Browsing by Author "Pillay, Che Sobashkar."

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Dynamics of the glutathione/glutaredoxin system.
(2014) Mashamaite, Lefentse Nelly.; Pillay, Che Sobashkar.; Rohwer, Johann M.
The glutathione/glutaredoxin system is made up of glutaredoxins, glutathione (GSH) and glutathione reductase (GLR). Glutaredoxins, which are involved in essential cellular functions such as DNA synthesis, iron metabolism and iron-sulfur cluster assembly, become oxidised during their catalytic cycle and are reduced by GSH and GLR. Glutaredoxins also play a critical role in regulating the glutathionylation/deglutathionylation cycle. Under oxidative stress conditions, protein thiols may be glutathionylated and glutaredoxin activity is important for restoring the functions of these proteins. While the individual components of this system have been studied extensively, the dynamics of the system as a whole has not been described despite its importance in the glutathionylation/deglutathionylation process. Computational systems biology approaches could be used to describe this type of regulation but the kinetic mechanism used by glutaredoxins for deglutathionylation is unclear as a monothiol and a dithiol mechanism have both been proposed for glutaredoxin activity. The in vitro data supporting these mechanisms have been contradictory with a number of discrepancies observed in the literature, including contrasting activities of mutant glutaredoxin Cxx(C→S) and wild-type glutaredoxins. Further, Lineweaver-Burk plots showed a curved line pattern in some studies, while other studies reported a linear pattern in response to GSH. Finally, analyses of the Lineweaver-Burk plots in two substrate kinetics experiments revealed both parallel line and intersecting initial velocity line patterns for deglutathionylation. Computational and mathematical models were used to resolve these discrepancies and we showed that the mono- and di- thiol mechanisms, are in fact identical. Mathematical models of mutant and wild-type glutaredoxin activities revealed that the GSH concentration and the rate constant for GSH oxidation significantly affected these relative activities which explained the contradictory data for wild-type and mutant glutaredoxins. The sigmoidal response to GSH was due to the kinetic order of this reaction and our results demonstrated that the resulting parallel and intersecting kinetic line patterns observed in some studies depended on the reversibility of the deglutathionylation reaction. Finally, fitting experiments showed that our models were able to accurately describe the in vitro data. Collectively, our results showed how deglutathionylation should be described in computational systems biology models and further revealed how the formation of oxidised glutaredoxin may play a vital role in the regulation of glutaredoxin activity.
The dynamics of thioredoxin-dependent reactions.
(2014) Photolo, Mampolelo M.; Pillay, Che Sobashkar.
The thioredoxin system, comprising thioredoxin (Trx), thioredoxin reductase (TrxR) and NADPH, is present in most organisms from prokaryotes to eukaryotes. The system plays a central role in the redox regulation of several key cellular processes including DNA synthesis, apoptosis, glycolysis and redox signal transduction and changes in this system are associated with the progression of a number of diseases including certain cancers, malaria and HIV. Understanding the regulation of this network from a systems perspective is therefore essential. Our lab developed the first computational model of the Escherichia coli thioredoxin system and analyzed this system using mathematical and core models. In contrast to the commonly held assumption of the thioredoxin network as an electrical circuit with no cross-talk, our analysis showed this system displayed ultrasensitivity, adaptability and was interconnected via the thioredoxin redox cycle. In this study, a model of the Saccharomyces cerevisiae thioredoxin system was developed and computational modeling showed that an increase in concentration of one of the substrates in the thioredoxin system could decrease the flux of another thioredoxin oxidation reaction in a concentration-dependent manner. To complement these computational analyses, yeast thioredoxin reductase and thioredoxin were cloned, expressed and purified. An in vitro kinetic assay using insulin as a substrate and immunoglobin Y (IgY) as a competing substrate was subsequently undertaken. Our results showed that in some cases an increase in IgY concentration affected the rate of insulin reduction as measured by turbidity at 650 nm confirming the computational model's prediction. However, unexpectedly, with an increase in IgY concentration, there was a decrease in apparent absorbance at 650 nm at longer time points. These in silico and in vitro analyses shed light on how the thioredoxin system connects seemingly unrelated parts of metabolism into an integrated network, but additional experiments are required in order to improve the kinetic analyses in this study.
