido consideradas el desecho tóxico del metabolismo celular y de diversas actividades enzimáticas. Sin embargo, recientemente se han identificado las múltiples dianas que estas especies coordinan y su estricta regulación en sistemas biológicos. Principalmente, EROS son responsables de modificaciones oxidativas en las proteínas las cuales son esenciales para señalización celular en células aeróbicas. El equilibrio de EROS entre su producción y su eliminación por los sistemas antioxidantes es mantenido bajo condiciones celulares normales y posibilita el correcto funcionamiento de procesos celulares. Sin embargo, una producción descontrolada de EROS genera estrés oxidativo, uno de los principales factores implicados en la enfermedad del cáncer. El proceso tumorigénico es descrito como una ganancia de características malignas las cuales desregulan la señalización celular incrementando proliferación, evadiendo muerte celular y dotando a células tumorales con nuevas características. Estas células pueden reactivar el proceso de transición epitelio-mesénquima (TEM) esencial para el desarrollo del organismo. Así que aquí es cuando las células tumorales llevan a cabo “el cambio en cadherinas”, ganan motilidad y desarrollan propiedades de degradación de matriz extracelular a través de metaloproteinasas (MMP) lo cual genera metástasis. Se conoce que las células malignas de cáncer poseen mayores niveles de EROS que células con un fenotipo normal ya que esto contribuye a la generación de cualidades protumorales. Sorprendentemente, estos descontrolados niveles de EROS que son dañinos para las células normales no conllevan efectos citotóxicos en células tumorales y participan en una activación de múltiples cascadas de señalización. Esto es solo posible debido a un incremento de antioxidantes en células tumorales controlando EROS y permitiendo alcanzar señalización protumorigénica y al mismo tiempo impidiendo la muerte celular. De hecho, el gran regulador de la defensa antioxidante NRF2 es descrito como un potencial contribuidor a todas las cualidades relacionadas con el fenotipo tumoral. Por tanto, la represión de estos sistemas de defensa antioxidante ha sido planteada como una estrategia antitumoral muy prometedora en cáncer y también en combinación con los tratamientos convencionales. Peroxirredoxinas son enzimas importantes en la defesa antioxidante celular cuya función consiste en la reducción de H2O2, peroxinitrito e hidroperóxidos acilados. Dentro de ellas se encuentra PRDX6 que a través de su actividad peroxidasa es la única que reduce hidroperóxidos de fosfolípidos derivados de peroxidación lipídica. Además, PRDX6 a través de sus actividades fosfolipasa A2 independiente de calcio (aiPLA2) y lisofosfatidilcolina acil transferasa (LPCAT) está involucrada en la reparación de membranas celulares. PRDX6 está sobreexpresada en cáncer. Aunque su papel en esta enfermedad queda aún por descifrar, PRDX6 protege contra efectos citotóxicos de EROS y contribuye en la síntesis de lipoquinas protumorales. Además, PRDX6 participa en la actividad prooxidante de NOX1 cuyos efectos parecen estar relacionados con la capacidad metastásica de ciertas células tumorales. El carcinoma hepatocelular (CHC) es el tipo más común entre los cánceres de hígado primarios donde sorafenib y otros fármacos representan los únicos tratamientos disponibles en estadios avanzados, pero cuentan con efectos reducidos ya que las células tumorales hepáticas rápidamente adquieren resistencia a estos fármacos lo cual muestra la necesidad urgente de encontrar nuevas opciones terapéuticas. El propósito de esta tesis es descifrar funciones protumorales de PRDX6 en CHC usando las líneas celulares epitelioide HepG2 y mesenquimal SNU475. Para ello, PRDX6 fue eliminada de estas líneas utilizadas a través de la metodología CRISPR/Cas9. Además, PRDX6 fue sobreexpresada en conjunción con el sistema NOX1 en células SNU475 para explorar los efectos metastásicos de esta asociación de proteínas en CHC. Después de la eliminación de PRDX6, ambas líneas celulares mostraron una disrupción del estatus redox celular, incrementaron los niveles de EROS, sin una inducción de NRF2 y los niveles de Grx1 fueron más bajos. El análisis del proteoma redox detectó 218 péptidos más oxidados y 36 más reducidos en células HepG2 después de la ausencia de PRDX6. Entre ellos, las dos cisteínas extras de Grx1, Cys79 y Cys83, que estuvieron más oxidadas lo cual puede tener consecuencias drásticas para la actividad de Grx1. En ambas líneas celulares sin PRDX6, el análisis “Seahorse“ mostró disfunción mitocondrial y glucolítica resultando en una reprogramación metabólica. Además, la eliminación de PRDX6 conllevó una disminución en proliferación celular sin efectos apoptóticos en ambas líneas celulares. Estudios de ciclo celular identificaron una parada en G2/M como la causa de proliferación ralentizada en ausencia de PRDX6 donde proteínas como PCNA y GSK3b podrían estar involucradas. La proteína de proliferación PCNA fue reprimida en las líneas celulares HepG2PRDX6-/- y SNU475PRDX6-/- por cambios oxidativos o por una expresión más baja, respectivamente. Las células SNU475PRDX6-/- presentaron una inactivación de GSK3b a través de la fosforilación de su residuo Ser9 producido por fosforilación de AKT. Estos niveles de PCNA y la activación GSK3b fueron recuperados de forma exitosa una vez que PRDX6 estuvo presente otra vez en la línea celular SNU475PRDX6-/-. El grado de malignidad de ambas líneas tumorales fue también afectado con la falta de PRDX6 presentando una reducida expresión de marcadores del proceso TEM a través del “cambio en cadherinas”. Adicionalmente, la línea celular SNU475 mostró una represión intracelular y extracelular de la actividad enzimática de MMP2 con bajas capacidades migratorias e invasivas después de la eliminación de PRDX6. Los efectos metastásicos de la actividad prooxidante de PRDX6 a través de NOX1 fueron también evaluados en la línea celular SNU475. Sorprendentemente, PRDX6 incrementó la actividad del sistema NOX1 a través de un posible mecanismo de estabilización del componente NOXA1. También se observó que las principales actividades de PRDX6, fosfolipasa y peroxidasa, fueron esenciales en la generación de EROS derivadas de NOX1 lo cual contribuyó activamente a un incremento de la expresión de marcadores del proceso TEM y las capacidades migratorias e invasivas de las células SNU475. Todos estos cambios apoyaron el papel de PRDX6 en el desarrollo tumoral lo cual fue demostrado en modelos de ratón a través de xenoinjertos ya que su ausencia redujo la capacidad de formación tumoral de estas líneas celulares en dichos modelos. Como consecuencia, estos resultados apuntan a PRDX6 como un buen candidato en estrategias antitumorales. De hecho, datos preliminares del uso de microARNs específicos contra PRDX6 encapsulados en nanopartículas mostraron una menor capacidad de formación tumoral de células HepG2 en dichos modelos de ratón. De este modo, esta estrategia contra los niveles de PRDX6 in vivo podría servir como un tratamiento antitumoral bastante prometedor o también combinando sus efectos con sorafenib en pacientes de CHC

