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Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002.

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Biochemistry. 5th edition.

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Section 23.2Protein Turnover Is Tightly Regulated

How can a cell distinguish proteins that are meant for degradation? Ubiq-uitin, a small (8.5-kd) protein present in all eukaryotic cells, is the tag that marks proteins for destruction (Figure 23.2). Ubiquitin is the cellular equivalent of the “black spot” of Robert Louis Stevenson's Treasure Island: the signal for death.

Figure 23.2. Ubiquitin.

Figure 23.2

Ubiquitin. Image mouse.jpg The structure of ubiquitin reveals an extended carboxyl terminus that is activated and linked to other proteins. Lysine residues also are shown, including lysine 48, the major site for linking additional ubiquitin molecules.

23.2.1. Ubiquitin Tags Proteins for Destruction

Ubiquitin is highly conserved in eukaryotes: yeast and human ubiquitin differ at only 3 of 76 residues. The carboxyl-terminal glycine residue of ubiquitin (Ub) becomes covalently attached to the ϵ-amino groups of several lysine residues on a protein destined to be degraded. The energy for the formation of these isopeptide bonds (iso because ϵ- rather than α-amino groups are targeted) comes from ATP hydrolysis.

Image ch23fu2.jpg

Three enzymes participate in the attachment of ubiquitin to each protein (Figure 23.3): ubiquitin-activating enzyme, or E1, ubiquitin-conjugating enzyme, or E2, and ubiquitin-protein ligase, or E3. First, the terminal carboxylate group of ubiquitin becomes linked to a sulfhydryl group of E1 by a thioester bond. This ATP-driven reaction is reminiscent of fatty acid activation (Section 22.4.1). An adenylate is linked to the C-terminal carboxylate of ubiquitin with the release of pyrophosphate, and the ubiquitin is transferred to a sulfhydryl group of a key cysteine residue in E1. Second, activated ubiquitin is shuttled to a sulfhydryl group of E2. Finally, E3 catalyzes the transfer of ubiquitin from E2 to an ϵ-amino group on the target protein.

Figure 23.3. Ubiquitin Conjugation.

Figure 23.3

Ubiquitin Conjugation. The ubiquitin-activating enzyme E1 adenylates ubiquitin (Ub) and transfers the ubiquitin to one of its own cysteine residues. Ubiquitin is then transferred to a cysteine residue in the ubiquitin-conjugating enzyme E2. Finally, the (more...)

The attachment of a single molecule of ubiquitin is only a weak signal for degradation. However, the ubiquitination reaction is processive: chains of ubiquitin can be generated by the linkage of the ϵ-amino group of lysine residue 48 of one ubiquitin molecule to the terminal carboxylate of another. Chains of four or more ubiquitin molecules are particularly effective in signaling degradation (Figure 23.4). The use of chains of ubiquitin molecules may have at least two advantages. First, the ubiquitin molecules interact with one another to form a binding surface distinct from that created by a single ubiquitin molecule. Second, individual ubiquitin molecules can be cleaved off without loss of the degradation signal.

Figure 23.4. Tetra Ubiquitin.

Figure 23.4

Tetra Ubiquitin. Image mouse.jpg Four ubiquitin molecules are linked by isopeptide bonds. This unit is the primary signal for degradation when linked to a target protein.

Although most eukaryotes have only one or a small number of distinct E1 enzymes, all eukaryotes have many distinct E2 and E3 enzymes. Moreover, there appears to be only a single family of evolutionarily related E2 proteins but many distinct families of E3 proteins. Although the E3 component provides most of the substrate specificity for ubiquitination, the multiple combinations of the E2–E3 complex allow for more finely tuned substrate discrimination.

