Catching K33 ubiquitin chains

Approximately 30% of newly generated proteins are turned over with a half-life of less than 10 minutes. Considering that 80% of cellular proteins are degraded via the ubiquitin-proteasome system, it is clear the important role this pathway plays in regulating multiple cellular processes. Moreover, with such diversity in ubiquitin substrates it is no wonder that this pathway has been implicated in numerous diseases, including neurodegenerative processes, viral disease and cancer.

Of the eight possible ubiquitin linkages, K48 linkages are associated with targeting to the proteasome for degradation and K63 is associated with cell signalling roles, such as DNA repair, DNA replication and signal transduction. While K48 and K63 are the most abundant ubiquitin chains within the cell, the others no doubt serve specific purposes as there specific E3 ligases and specific deubiquitinases are expressed to make and break these chains. Some studies have linked all chain types (K6, K11, K27, K29, K33 and K63) to some extent with targeting proteins to the proteasome. Yet deciphering the precise function of these atypical ubiquitin linkages has proven difficult as there have been no tools available to study these chains. There are no good antibodies available and so there has been no way of recognising, labelling or binding them.

Handily Affimer technology is now available as antibody alternatives for both the K6 and K33 di-ubiquitin linkages. They have been tested for cross-reactivity across all the different ubiquitin chains and will bind specifically, allowing researchers to detect and track these chains within the cell.


In the absence of our new Affimer reagents for use in this research field the Kommander group examined K33 ubiquitin linkages by using a selective enzymatic method and exploiting the intrinsic specificity of the ubiquitin system to capture one of its more elusive moieties. In this study they exploited the preference of the HECT E3 ligase AREL1 for assembling K33 chains to produce these chains in vitro, but only 36% of the chains produced using AREL1 are of the K33 type. To remove the other ubiquitin linkages contaminating the mixture, they used chain-specific deubiquitinases to disassemble all the other chains and leaving a K33 enriched sample, where K33 chains comprised over 80% of the ubiquitin chains present.


They characterised the K33-linked ubiquitin chains by crystallising the di-ubiquitin to demonstrate that they adopt an open linear conformation, similar to that of the K63 linkages. The also crystallised K33 chains in complex with the deubiquitinase TRABID, which is specific for K29 and K33 linkages. These structures reveal that TRABID interacts with K33 ubiquitin chains via the N-terminal NZF1 domain, whereby the ?30-aa NZF fold intercalates between and interacts with two ubiquitin molecules and each ubiquitin can bind two NFZ domains. In this manner the K33 ubiquitin chain and TRABID NZF domains form a helical filamentous structure, which could overcome the low affinity of the ubiquitin system by increasing avidity. This system offers the potential whereby an ubiquitin chain that acts as a scaffold could provide several ‘docking stations’ for specific ubiquitin binding domains through its linkage type and thus would allow the recruitment and assembly of multi-protein complexes through those binding domains.

Other additional questions are still to be addressed, such as the role of atypical ubiquitin chains in vivo, the dynamics of chain assembly and disassembly in response to different stimuli (cell-cycle phase, stress responses), and the identification of E3 ligases and DUBs specific for each chain type.

The recent study from the Kommander lab allows a first glimpse into the complex signalling networks of the atypical K33 ubiquitin chains and Affimer binders to these targets will certainly help in future studies addressing these and other questions in this exciting field.