The Toll-like Receptor 4 and Signalling through Ubiquitylation
FIG 15.15 Ubiquitin-binding proteins involved in substrate presentation to the proteasome.
There are numerous proteins that bind ubiquitins and which are involved in the presentation of substrate to the proteasome. They contain ubiquitinassociated (UBA) or ubiquitin-interacting motifs (UIM) (among others). The domain organization of two examples are illustrated. hHR23a is composed of two UBA sites and one ubiquitin-like domain (UBL). It binds ubiquitylated substrate at both UBA sites, and this may be one mechanism by which only poly-ubiquitylated proteins are selected for destruction. With its UBL domain it binds the ubiquitin-interacting motif from S5a, an integral component of the PA700 particle (1oqy,79 1aar70).
same target lysine residues, sumoylation can prevent ubiquitylation and can thus serve to protect proteins from degradation at the proteasome.81
The PIAS family of E3-ligases involved in the conjugation of SUMO represent a variant on RING-type E3 ligases.81 They are able to interact with some E2ubiquitin conjugating enzymes but not the E2-SUMO conjugation enzyme, Ubc9 being preferred (Figure 15.14). Sumoylation of c-Jun, Elk, c-Myb, and the androgen receptors by PIAS suppresses their activity.83 Sumoylation can also act as an activator, TCF-4 and CREB being good examples.84,85
Essay: the proteasome complex
The continuous recycling of cellular proteins requires a coherent process of synthesis and degradation. The numbers are striking. Of the total protein body mass of healthy human adults, 2%, about 50 g, is renewed every day. About 75% of the amino acid nitrogen is reused for de novo protein synthesis, the remaining 25% being eliminated as urea. It follows that about 200 g (wet weight!) of meat, eggs, or milk products are needed to compensate the loss.
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Signal Transduction
Only the nitrogen content is eliminated as urea or uric acid. The keto acids enter the Krebs cycle as pyruvate, which may be used to make fat or metabolized to release energy.
Dairy products, fish, meat, pulses, fruit, vegetables, nuts, all contain protein. Vegetarians have to eat more than carnivores to satisfy their needs. This applies to all animals. Grazers have to eat almost continuously, while meat-eating predators like lions and tigers eat infrequently and have the luxury of free time. Humans didn’t have much free time until they became large-scale meat eaters.
The rate of protein turnover in the liver is much greater, 40% per day. The enormous disparity is due to the difference between proteins that constitute the extracellular mass, with half-lives of days to months, and cellular proteins that exist for only a few hours.
Disposal of proteins occurs by either ubiquitylation and degradation in the proteasome or by degradation of proteins (or whole organelles) by lysosomes, or by autodestruction of cells by caspases (apoptosis). All these are subject to regulation. Many proteases are synthesized as inactive precursors, requiring partial proteolysis that occurs in specialized organelles. Access of substrate to the proteolytic activity may be dependent on post-translational modifications (as is the case of the proteasome which only recognizes ubiquitylated substrates).
The proteasome
Polyubiquitylated proteins, in particular the 48K-type, are recognized by receptors on the proteasome, effectively a large complex of proteases. The proteasome can be regarded as the functional opposite of the ribosome, which it also rivals with respect to size ( 30 nm) and complexity. It is composed of two major subunits, a 20S particle and a regulatory 11S (PA28) or 19S (PA700) particle (proteasome activators) (Figure 15.16). The 20S particle is highly conserved, being present in archaea, eubacteria, and all eukaryotes. The proteasome occurs in different configurations; single 20S, singly and doubly PA28-capped, singly PA700-capped, PA700 and PA28 capped (hybrid), and doubly PA700-capped. All these species are highly dynamic, with
rapid recruitment and exchange of regulatory caps. It is suggested that the association of different caps signifies different stages of the degradation process.86
20S particle
The 20S particle, of which numerous copies are present in both cytosol and nucleus, has a cylindrical form (15 11 nm). It is composed of 28 subunits; 2 copies each of subunits 1–7 and 1–7 (Figure 15.16). The proteolytic activity is situated on the inside of the cylinder and is associated with three different subunits, each targeting different amino acids. The caspase-like 1 subunit cuts after acidic residues, the trypsin-like 2 cuts after basic residues and the chymotrypsin-like 5 cuts after hydrophobic residues. Together they assure
the degradation of a large range of proteins into fragments of 3–25 residues.88
(Figure 15.17).
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The Toll-like Receptor 4 and Signalling through Ubiquitylation
FIG 15.16 Proteasome structure.
(a) The proteasome is composed to two functional entities, the actual protease complex (20S particle) and the activating subunits, the caps. Of these two variants exist, PA700 (19S particle) and PA28
(11S particle). Different combinations of 20S and the caps exist. They play
a role in binding, unfolding, and transport of the substrate. (b–d) The 20S particle is composed of - and-subunits, the -subunits forming the antechamber, the -subunits the catalytic chamber. (e) Not all -
subunits are functional proteases; this activity is restricted to 1, 2 and 5. Image in (a), courtesy of B. Baumeister, Martinsried, Germany. Image in (e), courtesy of K. Hendil, Copenhagen, Denmark (1ryp87).
FIG 15.17 Proteasome chain degradation.
Within the catalytic chamber, the unfolded substrate slides along the active domains of the proteases. 1 cuts after acidic residues, 2 after basic residues, and 5 after large hydrophobic residues. The polypeptide chain is cut up into small chunks
of 3–25 amino acids (1fnt89).
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