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Cooper GM. The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer Associates; 2000.

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The Cell: A Molecular Approach. 2nd edition.

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Protein Folding and Processing

Translation completes the flow of genetic information within the cell. The sequence of nucleotides in DNA has now been converted to the sequence of amino acids in a polypeptide chain. The synthesis of a polypeptide, however, is not equivalent to the production of a functional protein. To be useful, polypeptides must fold into distinct three-dimensional conformations, and in many cases multiple polypeptide chains must assemble into a functional complex. In addition, many proteins undergo further modifications, including cleavage and the covalent attachment of carbohydrates and lipids, that are critical for the function and correct localization of proteins within the cell.

Chaperones and Protein Folding

The three-dimensional conformations of proteins result from interactions between the side chains of their constituent amino acids, as reviewed in Chapter 2. The classic principle of protein folding is that all the information required for a protein to adopt the correct three-dimensional conformation is provided by its amino acid sequence. This was initially established by Christian Anfinsen’s experiments demonstrating that denatured RNase can spontaneously refold in vitro to its active conformation (see Figure 2.17). Protein folding thus appeared to be a self-assembly process that did not require additional cellular factors. More recent studies, however, have shown that this is not an adequate description of protein folding within the cell. The proper folding of proteins within cells is mediated by the activities of other proteins.

Proteins that facilitate the folding of other proteins are called molecular chaperones. The term “chaperone” was first used by Ron Laskey and his colleagues to describe a protein (nucleoplasmin) that is required for the assembly of nucleosomes from histones and DNA. Nucleoplasmin binds to histones and mediates their assembly into nucleosomes, but nucleoplasmin itself is not incorporated into the final nucleosome structure. Chaperones thus act as catalysts that facilitate assembly without being part of the assembled complex. Subsequent studies have extended the concept to include proteins that mediate a variety of other assembly processes, particularly protein folding.

It is important to note that chaperones do not convey additional information required for the folding of polypeptides into their correct three-dimensional conformations; the folded conformation of a protein is determined solely by its amino acid sequence. Rather, chaperones catalyze protein folding by assisting the self-assembly process. They appear to function by binding to and stabilizing unfolded or partially folded polypeptides that are intermediates along the pathway leading to the final correctly folded state. In the absence of chaperones, unfolded or partially folded polypeptide chains would be unstable within the cell, frequently folding incorrectly or aggregating into insoluble complexes. The binding of chaperones stabilizes these unfolded polypeptides, thereby preventing incorrect folding or aggregation and allowing the polypeptide chain to fold into its correct conformation.

A good example is provided by chaperones that bind to nascent polypeptide chains that are still being translated on ribosomes, thereby preventing incorrect folding or aggregation of the amino-terminal portion of the polypeptide before synthesis of the chain is finished (Figure 7.17). Presumably, this interaction is particularly important for proteins in which the carboxy terminus (the last to be synthesized) is required for correct folding of the amino terminus. In such cases, chaperone binding stabilizes the amino-terminal portion in an unfolded conformation until the rest of the polypeptide chain is synthesized and the completed protein can fold correctly. Chaperones also stabilize unfolded polypeptide chains during their transport into subcellular organelles—for example, during the transfer of proteins into mitochondria from the cytosol (Figure 7.18). Proteins are transported across the mitochondrial membrane in partially unfolded conformations that are stabilized by chaperones in the cytosol. Chaperones within the mitochondrion then facilitate transfer of the polypeptide chain across the membrane and its subsequent folding within the organelle. In addition, chaperones are involved in the assembly of proteins that consist of multiple polypeptide chains, in the assembly of macromolecular structures (e.g., nucleoplasmin), and (as discussed later in this chapter) in the regulation of protein degradation.

Figure 7.17. Action of chaperones during translation.

Figure 7.17

Action of chaperones during translation. Chaperones bind to the amino (N) terminus of the growing polypeptide chain, stabilizing it in an unfolded configuration until synthesis of the polypeptide is completed. The completed protein is then released from (more...)

