Design of artificial proteins containing unnatural amino acids, backbone linkages or cofactors have also been reported, making it possible to prepare proteins with structural and functional properties beyond those of native proteins.
* Techniques are available that allow the incorporation of unnatural moieties, such as unnatural amino acids, backbone linkages or cofactors, into proteins.
An important function of GSH is in phase II detoxification, in which GSH is conjugated with xenobiotics and metabolic by-products in reactions catalyzed by glutathione S-transferases , . We report here that mRNA levels of GstD1, a known antioxidant and detoxification response gene in Drosophila, , is expressed rhythmically in heads of wild type flies. This is consistent with previous microarray-based analyses which suggested that GstD1 and several other GSTs are expressed rhythmically in the adult Drosophila head , , . Interestingly, GstD1 expression peaks in mid-day, when GSH levels become significantly reduced (compare and ). Other GSTs also peak at this time , suggesting a scenario where GSH is depleted due to conjugation and then replenished later in the circadian cycle. It has been hypothesized that the clock may coordinate redox responses as part of a strategy to increase the potential for neutralization of toxins during the morning when flies are active . In agreement with this view, we showed that the circadian clock regulates susceptibility to pesticides as well as expression of specific genes that control xenobiotic metabolism , .
Rhythm in GSH biosynthesis could be important for many aspects of clock-controlled cellular homeostasis since this prevalent endogenous compound acts as a major antioxidant, regulates activity of detoxification enzymes, and mediates redox-sensitive signaling. GSH functions in the central nervous system also include maintenance of neurotransmitters, and membrane protection , . Our previous study suggested that ROS and oxidative damage levels fluctuate in heads of wild type flies raising a possibility that GSH rhythms may be linked to these phenotypes. However, the mechanism remains to be elucidated as GSH does not directly react with peroxides. The removal of hydrogen peroxide and other peroxides occurs in high-turnover reactions catalyzed by glutathione peroxidases and peroxiredoxins –. Interestingly, some of these enzymes display circadian oxidation-reduction cycles in model organisms across phyla, including Drosophila.
What is the biological advantage of adding a circadian level of regulation to GSH biosynthesis? While excessive ROS levels are detrimental to cell function, some levels of ROS are necessary in the organism, as these molecules are responsible for essential processes including cell signaling cascades and immune response. Thus, GSH acts not only as an antioxidant, but also plays a critical role in a plethora of redox-sensitive cellular functions (reviewed in ). While over-expression of GCLc in Drosophila neuronal tissue, and thus increased GSH levels, correlated with protection against oxidative stress and extension of lifespan , , recent findings suggests that GSH may rather function via affecting specific metabolic and defense pathways . An array of connections has been recently established between circadian clocks and metabolism in mammals , , and in flies . Our present study adds an important novel link to this array by demonstrating circadian control of glutathione, a compound that is critically involved in maintaining human health.
One of the hallmarks of cancer is the ability to generate and withstand unusual levels of oxidative stress. In part, this property of tumor cells is conferred by elevation of the cellular redox buffer glutathione. Though enzymes of the glutathione synthesis and salvage pathways have been characterized for several decades, we still lack a comprehensive understanding of their independent and coordinate regulatory mechanisms. Recent studies have further revealed that overall central metabolic pathways are frequently altered in various tumor types, resulting in significant increases in biosynthetic capacity, and feeding into glutathione synthesis. In this review, we will discuss the enzymes and pathways affecting glutathione flux in cancer, and summarize current models for regulating cellular glutathione through both de novo synthesis and efficient salvage. In addition, we examine the integration of glutathione metabolism with other altered fates of intermediary metabolites, and highlight remaining questions about molecular details of the accepted regulatory modes.
Organic acids (lactic, glutaminic, citric) form complexes with some toxicants. Alkaline earths and rare earths, as well as some heavy elements in the form of cations, are complexed also with organic oxy- and amino acids. All these complexes are usually diffusible and easily distributed in tissues and organs.
In addition to cellular detoxification, members of MAPEG (membrane-associated proteins in eicosanoid and glutathione metabolism) family, which are distantly related to glutathione transferases, contribute to eicosanoid biosynthesis (). Prostaglandin E synthase converts prostaglandin H2 to prostaglandin E2, using GSH as a cofactor. Leukotriene C4 synthase adds GSH to leukotriene A4 to produce leukotriene C4 (; ; ). The γ-glutamyl peptide bond of the attached glutathione is cleaved by another glutathione metabolic enzyme, γ-glutamyltranspeptidase (GGT5), to generate leukotriene D4 (). Glutathione is also used in the storage and transport of cysteine (). A different isozyme of γ-glutamyltranspeptidase (GGT1) initiates the enzymatic cleavage of extracellular glutathione, leading to the degradation of glutathione to its component amino acids (). Cysteine derived from extracellular glutathione is then imported into the cell and used for protein and intracellular glutathione production (discussed below).
This study advanced our understanding of the effects of circadian clocks on cellular homeostasis. We found that the circadian system regulates de novo synthesis of glutathione by direct transcriptional control of the genes encoding GCL subunits, as well as modulation of the activity of the GCL holoenzyme and hence, its end-point product, GSH. Given the conserved nature of the circadian clock and that many metabolites linked to redox show diurnal oscillations in mammals , the molecular connections we established here between the circadian clock and GSH biosynthesis may be conserved across different phyla.
Incorporation of unnatural amino acids and non-native metal cofactors into proteins is an emerging field in the area of protein design, as it offers the tantalizing prospect of introducing new functionality and provides exquisite probes for and fine-tuning of native protein properties.
Discussion of the techniques prominent for incorporation of unnatural amino acids and non-native cofactors is followed by some of the most interesting applications of these techniques reported to date.
Although many of the sites have been modified with success, the relative paucity of functional group availability within proteinogenic amino acids can sometimes leave open questions about specific functions of the metal binding ligands.