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Научни трудове на Съюза на учените в България-Пловдив Серия Г. Медицина, фармация и дентална медицина т.ХХ1. ISSN 1311-9427 (Print), ISSN 2534-9392 (On-line). 2017. Scientific works of the Union of Scientists in Bulgaria-Plovdiv, series G. Medicine, Pharmacy and Dental medicine, VoLXXI. ISSN 1311-9427 (Print), ISSN 534-9392 (On-line). 2017.
ТИОРЕДОКСИН, ТИОРЕДОКСИН - ВЗАИМОДЕЙСТВАЩ БЕЛТЪК И ПОСТТРАНСЛАЦИОННИТЕ ИММОДИФИКАЦИИ ПРИ ЗАХАДЕН рСПАЕТ ОСИ 2 Теодора Станкова1, РеАман Али Бяаг2, Еисма /Делчева1, Ана Манева1 1 Катедра„Хомия и Медиц идоки ундведеитет - Пловдив
2Студ ентхом ддидина,Мед оцинскиумдворситет-Пловдия
THIOREDOXIN, THIOREDOXIN-INTERACTING PROTEIN AND THEIN POSTTRANSLNIIONAL MODIFICATIONS IN DIABETES
MELLITUS NEPE A Teodora Stankova1, RehmanAH Daig2,Ginka Delcheva1, Ana Maneva1 1 Dtpartmen2 ofChemistry acd B^o^li^N^^trj', Medical University -Plovdiv 2 Studedi of Medicine, Medica1 Univershy - Plovdiv
It has been widely recognized that deregulation of redox responses at intra- and extracellular levels is one of the pivotal factors for the initiation and progression of multiple diseases. Thioredoxin (Trx) is an ubiquitously expressed protein in all forms of life, executing not only antioxidative and protein reducing activities, but also playing an important role in signal transducion, inflammatory modulation, anti-apoptosis, immune function and atherosclerosis. Diabetes mellitus (DM) and its complications have been associated with increased oxidative stress and alterations in the serum levels of Trx and its endogenous inhibitor - thioredoxin-interacting protein (TXNIP). This suggests their participation in disease pathogenesis, but the exact mechanism has not been fully elucidated. In this review, the biological properties of Trx, TXNIP and the regulation of their activity by various posttranslational modifications (PTM) such as glutathionylation, thiol-oxidation and S-nitrosylation are highlighted.
Keywords: thioredoxin, thioredoxin-interacting protein, diabetes mellitus
Oxidative stress is defined as an imbalance between the production of reactive oxygen species (ROS) and the biological system's ability to readily detoxify the reactive intermediates or easily repair the resulting damage (Valko et al., 2007). The biological systems are normally able to defend themselves against ROS by regulating the cellular reduction/oxidation (redox) status through the use of several antioxidant systems. Sulfhydryl biochemistry has been shown to play an important role in regulating cell function, since the redox state of cysteine sulfhydryls often determines the structure and activity of enzymes, transcription factors, and transport proteins required for cell viability. The thioredoxin (Trx) system and the glutathione (GSH) system are the two main ubiquitously expressed thiol-reducing antioxidant systems (Fernanades & Holmgren, 2004).
Diabetes mellitus per se is a state associated with increased oxidative stress, that may also worsen the clinical course of the disease and contribute to development of diabetic complications. On one hand glucose autoxidation, activation of hexosamine and PKC pathways, induction of
NADPH oxidase, advanced glycation end product/receptor interactions etc. may contribute to increased production of free radicals in diabetes. On the other hand the enzyme activity of Cu-Zn superoxide dismutase (SOD) and levels of antioxidants such as vitamin E, vitamin C, and reduced GSH have been reported to be decreased in diabetes (Matough et al., 2012). Trx has been suggested as a marker of oxidative stress in various diseases, as it is induced by oxidative stressors such as hydrogen peroxide, ultraviolet irradiation and inflammation (Nakamura et al., 1997). Therefore, evaluation of the possible role of Trx and TXNIP in diabetes has recently gained much interest. So the current review summarizes the biological properties of Trx, TXNIP and the regulation of Trx activity by some PTM in DM type 2 (DMT2).
