The Structure and Functions of Ubiquitin as a component of the Ubiquitin-Proteasome System (UPS), and the Role of the UPS in Alzheimer's Disease. Ubiquitin is a small protein that is present in almost all tissues of all eukaryotic organisms, hence acquiring the name 'ubiquitin' (from the Latin meaning 'everywhere'). It plays a particularly important role in the targeting and labelling of unwanted proteins for destruction within the cell, via the ubiquitin-proteasome system (UPS). As well as this, it is involved in a number of other cellular processes. Such a diverse range of roles is enabled through the ability of ubiquitin to attach to other proteins. In fact the functions of ubiquitin are so crucial to the cell that it is known to be one of the most highly conserved proteins across all eukaryotes.
The Discovery of Ubiquitin as a component of ATP-dependent Proteolysis
Ubiquitin was first described in 1975, as an 8.5kDa protein expressed in the cytosol and nucleus of eukaryotic cells. However, it was not until the early 1980s that the principle function of ubiquitin was determined, through the groundbreaking studies of Avram Hershko, Aaron Ciechanover and Irwin Rose - work for which they were rewarded with the Nobel Prize in Chemistry in 2004. Up until this point, other studies had shown that protein degradation within the cell did require energy, though the mechanism through which this was achieved and the components involved were unknown. Hershko and Ciechanover published a paper in 1978 using cell extracts from reticulocytes to study ATP-dependent proteolysis of denatured globin proteins . They found that the extract could be divided into two separate fractions which were inactive on their own, but when recombined the ATP-dependent proteolytic activity was restored. They reported that Fraction I contained an active heat-stable polypeptide with a molecular weight of approximately 9kDa, which they called APF-1 (active principle in fraction 1). APF-1 protein was later found to be ubiquitin.
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However, the actual function of APF-1/ubiquitin was still unknown until Hershko, Ciechanover and Rose produced two breakthrough reports in 1980. In the first paper, they discovered that APF-1 could bind covalently to various proteins in the extract by utilising ATP . It was determined that this stable covalent linkage of APF-1 to proteins was a part of the mechanism by which proteins were broken down. The second paper then showed that the chemical bond found previously was in fact an amide bond between APF-1 and the Îµ-NH2 group of lysine residues in proteins, and that this conjugation appeared to be enzyme-catalysed . In addition, they found that multiple molecules of APF-1 could bind to the same target protein - the process now known as polyubiquitination, which is vital in the triggering signal causing protein degradation. The discoveries the researchers made were revolutionary in the understanding of proteolysis, as it then opened the field for further research.
The Structure and Function of Ubiquitin
The primary structure of the protein was in fact identified before it was known to have a role in ATP-dependent proteolysis. It had been isolated and sequenced from a variety of sources, but one study in particular in 1975 showed how the primary structure of human ubiquitin was identical to that of bovine ubiquitin . Other reports had also shown this to be the case with other eukaryotic species. Through these studies it can be seen how highly ubiquitin is conserved across all eukaryotes, suggesting its functional importance for the survival of these organisms. Every residue within the polypeptide chain has an essential role in forming the overall ubiquitin structure that allows it to carry out its key function in protein degradation. For this reason, the genetic sequence is remarkably preserved throughout eukaryotes, without almost any modification.
Figure 1 - The amino acid sequence of ubiquitin. Figure obtained from Vijay-Kumar et al., 1985 .
Ubiquitin consists of a single 8565Da polypeptide chain of 76 amino acids. It was at first believed to be only 74 amino acids long, and that arginine-74 was the COOH-terminal residue . However, it was subsequently found that ubiquitin has an additional two glycine residues at the C-terminal, so the polypeptide chain ends Arg-Gly-Gly. This is the physiologically active form of ubiquitin, whereas the ubiquitin lacking the Gly-Gly terminal was likely to have been an 'in vitro proteolytic artefact' . UBQ cartoons - structure.bmp
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Figure 2 - Rasmol image showing the secondary structure of ubiquitin. Image was self-generated using 1UBQ.The actual three-dimensional conformation of ubiquitin was determined through two studies carried out by Vijay-Kumar et al. in the 1980s [5 & 6]. It was found that the secondary structure of ubiquitin is fairly basic. The single polypeptide chain consists of a five stranded mixed Î²-sheet, a 3.5 turn Î±-helix and a short piece of a 310 helix. This can be seen in Figure 2, where the Î²-strands are coloured yellow and the helices are coloured red. The core of the protein is organised in a Î²(2)-Î±-Î²(2) arrangement known as a Î²-grasp fold . This is simply the presence of two beta strands either side of an Î±-helix within the chain, in which the strands bend around the helix to form a compact structure. The Î²-sheet within ubiquitin demonstrates the characteristic left-handed twist. Two of the inner strands of the Î²-sheet, made up of residues 1-7 and 64-72 are parallel, whilst the other three strands (composed of residues 10-17, 40-45 and 48-50) run in antiparallel fashion. The Î±-helix involves the residues 23-34 forming 3.5 turns, whereas the 310 helix is very short and only includes 4 residues, 56-59. In addition, there are seven reverse turns also present within the molecule. These reverse turns are responsible for ubiquitin's extremely compact and globular structure. For the molecule to be maintained in this compact conformation, it has to be strongly stabilised by the presence of many hydrogen bonds. As well as the expected hydrogen bonding occurring in the Î±-helix and Î²-sheet, there are a number of unusual hydrogen bonds involving the two helices and two of the reverse turns . Vijay-Kumar et al. found that the first two residues of the Î±-helix and the 310 helix form hydrogen bonds with the second and fourth residues of separate type I reverse turns. Such a high number of hydrogen bonds are accountable for the preservation of ubiquitin in a tight compact structure. In relation to function, this stops ubiquitin unfolding whilst the protein it is attached to is being unfolded or modified in some way. Vijay-Kumar et al. state that 'approximately 87% of the polypeptide chain is involved in hydrogen-bonded secondary structure' .
