| The significance of the domains of protein disulfide isomerase for the different functions of the protein | ||
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Folding is the process whereby newly synthesized polypeptide chains acquire their three-dimensional conformation. Folding can happen spontaneously (Anfinsen 1973), however, uncatalyzed folding is slow and inefficient and therefore two classes of proteins have evolved to assist this event. Molecular chaperones (Ellis 1987) prevent off-pathway reactions, such as aggregation and misfolding, while protein folding catalysts (foldases) act by accelerating rate-limiting steps on the folding pathway (Gething & Sambrook 1992).
The three-dimensional structure of proteins can be stabilized by the formation of disulfide bonds between spatially adjacent cysteine residues. Native disulfide bond formation is not trivial and hence disulfide bonds are generally only found in proteins which pass beyond the carefully regulated intracellular environment, i.e. secreted proteins, outer membrane proteins or proteins which are destined to reside in organelles of the secretory pathway (Helenius et al. 1992). In eukaryotes all such proteins pass through the endoplasmic reticulum (ER) and it is here that native disulfide bonds are formed and the protein folds (Stevens & Argon 1999). Folding takes place even while the newly synthesized polypeptide chain is being translocated into the lumen of the ER (Bergman & Kuehl 1978, 1979), a fact which significantly increases the complexity of studying these essential processes. The primary enzyme responsible for catalyzing the formation of native disulfide bonds in proteins is the ubiquitously expressed enzyme protein disulfide isomerase.
Disulfide bonds in proteins usually serve to stabilize the correctly folded three-dimensional conformation of the protein. For some proteins the stabilization of the native
conformation by disulfide bonds is such that they can be unfolded simply by adding reducing agents into the buffer (Creighton 1986).
The formation of native disulfide bonds between cysteines is a process which requires suitably oxidizing conditions and catalysts. In eukaryotes, these criteria are provided in the lumen of the ER, into which newly produced polypeptide chains destined for the secretory pathway are translocated. The redox environment in the ER is determined by the abundant tripeptide glutathione (Hwang et al. 1992), with the redox potential being dependent on the total glutathione concentration and on the ratio of the reduced (GSH) and oxidized (GSSG) forms (Jones 2002). In the reducing environment of the cytosol the ratio of GSH/GSSG is estimated to be between 30/1 and 100/1, while in the oxidizing ER the GSH/GSSG ratio is about 1/1-3/1 (Hwang et al. 1992). How this ratio is maintained or how the redox potential is regulated is not known. In vitro oxidized glutathione is sufficient to provide oxidizing equivalents for native disulfide bond formation (Lyles & Gilbert 1991) and it has long been the assumption that glutathione would be the source of oxidizing equivalents needed for the catalysis of disulfide bond formation in vivo. However, recent evidence indicates that glutathione may not be directly involved in this pathway (Frand & Kaiser 1998, Cuozzo & Kaiser 1999).
Even though the presence of several oxidants of protein thiol-disulfides in the eukaryotic ER have been known for a long time, from small molecular weight compounds such as glutathione (Hwang et al. 1992) and cysteamine disulfides (Poulsen & Ziegler 1977), to protein catalysts such as the members of the PDI family and sulfhydryl oxidases (Thorpe et al. 2002), the exact mechanism and the flow of oxidizing equivalents is still poorly understood. What is clear is that multiple pathways exist for native disulfide bond formation. For example for protein thiol-disulfide oxidation there is evidence at least for four parallel pathways: Ero1p-mediated oxidation of substrate proteins (Cuozzo & Kaiser 1999, Frand & Kaiser 1999, Tu et al. 2000), oxidation by flavin-dependent sulphydryl oxidases (Suh et al. 1999, Suh & Robertus 2000, Sevier et al. 2001), oxidation by the members of the PDI family of proteins (PDI, ERp57, PDIp, ERp72, P5, ERp44, PDIr, ERdj5/JPDI and ERp18, for references see section 2.2.3.) and oxidation by oxidized glutathione (Hwang et al. 1992). Since both PDI and glutathione may be reoxidized, directly or indirectly, by Ero1p, there is clear potential for functional cross-talk between these pathways.
Ero1p is a membrane-associated ER protein which was originally identified in yeast as an essential protein for the oxidative protein folding process (Frand & Kaiser 1998, Pollard et al. 1998). It was shown that Ero1p has a distinct function from that of PDI and that the Ero1p-mediated pathway for oxidation is independent of the presence of oxidized glutathione (Frand & Kaiser 1998). The roles of Ero1p and PDI were further defined when it was shown that Ero1p can reoxidize PDI (Frand & Kaiser 1999) and that Ero1p and PDI were sufficient for the folding of RNase in vitro in the presence of FAD, which Ero1p strongly binds (Tu et al. 2000). The role of PDI as an oxidant in the Ero1p-PDI catalyzed dithiol-disulfide oxidation cycle was shown by an active-site mutation of PDI, with the second cysteine of both -CXXC- active sites mutated to alanine; -CXXA-. This mutated PDI is not able to catalyse refolding of reduced RNase in the presence of Ero1p. In addition, the mutant PDI in the presence of wild-type PDI and Ero1p inhibits the refolding of reduced RNase, resulting in mutant PDI-Ero1p-mixed disulfides (Tu et al. 2000). These studies potentially answer the question of how PDI is reoxidized after it acts as a protein dithiol-disulfide oxidant, but it still leaves open, what determines the other activities of PDI, such as protein disulfide isomerization.
The Ero1-mediated oxidation pathway is sensitive to cellular levels of free FAD, as high levels of free FAD stimulate the oxidation of protein thiols through Ero1p-PDI cycle and low levels slow, but do not completely halt it (Tu & Weissman 2002). What mechanism is behind this regulation is not known, but metabolic or nutrional control has been suggested by the authors. It is not yet known by what mechanism Ero1p is reoxidized, but the role of molecular oxygen as a final electron acceptor has been established in a recent study (Tu et al. 2002). Glutathione can also appear on the Ero1p pathway, as it was shown to be a substrate for oxidation by Ero1p (Cuozzo & Kaiser 1999). What role glutathione plays in vivo in this system is not known, but it has been suggested that glutathione may act as a buffer against hyperoxidizing conditions in the ER (Cuozzo & Kaiser 1999).
Whatever parallel pathways exist for native disulfide bond formation in proteins, PDIs are the only proteins shown to catalyse late-stage disulfide isomerization reactions, which in vitro (Weissman & Kim 1993) and in vivo (Molinari & Helenius 1999) are rate-limiting steps in folding.