Figure 7.18. Action of chaperones during protein transport.

Figure 7.18

Action of chaperones during protein transport. A partially unfolded polypeptide is transported from the cytosol to a mitochondrion. Cytosolic chaperones stabilize the unfolded configuration. Mitochondrial chaperones facilitate transport and subsequent (more...)

Many of the proteins now known to function as molecular chaperones (Table 7.2) were initially identified as heat-shock proteins, a group of proteins expressed in cells that have been subjected to elevated temperatures or other forms of environmental stress. The heat-shock proteins (abbreviated Hsp), which are highly conserved in both prokaryotic and eukaryotic cells, are thought to stabilize and facilitate the refolding of proteins that have been partially denatured as a result of exposure to elevated temperature. However, many members of the heat-shock protein family are expressed and have essential cellular functions under normal growth conditions. These proteins serve as molecular chaperones, which are needed for polypeptide folding and transport under normal conditions as well as in cells subjected to environmental stress.

Table 7.2. Molecular Chaperones.

Table 7.2

Molecular Chaperones.

The Hsp70 and Hsp60 families of heat-shock proteins appear to be particularly important in the general pathways of protein folding in both prokaryotic and eukaryotic cells. The proteins of both families function by binding to unfolded regions of polypeptide chains. Members of the Hsp70 family stabilize unfolded polypeptide chains during translation (see, for example, Figure 7.17) as well as during the transport of polypeptides into a variety of subcellular compartments, such as mitochondria and the endoplasmic reticulum. These proteins bind to short segments (seven or eight amino acid residues) of unfolded polypeptides, maintaining the polypeptide chain in an unfolded configuration and preventing aggregation.

Members of the Hsp60 family (also called chaperonins) facilitate the folding of proteins into their native conformations. Each chaperonin consists of 14 subunits of approximately 60 kilodaltons (kd) each, arranged in two stacked rings to form a “double doughnut” structure (Figure 7.19). Unfolded polypeptide chains are shielded from the cytosol by being bound within the central cavity of the chaperonin cylinder. In this isolated environment protein folding can proceed while aggregation of unfolded segments of the polypeptide chain is prevented by their binding to the chaperonin. The binding of unfolded polypeptides to the chaperonin is a reversible reaction that is coupled to the hydrolysis of ATP as a source of energy. ATP hydrolysis thus drives multiple rounds of release and rebinding of unfolded regions of the polypeptide chain to the chaperonin, allowing the polypeptide to fold gradually into the correct conformation.

Figure 7.19. Structure of a chaperonin.

Figure 7.19

Structure of a chaperonin. GroEL, a member of the Hsp60 family, is a porous cylinder composed of two stacked rings. Each ring consists of seven subunits. (Courtesy of Paul B. Sigler, Yale University.)

In some cases, members of the Hsp70 and Hsp60 families have been found to act together in a sequential fashion. For example, Hsp70 and Hsp60 family members act sequentially during the transport of proteins into mitochondria and during the folding of newly synthesized proteins in E. coli (Figure 7.20). First, an Hsp70 chaperone stabilizes nascent polypeptide chains until protein synthesis is completed. The unfolded polypeptide chain is then transferred to an Hsp60 chaperonin, within which protein folding takes place, yielding a protein correctly folded into its functional three-dimensional conformation. Members of the Hsp70 and Hsp60 families are found in the cytosol and in subcellular organelles (e.g., mitochondria) of eukaryotic cells, as well as in bacteria (see Table 7.2), so the sequential action of Hsp70 and Hsp60 appears to represent a general pathway of protein folding. An alternative pathway for the folding of some proteins in the cytosol and endoplasmic reticulum may involve the sequential actions of Hsp70 and Hsp90 family members, although the function of Hsp90 is not yet well understood.

Figure 7.20. Sequential actions of Hsp70 and Hsp60 chaperones.