Tomato expressing Arabidopsis glutaredoxin gene AtGRXS17 confers tolerance to chilling stress via modulating cold responsive components

Chilling stress is a production constraint of tomato, a tropical origin, chilling-sensitive horticultural crop. The development of chilling tolerant tomato thus has significant potential to impact tomato production. Glutaredoxins (GRXs) are ubiquitous oxidoreductases, which utilize the reducing power of glutathione to reduce disulfide bonds of substrate proteins and maintain cellular redox homeostasis. Here, we report that tomato expressing Arabidopsis GRX gene AtGRXS17 conferred tolerance to chilling stress without adverse effects on growth and development. AtGRXS17-expressing tomato plants displayed lower ion leakage, higher maximal photochemical efficiency of photosystem II (Fv/Fm) and increased accumulation of soluble sugar compared with wild-type plants after the chilling stress challenge. Furthermore, chilling tolerance was correlated with increased antioxidant enzyme activities and reduced H(2)O(2) accumulation. At the same time, temporal expression patterns of the endogenous C-repeat/DRE-binding factor 1 (SlCBF1) and CBF mediated-cold regulated genes were not altered in AtGRXS17-expressing plants when compared with wild-type plants, and proline concentrations remained unchanged relative to wild-type plants under chilling stress. Green fluorescent protein -AtGRXS17 fusion proteins, which were initially localized in the cytoplasm, migrated into the nucleus during chilling stress, reflecting a possible role of AtGRXS17 in nuclear signaling of chilling stress responses. Together, our findings demonstrate that genetically engineered tomato plants expressing AtGRXS17 can enhance chilling tolerance and suggest a genetic engineering strategy to improve chilling tolerance without yield penalty across different crop species

