Oxidative folding

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Oxidative protein folding is a process that is responsible for the formation of disulfide bonds between cysteine residues in proteins. The driving force behind this process is a redox reaction, in which electrons pass between several proteins and finally to a terminal electron acceptor.

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In prokaryotes

In prokaryotes, the mechanism of oxidative folding is best studied in Gram-negative bacteria. This process is catalysed by protein machinery residing in the periplasmic space of bacteria. The formation of disulfide bonds in a protein is made possible by two related pathways: an oxidative pathway, which is responsible for the formation of the disulfides, and an isomerization pathway that shuffles incorrectly formed disulfides.

Oxidative pathway

Oxidative pathway in Gram-negative bacteria Oxidative pathway in bacteria (mechanism).jpg
Oxidative pathway in Gram-negative bacteria

The oxidative pathway relies, just like the isomerization pathway, on a protein relay. The first member of this protein relay is a small periplasmic protein (21 kDa) called DsbA, which has two cysteine residues that must be oxidized for it to be active. When in its oxidized state, the protein is able to form disulfide bonds between cysteine residues in newly synthesized, and yet unfolded proteins by the transfer of its own disulfide bond onto the folding protein. After the transfer of this disulfide bond, DsbA is in a reduced state. For it to act catalytically again, it must be reoxidized. This is made possible by a 21 kDa inner membrane protein, called DsbB, which has two pairs of cysteine residues. A mixed disulfide is formed between a cysteine residue of DsbB and one of DsbA. Eventually, this cross-link between the two proteins is broken by a nucleophilic attack of the second cystein residue in the DsbA active site. On his turn, DsbB is reoxidized by transferring electrons to oxidized ubiquinone, which passes them to cytochrome oxidases, which finally reduce oxygen; this is in aerobic conditions. As molecular oxygen serves as the terminal electron acceptor in aerobic conditions, oxidative folding is conveniently coupled to it through the respiratory chain. In anaerobic conditions however, DsbB passes its electrons to menaquinone, followed by a transfer of electrons to fumarate reductase or nitrate reductase.

Isomerization pathway

Isomerization pathway in Gram-negative bacteria Isomerization pathway in bacteria (mechanism).jpg
Isomerization pathway in Gram-negative bacteria

Especially for proteins that contain more than one disulfide bond, it is important that incorrect disulfide bonds become rearranged. This is carried out in the isomerization pathway by the protein DsbC, that acts as a disulfide isomerase. DsbC is a dimer, consisting of two identical 23 kDa subunits and has four cysteine residues in each subunit. One of these cysteines (Cys-98) attacks an incorrect disulfide in a misfolded protein and a mixed disulfide is formed between DsbC and this protein. Next, the attack of a second cysteine residue results in the forming of a more stable disulfide in the refolded protein. This may be a cysteine residue either from the earlier misfolded protein or one from DsbC. In the last case, DsbC becomes oxidized and must be reduced in order to play another catalytic role. There is also a second isomerase that can reorganize incorrect disulfide bonds. This protein is called DsbG and it is also a dimer that serves as a chaperone. To fulfil their role as isomerases, DsbC and DsbG must be kept in a reduced state. This is carried out by DsbD, which must be reduced itself to be functional. Thioredoxin, which itself is reduced by thioredoxin reductase and NADPH, ensures the reduction of the DsbD protein.

Because these two pathways coexist next to each other in the same periplasmic compartment, there must be a mechanism to prevent oxidation of DsbC by DsbB. This mechanism indeed exists as DsbB can distinguish between DsbA and DsbC because this latter has the ability to dimerize.