Figure 7.20

Sequential actions of Hsp70 and Hsp60 chaperones. Chaperones of the Hsp70 family bind to and stabilize unfolded polypeptide chains during translation. The unfolded polypeptide is then transferred to chaperones of the Hsp60 family, within which protein (more...)

Enzymes and Protein Folding

In addition to chaperones, which facilitate protein folding by binding to and stabilizing partially folded intermediates, cells contain at least two types of enzymes that catalyze protein folding by breaking and re-forming covalent bonds. The formation of disulfide bonds between cysteine residues is important in stabilizing the folded structures of many proteins (see Figure 2.16). Protein disulfide isomerase, which was discovered by Christian Anfinsen in 1963, catalyzes the breakage and re-formation of these bonds (Figure 7.21). For proteins that contain multiple cysteine residues, protein disulfide isomerase (PDI) plays an important role by promoting rapid exchanges between paired disulfides, thereby allowing the protein to attain the pattern of disulfide bonds that is compatible with its stably folded conformation. Disulfide bonds are generally restricted to secreted proteins and some membrane proteins because the cytosol contains reducing agents that maintain cysteine residues in their reduced (—SH form), thereby preventing the formation of disulfide (S—S) linkages. In eukaryotic cells, disulfide bonds form in the endoplasmic reticulum, in which an oxidizing environment is maintained. Consistent with the role of disulfide bonds in stabilizing secreted proteins, the activity of PDI in the endoplasmic reticulum is correlated with the level of protein secretion in different types of cells.

Figure 7.21. The action of protein disulfide isomerase.

Figure 7.21

The action of protein disulfide isomerase. Protein disulfide isomerase (PDI) catalyzes the breakage and rejoining of disulfide bonds, resulting in exchanges between paired disulfides in a polypeptide chain. The enzyme forms a disulfide bond with a cysteine (more...)

The second enzyme that plays a role in protein folding catalyzes the isomerization of peptide bonds that involve proline residues (Figure 7.22). Proline is an unusual amino acid in that the equilibrium between the cis and trans conformations of peptide bonds that precede proline residues is only slightly in favor of the trans form. In contrast, peptide bonds between other amino acids are almost always in the trans form. Isomerization between the cis and trans configurations of prolyl peptide bonds, which could otherwise represent a rate-limiting step in protein folding, is catalyzed by the enzyme peptidyl prolyl isomerase. This enzyme is widely distributed in both prokaryotic and eukaryotic cells and can catalyze the refolding of at least some proteins. However, its physiologically important substrates and role within cells have not yet been determined.

Figure 7.22. The action of peptidyl prolyl isomerase.

Figure 7.22

The action of peptidyl prolyl isomerase. Peptidyl prolyl isomerase catalyzes the isomerization of peptide bonds that involve proline between the cis and trans conformations.

Protein Cleavage

Cleavage of the polypeptide chain (proteolysis) is an important step in the maturation of many proteins. A simple example is removal of the initiator methionine from the amino terminus of many polypeptides, which occurs soon after the amino terminus of the growing polypeptide chain emerges from the ribosome. Additional chemical groups, such as acetyl groups or fatty acid chains (discussed shortly), are then frequently added to the amino-terminal residues.

Proteolytic modifications of the amino terminus also play a part in the translocation of many proteins across membranes, including secreted proteins in both bacteria and eukaryotes as well as proteins destined for incorporation into the plasma membrane, lysosomes, mitochondria, and chloroplasts of eukaryotic cells. These proteins are targeted for transport to their destinations by amino-terminal sequences that are removed by proteolytic cleavage as the protein crosses the membrane. For example, amino-terminal signal sequences, usually about 20 amino acids long, target secreted proteins to the plasma membrane of bacteria or to the endoplasmic reticulum of eukaryotic cells while translation is still in progress (Figure 7.23). The signal sequence, which consists predominantly of hydrophobic amino acids, is inserted into the membrane as it emerges from the ribosome. The remainder of the polypeptide chain passes through a channel in the membrane as translation proceeds. The signal sequence is then cleaved by a specific membrane protease (signal peptidase), and the mature protein is released. In eukaryotic cells, the translocation of growing polypeptide chains into the endoplasmic reticulum is the first step in targeting proteins for secretion, incorporation into the plasma membrane, or incorporation into lysosomes. The mechanisms that direct the transport of proteins to these destinations, as well as the role of other targeting sequences in directing the import of proteins into mitochondria and chloroplasts, will be discussed in detail in Chapters 9 and 10.

