Polyubiquitin binding and disassembly by deubiquitinating enzymes.

Ubiquitin (Ub) is a highly conserved protein of 76 amino acids that is covalently linked to target proteins altering their localization, function, or stability 1-3. Proteins can be modified with a large number of different isoforms of ubiquitin and these different ubiquitins are thought to signal different outcomes. The question of how these different forms of ubiquitin are recognized is central to understanding the specificity of various types of ubiquitination 4-6. Ubiquitin acts as a signal by being conjugated to proteins through three sequential steps. In the first step, ubiquitin is activated by the ATP-dependent formation of a thiolester bond between the C-terminus of ubiquitin and the active site cysteine of an ubiquitin activating enzyme or E1. The second step involves the transfer of the ubiquitin molecule from the E1 to the active site cysteine of an ubiquitin-conjugating enzyme or E2. Finally, the ubiquitin is transferred to a lysine residue of the target protein in a reaction catalyzed by an ubiquitin ligase or E3. This last step occurs in a substrate-specific manner and it is highly regulated 7-9. Several rounds of ubiquitination can occur on ubiquitin itself, leading to the formation of a polyubiquitin chain. Any of seven lysines, or the amino terminus, of ubiquitin can be used to polymerize ubiquitin and so there are a huge number of differently linked polyubiquitin signals that can be formed. Chains can be linked by the same lysine on each ubiquitin (K29, K48, K63, etc.) to yield homogeneous chains, or utilize different lysines on some ubiquitins to yield heterogeneous chains. In the latter case, the lysine used can vary from ubiquitin to ubiquitin, or chains can be formed that are branched at a single ubiquitin by linking two ubiquitins to two different lysines at the branch point. It is commonly assumed that different polyubiquitin chains are associated with different cellular fates. Receptors are thought to recognize the different ubiquitin modifications (mono- and polyubiquitin) attached to the target proteins and to mediate the different signaling outcomes 4,10. These receptors have ubiquitin binding domains that interact with ubiquitin or polyubiquitin, and may also have domains that can also interact with the modified target proteins or other macromolecules. Like most posttranslational modifications, ubiquitination is reversible 11 and its removal is carried out by enzymes collectively known as deubiquitinating enzymes (DUBs) 12. DUBs are proteases that have been implicated in a wide variety biological processes 12,13. They are responsible for the removal of ubiquitin or polyubiquitin from target proteins, the processing of ubiquitin precursors, and the disassembly of unanchored polyubiquitin (a polyubiquitin chain not attached to another protein) that is either synthesized de novo, or released by the action of other DUBs. Thus, like the cellular targeting receptors they recognize the different forms of ubiquitin and polyubiquitin. For instance, the tumor suppressor CYLD acts exclusively on K63-linked chains 14, yeast OTU1 prefers long K48-linked chains 15, and USP5cleaves both linkages 16. Nearly 100 DUBs in five different protein families are encoded by the human genome. Several DUBs have been shown to bind or process polyubiquitin or polyubiquitinated substrates in vivo, and many DUBs have been shown to cleave polyubiquitin in vitro. This review will discuss the specificity of ubiquitin and polyubiquitin binding by DUBs. The DUBs discussed will be limited to those where binding and specificity have been directly demonstrated, either through structure determination or direct binding and catalytic studies. It will focus on the current body of knowledge regarding structure of ubiquitin binding domains of DUBs and the mechanisms by which these DUBs recognize and selectively disassemble different polyubiquitin chains. In addition to clarifying the mechanisms of chain recognition by DUBs, the conclusions gleaned from these proteins may well serve as a model for the recognition of these chains by other receptors.