If proteasomes are to rapidly degrade only those proteins that are either defective or scheduled to be removed, they must be able to distinguish between those proteins that need to be degraded and those that don’t. Cells mark proteins that should be degraded by covalently attaching to them a linear chain of multiple copies of a 76-residue polypeptide called ubiquitin (Ub) that is highly conserved from yeast to humans. This “polyubiquitin tail” serves as a cellular “kiss of death,” marking the protein for destruction in the proteasome. The ubiquitinylation process (Figure 3-31b, steps 1–3) involves three distinct steps:

Generally, following attachment of four or more ubiquitins in a polyubiquitin chain, the 19S regulatory cap of the 26S proteasome (sometimes with the help of accessory proteins) recognizes the polyubiquitin-labeled protein using its Ub receptors (see Figure 3-31a), uses ATPases to unfold it, and transports it into the proteasome core for degradation. As a polyubiquitinylated substrate is unfolded and passed into the core of the proteasome, enzymes called deubiquitinases (Dubs) hydrolyze the bonds between the individual ubiquitins and between the targeted protein and ubiquitin, recycling the ubiquitins for additional rounds of protein modification (see Figure 3-31b). Analysis of the human genome sequence indicates the presence of about 90 distinct Dubs, about 80 percent of which use cysteine in a catalytic triad similar to that in the serine proteases described earlier (the sulfhydryl in the cysteine side chain is used in place of the hydroxyl in the side chain of the serine). In some Dubs, zinc is a key participant in the catalytic reactions.

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Specificity of Degradation Targeting of specific proteins for proteasomal degradation is primarily achieved through the substrate specificity of E3 ligases (see Figure 3-31b, step 3). As a testament to their importance, there are an estimated 600 or more ubiquitin ligase genes in the human genome. The many E3 ligases in mammalian cells ensure that the wide variety of proteins to be polyubiquitinylated can be modified when necessary. Some E3 ligases are associated with chaperones that recognize unfolded or misfolded proteins; for example, the E3 ligase CHIP is a co-chaperone for Hsp70. These and other proteins (co-chaperones, escort factors, adapters) can mediate E3 ligase–catalyzed polyubiquitinylation of dysfunctional proteins that cannot be readily refolded properly and, consequently, mediate their delivery to proteasomes for degradation. In such cases, the chaperone-ubiquitinylation-proteasome system works in concert for protein quality control.

In addition to quality control, the ubiquitin-proteasome system can be used to regulate the activity of important cellular proteins. An example is the regulated degradation of proteins called cyclins, which control the cell cycle (see Chapter 19). Cyclins contain the internal sequence Arg-X-X-Leu-Gly-X-Ile-Gly-Asp/Asn (where X can be any amino acid), which is recognized by specific ubiquitinylating enzyme complexes. At a specific time in the cell cycle, each cyclin is phosphorylated by a cyclin kinase. This phosphorylation is thought to cause a conformational change that exposes the recognition sequence to the ubiquitinylating enzymes, leading to polyubiquitinylation and proteasomal degradation.

Other Functions of Ubiquitin and Ubiquitin-Related Molecules There are several close relatives of ubiquitin that employ similar E1-, E2-, and E3-dependent mechanisms of activation and transfer to acceptor substrates. These ubiquitin-like modifiers control processes as diverse as nuclear import, regulated by the ubiquitin-like modifier Sumo, and autophagy, regulated by the ubiquitin-like modifier Atg8/LC3 (see Chapter 14). Furthermore, the attachment of ubiquitin to a target protein can be used for purposes other than to mark the protein for degradation, as we will see later in this section, and some of these functions involve polyubiquitin linkages other than those via Lys-48.

Like ubiquitinylation, deubiquitinylation is involved in processes other than proteasome-mediated protein degradation. Large-scale, mass-spectrometry-based “proteomic” methods described later in this chapter, together with sophisticated computational approaches, have suggested that Dubs, which are often bound in multiprotein complexes, are involved in an extraordinarily wide range of cell processes. These processes vary from cell division and cell cycle control (see Chapter 19) to membrane trafficking (see Chapter 14) to cell signaling pathways (see Chapters 15 and 16).