In eukaryotes

Process of oxidative folding in eukaryotes Oxidative folding in eukaryotes (mechanism).jpg
Process of oxidative folding in eukaryotes

A very similar pathway is followed in eukaryotes, in which the protein relay consists of proteins with very analogous properties as those of the protein relay in Gram-negative bacteria. However, a major difference between prokaryotes and eukaryotes is found in the fact that the process of oxidative protein folding occurs in the endoplasmatic reticulum (ER) in eukaryotes. A second difference is that in eukaryotes, the use of molecular oxygen as a terminal electron acceptor is not linked to the process of oxidative folding through the respiratory chain as is the case in bacteria. In fact, one of the proteins involved in the oxidative folding process uses a flavin-dependent reaction to pass electrons directly to molecular oxygen.

A homolog of DsbA, called protein disulfide isomerase (PDI), is responsible for the formation of the disulfide bonds in unfolded eukaryotic proteins. This protein has two thioredoxine-like active sites, which both contain two cysteine residues. By transferring the disulfide bond between these two cysteine residues onto the folding protein it is responsible for the latter's oxidation. In contrast to bacteria, where the oxidative and isomerization pathways are carried out by different proteins, PDI is also responsible for the reduction and isomerization of the disulfide bonds. For PDI to catalyse the formation of disulfide bonds in unfolded proteins, it must be reoxidized. This is carried out by an ER membrane-associated protein, Ero1p, which is no homolog of DsbB. This Ero1p protein forms a mixed disulfide with PDI, which is resolved by a nucleophilic attack of the second cystein residue in one of the active sites of PDI. As result, oxidized PDI is obtained. Ero1p itself is oxidized by transferring electrons to molecular oxygen. As it is an FAD-binding protein, this transfer of electrons is strongly favoured when Ero1p is bound to FAD. Also a transport system that imports FAD into the ER lumen has been described in eukaryotes. Furthermore, it has been shown that the ability to reduce or rearrange incorrect disulfide bonds in missfolded proteins is provided by the oxidation of reduced glutathione (GSH) to oxidized glutathione (GSSG).

ROS and diseases

Because of the property of Ero1p to transfer electrons directly to molecular oxygen via a flavin-dependent reaction, its activity may produce reactive oxygen species (ROS). In bacteria, this problem is solved by coupling oxidative folding to the respiratory chain. There, the reduction of molecular oxygen to water is carried out by a complex series of proteins, which catalyse this reaction very efficiently. In eukaryotes, the respiratory chain is separated from oxidative folding since cellular respiration takes place in the mitochondria and the formation of disulfide bonds occurs in the ER. Because of this, there is much more risk that ROS are produced in eukaryotic cells during oxidative folding. As is known these ROS may cause many diseases such as atherosclerosis and some neurodegenerative diseases.

Examples

Classical examples of proteins in which the process of oxidative folding is well studied are bovine pancreatic trypsin inhibitor (BPTI) and ribonuclease A (RNaseA). These two proteins have multiple disulfide bonds and so they are very useful to follow and understand the process of oxidative folding. Another example is alkaline phosphatase, which contains two essential disulfides. It was used as an indicator protein to screen the effect of mutations in DsbA.

Related Research Articles

Oxidative phosphorylation The phosphorylation of ADP to ATP that accompanies the oxidation of a metabolite through the operation of the respiratory chain. Oxidation of compounds establishes a proton gradient across the membrane, providing the energy for ATP synthesis.

Oxidative phosphorylation or electron transport-linked phosphorylation or terminal oxidation is the metabolic pathway in which cells use enzymes to oxidize nutrients, thereby releasing the chemical energy stored within the nutrients in order to produce adenosine triphosphate (ATP). In eukaryotes, this takes place inside mitochondria. Almost all aerobic organisms carry out oxidative phosphorylation. This pathway is so pervasive because it releases more energy than alternative fermentation processes such as anaerobic glycolysis.

Cysteine Amino acid

Cysteine (symbol Cys or C; ) is a semiessential proteinogenic amino acid with the formula HOOC-CH-(NH2)-CH2-SH. The thiol side chain in cysteine often participates in enzymatic reactions as a nucleophile. The thiol is susceptible to oxidation to give the disulfide derivative cystine, which serves an important structural role in many proteins. When used as a food additive, it has the E number E920. It is encoded by the codons UGU and UGC.