It is noticeable that the COOH-terminal of ubiquitin is not in keeping with the rest of the molecule. It protrudes from the compact structure, which can clearly be seen in Figures 2 and 3. It does not form any hydrogen bonds or hydrophobic interactions with the rest of the molecule, so as a result is given substantial freedom of motion . This is extremely important in allowing ubiquitin to form conjugates with other proteins. Ubiquitin functions by forming isopeptide bonds between the carboxyl group of the terminal Glycine-76, and the Îµ-amino groups of lysine residues in other proteins. Therefore, the C-terminal of ubiquitin must be available for enzymes that catalyse the formation of this covalent interaction. In particular, enzymes E1 and E2 must be able to easily access it, as Gly-76 forms thioester bonds with cysteine residues in the active sites of these enzymes, which in turn transfers ubiquitin to the target protein. A study by Di Stefano and Wand (1987) showed that in solution, the C-terminal of ubiquitin can rotate freely, demonstrating its conformational flexibility .ubiquitin backbone comparison2.bmp
Figure 3 - Comparison of the Î±-carbon backbone of ubiquitin between a stereo drawing obtained from Vijay-Kumar et al. 1985 (left) , and a self-generated image using Rasmol (right) in which the final 4 residues (Leu-Arg-Gly-Gly) are coloured red.
Another important point to note regarding the physical structure of ubiquitin is the position of the N-terminus. Met-1 and the following six residues together make up one of the strands of the Î²-sheet. The two adjacent strands are responsible for tightly constraining the first seven residues in the molecule. The side chain of Met-1 is also hydrogen bonded to the backbone of Lys-63 . Consequently, the N-terminus is buried well within the interior of the molecule (which can be seen in Figure 3), making it inaccessible for enzymes (in contrast to the C-terminus). It is believed that this is to prevent degradation of ubiquitin as it enters the ubiquitin-proteasome pathway, in which its attached protein is degraded .
Ubiquitin also has an extremely stable structure. The molecule is resistant to digestion by trypsin, which is surprising considering that it contains four arginine and seven lysine residues. The protein also demonstrates high chemical and thermal stability. Lenkinski et al. reported that ubiquitin remained stable over a range of pH values, and showed no apparent denaturation between temperatures 23oC to 80oC . It is likely that ubiquitin's prominent hydrophobic core (formed from hydrophobic residues of the Î±-helix and Î²-sheet) contributes to this stability, as well as the high proportion of hydrogen bonding in the structure.
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Functionally, the most important of all the residues making up ubiquitin are the seven lysine residues. At least four of which are exposed on the surface of the molecule (Lys-63, Lys-48, Lys-29 and Lys-11), and can therefore act as potential binding sites for ubiquitin-ubiquitin conjugation to form polyubiquitin . The lysine residue of one ubiquitin molecule bonds to the Gly-76 residue of the next. Chains formed through different lysine linkages direct ubiquitin to perform different functions. However it is Lys-48 linked polyubiquitin in particular, that acts as the specific signal for degradation of the attached protein by the 26S proteasome (the protein complex that degrades the ubiquitin-tagged proteins). Studies have shown that at least four ubiquitin molecules linked in the chain is the minimum signal for effective targeting at the proteasome . The three-dimensional structure of Lys-48 linked polyubiquitin is much defined in exposing three key adjacent hydrophobic residues - Leu-8, Ile-44 and Val-70 on the surface of each ubiquitin molecule. These three residues together form a 'hydrophobic patch' that is functionally significant in enhancing proteolytic targeting. The repeating patches of the chain interact with a specific subunit (S5a) of the regulatory complex of the 26S proteasome , hence allowing binding of polyubiquitin to the proteasome resulting in the breakdown of the ubiquitin-tagged protein.
The Role of the Ubiquitin-Proteasome System in Alzheimer's Disease
Alzheimer's disease is a terminal neurodegenerative disorder that is characterised by memory loss and a decline in other cognitive skills, resulting in severe dementia. It affects a large proportion of elderly people, especially those over the age of 65. Although the definite causes of non-familial Alzheimer's disease (AD) is still currently not well understood, recent evidence suggests that aberrations in the Ubiquitin-Proteasome System (UPS) function is directly involved in AD pathogenesis . As seen previously, the UPS is important in protein quality control, tagging and then degrading misfolded and abnormal proteins. In AD however, there is the accumulation of abnormal ubiquitinated proteins in the brain, clearly implying the involvement of the UPS . Three proteins in particular have been found to accumulate in the AD brain: amyloid peptide, tau and UBB+1, and all affect the proteasomal pathway in some way.
The protein UBB+1 is perhaps the most relevant when considering the actual role of ubiquitin in AD pathogenesis. UBB+1 is a mutant of the ubiquitin protein, arising from a molecular misreading of the ubiquitin-B gene (one of the genes that code for ubiquitin) .