Figure 7.23. The role of signal sequences in membrane translocation.

Figure 7.23

The role of signal sequences in membrane translocation. Signal se-quences target the translocation of polypeptide chains across the plasma membrane of bacteria or into the endoplasmic reticulum of eukaryotic cells (shown here). The signal sequence, a (more...)

In other important instances of proteolytic processing, active enzymes or hormones form via cleavage of larger precursors. Insulin, which is synthesized as a longer precursor polypeptide, is a good example. Insulin forms by two cleavages. The initial precursor (preproinsulin) contains an amino-terminal signal sequence that targets the polypeptide chain to the endoplasmic reticulum (Figure 7.24). Removal of the signal sequence during transfer to the endoplasmic reticulum yields a second precursor, called proinsulin. This precursor is then converted to insulin, which consists of two chains held together by disulfide bonds, by proteolytic removal of an internal peptide. Other proteins activated by similar cleavage processes include digestive enzymes and the proteins involved in blood clotting.

Figure 7.24. Proteolytic processing of insulin.

Figure 7.24

Proteolytic processing of insulin. The mature insulin molecule consists of two polypeptide chains (A and B) joined by disulfide bonds. It is synthesized as a precursor polypeptide (preproinsulin) containing an aminoterminal signal sequence that is cleaved (more...)

It is interesting to note that the proteins of many animal viruses are derived from the cleavage of larger precursors. One particularly important example of the role of proteolysis in virus replication is provided by HIV. In the replication of HIV, a virus-encoded protease cleaves precursor polypeptides to form the viral structural proteins. Because of its central role in virus replication, the HIV protease (in addition to reverse transcriptase) is an important target for the development of drugs used for treating AIDS. Indeed, such protease inhibitors are now among the most effective agents available for combating this disease.

Glycosylation

Many proteins, particularly in eukaryotic cells, are modified by the addition of carbohydrates, a process called glycosylation. The proteins to which carbohydrate chains have been added (called glycoproteins) are usually secreted or localized to the cell surface, although some nuclear and cytosolic proteins are also glycosylated. The carbohydrate moieties of glycoproteins play important roles in protein folding in the endoplasmic reticulum, in the targeting of proteins for delivery to the appropriate intracellular compartments, and as recognition sites in cell-cell interactions.

Glycoproteins are classified as either N-linked or O-linked, depending on the site of attachment of the carbohydrate side chain (Figure 7.25). In N-linked glycoproteins, the carbohydrate is attached to the nitrogen atom in the side chain of asparagine. In O-linked glycoproteins, the oxygen atom in the side chain of serine or threonine is the site of carbohydrate attachment. The sugars directly attached to these positions are usually either N-acetylglucosamine or N-acetylgalactosamine, respectively.

Figure 7.25. Linkage of carbohydrate side chains to glycoproteins.

Figure 7.25

Linkage of carbohydrate side chains to glycoproteins. The carbohydrate chains of N-linked glycoproteins are attached to asparagine; those of O-linked glycoproteins are attached to either serine (shown) or threonine. The sugars joined to the amino acids (more...)

Most glycoproteins in eukaryotic cells are destined either for secretion or for incorporation into the plasma membrane. These proteins are usually transferred into the endoplasmic reticulum (with the cleavage of a signal sequence) while their translation is still in progress. Glycosylation is also initiated in the endoplasmic reticulum before translation is complete. The first step is the transfer of a common oligosaccharide consisting of 14 sugar residues (2 N-acetylglucosamine, 3 glucose, and 9 mannose) to an asparagine residue of the growing polypeptide chain (Figure 7.26). The oligosaccharide is assembled within the endoplasmic reticulum on a lipid carrier (dolichol phosphate). It is then transferred as an intact unit to an acceptor asparagine (Asn) residue within the sequence Asn-X-Ser or Asn-X-Thr (where X is any amino acid other than proline).