In biochemistry, a disulfide refers to a functional group with the structure R−S−S−R′. The linkage is also called an SS-bond or sometimes a disulfide bridge and is usually derived by the coupling of two thiol groups. In biology, disulfide bridges formed between thiol groups in two cysteine residues are an important component of the secondary and tertiary structure of proteins. Persulfide usually refers to R−S−S−H compounds.

Protein disulfide-isomerase

Protein disulfide isomerase, or PDI, is an enzyme in the endoplasmic reticulum (ER) in eukaryotes and the periplasm of bacteria that catalyzes the formation and breakage of disulfide bonds between cysteine residues within proteins as they fold. This allows proteins to quickly find the correct arrangement of disulfide bonds in their fully folded state, and therefore the enzyme acts to catalyze protein folding.

Flavin adenine dinucleotide Redox-active coenzyme

In biochemistry, flavin adenine dinucleotide (FAD) is a redox-active coenzyme associated with various proteins, which is involved with several enzymatic reactions in metabolism. A flavoprotein is a protein that contains a flavin group, which may be in the form of FAD or flavin mononucleotide (FMN). Many flavoproteins are known: components of the succinate dehydrogenase complex, α-ketoglutarate dehydrogenase, and a component of the pyruvate dehydrogenase complex.

Photosynthetic reaction centre

A photosynthetic reaction center is a complex of several proteins, pigments and other co-factors that together execute the primary energy conversion reactions of photosynthesis. Molecular excitations, either originating directly from sunlight or transferred as excitation energy via light-harvesting antenna systems, give rise to electron transfer reactions along the path of a series of protein-bound co-factors. These co-factors are light-absorbing molecules such as chlorophyll and phaeophytin, as well as quinones. The energy of the photon is used to excite an electron of a pigment. The free energy created is then used to reduce a chain of nearby electron acceptors, which have progressively higher redox-potentials. These electron transfer steps are the initial phase of a series of energy conversion reactions, ultimately resulting in the conversion of the energy of photons to the storage of that energy by the production of chemical bonds.

Copper proteins are proteins that contain one or more copper ions as prosthetic groups. Copper proteins are found in all forms of air-breathing life. These proteins are usually associated with electron-transfer with or without the involvement of oxygen (O2). Some organisms even use copper proteins to carry oxygen instead of iron proteins. A prominent copper proteins in humans is in cytochrome c oxidase (cco). The enzyme cco mediates the controlled combustion that produces ATP.

ER oxidoreductin

ER oxidoreductin 1 (Ero1) is an oxidoreductase enzyme that catalyses the formation and isomerization of protein disulfide bonds in the endoplasmic reticulum (ER) of eukaryotes. ER Oxidoreductin 1 (Ero1) is a conserved, luminal, glycoprotein that is tightly associated with the ER membrane, and is essential for the oxidation of protein dithiols. Since disulfide bond formation is an oxidative process, the major pathway of its catalysis has evolved to utilise oxidoreductases, which become reduced during the thiol-disulfide exchange reactions that oxidise the cysteine thiol groups of nascent polypeptides. Ero1 is required for the introduction of oxidising equivalents into the ER and their direct transfer to protein disulfide isomerase (PDI), thereby ensuring the correct folding and assembly of proteins that contain disulfide bonds in their native state.

PDIA3

Protein disulfide-isomerase A3 (PDIA3), also known as glucose-regulated protein, 58-kD (GRP58), is an isomerase enzyme. This protein localizes to the endoplasmic reticulum (ER) and interacts with lectin chaperones calreticulin and calnexin (CNX) to modulate folding of newly synthesized glycoproteins. It is thought that complexes of lectins and this protein mediate protein folding by promoting formation of disulfide bonds in their glycoprotein substrates.

