Sh Sh

Protein with incorrect disulfide bonds

▲ FIGURE 16-19 Formation and rearrangement of disulfide bonds by protein disulfide isomerase (PDI). PDI contains an active site with two closely 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

Protein with correct disulfide bonds

Protein with correct disulfide bonds

PDI-substrate protein intermediate. A second ionized thiol In the substrate then reacts with this intermediate, forming a disulfide bond within the substrate protein and releasing reduced PDI. (b) Reduced PDI can catalyze rearrangement of improperly formed disulfide bonds by similar thiol-disulfide transfer reactions. In this case, reduced PDI both initiates and is regenerated in the reaction pathway. These reactions are repeated until the most stable conformation of the protein is achieved. [See M. M. Lyles and H. F. Gilbert, 1991, Biochemistry 30:619.]

formed between cysteines that occur sequentially in the amino acid sequence while a polypeptide is still growing on the ribosome. Such sequential formation, however, sometimes yields disulfide bonds between the wrong cysteines. For example, proinsulin has three disulfide bonds that link cys-teines 1 and 4, 2 and 6, and 3 and 5. In this case, disulfide bonds initially formed sequentially (e.g., between cysteines 1 and 2) have to be rearranged for the protein to achieve its proper folded conformation. In cells, the rearrangement of disulfide bonds also is accelerated by PDI, which acts on a broad range of protein substrates, allowing them to reach their thermodynamically most stable conformations (Figure 16-19b). Disulfide bonds generally form in a specific order, first stabilizing small domains of a polypeptide, then stabilizing the interactions of more distant segments; this phenomenon is illustrated by the folding of influenza HA protein discussed in the next section.

Most proteins used for therapeutic purposes in humans or animals are secreted glycoproteins stabilized by disulfide bonds. When researchers first tried to synthesize such proteins using plasmid expression vectors in bacterial cells, the results were disappointing. In most cases, the proteins were not secreted (even when a bacterial signal sequence replaced the normal one); instead, they accumulated in the cytosol and often in a denatured state, in part owing to the lack of disulfide bonds. After it became clear that disulfide-bond formation occurs spontaneously only in the ER lumen, biotechnologists eventually developed expression vectors that can be used in animal cells (Chapter 9). Nowadays, such vectors and cultured animal cells are preferred for large-scale production of therapeutic proteins such as tissue plasminogen activator (an anticlotting agent) and erythropoietin, a hormone that stimulates production of red blood cells. I

(a) Eukaryotes

(b) Bacteria

(a) Eukaryotes

ER lumen

ER lumen

PDi V

Reduced substrate protein

Reduced substrate protein

wftiv ffllffiillft

▲ FIGURE 16-20 Pathways for the formation of disulfide bonds in eukaryotes and bacteria. Numbered red arrows indicate the sequence of electron flow. A disulfide bond (yellow bar) is formed by loss of a pair of electrons from cysteine thiol (-SH) groups. (a) In the ER lumen of eukaryotic cells, electrons from an ionized thiol in a newly synthesized substrate protein are transferred to a disulfide bond in the active site of PDI (see Figure 16-19). PDI, in turn, transfers electrons to a disulfide bond

(b) Bacteria

Periplasmic space

Periplasmic space v/V

Reduced substrate protein

Inner membrane

Inner membrane

Ubiquinone

In the luminal protein Ero1, thereby regenerating the oxidized form of PDI. The mechanism of reoxidation of Ero1 is not known. (b) In the periplasmic space of bacterial cells, the soluble protein DsbA functions like eukaryotic PDI. Reduced DsbA is reoxidized by DsbB, an inner-membrane protein. Finally, reduced DsbB is reoxidized by transferring electrons to oxidized ubiquinone, a lipid cofactor of the electron-transport chain in the inner membrane (see Chapter 8). [See A. R. Frand et al., 2000, Trends Cell Biol. 10:203.]

Chaperones and Other ER Proteins Facilitate Folding and Assembly of Proteins

Although many reduced, denatured proteins can spontaneously refold into their native state in vitro, such refolding usually requires hours to reach completion. Yet new soluble and membrane proteins produced in the ER generally fold into their proper conformation within minutes after their synthesis. The rapid folding of these newly synthesized proteins in cells depends on the sequential action of several proteins present within the ER lumen.

We have already seen how the chaperone BiP can drive post-translational translocation in yeast by binding fully synthesized polypeptides as they enter the ER (see Figure 16-9). BiP can also bind transiently to nascent chains as they enter the ER during cotranslational translocation. Bound BiP is thought to prevent segments of a nascent chain from mis-folding or forming aggregates, thereby promoting folding of the entire polypeptide into the proper conformation. Protein disulfide isomerase (PDI) also contributes to proper folding, which is stabilized by disulfide bonds in many proteins.

As illustrated in Figure 16-21, two other ER proteins, the homologous lectins (carbohydrate-binding proteins) calnexin and calreticulin, bind selectively to certain A-linked oligosaccharides on growing nascent chains. The ligand for these two lectins, which contains a single glucose residue, is generated by a specific glucosyltransferase in the ER lumen (see Figure 16-18, step 3a). This enzyme acts only on polypeptide chains that are unfolded or misfolded. Binding of calnexin and cal-reticulin to unfolded nascent chains prevents aggregation of adjacent segments of a protein as it is being made on the ER. Thus calnexin and calreticulin, like BiP, help prevent premature, incorrect folding of segments of a newly made protein.

Other important protein-folding catalysts in the ER lumen are peptidyl-prolyl isomerases, a family of enzymes that accelerate the rotation about peptidyl-prolyl bonds in unfolded segments of a polypeptide:

O NH

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