Ubiquitin C-terminus

The most extensively studied TRIAD is Parkin, shown schematically in Figure 4.5A. The N-terminus contains a region homologous to ubiquitin called the ubiq-uitin domain (UbD), which interacts directly with proteasomes. The C-terminus contains two RING fingers (Rl, R2) separated by a cysteine-rich in-between i ING (IBR) region. This TRIAD motif mediates E3 activity and interacts with molecular chaperones. The last three amino acids of Parkin interact with a PDZ domain and possibly function to anchor Parkin to lipid microdomains. 

Soon after it was shown that ubiquitin is conjugated to proteins, it was determined that this was a reversible process and deubiquitinating enzymes, or DUBs, could remove ubiquitin from ubiquitinated proteins [18, 19]. As the genes for ubiquitin and ubiquitin-like proteins were identified it became clear that all ubiquitin family members were synthesized as proproteins and processed to reveal the C-terminal glycylglycine of the active proteins [20]. Based on this information, DUBs were defined as proteases that cleave at the C-terminus of ubiquitin or ubiquitin-like proteins to reverse conjugation to target proteins and also process the proproteins. 

The central feature that defines all DUBs is that they recognize and act at the C-terminus of the ubiquitin or ubiquitin-like domain. All mature ubiquitin and ubiquitin-like proteins have a C-terminal gly-gly motif and DUB cleavage releases leaving groups attached to the carboxyl group of the C-terminal glycine. With the exception of the JAMM metalloproteases, DUB catalysis starts with the nucleophilic attack of the catalytic cysteine on the carbonyl carbon of the scissile bond to... 

The most promising tools developed for this sort of analysis are active-site-directed irreversible inhibitors of DUBs. These inhibitors are ubiquitin or ubiquitin-like proteins chemically modified at the C-terminus by an electrophilic moiety such as a Michael acceptor or alkyl halide. The modified ubiquitin can be incubated with a purified DUB or a cell lysate containing DUB activity. Ubiquitin vinyl sul-fone (UbVS) is one such irreversible inhibitor because the vinyl sulfone moiety reacts with the active-site cysteine of the DUB, forming a thioether linkage. The covalent adduct is stable and can be detected in a variety of ways. Labeling of DUBs is specific, as only a DUB active-site cysteine will efficiently react with the vinyl sulfone moiety. 

TapCT The C-terminus of the mammalian nuclear RNA export factor NXFl/2 (also known as Tap) contains a sequence region with significant similarity to UBA-like domains. This region is also found in the yeast RNA export factor Mex67. A three-dimensional structure of this domain is available and confirms its similarity to the UBA domain [68]. This UBA-like domain does not appear to bind to ubiquitin but rather to the Phe-Gly repeat motif found in a number of nu-cleoporins. The interaction surface of the UBA-like TapCT domain with a Phe-Gly-containing loop was mapped by an NMR/X-Ray combination technique and shown to be different from the ubiquitin-binding mode the Phe-Gly loop binds on the backside of the UBA-like domain and is in contact with helices a2 and a3 [68]. 

A related adapter family combines the UBA domain with a UBX domain, which is much more distantly related to ubiquitin than the true UbL domains [44, 45]. In these proteins, the UBA domain is frequently found at the N-terminus while the UBX domain forms the C-terminus of the protein an example is the yeast Shpl protein and its mammalian homolog p47. So far, there is limited data on the function of this protein family though they appear to shuttle ubiquitinated proteins to the Cdc48/p97 complex instead of to the proteasome [47, 106]. 

UCHs are cysteine proteases in that the critical residue in the catalytic site is a cysteine. In addition, histidine and aspartate residues are critical for catalytic activity. All UCHs contain these residues even if they do not share a high degree of homology elsewhere in the sequence. For example, the Aplysia UCH (Ap-uch) critical for the induction oflong-term facilitation has only 39% homology to its human counterpart UCH-Lf. Ap-uch and UCH-Ll both contain the catalytic cysteine, histidine, and aspartate residues at similar positions in the molecule. UCHs cleave small peptide chains linked to the C-terminus of ubiquitin. UBPs can cleave the isopeptide bond between ubiquitins in a polyubiquitin chain and the isopeptide bond between the ubiquitin and the substrate. 

The proteasome is a very large complex of at least 50 subunits. It is present in a wide variety of tissues and can constitute up to 1% of soluble protein in a cell. The catalysis occurs within the central core of the molecule and ATP hydrolysis is required to drive the protein into the core. Before the complex can break down proteins, the latter must first have been tagged by complexing with ubiquitin, a peptide of molecular mass 8.5 kDa. This attachment requires three enzymes and the hydrolysis of ATP and it results in a peptide link between the carboxylic group at the C-terminus of ubiquitin and the NH2 of a lysine side-chain in the condemned protein. Several ubiquitin molecules are attached to a single lysine so that a chain of four or more ubiquitin molecules is formed (Figure 8.2). It is. 

H2A.Bbd was discovered as an H2A-like protein encoded by human ESTs [77]. H2A.Bbd is only 42% identical to conventional H2A and is smaller due to a shortened C-terminus that lacks the ubiquitination site that is present in most other H2As (Fig. 6). H2A.Bbd appears to be associated with nucleosomes as indicated by the co-purification of an epitope-tagged H2A.Bbd with nucleosomal fragments in a sucrose gradient run at high ionic strength [77]. 

Orian, A., Gonen, H., Bercovich, B., Fajerman, I., Eytan, E., Israel, A., Mercurio, F., Iwai, K., Schwartz, A.L., and Ciechanover, A. (2000). SCFP-TrCP ubiquitin ligase-mediated processing of NF-kB pi05 requires phosphorylation of its C-terminus by IkB kinase. EMBO J. 19,2580-2591. 

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