Maleate isomerase

In enzymology, a maleate isomerase, or maleate cis-tran isomerase, is a member of the Asp/Glu racemase superfamily discovered in bacteria. It is responsible for catalyzing cis-trans isomerization of the C2-C3 double bond in maleate to produce fumarate, which is a critical intermediate in citric acid cycle. The presence of an exogenous mercaptan is required for catalysis to happen.

P4HB

Protein disulfide-isomerase, also known as the beta-subunit of prolyl 4-hydroxylase (P4HB), is an enzyme that in humans encoded by the P4HB gene. The human P4HB gene is localized in chromosome 17q25. Unlike other prolyl 4-hydroxylase family proteins, this protein is multifunctional and acts as an oxidoreductase for disulfide formation, breakage, and isomerization. The activity of P4HB is tightly regulated. Both dimer dissociation and substrate binding are likely to enhance its enzymatic activity during the catalysis process.

Disulfide bond formation protein B

Disulfide bond formation protein B (DsbB) is a protein component of the pathway that leads to disulfide bond formation in periplasmic proteins of Escherichia coli and other bacteria. In Bacillus subtilis it is known as BdbC.

Bacterial glutathione transferase

Bacterial glutathione transferases are part of a superfamily of enzymes that play a crucial role in cellular detoxification. The primary role of GSTs is to catalyze the conjugation of glutathione (GSH) with the electrophilic centers of a wide variety of molecules. The most commonly known substrates of GSTs are xenobiotic synthetic chemicals. There are also classes of GSTs that utilize glutathione as a cofactor rather than a substrate. Often these GSTs are involved in reduction of reactive oxidative species toxic to the bacterium. Conjugation with glutathione receptors reders toxic substances more soluble, and therefore more readily exocytosed from the cell.

DsbA

DsbA is a bacterial thiol disulfide oxidoreductase (TDOR). DsbA is a key component of the Dsb family of enzymes. DsbA catalyzes intrachain disulfide bond formation as peptides emerge into the cell's periplasm.

Thioredoxins are small disulfide-containing redox proteins that have been found in all the kingdoms of living organisms. Thioredoxin serves as a general protein disulfide oxidoreductase. It interacts with a broad range of proteins by a redox mechanism based on reversible oxidation of 2 cysteine thiol groups to a disulfide, accompanied by the transfer of 2 electrons and 2 protons. The net result is the covalent interconversion of a disulfide and a dithiol.

DsbC protein family

DsbC is a prokaryotic disulfide bond isomerase. The formation of native disulfide bonds play an important role in the proper folding of proteins and stabilize tertiary structures of the protein. DsbC is one of 6 proteins in the Dsb family in prokaryotes. The other proteins are DsbA, DsbB, DsbD, DsbE and DsbG. These enzymes work in tandem with each other to form disulfide bonds during the expression of proteins. DsbC and DsbG act as proofreaders of the disulfide bonds that are formed. They break non-native disulfide bonds that were formed and act as chaperones for the formation of native disulfide bonds. The isomerization of disulfide bonds occurs in the periplasm.

Ferredoxin-thioredoxin reductase

Ferredoxin-thioredoxin reductase EC 1.8.7.2, systematic name ferredoxin:thioredoxin disulfide oxidoreductase, is a [4Fe-4S] protein that plays an important role in the ferredoxin/thioredoxin regulatory chain. It catalyzes the following reaction:

Oxidation response is stimulated by a disturbance in the balance between the production of reactive oxygen species and antioxidant responses, known as oxidative stress. Active species of oxygen naturally occur in aerobic cells and have both intracellular and extracellular sources. These species, if not controlled, damage all components of the cell, including proteins, lipids and DNA. Hence cells need to maintain a strong defense against the damage. The following table gives an idea of the antioxidant defense system in bacterial system.

The Disulfide bond oxidoreductase D (DsbD) family is a member of the Lysine Exporter (LysE) Superfamily. A representative list of proteins belonging to the DsbD family can be found in the Transporter Classification Base.

PDIA2

Protein disulfide isomerase family A member 2 is a protein that in humans is encoded by the PDIA2 gene.

References

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