Endolysosomal proteolysis and its regulation.
(2003) Pillay, Che Sobashkar.; Dennison, Clive.
The endolysosomal system is a multifunctional system and is involved in catabolism, antigen presentation and regulation of hormones. The descriptions of, and functions assigned to organelles within the system are often applied using different criteria. Further, the properties of the hydrolases within the system, and the organelles that house them are usually studied in isolation from one another. Considering that the endolysosomal system may be extremely dynamic, an understanding of the system as an integrated whole is a necessary first step. Thus, a review of the endolysosomal system was undertaken. It was determined that the enzymes within the endolysosomal system are probably subject to 'organelle-dependent' regulation, i.e. the organelles create the appropriate luminal conditions for these enzymes to work. It is also likely that the effectors of these luminal conditions are regulated in a manner that is related to GTPase networks that control much of the cell's functions. The organisation of the endolysosomal system may be hierarchical, with proteases being downstream effectors of a system that is regulated at the whole body level. The main endoprotease class within the endolysosomal system are cysteine proteases. A literature review suggested that these enzymes may not be redox regulated within the endolysosomal system. However, the lysosomal endoprotease cathepsin B has been implicated in many pathologies where it is operating in pH and redox conditions different from the endolysosomal system. To study this, cathepsin B was isolated from bovine livers using a novel procedure that exploits the ability of tertiary butanol to temporarily inhibit protease interactions in tissue homogenates. This would prevent artefactual, as well as protease-inhibitor interactions. This novel procedure resulted in increased yields of cathepsin B. Cathepsin D, an aspartic protease, was isolated using established methods. In order to test the hypothesis that cathepsin B may be redox regulated in vivo, cathepsin B activity and stability were measured in cysteine and/or cystine-containing redox buffers. Cathepsin B activity in cysteine-containing buffers was similar at pH 6.0 and pH 7.0, over all thiol concentrations tested. In contrast, the stability of the enzyme was greater at pH 6.0 than at pH 7.0. This suggests that the enzyme's operational pH in vivo may be < pH 7.0. The activity of the enzyme was depressed in glutathione-containing buffers. When assessed in cysteine:cystine redox buffers (pH 6.0 - 7.0) cathepsin B was active over a broad redox potential range, suggesting that cathepsin B activity may not be redox regulated.
The glutaredoxin/glutathione post-stress recovery system is dependent on the availability of glutathione in the cell.
(2020) Blom, Erin Shelby.; Pillay, Che Sobashkar.
The cellular response to oxidative stress involves three interconnected processes: reactive oxygen species detoxification, adaptation and repair. Glutathionylation is an adaptive response in which glutathione binds to labile proteins protecting them from oxidative damage but also inactivating them. While it has been established that glutaredoxins play a crucial role in deglutathionylating these proteins, the kinetic regulation of this post-stress repair process is less clear. Intriguingly, aged cells have decreased glutathione levels, although the mechanistic significance of this decrease has not been well-understood. We hypothesized that in these cells, the lower glutathione levels reduced the efficiency of the glutaredoxin/glutathione system which impaired the recovery of the cell post-stress. To test this hypothesis, we used a validated computational model of the glutaredoxin/glutathione system to determine how perturbation of the glutaredoxin system affected the availability of active glutaredoxin as well as the rate of deglutathionylation. We separated the effects of the kinetic and thermodynamic components of glutaredoxin activity and found that the overall flux was primarily controlled by the kinetic effects and that the activity of the system was largely dependent on the availability of reduced glutathione. To test whether reduced deglutathionylation activity was a characteristic of aging, aging and glutathione determination experiments were undertaken in the fission yeast, Schizosaccharomyces pombe. In contrast to our hypothesis and data from other studies, fission yeast cells aged for five days were shown to have increased glutathione concentrations, from 36.62 μM to 43.09 μM in minimal media when compared with two-day old cells, except in the presence of additional glutathione or Lbuthionine sulfoximine, a glutathione synthesis inhibitor. Further, glutathionylation levels decreased or remained unchanged in the aged cultures which we speculate was due to an adaptive response by the glutathione synthesis pathway in these cells. Future experiments will need to measure both the glutaredoxin system and the metabolic pathways that provide reductive inputs into the system in order to understand the role of the glutathionylation cycle in post-stress recovery.