The role of Slc7a11 in controlling extracellular and intracellular redox environments of lung fibroblasts - potential targets for intervention in aging and idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by extracellular matrix deposition by fibroblasts. Aging and oxidative stress increase the susceptibility to IPF. Redox couples, cysteine/cystine (Cys/CySS) and glutathione/glutathione disulfide (GSH/GSSG), and their redox potentials (Eh) quantify oxidative stress. Fibroblasts from old mice maintain more oxidized extracellular Eh(Cys/CySS) than young mice. Microarray shows down-regulation of Slc7a11 potentially mediates this age-related oxidation. Slc7a11 is the key component of system Xc-, an antiporter that imports CySS and exports glutamate. The first aim of this dissertation is to investigate the mechanistic link between Slc7a11 expression and extracellular Eh(Cys/CySS). The second aim is to evaluate the effects of aging on the redox states of intracellular proteins and whether Slc7a11 contributes to the age-dependent effects. The last aim is to compare SLC7A11 expression, extracellular Eh(Cys/CySS) and intracellular Eh(GSH/GSSG) between human lung fibroblasts from IPF and non-IPF donors and to explore their association with pro-fibrotic gene expression. Slc7a11 expression was manipulated by pharmacological and genetic methods. Reduced and oxidized forms of Cys residues were labelled by Iodoacetyl Tandem Mass Tags. The ratio of oxidized/reduced forms (i.e., redox state) of a Cys residue was determined by multiplexed tandem mass spectrometry. Eh(Cys/CySS) and Eh(GSH/GSSG) were more oxidized in conditioned media of old fibroblasts. Up-regulation of Slc7a11 reduced extracellular Eh(Cys/CySS) for old fibroblasts. Inhibition of GSH synthesis had no effect on the ability of cells to restore their extracellular Eh(Cys/CySS). Redox states of 151 proteins changed with aging. Slc7a11 over-expression restored redox states of 104 proteins. Ingenuity Pathway Analysis showed these 104 proteins were involved in pathways of protein translation initiation, ubiquitin-proteasome-mediated degradation and integrin-cytoskeleton-associated signaling. Slc7a11 expression was lower in IPF fibroblasts. Extracellular Eh(Cys/CySS) was more oxidized and expression of pro-fibrotic genes was higher in IPF fibroblasts. In conclusion, Slc7a11 is the key regulator of extracellular Eh(Cys/CySS). Its effects are independent of GSH synthesis. Aging results in changes of redox states of proteins involved in protein turnover and cytoskeleton dynamics. Up-regulating Slc7a11 restores changes of protein redox states due to aging. Decreased SLC7A11 might represent a susceptibility factor for developing tissue disrepair and fibrosis in IPF

Identification and characterization of the localization and expression of CLIC4 under redox conditions in mammalian cells

Chloride intracellular ion channel-4 (CLIC4) is a member of the CLIC family of proteins which were originally identified as channels of intracellular membranes permeable to ion chloride ions. Its expression and localization to intracellular membrane is sensitive to oxidative stress. This sensitivity of CLIC4 is indicated with its physiological redox regulatory function either as an oxidoreductase enzyme or an ion channel in response to decrease the cellular glutathione level, ROS accumulation, and consequently non-native disulfide formation in the cells. The pathways for disulfide formation are well characterized. However, the understanding of redox state of CLIC4 and possibility to participate in reductive pathway to removing the non-native disulfide bond is still limited and whether CLIC4 as a membranebinding protein with oxidoreductase activity might be needed in the reduction pathway either in the cytosol or ER, are the questions we need to address. In this project the oxidative stress induced by TNF-α and CLIC4 in response to TNF-α can be re-localized to the ER from the cytosol. The ER is a host for disulfide formation within folding proteins entering the mammalian secretory pathway. The consequence of oxidative stress in the ER is the accumulation of misfolded and unfolded proteins. The mammalian cells have a family of oxidoreductase that is thought to be isomerised non-native disulfide bonds. This reductive catalytic activity of oxidoreductases is maintained via a reductive pathway. For CLIC4 to act as an oxidoreductase for performing isomerisation or reduction reactions, it must be preserved in a reduced position. Here, by mass spectrometry, using purified proteins and ER microsomal membrane following TNF-α induced oxidative stress, we illustrate CLIC4 is predominantly placed in a reduced state in the intact cells, demonstrating a reductive pathway is prepared in mammalian cells and CLIC4 can be involved with this pathway as an oxidoreductase through its either enzyme catalytic activity or ion channel activity. In this project, the glutathione has identified to be responsible for the reduction of CLIC4 during oxidative stress. Furthermore, when inhibitors of glutathione synthesis or reductase are added to the cells, CLIC4 is not reduced. The results demonstrate that glutathione plays a direct role in the isomerisation of disulfide bonds by maintaining CLIC4 in a reduced state. To confirm the reduction effect of GSH on CLIC4 and overall microsomal membrane protein in response to TNF-α, we have applied a cysteine-reactive tandem mass tag (Iodo-TMT) to differentially label cysteine residues and analyse the overall protein expression level and redox state into one-step analysis. The individually labeled samples have been pooled in differential combinations to create multiple six-plex samples to determine the effect of GSH on cysteine oxidation and overall protein expression in the microsomal membrane. The result highlights the redox status of CLIC4 under reduction of GSH was confirmed with MS/LC, and the TMT-labeling detected the redox state of the overall microsomal membrane proteins with respect to cysteine oxidation and protein expression. This study is important because CLIC4 as either a membrane binding protein or an ion channel can be considered as a mis-component in reductive pathway