Figure 7.26. Synthesis of N-linked glycoproteins.

Figure 7.26

Synthesis of N-linked glycoproteins. The first step in glycosylation is the addition of an oligosaccharide consisting of 14 sugar residues to a growing polypeptide chain in the endoplasmic reticulum (ER). The oligosaccharide (which consists of two N-acetylglucosamine, (more...)

In further processing, the common N-linked oligosaccharide is modified. Three glucose residues and one mannose are removed while the glycoprotein is in the endoplasmic reticulum. The oligosaccharide is then further modified in the Golgi apparatus, to which glycoproteins are transferred from the endoplasmic reticulum. These modifications (which will be discussed in Chapter 9) include both the removal and addition of carbohydrate residues as the glycoprotein is transported through the compartments of the Golgi (Figure 7.27). The N-linked oligosaccharides of different glycoproteins are processed to different extents, depending on both the enzymes present in different cells and on the accessibility of the oligosaccharide to the enzymes that catalyze its modification. Glycoproteins with inaccessible oligosaccharides do not have new sugars added to them in the Golgi. The relatively simple oligosaccharides of these glycoproteins are called high-mannose oligosaccharides because they contain a high proportion of mannose residues, similar to the common oligosaccharide originally added in the endoplasmic reticulum. In contrast, glycoproteins with accessible oligosaccharides are processed more extensively, resulting in the formation of a variety of complex oligosaccharides.

Figure 7.27. Examples of N-linked oligosaccharides.

Figure 7.27

Examples of N-linked oligosaccharides. Various oligosaccharides form from further modifications of the common 14-sugar unit initially added in the endoplasmic reticulum (see Figure 7.26). In high-mannose oligosaccharides, the glucose residues and some (more...)

O-linked oligosaccharides are also added within the Golgi apparatus. In contrast to the N-linked oligosaccharides, O-linked oligosaccharides are formed by the addition of one sugar at a time and usually consist of only a few residues (Figure 7.28). Many cytoplasmic and nuclear proteins, including a variety of transcription factors, are also modified by the addition of single O-linked N-acetylglucosamine residues, catalyzed by a different enzyme system. However, the roles of carbohydrates in the function of these cytoplasmic and nuclear glycoproteins are not yet understood.

Figure 7.28. Examples of O-linked oligosaccharides.

Figure 7.28

Examples of O-linked oligosaccharides. O-linked oligosaccharides usually consist of only a few carbohydrate residues, which are added one sugar at a time.

Attachment of Lipids

Some proteins in eukaryotic cells are modified by the attachment of lipids to the polypeptide chain. Such modifications frequently target and anchor these proteins to the plasma membrane, with which the hydrophobic lipid is able to interact (see Figure 2.48). Three general types of lipid additions—N-myristoylation, prenylation, and palmitoylation—are common in eukaryotic proteins associated with the cytosolic face of the plasma membrane. A fourth type of modification, the addition of glycolipids, plays an important role in anchoring some cell surface proteins to the extracellular face of the plasma membrane.

In some proteins, a fatty acid is attached to the amino terminus of the growing polypeptide chain during translation. In this process, called N-myristoylation, myristic acid (a 14-carbon fatty acid) is attached to an N-terminal glycine residue (Figure 7.29). The glycine is usually the second amino acid incorporated into the polypeptide chain; the initiator methionine is removed by proteolysis before fatty acid addition. Many proteins that are modified by N-myristoylation are associated with the inner face of the plasma membrane, and the role of the fatty acid in this association has been clearly demonstrated by analysis of mutant proteins in which the N-terminal glycine is changed to an alanine. This substitution prevents myristoylation and blocks the function of the mutant proteins by inhibiting their membrane association.