Modelling and analysis of peroxiredoxin kinetics for systems biology applications.
(2015) Eagling, Beatrice Demelza.; Pillay, Che Sobashkar.
Oxidative stress, caused by reactive oxygen species (ROS) such as hydrogen peroxide, can have harmful effects on important cellular components and processes which can lead to cell death. Cells have evolved extensive protein and non-protein antioxidant molecules to deal with hydrogen peroxide but it is now clear that hydrogen peroxide is also important signal molecule. It is not fully understood how cells maintain the balance between hydrogen peroxide detoxification and signal transduction. Peroxiredoxins are a ubiquitous family of antioxidant proteins that are the primary reductants of hydrogen peroxide and appear to be key molecules in mediating this balance. Using catalytic cysteines, peroxiredoxins reduce hydrogen peroxide and other ROS and in turn are reduced by thioredoxin and thioredoxin reductase. This coupled set of reactions collectively constitute the peroxiredoxin system and its precise role in redox signalling could be established using systems biology studies. However, there are some discrepancies on how peroxiredoxins should be described in these studies as three distinct kinetic models have been proposed for peroxiredoxin activity: the ping-pong enzyme, redox couple monomer and redox couple homodimer models. Further, different rate constants for hydrogen peroxide reduction by peroxiredoxins have been reported using steady state and competition assays and it is not clear which of these parameters should be used in computational models. In order to resolve these discrepancies, the three proposed peroxiredoxin kinetic models were simulated with core parameters and showed different responses to parameter changes. Computational modelling with in vitro datasets confirmed this result and also showed that many of the reported peroxiredoxin kinetic parameters have limited predictive value. Thus, the kinetic models for peroxiredoxin activity cannot be used interchangeably and computational models based on the reported peroxiredoxin kinetic parameters for hydrogen peroxide reduction should be viewed with caution. To confirm this result, the cytosolic peroxiredoxin thiolspecific antioxidant 1 (TSA1) from Saccharomyces cerevisiae was cloned, expressed and purified for in vitro analysis of this system. Data fitting of the peroxiredoxin kinetic models determined parameters that were able to predict independent datasets with increasing thioredoxin and peroxiredoxin concentrations using the ping-pong enzyme and redox couple monomer models but the redox couple homodimer model was unable to fit these datasets. A complex flux control pattern was also determined for the fitted models and whole system fitting to in vitro datasets is proposed to be a more accurate method for parameter determination for the peroxiredoxin system kinetic assays.
p38 MAPK and the C2C12 cell cycle : in vitro and in silico investigations.
(2011) Driscoll, Scott Robert Ellery.; Niesler, Carola Ulrike.; Pillay, Che Sobashkar.
The mammalian cell cycle and its points-of-entry are well characterized pathways. These points-of-entry are normally regulated via mitogens and include, amongst others, the ERK, JNK and p38 mitogen-activated protein kinase (MAPK) pathways. However, while the restriction point(R-point), the temporal switch-point at which a cell becomes irrevocably committed to division irrespective of mitogenic stimulus, is known among other cell types, its position within the murine myoblast line C2C12 is currently unknown. Similarly, while MAPK pathways, such as JNK and ERK, have been modeled computationally, no model yet exists of p38 MAPK as stimulated by mitogens. The aims of this dissertation, then, were to determine the R-point within the C2C12 cell cycle and construct a computational mitogen-stimulated p38 MAPK model. It was found that a synchronous C2C12 population, when stimulated to divide, took 7 to 9 hours to reach S-phase from G0, consistent with data from the literature. The R-point was determined to lie between 6 and 7 hours post G1-re-entry stimulation,which was consistent with studies in other cell types. Core modeling of the p38 MAPK pathway revealed that ultrasensitivitywas inherent within the pathway structure. Further, a branching/re-converging structure within the pathway imparted greater responsiveness to signal upon the pathway. A realistic p38 MAPK model demonstrated good responsiveness to signal, its output matched that of several other MAPK models, and it was capable of replicating previous in vitro data. This model can be used as a tool for further investigation of the mammalian cell cycle by linking it to other cell cycle models. The predictions by an expanded model may be better suited for understanding the effects of mitogen stimulus on the cell cycle in situ.
