EMBO reports paper uses unique ubiquitin Affimer to explore degradation substrates

A new paper published this week, from Mainz University, in EMBO reports examines the Valosin-containing protein (VCP)-specific ubiquitin-modified proteome and the substrate spectrum of this enzyme. Using Avacta’s unique Affimer specific to the K6 ubiquitin linkage the researchers were able to show that VCP acts on K6 ubiquitinated proteins within human cells, and to pull-down this range of substrates for identification via mass spectrometry.

VCP is an evolutionarily conserved ATPase that acts to structurally remodel ubiquitinated proteins prior to the degradation by the proteasome. It plays an essential role in extracting misfolded proteins from the ER as part of ERAD and has been suggested to work on protein remodelling of nuclear and outer mitochondrial membrane proteins unrelated to protein degradation. While inhibition of the ubiquitin-proteasome system has long been used as a treatment strategy for multiple myeloma, more recently the specific inhibition of VCP has been explored as a potential therapeutic target, with oral inhibitors currently in clinical trials for refractory multiple myeloma and solid tumours. However, the full scope and nature of the substrates of this enzyme have not been explored within human cells.

To investigate the nature of the ubiquitinated protein substrates of VCP, Professor Petra Beli’s team examined the specific ubiquitin linkages that showed an increase in abundance following inhibition of VCP. Surprisingly, in addition to the previously documented K48 and K11/K48 branched chains with which VCP interacts, a significant increase was seen in K6 ubiquitin linkages following VCP inhibition. This indicates that VCP selectively targets substrates ubiquitinated with K6 linked chains.

Using our K6 di-ubiquitin Affimer to pull-down ubiquitinated proteins from VCP inhibited lysates the researchers explored the range of K6-ubiquitinated VCP substrates. The exploration of non-canonical ubiquitin linkages has been stymied by the lack of available specific antibodies or affinity reagents in this area, with which to identify, bind and label them. Our Affimer binder to K6 ubiquitin linkages is the only available affinity reagent specific to this linkage, and within this study was successfully used to pull-down 83 protein substrates of VCP that are ubiquitinated with K6-linkages.

The K6-ubiquitinated protein substrates were identified via quantitative mass spectrometry and ranged in function, including receptor endocytosis, cell cycle, the ERAD pathway and proteasomal degradation, implicating a functional role of K6-linked ubiquitination and VCP in these cellular processes.

HUWE1, the ubiquitin E3 ligase, was the most prevalent ubiquitin ligase in the K6 pull-downs. Subsequent knockdown of this enzyme revealed a decrease in both K6- and K48-linkages, demonstrating that this ligase is able to assemble K6-ubiquitin chains and may be responsible for generating mixed K6/ K48 chains in vivo.

Further analysis of the proteins regulated by VCP inhibition showed a high abundance of ?-helices in their secondary structures, with less low complexity, disordered regions in their sequences compared to non-substrates or proteasomal substrates. VCP inhibition also resulted in the ubiquitination of a number of proteins that were not identified under conditions of proteasomal inhibition, such as many cell cycle regulators, indicating that VCP confers both proteasome-dependent and independent functions This was supported by knockdown of VCP resulting in cell cycle progression defects. Additionally, validation of the interaction between  VCP and the ubiquitinated transcription factor c-myc was suggestive of the control of c-myc turnover by VCP.

These exciting findings by Professor Beli’s group using Affimer reagents open the door to the investigation of atypical ubiquitin chains both in protein degradation and wider cellular roles, which has proven previously impossible in this way, with potential impact for drug discovery within this developing field.