Protein disulfide isomerases PDIs

The hydroxylation of specific Pro residues in procollagen, the precursor of collagen, requires the action of the enzyme prolyl 4-hydroxylase. This enzyme (Mt 240,000) is an a2/32 tetramer in all vertebrate sources. The proline-hydroxylating activity is found in the a subunits. (Researchers were surprised to find that the )3 subunits are identical to the enzyme protein disulfide isomerase (PDI p. 152) these subunits do not participate in the prolyl hydroxylation activity.) Each a subunit contains one atom of nonheme iron (Fe2+), and the enzyme is one of a class of hydroxylases that require a-ketoglutarate in their reactions. 

Finally, the folding pathways of a number of proteins require two enzymes that catalyze isomerization reactions. Protein disulfide isomerase (PDI) is a... 

Protein disulfide isomerase (PDI) was found to be important in catalyzing disulfide bond formation of antibody fragments, and it improved the efficiency of E. coli ribosome display of antibodies threefold when used during the in vitro translation reaction (Hanes and Pluckthun, 1997) (see also Section II, B). A fourfold improvement of ribosome display was observed when 1 OSa-RNA, which is involved in degradation of truncated proteins (see Section III, B, 2), was inhibited by using an antisense DNA oligonucleotide directed against the lOSa-RNA (Hanes and Pluckthun, 1997). 

Several of the identified proteins were known to be involved in signal transduction and platelet function, e.g., cortactin, myosin regulatory Hght chain (myosin RLG), and protein disulfide isomerase (PDI). 

Reoxidation of pro-a chains of types I and II procollagen into triple helical procollagen was studied using various concentrations of reduced (GSH) and oxidized (GSSG) glutathione as an oxidizing system in the reaction mixture. Pure protein disulfide-isomerase (PDI 5ng) was used in the reaction. 

Shuffle lest. An enzyme that catalyzes disulfidc-aiilfhydryl exchange reactions, called protein disulfide isomerase (PDI), has been isolated. PfM rapidly converts inactive scrambled ri bonuclcase into enzymatically active ribonucleasc. In contrast, insulin is rapidly inactivated by PDI. What does this important observation imply about the relation between the amino acid sequence ol insulin and its three-dimensional structure ... 

Disulfide bonds play an important part in the structure and stabilization of peptides and proteins. They are usually formed by spontaneous oxidation of the corresponding sulfide, but the reaction is catalyzed by a family of ubiquitous proteins called protein disulfide isomerases (PDIs) through a complex sequence of reactions that involves many partially folded intermediates [19]. This explains how the unfavored cisoid conformation may be represented for a given disulfide bond [20] though the transoid and trans isomers are much more frequently observed, as are intermediary rotamers [21]. 

A pathway analogous to the DsbA-DsbB system was recently shown to operate in the endoplasmic reticulum of eukaryotes (Frand and Kaiser, 1998 1999 Pollard et al., 1998). This pathway includes the membrane-bound protein EROl, which keeps protein disulfide isomerase (PDI) in an oxidized state in vivo. Consequently, PDI plays a role somewhat similar to that of DsbAin catalyzing the formation of disulfide bonds during the folding of secreted proteins. The mechanisms driving oxidative protein folding in eukaryotes are described in more detail in another chapter in this volume. 

The efficient formation of disulfide bonds In the lumen of the ER depends on the enzyme protein disulfide isomerase (PDI), which Is present In all eukaryotic cells. This enzyme Is especially abundant In the ER of secretory cells In such organs as the liver and pancreas, where large quantities of proteins that contain disulfide bonds are produced. As shown in Figure 16-19a, the disulfide bond in the active site of PDI can be readily transferred to a protein by two sequential thiol-disulfide transfer reactions. The reduced PDI generated by this reaction is returned to an oxidized form by the action of an ER-resident protein, called Erol, which carries a disulfide bond that can be transferred to PDI. It is not yet understood how Erol itself becomes oxidized. Figure 16-20 depicts the organization of the pathway for protein disulfide-bond formation In the ER lumen and the analogous pathway In bacteria. 

A FIGURE 16-19 Formation and rearrangement of disulfide bonds by protein disulfide isomerase (PDI). PDI contains an active site with two ciosely spaced cysteine residues that are easily interconverted between the reduced dithiol form and the oxidized disulfide form. Numbered red arrows indicate the sequence of electron transfers. Yellow bars represent disulfide bonds, (a) In the formation of disulfide bonds, the ionized (-S ) form of a cysteine thiol in the substrate protein reacts with the disulfide (S—S) bond in oxidized PDI to form a disulfide-bonded... 

Disulfide bonds are added to many secretory proteins and the exoplasmic domain of membrane proteins in the ER. Protein disulfide Isomerase (PDI), present in the ER lumen, catalyzes both the formation and the rearrangement of disulfide bonds (see Figure 16-19). 

Protein disulfide isomerase (PDI), a homo-dimeric protein (Mr 110 kDa) widely distributed across eukaryotic cells. PDI catalyzes the isomerization and rearrangement of disulfide bonds in the endoplasmic reticulum. Furthermore, S-denitrosation activity of PDI has been described. Recently, a direct, continuous, sensitive assay of PDI based on fluorescence self quenching has been described [B. Wflkin-son, H. F. Gilbert, Biochim. Biophys. Acta... 

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