Quantification of the thioredoxin system.
(2017) Letrisha, Padayachee.; Pillay, Che Sobashkar.
Abstract available in PDF file.
Redox properties of cathepsin B in relation to its activity in vivo.
(1999) Pillay, Che Sobashkar.; Dennison, Clive.
The main site for protein degradation along the endosomal pathway is believed to be the late endosome. Lysosomes are thought to be storage organelles that, when necessary, inject proteases into the late endosome. It was hypothesised that differences in the lumenal redox environments between the two organelles could be responsible for their functional differences. In an attempt to quantify this potential difference, the lysosomal cysteine protease cathepsin B was isolated by an improved purification procedure. Several intracellular reducing agents were used to activate cathepsin B, the most effective being cysteine. Cysteine was used to activate cathepsin B under various pH conditions in order to model endosomal conditions. An inverse relationship was found between the pH and the concentration of cysteine required to activate cathepsin B. This suggested that cathepsin B may have an optimal redox potential. In order to determine this potential, cysteinexystine redox buffers were made up and used in determination of the activity of the enzyme against a synthetic and a whole protein substrate (haemoglobin). No distinct redox potential could be determined using either substrate, but it was found that cystine stimulated proteolysis of haemoglobin. A similar stimulatory effect was observed for cathepsin D and papain hydrolysis of haemoglobin. This effect is possibly due to the ability of cystine to promote substrate structure, effectively increasing the substrate concentration. These findings and other results obtained from the literature have been used to create a model of how proteolysis may be regulated along the endosomal system.
Regulation of the thioredoxin system in Saccharomyces cerevisiae.
(2013) Padayachee, Letrisha.; Pillay, Che Sobashkar.
The thioredoxin system consisting of thioredoxin (Trx), thioredoxin reductase and NADPH plays a significant role in a large number of redox-dependent processes such as DNA synthesis and anti-oxidant defense. Elevated levels of this system have been associated with a number of diseases including cancer and HIV. Understanding the regulation of this network from a systems perspective is therefore essential. However, contradictory descriptions of thioredoxin as both an enzyme and redox couple have stifled the adoption of systems biology approaches within the field. Using kinetic modeling, this discrepancy was resolved by proposing that saturation of Trx activity could be due to the saturation of the Trx redox cycle which consequently allowed development of the first computational models of the thioredoxin system in Jurkat T-cells and Escherichia coli. While these models successfully described the network properties of the thioredoxin system in these organisms, further confirmatory studies were required before this modeling approach could be generally accepted. The aim of this study was to utilize computational and molecular methods to confirm or reject this proposed mechanism for thioredoxin activity. To determine if there is any difference in the kinetic models obtained when thioredoxin was modeled as an enzyme or as a redox couple, representative core models were developed. The data showed that when modeling Trx as a redox couple, the system was able to achieve steady state, there was a re-distribution of Trx into its oxidized form and, thioredoxin reductase affected the rates within the system. On the other hand, when Trx was modeled as an enzyme, the system could not reach a steady state, Trx remained in the reduced form and thioredoxin reductase concentration had no effect on the rates within the system. As these properties could be directly tested invitro, we sought to directly confirm which model was correct. The thioredoxin system from Saccharomyces cerevisiae was cloned, expressed and purified and substrate saturation curves were generated using insulin as a model substrate. The data showed that the system reached steady state and with increasing concentrations of insulin, the system saturated with a progressive re-distribution of the thioredoxin moiety into its oxidized form. Further, increasing the thioredoxin reductase concentration increased the flux through the system. Collectively, the results obtained through invitro analyses provided unambiguous support for the thioredoxin redox couple model. These results will enable the construction of a complete computational model of the yeast thioredoxin system and provide a basis for the analysis of this network in a number of pathologies.
The role of thioredoxin in the redox regulation of the Tpx1/Pap1 pathway in Schizosaccharomyces pombe.
(2022) Naidoo, Kelisa Cheyen.; Pillay, Che Sobashkar.