Thioredoxin (Trx) and Trx system
The thioredoxin system comprises thioredoxin (Trx), truncated Trx (Trx-80), thioredoxin reductase (TrxR), and NADPH, besides a natural Trx inhibitor, the thioredoxin-interacting protein (TXNIP). Three distinct forms of human Trx, encoded by separate genes, have been characterized so far: cytosolic Trx (Trxl), mitochondrial Trx (Trx2), and a Trx variant that is highly expressed in spermatozoa (SpTrx/Trx3) (Nordberg & Arner, 2001). The classical 12 kDa cytosolic Trxl is the most studied of the three forms of Trx and it is the subject of the present review.
Depending on its subcellular localization, Trx exerts different roles. In the extracellular environment, Trx exhibits chemokine-like activity, while in the cytoplasm, it regulates the cellular redox environment and also the activity of certain proteins. In the nucleus, Trx1 has been shown to interact with many transcription factors with implication on oxidative stress, inflammation, apoptosis and regulation of metabolism in DM such as redox effector factor 1 (Ref-1), hypoxia-inducible factor (HIF- 1a), nuclear factor kappa B (NF-kB), p53, activating protein-1 (AP-1), nuclear factor (erythroid-derived 2)-like 2 (Nrf-2), glucocorticoid receptor, estrogen receptor, and others (Go & Jones, 2010). Thereby Trx regulates their gene expression. Several factors induce nuclear translocation of Trx1, despite the lack of a nuclear localization signal, which suggests that Trx1 may be associated with signaling molecules that bridge the cytoplasmic and nuclear compartment. By contrast, the translocation of Trx1 to the membrane requires binding of TXNIP (Yoshihara et al., 2013).
The thioredoxin system reduces oxidized cysteine groups in proteins through an interaction with the redox-active center of thioredoxin (-Cys-Gly-Pro-Cys-). The oxidized Trx so formed is then reduced by the help of TrxR and NADPH. It is known that Trx performs most if its antioxidant functions through peroxiredoxins - Trx peroxidases, which aid in the direct reduction of peroxides such as H2O2 and different alkyl hydroperoxides (Arner & Holmgren, 2000).
Trx system is essential for maintaining the balance of the cellular redox status and it is involved in the regulation of redox signaling but also plays an important role in signal transduction, inflammatory modulation, anti-apoptosis, immune function and atherosclerosis (Yoshihara et al., 2013). Therefore, it has been suggested that deregulation of Trx system may be involved in DMT2 pathogenesis and its complications.
The major characteristics of DMT2 are peripheral insulin resistance and decompensation of the pancreas that can no longer keep up with the increased insulin requirements, resulting in hyperglycemia. The elevated glucose levels have detrimental effects on various tissues including the pancreatic p-cells. Glucose toxicity leads to progressive p-cell dysfunction, impaired insulin gene transcription and irreversible p-cell loss by apoptosis (Rhodes, 2005). Initiation and execution of apoptosis is triggered by changes in the redox environment, which suggests the participation of Trx and TXNIP in the stringently controlled process of cell death. Trx1 has been shown to be involved in apoptotic processes in at least two different ways. Firstly, the redox status of one of the key determinants of the apoptotic process, caspase-3, is maintained by Trx1 (Mitchell & Marletta, 2005). Secondly, Trx1 binds to and inhibits the apoptosis signal-regulating kinase-1 (ASK-1). This molecule, ASK-1, has been identified as mitogen-activated protein (MAP) kinase kinase kinase and oxidation of Trx1 causes the release of this binding followed by the induction of apoptosis via the activation of p38 MAP kinase and c-Jun-NH2-terminal kinase (Hsieh &
Papaconstantinou, 2006). Therefore, high levels of Trx1 protein could be effective in suppressing the progression of diabetes. In fact, transgenic overexpression of Trx1 ameliorates glucose intolerance, enhances pancreatic duodenal homeobox factor-1 (PDX-1) and Maf A expression and preserves p-cell functions, especially the insulin-secreting capacity. Its protective effects are also apparent in the early phase of p-cell failure, where it suppresses insulin hypersecretion induced by hyperglycemia and the ROS production associated with the glucotoxicity (Yamamoto et al, 2008). In contrast to Trx, TXNIP possesses proapoptotic activity, which is discussed below.
