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Background

The thioredoxin system consisting of NADP(H), thioredoxin reductase and thioredoxin provides reducing equivalents to a large and diverse array of cellular processes. Despite a great deal of information on the kinetics of individual thioredoxin-dependent reactions, the kinetic regulation of this system as an integrated whole is not known. We address this by using kinetic modeling to identify and describe kinetic behavioral motifs found within the system.
Results

Analysis of a realistic computational model of the Escherichia coli thioredoxin system revealed several modes of kinetic regulation in the system. In keeping with published findings, the model showed that thioredoxin-dependent reactions were adaptable (i.e. changes to the thioredoxin system affected the kinetic profiles of these reactions). Further and in contrast to other systems-level descriptions, analysis of the model showed that apparently unrelated thioredoxin oxidation reactions can affect each other via their combined effects on the thioredoxin redox cycle. However, the scale of these effects depended on the kinetics of the individual thioredoxin oxidation reactions with some reactions more sensitive to changes in the thioredoxin cycle and others, such as the Tpx-dependent reduction of hydrogen peroxide, less sensitive to these changes. The coupling of the thioredoxin and Tpx redox cycles also allowed for ultrasensitive changes in the thioredoxin concentration in response to changes in the thioredoxin reductase concentration. We were able to describe the kinetic mechanisms underlying these behaviors precisely with analytical solutions and core models.
Conclusions

Using kinetic modeling we have revealed the logic that underlies the functional organization and kinetic behavior of the thioredoxin system. The thioredoxin redox cycle and associated reactions allows for a system that is adaptable, interconnected and able to display differential sensitivities to changes in this redox cycle. This work provides a theoretical, systems-biological basis for an experimental analysis of the thioredoxin system and its associated reactions.

Systems biology approaches, such as kinetic modelling, could provide valuable insights into how thioredoxins, glutaredoxins and peroxiredoxins (here collectively called redoxins), and the systems that reduce these molecules are regulated. However, it is not clear whether redoxins should be described as redox couples (with redox potentials) or as enzymes (with Michaelis–Menten parameters) in such approaches. We show that in complete redoxin systems, redoxin substrate saturation and other purported enzymatic behaviours result from limitations in the redoxin redox cycles in these systems. Michaelis–Menten parameters are therefore inappropriate descriptors of redoxin activity; data from redoxin kinetic experiments should rather be interpreted in terms of the complete system of reactions under study. These findings were confirmed by fitting kinetic models of the thioredoxin and glutaredoxin systems to in vitro datasets. This systems approach clarifies the inconsistencies with the descriptions of redoxins and emphasizes the roles of redoxin systems in redox regulation.

The endolysosomal system comprises a unique environment for proteolysis, which is regulated in a manner that apparently does not involve protease inhibitors. The system comprises a series of membrane-bound intracellular compartments, within which endocytosed material and redundant cellular components are hydrolysed. Endocytosed material tends to flow vectorially through the system, proceeding through the early endosome, the endosome carrier vesicle, the late endosome and the lysosome. Phagocytosis and autophagy provide alternative entry points into the system. Late endosomes, lysosome/late endosome hybrid organelles, phagosomes and autophagosomes are the principal sites for proteolysis. In each case, hydrolytic competence is due to components of the endolysosomal system, i.e. proteases, lysosome-associated membrane proteins, H+-ATPases and possibly cysteine transporters. The view is emerging that lysosomes are organelles for the storage of hydrolases, perhaps in an inactivated form. Once a substrate has entered a proteolytically competent environment, the rate-limiting proteolytic steps are probably effected by cysteine endoproteinases. As these are affected by pH and possibly redox potential, they may be regulated by the organelle luminal environment. Regulation is probably also affected, among other factors, by organelle fusion reactions, whereby the meeting of enzyme and substrate may be controlled. Such systems would permit simultaneous regulation of a number of unrelated hydrolases.

In order to test the hypothesis that the lysosomal cysteine protease cathepsin B may be redox regulated in vivo, cathepsin B activity and stability were measured in cysteine- and/or cystine-containing 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. However, at pH 7.0, the stability of cathepsin B decreased with increasing reduction potential and ambient cystine concentration. This suggests that the stability of the enzyme at neutral pH is dependent on redox potential, and on the presence of oxidising agents.