What determines whether a protein becomes ubiquitinated? One signal turns out to be unexpectedly simple. The half-life of a cytosolic protein is determined to a large extent by its amino-terminal residue (see Table 23.1). This dependency is referred to as the N-terminal rule. A yeast protein with methionine at its N terminus typically has a half-life of more than 20 hours, whereas one with arginine at this position has a half-life of about 2 minutes. A highly destabilizing N-terminal residue such as arginine or leucine favors rapid ubiquitination, whereas a stabilizing residue such as methionine or proline does not. E3 enzymes are the readers of N-terminal residues. Other signals thought to identify proteins for degradation include cyclin destruction boxes, which are amino acid sequences that mark cell-cycle proteins for destruction, and proteins rich in proline, glutamic acid, serine, and threonine (PEST sequences).

Image caduceus.jpg Some pathological conditions vividly illustrate the importance of the regulation of protein turnover. For example, human papilloma virus (HPV) encodes a protein that activates a specific E3 enzyme. The enzyme ubiquitinates the tumor suppressor p53 and other proteins that control DNA repair, which are then destroyed. The activation of this E3 enzyme is observed in more than 90% of cervical carcinomas. Thus, the inappropriate marking of key regulatory proteins for destruction can trigger further events, leading to tumor formation.

23.2.2. The Proteasome Digests the Ubiquitin-Tagged Proteins

If ubiquitin is the mark of death, what is the executioner? A large protease complex called the proteasome or the 26S proteasome digests the ubiquitinated proteins. This ATP-driven multisubunit protease spares ubiq-uitin, which is then recycled. The 26S proteasome is a complex of two components: a 20S proteasome, which contains the catalytic activity, and a 19S regulatory subunit.

The 20S complex is constructed from two copies each of 14 subunits and has a mass of 700 kd (Figure 23.5). All 14 subunits are homologous and adopt the same overall structure. The subunits are arranged in four rings of 7 subunits that stack to form a structure resembling a barrel. The components of the two rings at the ends of the barrel are called the α subunits and those of the two central rings the β subunits. The active sites of the protease are located at the N-termini of certain β subunits on the interior of the barrel—specifically, those β chains having an N-terminal threonine or serine residue. The hydroxyl groups of these amino acids are converted into nucleophiles with the assistance of their own amino groups. These nucleophilic groups then attack the carbonyl groups of peptide bonds and form acyl-enzyme intermediates (Section 9.1.2). The structure of the complex sequesters the proteolytic active sites from potential substrates until they are directed into the barrel. Substrates are degraded in a processive manner without the release of degradation intermediates, until the substrate is reduced to peptides ranging in length from seven to nine residues.

Figure 23.5. 20S Proteasome.

Figure 23.5

20S Proteasome. Image mouse.jpg The 20S proteasome comprises 28 homologous subunits (α, red; β, blue), arranged in four rings of 7 subunits each. Some of the β subunits (highlighted in yellow) include protease active sites at the amino termini. (more...)

The 20S proteasome is a sealed barrel. Access to its interior is controlled by a 19S regulatory complex, itself a 700-kd complex made up of 20 subunits. This complex binds to both ends of the 20S proteasome core to form the complete 26S proteasome (Figure 23.6). The 19S subunit binds specifically to polyubiquitin chains. Key components of the 19S complex are six distinct ATPases of the AAA class (ATPase associated with various cellular activities) characterized by a conserved 230 amino acid ATP-binding domain of the P-loop NTPase family. This class of ATPase, found in all kingdoms, is associated with a variety of cell functions including cell-cycle regulation and organelle biogenesis. Although the exact role of the ATPase remains uncertain, ATP hydrolysis may assist the 19S complex to unfold the substrate and induce conformational changes in the 20S proteasome so that the substrate can be passed into the center of the complex. Finally, the 19S subunit also contains an isopeptidase that cleaves off intact ubiquitin molecules. Thus, the ubiquitinization pathway and the proteasome cooperate to degrade unwanted proteins. The ubiquitin is recycled and the peptide products are further degraded by other cellular proteases to yield individual amino acids.

Figure 23.6. 26S Proteasome.

Figure 23.6

26S Proteasome. A 19S cap is attached to each end of the 20S catalytic unit. [From W. Baumeister, J. Walz, F. Zuhl, and E. Seemuller. Cell 92(1998):367. Courtesy of Dr. Wolfgang Baumeister.]