Streptozotocin induced diabetic rat (SIDR) model is one of the best described animal models for diabetes mellitus. It was very recently shown that there was a significant increase in the Trx1 expression at the baseline level in the SIDR ischemia-reperfused (I/R) myocardium (Thirunavukkarasu et al., 2007). Kakisaka et al. also documented elevated serum levels of Trx in patients with DMT2 (Kakisaka et al., 2002). This could possibly be a compensatory mechanism against the hyperglycemia induced oxidative stress. Furthermore, Thirunavukkarasu et al. reported that resveratrol treatment in SIDRs, protected the myocardium from the adverse effects of I/R injury by the induction of VEGF expression through Trx1 mediated heme oxygenase-1 expression and Mn-SOD activity (Thirunavukkarasu et al., 2007). However, the Trx activity was found to be significantly reduced while the protein Trx levels did not differ in the SIDRs when compared to the normal control (Haendeler, 2005). On the contrary the expression of TXNIP dramatically increased in diabetic animals.
DMT2 is one of the major risk factors for cardiovascular diseases (CVD). Diabetes has been associated with the acceleration of atherosclerotic plaque formation (Giacco & Brownlee, 2010). The dichotomy of Trx is also evident from the involvement of Trx in atherosclerosis. Trx mRNA and TrxR mRNA have been reported to be increased in endothelial cells and macrophages of human atherosclerotic plaques (Zhang et al, 2007). The Trx2 system appears to have a more important role in preventing mitochondrial dysfunction than the mitochondrial GSH system in endothelial cells (Nordberg &Arner, 2001). Furthermore, Trx2 improves endothelial cell function and reduces atherosclerotic lesions in the apolipoprotein E-deficient mouse model. Yin et al. demonstrated that application of exogenous recombinant human Trx1 can be a promising novel strategy for the attenuation of DMT2 cardiac complications (Yin et al., 2010). However, Trx2 transgenic mice have shown higher levels of total antioxidants, reduced oxidative stress and increased nitric oxide levels when compared to the control (Zhang et al, 2007). But it still remains unclear whether the Trx system is beneficial or harmful with respect to the pathogenesis and progression of atherosclerosis.
Thioredoxin-interacting protein (TXNIP)
TXNIP, a multifunctional protein, which is also titled as Vitamin D3 up-regulating protein-1 or thioredoxin binding protein-2. It is a negative regulator of Trx1 function because it directly interacts with the active center of Trx1, thereby inhibiting its reducing activity. In addition to its inhibitory role, TXNIP plays important roles in lipid and glucose metabolism, inflammation, cardiac function, cell proliferation and apoptosis (Chung et al., 2006). TXNIP expression is ubiquitous and is induced by a variety of stresses, including UV light, y-rays, heat shock, and H2O2, as well as glucose.TXNIP overexpression renders cells more susceptible to oxidative stress and promotes apoptosis (Corbett, 2008). Furthermore, peroxisome proliferator-activated receptor gamma (PPARy) activation stimulates apoptosis in human macrophages by altering the cellular redox balance via regulation of TXNIP (Billiet et al., 2008). Under stress conditions, the TRX1/TXNIP complex dissociates. Thus, free TRX1 reduces oxidized proteins and scavenges free radicals. However, TXNIP binds to the cytosolic multiprotein complex NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome and changes its function from TRX1 repressor to NLRP3 activator. Upon stimulation, NLRP3 oligomerizes with the adaptor protein ASC (apoptosis associated speck-like protein containing a caspase-recruitment domain), which in turn activates caspase-1 and induces the cleavage and activation of the proinflammatory cytokine IL-ip. Induction of the active form of IL-ip mediates p-cell dysfunction and apoptosis (Zhou et
al., 2010). These evidences confirm the hypothesis that TXNIP is a key mediator of the DMT2 pathogenesis and links glucotoxicity with p-cells apoptosis.
Chronic hyperglycemia enhances the TXNIP expression, which inhibits Trxl glucose uptake through its arrestin domain independent of its binding to Trxl protein. Recent studies have also suggested that TXNIP expression is increased in skeletal muscle of human DMT2 and impaired glucose tolerance patients TXNIP aggravates hepatic glucose production and insulin sensitivity in skeletal muscle and adipose tissue (Parikh et al., 2007), while insulin reduces TXNIP expression (Chutkow et al., 2010). Considerable attention gained recently the role of TXNIP in the induction and progression of microvascular complications in diabetes such as diabetic retinopathy and nephropathy. TXNIP mRNA and protein are highly expressed in renal mesangial cells (Hamada & Fukagawa, 2007), neurons and retinal cells (Perrone et al., 2010). The blockade of TXNIP decreases oxidative stress and protects diabetic subjects from complications (Perrone et al., 2010).