Reactive oxygen species (ROS) can damage cellular components leading to dysfunction and cell death. Paradoxically, ROS, such as hydrogen peroxide, are also essential for a range of metabolic and signalling functions within cells. Given these opposing functions, cells have developed several redox signalling mechanisms to manage ROS within specific homeostatic limits. In bacterial cells, thiol-peroxidases (peroxiredoxins) and other enzymes detoxify ROS, while the antioxidant transcriptional response is induced by transcription factors directly oxidized by ROS. In many eukaryotes, these functions are combined with peroxiredoxins detoxifying ROS as well as activating redox-sensitive transcription factors. The relative benefits and disadvantages of such sensor-mediated redox signalling systems are unknown, and we aimed to understand the logic underlying this signalling mechanism using the Schizosaccharomyces pombe Tpx1/Pap1 pathway. In this pathway, the peroxiredoxin Tpx1 reduces hydrogen peroxide and oxidizes the redox transcription factor Pap1. Following a hydrogen peroxide perturbation, the Pap1 signal profile revealed a biphasic profile with a rapid initial increase followed by a relatively prolonged decrease in Pap1 oxidation. These dynamics were suggestive of an incoherent feedforward loop, and we hypothesized that the Trx1 protein was responsible for the incoherence as it could both dampen and increase the signal by reducing Pap1 and Tpx1, respectively. To test this hypothesis, we analyzed the effect of several oxidants (hydrogen peroxide, tert-butyl hydroperoxide, and diamide) on Pap1 activation to determine if we could selectively modulate signal duration. However, we could not quantitatively delineate the effects of these oxidants on the signal profiles obtained. We, therefore, utilized computational modelling to analyze the Tpx1/Pap1 pathway and found that excess Trx1 reduced Tpx1 faster, preventing the association of Tpx1 and Pap1. On the other hand, insufficient Trx1 allowed for Pap1 to be oxidized over a longer interval which increased the signal duration. Thus, our analysis showed that, in contrast to our hypothesis, Trx1 limitation, rather than incoherence, was responsible for the Pap1 oxidation profile. These results indicate that in the presence of ROS, Trx1 plays a vital role in determining the signal profile of Pap1.
Structural analysis of the mycobacterium tuberculosis redox defence network reveals a unique bi-fan motif design associated with hydrogen peroxide reduction.
(2017) John, Nolyn.; Pillay, Che Sobashkar.; Rohwer, Johann M.
Abstract available in PDF file.
The thioredoxin redox charge as a measure of cell redox homeostasis in Schizosaccharomyces pombe.
(2022) Bhagwandeen, Tejal.; Pillay, Che Sobashkar.
Thiol-based redox systems play essential roles in repairing oxidatively damaged proteins, deoxyribonucleotide synthesis, sulfur metabolism, protein folding, and oxidant detoxification and signaling. The principal thiol systems in most cells are the thioredoxin (Trx) and the glutathione/glutaredoxin (GSH/Grx) systems. In the thioredoxin system, reducing equivalents from NADPH are transferred by thioredoxin reductase to thioredoxin, resulting in reduced thioredoxin. Thioredoxin in the reduced form further reduces target proteins and is itself consequently oxidized. Given the system’s essential role in cellular physiology, inhibition of the thioredoxin system is an important drug target for communicable and non-communicable diseases. However, measuring the activity of the thioredoxin system in vivo is challenging. The thioredoxin redox charge (reduced thioredoxin/total thioredoxin) was proposed as a novel, surrogate measure of the thioredoxin system’s activity and could be used as a general measure of the cellular redox state. Indeed, published data showed that the thioredoxin redox charge and cell viability collapsed if a chemical inhibitor directly targeted thioredoxin reductase. To evaluate the utility of the thioredoxin redox charge as a generic indicator of redox stress, the fission yeast Schizosaccharomyces pombe, was subjected to various stressors including hydrogen peroxide, heat, cadmium sulfate and potassium ferricyanide and the thioredoxin redox charge and cell viability were measured over time. We found dynamic changes in the thioredoxin redox charge profiles, in response to these stressors, but only obtained weak, positive correlations between the thioredoxin redox charge and cell viability. Thus, and in contrast to our initial hypothesis, the thioredoxin redox charge appeared to be buffered in response to high-stress perturbations, even when cell viability was clearly inhibited. These results show that the redox poise of the thioredoxin system can presumably only be disrupted by direct inhibitors of the system. Future work should aim to elucidate the mechanisms underlying the preservation of the thioredoxin redox charge.