Redox compartmentalization in eukaryotic cells

Abstract

Diverse functions of eukaryotic cells are optimized by organization of compatible chemistries into distinct compartments defined by the structures of lipid-containing membranes, multiprotein complexes and oligomeric structures of saccharides and nucleic acids. This structural and chemical organization is coordinated, in part, through cysteine residues of proteins which undergo reversible oxidation-reduction and serve as chemical/structural transducing elements. The central thiol/disulfide redox couples, thioredoxin-1, thioredoxin-2, GSH/GSSG and cysteine/cystine (Cys/CySS), are not in equilibrium with each other and are maintained at distinct, non-equilibrium potentials in mitochondria, nuclei, the secretory pathway and the extracellular space. Mitochondria contain the most reducing compartment, have the highest rates of electron transfer and are highly sensitive to oxidation. Nuclei also have more reduced redox potentials but are relatively resistant to oxidation. The secretory pathway contains oxidative systems which introduce disulfides into proteins for export. The cytoplasm contains few metabolic oxidases and this maintains an environment for redox signaling dependent upon NADPH oxidases and NO synthases. Extracellular compartments are maintained at stable oxidizing potentials. Controlled changes in cytoplasmic GSH/GSSG redox potential are associated with functional state, varying with proliferation, differentiation and apoptosis. Variation in extracellular Cys/CySS redox potential is also associated with proliferation, cell adhesion and apoptosis. Thus, cellular redox biology is inseparable from redox compartmentalization. Further elucidation of the redox control networks within compartments will improve the mechanistic understanding of cell functions and their disruption in disease.

Figure 1

Mitochondrial Trx2 is more sensitive to oxidants than Trx1. Cells were exposed to arsenic (As), glucose and glutamine depletion (-Glc-Gln), or TNF-α. To provide reduced and oxidized controls, cells were treated with DTT (5 mM) and H₂O₂ (2 mM), respectively. Redox analyses of Trx2 and Trx1 were obtained by redox western blot methods [4, 5]. Briefly, the separation of Trx2 redox forms was obtained by difference in molecular weight due to alkylation of thiol in reduced Trx2 with AMS (4-acetoamido-4-maleimidylstilbene-2,2-disulphonic acid, 500 Da) under non-reduced condition of SDS-PAGE (Fig. 1, top). For Trx1 redox analysis, thiol alkylation of reduced Trx1 by IAA (iodoacetic acid) introduces extra negative charges, thereby giving rise to faster mobility in reduced form than oxidized form under native condition of PAGE (Fig.1, bottom). Using these methods, the results show that Trx2 with 100 µM As [72], Glc and Gln depletion [71], or TNF-α (20 ng/ml) [69] is significantly more oxidized than Trx1 (Fig. 1).

Figure 2

Nuclear redox system is distinct from cytoplasm and is protected from oxidation. A. Redox state of Trx1 shows that oxidation of cytosolic but not nuclear Trx1 occurs in Glc- and Gln-free media [71]. B. Cells transfected with vector control or nuclear-targeted Prx1 (NLS-Prx1) for 24 h were exposed to H₂O₂ for 20 min with indicated amounts. BIAM (biotinylated iodoacetamide) labeling followed by immunoprecipitation for p50 NF-κB was used to determine reduced p50 NF-κB [124]. NLS-Prx1 expression inhibited oxidation of p50 compared with vector control (Mock). C. Cells were transfected with vector control (Mock) or nuclear targeted-D-amino acid oxidase (NLS-DAO) and treated with N-acetyl-D-alanine (NADA, 0 – 1 mM) to stimulate H₂O₂ production in the nuclei of cells [89]. NLS-DAO/NADA-induced ROS caused significant oxidation of the nuclear but not the cytosolic GSH pool, as measured by protein-S-glutathionylation (Pr-SSG).

Figure 3

Independent redox regulation of cellular (cytoplasmic) GSH/GSSG and cytoplasmic Trx1 by EGF, As, and differentiation. Redox states of cellular GSH/GSSG (left, open symbols with solid lines) and Trx1 (right, filled symbols with dashed lines) were measured in cells treated with EGF (circle, [5]) and arsenic (As) (rectangle, [72]), and in cells progressing from proliferation to differentiation (triangle, [137]). EGF treatment caused significant oxidation of cytoplasmic Trx1 redox state (20 mV) but had no effect in GSH/GSSG redox state. Similarly, arsenic oxidized Trx1 but not GSH/GSSG. GSH/GSSG but not Trx1 redox state was significantly oxidized during cellular differentiation.

Figure 4

Extracellular Cys/CySS redox-controlled changes in cellular mechanisms. Cells exposed to Cys/CySS redox-controlled media, varying from most reduced (−150 mV) to most oxidized (0 mV) stimulated monocyte adhesion to endothelial cells (A, [142]), reduced proliferation in colonial epithelial cells, Caco2 (B, [157]), increased cell death due to oxidants (C, [143]), and stimulated fibroblast growth (D, [159]).

Figure 5

Non-equilibrium thermodynamics of biologic redox systems. A recent review of thiol/disulfide systems from the perspective of developing mathematical models for the dynamics of redox regulation identified three important areas for investigation [42]. The insulation of redox systems due to compartmentalization and macromolecular complexes identifies a need to understand redox communication between these compartments/complexes. Because there are many thiol/disulfide elements in proteins and their interaction rates are highly variable, effort is needed to develop a standardized means to define redox pathways. This problem may be addressed by functionally mapping protein cysteines according to their interactions with GSH and thioredoxin systems. Finally, there is a need to address the existence of orthogonal redox control elements. Many proteins and functional pathways have multiple redox-sensitive elements. Separate control by GSH and Trx allow different regulatory events to be controlled independently. Hence, different regulatory mechanisms can be considered to be orthogonal. Several examples are now known for such orthogonal regulation, but additional studies are needed to determine the generality of this type of regulation.