Posttranslational modifications (PTM) of Trx
PTMs are strategies routinely used by cells to expand their function, but can also reflect the status quo of struggled cellular functions under stressed conditions They play critical roles in cellular signaling, the impairment of which leads to many diseases (Cai et al., 2013). In the context of oxidative stress the redox regulation through the reversible oxidation of protein cysteines as in the case of glutathionylation, disulfide formation and S-nitrosylation have been regarded as protein damage. However, these PTMs are now considered as regulatory mechanisms. In addition to the two cysteine residues in the highly conserved active site sequence Cys32-Gly-Pro-Cys35 (CXXC), human Trxs contain 3 other, critical structural Cys residues, at positions -62, -69 and -73 (Niwa, 2007). They can undergo PTM, providing unique biological properties to Trx and regulating its activity.
Glutathionylation of proteins has been considered as a reversible means of storing GSH during oxidative stress and thus as a protective mechanism against irreversible protein thiol oxidation. This PTM occurs under normal physiological conditions, suggesting that it is not thus a characteristic modification during oxidative stress. Glutathionylation of Trx at Cys-73 reduces significantly Trx activity during oxidative stress, but Trx has shown to possess some means of degluthionylation and autoactivation (Niwa, 2007). Glutathionylation of Trx proves the existence of a balanced redox network and a crosstalk between GSH and Trx system, that were earlier considered to be clearly separate (Casagrande et al., 2002). Probably, the extent of this interaction between the two systems under conditions of oxidative stress may be an indicator of redox status of the cell.
Thiol-oxidation of Trx can affect not only Cys-32 and Cys- 35 in the active site, but also two other structural cysteines (Cys-62 and Cys-69) outside the redox regulatory domain. The oxidation of these two structural cysteines, involved in the maintenance of Trx tertiary structure, results in inhibition of the reduction of the two active site cysteines by TrxR (Niwa, 2007). In addition to this Trx has been reported to homodimerize via Cys-73 (Watson et al., 2003). Therefore, it can be speculated that the reversible oxidation of the conserved structural cysteines in Trx maybe a critical regulatory mechanism that can influence the accessibility of other proteins (e.g. TrxR) to Trx thereby regulating the function of these proteins.
S-Nitrosylation is the covalent addition of a NO moiety onto a cysteine thiol. S-nitrosylation of Trx at Cys-69 in endothelial cells increases the activity of Trx and also accounts for its anti-apoptotic capacity (Gaston et al., 2003). Available evidences imply S-nitrosylation in cardiovascular and neurological functions and impairments, which are one of the major complications of DM. Interestingly, Trx system can act as a major S-nitrothiol-caspase-3 denitrosylating protein, because denitrosylation by Trx1 alone in the absence of TrxR1 has been found ineffective (Mitchell & Marletta, 2005). The same authors documented that S-nitrosylation occurred not on Cys-69 but rather on Cys-73 of Trx. Thus it can be suggested that both Cys-69 and Cys-73 are required for S-nitrosylation and transferring NO to target proteins. S-nitrosylation of
Trx seems to be a potential therapeutic target, inducing the endogenous protective mechanism conferred by Trx. As a proof of the above statement, it was recently observed that the antioxidative effect of HMG-CoA reductase inhibitors (statins) via activation of endothelial NO synthesis was due to the S-nitrosylation of Trx (Haendeler et al., 2004).
The considerable evidence, presented in the current review, implies that the redox-sensitive signaling complex Trx/TXNIP is a key component in the redox regulation and the pathogenesis of DMT2. Therefore, it should be more profoundly studied and considered as a target for novel therapeutic approaches in diabetes mellitus and its complications.
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Corresponding author: Teodora Stankova, Department of Chemistry and Biochemistry, Medical University - Plovdiv, 15A Vasil Aprilov Blvd., Plovdiv 4002, e-mail: [email protected]