23.2.3. Protein Degradation Can Be Used to Regulate Biological Function

Image caduceus.jpg Table 23.2 lists a number of physiological processes that are con trolled at least in part by protein degradation. This control is exerted by dynamically altering the stability and abundance of regulatory proteins. Consider, for example, control of the inflammatory response. A transcription factor called NF-κB (NF for nuclear factor) initiates the expression of a number of the genes that take part in this response. This important factor is itself activated by the degradation of an inhibitory protein, I-κB. NF-κB is maintained in the cytoplasm in its inactive state by association with I-κB (I for inhibitor). In response to inflammatory signals, I-κβ is phosphorylated at two serine residues, creating an E3 binding site. The binding of E3 leads to the ubiquitination and degradation of I-κB and thereby disrupts the inhibitor's association with NF-κB. The liberated transcription factor migrates to the nucleus to stimulate transcription of the target genes. The NF-κB-I-κB system illustrates the interplay of several key regulatory motifs: receptor-mediated signal transduction, phosphorylation, compartmentalization, controlled and specific degradation, and selective gene expression.

Table 23.2. Processes regulated by protein degradation.

Table 23.2

Processes regulated by protein degradation.

23.2.4. The Ubiquitin Pathway and the Proteasome Have Prokaryotic Counterparts

Image tree.jpg Both the ubiquitin pathway and the proteasome appear to be pres- ent in all eukaryotes. Homologs of the proteasome are found in prokaryotes, although the physiological roles of these homologs have not been well established. The proteasomes of some archaea are quite similar in overall structure to their eukaryotic counterparts and similarly have 28 subunits (Figure 23.7). In the archaeal proteasome, however, all α subunits and all β subunits are identical; in eukaryotes, each α or β subunit is one of seven different isoforms. This specialization provides distinct substrate specificity.

Figure 23.7. Proteasome Evolution.

Figure 23.7

Proteasome Evolution. The archaeal proteasome consists of 14 identical α subunits and 14 identical β subunits. In the eukaryotic proteasome, gene duplication and specialization has led to 7 distinct subunits of each type. The overall architecture (more...)

Ubiquitin, however, has not been found in prokaryotes. Indeed, the high level of sequence similarity between the human and yeast proteins suggests that ubiquitin, in its present form, diverged relatively recently in evolutionary terms. Ubiquitin's molecular ancestors were recently identified in prokaryotes. Remarkably, these proteins take part not in protein modification but in coenzyme biosynthesis (Figure 23.8). The biosynthesis of thiamine (Section 8.6.1) begins with a sulfide ion derived from cysteine. This sulfide is added to the C-terminal carboxylate of the protein ThiS, which had been activated as an acyl adenylate. The activation of ThiS and the addition of sulfide are catalyzed by the enzyme ThiF. Human E1 includes two tandem regions of 160 amino acids that are 28% identical in amino acid sequence with a region of ThiF from E. coli. The evolutionary relationships between these two pathways were cemented by the determination of the three-dimensional structure of ThiS, which revealed a structure very similar to that of ubiquitin, despite being only 14% identical in amino acid sequence (Figure 23.9). Thus, a eukaryotic system for protein modification evolved from a preexisting prokaryotic pathway for coenzyme biosynthesis.

Figure 23.8. Biosynthesis of Thiamine.

Figure 23.8

Biosynthesis of Thiamine. The biosynthesis of thiamine begins with the addition of sulfide to the carboxyl terminus of the protein ThiS. This protein is activated by adenylation and conjugated in a manner analogous to the first steps in the ubiquitin (more...)

Figure 23.9. Structure of This.

Figure 23.9

Structure of This. Image mouse.jpg The determination of the structure of ThiS revealed it to be structurally similar to ubiquitin despite only 14% sequence identity. This observation suggests that a prokaryotic protein such as ThiS evolved into ubiquitin.

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

Copyright © 2002, W. H. Freeman and Company.
Bookshelf ID: NBK22397


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