Genes VII

What determines the stability of proteins? A cell contains many proteases, with varying specificities. We may divide them into three general groups:

• Some proteases are involved in specific processing events to generate mature proteins that are smaller than the precursors. Such proteases are involved in a variety of activities, including cleaving the signal sequence from a secreted protein, and cleaving cytosolic enzymes into their mature forms. The caspase group of proteases are involved a pathway that leads to cell death (see 27.13 A common pathway for apoptosis functions via caspases).
• Lysosomes are membrane-bounded organelles that degrade proteins imported into the cell; we discuss this process in the context of protein transport through membranes in 25 Protein trafficking.
• The proteasome is a large complex that degrades cytosolic proteins. It is involved in both general degradation (the complete conversion of a protein into small fragments) and in certain specific processing events. The major substrates for complete degradation are proteins that have been misfolded Xthis is basically a quality control system Xand certain proteins whose degradation is a regulatory event, for example, to allow progress through the cell cycle.

Degradation of a protein by a proteasome falls into two stages: first the protein is targeted; and then it is proteolysed. Targeting is illustrated in Figure 8.49. A small polypeptide called ubiquitin is connected by a covalent link to the substrate protein that is to be degraded. There are three components of the ubiquitination system. The ubiquitin-activating enzyme, E1, utilizes the cleavage of ATP to link itself via a high energy thiolester bond from a Cys residue to the C-terminal Gly residue of ubiquitin. The ubiquitin is then transferred to the ubiquitin-conjugating enzyme, E2, which in turn transfers the ubiquitin to form an isopeptide bond to the ε NH₂ group of a Lys in the substrate protein. The substrate protein has usually been previously bound to the ubiquitin protein ligase, E3. Ubiquitin is released from a degraded substrate by an isopeptidase (Ciechanover et al., 1980; Chau et al., 1989; for review see Jentsch, 1992; Ciechanover, 1994).

Responsibility for choosing substrate proteins to be ubiquitinated lies with both E2 and E3. In the simple scheme shown in Figure 8.49, E3 selects the substrate. A cell may contain several E3 proteins that use different criteria for selecting substrates. There are also multiple varieties of E2, and they also may play a role in targeting substrate proteins, sometimes independently of E3.

The addition of a single ubiquitin residue to a substrate protein is not sufficient to cause its degradation. Further ubiquitin residues are added to form a polyubiquitin chain, in which each additional ubiquitin is added to the Lys at position 46 of the preceding ubiquitin. The formation of polyubiquitin is a signal for the proteasome to degrade the protein.

The proteasome was originally discovered as a large complex that degrades proteins conjugated to ubiquitin. It exists in two forms. A 20S complex of ~700 kD has protease activity. Additional proteins convert the complex to a 26S form of ~2000 kD; they are regulatory subunits that confer specificity Xfor example, for binding to ubiquitin conjugates. ATP cleavage is required for the conversion from 20S to 26S, and is also required later in the reaction for cleaving peptide bonds, releasing the products, etc. (for review see Voges, Zwickl, and Baumeister, 1999).

The 20S complex takes the form of a hollow cylinder, and the additional components of the 26S complex are attached to the ends of the cylinder, making a dumbbell. Basically, the active sites are contained in the interior of a barrel, and access is obtained through relatively narrow channels, typically allowing only access only to unfolded proteins. This protects normal, mature proteins from adventitious degradation (Eytan et al., 1989; for review see Baumeister et al., 1998).

This general type of structure is common to ATP-dependent proteases. For example, the ClpAP protease in E. coli, which is not related by sequence to the proteasome, has a structure in which the ClpP protease forms two rings of 7 subunits each, with the proteolytic activities contained in a central cavity. ClpA provides the ATPase activity and translocates substrates into the cavity, where they are degraded. It is hexameric (which implies an interesting symmetry mismatch in the ClpAP complex). Degradation is processive; once a substrate has been admitted to the central cavity, the reaction proceeds to its end.

The simplest proteasome is found in the archaea. Figure 8.50 shows the top view of the crystal structure of the 20S assembly. It consists of two types of subunits, organized in the form α₇ Vβ₇ Vβ₇ Vα₇, where each heptamer forms a ring. Figure 8.51 shows the side view of the backbone. The α subunits form the two outer rings (on top and on the bottom), and the β subunits form the two inner rings. The β subunits have the protease activities, and the active sites are located at the N-terminal ends that project into the interior. The opening of ~20Å restricts the entrance for substrates. (A yet simpler structure is found in E. coli, where a protein related to the β subunit, HslV, forms a structure of two six-member rings with a proteolytic core (Lowe et al., 1995).)

The eukaryotic 20S proteasome is more complex, consisting of 7 different α subunits and 7 different β subunits. Figure 8.52 shows that it has the same general structure of α Vβ Vβ Vα rings. The rings in each half of the structure are organized in the opposite rotational sense. A significant structural difference with the archaeal proteasome is that the central hole is occluded, so that there is no obvious entrance from the ends of the cylinder. This probably means that the structure is rearranged at some point to allow entrance from the ends (Groll et al., 1997).

The eukaryotic 26S proteasome is formed when the 19S caps associate with the 20S core, binding to one or both ends, to form an elongated structure of ~45 nm in length. The 19S caps are found only in eukaryotic (not archaeal or bacterial) proteasomes. The caps recognize ubiquitinated proteins, and pass them to the 20S core for proteolysis. The 19S caps contain ~18 subunits, several of which are ATPases; presumably the hydrolysis of ATP provides energy for handling the substrate proteins (for review see Coux et al., 1996).

The hydrolytic mechanism of the proteasome is different from that of other proteases. The active site of a catalytic β-subunit is an N-terminal threonine; the hydroxyl group of the threonine attacks the peptide bond of the substrate. The proteasome contains several protease activities, with different specificities, for example, for cleaving after basic, acidic, or hydrophobic amino acids, allowing it to attack. a variety of types of targets. Proteolytic activities with different substrate specificities may be provided by different β subunits. More than one β subunit may be needed for a particular enzymatic activity. The peptide products typically are octa- and nona-peptides. Proteasomes function processively, that is, a substrate is degraded to completion within the cavity, without any intermediates being released. Basically the central chamber traps proteins until they have been degraded to fragments below a certain size.

Inhibitors of the proteasome block the degradation of most cellular proteins, showing that it is responsible for bulk degradation. In fact, a significant proportion of newly synthesized proteins are immediately degraded by the proteasome (which casts a light on the efficiency of the production of proteins) (Schubert et al., 2000; Reits et al., 2000). It is also responsible for cleaving antigens in cells of the immune system to generate the small peptides that are presented on the surface of the cell to provoke the immune response (see 24.10 T-cell receptors are related to immunoglobulins). The peptide fragments are then transported by TAP (the transporter associated with antigen processing) from teh cytosol into the ER, where they are bound by MHC molecules. Other reactions in which target proteins are completely degraded include the removal of cell cycle regulators; in particular, cyclins are degraded during mitosis (see 27 Cell cycle and growth regulation) and replication control proteins are degraded during the phase of DNA synthesis. In addition to these reactions, the proteasome may undertake specific processing events, for example, cleaving a precursor to a transcription factor to generate the active protein. The means by which these activities are regulated remain to be discovered.

6-19-2000