Figure 7.29. Addition of a fatty acid by N-myristoylation.

Figure 7.29

Addition of a fatty acid by N-myristoylation. The initiating methionine is removed, leaving glycine at the N terminus of the polypeptide chain. Myristic acid (a 14-carbon fatty acid) is then added.

Lipids can also be attached to the side chains of cysteine, serine, and threonine residues. One important example of this type of modification is prenylation, in which specific types of lipids (prenyl groups) are attached to the sulfur atoms in the side chains of cysteine residues located near the C terminus of the polypeptide chain (Figure 7.30). Many plasma membrane–associated proteins involved in the control of cell growth and differentiation are modified in this way, including the Ras oncogene proteins, which are responsible for the uncontrolled growth of many human cancers (see Chapter 15). Prenylation of these proteins proceeds by three steps. First, the prenyl group is added to a cysteine located three amino acids from the carboxy terminus of the polypeptide chain. The prenyl groups added in this reaction are either farnesyl (15 carbons, as shown in Figure 7.30) or geranylgeranyl (20 carbons). The amino acids following the cysteine residue are then removed, leaving cysteine at the carboxy terminus. Finally, a methyl group is added to the carboxyl group of the C-terminal cysteine residue.

Figure 7.30. Prenylation of a C-terminal cysteine residue.

Figure 7.30

Prenylation of a C-terminal cysteine residue. The type of prenylation shown affects Ras proteins and proteins of the nuclear envelope (nuclear lamins). These proteins terminate with a cysteine residue (Cys) followed by two aliphatic amino acids (A) and (more...)

The biological significance of prenylation is indicated by the fact that mutations of the critical cysteine block the membrane association and function of Ras proteins. Because farnesylation is a relatively rare modification of cellular proteins, interest in this reaction has been stimulated by the possibility that inhibitors of the key enzyme (farnesyl transferase) might prove useful as drugs for the treatment of cancers that involve Ras proteins. Such inhibitors of farnesylation have been found to interfere with the growth of cancer cells in experimental models and are undergoing evaluation of their efficacy against human tumors in clinical trials.

In the third type of fatty acid modification, palmitoylation, palmitic acid (a 16-carbon fatty acid) is added to sulfur atoms of the side chains of internal cysteine residues (Figure 7.31). Like N-myristoylation and prenylation, palmitoylation plays an important role in the association of some proteins with the cytosolic face of the plasma membrane.

Figure 7.31. Palmitoylation.

Figure 7.31

Palmitoylation. Palmitate (a 16-carbon fatty acid) is added to the side chain of an internal cysteine residue.

Finally, lipids linked to oligosaccharides (glycolipids) are added to the C-terminal carboxyl groups of some proteins, where they serve as anchors that attach the proteins to the external face of the plasma membrane. Because the glycolipids attached to these proteins contain phosphatidylinositol, they are usually called glycosylphosphatidylinositol, or GPI, anchors (Figure 7.32). The oligosaccharide portions of GPI anchors are attached to the terminal carboxyl group of polypeptide chains. The inositol head group of phosphatidylinositol is in turn attached to the oligosaccharide, so the carbohydrate serves as a bridge between the protein and the fatty acid chains of the phospholipid. The GPI anchors are synthesized and added to proteins as a preassembled unit within the endoplasmic reticulum. Their addition is accompanied by cleavage of a peptide consisting of about 20 amino acids from the C terminus of the polypeptide chain. The modified protein is then transported to the cell surface, where the fatty acid chains of the GPI anchor mediate its attachment to the plasma membrane.

Figure 7.32. Structure of a GPI anchor.

Figure 7.32

Structure of a GPI anchor. The GPI anchor, attached to the C terminus, anchors the protein in the plasma membrane. The anchor is joined to the C-terminal amino acid by an ethanolamine, which is linked to an oligosaccharide that consists of mannose, N (more...)

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By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2000, Geoffrey M Cooper.
Bookshelf ID: NBK9843

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